Advances in Tissue Banking, Vol. 7

Rduances in Tissue Banking Vol.7 Editor G l y n O P h i l l i p s The Chinese saying on the cover has been used as a...

Author: Glyn O. Philips

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Rduances in

Tissue Banking Vol.7

Editor G l y n O P h i l l i p s

The Chinese saying on the cover has been used as a motivation for the International Atomic Energy Agency radiation and tissue banking programme.

Rduances in


SERIES IN ADVANCES IN TISSUE BANKING Editor-in-Chief: G. 0. Phillips

Published Vol. 1

Advances in Tissue Banking edited by G. O. Phillips et al.

Vol. 2


Vol. 3


Vol. 4


Vol. 5

The Scientific Basis of Tissue Transplantation edited by A. Nather

Vol. 6

Advances in Tissue Banking edited by G. O. Phillips

Vol. 7

Advances in Tissue Banking edited by G. O. Phillips

Hduances in


Editor-in-Chief

Glyn O Phillips Research Transfer Ltd, Cardiff, Wales, UK

This is a special volume associated with the World Congress of Tissue Banking held in Boston, USA, to which all international Tissue Banking Associations contributed. The International Atomic Energy Agency provided from their extensive tissue banking programme. Thanks, in particular, are due to Dr Sam Doppelt and Jorge Morales for their support. Regional Editors

A Nather

(Asia Pacific) National University Hospital Tissue Bank, Singapore

D

M S t r o n g (North America) Puget Sound Blood Center, USA

R von Versen

(Europe) German Institute for Cell and Tissue Banking, Germany

YJ? World Scientific NEW JERSEY

• LONDON

• SINGAPORE

• SHANGHAI

• HONGKONG

• TAIPEI • C H E N N A I

Published by World Scientific Publishing Co. Pte. Ltd. 5 Toh Tuck Link, Singapore 596224 USA office: Suite 202, 1060 Main Street, River Edge, NJ 07661 UK office: 57 Shelton Street, Covent Garden, London WC2H 9HE

British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

ADVANCES IN TISSUE BANKING (Vol. 7) Copyright © 2004 by World Scientific Publishing Co. Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the Publisher.

For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher.

ISBN 981-238-723-4

Printed in Singapore by World Scientific Printers (S) Pte Ltd

ADVANCES IN TISSUE BANKING

International Advisory Board H. Burchardt, LISA A. Gross, Canada M. Itoman, Japan J. Kearney, UK J. Komender, Poland B. Loty, France P. Mericka, Czech Republic D.A.F. Morgan, Australia D. Pegg, UK M. Salai, Israel W.W. Tomford, USA Y. Vajaradul, Thailand H. Winkler, Austria N. Yusof, Malaysia N. Triantafyllou, Greece R. Capanna, Italy W.W. Boeckx, Belgium C.J. Yim, Korea

V

LIST OF CONTRIBUTORS

JORGE MORALES IAEA Department of Technical Cooperation Vienna, Austria GLYN O. PHILLIPS Phillips Hydrocolloids Research Ltd. 2 Plymouth Drive Radyr, Cardiff CF15 8BL, UK TED EASTLUND Division of Transfusion Medicine Department of Laboratory Medicine and Pathology University of Minnesota Medical School Minneapolis, Minnesota 55455, USA D. MICHAEL STRONG Puget Sound Blood Center/Northwest Tissue Centre 921 Terry Ave, Seattle, Washington 98104, USA JEROEN VAN BAARE, STEPHAN VEHMEIJER and ROLF BLOEM Netherlands Bone bank Foundation, Portgebouw Noord, Rijrisburgerweg 10 2333 AA Leiden, The Netherlands OCTAVIO V MARTINEZ University of Miami Tissue Bank Department of Orthopaedics (R-12) University of Miami, PO Box 016960 Miami, Florida, US 33101 Vll

vm

List of Contributors

AXEL PRUSS Institute for Transfusion Medicine, Tissue Bank University Hospital Charite Schumannstr. 20/21, D-10117, Berlin MOUJAHED KAO and GEORG PAULI Robert Koch-Institut, Retrovirology Norduter 20, D-13353, Berlin, Germany MARTELL WINTERS Bioburden Section Leader, Nelson Laboratories, Inc. 6280 South Redwood Road Salt Lake City, UT 82123 Y. YU, J.B. CHEN, J.-L. YANG and W.R. WALSH Orthopaedic Research Laboratories Prince of Wales Hospital University of New South Wales Sydney 2031 NSW, Australia R. VERHEUL, N. JOHNSON and D.A.F. MORGAN Queensland Bone Bank Princess Alexandra Hospital Health Service District Wooloongabba 4102 QLD, Australia LARS FROMMELT Institut fur Infektiologie klinische Mikrobiologie und Krankenhaushygiene ENDO-KLINIK Holstenstr. 2, D-22607 Hamburg LUTZ GURTLER Friedrich Loeffler Institut fur Medizinische Mikrobiologie Ernst Moritz Arndt Universitat, Matin-Luther-Strasse D-17487 Greifswald


THOMAS VON GARREL Klinik fur Unfall-, Wiederherstellungs- und Handchirurgie Philipps Universitat, Baldingerstrasse, D-35043 Marburg PAUL L. ROMAIN Department of Rheumatology The Cambridge Hospital 1493 Cambridge Street, Cambridge, MA 02139 JEROEN VAN BAARE Netherlands Bone bank Foundation Portgebouw Noord, Rijnsburgerweg 10 2333 AA Leiden, The Netherlands GER KROPMAN Euro Skin bank, Beverwijk, The Netherlands MARCO ANTONIO GARCES MORALES and CESER ALEJANDRO REYNAGA LUNA Plastic and Burns Department Hospital Nacional Arzobispo Loayza Av. Alfonso Ugarte s / n Lima, Peru HENRY J. MANKIN Orthopaedic Oncology Service Massachusetts General Hospital Harvard Medical School, Boston, MA 02114 WONG YONG SHON, CHANG YONG HUR and SOONG HYUN JUNG Department of Orthopaedic Surgery Guro Hospital, Korea University #80, Guro-Dong, Guro-Ku, Seoul, Korea 152-703

IX

X


PETR VISNA Traumatological Hospital Ponavka 6, 602 00 Brno, Czech Republic JIRI ADLER Tissue Bank, University Hospital Brno Czech Republic L. PASA and R. HART Traumatological Hospital Brno Czech Republic J. FOLVARSKY University Hospital, Hradec Kralove Czech Republic V.I. SAVELIEV, LA. KUZNETSOV, A.V. KALININ, A.A. BULATOV and LA. SOLODOV Russian Research Institute of Traumatology and Orthopaedics named after R.R. Vreden Baikov Str. 8, 195427 St. Petersburg, Russia A.V. KALININ, V.I. SAVELIEV and A.A. BULATOV Russian Research Institute of Traumatology and Orthopaedics, named after R.R. Vreden Baikov Str. 8, 195427 St. Petersburg, Russia LUCA DAINESE, GIANLUCA POLVANI, ANNA GUARINO and PAOLO BIGLIOLI Department of Cardiac Surgery University of Milan-Italian Homograft Bank (BIO) Centro Cardiologico Monzino, IRCCS Via Parea 4, 20138 Milan, Italy


MARILENA FORMATO Department of Physiological, Biochemical and Cellular Sciences, University of Sassari Via Muroni 25, 07100 Sassari, Italy HAN-KI PARK, YOUNG-HWAN PARK, SANG-HYUN LIM, JONG-HOON KIM, and BUM-KOO CHO Yonsei Cardiovascular Research Institute Cardiovascular Hospital Yonsei University College of Medicine 134 Shincheondong, Seodaemunku, Seoul, Korea SANG-HO CHO Department of Pathology Yonsei University, College of Medicine 134 Shincheondong, Seodaemunku, Seoul, Korea JONG-CHUL PARK and DONG-WOOK HAN Department of Medical Engineering Yonsei University, College of Medicine 134 Shincheondong, Seodaemunku, Seoul, Korea CHEE-SOON YOON Department of Thoracic and Cardiovascular Surgery College of Medicine, Konyang University 685 Gasuwondong, Seogu, Daejun, Korea SHI-HO KIM Department of Thoracic and Cardiovascular Surgery College of Medicine, Donga University 3-1 Dongdaesindong, Seogu, Pusan, Korea SAM-YOON LEE Department of Thoracic and Cardiovascular Surgery College of Medicine, Wonkwang University 344-2 Shinyongdong, Iksanshi, Junrabukdo, Korea

XI

Xll


DORIS A. TAYLOR Center for Cardiovascular Repair University of Minnesota BSBE 7-105 312 Church Street 5E Minneapolis, MN 55455 SITARAM EMANI, MATTHEW ELLIS and RICHARD B. THOMPSON Departments of Medicine and Surgery Duke University Medical Center Box 3345, DUMC Durham, NC 27710 DAGMAR HAVRANOVA, JIRI ADLER, JANA KOMARKOVA, ANNA TEJKALOVA and EVA HLAVACKOVA Tissue Bank, University Hospital Brno Jihlavska 20, 625 00 Brno, Czech Republic EVA VLKOVA, HANA HRUBA and MONIKA HORACKOVA Ophthalmology Department University Hospital Brno Jihlavska 20, 625 00 Brno, Czech Republic MAHMOOD FARAZDAGHI Tissue Banks International, 815 Park Avenue, Baltimore, Maryland 21201, USA SONJA GRUNEWALD, UWE PAASCH and HANS-JUERGEN GLANDER Department of Dermatology/Division of Andrology University of Leipzig, Stephanstrasse 11 D-04103 Leipzig, Germany


CORNELIA THIEME, UWE PAASCH and HANS-JUERGEN GLANDER Department of Andrology, University of Leipzig Stephanstrasse 11 04103 Leipzig, Germany YOUNG-HWAN PARK, DONG-WOOK HAN and JONGCHUL PARK Department of Thoracic and Cardiovascular Surgery Yonsei Cardiovascular Research Institute Cardiovascular Hospital Yonsei University College of Medicine 134 Shincheondong, Seodaemunku, Seoul, Korea SHI-HO KIM Department of Thoracic and Cardiovascular Surgery, Donga University College of Medicine 3-1 Dongdaesindong, Seogu, Pusan, Korea SCOTT A. BARBOUR and WARREN KING Palo Alto Medical Foundation Palo Alto, California

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PREFACE

With confidence I can say that this is the most comprehensive volume dealing with tissue banking presently available. There are 27 full chapter contributions from the most distinguished and experienced practitioners in the subject: surgeons, microbiologists, and tissue bankers. The volume deals with all the major facets of tissue banking and utilisation: procurement processing, and utilisation. Safety of Tissues (Section II) Safety of allografts is now a major concern due to microbial and viral contamination of tissues even in the most sophisticated centres. Thus publication here of the International Atomic Energy Agency's Code of Practice for the Radiation Sterilisation of Tissues is an important event, as is their guidance on Standards and Public Awareness of this often misunderstood technology. There is now convincing evidence that safety has been compromised by certain regimes used to prepare human tissues for transplantation. Drs Eastland and Strong document fully the diseases which have been transmitted though tissues, in some instances, leading to fatalities. Thereafter, Section 2 continues to provide an in-depth investigation of this problem, particularly in bone and a consideration of safe and effective methods to use such tissues. The University of Miami Tissue Bank has been a leader in methods of microbiological screening of donors, and the final tissues. Dr Martinez (Chapter 5) delivers this experience concisely to us. The potential presence of viruses poses a formidable challenge to the tissue banker, and even when introducing an end-sterilisation radiation step, the outcome is XV

XVI

Preface

not clear-cut. Dr Axel Pruss and colleagues (Chapter 6) have taken us forward in a giant step in their careful controlled study of this problem. All roads seem now to lead to the desirability of adopting an end-sterilisation process if safe tissue are to be guaranteed (Chapter 7). When ionising radiation is used as part of a standardised working system of a tissue bank, then a degree of sterility assurance can be achieved. The IAEA programme has devoted considerable resources to define such procedures and the Code of Practice (Chapter 8) and Standards (Chapter 9) within which such a Code should be employed. There are limits to such use of radiation for bone, depending on the effects of radiation on mechanical strength and osteoinductivity (Chapter 10) of the final product. It is necessary to evaluate this aspect as Dr Yu and colleagues have done. The Marburg Bone Sterilisation Process for femoral heads may not have a universal application, but its effectiveness when properly used cannot be denied, as demonstrated by Dr von Garrel and his colleagues (Chapter 11). Ethical and Social Attitudes (Section III) The social and legal problems which arose in the UK, as a result of the unlawful procurement of tissues, have adversely affected public attitudes towards the procurement of tissues for cadaveric donors. Dr Paul L. Romain (Chapter 12) sets out in stark terms the ethical challenges which this field now poses. The justification for the practice remains the health benefits which such donation permits. In particular, the availability of such tissues in the wake of disasters such as has been experienced first in the Netherlands (Chapter 13) and then in Peru (Chapter 14) is evaluated. These contributions provide the perfect justification for having a ready supply of safe tissue grafts for immediate treatment of burns or traumatic damage.

Preface

xvii

Tissue Grafts in Orthopaedics (Section IV) The main customers of tissue banks continue to be orthopaedic surgeons. In this volume we are privileged to have an outstanding contribution from the doyen of this valuable technology — Dr Henry Mankin (Chapter 15). What a contribution he has made, and how glad we all are that he continues to be active and inspire further his one-time students, who are now distinguished exponents in their own right. This section demonstrates again the value of using both fresh and processed allografts in revision arthroplasty, and knee defects (Chapters 16 and 17). For the first time in this series we are able to learn of the long Russian experience in this field (Chapter 18 and 19). Other Areas Covered (Sections V-VIII) Cardiovascular grafts sperm banking and controlled process for the cryopreservation of tissues (Chapters 24-27) are other subjects covered in this volume. The motivation for such a comprehensive volume came in the Congress held in Boston which drew together all the international associations of tissue banking: American, Asia Pacific, Latin American and European. The whole world has been harnessed to construct this outstanding and historic volume.

Glyn O. Phillips Editor-in-Chief


CONTENTS


vii

Preface

xv

Section I:

Chapter 1

Chapter 2

The Contribution of the International Atomic Energy Agency (IAEA) to Tissue Banking

1

The International Atomic Energy Agency (IAEA) Programme in Radiation and Tissue Banking: Past and Present

3

IAEA Public Awareness Strategies for Tissue Banks

13

Section II:

Safety of Tissue Allografts

49

Chapter 3

Infectious Disease Transmission through Tissue Transplantation

51

Chapter 4 Chapter 5 Chapter 6 Chapter 7

Bacterial Contamination of Bone Allografts in The Netherlands

133

Microbiological Screening of Cadaver Donors and Tissues for Transplantation

143

Safety of Virus Inactivation Methods for Allogeneic Avital Bone Tissue Transplants

157

Methods of Culturing, Problems Associated with Bacteriostasis, and Radiation Sterilisation Options

193

Contents

XX

Chapter 8

IAEA Code of Practice for the Radiation Sterilisation of Tissue Allografts: Requirements for Validation and Routine Control

211

IAEA International Standards for Tissue Banks

267

In Vivo Assessment of Gamma Irradiated Bone: Osteoconductivity and Osteoinductivity

321

Disinfection of Femoral Heads for Bone Grafting Using the Marburg Bone Bank System (Lobator Sd 1) — A Retrospective Evaluation of Quality Control in the Endo-Klinik Bone Bank

339

Ethical and Social Aspects of Tissue Banking

353

Tissue Banking and Transplantation: The Ethical Challenges

355

The Volendam Burn Disaster and the Importance of International Collaboration in Tissue Banking

369

The Need for a Tissue Bank in a Disaster: Experience in Arzobispo Loayza National Hospital after the Tragedy in "Mesa Redonda", Lima, Peru

375

Section IV:

Tissue Grafts in Orthopaedics

387

Chapter 15

Major Limb Reconstruction Using Massive Cadaveric Allografts

389

Revision Arthroplasty Using Fresh Frozen Allograft with Cemented Cup for Acetabular Bone Deficiency

417

Chapter 9 Chapter 10

Chapter 11

Section III: Chapter 12 Chapter 13

Chapter 14

Chapter 16

Contents

Chapter 17

Present Ways of Treating Chondral and Osteochondral Knee Defects

431

Knee Joint Ligament Alloplasty with Tendon Grafts Sterilised with Gaseous Ethylene Oxide

455

New Approaches to Comparative Evaluation of Allogenic and Autologous Bone Transplants Procured in Various Ways

467

Section V:

Cardiovascular Grafts

483

Chapter 20

Cryopreservation of Porcine Aortic Valve: Open Status of the Aortic Leaflets Results in Increased Matrix Glycosaminoglycans Structural Maintenance

485

Pathologic Changes of the Cryopreserved Carotid Artery and Jugular Vein Implanted at the Canine Carotid Artery

501

Cellular Therapy for Heart Failure: A Review of Skeletal Myoblast Transplantation into Infarcted Myocardium

519

Section VI:

Cornea Grafts

547

Chapter 23

Cornea Transplantation Strategy — Organ Culture versus Cold Storage

549

Chapter 18

Chapter 19

Chapter 21

Chapter 22

Section VII: Sperm Banking Chapter 24

Characterisation and Depletion of Membrane Deteriorated Human Spermatozoa After Cryopreservation

559

561

Contents

xxu

Chatper 25

A Repository System for Cryopreserved Semen Samples and Testicular Biopsies Embedded in a Workflow Management System

Section VIII: Cryopreservation Chapter 26

Chapter 27

575 591

Finding the Ideal Freezing Curve for Tissues through Indirect Themophysical Calculation

593

The Safe and Effective Use of Allograft Tissue: An update

611

SECTION I: THE CONTRIBUTION OF THE INTERNATIONAL ATOMIC ENERGY AGENCY (IAEA) TO TISSUE BANKING


Advances in Tissue Banking Vol. 7 © 2003 by World Scientific Publishing Co. Pte. Ltd.

1 THE INTERNATIONAL ATOMIC ENERGY AGENCY (IAEA) PROGRAMME IN RADIATION AND TISSUE BANKING: PAST AND PRESENT

JORGE MORALES IAEA D e p a r t m e n t of Technical Cooperation, Vienna, Austria GLYN O. PHILLIPS Phillips Hydrocolloids Research Ltd, Cardiff, Wales, UK

1. The Early Period The IAEA has gained more experience and success than any other international organisation in establishing Tissue Banks in developing countries and applying ionizing radiation for sterilising tissue grafts used in transplant surgery (in orthopaedic reconstruction, treatment for cancer, trauma and high velocity impact damage), the treatment of burns, leprosy and intractable skin wounds, and pressure sore ulcers. This extensive programme has its origins in an IAEA Expert group meeting in the Joliot Curie Radiobiology Institute in Budapest around 1971, when it was resolved that there were advantages to be gained by using ionizing radiations to sterilise human and animal tissue. Yet for the early years, these interests 3

4

/. Morales & G.O. Phillips

were pursued under a broad cover of the sterilisation of medical products. The first declaration of the IAEA's official move into this field came with the 1974 Symposium in Bombay on "Sterilisation of Medical Products and Biological Tissues". The first vehicle used by IAEA was the Research Coordination Programme. Interested parties were encouraged to collaborate to study the effects of radiation on tissues. The outcome of this work was the IAEA Advisory Group Meeting held in Athens in 1976 on "The Effects of Sterilising Radiation Doses upon the Antigenic Properties of Proteins and Biological Tissues". The Proceedings of the meeting have been published (Phillips et al., 1978). In retrospect, it can now be realized how historic this meeting was; with pioneers in the broader subject of Tissue Banking participating: Gary E. Friedlander, Kenneth W. Sell, D. Michael Strong, K. Ostrowski, Sandor Pellet, Rudolph Klen, A. Dziedic-Goclawska, Pe Khin and Nicholas Triantafyllou. Thereafter, the programme gained momentum. Meetings under the auspices of IAEA were held in the Republic of Korea (1978) and Czechoslovakia (1981). The experience of the Eastern European countries was particularly helpful over this early period. The first truly Tissue-Banking contract (BUR/7/004) provided by IAEA emerged shortly afterwards, and in March 1983 a Tissue Bank was established in Burma (now Myanmar). From the mid-1980's the name "Tissue Banking" began to appear in official IAEA titles of programmes. Among the first was Sri Lanka where the late Dr. Hudson Silva had already established his famous Sri Lanka Eye Donation Society. Thailand thereafter played a major role in stimulating the programme in Asia and the Pacific Region through the contribution of Dr. Y. Vajaradul. The first Regional Workshop was held in Bangkok in November 1989 under the auspices of the IAEA Regional Coordination Agreement (RCA). To coincide with this meeting was the foundation of the Asia Pacific Surgical Tissue Banking Association, of which Dr. Vajaradul was the first Secretary General.

IAEA: Past and Present

5

It was the RCA programme, which delivered the necessary impetus and financial support, with Dr. John Easy being the RCA Coordinator who supplied the push over a ten-year period. The other pioneering Technical Officers at IAEA were Dr. Ramen Mukherjee and Dr. Vitomir Markovic. It was their support and vision that initiated the development of the first Training Curriculum in Radiation and Tissue Banking, using distance learning anywhere in the world. More detail about the historical development is given in Phillips and Strong (1997 and 1999), Phillips and Tatsuzaki (1998), Phillips (1999), Phillips (2000) and Phillips and Morales (2002). 2. Economic and Health Care Impact In all, some 30 countries have subsequently been involved (but not all funded) in this initiative: in Asia/Pacific Region: Australia, Bangladesh, China, India, Indonesia, Japan, Republic of Korea, Malaysia, Pakistan, Philippines, Singapore, Sri Lanka, Thailand and Viet Nam; in Latin America: Argentina, Brazil, Chile, Cuba, Mexico, Peru and Uruguay; in Africa and Middle East: Algeria, Jordan, Libya, Zambia and Iraq and in Europe: Greece, Poland, Turkey and Slovakia. All these countries are using radiation sterilisation as the method of choice. Experts from Australia, Europe, Japan and USA have assisted the IAEA Radiation and Tissue Banking Programme. In the countries involved with the IAEA Radiation and Tissue Banking Programme, as experience has been gained, the growth had been exponential. To the end of the year 2000, more than 220,000 tissue allografts have been produced and used clinically. Taking the mean value of the cost levied by Tissue Banks in the USA and Europe, as a re-charge for the processing, the value of these grafts can be placed at US$51,768,553. The total expenditure associated with the IAEA Radiation and Tissue Banking Programme over the same period was US$6,313,335, which includes a training cost of US$2,036,034. Not only does these provide a benefit in health care to these

6

/. Morales & CO. Phillips

countries but also avoid the costly importation of grafts into the country. In Mexico, for example, when a US$400 graft is imported from USA, the cost to the patient is at least US$3,000, due to value added taxes, import, agent, hospital charges, etc. It costs US$10,000 to import a whole femur from USA into the Republic of Korea. The following tables summarise the main outcomes of the IAEA Radiation and Tissue Banking Programme in general, and in the Latin America region in particular. The graft production over the period 1988-end 2000 in 16 countries for which data has been reported is the following (Table 1): Table 1. Graft production and cost benefits. Cancellous bone

Massive bone allografts

Skin and Amnion

Others*

69,195

8,588

96,645

50,278

*Pig skin, dura mater, demineralised bone, xenografts, pericardium, tendon, ligaments, and fascia. The total graft production is 222,580, valued at US$51,768,553 (at mean European and USA Tissue Bank prices).

In Latin America, the IAEA supported the formation of 7 Tissue Banks and trained 66 Doctors, Tissue Bank Operators and Nurses. Table 2 shows the distribution of the trainees who have set out to establish Tissue Banks and used the country nuclear center to carry out the radiation sterilisation of their grafts. It summarises the position at the end of 2001. The main benefit, however, is undoubtedly the improvement in health care in the individual countries. There exist major problems in the use of autografts, which is grafts taken from the patient and then transferred to another site during surgery, as already noted. Also, the available amount of autograft bone, particularly in child or infants, is limited. The second lesion offers a site for potential infection, which is particularly relevant in a developing country. Skin and amnion offer immediate cover


7

Table 2. Catalysing the start up of new Tissue Banks in Latin America. Country

Number on Attended Number of Number National 1 year Fellowship of New Training Training Training Tissue Course Course Banks

Argentina Brazil Chile Cuba Mexico Peru Uruguay

3

10

16 4 4 5 6 2 2

Total

13

39

7 2 6 3 5 23

11 6 4 10 4 1 1

Date when joined IAEA Programme 1993 1998 1998 1994 1996 1994 2001

37

Note: Initial total Tissue Banks in the region is 7.

for burns, wounds or leprosy lesions, which can promote healing, prevent fluid and energy loss and the introduction of infections. Another major advantage of the IAEA Programme in these countries is the exposure that surgeons get to the newer methods of using allografts and creating a cultural change in their approach to surgical treatment in their country. In Western countries, the use of such grafts is now routine, and more than one million are used world-wide. The perceived need in a developing country is not always apparent, but as the technology is introduced, the health benefits become clear. When this occurs, the graft supply is far from sufficient. The IAEA Programme has inspired this revolution in many countries and the growth henceforth will be exponential. Finally, it is not possible to measure the benefits for giving more than 220,000 people restored limbs, prevention of amputation, repair of fractures and spinal defects, or to treat burnt children and people who have been burnt or suffer from diseases

8


like leprosy, or paraplegics who are vulnerable to pressure sores. This is the true measure of the benefits of the IAEA Programme. 3. Innovative Training Distance learning training for tissue banking operators, managers and doctors within the IAEA Radiation and Tissue Banking Programme started in 1995. Initiated in the Asia and Pacific Region, with the support of international experts, the Singapore Government and the National University of Singapore, the first comprehensive IAEA/NUS Curriculum in Radiation and Tissue Banking was produced. It is now a tangible asset. This Curriculum, now available in English, Spanish and Korean, is an unique vehicle for delivering training of tissue bank operators, managers and doctors worldwide. The University Diploma extends over one year, starting with a two week faceto-face contact in Singapore or Buenos Aires, is the first available anywhere in the world. In Asia/Pacific, Latin America, Africa and Europe, 296 tissue bank operators, managers and doctors have been trained under the IAEA Radiation and Tissue Banking Programme, out of which 65 have been successful graduated in the University of Singapore and 9 at the University of Buenos Aires. For the 81 trainees from 18 countries who attained the University Diploma level, the total cost was US$557,117 compared with US$972,000 in the UK for the same level course, using conventional training methods. 4. The On-going Programme A Global Programme has been identified, which will place all activities within an inter-regional and regional structure. There is now an unified management and evaluation system for all activities. The inter-regional programme provides the basic expert and technical services, which can then be transferred to Latin America, Asia/Pacific and Africa Regions for implementation through the regional projects. There has been recently

IAEA; Past and Present

9

requests from a group of European countries, including Ukraine and Russia, which indicate a more active participation of the European region in the IAEA Programme. The IAEA Programme will ensure that all tissue banks have access to the latest international standards, and supported with documentation to enable them to mount an effective public awareness and tissue production programmes to promote health and economic benefits within their region and country. The activities will all be designed to ensure better public acceptance of the benefits of radiation sterilised tissue grafts and ensure that the same uniform international standards are being used throughout Asia/Pacific, Africa, Latin America and eventually the European region. The key initiatives will be: (a) The IAEA International Standards for Tissue Bank: Presently there is a great deal of variation. Expert and regional groups will review the present adopted USA and European practice and the planned activities will ensure that these, when adopted, are compatible with regional circumstances. (b) The IAEA Code of Practice for the Radiation Sterilisation of Biological Tissues. No international body has undertaken this important function, which is within the IAEA's technical competence. This Code of Practice will then be introduced into the regions for practical evaluation. (c) The IAEA Handbook for Public and Professional Awareness: Public and professional awareness is the major obstacle to even further extension of the use of radiation sterilised grafts in individual countries. Education is needed at all levels. Documentation to assist workshops and national programmes will be prepare and adapted to the regions in several languages. (d) Internet training: Internet training will be introduced for all "Radiation and Tissue Banking" training courses all over the world. This is a logical extension and will build upon the IAEA Distance Learning Programmes now being used. The resources of the multi-media curriculum and its recent update, needs to be exploited in this way to give full benefits

10


to IAEA. Already the educational methods and technical needs have been analysed, and as a result an implementation strategy agreed. In April 2002 the first International Training Course for Tissue Bank Operators was held in Singapore with the participation of 23 students form 13 countries of Africa, Latin America, Asia and Europe. The Tissue Bank in Singapore will become the International Center for Training Tissue Bank Operators when a Memorandum of Understanding is signed with the National University of Singapore. Another training course at the regional level will be held in 2002 in Buenos Aires for the Latin American countries. Eight participants will be accepted from 7 countries. It is expected that a Memorandum of Understanding will be signed between the IAEA and the National Atomic Energy Commission of Argentina in the following months to allow the establishment of a Regional Training Center for tissue bank operators in the country for the Latin America Region. A Memorandum of Understanding was signed in May this year with the Musculoskeletal Transplant Foundation (MTF) in the USA to promote the co-operation with the IAEA in the field of tissue banking, in particular in the area of training medical doctors and transplant coordinators. The establishment of a Homepage on the Internet for the IAEA Programme was finalised in 2002 (www.tissuebanking.org). Areas for co-operation between the IAEA and WHO have been identified and a letter of intention have been prepared and submitted for approval to the competent authorities in both organisations. 5. Benefits to D e v e l o p e d Countries Before the IAEA Programme started using radiation to sterilise tissues, the situation elsewhere in the world was very mixed. There were misconceptions about the benefits of using this technology, which are considerable, such as:


11

• No significant temperature, physical and chemical changes are induced which influence the required function of the tissues. • The high penetration enables the bulk of the hard or soft tissues to be sterilised in final packaged form. The systematic use of radiation sterilisation in final packaging reduces the cost of investment by approximately 80% compared with alternative methods. • The effect is instantaneous and simultaneous for the whole target. The process control is precise and can be applied accurately to achieve sterility. Irradiation time is the only variable to achieve a sterility assurance level of 10~6. A series of cases, particularly in the USA, where infections were transmitted through tissues, which had not been endsterilised, drew fresh attention to the experience of the IAEA Radiation and Tissue Banking Programme. Now at least half of the grafts in the USA are either being sterilised or decontaminated using radiation. The method is mandatory in Austria and widely practised in UK, Germany, Belgium, Slovakia, Poland and France. At a conservative estimate, it can be demonstrated that at least 600,000 tissues grafts in developed countries are now being annually radiation sterilised directly as a result of the IAEA Programme, through a type of reverse educational transfer to developed countries. Experts from developed countries are supporting the IAEA Programme. This is a confirmation that the benefits of collaboration between developed and developing countries are not all one-way. 6. References PHILLIPS, C O . , TALLENTIRE, A. and TRIANTAFYLOU, N. (eds.) (1978). Radiation Sterilisation of Irradiated Tissues and their Potential Uses, The North East Wales Institute, Wales, UK. PHILLIPS, G.O. and STRONG, D.M. (1997 and 1999). The Contribution of the International Atomic Energy Agency to Tissue

12


Banking, American Association of Tissue Banks: Tissue and Cell Report, 1997, 4(1), 5-10, and Advances in Tissue Banking (ed. G.O. Phillips), 3(1999), 357-397. PHILLIPS, G.O. and TATSUZAKI, H. (1998). The Tissue Banking Programme supported by the International Atomic Energy Agency (IAEA), Transfusion Today, 37 (December), 24-25. PHILLIPS, G.O. (1999). Tissue Banking in the Asia Pacific Region, Advances in Tissue Banking (ed. G.O. Phillips), 3(1999), 399-402. PHILLIPS, G.O. (2000). The future role of the International Atomic Energy Authority (IAEA) in Tissue Banking, Cell and Tissue Banking, 1, 27-40. PHILLIPS, G.O. and MORALES, J. (2002). Catalysts of Better Health Care: Medical Tissue Banks Bring Multiple Benefits to Countries, International Atomic Energy Agency Bulletin, 44, 17-20.


2 IAEA PUBLIC AWARENESS STRATEGIES FOR TISSUE BANKS

A N IAEA CONSULTATION D O C U M E N T

1. Public Awareness as a Component w i t h i n the Total Tissue Banking System The aim of this paper is to provide guidance on organising and running awareness campaigns and detail some promotional activities that have proved to be successful. It is not exhaustive and it cannot provide universal answers. Local conditions will dictate the appropriate approach and might also provoke innovative solutions. But it is a collection of ideas, contributed by people working against wide ranging ethnic and cultural backgrounds, which have been proved to work. Tissue Banking requires interaction between the public, the professional health care staff and the Tissue Bank. This interrelationship is shown in the following flow chart (Fig. 1). Recognising this integrated relationship, IAEA has addressed the following component needs: • Public awareness strategies for tissue banks. • International standards on tissue banks. • Code of practice for the radiation sterilisation of biological tissues. Additionally, training programs have been identified which support the activities and quality management within the tissue 13

IAEA Public Awareness Stategies for Tissue Banks

14

bank, and the professional education of surgeons and health care personnel who use the tissues and have a role to play in promoting donor availability. In this paper, strategies for public awareness are identified. But it is important to understand that public awareness alone \*i&m&®MW&e&

Public Awareness Strategies for Tissue Banks

Donor Source

•

National Transplant Systems > Specific Strategics

• •

Hospitals Coroner S> stems

•

Otlrci

Honor Referral .mil Transplant Cu-urdiimlion

Professional Education Doctors Health (.'we staff

• iJJ.

Surgical L'scrs

Tissue Banks Recovery I'rocessmj: Sterilisation Storage Labelling Distribution

Standaid* OIL I'lssue Ranks

~^

1AKA 1 raining Programs

Code of I'racuvc loi filiation SlciilisiitJOEi of Uiologkal Tissues

Fig. 1. A general tissue banking system.

•


15

will not be successful unless it is a part of an integrated system as illustrated in Fig. 1. 1.1. Donor referral and transplant coordination systems A critical link between tissue banks and donor occurs within the donor referral and transplant coordination system. Public awareness and professional education activities aim to increase and facilitate tissue donation within the donor referral and transplant coordination system. This system is composed to two interdependent processes: donor referral and transplant coordination. These activities may not be distinct and often overlap, but in any event, the two functions need to be achieved. The tissue bank can either be involved in the control of the activities or contract out to another organisation or individuals. This paper deals with an activity that precedes both donor referral and transplant coordination with the objective of providing information to potential donors and their families. 1.1.1. Donor referral Donor referral is the process by which the tissue bank is informed or notified of potential donors when a death occurs at the donor sources (hospital, coroners system, organ procurement agencies, funeral homes, others). Donor referrals are made using some basic donor suitability criteria, which have been previously provided to the donor source staff as part of a professional education and awareness program. Every tissue bank will need to establish processes to ensure suitable potential donors are identified and referred to the tissue bank. This notification may be made because of a mandated requirement or it may be voluntary. Regardless of whether this system of referral is under the control of the tissue bank or not, it will require liaison with, and education of other healthcare personnel.

16


In order to establish a donor referral system, the tissue bank will need to consider: • Identification and prioritization of referral sources (these may be external or internal). General criteria on which donor suitability is based may include: age, contra indications to donation, time limits between death and donation. • Establishment of relationships and agreements with those donor sources. • Identification and training of donor source staff in order to participate in the identification and referral of potential donors: explain the referral and donation process and the role of the staff members in the process; outline timelines for referral and recovery processes; provide basic donor criteria; explain tools necessary to make referrals (phone numbers, forms, etc.); and establish professional education that includes routine follow-up and feedback to the donor source staff. 1.1.2. Transplant coordination Once the donor referral is made to the tissue bank, the transplant coordination process can begin. Transplant coordination is the responsibility of suitably qualified personnel who have been trained in performing the various elements of the transplant coordination process. The transplant coordination process may include: • Confirming the suitability of the potential donor with the referral source. • Obtaining contact information for the next-of-kin. • Approaching next-of-kin to discuss donation options: tissue that can be donated; uses of the donated tissue; description of the recovery, testing, processing, storage, and distribution of tissue; consent process; medical social history; services available to the donor family. • Coordinating the recovery team. • Performing tissue recovery. • Completing documentation.


17

• Following up with family (thank you letters, donor family services, etc.). • Following up with donor referral source (feedback on the process). 2. Planning a Public Awareness Campaign Tissue banking is the recovery, processing, sterilisation, storage, labelling and distribution of tissues for transplantation. Although public awareness about all these activities might not seem to be a core activity of tissue banking, without donors, users and recipients, banking itself will have no value. Therefore, it is vital to communicate what tissue banking is, why availability of tissues for transplantation is important, its role in the community and how individuals can benefit from its existence, and finally, how its services can be accessed. Some tissue banks have public awareness programs permanently in place. This strategy can be very successful. In Thailand, for example, the promotion of the social importance of tissue donation has been introduced into the ethos of the Scout movement which itself is a mandatory part of the education system. The Rotary movement has also been convinced of the importance of tissue banking and promotes the principle as part of its social development policy. Religious and cultural leaders can play an important role in promoting tissue banking in certain communities. In developed countries, for example the USA, it is the role of central government to promote awareness amongst the public. Although the major emphasis is on organ donation, it is now also being extended to tissue donation. Such initiatives take time to set up and require continuing effort, but the results more than justify the resources employed. However, persisting with a routine in communication with the public that might have been previously successful may have drawbacks. A familiar approach can lose impact. Over several years, there might be demographic or cultural changes that will make the approach less effective. It is also difficult to assess the

18


result of the effort — are people responding because of the communication or would they respond anyway? And if it is not known exactly who is responding and why, it becomes impossible to decide who is not being reached and to find ways of getting the message to them. A coordinated public awareness program, run over a specific time scale, will enable those operating it to: • • • • •

Set targets. Assess results against the targets that were set. Work out how many hours and the budget to be spent. Decide priorities. Decide what is possible, given staff, time and cash restraints.

The key to a successful public awareness program is planning the communication — deciding what needs to be said, who needs to hear the message and how that message will be delivered. 3. Communication Strategy Although each tissue bank will have to establish its own communication strategy taking into account its geographical area, social reality, religious beliefs and specific needs, there will be some common elements in every communication strategy for every tissue bank. The following ten-point plan will provide an overview of how to develop a Communication Strategy and some of the elements that need to be considered: (1) goals; (2) situation analysis; (3) success factors; (4) objectives; (5) target audiences; (6) selection of media; (7) strategic messages; (8) delivering strategic messages; (9) feedback and evaluation; and (10) crisis management. 3.1. Goals Each tissue bank should identify the goals of its communication strategy and how it will achieve them. Goals may vary and might include:


19

• To enhance the public acceptability of allografts. • To promote the clinical application of allografts among medical professionals. • To ensure a high level of accessibility of allografts. • To increase the number of tissue donors. • To ensure the production of high quality and safe allografts. • To maintain high ethical standards. Once established, these goals would set the foundations upon which the communication strategy will be built. The first step, however, is to evaluate the present position. The progress of any journey can best be gauged by looking back at the starting point. Just as important, it will ensure the strategy is based on current reality. 3.2. Situation analysis A thorough analysis of the situation upon which the communication strategy will be built is vital to understand the environment in which the work will take place. This will help to ensure its effectiveness. If the situation is not fully analysed during the planning stage of the communication strategy, a vital component may be overlooked leading to the failure of the entire plan. In order to understand the situation it might be advisable: (a) To analyse the Strengths, Weaknesses, Opportunities and Threats (SWOT analysis) of the operation. For example: Strength

— the tissue bank is run by the orthopedic department of which the assistants are supportive of its activities. Weakness — the tissue bank does not have access to cardiac surgeons. Opportunity — the scope of activities of the tissue bank can be widened by establishing relationships with cardiac surgeons. Threat — the cardiac surgeons might decide to use imported or artificial cardiac valves.

20


(b) To conduct surveys (e.g. send out questionnaires to key stakeholders, etc). (c) To interview key people (e.g. surgeons, hospital administrators, government authorities, donor families, recipients, religious leaders, community leaders, etc). (d) To conduct workshops with key personnel. (e) To review the results of previous campaigns and experiences of other tissue banks. (f) To analyse already available data (e.g. number of potential users, tissue demand, numbers of imported tissues, etc). (g) To prepare a budget so that the financial implications of developing the communication strategy can be fully understood. 3.3. Success factors From the situation analysis it is necessary to identify factors that will be critical to the success of the communication strategy. These will then become targets in the communication strategy. Some examples are found in the Table 1.

Table 1. Success factor

Target

Low number of donations among a particular ethnic or religious group.

To gain the support of these communities or religious leaders for donation of tissues.

Access to surgeons.

To ensure that surgeons are aware of the safety, relative low costs and availability of allografts.

Low awareness about tissue transplantation in the general public.

To increase community acceptance and awareness of tissue donation by publicly recognising the contribution of donor families.


21

3.4. Objectives While the goals of a tissue bank will reflect its general purposes, objectives are the means by which these goals will be achieved. Objectives that could be considered are: • To raise awareness on tissue banking in the community and among medical practitioners, funding bodies and sponsors. • To provide high quality information on the issues surrounding tissue transplantation to facilitate decision making by potential recipients, donors and their families. • To reassure medical professionals about the safety and clinical utility of allografts. • To publicise the availability of allografts. • To cultivate an ethical, professional and caring image of the tissue bank. • To provide donor families with a supportive communications network. • To make tissue donation a natural consideration at the time of death. • To form a strategic alliance with partners such as community groups, health authorities, corporations, etc., to promote tissue donation. • To keep government authorities informed of tissue banking activities. • To assist government authorities in promoting tissue banking. • To assist government authorities in developing regulatory systems for tissue banking. • To enlist government support for tissue banking. 3.5. Target audiences If the communication strategy is to be successful, the audiences that need to be addressed must be identified. This means recognising all relevant groups of people. This will help in establishing priorities within the limits of the budget or

22


resources. It will also help in the selection of the appropriate media or techniques that will be effective with the audiences and the messages that each audience will be receptive to. But whatever the resources available, the principles set out here should be followed. If the audiences are not carefully selected, funds and resources will be wasted trying to reach everyone. The same message will be delivered universally. At best it will be ineffective for many groups; at worst it could be unsuitable or even objectionable. Depending upon the objectives, the audiences might include: (a) Donor families There would be no tissue donation without the consent of donor families. Consequently donor families should be given special prominence in a communication strategy when an objective focuses on donors. Research indicates that most families want the option of obtaining information, even if they do not read it until well after they have made their decision. Because of their personal experience donor families are in the best position to encourage community support for tissue donation. (b) Recipients Tissue banks need the assistance of recipients to promote the benefits they have received from tissue donation. The provision of high quality information to surgeons for distribution to recipients may be a useful tool in gaining their support for the activities of the tissue bank. (c) User surgeons Well-informed surgeons can greatly assist the tissue bank by talking to their colleagues about the high quality of allografts and service supplied. They are also responsible for informing recipients. Surgeons thus need to know about the services of the tissue bank, the kind of tissue available and their clinical usefulness. They also need to know the safety protocols of the tissue banks and their impact on costs.


23

(d) Bereaved families Effort should be made to raise community awareness of tissue donation among bereaved families. (e) Hospital staff Finance managers, medical directors and chief executive officers may be involved in the administration of tissue transplantation. Intensive care and specialty directors may be responsible for developing and implementing policies on tissue transplantation. They may also refer potential donors and deal with their families. They therefore need to be kept informed of all the developments and requirements of the tissue bank. (f) The media While the media are a means by which strategic messages are communicated to target audiences, it is legitimate to regard them as an audience in its own right. Medical and science journalists and producers for radio and television are in a position to reflect and influence public opinion and must be appraised of the benefits of tissue transplantation, the status of tissue donation and advances in the field. (g) Tissue bank staff Anecdotal evidence suggests that personal contact with donor families with the back up of a strong corporate identity would help individuals to make a commitment to donation. Tissue bank staffs are potential ambassadors for tissue donation. (h) Community groups Community groups (e.g. Rotary, churches, scouting, scientific societies, etc.) can be important in raising public awareness about the activities related to tissue banking and transplantation. They can also be supportive of these activities through spreading the word a n d / o r fundraising. (i) Other transplant groups The cooperation of other transplant groups is essential to elevate the status of tissue donation to that of organ donation.

24


(j) Government departments In some countries, government departments and other regulatory authorities may set the policies for donation and transplantation programs. They may also be responsible for budget allocation and regulation of activities. For these reasons, government departments and regulatory authorities should be kept informed about the activities of tissue banks and their achievements. 3.6. Selection of media Having agreed on the objectives and the target audiences to be addressed, the communication media must be selected. This is another area where choice will be dictated by local circumstances. To many people in the developed world, "the media" means the established channels of mass communication — radio, television, the press and, perhaps, cinema. However, in a far larger part of the world, television is regarded as an elitist and minority medium. The impact of newspapers might be restricted by low literacy, low purchasing power, distribution problems or a shortage of newsprint. Even where newspapers have few of these problems, they might have to serve several language groups and so the circulations of the different editions will be small. Radio might be widespread but it is patchy in areas without reliable electricity. Where such situations exist, public information messages have been successfully distributed by taking the message to the people by means of traveling cinemas and video shows, exhibitions and traditional or folk media. However, it would be well to regularly assess the scope and influence of the various media to ensure that the most effective means is being used to contact the audience selected. The media "balance" will change if there are increases in literacy, improvements in prosperity or technological advances. Political change can very quickly alter the way people receive information. Areas where controls on free expression have been


25

relaxed have seen a huge and rapid increase in the number of newspapers published, all reflecting a wide variety of political and social attitudes. As purchasing a newspaper is a voluntary act, that is, people choose to pay for the product rather than take what is given as in radio or television, it follows that people buy a newspaper because it reflects their views. This is significant because the growth of the press offers an opportunity to target specific groups in a way that is difficult with any other media. In areas where controls on free expression have been introduced, individuals have used the World Wide Web to create newspapers and even run live radio stations in opposition. This creative use of the media is instructional. It is now possible to "webcast" live video and the technology that allows this is becoming cheaper and more reliable. The "new media" should at least be given consideration in any media communication program. 3.7. Strategic messages The key messages form the central core of the communication strategy. They should be reflected explicitly or implicitly in all communications with stakeholders so as to build within the target audience a broad appreciation of subjects which will ultimately lead to the fulfillment of the objectives. The following are suggestions that could be considered as primary and subordinate strategic messages: (a) Value of tissue transplants to community health One donor can benefit many lives; possibility of someone in the audience benefiting from a future transplant; types of tissue that can be donated; tissue donation can be possible even when organ donation is not; advantages of tissue transplants over alternatives; e.g. cost benefits, efficacy, etc.; leading edge medical technology.

26


(b) Safety is the first priority There is a small but nonzero risk associated with all biological material; there is comprehensive quality control, including irradiation of tissue; need to fully inform recipients. (c) Focus on donor family Donor families could be important in raising community awareness; donor families may seek the support of the tissue bank; the body is respected and left intact. (d) Highly professional and ethical tissue bank Leading authority in tissue donation; fully accredited; fees are based on the cost of producing, storing and supplying safe tissue; user surgeons contribute to policy development; professional advice on all aspects of tissue donation and banking. (e) Technical aspects of tissue donation Suitability of potential donor; time frame; ethical and responsible disposal of tissue; restoration of the body after tissue removal; administrative procedures. (f) Technical aspects of tissue transplants Types and availability of tissues; procedure for obtaining and using tissues; procedure for sending tissues to the tissue bank. 3.8. Delivering strategic messages Strategic messages operate much like a mission statement. They reflect the value of what the tissue bank does, its technical and professional competence, compassion, sensitivity and ethical standards. Unlike a mission statement, however, strategic messages are not static but active. They can be used to project your values to audiences with differing needs. Finally, the strategic messages can be presented in different ways to make the message easily acceptable to a target audience. The delivery of a strategic message is the matching of an audience, the message and the resource, or delivery vehicle. For


27

example, the strategic messages for user surgeons might be: safety is the first priority; highly professional and ethical tissue bank; the technical aspects of tissue donation. It might be decided that the most persuasive way of delivering these strategic messages to this group is by: recipient information booklet; articles in medical journals; professional development of medical specialists; or displays or posters at medical conferences. Strategic Messages might be part of a public awareness campaign but they will generally be used in a different way and for a different reason. They deliver a specific message to a selected group consistently and over a long period. They aim to change attitudes and maintain that change by constant reinforcement. Communication with user surgeons, for example, will always contain the strategic messages of safety, professionalism and technical information regardless of the main subject. As such they should be part of continuing effort of a tissue bank. The following (Table 2) shows how the three elements — target audience, strategic message and resources can be matched to ensure success in reaching the proposed objective. Public awareness campaigns are generally more focused, shorter term and aim to educate to change long-term behavior. This subject is discussed in more detail in the public awareness campaign section. 3.9. Feedback and evaluation There is a need for constant re-evaluation of the communication strategy employed in pursuing established objectives through a feedback system. Communication strategies are of a dynamic nature and must be modified and adapted in response to identified successes, failures or even subtle changes in the initial objectives. The outcomes from the communication strategy should be evaluated at regular time intervals established in the initial

28


Table 2. Target audience

Strategic message(s)

Resource(s)

Donor families

Value of tissue transplants to community health. Safety is the first priority. Focus on the donor family. Highly professional and ethical tissue bank. Technical aspects of tissue donation.

High quality donor information/help kit. Establish "Friends" to provide grief support and raise community awareness. Quarterly newsletter. Thanksgiving service.

Recipients

Value of tissue transplants to community health. Safety is the first priority. Highly professional and ethical tissue bank.

Booklet for distribution through surgeon. Quarterly newsletter. Thanksgiving service. Media coverage. Outward focused brief annual report.

User surgeons

Safety is the first priority. Highly professional and ethical tissue bank. Technical aspects of tissue donation.

Recipient booklet. Articles in medical journals. Professional development of medical specialists. Displays/posters at medical conferences.

Bereaved families

Value of tissue transplants General information on tissue donation. to community health. Focus on the donor family. Highly professional and ethical tissue bank. Technical aspects of tissue donation.

Media

Value of tissue transplants to community health. Safety is the first priority.

Establish contacts. Invite to tissue bank seminars.

29


Table 2 (Continued) Target audience


Resource(s)

Focus on the donor family. Highly professional and ethical tissue bank. Technical aspects of tissue donation. Technical aspects of tissue transplantation.

Provide newsletters and annual report. Invite to "Friends" events and thanksgiving service.

Tissue bank staff

Value of tissue transplants Regular presentations to tissue bank to community health. seminars. Safety if the first priority. Focus on the donor family. Encourage staff to join "Friends". Highly professional and Provide newsletter, ethical tissue Bank. donor registry forms Technical aspects of tissue and annual report. donation. Technical aspects of tissue transplantation. Role in promoting activities and professionalism of tissue bank.

Hospital staff

Safety is the first priority. Highly professional and ethical tissue bank. Technical aspects of tissue donation.

Liaison with tissue bank. In service seminars. Newsletter. Invitation to tissue bank seminars. Provide information booklets.

Community groups

Value of tissue transplants to community health. Technical aspects of tissue donation.

Seminars, presentations, promotional activities, etc.

30

IAEA Public Awareness Stategies for Tissue Banks Table 2 (Continued)

Target audience


Resource(s)

Other transplant groups

Safety is the first priority. Establish personal contact. Focus on the donor family. Invite to seminars. Highly professional and Market the advantages ethical tissue bank. of greater Technical aspects of tissue collaboration. donation. Negotiate inclusion of tissue donation in organ donation information. Send newsletter and annual report.

Government departments

Value of tissue transplants to community health. Safety is the first priority. Highly professional and ethical tissue bank. Technical aspects of tissue donation and transplantation.

Meetings with authorities. Provision of information. Participation in the medical advisory board.

planning process. This will require feedback from key stakeholders, using the same tools that were employed for the initial Situation Analysis. The evaluation may reveal whether there have been advances in certain areas of the communication strategy and also other areas where the communication strategy has had limited success. Adaptation of the communication strategy so as to incorporate the information obtained through feedback information and new Situation Analysis will ensure that the communication strategy remains effective.


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3.10. Crisis management The success of tissue banks relies upon the trust and goodwill of the public as well as the confidence of medical professionals. These are based upon a positive perception of such things as the contribution of a tissue bank to society, its procedures, the competence of its professionals, its ethical code and safety standards. If, because of something that is done, something that is not done, misinformation or malicious rumor, that positive perception is damaged, then years of education and promotion could be undone within days. The tissue bank would be facing a crisis. 3.10.1. Features of a crisis (a) Someone is to blame An incident that could not have been anticipated and was a result of natural forces will not usually cause a crisis for a tissue bank. However, if it is the result of someone's negligence, then the tissue bank will become the focus of public and, therefore, media attention and anger. (b) Something is at stake There is no crisis if there is nothing that can be damaged by the public anger and media exposure. In the case of a tissue bank, what is at stake might be its donor base or the cooperation of other medical professionals. (c) Someone finds out A crisis only begins when it becomes public. 3.10.2. Usual reaction The usual reaction to a crisis is characterised by panic and inaction. It is tempting to think that the crisis will go away if nothing is said or done — it won't.

32


3.10.3. Result of inaction As a consequence of inaction, public anger grows; rumor replaces fact; regulatory bodies, politicians, etc., become involved; confidence collapses and the crisis spirals out of control. 3.10.4. Handling of the crisis (a) Stop whatever is causing the problem This might be costly and inconvenient, but it removes the cause of the emotion and shows the tissue bank to be sensitive and considerate of its public responsibilities. It can also demonstrate a commitment to safety, quality or whatever else the tissue bank's "brand" represents. (b) Put out holding statement Say something to show the problem has been identified, it is being dealt with, there is no cause for public alarm and, most importantly, that the tissue bank is a source of information on the subject. This will allow rumors to be stopped before they get into the media or into general circulation. (c) Assemble crisis team Unless care is taken, the crisis will take over the entire tissue bank. A small team must be assigned to handle the crisis while others get on with the day to day job of running things. (d) Decide on audiences These might be the general public, regulatory bodies, medical professionals, politicians, people living close to the building, professional bodies and associations, etc. And do not forget to inform the staff what is happening; they are an audience too! (e) Decide what will be said Separate messages will probably be needed for audiences.

different


33

Your audiences will fall into one or more of three groups: Passive — they do not know. Informed — they know but are not active. Active — they know and they are taking action. The purpose of communication in a crisis is to: prevent the Passive from receiving misleading or emotive information and moving from Passive to Active. Persuade the Informed audience that there is no need to take action. Reassure the Active that the crisis is being dealt with and they no longer need to become directly involved. 3.10.5. Planning for crisis A crisis should not catch a tissue bank unprepared. Almost all eventualities can be anticipated and planned for. The first step is communication. If you have tissue banks or individuals that are not supportive of tissue banking talk to them and explain what is done and why. They might never be convinced but understanding might prevent them from moving from being an informed audience to an active audience in a crisis. The second step is anticipation. Decide NOW what defines a crisis for the tissue bank. There are only a handful of things that are likely to cause problems. Work out what they are. Ask: what might go wrong; what outside events might affect us; and which response should be made in each case. 3.10.6. Create a crisis team Decide NOW who will run the crisis, who will deal with the media, government, regulatory bodies, etc., and who will continue to run the tissue bank. 3.10.7. Identify your audiences Decide who will need to know.

34


3.10.8. Work out what you will communicate What must be said to each audience. 3.10.9. Write it d o w n Create a document that includes contact numbers for all the audiences as well as home, mobile and holiday numbers for key staff and "friendly" journalists. Keep it up to date. 4. Public Awareness Campaigns Public awareness campaigns aim at creating a climate in which the immediate goals of the tissue bank — increasing donor registration numbers, obtaining corporate sponsorship, encouraging family discussion, etc. — become acceptable and result in increased donation. Unlike strategic messages, which are a statement of the worth and values of a tissue bank, public awareness campaigns are usually very focused. They set out to achieve a desired outcome, they often include the use of several different media to influence the target audience and they are finite, so the results can be measured against effort and expenditure. For example, in a program to increase enrollment of university students as donors the campaign organisers would select the target audience — in this case both students and their families. They would decide on the messages, which would include reassuring and educating parents, persuading the young people to apply for a donor card and impressing upon them the importance of informing their families. The organisers would set a target, perhaps how many more students would carry a donor card after a specific effort over a set time period. Finally, they would decide on the communication tools to be used. Given that the principal audience is the young people, the Internet might be selected together with videos. Within the university, students are a captive audience and so displays or


35

exhibitions could be set up and lectures organised linked to the distribution of brochures. If resources are available, the communication tools might be extended to include information for would-be students and their families delivered with university entrance materials; advertisements on local and university radio stations or even a donor disco; the possibilities are limited only by money, effort and the imagination of the organising team. Examples that have already been found to work by other tissue banks worldwide are many and various. 4.1. Potential public awareness activities Potential public awareness activities include: a school program "teaching the teachers" workshop; a scout program; a kids club; a donor registry; a university/college student enrolment program; a community program; a corporate and community tissue banks program; donor and transplant recipient services; a drivers licensing bureau program; and an old folks' home. There should be constant monitoring of progress throughout the campaign so that, if necessary, effort can be redirected to ensure the objectives are met. Finally, there should be a careful appraisal of the results to identify what worked well, what could have worked better and what lessons have been learned. To recap, a successful public awareness campaign should include the following: an assessment of the problem or need; the setting of realistic targets or desired outcome; assessment of resources; the choice of target audience; choice of messages; selection of tools; a distinct start — possibly a public launch; constant monitoring; an agreed end; and appraisal of results. 4.2. Tables The following tables contain details of the potential public awareness campaigns including their desired outcome, messages and communications tools.

36


Method of practice

School program "teaching the teachers" workshop

Target audience

Primary target: elementary and high school teachers, Secondary target: students and families.

Desired outcome

Train teachers to educate students about organ and tissue donation. To encourage a family discussion about a donation decision.

Messages

Overview of organ and tissue donation. Importance of sharing your decision with your family. Concept of giving, sharing and receiving.

Tools

Two-hour workshop.

Recomendations

Seek support from the education authority, promoting joint programs among the relevant authorities.

Teacher curriculum. Pre-test. Video (highlights recipients). Brochure.

Scout program

Method of practice Target audience

Scouts,

Desired outcome

Educate scouts about donation so they can promote and discuss the donation decision with their families and the community.

Messages

"Serving Life". Provide information and promotion of donation to their community through the scouts service commitment.

Tools

Brochures/leaflets. Video (transplant recipients). Lecture.

Recomendations

Seek scout leaders' support to implement a program with reward (badges).


Method of practice

37

Kids club

Target audience

Children (ages 4-12 years old) and parents.

Desired outcome

To promote donation discussion among the child's family, classmates and their families.

Messages

Benefits of donation and transplantation.

Tools

Videos and posters. Donor cards ("donation promoter card"). Stickers. Always stress importance of child's involvement in donation discussion.

Recomendations Method of practice

Donor registry

Target audience

General Public (age 18 and older).

Desired outcome

Develop a database to record individual donation decision to facilitate the donation process at the time of death.

Messages

Make your decision now, so your family can follow through with your wishes later.

Tools

Brochures. Form with appropriate signatures. Data base system.

Recomendations

Seek government legislation to implement and maintain a donor register.

38


Method of practice

University/College Students/Enrolment Program

Target audience

Students and their families.

Desired outcome

Every student within the university/college will make a decision about donation and carry a donor card.

Messages

Educate the students about donation and the importance of making a donation decision and communicating it to their families.

Tools

Video. Lecture. Brochure/donor card. Internet. Display/exhibit.

Recomendations

Seek support from the university or college authorities.

Method of practice

Community program

Target audience

General public.

Desired outcome

Educate the general public about donating, the importance of making a donation decision and communicating that decision to their family.

Messages

Importance and benefits of donation and transplantation. Make a decision now, so your family can carry out your wishes later.

Tools

Media campaign. Special events. Celebrity endorsement. Displays/health fairs. Internet. Donor cards. Drivers License Program (and other official identification documents). Toll free number. Brochure.

Recomendations

Keep message simple and consistent.


Method of practice

Corporate and community tissue banks programs

Target audience

Corporations (executives, staff). Service groups. Community groups.

Desired outcome

Educate members, staff or customers about donation. Seek sponsorship. Make a decision now, so your family can carry out your wishes later.

Messages

Benefits of donation and transplantation.

Tools

Brochures. Video. Internet. Program materials with recognition of sponsorship. Lectures. Scholarships.

Recomendations

Keep message simple and consistent.

Method of practice

39

Donor and transplant recipient services

Target audience

Donors, recipients and their families, General public.

Desired outcome

Increase donation through recognition of the donor and support of the family who generously donated. Highlighting the improved quality of life of the recipients.

Messages

"Celebrate life".

Tools

Transplant recipient Olympic games. Donor memorial services. Donor recognition (day). Donor medal/certificate. Donor quilt.

Recomendations

Enlist volunteer donor families and recipients to plan coordinate and participate.

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Method of practice

Drivers licensing bureau program (or official identification document)

Target audience

Driver License staff. Drivers/general public.

Desired outcome

Drivers License staff consistently offers the option of a donor designation to each individual who renews or applies for Drivers License or official identification document.

Messages

Educate the Drivers License Bureau staff about donation and importance of the donation decision and the family discussion. Ensure Drivers License staff gives a donation brochure to individuals who have questions.

Tools

Posters (posted in the Drivers License Bureau office). Lectures. Videos (donor families and transplanted recipients). Brochures (staff and public).

Recomendations Provide information/materials related to donation to Drivers License Bureau staff. Support of the Bureau staff is key in offering drivers the option of donor designation. Method of practice

Old folks' home

Target audience Senior public. Desired outcome

Educate senior people to participate in promotion of program. Promote tissue (e.g. Cornea) donation.

Messages

Nobody is too old to participate in promotion of donation program.

Tools

Media campaign. Special events. Posters. Brochure.

Recomendations Keep message simple and consistent.


41

5. U s i n g the Media Most public awareness programs include some attempt to involve the media. Sometimes the attempt results in an enthusiastic response from the journalists and sensitive, valuable coverage. Sometimes it results in disinterest and little or no coverage. Occasionally it results in inaccurate or sensational coverage of one unimportant aspect of the program, which misinforms the public and damages what is being attempted. Many people who have occasionally had to deal with the media — medical professionals, academics, businessmen, scientists, public service workers and charities — have experienced one or more of these responses. One factor that unites them all is they don't know why. Those who have had a good experience are pleased, but those who have had a bad experience are angry. Frequently both groups are mystified by the response. It is true that taking the media as a whole, newspapers, radio and television, it does seems to have contradictory and conflicting needs. This section of the document aims to explain why the media acts as it does and how to anticipate that response. From this it will become easier to identify the media most suitable for the Target Audience. First, the media is not there to right wrongs, to shine light into dark places or to highlight those things that are important in life although it might do all of those things. It exists principally to make money and occasionally, also, to extend influence. To do this it must generate an audience. Moreover the media creates readers, viewers or listeners by identifying a target group or groups and feeding them what they want. 5.1. The audience A large national broadcaster will produce a range of programs to appeal to a selection of audiences. But the more media

42


competition there is in any sector, the more specific the media becomes in the audience it targets. Where there is a lot of competition, typically in newspapers, both national and local, cable television and local radio, the media outlets will target a very narrow group. They will know the lifestyle of that group very well indeed and know their hopes, fears, beliefs prejudices, drives and aspirations. They also know that people will read the newspapers and listen to programs that reflect their values and beliefs. Thus, they will provide their Target Audience with information that reflects the world as that audience sees it. There are two significant points arising from this. Firstly, it is usually a waste of time sending the same information to all sections of the media. Secondly, even when a Target Audience has been identified, the media that appeals to that Target Audience might not be interested in what you want to say. For example, let us suppose that an objective is to persuade more 18-25 year-olds to opt to be tissue donors. Possibly the group is being targeted because it has been identified as having a general lack of awareness and interest in the subject. However, if that is the case, why should a magazine or radio station run a story or feature on something they know their readers or listeners are unaware of and probably not interested in! 5.2. Give them what they want! The answer is that the information must be linked with something that the Target Audience will find interesting. This might sound like a lot of effort for no guaranteed return but trying to sell the media something they don't want is effort for no return. 5.3. Case study one — Quit and W i n A public health organisation decided to run a campaign to try and persuade young people — teenagers and early twenties — to


43

give up smoking. The group was chosen because it was considered that although many would have been smoking for several years, this habit might be easier to break and the health benefits would be easier to "sell" and more readily recognised. For economy, the campaign had to be run largely through the news columns of local newspapers and the news programs of local TV and radio rather than by advertising. The campaign organisers looked at what the young people in that group valued. It was recognised that among the few things that this group felt important enough to save money were for cars and holidays. A car distributor was persuaded to give a car as a first prize on the promise of publicity and several holiday companies gave holidays. The campaign became "Quit and Win". People pledged to give up smoking by filling in a form, signed by a friend. After three months they became eligible to take part in a free draw for a holiday. The campaign ended after six months with the final draw for the car. Pictures and interviews with the happy winners were regular features in the media in months four, five and six while the car was handed over by the Minister for Health with great publicity. But what of those who didn't win? A subsidiary message of the campaign was how much could be saved by not spending money on cigarettes — about the equivalent of $7 US a packet in the United Kingdom. There were other interviews at regular intervals about people who had saved for their own holidays or other consumer goods by giving up smoking. There were some up-front costs. There were posters to be printed for the workplace, entry forms and administrations costs. But the value of the publicity was many times the fixed costs. More importantly, the campaign succeeded in its three aims: a number of people stopped smoking; they became role models — their success was likely to influence others;

44


giving up became a "smart" thing to do — not something to be mocked. 5.4. Case study t w o — Medic Alert Medic Alert is a charity that was founded by a parent whose child almost died of an allergic reaction to a food while at a playgroup. The child's allergy had been explained but the information was not passed on. Consequently, when the child became unconscious, no one recognised the cause and it was only by chance that the child was saved. Medic Alert provides a database of the medical conditions and current treatment for hundreds of thousands of people. People at risk wear a disc bearing a personal identification number and a 24-hour emergency telephone number. It ensures that if that person is taken ill or is injured vital information can be given which might save their lives. Since its inception, hundreds of lives have been saved. Obviously, the more the people who know about it, the more the people that can benefit. The charity decided that television was the ideal media for spreading the message. They approached the producers of several popular drama series with true stories of people who would have been unwittingly killed by routine treatment when they were unconscious but at the last minute someone noticed the badge and called the emergency number. They stressed the dramatic nature of this — moments away from death. The result was that Medic Alert was featured in three television series — a hospital drama and two police dramas all with an audience of several million. Very different from the first case but again, the charity recognised their product had something television producers wanted — dramatic impact. The advantage of using fictional television or radio series is not only publicity but, in cases where there are ethical or religious objections, the argument itself can become part of the program story line.


45

5.5. D e a l i n g w i t h journalists Start by building a relationship with journalists. Getting material into a newspaper or on local television and radio is often as much about personal contact as the subject matter. Each day in a newsroom, hundreds of pieces of information jostle for attention. If the journalist making a decision about what to use knows and trusts the tissue bank and believes that what it is trying to do is a good thing, they are more likely to use the information when they come across it. Remember, journalists are not necessarily friends — they have a job to do. But a relationship can be built up from which both sides benefit. The Quit and Win campaign was a good one but it got more publicity than it might have done because the people running it were on first name terms with a large number of news journalists and health correspondents. 5.6. Getting in touch The most common way of giving journalists information is the news release, mailed or e-mailed to interested journalists and news desks. Bearing in mind the previous section it should be directed to a journalist contact that has been cultivated. However, there are a few things you can do to help it further. (a) Write an eye-catching headline The headline you write is unlikely to be the headline that appears in the newspaper — the object is to make the journalist receiving it read on. (b) Start with the most important points What is actually happening? Where is it happening? Why? When? How? Answering as many of these questions in the first paragraph will usually have the effect of ensuring the most important information comes first.

46


(c) Explain how it will benefit people Events are only news because they affect people. The event might be agreement to receive hundreds of corneas from the tissue bank of another country. But the effect will be the gift of sight for hundreds of people who would otherwise be blind. That makes a much more emotive and stronger story for the media. (d) Add a strong "quote" from a senior figure Quotations can make a story more personal and, if from a prominent person, more authoritative. (e) Use one side of a standard sheet of paper If a news release looks long and complicated a journalist might not begin reading it. If more information is wanted, they will call. (f) End with a contact number Make sure someone who has authority to speak to the media is available at that number. 5.7. The interview It is flattering to be approached to give a media interview, but anyone approached should ask themselves honestly if they are the right person to be interviewed. If not, who should be? Then the journalist should be asked: (a) Which program/publication? This will indicate the sort of audience — readers, viewers or listeners — that will receive the message. (b) What do you want to talk about? This is necessary anticipated questions.

to allow preparation

of answers

to


47

(c) Are you talking to anyone else? This might be the only opportunity to learn if the journalist is also talking to someone critical of tissue banking. Then decide, is it in the interests of the tissue bank to do this interview? If so, prepare. Work out what the journalist is likely to ask; and what messages must be put out in the interview? Remember, an interview is an opportunity to put over positive points about the tissue bank and what it is trying to do. Don't put an opposing point of view to be "reasonable". If there are two sides to the issue, there will be plenty of others willing to make the opposing point of view and the journalist has probably found them. Another key to preparation is being clear about what the interview should achieve. Is it to inform? To encourage people to take a course of action? To calm people's fears? Think of two or three main points; things that are at the heart of what must be achieved and make sure that they are used. They must be delivered with enthusiasm and energy — broadcast journalists like interviews that sound good but it will also encourage a press journalist to use that as a "quote". Do not be nervous. They need your expert knowledge. In some cases, a journalist will arrive having had very little opportunity to learn about your tissue bank or its work. This is an opportunity to brief them and even suggest some questions they might want to ask. Finally, will journalists always be open and honest about what they want from the interview? The answer is no. Interviewees must try and work that out for themselves, which returns to the beginning and the audience. What audience this journalist is writing or broadcasting for; and what does that audience think of the tissue bank and tissue banking in general? Remember, journalists are not seekers after truth — they're seekers after stories! And that's not necessarily the same thing.

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6. Promotional Tools A reference section has been compiled to illustrate how a database can be developed and provide a resource for tissue bank and organ transplant professionals. Through it, they can access examples of good practice, publicity, promotional and advertising materials that have proven to be successful in programs throughout the world. It is by no means exhaustive. Users may access information via the IAEA website or by contacting the relevant institutions that have contributed to that database. The information has been categorised as follows: Public Education; Professional Education; Donor Appeal and Donor Management. The material is further grouped into the following sub-categories: fliers; posters; stickers; audio-visual aids; newsletter; calendar themes, etc. The reference section can be obtained from IAEA on request.

SECTION II: SAFETY OF TISSUE ALLOGRAFTS



3 INFECTIOUS DISEASE TRANSMISSION THROUGH TISSUE TRANSPLANTATION

TED EASTLUND Division of Transfusion Medicine Department of Laboratory Medicine and Pathology University of Minnesota Medical School Minneapolis, Minnesota 55455, USA D. MICHAEL STRONG Puget Sound Blood Center/Northwest Tissue Centre Seattle, Washington 98104, USA

Abstract The incidence of tissue allograft-transmitted infection is unknown and can best be inferred from prospective studies — that have not yet been performed and reported. Viral, bacterial and fungal infections have been transmitted via tissue allografts such as bone, skin, cornea and heart valves. Bone allografts have transmitted hepatitis C, human immunodeficiency virus (HIV-1), human T-cell leukaemia virus (HTLV), tuberculosis and other bacteria. Corneas have transmitted rabies, Creutzfeldt-Jakob disease (CJD), hepatitis B virus, cytomegalovirus (CMV), herpes simplex virus, bacteria and fungi. Heart valves have been

51

52

T. Eastlund & D.M. Strong

implicated in the transmission of tuberculosis, hepatitis B, bacteria and fungi. HIV-1, CMV and bacteria have been transmitted by skin allografts. CJD has also been transmitted by dura mater and pericardium transplants. Over the past several years, improvements in donor screening criteria, such as excluding potential donors with "risky lifestyles" for HIV-1 and hepatitis infection, and donor blood testing, have greatly reduced the risk. The addition of viral nucleic acid testing of the donor will further enhance the ability to detect pathogens and reduce the risk of disease transmission. During tissue processing, many allografts are exposed to antibiotics, disinfectants and terminal sterilisation such as irradiation, which further reduce or remove the risk of transmitting disease. Because the effectiveness of some tissue grafts depends on cellular viability, not all can be subjected to sterilisation steps, and, therefore, the risk of infectious disease transmission remains. For these situations, preventing the transmission of infection with the graft depends on careful donor selection and testing; aseptic technique during surgical removal of the tissue from the donor; aseptic processing; microbiological testing of the allograft to ensure that virulent organisms are not acquired during processing, or if present, are identified and eliminated as much as possible. To further ensure safety in the use of allografts, the physician and hospital should select tissue banks that follow national professional standards, as their source. 1. Introduction Tissue transplantation therapy, which has been utilised for over 50 years (Strong, 2000), is a rapidly developing field carrying with it great promise for ameliorating or curing many diseases. One of its drawbacks, however, is the potential for donor-torecipient disease transmission. This risk is greatly reduced by excluding donors at risk of carrying infection, and by testing the

53

Infectious Disease Transmission Through Tissue Transplantation

donor for transmissible infectious disease. Aseptic surgical technique in a quality environment, when removing the tissue from the donor, when processing and storing the tissue, and during implantation, is critically important to prevent bacterial and fungal contamination. Non-viable tissue grafts such as bone can undergo disinfection and sterilisation steps. During the past two decades, the disease transmission risk associated with tissue transplantation has been greatly reduced by implementation of standards set by professional organisations, such as the American Association of Tissue Banks (AATB, 2001), the European Association of Tissue Banks (EATB), the Eye Bank Association of America (EBAA, 2001), and governmental regulations. However, the incidence of transplant-transmitted infection is uncharted, and the studies needed to determine this have hitherto not been performed. Cadaveric donations (Table 1) and clinical transplants (Table 2) of cornea, bone, skin, heart valve and other tissue allografts in the United States greatly exceed that of organs (United Network for Organ Sharing, 2001). Organ transplantation flourished in the early 1980s following the discovery and introduction of cyclosporin as an effective immune suppressant. This brought a large supply of cadaveric donors that could also be used for tissue donation. Unlike the limitations of organ transplants, tissue transplantation generally is not limited by HLA histocompatibility barriers (Choo and Eastlund, 1996) or by ABO Table 1. Cadaveric organ and tissue donation in the United States. Type of donor tissue

Donations per year

Cornea donors* Bone, skin, or other tissue donor* Organ donor*

46,729 18,021 6,082

*Eye Bank Association of America, 2001. +American Association of Tissue Banks, 2001. Ûnited Network for Organ Sharing, 2001.

54


Table 2. Estimated number of allografts transplanted annually in the United States. Tissue CADAVER TISSUE* Bone Corneat Skin (sq. ft.) Heart Valve Vessels Pericardium CADAVER ORGAN ALLOGRAFTS* Kidney§ HeartH Liver Pancreas Lung Intestine LIVING DONOR TISSUE AND CELLS Red Blood Cells" Unrelated Bone Marrow Stem Cells*** Peripheral Blood Stem Cells Cord Blood Stem Cells

Transplants 675,370 50,868 11,222 5,500 433 5,327 17,601 9,094 2,210 4,667 465 1,034 113

13,361,000 1,160 524 59

'American Association of Tissue Banks — 2000; Office of the Inspector General — 2001. tEye Bank Association of America — 2001. Ûnited Network for Organ Sharing — 2001. Încludes 884 combined kidney and pancreas transplants. Încludes 27 combined heart and lung transplants. ''National Blood Data Resource Centre — 2002. '"National Marrow Donor Program — 2001. blood group incompatibility (Eastlund, 1998a). No longer being a scarce resource, the widened availability of tissue allografts encouraged new clinical use and brought attention not only to their effectiveness and advantages over autografts, but also to


55

their drawbacks, side effects and complications. One complication of tissue transplantation has been transmission of disease of donor origin to the recipient (Gottesdiener, 1989; Eastlund, 1995). Although transmission of malignant tumour of donor origin is a recognised complication of organ transplantation in immunosuppressed recipients (Perm, 1993), there has been only one report of a tissue graft transmitting a malignancy: over 50 years ago, a single case of a retinoblastoma transplanted with a cornea was reported (O'Day, 1989). On the other hand, bacterial, fungal and viral infectious diseases have been transmitted more often and by several types of tissue. Most reported tissue transplant-transmitted infections have been of donor origin. Viral infections can be transmitted if the donor has a viral infection with viral levels too low for detection. In asymptomatic donors who were recently infected, a transient viremic phase can exist prior to development of a positive donor screening test for antibodies. Preventing donorto-recipient infectious disease transmission relies heavily on selecting safe donors not only through testing, but also by medical and social behaviour screening, to select donors more likely to be free of transmissible infections. Bacteria and fungi can be introduced into the tissue allograft as a contaminant during surgical removal of the tissue from the donor, or from processing, storage or implantation. During allograft processing, the use of sterile supplies and aseptic technique in an environment as free as possible of microbial contamination is important. Environment monitoring, adequate sampling techniques and final sterility testing are also essential. The characteristics of each tissue allograft, and whether it can be exposed to disinfection and sterilisation, have a large bearing on whether disease transmission is likely. Some tissue allografts, e.g. corneas, heart valves and skin, need to remain viable and cannot be exposed to disinfectants or sterilisation without an unacceptable loss of viability. Other grafts are non-viable, largely comprised of acellular connective tissue, and can be disinfected or sterilised more freely resulting in a greater assurance of

T. Eastlund & DM. Strong

56

Table 3. Allograft characteristics affecting ability to transmit disease. Nonviable allograft

Viable allograft Type Heart valve and vessels Cornea Skin Marrow Blood stem cells Vascularized organs Semen and oocyte Foetal tissue

Bone Dura mater Pericardium Tendon Costal cartilage Fascia Ear ossicles Characteristics Non-viable Acellular Connective tissue Can be processed, sterilised

Contains viable cells May be antibiotic treated Cannot be sterilised

sterility (Table 3). This review focuses on infectious disease transmission through transplanted tissues, and the steps taken for its prevention. Disease transmitted through organ and cellular transplantation and through tissue and cells donated by living individuals (i.e. blood, marrow, semen, and oocytes) will not be addressed. 2. Infections Transmitted by Cornea Allograft 2.1. H u m a n immunodeficiency virus As thin avascular tissue, cornea comprises a well-hydrated transparent layer of connective tissue, and a single-cell layer of viable endothelial cells. Consequently, it is not very immunogenic; nor is it often rejected by the recipient unless it becomes vascularised. Similarly, it is not very efficient in transmitting


Table 4. Infectious disease transmitted by tissue allografts. Allograft

Infectious disease

Bone

Hepatitis C Hepatitis, unspecified type HIV Bacteria Tuberculosis HTLV

Tendon

Bacteria Hepatitis C HIV

Cartilage

Bacteria

Cornea

Hepatitis B Rabies Herpes simplex virus Creutzfeldt-Jakob disease Cytomegalovirus (?) Bacteria Fungus

Dura

Creutzfeldt-Jakob disease Bacteria

Heart valve

Hepatitis B Tuberculosis Fungus Bacteria

Skin

HIV (?) Bacteria Cytomegalovirus (?) HCV (?)

Pericardium

Creutzfeldt-Jakob disease Bacteria

57

58


viral infectious disease from the donor. Diseases transmitted through corneal transplants are listed in Table 4. The cornea is not efficient in carrying or transmitting HIV. Based on assumptions about HIV antibody test sensitivity, Goode et al. estimated that 3 per 10,000 cornea allografts will be from HIV-infected donors despite HIV antibody testing (Goode et al, 1988). There have been several documented cases in which cadaveric organ and tissue donors were infected with HIV, but the cornea recipients did not become infected. Although HIV has been isolated from tears, retina, cornea, aqueous humour, iris, and conjunctiva (Cantrill et al, 1988; Fujikawa et al., 1985a; Fujikawa et al, 1985b; Heck et al, 1989; Salahuddin et al, 1986), HIV from infected cadaveric donors has not been transmitted to cornea recipients (Pepose et al, 1987; Schwarz et al, 1987). This should not be surprising because the inoculum of HIV is small in the relatively avascular, hypocellular cornea as compared to that in an organ transplant or a blood transfusion from an HIV infected donor.

2.2. Hepatitis B virus Failure to transmit viral infection from hepatitis B surface antigen (HBsAg)-positive donors has been reported in two recipients of corneas. This suggests that the cornea is inefficient as a mode of HBV transmission, although in these cases the administration of hepatitis B immune globulin and vaccine to the recipient may have prevented infection (Raber and Friedman, 1987). Khalil et al. assessed the presence of HBsAg and HBV DNA in corneal buttons taken from HBsAg-positive donors (Khalil et al, 1995). They found HBV in a small percentage of corneas. Others studied 31 donors infected with HBV or HCV but were unable to detect HBV DNA or HCV RNA in the corneas (Sengler et al, 2001). Despite this inefficiency, HBV transmission by corneal transplantation has been reported. In earlier reviews by O'Day (O'Day, 1989) and Raber and Friedman (Raber and Friedman, 1987), there were brief reports of hepatitis


59

B transmission to cornea recipients from HbsAg-positive donors. Two cases of recipient HBV infection after transplants from two different HBsAg-positive donors were eventually reported (Hoft et al, 1997). Corneal donations took place from two donors; one in 1984 from an alcoholic man, and one in 1985 from an injecting drug user. Tests for HBV were performed on the donors retrospectively after recipients developed HBV infections. Both donors were positive for HBsAg. Only one of the two recipients of corneas from each of the two donors developed symptomatic HBV infection. The use of current professional standards and federal regulations would have prevented these cases since exclusion of donors with hepatitis risk behaviours, and testing for HBsAg, are now required. 2.3. Fungal disease Cornea transplants have transmitted fungal and bacterial infections (O'Day, 1989). Fungal growth can develop during storage of corneas in refrigerated liquid media containing bacterial antibiotics. Fungal contamination does not arise from a clinicallyapparent eye infection of the donor, but may originate as a contaminant on the eye surface, or acquired during collection, processing or storage. Clinically important fungal infections in recipients are rare but can be serious (Pels and Vrensen, 1999). Donor-to-recipient transmission of Torulopsis glabrata (Larsen et al, 1978; Cameron et al, 1991; 1998), Exophilia (Benaoudia et al, 1999), Cryptococcus neoformans (Beyl and Waltman, 1978), Aspergillus flavus (Cameron et al, 1991), Candida tropicalis (Behrens-Baumann et al, 1991), and Candida albicans (Insler and Urso, 1987; Stuart and Linn, 1984; Merchant et al, 2001; Sutphin et al, 2002; Cameron et al, 1991) have been reported. Merchant et al. reported that, in 40 of 44 cases, Candida albicans was cultured from both the recipient and the donor corneal rim (Merchant, 2001). In many of these cases, current donor selection practices would have excluded these donors, including a donor who died of polymyositis and was being treated with

60


immunosuppressive drugs and who transmitted Cryptococcus neoformans; and an alcoholic donor with disseminated Candida tropicalis, bronchopneumonia, and pancreatitis who had the same yeast cultured from his throat and from his organs postmortem (Behrens-Baumann et al, 1991). 2.4. Bacterial disease Post-transplant bacterial infection of the cornea can be serious and can lead to wound dehiscence, corneal perforation and emergency regrafting. In cases where the infecting organism is found in corneal tissue, or in the corneal storage medium, or where both corneas of one donor had transmitted the same bacterial infection to two different recipients, the likelihood that the bacterial infection was from the transplanted cornea is high. In two reported cases, both implicated donors would not have been accepted as donors using today's donor eligibility criteria, because of the presence of bilateral bronchopneumonia in one, and Hodgkin's disease, small bowel perforation, peritonitis, and high fever in the other (Gandhi et al., 1981). Leveille et al. reported four cases of bacterial endophthalmitis in 1,876 recipients of corneal transplants (Leveille et al., 1983). Three of these infections were from an organism that was also isolated from the donor cornea. It was found that the presence of sepsis in the cornea donor appeared to be a risk factor for bacterial transmission. Others found that mechanical ventilatory support of the corneal donor was not a risk factor by itself (Seedor et al., 1987). Some studies have not found a correlation between the use of a septic donor and recipient corneal infection, and propose that corneal grafts from septic donors be available for transplantation (Spelsburg et al., 2002). This proposal was supported by others who found that external bacteria on donor cornea are mainly skin bacteria; and internal bacteria are mainly gut bacteria. It is speculated that these gut bacteria arose from peri-mortem bacteraemia and not from an underlying infection of the donor (Robert et al, 2002). They observed that bacterial


61

infection in the donor at the time of death had no effect on the incidence of endophthalmitis in cornea transplant recipients. In some cases of bacterial endophthalmitis, it was difficult to determine whether the bacteria were from the donor, or acquired during surgical removal of the cornea (Moore et ah, 1989). Bacterial contamination is more likely with the use of suboptimal techniques during the surgical removal of the tissue from the cadaveric donor. In one programme, the occurrence of several post-transplant infections by Streptococcus pneumoniae prompted an investigation. After collection techniques were modified to reduce contamination, there were no further infections by the same microbe during the following year (Moore et ah, 1989). A corneal disinfection step with povidone-iodine can lessen the risk (Pels and Vrensen, 1999). Antonios et ah, reported that 0.23% of corneal transplants became infected due to bacterial contamination of the allograft during storage, and that the risk was highest if the cornea had been stored for five or more days prior to use (Antonios et ah, 1991). Bacteria on contaminated corneas can survive refrigerated storage in media containing antibiotics (Baer et ah, 1989; Glaros et ah, 1991). Recently, gentamicin has become a widely-used antibiotic for storage (Gopinathan et ah, 1994). Gentamicinresistant bacteria have been found in 14% of stored corneas, but generally have not resulted in recipient infections (Farrell et ah, 1991). Streptococcus, Propionibacterium, Staphylococcus and diphtheroids were commonly found. Recently, Khokhar et ah reported a bacterial infection from a corneal transplant from an organism which was resistant to gentamicin, which was the only antibiotic in the storage medium (Khokhar et ah, 2002). Failure of corneas to transmit the syphilis spirochete has been demonstrated in animal experiments in which corneas were transplanted from rabbits with latent or active syphilis (Randolph, 1952). The spirochete causing Lyme disease has been isolated from human cornea, but it is not expected to be a risk to cornea recipients because prospective corneal donors with active Lyme disease would be excluded. In addition, infected

62


donor corneas show infiltrative birdshot keratitis, and this would be discovered and be a cause for discard prior to use (SuttorpSchulten et al, 1993). 2.5. Other viruses Cytomegalovirus does not appear to be readily transmitted by cornea transplantation from seropositive cadaveric donors to seronegative recipients. Of the 25 seronegative patients receiving a corneal graft from a seropositive donor, only two seroconverted (Holland et al, 1988). Herpes simplex virus, type 1, has been found to be widespread in corneal stromal cells but only one case of transmission by a cornea allograft has been reported. The infection caused corneal deterioration in the recipient by the fifth day after transplant (Cleator et al, 1994). HSV DNA was found in two of five cornea allografts from other donors (Tullo et al., 1990). 2.6. Rabies Rabies virus infection in humans is often found in the cornea. Because of this, a cornea impression test has been useful for early diagnosis (Koch et al., 1975). Corneal allografts are also capable of transmitting rabies. Seven cases of fatal rabies transmission from cornea transplantation had been reported in the US, France, Thailand and India during 1979-1988, and in Iran in 1994 (Gode and Bhide, 1988; Centres for Disease Control, 1979-1981; Houff et al, 1979; Baer et al, 1982; Javadi et al, 1996). The first case involved a 39 year old man in the US with ascending paralysis (Centers for Disease Control, 1979), and the second involved a donor in France who died from paraplegia, encephalitis and myocarditis (Centers for Disease Control, 1980). In 1997 Javadi et al, and Gode and Bhide each reported rabies developing in two patients who received corneal transplants from the same donor. (Javadi et al, 1996; Gode and Bhide, 1988). Each of these cadaveric donors had an obvious acute neurological illness


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clinically consistent with rabies. National professional standards used by tissue banks today prohibit the use of these donors and would have prevented these cases of rabies transmission. 2.7. Creutzfeldt-Jakob disease The ability of the cornea to transmit Creutzfeldt-Jakob disease (CJD) has been reported in humans (Duffy et al, 1974), and confirmed experimentally using hamsters and guinea pigs (Manuelidis E. et al, 1977). CJD was transmitted by a cornea donated by a 55 year old man who died with bronchopneumonia following a two-month history of incoordination, myoclonic jerks, and neurological decline (Duffy et al, 1974). Cornea donors implicated in CJD or in rabies transmission had acute antemortem neurological illnesses with signs and symptoms such as fever, asymmetric weakness or paralysis, swallowing difficulties, ophthalmoplegia, diplopia, absent reflexes, abnormal sensation or coma. In all cases of rabies and CJD transmission, each donor was clearly not eligible to donate under criteria in place today, which exclude persons with acute and chronic neurological illnesses (AATB, 2001; Campagnari and O'Malley, 1994; Eye Bank Association of America, EBAA, 2001). Recent reviews again emphasise the importance of excluding prospective donors with neurological illness as a measure of preventing CJD (Hogan and Cavanaugh, 1995; Hogan et al, 1999). Overall, the risk of transmitting CJD by corneas is very low. 3. Infections Transmitted by Bone Allograft 3.1. Bacterial disease Bacterial infection due to use of a contaminated bone allograft has rarely been reported. Lord and associates (Lord et al., 1988) observed one of 283 massive frozen bone allografts to be a cause of a postoperative infection. Tomford and colleagues (Tomford et al, 1981) found one of 303 small freeze-dried bone

64


grafts from the Navy Tissue Bank to be implicated in a recipient Staphylococcus epidermis infection. These bacteria may have been donor-derived or may have been acquired at the time of procurement. Tomford and co-workers (Tomford et al., 1990) reported an infection rate of about 4 to 5% in use of 324 culturenegative, non-sterilised unprocessed frozen bone allografts at Massachusetts General Hospital. These rates of infection were not different from those found following surgery using prosthetic devices. Bone allograft appeared responsible for infections in only three patients, all of whom developed infection with Serratia marcescens and received bone from the same donor. Others have also reported bacterial infection as a complication of spinal surgery using bone allograft but, as a whole, the incidence was similar to that when using autograft (Aurori et al., 1985; Knapp and Jones, 1988; McCarthy et at., 1986; Transfeldt et at., 1985). Aho et al., reported two deep bacterial infections during use of 63 large allografts, apparently caused by transplantation of the unprocessed frozen large bone allografts (Aho et al., 1998). The same bacteria (Pseudomonas aeruginosa and Staphylococcus epidermidis) were isolated from the allograft prior to surgery and from the recipient site of infection. These two allografts had no growth on bacterial testing at the time of procurement but the bacteria were identified when the frozen allograft was thawed and retested at the time of surgery. These cases demonstrate the potential risk: even though the cadaveric bone donor may appear free of infection, bacterial contamination missed due to inadequate bacterial sampling or testing technique, can be a cause of infection. The bacterial infection risk appears to be similar to that of other major orthopaedic surgeries not using allografts. About 40 years ago, several cases of tuberculosis of the bone in patients undergoing spinal fusion were reported from the use of frozen rib allografts (James, 1953). The source of bone allograft was the rib resected during chest surgery from patients with active pulmonary tuberculosis. As one of the current donor screening criteria, individuals with active or previously treated tuberculosis are excluded from donating (AATB,


65

2001; Campagnari and O'Malley, 1994). This exclusionary criterion is important because there is no blood screening test for tuberculosis that offers a good predictive value.

3.2. Hepatitis Hepatitis has been reported from use of unprocessed refrigerated and frozen bone allografts, but not from bone grafts that were cleaned of cells and fat with water jetting and ethanol soaks prior to being freeze-dried or treated with sterilants such as gamma irradiation or ethylene oxide. In 1954, prior to the availability of viral hepatitis testing of donors, a Yale medical student received a refrigerated bone graft to treat a depressed fracture of the proximal tibia, and developed hepatitis with jaundice 10 weeks later (Shurkin, 1954). The bone graft was obtained from the amputated leg of a patient with occlusive vascular disease and gangrene. Otherwise, the donor was in good health, with normal liver function and without a history of jaundice or liver disease. The donor had received blood transfusions three years previously. Three reports from nearly a decade ago documented that hepatitis C virus (HCV) can be transmitted from donor-torecipient through the use of frozen, unprocessed bone allografts (Eggen and Nordbo, 1992; Pereira et ah, 1993; Conrad et al.r 1995). In the first case, donor testing for HCV antibodies was not available. HCV was transmitted by the use of a femoral head allograft after it was donated by a donor undergoing hip arthroplasty and stored frozen for two months (Eggen and Nordbo, 1992). In a second report, HCV was transmitted from an infected cadaveric tissue donor through frozen, unprocessed bone and tendon grafts, but not through freeze-dried bone allografts that were treated with gamma irradiation (Conrad et ah, 1995). In this study, the cadaver bone donor tested negative for HCV antibodies using the first generation test available at the time of donation in 1990, but stored serum tested positive when

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a new, more sensitive test was introduced in 1992. Testing for HCV RNA by polymerase chain reaction (PCR) was also positive. In a third brief report involving five HCV-infected organ and tissue cadaveric donors, a minority of the recipients of frozen bone allografts became infected with HCV (Pereira et al, 1993). In a more recent case an HCV-infected organ and tissue donor was tested and found negative for HCV antibodies (Tugwell et al, 2002). Despite this negative screening test for HCV, several organ and tissue recipients became infected. When blood samples from the donor were tested later for HCV,RNA, the results were positive, and this confirmed the link between the donor and multiple infected recipients. The donor had been recently infected and was viremic but had not yet produced detectable antibodies. Bone allografts from the same donor that had been treated with gamma irradiation did not transmit HCV. With the implementation of HCV RNA as a donor screening test in the future, cases such as this will be prevented. There have been no reports of HBV transmission through bone transplantation, although it has been recognized as a complication of organ, cornea, and heart valve transplantation. It is quite probable that there have been transmissions, but none have been recognised and published. 3.3. H u m a n immunodeficiency virus HIV-1 has been transmitted through blood, tissues, and organs (Petersen et al., 1993; Simonds et al, 1992). Viable HIV-1 can be recovered from bone, marrow, and tendons of patients with acquired immunodeficiency syndrome (Buck et al, 1990; Merz et al, 1991; Nyberg et al, 1990; Marthy and Richter, 1998). In 1984, a fatal HIV-1 infection was transmitted to a woman undergoing spinal fusion for scoliosis, through the use of a frozen femoral head allograft several weeks after it had been donated during hip arthroplasty from a donor who had a history of


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intravenous drug abuse and who had an enlarged lymph node that had been biopsied the previous year (CDC, 1988b). Both the donor and the bone allograft recipient subsequently died of AIDS. A test for HIV-1 antibody was not available at the time of donation. This donor would not have been eligible to be a donor today due to his history of intravenous drug abuse and lymphadenopathy. There have been other cases of HIV infection in recipients of bone allograft, derived from HIV-infected donors who were not tested for HIV at the time of donation. Prior to HIV antibody test availability in Germany, 12 recipients had had frozen bone allografts from an infected cadaveric donor during November 1984 through January 1985 (Schratt et al, 1996). Only four of these recipients became HIV-positive. Seven remained HIVnegative. In Taiwan a man donated a femoral head during hip replacement surgery but was not tested for HIV. The bone allograft was used in a 34 year old woman in 1996 during knee reconstructive surgery. She seroconverted with HIV antibodies when tested five months later (Li et al, 2001). Another reported case of HIV transmission through the use of frozen bone allograft involved a seronegative but infected cadaveric donor but the test was new and not very sensitive. Multiple organs, corneas, bones, and connective tissues were transplanted (Simonds et al, 1992). Three organ recipients and three recipients of frozen bone and tendon allografts became HIV-infected. These allografts had not been sterilised with gamma irradiation or ethylene oxide gas prior to use. The donor tested negative for HIV antibodies at the time of donation in October 1985, which was a few months after the first, relatively insensitive HIV-antibody testing kits became available. Between 1985 and 1991, several modifications greatly improved test sensitivity. Prior to 1989, HIV antibodies were detectable after a median of 63 days from initial infection (Bowen et al, 1988; Horsburgh et al, 1989). A study of HIV-infected blood donors between March 1987 and 1991, when whole viral lysate enzyme

68


immunoassays were used to detect HIV antibodies, showed an average seronegative window period of 45 days (Petersen et al, 1994). A report in 1992 showed that HIV-antibody test kits in use at that time detected twice as many infected individuals as did the test kits available in 1985 (Nowicki, 1992). Since 1992, HIV-antibody tests have become even more sensitive, detecting IgM, the earliest form of antibodies, earlier by an average of 8 20 days (Stramer et al, 1989; Zaaiger et al., 1992) and resulting in a seronegative window period of approximately 22 days (Busch, 1994; Busch et al., 1995). Since then, blood donor testing for HIV RNA by nucleic acid testing (NAT) has been implemented, and has further reduced the risk of a transfusion; and when validated and implemented for cadaver tissue donors, will reduce the risk in tissue transplantation (Stramer et al., 2000). The prevalence of HIV antibodies in bone donors is low, and when medical history screening and selection processes are applied vigorously, it should not be greatly different from that of voluntary blood donors. This may be true for living bone donors (Hamilton et al, 1990; Scofield et al, 1993a; 1993b) but not necessarily for cadaveric donors. Of 9,000 living bone donors who donated femoral heads at the time of hip arthroplasty surgery, none were found to have confirmed positive tests for HIV-1 antibodies at the time of donation (Hamilton et al, 1990). Prevalence of infectious disease markers in surgical bone donors was not different from that of blood donors, except for a higher prevalence of false positive syphilis tests and antibodies to HBV core protein (Scofield et al, 1993a). Retesting of 1,608 living bone donors 180 days later yielded none with confirmed positive HIV or HCV tests (Scofield et al, 1993b). Of 5,513 cadaver bone donors tested throughout the United States in 1992, three confirmed positive for HIV antibodies (Eastlund et al, 1994), but these three were from a single tissue bank that later disclosed accepting donors with risk factors for HIV. A more recent survey by AATB (AATB, 2000) revealed a higher prevalence rate of infectious disease markers than have been reported for blood donors, with rates ranging from two to 40

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Table 5. Positive infectious disease markers in tissue donors and blood donors. Test

Tissue donors*

Blood donors'1'

Anti HIV-1/2 HBsAg Anti-HCV Anti-HTLV-I/II Syphilis

0.4% 1.2% 1.5% 0.8% 1.0%

0.05% 0.03% 0.12% 0.12% 0.5%

'American Association of Tissue Banks. 18,021 cadaveric donors in 2000. (AATB, 2000) tPuget Sound Blood Centre. 182,138 blood donors in 2001. (Strong, 2002)

times higher (Table 5). The addition of viral nucleic acid testing since 1999, in screening blood donors for HIV and HCV, has further reduced the risk from blood donors. Among first time blood donors, there is a well known higher infectious disease marker rate, and such donors may be more similar to organ and tissue donors (Schreiber et ah, 2003). Because prospective tissue donors with HIV risk behaviours and positive tests for HIV are excluded, and most bone graft processing removes blood and marrow cells and applies disinfectants and sterilants, the risk of HIV transmission by bone transplantation is now very remote, or nearly absent (Asselmeier et at, 1993). The risk of transmitting HIV through bone grafting has been calculated to be less than one in a million grafts (Buck et ah, 1989; Carlson et ah, 1995), and is even less if the graft has been subjected to processing and sterilisation steps using gamma irradiation or ethylene oxide. However, the HIV transmission risk is higher in the less frequently used frozen unprocessed bone allograft. An accurate estimate of the risk cannot be made until a more accurate determination of the prevalence of HIV infection in the donor and recipient population is available, and prospective studies have been done on recipients.

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3.4. H u m a n T-Lymphotrophic virus Asymptomatic HTLV-I infection has been transmitted by transplantation of a fresh-frozen unprocessed femoral head bone allograft (Sanzen and Carlsson, 1997). A 62 year old man became infected by HTLV from a blood transfusion in 1987 during hip surgery. One month later he developed fever, a rash and a transient right-sided radial nerve palsy. Frozen sera obtained during this illness (but tested later) demonstrated HTLV seroconversion. In 1991 he donated a femoral head without antiHTLV testing during a second surgery for a hip prosthesis. The unprocessed frozen femoral head was used as a graft in a different patient one month later. This bone graft recipient developed HTLV-I antibodies but had no HTLV-I associated disease. 4. Infections from Cartilage and Osteochondral Allografts Costal cartilage allografts are routinely disinfected or sterilised prior to their use as allografts, and, provided in a freeze-dried or frozen form. There have been no reports of processed costal cartilage transmitting infection from the donor to the recipient. Donald and Cole surveyed 312 surgeons who used cartilage allografts preserved by eight different methods for facial reconstructive surgery (Donald and Cole, 1982). They found a postoperative bacterial infection rate of 19% that was similar to the 16% reported following use of autologous cartilage. 4.1. Bacterial infection Until recently, the use of articular cartilage, e.g. in the form of a fresh or frozen unprocessed femoral condyle, has not been a significant risk of transmitting bacterial infections. An articular cartilage allograft, used in knee surgery, is minimally processed to maintain cartilage mechanical properties and chondrocyte


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viability, although it is not known whether chondrocyte viability is important. Thus, the allograft is often only briefly exposed to antibiotic solutions, and frozen with or without a cryoprotectant such as dimethyl sulfoxide. These cartilage allografts that have not been exposed to disinfectants or sterilisation as a final step, are capable of transmitting bacterial infection. In the US during 2001, a fatal bacterial sepsis case involved an osteochondral allograft (femoral condyle) and led to an investigation linking the fatality to an allograft contaminated by Clostridium (CDC, 2001a; 2001b; 2002a). In this case a 23year old male patient had surgical-site pain three days after a knee allograft implant procedure, leading rapidly to hypotension and death 24 hours later. The cartilage allograft had been aseptically processed, packaged and distributed by a for-profit tissue processing facility that is not accredited by AATB. A thorough investigation by the CDC and US FDA revealed several factors that contributed to the distribution and use of the contaminated allograft (CDC, 2002a) (Table 6). Bacterial testing at Table 6. Contributing factors leading to contaminated osteochondral allograft and fatal Clostridium sepsis.* Donor body not refrigerated until 19 hours after death — procurement 23.5 hours after death 1) The tissue processing facility did not obtain pre-processing bacterial testing of tissues. 2) The tissue was processed aseptically without use of disinfectants or terminal sterilisation. 3) Antibiotic soaks were used, and antibiotic residue may have interfered with detection of Clostridium. 4) No bacteriostasis testing was performed at the time of final sterility testing. 5) Processing steps were not validated to achieve an acceptable level of sterility assurance. 'Centres for Disease Control, MMWR 2002, 51 (March 15), 207-210.

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the time of removal from the cadaveric tissue donor, and prior to processing in the tissue facility, was not performed. AATB standards require this step to identify virulent microbes that might not be eliminated despite antibiotic soaking during processing (AATB, 2001). The cadaveric donor body was stored at room temperature for 19 hours, and at refrigerated temperatures for an additional 4.5 hours prior to tissue removal. National professional standards limit the storage of the cadaveric body to 12 hours or less if stored at room temperature (AATB, 2001). If refrigerated, the tissue can be surgically removed from the donor u p to 24 hours after death. C. sordelli was also found in fluid bathing the tissues during processing. The tissue was processed aseptically with antibiotic soaks but included no other disinfectants or sterilants. Antibiotic residues may have interfered with detection of C. sordelli during final sterility testing. No bacteriostasis testing was performed by the tissue processing facility to determine whether antibiotic residues caused falsely-negative final sterility test results. Tissue processing had not been validated to achieve an acceptable level of sterility assurance. C. sordelli was found in two of 19 other unused tissues from the same donor. Two of nine patients who received other tissue allografts from the same donor became infected (CDC, 2002a). The tissue processing facility was prohibited by the US FDA from further processing and distribution until it agreed to make required changes, including following AATB standards. The investigation of this case led to the discovery of Clostridium infections in several other recipients of frozen tendons provided by the same tissue processing facility (CDC, 2002a). 4.2. Ear ossicles Tympanic membrane and middle ear ossicles are transplanted to restore hearing, but there have been no reports of disease transmission (Lesinski, 1977). The bacteriologic sterility of ear ossicles after extraction from a cadaveric donor, is unlikely. These tissues


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are frequently obtained after an intracranial autopsy, and the tympanic membrane is normally exposed to the external environment and frequently colonised with bacteria. Consequently, treatment with disinfectants or sterilants during processing is standard practice (Glassock et al., 1988). 5. Infections Transmitted by T e n d o n Allograft 5.1. Viral diseases The use of the patellar tendon allograft to replace the knee's injured anterior cruciate ligament has become commonplace (Noyes et al., 1990). HIV has been isolated from tendons in HIV-infected persons (Buck et al, 1990; Buck and Malinin, 1994) and has been transmitted from a seronegative cadaveric tissue donor through a donated patellar tendon used in knee surgery (Simonds et al, 1992). HCV was transmitted to recipients of frozen tendon allografts from an anti-HCV positive cadaveric donor (Conrad et al, 1995). It is possible that HIV and HCV were harboured in the unprocessed bone blocks at either end of the tendon allograft. These allografts had not been processed to remove blood and marrow cells. Despite these cases, the risk to recipients is presumably low as long as donor screening steps are applied as required by national standards (AATB, 2001; Campagnari and O'Malley, 1994) and federal regulations (FDA, 1993; 1997; 1999). In addition, tendons can be treated with gamma irradiation to further reduce the risk of disease transmission. Selecting donors without risk factors and without HCV antibodies, makes the risk of spreading HCV by transplant an exceedingly rare event. However, an early HCV infection in a cadaveric organ and tissue donor not yet producing antibodies, was reported recently (Tugwell et al., 2002). A patellar tendon allograft recipient developed acute, symptomatic hepatitis C in May 2002, six weeks after transplantation. No other potential sources of infection were identified. The tissue donor was

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anti-HCV negative but stored serum showed HCV RNA when tested later. Thirty-nine other persons received tissues or organs from this same donor. Early results of a partially completed investigation showed that of 18 recipients tested, six showed HCV infection, including a lung recipient who became HCV RNA positive on day 4 and died of liver failure 14 months later. Presumably, the cadaveric donor was in a viremic stage early in infection prior to antibody development. To date, cadaveric testing for HCV RNA is not available for routine use. HCV RNA should be considered for cadaveric tissue donors as soon as test reliability has been evaluated, particularly using cadaveric samples obtained up to 24 hours after death. 5.2. Bacterial infection Recently, there have been several reported cases of bacterial infection arising from the use of contaminated patellar and Achilles tendon allografts. These cases were primarily a cluster of Clostridium infections in connective tissue provided by a single tissue processing facility. After a fatality due to contaminated cartilage allograft (CDC, 2002a) the resulting investigation of the tissue bank and tissue recipients found Clostridium infections in several other tendon recipients, thus prompting changes within the tissue processing facility to prevent further cases. Several other non-clostridial infections were subsequently discovered in patients who had received tendons from other tissue banks, but only a few were demonstrated to be caused by the tendon allograft (CDC, 2002a). Two cases involved non-clostridial bacterial infections in patients who received frozen tendon allografts for anterior cruciate ligament repair, and in these cases a tissue bank distributed the contaminated allografts due to human error (CDC, 2001c). The intended terminal sterilisation by gamma irradiation did not take place. Another case involved pseudomonas infection in two recipients using frozen tendon allografts derived from the same donor, suggesting that the tendon caused the infection


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(CDC, 2001c). However, the source of the bacteria was not determined. An independent investigation later revealed that the tendons had received sterilisation treatment with 1.8 Mrad gamma irradiation (Eastlund et ah, 2003). Because the pseudomonas bacterium is exquisitely sensitive to gamma irradiation, the tendon seemed unlikely to be the source, and acquisition of the contaminant during the surgical procedure needed to be more thoroughly considered as the potential cause. A recent investigation of suspected tendon allograft-related infections was made at six US tissue banks that process bone and connective tissue allografts (Eastlund et ah, 2003). Of the 27 suspected cases, only five bacterial infections were determined to be probably caused by the allograft: a contaminated patellar or Achilles tendon in three cases; an osteochondral allograft in one case; and aseptically processed cancellous bone chips in another case. In each of these five cases, investigators found evidence of the same infecting organism either in samples of tissue taken at the time of surgical removal from the cadaveric donor, or in sampling during final sterility testing at the end of tissue processing by the tissue bank. Subsequently, these tissue banks implemented procedures to prevent use of tissue with evidence of virulent organisms found at the time of tissue procurement or at final sterility testing. Of 26 other cases of musculoskeletal allograft-associated bacterial infections investigated by the CDC, data was not provided to confirm that the allograft was the cause. Thirteen cases involved Clostridium infections, and eleven of the implicated allografts — mostly frozen tendons — were distributed by a single tissue processing facility (CDC, 2002a). As a consequence of this, the US FDA implemented an emergency federal regulation requiring that allograft processing facilities have written procedures that are validated for the prevention of infectious disease contamination (or cross-contamination) by tissue during processing (FDA, 2002). It also required maintenance of records concurrently with the performance of each significant step.

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Validation data must demonstrate that procedures reliably prevent infectious disease contamination during processing. 6. Infections Transmitted by Cardiovascular Allograft 6.1. Viral disease The capacity of human heart-valve allografts to transmit HBV was demonstrated in a study of 31 patients who received heart valve allografts from HBsAg-positive donors. Twentytwo recipients were HBsAg-positive prior to transplant, or were immune to HBV and not susceptible to HBV infection. Of the nine recipients susceptible to HBV infection, only one developed HBV viral markers. None developed clinically apparent hepatitis. However, four susceptible recipients received hepatitis B immune globulin, and one received HBV vaccine following transplant, which may have prevented infection (Morris et al., 1990). Currently, all donors are tested for HBsAg, and if positive, are excluded. Despite testing of donors for HBsAg and anti-HBc, HBV transmission can still occur because some donors can have circulating HBV at levels not detectable in routine tests. Thijssen et al., found one of 676 heart valve allograft donors to have HBsAg detectable with routine screening tests (Thjissen et al, 1993). In addition, they found 10% to have anti-HBc. Fifty-two of 63 donors with anti-HBc also had antibodies to HBV surface protein (anti-HBs), indicating a resolved HBV infection and a recovered, immune non-infectious status. Three of those with anti-HBc but without anti-HBs were positive for HBV DNA via a more sensitive liquid-phase DNA hybridisation assay. This would suggest a possible use of anti-HBc donor testing to prevent transmission of HBV; however, one study of blood donors has shown a lack of predictive value in preventing post-transfusion hepatitis (Blajchman et al, 1993). More recently, however, several reports have confirmed that some anti-HBc positive donors will be positive for HBV DNA and will transmit HBV (Roth et al, 2002).


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6.2. Mycobacterium and other bacterial disease Postoperative bacterial endocarditis is a complication of cardiac valve replacement surgery, occurring at similar rates for valves of human, porcine or mechanical origin. There have been several reports that implanted heart valves may be capable of transmitting mycobacterium of donor origin. Several cases were reported of the transmission of non-tuberculous mycobacterium by use of contaminated non-human prosthetic heart valves. In one report, a patient manifested a Mycobacterium chelonei infection five months after implantation of a porcine xenograft heart valve (Tyras et ah, 1978). The valves used in this patient, and in seven others, were sampled prior to implantation, and the same bacteria grew from all. Only one patient had a clinical infection. These porcine xenograft valves apparently became contaminated with M. chelonei during production at the manufacturer. Another patient who received a prosthetic valve from the same manufacturer during the same time period also developed M. chelonei endocarditis, but the infection was not evident until three years later (Rumisek et ah, 1985). A total of four patients were reported as getting infected by M. chelonei from the same manufacturer's contaminated prosthetic valves. Khanna et ah, reported a case of miliary tuberculosis developing 8 months after receiving a human valve allograft, and concluded that the infection arose from the transplanted valve (Khanna and Monro, 1981). Anyanwu et ah, reported seven cases of miliary tuberculosis among 934 patients receiving human heart valve allografts (Anyanwu et ah, 1976). There was unconfirmed suspicion that the source of the infection was from the implanted valves. Of the seven cadaveric donors implicated, all were over 60 years of age; two had a history of treated tuberculosis, and one had active pneumonia at the time of death. Each of these donors would have been excluded from cadaveric donation of heart valves due to age or medical history, under today's donor criteria (AATB, 2001; Campagnari and O'Malley, 1994).

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The addition of antibiotics effective against tuberculosis in the processing steps, along with routine culturing of donor heart tissue for Mycobacterium, and excluding prospective donors with a history of treated tuberculosis, all contribute to today's low risk of transmitting tuberculosis by heart valve transplantation (Hopkins, 1989; Yankah et al, 1988). 6.3. Fungal disease There has been a recent well-documented case of fungal infection transmitted by heart valve allograft (Kuehnert et al, 1998). A 61 year old male with long standing aortic insufficiency was given a human cryopreserved aortic valve and was discharged doing well after five days. Sixteen days later, he had high fevers, nausea and diarrhoea. An infected valve allograft was removed. Candida albicans was found in his blood, on valvular vegetations and from an intramyocardial abscess (Kuehnert et al, 1998). An investigation of the tissue processing facility's records revealed that the same yeast with similar DNA fingerprinting had been identified from a sample obtained prior to processing the valve. The heart valve had been temporarily soaked in antibacterial solutions (imipenem, netilmicin, vancomycin) and antifungal solutions (amphotericin B, fluconazole) during processing. After processing of the valve, a final sterility test showed no growth but fungistasis procedures were not performed to assure that residues of antifungal solutions did not interfere with identifying microbial contamination (Kuehnert et al, 1998). Although the fungus isolated at the tissue bank was identical to that from the patient, the latter was more resistant to amphotericin and fluconazole. According to Joly and Carbon (Joly and Carbon, 1998) the resistance was induced during in vitro disinfection at the tissue bank that was using these two antifungals. These authors concluded that since fungal endocarditis is severe, and that the disinfection process of soaking the valve in an antimicrobial solution containing amphotericin B and fluconazole can be ineffective, it is necessary to discard the


79

allograft if fungal contamination is detected from samples taken prior to processing. This underscores the importance of performing tests for fungal contamination prior to processing, a practice routinely carried out by AATB-accredited heart valve banks, but not by the tissue processing facility that was involved with this patient's Candida endocarditis. In addition, the authors recommended avoiding disinfection of fungal contamination because of the lack of effectiveness; the severity of fungal infection in the presence of a foreign body; and the risk of inducing resistance to the anti-fungals used (Joly and Carbon, 1998). 7. Infections Transmitted b y Pericardium Allograft There was one case of CJD reported in a recipient of human pericardium allograft used to reconstruct a tympanic membrane (Tange et al, 1990). Transmission from the donor was not confirmed. Three cases of post-transplant bacterial meningitis due to Ochrobactrum anthropi were caused by the use of human pericardium allograft to patch dural defect (CDC, 1995; Chang et al, 1996; Christenson et al, 1997). The bacteria was not of donor origin but was acquired during processing. The organism was also found in an unused pericardium allograft from the same donor, and in an unused bottle of Hanks Balanced Salt Solution from the same lot that was used for processing. 8. Infections Transmitted by Dura Mater Allograft Freeze-dried dura mater allografts have been used for closure of dural defects caused by resection of brain tumour, or repair of traumatic injury. Long-term follow-up of 1,364 dura allografts donated, processed and used in the United States and sterilised either with ethylene oxide (Malinin et al, 1978; Prolo, 1981; Rosomoff and Malinin, 1976) and 804 dura grafts sterilised with gamma irradiation (Cantore et al, 1987) revealed no infections attributable to the graft. Surgeons have reduced their use of dura

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allograft in favour of autologous fascia because of concern for transmitting CJD. 8.1. Creutzfeldt-Jakob disease Creutzfeldt-Jakob Disease (CJD) occurs at a rate of one per million in the general population, and is a fatal, transmissible, dementing spongiform encephalopathy usually accompanied by myoclonus and characteristic electroencephalographic abnormalities. It is caused by a prion, a small proteinaceous infectious particle. CJD has been transmitted from cadaveric donors through the use of pituitary-derived growth hormone and gonadotrophin (Brown, 1990; Brown et al, 1985; Cochius et al, 1990), cornea (Duffy et al, 1974), pericardium (Tange et al., 1990), and dura mater allografts (CDC, 1987b; 1987c; 1989; 1993; Lane et al, 1994; Masullo et al, 1989; Thadani et al, 1988; Yamada et al, 1994). Many cases of CJD were transmitted by use of dura allograft that was obtained from the same German medical device manufacturer, and transplanted worldwide mainly in 1984 and 1985 (Yamada et al, 1994; Lang et al, 1995). Several factors may have contributed to this outbreak, such as pooling of dura from many different cadavers during processing, and possibly obtaining dura from individuals who may have been demented. Other than dura, cornea and possibly pericardium, there have been no reports of CJD from use of other cell, tissue, or organ allografts. Because CJD is resistant to the usual sterilants, and there is no blood test for CJD, tissue banks have adopted donor selection procedures to exclude those who have received pituitary-derived growth hormone or dura allograft, or have had dementia or other symptoms of neurological disease, or have a family history of CJD, and those who have been diagnosed with or are suspected to have CJD (Table 7). The importance of excluding prospective donors with dementia alone, is demonstrated by studies showing that 18% of 230 patients with CJD initially presented with dementia as an isolated symptom (Brown et al., 1986), and

Infectious Disease Transmission Through Tissue Transplantation Table 7. Donor exclusions for CJD and nvCJD risk behaviour. 1) Persons with a diagnosis of CJD or known family history (blood relative) of a person with non-iatrogenic CJD. 2) Persons with a history of dementia or degenerative neurological disorder of viral or unknown cause. 3) Persons who have received injections of human pituitary derived growth or gonadotrophin hormone. 4) Persons who received transplants of human dura mater.

5% of those diagnosed with Alzheimer's disease were shown to have CJD at autopsy (Boiler et ah, 1989). National professional standards in the United States prohibit pooling of tissue from multiple donors and during processing, and prohibit accepting donors who have received human pituitary derived growth hormone (AATB, 2001). This is important because the hormone has transmitted CJD when used to treat short stature, and because the latency period between acquisition of the infection and development of the disease can be over a decade (Preece, 1983). With the current donor screening practices used by tissue banks, it seems unlikely that CJD will pose a serious threat to the safety of organ, tissue, or cell allograft use. The CJD agent is surprisingly resistant to the usual sterilants and disinfectants, but is fairly susceptible to sodium hydroxide (Brown et ah, 1984; Kearney JN and Johnson C, 1991). Some tissue banks have added sodium hydroxide exposure during dura processing, but have not adopted it for bone or other tissue allografts. The use of sodium hydroxide has not been widely adopted because the prevalence of CJD is very low in the general population, and even lower in the carefully screened tissue donor selected to be free of dementia, other neurological symptoms, or growth hormone treatment. Sodium hydroxide solubilises proteins, and it is not known whether it penetrates dense bone, impairs mechanical or osteoinductive properties of bone allograft, or persists in bone after processing (Kearney and

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T. Eastlund & DM.

Strong

Johnson, 1991). Due to its toxicity to cell viability, sodium hydroxide treatment is not suitable for viable tissue, cell or organ transplants. 9. Infections Transmitted by Skin Allograft 9.1. Bacterial infection Skin allograft — cryopreserved in glycerol — has been reported to carry bacterial infection (Monafo et ah, 1976). A frozen skin allograft contaminated with Pseudomonas aeruginosa was applied to a 5-year old burn patient, causing a severe infection with high fevers and Pseudomonas sepsis. In two other patients, contaminated skin allograft caused Herellea burn wound infection. National professional standards currently in place require bacteriologic sterility testing of frozen skin allografts, mandating disposal if virulent bacteria are found (AATB, 2001). In contrast to cryopreservation procedures, where skin is usually processed within 24 hours of recovery and stored at -80 degrees C or colder temperatures, skin allograft can also be stored for several days in liquid antibiotic solutions at refrigerated temperatures. At the end of the storage period, and prior to application on burned patients, the skin is often bacterially tested. Kealey reported that during 10 years of a skin bank's operation, positive results arose in only two samples of skin stored for many days at a cold temperature, in a solution of gentamicin and penicillin (Kealey, 1997). These test results, available after clinical use of the skin allograft, have sometimes shown the presence of virulent bacteria. Despite occasionally transplanting skin with virulent bacteria, clinically significant infections caused by the allograft are rare. In one small study, refrigerator-stored skin was cultured immediately prior to use, and virulent bacteria was found in a few. No adverse clinical outcome was found in recipients (Clayton et al.r 1995). Others store skin allografts in 85% glycerol solution. Of these skin allografts, 10% initially had bacterial contamination, mostly due to Staphylococcus epidermidis; but after


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prolonged storage in glycerol, bacteria were no longer detectable (Van Baare et al, 1998). 9.2. Viral infection Viral disease transmission by skin allografts has been reported. Epidermal cells can be infected with HIV-1, and the epidermis of HIV-infected individuals can transmit HIV to white cells in vitro (Berger et al, 1992; Gala et al, 1997). In one study, HIV RNA was found in only one of 12 infected patients (Kanitakis et al, 1982). Clarke reported, in a brief letter, a weakly positive test for antibody to HIV-1 in a burn patient after receipt of skin from an HIV-positive donor (Clarke, 1987). The results of donor testing were not known before the skin was used. The authors did not report whether other recipient risk factors were present, or supply the results of confirmation testing. HIV transmission from skin allograft has been recently reviewed (Van Baare et al, 1997; Pirnay et al, 1997). Transmission of hepatitis from skin allograft has not been reported although HCV nucleic acid has been demonstrated in skin from infected donors (Conrad et al., 1995). There are recent reports implying transmissibility of human cytomegalovirus (CMV). Animal models clearly demonstrate that skin grafts are capable of transmitting CMV (Abecassis, 1994; Abecassis et al., 1993; Shelby et al, 1988; Shelby and Stanley, 1987). Earlier studies by Kealey et al, showed that burn patients acquire CMV during hospitalisation, and that blood transfusions may be a contributing factor (Kealey et al, 1987). A subsequent study eliminated blood transfusion as a contributor, by studying patients who received skin allografts but no CMV-positive blood. The study showed that CMV-negative burn patients who receive skin allografts from CMV-positive donors can seroconvert to become CMV-positive (Cederna et al, 1994). CMV resides in peripheral blood leukocytes in asymptomatic CMV antibody-positive donors, long after their initial infection. Asymptomatic CMV-positive donors can transmit CMV infection

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through transfusion and transplantation if the recipient is CMV negative. Most healthy adult prospective donors have CMV antibodies: therefore, excluding CMV positive donors to prevent CMV transmission would exacerbate the already existing shortage of skin allografts, and would not be practical. Testing donors for CMV antibody is not required by national professional standards. Immunosuppressed individuals such as organ recipients have a high mortality and morbidity rate from transplant-transmittedCMV of donor origin. The burned patient also acquires CMV infection but does not generally experience serious complications as regularly as organ recipients, perhaps because burn-related immunosuppression may be less profound than that produced by drugs used to prevent organ rejection. As burn patients begin to receive potent immunosuppressants (such as cyclosporine) to block rejection of skin allografts, CMV may become a more serious complication of burn care and related blood transfusion and skin allografting. Further studies of skin allograft recipients are needed to determine whether transmission of CMV by skin allograft is associated with symptomatic disease as seen in organ transplantation recipients, or whether the infection is asymptomatic, as generally seen in transfusion-transmitted CMV infections in immunocompetent blood transfusion recipients. Prior to knowing the outcome of these studies, it is premature to assume that it is beneficial to base selection of skin donors on CMV antibody testing. 10. Risk of Transmitting Other Diseases 10.1. Syphilis There have been no reports of syphilis transmission via organ, tissue or cell transplants — probably due to the low prevalence of infection in the screened donor population; frequent use of antibiotics during tissue processing and storage; the absence of the spirochete in many tissues and cells; and low temperature


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storage of allografts, which kills or suppresses growth of the spirochete. Because Treponema pallidum, the agent causing syphilis, does not survive when stored at 4° beyond 48-72 hours (Barnes, 1992), transmission of syphilis by tissue allografts is not expected. Most transplanted tissues are initially stored refrigerated or frozen for several days prior to processing and use. Syphilis could possibly be transmitted if the tissue allografts were used shortly after donation, and if the donor were spirochetemic. Donor testing for syphilis has major limitations, and will not reliably detect spirochetemia. During the short early period of spirochetemia at initial infection with syphilis, the syphilis screening test is negative. Most, but not all, tissue banks perform the syphilis screening tests on donors despite its uncertain value (AATB, 2001; Campagnari and O'Malley, 1994; Strong et al., 1991). Most positive syphilis screening tests in donors are falsely positive. Organs and tissues have been transplanted in the face of positive donor syphilis tests without transmitting the disease (Gibel et al., 1987). Professional tissue banking standards permit use of tissues from donors with falsely positive syphilis screening tests: reactive nontreponemal tests with nonreactive treponemal confirmation tests (AATB, 2001; Campangnari and O'Malley, 1994). 11. Emerging Infection Risks Infection risks of tissue transplantation have usually been identified after first being recognised as blood transfusion-transmitted infections. Many real or theoretical risks of tissue transplantation can be considered by looking at the emerging infections that threaten to affect transfusions (Chamberland, 2002; Strong and Katz, 2002). Most recently, West Nile Virus (WNV) infection has swept through the United States with nearly 4,000 human cases identified, and 254 deaths in 2002 (CDC, 2002b). In addition to being mosquito-borne, WNV has been transmitted through organ transplantation, blood transfusions, transplacental intrauterine spread, and percutaneous route from laceration and needlestick


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(CDC, 2002b). There are, on average, four malaria transmissions annually in the US (Mungai et al, 2001). Approximately 40 cases of transfusion transmitted babesiosis have been reported from parasitemic blood donors (Dobroszycki et al., 1999). Trypanosoma cruzi transmission from transfusion has been newly reported (Leiby et al., 1999). Parvovirus B19 has been transmitted to a few recipients of plasma derivatives, red cells, platelets and solvent-detergent-treated plasma (Koenigbauer et al., 2000). 12. Reducing the Risk Through Donor Selection To minimize the risk of transmitting infectious disease, several important approaches are taken by transplanting surgeons and tissue banks. An initial approach by the surgeon is to judiciously use tissue allografts, and from accredited organisations; use sterilised allografts whenever possible; and consider use of autografts and alternative non-human graft material. However, the most important steps are exercised by the tissue bank in excluding those prospective donors suspected to be at risk for HIV, hepatitis (Table 8), CJD (Table 7) and bacteriologic and Table 8. Donor exclusions for Hepatitis and HIV risk behaviour. HIV and Hepatitis Risk Behaviour Exclusions 1) 2) 3) 4) 5) 6) 7)

8) 9)

Persons with clinical or laboratory evidence of HIV infection. Men who have had sex with other men even once in past five years. Nonmedical injections of drugs in past five years. Persons with haemophilia or related clotting disorders, who have received human-derived clotting factor concentrates. Persons who engaged in sex for money or drugs in past five years. Persons who have had sex with any of the above in past 12 months. Exposure to blood that is suspected to be HIV- or hepatitis-infected through percutaneous inoculation of open-wound or mucous membrane contact in past 12 months. Inmates of prisons for at least seven days in past 12 months. Tattoo received in past 12 months.


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fungal infections. Tissue transplantation is generally considered a non-urgent surgical procedure, permitting a careful tissue donor selection process. Tissue donor selection by tissue banks has evolved to include a direct interview with the donor's next-ofkin concerning the donor's medical history and risk behaviours for HIV and hepatitis, along with blood infectious disease testing, a physical examination, and the results of an autopsy examination, if performed (Table 9). These donor selection steps are essential activities that result in a low risk of transmitting disease. In addition, many tissue allografts can undergo further processing and exposure to antibiotics, disinfectants or sterilants (Table 10), all of which further reduce the hazard of disease transmission (Asselmeier et ah, 1993). Although there have been no carefully-controlled prospective studies of allograft recipients to determine the incidence of disease transmission, there is good reason to believe that established donor screening procedures, infectious disease testing and the effectiveness of processing and sterilisation to reduce or eliminate bacteria and virus, results in a very low risk of transmitting disease. Donor blood testing, physical examination, autopsy reports, and surgical removal through aseptic technique and use of sterile disposable supplies, are further aids in providing tissue with a low risk of transmitting disease. Lastly, sterilisation of certain tissues can be performed, and is very effective. The bone graft disinfection and sterilisation step most often used by bone banks in the United States are gamma irradiation at doses of 15 to 30 kGy (Strong et ah, 1995). 12.1. Donor selection One important contribution to recipient safety is to seek voluntary, non-remunerated donors. Monetary inducement to the nextof-kin of cadaveric tissue donors is prohibited by professional standards (AATB, 2001), but it is being considered as a means of reducing the severe organ supply shortage in the United States (Peters, 1991). Monetary reimbursement of semen and oocyte

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donors is not prohibited by national professional standards (AATB, 2001) or governmental guidelines (CDC, 1988a; 1994) or regulations (FDA 1993; 1997; 1999), but is considered necessary by most US reproductive tissue banks in the United States. Monetary payment to donors for blood donation increases the risk of disease transmission (Eastlund, 1998b). Monetary incentives to donate may cause donors or their next of kin to be untruthful about the donor's health history information, and to donate when they should not. Published data clearly show a 5-10 times increase in incidence of donor-to-recipient posttransfusion hepatitis B virus infection with the use of paid blood donors (Eastlund, 1998b). In addition, there is an 11-15 times increase in prevalence of HCV antibodies, and 3-14 times increase in prevalence of HIV antibodies in paid blood donors compared to voluntary blood donors (Eastlund, 1998b). HCV RNA was detected more often in clotting factor concentrates derived from paid donor plasma than volunteer donor plasma (Markris et al., 1993). Transfusion transmitted malaria has been documented from a paid donor who lied about a history of malaria to sell his blood (Guerrero et al., 1983). To minimize the risk of transmitting infectious disease, tissue donor eligibility requirements have been set by national professional standards (AATB, 2001; Campagnari and O'Malley, 1994; EBAA, 1994; EATB, 2000), United States Public Health Service guidelines (CDC, 1988a; 1994), and United States federal regulations (FDA, 1993; 1997; 1999). Donor selection is an important first step taken to ensure that the resulting allograft is safe and effective. The cadaver tissue donor selection process includes a donor's medical and social history obtained from the next-of-kin and medical care providers; blood tests; a physical examination performed by tissue bank personnel; and an autopsy, if performed (Table 9). Preliminary donor selection is based on the donor's medical history and circumstances surrounding death. Donors are excluded if elements of the past medical and social history or recent hospitalisation indicate a risk of infection, malignant disease, or inadequate quality of donated organ or

Infectious Disease Transmission Through Tissue Transplantation Table 9. Selection steps to prevent disease transmission from cadaver tissue donor. Voluntary donation without monetary inducement Health History Review • Review of medical records. • Interview of next of kin. • Exclusion of those with infection, malignancy. • No human pituitary-derived growth hormone. • No HIV, hepatitis risk behaviour. • No nvCJD risk behaviour. Blood Tests • Hepatitis B surface antigen. • Antibody to HIV-1, HIV-2, HCV. • Antibody to HTLV-I, HTLV-II, syphilis.* • Blood culture (optional). • Antibody to HBV Core Antigen (for living donors only).* Physical Examination • Unexplained jaundice. • Evidence of injectable drug use. • External signs of infection, including HIV. Autopsy Examination (if performed) • Exclude those with infection, malignancy. Maternal HIV testing and risk factor exclusion if donor < 18 months old (56). *AATB, EBAA, United Network for Organ Sharing and American Red Cross requirements. *AATB requirement.

tissue. The living donor, the legal c o n s e n t i n g next-of-kin or life p a r t n e r of a cadaveric donor, or both, m u s t be directly interviewed to d e t e r m i n e w h e t h e r HIV, hepatitis or CJD risk b e h a v i o u r s are p r e s e n t (Tables 7 a n d 8). Persons w i t h HIV a n d hepatitis risk b e h a v i o u r s are e x c l u d e d from d o n a t i o n . A g e criteria are set to e n s u r e functional quality of the d o n a t e d graft.

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For example, donated bone from a very young donor may not be satisfactory for use in load bearing graft applications due to the presence of nonmineralized epiphyseal growth plates. The presence of many other tissue and organ specific exclusionary criteria, such as chronic corticosteroid use and advanced age (which can weaken donated bone), or diabetes indicating a non-functional pancreas, will exclude donation of that part, but not necessarily other grafts from the same donor. The tissue bank physician makes the final determination of suitability of a cadaveric tissue donor as required by national professional standards (AATB, 2001; Campagnari and O'Malley, 1994). 12.2. Physical examination of donor The next screening step is a limited physical examination of the tissue donor by procurement staff at the time of cadaveric donation (AATB, 2001; Campagnari and O'Malley, 1994). The body is examined for signs of injecting drug use, and signs of HIV, hepatitis or other infection or trauma over bodily sites that can affect the quality of donated tissue. 12.3. Blood testing of donor Donor blood testing for disease markers plays an important role in reducing the risk of disease transmission. By eliminating prospective donors with infectious disease risk factors prior to blood testing, the risk of a seronegative but infected donor is minimized. Testing for HBsAg, anti-HIV and anti-HCV is required by federal regulations (FDA, 1993; 1994; 1999) and national professional standard setting organisations (AATB, 2001; Campagnari and O'Malley, 2002; EBAA, 2002). Other tests required by standard setting organisations are syphilis and anti-HTLV-I/ II (AATB, 2001; Campagnari and O'Malley, 1994; EBAA, 2002). Blood cultures are not required, but are commonly performed for cadaveric tissue donors to aid in determining whether the donor is infected, particularly if the donor was hospitalised, and


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ventilation was mechanically assisted. Their predictive value is very uncertain, but can be used to augment bacterial testing of tissues. HIV antigen (p24 antigen) testing of the donor is not performed by most tissue or organ banks. Large scale studies of low risk (Alter et al, 1990) and high risk (Busch et al, 1990) blood donor populations demonstrated a lack of utility for HIV antigen screening. These blood donor studies, and similar studies on smaller numbers of cadaver bone (Harrell et al, 1993) and cornea (Pepose et al, 1992) donors did not detect HIV infected donors beyond those already detected by testing for HIV antibodies. Studies are underway to determine whether testing donors for HIV RNA and HCV RNA by nucleic acid testing is practical. One study of 1,424 cadaver bone donors showed that the use of HIV DNA (not HIV RNA) and p24 antigen blood testing did not detect additional HIV infected cadaveric bone donors (Harrell et al, 1993). All 1,424 donors negative for HIV-1 antibodies were also negative for HIV DNA. Although viral nucleic acid testing is more sensitive than antibody assays, it may be premature to apply it routinely to cadaver donor testing due to the low HIV prevalence in the donor population, its uncertain predictive value, its false positive rate, and its false negative rate due to haemoglobin contamination and other interfering substances in cadaveric post-mortem blood samples. Testing of living blood donors for HIV and HCV RNA has markedly improved the safety of the blood supply even though screening has been done using pools of 16 to 24 samples (Stramer et al., 2000). Initially, viral nucleic acid testing was not feasible in blood donor screening applications due to lack of automation, time and space restrictions and cost. Recently, however, two test systems are being used to test over 13 million blood donations annually in the United States: the Roche Molecular Systems COBAS AMPLISCREEN tests for HCV and HIV, and the Gen-Probe/ Chiron Pooled Plasma HIV-1/HCV Amplified Assay. Testing is being done on pooled samples using pools of 24 or 16. Testing of pooled samples reduces the number of tests required on a

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daily basis, and also the time to perform testing and the cost. It also takes into account the rapid rise of viral nucleic acid in recently infected individuals, so that pooling has a minimal impact on the sensitivity of these assays. The increased sensitivity of these two systems over previously available PCR tests has also made this possible. Both systems have now been licenced for blood donor screening, and efforts are underway to qualify them for organ and tissue donor screening. The same approach is now being tested in trials for HBV, and soon, for the West Nile Virus. Many bone banks test donors for anti-HBc, a test originally introduced for blood donors as a surrogate for detecting non-A, non-B hepatitis carriers. The utility of this test as a surrogate has been diminished since the addition of specific tests for HCV (HCV antibodies and HCV RNA), the major cause of non-A, non-B hepatitis (Blajchman et ah, 1993). Although not required by AATB Standards, and in the absence of HBV DNA testing, the use of anti-HBc in donor testing likely had a safety benefit in reducing HBV infections. Several reports have documented the presence of HBV in the sera of HBsAg-negative, anti-HBcpositive blood donors (Yotsuyanagi et ah, 2001; Roth et ah, 2002; Wang et ah, 2002). These reports also suggest that the addition of HBV DNA testing will increase the sensitivity of HBV detection, but may not entirely replace the need to test for anti-HBc or HBsAg. For example, recipient directed lookback procedures have revealed recipients of HBsAg negative, NAT negative, anti HBc-positive blood components to have been infected with HBV (Roth et ah, 2002). 12.4. Hemodilution of donor b l o o d sample Massive blood loss and intravascular volume replacement by transfusion of blood, colloid, and crystalloid solutions can cause hemodilution, and result in unreliable donor test results for infectious diseases (Eastlund, 2000). In 1987, a case of HIV transmission to multiple recipients of organs derived from an infected


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donor was reported. Testing of the donor was negative when blood was sampled immediately after receiving blood transfusions amounting to two to three total blood volumes, and an additional large volume of crystalloid solution over an elevenhour period (CDC, 1987). When a blood sample was obtained 48 hours later and tested for anti-HIV, it was positive due to intravascular replenishment of immunoglobulin from extravascular sites. In 1993, US federal regulations (FDA, 1993) were first published, with subsequent modification (FDA, 1997; 1999), requiring quarantine of tissue from adult donors who had blood loss and received greater than two litres of blood and colloids within 48 hours of blood sampling; or greater than one litre of crystalloid within a one hour of sampling. The donated tissue was not to be used unless a pre-transfusion sample was available for testing, or an algorithm was used to evaluate blood and colloid volumes administered in the 48 hours prior to blood sampling, to ensure that any plasma dilution sufficient to alter test results, had not occurred. AATB Standards also require tissue banks to follow written procedures the set hemodilution limits to prevent use of falselynegative results when testing post-transfusion blood samples for infectious disease (AATB, 2001). Acceptability limits must be part of written procedures. Standards of the American Red Cross Tissue Services require that in the case of blood loss and transfusion within 48 hours of death, a pre-infusion sample must be used (Campagnari and O'Malley, 1994). A post-infusion sample may be used in patients with major clinical blood loss, provided that the tissue bank physician has evaluated whether blood and crystalloid infusions have compensated for blood loss; estimated hemodilution is 50 percent or less of the total blood volume; and the tissue bank physician has given written approval. The estimated amount of hemodilution depends upon the type of fluid infused, and the amount of time elapsed since infusion.

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12.5. Quality of cadaver donor b l o o d samples The testing of cadaveric tissue donor serum for viral markers may be complicated by false positive tests when sampling is delayed after death (Pepose et al, 1992) or when there is haemolysis (Novick et al, 1993; Pepose et al, 1992). False positive results for HBsAg and p24 antigen due to haemolysis may be found depending upon which manufacturer's test kit is used (Novick et al, 1993). Sample quality and presence of haemoglobin can also cause false negative results during testing for HIV and HCV by NAT (Adams et al, 1993; Comeau et al, 1992). Frozen storage and multiple freeze-thaws do not have a major effect on detectability of antibodies to infectious agents in serum, but they may reduce the reliability of testing for microbial nucleic acids by NAT. Busch et al, showed that multiple freeze thaws can reduce the detectability of HCV RNA by NAT (Busch et al, 1992). Published studies are too few for any firm conclusions to be drawn, other than a possible deleterious effect of frozen storage and freeze thawing on tests of serum for HCV RNA, and tests of peripheral blood white cells for HIV DNA, by NAT. Despite this, NAT was used to detect and retrospectively diagnose HIV infection using marrow specimens from an organ and tissue donor, and frozen sera from organ recipients five years after donation and transplantation (Simonds et al, 1992). HCV testing by NAT was also useful in confirming HCV infection in one cadaveric donor (Conrad et al, 1995) and was essential in another (Tugwell et al, 2002). Although NAT for hepatitis and HIV will be very useful, the tests are under development for organ and tissue donor testing. 12.6. Autopsy Autopsies of donors are not generally required, tissues that can be stored, a final donor suitability is not made by the tissue bank physician until the autopsy, if performed, have been reviewed

but for those determination the results of (AATB, 2001;


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Campagnari and O'Malley, 1994). Autopsy findings that have disqualified donors, include previously undiagnosed malignancy, widespread granulomas, abscess, and pneumonia. 12.7. Living bone donor selection Femoral heads can be donated by persons undergoing hip arthroplasty. Donor medical history screening and testing requirements are similar to those for cadaver bone donors, except that the donor medical history interview can be made directly, and a retest for HIV and HCV antibodies is required 180 days following donation (AATB, 2001; Campagnari and O'Malley, 1994; CDC, 1988a; 1994). The retest aims to identify recently infected donors who were seronegative for HIV and HCV antibodies at the time of donation. This 180-day retest is required for semen and bone donors but not for living donors of blood, marrow, amnion, umbilical veins, or foetal tissue. Retesting the low risk voluntary living bone donors has not detected any additional infections despite thousands of donations and retests (Scofield et al., 1993b), whereas it may have utility in the higher risk population of paid semen donors. Most tissue banks have ceased collecting surgical femoral heads, partly due to lower quality, but also due to the difficulties of acquiring the 180-day sample for retesting. Testing of samples for HCV and HIV RNA from living donors at the time of donation would enhance safety and could eliminate the need for a 180-day retest. 13. Reducing the Risk During Cadaveric Tissue Collection Removal of tissue allografts takes place up to 24 hours after the death of a cadaveric donor. Most transplantable tissue such as corneas, bone, tendon and skin are recovered from donors in whom there has been complete cardiac and respiratory cessation for some time period. The collection of tissue allografts from

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the cadaveric donor begins as soon as possible after death, to reduce the risk of endogenous or exogenous bacterial contamination, and to maintain functional characteristics or viability of the tissue. National professional standards require bone, tendon and heart valve recovery to be completed within 12 hours of death, or within 24 hours of death if the body is refrigerated (AATB, 2001; Campagnari and O'Malley, 1994). Recovery of corneas is preferred within four to six hours of death, to ensure endothelial cell viability. Viable skin allografts are usually collected within six hours of death if the body is not refrigerated but within 24 hours otherwise (AATB, 2001; Campagnari and O'Malley, 1994). Delayed removal of tissue from the cadaver donor can possibly result in an increase of anaerobic and spore forming bacterial pathogens which are more resistant to disinfecting and sterilisation procedures. The majority of tissue banks which procure hearts for heart valves, culture the transport media which is filtered prior to processing; the filter is then cultured, thus increasing the sensitivity of detection. The results are used to determine whether to release the allograft for clinical use. Physical manipulation, environmental exposure and storage conditions of the body prior to tissue removal can contribute to bacterial or fungal contamination of the tissue allograft. It is possible that in some of these donors there is post-mortem translocation or transmigration of bacteria from the intestines or respiratory system to other parts of the body, and to the tissue removed for transplant purposes. The epithelial mucous membrane of the intestine is a very fragile barrier protecting a person from intestinal bacteria. This intestinal barrier is particularly sensitive to ischemia and reperfusion, and a second insult — even mild hypotension — can affect intestinal barrier function, leading to loss of mucosal defence, and consequently, escape and spread of luminal bacteria to other organs. This sequence is called bacterial translocation (Fukasima et ah, 1992; Steffen and Berg, 1983; Mejima et ah, 1984). It is unknown whether bacterial


Table 10. Additional steps taken to control microbial contamination of tissue allografts. Aseptic Procurement • Monitoring and control of microbial contamination of air, surfaces. • Sterile barrier dress for personnel. • Aseptic technique. • Sterile instruments and supplies. • Cleaning, disinfecting donor skin. • Procurement cultures. Aseptic Processing • Monitoring and control of microbial contamination in air, surfaces. • Sterile water, reagents, supplies, instruments, equipment. • Aseptic Technique. • Sterile barrier dress for personnel. • Removal of extraneous tissue and blood. • Exposure to disinfectants, antibiotics. • Sterilisation. • Sterility Testing. Final Packaging • Sterile supplies, sealed impervious packaging.

translocation occurs consequent to cardiopulmonary resuscitative attempts, autopsies or other post-mortem manipulation of the cadaveric tissue donor. A surgical autopsy is performed in approximately half of cadaveric tissue donors prior to tissue allograft removal, and this can contribute to bacterial contamination. The environment in which the tissue allograft is removed may also have an impact of whether the tissue contains microbial contamination. Tissues are removed at various environmental sites: hospital operating rooms, autopsy suites in hospital morgues and regional forensic medical examiner facilities; funeral homes, and in dedicated tissue procurement facilities at medical examiner facilities or tissue banks. To reduce the risk of bacterial contamination, the room and table top is cleansed;

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Table 11. Surgical removal of tissue — Cadaver tissue donor. Operating room or morgue. Cleaning and preparing of room. Cleaning and preparing of body. Sterile drapes and equipment. Aseptic surgical technique. Dissecting and removing of tissue. Preserving blood vessels for embalmer, Funeral Director. Bacteriologic testing. Restoring anatomic shape of body.

sterile drapes are used; donor excision sites are disinfected; sterile equipment and supplies are used; and aseptic technique is practised (Table 11). One study reported that prior organ donation and the location of the procurement site have been correlated with the discover of virulent bacteria on tissue allografts removed from cadaveric donors (Johnson et al, 2002). They found virulent bacteria on at least one tissue in 58 (21%) of 275 donors, and on 125 (3.5%) of 3586 individual tissues. In contrast, non-pathogenic bacteria were found on 622 (17.3%) of tissues. Bacterial contamination is a common finding of tissues freshly removed for purposes of transplantation (Bettin et al, 1998; Deijkers et al., 1997; Martinez et al, 1985; Vehmeyer et al, 1999; Vehmeyer and Bloem, 1999; Farrington et al, 1998; Chapman and Villar, 1992; Sutherland et al, 1997; Novick et al, 1991; Bennett et al, 1991; Scofield et al, 1994; Journeaux et al, 1999). Swabbing of the bone allograft surface is routinely performed by most tissue banks at the time of procurement, but the rate of bacterial contamination — when swab culturing the entire allograft — was found by Veen et al, to be one tenth that of entire bone that had been immersed in culture media (Veen et al, 1994). The limited sensitivity of swab culturing techniques has been confirmed (Vehmeyer et al, 2001).


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14. Reducing the Risk During Cadaveric Tissue Processing Tissue allografts can also be contaminated during processing from air and environment surfaces, from personnel, and from contaminated reagents, surgical instruments, supplies and processing equipment (Table 12). In rare cases, contamination has been acquired during liquid nitrogen storage (Hawkins et al., 1996). An additional site of acquired contamination can be the hospital or surgical centre operating room where microbial contamination can develop during opening of the container, handling of the allografts, and exposure to the air in the operating room. Table 12. Possible sources of bacterial contamination. Donor infection prior to death. Postmortem translocation of bacteria in body to the tissue donated. Effect of prior autopsy. Length of time and temperature during body storage prior to procurement. External contamination during procurement • Morgue versus OR versus special facility. • Surgical technique. Contamination during processing • Air (particulate and bacteria monitoring). • Water (bacteria monitoring). • Surfaces: (tables, walls: bacterial testing). • Equipment (sterilised). • Supplies (sterilised). • Reagents, solutions (sterilised). • Staff (aseptic technique, sterile barrier clothing, bacterial testing). Contaminated during storage • Liquid nitrogen storage.

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In cases of HIV and HCV transmission by tissue allografts, the origin of the virus has been from an infected donor. In contrast, bacterial contamination of tissue allografts can arise from infected donors; or contamination may occur during surgical removal of tissue from the donor, or from contaminated environments, equipment or supplies during processing at a tissue processing facility; or from implantation at the time of surgery. Electrolyte solutions purchased commercially (CDC, 1995), or deionised water prepared by the tissue bank (Farrington et ah, 1996), can be contaminated by bacteria which consequently contaminate the tissue allograft when used for processing. Some tissue banks have been able to demonstrate removal of low virulence bacteria during processing of heart valves, by soaking the allograft in antibiotics (Lau and Lazaro, 2002). Others processing and storing corneas (Antonios et ah, 1991) and skin (Clayton et ah, 1995) have demonstrated that bacteria can survive in refrigerated antibiotic solutions. The recent outbreak of Clostridium infections from minimally-processed cartilage and tendon allografts, and fungal infection from a heart valve from a single facility, demonstrate that antibiotic soaking is not effective against all virulent microbes, and that residual antibiotics can interfere with, and existing sampling techniques can miss, detection of the microbe during final sterility testing (CDC, 2002; Kuehnert et al., 1998). This is particularly true for spore-forming organisms where the vegetative forms can be eliminated by antibiotics, but the spores are not. Complete absence of bacterial and fungal contamination is far greater in importance for bone, cartilage, tendon, and heart valve allografts that are surgically implanted within the body, than for cornea and skin allografts that are implanted at non-sterile sites on the surface of the body. 15. Recipient Safety and Federal Regulations In 1993 the US Food and Drug Administration (FDA) investigated the importation of human tissue allograft from foreign


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countries (FDA, 1993). The FDA documented that full medical history screening and interviews with the next-of-kin (to exclude those with risk behaviours for HIV and hepatitis), had not been performed. Donor medical records were not available, and consent to donate had not been obtained. Although donor blood testing for HIV and hepatitis were alleged to be negative, the FDA retested samples and found at least one to be markedly positive for HBsAg. FDA also found that imported tissue from some foreign tissue banks were recovered without aseptic precautions (FDA, 1993). It was not ascertained that imported tissue allografts caused hepatitis or HIV infection in recipients. However, a decade earlier many human dura mater allografts that had been processed in pools, were imported into the US by a German medical device manufacturing firm, and these were implicated in the spread of CJD to recipients. This raised the issue of pooling, and the increased risk of contaminating all tissue in a batch. In addition, the ability to trace recipients of a particular donor's tissue becomes nearly impossible if multiple donors are involved. Although the US Public Health Service Centres for Disease Control had published recommendations for screening organ, tissue, and semen donors (CDC, 1988a; 1988b; 1994), the FDA added legal requirements to provide further protection for the public from HIV and hepatitis infection through transplantation of imported tissue. The FDA issued an "Important Alert" to health professionals in December 1993, so that tissue allografts would not be imported without prior notification to the FDA. In addition, the FDA published an emergency regulation on December 14, 1993, which took effect immediately (FDA, 1993). The regulation, entitled "Human Tissue Intended for Transplantation" applies to bone, tendon, skin and cornea allografts, but not semen or organs. The regulation required tissue donor testing for HBsAg and antibodies to HIV and HCV, by a CLIA-registered laboratory using FDA licensed test kits, and using a blood sample that has not been diluted enough from recent transfusion

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to alter test results. Other requirements include donor medical history screening to assure absence of risk factors or clinical evidence of HBV, HCV, or HIV infection; written operating procedures; and record keeping to ensure traceability (FDA, 1997; 1999). The FDA has proposed requirements for Good Tissue Banking Practices modelled after GMP requirements for biologies, drugs and blood transfusion (FDA, 1997; 2001a; 2001b). In November 2001, the sudden death of a man who had received a cartilage allograft contaminated with Clostridium led to an investigation by CDC and FDA. In 2002, the US FDA enacted emergency regulations to require allograft processing facilities to observe written procedures that are validated and certified to reliably prevent infectious disease contamination and cross-contamination during processing (FDA, 2002). Equivalent regulation exists in Europe. In addition to federal regulations stipulating donor screening requirements to ensure safety, there are other federal regulations for cellular transplants and tissue composites, to ensure clinical effectiveness and to require compliance with good manufacturing practices for medical devices (Kessler et ah, 1993; FDA, 2001b). These regulations cover extensively processed cells, and gene therapy. 16. Conclusion Transplantation of tissues has resulted in the transmission of bacterial, viral, fungal and prion diseases from donor to recipient. When the first truly effective immunosuppressant, cyclosporin, became available in 1981, organ transplantation flourished. The numbers of organs transplanted each year grew rapidly, leading to implementation of effective programmes to develop public support for organ donation. As organ donation grew, so did tissue donation. With the rapid growth of transplantation, early cases of transplant transmitted infection arose from infections of the donor, but these could have been


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prevented if tests were available. Donor testing only reduced the risk. Early cases of transmitted viral infection involved donors who had HIV and hepatitis risk behaviours. Exclusion of these as prospective donors has played an important role in reducing the risk of transmission. More recently, several cases have been reported, wherein the infecting organism is bacterial and fungal instead of viral, and the microbes did not arise from the donor but was a contaminant acquired during the procurement, processing or storage of the tissue. Newer national professional standards and US FDA regulations are addressing this area. To prevent, or at least minimise, the risk of transmission of infectious disease, several approaches are important. Surgeons should use allografts only when needed, and obtain them from accredited tissue banks. They should consider use of autografts, alternative nonhuman graft material, or processed and sterilised tissue allografts. Whenever possible tissue banks reduce the risk by rejecting those donors suspected to be susceptible to HIV, hepatitis, or other viral or bacterial infections; performing physical examinations of donors, reviewing autopsy reports, and using aseptic technique during surgical removal of donated tissue. Bacterial testing of tissues donated and other laboratory testing procedures, help identify potentially infectious donors. Preventing the acquisition of microbial contamination and of cross-contamination during processing of tissues is required. Final sterility testing needs to be sufficiently sensitive to provide assurance of sterility. Lastly, sterilisation of certain tissues can be very effective, but it is not universally applicable, because some infectious agents such as CJD are impervious to sterilants, and because the clinical effectiveness of many tissues (such as skin, cornea, valves, veins) can be altered by sterilisation procedures (Pruss et ah, 2001). Application of all these steps will ensure a very low risk of transmitting disease from the donor to the recipient.

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17. References ABECASSIS, M. (1994). Transmission of CMV by skin allografts — A review, Tissue Cell Rep. 2, 15-20. ABECASSIS, M., KAUFMAN, D. and STUART, F. (1993). Direct evidence that murine cytomegalovirus (CMV) can be transferred via skin grafting using novel oligonucleotides in the polymerase chain reaction (PCR). Proceedings 19th Annual Meeting, American Society of Transplant Surgeons. Houston, TX. ADAMS, M., LEE, T.H., BUSCH, M.P., HEITMAN, J., MARSH, G.J., GJERSET, G.F. and MOSELY, J.W. (1993). Rapid freezing of whole blood or buffy coat sample for polymerase chain reaction and cell culture analysis: Application to detection of human immunodeficiency virus in blood donor and recipient repositories, Transfusion 33, 504-508. AHO, A.J., HIRN, M., ARO, H.T., HEIKKILA, J.T. and MEURMAN, O. (1998). Bone bank service in Finland. Experience of bacteriologic, serologic and clinical results of the Turku bone bank 1972-1995, Acta Orthop. Scand. 69, 559-565. ALTER, H.J., EPSTEIN, J.S., SWENSON, S.G., VAN RADEN, M.J., WARD, J.W., KASLOW, R.A., MENITOVE, J.E., KLEIN, H.G., SANDLER, S.G. and SAYERS, M.H. (1990). Prevalence of human immunodeficiency virus type 1 p24 antigen in US blood donors — An assessment of the efficacy of testing in donor screening, N. Engl. J. Med. 323, 1312-1317. AMERICAN ASSOCIATION OF TISSUE BANKS (2000). Annual Registration Survey of Accredited Tissue Banks. McLean VA: American Association of Tissue Banks. AMERICAN ASSOCIATION OF TISSUE BANKS (2001). Standards for tissue banking. McLean VA: American Association of Tissue Banks.


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(1993). Birdshot chorioretinitis and Lyme Borreliosis, Am. J. Opththalmol. 115, 149-153. TANGE, R.A., TROOST, D. and LIMBURG, M. (1990). Progressive fatal dementia (Creutzfeldt-Jakob disease) in a patient who received homograft tissue for tympanic membrane closure, Eur. Arch. Otorhinolaryngol. 247, 199-201. THADANI, V., PENAR, P.L., PARTINGTON, J., KALB, R., JANSSEN, R., SCHONBERGER, L.B., RABKIN, C.S. and PRICHARD, J.W. (1988). Creutzfeldt-Jakob disease probably acquired from a cadaveric dura mater graft, /. Neurosurg. 69, 766-769. THIJSSEN, E.J., KROES, A.C., BOX, E., PERSIJN, G.G. and ROTHBARTH, P.H. (1993). The significance of complete serological testing for hepatitis B in heart valve banking, Transplantation 56, 82-84. TOMFORD, W.W., STARKWEATHER, R.J. and GOLDMAN, M.H. (1981). A study of the clinical incidence of infection in the use of banked allograft bone, /. Bone Joint Surg. 63A, 244-248. TOMFORD, W.W., THONGPHASUK, J., MANKIN, H.J. and FERARO, M.J. (1990). Frozen musculoskeletal allografts. A study of the clinical incidence and causes of infection associated with their use, /. Bone Joint Surg. 72A, 1137-1143. TRANSFELDT, E.E., LONSTEIN, R., WINTER, D. and BRADFORD, D. (1985). Wound infections in reconstructive spinal surgery, Orthop. Trans. 9, 128-129. TUGWELL, B.D., PATEL, P.R., WILLIAMS, I.T., THOMAS, A., HOMAN, H., HEDBERG, K. and CIESLAK, P.R. (2002). Hepatitis C Virus (HCV) Transmission to Tissue and Organ Recipients from an antibody negative donor — United States, 2002. In: 42nd Ann. Interscience Conf. Antimicrob. Agents

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Strong

Chemother. (ICAAC) (September 28), San Diego, CA, Poster presentation LB-17. TULLO, A.B., MARCYNIUK, B., BONSHEK, R., DENNETT, C , CLEATOR, G.M., LEWIS, A.G. and KLAPPER, P.E. (1990). Herpes virus in a corneal donor, Eye 4, 766-767. TYRAS, D.H., KAISER, G.C., BARNER, H.B., LASKOWSKI, L.F. and MARR, J.J. (1978). Atypical mycobacteria and the xenograft valve, /. Thorac. Cardiovasc. Surg. 75, 331-337. UNITED NETWORK FOR ORGAN SHARING (2001). Cadaveric donors and cadaveric organ transplantation in the US, UNOS Update 10, 29. VANBAARE, J., MACKIE, D.P. and MIDDELKOOP, E. (1997). HIV transmission by transplant of allograft skin: A review of the literature (letter), Burns 23, 460. VEEN, M.R., BLOEM, R.M. and PETIT, P.L.C. (1994). Sensitivity and negative predictive value of swab cultures in musculoskeletal allograft procurement, Clin. Orthop. 300, 259263. VANBAARE, J., LIGTVOET, E.E. and MIDDELKOOP, E. (1998). Microbiological evaluation of glycerolised cadaveric donor skin, Transplantation 65, 966-970. VEHMEYER, S.B.W. and BLOEM, R.M. (1999). Bacterial contamination of post-mortal bone allografts. In: Advances in Tissue Banking, G.O. Phillips, J.M. Kearney, D.M. Strong, R. VonVersen and A. Nather, eds., World Scientific, Singapore, Vol. 3, pp. 33-41. VEHMEYER, S.B.W., BLOEM, R.M., DEIJKERS, R.L.M., VEEN, M.R. and PETIT, P.L.C. (1999). A comparative study of blood and bone marrow cultures in cadaveric bone donation, /. Hosp. Infect. 43, 305-308.


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VEHMEYER, S.B., BLOEM, R.M. and PETIT, RL. (2001). Microbiological screening of post-mortem donors — two case reports, /. Hosp. Infect. 47, 193-197. WANG, J., LEE, C , CHEN, P., WANG, T. and CHEN, D. (2002). Transfusion-transmitted HBV infection in an endemic area: The necessity of more sensitive screening for HBV carriers, Transfusion 42, 1592-1597. YAMADA, S., AIBA, T., ENDO, Y., HARA, M., KITAMOTO, T. and TATEISHI, J. (1994). Creutzfeldt-Jakob disease transmitted by a cadaveric dura mater graft, Neurosurgery 34, 740744. YANKAH, A.C., HETZER, R., MILLER, D.C., ROSS, D.N., SOMERVILLE, J. and YACOUB, M.H. (eds.) (1988). In: Cardiac valve allografts 1962-1987, Current concepts on the use of aortic and pulmonary allografts for heart valve substitutes. SpringerVerlag, New York. YOTSUYANAGI, H., YASUDA, K., MERIYA, K., SHINTANI, Y., FUJIE, H., TSUTSUMI, T., NORJIRI, N., JUJI, T., HOSHINO, H., SHIMODA, K., HIRO, K., ILINO, S. and KOIKE, K. (2001). Frequent presence of HBV in the sera of HBsAg-negative antiHBc-positive blood donors, Transfusion 41, 1093-1099. ZAAIJER, H.L., EXEL OEHLERS, P., KRAAIJEVELD, T., ALTENA, E. and LELIE, P.N. (1992). Early detection of HIV-1 by third generation assay, Lancet 340, 770-772.



4 BACTERIAL CONTAMINATION OF BONE ALLOGRAFTS IN THE NETHERLANDS

JEROEN VAN BAARE Netherlands Bone bank Foundation Leiden, The Netherlands STEPHAN VEHMEIJER Department of Orthopaedics Leiden University Medical Centre Leiden, The Netherlands ROLF BLOEM Department of Orthopaedics, Reinier de Graafgasthuis Delft, The Netherlands

1. Introduction The increased need for allograft bone tissue in The Netherlands resulted in the establishment of the Leiden Bone bank Foundation in 1988. This was initiated by the Department of Orthopaedics, Leiden University Medical Centre and Bio This paper is a short summary of the recently published PhD. thesis of Dr. Stephan Vehmeijer (Vehmeijer, 2002). A hard copy of this thesis is available on request. 133

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/. van Baare, S. Vehmeijer & R. Bloem

Implant Services, a daughter organisation of Euro Transplant, Leiden. In 1998, the name of the bone bank was changed to Netherlands Bone bank Foundation (NBF), as a result of the implementation of two new laws on tissue banking. The Act on Organ Donation, July 1, 1998, concerns the central registration of consent for the donation of organs and tissues. In addition, the Royal Degree on Quality Requirements for Organ Banks was implemented on November 1, 1998. This Degree regulates tissue banking in The Netherlands and should further improve the quality and safety of human allograft tissue by specific regulations, accreditation of tissue banks by the government and the introduction of a certified quality system for tissue banks, such as ISO 9001. 2. Investigations by NBF NBF has been retrieving musculoskeletal tissues from living donors as well as from post-mortem donors (see Figs. 1 and 2). Currently, approximately 1,500 femoral heads are donated each year by patients undergoing primary total hip arthroplasty. The allograft femoral heads are stored as unprocessed deep-frozen tissue. The use of femoral head allografts in orthopaedic surgery

Femoral Heads from Living D o n o r s

•g 1500 £ 1200 « 900 | 600 £• 300

L UL11 1997

1998

1999

2000

2001

Year

Fig. 1. Number of Femoral heads received by NBF.

Bacterial Contamination of Bone Allografts in the Netherlands

Post-mortem Donors 120 -|

ilixiil 1997

1998

1999

2000

2001

Year

Fig. 2. Number of post-mortem donors received at NBF.

is widely accepted, particularly in revision hip and knee arthroplasty. Favourable results of the impaction grafting technique have increased the demand for this type of allograft (Sloof, 1996; Gie, 1993; van Donk, 2002). Also, NBF has been receiving approximately 100 post-mortem bone donors each year. Large bone segments can be retrieved from these donors, and used for reconstructive surgery in orthopaedic oncology or in revision arthroplasty. Grafts can also be processed into smaller units and applied in spinal fusions, or used for the filling of bone defects, for example, defects associated with bone cysts. 3. The Role of Tissue Banks Tissue banks play an important role in the safety and quality of allograft tissue. To ensure safety of bone allografts, many procedures are carried out to minimise the risk of transmission of infectious diseases. The medical and social history of potential donors is extensively screened. The procurement of donors is performed in the operating theatre with strict aseptic procedures; and serology and bacteriology tests are performed. Despite all these precautions, complications after bone transplantation may occur. Non-union, fracture and infection

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are relatively common, particularly in reconstructive surgery following resection of bony tumours. Infection is the most devastating complication, often leading to failure and resection of the graft. Infection can be caused either during surgery or through graft contamination. Tissue banks can only control the possible contamination of allografts, which can be divided into two main sources. Contamination can be superficial, e.g., caused by the procurement team during retrieval of the grafts, or through the skin of the donor. On the other hand, the contamination can be of deep origin, e.g. caused by endogenous donor sources. To detect possible contamination of each individual transplant, several culture techniques can be used. NBF has been using the swab technique, in which the entire surface area of each individual graft is swabbed carefully. The swab is taken directly after retrieval, and before any other processing procedures. The swab stick is inoculated onto culture plates and the swab stick itself is incubated in broth, which is then subcultured. This method allows for a semi-quantitative assessment of the bacterial load of the graft. A major disadvantage of the swab culture technique is that only the external surface is sampled. Although currently not implemented in the procedures at NBF, tissue banks may perform tissue cultures, as they consider this a more direct method to determine the bacterial load. Samples from representative areas of the graft are incubated in broth, which is then subcultured. This method is highly sensitive in cases where samples are contaminated with micro-organisms. It is, however, unlikely that micro-organisms present on an allograft are evenly distributed on the surface. As samples are taken from a limited number of sites on the graft, sampling error is evident. Micro-organisms inside the graft can remain unnoticed. To detect these micro-organisms, NBF has been performing post-mortem blood cultures. Although the value of these cultures remains unclear, and the interpretation of the results is difficult, it is the opinion of NBF that contamination by endogenous


137

Table 1. Analysis of blood cultures of 550 bone donors at Netherlands Bone bank Foundation. 550 donors

1

i

1 140 donors blood culture +

410 donors blood culture -

1 70 donors skin contaminants

1

70 donors non-skin contaminants

1 16 donors swab & blood culture with identical micro-organism

donor sources can be detected through this technique. Therefore the blood culture of 550 bone donors were reviewed. The results are given in Table 1. Prior to bone retrieval, blood was taken from the subclavian vein or artery under aseptic conditions. For multi-organ donors, samples were taken through venal puncture before organ perfusion; and if post-mortem heart valve procurement preceded bone procurement, blood samples were taken after mid-sternal thoractomy from the interior cava vein under aseptic conditions. Blood samples were then cultured for aerobic and anaerobic micro-organisms for seven days. The swabs were inoculated within 24 hours onto blood agar and chocolate plates, and then cultured under aerobic and anaerobic conditions. The swab sticks themselves were incubated in broth which was inoculated after five days, onto blood agar and chocolate plates, and cultured for 48 hours. A positive blood culture was detected in 140 donors. From these donors, 70 cultures were tainted with skin contaminants. This can be explained by the fact that samples were contaminated during the blood retrieval procedure. The other

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Table 2. Details of a donor with positive swab & blood culture of same micro-organism. Donor A > Age: 28 years > Cause of death: drowning 15 minutes in water attempt to resuscitate failed > No open wounds nor fractures, no autopsy > No prior illness > No other procurement procedures before bone retrieval > Procurement 13 hours after circulatory arrest, completed within two hours > Total number of grafts: 12

70 donors showed contamination of the blood culture with non-skin contaminants, which is difficult to explain by the retrieval procedure. From these donors 16 donors were identified with the same micro-organism for both the swab culture and blood culture. These 16 donors could be divided into the following groups: a traumatic cause of death was observed in 12 donors; three donors received pre-mortem streptokinase; and one donor died by drowning. To stress the importance of the blood culture as an additional safety measurement, one donor will be described below in detail (Table 2). 4. Blood Cultures as an Additional Safety Measurement The blood culture of the donor was positive for Aeromonas species. Aeromonas is an organism of high pathogenic potential, and known for its presence in water. As the donor was discarded for this reason, there was a possibility to culture the entire grafts individually in toto and compare these results with the swab culture results. The results are shown in Table 3. The swab


139

Table 3. Culture results of grafts obtained from donor mentioned in Table 2. Graft

Swab Culture

Culture of Entire Graft

Femur proximal left

-

CNS

Femur distal left

-

CNS

Tibia proximal left

-

Fibula proximal left

CNS

CNS

Achilles tendon left

-

Not cultured

Fascia lata left

-

CNS

Aeromonas spp CNS

CNS

CNS

Aeromonas spp

Tibia proximal right

-

CNS

Fibula proximal right

CNS

Staphylococcus aureus

Achilles tendon right

CNS

Aeromonas spp

-

CNS

Femur proximal right Femur distal right

Hemipelvis right

Staphylococci schleiferi

CNS = Coagulase Negative Staphylococci - = no growth culture showed only one graft positive for Aeromonas species and some grafts had no bacterial contamination. Cultures of the grafts in toto resulted in four positive grafts contaminated with Aeromonas species. However, also with this technique, many grafts showed only positive contamination results for Coagulase Negative Staphylococci, a frequently observed skin-contaminant in bone grafts. The pulmonary and heart valves were procured after the bone retrieval. The culture results of the heart valves were also positive for Aeromonas species. Therefore, considering the blood culture results; the cultures of the entire graft; and also

140


the cultures of the heart valves, it is very likely that a haematogenous spread of Aeromonas species occurred pre-mortem, contaminating most organs, including the bones. One of the latest reports of infection after bone transplantation was published in the Morbidity and Morbidity weekly Report (4). One patient underwent reconstructive knee surgery with a femoral condyle allograft. The patient died the next morning. Pre-mortem blood cultures grew Clostridium sordelli. Another patient also underwent reconstructive knee surgery with an allograft femoral condyle and meniscus. He developed high fever the next day (39.7°C) but recovered with antibiotics. Cultures were not obtained. The allograft tissue was, however from the same tissue donor. Other bone allograft tissue at the tissue bank, not yet released for clinical use, also showed positive cultures for Clostridium sordelli. Reviewing the 550 bone donors at NBF for any Clostridium cases, three donors were found to be positive for Clostridium sordelli. However, none of the swab cultures of any graft showed contamination for Clostridium sordelli. All grafts of these donors were discarded due to the positive blood culture. 5. Conclusions Blood cultures can help to identify unnoticed haematogenous spread of (high pathogenic) micro-organisms. Grafts with positive swab cultures of non-skin contaminants, or, grafts obtained from donors with positive blood cultures from which non-skin contaminants are isolated, should not be used as large minimally-processed grafts. 6. References GIE, G.A., LINDER, L. and LING, R.S.M. et al. (1993). Impacted cancellous allografts and cement for revision total hip arthroplasty, /. Bone Joint Surg. (Br.) 75-B, 14-21.


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MMWR, November 23 (2001), 50(46); and March 15 (2002), 51(10). SLOOFF, T.J., BUMA P. and SCHREURS, B.W. et al. (1996). OAcetabular and femoral reconstruction with impacted graft and cement, Clin. Orthop. 324, 108-15. VAN DONK, S. (2002). Experimental and clinical data on the incorporation of impacted morsellised bone grafts. Nijmegen. ISBN 90-9015620-8. VEHMEIJER, S.B.W. (2002). Bacterial contamination of bone allografts. Leiden. ISBN 90-9015577-5.



5 MICROBIOLOGICAL SCREENING OF CADAVER DONORS AND TISSUES FOR TRANSPLANTATION

OCTAVIO V. MARTINEZ University of Miami Tissue Bank

1. Introduction The risk of transmission of infectious agents by means of cadaver-derived osteoarticular and other tissues, has been the subject of concern since the earliest reports of the use of human tissues for transplantation. Over the years, tissue banks have developed multiple interdisciplinary approaches for screening prospective cadaver donors for the presence of infectious agents, as well as for the examination of the tissues and grafts derived from the same. While some form of a microbiological culture protocol is applied routinely to tissues obtained from cadavers or from living donors at the time of tissue retrieval, it is to be noted that such cultures alone are insufficient to reliably identify all non-septic donors, or to detect all contaminated tissues derived from these sources. Microbiologic screening is part of a collective of tests and procedures designed to minimise the likelihood of processing tissues harbouring infectious agents. Complementary procedures may include obtaining a medical and social history on the donor; performing serologic or molecular amplification tests for bacterial and viral pathogens; 143

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O.V. Martinez

performing histopathological studies of lymph nodes or other organs; or a diagnostic autopsy; and implementing an effective strategy for tracking graft recipients. There is a two-fold purpose to the performance microbiological cultures of the blood and tissues of cadaver donors. First, to search for evidence of clinically unapparent sepsis in donors judged to be non-septic by medical history and physical examination or autopsy. The guidelines of the American Association of Tissue Banks state that tissues from donors exhibiting evidence of significant active infection, including septicemia at the time of donation, are not to be released for transplantation (AATB, 2001). Second, cultures are intended to identify specific tissues contaminated with bacteria or fungi at the time of excision. Microbiological cultures at the time of tissue retrieval influence the subsequent processing of tissues in other ways. Some tissue banks utilise the results of these cultures to determine preprocessing treatment of bones to reduce bioburden, or to determine the processing method, ascertain the need for endotoxin testing, and monitor quality assurance for procedures and processing personnel (Anderson and Vessey, 1996). 2. Sampling Methods The most common method for culture of the bones, cartilage or tendons employs the use of cotton swabs to sample both the surface, and whenever possible, the medullar canal of long bones. Other methods include the immersion of whole tissues or small segments of bone in a liquid culture medium. Occasionally, tissues are immersed in a diluent solution such as physiological saline, agitated to release any microbial contaminants, then a small portion of the diluent is cultured in liquid or agar medium or both under aerobic and anaerobic conditions (Farrington et al, 1998; LaPraire and Gross, 1991). There are legitimate concerns regarding the sensitivity of swab cultures for the detection of low numbers of microorganisms in the tissues. Veen et al. (1994) compared the results

Microbiological Screening of Cadaver Donors

145

of three types of cultures from 75 fibular segments. These included surface swabs inoculated directly on agar medium; surface swabs inoculated into agar medium followed by immersion in a culture broth; and full immersion of bone segments in broth medium. The results showed a significantly lower rate of recovery of microorganisms by the direct agar plating method and the combined agar-broth method (culture sensitivity of 10% and 39% respectively) compared to the full broth immersion procedure. Mills and Roberts (2001) reported the recovery of microorganisms from 5.9% of surface swabs cultures from processed bone grafts (sensitivity, 22.1%) compared to 12.7% of tissue segments from grafts cultured by broth immersion. These investigators concluded that the destructive culture method based on the immersion technique, was approximately 3.5 times more sensitive than methods that relied on surface swab cultures. It is not known whether the sensitivity of a method that relies on single swab cultures can be increased by performing multiple cultures from each of the tissues. 3. Culture Media and Length of Incubation There is little agreement on the optimal choice of culture medium and the length of incubation for cultures obtained from cadaver musculoskeletal tissues at the time of procurement or from prepared allografts. Several investigators have reported the use of liquid thioglycollate medium (Barrios et al, 1994), brain heart infusion broth (Deijkers et al, 1994) or a combination of agar and broth culture media (Vehmeyer et al, 2001). The reported length of incubation ranges from 48 hours (Barrios et al, 1994) to as long as 10 to 14 days (Bettin et al, 1998). Bennett et al (1991) analysed the results of cultures of cadaver connective tissues and bone from living and cadaver donors, and reported that an incubation time of 14 days allowed for the detection of an additional 6% of contaminated tissues compared to a seven-day incubation protocol. In contrast, other investigators have reported that seven days of incubation were sufficient for

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detecting most organisms considered by the authors to be significant pathogens; therefore, extending the incubation period beyond seven days was judged to be unwarranted (Reik et ah, 1998). The microbiological screening protocol at the University of Miami Tissue Bank (UMTB) specifies the use of swab cultures of the surface and, when appropriate, marrow canal of bones and soft tissues obtained at procurement. The swabs are inoculated into tubes of thioglycollate medium previously degassed by boiling, and incubated at 35°C until microbial growth is evident or, if negative, for up to 14 days. The organisms most commonly recovered from these sources include species of coagulase-negative staphylococci, alpha-haemolytic streptococci and species of aerobic and anaerobic diphteroids (Table 1). Most isolates of organisms considered to be of significant pathogenic potential such as beta-haemolytic streptococci, Clostridia, Staphylococcus aureus and gram-negative enteric organisms, were

Table 1. Microorganisms isolated from the bones of 649 cadaver tissue donors at the time of tissue retrieval. Organism

Donors

% Positive

Cultures

% Positive

279

42.9

734

3.33

151

23.2

342

1.56

Streptococcus species

91

14.0

249

1.13

Corynebacterium species

69

10.6

97

0.44

Aerobic, Gram Negative Bacilli Clostridium species

54 30

8.3 4.6

192 173

0.87 0.79

Staphylococcus species Propionibacterium species

Total 21,971 cultures. Mean 33.8 cultures/donor. Total positive cultures = 1,946 (8.9%).


147

Table 2. Time of detection by culture of microorganisms recovered at procurement from cadaver musculoskeletal tissues. No.

Organism

Positive

Percent detected on culture days 7

Clostridium species

173

97

3

Staphylococcus species

734

44

28

14

14

Streptococcus species

249

69

23

4

4

Corynebacterium species

96

5

22

29

44

Propionibacterium species

342

1

11

28

59

1,917

47

20

14

20

All isolates

detected within 48 hours of incubation. However, up to 40% to 60% of corynebacteria and propionibacteria required seven or more days of incubation for detection (Table 2). It is to be noted that the time of detection of microorganisms in culture is, to an extent, dependent on the technology employed. Kostiak and Michlowski (2002) reported that the application of an automated microbial detection system (for blood cultures) to cultures of musculoskeletal tissues, allowed for the implementation of a 7-day incubation protocol with results comparable to those obtained with a reference 14-day incubation conventional culture system. In summary, the establishment of an optimal incubation period for cultures from cadaver tissue donors requires consideration of the need for a rapid turn around time, the type of microorganisms targeted for detection, and the type of detection system employed.

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4. Blood Cultures The role of postmortem blood cultures as indicators of sepsis and tissue contamination in cadaver donors of organs and tissues, has been the subject of extensive debate. A number of investigators have stated that postmortem blood cultures can yield useful information (Silver and Sonnerwirth, 1969) with a degree of reliability equivalent to that of antemortem cultures (Hove and Pencil, 1998) provided specific techniques were utilised for the collection of the blood. Others have suggested that postmortem blood cultures rarely, if ever, provide information that is not already known, or can be interpreted from other sources (Wilson et ah, 1993). One study (Gocke et al., 1998), addressing the use of blood cultures as predictors of tissue contamination in cadaver bone donors, revealed little concordance between blood culture results and the outcome of cultures of the tissues from the same donors. In that study, less than onehalf of donors with positive blood cultures also had positive tissue cultures, and only 13% of positive tissue cultures yielded matching isolates from the blood. The interpretation of blood culture results from cadaver tissue donors can be difficult not only because of the unavailability of clinical findings suggestive of sepsis, but also because the results can be influenced by a variety of perimortem and postmortem events. Lengthening postmortem interval, and a traumatic manner of death, have been reported to increase the likelihood of obtaining positive findings from cultures in cadavers judged to be non-septic a the time of death (Martinez and Malinin, 1996). Harvesting of viable organs also influences the outcome of blood cultures. A review of blood culture results from 1,923 donors examined at the UMTB in a five-year period revealed that only 20 of 501 (4%) donors of viable organs had microorganisms isolated from the blood, compared to 442 of 1,422 (31.1%) donors of devitalised tissues only. These differences in the rate of positive blood cultures between the two donor populations may be the result of significant differences in the timing of blood

149

Microbiological Screening of Cadaver Donors Bone and Organ Donors

Bone donors

Brain Death

Organ Retrieval

Physical Death

Physical Death

Blood cultures Tissue Cultures •*-

Tissue Retrieval

Fig. 1. Timing of the collection of blood and tissue specimens for culture from cadaver donors of bones and organs.

collection. Among donors of musculoskeletal tissues only, blood is obtained for culture following a postmortem period of variable length. In contrast, blood cultures are performed on beatingheart cadaver organ donors following the determination of brain death but prior to the excision of physiologically viable organs (Fig. 1) when the natural clearing mechanisms of the blood may still be functional. It is of interest that, in contrast to the effect that organ donation exerts on the outcome of blood cultures, the excision of viable organs does not seem to have a significant effect on the results of cultures of the bones. In the experience of the UMTB, the rate of positive tissue cultures among donors of musculoskeletal tissues only ranged from 9% for donors with negative blood cultures, to 20% for those with positive findings in the blood. The corresponding rates for combined donors of organs and tissues were 6% and 17% for donors with negative

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and positive blood cultures respectively. Although the organisms recovered from the blood did not always match the identity of those isolated from the tissues, donors with positive blood cultures were more likely to yield positive cultures from the tissues than those with negative blood culture results. 5. Correlation of Blood and Tissue Culture Results Efforts to relate the results of postmortem blood cultures to those of tissues as indicators of disseminated sepsis in prospective donors, have not yielded encouraging results. Martinez et al. (1985) evaluated the culture results from 297 cadaver donors and concluded that blood cultures alone were unreliable predictors of tissue sterility. A more recent study, however, suggests a role for bone marrow aspirate cultures as an aid in the interpretation of blood cultures from clinically non-septic cadavers (Martinez et al., 2003). A retrospective review of the records of 185 selected cadaver donors of musculoskeletal tissues only with positive blood cultures or positive cultures of a bone marrow aspirate from the iliac crest, showed that a majority of the subjects (100 donors; 54.1%) had cultures positive with one or more organisms from both sites. The positive predictive value (PV+) of blood cultures alone for the recovery of the same type of microorganisms from the bones was 38%. For bone marrow aspirates alone, PV+ was 42%. Yet, the recovery of the same type of organisms from both the blood and the bone marrow aspirate had a PV+ of 72%. It is reasonable to assume that not all microbial contaminants of tissues from clinically non-septic donors are the result of haematogenous dissemination. The high prevalence of organisms from the skin or mucous surfaces normally recovered from the tissues, suggests that to a significant extent, contamination can occur at the time of tissue excision in spite of the use of sterile techniques. This data limits the use of blood or bone marrow aspirate cultures as absolute predictors of tissue contamination. As many as 21% of the donors studied had potential pathogens in the tissues that were not recovered


151

from the blood or marrow aspirates. Similarly, pathogens were cultured from the tissues only in 14% of donors from a control group of 185 subjects with negative blood and marrow aspirate cultures (Martinez et al, 2003). These results suggest that neither blood nor marrow aspirates can be considered reliable substitutes for cultures of the tissues, but their combined results may serve as a complementary diagnostic modality for the evaluation of the donor. 6. Interpretation of Culture Results The interpretation of culture results from blood and tissues of cadaver donors has a direct impact on the disposition of the donor, as well as the subsequent processing of the grafts. A number of authors have reported on the various criteria used for donor deferral and tissue acceptance or rejection. The basis of the selection criteria depends on diverse factors such as the presence or absence of micro-organisms of high pathogenic potential in the blood or tissues; the presumed source and distribution of the isolates; identity among isolates from the tissues and the blood; or the magnitude of the microbial load in the tissues. Malinin et al. (1985) recommended rejection of donors who harboured identical species of micro-organisms in the bloodstream and the bone marrow of two or more anatomically unrelated bones. Recovery of multiple organisms from the internal or external flora, or the isolation of highly pathogenic organisms from one or more sites, also justified rejection of the donor. Other investigators accepted tissues for subsequent processing and allograft preparation only if cultures of the blood and surface swab cultures of the tissues were negative for growth after seven days of incubation (Tomford et al, 1990). La Praire and Gross (1991) described the culture of segments of femoral heads obtained from patients undergoing total hip arthroplasty, as a screening procedure for banked bone. No blood cultures were performed. The segments were incubated in broth medium for up to six days, and any evidence of microbial growth was interpreted as

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an indication for discarding the bone. Jackson (1987) recommended the combined use of blood and tissue culture results as a guide for acceptance or rejection of tissues. The excised bones were cultured in liquid medium at 30°C to 32°C and at 20°C to 25°C for 14 days. If the blood cultures were positive, the donor was assumed to be septic, and deferred for donation. Infection also was assumed to have occurred if 20% or more of the tissues yielded the same organisms. Tissues contaminated with organisms common to the skin flora were judged suitable for processing by secondary sterilisation methods. A quantitative approach to the interpretation of tissue culture results has been described (Farrington et ah, 1998). This method involved the use of an immersion technique for the determination of the microbial burden in femoral heads and other skeletal tissues obtained from living and cadaver donors. Each tissue was immersed in sterile saline solution and small samples of the diluent were cultured for the determination of microbial counts. Tissues were rejected if the microbial load of various selected pathogens (Staphylococcus aureus, Clostridium species, Pseudomonas species, beta-haemolytic streptococci and the Enterobacteriaceae) exceeded 50 colony-forming units (cfu) per mL. Tissues also were rejected if cultures yielded > 100 cfu/mL of any other organisms. All tissues with lower bioburden were considered acceptable for further processing and secondary sterilisation. 7. Conclusions The establishment of criteria for the interpretation of cultures from cadaver tissue donors is dependent on a number of factors. Tissue banks select suitable donors and obtain and process bone tissues in a variety of ways. Acceptance or rejection of such tissues is ultimately a risk-benefit judgment. The foremost objective of a microbiological screening protocol for prospective donors should be the exclusion of high-risk subjects from the donor pool. To this end, it may be prudent to consider the distribution


153

of certain microorganisms in the blood and tissues as evidence suggestive of haematogenous dissemination, hence, a cryptic septic state. The selection of specific tissues for continued sterile processing, or the application of sterilisation methods, could be based, in part, on an assessment of the pathogenic potential of the microorganisms recovered by culture. Finally, the reliability of available sterilisation methods also needs to be considered in assessing risk. 8. References AMERICAN ASSOCIATION OF TISSUE BANKS (2001). Standards for Tissue Banking. McLean, Virginia, pp. 35-36. ANDERSON, M. and VESSEY, A. (1996). Cadaveric procurement cultures — What do they mean and how they are used. Abstr. 20th Meet. Amer. Assoc. Tisssue Banks. Washington, D.C. Abstract S-4. BARRIOS, R., LEYES, M., AMILLO, S. and OTEIZA, C. (1994). Bacterial contamination of allografts, Acta Orthop. Belg. 60, 293-295. BENNET, M., JOHNSON, J., NOVICK, S., HILGREN, J., RABE, F. and EASTLUND, T. (1991). Prevalence and growth rate of microbes found at procurement of cadaveric and living donor bone and connective tissue. Abstr. 15th Ann. Meet. Amer. Assoc. Tissue Banks. Clearwater, Florida, p. 35. BETTIN, D., HARMS, C , POLSTER, J. and NIEMEYER, T. (1998). High incidence of pathogenic microorganisms in bone allografts explanted in the morgue, Acta Orthop. Scand. 69, 311314. DEIJKERS, R., BLOEM, R., PETIT, P., BRAND, R., VEHMEYER, S. and VEEN, M. (1997) Contamination of bone allografts: Analysis of incidence and predisposing factors, J. Bone Joint Surg. 79B, 161-165.

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FARRINGTON, M., MATTHEWS, L, FOREMAN, J., RICHARDSON, K. and CAFFEY, E. (1998). Microbiological monitoring of bone grafts: Two years' experience at a tissue bank, /. Hosp. Infec. 38, 261-271. GOCKE, D., YEAGER, J., DOUGHRTY, C. and OSBORNE, J. (1998). Lack of correlation of blood culture and tissue culture in cadaver donors: The MTF experience. Abstr. 22nd Meet. Amer Assoc. Tissue Banks. New Orleans, Louisiana. Abstract S-16, p. 64. HOVE, M. and PENCIL, S. (1998). Effect of postmortem sampling technique on the clinical significance of autopsy blood cultures, Hum. Pathol. 29, 137-139. JACKSON, B. (1987). Bone banking: An overview, Lab. Med. 18, 830-833. KOSTIAK, P. and MICHLOWSKI, M. (2002). Validation of an automated seven-day culturing methodology for the detection of aerobic and anaerobic microorganisms from retrieved musculoskeletal tissues. Abstr. 26th Ann. Meet Amer. Assoc. Tissue Banks. Boston, Massachusetts. Abstract PR-11, p. 63 LaPRAIRIE, A. and GROSS, M. (1991). A simplified protocol for banking bone from surgical donors requiring a 90-day quarantine and an HIV-1 antibody test, Can. J. Surg. 34, 41-48. MALININ, T., MARTINEZ, O. and BROWN, M. (1985). Banking of massive osteoarticular and intercalary bone allografts — 12 years, experience, Clin. Orthop. Rel. Res. 197, 44-54. MARTINEZ, O., MALININ, M., VALLA, P. and FLORES, A. (1985). Postmortem bacteriology of cadaver tissue donors: An evaluation of blood cultures as an index of tissue sterility, Diag. Microbiol. Infec. Dis. 3, 193-200. MARTINEZ, O. and MALININ, T. (1996). The effect of postmortem interval and manner of death on blood and bone


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marrow cultures from non-septic cadaver donors of tissues for transplantation. Abstr. 96th Gen. Meet Amer. Soc. Microbiol. New Orleans, Lousiana. Abstract C-84, p. 96. MARTINEZ, O., BUCK, B., HERNANDEZ, M. and MALININ, T. (2003). Blood and marrow cultures as indicators of bone contamination in cadaver donors, Clin. Orthop. Rel. Res. 409, 317-324 MILLS, A. and ROBERTS, M. (2001). Evaluation of culturing methods at predicting allograft sterility for aseptically processed tissue. Abstr. 25th Ann. Meet. Amer. Assoc. Tissue Banks. Washington, D.C. Abstract S-9, p. 49. REIK, R., WOWK, S., MEADE, D. and BLAIR, K. (1998). Procurement culture study. Abstr. 22nd Meet. Amer. Assoc. Tissue Banks. New Orleans, Louisiana. Abstract S-22, p. 70. SILVER, H. and SONNERWIRTH, A. (1969). A practical and efficacious method for obtaining significant postmortem blood cultures, Amer. J. Clin. Pathol. 52, 433-437. TOMFORD, W., THONGPHASUK, J., MANKTN, H. and FERRARO, M. (1990). Frozen musculoskeletal allografts. A study of the clinical incidence and causes of infection associated with their use, /. Bone Joint Surg. 72A, 1137-1143. VEEN, M., BLOEM, R. and PETIT, P. (1994). Sensitivity and negative predictive value of swab cultures in musculoskeletal allograft procurement, Clin. Orthop. Rel. Res. 300, 259-263. VEHMEYER, S., BLOEM, R. and PETIT, P. (2001). Microbiological screening of post-mortem bone donors — Two case reports, J. Hosp. Infec. 47, 193-197. WILSON, S., WILSON, M. and RELLER, B. Diagnostic utility of postmortem blood cultures, Arch. Pathol. Lab. Med. 117, 986988.



6 SAFETY OF VIRUS INACTIVATION METHODS FOR ALLOGENEIC AVITAL BONE TISSUE TRANSPLANTS

AXEL PRUSS Institute for Transfusion Medicine (Tissue Bank) University Hospital Charite, Berlin M O U J A H E D K A O a n d GEORG PAULI Robert Koch-Institut, Berlin, G e r m a n y

1. Introduction At present, several procedures are used for the inactivation of viruses in the production of avital allogeneic bone transplants. Among these are gamma irradiation (Bright, 1987; Ostrowski, 1968; Sautin, 1963), thermal treatment with moist heat (Hofmann et al, 1996; Knaepler et al, 1994; von Garrel et al, 1997) and peracetic acid-ethanol treatment combined with negative pressure (Starke and von Versen, 1984; von Versen et al., 1992). Apart from the varying irradiation doses reported in the literature, ranging between 15 and 40 kGy, the problems with gamma irradiation appear to be, above all, the development of toxic radicals, in particular with non-processed transplants (Moreau et al., 2000), and the negative effect on biomechanical parameters (Rock, 1991). Additionally, logistics management is complex. Definite assessments regarding the efficiency of the 157

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A. Pruss, M. Kao & G. Pauli

sterilisation procedure of contaminated bone tissue, so far exist only in sporadic reports, and are limited to the human immunodeficiency virus (Campbell and Li, 1999; Hernigou et al, 2000). Experiences with the irradiation of medical products have led to the statement that an irradiation dose of 25 kGy is obviously sufficient for inactivating relevant pathogens, including viruses (Botzenhardt and Thofern, 1988). A reduction of infectivity titres by 6 logio for bacteria, fungi, and spores is recommended for the "industrial sterilisation of medical products" (IAEA, 1990; European Committee for Standardization, 2000) in order to reach the "sterility assurance level" (SAL) (Gaughran, 1985). For example, an SAL of 10" 6 means that the probability of a single viable micro-organism being present on a product unit is one in one million after the unit has undergone a terminal sterilisation process validated to this SAL. However, the concept of SAL is not applicable for assessing the viral safety of human tissue because of the difficulty in standardising such materials. Thermal treatment by means of the Lobator sd-2 system (telos, Marburg/Germany), which is widespread in Germany, is used to disinfect femoral heads which are collected in the context of total endoprosthesis operations of the hip joint. Validation methods described for this procedure (Knaepler et al, 1994; von Garrel et al., 1997) meet the requirements of national and international standards (Paul-Ehrlich-Institut and Bundesinstitut fur Arzneimittel und Medizinprodukte, 1994; CEN, 1994; EMEA, 1996) with slight theoretical reservations. Due to its carcinogenic and mutagenic effect, sterilisation with ethylene oxide was discontinued in Germany (Bundesgesundheitsamt, 1986). Recent investigations, however, have documented the suitability of the procedure if performed in combination with thermal treatment, centrifugation, ultrasonic treatment and washing steps, in the case of human femoral heads (Lomas et al., 2000). A possible reduction of the osteoinductive potential by sterilisation with ethylene oxide is discussed controversially (Thoren and Aspenberg, 1995; Aspenberg and Lindqvist, 1998; Zhang et al, 1997). The use of beta-propiolactone (Lo Grippo,

Safety of Virus lnactivation Methods

159

1987) and formaldehyde also have their limitations and, in addition, clearly reduce the osteoinductive effect (Munting et al, 1988). Since the 1980s, ethanol and peracetic acid (Sprossig and Mucke, 1969; Wutzler and Sauerbrei, 2001) have increasingly been used for tissue sterilisation (von Versen et al, 1992; Pruss et al, 1999; Pruss et al., 2001a). A prerequisite for effective sterilisation of spongiosa bone tissue is the preceding defatting step (Thoren et al., 1995) as well as to observe a maximal transplant thickness of 15 mm (Pruss et al., 2001a). No significant reduction of osteoinductivity or biomechanical properties after PES sterilisation were reported by Haynert (1990) and Thielicke et al. (1990). So far, only incomplete data existed for validating the various methods regarding their inactivating efficiency against clinically relevant pathogens. Considering the demands of clinicians and patients for maximal protection from infections, the following procedures were to be investigated scientifically, in accordance with the guidelines currently in force: • Peracetic acid-ethanol sterilisation (PES model). • Gamma irradiation (irradiation model). • Disinfection by moist heat with the Lobator sd-2 system (thermal treatment model). The panel of pathogens investigated included the majority of potentially possible in-vivo infections of bone donors. The results presented are based on investigations which have already been published in part (Pruss et al, 1999; 2001b; 2002). 2. Material and Methods 2.1. Selection of bone tissue donors Potential bone tissue donors underwent clinical examination for a variety of infectious diseases: virus hepatitis, tuberculosis, syphilis, septicaemia, systemic viral disease, and mycosis, demonstrable at time of death. Excluded were donors with the above-mentioned diseases; those with malignoma; those who

160


had received human growth factor preparations or dura mater transplants; and those with other exclusion criteria by the European Association of Tissue Banks (EATB, 1999). Every bone tissue donor was tested ante or post mortem for hepatitis B virus surface antigen (HBsAg) and for antibodies against HIV-1/-2/ -gO, HCV, and Treponema pallidum. Only individuals negative for these markers were considered as donors. 2.2. PES model 2.2.1. Spongiosa cube specimen As source material for these transplants, spongiosa tissue was collected under sterile conditions from the columna vertebralis and from the epiphyses of femur and tibia, respectively. Fat and connective tissue were carefully removed under aseptic conditions, using scalpel and surgical tweezers. The processchallenge devices (15 x 15 x 15 mm) were cut on the belt saw (Bizerba, Balingen/Germany). Then they were rinsed for 30 minutes using sterile water at 37°C under high pressure to remove the blood completely from the bone tissue. Any remaining fat was removed by placing the tissue into a defatting mixture of chloroform-methanol (two volumes of chloroform and one volume of methanol) under constant agitation (laboratory shaker THYS 2, [MLW, Leipzig/Germany]) over a period of two hours (change of defatting medium after every 30 minutes). Subsequently, the tissues were flushed with methanol eight times (a 15-minute ultrasonic bath treatment) to completely remove any residual chloroform. Methanol was removed by flushing the tissues twice with sterile deionised water. 2.2.2. Virus contamination, sterilisation and homogenisation The defatted and air-dried spongiosa cuboid was placed in a sterile 50 mL Falcon polyethylene cell culture tube (BectonDickinson, Heidelberg/Germany) with a screw-type cap, and covered in 15 mL of the virus suspension. The tube was closed

Safety of Virus Inactivation Methods

161

Fig. 1. Peracetic acid-ethanol sterilisation method.

with a cap that had been perforated eight times to allow equilibration of pressure, placed in an exsikkator, and subjected to negative pressure (200 mbr) by means of a vacuum pump (Meintrup Labortechnik, Lahden-Holte/ Germany), see Fig. 1. After 15 minutes the cuboids were completely penetrated. The remaining suspension was decanted and the tube overlaid with 15 mL of the respective sterilisation solution or, as control, with 15 mL medium. The following three test assays were set up per virus — sample I (SI): contaminated spongiosa cuboid +15 mL peracetic acid-ethanol mixture; sample II (S2): contaminated spongiosa cuboid + 15 mL medium inside the exsikkator; sample III (S3): contaminated spongiosa cuboid + 15 mL medium outside the exsikkator. The sample groups were placed in an exsikkator with a vacuum of 200 mbr, and incubated under continuous agitation at room temperature for four hours. Afterwards, the virus titres in the supernatants (neutralised with sodium thiosulfate 1%; 1/1, v/v) of all three samples were determined. The spongiosa cuboids (volume 2 ml) were transferred to sterile steel beakers of an Omni-Mixer (type OM, Ivan Sorvall

162


Inc., Norwalk, CT, USA), and after addition of 10 mL of 1% sodium thiosulfate, the cuboids were homogenised under cooling in a water ice bath at 1,500 rpm at 4°C for two minutes. Homogenates were centrifuged at 3,000 rpm for 10 minutes at 4°C to collect all virus in the supernatant. Virus titres in the supernatant of the homogenates were determined. Penetration of the bone tissue up into the centre of the cuboid had been verified in preliminary tests. Here, 1 /im flow cytometer (FACS, Becton Dickinson) calibration particles were used as virus analogue. The size of the particles was definitely larger than that of the virus species investigated (PRV 170 nm, HIV about 100 nm, BVDV about 50 nm, HAV about 30 nm, FV-1 about 30 nm, PPV/BPV about 20 nm; Modrow and Falke, 1998). Under the terms of the assay described here, the calibration particles were brought in contact with the cuboid subjected to negative pressure. Subsequently, the central part of the cuboid (a cylinder which was earlier punched from the cuboid, with the upper and lower sections removed, the middle part of the central cylinder repositioned, and the cavities filled and sealed with bone wax [Ethic-R bone wax™]) was collected and centrifuged. From the supernatant, the recovery rate for the particles in the centre of the bone cuboid was determined by flow cytometric analysis and calculations of the concentration. 2.3. Irradiation model 2.3.1. Femoral diaphyses specimen, virus contamination The manufacturing process for diaphysis transplants included the following steps: preparation of diaphyses from human femurs after removing all attached muscles and connective tissue from the bone surface; sawing of diaphyses into segments of 75 mm length (belt saw, Bizerba); washing of the bone marrow canal several times with physiological salt solution (0.9% NaCl, B-Braun, Melsungen/Germany) in order to remove blood from the tissue.


163

It is known that irradiation at low temperatures positively affects the biomedical properties of implants (Ascherl et al, 1986). To use an experimental design as close as possible to that of the production process, all inactivation experiments by irradiation were therefore performed in a Styrofoam box filled with dry ice. Temperature validation (frozen diaphysis on dry ice with medium outside the irradiation facility) showed that the virus suspension inside the diaphysis reached a temperature of - 3 0 ± 5 ° C during irradiation. For the inactivation experiments, the lower open end of each diaphysis was tightly sealed with bone cement (Palacos R™, Heraeus Kulzer, Wehrheim/Germany). The exterior surface of the diatheses was coated with a film of Ethic-R bone wax (Ethicon; thickness approx. 0.1 mm) in order to close any small canals or holes on the bone surface. That the bone was watertight was confirmed by filling the bone marrow canal with 5 ml aqua ad iniectahilia for 24 hours. After removing the water, the diaphyses were transferred into a plastic vessel containing 0.251 of water, fixed with styroflex in an upright position and frozen at -21°C. Then 5 ml of a suspension of cell-free virus were pipetted into the cavity of the diaphyses; the open end was

1^ ^ ^ Fig. 2. Irradiation model (femoral diaphyses).

164


sealed with bone wax, and the diaphyses stored at -21°C (Fig. 2). All steps were carried out under sterile conditions and safety precautions in a biological safety cabinet. The virus-contaminated diaphyses were transported to the gamma irradiation facility of Gamma Service Produktbestrahlung GmbH (Radeberg/Germany), irradiated (see 2.3.2.) with doses ranging from 30.6-35.4 kGy deduced from the D 10 values of the most resistant viruses (BPV, HIV-2, PV-1). One contaminated but not irradiated diaphysis per virus investigated was retained as control to monitor storing and transportation conditions. The titre of viruses in the control sample was used as reference for calculating the experimentally determined inactivation factors. After irradiation all samples were returned to the virological laboratory under the cooling conditions described above. After thawing of the samples the virus titres were determined. 2.3.2. Determination of gamma ray dose distribution, irradiation procedure For technical reasons, it is difficult to measure the X-ray dose directly in specimens, i.e., in the experimentally contaminated diaphyses. Therefore, a dose-distribution study within and on the surfaces of the box used in the irradiation experiments, was performed in the gamma irradiation facility, using a cobalt source 60 Co. Validation took place in a Styrofoam box (length: 34 cm, width: 25 cm, height: 34 cm, weight: 1.46 kg, sample load: 6 plastic flasks with diaphyses or paraffin phantoms) at room temperature and a dose of 1 kGy/h. The following measure points were chosen to determine the dose distribution: two diaphyses and four paraffin phantoms corresponding in size and shape to diaphyses, were used to study the dose distribution inside the box and the influence of mass variation. Dosimeters were placed (i) inside the diaphyses or phantoms (an alanine pellet dosimeter at each end); (ii) on the surface of samples (four alanine foil dosimeters for each sample); (iii) on the inner surface of the box (four alanine foil dosimeters); and (iv) on the outer


165

surface of the box (four alanine foil dosimeters). All procedures followed EN 552 (Deutsches Institut fur Normung, 2001) without major changes. 2.3.3. Determination of virus inactivation kinetics and the decimal reduction value (D 10 value) Eight 15 mL Nunc tubes were filled with 5 mL of cell-free virus suspension each, transferred into a plastic vessel, fixed with styroflex, and placed into a Styrofoam box containing dry ice, which was transported to the irradiation facility in a transport container according to EN 829 (Deutsches Institut fur Normung, 1996). One tube was removed from each group to be used as control, and placed into a plastic vessel on dry ice. The remaining seven vials in the box were exposed to the 60 Co source. At defined intervals (1 kGy/h), one vial each was removed from the box and stored in a plastic vessel on dry ice (temperature in the virus suspension -30±5°C) until virus titration, which took place in the virological laboratory after transportation and thawing as described above. The results of the inactivation kinetics of the different viruses showed a linear relationship between the logarithm of the infectivity titres and the radiation dose applied. To evaluate the results of irradiation, the following equation was used: N(D) = N 0 xlO("D/Dio)" N 0 is the virus titre (log) before and N(D) after irradiation with the dose D; D 10 is the dose necessary to reduce the titre of the infectious agent by a factor of 1 logio; and n is the correction factor introduced to avoid a systematic error (Fritz-Niggli, 1997). 2.4. Thermal treatment model The investigations have not been completed yet (experiments with HIV-2 to be performed yet, control experiments at 4°C). Therefore preliminary results are represented.

166


2.4.1. Femoral head specimen Human femoral heads ( 0 55 ± 1 mm) from donors were used, all of whom had been tested serologically, and whose medical history did not feature exclusion criteria. Subsequently, cartilage was removed from femoral heads by means of a cartilage milling cutter (Aesculap, Tuttlingen/Germany). A central cylinder ( 0 11 mm) was then drilled by means of a keyhole saw from the sagittal plane of the femoral head. Into this defect an exactly matching polypropylene tube (Falcon tube 15 ml) was positioned. The lower end of the tube was equipped with one of the 5 mm corticospongiod cylinder segments, tightly closed with a rubber plug, and sealed with Palacos-R bone cement (Heraeus Kulzer, Wehrheim/Germany). The central cylinder was provided with a 6 mm drillhole, divided into four segments, indicated by the dotted lines (see Fig. 3), and placed into the polypropylene tube. Subsequently, 1 mL virus suspension was pipetted into the central drilling channel. Then the upper corticospongious cylinder segment (5 mm) was fitted-in properly; a cover was screwed onto the tube and fixed with Palacos. The femoral head thus prepared for the experiments was placed into the sterile transplant container and treated thermally according to the manufacturer's instructions (see below) in Ringer solution (B. Braun, Melsungen/Germany). After the cooling phase the incubation fluid was removed under sterile conditions. After the automatic thermal disinfection procedure with the Lobator sd-2 was completed, the central cylinder, the slices of 5 mm spongiosa with corticalis polypropylene tube

-^-^Y

~*V-^——.

central cylinder with drilled hole and virus suspension (segm.) cap

rubber plug/sealing (Palacos) sealing (Palacos)

Fig. 3. Sketch of process-challenge device in diagram form.


167

corticalis, as well as the suspension still in the tube, were removed, and 9 mL of cell culture medium were added (1:10 predilution). Bone material and medium were homogenised in an autoclaved stainless steel container of an Omni mixer (type OM, Sorvall) in the ice bath at 1.500 U/min, and afterwards centrifuged (4°C, 3,000 x g). The supernatant obtained, which is considered to be a 1:10 starting dilution, was titrated. In order to adequately verify that a core temperature of at least 82.5°C was maintained for at least 15 minutes, femoral heads were used in three series for this investigation: native femoral heads, femoral heads with the cartilage removed, and the virus model (femoral head with cartilage removed plus tube). For safety reasons, and to protect the investigator, no parallel temperature measurements were taken in the virus experiments, so that the measurement of comparative core temperatures served to validate the above-mentioned virus model. A central drill hole (0.5 x 27.5 mm) was made at the onset of the Ligamentum capitis femoris into the centre of the femoral head (native or decartilaged), into which the sensor was fitted properly. The temperature sensor (Therm 2281-8, AMR, Holzkirchen/Germany) was run hermetically through the screw-type cap of the disinfection container, and fixed in the bone at the position 27.5 mm with Palacos-R bone cement (Heraeus Kulzer, Germany). Because the sensor takes only measures at its end point, the central temperature gradient was recorded. The starting temperature within the femoral head ranged between 24°C and 26°C. Measurements (temperature/ time) were taken every minute and entered in a computer program (off-line version 4.32/DOS-7.10, DEMA-soft GmbH, Holzkirchen/Germany). The result logs were saved, printed out and evaluated. 2.4.2. "Lobator sd-2" thermodisinfection system (Marburg bone bank system) The lobator sd-2 (see Fig. 4) was developed by the company telos H+V GmbH (Marburg/Germany) for thermal disinfection


168

Fig. 4. Marburg bone bank system (Lobator sd-2).

of allogeneic femoral head bone grafts for clinical application. At present, the procedure has been implemented in Germany exclusively in bone banks affiliated with hospitals. This thermophysical disinfection system guarantees a temperature of 82.5°C for at least 15 minutes, in the centre of femoral heads with a diameter of < 56 mm. The temperature gradients (heating phase, plateau, cooling phase; total duration: 94 minutes) are predetermined by programming of the device, and are not alterable. 2.5. Viruses The viruses investigated were obtained from stocks by the Robert Koch-Institut Berlin, and are registered there and documented. 2.5.1. Enveloped viruses • Human immunodeficiency virus Type 2 (HIV-2), ssRNA, retroviridae genus lentivirus, isolate SBL6669, only tested in PES and irradiation model.

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• Bovine Virus Diarrhoe Virus (BVDV), ssRNA, flaviviridae genus pestivirus, strain Ug 59/Denmark as model virus for hepatitis C virus (HCV). • Pseudorabies virus (PRV; Aujeszky's disease virus), dsDNA, herpesviridae genus varicellovirus, strain Bartha.

2.5.2. Non-enveloped viruses • Hepatitis A virus (HAV), ssRNA, picornaviridae genus hepatovirus, strain HM 175cyt. Table 1. Overview of the cell lines used, and parameters of cell cultivation. Virus

Cell line

Cell cultivation

HIV-2

Lymphoma cells (Molt 4 clone 8)

Cell culture medium: RPMI 1640 reading: 10-14 days pi.*

BVDV

Calf lung cells

Cell culture medium: DMEM f with anti-BVDV-free serum reading: 5 days p.i.

PRV

Mink lung cells

Cell culture medium: DMEM reading: 3 days p.i.

HAV

Embryonal rhesus monkey kidney CRL 1688

Cell culture medium: DMEM reading: 10-14 days p.i.

PV-1

Fetal lung cells


PPV

Fetal porcine testis cells CRL 1746


BPV

Calf lung cells


*p.i. = post infection tDulbecco's Modified Eagle Medium high glucose, fetal calf serum 5%, glutamine (0.5 mg/ml), Penicillin (40 E/ml), Streptomycin (0.04 mg/ml).

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• Poliomyelitis virus type 1 (PV-1), ssRNA, picornaviridae genus enterovirus, vaccine strain PI 18. • Porcine parvovirus (PPV)/Bovine parvovirus (BPV), ssDNA, parvoviridae genus parvovirus. 2.6. Cell cultures/cell lines The cell lines investigated were obtained from stocks by the Robert Koch-Institut Berlin and are registered there and documented (Table 1). The respective viruses were obtained from the supernatants of cultivated infected cells, following procedures reported elsewhere (Scheidler et ah, 1998). The cell debris was discarded after centrifugation, and the viruses obtained were frozen in aliquots at -70°C. 2.7. Virus titrations, virus titre calculation 2.7.1. Preparation of the micro titre plates After preparing 10-fold dilutions of the virus suspensions (supernatants/homogenates) with cell culture medium, 100 jA of each dilution were pipetted into each of four or eight wells of a 96-well micro titre plate. Each of these contained 100 |il medium with 1-5 x 104 cells suitable for the cultivation of the respective virus (Table 1). The micro titre plate was covered, and incubated at 37°C until the virus control showed a cytopathogenic effect (CPE). 2.7.2. Virus titration, CPE, reduction factor Determination of the virus content was done by end-point titration (micro titre plate, four- or eightfold preparation). Regarding the procedure of titration or the methodology of the reduction factor calculation, see the specifications of the publication "Requirements of validation studies as evidence of the virus


171

Table 2. Criteria for the determination of the cytopathogenic effect (CPE). Virus

CPE

HIV-2 BVDV PRV HAV PV-1 PPV, BPV

Syncytia formation Formation of vacuoles, granulation of the cytoplasm Giant cells, polycaryocytes Formation of vacuoles, cell degeneration, no cell lysis Rounding off of cells, decentralisation of the nucleus Plaque formation, cell lysis

safety of drugs from human blood or plasma" (Paul-EhrlichInstitute and Bundesinstitut filr Arzneimittel und Medizinprodukte, 1994). Viruses can cause different forms of the CPE (cell lysis, cell fusion, inclusion bodies, syncytia formation, transformation, etc.). Table 2 gives an overview of the CPEs relevant to the present study. The cytopathogenic effects were observed over several days by means of inverse transmitted light microscopy, always from the same investigator, while a second person read and confirmed them before the concluding evaluation. When toxic effects occurred in the supernatant examined, the appropriate suspension dilution was assessed as "inactivated" in the sense of a detection limit. A quantitative measurement of the virus reduction, using the decrease of the virus genome in the experimental preparation, is problematic due to false positive results which are to be expected (cross-reactivity with genome from blood cells or bone tissue cells) as well as the missing information regarding infectiosity (Willkommen, 2001). The titre reduction indicates the degree of virus inactivation. The titre was given as the virus dilution when 50% of the cell culture showed a cytopathogenic effect (TCID50 = tissue culture infectious dose 50%). The titre was calculated according to Reed and Munch (1938) a n d / o r Spearman and Karber (1974).

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3. Results 3.1. PES m o d e l 3.1.1. Suspension test (kinetics) Using the PES treatment in the suspension tests, it could be shown that most viruses (PV-1, BVDV, HIV-2, PRV) were inactivated under the level of detection as early as after an exposure time of five minutes. In the experimental design described, the reduction for these viruses was associated with a depletion by more than 4 logio gradates. Due to the high toxicity of PES on the HIV-2 cell system, as well as the relatively low starting titre, the exact reduction factor (R,) could not be determined accurately. HAV and PPV showed higher resistance against PES, and even after four hours HAV was not completely inactivated. Here the inactivating kinetics showed a biphasic course, i.e., a rapid initial inactivation by approx. 2.6 log 10 after the first five minutes was followed by a depletion by around 1 logio gradates over the following four hours. Therefore the reduction factor was R = 3.4 logio after 1 hour, and R = 3.7 logio after four hours. The kinetics for PPV also showed a rapid Table 3. Inactivation kinetics of the viruses in the suspension test. Virus concentration (log TCID.50/ml)

Exposure time (min)

PV-1

BVDV

HIV-2

PRV

HAV

PPV

0 5 10 20 30 60 120 240

7.76 2.3

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iR3

Fig. 2. Burn speed of textile.

highly flammable clothes, mainly consisting of acrylic a n d / o r polyester material (Fig. 2). The fire was caused by some small fireworks which came into contact with the decoration on the ceiling. The fire lasted only 10 seconds. Directly after the fire, 203 patients were admitted to 27 hospitals, mainly injured with burns and inhalation trauma. During the fire three people died. The first week after the fire, another seven people died due to burns and inhalation trauma, and 90 burn patients were in the hospital. In the following months, four patients subsequently died. The day after the disaster it was unclear how many burn patients were admitted to hospitals. As all Intensive Care units were occupied, patients were also transported to hospitals outside The Netherlands. Euro Skin Bank had 75,000 cm 2 of glycerol-preserved allograft skin in storage. It was obvious that more allograft skin was needed than the skin bank had in stock. Also, the "regular" burn patients were being admitted to hospitals in Europe and these burn centres also needed allograft skin to treat them.

The Volendam Burn Disaster

373

Therefore, a number of skin banks were contacted in order to procure a big supply of allograft skin. Sam Whyatt of the Stephen Kirby skin bank in London, UK, was contacted, and shipped 10,000 cm2. Daniel Lismont of the tissue bank in Leuven, Belgium, shipped 5,000 cm2. Dr. Jiri Adler from the tissue bank in Brno, Czech Republic, shipped 20,000 cm2. Also, Glenn Greenleaf from LifeCell in New Jersey, USA, was contacted. Glenn Greenleaf is one of the most dedicated persons in skin banking and burn wound treatment over the last decade, with many national and international contacts. He called a number of skin banks in the USA and found LifeNet prepared to ship another 20,000 cm2 of allograft skin to Euro Skin Bank. In total, 200,000 cm2 was needed to treat all burn patients of the Volendam burn disaster. As already mentioned previously, disasters are seldom expected. This was also the case with the terrorist attack on the World Trade Centre in New York on September 11, 2001 (Fig. 3). Euro Skin Bank contacted Glenn Greenleaf immediately to offer

Fig. 3. World Trade Centre on September 11, 2001 (from CNN exclusive).

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/. van Baare & G. Kropman

any help and assistance where possible. As with many disasters there was no good overview of the extent of casualties, and of how much skin would be needed. Eventually, no extra skin was needed as, unfor-tunately, burn-victim mortality was high. The American Association of Tissue Banks has now already set up an Emergency Preparedness Plan to have a quick overview of where and how much allograft tissue is available. International collaboration is therefore very important, but is only possible through long-term relationships. All aspects of tissue banking should be considered when retrieving allograft tissue from other tissue banks, to ensure the tissue is safe and of high quality, including prior consent, screening procedures, disease testing, good lab storage and reliability. These cases demonstrate the importance of international collaboration well before disasters and terrorist attacks ever occur.


14 THE NEED FOR A TISSUE BANK IN A DISASTER: EXPERIENCE IN ARZOBISPO LOAYZA NATIONAL HOSPITAL AFTER THE TRAGEDY IN "MESA REDONDA", LIMA, PERU

MARCO ANTONIO GARCES MORALES and CESER ALEJANDRO REYNAGA LUNA Plastic and Burns Department of Loayza Hospital Lima, Peru

"When we know what do we have to do in order to save a life, we don't argue ... we do it" ... Peter Safar, 1987

1. Introduction This experience is based on the participation of a human group, which was responsible for the plastic surgery and burns treatment service of Loayza Hospital during the disaster in Mesa Redonda on December 29, 2001. At 7:10 p.m. on a Saturday, a great tragedy occurred in central Lima. It started in the cradle of a flea market, where there was a minimum of safety regulations, and the incident lasted for five hours, leaving a macabre scene of death and desolation. 375

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M.A.G. Morales & C.A.R. Luna

It all began with the explosion of 90 tons of gunpowder, generating temperatures of 1,200°C that claimed more than 660 victims: 300 dead, 352 who disappeared, and caused a loss of 100 million dollars. 2. General Analysis To be able to understand our task, we needed to define the basic concepts. A disaster is a big catastrophe, and an unfortunate and mournful event. To be able to confront it, it is necessary to have a plan in order to respond to the great number of deaths and injuries, and be prepared to attend to them with efficiency, and to adopt the necessary security measures, which will contribute to its future prevention. The tragedy in Mesa Redonda was very complicated. Its root cause was human and technical failure. Based on the number of victims, this tragedy was defined as very serious by Mabrouk, 1981 and Gomez, 1988. The victims' injuries were burns, inhalation and other bodily damage produced in different parts of the body by fire, gunpowder explosions, building collapses, physical crushing, toxic gases and electric discharges. 3. On-Site Management of the Mesa Redonda Tragedy Priority in the tragedy area was given to the evacuation of victims who could receive immediate treatment in an adequate location, with the possibility of saving their lives. This first intervention was handled by the emergency systems of the Peruvian civil defense: firemen, policemen, health ministry and social insurance department, and civilians. There were more than 5,000 people involved, with no escape paths, trapped in traffic jams, dodging fireworks all over the streets. The lack of a fireproof system, and constructions that lacked security measures, formed a fatal urban trap, as found in most cosmopolitan cities of Third World countries.

The Need for a Tissue Bank in a Disaster

377

During the first 15 minutes, a fireball formed, with the temperature reaching between 700-1,200 degrees Celsius. This is an important point, since it explains the evaporation of the bodies. For every three bodies present, one evaporated. There was chaos, panic and confusion surrounding the area. Hundreds of phone calls were received and needed to be responded to immediately. The first evaluation suggested that 300 people were trapped. Evacuation of the wounded was started but the situation became uncontrollable. Later, defensive actions were taken against the fire; security perimeters were established, and the electricity supply was cut. There was an increase of water pressure directed to the area, and water trucks started to arrive. An incident command center was installed, along with the opening of sectors for medical assistance, search and rescue, fireproofing, logistics, security and public relations. The coordination of assistance to patients within Loayza Hospital started towards the daily changing of shift, and because of the holiday period, there were many beds available. There was great solidarity perceived among members of the health team; personnel on vacation and those on leave returned immediately to their working positions. 4. Management of the Tragedy in the Emergency R o o m The first check-up took place in the tragedy area. It was fast and simple, based on the tragedy classification 919970, which is a simplified variant of the start method. Respiration, circulation and consciousness were evaluated, with control of the unconscious victims and haemorrhage control. The second check-up was undertaken in the emergency room, taking into account the age, sex, extension and depth of the burns and inhalation harm suffered by the patient. The victims were later taken to the medical area where the third and most important check-up was made. Of the 147 patients, 55 were hospitalised in the medical area, which had been adapted for the burns, plastic surgery and general treatment.

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5. The Plastic Surgery and Burns Service An operational command centre was installed to manage the circumstances of the tragedy, and to appraise the physical resources, equipment, logistics (skin substitutes), communication, and transportation. The Peruvian Government ordered, by supreme resolution, free treatment in the hospitals for those people with burns and related injuries. Of the 147 patients in the emergency room, 92 received outpatient treatment (analgesics, oral hydro reposition, sedatives) and 55 were hospitalised. The treatment started with the participation of a multidisciplinary team to achieve an effective union between the medical and paramedical teams for the treatment of the burn patients according to the following scheme: LOCALISATION

EVOLUTIVE STATE

\ / ATMOSPHERE CARE

t I

BURNED PATIENT ,

HEALTH SYSTEM

CAUSE-BURN

/ \ BURNS EXTENT

6. Materials and Methods There were more female patients (60.71%) than males (39.29%). The proportion by age was greater among those 21-40 year old patients (75%) and among 0-20 year old patients (10.71%) and those from the 41-60 age group (14.29%). The burns extent (ruler of 9 and with the palm of the hand) of less than 10% was 48.15% in patients, and greater than 10% in 51.95% of patients. In terms of depth of burn (using the Benaim and American classification) the patients had second to third degree burn degrees in relation to seriousness (Garces abbreviated index of the burninjury). Other diagnoses were: inhalation damage, thermal keratoconjunctivitis, diabetes, cervical concussion, bronchial asthma, and fractures.


379

The treatment started with the removal of burnt cloth; cooling of the burn in patients with less than 10% burns; cleaning of the burned areas with iodine; administration of 1% silver diazine, and the application of the occlusive method with gauze. This was necessary because of the lack of sufficient supplies from an available tissue bank, which would have allowed sufficient temporary biological dressings. This procedure was followed every day in patients with greater than 10% of burns. Also, resuscitation isotonic solutions were applied. The patients with inhalation damage were treated with oxygen, hyperbolic oxygen, artificial respirators and treatment for respiratory rehabilitation. Nevertheless, there was a need for a bronco-fibrescope to diagnose and treat the lower and upper respiratory paths. The surgical treatment proceeded with the elimination of crust (dead skin). From the second to fifth day, the surgical teams worked double shifts from Monday to Sunday. They made eight interventions per day with the help of doctors from Israel and China. They used manual and electric dermatoms, compressed air and skin meshes, and also the escarectomy technique according to the depth and place of the burn, including tangential escarectomy, tangential debridement, aponeurotic escarectomy and the following surgeries: surgery heels + escarectomy; escarectomy + autograft; escarectomy + heterograft; escarectomy + autograft + heterograft; and local flaps. The skin substitutes used were autografts (microcrafts, stamps, laminates, and meshes). For the transitory patients cadaveric skin (from the USA), pig skin in laminates of 10 x 10 cm, rings in laminates of 30 x 30 cm were used. In the post operative stage, sulfamylon solution in 5% and silver nitrate in 0.5% were used. 7. Discussion Accidents in the home, during transportation, at work, at entertainment centres, and during civil violence or wars, produce great numbers of victims. The cost is more than 550 million dollars and in the United States these accidents cause 33% of the

380


hospitalisations, with each hospitalisation costing US$34,000 and up to US$317,000 for severe cases. For this reason, it is important to take all possible preventive measures. The medical management of the Mesa Redonda tragedy was carried out under the unified temporary command system, due to the lack of resources. This included an incident action plan. The transportation of the patients was carried out as quickly as possible because the tragedy was out of control. In the hospital the treatment needed was determined in three check ups, according to the patient's condition. For these reasons, it was necessary to have human resources which can work in motivated and compact multidisciplinary teams to provide adequate diagnoses, and to start the respective treatments promptly. The treatment was undertaken, using a simple protocol starting with a resuscitation period (parlank formula). For local treatment, only silver diazine at 1% was used. Surgical treatment started between the second and fifth day according to the depth, extension and localisation of the burn. In patients with extended burn areas, skin substitutes were used, which had been supplied by the tissue bank located at the Children Health Institute (Insititut de Nino), Lima. Donations of pig skin came from China, allografts from the United States and fresh and refrigerated tissue rings prepared by our service. The treatments utilised 661,500 cm 2 of skin substitutes. The actual needs were greater due to the escarectomy and need for autografts after the escarectomy. It is known that the preparation of the bed is very important for the grafts, especially for the patients with large burn areas and where only small areas of donated skin are available. 8. Conclusions (1) To deal with a tragedy involving a high number of casualties, the common areas at the hospital should be converted into restricted areas.


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(2) The initial patient treatment must be undertaken using a simple protocol. (3) Escarectomy must be carried out between the second and fifth days. (4) There is a need for a multi-disciplinary and trans-disciplinary team. (5) Use of skin substitutes available (pig skin, anionic membrane and the products of tissue banks) is urgently required and logistically planned for, before contingencies. (6) The creation of tissue banks in all the countries is necessary so that they can together contribute when there is such an emergency. The value of a man should be judged according to what he gives and not according to what he is able to receive. Albert Einstein

9. References AMERICAN BURN ASSOCIATION (1984). Guidelines for service standards and severity classification in the treatment of burn injuries, Am. Col. Surg. Bull. 69, 24-28. ARMSTRONG, C.E., Jr, SCHAEFFER Y C.P. Y C. P. ARTZ (1984). Treatment of. ARTIGAS, N. Y COL. (1984). Normas Medico Quirurgicas Para El Tratamiento De Las Quemaduras. Editorial Andres Bello, Santiago De Chile. ARTURSON, G. (1981). The Los Alfaques Disaster: A boiling liquid expanding — Vapour Explosion, Burns 7, 233-251. ARTURSON, G. (1987). The Tragedy of San Juanica: "The Most Severe LPG Disaster in History", Burns 13, 87-102.

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ASISTENCIA SANITARIA INTERNACIONAL (1989). Medicina De Catastrofe. Boletin Del Dpto De Med De Catast. Dir. Nac. Defensa Civil Min. De Defensa. Buenos Aires 2(9), 10. BABCOCK. C.L (1974). High Rise. Building Fires and Fire Safety. NEPA, pp. 3. BACKES, A.T. (1973). Great Fire of America. Wankesha Country Beautiful Corp., pp. 124-130. BECK, A.T. Y COL (1961). An inventory for measuring depression (B. D. I), Arch. Gen. Psychiatry 4, 561. BENAIM, F. (1956). "Diagnostico De Gravedad De Las Quemaduras", Bol. Soc. Arg. Cir. Plast. 1. BENAIM, F. (1959). Diagnostico De Gravedad De Las Quemaduras, Bol. Soc. Arg. Plast. 1. BENAIM, F. (1962). Tratamiento De Urgencia De Las Quemaduras Graves. Vol. 1, pp. 13-34. Buenos Aires: Edic. Fanetti. BENAIM, F. (1968). Quemaduras. En Michans, J.R. (Ed.) Patologia Quirurgica, 2a. Edic, Vol. 1, Cap. 6. Buenos Aires. El Ateneo, pp. 134. BENAIM, F. (1971a). Burn Centers And Prevention Campaigns in Latin America Countries. En Matter, P., T.L. Barclay Y Z. Koniciova (Eds- Transaction of the thrid international congress on research in burns, Bern: Hans Huber Publishers, pp. 39. BENAIM, F. (1971b). Personal opinion on a uniform classification of the depth of burns. Transaction of the third international congress on research in burns. Bern: Hans Huber Publishers, p. 715. BENAIM, F. (1978). Cuidado Progresivo Y Equipamiento Especializado En Las Unidades De Quemados, Cir. Plast. Arg. 2, 28-35.


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BENAIM, F. (1982). Evolution De La Asistencia Al Quemado En La Argentina, Rev. Arg. De. Cir. 43, 32-40. BENAIM, F. (1984a). El Especialista En Quemados, Rev. Arg. Quern. 2(3). BENAIM, F. (1984b). Qeumaduras. En Torres, R. Tratado De Cirugia, Cap. 10. Interamericana, Mexico, pp. 259-304. BENAIM, F. (1984C). Plan De Regionalizacion Para La Atencion Del Paciente Quemado En La Republica Argentina, Rev. Ar. De. Cir. 46, 170-175. BENAIM, F. (1984d). Asistencia Interdisciplinaria Para El Paciente Quemado, Rev. Arg. Quern. 2(2). BENAIM, F. (1986). Quemaduras. En Coiffman, F. Y Col, ed., Texto De Cirugia Plastca, Reconstructiva Y Estetica, Tomo 1, Cap. 3. Salvat, Barcelona, pp. 243-282. BENAIM, F. (1989a) Catastrophe En Niteroi, Brazil 1961. Fire in a Circus. Argentinian Help to the Burn Victims. Ludwingshafen International Symposium on Disaster Management of Burns. September 1-2, Ludwingshafen, W. Germani. BENAIM, F. (1989b). Quemaduras. En Boretti, J.J. Y C. Lovesio, eds., Cirugia, Cap. 15. El Ateneo, Buenos Aires, pp. 202-216. BENAIM, F. Y D. SCHNEERSON (1964). Conducta Medica En Caso De Catastrofe De Quemaduras. Symposium Conducta Medica En Caso De Catastrofe. Academia Nacional De Medicina, Buenos Aires, pp. 313-324. BENAIM, F. Y COL (1964). Incendios Y Exlposiones. Symposium Conducta Medica En Caso De Catastrofe. Academic Nacional De Medicina, Buenos Aires, pp. 481-487. BEN-HUR, N. Y H. SOROFF (1975). Quemados Y Sindrome De Quemaduras Por Cohetes Antitanque, Cir. Plas. lb. Lat. 1, 77-84.

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BENNETT, C. Y H. BRISTOL (1970). Controlled Survery of Effects on Health of Local Community Disaster, Br. Med. }. 3, 454. BENZAQUIN, P. (1976). Fire in Boston's Coconut Grove. Braden Press, Boston, pp. 204-224. BEST, R. (1978). Reconstruction of a tragedy: The Beverly Hills Supper Club Fire. NFPA, Boston, p. 314. BONTE, F. (1988). Chernobyl Retrospective, Semin. Nucl. Med. 18, 16-24. BOSWICK, J. (1987). Initial care of burn wounds. En Aspen Publishers Inc. (Ed.) The art and science of burn care. Rockville Maryland, Royal Tunbridge Wells. Botha, C. (1986). The dental identification of fire victims, /. Forensic Odontostomatol. 4, 67-75. BUCEK, S. Y V. PINMCOVA (1987). Micridisastres in the Prague Burn Centre. II Congress On Burn Treatment. Kosice-Saca, Czeschoslovakia, p. 69. BURTON, H. (1973). The Morro Castle. Viking Press, New York, pp. 92-103. BUTMAN, A. (1982). Responding to the mass casuality incident. A guide for EMS personnel. Emergency training, Akron, Ohio. CARSWELL, J. Y A. HATHAWAY (1976). A fire at Nakivubo, Kampala: A case Report. II — Infection in a group of burned patients, Burns 2, 184-190. CHAMPION, H. Y COL (1981). The Trauma Score, Ann. Med. 9, 672. CHURCHILL, B. (1953). Panic in Disaster, Ann. Surg. 138, 395. CLASS, A. (1959). Physiological aspects of disaster, JAMA 171, 222. COBB, S. Y E. LINDEMANN (1943). Neuropsychiatric observation during the Coconut Grove fire, Ann. Surg. 117, 814.


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COLE, M. Y COL (1986). Gasoline explosions, gasoline snifting: An epidemic in young adolescent? JBCR 7, 532-534. COMMITTEE ON TRAUMA OF THE AMERICAN COLLEGE OF SURGEONS (1986). Qualification of trauma-care personnel, Am. Col. Surg. Bull. 71, 13-38. COMMITTEE ON TRAUMA OF THE AMERICAN COLLEGE OF SURGEONS (1983). Hospital and pre-hospital resources of optimal care of injured patients, Am. Col. Surg. Bull. 68,11-21. CORNELL, J. (1979). The great International Disaster Book. New York Gulf and Western Corp., pp. 290-292. DAS, R.A. (1984). Circus Fire Disaster In India (Feb. 7,1981), Bull. Clin. Rev. 1, 26-31. DAS, R.A. (1983). 1981 Circus Fire Disaster in Bangalope, India: Causes, management of burn patients and possible presentation, Burns 10, 17-29. DE LEONE, H. (1988). Categorizacion De Pacientes En Caso De Catastrofe. Circulacion. Medic. De Catastrofe. Boletin Del Dpto Med De Catast, Dir. Nac. Defensa Civil. Min. De Defensa. Buenos Aires 1(2), 1. DICKINSON, W., D. SHARPE Y A. ROBERTS (1988). Tangencial excision of scalp burns: Experience from the Brodford fire, Burns 14, 151-155. EDLICH, R. Y COL. (1983). Firefighter's guide to emergency rescue and care of victim burned in structural fire, JBCR 4, 367-373. FREY, R. Y P. (1980). Types and Events of Disaster. Springer, Verlag, New York, pp. 282-285. GOMEZ, M. (1988). Categorizacion De Pacientes En Caso De Catastrofe. Respiracion. Medic. De Catastrofe. Boletin Del Dpto Med De Catastr. Dir. Nac. Defensa Civil. Min. De Defensa. Buenos Aires 1(2), 4.

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GORMICAN, S. (1982). CRAMS Scale: Field triage of trauma victims, Emerg. Med. 11, 132. GUNN, S. (1989). Disaster Medicine and International Relief Multilingual Dictionary. English, French , Spanish, Arabic. Kluwer Academic Publishers, Dordrecht, Holland. GRUPTA, R. Y A. SRIVASTAVA (1988). Study fatal burns cases in Kanpur (India), Forensic Sci. Int. 37, 81-89. HADDEN, W., W. RUTJERFORD Y J. MERRET (1978). The injuries of terrorist bombing in a study of 1532 consecutive patients, Br. J. Surg. 62, 525. HAMILTON, M. (1960). Depression Rating Scale (HDRS), /. Neural Neurosurg. Psychiatry 23, 56.

SECTION IV: TISSUE GRAFTS IN ORTHOPAEDICS



15 MAJOR LIMB RECONSTRUCTION USING MASSIVE CADAVERIC ALLOGRAFTS

HENRY J. M A N K I N Orthopaedic Oncology Service Massachusetts General Hospital H a r v a r d Medical School, Boston, M A 02114

1. Introduction Over the past 31 years, limb-sparing bone tumour treatment has depended heavily on the replacement of the resected segment of bone with a cadaveric allograft. Since the pioneer efforts of Frank Parrish in Houston (Parrish et al, 1966; 1973) and Carlos Ottolenghi in Buenos Aires (Ottolenghi et al, 1996), a number of orthopaedic oncologists have utilised the system and reported their findings (Alho et al, 1998; Delloye et aZ.,1988; Dick et al, 1985; Friedlaender et al, 1999; Gouin et al, 1996; Hejna et al, 1997; Hornicek et al, 1998; 1999; Jofe et al, 1988; Makley et al, 1985; Mankin et al, 1982; 1987; 1996; Mnaymneh et al, 1986; 1989; Ortiz-Cruz et al, 1997). Bone banks have appeared, and through the efforts of the American Association of Tissue Banks and their leaders, have developed a set of guidelines to maintain the safety and competence of the parts (Buck et al, 1989; Conway et al, 1990;

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H.J. Mankin

Schachar et al, 1991; Strong et al, 1996; Tomford et al, 1983; 1989; 1999). Special attention has been paid to cryopreservation of cartilage (Schachar et al, 1991; 1994; Tomford et al, 1983); prevention of transfer of infection (Buck et al, 1989; Hernigou et al, 1991; Lord et al, 1988; Strong et al, 1991; Tan et al, 1997; Tomford et al, 1990); banking methodology (Tomford et al, 1989; 1999); use of radiation sterilisation techniques (Conway et al, 1990; Loty et al, 1990); and most recently genetic structure of the graft in correlation with the host recipient (Alho et al, 1998; Friedlaender et al, 1976; 1999; Muscolo et al, 1996; Strong et al, 1996). Since 1971, the author and a group of colleagues in the Othopaedic Oncology Service at the Massachusetts General Hospital have performed almost 1,200 operative procedures in which a segment of frozen allograft bone has been introduced into a patient, most frequently after wide or marginal resection of a malignant or aggressive bone tumour. Since 1976, the bone segments have come from a local bone bank, which is now headed by William W. Tomford, a respected member of the AATB and a leader in the field of bone banking (Tomford et al, 1989; 1999). A careful review of the results of the allograft procedures was performed, and the purpose of this presentation is to analyse the outcomes and determine the factors which seem to play a role in the success or failure of the procedure. It should be noted that of the almost 1,200 procedures 75 were hemipelvis grafts, which are considerably different from those for more peripheral parts. The indications, clinical management, complications and outcomes for this group differ from the other procedures, and are therefore not included in this review. The study is limited to a description of the results for 1,098 patients who received their peripheral part alloimplants between November of 1971 and June of 2002. The outcome studies require that only patients followed for two or more years are included, and this number is 1,052.


391

2. Materials and Methods Data for the entire allograft series were collected in a Microsoft Foxpro system (Microsoft, Redmond, WA) (Mankin et al, 2002) which houses information for almost 15,000 patients treated by the Orthopaedic Oncology Group since 1972. A special file in this system is used to maintain all of the data regarding the 1,098 allografts performed by the group since 1971. Information included consists of the patient's demographics, diagnosis, MSTS stage (Enneking et al., 1980), anatomical site, resection margin, use of adjunctive treatment modalities, tumour complications, allograft complications, additional operative procedures and outcome. The statistics are in part performed by the Foxpro system, but in addition, a statistical program was used for study of data using Cox regressions (Cox et al, 1972) and KaplanMeier outcome studies (Kaplan et al, 1958) (BMDP System, Los Angeles, CA). The 1,098-patient group consists of 573 males and 525 females with a followup ranging from two to 367 months, with a mean figure of 86 ± 72 months. The average age for the patients was 32 ± 18 years, with a range from two to 86 years. An analysis of the diagnoses for which the procedure was performed, shows that the majority were done for malignant tumours including osteosarcoma (285 patients), chondrosarcoma (148 patients), parosteal osteosarcoma (62 patients), fibrosarcoma or MFH (51 patients), Ewing's sarcoma (50 patients), metastatic carcinoma (47 patients), soft tissue sarcomas (34 patients), and adamantinoma (32 patients). In addition, in the early days of the system, 139 patients with giant cell tumours were also treated with resection and allograft implantation, as were 56 patients with other neoplasms. There were in addition, a total of 182 patients treated for non-tumourous conditions including failed allografts or total joint replacements (90 patients), massive osteonecrosis or traumatic loss (58 patients), fibrous dysplasia (14 patients) and 20 others.

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Table 1. Anatomical sites for 1,098 allograft procedures performed between 11/1971 and 6/2002. Osteoarticular (583) Distal femur Proximal tibia Proximal humerus Proximal femur Distal radius Distal humerus Scapula Distal tibia Proximal ulna Talus Elbow Phalanx

268 116 84 41 27 15 10 8 6 3 3 2

Intercalary (295) Femur Tibia Humerus Fibula Ulna Radius Scapula Hand bone Foot bone Sternum Vertebra

122 102 49 6 5 5 2 1 1 1 1

Allograft-prosthesis (138) Proximal femur Distal femur Proximal tibia Entire femur Proximal humerus Scapula Elbow

72 35 16 10 2 1 1

Allograft arthrodesis (82) Distal femur Proximal humerus Proximal Femur Distal Tibia Proximal tibia Distal radius

36 28 8 6 3 1


393

Five hundred and eighty-three of the procedures were osteoartieular (involving a joint); 295 intercalary (introduced to fill a gap in the host bone); 138 were allograft plus prosthesis and 82 were allograft-arthrodesis. The anatomical locations for these segments are shown in Table 1. The outcome for these patients was assessed according to a scheme originally proposed in the 1980,s (Mankin et ah, 1982). The results, or more appropriately, the "outcome" of the procedure, was determined by scoring the result as excellent, good, fair or failure. An excellent outcome is defined as a functionally normal result with basically no limitations or disability, no local recurrence or pain. A good outcome is defined as a pain-free functionally useful limb but limited in terms of agility and sports participation. Patients described as fair are limited in employability, have discomfort and limitations, which require support

(A)

(B)

Fig. 1. A 19 year old woman presented with a painful right humerus in June, 1974. A fibrosarcoma was discovered [Fig. 1(A)] and the proximal humerus resected and replaced with an allograft segment [Fig. 1(B)]. At the age of 47, she remains partially limited in abduction and forward flexion, but otherwise enjoys normal movement.

H.J. Mankin

394

for ambulation. The term failure was reserved for patients who required removal of the graft or amputation. For the statistical studies reported in this presentation, an "excellent" or "good" result is considered to be a successful outcome while a "fair" or "failure" is designated as a failure. Of note is the fact that the failure category includes patients who have had a local recurrence, which led to removal of the graft or amputation. If one is determining the outcome of the grafting procedure rather than the tumour treatment, it is appropriate to remove these cases from the series. Examples of allograft procedures are shown in Fig. 1 through 3. The results of all three of these cases were considered to be "good", and hence their designation as successful outcomes. All three of these patients remain free of disease, have no pain and although two are moderately restricted, they remain quite functional. The patient described as Case 1 has remained a successful outcome for 31 years.

(A)

(B)

(C)

Fig. 2. A 16 year old female was found to have an adamantinoma of the right tibial shaft in September 1988 [Fig. 2(A)]. A resection was performed and an allograft implanted [Fig. 2(B)]. A recent X-ray [Fig. 2(C)] shows excellent healing of the graft. The patient is now 31 years of age and is fully functional.


(A)

(B)

(D)

395

(C)

(E)

Fig. 3. A 16 year old female presented in September 1980 with a painful proximal left tibia. She had two previous operative procedures for a giant cell tumour and each time had a prompt and aggressive recurrence. [Fig. 3(A)]. After complete resection of the proximal end of the tibia an allograft was implanted [Fig. 3(B)] and fixed with a plate and screws. The recipient's patellar tendon was sutured to the donor's tibial tubercle. A recent X-ray [Fig. 3(C)] shows good healing of the graft and preservation of the joint. At the age 38, her function remains good with full extension [Fig. 3(D)] and 90 degrees of flexion [Fig. 3(E)].

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H.].

Mankin

LIFE TABLE FOR ALLOGRAFT TRANSPLANTATION 77% _j

1

1

1

1

1

1_

12 16 20 GRAFT SURVIVAL IN YEARS

Fig. 4. A Kaplan-Meier plot for the entire series allografts followed at least two years. The excessive fall-off in the first three years is noticeable but the overall figure for the entire series is 77% good or excellent.

3.

Results

The overall level of success for patients treated between November 1971 and September 2000 is shown in the Kaplan-Meier graphic in Fig. 4. The success rate for this series is a mean value of 77% according to the described definition of "excellent" and "good" results defined as successful, and "fair" and "failure" defined as failure. The numerical values for the total number of cases in this group is 73% (769/1,052), but if as described above, the 49 local recurrences which led to removal of the graft or amputation are deleted, the value becomes 769/1,003 or 77%. 3.1.

Date of performance

Analysis of the effect of date of performance showed no statistical difference for 131 patients whose surgery was performed


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prior to 1982, from the 520 performed between 1982 and 1991, and the 352 procedures performed from 1992 to 2000. 3.2. Gender The statistical study comparing the results for 485 female patients (75% success rate) and 518 males (79% success rate) failed to show a significant difference between the two groups. 3.3. Age Four age groups were studied. These included 326 patients whose ages were less than 20, and for whom the result was a 73% success rate; 381 patients between the ages of 20 and 39, for whom the success rate was 78%; 195 patients whose ages fell between 40 and 59, whose success rate was 79%; and 101 patients whose ages were greater than 60 years and whose success rate for the allograft segments was also 79%. Although a slight difference was noted for the younger patients, these data are not statistically significant. 3.4. Anatomical site The variation in anatomical site was statistically significant. Of 353 implanted distal femoral segments, 242 remained successful (69%). Proximal tibial segments were somewhat better in terms of outcome with 112 of the 150 grafts remaining successful (75%). Proximal femoral grafts had a success rate of 79% (108 of 136) and the best of all were the 122 proximal humeral grafts with a success rate of 82% for 122 implants (p < 0.01 by chi square). 3.5. Types of grafts Figure 5 displays the results for the type of graft. As can be noted, the best results are with the intercalary grafts which have

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a success rate of 86%. The allograft prostheses and osteoarticular grafts are similar in result at approximately 75% excellent or good. Poorest of all are the allograft-arthrodeses, which have a failure rate of almost 40% (Donati et al., 2002). These data are highly significant according to the Kaplan-Meier plotting system at p < 0.00001. 3.6. Effect of diagnosis, stage and adjuvant therapy Diagnosis and stage of disease had a significant effect on graft survival. These data are clearly evident in Fig. 6 which compares the results for the three major high grade tumours, osteosarcoma (66% success), chondrosarcoma and Ewing's sarcoma (both at over 80% successful). Chondrosarcomas are usually not treated

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EFFECT OF MSTS STAGE ON ALLOGRAFT SURVIVAL (P 0.05), such as progressive motile spermatozoa 32.5 ± 4.7% versus 31.5 ± 4.9%, spermatozoa with intact acrosome 16.7 + 2.0% versus 15.4 ± 1.9%, spermatozoa stainable by eosin 29.2 + 6.4%

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versus 29.6 + 6.7%. The passage through the column led to a sperm loss of 0.8 + 1.2%. 4. Discussion The term apoptosis defines a cell death programme, which is molecularly and morphologically distinct from necrosis. Intense research gave a remarkable insight into that signalling cascades through the network of pathways. While apoptosis is extensively described for somatic cells, there is sadly, a lack of data in male germ cells. Recent models of apoptosis include receptormediated pathways and intrinsic triggered apoptosis besides cytotoxic or stress-induced forms. Binding of ligands like Fas-L activates the death receptors (for example, CD95, Fas) resulting in trimerisation and coupling with adapter proteins to form a death-inducing signalling complex (DISC). The Fas-receptor (CD95) is a glycosylated cell surface molecule of about 45 to

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52 kDa (Walczak and Krammer, 2000). DISC transduces the apoptotic signal via activation of caspases. Caspases (cytosolic cysteine containing aspartate specific proteases, CP) are a family of highly specific proteases that contain an amino acid, cysteine, in their active sites. After proteolytic activation in a cascade (Thornberry and Lazebnik, 1998; Wolf and Green, 1999) their targets are cleaved after the amino acid aspartate (Alnemri, 1997). Depending on their function, two groups of caspases could be differentiated: initiator (CP 8, 9, 2, 10), and effector or downstream (CP 3, 6, 7) caspases. Caspase 8 was identified to be the most important initiator enzyme triggered by DISC (Kischkel et al., 1995). CP9 realises, together with many other regulators and transductors (for example, cytochrome c released from disintegrated mitochondria), intrinsic apoptosis. Both enzymes are highly effective activators of downstream caspases. CP3, the most important among them, executes the final disassembling of the cell by cleaving of a variety of cell structure proteins. On the other hand, activation of protein kinase C5 which is involved in the phosphatidylserine redistribution and endogenous endonucleases is known to be CP3 triggered (Frasch et al., 2000). Finally, the caspase-activated DNAse generates DNA strand breaks resulting in decreased male fertility (Enari et al., 1998; Zini et al., 2001). Therefore, the usage of spermatozoa that are about to absolve apoptotic processes, should be minimised in assisted fertilization techniques. After cooling biological membranes, a phase transition of the lipids in the membrane of spermatozoa takes place (Hammerstedt et al., 1990). This reordering of membrane components may lead to a loss of stability of the lipid bilayer (Schiller et al., 2000) and exposure of phosphatidylserine on the sperm surface, which occurs in nucleated cells during early phases of apoptosis (Fadok et al., 1992). The location of phosphatidylserine on the outer leaflet of the plasma membrane can be identified by the phosphatidylserine-binding ligand Annexin V, a 3 5 36 KD protein (van Heerde et al., 1995). The specifity of Annexin V-binding to phosphatidylserine has been demonstrated (Martin

570


et ah, 1995) and was applied in our experiments for immunomagnetic separation of spermatozoa after cryopreservation. Both the death receptor CD95 and the activation of caspases 1-9 were predominantly found within the ANMB positive spermatozoa. Cryopreservation significantly raised the number of cells showing externalised phosphatidylserine. The elevation in CD95 positive cells after cryopreservation was not significant, but there was a marked increase of Pan-caspase positive sperm. The process of freezing and thawing increased the levels of spermatozoa presenting with those activated enzymes in the ANMB positive fraction only. This indicates induction, transmission and execution of apoptosis due to cryopreservation. Obviously, cryopreservation has had no significant impact on CD95. These results imply that apoptosis due to cryopreservation is not triggered by Fas-Ligand — Fas-Receptor (CD95) interaction, but involves the caspase cascade. The association between externalised phosphatidylserine and CD95 may represent a significance of the death receptor in different settings. Our investigations revealed a highly significant correlation between externalisation of phosphatidylserine and CD95 at the spermatozoal surface, as well as activation of Caspases 1-9 in the cytosol. The Annexin V-MACS technique was able to strongly reduce spermatozoa that exhibit these features of apoptosis, and might be a very useful tool to achieve vital, non-apoptotic spermatozoa in order to improve the fertilisation rates in assisted reproduction. Furthermore, the separation columns and their magnetic field did not exert any detectable effect on the spermatozoa in our experiments — a prerequisite for application in the clinical laboratory. MACS may be used not only for providing a high quality sperm fraction. The technique may also be applied to the evaluation of semen sample quality by detection of the ratio between ANMB" and ANMB+ — sperm fractions. Thus, the known feasibility and safety of the MACS enrichment procedure in patients with autologous transplantation of peripheral blood stem cells (Despres et ah, 2000) was confirmed by andrological examination methods.


571

Taken together, the binding of superparamagnetic Annexin V-conjugated microbeads is an excellent method with which to deplete apoptotic spermatozoa from cryopreserved semen samples. 5. Summary Cryopreservation increases the rate of spermatozoa with decreased capability to fertilise oocytes. In order to optimise the fertilisation rates, a better understanding of the underlying biochemical process is necessary. Externalisation of phosphatidylserine at the outer membrane as a feature of apoptosis in somatic cells, was observed after cryopreservation. It was the objective of our study to evaluate apoptosis in terms of CD95 (Fas-receptor) on the sperm surface, and activated caspases in the cytosol of spermatozoa showing cryopreservation-induced membrane damage. Using the high affinity of Annexin V to phosphatidylserine 30, neat and cryopreserved semen samples were treated by immunomagnetic cell sorting (MACS) with Annexin V microbeads (ANMB) to deplete cells with deteriorated membranes from those cells with intact ones. Cryopreservation increased the number of cells with externalised phosphatidylserine and activated caspases, but not those having CD95 on their surface — indicating an important role of the caspase cascade in cryopreservation-induced apoptosis. The Annexin V-MACS was able to remove sufficient CD95-positive and caspase-positive spermatozoa from neat as well as from cryopreserved semen samples. There was no significant influence of the separation column and the magnetic field on the sperm functions, and a very slight sperm loss by the Annexin V-MACS. Therefore, this technique is an excellent method with which to eliminate spermatozoa at early apoptotic stages, from the cryopreserved semen samples, and might be a useful tool in future assisted reproduction programmes.

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6. References ALNEMRI, E.S. (1997). Mammalian cell death proteases: A family of highly conserved aspartate specific cysteine proteases, /. Cell Biochem. 64, 33-42. CROSS, N.L., MORALES, P., OVERSTREET, J.W. and HANSON, F.W. (1986). Two simple methods for detecting acrosomereacted human sperm, Gamete Res. 15, 213-226. DESPRES, D., FLOHR, T., UPPENKAMP, M., BALDUS, M., HOFFMANN, M., HUBER, C. and DERIGS, H.G. (2000). CD34 + cell enrichment for autologous peripheral blood stem cell transplantation by use of the CliniMACs device, /. Hematother. Stem Cell Res. 9, 557-564. EKERT, P.G., SILKE, J. and VAUX, D.L. (1999). Caspase inhibitors, Cell Death. Differ. 6, 1081-1086. ENARI, M., SAKAHIRA, H., YOKOYAMA, H., OKAWA, K., IWAMATSU, A. and NAGATA, S. (1998). A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD, Nature 391, 43-50. FADOK, V.A., SAVILL, J.S., HASLETT, C , BRATTON, D.L., DOHERTY, D.E., CAMPBELL, P.A. and HENSON, P.M. (1992). Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognise and remove apoptotic cells, /. Immunol. 149, 40294035. FRASCH, S.C., HENSON, P.M., KAILEY, J.M., RICHTER, D.A., JANES, M.S., FADOK, V.A. and BRATTON, D.L. (2000). Regulation of phospholipid scramblase activity during apoptosis and cell activation by protein kinase Cdelta, /. Biol. Chem. 275, 23065-23073. GLANDER, H.J. and SCHALLER, J. (1999), Binding of Annexin V to plasma membranes of human spermatozoa: A rapid assay


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for detection of membrane changes after cryostorage, Mol. Hum. Reprod. 5, 109-115. HAMMERSTEDT, R.H., GRAHAM, J.K. and NOLAN, J.P. (1990). Cryopreservation of mammalian sperm: What we ask them to survive, /. Androl. 11, 73-88. KISCHKEL, F.C., HELLBARDT, S., BEHRMANN, I., GERMER, M., PAWLITA, M., KRAMMER, P.H. and PETER, M.E. (1995). Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor, EMBO J. 14, 5579-5588. MARTIN, S.J., REUTELINGSPERGER, C.P., MCGAHON, A.J., RADER, J.A., VAN SCHIE, R.C., LAFACE, D.M. and GREEN, D.R. (1995). Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: Inhibition by overexpression of Bcl-2 and Abl, /. Exp. Med. 182, 1545-1556. MCLAUGHLIN, E.A., FORD, W.C. and HULL, M.G. (1990). A comparison of the freezing of human semen in the uncirculated vapour above liquid nitrogen and in a commercial semi-programmable freezer, Hum. Reprod. 5, 724-728. SCHILLER, J., ARNHOLD, J., GLANDER, H.J. and ARNOLD, K. (2000). Lipid analysis of human spermatozoa and seminal plasma by MALDI-TOF mass spectrometry and NMR spectroscopy — Effects of freezing and thawing, Chem. Phys. Lipids 106, 145-156. TESARIK, J., MENDOZA, C. and CARRERAS, A. (1993). Fast acrosome reaction measure: A highly sensitive method for evaluating stimulus-induced acrosome reaction, Fertil. Steril. 59, 424-430. THORNBERRY, N.A. and LAZEBNIK, Y. (1998). Caspases: Enemies within, Science 281, 1312-1316.

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VAN HEERDE, W.L., DE GROOT, P.G. and REUTELINGSPERGER, C.P. (1995). The complexity of the phospholipid binding protein Annexin V, Thromb. Haemost. 73, 172-179. VAUX, D.L. and KORSMEYER, S.J. (1999). Cell death in development, Cell 96, 245-254. WALCZAK, H. and KRAMMER, P.H. (2000). The CD95 (APO-1/ Fas) and the TRAIL (APO-2L) apoptosis systems, Exp. Cell Res. 256, 58-66. WHO (1999). WHO Laboratory Manual for the Examination of Human Semen and Sperm — Cervical Mucus Interaction. Cambridge University Press. WOLF, B.B. and GREEN, D.R. (1999). Suicidal tendencies: Apoptotic cell death by caspase family proteinases, /. Biol. Chem. 274, 20049-20052. ZINI, A., BIELECKI, R., PHANG, D. and ZENZES, M.T. (2001). Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men, Fertil. Steril. 75, 674-677.


25 A REPOSITORY SYSTEM FOR CRYOPRESERVED SEMEN SAMPLES AND TESTICULAR BIOPSIES EMBEDDED IN A WORKFLOW MANAGEMENT SYSTEM

CORNELIA THIEME, UWE P A A S C H a n d HANS-JUERGEN G L A N D E R D e p a r t m e n t of Andrology, University of Leipzig Stephanstrasse 11, 04103 Leipzig, G e r m a n y

1. Introduction High levels of quality standards, multi-disciplinary and multicentre co-operation are demanded in the management of patients in andrology (ESHRE Andrology Special Interest Group, 1998). Huge amounts of data originating from several workplaces and units are acquired during the diagnostic and treatment of infertile couples. In addition, cryopreservation of ejaculated spermatozoa and testicular biopsies as a standard therapeutic procedure, has to be organised in a reliable and safe way. Therefore, fast electronic recording systems and standardised data management tools would facilitate the treatment approaches in reproductive medicine (Evidence-Based Medicine Working Group, 1992). However, the paper-based medical record is commonly used for documentation in spite of the advantages of computerisation

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(Moritz et at, 1995). These situations originated from the lack of specialised software and interfaces which would allow the automated processing of information, and provide active support to control the process of diagnosis and treatment (Ludwig et ah, 2001). On the other hand, the advantages of computerisation for a repository is well known. Therefore, "standalone" data systems for those tasks in andrology are available. The disadvantage of these systems lies in the missing connection to all other clinical data. This type of data management limits the value and accessibility of that data (Paasch and Glander, 1997). The integration of all systems to a workflow supporting system is restricted due to the very basic specification of interfaces and standards of data exchange (HL7, Health Level 7, Internet: http://www.hl7.de/) used in health care. Therefore, new concepts of integration and workflow managment systems are required to support the process of medical care and repository management according to the latest guidelines, and to assure the highest level of quality (Mortimer and Fraser, 1996; Evidence-Based Medicine Working Group, 1992).

2. Methods A concept of an electronic database programme with an embedded repository system for cryopreservation has been developed from December 1995 to June 2002 (Winsperm®, WSP). 2.1. Software and database m o d e l Out of a long list of available products with their own strengths and weaknesses (Oracle, SQL-Server, Sybase, Informix, Interbase, Delphi, Paradox, Dbase, Approach, 4th Dimension, File Maker, FoxPro ...) the relational database management system (RDBMS) Microsoft® Access® (MSA) integrated in the software package Microsoft® Office® for Windows 9x/NT/2000® was chosen because of its wide distribution at andrology centres in Germany.

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MSA is a powerful RDBMS that incorporates a client-server architecture and allows the portability to the hard- and software used at the infertility centres. On the other hand, this system causes a few problems. Firstly, there is no downstream compatibility (for example, Access 2.0, 95 (7.0), 97 (8.0)). Secondly, it is difficult to set up a management system for real time processing of high amounts of data under remote area conditions. All data are stored in four backend systems, 114 tables, more than 100 reports and approximately 1,000 database fields. User Access is given by two FrontEnds: WSP Client and WSP Work. The first realises a structured data entry system while the latter gives unlimited access for scientific evaluation. The CryoBase is defined as a completely integrated repository system which offers all the features needed for the administration of cryopreservation-related data. The software development was organised according to the principles of evolutionary system development in co-operation with six university-based centres of andrology (Leipzig, Muenster, Marburg, Frankfurt, Munich and Jena (Floyd et al., 1997; Kushniruk, 2002). This type of development realised a close integration of the users from the outset, and resulted in an enhanced quality of process management during software development. 2.2. The w o r k f l o w — M o d e l The couple orientated view entails the integration of all logistic, administrative, evaluation and execution of diagnosis and treatment into a complete module within a electronic system. Computer systems designed to support those tasks are called electronic Workflow Management Systems (WMS). WMS are designed for fast and structured acquisition of data at the place and time at which they are produced. In addition, the quality of data is improved if the owner or producer of the data practises reliable methods for storage after-control (Hastedt-Marckwardt,

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1999). Electronic signatures and authorisation settings had to be implemented to achieve high quality results. Working environments supported by WMS are suitable for handling even large amounts of data — as is the case for repository systems for cryopreserved specimens — which are necessary for andrologists. An optimal solution offers the complete integration of those functions into the WMS. The first level of workflow management control is realised in WSP by 12 separate waiting room lists (WRL) according to the diagnostic and treatment regimen. The workflow tasks are generated at the moment of a patient's registration at the desk. However, at each point of consultation within the workflow process, new tasks can be created by the medical staff. First of all, the simple generation of WRL avoids the time consuming search for the recordset needed to start the next task. By clicking at one of the list entries out of the appropriate WRL, the recordset needed is instantly selected, and all data can be entered into several of the Task Office (TAO) data forms in a structured manner. After finishing the data entry, a double-click on the WRL item terminates the given workflow process and deletes the item from the WRL. The couple then can be notified for another WRL which ensures the proper adjustment of the whole process of diagnosis and treatment (Leymann, 1997). 2.2.1. The Control Centre The screen which is seen at the start of the system is called Control Centre (COC, Fig. 1). It forms a complete navigation and acquisition system for demographic data (patient navigator, PAN), the waiting room list (WRL) as a part of the workflow management system, and the access to all data forms by means of the Task Office. In addition, a centrally-placed menu bar houses icons of the CryoBase; the report manger (REM, Fig. 2); forms which display all important data of the couple at a glance; parameter settings; password change function, and the statistics as well as a quality control module.

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2.2.2. Patient navigator The upper third of the COC is defined as PAN. There the desired couple, in terms of demographic data as well as information about health care insurance, the general practitioner, the date of first admission and the main reason for consultation, can be easily selected. Almost all people in Germany who are insured for health care are equipped with an electronic smart card which stores demographic data provided by the insurance company. Therefore WSP is equipped with an smart card reader. Reading the insurance company smart card (ICC) is the probably best way to search for a male or a female partner within WSP. Alternatively, the database can be searched using the couple number, or by jumping to the first or final recordset. Finally, one can move throughout the database by incremental or decremental search of the couple number. Once the desired recordset of a couple is selected, all demographic data might be rapidly updated using the form demographic data. Also, the notices to general practitioners are accessible here. If the first name, name and date of birth entered by hand or smart card do not match with any entries for male or female demographics, a new recordset of demographic, data for a couple is created automatically by increasing the couple number. 2.2.3. Workflow management system To initiate a work flow the actual within the PAN, the selected couple must be simply notified for a WRL. WSP is developed with 12 WRL: spermatology, consulting room 1-5, psychologist, blood test, secretary, ultrasound, operation and genetic council. According to the nature of the WRL, the male alone, or both partners, might be notified separately. The workflow is then continued by selecting the appropriate WRL at the workplace. A single click on a WRL list entry selects the couple within the PAN and all data can be acquired using the TAO. The TAO also provides direct access to the cryopreservation repository system

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(CryoBase) via the form microsurgery and spermatology. Once all data are stored, the workflow is terminated by a double-click on the WRL list entry. List entries can be recovered even when deleted by accident. According to the process of diagnosis and treatment, a new WRL entry might be set. 2.2.4. Task Office According to the appropriate data relations and the process of data creation in diagnosis and treatment, the TAO of WSP provides the access to standardised forms for data entry (Fig. 3). At the time of data entry the data's correctness, completeness and plausibility is checked if possible. To avoid wrong entries because of misinterpretations, a small tip-text appears for each field if selected by the cursor. All forms are designed in a consistent manner, and consist of a head and a body. The head contains control buttons and fields to display the couple selected, in conjunction with optional buttons (for example, CryoBase at spermatology). 2.2.5. Data evaluation There are many ways to evaluate data in WSP. A variety of reports are supplied by the REM of WSP and its optional modules. Alternatively, queries might be used for scientific evaluation using WSP Work. In addition, the open database connectivity (ODBC) interface can be used by programmes equipped with it to directly extract the desired data out of WSP. 2.2.6. WSP Client Reports First, the report manager (REM, Fig. 2) allows a rapid printout of 51 differently designed reports of all areas of routine diagnosis and treatment. These reports can be previewed separately for women and men; instantly printed or exported to Microsoft® Word®. The address of the general practitioner and a second

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undersigning officer may be added optionally. The integrated repository system CryoBase and the module for Statistics are equipped with their own REM and together provide an additional 44 reports. Apart from routine documentation for patients, WSP is equipped to provide laboratory documentation (log files). Another tool for rapid communication is the set of two serial letter modules and an epicrisis writing system integrated into the central menu bar. The first serial letter module designed to create letters to referring colleagues; the second serial letter module is to send letters to patients; and the latter writing system is used to create rapid documentation. All modules are equipped with a SLL containing standard sentences. After those have been selected, free text entry is allowed to adapt the text to special needs. The same time of report creation possibilities is integrated into the histology form. 2.2.7. Query formulation in WSP Work The use of queries is the most powerful way for sorting, calculating, and organising data from any of the incorporated tables of the database. Queries produce a temporary collection of fields of the selected recordsets with the latest data every time they are run. Queries on this database can be independently designed by the users. A query assistant implemented in Microsoft® Access® allows users to create queries easily, without any knowledge of the Structured Query Language (SQL) which has to be used. Alternatively, the SQL-syntax may be used. 3. Cryopreservation Repository System — CryoBase Evidence based medicine in reproductive medicine comprises deep temperature cryopreservation in close conjunction with all other features of clinical procedures. Therefore it is not only necessary to be in charge of a sufficient repository system — a close connection to all other patient's data has to be established.

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Therefore WSP contains a completely integrated repository system called CryoBase. CryoBase consists of a standard repository system in conjunction with a powerful search engine and also the German version of a system for billing and dunning, as well as an automatic alert function (Fig. 4). The interrelation of the repository system with the workflow of WSP, starts at the level of data acquisition for ejaculates or microsurgery. Both forms are equipped with the appropriate button for the CryoBase. Therefore, not only data as used for the repository are accessible by the system — all related data to a frozen specimen can be extracted out of the system. Even if a sample has been used for therapeutic interventions, the interrelations are not deleted. This concept allows clinical and scientific data access at any time. Reports and contracts of the CryoBase (n = 15) can be created as known from the standard REM. In addition, samples for scientific purposes can also be administrated by that repository system. 4. Results WSP was first introduced at a single workplace in 1997. Real time networking and perfection of the WMS began in 1998. At present WSP has been migrated from Microsoft® Access® 95 over to Access 97 and then Access 2000. The computing power of a standard office computer is found to be sufficient to run WSP smoothly. Network configuration and data transmission bandwidths are very important to support (instead of hinder) the daily clinical work. To date, there are no reports from users about any instability of the software which is used an different platforms (Microsoft® Windows 9x/NT/2000®), network operating systems (Microsoft® Windows NT/2000 Server®) and network facilities (twisted pair, glass fibre). Compromised databases are often the result of disrupted communication to the server during the writing of recordsets. However, administrators are able to fix a damaged backend with the integrated repair


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module (Paasch and Glander, 1999a; Paasch and Glander, 1999b; Paasch et ah, 2000). WSP is at present used at six university centres in Germany, and another three centres are going to implement it. Up to autumn 2002, the DWH in our centre contains approximately 5,500 couples and approximately 40,000 clinical record sets. Almost 6,500 spermiograms with 1,460 related CASA results are stored in the proper relation to all other data within WSP. Time is saved in daily routine work by the usage of the three REM with 95 templates to create letters, reports, log files and statistics. Of additional advantage is the Statistics module which instantly provides the latest results of quality control by computing the so-called monthly mean. This module provides, in addition, the easiest way to detect errors due to wrong data entry. Presently, the 380 semen samples or testicular biopsies stored within the CryoBase are also related to all other information of the system. This close integration of a repository system into a workflow management system provides unique access to all data never before realised (to our knowledge). As a consequence, the system has been used to identify trends of classical spermiogram variables, differences in subpopulations of patients, and of testicular cancer (Paasch et ah, 2001). The German Society of Andrology (Deutschen Gesellschaft fiir Andrologie) has classified WSP as a sufficient tool for evaluation of epidemiological trends (Krause et ah, 2002). A German or English trial version can be obtained on request. 5. Conclusion WSP realises the first step toward an andrological DWH with an integrated, complete repository system for cryopreserved specimens. Electronic workflow management systems support the administrative and scientific decision-making at present, and will be an important fact of competition in future (Ludwig,

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2002). The integrated CryoBase achieves the close connection of all cryopreservation-related information in a repository system to any data of clinical and research origin stored in WSP. Furthermore the real time online administration capabilities help staff to achieve better internal quality control in the cryobank. 6. Summary Real time acquisition of all clinical and logistic data concerning cryopreserved semen or testicular tissue combined with "Datamining (DMG)" within a "Data Warehouse (DWH)" for andrology realises a step towards better handling and internal quality control of a cryobank. The DWH was setup on operational databases which was not available for andrological settings u p to the introduction of Winsperm® in 1997. This relational database management system accomplish a specialised workflow database to store, retrieve and evaluate all data of the samples cryopreserved in term of a repository system. Our objective is the presentation of the repository system CryoBase implemented in Winsperm®2003 (WSP), a specialised workflow database for standardized acquisition and evaluation of data of infertile couples as a prerequisite of an evidence based medicine according to the guidelines of the European Academy of Andrology and European Society of Human Reproduction and Endocrinology (EAA and ESHRE). 7. References ESHRE ANDROLOGY SPECIAL INTEREST GROUP (1998). Guidelines on the application of CASA technology in the analysis of spermatozoa, Hum. Reprod. 13, 142-145. EVIDENCE-BASED MEDICINE WORKING GROUP (1992). Evidence-based medicine. A new approach to teaching the practice of medicine, JAMA 268, 2420-2425.


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FLOYD, C , KRABBEL, A., RATUSKI, S. and WETZELL, I. (1997). Zur Evolution der evolutionaren Systementwicklung: Erfahrungen aus einem Krankenhausprojekt, Informatik-Spektrum 20, 13-20. HASTEDT-MARCKWARDT, C. (1999). Workflow-ManagementSysteme, Informatik-Spektrum 22, 99-109. KRAUSE, W., BEHRE, H.M. and NIESCHLAG, E. (2002). Bestandsaufnahme der Entwicklung der Andrologie in Deutschland, Reproduktionsmedizin 18, 89-91. KUSHNIRUK, A. (2002). Evaluation in the design of health information systems: Application of approaches emerging from usability engineering, Comput. Biol. Med. 32, 141-149. LEYMANN, F. (1997). Transaktionsunterstuetzung fuer Workflows, Informatik Forsch. Entw. 12, 82-90. LUDWIG, C.A. (2002). Qualitatsstandards fur das computerbasierte Patientendossier, Schweizerische Arztezeitung 82, 291-292. LUDWIG, C.A., BUERKI, L., ECKHARDT, A. and CAMEY, B. (2001). Strategiegesteuerte Informationssysteme fur das Gesundheitswesen, Swiss Medical Informatics 48, 6-9. MORITZ, V.A., MCMASTER, R., DILLON, T. and MAYALL, B. (1995). Selection and implementation of a laboratory computer system, Pathology 27, 260-267. MORTIMER, D. and FRASER, L. (1996). Consensus workshop on advanced diagnostic andrology techniques, Hum. Reprod. 11, 1463-1479. PAASCH, U. and GLANDER, H.J. (1997). Contribution to a standardised computer assisted sperm motion analysis, CASA (System Stroemberg Mika), Z. Hautkr. 72, 29-34. PAASCH, U. and GLANDER, H.J. (1999). Principle network architectures using the andrological database Winsperm 99.x, Andrologia 31, 383.

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PAASCH, U. and GLANDER, H.J. (1999). The andrological Database Winsperm® 99.x — Techniques of network architecture and remote support. 1st European symposium on Teledermatology, Jena 19-20 November 1999, Allergologie 22(11), 682-683. PAASCH, U., THIEME, C. and GLANDER, H.J. (2000). Winsperm — Elektronisches Datenmanagement in der Andrologie, /. Pert. Reprod. 10, 13-25. PAASCH, U., THIEME, C. and GLANDER, H.J. (2001). Analyses of sperm quality in large populations over decades by "Datamining" and "Datawarehousing" (Winsperm®). Andrology in the 21st Century, /. Androl. 22(Suppl), 188.

SECTION VIII: CRYOPRESERVATION



26 FINDING THE IDEAL FREEZING CURVE FOR TISSUES THROUGH INDIRECT THEMOPHYSICAL CALCULATION

YOUNG-HWAN PARK, WOONG-SUB YOON 1 , TAEK-SOO KIM1, CHEE-SOON YOON 2 , SHI-HO KIM3, HAN-KI PARK, JONG-HOON KIM, DONG-WOOK HAN, JONG-CHUL PARK, BUM-KOO CHO Department of Thoracic and Cardiovascular Surgery, Yonsei Cardiovascular Research Institute, Cardiovascular Hospital, Yonsei University College of Medicine 1 Yonsei Propulsion-Combustion Laboratory, Yonsei University College of Mechanical Engineering, Seoul 2 Department of Thoracic and Cardiovascular Surgery, Konyang University College of Medicine, Daejun 3 Department of Thoracic and Cardiovascular Surgery, Donga University College of Medicine, Busan, Korea

1. Introduction The human cardiac valve allograft is the choice replacement for a damaged cardiac valve due to valvular heart disease or degeneration. The cardiac valve can be harvested from braindeath donors, and tissue preservation is the most important process. These tissues must be treated by chemical agents, 593

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radiation and refrigeration. However, the duration of preservation is limited; therefore these valves must be frozen. The freezing method presents several problems. Firstly, the latent heat of fusion changes the crystal size of water during freezing, and will damage the cellular structure. Secondly, there is the problem of variability of crystal size below -80°C. Angel et al, reported his experience with the cryopreservation method. (Angel, 1976). Below -80°C, the frozen tissue will be stable in a so-called "glassy state". At present, most homografts are stored in liquid nitrogen at about -196°C. Computer-controlled freezing with liquid nitrogen solves the problem caused by the latent heat of fusion. The freezing programme for valved homografts is well known and is widely used in most tissue banks. This freezing programme will not be applied to the saphenous vein, carotid artery or tissue engineering products. So, studies are needed to determine new ideal freezing curves for various tissues, using a simulation programme. 2. Materials and Methods 2.1. Freezing process The physiochemical behavior of materials during freezing is shown in Fig. 1. In pure water (ABCDE) and aqueous solutions (A'B'C'D'E'), the chart shows the temperature change with time. Before the process of crystallisation, the temperature drops below the freezing temperature. This is the so-called super-cooling that provides the activation energy during the nucleation process as a non-equilibrium state. In the case of pure water, before crystallisation, several degrees of super-cooling are observed. When the water is frozen, air bubbles, particles or rough surfaces will be needed for nucleation. If these are absent, water will be super-cooled. After the development of small crystals, the freezing process occur rapidly. This phenomenon is observed in ampoule freezing, but not in a freezing solution. In aqueous solution, the starting temperature

Finding the Ideal Freezing Curve for Tissues

595

Time Fig. 1. Freezing curve.

of freezing (B') is higher than that of pure water (B) because the solute acts as the nucleation point. Because the freezing process of the freezing solution is very similar to that of the aqueous solution, and the concentration of the solution is high, supercooling is not observed. Freezing time is defined as the time interval during which the system begins the crystallisation process and releases the latent heat of the system. Modelling this interval is generally a difficult task, so some assumptions were made, and therefore the results may be inaccurate. 2.2. Thermophysical m o d e l i n g Here, the lumped thermal capacity model is defined: • • • • •

the interaction between the system and its surroundings; assume uniformity of temperature; no temperature gradient in the system; no heat transfer within the system; and quiescent environment.

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These assumptions are relatively simple, but they allow easy calculations. Freezing can be divided into three phases: cooling (above freezing), freezing, and cooling (below freezing). The whole freezing process of about 100 minutes was monitored every one second, and thermophysical responses were calculated over 6,000 divisions. For this purpose, we use the equations below to find solutions, which were used to calculate the time constant. During the cooling process, we used the lumped thermal capacity model, and during the freezing process, we assumed that system temperature was constant during the release of the heat of fusion, and after this was released, the freezing process was deemed to have been completed. Releasing heat of fusion was calculated as the summation of thermal flow in each step. 3. Equations 3.1. Cooling process Energy equation: dt : internal energy,

6': heat transfer,

0'v: heat generation.

dt For fixed mass (pV) dU - pVdu, du = CdT, VC* — = 6' p H dt PVc^

at

=

-hcA(T-Te).

Where, Te is the environment temperature. Solution of the temperature response assumes:

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(a) Bath (chamber) is large. (b) hcA/pVC is approximated by a constant value independent of temperature. The solution is: T-T T0-Te

= g-t/tc

tc =

pVc hrA'

For the direct calculation approach, every thermodynamic property of the system needs to be known before we can find

f

Start J I Density, Latent heat of fussion A Specific heat, Volume, Area, etc.

Read thermophysical properties

Input chamber temp, profile

Cooling process Depression of freezing point

Freezing process

Cooling process

Result out

(

End

")

Fig. 2. Algorithm for direct calculation.

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the thermal response, whereas the indirect approach needs only the time constant from experiments. 4. Direct Calculation Firstly, the programme reads thermophysical properties like density, latent heat of fusion, specific heat and volume, etc. Thereafter, the chamber temperature profile is input. The freezing process is divided into three phases. The first is prefreezing cooling; the second is the freezing process, and the third is the post-freezing cooling. The computer calculates the result and draws the freezing curve (Fig. 2). C Start i

'

Read experimental data • '

Polynomial regression analysis r •

Time constant calculation '' Thermal response calculation

Chamber temp, calculation

Sample temp, calculation

Result out

(^

End

^)

Fig. 3. Algorithm for indirect calculation.

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5. Indirect Calculation After reading experimental data of specific tissue freezing, the data is approximately fitted with polynomial curves and the time constants are calculated (Fig. 3). Based on the indirect calculation theory, we developed the freezing curve programme. This programme froze four different tissue engineered products. We observed the cellular viability depending on different freezing curves and different freezing methods. 5.1. Results By the direct calculation method, samples of 3.6 cc ampoules, 180 cc saphenous vein, and 200 cc cardiac valve homografts were calculated (Figs. 4-6). There were several differences between the calculated freezing curve and the experimental curve. This

•

Direct Calculation Results (1) 3.6 cc ampoule (solvent)

lv-»8

mperat ure (deg)

—•—Chamber temp. I -•— Sample temp, f

"

J

A.A.L..

.^^

jj)

I

-

-SO

—•— Chamber temp. -•— Sample temp.

i

Si,

8 . -100

V-

E

-

-150

^ Time (min)

Experiment

Time (min)

Calculation

Fig. 4. Result of direct calculation for 3.6 cc ampoule. Left graph shows actual experiment data, and right graph is calculated.

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• Direct Calculation Results (2) 180 cc saphenous vein (solution) 50 — * - j - Chamber temp.

ure (deg]

* ! Sample temp.

h

[ ÊJ^ \ i 1 "\

1J

i-

40

60

80

100

120

Time (min)

Time (min)

Calculation

Experiment

Fig. 5. Result of direct calculation for 180 cc saphenous vein. Left graph shows actual experiment data, and right graph is calculated.

•

Direct Calculation Results (3) 200 cc cardiac homograft (solution) 1

•

I

'

'

'

! 0

V

:

— • — C h a m b e r temp. - • — Sample temp.

• r Chamber temp. —»-j- Sample temp.

-—».-_

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i :

-100

E

!

•

~

, , , i , , , i , , , i , 100

Time (min)

Experiment

120

100

120

Time (min)

Calculation

Fig. 6. Result of direct calculation for 200 cc cardiac homograft. Left graph shows actual experiment data and right graph is calculated.

601


• Indirect Calculation Results (1) Inverse operation of the time constant —•—180cc -•— 200cc

insta nt(se

fi

o

CJ

310 4

:

.

•

f

-

\*

-

\^t)}% •

5000 0 -60

-40

-20

0

Sample temp, (deg)

Fig. 7. Inverse operation of the time constant by the indirect calculation method. This graph shows that the time constants from experimental data are different according to the size (volume) and the thermal properties of the tissues.

occurred because the factors working in the equations were incorrect or impossible to be calculated, and there were too many conditions to calculate. By the indirect calculation method, samples of 180 cc saphenous vein, 200 cc cardiac valve homograft were frozen by the usual homograft freezing curve, and the time constant was calculated. This time constant is the time required to reach the same temperature, and there are many differences between two samples (Fig. 7). This graph shows that the inverse operation of the time constant from the experimental data, is different according to the size (volume) and the thermal properties of tissues. Based on the time constant, temperature changes in the chamber with this time constant define the temperature change of the sample tissues (Fig. 8). In the left graph, the freezing curve is the cardiac homograft freezing curve, and if

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•

Indirect Calculation Results (2) Analogy (Time constant)

50 — • — Chamber temp. - • — Sample temp.

— • — Chamber temp. - • — sample torn p. ^

0

o g)

-;

g -50 3

-50

\ \

"3

i

Q.-100

E

-

-150

20

40

60

Time (mi

80

100

1 0

-200

20

40

60

80

100

12

Time (min)

Fig. 8. Example graph for showing analogy by time constant. Temperature change in the chamber with this time constant causes the temperature changes in the sample tissue. So two graphs are the same, depending on the time constant. In the left graph, the freezing curve is the cardiac homograft-freezing curve. If the chamber temperature profile is changed like the graph on the right, the sample temperature will follow as dotted line.

the chamber temperature profile is changed as shown on the graph to the right, the sample temperature will follow as a red line. For the linear temperature response of the sample tissue, the rate of change of the chamber temperature should be infinite (Fig. 9). It is impossible in nature. So the chamber temperature should be controlled in the range of not increasing sample temperature (Fig. 10). The right chamber temperature profile will prevent latent heat damage and reduce the fluctuation of crystal size with temperature variations. This computer programme reads the experimental data through a digitiser, and we can get the data at every turning point. If chamber data is to be changed, the computer calculates the sample temperature curve, and the sample data needs to be changed, the computer calculates the chamber temperature curve. This programme will be plotted


603

• Indirect Calculation Results (3) Inverse calculation of the chamber temperature

— • — Sample temp. -•— Chamber lemp.

Sample temperature

:

Inverse "

Chamber temperature

X >

:

~ n 20

40

Time (min)

Fig. 9. Inverse calculation of the chamber temperature. For the linear temperature response of the sample tissue, the rate of change of the chamber temperature should be infinitely large.

• Indirect Calculation Results (4) —"— Chamber temp. ~*— Sample lemp.

20

40

60

Time (min)

100

120

20

40

60

80

100

120

Time (min)

Fig. 10. End results of the freezing curve. The right chamber temperature profile will prevent latent heat damage and reduce the fluctuation of the crystal size with temperature variations.

Y.-H. Park et d.

604

~m: Fig. 11. After freezing of the saphenous vein with a cardiac homograft freezing curve, the programme found a new ideal freezing curve, and this graph shows actual chamber and sample temperature curves by new program. (The black line is the chamber temperature and the gray line is the sample temperature.)

as combinations of several kinds of curves. After the sample is frozen, the freezing rate of l°C/minute is applied until the sample temperature reaches -80°C. Figure 11 shows the new ideal freezing curve for a saphenous vein. By this method, we found the new ideal freezing curve specific to a sapheous vein (Fig. 11). We found the new curves for various tissue engineered products (Fig. 12). (a) PLGA 75:25 with hMCS (PLGA 75:25 scaffolds cultured with human mesenchymal stem cells). (b) hBone Chip with OBC (human bone chips cultured with osteoblast cells derived from mesenchymal stem cells). (c) FLGA with Rb Chondrocyte (PLGA scaffolds cultured with rabbit chondrocyte).


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Specimen II

PLGA 75:25 with hMCS

hBone Chip with OBC

PLGA with Rb Chondrocyte

CAp-AtCol+PLLA with RCO

H

U

r

. . . , .

Specimen III

.,,

-,

.

**-,...

-.,..

^ i . ^ , -

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Specimen IV

Specimen I:

PLGA 75:25 with hMCS (PLGA 75:25 scaffolds cultured with human mesenchymal stem cells) Specimen II: hBone Chip with OBC (human bone chips cultured with osteoblast cell derived from MSC) Specimen III: PLGA with Rb Chondrocyte (PLGA scaffolds cultured with rabbit chondrocytes) Specimen IV: Cap__AtCol + PLLA with RCO (Carbonate apatite-atelocollagen + PLLA composites cultured with rat calvarial osteoblasts) Fig. 12. New ideal freezing curve according to various tissue-engineering products.

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(d) Cap_AtCol + PLLA with RCO (Carbonate apatite-atelocollagen + PLLA composites cultured with rat calvarial osteoblasts). These four tissues were frozen by the simple freezing method as well as by the ideal freezing method, and compared with a no-freezing group. The viability using rate-controlled freezing is better than that using mechanical freezing; however, there were no significant differences between the simple freezing group

120

O

u. >.

• PLGA 75:25 with hMCS

3 hBone Chip with OBC

• PLGA with Rb Chondrocyte

I Cap-AtCol+PLLA with RCO

100

80

m

60

i*B

n

.55 40 > 0)

O

20

non-treated control

mechanical freezing

rate-controlled freezing

Freezing method •

PLGA 75:25 with hMCS: PLGA 75:25 scaffolds cultured with human mesenchymal stem cells H hBone Chip with OBC: human bone chips cultured with osteoblast cell derived from mesenchymal stem cells • PLGA with Rb Chondrocyte: PLGA scaffolds cultured with rabbit chondrocytes • Cap_AtCol + PLLA with RCO: Carbonate apatite-atelocollagen + PLLA composites cultured with rat calvarial osteoblasts Fig. 13. Viability of cells according to freezing methods. Mechanical freezing means freezing at -70°C in a deep freezer. In rate-controlled freezing, each sample is frozen by individual ideal freezing curves.


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and the ideal freezing group. The rate-controlled freezing means that the specimens are frozen by the new, ideal freezing curve respectively. (Fig. 13) 6. Discussion Cryopreservation methods are well known as an ideal preservation method for cardiac homograft valves. The survival of fibroblast in the extracellular matrix is important for improvement of durability. Each process of harvesting, processing, freezing, storing and thawing is important for cellular survival. Lessening the warm and cold ischemic time, and dissecting the heart on the ice, are essential for cellular survival. During soaking in the antibiotic solution, the temperature and the duration will affect cellular survival. During thawing, the rapid increase of temperature will damage the tissues less than a slow increase. Every process will affect the cellular survival, and should be strictly managed in the tissue bank. The reason for freezing is to achieve a longer preservation period. In the frozen state, the metabolic rate decreases rapidly, and the cell can survive without much of an energy supply. But between -15°C and -80°C, ice crystals will grow and shrink inside the tissue depending on the temperature. Below -130°C, liquid water that is still not frozen, changes into a glassy state. Its viscosity is infinite, and the water cannot be moved. Below -196°C, there is insufficient thermal energy for chemical reactions. Therefore the cardiac valve should ideally be stored in liquid nitrogen. In this study, we want to find an ideal freezing curve that may be applied to various kinds of tissues. Mazur emphasised that intracellular ice crystals are the most harmful factor during freezing and thawing (Mazur, 1984). If the freezing is too fast, the tissue will be super-cooled because intracellular water does not go out; and if the freezing is too slow, the tissue will be damaged because intracellular water is lost due to high oncotic pressure outside the cell, and the cell

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will shrink. For this purpose, the cryoprotectants (DMSO; DimethylSulfoxide) 1M are useful to prevent cellular shrinkage by maintaining the cellular oncotic pressure. Around the temperature of latent heat, the computer-controlled programme is necessary to ensure that the tissue temperature will not increase during freezing. Mazur reported that the freezing rate of 1°C/ minute is ideal to minimise intracellular ice crystallisation after freezing (Mazur, 1984). Takamatsu observed the compression damage between forming ice crystals during rapid freezing, by using a cryomicroscope (Takamatsu, 1999). Most damage will occur around -1.8°C. Brockbank feels that intercellular ice formation would damage the extracellular matrix, and the debris will evoke calcification (Brockbank, 2000). In the carotid artery, Song reported that the vascular function was maintained well when adequate DMSO concentration was below 15%, and the freezing rate was about 0.69°C/minute (Song, 1995). He also developed the vitrification method (socalled ice-free freezing method) which was applied to vascular freezing. He observed that the smooth muscle cells and endothelial cells were functioning well and the structures were well maintained after thawing (Song, 2000). During thawing, Pegg suggested that from -196°C to -100°C the tissue should be thawed slowly so that the fracture of tissue might be prevented (Pegg, 1997). In a tissue bank, several kinds of tissues will be treated, and various ideal freezing curves will be necessary for various tissues. Products of tissue engineering techniques should be frozen by the ideal freezing curve. So, this indirect calculation method can be an alternative method to finding ideal freezing curve. 7. Conclusion Because we cannot know the thermophysical properties of all tissues and solutions, the direct calculation approach is almost


609

impossible. In the indirect calculation approach, data taken from experiments are approximately fitted with the polynomial curve. By the results of experiments with new ideal freezing curves, we found that there are no significant differences among four samples; however, we think that there is a significant difference if the sample size is larger. This method will lower the cost and time, and can be applied to all kinds of tissues via computer simulation. 8. Summary Liquid nitrogen freezing techniques have already met with widespread success in biology and medicine as a means of long-term storage for cells and tissues. The use of cryoprotectants such as glycerol and dimethylsulphoxide to prevent ice crystal formation, with carefully controlled rates of freezing and thawing, allows both structure and viability to be retained almost indefinitely. Cryopreservation of various tissues has various controlled rates of freezing. To find the optimal freezing curve, we investigated two methods of thermodynamic calculation of tissues freezing curves. One is the direct calculation method. We should know the thermophysical characteristics of all components: latent heat of fusion, area, density and volume, etc. This kind of calculation is highly sophisticated, and some variables cannot be determined. The other is the indirect calculation method. We performed the tissue freezing with already used freezing curves and observed the actual freezing curve of that tissue. We also modified the freezing curve by several steps of calculation, polynomial regression analysis, time constant calculation, thermal response calculation and inverse calculation of chamber temperature. We then applied that freezing programme on mesenchymal stem cells, chondrocytes and osteoblasts, but could not find any differences in tissue survival. The reason could be that the freezing material is small and contains cellular components. We expect a significant difference of cellular viability, if the freezing curve is applied to tissues on a large scale.

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We conclude that this programme would be helpful for determining the ideal freezing curve easily. 9. A c k n o w l e d g e m e n t This study was supported by a grant (HMP-98-G-2-052) of the HAN (Highly Advanced National) Project, Ministry of Health & Welfare, Republic of Korea. 10. References ANGEL, J.D., CHRISTOPHER, B.S., HAWTREY, O. and ANGEL, W.M. (1976). Fresh, viable human heart valve bank: Sterilisation sterility test and cryogenic preservation, Transplant Proc. 8 (Suppl. 1), 139 BROCKBANK, K.G., LIGHTFOOT, F.G., SONG, Y.C. and TAYLOR, M.J. (2000). Interstitial ice formation in cryopreserved homografts: A possible cause of tissue deterioration and calcification in vivo, }. Heart. Valve Dis. 9, 200-206. TAKAMATSU, H. and RUBINSKY, B. (1999). Viability of deformed cells, Cryobiology 39, 243-251. MAZUR, P. (1984). Freezing of living cells: Mechanisms and implications, Am. J. Physiol. 247, C125-142. PEGG, D.E., WUSTEMAN, M.C. and BOYLAN, S. (1997). Fractures in cryopreserved elastic arteries, Cryobiology 34, 183-192. SONG, Y.C., PEGG, D.E. and HUNT, C.J. (1995). Cryopreservation of the common carotid artery of the rabbit: Optimisation of dimethy sulfoxide concentration and cooling rate, Cryobiology 32, 405-421. SONG, Y.C, HAGEN, P.O., LIGHTFOOT, F.G., TAYLOR, M.J., SMITH, A.C. and BROCKBANK, K.G. (2000). In vivo evaluation of the effects of a new ice-free cryopreservation process on autologous vascular grafts, /. Invest. Surg. 13, 279-288.


27 THE SAFE AND EFFECTIVE USE OF ALLOGRAFT TISSUE: AN UPDATE

SCOTT A. BARBOUR a n d W A R R E N KING Palo Alto Medical F o u n d a t i o n Palo Alto, California

1. Introduction The use of allograft tissue in orthopaedic surgery has been a tremendous advance in the management of a variety of orthopaedic problems. Today, allograft tissues are commonly used for a variety of procedures, including limb-salvage, ligament reconstructions, cartilage resurfacing, and as osteoconductive and inductive substrates, to name a few. The role for allograft tissue is constantly expanding, and becoming more common. In 1999 alone, US tissue banks distributed over 750,000 allografts (Gadzag and Lane, 1995). The use of allograft tissue has many advantages over autografts including unlimited size, lack of donor site morbidity and availability for revision surgery. However, allograft is not without some disadvantages. It has decreased osteoinductive and osteoconductive characteristics, as well as increased incorporation times when compared to autograft tissues (Gadzag and Lane, 1995). But the most significant disadvantage is the risk of disease transmission.

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Currently, the risk of viral transmission through allograft tissue transplantation is extremely low. Proper donor screening and tissue processing help prevent the transmission of viral disease. However, window periods of infection where detection is missed through serological tests, and human error, make transmission still possible (Tomford, 1983). As of 1995, two documented case reports of HIV, one case of Hepatitis B and three cases of Hepatitis C had described infected donors being involved in transplantation of musculoskeletal allografts, resulting in several cases of disease transmission (Tomford, 1983). It is important to note that with steadfast adherence to the screening methods available today, most if not all of these infections would have been avoided (Tomford, 1983). The risk of bacterial infection from allograft tissue is unknown (CDC, 2001). This is in part due to several confounding factors. The lack of a standardised procurement protocol for tissue harvesting; failure of surgeons to recognise or confirm the allograft as the source of infection; and the lack reporting when this complication does occur. We present four cases of Clostridium septicum infection, which occurred following Anterior Cruciate Ligament (ACL) reconstruction with contaminated allografts from the tissue bank. 2. Case 1 A 50-year-old male sustained a torn right anterior cruciate ligament while playing baseball, and received a hemipatellar tendon allograft reconstruction in March, 1998. The fresh frozen graft was removed from the package on the back table, and a sample of the bone and tendon was sent for aerobic and anaerobic culture prior to implantation. The patient was seen in follow up four days later, and was doing well; physical therapy was prescribed. Seven days later the patient was evaluated in the clinic and found to have an erythematous, warm right knee. The patient complained of a significant increase in pain, and reported feeling

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febrile. Culture results of purulent fluid aspirated from his right knee identified the gram-positive rod, Clostridium septicum. The patient was immediately taken to the operating room where an arthroscopic debridement and removal of the graft and bioabsorbable screws was performed. He was admitted to the hospital and placed on intravenous (IV) antibiotics and his right knee was subsequently debrided multiple times in the operating room. Several intraoperative cultures confirmed Clostridium septicum infection. The patient completed a four-week course of IV antibiotics followed by a two-week course of oral antibiotics as well as physical therapy. His case was further complicated by arthrofibrosis with a range of motion of 15-90 degrees. Three months following the initial operation he was again taken to the operating room for an arthroscopic lysis of adhesions and manipulation under anaesthesia, improving his range of motion to 5-100 degrees. The primary surgeon routinely sends allograft tissue for culture prior to implantation, to determine if any subsequent postoperative infections were from contaminated tissue. In this case, the preoperative tissue culture results correlated with the rare organism cultured from the patient's knee aspirate, indicating that the tissue came from the tissue bank already contaminated. For this reason, the tissue bank was contacted and informed of the incident. They responded that their testing revealed no such contamination of the allograft tissue, and held the position that the infection was the result of contamination at the time of surgery. The tissue bank was also unwilling to share any information regarding methods used to harvest the tissue, or the circumstances of the cadaver donor's death and medical history. Our patient, however, filed a lawsuit against the tissue bank, allowing us access to the cadaver donor's autopsy report and harvesting methods of the tissue bank. From these documents we were able to track other tissues from the same donor to other recipients around the country. It was learned that a patient in another part of the country received the other half of the

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hemipatellar tendon allograft from this donor, and contracted a post-operative infection with the same rare organism, Clostridium septicum. Despite the findings, the tissue bank still held the position that the infections were separate complications, resulting from perioperative tissue contamination. The tissue bank ultimately reached an out-of-court settlement with the patient that included a confidentiality clause prohibiting disclosure of the facts of this case. 3. Case 2 A 50-year-old male was taken to the operating room by a surgeon on April 1, 1998, where a bone patella tendon bone allograft right ACL reconstruction was performed without complication. The patient did well initially; however, approximately 10 days post operatively the patient began to experience increasing pain and swelling of his right knee, as well as high fevers. He was re-admitted to the hospital and taken to the operating room for an arthroscopic debridement of his right knee. Cultures taken intraoperatively eventually grew out Clostridium septicum. On May 5, 1998 the patient was again taken to the operating room where he had a debridement and removal of the interference screws and graft. Intraoperative findings included lysis of the graft and separation of both the tibial and femoral bone plugs from the tendon. On May 11, 1998, the patient had a colonoscopy to rule out a colonic lesion. The findings of that study were negative. The patient was treated with six weeks of appropriate antibiotics, and the infection eventually resolved. Medical records were not available to know the total number of debridements required, or the patient's final condition. The senior author, and the surgeon of this patient, were later discussing the unusual findings of the first two cases at an orthopaedic meeting. Subsequent investigation lead to the realisation that this patient received his graft from the same cadaver donor as the patient in Case 1.


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The tissue bank was informed of these findings, and following investigation, the tissue bank concluded that the two cases were unrelated and no further action was taken. 4. Case 3 On April 27, 1998 a 16-year-old female had her right ACL reconstructed with a bone patella tendon bone allograft by a surgeon. On May 3, 1998 the patient began to experience increasing pain and swelling of her right knee, as well as fevers to 103°F. Her knee was aspirated and sent for culture, and she was admitted to the hospital for intravenous antibiotic treatment. Cultures were negative at five days; however, the patient's condition failed to improve, and she was ultimately taken to the operating room for debridement and removal of the graft on May 8, 1998. Six days later, culture results confirmed infection with the Clostridium septicum species. She was treated with a six-week course of appropriate antibiotics, and a repeat debridement on June 17, 1998, and her condition eventually resolved. She regained full range of motion of her right knee and eventually had a revision autograft bone patella tendon bone ACL reconstruction that proceeded without complication. The patient eventually filed a lawsuit against the tissue bank that was settled out of court with a monetary award to the patient, with an associated gag order. 5. Case 4 A 51-year-old male, an avid runner was treated with a left bone patella tendon bone allograft ACL reconstruction on April 20, 2001 by a surgeon. He had an unremarkable post operative course initially; however, he later began to experience increasing pain and swelling of his left knee, with associated fever to 102°F, and was readmitted to the hospital on May 2, 2001. He was taken to the operating room where an irrigation and

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debridement was performed. The patient had culture results that were positive for Clostridium septicum infection, and he was placed on oral antibiotics that alleviated his symptoms. He was discharged from the hospital on oral antibiotics. Following his discharge the patient suffered a loss of knee motion and was taken to the operating room again on May 25, 2001 for a manipulation under anaesthesia. The patient seemed to improve following the manipulation; however, he again began to experience high fevers to 102°F and was readmitted to the hospital on June 15, 2001. X-ray evaluation of his left knee revealed a distal thigh abscess consistent with a gas-producing organism. He was taken to the operating room on the same day for debridement of his left distal thigh, and cultures again grew out Clostridium septicum. He was subsequently treated with appropriate antibiotics, and his clinical course improved. Unfortunately, he developed a flexion contracture of his left knee, and when he was seen at our institution, he had a left knee range of motion from 40° to 45°. The senior author treated the patient with an arthroscopic lysis of adhesions and manipulation under anaesthesia, ultimately improving his range of motion to 5° to 80°. His legal case is currently in litigation.

6. Discussion Clostridia species are gram-positive rods found in soil, decaying vegetation, marine sediment, and in the intestinal tract of humans, insects and other vertebrates, and most of the species possess the ability to sporulate when exposed to unfavourable environmental conditions (Bennett, 2000). Clostridium septicum has been identified in 2% of stool cultures in previous normal flora studies of humans, and carriage rates of 10-63%, most commonly in the appendix, have been reported in the literature (Godette and Kapta, 1996). Clostridium septicum is most often encountered clinically as bacteraemia associated with intestinal malignancy, or a relapse of leukaemia (Larson and Bubrick, 1995).


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However, careful review of the literature identifies agonal bacteraemia in potential cadaver donors as a significant finding, as it pertains to the procurement of allograft tissues (Isenberg and D'Amato, 1995). MacEwen reported the first use of musculoskeletal allograft in 1880 (Dolano and Kapta, 1991). Lexer reported on 23 cases of articular cartilage transplantation between 1908 and 1925. These reports included phalangeal, elbow and knee joints, and he claimed a 50% success rate (Lexer, 1925). Noyes et ah, reported allograft ligament reconstructions in 1981 (Noyes and Barber, 1990) and Milachowski reported on transplanted menisci in 1984 (Milachowaki and Wermeier, 1989). Today the use of allograft tissues is extremely common, leading to an increasing demand for tissue, and requiring surgeons to keep informed of the risks and benefits of this ever-expanding technology. Musculoskeletal allografts are processed using a variety of methods depending on the tissue being transplanted, and on the requirements of the surgeon. Fresh allograft refers to tissue that is harvested under sterile conditions, that is transplanted directly from the host to the recipient. The tissue is normally maintained in lactated Ringer's solution at 2°C-4°C for a period up to seven days. Studies show that viable cartilage cells diminish substantially in culture media after 24 hours, and are virtually nonexistent after seven days (Shelton and Treacy, 1998). This type of tissue is necessary for successful articular cartilage and meniscal cartilage transplants to preserve viable cartilage cells. Although these tissues are thoroughly washed prior to transplantation, they still contain some marrow elements and viable cells from the donor; thus, an immune response does occur (Langer and Czitron, 1975). Currently, no standard of processing these tissues with regard to washing, antibiotic treatments, or sterilisation treatments (gamma radiation, ethylene oxide, etc.) exists (Oversight Committee on Tissue Banking, 2001). Cryopreservation is a process of controlled-rate freezing using DMSO and glycerol to remove water during the freezing process in an attempt to preserve viable cells. The process works by

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altering water crystallisation during the freezing process, which preserves up to 80% of cells (Shelton and Treacy, 1998). Grafts are cooled to 0°C and processed within 48 hours. They are then incubated in an antibiotic solution for 24 hours at 37°C, and frozen to -135°C and packed in a cryoprotectant solution for up to 10 years (Shelton and Treacy, 1998). This technique works well for meniscal cartilage; however, the process damages articular cartilage. The increased cost prohibits its use for other types of tissue, as other cheaper techniques work just as well (Shelton and Treacy, 1998). Fresh frozen tissue is the most common technique for processing ligament allografts. After tissue harvest under sterile conditions, the tissue is usually cultured and frozen while serologic test results are carried out in a process that takes two to four weeks. It is then soaked in an antibiotic solution at room temperature for one hour, and then packaged and frozen without solution for up to five years (Shelton and Treacy, 1998). No viable cells reliably survive this process (Shelton and Treacy, 1998), which has several important clinical ramifications. Loss of cells decreases the likelihood of immune reactions or disease transmission; however, the effectiveness of this treatment is controversial. Finally, freeze-dried allograft tissues are also commonly used in ligament reconstruction. After harvest under sterile conditions, the tissue is frozen pending serologic and bacterial culture results. The tissues are then subjected to a one-hour bacterial solution soak at room temperature. A process of refreezing and lyophilisation to residual moisture of less than 5%, followed by packaging and storage for up to five years is then carried out. The colour and strength of the tissues are altered; however, studies have shown no deleterious effects on clinical outcomes of ligament reconstruction (Shelton and Treacy, 1998). Allogeneic tissues function as a scaffold that is eventually incorporated into the host. Incorporation generally occurs through three stages. First, death of cells from the donor tissue occurs. This stage is usually considered to be a characteristic of fresh, or

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cryopreserved tissues, because other processing methods (fresh frozen or freeze-dried) are considered to have already caused cell death in donor tissues. The second stage includes revascularisation of the donor tissue, with repopulation of the donor tissue by host cells (Shelton and Treacy, 1998). These first two stages occur relatively rapidly compared to the final stage — remodelling — that occurs more slowly. Jackson et al., demonstrated complete replacement of donor cells by host cells in goat ACL's in four weeks (Backson and Simon, 1963). It has been shown that complete remodelling of allograft tissue may take one-and-a-half times as long as autograft tissue to regain comparable strength (Cordrey and McKorkle, 1963). Prolongation of the remodelling phase may be the result of tissue-antigen mismatch, resulting in a subclinical immune response (Shelton and Treacy, 1998). Several studies in dogs and goats have noted similar gross, and histologic patterns of tissue, between native and allograft ligaments at six months to 1-year post transplantation (Garrett, 1996; Mankin and Doppelt, 1983; Zukor and Paitich, 1989). In addition to proper donor screening, adherence to sterile techniques during procurement and processing, and appropriate serologic and bacteriologic testing and post harvest sterilisation, can improve the safety of allograft tissue. Currently, the two most common methods of sterilisation are ethylene oxide treatment and gamma irradiation. Unfortunately, both of these treatments can have deleterious side effects, and are therefore not universally employed. Ethylene oxide leaves behind a chemical residue that may cause chronic synovitis and graft failure (Jackson and Windier, 1990), and gamma irradiation greater than 3.0 Mrad weakens collagen tissue (Pelker and Friedlaender, 1987). Tissues are weakened by irradiation due to the destruction of collagen chains, probably mediated by oxygen free radicals (Hamer and Stockley, 1999). Recent literature suggests that gamma radiation can be efficaciously employed to sterilise allograft material without a deleterious clinical effect. In 1951 Meeker and Gross reported that 1.5 Mrad destroyed 95% of bacterial organisms, but that 3.0 Mrad

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caused significant tissue damage. Fideler et al, showed that 30 kGy was necessary to eradicate HIV in bone-patella tendonbone allograft at -70°C (Fideler and Vangsness, 1995). In 1956 Turner et al, reported that bone grafts sterilised at 2.0 Mrad underwent abnormal absorption and fragmentation, but reported only two infections in 100 patients receiving 189 allografts, demonstrating the effective sterilising properties of gamma radiation. In 1959, Bassett and Packard reported a less than 1% infection rate in 1,037 patients receiving bone graft sterilised with 2.0 Mrad of gamma radiation. Fideler showed in 1995, that the initial biomechanical strength of fresh frozen bone-patellar tendon bone allograft was reduced 15% after irradiation with 2.0 Mrad of gamma radiation, but that stiffness, elongation, and strain were not reduced with statistical significance. Goertzen reported in JBJS (Br) 1995, that canine BPT-B allograft treated with 2.0 Mrad gamma radiation protected with Argon gas, compared favourably with non-irradiated controls with regard to maximum load to failure at 12 months. The irradiated group failed at 718.3 N (63.8% of normal ACL's), and the control group failed at 780.1N (69.1% of normal). In addition, histologic studies showed no difference between the groups with regard to collagen structure. Silver staining showed the presence of Golgi tendon organs and free nerve endings in both groups, and only a slight hypervascularity was noted in the controls compared to the irradiated group. The literature on sterilisation demonstrates that no technique is 100% effective at rendering allogeneic tissues sterile, but that improved sterility with certain treatment protocols can be employed to improve the quality of these tissues without adversely effecting clinical outcomes. Torisuka et al., studied the effect of 25 kGy of gamma irradiation, and freeze-drying on patella tendon graft remodelling after transplantation in the rat model (Toritsuka and Shino, 1997). They studied four groups, including fresh-frozen, freezedried, fresh-frozen gamma-irradiated, and freeze-dried gamma irradiated. They discovered that freeze drying and gamma irradiation temporarily accelerated graft remodelling in the early


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phase (first 12 weeks), accompanied by an increase in newly synthesised collagen, and a decrease in donor collagen (Toritsuka and Shino, 1997). Histologically, no difference in the progression of cellular repopulation was observed between the four groups. It was thus assumed that the chemotactic properties of the graft materials were unaffected, and that accelerated graft remodelling was potentiated. Although it can be assumed that the rapid decrease in donor collagen would initially weaken the grafts treated in this manner, further studies would be required to determine if this phenomenon has any clinical significance requiring a modification of rehabilitation protocols. One can conclude that grafts treated in this manner would be safer, less likely to promote immunologic reactions, and accelerate incorporation of the grafts. Our current knowledge of the safe procurement of allogeneic tissues was originally adapted from procedures developed by the United States Navy Tissue Bank, and has been modified through clinical experience over many years. As our understanding of the risks and means by which disease is transmitted is further delineated, efforts have been made to modify techniques in order to insure the highest possible safety of allograft tissue. Current understanding of safe tissue banking procedures has lead to the development of a comprehensive screening process for potential donors. These include a detailed medical and social history; serologic tests for HIV I/II antibodies, HIV antigen, PCR HIV, hepatitis B surface antigen, hepatitis B surface antibodies, hepatitis B core antibodies, hepatitis C virus antibodies, and the RPR test for syphilis. In addition, an autopsy of potential donors, with a separate study of lymph nodes, completes the screening process. Currently, only a medical history, social history, and serologic testing are required by the FDA. It is also pointed out by Malinin et ah, in 1985, that procurement of tissue is a surgical skill requiring strict adherence to sterile technique. Procurement is also a clinical service, which carries with it the responsibilities incumbent on any physician providing medical care. As such, it

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would seem logical that personnel who procure tissue be held to the same standard as other medical professionals. Buck and Malinin reported on their experience at the University of Miami Tissue Bank in 1994. They discussed the reasons for exclusion of 187 of 1,000 consecutive donors over a six-andone-half-year period. Eighty-five were excluded on the basis of bacteriologic criteria alone. Seventeen had bacteriological exclusionary criteria as well as serologic a n d / o r morphologic exclusionary criteria. Sixty-eight cases were excluded for hepatitis detected using serologic markers, or morphologic changes, and one case based on histologic changes alone (Buck and Malinin, 1994). Documented or suspected HIV accounted for 10 exclusions. In one case a donor was excluded based on classic nonspecific changes of HIV noted in the lymph nodes (Murchadha and Wolfe, 1987; Racz and Tenner-Racz, 1986). Another donor was excluded based on birefringent material consistent with drug abuse, noted in granulomas located in the liver and lungs (Tomashefski and Hirsch, 1989). Seven donors were rejected for granulomatous disease; another four for unsuspected malignancies, and two for myocarditis. The experience of Buck and Malinin clearly demonstrates the need for screening beyond serologic tests to insure the safety of allograft tissue. In order to understand the potential risks of bacterial infection with the use of allograft tissue, one must first identify the manner in which these tissues become contaminated. A review of the literature reports a 5% to 44% rate of bacterial contamination of allograft tissue. Due to variability of the culture techniques employed it is not possible to compare the contamination rates. Diejkers et al., analysed the incidence and predisposing factors of bacterial contamination of allograft tissue in JBJS [Br] 1996. They evaluated 1,999 bone and soft tissue grafts from 200 cadaver donors under sterile operating conditions. After removal of the grafts from the cadaver donors, they were rinsed with an antibiotic solution in the first 150 donors, and with saline only in the last 50 donors. Swabs were then taken from the entire


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graft surface and placed in a transport medium. The swabs were subsequently inoculated onto blood agar and chocolate agar plates, and cultured under aerobic and anaerobic conditions. The entire swab sticks were then placed in a brain heart infusion broth and cultured for another 72 hours under both aerobic and anaerobic conditions. Blood samples from the cadaver donors were cultured aerobically and anaerobically for seven days. Microbial load was considered low if the microorganisms grew only in the broth, and high if growth occurred directly on the plates. They found that 50% of the grafts cultured positive for organisms of low pathogenicity, and 69% were of a low microbial load. Three percent of the grafts grew out organisms of high pathogenicity. The authors determined that organisms of low pathogenicity (for example, coagulase-negative staphylococci, Corynebacterium, Propionibacterium acnes) were likely to have contaminated the grafts exogenously during procurement. Organisms of high pathogenicity originated endogenously from the donor, and were usually contaminants from the gastrointestinal tract or the upper respiratory tract, and were more likely to cause a clinically significant infection in the recipient. They found that contamination with organisms of high pathogenicity was 3.4 times higher in donors with a traumatic cause of death, and 5.2 times higher in those with positive blood cultures. It was also noted that while washing the grafts with antibiotic solution reduced the organisms of low pathogenicity by a factor of two, organisms of high pathogenicity were not reduced with antibiotic soaks. The authors concluded that exogenous contamination was most affected by the procurement team, and that endogenous contamination was best controlled through careful donor selection (Diejkers and Bloem, 1997). Martinez et al., reported on the microbiologic cultures of blood and bone, of 239 cadaver donors, and 58 "beating heart donors" who had been aseptic prior to tissue harvest. The incidence of positive blood cultures was significantly lower in

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the "beating heart donors" (8.6%) compared to the cadaver donors (38%) (Martinez and Malinin, 1985). Agonal bacteraemia is a well-described process whereby endogenous bacteria, such as normal intestinal flora, are disseminated throughout the body after death in cadaver donors (Isenberg and D'Amato, 1995). This process is thought to be facilitated by the loss of barrier function of gut capillaries, and may also result from trauma or manipulation procedures during resuscitation attempts around the time of death (Diejkers and Bloem, 1997; Veen, 1994). The difference in blood culture results between the two groups of this study was attributed to the persistence of anatomic barriers to microbial invasion, and competent microbial clearing mechanisms in the "beating heart donor" group. Microorganisms were isolated from the tissues of 55.4% of the cadaver donors, and 67.9% of the "beating heart donors" that had negative blood cultures. Clostridium species were the second most commonly isolated group behind coagulase negative staphylococcus. It was also noted that 60% of donors with positive blood cultures for Clostridium species also cultured positive from tissue samples (Martinez and Malinin, 1985). They found the predictive value of positive blood cultures to be 83.5%; however, the predictive value of negative blood cultures was only 44.5% in the two groups. These results demonstrate that blood cultures are ineffective in confirming the sterility of allograft tissue, and that the Clostridium species seem to be disseminated at the time of death in the cadaver donors, and often seed tissues prior to harvest. The tremendous increase in allograft tissue applications, and thus, demand, has resulted in a failure to adequately supervise the safety of this industry. Generally, allograft tissues are extremely safe, and complications resulting from disease transmission are extremely small. However, when they do occur, the consequences are often catastrophic, and potentially avoidable with strict adherence to known protocols designed to insure safety.


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In January 2001, the Office of the Inspector General, Department of Health and Human Services, convened an Oversight committee to profile the current state of tissue banking in this country. It was noted that oversight of tissue banking practices occurs at three levels; the Food and Drug Administration (FDA), the American Association of Tissue Banks, and at the state level. The Food and Drug Administration requires donor screening and testing to prevent the transmission of communicable diseases. The FDA had conducted 188 inspections of 118 tissue banks known to the government at that time, since 1993. The American Association of Tissue Banks (AATB) conducts a voluntary accreditation program that evaluates the procurement practices of each tissue bank. As of January 2001, 58 tissue banks had been accredited, and another 90 identified banks were not. According to the oversight committee's report, only New York and Florida require licensing and inspection of tissue banks, although authorities in California report that AATB certification is required in that state. Furthermore, the Oversight Committee points out that many tissue banks do not seek AATB accreditation because there is no incentive to do so. The committee reported that no cases of disease transmission have been identified since the FDA's regulation regarding donor screening and testing in 1993. However, it is our contention that bacterial infections have occurred as the result of contaminated allograft tissue as demonstrated by our current Case 1 report. We are treating two other patients who had ACL allograft transplants by other surgeons, complicated by postoperative Clostridia infections. In addition, we are aware of a fourth patient who received the other half of the hemi-patellar tendon graft that caused our patient's infection, and contracted the same rare Clostridium septicum infection. In these particular cases, no preoperative graft cultures were taken to confirm the graft was the source of infection, but three patients received a legal settlement from the tissue bank with a nondisclosure clause, and a fourth is currently pending a legal action. Despite the lack preoperative culture results from three of these cases, Clostridium is an

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extremely rare organism in post operative wound infections, and it seems likely that the graft material was the source of infection. We believe that the rarity of contamination, failure of surgeons to recognise allografts as a source of infection, and the tissue bank's failure to properly investigate these complications, has resulted subsequent infections that may have been otherwise avoided. The oversight committee found 36 tissue banks that have never been inspected, out of 154 identified tissue banks, and notes that the actual number and location of all tissue banks is unknown. While the FDA does regulate donor screening and testing, no regulations exist for quality and handling of tissue. Of the 118 tissue banks inspected by the FDA since 1993, 26 notices of official action requiring the banks to take corrective action were issued. In 72 others, notices were issued that suggested changes to improve quality (Department of Health and Human Services, 2001). The FDA reported a list of examples of safety and qualities problems found in those tissue banks inspected. These included "lack of adequate controls to assure product sterility, lack of standard operating procedures to prevent cross contamination of human tissue during manufacture, and distribution and implantation of soft tissue grafts from a single donor with possible bacterial contamination," just to name a few. Clearly, the current supervision and accountability of the tissue banking industry is woefully inadequate and poses an unnecessary risk to recipients of allograft tissue. 7. Summary The use of allogeneic tissue in orthopaedic surgery is generally safe and efficacious. The indications for these tissues continue to expand, and represent a marked advance in the implementation of medical care. Recently, the risks of bacterial infection from contaminated grafts, and the devastating consequences, have become recognised, prompting the CDC and other government agencies to scrutinise the tissue banking industry.


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Several changes in the practice of tissue banking, and the use of allograft tissue, will be required to improve the quality of allogeneic tissue. First it is imperative that tissue banks be closely regulated to insure that proper practices for safe tissue banking are being followed. Furthermore, research needs to be conducted to develop safer methods for tissue procurement. A review of the literature demonstrates that blood and swab cultures, and antibiotic washes, are ineffective in confirming the sterility of donor tissue. In addition, current literature on known methods of sterilisation such as gamma irradiation, demonstrate efficacious employment, demanding that standard protocols be implemented for all tissue banks. An argument can also be made to require that only trained professionals, held to the same standards as other medical professionals, be allowed to harvest tissue. It is also necessary to make donor medical records available to surgeons using their graft material, to aid in clinical decision-making. Finally, reporting of bacterial infections to a central agency to monitor future outbreaks, should be mandatory. When complications do occur, these cases need to be investigated, rather than buried by legal sanctions, to improve tissue-banking techniques. Based on these case reports, we would recommend that the implanting surgeon send cultures of allograft tissue, so that appropriate action may be taken should high pathogenic bacteria such as Clostridia be encountered. These changes are likely to increase the cost of tissue banking, and may create difficulty in attaining graft materials. However, even though the complication rate associated with the use of allograft tissue is extremely low, the devastating impact on morbidity and mortality under the current system is unacceptable. Additionally, it is incumbent on the orthopaedic surgeon to keep informed on the various tissue types and indicated uses. Understanding these factors will allow orthopaedic surgeons to inform their patients more knowledgeably on the potential risks, and to use the various graft types available in the most efficacious manner.

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8. References BARRIOS, R.H., LEYES, M , AMILLO, S. and OTEIZA, C. (1994). Bacterial contamination of allografts, Acta Orthopaedica Begica 60-2, 152-154. BECHTOLD, J.E., EASTLUND, T.D., BUTTS, M.K., LAGERBORG, D.F. and KYLE, R.F. (1994). The effects of freeze-drying and ethylene oxide sterilisation on the mechanical properties of human patellar tendon, Am. }. Sports Med. 22, 562-566. BENNETT, L. (2000). Gas gangrene and other clostridium-associated diseases. In: Mandell, Douglas, and Bennett's Principles and Practice of Infectious Disease, 5th Ed. Churchill Livingstone, Philadelphia, PA, pp. 2549-2561. BETTEN, D., DETHLOFF, M., STEINBECK, J. and POLSTER, J. (1994). Organisation of a bone and tissue bank, Z. Orhtop. Ihre. Grengeb. 132, 453-458. BOLANO, L. and KOPTA, J.A. (1991). The immunology of bone and cartilage transplantation, Orthopaedics 14, 987-996. BRIGHT, R.W., FRIEDLAENDER, G.E. and SELL, K.W. (1977). Current concepts: Tissue banking: The United States Navy tissue bank, Milit. Med. 142, 503. BUCK, B.E. and MALININ, T.I. (1994). Human bone and tissue allografts, preparation and safety, Clin. Orthop. 303, 8-17. BUCK, B.E., MALININ, T.I. and BROWN, M.D. (1989). Bone transplantation and human immunodeficiency virus: An estimate of risk of acquired immunodeficiency syndrome (AIDS), Clin. Orthop. 240, 129-136. CENTER FOR DISEASE CONTROL (CDC) (2001). Septic arthritis following anterior cruciate ligament reconstruction using tendon allografts, MMWR 50, 1081-1083.


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CENTER FOR DISEASE CONTROL (CDC) (2001). Unexplained deaths following knee surgery, Minnesota, MMWR 50, 10351036. CORDREY, L.I., MCCORKLE, H. and HILTON, E. (1963). A comparative study of fresh autogenous and preserved homogenous tendon grafts in rabbits, /. Bone Joint Surg. [Am.] 45, 182-195. DEPARTMENT OF HEALTH AND HUMAN SERVICES, OFFICE OF INSPECTOR GENERAL, OVERSIGHT OF TISSUE BANKING (2001), January. DIEJKERS, R.L.M., BLOEM, R.M., PETIT, P.L.C., BRAND, R., VEHMEYER, S.B.W. and VEEN, M.R. (1997). Contamination of bone allografts, analysis of incidence and predisposing factors, /. Bone Joint Surg. [Br.] 79-B, 161-166. FARRINGTON, M., MATTHEWS, I., FOREMAN, J., RICHARDSON, K.M. and CAFFREY, E. (1998). Microbial monitoring of bone grafts: Two years' experience at a tissue bank, /. Hosp. Infect. 38, 261-271. FIDELER, B.M., VANGSNESS, C.T. Jr., MOORE, T., LI, Z. and RASHEED, S. (1994). Effects of gamma irradiation on the human immunodeficiency virus: A study in frozen human bonepatellar tendon-bone grafts obtained from infected cadavera, /. Bone Joint Surg. [Am.] 76-A, 1032-1035. FIDELER, B.M., VANGSNESS, C.T. Jr., LU, B., ORLANDO, C. and MOORE, T. (1995). Gamma irradiation: Effects on biomechanical properties of human bone-patellar tendon-bone allografts, Am. J. Sports Med. 23, 643-646. FIDELER, B.M., VANGSNESS, C.T. Jr, LU, B., ORLANDO, C , MOORE, T., FINEGOLD, S.M., ATTEBERY, H.R. and SUTTER, V.L. (1974). Effect of diet on human fecal flora: Comparison of Japanese and American diets, Am. J. Clin. Nutr. 27,1456-1469.

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GARRETT, J.C. (1996). Osteochondral allografts for reconstruction of articular defects. In: Operative Arthroscopy, 2nd Ed. R.B. Caspari, R.W. Jackson, J.B. McGinty, and G.G. Poehling, eds., Lippincott-Raven, Philadelphia, pp. 395-403. GAZDAG, A.R., LANE, J.M., GLASER, D. and FORSTER, R.A. (1995). Alternatives to autogenous bone graft: Efficacy and indications, /. Am. Acad. Orthop. Surg. 3, 1-8. GEORGE, W.L. and FINEGOLD, S.M. (1985). Clostridia in the human gastrointestinal flora. In: Clostridia in Gastrointestinal Disease, S.P. Boriello, ed., CRC, Boca Raton, Fla., pp. 1-37. GODETTE, G.A., KOPTA, J.A., and EGLE, D.M. (1996). Biomechanical effects of gamma irradiation on fresh frozen allografts in vivo, Orthopaedics 19(8), 649-653. GOERTZEN, M.J., CLAHSEN, H., BURRIG, K.F. and SCHULITZ, K.P. (1995). Sterilisation of canine anterior cruciate allografts by gamma irradiation in argon, /. Bone Joint Surg. [Br.] 77-B, 205-212. HAMER, A.J., STOCKLEY, I. and ELSON, R.A. (1999). Changes in allograft bone irradiated at different temperatures, /. Bone Joint Surg. [Br.] 81-B, 342-344. HIRN, M.Y., SALMELA, M. and VUENTO, R.E. (2001). Highpressure saline washing of allografts reduces bacterial contamination, Acta Orthop. Scand. 72, 83-85. ISENBERG, H.D. and D'AMATO, R.F. (1995). Indigenous and pathogenic microorganisms in humans. In: Manual of Clinical Microbiology, 6th Ed. E.J. Baron, P.R. Murray, M.A. Pfaller, F.C. Tenover and R.H. Yolken, eds., ASM Press, Washington, D.C., pp. 5-18. IVORY, J.P. and THOMAS, I.H. (1993). Audit of a bone bank, /. Bone Joint Surg. [Br.J 75-B, 355-357.


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Rduances in


This is the most comprehensive volume dealing with tissue banking presently available, with 27 contributions from the most distinguished and experienced practitioners in the field: surgeons, microbiologists and tissue bankers. Safety of allografts is now a major concern due to possible microbial and viral contamination of tissues, even in the most sophisticated centres. Thus, publication here of the International Atomic Energy Agency's Code of Practice for the Radiation Sterilisation of Tissues is important, as is their guidance on Standards and Public Awareness regarding this often misunderstood technology. The volume spans all the methodologies used in the field and covers a spectrum of tissues: bone, skin, cardiovascular grafts, corneal grafts and sperm banking. Of particular interest in these days of gigantic disasters is the evaluation of the value of an effective tissue bank during the Volendam burns disaster in the Netherlands and the horrific disaster in "Messa Redonda" Peru. Orthopaedics, as usual, has the premier usage of tissues and this volume is graced by a landmark contribution from that doyen of massive allograft surgery, Henry Mankln. Balancing out the US experience is a contribution from Russia, which outlines new approaches to using allograft and autograft bone. The motivation for such a comprehensive volume came at the congress held In Boston, which drew together all the international associations of tissue banking: American, AsiaPacific, Latin American and European.The whole world has been harnessed to construct this outstanding and historic volume. ISBN 981-238-723-4

World Scientific www.worldscientific.com 5445 he

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Advances in Tissue Banking, Vol. 7

The Scientific Basis of Tissue Transplantation (Advances in Tissue Banking)

Advances In Tissue Engineering

Reproductive Tissue Banking: Scientific Principles

Reproductive Tissue Banking: Scientific Principles

ADVANCES IN CATALYSIS VOLUME 7, Volume 7

Advances in Computers, Volume 7

Advances in Agronomy, Volume 7

Advances in Computers, Volume 7

Advances in Geophysics, Volume 7

Advances in Genetics Volume 7

Advances in Immunology Volume 7

Eye Banking (Developments in Ophthalmology, Vol. 43)

Free Banking (Vol. II) History

Radiation in Tissue Banking: Basic Science and Clinical Applications of Irradiated Tissue Allografts

Advances in Geophysics, Vol. 23

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Advances in Agronomy, Vol. 86

Advances in Photochemistry, Vol 27

Advances in Geophysics, Vol. 31

Advances in Geophysics, Vol. 26

Grantville Gazette Vol 7

Careers in commercial banking, corporate banking, investment banking

Frontiers in Tissue Engineering

Frontiers in Tissue Engineering

Scaffolding In Tissue Engineering

Advances in Applied Mechanics, Volume 7

Advances in Microbial Physiology, Volume 7

Advances in Business and Management Forecasting, Volume 7 volume 7

Advances in Clinical Chemistry Volume 7

Advances in Tissue Banking, Vol. 7

The Scientific Basis of Tissue Transplantation (Advances in Tissue Banking)

Advances In Tissue Engineering

Reproductive Tissue Banking: Scientific Principles

Reproductive Tissue Banking: Scientific Principles

ADVANCES IN CATALYSIS VOLUME 7, Volume 7

Advances in Computers, Volume 7

Advances in Agronomy, Volume 7

Advances in Computers, Volume 7

Advances in Geophysics, Volume 7

Advances in Genetics Volume 7

Advances in Immunology Volume 7

Eye Banking (Developments in Ophthalmology, Vol. 43)

Free Banking (Vol. II) History

Radiation in Tissue Banking: Basic Science and Clinical Applications of Irradiated Tissue Allografts

Advances in Geophysics, Vol. 23

Advances in Photochemistry, Vol 27

Advances in Agronomy, Vol. 86

Advances in Photochemistry, Vol 27

Advances in Geophysics, Vol. 31

Advances in Geophysics, Vol. 26

Grantville Gazette Vol 7

Careers in commercial banking, corporate banking, investment banking

Frontiers in Tissue Engineering

Frontiers in Tissue Engineering

Scaffolding In Tissue Engineering

Advances in Applied Mechanics, Volume 7

Advances in Microbial Physiology, Volume 7

Advances in Business and Management Forecasting, Volume 7 volume 7

Advances in Clinical Chemistry Volume 7

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