Practical Transfusion Medicine 2nd ed by stikeshi

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									Practical
Transfusion
Medicine
EDITED BY

Michael F. Murphy
MD, FRCP, FRCPath
Professor of Blood Transfusion Medicine
University of Oxford
Consultant Haematologist
National Blood Service and Department of Haematology
The John Radcliffe Hospital, Oxford



Derwood H. Pamphilon
MD, MRCPCH, FRCP, FRCPath
Consultant Haematologist
Institute for Transfusion Sciences
National Blood Service
Bristol



FOREWORD BY

D.J. Weatherall

Second edition
Practical Transfusion Medicine
Practical
Transfusion
Medicine
EDITED BY

Michael F. Murphy
MD, FRCP, FRCPath
Professor of Blood Transfusion Medicine
University of Oxford
Consultant Haematologist
National Blood Service and Department of Haematology
The John Radcliffe Hospital, Oxford



Derwood H. Pamphilon
MD, MRCPCH, FRCP, FRCPath
Consultant Haematologist
Institute for Transfusion Sciences
National Blood Service
Bristol



FOREWORD BY

D.J. Weatherall

Second edition
© 2005 by Blackwell Publishing Ltd
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First published 2001
Second edition 2005

Library of Congress Cataloging-in-Publication Data
Practical transfusion medicine / edited by Michael F. Murphy, Derwood H. Pamphilon. — 2nd ed.
       p. ; cm.
Includes bibliographical references and index.
ISBN 1-4051-1844-X
1. Blood— Transfusion.
[DNLM: 1. Blood Transfusion. 2. Blood Grouping and Crossmatching. 3. Communicable
Disease Control. 4. Specimen Handling. WB 356 P8957 2005] I. Murphy, Michael F.
(Michael Furber) II. Pamphilon, Derwood H.
RM171.P727 2005
615¢.39— dc22
                                                                                2004016676

ISBN-13: 978-1-4051-184-46
ISBN-10: 1-4051-184-4X

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Contents


   List of Contributors, vii                      Part 3 Complications of transfusion
   Foreword, xi
                                                  13 Haemolytic transfusion reactions Sue Knowles
   Preface to the second edition, xiii
                                                     and Geoff Poole, 161
   Preface to the first edition, xv
                                                  14 Febrile reactions and transfusion-related acute
                                                     lung injury Michael F. Murphy and
Part 1 Basic principles of transfusion               Sheila MacLennan, 171
                                                  15 Urticarial and anaphylactic reactions
 1 Introduction Ian M. Franklin, 3                   David J. Unsworth, 179
 2 Essential immunology for transfusion           16 Bacterial contamination Patricia E. Hewitt,
   medicine Willem H. Ouwehand and                   184
   Tim B. Wallington, 13                          17 Post-transfusion purpura Michael F. Murphy,
 3 Human blood group systems                         191
   Geoff Daniels, 24                              18 Immunomodulation and graft-versus host
 4 Human leucocyte antigens                          disease Lorna M. Williamson and
   Cristina V. Navarrete, 34                         Cristina V. Navarrete, 195
 5 Platelet and neutrophil antigens David L.      19 Transfusion-transmitted infections
   Allen, Geoffrey F. Lucas, Willem H.               Alan D. Kitchen and John A.J. Barbara, 208
   Ouwehand and Michael F. Murphy, 50             20 Variant Creutzfeldt–Jakob disease
                                                     Marc L. Turner, 229

Part 2 Clinical transfusion practice
                                                  Part 4 Practice in blood centres and hospitals
 6 The effective and safe use of blood
   components Brian McClelland and                21 Donors and blood collection Liz Caffrey and
   Tim Walsh, 67                                     Moji Gesinde, 241
 7 Bleeding associated with trauma and surgery    22 Blood donation testing and the safety
   Beverley J. Hunt, 86                              of the blood supply David Wenham and
 8 Prenatal and childhood transfusions               Simon J. Stanworth, 250
   Irene Roberts, 97                              23 Production and storage of blood components
 9 Haematological disease Michael F. Murphy          Lorna M. Williamson and Rebecca
   and Simon J. Stanworth, 119                       Cardigan, 259
10 Transfusion strategies in organ transplant     24 Medicolegal aspects Patricia E. Hewitt, 274
   patients Derwood H. Pamphilon, 132             25 Blood transfusion in hospitals Sue Knowles
11 Inherited and acquired coagulation disorders      and Geoff Poole, 280
   Joanne E. Joseph and Samuel J. Machin, 138     26 Autologous transfusion Dafydd Thomas, 298
12 Uses of intravenous immunoglobulin             27 Tissue banking Deirdre Fehily and
   David J. Unsworth and Tim B. Wallington, 151      Ruth M. Warwick, 309


                                                                                                   v
Contents


28 Cord blood banking Ruth M. Warwick,             35 Recombinant antibodies and other proteins
   Sue Armitage and Deidre Fehily, 320                Marion Scott, 403
29 Therapeutic apheresis Tim B. Wallington and     36 Blood transfusion in a global context
   David J. Unsworth, 328                             David Roberts, Jean-Pierre Allain, Alan
                                                      Kitchen, Stephen Field and Imelda Bates, 415
                                                   37 The design of interventional trials in
Part 5 Developments in transfusion medicine
                                                      transfusion medicine Paul Hébert, Alan
30 Blood substitutes Chris V. Prowse and              Tinmouth and Dean Fergusson, 424
   David J. Roberts, 341                           38 Getting the most out of the evidence for trans-
31 Cytokines in transfusion practice Derwood H.       fusion medicine Simon J. Stanworth, Susan J.
   Pamphilon, 350                                     Brunskill and Chris J. Hyde, 436
32 Haemopoietic stem cell processing and storage   39 The future of transfusion medicine Walter
   David H. McKenna and Mary E. Clay, 357             Sunny Dzik, 445
33 Haemopoietic stem cell transplantation and
                                                      Index, 457
   immunotherapy Ian M. Franklin, 369
34 Gene therapy Colin G. Steward and                  Colour plates are found between pp. 304 and
   Marina Cavazzana-Calvo, 390                        305




vi
List of Contributors


Jean-Pierre Allain                      Rebecca Cardigan                             Dean Fergusson
Division of Transfusion Medicine        National Blood Service                       Centre for Transfusion Research
National Blood Service                  Cresent Drive                                University of Ottawa
University of Cambridge                 Brentwood                                    Ottawa, Ontario K1M 8L6
Long Road                               Essex                                        Canada
Cambridge, CB2 2PT                      CM15 8DP

                                                                                     Stephen Field
David L. Allen                          Marina Cavazzano-Calvo                       National Blood Service
National Blood Service                  Director of Biotherapy                       North London
John Radcliffe Hospital                 Hopital Necker-Enfants Malades               Colindale Avenue
Headley Way                             149 Rue des Sevres                           Colindale
Headington                              Cedex 15                                     London, NW9 5BG
Oxford, OX3 9DU                         Paris 75743 France

                                                                                     Ian M. Franklin
Susan Armitage                          Mary E. Clay                                 Department of Medicine
National Blood Service                  Department of Laboratory Medicine &          University of Glasgow
Cord Blood Bank                           Pathology                                  Royal Infirmary
Deansbrook Road                         MMC 198 Room D-251 Mayo Building             Glasgow
Edgeware                                University of Minnesota Medical School       G31 2ER
Middlesex, HA8 9DB                      420 Delaware Street SE
                                        Minneapolis, MN 55455 USA
                                                                                     Moji Gesinde
John A.J. Barbara                                                                    National Blood Service
National Blood Service                  Geoff Daniels                                Leeds Blood Centre
North London                            Bristol Institute for Transfusion Sciences   Bridle Path
Colindale Avenue                        National Blood Service                       Leeds
Colindale                               Southmead Road                               LS15 7TW
London, NW9 5BG                         Bristol
                                        BS10 5ND
                                                                                     Paul Hébert
Imelda Bates                                                                         Department of Medicine
Liverpool School of Tropical Medicine   Walter Sunny Dzik                            The Ottawa Hospital/General Campus
Pembroke Place                          Massachusetts General Hospital               501 Smyth Road
Liverpool                               Blood Transfusion Service, J-224             Room 1812H, Box 201
L3 5QA                                  55 Fruit Street                              Ottawa, ON K1H 8L6 Canada
                                        Boston, MA 02446
                                        USA
Susan Brunskill                                                                      Patricia E. Hewitt
National Blood Service                                                               National Blood Service
John Radcliffe Hospital                 Deirdre Fehily                               North London
Headley Way                             National Transplant Centre                   Colindale Avenue
Headington                              Via Giano della Bella                        Colindale
Oxford, OX3 9BQ                         00161 Rome                                   London, NW9 5BG
                                        Italy

Elizabeth Caffrey                                                                    Beverly J. Hunt
National Blood Service                                                               Department of Haematology &
University of Cambridge                                                                Rheumatology
Long Road                                                                            Guy’s and St Thomas’ Foundation Trust
Cambridge                                                                            Lambeth Palace Road
CB2 2PT                                                                              London
                                                                                     SE1 7EH




                                                                                                                             vii
List of Contributors

Christopher J. Hyde                        David H. McKenna                              David J. Roberts
National Blood Service                     Clinical Cell Therapy Laboratory              National Blood Service
John Radcliffe Hospital                    University of Minnesota Medical School        John Radcliffe Hospital
Headley Way                                1900 Fitch Avenue                             Headley Way
Headington                                 St Paul, MN 55108                             Headington
Oxford, OX3 9BQ                            USA                                           Oxford, OX3 9BQ


Joanne E. Joseph                           Sheila MacLennan                              Irene A.G. Roberts
Department of Haematology and Stem Cell    National Blood Service                        Department of Haematology
   Transplantation                         Leeds Blood Centre                            Commonwealth Building, 4th Floor
St Vincent’s Hospital                      Bridle Path                                   Imperial College Hammersmith Campus
Victoria Street                            Leeds                                         Du Cane Road
Darlinghurst, NSW 2010                     LS15 7TW                                      London, W12 0NN
Australia

                                           Michael F. Murphy                             Marion Scott
Alan D. Kitchen                            National Blood Service                        Bristol Institute for Transfusion Sciences
National Blood Service                     John Radcliffe Hospital                       National Blood Service
North London                               Headley Way                                   Southmead Road
Colindale Avenue                           Headington                                    Bristol
Colindale                                  Oxford, OX3 9BQ                               BS10 5ND
London, NW9 5BG

                                           Cristina V. Navarrete                         Simon J. Stanworth
Susan Knowles                              National Blood Service                        National Blood Service
Department of Haematology                  North London                                  John Radcliffe Hospital
Epson & St Helier University Hospitals     Colindale Avenue                              Headley Way
  NHS Trust                                Colindale                                     Headington
Wrythe Lane                                London, NW9 5BG                               Oxford, OX3 9BQ
Carshalton
Surrey, SM5 1AA
                                           Willem H. Ouwehand                            Colin G. Steward
                                           National Blood Service                        BMT Unit
Geoffrey F. Lucas                          University of Cambridge                       Royal Hospital for Sick Children
National Blood Service                     Long Road                                     Upper Maudlin Street
Southmead Road                             Cambridge                                     Bristol
Bristol, BS10 5ND                          CB2 2PT                                       BS2 8BJ


Samuel J Machin                            Derwood H. Pamphilon                          Dafydd Thomas
Department of Haematology                  Bristol Institute for Transfusion Sciences    Morriston Intensive Care Unit
University College London Medical School   National Blood Service                        Swansea NHS Trust
3rd Floor Cecil Fleming House              Southmead Road                                Morriston Hospital
Grafton Way                                Bristol                                       Swansea
London, WC1E 6DB                           BS10 5ND

                                                                                         Alan Tinmouth
Brian McClelland                           Geoff Poole                                   Centre for Transfusion Research
Edinburgh and SE Scotland Blood            National Blood Service                        University of Ottawa
  Transfusion Centre                       Southmead Road                                Ottawa, Ontario K1H 8L6
National Science Laboratory                Bristol                                       Canada
21 Ellen’s Glen Road                       BS10 5ND
Edinburgh, EH17 7QT

                                           Christopher V. Prowse
                                           Scottish National Blood Transfusion Service
                                           National Science Laboratory
                                           21 Ellen’s Glen Road
                                           Edinburgh
                                           EH17 7QT



viii
                                                                                             List of Contributors

Marc L. Turner                    Tim Walsh                         David Wenham
Edinburgh and SE Scotland Blood   Department of Anaesthetics        National Blood Service
  Transfusion Centre              Critical Care and Pain Medicine   North London
Royal Infirmary of Edinburgh       New Royal Infirmary of Edinburgh   Colindale Avenue
51 Little France Crescent         Little France                     Colindale
Old Dalkeith Road                 Edinburgh EH16 4SU                London, NW9 5BG
Edinburgh, EH16 4SA Scotland

                                  Ruth M. Warwick                   Lorna M. Williamson
David J. Unsworth                 National Blood Service            Division of Transfusion Medicine
National Blood Service            Tissue Services                   National Blood Service
Southmead Road                    Deansbrook Road                   University of Cambridge
Bristol                           Edgeware                          Long Road
BS10 5ND                          Middlesex, HA8 9DB                Cambridge, CB2 2PT


Tim B. Wallington
National Blood Service
Southmead Road
Bristol
BS10 5ND




                                                                                                              ix
Foreword



Although we now take blood transfusion very              facing legal proceedings for a mismatched transfu-
much for granted, and it is an integral part of clini-   sion.
cal practice, the early days of its development were        Although over the next two centuries there was
anything but smooth. Indeed, it seems likely that it     considerable progress in developing better ways of
spawned two of the earliest documented cases of          transferring blood from one individual to another,
scientific fraud and medical malpractice.                 it was the beautifully elegant studies of Karl Land-
   In 1654 a Florentine physician, Francesco Folli,      steiner, carried out just over 100 years ago, that
claimed that he had invented blood transfusion           formed the basis for modern immunohaematology
and even published a book many years later to            and the successful development of blood transfu-
illustrate the complex equipment which he had            sion. It is a remarkable fact that some of the most
used. He subsequently confessed that he had not          important discoveries that have changed medical
yet done the experiment and, as far as is known, he      practice have been based on extremely small-scale
never did! The first well-documented blood trans-         and simple experiments; the demonstration of the
fusions were carried out in 1667, in Oxford and          efficacy of penicillin required only eight mice, four
Paris. The Oxford experiments were the work of           treated and four controls. Landsteiner described
the physician, Richard Lower, who was stimulated         blood groups A, B and C (later called O), using
by the studies of the architect, astronomer and          serum and red cells from six healthy males; the
polymath Christopher Wren, who had invented a            results were confirmed with sera from 16 other
series of cannulas for injecting drugs into the veins    healthy individuals. Group AB was discovered a
of animals. Lower’s first successful transfusion was      year after Landsteiner’s classical experiment, the
from the cervical artery of one dog into the jugular     M, N and P groups were reported by Landsteiner
vein of another, previously exsanguinated. Perhaps       and Levine in 1927, and the rhesus system was
stimulated by news of the first transfusion involv-       characterized by Levine and Weiner in the early
ing a human being in Paris in the same year, two         1940s. More than 250 red cell antigens have now
years later Lower injected a small amount of sheep       been described and most belong to one of 29
blood into a mildly deranged clergyman before an         systems. Many of the genes that regulate these
admiring audience at the Royal Society. The              systems have been identified and much is known
patient survived and claimed to feel better. In the      about the structure of the blood group antigens
same year a French physician, Jean-Baptiste Denis,       and the molecular basis for their diversity. Yet
began a series of experiments in which he trans-         despite all this sophisticated knowledge, and
fused varying amounts of animal blood into               equally tantalising information from studies of
patients with mental illnesses. Things seemed to go      varying susceptibility of individuals with different
well until he gave repeated injections of the blood      blood groups to a wide variety of diseases, we still
of ‘a gentle calf’ to a lunatic; the patient had a       don’t know why we have blood groups and in
typical transfusion reaction and, although he            many cases have little understanding of their bio-
recovered temporarily, died two months later.            logical function.
Denis’s enemies persuaded the patient’s wife to             Since the Second World War blood transfusion
bring a legal action against him but, in the event,      medicine has changed dramatically and is likely to
the defence was successful in proving that the man       undergo even more dramatic developments as the
had been poisoned with arsenic by his wife!              new millennium evolves. Rapid progress towards
Readers of this book might wish to remind their          the definition of subpopulations of stem cells, an
counsels of this possibility next time they are          increasing ability to alter the properties of cell pop-


                                                                                                              xi
Foreword


ulations by recombinant DNA technology and the           which are occurring at the fringes of the speciality,
vista, if distant, of specific organ therapy based on     it focuses mainly on the problems that are encoun-
work on human embryonic stem cells all point to          tered daily in transfusion medicine. Because this
an extremely exciting future for the field. There         field abuts on almost every aspect of clinical prac-
seems little doubt that, given their expertise in han-   tice it should be of value to a wide range of clini-
dling and storing cells, blood transfusion special-      cians as well as to students and practitioners of
ists will play an increasing role in the practical       transfusion medicine. Whether by accident or
applications of these new advances in cell biology.      intent the first edition of this book appeared
Any young person who enters the field over the            exactly 100 years after Karl Landsteiner made his
next few years can be guaranteed an exciting             seminal observations. What better tribute could
future.                                                  there have been to one of the most beautifully
   The second edition of this fine book provides a        simple and clinically important experiments in the
comprehensive and practical account of modern            history of medicine, a view confirmed by the early
blood transfusion practice. While encompassing           appearance of this second edition. I wish it all the
descriptions of some of the scientific developments       success it deserves.

                                                                                            D.J. Weatherall
                                                                                          Oxford, July 2004




xii
Preface to the second edition



This second edition has become necessary because       principles of transfusion medicine, the use of trans-
of rapid changes in transfusion medicine over the      fusion in specific clinical areas, its practical aspects
last three years. The pace of change seems likely to   in blood centres and hospitals, the complications
increase with new scientific and technologi-            of transfusion and potential advances. This latter
cal developments, the challenge of ‘emerging’          section in the first edition was particularly well
pathogens, and renewed efforts to improve clinical     received, and it is expanded in the second edition
transfusion practice. In the UK, the implications      with new chapters on stem cell processing, recom-
of the probable transmission of variant                binant antibodies/proteins, transfusion in the
Creutzfeldt–Jakob disease by blood transfusion         tropics, design of clinical trials in transfusion med-
are wide-ranging across the whole transfusion          icine, and a final chapter reviewing advances since
chain from donor to patient.                           1995 and ‘horizon scanning’ about likely future
   The primary aim of the second edition remains       developments up to 2010 and beyond.
the same as the first, that is to provide a compre-        We are very grateful to the colleagues who have
hensive guide to transfusion medicine. The book        contributed to this book at a time of continuing
includes information in more depth than con-           change. Although, as with the first edition, most
tained within handbooks of transfusion medicine,       authors work in the blood services in the UK, con-
and is presented in a more concise and ‘user-          tributors for the second edition include those in
friendly’ manner than standard reference texts.        full-time clinical practice, and colleagues from
The feedback we received on the first edition from      outside the UK to provide a broader perspective.
reviews and colleagues was that this objective was     We acknowlege the contribution to Practical
achieved, and that we had provided a consistent        Transfusion Medicine of two colleagues, Cynthia
style and format throughout the book. We have          Beatty and Gail Williams, who were unable to
strived to maintain this to provide a text that will   update their chapters because of new commit-
be useful to the many clinical and scientific staff,    ments. We are grateful to Janet Birchall and Simon
both established practitioners and trainees, who       Stanworth for providing critical comments on
are involved in some aspect of transfusion medi-       several chapters, and to Helen Williams for her
cine and who require an accessible text.               invaluable assistance. We have again received enor-
   The book is again divided into five sections         mous support from our publishers, particularly
which systematically take the reader through the       Maria Khan, Rebecca Huxley and Claire Bonnett.

                                                                                          Michael Murphy
                                                                                       Derwood Pamphilon
                                                                                                    2004




                                                                                                          xiii
Preface to the first edition



Blood transfusion continues to enjoy an ever                We have endeavoured to provide information
increasing public profile. This has occurred in part      that defines practical approaches to the problems
because of the emergence of new pathogens which          that are encountered in transfusion medicine. To
have posed a significant threat to the safety of the      this end we have used a consistent format to make
blood supply, and also due to major scientific            access to information easy, irrespective of whether
developments. In the new millennium advances in          the book is read cover to cover by haematologists
technology have facilitated the provision of high-       updating or revising for exams, or used as a refer-
quality blood components and a range of sophisti-        ence book by clinical or laboratory staff faced with
cated diagnostic and specialist services within          specific problems. To facilitate this approach the
modern blood centres. There has been enormous            book is divided into five sections which systemati-
progress in transfusion medicine which has devel-        cally take the reader through the principles of
oped into a specialist area of its own in the last       transfusion medicine, the use of transfusion in spe-
decade. It now encompasses many important areas          cific clinical areas, its practical aspects in blood
of medicine including haematology, immunology,           centres and hospitals, the complications of trans-
transplantation science, microbiology, epide-            fusion and potential advances, some of which are
miology, clinical practice and research and              already with us and some of which will continue to
development.                                             impact significantly on transfusion services in the
   In this book we have aimed to provide a compre-       future.
hensive guide to transfusion medicine. This                 We are grateful to the colleagues who have con-
includes information in more depth than con-             tributed to this book at a time of rapid develop-
tained within handbooks of transfusion medicine,         ment and considerable organizational change in
but at the same time presented in a more concise         healthcare as a whole but specifically within blood
and ‘user-friendly’ manner than standard reference       services in the UK. We are indebted to Bridget
texts. Ably assisted by many expert colleagues, we       Hunt and Susan Sugden for their patience and for-
have compiled a text which should prove invalu-          bearance; without their invaluable assistance in
able to haematologists in training as well as con-       compiling the text this book would not have been
sultants in established practice. We have also           possible. We have received enormous support
aimed to provide useful information to oncolo-           from our publishers, particularly Andrew Robin-
gists, surgeons, anaesthetists and other clinicians,     son, who gave us considerable assistance at a time
nursing staff in general and specialist units and sci-   when this book was at its early conceptual stages,
entific and technical staff in haematology and            and Marcela Holmes whose wisdom and expertise
blood transfusion.                                       have been invaluable in its completion.

                                                                                          Michael Murphy
                                                                                       Derwood Pamphilon
                                                                                                    2001




                                                                                                          xv
Part 1


Basic principles of transfusion
Chapter 1
Introduction

Ian M. Franklin




Nearly 4 years have elapsed since the first edition    oped and died of vCJD in 2003, 7 years after
of this book. Has anything occurred in the world      receiving the blood. The donor was healthy at
of transfusion medicine to alter the concepts that    the time of donation in 1996, but became unwell
were important then? In the past 12 months, a         and died of vCJD in 2000. In the absence of a
number of crucial events have occurred that have      blood test for vCJD, and with no way of confirm-
again acted to increase global anxiety about the      ing that the two patients had the same or different
safety of blood transfusion. The arrival of severe    ‘strains’ of vCJD, this is not conclusive evidence
acute respiratory syndrome (SARS) and its prompt      for transmission. On the balance of probabilities,
recognition as a novel coronavirus in early 2003      however, it seems likely that the transfusion recipi-
focused attention on the difficulties of maintaining   ent acquired vCJD from the blood transfusion.
blood safety in the face of an unknown emerging       This event has triggered a further round of new
infection. In the absence of any knowledge of the     initiatives in the UK to protect blood safety and
epidemiology of the infection, it had to be assumed   retain confidence in the transfusion of blood. In
that there was the potential for SARS to be trans-    addition to leucocyte depletion of blood compon-
mitted by blood. This remains an unresolved issue     ents and importing both plasma for fractionation
that will have to await a better understanding of     and fresh frozen plasma (FFP) for those born
the virus, which perhaps may be obtained in any       after 31 December 1995, it appears likely at
new outbreak in 2004 or later. Anxieties over         present (January 2004) that the exclusion of
SARS were followed quickly by the expected US         donors who have received a transfusion in the UK
summer epidemic of West Nile virus (WNV),             since 1980 will be added to this list. Renewed
known to be a transfusion-transmitted infection,      efforts to reduce inappropriate transfusion
and for which precautions in the USA and Europe       because of concerns about the impact of this new
were urgent and needed to be robust. These            measure on the sufficiency of the blood supply is
included the use of nucleic acid testing for WNV      also probable. Perhaps the one cause for optimism
genome in all donations in the USA, Canada and        comes from the failure of a massive epidemic of
Mexico. In Europe, recent visitors to North           vCJD to develop in the UK, at least to date,
America were not accepted as donors for 4 weeks       and most estimates of the ultimate size of the
after return.                                         epidemic have been reduced considerably. This
   Most recently, after a few years in which the      makes it even more important to minimize sec-
expected major epidemic failed to materialize, the    ondary cases acquired from blood transfusion. As
possibility that variant Creutzfeldt–Jakob disease    the UK blood services prepare for additional pre-
(vCJD) may well be a transfusion-transmitted          cautions to prevent vCJD, through deferral of
infection in humans became more likely, following     transfused persons as donors, fears over ‘chicken
worrying results in sheep transfusion studies some    flu’ are beginning to dominate the headlines and
years ago. A patient, one of only 48 known to be at   once more pictures from Asia show citizens
risk through receiving a labile blood component       wearing masks as they go about their daily lives.
from a donor who later developed vCJD, devel-         Although this is currently topical, it may appear


                                                                                                         3
Chapter 1


out of date later in the year and over the next         and for providing the clear logistical base from
2–3 years.                                              which they might be organized. The early trans-
   Other crucial events have included the relentless    fusion services, certainly in the UK, were often
march of two new technologies aimed at improv-          related to military practice and modern practition-
ing blood safety. The first, nucleic acid testing        ers might be forgiven for believing that the sole
(NAT) for viral pathogens, is already established,      objectives were collection, process and supply of
although concerns over cost–benefit analyses, at         (at that time) bottled blood and plasma. There also
least where NAT is a second-line test to a highly       seems little doubt in retrospect that there was great
effective antibody detection system, may lead to        profligacy in the use of blood and in particular
review. The second, pathogen inactivation (PI) also     plasma. Some of this stemmed, no doubt, from
appeared to be heading for implementation, and          inadequacies in surgical practice and an equivalent
one system, Intercept, had been licensed for treat-     lack of understanding of blood coagulation, but
ment of plasma in the EU. However, after a few          the failure to collect even the most basic evidence
patients developed antibodies to aspects of the         of any benefits of blood or plasma transfusions has
agents of the system, a delay in further trials is      bedevilled the field ever since. Following a consis-
inevitable until the safety profile can be assessed      tent increase from the 1950s, blood usage in the
further. Another, different, PI system has devel-       USA has shown a downward trend in the past
oped similar problems with neoantigen formation.        decade from a peak in 1986, and demand has been
Although other PI systems are continuing to be          decreasing for the last 3 years in the UK. The
developed, all of these work by using a chemical        reasons for this are probably multifactorial, but
agent to prevent nucleic acid replication, and so       include improved surgical techniques as well as
each must have a potential for antigenicity that        concerns about blood safety. Despite this, there is
will require extensive study before any such system     evidence for disparities in blood usage between
could be introduced for large-scale use.                surgeons and between hospitals, for similar activi-
   The above events continue to make safety and         ties. There is also wide variation in the use of blood
supply the main priorities of blood services and        avoidance strategies such as autologous transfu-
this has changed little from where they were 5 or       sion using cell salvage and preoperative deposit.
even 20 years ago. Therefore, the four key areas        There is, in the UK, little use of preoperative clinics
considered in the previous edition still appear to be   to enable haemoglobin correction with iron, other
as relevant now as then and are listed below. The       haematinics or erythropoietin. In the UK, these
four principal areas to be considered are:              issues will be addressed over the next few years by
• blood safety;                                         ‘Better Blood Transfusion’ initiatives.
• the appropriate and effective use of blood and
blood products;
• donor recruitment and retention; and                  Blood safety
• informing patients about blood transfusion.
The opinions expressed in this introduction are         Trends in transfusion practice in the past two
those of the author alone.                              decades, since the identification of acquired
   Blood has been assumed to have mystical quali-       immunodeficiency syndrome (AIDS), have been in
ties from the early days of transfusion experiments     the general direction of enhanced safety of plasma
in the seventeenth century by Lower in England          products and cellular components, as well as
and Denis in France. A number of predictable dis-       improved purity. With pooled fractionated plasma
asters caused the subject to fall into disrepute, and   products there was a shift from low, then to inter-
progress in transfusion had to wait until there was     mediate and eventually to highly purified factor
adequate understanding of blood groups to enable        VIII, for example, which provided many benefits in
safe transfusions between individuals. The impera-      safety and specificity of treatment. Together with
tives of the Second World War were also important       the development of the necessary technology, these
in emphasizing the need for transfusion services        advances led to the realization that recombinant


4
                                                                                                      Introduction


products, ideally free of any added human or             One obstacle to early implementation was the
animal proteins such as albumin, were the ultimate       desire to have the whole of the UK introducing the
expression of the drive towards total safety and         test at the same time, so that there would be no
absolute purity.                                         difference in quality of component anywhere.
   This success in improving the safety of plasma        Although in an ideal world all parts of an individ-
products by eliminating donor-derived material           ual blood service would implement testing of a
seemed to have encouraged the view that the goal         new agent at the same time, this seems inappropri-
of zero risk from transfusion was to be required by      ate when a significant delay is introduced thereby.
regulators and governments. Although there have          It would seem to be preferable for larger blood ser-
been no specific statements to change this, a trend       vices to begin testing as soon as possible in some
seems to be emerging in favour of a ‘balance of          parts of the service, even if others are not yet ready.
risk’ approach. In the Netherlands, the health min-      At least in this way some donations would be pro-
ister has made clear that optimal, not maximal,          tected. In countries where there is no single author-
safety is the goal. Although it is not clear what this   ity managing blood services, this already happens.
means precisely, the inference is that some form         In the UK, leucocyte depletion of all labile blood
of cost–benefit judgement must be included in             components was implemented to prevent the then
the equation for achieving blood safety. The             ‘theoretical’ risk that vCJD might be transmitted
European Union (EU) Commissioner for Health              by blood transfusion. Leucocyte depletion was
and Consumer Protection, David Byrne, who has a          phased in as soon as it was possible operationally
portfolio that includes food and blood safety,           – there was no ‘big bang’ before which compon-
stated in a speech entitled ‘Irrational Fears or         ents were not leucocyte depleted and after which
Legitimate Concerns’ on 3 December 2003 that             they were. New virus or other tests and safety mea-
zero risk cannot be achieved. And in the UK, min-        sures should be managed similarly in future.
isters have begun to question how much must be              The other obstacle to new tests or safety mea-
spent on the safety of the railways before this          sures is the impact on supply, i.e. on donors. There
becomes excessive. The inference is that other           is no doubt that blood donors are the essential cor-
areas of public life must achieve a balance between      nerstones of a transfusion service. Nevertheless
delivering an effective service without crippling        patients expect and believe that the transfusions or
costs arising from chasing absolute safety.              tissues they receive will be as safe as possible, and
   Prior to these public statements, it appeared that    that those donors who may pose an additional risk
the provision of blood by national blood services        to safety should not be accepted. The range of risks
was almost unique in the political imperative that       for which donors are deferred continues to
required total safety, at whatever cost. This obses-     increase, and significant numbers are turned away
sion with reducing risks to zero led to there being a    because of recent tattoos or body piercings, or
perception that there are problems with the safety       travel to areas where there are concerns about old
of blood. Some of this came about because of later       or emerging agents, such as WNV, malaria or
criticism of earlier decisions, in particular in the     Trypanosoma cruzi exposure. Testing is becoming
UK over delays in implementing hepatitis C virus         more complex and extensive and the prospect that
(HCV) testing (discussed in detail in the first           PI might achieve the same result as more testing, in
edition). The failure to introduce the first-             a single manufacturing process, is most attractive.
generation test for HCV antibody led to a delay in       On the face of it, PI of cellular components, e.g. of
the effective testing for this known transfusion-        platelet concentrates using the psoralen S-59 and
transmitted virus, and there is no question that         ultraviolet A light, holds great promise by prevent-
some patients acquired HCV during this period.           ing virus and bacterial replication. Removing the
This delay was strongly criticized in the judgement      risk of transfusion-transmitted graft-versus-host
in the English courts by Justice Burton, who con-        disease by preventing T-cell replication would be
sidered that testing should have been introduced in      an added bonus. Unfortunately, the occurrence of
January 1991 and not September as happened.              antibodies to blood cells produced by two of these


                                                                                                                5
Chapter 1


systems may well prove a major, if not fatal, blow       between 1 in 300 000 and 1 in 2 000 000. These
to this approach for the time being.                     risks are now perceived to be politically and eco-
                                                         nomically worth preventing. Sir Kenneth Calman,
                                                         the former Chief Medical Officer of the Depart-
Regulation of blood services
                                                         ment of Health in the UK, addressed issues of risk
Blood service regulation developed following a           in a series of articles. He provided examples of
number of episodes of transfusion-transmitted            activities associated with moderate risk, such as
infections that occurred in the 1960s and 1970s. In      smoking 10 cigarettes a day (1 in 200 chance of
the USA the responsible body is the Food and Drug        death in any one year), to infinitesimal risks, such
Administration (FDA), through its Center for             as being struck by lightning. Schreiber and col-
Biologics and Research (CBER) division. In the UK        leagues, writing on behalf of the US Retrovirus
the regulator has been the Medicines Control             Epidemiology Donor Study, estimated the risk
Agency, mainly for the production of pharmaceuti-        for transfusion-transmitted virus infections at
cals from plasma, as cellular components were not        between 1 in 63 000 for hepatitis B virus (Calman
considered to meet the requirement for a ‘product’.      risk level, very low) and 1 in 493 000 for human
This latter nicety was dealt with by Justice Burton      immunodeficiency virus (HIV) (Calman risk level,
in his HCV judgement, which confirmed that                minimal) (Table 1.1). There are no equivalent
cellular blood components were indeed products.          figures for the UK, although an estimate for HIV
The Medicines Control Agency merged with the             can be made from evidence of only two known
Medical Devices Agency in 2003 to form the               transmissions of HIV since the introduction of
Medicines and Healthcare products Regulatory             testing in 1985. During that time some 30 million
Agency (MHRA). For Europe, there is an over-             donations have been transfused. In the first year of
arching medicines safety body, the European              testing for HCV RNA using PCR, only one true
Agency for the Evaluation of Medicinal Products          PCR-positive, HCV antibody-negative donation
(EMEA), but no unifying EU legislation until the         has been detected, during a period when about
EU Blood Directive entered EU law in January             3 million donations were tested. Although PCR
2003. This will be implemented by the end of 2004        testing for HCV RNA was initially introduced for
by EU member states, and requires defined stand-          testing plasma donations, it has been a mandatory
ards for all aspects of the blood supply chain. For      release criterion for cellular components since
the first time in the UK there will be a legal require-   2000, in order to remove a risk of around 1 in
ment to trace blood donations to the recipient,          2 000 000 or less. The number of NAT-positive,
which will take regulators into hospitals for the        HCV antibody-negative donations has been very
first time. There are still no legal requirements to      small since then, and the cost of each transfusion-
consider transfusion alternatives or to implement        transmitted case avoided has been immense.
optimal blood-use programmes.                               Why should such minimal or even infinitesimal
                                                         risks be unacceptable in blood transfusion? There
                                                         is no doubt that the appalling stigmatization of
Risk management
                                                         individuals that occurred during the development
Awareness of the importance of protecting                of the AIDS epidemic in the USA and Europe has
patients from potential risk following transfusion       some part to play. Descriptions of transfusion-
has taken a much higher profile recently. Ten years       transmitted infections in the media invariably use
ago there were delays in introducing tests that          words such as ‘tainted’ and ‘contaminated’ in rela-
would clearly have impacted significantly at the 1        tion to the blood supply. The invasion of the body
in 1000 or 1 in 10 000 level for HCV transmission.       by an unseen, unknown and unwelcome virus or
Now, blood services in Europe and the USA are            other agent may explain some of the psychological
implementing tests using nucleic acid amplification       revulsion. Commissioner Byrne alluded to this
by polymerase chain reaction (PCR) where it is           issue in his 3 December speech, and suggested that
possible to detect events with an incidence of           the control that individuals can exert over a risk is


6
                                                                                                                           Introduction

Table 1.1 Descriptions of risk in relation to the risk of an individual dying (D) in any one year or developing an adverse
response (A). (From Calman 1996 with permission.)

Term used     Risk range                  Example                                                                        Risk estimate

High          > 1 : 100                   (A) Transmission to susceptible household contacts of measles and chickenpox   1 : 1–1 : 2
                                          (A) Transmission of HIV from mother to child (Europe)                          1:6
                                          (A) Gastrointestinal effects of antibiotics                                    1 : 10–1 : 20
Moderate      1 : 100–1 : 1000            (D) Smoking 10 cigarettes a day                                                1 : 200
                                          (D) All natural causes, age 40                                                 1 : 850
Low           1 : 1000–1 : 10 000         (D) All kinds of violence and poisoning                                        1 : 3300
                                          (D) Influenza                                                                   1 : 5000
                                          (D) Accident on road                                                           1 : 8000
Very low      1 : 10 000–1 : 100 000      (D) Leukaemia                                                                  1 : 12 000
                                          (D) Playing soccer                                                             1 : 25 000
                                          (D) Accident at home                                                           1 : 26 000
                                          (D) Accident at work                                                           1 : 43 000
                                          (D) Homicide                                                                   1 : 100 000
Minimal       1 : 100 000–1 : 1 000 000   (D) Accident on railway                                                        1 : 500 000
                                          (A) Vaccination-associated polio                                               1 : 1 000 000
Negligible    < 1 : 1 000 000             (D) Hit by lightning                                                           1 : 10 000 000
                                          (D) Release of radiation by nuclear power station                              1 : 10 000 000




crucial in the acceptance of that risk. So accepting                 be introduced might be interesting and educational
a lift in a car enables a person to decide whether                   for all concerned.
the car seems roadworthy, the driver sober and                          Issues to be considered when deciding whether
likely to be a safe choice. Similarly perhaps with                   to implement new testing or other safety measures
issues such as home versus hospital child birth,                     for a transfusion-transmitted infection include the
where a significant minority of women choose                          following.
home delivery despite evidence of greater risk.                      1 Nature of agent being tested for, and the disease
With a blood transfusion, or a food additive, no                     it causes.
such choice is possible. The recipient or consumer                   2 Is there effective treatment?
must accept the safety of the blood or food at face                  3 How much does that treatment cost?
value. If that acceptance is later found to have led                 4 Is there perceived stigmatization or implications
to a transfusion-transmitted infection, then anger,                  for subsequent lifestyle, e.g. sexually transmissible.
compensation claims and litigation are the                           5 What compensation might be payable if no
common responses.                                                    testing is implemented?
   Unfortunately, there is no arena in which a dis-                  6 What is the potential loss of reputation to the
passionate discussion about blood safety can be                      blood service?
held. It would surely be helpful to discuss these                    7 How much does the test or intervention cost?
important issues in a forum in which patient                         8 How effective is the test or intervention at pre-
groups, transfusion professionals, clinical users of                 venting future transmission?
blood and those responsible for funding new
developments could consider the issues outside the
                                                                     vCJD precautions
blame culture that follows a perceived transfusion
problem. Involvement of ‘stakeholders’ in deciding                   The publication of the results of experiments in
the principles by which new, expensive and                           sheep which showed that vCJD could be transmit-
perhaps only moderately effective measures should                    ted by whole blood transfusion suggested that it


                                                                                                                                     7
Chapter 1


would be only a matter of time before vCJD was          in fact carry risks. A 1999 randomized trial of red
transmitted by blood transfusion between                cell transfusion thresholds in the setting of inten-
humans. Although the case reported in December          sive care suggested that less was best, and a system-
2003 remains only ‘possible’, the acquisition of        atic review of albumin use in critically ill patients
vCJD by one of only 48 patients being followed          strongly suggested an adverse outcome in those
who were known to be at transfusion risk is highly      patients who received albumin rather than crystal-
suggestive. The extension of the risk reduction         loids. The vested interests of those on either side of
measures already introduced (importation of             the albumin controversy demonstrated the diffi-
plasma from countries with no or low vCJD, uni-         culty of both collecting evidence that would be
versal leucocyte depletion of fresh components          believed universally and in the acceptance by clini-
and importation of FFP for children born after 31       cians of the possibility that they may have been
December 1995) to include the deferral of all pre-      wrong all along. It has been well known for some
viously transfused donors is imminent. This will        time that individuals who reject blood transfusion
put pressure on supplies at a time when donor           for religious reasons, such as Jehovah’s Witnesses,
attendance seems to be falling.                         can undergo open heart surgery with a reasonably
   As was the case 4 years ago, a more appropriate      high degree of safety. This in itself might suggest
move in terms of addressing concerns about safety       that for many years there has been a greatly exces-
would be a more rigorous process of thought for         sive use of blood (as perioperative red cell transfu-
each and every transfusion, especially in those         sions). This is not to say that blood transfusion has
individuals who are likely to have a long survival      not enabled new and innovative surgical proce-
after it. This would include all children and those     dures to be initiated. Blood remains essential for
adults who do not have life-threatening diseases,       many cardiac surgery operations and for liver
such as candidates for replacement hip surgery.         surgery, to cite but two, and of course many
The very large sums of money allocated to vCJD          patients with malignancy could not receive
prevention in the UK (£70–100 million per year)         chemotherapy without the use of blood compon-
might have been better spent, for example, by           ents to support them. Even in situations where
investing in an educational programme for hospi-        blood transfusion is life-saving, risks remain from
tal workers at all levels. The introduction of hospi-   errors in the transfusion process, leading to the
tal transfusion teams to ensure that patients get the   ‘wrong blood [being] given’, issues highlighted in
right blood and are not excessively or unnecessar-      the UK Serious Hazards of Transfusion (SHOT)
ily transfused would have been another approach         reports.
that should have been considered.                          In the face of an increasing body of evidence sug-
                                                        gesting that blood transfusion carries both known
                                                        and unknown risks, surely we should be seeking to
Appropriate and effective use of blood and              eliminate unnecessary transfusion. Evidence of the
blood products                                          clear benefits of red cell transfusion from good
                                                        randomized trials is lacking, although there is now
Recent studies indicate that the most important         evidence that patients with cardiac decompensa-
effect on the effective use of blood within a hospi-    tion tolerate anaemia badly and do benefit from
tal or group of hospitals seems to be its culture of    transfusion to higher haemoglobin levels. It is
transfusion. It has been known for some years that      therefore incumbent upon clinicians to think once,
blood transfusion activity is based more on local       twice and three times before transfusing patients
custom and practice than on evidence. Clear differ-     and serious consideration must be given to involv-
ences exist in transfusion practice and blood usage     ing patients in these decisions (see below). One
between individuals and between hospitals. Al-          thing that does seem clear is that now vCJD seems
though there has long been an assumption that           likely to be transmissible through blood transfu-
blood must be a good thing, recent evidence sug-        sion then there will be an interest in each and every
gests that even moderate transfusion practices may      transfusion received by a person who contracts


8
                                                                                                      Introduction


vCJD (or even tests ‘positive’ for it if and when        known to be associated with a higher risk (e.g.
there is a test). Clinicians responsible for prescrib-   transmissible infectious disease) and other tempo-
ing blood must be able to justify each transfusion.      rary reasons for deferral such as body piercing,
   The appropriate and inappropriate indications         tattooing and international travel. Technical diffi-
for transfusion are covered elsewhere in this book.      culties in the process of donation may also impair
However, the evidence continues to accumulate            the percentage of units going forward for patients,
that there are still hazards of blood transfusion        such as low volume donations, long donation time
that it is not possible to avoid, and that blood         and technical problems with leucocyte filtration,
transfusion will never be zero risk. The time is         to give some examples.
overdue for a concerted effort to reduce the use of         Where donors would find wastage unacceptable
allogeneic blood to those situations where it is         would be if they were aware that their donation
essential to saving or prolonging life, or the quality   might simply go out of date because nobody had
of life. Since the previous edition of this book, the    used it or because it had been left carelessly out of a
four UK Chief Medical Officers convened a further         blood refrigerator. Improvements in crossmatch to
seminar in September 2001 to consider the issue of       transfusion ratios are continuing all the time but
‘Better Blood Transfusion’. This was followed in         much more needs to be done because it is impera-
2002 by a further Health Service Circular (HSC) to       tive that blood is not ‘tied up’ waiting for patients
the chief executives of hospitals in the UK, setting     who are very unlikely to need it, and so unavail-
out an agenda for hospitals to follow. This second       able to those who do. In this way so-called ‘elec-
seminar was held partly because of a generally dis-      tronic crossmatching’ holds out much hope and is
appointing response to the first in 1998! Although        already implemented safely in many parts of the
the second HSC provides a clear toolkit for imple-       world. Innate conservatism and lack of investment
mentation of better transfusion practice, hospitals      seem to have inhibited its more widespread accep-
have many competing priorities, and it is still diffi-    tance. Many of these measures can be imple-
cult to maintain blood transfusion at a high             mented if only there was a sufficient will to do so.
enough level of urgency for hospitals to respond in
a consistent way. The cynic might be forgiven for
believing that only if there is a blood shortage, suf-   Donor recruitment
ficient to impact on surgical activity, will hospitals
really tackle the issues of best transfusion practice.   After the end of the Second World War there was a
                                                         strong sense of community, and in addition many
                                                         people worked in large industrial settings with a
Reducing wastage
                                                         strong sense of identity. This made blood col-
A discussion about the disparity between the             lection easy, since workplace sessions readily
demands for blood placed on transfusion services         recruited large numbers of willing donors. Gradu-
by clinicians and the true needs of the patients         ally, many of the large industries have disappeared,
being treated is beyond the scope of this chapter.       and in their place service sector jobs more widely
However, one good first step towards ensuring that        dispersed geographically have arisen. Competi-
there is always sufficient blood would be to check        tion, the changes in the place of women in society
that no blood donation is wasted. Unfortunately,         (most now work) and a perception that everyone
this is far from the case and figures of between 5        now has less free time have provided challenges to
and 40% are quoted informally for different              which blood services have had to adjust. Some-
regions, hospitals or blood groups. The loss of          times these responses have been slow. For too long
potential donations begins as soon as a prospective      the premise seemed to be that individuals would
donor arrives to offer a donation. An increasing         tolerate a wait of many hours to donate, and the
proportion of individuals who come forward               whole process was very centred towards the blood
offering themselves as donors are unsuitable for         service collection system rather than donor
reasons of low haemoglobin, lifestyle issues             requirements. Only recently has this been fully


                                                                                                                9
Chapter 1


acknowledged as inappropriate and moves                 sion as part of the consent process. In the UK the
towards donation by appointment, improving the          consent issue has been considered repeatedly over
processing of donors through the session (‘donor        the past few years and is one area where medical
flow’) and an increased emphasis on the profes-          care is lagging well behind what is likely to be con-
sionalism of donor staff have all helped to main-       sidered acceptable in the event of a legal challenge.
tain the donor base.                                    The biggest difficulty appears to be dissecting the
   It is essential that transfusion services continue   need to obtain informed consent and the resources
to make it easier and more convenient for individ-      required to provide staff with the time and exper-
uals to donate. There is now a more mature and          tise to discuss the issues. In the absence of any sig-
active relationship developing between donors and       nificant momentum to obtain consent as a matter
the blood services, and this process should con-        of good practice, perhaps concerns over shortages
tinue since it appears that donors are not solely       of blood, the need to consider alternatives, and
motivated by general altruism – a non-specific           potential litigation might encourage some form of
wish to do a good thing – but are aware of spe-         dialogue between recipient and the healthcare
cific issues. More might be done to strengthen this,     team. In an era of potential blood shortage, blood
perhaps by using advertising more targeted to pro-      conservation measures might achieve importance
viding information about the uses of blood and          and preadmission clinics, which would need to be
how it makes a specific difference, over and above       a minimum of 3 weeks prior to surgery, might be
the general exhortations such as ‘we can’t operate      one way for this to occur. Discussion of alterna-
without you’. Is there really a reduction in altruism   tives to transfusion such as correction of anaemia,
in the UK, as has been suggested? There may be a        perioperative salvage or predeposit donation all
change, particularly in young people who perhaps        need time and could be combined with a formal
appear rather more self-obsessed than previous          agreement by the patient to receive allogeneic
generations. The lack of major conflicts such as         blood if that proved necessary.
wars and other common adverse circumstances,               Certainly the current situation where most
while most welcome, tends to reduce the opportu-        patients receive little or no pretransfusion infor-
nities for building community spirit. However, on       mation or advice cannot be allowed to continue
reflection and reviewing some of the literature in       for much longer without a real risk of litigation in
the area, it is more likely that it is the change in    the future. Also, the lack of information makes it
society in terms of longer working hours and more       impossible to discover the true opinion of individ-
commitment to careers in early adulthood causing        uals about to have a transfusion, or the likely
less time to consider or attend for donation that is    interest in alternative strategies such as autolog-
important. It is up to transfusion services, the        ous transfusion or other blood conservation
healthcare industry in general and government in        approaches, or to deliver them nationally with
addition to generate and maintain interest in and       equity. At present, well-informed individuals in
awareness of the need for blood donation.               major cities probably have a chance of accessing
                                                        an autologous blood programme, but certainly not
                                                        the great majority of potential recipients. The chal-
Informing patients about blood transfusion              lenge for the transfusion services is to convince
                                                        themselves and colleagues that delivering informa-
In many countries it has been a specific require-        tion about transfusion really is an imperative.
ment that informed consent is obtained from each        What else might be done in the interim? Literature
patient prior to blood or plasma transfusion. Diffi-     for patients already exists about blood transfusion
culties in defining what constitutes informed            and its risks, but these do not always reach the
consent and what information must be imparted           parts of the health service that most need them, i.e.
are considerable. In the USA, a legal decision has      the medical and surgical wards and clinics.
meant that recipients must be given information         Perhaps, rather like package inserts for pharma-
about the alternatives to allogeneic blood transfu-     ceutical products that must contain a patient infor-


10
                                                                                                         Introduction


mation leaflet, a leaflet should be issued with each      transfer, tissue engineering and tissue and organ
unit of blood, plasma or platelets and handed to        banking. Exciting developments in virus inactiva-
the recipient. This might become tedious for blood      tion and in blood cell substitutes continue to
and marrow transplant units with recipients of          provide research opportunities at the clinical inter-
multiple transfusions but may be useful for the         face, and improving the education of donors and
majority of patients, or their relatives, who receive   patients will provide great opportunities for those
blood for major surgery as a single event.              in donor and patient care services. Transfusion
                                                        medicine will continue to be a little like walking
                                                        through a tropical rainforest, where the known
Conclusion                                              paths are clear but still require careful navigation,
                                                        and new and unseen threats may still lurk around
The past two decades have seen blood transfusion        the next corner to trap the unwary. But just as the
services in developed nations trying desperately to     rainforest contains a huge biodiversity to keep
minimize the risk of the next transfusion-transmit-     the most jaded traveller interested, so the field of
ted infection, one of which seems to appear every 5     transfusion medicine can never be anything other
years or so. Douglas Starr, in his book Blood: an       than a fascinating and rewarding area in which to
Epic History of Medicine and Commerce, spells           work.
out most forcibly the errors of omission and com-
mission made over the years. These were more
usually due to a combination of denial and naivety      Further reading
rather than gross negligence. Five years on, and it
still makes compulsory reading for anyone               Bird SM. Recipients of blood or blood products ‘at vCJD
working in a senior position in a blood service (see      risk’. Br Med J 2004; 328: 118–19.
Further reading). Attempts to educate the public        Calman KC. Cancer: science and society and the
                                                          communication of risk. Br Med J 1996; 313: 799–802.
about risk will fail as long as blood transfusion
                                                        Calman KC, Royston G. Personal paper: risk language and
mishaps are newsworthy, even where they occur             dialects. Br Med J 1997; 313: 939–42.
by chance in an otherwise effectively functioning       Chalmers I. Human albumin administration in critically ill
system. The only realistic way forward is to engage       patients. I would not want an albumin transfusion (letter,
all participants in the blood transfusion process in      comment). Br Med J 1998; 317: 885.
active discussion. The most obvious way to begin        Cochrane IGAR. Human albumin administration in
such a dialogue would be through a pretransfusion         critically ill patients: systematic review of randomised
                                                          controlled trials (see comments). Br Med J 1998; 317:
interview that would bring physician/surgeon
                                                          235–40.
together with the patient to discuss blood safety,      Goodnough LT, Shander A, Brecher MA. Lancet 2003; 361:
and as an obvious prerequisite would require the          161–9.
blood services to provide training and information      Hebert PC, Wells G, Blajchman MA et al. A multicentre,
for colleagues in hospitals. Such an innovation           randomized, controlled clinical trial of transfusion
might just pave the way for a realistic debate about      requirements in critical care. Transfusion requirements in
the wisdom of further attempts to reduce the risks        Critical Care Investigators, Canadian Critical Care Trials
                                                          Group (see comments). N Engl J Med 1999; 340:
of transmission of known viruses by blood transfu-
                                                          409–17.
sion to an unattainable singularity of zero risk.       Hunter N. Scrapie and experimental BSE in sheep. Br Med
   Much of this introduction has focused on the           Bull 2003; 66: 171–83.
problems and challenges that face blood services as     Llewelyn CA, Hewitt P, Knight RSG et al. Possible
we enter the new millennium. That there are plenty        transmission of variant Creutzfeldt–Jakob disease by
of opportunities as well as threats is certain, and       blood transfusion. Lancet 2004; 363: 417–21.
the very dependence of blood services on good           McClelland B. Albumin: don’t confuse us with the facts.
                                                          Rather than fulminating, seek to answer the questions
manufacturing practice and good laboratory prac-
                                                          raised (editorial, comment). Br Med J 1998; 317:
tice is opening doors for crucial collaborations in       829–30.
the related fields of cellular immunotherapy, gene


                                                                                                                 11
Chapter 1

Schreiber GB, Busch MP, Kleinman SH, Korelitz JJ. The risk      Division, Central Office of Information, November 1998,
  of transfusion-transmitted virus infections. N Engl J Med     Report No. SR0015.
  1996; 334: 1685–90.                                         Starr D. Blood: An Epic History of Medicine and
Serious Hazards of Transfusion. Annual Report                   Commerce. London: Little, Brown, 1999.
  2001–2002. www.shotuk.org                                   Wu YY, Snyder EL. Safety of the blood supply. Role of
Sharp Research Ltd. Altruism and Blood Donation.                pathogen reduction. Blood Reviews 2003; 17: 111–22.
  Qualitative Research Report. London: Research




12
Chapter 2
Essential immunology for transfusion medicine

Willem H. Ouwehand and Tim B. Wallington




The immune system is a sophisticated and multi-         Cellular basis of the immune response
layered defence against infection. It is based on the
recognition of non-self in any potential pathogen.      The key effector cells are T cells, B cells and
Since donor organs are not self, their transplanta-     natural killer (NK) cells. The progenitors of T
tion from one individual to another is only             cells, B cells and NK cells are derived from the
possible if the obstacles inherent in the recipient’s   same haematopoietic stem cells (HSC) that give
immune system can be overcome. The transfer of          rise to other types of blood cell. Cells of the mono-
blood components (either as therapy or during the       cyte–macrophage series, including Langerhans’
course of pregnancy) is a form of transplantation.      cells and dendritic cells, process and present
This discussion concentrates on the immunobiol-         antigen to both T and B cells. Progenitor cells
ogy of clinical problems that are encountered as        migrate from the circulation into the epithelial
a result. There are excellent texts which discuss       thymus to become T cells. There they interact with
human immunobiology in detail (see Further              the stromal cells and their soluble products to
reading).                                               undergo cell division, clonal selection and matura-
   The immune system has evolved distinct mech-         tion. In addition they acquire their antigen recep-
anisms for coping with extracellular pathogens,         tor (T-cell receptor or TCR) and other surface
such as bacteria, based on the production of anti-      molecules which will determine their function,
body and with intracellular pathogens, such as          CD8 on cytotoxic T cells, CD4 on helper T cells.
viruses, based on the activity of effector T cells.     Immature T cells initially express both CD4 and
Two essential layers of defence are utilized:           CD8 molecules, which interact respectively with
• innate immunity, which is primitive in evolu-         major histocompatibility complex (MHC) class II
tion and not single pathogen specific (e.g. man-         or class I molecules on thymic stromal cells to
nan-binding protein which binds microbial               influence their maturation into CD4 or CD8 T
cell-wall saccharides and Toll-like receptors which     cells. Through this process self-reactive T cells are
can be directly involved in macrophage activa-          removed. Later, when they migrate to the periph-
tion); and                                              ery, T cells may undergo selective clonal activation
• adaptive immunity in higher animals, which            triggered by antigen, which leads to proliferation
adding to innate mechanisms brings specificity and       and maturation. Subsequent function depends on
memory to immune responses (e.g. antibody for-          whether the cells carry the CD4 or CD8 antigens.
mation in defence against bacteria).                       B-cell development is a multifocal process that is
   Most problems encountered in transfusion             concentrated in fetal liver before bone marrow
medicine are antibody based, i.e. the humoral           becomes the major haematopoietic organ.
immune response, and this will be considered in         Progenitor cells receiving signals from local
greater detail below.                                   stromal cells begin to divide and begin the process
                                                        that will provide an antigen receptor, in this case
                                                        surface immunoglobulin (SIg). Like T cells, im-
                                                        mature B cells are easily tolerated or killed by


                                                                                                          13
Chapter 2


premature stimulation via their antigen receptors             a constant region (Fc), which allows the molecule
to prevent damage to self. After migrating from the           to bind target antigen via Fab and trigger effector
bone marrow, B cells mature, express SIg antigen              functions through the Fc portion. These molecules
receptors, and respond to antigens together with              are more generally called immunoglobulins. They
T-cell help from CD4 cells by undergoing prolifer-            also serve as antigen receptors on B cells.
ation and plasma cell differentiation.
   NK cells are non-T, non-B lymphoid cells
                                                              Antibody effector functions
capable of killing virus-infected cells either specifi-
cally targeted by the presence of antibody on their           The constant regions of the heavy (H) chain of
surface (antibody-dependent cell-mediated cyto-               immunoglobulins are responsible for the triggering
toxicity, ADCC) or through the recognition of                 of an effector pathway. This occurs either:
changes in the infected cells surface that allow NK           • by binding to appropriate Fc receptors on effec-
cell attack. This mechanism is greatly enhanced by            tor cells, such as leucocytes and mast cells; or
the cytokine interferon (IFN)-g, illustrating the fact        • by activation of the complement cascade.
that these key effector cells usually act in concert in          There are five immunoglobulin isotypes based
the defence against infection.                                on different genes for the C domains of the H chain
                                                              (Table 2.1). Immunoglobulin G (IgG) and IgA
                                                              have four and two subclasses, respectively. The
Humoral immune response                                       immunoglobulin isotypes and subtypes differ sig-
                                                              nificantly in their ability to recruit effector func-
Antibody
                                                              tions (see Table 2.1). This is of clinical significance
The specific effector molecule is an antibody which            in transfusion as the ability of antibodies to bring
is secreted into the extracellular space from plasma          about erythrocyte or platelet destruction varies
cells. It is a unique tetramer made up of two identi-         according to their isotype and IgG subclass.
cal heavy and two identical light chains (Fig. 2.1).
These combine variability of amino acid sequence
                                                              Basis of antibody variability
and thus variability of tertiary structure (Fab) with
                                                              The molecular biology of this is complex. The
                                                   N
                                        VH                    genes for the five heavy chains and the l and k light
                                                   terminal
                                                              chains are found on separate chromosomes, at
                                                              band 14q32, 22q11 and 2p11, respectively. Each
                  Hinge region   CH1                          chain is separately synthesized before being assem-
                                                   VL         bled into an antibody molecule. On chromosome
     CH3            CH2
                                                              14, which carries the H-chain genes, there are
       S
                                             CL               three clusters: approximately 50 variable (V)
C terminal                   S
                                                              region genes, which encode the first 95 amino
                                                              acids of the V portion, more than 20 diversity (D)
                                                   VL
                                                              region genes and six joining (J) region genes.
             Fc
                                                              Together these genes encode for H-chain V
                                                              regions. Like letters of the alphabet they can be
                                                              joined at random into three-letter ‘words’, thus
                                        VH                    providing much variability in the receptor portion
                                  Fab
                                                              of the H chain. Similarly, 22q11 and 2p11 have
                                                              two clusters of genes for the V portion of the k and
Fig. 2.1 Basic structure of an immunoglobulin molecule.
                                                              l light chains, respectively, which can recombine
Domains are held in shape by disulphide bonds, though
only one is shown. CH1–3, constant domain of an H chain;      in this way. The incredible diversity of antibody
CL, constant domain of a light chain; VH, variable domain     specificity, found even at the level of the germline,
of an H chain; VL, variable domain of a light chain.          is the result of these events, coming together in


14
                                                                                                                                        Essential immunology

Table 2.1 Immunoglobulin classes and their functions.

                  Structure                                     Function

                                                                                              Complement                Reaction with              Placental
Isotype           Heavy chain            Light chain            Configuration*                 fixation†                  FcR                        passage

IgM               m                      k, l                   Pentamer                      +++                       L                          –
IgG1              g1                     k, l                   Monomer                       +++                       M, N, P, L, E              ++
IgG2              g2                     k, l                   Monomer                       +                         P, L                       +/–
IgG3              g3                     k, l                   Monomer                       +++                       M, N, P, L, E              ++
IgG4              g4                     k, l                   Monomer                       –                         N, L, P                    +
IgA1              a1                     k, l                   Monomer                       –                         —                          –
IgA2              a2                     k, l                   Dimer in secretion            –                         —                          –
IgD               d                      k, l                   Monomer                       –                         —                          –
IgE               e                      k, l                   Monomer                       –                         B, E, L                    –

* Pentamer, five basic tetrameric units (in vitro good agglutination); dimer, two basic units; monomer, one basic unit.Two or more basic units are held
together by a J chain.
† Classical pathway.
B, basophils/mast cells; E, eosinophils; L, lymphocytes; M, macrophages; N, neutrophils; P, platelets.
                                                                 me
                                                                so




                                                                            'Silent' area = intron
                                                            mo
                                                           ro
                                                         Ch




                                                                     (V)n     D       J     Cm         Cd Cg3 Cg1 Ca1 Cg2 Cg4 Ce Ca2
                                                       14 H

                                                                                                     The product is VHCm, i.e. an IgM heavy
                                                                              VDJCm                  chain with a particular variable region        Final
                                                                     (V)n      J      Ck                                                            product
                                                                                                                                                    IgMk
                                                         2 k                                                                                        or
                                                                                                                                                    IgMl
                                                                            VJCk
                                                                     (V)n      J      Cl
                                                       22 l

Fig. 2.2 Genes encoding antibodies (see
text for explanation).                                                      VJCl


the tertiary structure of the Fab2 portion of the                               • There is switching in the immunoglobulin
immunoglobulin molecule (Fig. 2.2). The variabil-                               isotype mix to molecules that are more effective in
ity of the receptor for antigen on T lymphocytes                                neutralizing the wide variety of pathogen types
(TCR) is the product of similar mechanisms.                                     that may be encountered; this process is controlled
                                                                                and driven by helper T lymphocytes.
                                                                                • Mutation at hotspots where the V, D and J genes
Somatic mutation
                                                                                join and similarly within the V portion of light
Antibody function is further refined in the sequen-                              chains, which refine the shape of the receptor
tial production of immunoglobulin isotypes as the                               area and thus the specificity of the antibodies
adaptive immune response matures, as follows.                                   produced.


                                                                                                                                                         15
Chapter 2


Blood cell antibodies illustrating the                Antigen in the adaptive humoral immune response
above principles
                                                      The immune response is driven by antigens which
These mechanisms at work are well illustrated by      select the lymphocytes that are able to participate.
the behaviour of commonly found blood cell anti-      Therefore selective use of V genes in antibody
bodies. In a typical T-cell-dependent antiprotein     production against a certain antigen might be
(e.g. anti-D) immune response, the switching of the   expected. Studies on the V-gene use of blood cell
immunoglobulin isotype from IgM to IgG is asso-       antibodies support this and have thrown light on
ciated with an increase in the affinity of the anti-   certain serological anomalies. Most evidence has
body. In the early phase of the response the V        been acquired by studies on the molecular struc-
region is encoded by genes from the germline and      ture of the V domains of monoclonal antibodies
antibody affinity is low. As a result, in the early    against the carbohydrate antigen I and the protein
phase of an anti-D response, a panreactive anti-      antigen RhD on the red cell membrane. These
body, reactive with several blood groups, might       studies suggest that:
be detected in panel studies. This reaction is        • there is preferential but not exclusive use
most likely caused by low-affinity IgM antibodies      of certain V genes in the generation of these
which, although responding to RhD, are able, par-     specificities;
ticularly at 4°C, to react with other blood groups    • there is a significant overlap in the amino acid
because of an avidity effect. Maturation of the       sequence of the V domains of cold agglutinins
immune response with the selection of B and T         against the lactosylceramide I and anti-D
cells with greater specificity for RhD results in      antibodies;
improved antibody affinity and the disappearance       • pathological anti-I cold agglutinins, as observed
of cross-reactivity. This is the consequence of       in the majority of patients with cold haemagglu-
somatic mutation of the rearranged V gene. In         tinin disease, uniquely use the VH gene segment
high-affinity anti-D antibodies, up to 20 of the 90    DP63 (V-4.34);
codons encoding the V region have changed from        • postinfectious polyclonal anti-I antibodies seem
germline.                                             to make use of the same VH gene segment, while
   Temperature      dependency      of    antibody–   cold agglutinins with other specificities do not; and
erythrocyte interactions is used as a surrogate       • in over 50% of monoclonal IgM anti-D anti-
marker for antibody affinity.                          bodies the VH domain is encoded by the DP63 VH
• Low-affinity antibodies generally do not bind        gene, the same as that encountered in pathological
sufficiently strongly at 37°C to be detected by        anti-I cold agglutinins.
agglutination but they do at lower temperatures.         It is attractive to speculate that RhD-specific B
They are also generally of no clinical significance.   cells evolve from B cells with anti-I specificity. This
• Antibodies of intermediate and high affinity do      suggests that in the germline these cells provide an
remain bound and are detected in the antiglobulin     SIg receptor which is best fit at that stage in the
test.                                                 immune response for the tertiary structure pre-
   Pretreatment of red cells with proteases is also   sented by D. In this scenario the drift in anti-
an effective method to reveal the presence of low-    body specificity from anticarbohydrate (anti-I) to
affinity antibodies against, for example, RhD as       antiprotein (anti-D) is best explained by minor
this reduces the strength of interaction between      changes in the amino acid sequence of the V
antibody and cell required for agglutination.         domains of anti-I antibodies brought about by
Other circumstances can also favour the binding of    somatic mutation. Ultimately the low-affinity
low-affinity antibodies. Some of the isotypes able     binding for I is lost. This is also influenced by the
to activate complement are detected in haemolysin     switch from IgM to IgG. In serology these struc-
tests if the antibody is present in excess and the    tural observations are supported by the functional
activation of complement is facilitated by lowering   observations on low-affinity interactions men-
the pH of the reaction medium.                        tioned earlier and by the fact that certain IgM


16
                                                                                                Essential immunology


monoclonal antibodies used for D typing show              cells carrying these antigens. The response is
reactivity at 4°C with protease-treated RhD-              essentially limited to IgM because T cells are not
negative red cells.                                       involved and are not available to induce isotype
   The isotype and subclass of blood cell antibodies      switching, although there is evidence that some
are at least partly determined by the chemical            switching can occur in the absence of T cells. The
nature of the antigen which had stimulated their          repetitive carbohydrate structures (epitopes) pre-
production.                                               sented by the A- and B-determining portions of the
• Blood cell antibodies against carbohydrate              relevant cell surface glycolipids and glycoproteins
antigens are generally IgM or IgG2 and IgG4 or a          are structurally the same as bacterial polysaccha-
combination of these.                                     ride and indeed it is antigens from gastrointestinal
• Antibodies against protein blood group antigens         bacteria that trigger isoagglutinin production. The
are typically of the IgG class, predominantly IgG1        isoagglutinins are present from the first months of
and IgG3, although autoantibodies can be of the           life. IgG anti-A or anti-B antibodies can also be
IgA type. This suggests a direct involvement of the       formed to this stimulus, usually of the IgG2 or
antigen.                                                  IgG4 type.
   The source of antigen might not of course be red
cells if the same structure is shared, so-called cross-
                                                          T-cell-dependent antibody formation
reactivity. An increase in the titre of anti-I anti-
bodies occurs after infection with Mycoplasma             Unlike glycolipids and glycoproteins, the forma-
pneumoniae. Some preparations of the vaccine              tion of antibodies against blood cell membrane
TAB (typhoid, paratyphoid A and paratyphoid B)            proteins is always dependent on interaction with
stimulate anti-A and anti-B and cause not only a          T-helper cells. The immune response to RhD is an
rise in agglutinin titre but also the development of      example.
immune characteristics. Many of the low-affinity           • RhD is the most immunogenic red cell mem-
reactions seen in red cell serology reflect part of the    brane protein antigen. RhD is a 30-kDa non-
response to bacterial antigens, usually carbohy-          glycosylated membrane protein.
drate. When the affinity and concentration of such         • Analysis suggests that only short peptide loops,
antibodies increases above certain thresholds com-        part of the molecule, are displayed on the cell
plement-mediated haemolysis can occur, making             surface.
the phenomenon of clinical significance.                   • Ample evidence indicates that anti-D antibodies
                                                          recognize discontinuous amino acid sequences
                                                          derived from several of the extracellular RhD
T-cell-independent antibody formation
                                                          loops.
As we have seen, the formation of antibodies by           • These discontinuous residues come together in
B cells is dependent on interaction with helper           the tertiary structure of the RhD protein. There-
T cells. However, some antigens can stimulate             fore isolation of RhD from the membrane disrupts
certain subsets of B cells (B1 and marginal zone B        the majority of B-cell epitopes and reactivity with
cells) directly, independent of T-cell help. These        anti-D antibodies.
cells provide a first line of defence to bacteria by       This is not a repetitive structure as with ABO and
producing antibody specific to bacterial polysac-          the B-cell response requires T-cell help.
charide. This route to antibody production is also           The response of T-helper cells, like B cells, is
important in the response to certain red cell anti-       antigen specific but triggered in a totally different
gens. The presence of naturally occurring IgM             way. T-helper cells recognize short linear segments
antibodies against A and B is an excellent example        of amino acids derived by intracellular digestion
of T-cell-independent antibody formation. In the          from the RhD protein and presented to the helper
presence of an intact immune system isoagglu-             T cell by the human leucocyte antigen (HLA) class
tinins to the missing A or B antigens are always          II molecule, which is a cell surface molecule. This is
found, although there has been no exposure to red         achieved most effectively by professional antigen-


                                                                                                                17
Chapter 2


presenting cells (APCs). APCs belong to a family       cells will drive the expansion of HPA-1a-specific B
of cells with diverse anatomical locations and of      cells responding to intact HPA-1. With time the
diverse ontogeny:                                      process of antigen take-up, processing and presen-
• Langerhans’ cells in the skin;                       tation will pass from the classical APCs to HPA-
• interdigitating, follicular and germinal centre      1a-specific B cells. This helps to bring together the
dendritic cells in lymph nodes and spleen; and         complex interaction of cells needed for a mature
• B cells and macrophages.                             antibody response, as both the surface molecules
   The antibody response to a red cell antigen like    needed for antigen presentation to helper T cells
RhD involves the collaboration of at least three       and for interaction with intact HPA-1 are present
cell types and of antigen in two forms, both intact    on the same cell set.
and processed. This enables two important fea-
tures of the immune response:
                                                       HLA class II restriction of antibody response
• an efficient mechanism for tolerance to self
antigens enables prevention of the failure to dis-     We have seen that there must be a genetic element
criminate self from non-self that is the basis of      to an individual’s immune response in that the
autoimmunity; and                                      first encounter with antigen is dependent on the
• the maturation of the antibody response,             germline V-region genes, which show differences
through isotype switching, is facilitated.             between individuals. Whether or not processed
This is illustrated in detail by the specific example   peptide from a particular alloantigen can interact
from transfusion medicine described below.             with a particular HLA class II molecule to trigger
                                                       a T cell is also dependent on genetic variability.
                                                       In the immune response this is important to the
Human platelet antigen-1a presentation via the
                                                       immunogenicity of antigens in individuals.
HLA class II route
                                                       Sometimes the peptide is presented exclusively by
If a human platelet antigen (HPA-1a)-negative          a certain HLA class II molecule and a linkage
mother is carrying an HPA-1a-positive fetus,           between HLA class II type and antibody response
platelets may enter the maternal circulation and       can be observed. The HLA DRB3*0101 restricted
immunize her against HPA-1a. This is the end           response against HPA-1a (GPIIIa, leucine 33) is the
result of quite complex events and can have disas-     best example of an HLA class II restricted response
trous consequences for the fetus. Fetal HPA-1a         in humans. The very much lower immunogenicity
may be ingested by maternal APCs and degraded          of the antithetical antigen HPA-1b (HPA-1b,
by endosomal enzymes like cathepsin G. Short           proline 33) is most likely explained by a less good
fragments of 12–15 amino acids will be produced        fit of the peptide containing the proline at position
in endosomes, which in the trans-Golgi network         33 when compared with the one containing leucine
fuse with HLA class II-containing vesicles. The        at that position.
fusion results in a downwards pH shift, which
results in the removal of the invariant chain from
                                                       Antibody-mediated blood cell destruction
the HLA class II molecule (this chain prevents the
premature loading of the cleft in the molecule that    Most red blood cell alloantibodies and autoanti-
is used for presentation of the digested antigen). A   bodies of the IgG isotype bring about destruction
plethora of peptides will be bound in the HLA          in the extravascular compartment via the interac-
class II groove, of which some will have been          tion of the IgG constant domain with Fcg receptors
derived from the fetal GPIIIa. Once migrated to        on cells of the mononuclear phagocytic system.
the surface of the APC, specific helper T cells then    Several receptor types are described.
recognize the change in the HLA class II molecule      • FcgRI is the most important in blood cell
produced by the peptides to which they are specific     destruction. This is a relatively high-affinity recep-
and T-cell proliferation will ensue. Cytokines pro-    tor found predominantly on monocytes. The con-
duced by expanding clones of reactive T-helper         sequence of adherence of IgG-coated red cells to


18
                                                                                             Essential immunology


FcgRI-positive cells is phagocytosis and lysis. This    often of low affinity and do not bind to red cells at
is usually extravascular and takes place in the         37°C. However, when ABO blood group anti-
spleen. The lysis can be demonstrated in vitro as       bodies do bind at 37°C, there will be rapid comple-
ADCC.                                                   mant-mediated destruction of incompatible red
• FcgRII is a lower affinity receptor found on           cells where there is a major A to O or B to O mis-
monocytes, neutrophils, eosinophils, platelets and      match. This may result from a transfusion error,
B cells.                                                and remains an important cause of transfusion-
• FcgRIII is also relatively low affinity and found      related mortality and morbidity.
on macrophages, neutrophils, eosinophils and NK            Blood cell antibodies that can activate comple-
cells. It is responsible for the ADCC demonstrable      ment are more effective in achieving cell destruc-
in vitro with NK cells.                                 tion than non-complement-activating antibodies.
• There is also an FcgR on the placenta of a differ-    In contrast to extravascular FcgR-mediated
ent molecular family which mediates the transfer        destruction, complement-mediated lysis occurs
of IgG into the fetus.                                  in the intravascular compartment. The ensuing
   The severity of red blood cell sequestration by      release of anaphylatoxins such as C3a and C5a
IgG antibodies is determined by the concentration       contributes to the acute systemic effects that occur.
of antibody, its affinity for the antigen, antigen       IgM, IgG1 and IgG3 antibodies are the most effec-
density and the IgG subclass. IgG2 and IgG4 anti-       tive isotypes in binding C1q and initiating activa-
bodies are generally unable to reduce red cell sur-     tion of the complement cascade via the classical
vival, while IgG1 and IgG3 are. There is ample          pathway. However, they are dependent on aggre-
evidence in patients with warm-type autoimmune          gation for a sufficiently high antibody density to
haemolytic anaemia that IgG1 and IgG3 are more          trigger C1q and overcome the regulators of com-
effective in causing red cell destruction than IgG2     plement activation that are present. The concen-
and IgG4. The level of IgG1 coating of red cells        tration of antibody may be too low to achieve the
needs to exceed a threshold of approximately 1000       density necessary. The antigen topography (e.g. of
molecules per red cell to cause cell destruction. For   RhD) can prevent the binding and activation of the
a long time it has been speculated that polymor-        C1q molecule.
phisms in the genes of the family of Fcg receptors         Complement is a complex system of plasma
might be significant in causing differences of sever-    proteins, both part of innate immunity and vital
ity of blood cell destruction observed between          to the effector functions of complement-fixing
patients with apparently similar levels of IgG          immunoglobulin isotypes. Central to comple-
coating. So far, firm evidence for such polymor-         ment’s function is the activation of C3 as this
phisms has been lacking, although a single amino        leads to the opsonization of bacteria (Fig. 2.3). C3
acid polymorphism of the FcgRIIa receptor dra-          can be activated by three routes: the classical
matically alters the affinity for human IgG2 and         pathway, the alternate pathway and lectin binding.
additional polymorphisms might have an effect on        Lysis is dependent on activation, downstream
the interaction with IgG1 and IgG3.                     from C3, of components of the membrane attack
                                                        pathway.
                                                           The classical complement pathway consists of:
Complement system                                       • four numbered components (C1–C4); and
                                                        • two regulatory proteins (C1 inhibitor, C4-
The complement system, either working alone or          binding protein).
in concert with antibody, is important for effective    The first component (C1) comprises three subcom-
immunity to many extracellular pathogens. It also       ponents, C1q, C1r and C1s. It is the interaction
often plays an important part in immune red cell        between C1q and aggregated IgG or IgM bound to
destruction and can be the reason for important         antigen that initiates activation of the classical
systemic complications of haemolysis. Naturally         complement sequence. The fixation of C1q acti-
occurring IgM antibodies against A and B are            vates C1r and C1s. C1s cleaves C4 and C2, whose


                                                                                                             19
Chapter 2


               Classical                             Alternate

                  Clqrs                               C3b
                           MBL - MASP
                                                 +
               C4b    C2b                                    Factor D
                                                             Factor B
                           C3
Opsonization    C3b4b2b                              C3bBb

                     Properdin                               Mediators of
                                                             inflammation
  Key enzymes                             C5
  C3 Convertases                                                        Ba
   C4b2a—classical                        C6
   C3bBb—alternate                                                C2 Kinin
                                  Lysis




                                          C7
  C5 Convertases                                                        C3a
   C3b4b2a—classical                      C8
                                                                        C5a     Fig. 2.3 The different pathways for
   C3bBbP—alternate
                                                                                complement activation. MBL, mannan-
                                          C9
                                                                    C567        binding lectin; MASP, MBL-associated
                                   Final lytic                                  serine protease.



active fragments C4b and C2a form the classical                  C5 convertase, which cleaves C5 to two fragments
pathway C3 convertase.                                           C5a and C5b. The former is a potent anaphyla-
   The alternative pathway to C3 activation con-                 toxin. C5b forms a complex with C6, C7 and C8,
sists of:                                                        which facilitates the insertion of a number of C9
• C3b, factor B and factor D; and                                molecules in the membrane. The C5b-8 and the
• the regulatory proteins, properdin and factors H               multimeric C9 molecules form the membrane
and I.                                                           attack complex (MAC), creating a lytic pore in the
Factor B binds to a cleavage fragment of C3, C3b,                membrane and lysing the target cell. Cells not
to form C3bB. Factor D cleaves the bound factor B                immediately involved in the process but close to it
to form the alternative pathway C3 convertase                    can also be lysed by seeded MAC, the so-called
(C3bBb). It activates C3 in a fashion similar to the             bystander lysis.
C3 convertase of the classical pathway, C4b2a.                      Blood cells coated with C3b will bind to cells
Properdin acts to stabilize this alternative pathway             carrying receptors for C3b (CR1 or CD35). This
C3 convertase, as do carbohydrate-rich cell sur-                 adherence can lead to extravascular cell destruc-
faces, by partially shielding the convertase from                tion, mainly in the liver, but if the bound C3b
inhibitors. Activation via the alternative pathway               degrades to its inactive components iC3b and
would otherwise be unchecked if it were not                      C3dg before the cell is lysed then the cell is pro-
inhibited, as it requires no specific stimulus.                   tected from lysis. Membrane-bound molecules
   The lectin pathway is initiated by mannan-                    such as decay accelerating factor (DAF) and mem-
binding lectin. This is structurally related to C1q              brane inhibitor of reactive lysis (MIRL) protect red
and binds avidly to carbohydrate on the surface of               cells from lysis in this way. They are of clinical
microorganisms. It activates C4 through a serine                 importance as:
protease, which is similar to C1r and C1s with the               • DAF (CD55) and MIRL (CD59) are linked to
same outcome.                                                    the blood cell membrane via a glycosylphos-
   The attack pathway is dependent on the forma-                 phatidylinositol (GPI) anchor;
tion of the trimolecular complex of C4b2a3b or                   • patients with paroxysmal nocturnal haemoglo-


20
                                                                                               Essential immunology


binuria (PNH) have an acquired mutation in the           crucial role for helper T cells. It is dependent on the
PIG-A gene in a subset of their HSC which pre-           secretion of the cytokines interleukin (IL)-1, IL-2,
vents synthesis of the anchor;                           tumour necrosis factor (TNF) and IFN-g, the so-
• progeny from the affected stem cells lack GPI-         called proinflammatory or Th1-type cytokines.
linked membrane proteins; and                            These recruit inflammatory cells, in particular
• the absence of DAF and MIRL from red                   macrophages, to sites of infection and arm them to
cells increases the sensitivity for complement-          kill certain bacteria, e.g. Mycobacterium tubercu-
mediated lysis which occurs when the pH is               losis, which normally proliferates inside cells and
marginally lowered during sleep, resulting in            is resistant to killing after phagocytosis. Helper
haemoglobinuria.                                         T cells also function in a Th2 manner, releasing
   In vitro acidification is used in Ham’s acid test to   cytokines that promote antibody formation
reveal the presence of a population of erythrocytes      including production of IgE, which is important
with increased sensitivity for complement-               in protection against parasites. The core Th2
mediated lysis. Flow cytometric analysis looking         cytokine profile is IL-4, IL-5, IL-10 and IL-13.
for the absence of GPI-linked proteins on a subset
of leucocytes derived from mutated stem cells is an
                                                         Cell-mediated immunity in transfusion medicine
alternative test for the diagnosis of PNH.
                                                         Cell-mediated immunity is of much less impor-
                                                         tance to the transfusion of blood cells than
Cell-mediated immunity                                   humoral immunity. It is important in the defence
                                                         against blood-borne virus (discussed below). Th1-
Antigen-specific cell-mediated responses are              type cytokines released from leucocytes in stored
carried out by T cells. They provide the immune          blood are the main cause of non-allergic febrile
system’s main defence against intracellular micro-       transfusion reactions in susceptible individuals.
organisms and can lyse cells expressing specific             As we have seen, virally infected target cells are
antigens (i.e. cytotoxicity). In addition they release   marked for recognition by cytotoxic T cells by the
cytokines that can trigger inflammation and are           presence of oligopeptides derived from viral pro-
responsible for delayed hypersensitivity and symp-       teins in the cleft of the HLA class I molecule. This
toms usually associated with infection such as           is a process analogous to that which occurs for
fever, myalgia and fatigue.                              antigen presentation on class II HLA by APCs.
                                                         The cytotoxic T-cell response to this antigen is
                                                         very intense. Recent studies with cytomegalovirus
Cytotoxic T cells
                                                         (CMV)-derived peptides captured in HLA class I
Cytotoxicity is the job of cytotoxic T cells, which      tetramers have revealed that up to 8% of CD8+ T
are distinguished by the presence of CD8 on the          cells are CMV specific during CMV infection and
cell surface. This facilitates their interaction with    similar results have been obtained with peptides
HLA class I on the surface of cells altered by the       derived from other viruses. Biologically, the
presence of antigen, usually as the result of virus      antibody response can afford to lag behind this
infection. Cytotoxic T cells are the main means of       response and usually does. This is of importance to
protection against virus infection. They are also        transfusion practice.
important mediators of allograft rejection. Like            Prevention of the transmission of hepatitis B,
the B-cell response, the cytotoxic T-cell response       hepatitis C, human immunodeficiency virus (HIV)
requires help from T-helper cells and is regulated       types 1 and 2, and CMV by blood transfusion is
by these cells.                                          one of the major challenges of transfusion medi-
                                                         cine. Counselling of donors, together with the
                                                         detection of viral antigens and antibodies and the
Delayed hypersensitivity
                                                         development of tests for virally derived nucleic
Delayed hypersensitivity is an example of another        acid, is the bedrock for the prevention of viral


                                                                                                               21
Chapter 2


transmission. Antibody-based immunoassays are               Antigen presentation and its
the mainstay but after a first encounter with a virus        clinical implications
the formation of viral antibodies will require time;
they are not the first line of defence or even the           The interaction between APCs and helper T cells is
means to recovery from infection for many of the            complex (Fig. 2.4). We now understand which of
pathogens concerned but the basis of immunity               the many interactions are prominent in turning
against subsequent infection. There is a critical           on T-cell activity. This knowledge may provide
window, which may be several months, in which               the means of specific manipulation of immune
the donor carries the virus but is still antibody           responses.
negative.                                                      The requirement is for concurrent signalling
   The sequence of immunodominant oligopep-                 over two independent pathways, one antigen
tides which appear in the HLA class I molecule for          specific via TCR and HLA/peptide and the other
all four main blood-borne viruses and several               non-specific, the CD28/CD80 pathway being
others of clinical significance has been defined and          prominent. This interaction also provides control
applied to treatment. Such short oligopeptides can          to prevent inappropriate T-cell proliferation.
be used to load APCs for in vitro education and             Initial triggering via CD28 results in T-cell pro-
proliferation of virus-specific cytotoxic T cells.           liferation and IL-2 production, which in turn
These educated T cells can be clinically used for the       induces the expression of cytotoxic T-lymphocyte
prevention of viral infection in immunosuppressed           antigen (CTLA)-4 on the expanding T-cell clone.
transplant patients.                                        CTLA-4 is a competitive inhibitor of proliferation
• Adoptive immunotherapy for prevention of                  and competes with CD28 for binding with CD80
post-transplant Epstein–Barr virus-associated lym-          and CD86. This molecular competition in the
phomas by infusion of virus-specific cytotoxic T             control of T-cell proliferation results in a balanced
lymphocytes has been successfully applied in allo-          expansion of antigen-specific T-cell clones. The
geneic bone marrow transplantation in children.             absence of initial signalling through B7 from the
• Preliminary data suggest that CMV infection in            APC when antigen is appropriately presented leads
allogeneic bone marrow transplant patients can be           to apoptosis of the helper T cell. This can be
prevented in a similar way.                                 exploited in the modulation of immune responses
   As blood services are adept at handling cells in         through molecules which block the interaction.
vitro, they are increasingly supporting the clinical        The following are examples which are of relevance
development of these novel cellular therapies,              to transfusion medicine.
which are a direct application of the underlying               Clinical studies in HSC transplantation suggest
immunobiology.                                              that donor lymphocytes can be tolerated for

            Cell membrane                                 Cell membrane



                                           TCR
                      MHC class I or II
                                           CD4 or CD8

                       (ICAM-1) CD54       (LFA-1 ) CD11a/CD18
 APC/virus
  infected                  (LFA-3) CD58   CD2                       T cell
target cell
                            (B7.1) CD80    CTLA-4

                            (B7.2) CD86    CD28

                                  CD40     CD40L
                                                                              Fig. 2.4 Adhesion molecules and
                                                                              signalling pathways in T-cell activation.


22
                                                                                                Essential immunology


incompatible HLA alloantigens on host cells by ex        effector T-cell function. Again there is the potential
vivo exposure to them in the presence of a recom-        for clinical exploitation. Anti-CD40 antibodies
binant CTLA-4–IgG fusion protein which blocks            might be powerful therapeutic reagents in kidney
B7.                                                      transplantation. HLA-incompatible transplants
  In platelet transfusion, alloimmunization to           can be achieved successfully in monkeys if pre-
HLA antigens with subsequent failure to incre-           treated with anti-CD40 antibodies. The ability of
ment after further platelet transfusions is a clinical   anti-CD40 antibodies to control autoreactive B
problem. This may be reduced by leucocyte-               cells in treatment-refractory autoimmune throm-
depleting blood components, thus removing APCs           bocytopenia looks promising. Such designer thera-
along with other white cells. A similar effect is        peutics look likely to replace less specific therapies,
obtained by treatment which modifies cell mem-            such as polyvalent intravenous immunoglobulin.
branes and particularly the B7 signal, such as              The amount of antigen required to activate B
ultraviolet irradiation.                                 cells is reduced if C3d is covalently bound to
  Conversely, immune responses can be deliber-           antigen as this leads to the concurrent signalling of
ately induced by priming separated dendritic cells       CD21 (the complement receptor type 2 on B cells)
with immunogenic peptide derived from the rele-          as well as SIg by antigen. This is also a pathway
vant antigen. This approach is under investigation       that is open to manipulation.
in cancer therapy and again blood services are
involved due to their familiarity with the safe
handling of such cells under conditions of good          Further reading
manufacturing practice.
  Similar molecular switches and controls influ-          Chapel H, Haeney M, Misbah S, Snowden N. Essentials of
ence the B-cell response to antigen. In the inter-         Clinical Immunology, 4th edn. Oxford: Blackwell
action between B cells and T-helper cells the              Science, 1999.
                                                         Janeway CA, Travers P, Walport M, Capra JD. Immuno
CD40/CD154 (CD40-ligand) pathway exercises
                                                           Biology. The Immune System in Health and Disease, 5th
control over B-cell isotype switching and subse-           edn. London: Churchill Livingstone, 2004.
quent maturation of the antibody response. This
pathway is also important in certain aspects of




                                                                                                                23
Chapter 3
Human blood group systems

Geoff Daniels




A blood group may be defined as an inherited char-     They also provide good models for contrasting
acter of the red cell surface detected by a specific   carbohydrate- and protein-based blood group
alloantibody. This definition would not receive        systems.
universal acceptance as cell surface antigens on
platelet and leucocytes might also be considered
blood groups, as might uninherited characters on      The ABO system
red cells defined by autoantibodies or xenoanti-
bodies. However, the definition is suitable for the    ABO is often referred to as a histo-blood group
purposes of this chapter.                             system because, in addition to being expressed on
   Most blood groups are organized into blood         red cells, ABO antigens are present on most tissues
group systems. Each system represents a single        and in soluble form in secretions. At its most basic
gene or a cluster of two or more closely linked       level, the ABO system consists of two antigens, A
homologous genes. Of the 285 blood group speci-       and B, indirectly encoded by two alleles, A and B,
ficities recognized by the International Society       of the ABO gene. A third allele, O, produces
for Blood Transfusion, 245 belong to one of 29        neither A nor B. These three alleles combine to
systems (Table 3.1). All these systems represent a    effect four phenotypes: A, B, AB and O (Table 3.2).
single gene, apart from Rh, Xg and Chido/
Rodgers, which have two closely linked homolo-
                                                      Clinical significance
gous genes, and MNS with three genes.
   Most blood group antigens are proteins or          Two key factors make ABO the most important
glycoproteins, with the blood group specificity        blood group system in transfusion medicine.
determined primarily by the amino acid sequence,      Firstly, almost without exception, the blood of
and most of the blood group polymorphisms result      adults contains antibodies to those ABO antigens
from single amino acid substitutions, though there    lacking from their red cells (see Table 3.2). In addi-
are many exceptions. The four types of red cell       tion to anti-A and anti-B, group O individuals
surface glycoproteins, based on their integration     have an antibody, called anti-A,B, to a determinant
into the red cell membrane, are shown in Fig. 3.1.    common to A and B. Secondly, ABO antibodies are
Some blood group antigens, including those of the     invariably IgM, though they may also have an
ABO, P, Lewis and H systems, are carbohydrate         IgG component, activate complement, and cause
structures on glycoproteins and glycolipids. These    immediate intravascular red cell destruction,
antigens are not produced directly by the genes       which can give rise to severe and often fatal
controlling their polymorphisms — the genes           haemolytic transfusion reactions (see Chapter 13).
encode transferase enzymes that catalyse the          Major ABO incompatibility (i.e. donor red cells
final stage in the synthesis of an oligosaccharide     with an ABO antigen not possessed by the recipi-
chain.                                                ent) must be avoided in transfusion and, ideally,
   The two most important blood group systems         ABO matched blood (i.e. of the same ABO group)
from the clinical point of view are ABO and Rh.       should be provided.


24
                                                                                                                      Human blood group systems

Table 3.1 Human blood group systems.

No.            Name                     Symbol                    No. of antigens               Gene name(s)                        Chromosome

001            ABO                      ABO                        4                            ABO                                  9
002            MNS                      MNS                       43                            GYPA, GYPB, GYPE                     4
003            P                        P1                         1                            P1                                  22
004            Rh                       RH                        48                            RHD, RHCE                            1
005            Lutheran                 LU                        20                            LU                                  19
006            Kell                     KEL                       25                            KEL                                  7
007            Lewis                    LE                         6                            FUT3                                19
008            Duffy                    FY                         6                            FY                                   1
009            Kidd                     JK                         3                            SLC14A1                             18
010            Diego                    DI                        21                            SLC4AE1 (AE1)                       17
011            Yt                       YT                         2                            ACHE                                 7
012            Xg                       XG                         2                            XG, MIC2                            X/Y
013            Scianna                  SC                         5                            SC                                   1
014            Dombrock                 DO                         5                            DO                                  12
015            Colton                   CO                         3                            AQP1                                 7
016            Landsteiner-Wiener       LW                         3                            LW                                  19
017            Chido/Rodgers            CH/RG                      9                            C4A, C4B                             6
018            H                        H                          1                            FUT1                                19
019            Kx                       XK                         1                            XK                                  X
020            Gerbich                  GE                         8                            GYPC                                 2
021            Cromer                   CROM                      12                            DAF                                  1
022            Knops                    KN                         8                            CR1                                  1
023            Indian                   IN                         2                            CD44                                11
024            Ok                       OK                         1                            CD147                               19
025            Raph                     RAPH                       1                            CD151                               11
026            John Milton Hagen        JMH                        1                            SEMA7A                              15
027            I                        I                          1                            GCNT2                                6
028            Globoside                GLOB                       1                            B3GALT3                              3
029            Gill                     GIL                        1                            AQP3                                 9




                                                Single-pass                                   Polytopic                      GPI-anchored
                                            Type 1     Type 2                                (multi-pass)                         Type 5
                                               N              C                                 Type 3
                                                                                                                                    N
                                                                                                                N

                                                                                                                                           C



Fig. 3.1 Diagrammatic representation                                          N                             C
                                        C                         N                                                           C
of the four types of glycoproteins
of the red cell surface membrane,       Glycophorins          Kell                  RhD, RhCcEe, RhAG               Duffy        Yt(AChE),
with examples of blood group               A to D,                                  Kidd, Diego (band 3),                     Dombrock Cromer
antigens expressed on those types      Lutheran, LW,                                   Colton (AQP1),                           (CD55) JMH
of glycoproteins. GPI,                 Knops (CD35),                                   Gill (AQP3), Kx                           (CDw108)
glycosylphosphatidylinositol.          Indian (CD44)


                                                                                                                                               25
Chapter 3

Table 3.2 ABO system.

                                            Frequency

Phenotype              Genotypes            Europeans*        Africans†            Indians‡          Antibodies present

O                      O/O                  43%               51%                  31%               Anti-A, -B, -A,B
A1                     A1/A1, A1/O, A1/A2   35%               18%                  26%               Anti-B
A2                     A2/A2, A2/O          10%                5%                   3%               Sometimes anti-A1
B                      B/B, B/O              9%               21%                  30%               Anti-A
A1B                    A1/B                  3%                2%                   9%               None
A2B                    A2/B                  1%                1%                   1%               Sometimes anti-A1

* English people.
†
  Donors from Kinshasa, Congo.
‡ Makar from Mumbai.




   ABO antibodies seldom cause haemolytic                membrane glycoproteins, mainly the anion trans-
disease of the newborn and when they do it is            porter band 3 and the glucose transporter GLUT1,
usually mild. The prime reasons for this are (i) IgM     but are also on glycosphingolipids embedded in
antibodies do not cross the placenta; (ii) IgG ABO       the membrane. The tetrasaccharides that represent
antibodies are often IgG2, which do not activate         the predominant form of A and B antigens on red
complement or facilitate phagocytosis; and (iii)         cells are shown in Fig. 3.2, together with their
ABO antigens are present on many fetal tissues           biosynthetic precursor, the H antigen, which is
and even in body fluids, so the haemolytic poten-         abundant on group O red cells. The product of the
tial of the antibody is greatly reduced.                 A allele is a glycosyltransferase that catalyses the
                                                         transfer of N-acetylgalactosamine (GalNAc) from
A and B subgroups
The A (and AB) phenotype can be subdivided into
A1 and A2 (and A1B and A2B). In a European                                                 Gal         GlcNAc c       R
population, about 80% of group A individuals are         O      (H)
A1 and 20% A2 (see Table 3.2). A1 and A2 differ
                                                                                     Fuc
quantitatively and qualitatively. A1 red cells react
more strongly with anti-A than A2 cells. In addi-
tion, A2 red cells lack a component of the A antigen
present on A1 cells and some individuals with the                                          Gal         GlcNAc         R
A2 or A2B phenotype produce an antibody, anti-A1,        A                GalNAc
which agglutinates A1 and A1B cells but not A2 or                                    Fuc
A2B cells. Anti-A1 is seldom reactive at 37°C and
generally considered clinically insignificant.
   There are numerous other ABO variants, involv-
ing weakened expression of A or B antigens (A3,                                            Gal         GlcNAc         R
Ax, Am, Ael, B3, Bx, Bm, Bel), but all are rare.         B                 Gal

                                                                                     Fuc
Biosynthesis and molecular genetics
                                                         Fig. 3.2 Tetrasaccharides representing A and B antigens,
Red cell A and B antigens are expressed predom-          and their biosynthetic precursor (H), which is abundant
inantly on oligosaccharide structures on integral        in group O. R, remainder of molecule.


26
                                                                                               Human blood group systems


a nucleotide donor substrate, UDP-GalNAc, to                    product, but with a charged arginine residue
the fucosylated galactose (Gal) residue of the H                instead of a neutral glycine (A) or alanine (B) at
antigen, the acceptor substrate. The product of the             position 268. This amino acid change at a vital
B allele catalyses the transfer of Gal from UDP-Gal             position inactivates enzyme activity. The A2 allele
to the fucosylated Gal residue of the H antigen.                has a sequence almost identical to A1, but has a
GalNAc and Gal are the immunodominant sugars                    single nucleotide deletion immediately before the
of A and B antigens, respectively. The O allele pro-            translation stop codon. The resultant frameshift
duces no transferase, so the H antigen remains                  abolishes the stop codon, so the protein product
unmodified.                                                      has an extra 21 amino acids at its C-terminus,
   The ABO gene on chromosome 9 consists of                     which reduces the efficiency of its GalNAc-
seven exons. The A1 and B alleles differ by seven               transferase activity and might alter its acceptor
nucleotides in exons 6 and 7, which encode a total              substrate specificity.
of four amino acid substitutions at positions 176,
235, 266 and 268 of their glycosyltransferase
                                                                Biochemically related blood group systems:
products (Fig. 3.3). It is primarily the amino acids
                                                                H, Lewis and I
at positions 266 and 268 that determine whether
the gene product is a GalNAc-transferase (A) or                 H antigen is the biochemical precursor of A and
Gal-transferase (B). The most common O allele                   B (see Fig. 3.2). It is synthesized by an a1,2-
(O1) has an identical sequence to A1, apart from a              fucosyltransferase, which catalyses the transfer of
single nucleotide deletion in exon 6, which shifts              fucose from its donor substrate to the terminal Gal
the reading frame and introduces a translation                  residue of its acceptor substrate. Without this
stop codon before the region of the catalytic site,             fucosylation neither A nor B antigens can be made.
so that any protein produced would be truncated                 Two genes, active in different tissues, produce
and have no enzyme activity. Another common O                   a1,2-fucosyltransferases: FUT1, active in meso-
allele, called O1v, differs from O1 by at least nine            dermally derived tissues and responsible for H
nucleotides, but has the same single nucleotide                 on red cells, and FUT2, active in endodermally
deletion as that in O1 and so cannot produce any                derived tissues and responsible for H in many
functional enzyme. O2, which represents about                   other tissues and in secretions. Homozygosity
3% of O alleles in a European population, does                  for inactivating mutations in FUT1 leads to an
not have the nucleotide deletion characteristic of              absence of H from red cells and therefore an
most O alleles and encodes a complete protein                   absence of red cell A or B, regardless of ABO geno-
                                                                type. Such mutations are rare, as are red cell
                                                                H-deficient phenotypes. In contrast, inactivating
        D                                       D               mutations in FUT2 are relatively common and
       261               526        703 796 803 1059
                                                                about 20% of Caucasians (non-secretors) lack H,
       exon 6                   exon7
                                                                A and B from body secretions despite expressing
                        176         235 266 268
                        Arg         Gly Leu Gly                 those antigens on their red cells. Very rare indi-
                                                           A1
                                                                viduals who have H-deficient red cells and are also
                         Arg        Gly Leu Gly
                                                           A2   H non-secretors (Bombay phenotype) produce
                         Gly        Ser Met Ala                 anti-H together with anti-A and -B and create a
                                                           B
                                                                severe transfusion problem.
                                                           O
                                                                   Antigens of the Lewis system are not produced
Fig. 3.3 Diagrammatic representation of exons 6 and 7 of        by erythroid cells, but become incorporated into
the ABO gene showing the position of the nucleotide
                                                                the red cell membrane from the plasma. Their
deletions (D) responsible for the common form of O (exon
6) and for A2 (exon 7), and the positions of the four
                                                                corresponding antibodies are not usually active at
nucleotide changes in exon 7 responsible for the amino acid     37°C and are not generally considered clinically
residues that are characteristic of A- and B-transferases.      significant. Lea and Leb are not the products of
Below are representations of the encoded transferases.          alleles. The Lewis gene (FUT3) product is an


                                                                                                                     27
Chapter 3


                                                                                  RHD                         RHCE
  Precursor                        Gal         GlcNAc        R           5´                   3´   3´                        5´
 of H and Lea

                                                                 exons        1             10     10                    1
                                Gal            GlcNAc        R                     D                           E/e
                                                                                                        C/c
       H
precursor of Leb             Fuc



                                Gal            GlcNAc        R
                                                                          N                      C N                     C
      Lea
                                                                 Fig. 3.5 Diagrammatic representation of the Rh genes,
                                             Fuc
                                                                 RHD and RHCE, shown in opposite orientation as they
                                                                 appear on the chromosome, and of the two Rh proteins
                                                                 in their probable membrane conformation, with 12
                                   Gal         GlcNAc        R
                                                                 membrane-spanning domains and six extracellular loops
      Leb
                                                                 expressing D, C/c and E/e antigens.
                             Fuc             Fuc

Fig. 3.4 Oligosaccharide structures representing Lea and Leb
expression and their biosynthetic precursors. R, remainder       systems, with 49 specificities. The most important
of molecule.                                                     of these is D, and then C, c, E and e.

a1,3/4-fucosyltransferase that transfers fucose to
                                                                 Rh genes and proteins
the GlcNAc residue of the secreted H precursor in
non-secretors to produce Lea and to secreted H in                The antigens of the Rh system are encoded by two
secretors to produce Leb (Fig. 3.4). Consequently,               genes, RHD and RHCE, which produce D and
H secretors are Le(a–b+) or Le(a+b+), H non-                     CcEe antigens respectively. The genes are highly
secretors are Le(a+b–) and individuals homozy-                   homologous, each consisting of 10 exons. They are
gous for FUT3 inactivating mutations (secretors or               closely linked, but in opposite orientation, on
non-secretors) are Le(a–b–).                                     chromosome 1 (Fig. 3.5). Each gene encodes a 417
   I antigen represents branched N-acetyl-                       amino acid polypeptide that differ by only 31–35
lactosamine (Galb1–4GlcNAc) structures in the                    amino acids, according to Rh genotype. The Rh
complex carbohydrates that also express H, A                     proteins are palmitoylated, but not glycosylated,
and B antigens. The I gene (GCNT2) encodes a                     and span the red cell membrane 12 times, with
branching enzyme, which only becomes active                      both termini inside the cytosol and with six exter-
during the first months of life. Consequently, red                nal loops, the potential sites of antigenic activity
cells of neonates are I-negative. Rare individuals               (see Fig. 3.5).
are homozygous for inactivating mutations in
GCNT2 and never form I on their red cells. This
                                                                 D antigen
phenotype, called adult i, is associated with pro-
duction of anti-I, which is usually only active                  The most significant Rh antigen from the clinical
below 37°C, but may occasionally be haemolytic                   point of view is D. About 85% of Caucasians are
at body temperature.                                             D+ (Rh-positive) and 15% are D– (Rh-negative).
                                                                 In Africans only about 3–5% are D– and in the Far
                                                                 East D– is rare.
The Rh system                                                       The D– phenotype is usually associated with
                                                                 absence of the whole D protein from the red cell
Rh is the most complex of the blood group                        membrane. This explains why D is so immuno-


28
                                                                                       Human blood group systems


genic, as the D antigen comprises numerous             In 1970, at the beginning of the anti-D prophylaxis
epitopes on the external domains of the D protein.     programme, there were 1.2 deaths per 1000 births
In Caucasians, the D– phenotype almost always          in England and Wales due to HDN caused by anti-
results from homozygosity for a complete deletion      D; by 1989 this figure had been reduced to 0.02.
of RHD. D+ individuals are either homozygous
or heterozygous for the presence of RHD. In
                                                       Prediction of fetal Rh genotype by
Africans, in addition to the deletion of RHD,
                                                       molecular methods
D– often results from an inactive RHD (called
RHDY) containing translation stop codons within        Knowledge of the molecular bases for D– pheno-
the reading frame. Other genes containing inacti-      type has made it possible to devise tests for pre-
vating mutations are also found in D– Africans         dicting fetal D type from fetal DNA. This is a
and in D– Asians.                                      valuable tool in assessing whether the fetus of a
  Weak forms of D (previously known as Du)             woman with anti-D is at risk from HDN. Most
result from amino acid substitutions in the            methods involve polymerase chain reaction (PCR)
membrane-spanning or cytosolic regions of the D        tests that detect the presence or absence of RHD. It
protein.                                               is important to test for more than one region of
  Red cells of some D+ individuals lack some or        RHD, so that hybrid genes responsible for partial
most of the D epitopes and, if immunized by a          D antigens do not give a false result, and to test for
complete D antigen, can make antibodies to the         RHDy, so that this does not give rise to a false-
epitopes they lack. There are numerous types of        positive result. Until recently the usual source of
these partial D antigens. They result from amino       fetal DNA has been amniocytes. These are
acid changes in the external loops of the D protein.   obtained by amniocentesis, which has an inherent
Usually this is due to one or more exons of RHD        risk of fetal loss and of fetomaternal haemorrhage.
being exchanged for the equivalent exons of            It is now possible to use sensitive PCR technology
RHCE in a process called gene conversion, but          to determine fetal D type from the small quantity
sometimes straightforward missense mutations are       of free fetal DNA present in maternal plasma, as
responsible.                                           early as 12 weeks into the pregnancy. This non-
                                                       invasive form of fetal D typing is now provided as
                                                       a reference service in a few countries.
Anti-D
Anti-D is almost never produced in D– individuals
                                                       C and c, E and e
without immunization by D+ red cells. However,
D is highly immunogenic and about 85% of D–            C/c and E/e are two pairs of allelic antigens pro-
individuals will make anti-D following infusion of     duced by RHCE. The fundamental difference
200 mL or more of D+ red cells. Anti-D can cause       between C and c is a serine–proline substitution at
severe immediate or delayed haemolytic transfu-        position 103 in the second external loop of the
sion reactions and D+ blood must never be trans-       CcEe protein (see Fig. 3.5), although the situation
fused to a patient with anti-D.                        is more complex than that. E and e represent a
   Anti-D is the most common cause of severe           proline–alanine substitution at position 226 in
haemolytic disease of the fetus and newborn            the fourth external loop. Taking into account the
(HDN). The effects of HDN caused by anti-D are,        presence and absence of D, and of the C/c and E/e
at its most severe, fetal death at about week 17 of    polymorphisms, eight different haplotypes can be
pregnancy. If the infant is born alive, the disease    recognized. The frequencies of these haplotypes
can result in hydrops and jaundice. If the jaundice    and the shorthand symbols often used to describe
leads to kernicterus, this usually results in infant   them are shown in Table 3.3.
death or permanent cerebral damage. The preva-            Anti-c is clinically the most important Rh
lence of HDN due to anti-D has been substantially      antigen after anti-D and may cause severe HDN.
reduced by anti-D immunoglobulin prophylaxis.          On the other hand, anti-C, anti-E and anti-e rarely


                                                                                                             29
Chapter 3

Table 3.3 Rh phenotypes and the genotypes that produce them (presented in DCE and shorthand terminology).

Phenotype                                        Frequency (%)

D           C         c      E         e         Europeans*       Africans†       Asians‡        Genotypes

+           +         -      -         +         18.5               0.7           56.0           DCe/Dce     R1/R1
                                                                                                 DCe/dCe     R1r¢
+           -         +      +         -          2.3               1.3            3.5           DcE/DcE     R2R2
                                                                                                 DcE/dcE     R2r≤
+           -         +      -         +          2.1             58.9             0.2           Dce/dce     Ror
                                                                                                 Dce/Dce     RoRo
+           +         -      +         -         Rare             Rare            Rare           DCE/DCE     RzRz
                                                                                                 DCE/dCE     Rzry
+           +         +      -         +         34.9             13.2             8.4           DCe/dce     R1r
                                                                                                 DCe/Dce     R1Ro
                                                                                                 Dce/dCe     Ror¢
+           -         +      +         +         11.8             18.3             2.1           DcE/dce     R2r
                                                                                                 DcE/Dce     R2Ro
                                                                                                 Dce/dcE     Ror≤
+           +         -      +         +          0.2             Rare             1.1           DCe/DCE     R1Rz
                                                                                                 DCE/dCe     Rzr¢
                                                                                                 DCe/dCE     R1ry
+           +         +      +         -          0.1             Rare             0.3           DcE/DCE     R2Rz
                                                                                                 DCE/dcE     Rzr≤
                                                                                                 DcE/dCE     R2ry
+           +         +      +         +         13.4               2.1           28.1           DCe/DcE     R1R2
                                                                                                 DCe/dcE     R1r≤
                                                                                                 DcE/dCe     R2r¢
                                                                                                 DCE/dce     Rzr
                                                                                                 Dce/DCE     RoRz
                                                                                                 Dce/dCE     Rory
-           +         -      -         +         Rare              0.1            Rare           dCe/dCe     r¢r¢
-           -         +      +         -         Rare             Rare            Rare           dcE/dcE     r≤r≤
-           -         +      -         +         15.1              4.1             0.1           dce/dce     rr
-           +         -      +         -         Rare             Rare            Rare           dCE/dCE     ryry
-           +         +      -         +          0.1              1.3             0.1           dCe/dce     r¢r
-           -         +      +         +          0.1             Rare            Rare           dcE/dce     r≤r
-           +         -      +         +         Rare             Rare            Rare           dCe/dCE     r¢ry
-           +         +      +         -         Rare             Rare            Rare           dcE/dCE     r≤ry
-           +         +      +         +         Rare             Rare            Rare           dcE/dCe     r≤r¢
                                                                                                 dCE/dce     ryr

* English donors.
† Yoruba of Nigeria.
‡
  Cantonese of Hong Kong.




30
                                                                                               Human blood group systems


cause HDN and when they do the disease is gener-           The Kell antigens are located on a large glyco-
ally mild, though all have caused severe disease.        protein that crosses the cell membrane once
                                                         and has a glycosylated C-terminal extracellular
                                                         domain, maintained in a folded conformation by
Other Rh antigens
                                                         multiple disulphide bonds. The Kell glycoprotein
Of the 49 Rh antigens, 20 are polymorphic (i.e.          belongs to a family of endopeptidases, which
have a frequency between 1 and 99% in at least           process biologically important peptides, and are
one major ethnic group), 21 are rare antigens and        able to cleave the biologically inactive peptide big
eight are very common antigens. Antibodies to            endothelin-3 to produce endothelin-3, an active
many of these antigens have shown themselves to          vasoconstrictor.
be clinically important and it is prudent to treat
all Rh antibodies as being potentially clinically
                                                         Duffy system
significant.
                                                         Fya and Fyb represent a single amino acid substitu-
                                                         tion in the extracellular N-terminal domain of the
Other blood group systems                                Duffy glycoprotein. Their incidence in Caucasians
                                                         is 66% Fya and 80% Fyb. About 70% of African-
Of the remaining blood group systems (see Table          Americans and close to 100% of West Africans are
3.1), the most important clinically are Kell, Duffy,     Fy(a–b–) (Table 3.4). They are homozygous for an
Kidd and MNS, and are described below.                   Fyb allele containing a mutation in a binding site
                                                         for the erythroid-specific GATA-1 transcription
                                                         factor, which means that Duffy glycoprotein is not
Kell system
                                                         expressed in red cells, although it is present in
The original Kell antigen, K (KEL1), has a fre-          other tissues (Table 3.5). The Duffy glycoprotein is
quency of about 9% in Caucasians, but is rare in         the receptor exploited by Plasmodium vivax mero-
other ethnic groups. Its allelic antigen, k (KEL2), is   zoites for penetration of erythroid cells. Conse-
common in all populations. The remainder of the          quently, the Fy(a–b–) phenotype confers resistance
Kell system consists of one triplet and three pairs      to P. vivax malaria. The Duffy glycoprotein (also
of allelic antigens: Kpa, Kpb and Kpc; Jsa and Jsb;      called Duffy antigen chemokine receptor, DARC)
K11 and K17; K14 and K24; plus 11 high-                  is a red cell receptor for a variety of chemokines,
frequency and four low-frequency antigens. All           including interleukin-8.
represent single amino acid substitutions in the            Anti-Fya is not infrequent and is found in pre-
Kell glycoprotein.                                       viously transfused patients who have usually
   Anti-K can cause severe haemolytic transfusion        already made other antibodies. It can cause
reactions and HDN. About 10% of K-negative               haemolytic transfusion reactions, but seldom
patients who are given one unit of K-positive            causes HDN. Anti-Fyb is very rare.
blood produce anti-K, making K the next most
immunogenic antigen after D. About 0.1% of all
cases of HDN are caused by anti-K; most of the           Table 3.4 Duffy system: phenotypes and genotypes.
mothers will have had previous blood transfu-
sions. HDN caused by anti-K differs from Rh                                                    Frequency (%)
HDN in that anti-K appears to cause fetal anaemia
by suppression of erythropoiesis, rather than            Phenotype      Genotype               Europeans        Africans
immune destruction of mature fetal erythrocytes.
                                                         Fy(a+b-)       Fy a/Fy a or Fy a/Fy   20               10
Anti-K is a very rare antibody. It is always immune
                                                         Fy(a+b+)       Fy a/Fy b              48                3
and has been incriminated in some cases of mild
                                                         Fy(a-b+)       Fy b/Fy b or Fy b/Fy   32               20
HDN. Most other Kell-system antibodies are rare          Fy(a-b-)       Fy/Fy                   0               67
and best detected by an antiglobulin test.


                                                                                                                     31
Chapter 3

                                                                                      Table 3.5 Nucleotide polymorphisms
            GATA box sequence                                                         in the promoter region and in exon 2
Allele      -64 to -69 (promoter)   Codon 42 (exon 2)   Antigen                       of the three common alleles of the
                                                                                      Duffy gene.
Fya         TTATCT                  GGT (Gly)           Fya
Fyb         TTATCT                  GAT (Asp)           Fyb
Fy          TTACCT                  GAT (Asp)           Red cells: none
                                                        Other tissues: probably Fyb




Kidd system                                                  MNS system
Kidd has two alleles, Jka and Jkb, which represent a         MNS, with a total of 43 antigens, is second only to
single amino acid change in the Kidd glycoprotein.           Rh in complexity. These antigens are present on
Jka and Jkb antigens both have frequencies of                one or both of two red cell membrane glyco-
about 75% in Caucasian populations. A Kidd-null              proteins, glycophorin A (GPA) and glycophorin
phenotype, Jk(a–b–), results from homozygosity               B (GPB). They are encoded by two homologous
for inactivating mutations in the Kidd gene,                 genes, GYPA and GYPB, on chromosome 4.
SLC14A1. It is very rare in most populations, but               The M and N antigens, both with frequencies of
reaches an incidence of greater than 1% in Polyne-           about 75%, differ by amino acids at positions 1
sians. The Kidd glycoprotein is a urea transporter           and 5 of the external N-terminus of GPA. S and s
in red cells and in renal endothelial cells.                 have frequencies of about 55 and 90%, respec-
   Anti-Jka is uncommon and anti-Jkb is very rare,           tively, in a Caucasian population, and represent
but they both cause severe transfusion reactions             an amino acid substitution in GPB. About 2% of
and, to a lesser extent, HDN. Kidd antibodies have           black West Africans and 1.5% of African-
often been implicated in delayed haemolytic trans-           Americans are S– s–, a phenotype virtually
fusion reactions. They are often difficult to detect          unknown in other ethnic groups, and most of these
serologically and tend to disappear rapidly after            lack the U antigen, which is present when either S
stimulation.                                                 or s is expressed. The numerous MNS variants
                                                             mostly result from amino acid substitutions in
                                                             GPA or GPB and from the formation of hybrid
Diego system
                                                             GPA–GPB molecules, resulting from intergenic
Diego is a large system of 21 antigens: two pairs of         recombination between GYPA and GYPB. GPA
allelic antigens (Dia and Dib, Wra and Wrb) plus 17          and GPB are exploited as receptors by the malaria
antigens of very low frequency. All represent single         parasite Plasmodium falciparum.
amino acid substitutions in band 3, the red cell                Anti-M and anti-N are not generally clini-
anion exchanger. The original Diego antigen, Dia,            cally significant, though anti-M is occasionally
is very rare in Caucasians and black people, but             haemolytic. Anti-S, the rarer anti-s, and anti-U
relatively common in Mongoloid people, with fre-             can cause HDN and have been implicated in
quencies varying between 1% in Japanese and                  haemolytic transfusion reactions.
50% in some native South Americans. Anti-Dia
and anti-Dib are immune and rare, but can cause
HDN. Wra has a frequency of about 0.1%. Its                  Biological significance of blood
high-frequency allelic antigen, Wrb, is dependent            group antigens
on an interaction of band 3 with glycophorin A
for its expression. Naturally occurring anti-Wra is          The functions of several red cell membrane protein
present in approximately 1% of blood donors.                 structures bearing blood group antigenic determ-
Very rarely, anti-Wra causes HDN.                            inants are known, or can be deduced, from their


32
                                                                                             Human blood group systems


structure. Some are membrane transporters, facili-      Summary
tating the transport of biologically important mol-
ecules through the lipid bilayer: band 3 membrane       Blood groups are inherited characters of the red
glycoprotein, the Diego antigen, provides an anion      cell surface detected by specific alloantibodies.
exchange channel for HCO3– and Cl– ions; the Kidd       Most of the authenticated 285 blood group speci-
glycoprotein is a urea transporter; the Colton glyco-   ficities are organized into 29 blood group systems,
protein is aquaporin 1, a water channel; the GIL        each representing a single gene or a cluster of
antigen is aquaporin 3, a glycerol transporter; and     closely linked homologous genes. Blood group
the Rh protein complex might function as an             antibodies are of clinical importance in transfusion
ammonium transporter or a CO2 channel. The              medicine as they can cause haemolytic transfusion
Lutheran, LW and Indian (CD44) glycoproteins are        reactions and HDN. The carbohydrate ABO anti-
adhesion molecules, possibly serving their primary      gens and protein Rh antigens are the most impor-
functions during erythropoiesis. The Duffy glyco-       tant from the clinical aspect. Blood group proteins
protein is a chemokine receptor and could function      serve a variety of functions, though little is known
as a ‘sink’ or scavenger for unwanted chemokines.       about the biological significance of blood group
The Cromer and Knops antigens are markers for           polymorphism.
decay accelerating factor (CD55) and complement
receptor 1 (CD35), respectively, which protect the
cells from destruction by autologous complement.        Further reading
Some blood group glycoproteins have enzyme
activity: the Yt antigen is acetylcholinesterase and    Avent ND. Fetal genotyping. In: Hadley A, Soothill P, eds.
the Kell antigen is an endopeptidase, though their        Alloimmune Disorders of Pregnancy. Cambridge:
functions on red cells are not known. The C-              Cambridge University Press, 2002: 121–39.
                                                        Avent ND, Reid ME. The Rh blood group system: a review.
terminal domains of the Gerbich antigens, GPC and
                                                          Blood 2000; 95: 375–87.
GPD, and the N-terminal domain of the Diego
                                                        Chester AM, Olsson ML. The ABO blood group gene: a
glycoprotein, band 3, are attached to components          locus of considerable genetic diversity. Transfus Med Rev
of the cytoskeleton and function to anchor it to          2001; 15: 177–200.
the external membrane. The carbohydrate moieties        Daniels G. Functional aspects of red cell antigens. Blood
of the membrane glycoproteins and glycolipids,            Rev 1999; 13: 14–35.
especially those of the most abundant glycoproteins     Daniels G. Human Blood Groups, 2nd edn. Oxford:
                                                          Blackwell Science, 2002.
(band 3 and GPA), constitute the glycocalyx, an
                                                        Daniels G, Poole J, de Silva M et al. The clinical significance
extracellular coat that protects the cell from
                                                          of blood group antibodies. Transfus Med 2002; 12:
mechanical damage and microbial attack.                   287–95.
   The structural differences between allelic red       Daniels GL, Fletcher A, Garratty G et al. Blood group
cell antigens (e.g. A and B, K and k, Fya and Fyb)        terminology 2004. Vox Sang 2004, in press.
are small, often being just one monosaccharide or       Denomme GA. The structure and function of the molecules
one amino acid. The biological importance of              that carry human red blood cell and platelet antigens.
                                                          Transfus Med Rev 2004; 18: 203–31.
these differences is unknown and there is little
                                                        Henry S, Samuelsson B. ABO polymorphisms and their
evidence to suggest that the product of one allele
                                                          putative biological relationships with disease. In: King
confers any significant advantage over the other.          M-J, ed. Human Blood Cells: Consequences of Genetic
Some blood group antigens are exploited by                Polymorphism and Variations. London: Imperial College
pathological microorganisms as receptors for              Press, 2000: 1–103.
attaching and entering cells, so in some cases          Reid ME, Lomas-Francis C. The Blood Group Antigen
absence or changes in these antigens could be             Facts Book. San Diego: Academic Press, 1997.
                                                        Watkins WM (ed.) Commemoration of the centenary of the
beneficial. It is likely that interaction between cell
                                                          discovery of the ABO blood group system. Transfus Med
surface molecules and pathological micro-
                                                          2001; 11: 239–351.
organisms has been a major factor in the evolution
of blood group polymorphism.


                                                                                                                   33
Chapter 4
Human leucocyte antigens

Cristina V. Navarrete




The human leucocyte antigen (HLA) system con-            been mapped to a locus located 4 Mb telomeric to
sists of a family of cell surface polymorphic mol-       HLA-F. Mutations in this gene have been shown to
ecules involved in the presentation of antigen to T      be responsible for the development of hereditary
cells and therefore plays a central role in the induc-   haemochromatosis.
tion and regulation of immune responses. HLA               Following the development of recombinant
molecules are also known to be involved in the           DNA technology, it has been possible to perform a
pathogenesis of certain autoimmune and infec-            detailed analysis of the HLA region, leading to the
tious diseases and they have an important influ-          unravelling of the genetic complexity and structure
ence on the outcome of solid organ and                   of its genes and molecules. These findings, com-
haemopoietic stem cell transplantation. Further-         bined with parallel studies on their function genes,
more, HLA antigens present in blood cells are            have led to a better understanding of the role of the
responsible for some of the serious clinical compli-     HLA system in clinical medicine.
cations of blood transfusion.
   The genes coding for the HLA molecules are
located on the short arm of chromosome 6 and             HLA class I genes
span a distance of approximately 4 Mb. This
region is divided into three subregions.                 The HLA class I genes have been classified accord-
• Class I subregion contains genes coding for the        ing to their structure, expression and function as
heavy chain of the classical (HLA-A, -B and -C)          classical (HLA-A, -B and -C) and non-classical
and non-classical (HLA-E, -F and -G) HLA class I         (HLA-E, -F and -G). Both classical and non-classi-
molecules;                                               cal HLA class I genes code for a heavy (a) chain, of
• Class II subregion contains genes coding for the       approximately 43 kDa, non-covalently linked to a
HLA class II molecules (DR, DQ and DP) and               non-polymorphic light chain, the b2-microglobulin
genes involved in the processing and transport of        of 12 kDa, which is coded for by a gene on chro-
antigenic peptides.                                      mosome 15. The extracellular portion the heavy
• Class III subregion lies between the other two         chain has three domains (a1, a2 and a3) approxi-
subregions and contains genes coding for a diverse       mately 90 amino acids long. These domains are
group of proteins, including complement com-             encoded by exons 2, 3 and 4 of the class I gene,
ponents (C4Bf), tumour necrosis factor (TNF) and         respectively. The a1 and a2 domains are the most
heat-shock proteins.                                     polymorphic domains of the molecule and they
   A number of additional genes, including the           form a peptide-binding groove that can accommo-
major histocompatibility complex (MHC) class I           date antigenic peptides approximately eight to
chain-related gene A (MICA) and MHC class I              nine amino acids long.
chain-related gene B (MICB) also involved in                The exon/intron organization of the non-classi-
immune responses, have been mapped between the           cal HLA class I genes (E, F and G) is very similar to
class I and class III subregions (Fig. 4.1). In addi-    the classical class I genes but they have a more
tion, the non-classical class I-like gene HFE has        restricted polymorphism.


34
                                                                                                                                    Human leucocyte antigens


                                                                                              HLA class III
                               HLA class II subregion                                          subregion                         HLA class I subregion

                         DMB       LMP7                                                            MICB MICA
                                 TAP1   TAP2                                                     TNF
      DP           DOA DMA      LMP2     DOB            DQ                   DR              HSP70        B C                       E       A H G F




   B2 A2   B1 A1                                B2 A2 B3 B1 A1     B1 B2 B5 B3 B4        A         A   B


                                Class II           Class II              ABC                   Proteosome-                 HSP70
                                genes              genes                 transporter           like genes
                                pseudogenes                              genes

                                TNF                Classical             Non-classical            MICA and
                                                   class                 class I                  MICB
                                                   I genes               genes

Fig. 4.1 Map of the human leucocyte antigen complex. HSP, heat-shock protein; TNF, tumour necrosis factor.




                                                               Peptide                   α1                         α2
                                                               binding
                                                                                                       S
                                                                region
                                                                                                               S

                                                                                                   N
                                                   Immunoglobulin-                                 S            S
                                                                                β2-m                                        α3
                                                        like region                                S            S

                                                                                                   C
                                                    Transmembrane
                                                            region
                                                                                                                     P
                                                                                                                     P
                                                         Cytoplasmic                                                 P
                                                              region
                                                                                                                C




                                                                                   L         α1            α2               α3      TM       CYT         3'UT
                                                  HLA class
                                                    A gene         Regulatory
                                                                   sequences       1          2            3                4         5     6 7           8
Fig. 4.2 HLA class I molecule. b2-m,
b2-microglobulin.                                                                                                        Exons


   A schematic representation of the classical HLA                        code for a heterodimer formed by two non-
class I gene and molecule is shown in Fig. 4.2.                           covalently associated a and b chains of approxi-
                                                                          mately 34 and 28 kDa respectively. The expressed
                                                                          a and b chains consist of two extracellular
HLA class II genes                                                        domains as well as transmembrane and cytoplas-
                                                                          mic domains. The a1/b1 and a2/b2 domains are
The class II DR, DQ and DPA and DPB genes                                 encoded by exon 2 and exon 3 of the class II gene


                                                                                                                                                              35
Chapter 4


respectively. The majority of the polymorphism is                                 individual varies according to the DRB1 allele
located in the b1 domain of the DR molecules and                                  expressed, e.g. HLA-DR1, -DR103, -DR10 and
in the a1 and b1 domains of the DQ and DP mol-                                    -DR8 alleles express the DRB1 gene only. DR15
ecules. Similarly to the class I molecules, these                                 and DR16 alleles express the DRB1 and DRB5
domains also form a peptide-binding groove.                                       genes, which code for the DR51 product; HLA-
However, in the case of the class II molecules (DR),                              DR17, -DR18, -DR11, -DR12, -DR13 and -DR14
the groove is open at both sides and it can accom-                                alleles express the DRB1 and the DRB3 genes,
modate antigenic peptides of varying size,                                        which code for the DR52 specificity; and, finally,
although most of them are approximately 13–25                                     the HLA-DR4, -DR7 and -DR9 alleles express the
amino acids long. A schematic representation of                                   DRB1 and the DRB4 genes coding for the DR53
the HLA class II gene and molecule is shown in                                    product (Fig. 4.4). There are a few exceptions to
Fig. 4.3.                                                                         this pattern of gene expression, e.g. a DRB5 gene
                                                                                  has been found to be expressed with some DR1
                                                                                  alleles and non-expressed or null DRB5 and
Expression of HLA class II genes                                                  DRB4 genes have also been identified.
                                                                                     In contrast to the DRB genes, there are two
There is one non-polymorphic DRA and nine                                         DQA and three DQB genes, of which only the
DRB genes, of which B1, B3, B4 and B5 are highly                                  DQA1 and DQB1 are expressed and both are
polymorphic and B2, B6 and B9 are pseudogenes.                                    polymorphic. Similarly, there are two DPA and
The main DR specificities (DR1–DR18) are deter-                                    two DPB genes, of which only the DPA1 and
mined by the polymorphic DRB1 gene. Further-                                      DPB1 are expressed and both are polymorphic.
more, the number of DRB genes expressed in each                                      There are additional genes located in the class II




            Peptide               α1                        β1
            binding
                                                NN S
             region
                                                        S




 Immunoglobulin-                      S                 S
                       α2                                             β2
      like region                     S       Papain S
                                          cleavage sites

  Transmembrane
          region
                                  C                         C
      Cytoplasmic
           region


                                                                 Exons
                              1                        2               3         4        5
                              L                        α1             α2       TM/CYT   3'UT
HLA class
  A gene         Regulatory
                 sequences
HLA class
  B gene
                              L            β1                    β2      TM CYT 3'UT
                              1            2                      3       4 5 6
                                                        Exons                                   Fig. 4.3 HLA class II molecule.


36
                                                                                             Human leucocyte antigens


                                                 Specifications
                                                                  DRB1           DRB6            DRB9      DRA
                                         Dr1, DR10, DR103, DR15                   Y               Y


                                                                  DRB1           DRB6    DRB5    DRB9      DRA
                                               DR15, DR16, DR1                    Y               Y
                                                                                         DR51
                                         DR17, DR18, DR11, DR12   DRB1           DRB2    DRB3    DRB9      DRA
                                           DR13, DR14, DR1403,                    Y               Y
                                                        DR1404                           DR52
                                                                  DRB1                           DRB9      DRA
                                                           DR8                                    Y

                                                                  DRB1   DRB7    DRB8    DRB4    DRB9      DRA
                                                  DR4, DR7, DR9            Y      Y               Y
Fig. 4.4 Expression of HLA-DRB genes.                                                    DR53



region which are involved in the MHC class I                detected in endocrine tissue, skeletal muscle and
antigen presentation pathway. These include the             cells of the central nervous system. HLA-E and -F
low-molecular-mass polypeptide genes (LMP2                  are also expressed on most tissues tested but HLA-
and LMP7) and the transporter associated with               G shows a more restricted tissue distribution and
antigen-processing genes (TAP1 and TAP2). The               to date the HLA-G product has only been found to
LMP2 and LMP7 genes are thought to improve                  be expressed on extravillous cytotrophoblasts of
the capacity of the proteosomes to generate                 the placenta and mononuclear phagocytes.
peptides of the appropriate size and specificity to             The HLA class II molecules are constitutively
associate with the class I molecules. Conversely,           expressed on B lymphocytes, monocytes and den-
the TAP1 and TAP2 genes are primarily involved              dritic cells but can also be detected on activated T
in the transport of the proteosome-generated pep-           lymphocytes and activated granulocytes. It is not
tides to the endoplasmic reticulum, where they              clear whether they are also present on activated
associate with the class I molecules.                       platelets.
   In addition, the DMA and DMB genes, which                   HLA class II expression can be also be induced
code for a heterodimer involved in the loading of           on a number of cells and tissues such as fibroblasts
peptides presented by HLA class II molecules, are           and endothelial cells as the result of activation
also located in this subregion (see Fig. 4.1). The          and/or the effect of certain inflammatory
main function of the DM molecules is to facilitate          cytokines, such as interferon (IFN)-g, TNF and
the release of the class II-associated invariant chain      interleukin (IL)-10.
peptide from the peptide-binding groove of the
HLA class II molecule so that it can be exchanged
for the relevant antigenic peptide.                         Genetics

                                                            One of the main features of the HLA genes is their
Distribution of HLA molecules                               high degree of polymorphism and the strong
                                                            linkage disequilibrium (LD) in which they segre-
The classical HLA class I molecules (A, B, C) are           gate. LD is a phenomenon where the observed
expressed on the majority of tissues and cells,             frequency of alleles of different loci segregating
including T and B lymphocytes, granulocytes and             together is greater than the frequency expected by
platelets. Low levels of expression have been               random association. The difference between the


                                                                                                                 37
Chapter 4


observed and the expected frequencies for a par-      (KIRs), and the C-type lectin superfamily CD94-
ticular combination of alleles is called the delta    NKG2. The interaction between the inhibitory
value and is positive for alleles in LD with each     receptors and the relevant HLA ligand results in
other. Whereas some of the polymorphism and the       the prevention of NK lysis of the target cell. Thus
patterns of LD are expressed with similar frequen-    NK cells from any given individual will be allo-
cies in all populations, others are unique to some    reactive towards cells lacking their corresponding
population groups. For example, HLA-A2 is             inhibitory KIR ligands, e.g. tumour or allogeneic
expressed at a relatively high frequency in most      cells. In contrast NK cells will be tolerant to cells
population groups studied so far, whereas B53 is      from individuals who express the corresponding
found predominantly in black people.                  KIR ligands. This information is currently being
   In addition, all HLA genes are codominantly        exploited in the clinical setting in order to promote
expressed and are inherited in a mendelian fashion    the graft-versus-leukaemia (GVL) effect mediated
and the genetic region containing all HLA genes on    by NK cells.
each chromosome is termed the haplotype. Some            HLA class II molecules are mostly involved in
haplotypes are also found across different ethnic     the presentation of exogenous antigenic peptides
groups, e.g. HLA-B44-DR7, whereas others are          to CD4+ helper T cells. Once activated, these CD4+
unique to a particular population, e.g. HLA-B42-      cells can initiate and regulate a variety of processes
DR18 in black Africans. This characteristic is        leading to the maturation and differentiation of
particularly relevant for the selection of HLA-       cellular (CD8+ cytotoxic T cells) and humoral
compatible family donors for patients requiring       effectors by the secretion of proinflammatory
solid organ or bone marrow transplantation            cytokines (IL-2, IFN-g, TNF-a) and regulatory
(BMT).                                                cytokines (IL-4, IL-10 and transforming growth
                                                      factor-b). The nature of the peptide presented by
                                                      class I and II molecules is largely dependent on the
Function of HLA molecules                             location of the peptide within the processing
                                                      machinery of the cells.
HLA molecules are directly involved in the presen-
tation of antigenic peptides to T cells. This is a
highly regulated process and requires a fine inter-    Identification of HLA gene polymorphism
action between the HLA molecules, the antigenic
peptide, the T-cell receptor and a number of          Traditionally, characterization of HLA polymor-
costimulatory molecules (e.g. CD80, CD86) and         phisms has been carried out using serological and
adhesion molecules such ICAM-1(CD54) and              cellular techniques. There are, however, several
LFA-3 (CD58).                                         limiting factors in the use of serological typing
   The HLA class I molecules are primarily but not    methods:
exclusively involved in the presentation of endoge-   • it is often difficult to obtain antibody of suffi-
nous antigenic peptides to CD8+ cytotoxic T cells.    cient titre and specificity to distinguish between all
However, it has now been shown that both classi-      described HLA types;
cal and non-classical HLA class I molecules also      • the antibodies available are very often of
interact with a new family of receptors present       Caucasian origin, making it difficult to HLA type
on natural killer (NK) cells. Some of these recep-    patients from other population groups;
tors, which are polymorphic and differentially        • patients undergoing chemotherapy normally
expressed, have an inhibitory role whereas others     have low white cell counts and cell antigen expres-
are involved in NK cell activation. The killer-       sion can be affected by the chemotherapy;
activating and killer-inhibitory receptors belong     • the requirement for viable cells, ideally 24 h
to two distinct families: the immunoglobulin          after venepuncture, means that serological HLA
superfamily called killer immunglobulin receptors     typing results are not always reliable; and



38
                                                                                            Human leucocyte antigens


• because class II molecules are expressed on B        Table 4.1 Number of recognized HLA antigens/alleles.
cells and not (resting) T cells, the low number of B   (From Marsh et al. 2002 with permission.)
cells in a sample makes serological class II typing
                                                                                  Alleles                  Antigens
difficult.
   However, with the development of gene cloning
                                                       HLA class I
and DNA sequencing it has been possible to             HLA-A                      250                      24
perform a detailed analysis of these genes at the      HLA-B                      490                      50
single nucleotide level. This analysis has shown the   HLA-C119                     9
existence of shared nucleotide sequences between       HLA-E                        6
alleles of the same and/or different loci and the      HLA-F                        1
existence of certain locus-specific nucleotide          HLA-G                       15
sequences in both the coding (exons) and non-          HLA class II
coding (introns) regions of the genes. The DNA         HLA-DRB1                   315                      17
sequencing of a number of alleles of various loci      HLA-DRA1                     3                      —
has also demonstrated that the majority of the         HLA-DRB3                    38                       1
polymorphism is located in regions of the a1 and       HLA-DRB4                    12                       1
a2 domain of the class I molecules and of the a1       HLA-DRB5                   150                       1
and b1 domain of the class II molecules. These are     HLA-DQB1                    53                       6
called hypervariable regions.                          HLA-DQA1                    22                      —
                                                       HLA-DPB1                    99                       6*
   Based on this information, a number of tech-
                                                       HLA-DPA1                    20                      —
niques have been developed to characterize HLA
polymorphisms. Most of the described techniques        * Cellularly defined.
make use of the polymerase chain reaction (PCR)
to amplify the specific genes or region to be           reverse blot, involves the addition of the PCR-
analysed. These techniques include PCR-SSOP            amplified product to labelled probes immobilized
(sequence-specific oligonucleotide probing), PCR-       on membranes (strips) or plates. PCR-SSOP is
SSP (sequence-specific priming), and conforma-          useful when large numbers of samples need to be
tional methods including reference strand              HLA typed, e.g. bone marrow or cord blood
conformational analysis (RSCA) and sequencing-         donors.
based typing (SBT). The number of recognized
serologically defined antigens and DNA-identified
                                                       Sequence-specific priming
HLA alleles is shown in Table 4.1.
                                                       This technique involves the use of sequence-
                                                       specific primers in the PCR step, i.e. primers
Sequence-specific oligonucleotide probing
                                                       designed to anneal with DNA sequences unique to
In this technique the gene of interest is amplified     each allele and locus. The detection of the PCR-
using generic primers, i.e. primers designed to        amplified product is then carried out by running
anneal with DNA sequences common to all alleles        the product on an agarose gel. This technique
of the loci of interest. The amplified PCR product      allows the rapid identification of the HLA alleles
is then immobilized onto support (e.g. nylon)          in individual samples since the readout of this
membranes and the specificity of the products           method is the presence or absence of the product
analysed by reacting the membranes with labelled       for which specific primers were used. This tech-
oligonucleotides designed to anneal with polymor-      nique is therefore ideally suited to HLA typing
phic sequences present in each allele. By scoring      individual samples, e.g. patients requiring HLA-
the probes that bind to specific regions, it is pos-    matched platelets. However, although this is a very
sible to assign the HLA type.                          rapid procedure, many PCR reactions have to be
   A recent modification of this technique, called      set up per sample, e.g. at least 24 reactions for low-



                                                                                                                 39
Chapter 4


resolution DR typing. Furthermore, for PCR-SSP        technique based on the fact that mismatched DNA
typing the target sequence of the alleles must be     hybrids (heteroduplexes) migrate at a slower rate
known since novel unknown sequences may not           through gels (due to the presence of single-
always be detected.                                   stranded loops) than fully matched DNA
                                                      duplexes.
                                                         Heteroduplexes are formed during the anneal-
Conformational analysis methods
                                                      ing stage of the PCR when the sense strand of an
Conformational methods depend on the mobility         allele binds to the antisense strand of a different
of PCR products in the gels. A number of vari-        allele. The banding pattern of these products fol-
ations of this technique have been described,         lowing electrophoresis through a gel can be used to
including single-strand conformational polymor-       identify the alleles present. This technique is par-
phism analysis (SSCP), double-strand conforma-        ticularly useful for performing DNA-based ‘cross-
tional polymorphism analysis (DSCA), RSCA and         matching’. In this case, HLA genes from the
heteroduplex formation. In the SSCP technique,        patient and potential donor are amplified, dena-
PCR-generated DNA products are denatured by           tured by heating to about 95°C for several minutes
heating and rapid cooling to prevent reannealing      and then mixed together under conditions which
of the strands. The products are run on a polyacry-   promote reannealing. Where the donor and
lamide gel with the mobility depending upon the       patient alleles are similar but not identical, then,
secondary structure of the single-stranded DNA.       as in RSCA, homoduplexes are formed together
The major disadvantage of this approach for HLA       with chimeric or heteroduplexes where sense
typing is the tendency of single-stranded DNA to      strands from the patient anneal to antisense
adopt many conformational forms under the same        strands from the donor and vice versa. These
electrophoretic conditions, resulting in the pres-    heteroduplexes would have different migration
ence of several bands from a single product.          rates through gels when compared with the
   A modification of this technique that compares      homoduplexes.
the mobility in polyacrylamide of duplex mol-            When this mix is electrophoresed through a gel,
ecules generated by mixing PCR products is called     the banding pattern observed would contain all the
DSCA. In this case, the mobility depends on the       bands that would be observed if PCR products
mismatching of the sequence and the formation of      from the patient and donor were to be electro-
heteroduplex molecules. A further modification of      phoresed separately. However, in addition to
this technique, the RSCA, has recently been devel-    these there will be extra bands that are due to the
oped and successfully applied to HLA class I and II   heteroduplexes. The presence of extra bands in a
(DP) typing. In this technique the PCR amplifica-      heteroduplex ‘crossmatch’ would therefore indi-
tion is carried out on the test DNA and on a refer-   cate a difference in HLA type between the patient
ence DNA of known sequence, using fluorescently        and donor. These extra bands would not be
labelled primers for the PCR of the reference         observed if the HLA alleles of the patient and
DNA. The PCR products are then mixed, allowed         donor were identical. The sensitivity of the tech-
to anneal and run in an automated sequencer. Only     nique can be increased by adding DNA from a
those duplexes containing a labelled strand are       known HLA allele that is not present in the patient
detected, i.e. the homoduplex of reference DNA        or donor.
and the heteroduplexes of reference and test DNA.        One advantage of typing by conformational
The mobility of every known allele with the refer-    analysis when compared with methods such as
ence DNA is then established and used to compare      PCR-SSP and PCR-SSOP is that new mutations
with that of the DNA under test.                      not previously described can be readily picked up.
                                                         A disadvantage of the technique is that, like
                                                      SSCP, it cannot detect some HLA class II combina-
Heteroduplexing
                                                      tions while detecting too many silent mutations.
Heteroduplexing is another DNA conformational         Sometimes the banding patterns observed are


40
                                                                                         Human leucocyte antigens


complex and difficult to interpret. It also suffers in     The advantages and disadvantages of the
that it provides no actual HLA typing information.      various techniques described above are given in
  One of the main disadvantages of all the above        Table 4.2.
described techniques is that they require DNA
sequence data in order to design primers and
probes.                                                 HLA antibodies

                                                        HLA-specific antibodies may be produced in any
Sequencing-based typing
                                                        situation that exposes the host to these alloanti-
The principle of DNA sequencing is relatively           gens, including pregnancy, transplantation, blood
straightforward and involves the denaturation of        transfusions and planned immunizations. How-
the DNA to be analysed to provide a single-strand       ever, the affinity, avidity and class of the antibody
template. A sequencing primer is then added and         produced will depend on various factors, including
the DNA extension is performed by the addition          the route of immunization, the persistence and
of Taq polymerase in the presence of excess             type of immunological challenge and the immune
nucleotides. The sequencing mixture is divided          status of the host. Cytotoxic HLA antibodies can
into four tubes, each of which contains specific         be identified in approximately 20% of human
dideoxyribonucleoside triphosphate (ddATP).             pregnancies. The antibodies produced are nor-
When this is incorporated into the DNA synthesis,       mally multispecific, high titre, high affinity and of
elongation is interrupted with chain-terminating        the IgG class. Although these IgG antibodies can
inhibitors. In each reaction there is random            cross the placenta, they have not been shown to be
incorporation of the chain terminators and              harmful to the fetus. Conversely, the antibodies
therefore products of all sizes are generated.          produced following transplantation seem to be
The products of the four reactions are then             largely dependent on the degree of HLA mismatch
analysed by electrophoresis in parallel lanes of a      between donor and recipient and the majority of
polyacrylamide–urea gel and the sequence is read        these antibodies formed are IgG, although a few
by combining the results of each lane using an          IgM antibodies have also been identified.
automated DNA sequencer. The sequencing prod-              In contrast, the majority of HLA antibodies
ucts are detected by labelling the nucleotide chain     found in multitransfused patients are multispecific
inhibitors with radioisotopes and, more recently,       IgM and IgG and are mostly directed at public
with fluorescent dyes. In HLA SBT, some ambigu-          epitopes. The recent introduction of leucocyte-
ous results can be obtained with heterozygous           depleted blood components (see Chapter 22) may
samples and these may need to be retested by            lead to a reduction in alloimmunization in naive
PCR-SSP.                                                recipients but it may not be very effective in pre-
   HLA typing using the RSCA and SBT techniques         venting alloimmunization in already sensitized
permit high-resolution HLA typing, which is             recipients, i.e. women who have become immu-
known to be important in the selection of HLA-          nized as a result of pregnancy.
matched unrelated donors. A major advantage of             Finally, normal individuals were previously
all DNA-based techniques is that no viable cells        deliberately immunized with HLA-mismatched
are required to perform detailed HLA class I and II     cells in order to produce potent HLA-specific
typing. Furthermore, since all the probes and           reagents, but the deliberate immunization of these
primer are synthesized to order, there is a consist-    healthy individuals is nowadays difficult to justify
ency of reagents used, allowing the comparison of       ethically. However, at present, planned HLA
HLA types from different laboratories. However,         immunization is still carried out as a form of treat-
although serological typing is being rapidly            ment for women with a history of recurrent spon-
replaced by DNA-based typing techniques, sero-          taneous abortion. These women are immunized
logical reagents may still be required for antigen      with lymphocytes from their partners or a third
expression studies.                                     party to attempt to induce an immunomodulatory


                                                                                                             41
Chapter 4

Table 4.2 Advantages and disadvantages of DNA-based techniques.

Technique                   Advantages                                                          Disadvantages

Sequence-specific            Needs only one pair of genetic primers, fewer reactions to set up   Different temperatures required for each probe
  oligonucleotide probing   Larger number of samples can be processed simultaneously            Probes can cross-react with different alleles
  (SSOP)                    Requires small amount of DNA                                        Large numbers of probes required to identify
                            Cheap                                                                  specificity
                                                                                                Difficult to interpret pattern of reactions

Sequence-specific priming    Provides rapid typing with higher resolution than SSOP              Too many sets of primers are needed to fully
  (SSP)                     All PCR amplifications are carried out at same time, temperature       HLA type
                               and conditions                                                   Requires a two-stage amplification to provide
                            Fast and simple to read and interpret                                 HR typing

Reference strand            Easy to perform                                                     Requires expensive equipment
  conformational analysis   Provides higher resolution than SSOP and SSP                        Requires established data on viability value of
  (RSCA)                                                                                          each allele studied

Sequencing-based typing     Provides the highest level of resolution                            Not easy to perform
  (SBT)                     Able to identify new alleles                                        Requires expensive reagents and equipment
                            Does not require previous sequence data to identify new allele      Difficult to interpret
                                                                                                Requires DNA sequence data to compare results
                                                                                                Slower than SSOP (reverse blot),
                                                                                                SSP and RSCA




response that results in the maintenance of the                            the cell surface leads to the activation of comple-
pregnancy.                                                                 ment via the classical pathway and results in the
                                                                           disruption of the cell membrane. The lysed cells
                                                                           are then detected by adding ethidium bromide (EB)
Detection of HLA antibodies                                                and acridine orange (AO) at the end of the incuba-
                                                                           tion period. Live cells actively take up AO and
Over the years, a number of techniques to detect                           under ultraviolet (UV) light they appear green,
HLA antibodies have been described. These                                  whereas lysed cells allow the entry of EB, which
include the complement-dependent lymphocyto-                               binds to DNA, and they appear red under UV
toxicity (LCT) test, enzyme-linked immuno-                                 light. There are a number of alternatives to EB and
sorbent assay (ELISA) and flow cytometry. More                              AO including carboxyfluorescein diacetate and
recently, a new method to detect HLA antibodies,                           EB, eosin or trypan blue. The reactions are scored
Luminex, has been described.                                               by estimation of the percentage of dead cells in
                                                                           each well after establishing baseline values in the
                                                                           negative and positive controls (Table 4.3).
Lymphocytotoxicity
                                                                              One of the main disadvantages of the LCT assay
The LCT assay, developed by Terasaki and                                   is that it does not discriminate between HLA and
McClelland (1964), is the most commonly used. In                           non-HLA cytotoxic lymphocyte-reactive anti-
this technique, equal volumes of serum and cells                           bodies including autoantibodies, which are
are mixed and incubated to allow the binding of                            common in thrombocytopenic patients, particu-
the specific antibody to the target cell. This is fol-                      larly in post-BMT patients, in whom there is a dys-
lowed by the addition of rabbit complement and a                           function of the immune system. Fortunately, the
further incubation step. Complement-fixing anti-                            majority of these lymphocytotoxic autoantibodies
bodies reacting with the HLA antigen present on                            are IgM and can be identified by screening the


42
                                                                                                  Human leucocyte antigens

Table 4.3 Lymphocytotoxicity grading.                               The basic principle of the technique is as
                                                                 follows. HLA antigens are purified and immobil-
Cell death (%)    Score        Interpretation                    ized on a microwell plate, directly or via an anti-
                                                                 body directed against a non-polymorphic region of
 0–10             1            Background cell death, negative
                                                                 the HLA antigen. HLA-specific antibodies bound
11–20             2            Doubtful negative
21–50             4            Weak positive
                                                                 to the immobilized antigen can be detected with an
51–80             6            Positive                          enzyme-linked secondary antibody which, upon
81–100            8            Strong positive                   addition of specific substrate, catalyses a colour
                  0            Unreadable/invalid                change reaction that is detected in an ELISA reader
                                                                 (GTI QuickScreen).
                                                                    This ELISA test can only be used to determine
serum with and without dithiothreitol (DTT). The                 the presence or absence of HLA-specific antibody.
addition of DTT to the serum results in the break-               In order to detect HLA specificity, an alternative
down of the intersubunit disulphide bonds in the                 technique is used. In this case, HLA antigen is
IgM molecule, leading to the loss of cytotoxicity                isolated from a selected panel of cell donors or
due to IgM. Prolonged exposure or excess DTT                     cell lines derived from these donors. Antibodies
can lead to the breakdown of intramolecular disul-               directed against the non-polymorphic region of the
phide bonds in the IgG molecules and also inacti-                HLA class I molecule, i.e. the a3 domain, are used
vate complement, but this can be inhibited by the                to immobilize the specific HLA antigen to the
addition of cystine.                                             microwell, ensuring that the more polymorphic
   In our laboratory, we tested 104 serum samples                a1 and a2 domains are available for antibody
from immunologically refractory patients receiv-                 binding. The test is designed to cover all the major
ing HLA-matched platelet transfusions, 50% of                    HLA specificities at least once; thus it should be
the samples being positive by LCT but only 40%                   possible to determine antibody specificity in those
being positive when screened with DTT, illustrat-                sera which show a restricted panel reactivity.
ing the importance of using DTT to remove the                    Three commercial kits are now available to
reactivity due to IgM, non-HLA lymphocytotoxic                   perform the specificity screening, SangStat.-PRA-
antibodies (10%).                                                STAT, GTI QuickID and Lambda Antigen Tray
   The presence of lymphocytotoxic autoreactive                  LAT.
antibodies in itself is not thought to be of clinical               One of the main advantages of the ELISA tech-
significance in solid organ transplant recipients;                nique is that the non-cytotoxic antibodies detected
however, the clinical relevance of these antibodies              are HLA specific since it relies on the binding of the
in immunological refractoriness to random                        antibodies to wells coated with pools of solubilized
platelet transfusions is not yet clear.                          HLA antigens. It is also more sensitive and in our
   Another disadvantage of the LCT test is that it               laboratory the ELISA test achieves a 7% increase
only detects cytotoxic HLA-specific antibodies                    in sensitivity over the LCT test (49% vs. 42%).
and therefore the presence of non-cytotoxic HLA-                    The precise identification of the type, specificity
specific antibodies may be missed. Non-cytotoxic                  and titre of antibodies is important not only for the
antibodies are best detected by using an ELISA or                diagnosis but also to establish the best course of
by flow cytometry, as described below.                            treatment, since in the case of patients immuno-
                                                                 logically refractory to platelet transfusions, the
                                                                 majority of these patients can benefit from the pro-
Enzyme-linked immunosorbent assay
                                                                 vision of HLA matched or crossmatched negative
ELISA-based methods have often been the tech-                    platelets (see below).
nique of choice for antibody detection for a
number of antigen systems, particularly where
                                                                 Luminex
there has been a requirement for testing large
numbers of samples.                                              This technique uses fluorochrome-dyed poly-


                                                                                                                      43
Chapter 4


styrene beads coated with specific antigens. The                            sera with median fluorescence values greater than
precise ratio of these fluorochromes creates 100                            the mean + 3SD of the negative controls are con-
distinctly coloured beads, each of them coated                             sidered positive. A modification of the above pro-
with a different antigen. The beads are then incu-                         cedure can be used to detect antibodies reacting
bated with the patient’s serum and the reaction is                         with T or B cells.
developed using a PE-conjugated antihuman IgG                                 The main advantages of flow cytometric
(Fc specific) antibody.                                                     techniques are the increased sensitivity when
                                                                           compared with LCT and the detection of non-
                                                                           complement fixing antibodies, allowing the early
Flow cytometry
                                                                           detection of sensitization. However, one of the dis-
The use of flow cytometric techniques was initially                         advantages is that it also detects non-HLA and
investigated as an alternative crossmatch tech-                            lymphocyte-reactive antibodies, and the clinical
nique and was shown to be more sensitive than                              relevance of these antibodies is unclear.
LCT. The increased sensitivity may be attributed to                           The relative advantages and disadvantages of
a number of factors, one of which is the additional                        each of these techniques are given in Table 4.4.
reactivity due to the detection of non-cytotoxic
antibodies, some of which may be HLA specific.
As with LCT screening, cells and serum are mixed                           Clinical relevance of HLA antigens
and incubated to allow the binding of the antibody                         and antibodies
to the target antigen. The bound antibody is
detected by using an antibody labelled with a fluo-                         Although the main role of the HLA molecules is to
rescent marker such as fluorescein isothiocyanate                           present antigens to T cells, HLA molecules can
or R-phycoerythrin against human immunoglobu-                              themselves be recognized as foreign by host T cells
lin. The flow cytometer can then be used to identify                        by a mechanism known as allorecognition. Two
the different cell populations based on their mor-                         pathways of allorecognition have been identified,
phology/granularity and on the fluorescence. Test                           direct and indirect.


Table 4.4 HLA antibody screening techniques.

                          Advantages                                                 Disadvantages

Lymphocytotoxicity test   Well established                                           Viable cells required
  (LCT)                   Robust                                                     Needs separated T and B cells for class I and class II antibody
                          Requires small amount of serum                                screening
                          Used for antibody screening and crossmatching              Large and selected panel of cells required
                          Low cost                                                   Detects non-HLA cytotoxic antibodies, e.g. autoantibodies

Enzyme-linked             Easy to standardize                                        Not yet well established to define class I and class II
  immunosorbent assay     Objective readout                                             specificities
  (ELISA)                 Suitable for bulk testing                                  Large amounts of serum required
                          Detects cytotoxic and non-cytotoxic                        Cannot be used for crossmatching
                          HLA-specific antibodies
                          More sensitive than LCT
                          Medium cost

Flow cytometry            Highly sensitive                                           Not well standardized
                          Detects weak and early sensitization                       Large panel of cells required to establish antibody specificity
                          Detects cytotoxic and non-cytotoxic antibodies             Expensive
                          Can define class I and class II antibodies simultaneously   Detects non-HLA antibodies



44
                                                                                         Human leucocyte antigens


   In the direct allorecognition pathway, the host’s       The role of HLA antibodies in solid organ trans-
T cells recognize HLA molecules (primarily class        plantation is well established. The presence of cir-
II) expressed on donor tissues, e.g. tissue dendritic   culating HLA-specific antibodies directed against
cells and endothelial cells. Indirect allorecognition   donor antigens in renal and cardiac recipients has
involves the recognition of donor-derived HLA           been associated with hyperacute rejection of the
class I and II antigenic peptides presented by the      graft. It is therefore important that these anti-
host’s own antigen-presenting cells. Because of this    bodies are detected and identified as soon as the
mechanism, HLA antigens are therefore one of the        patient is registered on the transplant waiting list
main barriers to the success of solid organ trans-      to ensure that incompatible donors are not consid-
plantation or BMT and are responsible for the           ered for crossmatching. Furthermore, recent data
strong alloimmunization seen in patients following      have also suggested that the appearance of donor-
transplantation or blood transfusion.                   specific antibodies after transplantation may be a
                                                        sign of rejection, indicating the importance of
                                                        post-transplant monitoring for some groups of
Solid organ transplantation
                                                        patients.
Several studies have confirmed the importance of
matching for HLA-A, -B and -DR antigens. Results
                                                        HLA and BMT
published by the United Network for Organ
Sharing (UNOS) registry have shown that 1-year          The main risk factors affecting the survival of
graft survival in recipients of fully HLA-A, -B and     patients undergoing BMT are graft-versus-host
-DR matched kidneys was 94%, as opposed to the          disease (GVHD), leukaemia relapse, and graft
89% and 90% survival rate observed in recipients        rejection or graft failure.
of one HLA haplotype-matched kidney from a                 The probability of developing acute GVHD is
parent or a sibling, respectively. The differences      directly related to the degree of HLA incompatibil-
between match grades, which are minimal at 1            ity. Although BMT between HLA-identical sib-
year after transplant, become more apparent with        lings ensures matching for the classical HLA genes,
increased follow-up half-life figures.                   acute GVHD still develops in about 20–30% of
   Studies on the relative contribution of HLA-A,       patients transplanted with an HLA-identical
-B and -DR compatibility on the outcome of graft        sibling. This is probably due to the effect of
survival have shown that HLA-DR has the                 untested HLA antigens, e.g. DP, or minor histo-
strongest effect. When grafts from cadaver donors       compatibility antigens in the activation of donor T
are analysed, the 1-year graft survival rate is 88%     cells. However, patients receiving grafts from
for HLA-A, -B and -DR matched and 79% for mis-          HLA-matched unrelated donors have a higher risk
matched kidneys.                                        of developing GVHD than those transplanted
   With the application of the PCR-based tech-          using an HLA-identical sibling.
niques, it is now possible to identify molecular           Original studies on the role of HLA in the
differences between otherwise serologically identi-     outcome of BMT indicated that HLA-DR incom-
cal HLA types of donor and recipient pairs. Corre-      patibility was one of the main risk factors associ-
lation of these results with graft survival has         ated with the development of GVHD. However,
shown a higher graft survival rate when recipients      more recent studies have shown that mismatches
and donors are HLA-DR identical by serological          at the HLA-A alleles, and to a lesser extent HLA-C
and molecular techniques compared with when             alleles, are independent risk factors for death in
they are HLA-DR identical by serological but not        patients following haemopoietic stem cell trans-
molecular methods (87% vs. 69%). The use of             plantation from an unrelated donor. Furthermore,
DNA-based techniques allows the identification           mismatches at the HLA-C locus, as detected by
of ‘minor’ mismatches that were previously unde-        DNA sequencing, has been shown to be associated
tected by serological typing, particularly in the       with graft failure in patients receiving an unrelated
DRb1 chain.                                             BMT.


                                                                                                             45
Chapter 4


   The use of DNA-based methods for the detec-          receiving multiple transfusions can experience
tion of the HLA polymorphisms mentioned above           immunological refractoriness to random platelet
has provided a unique opportunity to improve            transfusions due to the presence of HLA anti-
HLA matching of patients and unrelated donors           bodies. These patients require transfusions of
and to reduce the development of GVHD.                  HLA-matched platelets (see Chapter 9).
However, it has been shown that the increased
GVHD seen as a result of HLA mismatch may
                                                        Blood transfusion
result in a lower relapse rate, probably due to a
GVL response associated with the graft-versus-          The clinical relevance of HLA antigens and anti-
host response. Furthermore, the use of T-cell-          bodies in blood transfusion has been well docu-
depleted marrow, which has successfully decreased       mented. White cells present in transfused products
the incidence of GVHD, also resulted in an              express antigens which, if not identical to those
increased incidence of leukaemia relapse. Thus it       present in the recipient, are able to activate T cells
appears that mature T cells in the marrow, which        and lead to the production of antibodies and/or
may be responsible for GVHD, may also be                effector cells responsible for some of the serious
involved in the elimination of residual leukaemic       complications of blood transfusion. On the other
cells.                                                  hand, antibodies (and sometimes cells) present in
   More recently, cord blood has been successfully      the transfused product may react directly with the
used as a source for haemopoietic stem cells for        relevant antigens in the recipient and in this way
patients requiring marrow reconstitution. Prelim-       provoke a transfusion reaction. The immunologi-
inary clinical data have shown reduced risk and         cal reactions due to the passive transfer of HLA
severity of GVHD following HLA-matched and              antibodies or HLA-specific effector cells include
HLA-mismatched cord blood transplantation. It           transfusion-related acute lung injury (TRALI) and
is possible that the immunological immaturity of        transfusion-associated graft-versus-host disease
cord blood mononuclear cells compared with              (TA-GVHD).
mononuclear cells present in adult bone marrow             TRALI is a relatively under-reported complica-
may result in a reduced GVHD effect. The impact         tion of blood transfusion, characterized by acute
of this on the relapse rates is not yet clear.          respiratory distress, pulmonary oedema and severe
   Conversely, the rate of graft rejection is signifi-   hypoxia. The development of TRALI has been
cantly higher in recipients of an HLA-mismatched        associated with the transfusion of blood com-
transplant than in those receiving a transplant         ponents containing HLA and HNA antibodies
from an HLA-identical sibling (12.3% vs. 2.0%).         reacting with the recipient white cells, causing
Graft failure, which is thought to be mediated          complement activation, accumulation of neutro-
by residual recipient T and/or NK cells reacting        phils in the lungs and oedema. TRALI cases have
with major or minor histocompatibility antigens         also been associated with the presence of white cell
present in the donor marrow cells, has also been        antibodies in recipients reacting with transfused
shown to be due to antibodies reacting with             leucocytes and/or to inter-donor antigen–antibody
donor’s HLA antigens. Thus, rejection is par-           reactions in pooled platelets.
ticularly high in HLA-alloimmunized patients.              TA-GVHD is a rare but often severe and fatal
Furthermore, studies on leukaemic patients with         reaction associated with the transfusion of cellular
donor-specific antibodies and with a positive T- or      blood components. It occurs primarily in immuno-
B-cell cytotoxic crossmatch have a high incidence       suppressed individuals, although it can also occur
of graft failure compared with those with a nega-       in immunocompetent recipients. In this condition,
tive crossmatch. Preformed cytotoxic antibodies         immunocompetent T lymphocytes present in
can also increase the incidence of graft failure in     blood or blood products are able to recognize
patients with aplastic anaemia. However, in spite       HLA and/or minor histocompatibility antigens
of these reports, HLA antibodies are particularly       present on the recipient cells and induce a GHVD
relevant in the post-BMT setting, where patients        reaction similar to that seen following haemopoi-


46
                                                                                              Human leucocyte antigens


etic stem cell transplantation. Diagnosis depends      different mechanisms to explain this association
on finding evidence of donor-derived cells, chro-       have been postulated, including LD with the rele-
mosomes or DNA in the blood and/or affected            vant disease susceptibility gene, the preferential
tissues of the recipient. CD4+ and CD8+ cytotoxic      presentation of the pathogenic peptide by certain
as well as CD4+ T-cell clones lacking direct cyto-     HLA molecules, and molecular mimicry between
toxicity but with supernatants containing TNF-b        certain pathogenic peptides and host-derived
have been isolated from the lesion of patients with    peptides.
TA-GVHD.                                                  Among those shown to be due to linkage with
   Immunological reactions due to antibodies           the relevant HLA-related genes is hereditary
present in the recipient include non-haemolytic        haemochromatosis (HH) and among those dis-
febrile transfusion reaction (NHFTR) and               eases in which HLA antigens seem to be involved
immunological refractoriness to random platelet        in the preferential presentation of peptides to anti-
transfusions. The occurrence of NHFTR has been         genic T cells is neonatal alloimmune thrombocy-
commonly associated with the presence of HLA           topenia. Diseases in which the molecular mimicry
antibodies in the recipient reacting with white        mechanism has been postulated include ankylos-
blood cells present in the transfused product.         ing spondylitis and Klebsiella infection. However,
However more recently, and particularly in coun-       the precise pathogenic mechanisms involved
tries that have introduced universal leucodeple-       remain unknown.
tion, it has been observed that NHFTR can also be         A number of diseases associated with both HLA
triggered by the direct action of cytokines such as    class I and II have been described (Table 4.5).
IL-1b, TNF-a, IL-6 and/or by chemokines such as
IL-8 which are found in transfused products.
   On the other hand, immunological refractori-
ness to random platelet transfusions is primarily
                                                       Table 4.5 HLA-associated diseases.
due to HLA and, to a lesser extent, HPA and high-
titre ABO alloantibodies present in the patient and    HLA class I genes
destroying the transfused platelets. This results in   Birdshot chorioretinopathy: HLA-A29
the lack of platelet increments following the trans-   Behçet’s disease: HLA-B51
fusion of random donor platelets. Although HLA         Ankylosing spondylitis: HLA-B27
antibodies are found in approximately 20–50%           Psoriasis: HLA-Cw6
of multitransfused patients, only approximately        Malaria: HLA-B53
10–20% of them become immunologically re-              HLA class II genes
fractory and require HLA-matched or HLA-               Rheumatoid arthritis
compatible platelet transfusions. Following the           HLA-DRB1*0401
introduction of universal leucodepletion, the pro-        HLA-DRB1*0404
portion of multitransfused patients with HLA              HLA-DRB1*0405
antibodies seems to have decreased to approxi-            HLA-DRB1*0408
mately 10–20% and only about 3–5% of these                HLA-DRB1*0101/0102
                                                          HLA-DRB1*1402
patients are immunologically refractory. These are
                                                          HLA-DRB1*1001
previously sensitized transplanted or transfused
                                                       Narcolepsy: HLA-DQB1*0602/DQA1*0102
recipients including multiparous women.                Coeliac disease: HLA-DQB1*0201/DQA1*0501
                                                       Neonatal alloimmune thrombocytopenia: HLA-DRB3*0101
                                                       Malaria: HLA-DRB1*1302/DQB1*0501
HLA and disease                                        Insulin-dependent diabetes mellitus: HLA-DQB1*0302/DQA1*0301

                                                       HLA-linked diseases
HLA genes are known to be associated with a
                                                       Haemochromatosis: (HLA-A3) HFE gene C282Y, H63D and S65C
variety of autoimmune, non-autoimmune and,             21-OH deficiency: (HLA-B47) 21-OH gene
more recently, infectious diseases. A number of


                                                                                                                  47
Chapter 4


                                                                               H63        D
Hereditary haemochromatosis
HH is a common genetic disorder in northern
Europe, where between 1 in 200 and 1 in 400 indi-                             a1              a2
viduals suffer from the disease, with an estimated
carrier frequency of between 1 in 8 and 1 in 10.                                   NH2
Clinical manifestations include cirrhosis of the                           NH2
liver, diabetes and cardiomyopathy. Detection of
asymptomatic iron overload is important since
removal of excess iron by phlebotomy can prevent                        b2m                        a3
organ damage. Previous screening methods relied
on the measurement of iron saturation, confirmed
                                                                          COOH                          C282   Y
with a fasting sample. Confirmation of HH was
by liver biopsy. Hence a non-invasive screening
method would be an advantage. Previously, a close
association between HLA-A3 and HH had been
described and, until recently, HLA-A3 was the
only test available to aid diagnosis, although this                                      COOH
was not very specific since the majority of HLA-          Fig. 4.5 HFE molecule. b2-m, b2-microglobulin.
A3-positive individuals do not have HH.
   A number of mutations have been identified in
the HFE gene, which is located 3 Mb telomeric            1a antibodies, about 15% of cases are due to
of the HLA region. Clinical data indicate that at        anti-HPA-5b.
least three of these mutations (C282Y, H63D                 Early studies indicated that the production of
and S65C) may predispose and affect the clinical         HPA-1a antibodies is strongly associated with the
outcome of this condition. Over 90% of HH                HLA-DRB3*0101 allele. However, only approxi-
patients in the UK are homozygous for the muta-          mately 35% of HPA-1a-negative, DRB3*0101-
tion that replaces a cysteine (C) with a tyrosine (Y)    positive women develop antibodies upon exposure
at codon 282 in the HFE gene. The second and             to the antigen, suggesting that other genes or
third mutations (H63D and S65C) are thought to           factors may be involved in the development of
be less important, although it may have an additive      alloimmunization against HPA-1a.
effect if inherited with the first mutation (Fig. 4.5).      It has been shown that the amino acid substitu-
Recent studies on blood donors have shown that           tion of leucine for proline at position 33 on the
approximately 1 in 280 donors are homozygous             GPIIIa chain is responsible for the production of
for the mutations.                                       alloantibodies and, more recently, T cells respond-
   A DNA-based technique to detect these three           ing to a peptide containing this residue have been
mutations simultaneously has now been developed          identified in an HPA-1b/b patient with an affected
in our laboratories and provides a simple, rapid         child.
and unambiguous definition of these mutations.

                                                         Further reading
Neonatal alloimmune thrombocytopenia
Neonatal alloimmune thrombocytopenia is due to           Brown C, Navarrete C. HLA antibody screening by LCT,
                                                           LIFT and ELISA. In: Bidwell J, Navarrete C, eds.
fetomaternal incompatibility for human platelet
                                                           Histocompatibility Testing. London: Imperial College
antigens (see also Chapters 5 and 8). More than            Press, 2000: 65–98.
80% of cases occur in women who are homozy-              Campbell RD. The human major histocompatibility
gous for the HPA-1b allele. Although the majority          complex: a 4000-kb segment of the human genome
of cases are associated with the presence of HPA-          replete with genes. In: Davies KE, Tilghman SM, eds.


48
                                                                                                    Human leucocyte antigens

  Genome Analysis, Vol. 5. Regional Physical Mapping.          Madrigal JA, Scott I, Arguello R, Szydlo R, Little A-M,
  New York: Cold Spring Harbor Laboratory Press, 1993:           Goldman JM. Factors influencing the outcome of bone
  1–33.                                                          marrow transplants using unrelated donors. Immunol
Dyer PA, Claas FHJ. A future for HLA matching in clinical        Rev 1997; 157: 153–66.
  transplantation. Eur J Immunogenet 1997; 24: 17–28.          Marsh SG, Albert ED, Bodmer WF et al. Nomenclature for
Gruen JR, Weissman SM. Evolving views of the major               factors of the HLA system. Tissue Antigens 2002; 60:
  histocompatibility complex. Blood 1997; 90: 4252–65.           407–64.
Harrison J, Navarrete C. Selection of platelet donors and      Murac, Raguenes O, Ferec C. HFE mutations analysis in
  provision of HLA matched platelets. In: Bidwell J,             711 haemochromatosis probands: evidence for S65C
  Navarrete C, eds. Histocompatibility Testing. London:          implication in mild form of hemochromatosis. Blood
  Imperial College Press, 2000: 379–90.                          1999; 93: 2502–5.
Howell M, Navarrete C. The HLA system: an update and           Parham P, McQueen KL. Alloreactive killer cells: hindrance
  relevance to patient–donor matching strategies in clinical     and help for haematopoietic transplants. Nat Rev
  transplantation. Vox Sang 1996; 71: 6–12.                      Immunol 2003; 3: 108–21.
Lardy NM, Van Der Hjorst AR, Ten Berge IJM et al.              Suthanthiran M, Strom TB. Renal transplantation. N Engl
  Influence of HLA-DRB1* incompatibility on the                   Med J 1994; 331: 365–76.
  occurrence of rejection episodes and graft survival in       Terasaki PL, McClelland JD. Microdroplet assay of human
  serologically HLA-DR-matched renal transplant                  serum cytokines. Nature 2000; 204: 998–1000.
  combinations. Transplantation 1997; 64: 612–16.              Thorsby E. HLA associated diseases. Hum Immunol 1997;
Madrigal JA, Arguello R, Scott I, Avakian H. Molecular           53: 1–11.
  histocompatibility typing in unrelated donor bone
  marrow transplantation. Blood Rev 1997; 11: 105–17.




                                                                                                                        49
Chapter 5
Platelet and neutrophil antigens

David L. Allen, Geoffrey F. Lucas, Willem H. Ouwehand and Michael F. Murphy




Human platelet antigens                                through interactions with extracellular matrix
                                                       proteins in the vascular endothelium and with
As on red cells, antigens on human platelets can be    plasma coagulation proteins. The majority of these
categorized according to their biochemical nature.     antigens are on the GPIIb/IIIa complex, which
• Carbohydrate antigens on glycolipids and glyco-      plays a central role in platelet aggregation as a
proteins (GPs): A, B and O antigens, P, Le.            receptor for fibrinogen, fibronectin, vitronectin
• Protein antigens: human leucocyte antigen            and von Willebrand factor. Other important
(HLA) class I A, B and C, GPIIb/IIIa, GPIb/IX/V,       GPs are GPIb/IX/V, the main receptor for von
etc.                                                   Willebrand factor, which is involved in platelet
• Haptens: quinine, quinidine; heparin; some           adhesion to damaged vascular endothelium;
antibiotics, e.g. penicillins and cephalosporins.      GPIa/IIa, which is involved in adhesion to colla-
These antigens can be targeted by some or all of       gen; and CD109, which also appears to be a colla-
the following types of antibodies:                     gen receptor. Congenital deficiency of these GPs
• autoantibodies;                                      results in bleeding disorders, e.g. lack of GPIIb/IIIa
• alloantibodies;                                      causes Glanzmann’s thrombasthenia and absence
• isoantibodies; and                                   of GPIb/IX/V results in Bernard–Soulier syn-
• drug-dependent antibodies.                           drome. The expression of platelet alloantigens
   Many platelet antigens are shared with other        located on these GPs may be altered in these disor-
cells, e.g. ABO and HLA class I (Table 5.1). This      ders and HPA typing performed by serological
section focuses on protein alloantigens expressed      assays (‘phenotyping’) may give discrepant results
predominantly on platelets, although some of           when compared with results obtained by molecu-
these are also present to a lesser extent on some      lar typing (‘genotyping’).
other blood cells, e.g. human platelet antigen
(HPA)-5 on activated T lymphocytes. These anti-
                                                       Inheritance and nomenclature
gens are commonly referred to as platelet-specific
alloantigens or human platelet alloantigens.           Most of the platelet alloantigen systems reported
                                                       to date have been shown to be biallelic, with
                                                       each allele being codominant. Historically, systems
Human platelet alloantigens
                                                       were named by the authors first reporting the
There are a number of well-characterized biallelic     system, usually using an abbreviation of the name
platelet alloantigen systems, and a number of rare,    of the patient in whom the antibody was detected.
private or low-frequency antigens have also been       Some systems were published simultaneously by
described (Table 5.2). Most of these antigens were     different laboratories and with different names,
first discovered during the investigation of cases of   e.g. Zw and PlA or Zav/Br/Hc, and only later were
neonatal alloimmune thrombocytopenia (NAIT).           they found to be the same polymorphism. In 1990,
  Platelet-specific alloantigens are located on         a working party for platelet immunology of the
platelet membrane GPs involved in haemostasis          International Society of Blood Transfusion (ISBT)


50
                                                                                                            Platelet and neutrophil antigens

Table 5.1 Antigen expression on peripheral blood cells.

Antigens             Erythrocytes           Platelets            Neutrophils          B lymphocytes     T lymphocytes           Monocytes

A, B, H              +++                    (+)/++               -                    -                 -                       -
I                    +++                    ++                   ++                   -                 -                       -
Rh*                  +++                    -                    -                    -                 -                       -
K                    +++                    -                    -                    -                 -                       -
HLA class I          -/(+)                  +++                  +++                  +++               +++                     +++
HLA class II         -                      -                    -/+++§               +++               -/+++§                  +++
GPIIb/IIIa           -                      +++                  (+)‡                 -                 -                       -
GPIa/IIa             -                      +++                  -                    -                 ++§                     -
GPIb/IX/V            -                      +++                  -                    -                 -                       -
CD109                -                      (+)/++§              -                    -                 -/++§                   +++D

* Non-glycosylated.
§ On activated cells.
‡
  GPIIIa (b3) in association with an alternative a chain (av).
D Dependent on monoclonal antibody used to assess expression.


agreed a new nomenclature for platelet poly-                                   quencies vary between populations, e.g. GPIIIa-
morphisms, the HPA nomenclature. The recently                                  proline-33 (HPA-1b) is extremely rare or absent in
founded international Platelet Nomenclature                                    the Far East, while GPIIIa-glutamine-143 (HPA-
Committee has published guidelines for accept-                                 4b) does not occur in Caucasians. These differ-
ance and naming of newly discovered platelet GP                                ences are important when investigating cases of
alloantigens.                                                                  suspected platelet alloimmunity in different ethnic
   In the HPA nomenclature, each system is num-                                groups.
bered consecutively (HPA-1, -2, -3 and so on) (see                                Until the early 1990s, platelet typing was
Table 5.2) according to its date of discovery, with                            performed by serological assays. These assays
the high-frequency allele in each system being des-                            required the use of monospecific antisera, which
ignated ‘a’ and the low-frequency allele ‘b’. Newly                            were relatively uncommon as the majority of
discovered systems are only officially included                                 immunized individuals produced HLA class I anti-
when confirmed by a second party and approved                                   bodies in addition to the platelet-specific anti-
by the nomenclature committee. If an antibody                                  bodies. The typing that could be performed
against only one allele has been reported, a ‘w’ (for                          therefore was limited, and many laboratories were
workshop) is added after the antigen name, e.g.                                only able to phenotype for HPA-1a. The publica-
HPA-10bw. One possible reason why antibodies                                   tion of more advanced assays, such as monoclonal
against the ‘a’ antigen have not yet been reported                             antibody-specific immobilization of platelet anti-
for many of the recently discovered systems is that                            gens (MAIPA) (Fig. 5.1) permitted more extensive
the ‘b’ allele is of such low frequency that ‘bb’                              phenotyping but some antisera were simply not
homozygous individuals either do not exist or are                              available.
extremely rare.                                                                   With the advent of techniques such as immuno-
   In Caucasian populations, the allele frequency                              precipitation of radioactively labelled platelet
for the majority of HPA systems is skewed towards                              membrane proteins, MAIPA and the polymerase
the ‘a’ allele and homozygosity for the ‘b’ allele is                          chain reaction (PCR), the molecular basis of the
below 3%. This places significant pressures on the                              majority of clinically relevant platelet-specific
blood services in the management of alloimmu-                                  alloantigen systems was elucidated (Fig. 5.1 and
nized patients since compatible red cells and                                  see Table 5.2). The molecular basis for all the HPA
platelets are difficult to obtain for patients with                             alloantigen systems has been determined and in all
antibodies against the ‘a’ alloantigen. Allele fre-                            but one (HPA-14bw) the difference between the


                                                                                                                                        51
Chapter 5

Table 5.2 Platelet-specific alloantigen systems.

System      Antigen         Alternative names   Phenotype frequency* (%)   Glycoprotein (GP)   Nucleotide change   Amino acid change

HPA-1       HPA-1a          Zwa, P1A1            97.9                      GPIIIa              T196                Leucine33
            HPA-1b          Zwb, P1A2            28.8                                          C196                Proline33

HPA-2       HPA-2a          Kob                 >99.9                      GPIba               C524                Threonine145
            HPA-2b          Koa, Siba            13.2                                          T524                Methionine145

HPA-3       HPA-3a          Baka, Leka           80.95                     GPIIb               T2622               Isoleucine843
            HPA-3b          Bakb                 69.8                                          G2622               Serine843

HPA-4       HPA-4a          Yukb, Pena          >99.9                      GPIIIa              G526                Arginine143
            HPA-4b          Yuka, Penb           <0.1                                          A526                Glutamine143

HPA-5       HPA-5a          Brb, Zavb            99.0                      GPIa                G1648               Glutamic acid505
            HPA-5b          Bra, Zava, Hca       19.7                                          A1648               Lysine505

HPA-6       HPA-6bw         Caa, Tua              0.7                      GPIIIa              G1564               Arginine489
                                                                                               A1564               Glutamine489

HPA-7       HPA-7bw         Mo                    0.2                      GPIIIa              C1267               Proline407
                                                                                               G1267               Alanine407

HPA-8       HPA-8bw         Sra                  <0.01                     GPIIIa              C2004               Arginine636
                                                                                               T2004               Cysteine636

HPA-9       HPA-9bw         Maxa                  0.6                      GPIIb               G2603               Valine837
                                                                                               A2603               Methionine837

HPA-10      HPA-10bw        Laa                  <1.6                      GPIIIa              G281                Arginine62
                                                                                               A281                Glutamine62

HPA-11      HPA-11bw        Groa                 <0.25                     GPIIIa              G1996               Arginine633
                                                                                               A1996               Histidine633

HPA-12      HPA-12bw        Iya                   0.4                      GPIbb               G141                Glycine15
                                                                                               A141                Glutamic acid15

HPA-13      HPA-13bw        Sita                  0.25                     GPIa                C2531               Threonine799
                                                                                               T2531               Methionine799

HPA-14      HPA-14bw        Oea                  <0.17                     GPIIIa              D AAG1929–1931      D Lysine611

HPA-15      HPA-15a         Govb                 74                        CD109               C2108               Serine703
            HPA-15b         Gova                 81                                            A2108               Tyrosine703

HPA-16      HPA-16bw        Duva                 <1                        GPIIIa              C517                Threonine140
                                                                                               T517                Isoleucine140
                            Vaa                  <0.4                      GPIIb/IIIa
                            PlT                 >99.9                      GPV
                            Vis                                            GPIV
                            Pea                                            GPIba
                            Dya                                            38 kDa GP
                            Moua                 26                        Unknown

* Frequencies based on studies in Caucasians.




52
                                                                                                                        Platelet and neutrophil antigens


                                                Patient serum
                                                  containing
                                                                                        (a)                                                                        (b)
                                                                                                                             Inert serum
                                                 anti-HPA-1a
                                                                                                                             (negative control)




                                           1.
                                                    Hu                 +          MoMab                                                         +            MoMab

                                                 GPIIb/IIIa                     HLA class-1                               GPIIb/IIIa

                                                              Donor platelets                                                          Donor platelets
                                                                                       GPIa/IIa
                                                                                                           Microplate


                                                  Hu                            MoMab                                                                      MoMab
Fig. 5.1 Monoclonal antibody-specific
immobilization of platelet antigens.       2.
                                                              Donor platelets                                                            Donor platelets

(1) A cocktail of target platelets,
murine monoclonal antibody
(MoMab) directed against the
glycoprotein being studied, e.g.                      Hu                                                                                                   MoMab
GPIIb/IIIa and human serum is              3.                                       MoMab

prepared: (a) the test serum contains
anti-HPA-1a; (b) no anti-platelet
antibodies are present. (2) After
                                                       Hu
incubation a trimeric (a) or dimeric       4.
(b) complex is formed. Excess serum
and MoMab is removed by washing.                        MoMab                                                            MoMab
(3) The platelet membrane is                                                    Goat anti-mouse                                                 Goat anti-mouse
solubilized in a non-ionic detergent,
releasing the complexes into the fluid
                                                                        Enzyme-conjugated goat anti-human Ig
phase and particulate matter is
removed by centrifugation. (4) The                     Hu
lysates containing the
glycoprotein–antibody complexes are        5.
                                                        MoMab                                                            MoMab
added to the wells of a microtitre plate
                                                                                Goat anti-mouse                                                 Goat anti-mouse
previously coated with goat anti-
mouse antibody. (5) Unbound lysate is
removed by washing and an enzyme-
conjugated goat anti-human antibody
added. (6) Excess conjugate is
                                                       Hu
removed by washing and a substrate
solution added. Cleavage of the
substrate, i.e. a colour reaction,         6.           MoMab                                                            MoMab

indicates binding of human antibody                                             Goat anti-mouse                                                 Goat anti-mouse
to the target platelets



two alleles is based on a single nucleotide differ-                                minimal post-PCR handling and has become one
ence that results in a single amino acid substitu-                                 of the cornerstone techniques in HLA typing and
tion. Most polymorphisms have been found on                                        is widely used for HPA genotyping (Fig. 5.3).
GPIIb/IIIa (CD61/CD41) (see Table 5.2 and Fig.                                     High-throughput DNA-based typing techniques
5.2).                                                                              with automated readout are under development
   Based on this knowledge, many molecular                                         and will be used in platelet immunology reference
typing techniques have been developed over the                                     laboratories in the near future.
last decade and these have largely overcome prob-                                     Knowledge of the genetic basis of platelet-
lems in platelet typing. One such assay is PCR                                     specific antigens makes it possible to carry out
using sequence-specific primers (PCR-SSP). This is                                  molecular genotyping on whatever DNA-
a fast and reliable molecular typing technique with                                containing material is available, e.g. platelet typing


                                                                                                                                                                         53
Chapter 5


     GPIIb/IIIa                                                           Fibrinogen binding site
     aIIbb3                                                               RGD binding site
     CD61/CD41
                            GPIIIa                                                                  Fig. 5.2 Schematic representation of
       HPA-4                                                                                        platelet GPIIb/IIIa or the aIIbb3
                                                              GPIIba
       Arg/Gln143                                  Ca++                                             integrin. GPIIIa is recognized by
               HPA-7                                                                                murine monoclonal antibodies of the
               Pro/Ala407                                                                           CD61 cluster and the heterodimer by
        HPA-16                                    Ca++                      HPA-9
        Thr/Ile140                                                                                  antibodies of the CD41 cluster. The
     HPA-10                                                                 Val/Met837              amino acid substitutions arising from
                                           Ca++
     Arg/Gln62                                                                                      the allelic variation of the GPIIb and
  HPA-6                                    Ca++                                                     GPIIIa genes are depicted by white
  Arg/Gln489                                                                                        circles and the name of the HPA
HPA-1                                                                                               system is noted. Amino acids are given
Leu/Pro33                                                 S
                                                                                                    in three-letter acronyms. The
                                                          S                                         fibrinogen-binding sites are in light
                                                                                                    grey and the Arg/Gly/Asp (RGD)-
                                                                                                    binding site is in dark grey. The RGD
 HPA-14                                                                               HPA-3
 Lys611 deletion                     HPA-11                      GPIIbb               Ile/Ser843    peptide is the minimal fibrinogen-
                                     Arg/His633
    HPA-8                   COOH                          COOH
                                                                                                    derived peptide which binds
    Arg/Cys636                                                                                      GPIIb/IIIa.



                                     —                                         platelets through pregnancy or transfusion they
                                                                               may produce anti-GPIV isoantibodies. These
                                                                               isoantibodies are of clinical significance as the sur-
                                                                               vival of transfused normal donor platelets will be
                                                                               reduced or, in pregnancy, NAIT may ensue. Simi-
                     —
                                                                               larly, formation of isoantibodies might complicate
Fig. 5.3 PCR-SSP determination of HPA-1–5 genotypes.                           the transfusion support of patients with congenital
The upper band present in all lanes is the 429-bp product of
                                                                               deficiencies of platelet GPs, such as GPIIb/IIIa
human growth hormone. The lower bands are the products
of sequence-specific primers. The results are read from left
                                                                               (Glanzmann’s        thrombasthenia),      GPIb/IX/V
to right, i.e. lane 1 HPA-1a, lane 2 HPA-1b, etc. The HPA                      (Bernard–Soulier syndrome) or GPVI.
genotype in this case is 1b/1b, 2a/2a, 3a/3b, 4a/4a, 5a/5a.                       Many platelet membrane glycoproteins are the
Courtesy of Dr Paul Metcalfe.                                                  target of autoantibodies in autoimmune thrombo-
                                                                               cytopenia. Such autoantibodies will bind to the
                                                                               platelets of all individuals regardless of their HPA
using fetal DNA from amniocytes or from chori-                                 type. Platelet autoimmunity is frequently associ-
onic villous biopsy samples. However, when used                                ated with B-cell malignancies and autoantibody
in the setting of first-trimester fetal HPA typing,                             formation is not infrequent in the period after
extreme caution is required to exclude possible                                haematopoietic stem cell transplantation during
contamination with maternal cells and consequent                               immune cell re-engraftment. In both situations, the
erroneous typing.                                                              presence of autoantibodies might contribute to the
                                                                               refractoriness to donor platelets.
                                                                                  Some drugs too small to elicit an immune
Platelet isoantigens, autoantigens and haptens
                                                                               response may bind as a hapten to platelet GPs in
In some individuals, certain GPs may be absent                                 vivo. In some patients, the haptenized platelet GP
from the platelet surface, e.g. approximately 4%                               can trigger the formation of antibodies that only
of black and 10% of Japanese individuals do not                                bind to the GP in the presence of the hapten. A
express GPIV (CD36) on their platelets. If these                               classic example is quinine and its stereoisomer
individuals are exposed to normal, GPIV-positive                               quinidine. Typically, quinine-dependent antibodies


54
                                                                                       Platelet and neutrophil antigens


are either anti-GPIIb/IIIa and/or anti-GPIb/IX/V.       sensitivity of these assays is not satisfactory for all
Similarly, the interaction of heparin with platelet     alloantigen systems and some systems (e.g. HPA-
factor 4 can cause antibody formation and lead to       15) are not included.
a drug-dependent thrombocytopenia.
  Besides the more classic examples of quinine and
                                                        Clinical significance of HPA alloantibodies
heparin, many other categories of drugs including
several antibiotics have been associated with           HPA alloantibodies are responsible for the follow-
hapten-mediated platelet antibody formation. In         ing clinical conditions:
haemato-oncology patients, who often receive a          • NAIT (this condition is described in detail
spectrum of drugs, the unravelling of the causes of     below; see also Chapter 8);
persistent thrombocytopenia or poor response to         • refractoriness to platelet transfusions (described
platelet transfusions can be complex and some-          in detail in Chapter 9); and
times frustrating. However, resolution of such a        • post-transfusion purpura (described in detail in
problem, for example the identification of drug-         Chapter 17).
dependent antibodies such as anti-vancomycin
antibodies as the culprit of poor response to
                                                        Neonatal alloimmune thrombocytopenia
platelet transfusions, can be important in improv-
ing patient care.                                       History
                                                        The first case of NAIT was described by van
                                                        Loghem in 1959. The existence of the platelet
Detection of platelet alloantibodies
                                                        equivalent of haemolytic disease of the newborn
Over the last four decades, tests for the detection     had long been suspected, but its recognition was
of platelet-specific antibodies have evolved from        delayed because the detection of platelet anti-
being non-specific and insensitive, e.g. the platelet    bodies was more technically demanding than that
agglutination test and chromium release assay, to       of red cell antibodies.
more sensitive but still non-specific techniques           NAIT is now a well-recognized clinical entity
such as the platelet immunofluorescence test             with an estimated incidence of severe thrombocy-
(PIFT), which is unable to distinguish between          topenia due to maternal alloantibodies of 1 in
platelet-specific and HLA class I antibodies, and to     1000 to 1200 live births. Unlike haemolytic
state-of-the-art GP capture assays such as the          disease of the newborn, about 50% of cases of
MAIPA assay. Despite the limitation described           NAIT occur in first pregnancies.
above, the PIFT remains the most widely used
assay and, when results are analysed by a flow           Definition
cytometer, is one of the most sensitive assays avail-   NAIT is due to maternal HPA alloimmunization
able. The principles of PIFT are shown in Plate 5.1     caused by fetomaternal incompatibility for a fetal
(shown in colour between pp. 304 and 305).              human platelet alloantigen inherited from the
However, it is the MAIPA assay that has become          father but which is absent in the mother. Fetal and
the modern cornerstone for the identification of         neonatal thrombocytopenia may result from pla-
platelet-specific antibodies. This assay captures        cental transfer of IgG antibodies against HPAs.
specific GPs using platelet glycoprotein-specific
monoclonal antibodies and can be used to analyse        Pathophysiology
complex mixtures of antibodies in patient sera.         Maternal IgG alloantibodies against a fetal HPA
The principle of this assay is shown in Fig. 5.1.       alloantigen cross the placenta and bind to fetal
   Detailed knowledge of the molecular basis of         platelets. Dependent on the quantity, affinity and
platelet-specific alloantigens has enabled third-        subclass of the IgG antibodies and the density of
generation antibody detection assays with purified       the target antigen, platelet survival will be reduced.
or recombinant platelet GPs to be developed.            Severe thrombocytopenia and haemorrhage are
However, there remains a disadvantage that the          generally caused by anti-HPA-1a. Anti-HPA-5b


                                                                                                                   55
Chapter 5


tends to cause mild thrombocytopenia, although             The ability of an HPA-1a-negative mother to
intracranial cerebral haemorrhage (ICH) has been        form anti-HPA-1a is controlled by the HLA
reported.                                               DRB3*0101 allele. The chance of antibody forma-
   NAIT due to alloantibodies against the other         tion in an HLA DRB3*0101-negative individual is
HPA alloantigens is infrequent. HLA class I anti-       small when compared with a DRB3*0101-positive
bodies, which are present in 15–25% of multi-           individual, with an odds ratio of 140. Such an
parous women, are not thought to be responsible         explicit linkage between an HLA class II type
for fetal thrombocytopenia as the majority of           and the formation of alloantibodies has not been
antibody is absorbed in the placenta, and if            observed for any of the other platelet-specific
transferred to the fetal compartment will dis-          alloantigen systems.
tribute over all HLA class I positive cells. Destruc-      Although the negative predictive value of the
tion of IgG-coated fetal platelets takes place in the   absence of HLA-DRB3*0101 for HPA-1a alloim-
spleen through interaction with mononuclear             munization in HPA-1a-negative women is greater
cells bearing receptors for the constant domain of      than 90%, its positive predictive value is only
IgG-Fc.                                                 35%, limiting its potential usefulness in an antena-
   Platelet antigens are known to be expressed          tal screening programme. About 10% of HPA-1a-
from 16 weeks’ gestation, and placental transfer        negative pregnant women develop anti-HPA-1a,
of IgG antibodies can occur from 14 weeks, so           and about 30% of these have an affected
thrombocytopenia can occur very early in preg-          fetus/neonate with a platelet count less than
nancy. There have been reports of ICH before 20         50 ¥ 109/L.
weeks’ gestation.
                                                        Clinical features
Incidence                                               A typical case of NAIT presents with skin bleeding
Prospective screening of pregnant Caucasian             (purpura, petechiae and/or ecchymoses) or more
women for HPA-1a alloimmunization has shown             serious haemorrhage, such as ICH, in a full-term
that about 1 in 1100 neonates have severe throm-        and otherwise healthy newborn with a normal
bocytopenia (< 50 ¥ 109/L) because of anti-HPA-         coagulation screen and isolated thrombocytopen-
1a. Anti-HPA-5b is the platelet alloantibody that       ia. There are less common presentations in utero,
most frequently occurs in pregnancy, but it causes      including ventriculomegaly, cerebral cysts and
clinically significant platelet destruction much less    hydrocephalus. Although rare, hydrops fetalis has
frequently than anti-HPA-1a.                            been reported in association with NAIT and this
                                                        diagnosis should be considered if there are no
Relative immunogenicity                                 other obvious reasons for the hydrops.
HPA-1a and HPA-5b are the most immunogenic                 Nearly 50% of severe ICHs occur in utero,
platelet alloantigens and are implicated in about       usually between 30 and 35 weeks’ gestation, but
85% and 10%, respectively, of clinically diag-          sometimes even before 20 weeks. At the other end
nosed cases of NAIT. Alloantibodies in the other        of the clinical spectrum, NAIT can be discovered
systems are less frequently responsible for NAIT.       incidentally when a blood count is performed for
The HPA-15 system was described a decade ago,           other reasons.
but its clinical relevance has only recently been          Severe NAIT with a platelet count below 20 ¥
demonstrated. HPA-15 has now been shown to be           109/L in a neonate is a serious condition. Rapid cor-
the third most commonly encountered alloanti-           rection of the platelet count with HPA-compatible
body specificity and, more importantly, these anti-      platelets is essential to prevent ICH and the possi-
bodies were the only possible explanation for           bility of lifelong disability. This management
the thrombocytopenia in several cases of NAIT.          should be combined with laboratory investigations
Therefore, testing for HPA-15 alloantibodies            to confirm the clinical diagnosis, but platelet trans-
should be included in the routine investigation of      fusion of compatible platelets should not be delayed
suspected NAIT cases.                                   while waiting for the laboratory results.


56
                                                                                      Platelet and neutrophil antigens


Differential diagnosis                                   case, the platelet count should recover to normal
Other causes of neonatal thrombocytopenia are            within a week, although a more protracted recov-
infection, prematurity, intrauterine growth retar-       ery can occur.
dation, maternal platelet autoimmunity and,
rarely, inadequate megakaryocytopoiesis.                 Antenatal management
   Precise figures on the incidence of neonatal           In a subsequent pregnancy of a mother with a pre-
thrombocytopenia caused by viral infection are           viously affected pregnancy with NAIT, a decision
unavailable. Maternal platelet autoimmunity is           needs to be taken on the approach to patient
rarely associated with severe thrombocytopenia in        management in collaboration with a fetal medicine
the neonate, but should be considered in women           unit. Treatment during pregnancy is indicated
with a history of autoimmune thrombocytopenia.           for those cases in which the estimated risk of
   Platelet-type von Willebrand’s disease, in which      severe fetal/neonatal thrombocytopenia is consid-
mutations in the GPIba gene are associated with a        erable and this is based on the history of haem-
propensity for in vitro platelet aggregation, can        orrhage and thrombocytopenia in previous
lead to falsely low platelet counts.                     pregnancies.
                                                            The possible treatments during pregnancy are:
Laboratory investigations                                • intrauterine intravascular transfusion of com-
Only antibodies against platelet-specific alloanti-       patible platelets combined with fetal blood sam-
gens are thought to cause immune thrombocyto-            pling (FBS) at weekly intervals or just before
penia in the fetus and neonate. For appropriate          delivery;
clinical management, the cause of severe thrombo-        • intravenous immunoglobulin (IVIG) and/or cor-
cytopenia in an otherwise healthy neonate should         ticosteroids administered to the mother; or
be determined with urgency. Detection of maternal        • a combination of the above.
platelet-specific antibodies is usually carried out by       As few trials have been performed in this field, it
two techniques, the indirect PIFT and the MAIPA          is difficult to make definite recommendations.
assay, using a panel of HPA-typed platelets.             Weekly platelet transfusions by FBS have been
   The most frequently detected antibodies are           shown to prevent ICH even in high-risk cases (Fig.
anti-HPA-1a, anti-HPA-5b and anti-HPA-15b.               5.4), but are technically demanding, invasive and
The serum of the mother is also tested against           associated with a high mortality due to haemor-
paternal platelets by both tests so that any alloanti-   rhage, cardiac dysrhythmias and premature
bodies against low-frequency alloantigens and            labour. The high incidence of complications asso-
private antigens can be detected. The parents are        ciated with FBS and the effectiveness of maternal
typed for the HPA-1, HPA-2, HPA-3, HPA-5                 therapy in about 67% of cases suggest that treat-
and HPA-15 alloantigens using PCR-SSP. In                ment with IVIG (1 g/kg per week) without an
Caucasians, typing is usually confined to the afore-      initial FBS should be used as initial management.
mentioned five systems as the immunogenicity of           This should be considered from or before 16
the other alloantigen systems is comparatively low.      weeks’ gestation in those cases where there is a
                                                         history of antenatal ICH in previous pregnancies,
Neonatal management                                      because the earliest reports of ICH are at 16 weeks.
A neonatal platelet count below 20 ¥ 109/L should        IVIG could be started a few weeks later (20–22
be corrected immediately, preferably with HPA-           weeks) for those fetuses with a sibling history of
1a- and HPA-5b-negative donor platelets, as these        severe thrombocytopenia but no antenatal ICH.
will be compatible with the maternal HPA alloan-         FBS may be used to monitor the effect of maternal
tibody in over 95% of cases. The results of the lab-     therapy (4–8 weeks after starting IVIG) and to
oratory investigations should not delay immediate        indicate if alternative therapy is required. Failure
platelet transfusion, as full investigation may be       of maternal therapy may potentially be rescued by
time-consuming and the risk of cerebral bleeds is        the addition of oral prednisolone (0.5 mg/kg daily)
highest in the first 48 h after delivery. In a typical    and/or an increased dose of IVIG (2 g/kg per


                                                                                                                  57
Chapter 5



                                          10 000



                                           1 000



                      Platelets x 109/L
                                                                                                                        300

                                            100
                                                                                                 Platelet transfusion
                                                                                                                        30

                                             10



                                              1
                                                   25   26   27   28 29      30 31    32    33      5     12    19
                                                                    Weeks gestation                Days postnatal
                                                                                              CS

Fig. 5.4 Seventh pregnancy of a patient who has had five                        until delivery. The figure shows pre-transfusion and post-
miscarriages. The last of these was shown to have hydrops                      transfusion platelet counts following serial FBS and platelet
and hydrocephalus and a platelet count of only 17 ¥ 109/L,                     transfusions. The fetal platelet count was less than
and the serological findings supported a diagnosis of                           10 ¥ 109/L at 26 weeks. The aim was to maintain the fetal
neonatal alloimmune thrombocytopenia due to anti-HPA-                          platelet count above 30 ¥ 109/L by raising the immediate
1a. The fetal platelet count was less than 10 ¥ 109/L at 25                    post-transfusion platelet count to above 300 ¥ 109/L after
weeks’ gestation in the sixth pregnancy, and a cord                            each transfusion. The fetal platelet count fell below
haematoma developed during fetal blood sampling (FBS)                          10 ¥ 109/L on one occasion when there were problems in
resulting in fetal death. In the seventh pregnancy,                            preparing the fetal platelet concentrate and the dose of
prednisolone 20 mg daily and intravenous IVIG 1 g/kg                           platelets was inadequate. CS, Caesarean section.
weekly were administered to the mother from 16 weeks


week), serial fetal platelet transfusions or early                             antenatal intracranial hemorrhage and severe
delivery at an acceptable gestation. Further studies                           thrombocytopenia (platelet count < 20 ¥ 109/L) in
are required to determine the optimal antenatal                                a previous pregnancy;
management for NAIT.                                                           • antibody specificity and titre do not reliably
  The delivery also needs careful planning                                     correlate with the severity of NAIT, and are cur-
between obstetric and paediatric teams in close                                rently of no value in the management of individual
consultation with the consultant haematologist.                                cases;
                                                                               • the zygosity of the partner.
Counselling
Counselling of couples with an index case about                                HPA-typed donor panels
the risks of severe fetal/neonatal thrombocytopen-                             Establishing donor panels for fetal and neonatal
ia in a subsequent pregnancy needs to be based on                              platelet transfusion requires a major commitment
the severity of disease in the index case and the                              from blood services. The frequency of HPA-1a-
outcome of immunological investigations. The fol-                              negative donors in Caucasians is 2.5%. Therefore,
lowing should be taken into account:                                           identification of suitable donors requires simple,
• thrombocytopenia in subsequent cases is as                                   affordable and high-throughput typing tech-
severe or, generally, more severe;                                             niques, as available for red cell phenotyping.
• the best predictors of severe fetal thrombocy-                               Recently such techniques have been developed
topenia in a future pregnancy are the occurrence of                            for HPA-1a phenotyping. Besides donors being


58
                                                                                                                  Platelet and neutrophil antigens


negative for the mandatory microbiological tests,                       antigens, e.g. HNA-1a, HNA-1b, HNA-1c poly-
they should also test negative for antibodies                           morphisms on CD16. The current nomenclature
against red cells, platelets and leucocytes, and be                     for the HNA systems includes polymorphisms that
cytomegalovirus seronegative. It has been calcu-                        are both cell specific and ‘shared’ (Table 5.3).
lated that in order to recruit one HPA-1a-negative
donor who is able to meet all of the above criteria,
                                                                        HNA-1 system
approximately 1500–2000 donors will have to be
phenotyped for HPA-1a. In addition, therapeutic                         The most immunogenic polymorphisms, the trial-
platelets should be RhD matched, as small                               lelic HNA-1 alloantigen system, are localized on
amounts of red cells present in platelet concen-                        neutrophil FcgRIIIb (CD16), which is one of the
trates may immunize RhD-negative recipients,                            two low-affinity receptors (R) for the constant
and negative for high-titre anti-A and anti-B anti-                     domain (Fc) of human IgG (g). Four amino acid
bodies. In order to recruit a single O RhD-negative                     changes with arginine/serine, asparagine/serine,
HPA-1a-negative donor whose platelets will be                           aspartic acid/asparagine and valine/isoleucine sub-
suitable for a first fetal or neonatal platelet transfu-                 stitutions at positions 36, 65, 82 and 106 respec-
sion, where the fetal/neonatal blood group is                           tively define the difference between HNA-1a and
unknown, approximately 15 000–20 000 donors                             -1b, and a single amino acid substitution
need to be phenotyped for HPA-1a.                                       (alanine/asparagine) at position 78 defines the
                                                                        HNA-1c polymorphism (Fig. 5.5). The expression
                                                                        of HNA-1c is frequently associated with the pres-
Human neutrophil antigens                                               ence of an additional FcgRIIIb gene and increased
                                                                        expression of FcgRIIIb.
The antigens on the surface of human neutrophils                           Two other FcgRIIIb-associated high-frequency
can, as with platelets, be divided into different cat-                  alloantigens have been reported: the LAN antigen
egories. There are common antigens that have a                          and SAR antigen. The FcgRIIIb ‘null’ phenotype is
wider distribution on other blood cells and tissues,                    rare and is based on a double deletion of the
e.g. I and P blood group systems and HLA class I.                       FcgRIIIb gene and is in some cases associated with
There are ‘shared’ antigens which have a limited                        a deletion of the FcgRIIc gene. The deficiency for
distribution among other cell types, e.g. human                         the most abundant FcgR on neutrophils can cause
neutrophil alloantigen (HNA)-4a and -5a poly-                           immune neutropenia in the newborn due to mater-
morphisms associated with CD11/18. There are                            nal isoantibodies against FcgRIIIb.
also a limited number of truly granulocyte-specific                         PCR-SSP can be used to determine the HNA-1a,


                                                              FcgRIIIbHNA-1a               FcgRIIIbHNA-1b                    FcgRIIIbHNA-1c

                                                         Arg (36)                          Ser (36)                              Ser (36)
                                                                    S                                 S                                     S
Fig. 5.5 Representation of the amino          Asn (65)                          Ser (65)                          Ser (65)
acid substitutions resulting in the            Ala (78)             S             Ala (78)            S               Asp (78)              S
HNA-1a, HNA-1b and HNA-1c                          Asp (82)
                                                                        Val (106)      Asn (82)           Ile (106)      Asn (82)               Ile (106)
forms of FcgRIIIb. The positions of the
amino acid substitutions arising from                               S                                 S                                     S
allelic variation of the FcgRIIIb gene
are depicted by black dots. Amino
                                                                    S                                 S                                     S
acids are given in three-letter
                                                                           Phosphatidylinositol
acronyms. The intrachain disulphide
bonds create two domains which are        Neutrophil
closely related to the C-terminal         surface
heavy-chain domains of IgG.               membrane


                                                                                                                                                     59
Chapter 5

Table 5.3 Neutrophil-specific alloantigen systems.

                             Original acronym       Caucasian phenotype                    Nucleotide    Amino acid
System           Antigen     for antigen            frequency (%)           Glycoprotein   change        change

HNA-1            HNA-1a      NA1                     46                     FcgRIIIb       G108          Arginine36
                                                                                           C114          None
                                                                                           A197          Asparagine65
                                                                                           G247          Aspartic
                                                                                           G319          acid82
                                                                                                         Valine106
                 HNA-1b      NA2                     88                     FcgRIIIb       C108          Serine36
                                                                                           T114          None
                                                                                           G197          Serine65
                                                                                           A247          Asparagine82
                                                                                           A319          Isoleucine106
                 HNA-1c      SH+                      5                     FcgRIIIb       A266          Aspartic
                                                                                                         acid78
                             SH-                                                           C266          Alanine78

HNA-2            HNA-2a      NB1                     97                     CD177          nk            nk
                             NB2                     32                     nk             nk            nk
                 ND          ND1                     98.5                   nk             nk            nk
                 NE          NE1                     23                     nk             nk            nk
                 LAN         LANa                   >99                     FcgRIIIb       nk            nk
                 SAR         SARa                   >99                     FcgRIIIb       nk            nk
                 Five        5a                      —                      nk             nk            nk

HNA-3a           Five        5b                       —                     70–95 kDa      nk            nk

HNA-4a           Mart        Marta(+)                99.1                   CD11b          G302          Arginine61
                             Marta(-)                                       CD11b          A302          Histidine61

HNA-5a           Ond         Onda(+)                >99                     CD11a          G2466         Arginine766
                             Onda(-)                                        CD11a          C2466         Threonine766

nk, not known.




-1b, -1c, -4a, -4b and FcgRIIIb null genotypes, and               viduals and HNA-2a alloantibodies typically give
transfected cells expressing the FcgRIIIb HNA-1a,                 a bimodal fluorescence profile with granulocytes
-1b and -1c allotypes have been used for alloanti-                from HNA-2a-positive donors. The gene encoding
body detection.                                                   the HNA-2a protein has recently been cloned and
                                                                  HNA-2a alloantigen status can be determined by
                                                                  phenotyping with polyclonal or monoclonal anti-
HNA-2 alloantigen
                                                                  bodies. The HNA-2a-negative phenotype is the
HNA-2a, formerly known as NB1, is localized on                    result of a point mutation in the mRNA and there
a 58–64 kDa GP (CD177) expressed as a glyco-                      is no antithetical antigen to HNA-2a. An anti-NB2
sylphosphatidylinositol-anchored membrane GP                      antiserum was originally thought to define the
found both on neutrophil surface membranes and                    antithetical antigen to HNA-2a (NB1) but this
on secondary granules. The percentage of neu-                     serum may instead recognize human monocyte
trophils expressing HNA-2a varies between indi-                   antigen 1.


60
                                                                                     Platelet and neutrophil antigens


                                                        based on the use of monospecific alloantisera
Alloantigens on CD11a and CD11b
                                                        derived from immunized patients. However, for
The genes encoding the aM and aL subunits of the        some HNA, e.g. HNA-1a, HNA-1b and HNA-2a,
b2 integrin or CD11b and CD11a are polymorphic          murine monoclonal antibodies with allele-specific
and are associated with HNA-4a and HNA-5a               reactivity are available. Furthermore, for some of
respectively. Alloantibody formation against these      the neutrophil alloantigen systems (HNA-1a, -1b,
two polymorphisms has been observed in transfu-         -1c, -4a, -4b) the molecular basis has been deter-
sion recipients, and recently a case of neonatal        mined and PCR-based genotyping can be used.
neutropenia due to anti-HNA-4a alloantibodies
has been described. The low incidence of neonatal
                                                        Clinical significance of HNA antibodies
neutropenia associated with these antibodies is
probably explained by the ubiquitous tissue distri-     Neutrophil-specific antibodies are implicated in:
bution of these proteins.                               • neonatal alloimmune neutropenia;
                                                        • non-haemolytic febrile transfusion reactions
                                                        (FNHTR) (also see Chapter 14);
Detection of neutrophil antibodies
                                                        • transfusion-related acute lung injury (TRALI)
The detection of neutrophil antibodies is difficult.     (also see Chapter 14);
The main problems are the abundant expression of        • transfusion-related alloimmune neutropenia;
the two low-affinity receptors for human IgG or          • autoimmune neutropenia;
FcgR, which results in increased binding of circu-      • persistent post-bone marrow transplant
lating immunoglubulins in normal sera and the           neutropenia.
requirement for fresh and typed donor neutrophils
as panel cells. The incidence of antibody-mediated
                                                        Neonatal alloimmune neutropenia
neutropenias is comparatively rare and therefore
the best strategy for investigation of clinical cases   Maternal alloimmunization against neutrophil-
is in a centralized laboratory so that adequate         specific alloantigens on fetal/neonatal neutrophils
technical expertise and the required reagents are       is rare as a clinically significant entity. The inci-
available.                                              dence of the disorder is estimated at 0.1–0.2% of
   Many techniques have been evaluated over the         live births but there are no reliable prevalence
years for their suitability for neutrophil antibody     figures. Clinical presentation is one of mainly bac-
detection. First-generation assays such as the gran-    terial infection with a selective neutropenia on a
ulocyte cytotoxicity and agglutination tests had a      whole blood count. Severe but reversible neutro-
very low specificity. The neutrophil immunofluo-          penia in the newborn may require treatment with
rescence and chemiluminescence tests have the           antibiotics and/or granulocyte colony-stimulating
advantage of good sensitivity but also detect HLA       factor (GCSF) to control bacterial infection and
class I antibodies. For some human neutrophil           hasten development of a normal neutrophil count.
alloantigen systems, e.g. antigens expressed on         Left untreated, the neutropenia in some cases has
CD16, CD177 and C11/18, assays comparable               been reported to extend up to 28 weeks, presum-
with the MAIPA can be applied but otherwise             ably because the target antigen is restricted to a
immunoprecipitation of surface radioactive-             small compartment of antigen-positive cells.
labelled neutrophils remains the only reliable tech-
nique to determine the nature of the antigen. The
                                                        FNHTR and TRALI (see Chapter 14)
principles of the granulocyte immunofluorescence
test and the monoclonal antibody immobilization         FNHTR are occasionally associated with the
of granulocyte antigens (MAIGA) assay are analo-        presence of leucocyte (HLA and neutrophil-
gous to the equivalent platelet tests described in      specific) alloantibodies in the recipient. Serological
Plate 5.1 and Fig. 5.1 respectively.                    investigations are of limited clinical value as
   Typing for neutrophil alloantigens is generally      the diagnostic specificity of tests for leucocyte


                                                                                                                 61
Chapter 5


antibodies is low. Moreover, the clinical manage-       sample with a panel of typed neutrophils in the
ment of FNHTR is to alter product specification if       indirect immunofluorescence and chemilumines-
premedication with corticosteroids is not effective.    cence tests is a suitable alternative and, in some
In the UK, where there is universal leucocyte deple-    studies, has been found to be only slightly less sen-
tion of blood, investigations for other causes of       sitive than the direct test.
fever associated with transfusion should be carried
out, e.g. tests for bacterial contamination. Testing
                                                        Persistent post-bone marrow transplant
for neutrophil-specific antibodies might be
                                                        neutropenia
required in the rare cases in which a severe
FNHTR cannot be otherwise explained.                    Antibody mediated neutropenia may be a serious
   TRALI is a severe and sometimes life-threaten-       complication of bone marrow transplantation. In
ing transfusion reaction. The majority of cases are     this context, the neutrophil antibodies may be
caused by donor leucocyte alloantibodies against        antoimmune and/or alloimmune in nature and
alloantigens present on the patient’s leucocytes        laboratory investigation requires serological and
although patient alloantibodies might be involved       typing studies to elucidate the nature of the anti-
in some cases. Investigations for TRALI are com-        bodies involved.
plex and need to include a screen for neutrophil-
specific and HLA alloantibodies in samples from
donors and the patient.                                 Further reading

                                                        Birchall JE, Murphy MF, Kaplan C, Kroll H on behalf of the
Transfusion-related alloimmune neutropenia                European FMAIT Study Group. European collaborative
The first case of transfusion-related alloimmune           study for the antenatal management of fetomaternal
                                                          alloimmune thrombocytopenia. Br J Haematol 2003;
neutropenia was recently described following the
                                                          122: 275–88.
infusion of plasma-reduced blood into a 4-week          Bussel JB, Zabusky MR, Berkowitz RL, McFarland JG.
old infant. The blood contained approximately             Fetal alloimmune thrombocytopenia. N Engl J Med
28 mL of plasma containing granulocyte-specific            1997; 337: 22–6.
HNA-1b antibodies and resulted in an absolute           Bux J, Jung KD, Kauth T, Mueller-Eckhardt C. Serological
neutropenia in the infant, which was resolved after       and clinical aspects of granulocyte antibodies leading to
treatment with GCSF after 7 days. The case is of          alloimmune neonatal neutropenia. Transfus Med 1992;
                                                          2: 143–9.
interest since it demonstrates that in some circum-
                                                        Bux J, Behrens G, Jaeger G, Welte K. Diagnosis and clinical
stances infused granulocyte-specific antibodies can        course of autoimmune neutropenia in infancy: analysis of
trigger neutropenia rather than TRALI.                    240 cases. Blood 1998; 91: 181–6.
                                                        Garner SF, Smethurst PA, Merienx, Y et al. A rapid one
                                                          stage whole blood HPA-1a phenotyping assay using a
Autoimmune neutropenia                                    recombinant monoclonal IgG1 anti-HPA-1a. Br J
Autoimmune neutropenia is a rare condition that           Haematol 2000; 108: 440–7.
                                                        Griffin HM, Ouwehand WH. A human monoclonal
can occur as a transient self-limiting autoimmunity
                                                          antibody specific for the leucine-33 (P1A1, HPA-1a) form
in young children or in a chronic form in adults.         of platelet glycoprotein IIIa from a V gene phage display
The autoantibodies tend to target the FcgRIIIb            library. Blood 1995; 86: 4430–6.
(CD16), CD177 or CD11/18 molecules but can              Lucas GF, Metcalfe P. Platelet and granulocyte
also be HNA specific, especially in children. The          polymorphisms. Transfus Med 2000; 10: 157–74.
most sensitive method for the detection of auto-        Maslanka K, Yassai M, Gorski J. Molecular identification
antibodies is to test the patient’s neutrophils using     of T cells that respond in a primary bulk culture to a
                                                          peptide derived from a platelet glycoprotein implicated in
the direct immunofluorescence test. However, the
                                                          neonatal alloimmune thrombocytopenia. J Clin Invest
combination of severe neutropenia and the                 1996; 98: 1802–8.
requirement for a fresh sample limits the applica-
bility of this test. Screening of a patient’s serum


62
                                                                                              Platelet and neutrophil antigens

Metcalfe P, Watkins NA, Ouwehand WH et al.                   Von dem Borne AE, de Haas M, Roos D, Homburg CH,
  Nomenclature of human platelet antigens. Vox Sang            van der Schoot CE. Neutrophil antigens, from bench to
  2003; 85: 240–5.                                             bedside. Immunol Invest 1995; 24: 245–72.
Murphy MF, Waters AH, Doughty HA et al. Antenatal            Warkentin TE, Smith JW. The alloimmune
  management of fetal alloimmune thrombocytopenia:             thrombocytopenic syndromes. Transfus Med Rev 1997;
  report of 15 affected pregnancies. Transfus Med 1994; 4:     11: 296–307.
  281–92.                                                    Watkins NA, Smethurst PA, Allen D, Smith GA, Ouwehand
Newman PJ, Valentin N. Human platelet alloantigens:            WH. Platelet alphaIIb/beta3 recombinant autoantibodies
  recent findings, new perspectives. Thromb Haemost             from the B-cell repertoire of a post-transfusion purpura
  1995; 74: 234–9.                                             patient. Br J Haematol 2002; 116: 677–85.
Shastri KA, Logue GL. Autoimmune neutropenia. Blood          Williamson LM, Hackett G, Rennie J et al. The natural
  1993; 81: 1984–95.                                           history of fetomaternal alloimmunization to the platelet-
Van Loghem JJ, Hanny Dorfmeijer Jr, van der Hart,              specific antigen HPA-1a (PlA1, Zwa) as determined by
  Schreuder F. Serological and genetical studies on a          antenatal screening. Blood 1998; 92: 2280–7.
  platelet antigen (Zw). Vox Sang 1959; 4: 161–9.
Von dem Borne AE, Ouwehand WH. Immunology of
  platelet disorders. Baillieres Clin Haematol 1989; 2:
  749–81.




                                                                                                                          63
Part 2


Clinical transfusion practice
Chapter 6
The effective and safe use of blood components

Brian McClelland and Tim Walsh




Principles                                                  The value of systematic review evidence as a
                                                         guide to best practice depends on the quality of the
This chapter focuses on clinical decisions about the     original trials and also on whether they reflect
transfusion of blood components and emphasizes           current practice. Systematic reviews may clearly
the opportunities for minimizing the need to trans-      identify therapies that are effective, ineffective or
fuse and safe practice. The reasons for taking this      harmful but often show that the available trials are
approach will be evident from the chapters on            simply inadequate to identify the clinical situations
adverse effects of transfusion. There is a high level    in which a blood component or product is benefi-
of concern to avoid the well-publicized risks of         cial or harmful. For example, Cochrane reviews of
transfusion. Although transfusion is very safe in        the effects of various intravenous replacement
the context of all the risks of hospital care, it is     fluids on mortality in severely ill patients are sum-
important that patients, and the wider public,           marized in Table 6.1. These led to considerable
understand that like all medical interventions it        controversy, recently resolved by a large clinical
can never be entirely free of risks.                     trial in Australia that has established that albumin
                                                         and saline solutions are effectively equivalent in
                                                         terms of safety and effectiveness when used for
Evidence for effectiveness
                                                         resuscitation of critically ill patients.
Clinical effectiveness, i.e. the measure of how
much a treatment can help the patient, is a balance
                                                         Information and consent
of benefits and risks. Many of the conventional
and widely taught indications for transfusing            Patients must be informed about the benefits, risks
blood components are not supported by reliable           and choices for their treatment whenever possible,
evidence of clinical benefit. It is important to take a   although this obviously cannot be done if the
critical approach to prescribing blood compon-           patient is unconscious or too sick to communicate.
ents. Guidelines for practice should, but do not         For patients awaiting elective surgery, the pre-
always, reflect the best available evidence for clini-    admission clinic is an ideal opportunity to include
cal effectiveness.                                       information about transfusion as part of the
   There are few good randomized, controlled,            information given to the patient about the whole
clinical trials of some of the main uses of transfu-     process of care. Formal consent for transfusion is
sion. Some important recent trials challenge con-        not required in the UK, but the prescriber has a
ventional teaching (see Chapter 36). Systematic          professional duty to make sure the patient knows
review (usually with analysis of the pooled results)     if transfusion is intended. The patient should be
of clinical trials or other studies is an important      made aware of the risks of receiving blood (and of
way of examining the basis of current practice, and      not receiving it) and of ways in which transfusion
often reveals that conventional current practice is      could be avoided, if this is an option. Whatever
based on small studies that are not well designed        method is used to give this information, the
(see Chapter 37).                                        patient’s notes should record that it has been


                                                                                                           67
Chapter 6

Table 6.1 Clinical effectiveness of conventional transfusion                    disabling anaemia. However, many transfusions
practices: the evidence may challenge conventional wisdom.                      are given in situations where it is very difficult
                                                                                to estimate the probability that the patient will
Human albumin and artificial colloids for infusion in critically ill patients*
                                                                                benefit. The challenge in clinical practice is to
Meta-analysis of clinical trials shows no evidence to suggest benefit
                                                                                make this judgement in the many clinical situa-
from the use of albumin vs. use of crystalloid, or the use of artificial
colloids vs. use of crystalloid. Controversy about the suggestion that the
                                                                                tions where there is real uncertainty. An example
use of albumin may increase mortality has been resolved with the                is the decision whether or not to transfuse an
publication of the SAFE study                                                   elderly patient with a moderately low haemoglo-
                                                                                bin (Hb) level in the postoperative period. Retro-
Red cell transfusion†
                                                                                spective studies of patients operated on for hip
Study of 838 sick patients in intensive care unit. Randomized to
transfusion to maintain Hb at 7–9 g/dL or 10–12 g/dL. Mortality at 30
                                                                                fracture reveal great variability in the use of peri-
days:                                                                           operative red cell transfusion. There is at present
   7–9 g/dL, 19%                                                                no evidence whether such patients are likely to
   10–12 g/dL, 23%                                                              benefit if they are ‘transfused up’ or if they are
                                                                                allowed to remain moderately anaemic. Two pilot
* Cochrane Injuries Group Albumin Reviewers (1998); Schierhout &                randomized controlled trials in hip fracture
Roberts (1998); SAFE Study Investigators (2004).                                patients examined the effect of alternative transfu-
†
  Hébert et al. (1999).                                                         sion protocols (transfuse at a ‘trigger’ level of
                                                                                10 g/dL, transfuse only for symptoms). The use of
given and that the patient’s questions have been                                the 10 g/dL trigger resulted in much more transfu-
answered. Suitable information leaflets are avail-                               sion (Table 6.2).
able on the websites of the various blood services.                                From a public health perspective, good blood
                                                                                management embraces wider issues such as
                                                                                anaemia prevention through programmes on
Surgical and medical use of blood                                               nutrition, malaria, human immunodeficiency virus
                                                                                (HIV) infection, parasite infestation, and so on.
Recent studies in the UK show that about half of
all red cell transfusions are given to surgical
                                                                                Blood management in elective surgery
patients and about half for medical indications.
The following sections outline some general prin-                               In elective surgery, transfusion cannot be consid-
ciples that apply to all patients who may need                                  ered in isolation as attention to the many individ-
transfusion and some that relate to specific cat-                                ual elements of management can greatly influence
egories of patients.                                                            the need to transfuse. This probably explains the
                                                                                low blood requirements of some surgical teams
                                                                                compared with others. Many studies have revealed
Good blood management: avoiding the need
                                                                                large differences in the use of blood components
for transfusion
                                                                                for a given surgical procedure in apparently similar
The guiding principle is that allogeneic (donor)                                patient populations managed in different hospitals
blood should be prescribed only when there are                                  (Fig. 6.1). This can only partly be explained by
good grounds for believing that the benefits of                                  obvious factors such as the patient’s age, gender
transfusing outweigh the risks. Put another way,                                and preoperative Hb status or by reported surgical
blood should only be prescribed when the clinician                              variables such as blood loss or duration of
is satisfied that the risk of not transfusing is likely                          operation.
to be greater than the risk of transfusing. The deci-                              The use of specific blood conservation tech-
sion is straightforward in some situations, for                                 nologies can, for some patients, further reduce the
example when a patient has major haemorrhage                                    need for allogeneic transfusion and may offer some
associated with profound thrombocytopenia, or                                   other benefits (see Chapter 26). However, when
when a patient being treated for cancer has severe                              these technologies are introduced they can be a


68
                                                                                                         Effective use of blood components

Table 6.2 Influence of changing the
‘transfusion trigger’ on blood use in                                              Scotland †                     USA
hip fracture patients: results from two
pilot clinical trials. Trial design:      Number eligible                          73                             192
patients admitted for repair of hip       Number consented                         61                             143
fracture (mean age 85 years) were         Postoperative Hb < 10 g/dL > 8 g/dL      23                              96
randomized to either ‘Symptomatic’
(transfuse if symptoms or if Hb           Randomized                                 9            9                 40           40
< 8 g/dL and clinician wishes to          Arm of trial                             Trigger        Symptom         Trigger        Symptom
transfuse) vs. ‘Trigger’ (transfuse to    Completed protocol                         9            9                 37           35
maintain Hb just > 10 g/dL)*.             Transfused: n (%)                          9 (100)      1 (11)            39 (98)      19 (45)

                                          * Data from Carson et al. (1998) and Palmer et al. (1998).
                                          †
                                            Two patients randomized in this series are included among the 80 randomized patients in the USA
                                          study.




Fig. 6.1 Percentage (mean ± SE) of              Total hip replacement                                    Right and left hemicolectomy
operated patients perioperatively
                                                100
transfused with red cell units in each
hospital, after adjustment for age,              80
gender, preoperative haematocrit and             60
blood loss, in total hip replacement             40
and right and left hemicolectomy.
Each hospital is identified by a                  20
country code followed by a letter, e.g.           0
NL (Netherlands), hospital A (NLA).                   DKA DKB GBC GBF GBH GBI NLA NLB                      BB    DB EA NLA NLB
(From Sanguis Study Group 1994                                     Hospital                                        Hospital
with permission.)




focus of attention for patients and professionals,                  tions in practice. Recorded Hb values and transfu-
with strong encouragement from the companies                        sion information provide objective data for clinical
that develop and market them (Table 6.3). This                      quality assurance.
should not distract attention from the need for
very critical evaluation of their ability to improve
                                                                    Blood management targets and interventions:
some important outcomes. A recent systematic
                                                                    elective surgery and emergencies
review of autologous transfusion techniques raises
considerable doubt about their effectiveness.                       1 Optimize the Hb level before planned surgery.
                                                                      (a) Detect, identify cause and treat anaemia.
                                                                      (b) Erythropoietin with iron.
Transfusion protocols or local practice guidelines
                                                                    2 Optimize iron stores before surgery, even if
A systematic review of the effects of clinical trans-               patient not anaemic.
fusion protocols indicates that the use of a proto-                 3 Optimize haemostasis before planned surgery.
col can reduce the use of blood. As an example, a                     (a) Identify congenital coagulation disorders.
protocol that specifies guideline Hb concentrations                    (b) Withdraw drugs that impair haemostasis
for transfusion and a plan for routine measure-                       (if safe to do so).
ment of Hb and possibly haemostasis at relevant                     4 Collect patient’s own blood before surgery.
stages can reduce large and unexplainable varia-                    5 Improve haemostasis during surgery.


                                                                                                                                        69
Chapter 6

Table 6.3 In elective surgery, the use of autologous blood and/or drugs reduces bleeding and erythropoietin reduces the use of
allogeneic transfusion. (From Laupacis & Fergusson 1997 with permission.)

                                                                                                                Exposure to allogeneic
                                                                                                                (donor) blood. Odds ratio
Intervention                 Type of surgery                               No. of trials      No. of patients   (95% confidence interval)

Autologous techniques
PAD                          Miscellaneous                                  6                  933              0.17 (0.08–0.32)
ANH                          Miscellaneous                                 16                  615              0.31 (0.15–0.62)
ANH                          Miscellaneous, methodologically sound          8                 NR                0.64 (0.31–1.31)

Cell salvage
Washed                       Orthopaedic                                    7                  429              0.39 (0.30–0.51)
Unwashed                     Orthopaedic                                    9                  733              0.35 (0.26–0.46)
Unwashed                     Cardiac                                       12                  899              0.85 (0.79–0.92)

Drugs
Aprotinin                    Cardiac                                       45                 5808              0.31 (0.25–0.39)
Desmopressin                 Cardiac                                       12                  793              0.98 (0.64–1.50)
Tranexamic acid              Cardiac                                       12                  882              0.50 (0.34–0.76)
Aminocaproic acid            Cardiac                                        3                  118              0.20 (0.004–1.12)
Erythropoietin + PAD         Orthopaedic                                   11                  825              0.42 (0.28–0.62)
Erythropoietin + PAD         Cardiac                                        5                  224              0.25 (0.08–0.82)
Erythropoietin alone         Orthopaedic                                    3                  684              0.36 (0.24–0.56)
Erythropoietin alone         Cardiac                                        2                  245              0.25 (0.06–1.04)

ANH, acute normovolaemic haemodilution; NR, not recorded; PAD, preoperative autologous blood donation.



   (a) Anaesthetic techniques.                                           potent stimulator of erythropoiesis, although some
   (b) Surgical techniques.                                              recent evidence suggests it may also have other
   (c) Positioning.                                                      physiological effects. Effective use in the surgical
   (d) Temperature control.                                              context may require parenteral iron repletion.
6 Collect and reinfuse blood lost during surgery:                           Erythropoietin, like red cell transfusion, has its
intraoperative blood salvage.                                            risks. If the Hb level is raised too rapidly, or too
7 Collect and reinfuse blood after surgery: post-                        high, the risk of hypertension and thrombosis
operative blood salvage.                                                 increase. Some patients receiving long-term ery-
  Autologous       transfusion  techniques    are                        thropoietin treatment by the subcutaneous route
described in Chapter 26.                                                 have developed red cell aplasia. In at least some
                                                                         cases this appears to reflect an immunological
                                                                         response to the drug and is a reminder that even
Erythropoietin (EPO, epoetin, human
                                                                         infrequent adverse events due to alternatives to
recombinant erythropoietin)
                                                                         conventional transfusion must not be ignored.
This is a peptide hormone, normally made in the                             Newer parenteral iron preparations, often used
kidney (see Chapter 35). The therapeutic product                         with erythropoietin to deliver the iron required for
is made by genetically engineered expression of the                      rapid erythropoiesis, still have an appreciable risk
human erythropoietin gene. There are currently                           of moderate and severe acute reactions, especially
three products licensed in the UK, with some dif-                        if the manufacturer’s dose and administration
ferences in the indications. Erythropoietin is a                         instructions are not closely followed.



70
                                                                                  Effective use of blood components


Pharmacological agents used to reduce                 Effectiveness of blood management interventions:
surgical bleeding                                     understanding the systematic reviews
These include aprotinin, which affects platelet       There are two ways of expressing the size of the
function and fibrinolysis, tranexamic acid and         effect that an intervention has on an outcome (see
e-aminocaproic acid (inhibitors of fibrinolysis),      also Chapter 37). If the intervention has no effect,
and desmopressin (1-deamino-8-D -arginine vaso-       the ratio between treatment and control groups is
pressin, DDAVP), which acts centrally to increase     1.0. In Table 6.3, the outcome of interest is the use
plasma factor VIII levels. These agents are de-       of allogeneic red cells. If the intervention reduces
scribed in detail in Chapter 7.                       the use of allogeneic red cells, the odds ratio or
                                                      relative risk is less than 1.0. If the intervention
                                                      were to increase transfusion, the ratios would be
Recombinant factor VIIa
                                                      greater than 1.0.
This product was developed to assist in managing         The 95% confidence intervals (CI) are one way
haemophilia patients with inhibitors, for which it    of showing the statistical strength of the result.
has proved effective. It has also been used to        Take the first example in Table 6.3, i.e. pre-
control massive surgical, traumatic or obstetric      operative autologous donation: the 95% CI is
bleeding, although it is not licensed for these       0.08–0.32. This indicates that there is a strong
indications. Formal trials of its effectiveness are   effect on the outcome and that there is less than a
needed in these indications. Because there may be     5% chance that the intervention is not effective. In
risks of thrombotic complications and because the     contrast, in the third example in Table 6.3 (acute
drug is currently extremely expensive, hospitals      isovolaemic haemodilution) the upper 95% CI is
have special procedures for making it available,      greater than 1.0. It cannot be concluded in this
e.g. consultation with a haematologist with an        case that the treatment is effective.
interest in haemostasis.
                                                      A ‘total quality management’ approach for
Do these interventions reduce the need for            minimizing anaemia, bleeding and the need
allogeneic blood transfusion in surgical patients?    for transfusion
Clinical trials to answer this question have been     Individual surgical teams can manage their
subject to systematic reviews with meta-analysis.     patients so as to minimize the need for transfusion
A number of recent papers are summarized in           without recourse to specific blood-sparing tech-
Table 6.3. These methods reduce the use of            nologies. This may reflect the use of protocols to
allogeneic transfusion but may have other             guide transfusion decisions and a commitment
consequences. For example, predeposit auto-           to attend to the many individual details of
logous transfusion usually increases the total        management that can reduce the need for blood
amount of red cell units transfused when both         replacement.
autologous and allogeneic units are counted. It is       A quality assurance system for management of
also important to note that reductions in red cell    anaemia and bleeding in surgery could be defined
transfusion are much more evident in studies          as an integrated approach that covers all aspects
that record a high blood use in the control group.    of management that influence the quality of care
In other words, some surgical teams manage            provided for the patient and ensures that they are
their patients using transfusion infrequently and     consistently and correctly handled. Figure 6.2
without recourse to any of the above blood-           illustrates aspects of care of a surgical patient that
sparing methods.                                      individually and collectively have an influence on
                                                      the use of transfusion during an episode of care.




                                                                                                               71
Chapter 6



     Preoperative                                             Intraoperative                                         Postoperative
      Preoperative                            Non-emergency                  Trauma/                                  Postoperative
      management                              surgery                        haemorrhage                              management

        Assess patient for conditions           Plan surgical and               Assess and resuscitate:                  Monitor, control and replace
        that may reduce oxygen delivery,        anaesthetic                       clear airway                           fluid and red cell losses;
        increase blood loss or reduce           management to:                    insert oropharyngeal airway            optimize oxygen supply and
        red cell production:                      Minimize blood loss                or endotracheal tube                temperature and analgesia
          anaemia                                 anaesthetic technique           give oxygen supplement
          iron deficiency                         vasoconstrictors                check for chest injury, seal           Monitor blood losses:
          blood coagulation problems              tourniquets                        open wound                           drains
          dehydration                             control of bleeding             if pneumothorax, insert drain           wounds
          malaria                                   points                           with underwater seal
          infections                              support circulation             control bleeding; local                Surgical re-exploration of
          cardiorespiratory disease                 posture                       pressure at bleeding site              continued bleeding

        Diagnose anaemia                        Fluid replacement               Monitor:                                 Give oxygen supplement
                                                                                  pulse, blood pressure,
        Treat anaemia                           Salvage and reinfuse blood        capillary refill                       Maintain fluid and electrolyte
                                                                                  conscious level                        balance
        Replenish iron stores                   Replace red cell losses by      Estimate blood/fluid loss
                                                transfusion if clinically                                                Maintain temperature control
        Stop drugs that impair blood            required                        Set up intravenous line and
        clotting                                                                infuse normal saline 3x                  Control pain
                                                                                estimated blood loss rapidly
        Correct fluid and electrolyte                                                                                    Treat infection
        balance                                                                 Decide if transfusion needed:
                                                                                 very urgent group O or                  Decide if transfusion is needed
        Recognize medical conditions:                                               patient's group
          treat or other necessary action                                        less urgent: ensure grouped             Treat postoperative anaemia:
          hepatitis, other virus                                                    and compatible blood is                ferrous sulphate
            infections                                                              available                              folic acid
          bacterial infection
          cardiovascular disease                                                Reassess                                 Treat other medical conditions
          respiratory disease
          others                                                                Operate to stop bleeding

        Decide if transfusion needed:                                           Replace red cell losses by
         autologous blood                                                       transfusion if clinically required
         donor blood

        Order blood, if clinically required


Fig. 6.2 Framework for minimizing the need for transfusion in perioperative management.


                                                                               On this basis, transfusion is only indicated if
Indications for the use of
                                                                               reduction in oxygen supply causes a clinically sig-
blood components
                                                                               nificant problem, and if it is likely that oxygen
Red cells and whole blood (Table 6.4)                                          supply will be improved by a rapid increase in red
                                                                               cell mass. Current practice in surgery and critical
Acute anaemia and bleeding: clinical assessment
                                                                               care in developed countries, influenced by one
and the decision to transfuse
                                                                               large trial on intensive care unit (ICU) patients (see
The generally accepted reason for transfusing red                              Table 6.1), is to adopt Hb concentrations of
cells is to increase the circulating red cell mass as a                        7–8 g/dL as the threshold below which transfusion
means of improving oxygen supply to the tissues.                               is indicated.


72
                                                                                                                        Effective use of blood components

Table 6.4 Use of red cell components. (From Sanguis Study Group 1994 with permission.)

To increase circulating red cell mass with the intention to relieve clinical features caused by insufficient oxygen delivery
In special situations such as sickle cell disease, thalassaemia (see Chapter 9)

Examples of clinical quality assurance indicators: red cell prescription indicator
                                                                                                   Observed

Patient records contain correct details of red cell transfusion                                    95% of records
Hb value recorded before transfusion                                                               90% of records
Patient records contain a stated reason for transfusion                                            23% of records
‘Avoidable’ red cell transfusions                                                                  % of red cell transfusions
Percentage of red cell units given that result in discharge haematocrit > 33%                      23% (lowest clinical unit), 82% (highest clinical unit)

Red cell transfusion during patient admission for specified procedure
                                                                                                   Pt/Po                                        u/Pt

Colectomy                                                                                           0–79 (41)                                   0.3 (1)
Transurethral resection of the prostate                                                             0–46 (17)                                   0–1 (0.5)
Coronary artery bypass graft                                                                       17–100 (83)                                  0–6 (3)
AAA                                                                                                64–100 (83)                                  0–6 (3)
Total hip replacement                                                                              29–100 (81)                                  0.5 (2)

Po, patients operated; Pt, patients transfused; Pt/Po, percentage of patients who are transfused; u/Pt, number of units received by transfused patients.

   Recent studies raise some questions about both                                    decision to transfuse is also influenced by known
the above rationale for red cell transfusion and the                                 and estimated blood loss and by the clinical judge-
adoption of this threshold Hb level. When resting                                    ment of the risk of further bleeding.
subjects are intentionally haemodiluted to very
low Hb concentrations, evidence of myocardial
                                                                                     Chronic anaemia
ischaemia is rare and only appears at Hb concen-
trations of 5–6 g/dL. When stable ICU patients                                       Patients with chronic renal failure have improved
were transfused with 2 units of red cells at an Hb                                   clinical outcomes if Hb concentrations are main-
level of 8–9 g/dL, there was no observable                                           tained near normal. The same may be true of some
improvement in any of the ICU measures of                                            patients with heart failure. Clinical experience,
systemic or regional oxygenation. These findings                                      backed by at least one study, is that many chroni-
appear to suggest that even lower Hb thresholds                                      cally anaemic patients including those with malig-
could be appropriate for red cell transfusion, at                                    nant disease have an improved quality of life if Hb
least in some patient groups. On the other side of                                   concentrations are kept nearer the normal range.
the debate, it is known that the performance of                                      Clinical trials have not been done to determine
athletes improves objectively when Hb concentra-                                     whether such patients benefit equally when the Hb
tions are raised to the high normal range by                                         concentration is maintained by erythropoietin
autologous transfusion or erythropoietin.                                            treatment or by red cell transfusion.
   In some clinical situations, the Hb concentration                                    The available evidence does not indicate that the
may be a poor indicator of the need to restore red                                   conservative transfusion guidelines currently rec-
cell mass by transfusion. After some types of major                                  ommended for surgical and critically ill patients
surgery, notably involving cardiopulmonary by-                                       are suitable for patients with chronic anaemia due
pass, there may be a period when Hb concentra-                                       to a serious underlying disorder. Current guide-
tion is lowered by the effects of haemodilution as                                   lines therefore advise that for surgical or critically
well as blood loss and must be interpreted in the                                    ill patients with evidence of heart disease, trans-
light of the patient’s fluid status. In practice, the                                 fusion at a Hb concentration around 10 g/dL is a


                                                                                                                                                       73
Chapter 6


reasonable compromise until better evidence is                            making the decision into a simple checklist or algo-
available.                                                                rithm that can be used (on paper or in the head) to
                                                                          help to focus the decision on whether or not to
                                                                          transfuse. Figure 6.3 is an illustration of this
Clinical factors relevant to the decision to
                                                                          approach.
transfuse (Table 6.5)
One way to aid clinical decision-making is to sum-
                                                                          Single-unit transfusion of red cells?
marize the factors that are most important in
                                                                          Dogmatic statements have often been made that
                                                                          there is no case for giving a single-unit transfusion.
Table 6.5 Factors in deciding whether a patient needs                     This dogma should be ignored. For example, in the
transfusion.                                                              case of a 45-kg patient with hypoxic signs or symp-
                                                                          toms attributed to a Hb concentration of 7 g/dL, a
Blood loss
                                                                          single unit of red cells may be quite sufficient to
External bleeding
                                                                          relieve symptoms (and raise the Hb concentration
Internal bleeding: non-traumatic, e.g. peptic ulcer, varices, ectopic
                                                                          by 1–2 g/dL).
   pregnancy, antepartum haemorrhage, ruptured uterus
Internal bleeding: traumatic, e.g. chest, spleen, pelvis, femur(s)
Red cell destruction, e.g. malaria, sepsis, HIV                           Choice of blood components
Haemolyis
                                                                          Whole blood versus red cell concentrate (Table 6.6)
Malaria
Sepsis                                                                    The doctrine of blood component therapy
HIV                                                                       (together with the requirement for plasma for frac-
Cardiorespiratory state and tissue oxygenation                            tionation) has encouraged the almost universal use
Pulse rate                                                                of red cell concentrates in most developed coun-
Blood pressure                                                            tries. In the UK, there is a case to review the
Respiratory rate                                                          relative merits of whole blood versus red cell
Capillary refill                                                           concentrates, since UK plasma is not currently
Peripheral pulses                                                         used for fractionation as a precautionary mea-
Temperature of extremities                                                sure against the risk of transmitting variant
Dyspnoea
                                                                          Creutzfeldt–Jakob disease (vCJD) (although
Cardiac failure
                                                                          current thinking is that minimizing the plasma
Angina
                                                                          content of red cell products may offer some reduc-
Conscious level
Urine output                                                              tion of any vCJD risk) (see Chapter 20). Whole
                                                                          blood may be entirely appropriate for a patient
Haemoglobin estimate                                                      with acute bleeding who requires both red cells
Clinical: tongue, palms, eyes, nails
                                                                          and expansion of plasma volume. In cases when
Laboratory: haemoglobin or haematocrit (PCV)
                                                                          disseminated intravascular coagulation (DIC) con-
Patient’s tolerance of blood loss and anaemia                             tributes to blood loss, it may be entirely logical to
Age                                                                       use whole blood (or leucocyte-depleted whole
Other conditions, e.g. diabetes, pre-eclampsic toxaemia, renal failure,   blood) since it contains at least part of the total
   cardiorespiratory disease, chronic lung disease, acute infection,
                                                                          dose of fibrinogen and stable clotting factors that
   treatment with b-blockers
                                                                          the patient requires and can reduce the need for
Anticipated need for blood                                                plasma units from other donors. The suggestion
Is surgery or anaesthesia anticipated?                                    that whole blood may be appropriate for some
Is bleeding continuing, stopped or likely to recur?                       patients will be seen by some as highly controver-
Is haemolysis continuing?                                                 sial, although in many parts of the world it is
                                                                          widely used.
HIV, human immunodeficiency virus; PCV, packed cell volume.


74
                                                                                                     Effective use of blood components



                                        Patient
                                        Name                                   Age                              Gender
                                        Hospital reference no.                 Date of assessment               Time


                                       No                   Hb < 11


                                                               Yes

                                                    Signs and/or symptoms of
                                       No             inadequate O2 supply
                                                            to tissues                            Pale
                                                                                                  Breathless
                                                               Yes                                Tachycardia
                                                                                                  Other

                                       No                  Comorbidity

                                                               Yes                                Malaria
                                                                                                  Sepsis
                                                                                                  Fever
                                                     Expected                                     Haemolysis
                                                       Delivery                                   Leukaemia
                                                       Bleeding                                   Ischaemic heart disease
                                                       Surgery                                    Other

                                       No              Haemolysis
                                                       Bone marrow failure
                                                       Other

                                                     Hb g/dL
                                                      Sample date

                                        Action (based on the information you have recorded above)

                                        Doses of red cell concentrate to raise Hb by 1g/dL:
                                        Adult: 1 unit (250 mL)/5O kg                   Infant/child: 3 mL/kg

                                        Decision:            Transfuse                 No
                                                                                       Yes                Units/mL
                                        Intended result:     Clinical
                                                             [Hb] raise to:            g/dL

                                        Review of result     Date:                     Time:
Fig. 6.3 Example of a transfusion
                                        Clinical                                       [Hb]
decision chart.



                                                                     and so receiving less transfusion, may have
Fresh or stored red cells for transfusion?
                                                                     improved outcomes. One interpretation of this
A widely quoted clinical study has suggested that                    observation is that this could be associated with
transfusion of stored red cells could actually                       some adverse effect of transfusing stored red cells.
impair regional oxygenation. The clinical trial                      This could not be confirmed in a recent blinded,
summarized in Table 6.1 suggested that some ICU                      randomized, controlled study comparing the effect
patients maintained at a lower Hb concentration,                     of fresh versus stored leucodepleted red cells


                                                                                                                                  75
Chapter 6

Table 6.6 Whole blood or red cell concentrates?

Whole blood                                                                   Red cell concentrates

Contains plasma                                                               Minimal plasma content
Replaces fibrinogen and other stable coagulation factors                       Does not replace coagulation factors
Volume-expanding effect of plasma may be an advantage in a                    If colloid volume expansion is shown to be harmful, the absence of
  hypovolaemic patient                                                            plasma could be a benefit
Red cells and plasma from the same donor                                      If both red cells and plasma are required, the patient is exposed to
                                                                                  more donors
Colloid volume expansion effect could cause volume overload                   Less volume load per dose of red cells, so safer if the patient is
                                                                                  normovolaemic
Does not produce plasma                                                       Provides plasma (FFP is likely to be used inappropriately if available!)
Does not contain platelets or factor VIII
In UK and some other countries supplied as leucocyte-depleted
Little or no good clinical trial evidence to compare the effectiveness of
   whole blood vs. red cell concentrates
Whole blood is intrinsically simpler and cheaper to prepare.This may be
   extremely important in countries with restricted health budgets

FFP, fresh frozen plasma.



on systemic and regional oxygenation in ICU                                 Table 6.7 Use of fresh frozen plasma (FFP) and cryoprecipitate.
patients. At present there is no clear evidence to
support the selection of fresh red cells for critically                     Replacement of plasma coagulation factors if a suitable licensed virus-
                                                                              inactivated product is not available
ill patients, although the ‘fresh versus stored’
                                                                            Some special indications, e.g. thrombotic thrombocytopenia purpura:
question cannot yet be considered to be fully
                                                                              infusion of plasma or plasma exchange with FFP (see Chapter 11)
answered.
                                                                            Contraindications
                                                                            To replace circulatory fluid volume
Fresh frozen plasma (Table 6.7)                                             To raise plasma albumin level
                                                                            As an alternative to total parenteral nutrition
Worldwide, the largest avoidable risk to patients
from transfusion is probably due to the trans-                              Example of clinical quality assurance indicator: FFP prescription
fusion of fresh frozen plasma (FFP) for unproven                                                               Observed*
clinical indications. In any area where blood
safety testing may be unreliable, transfusion of FFP                                                           Pre-transfusion        Post-transfusion
                                                                            Indicator                          (%)                    (%)
can be an important source of transmission of
these infections. A recent systematic review sug-
                                                                            Prothrombin ratio recorded         94                     94
gests that there are few well-supported indica-
                                                                            Prothrombin ratio > 2              68                     11
tions for transfusing FFP and this is reflected in
the recent UK clinical guideline. Plasma is just                            * 19 FFP transfusion episodes in 12 consecutive patients (unpublished
as likely as whole blood to transmit HIV or                                 local audit report).
hepatitis.
   FFP should be used only to replace rare clotting
factor deficiencies for which no virus-safe fraction-                        are the management of thrombotic thrombocy-
ated plasma product is available or when there is a                         topenic purpura (TTP) and haemolytic–uraemic
multifactor deficiency due to severe bleeding and                            syndrome, in which plasma infusion or plasma
DIC. Other indications for FFP (see Chapter 11)                             exchange with FFP is effective. A recent systematic



76
                                                                                                Effective use of blood components


review concludes that there is little sound evidence     should be used for patients born after 1996
for other uses of FFP.                                   according to Department of Health guidance.

Does FFP have to be used immediately
                                                         Platelets (Table 6.8)
after thawing?
After thawing, the level of factor VIII falls rapidly.   Use of platelet transfusions continues to grow
Factor V also falls, but levels of fibrinogen and the     annually. Audit against current guidelines may
other haemostatic proteins are maintained. New           reveal possible ways of reducing prescribing
UK guidelines permit the use of plasma that has          within current guidelines. A brief summary of indi-
been stored in the bloodbank for up to 24 h after        cations for platelet transfusion follows (see also
thawing. This has the advantage that plasma can          Chapter 9).
be released quickly when required for urgent man-
agement of massive bleeding.                             Prophylaxis of bleeding due to bone marrow failure
                                                         with thrombocytopenia
Minimizing vCJD risk in the UK: importation of           Recent studies indicate that the clinically stable
plasma and pathogen-reduced plasma                       patient is unlikely to benefit from prophylactic
In the UK, because of concerns about the risks of        platelet transfusion if the platelet count is greater
transmitting vCJD (see Chapter 20), the Depart-          than 10 ¥ 109/L. A higher threshold for transfusion
ment of Health has recommended that plasma               is appropriate with sepsis and other complications.
imported from countries not affected by bovine           Trials in progress are assessing the benefits of pro-
spongiform encephalopathy (BSE) be used for all          phylactic platelets versus platelets given only for
patients born after 1 January 1996 for whom FFP          bleeding.
is indicated. To safeguard against viral infections
undetected by testing, this plasma is treated to         Surgery in the thrombocytopenic patient
further reduce the risk of infectivity and is termed     UK guidelines provide recommendations to mini-
‘pathogen-reduced plasma’. This additional pro-          mize the risks due to bleeding in critical surgical
cessing causes reduced levels of plasma procoagu-        sites such as head, neck and spinal canal.
lants such as fibrinogen, possibly necessitating
higher volumes of plasma to achieve a given effect.      Table 6.8 Use of platelets (platelet concentrate).
In one pathogen-reduced plasma product, the
levels of natural anticoagulants such as protein         Treat bleeding due to thrombocytopenia, for example
S were reduced. This was associated with an                 platelet count < 10 ¥ 109/L due to bone marrow failure
increased risk of thrombosis in patients undergo-           platelet count < 50 ¥ 109/L prior to surgery in critical area (head and
ing liver transplantation. A similar problem may               neck) or invasive procedure (see Chapter 9)
                                                            in management of haemorrhage (‘massive transfusion’)
have occurred in patients undergoing plasma
                                                            during/after surgery on cardiopulmonary bypass, where ‘pump’
exchange for TTP.
                                                               damages platelets (see Chapters 9 and 11)

                                                         Example of clinical quality assurance indicator: platelet prescription
Cryoprecipitate (see Table 6.7)                          Indicator (platelet count recorded)           Observed (% of transfusion
                                                                                                       episodes)*
If virus-inactivated plasma fractions are available,
cryoprecipitate is only indicated as a source of
                                                         Within guideline                             70
fibrinogen in the management of DIC, e.g. in
                                                         Pre-transfusion                              89
obstetric haemorrhage. If no virus-inactivated           Post-transfusion                             85
plasma fraction is available, cryoprecipitate is used    Increased by at least 20 ¥ 109/L             70
to replace factor VIII in haemophilia A and von
Willebrand’s disease (see Chapter 11). Pathogen-         * 1701 episodes of platelet transfusion in 138 patients (unpublished
reduced cryoprecipitate from non-UK plasma               local audit report).




                                                                                                                                77
Chapter 6


Urgent and emergency transfusion                                    and discussed in detail in other chapters. This
                                                                    section concludes by reviewing some of the issues
Examples of clinical scenarios in which transfu-                    that arise in clinical situations where there is acute
sion may be needed are summarized in Table 6.9,                     major bleeding, or the expectation of it, so that
                                                                    blood is required very rapidly. There is little or no
Table 6.9 Some clinical situations where component                  systematic evidence for the effectiveness of the
transfusion may be needed.                                          measures suggested and trials to evaluate them are
                                                                    required, although they will be extremely difficult
                                    Factors that may influence the
                                                                    to do.
Situation                           decision to transfuse
                                                                      Clinical and blood bank experience is that
Emergencies                                                         delays in providing blood in a life-threatening
Obstetric haemorrhage               Clinical assessment during      emergency can put patients at risk, although there
                                       resuscitation                are few published reports that adequately docu-
Trauma                                                              ment this. Consistent use of a simple, agreed ter-
Ruptured aortic aneurysm                                            minology and for urgent contacts between, for
Upper gastrointestinal bleeding                                     example, the labour ward and the blood bank may
Elective surgery                                                    save lives.
Cardiac                             Previously fit patient
Orthopaedic                         Elderly, cardiovascular
                                                                    Hospital Major Haemorrhage Protocol
                                       disease
Solid tumours                       Liver disease                   The use of a Hospital Major Haemorrhage Proto-
                                    Other comorbidity               col communicated effectively to all relevant staff
                                    Pre-existing anaemia            is a sensible measure to improve the manage-
                                    Bleeding tendency               ment of such cases. Occasional ‘fire drills’ to fami-
                                    Jehovah’s Witness
                                                                    larize staff and test the protocol have been found
Bone marrow failure                                                 useful by staff in at least one large teaching
Malignant                           Stable                          hospital. Analysis by national haemovigilance
Myelodysplastic                     Fever                           schemes of ‘near miss’ incidents in supplying
Drugs or chemicals                  Splenomegaly                    blood in emergencies would be a valuable way of
Infective                           Platelet count
                                                                    sharing experience and building knowledge of
                                    Stage of treatment
                                                                    effective strategies for providing blood safely in
ITU patient                                                         emergencies.
Postoperative recovery              Oxygenation measurements
Sepsis syndrome                     Haemoglobin
                                    Platelet count                  Dealing with emergencies

Inherited haemoglobin disorders                                     A single patient with catastrophic bleeding is a
Infections                                                          major challenge for the clinical team. Road traffic
Malaria                             Age                             accidents and other less common disasters may
HIV                                 Pregnancy, delivery             bring several desperately ill patients in quick
                                    Surgery                         succession, adding the risks that can result
Immunological disorders                                             from uncertainties in patient identification. These
Immune haemolysis                                                   are situations when it is vital for all the team to
Immune cytopenias                                                   know and use the established local emergency
Neonatal problems                                                   procedures for:
Haemolytic disease of the newborn                                   • requesting blood;
Blood sampling                                                      • completing request forms and labelling samples;
                                                                    • communicating precisely with the transfusion
HIV, human immunodeficiency virus.                                   laboratory; and


78
                                                                                     Effective use of blood components


• checking blood components before collection             7 Tell the blood bank how quickly the blood is
and transfusion.                                        needed for each patient. Communicate using
It is extremely important to have clear communi-        words that have been previously agreed with the
cation between clinicians and the blood bank, and       blood bank to explain how urgently blood is
essential to have a simple standard procedure, such     needed.
as that described below. All staff (medical, nursing,    8 Do not ask for crossmatched blood in an emer-
laboratory and transport) play a very important         gency. Ask the blood bank to supply what can be
part and should be trained in the use of this           provided most quickly with reasonable safety
procedure, including an understanding of the            according to the local policy.
importance of their role.                                 9 Make sure that both you and the blood bank
                                                        staff know:
                                                           (a) who is going to bring the blood to the
Emergency transfusion procedures
                                                           patient; and
Resuscitation and management of massive blood              (b) where the patient will be, e.g. if your patient
loss are dealt with in Chapters 7 and 11 and more          is about to be transferred to another part of the
information about hospital transfusion procedures          hospital for an X-ray, make sure the blood will
can be found in Chapter 24. An example of a pro-           be delivered to the X-ray room.
tocol for massive blood transfusion is shown in         10 The blood bank may send group O RhD nega-
Table 6.10, and some key principles are described       tive blood, especially if there is any risk of errors in
below.                                                  patient identification. In an emergency, this may be
 1 In an emergency, insert an intravenous cannula,      the safest way to avoid a serious mismatched
use it to take the blood sample for crossmatching,      transfusion.
set up the intravenous infusion, and get the blood
sample and blood request form to the blood bank
as quickly as possible.                                 Avoiding the biggest risks of transfusion
  2 Make sure yourself that the blood bank staff
know when the blood is required and why.                A full review of transfusion risks is given in Chap-
  3 For each patient, the crossmatch sample tube        ters 13–20. This section deals with risks that are
and the blood request form must be clearly              common and which clinical staff can prevent by
labelled. If the patient is unidentified, some form of   adhering to simple procedures. The first is the risk
emergency admission number should be used. Use          of patients receiving a blood component that was
the patient’s name only if you are sure you have        intended for someone else, and the second is that
correct information.                                    a special requirement, e.g. gamma-irradiated or
  4 If another request for blood is needed for the      cytomegalovirus (CMV)-seronegative blood, is
same patient within a short period, use the same        not specified when blood is ordered. Following the
identifiers as on the first request form and blood        simple rules shown in Fig. 6.4 and below will go a
sample so that blood bank staff will know it is the     long way to preventing avoidable harm to patients.
same patient.                                           • When completing the blood request form, make
  5 If there are several staff working with emer-       sure the need for the transfusion and any special
gency cases, one person should take charge of           product, such as CMV-seronegative or gamma-
ordering blood and communicating with the blood         irradiated blood components, is clearly stated.
bank about the incident. This is especially impor-      • Recording your decision to prescribe and
tant if several injured patients are involved at the    administer a transfusion. There are two important
same time.                                              reasons for writing in the patient’s notes the reason
 6 If there is a special stock of ‘emergency O nega-    for giving a transfusion. First, it concentrates the
tive’ blood, e.g. in the labour ward, use this first     mind to have to write and sign a permanent record
in an emergency. Do not wait for crossmatched           of the basis of your clinical decision. Second,
blood if the patient is exsanguinating.                 should the patient have a problem related to the


                                                                                                                  79
Table 6.10 Example of a hospital policy for massive blood loss. (Adapted from Stainsby et al. 2000 with permission.)

Immediate actions                      Key points                                                Other considerations

Arrest bleeding                        Early surgical or obstetric intervention
                                       Upper gastrointestinal tract procedures
                                       Interventional radiology

Contact key personnel                  Most appropriate surgical team
                                       Duty anaesthetist
                                       Blood transfusion laboratory

Restore circulating volume             Insert wide-bore peripheral cannulae                      Blood loss is often underestimated
  NB In patients with major            Give adequate volumes of crystalloid/blood                Refer to local guidelines for the resuscitation of
  vessel or cardiac injury, it         Aim to maintain normal blood pressure and urine              trauma patients and for red cell transfusion
  may be appropriate to restrict          output > 30 mL/h in adults (0.5 mL/kg per h)           Monitor CVP if haemodynamically unstable
  volume replacement after
  discussion with surgical team

Request laboratory                     FBC, PT,APTT, fibrinogen; blood bank sample,               Take samples at earliest opportunity as results
  investigations                         biochemical profile, blood gases                            may be affected by colloid infusion
                                       Ensure correct sample identity and use of red label       Misidentification is commonest transfusion risk
                                         for transfusion samples                                 May need to give FFP and platelets before the
                                       Repeat FBC, PT, APTT, fibrinogen every 4 h, or after          FBC and coagulation results available
                                         one-third blood volume replacement, or after
                                         infusion of FFP

Request suitable red cells             Blood needed immediately                                  Contact blood transfusion laboratory or on-call
  NB All red cells are now             Use ‘emergency stock’ group O RhD negative                   laboratory scientist and provide relevant details
  leucocyte depleted. Volume           Blood needed in 15–60 min                                 Collect sample for group and crossmatch before
  is provided on each pack, and        Uncrossmatched ABO group specific will be                     using emergency stock
  is in the range 190–420 mL              provided when blood group known (15–60 min             Emergency use of RhD positive blood is acceptable
                                          from receipt of sample in laboratory)                     if patient is male or postmenopausal female
                                       Blood needed in 60 min or longer                          Blood warmer indicated if large volumes are
                                       Fully crossmatched blood will be provided                    transfused rapidly
                                                                                                 Consider use of cell salvage

Consider use of platelets              Anticipate platelet count < 50 ¥ 109/L after              Target platelet count > 100 ¥ 109/L for multiple/
                                         1.5–2¥ blood volume replacement                            CNS trauma, > 50 ¥ 109/L for other situations
                                       Dose: 10 ml/kg body weight for a neonate or small         May need to use platelets before laboratory
                                         child, otherwise one ‘adult therapeutic dose’              results available: take FBC sample before
                                         (one pack)                                                 platelets transfused

Consider use of FFP                    Anticipate coagulation factor deficiency after             PT/APTT > 1.5¥ mean control correlates with
                                          blood loss of 1.5¥ blood volume                          increased surgical bleeding
                                       Aim for PT/APTT < 1.5¥ mean control                       May need to use FFP before laboratory results
                                       Allow for 30 min thawing time                               available: take sample for PT, APTT, fibrinogen
                                       Dose: 12–15 mL/kg body weight (1 L or 4 units for           before FFP transfused
                                          an adult)

Consider use of cryoprecipitate        To replace fibrinogen and factor VIII                      Fibrinogen < 0.5 g/L strongly associated with
                                       Aim for fibrinogen > 1.0 g/L                                  microvascular bleeding
                                       Allow for 30 min thawing time
                                       Dose: 1 pack/10 kg body weight

Suspect DIC                            Treat underlying cause if possible                        Shock, hypothermia, acidosis: increased risk of DIC
                                       Mortality of DIC is high

APTT, activated partial thromboplastin time; CNS, central nervous system; CVP, central venous pressure; DIC, disseminated intravascular coagulation;
FBC, full blood count; FFP, fresh frozen plasma; PT, prothrombin time.
80
                                                                                                                                                                                                                                           Effective use of blood components


                         Step 1: Ask the patient to tell you their
                                 full name and date of birth
                                                                                                                                                                                     9              8E
                                                                                                                                                                                  01
                                                                                                                                                                       G ALD 10
                                                                                                                                                                     RA ON No. 56
                                                                                                                                                                   MO ACD AL 7/19
                                                                                                                                                                    M SPIT 1/0
                         Step 2: Check these details against the patient’s                                                                                            HO B 1
                                                                                                                                                                        DO

                                 wristband

                         Step 3: Check the hospital ID number on the
                                 patient’s wristband against documentation
                                 e.g. patient case notes or request form

                                                                                                                                                                                                                                                 (a)

                                  Step 1: Complete the blood collection form (or follow the
                                          local collection procedure) with the following information:

                                                                                                                          Any NHS Trust
                                             • Forename                                                         Blood and Blood Products Collection
                                                                                                                               Form
                                                                                                              To be completd for each unit collected
                                             • Surname
                                                                                                              Patient Name: MORAG McDONALD…..
                                             • Date of birth
                                                                                                              DOB: ………….1/07/56…………………….
                                             • Hospital number
                                                                                                              Hospital Number:…100198E…………

                                                                                                              Addressograph label may be used
                                                                                                              Please tick
                                                                                                              Red cells: … ……….
                                                                                                              Fresh frozen Plasma: ….
                                                                                                              Platetes: …….…..
                                                                                                              Other ( specify: ………..




                                  For each blood component collected

                                 Step 2: Check patient ID details
                                         against compatibility label
                                         attached to the blood                                                                          Ma c o
                                                                                                                                         ac
                                                                                                                                    P h a rm a
                                                                                                                                                                                                                       B




                                         component                                          DON
                                                                                            PAC O R
                                                                                           No. K
                                                                                                   CO M
                                                                                                           G10
                                                                                                              PA TIBL
                                                                                                                                    E W IT
                                                                                                                                          H
                                                                                           SUR NAM               1 602
                                                                                                      E                     5 97
                                                                                                                                   22 9
                                                                                                                                          N
                                                                                                            MA CD
                                                                                         FOR ENA                        ON AL                            G101 602 597 229 N
                                                                                                   ME                               D
                                                                                    HOS                      MORA
                                                                                    No. PIT AL                              G
                                                                                                           1001
                                                                                  ADD RES                 98E
                                                                                         S
                                                                                                25 HI

                                 Step 3: Document removal of                                           LL
                                                                                                TOWN ST RE
                                                                                                                  RED CELLS IN AD D ITIVE SO LU TIO N ,




                                                                                                                                                                                                         O
                                                                               D.O .B.                  CE NT ET LEU CO CYTE D EPLETED
                                                                                                              RE STORE AT 4OC ± 2O C     (SAGM)
                                                                                              11/07
                                                                                    ABO             /1956


                                         unit on blood fridge
                                                                                                                                                                  Volume 263 ml
                                                                                   O                      RHE SUS
                                                                                                                                              0 43 33
                                                                                                                            WA RD
                                                                          DER ESE                       PO S                        INS TRUC TION
                                                                                 RVE
                                                                                         DAT E                              ITU Alw ays check patient / component                                   Rh D PO SITIVE
                                                                                                                                                                                   CT 664/2




                                                                                                                                    compatibility / identity.                                            Do Not Use After:


                                         register or electronic
                                                                                                                     NUM
                                                                                                                         BE
                                                                                                                    OF UNIT R
                                                                                                                    IS SU S
                                                                                                                                    Inspect pack for signs of deterioration or                           03 DEC 2002 23:59
                                                                                                                         ED
                                                                         WEST STO RE AT o                                           damage.
                                                                              GLA         4 C.                          1           Risk of adverse reaction / infection.
                                                                          UNIVER SGOW
                                                                                SIT Y   HOS PIT
                                                                                      NHS       ALS                 OF
                                                                                          TRU ST


                                         release system                                                             2                                                                          CM V Ne ga ti ve
                                                                                                                                                                                                                             E TEGB 00 5




                                                                                                                                                  LOT    B1 08 0 21 0 62 0 + 6 B
                                                                                                                                                                                              LOT             S NBTS

                                                                                                                                                                                   REF
                                                                                                                                                                                                Da te B led 29 Oct 2 002
                                                                                                                                                   REF       C00105107B




                                                                                                                                                                                                                                           (b)
Fig. 6.4 Hospital procedures for safe transfusion. (a) Collection of blood sample for pretransfusion testing. Be extra vigilant
when checking the identity of the unconscious/compromised patient. (b) Procedure for collection of blood from the blood
refrigerator. NB Follow procedure for each blood component collected.




                                                                                                                                                                                                                                                                        81
Chapter 6


                                  Step1: Check the blood component has been prescribed
                                  Step2: Undertake baseline observations
                                  Step3: Before approaching the patient check the component for:




                                                                      Ma
                                                                      M    co                                                                            B
                                                                    P h a rm a
                                                                             a



                                COM
                                           P A TIB
                          DO NO
                         P ACK R                               LE W
                         No .
                                        G 10
                                               1 6 02
                                                                            IT H
                        SU RN

                                                                                                                                                                          Leaks
                                AM E                  59 7
                                                                 229
                                                                           N
                                         M AC
                      FO RE                         DONA
                           NA ME                        LD                                 G101 602 597 229 N
                 HO SP
                 No . ITA L

               AD DR
                                           M OR

                                        10 01
                                                98E
                                                        AG
                                                                                                                                                                          Discoloration
                       ESS
                                25 HI
                                      LL ST      RED CELLS IN AD DITIVE SOLU TION ,
                                                                                                                                                                          Clumping
                                                                                                                                        O
                                TO W
            D.O .B.                  N CE RE ET LEUCOCYTE D EPLETED
                                          NT RE
                                                STORE AT 4 OC ± 2O C   (SAGM)
                             11 /07
                 AB O
                                       /1956
                                                                                                    Volume 263 ml
                O                      RH ES
                                               US                              0 4 33 3
                                                            WA RD
        DE RE
             SERV
                                   POS                              IN STR UCTION
                 E DA                                       IT U Always check patient / component                                     Rh D POSITIVE
                      TE

                                                                                                                                                                                 Expiry date
                                                                                                                    CT 664/ 2




                                                     NU MB          compatibility / identity.                                           Do Not Use After:
                                                    O F UNIER       Inspect pack for signs of deterioration or                          03 D EC 2002 23:59
                                                    I SS UE TS
      W EST ST OR E AT o
                                                            D       damage.
            GL
       UN IVE AS GO W
                        4 C.                            1           Risk of adverse reaction / infection.
             RSITY    HOSP
                   NH S      ITA                    OF
                        TR US LS
                               T
                                                    2                                                                             CMV Negative
                                                                                                                                                              ETEG B005




                                                                                   LOT     B1 0 80 2 10 6 2 0+ 6B               LOT            SNBTS

                                                                                                                    REF
                                                                                                                                   Date Bled 2 9 O ct 200 2
                                                                                     REF       C00105107B




                                                                                                                                                                                               Fig. 6.4 (c) Procedure for the administration
(c)                                             If there is any discrepancy, do not transfuse                                                                                                  of blood.




transfusion, such as development of hepatitis in
                                                                                                                                                                               Before prescribing blood or blood products
the future, the record of the clinical decision may
                                                                                                                                                                               for a patient
prove to be important medicolegal evidence. A
record that the patient has been given information                                                                                                                             • What improvement in the patient’s clinical con-
about the transfusion (see above) may equally be                                                                                                                               dition am I aiming to achieve?
important medicolegal evidence, as doctors have                                                                                                                                • Are there any other treatments I should give
been criticized for alleged past failures to inform                                                                                                                            before making the decision to transfuse, such as
patients about the risks of blood products.                                                                                                                                    intravenous replacement fluids or oxygen?
                                                                                                                                                                               • What are the specific clinical or laboratory indi-
                                                                                                                                                                               cations of transfusion for this patient?
Conclusions
                                                                                                                                                                               • Do the benefits of transfusion outweigh the risks
It may be useful to think of a blood component                                                                                                                                 for this particular patient?
transfusion as a tissue transplant to emphasize that                                                                                                                           • What other options are there if no blood is avail-
prescribing is not a trivial decision. When you are                                                                                                                            able in time?
uncertain if transfusion is likely to give clinical                                                                                                                            • Will a trained person monitor this patient and
benefit to a patient, it may be helpful to ask your-                                                                                                                            respond immediately if any acute transfusion reac-
self some critical questions.                                                                                                                                                  tions occur?
                                                                                                                                                                               • Have I recorded my decision and reasons for
                                                                                                                                                                               transfusion on the patient’s chart and the blood
Before ordering blood in preparation for
                                                                                                                                                                               request form?
planned surgery
                                                                                                                                                                               • Finally, ask yourself ‘If this was myself or my
Can I reduce this patient’s need for transfusion by                                                                                                                            child, would I agree to the transfusion?’
correcting anaemia, stopping warfarin or aspirin,
checking for a coagulation disorder or arranging
in advance for intraoperative cell salvage to be
available in theatre?



82
                                                                                                                                                                                                                                             Effective use of blood components


                Step1: Ask the patient to tell you their full name and date of birth (where possible)
                Step2: Check ID details against the patient’s wristband and compatibility label

                   Compatibility label

                                 COMPATIBLE WITH
                         DONOR
                         PACK
                         No.                 G101 602 597 229 N                                                                         Surname
                         SURNAME
                                                                                                                                                                                                           8E
                                             MACDONALD                                                                                                                                            0   19

                         FORENAME                                                                                                 Forename                                         G    A
                                                                                                                                                                                         LD o.
                                                                                                                                                                                               10
                                                                                                                                                                                            N 56
                                                                                                                                                                                  A   N
                                                         MORAG                                                                                                                   R D O A L /1 9
                                                                                                                                                                               O       T     7
                                                                                                                                                                              M A C P I 1 /0
                         HOSPITAL
                         No.
                                                                                                                                Hospital number                                M OS 1
                                                                                                                                                                                  H OB
                                                         100198E                                                                                                                    D
                         ADDRESS
                                             25 HILL STREET
                                             TOWN CENTRE
                         D.O.B.
                                                11/07/1956                                                                              Date of birth
                                         ABO                             Rh                      WARD

                                      O                                  POS                   ITU
                         DERESERVE DATE                                                         NUMBER
                                                                                                OF UNITS
                                                                                                 ISSUED

                                                                                                    1
                                 STORE AT 4oC.                                                      OF
                           ANY NHS TRUST
                                                                                                    2




                                                                          Ma c o                                                                      B
                                                                        Ph a rm a
                                                                                                                                                                                                                   COMPATIBLE WITH
                                          COMP                                                                                                                          Donor component                         DONOR
                                              A              TIB LE                                                                                                                                             PACK
                                     DONO
                                                                                                                                                                                                                               G101 602 597 229 N
                                    PACK R
                                    No.
                                   SU RN
                                        AM E
                                                G101
                                                         602 59
                                                                7
                                                                    WITH
                                                                     229
                                                                                                                                                                            number                              No.

                                                 M AC
                                                                             N                                                                                                                                  SURNAME
                                                     DONA
                                 FORE
                                     NAME                           LD                       G101 602 597 229 N                                                                                                                 MACDONALD
                            HOSP                  M OR
                                                             AG
                            No. ITAL                                                                                                                                                                            FORENAME
                                                10 01
                           ADDR
                                ES   S
                                                     98 E                                                                                                                                                                          MORAG
                                          25 HI
                                               LL ST           LS
                                                         RED CE L IN ADDITIVESOLUTION,
                                          TOW




                                                                                                                                         O
                        D.O.B.                N CE REET L UCOCYTE DE TE
                                                          E           PLE D                                                                                                                                     HOSPITAL
                                                   NT RE
                                                                                                                                                                                                                No.
                                         11 /07/
                                                1956
                                                                        STORE AT 4 OC ± 2OC             (SAGM)                                                                                                                    100198E
                            ABO
                                                                                                    Volume 263 ml
                            O
                                               RHES
                                                    US                           0 4 3 33
                                                                                                                                                                                                                ADDRESS
                                                                                                                                                                            Blood group
                                                                 WARD
                                             POS
                    DE RE
                         SE RV
                                 E DATE
                                                                     INSTRUCTION
                                                                 ITU Always check patient / component                                  Rh D POSITIVE                                                                           25 HILL STREET
                                                                                                                     CT 664/2




                                                                                                                                         Do Not Use After:
                                                          NUMB
                                                         OF UN ER
                                                         ISSUEITS
                                                              D
                                                                        compatibility / identity.
                                                                        Inspect pack for signs of deterioration or                      03 DEC 2002 23:59                                                                      TOWN CENTRE
                   WE ST ST ORE AT                                      damage.
                        GL
                    UNIVE AS GOW HO
                                   4 oC.                     1          Risk of adverse reaction / infection.                                                                                                   D.O.B.
                         RSITY        SP
                               NHS TR ITALS
                                       UST
                                                          OF                                                                                                                                                                    11/07/1956
                                                         2                                                                        CMV Negative
                                                                                                                                                                                                                         ABO         Rh        WARD
                                                                                                                                                                         RhD group
                                                                                                                                                             ETEGB005




                                                                                     LOT     B1080210620+ 6B
                                                                                                                                LO T                                                                                      O
                                                                                                                                              SNBTS
                                                                                                                                                                                                                                     POS       ITU
                                                                                                                     REF
                                                                                                                                   Date Bled 29 Oct 2002
                                                                                       REF      C0 0105 107B                                                                                                                                  NUMBER
                                                                                                                                                                                                                DERESERVE DATE                OF UNITS
                                                                                                                                                                                                                                               ISSUED
                                                                                                                                                                                                                                                 1
                                                                                                                                                                                                                       STORE AT 4o C.           OF
                                                                                                                                                                                                                 ANY NHS TRUST
                                                                                                                                                                                                                                                 2

                                     If there is any discrepancy, do not transfuse                                                                                                                                                                       (d)
Fig. 6.4 (d) Identification checks at the bedside before transfusion. Be extra vigilant when checking the identity of the
unconscious/compromised patient.




                                                                                                                                                                                                                                                                          83
Chapter 6


                                                                                                         Example compatibility report
                                                                                                                   form

                                                                                                                           SCOTTISH NATIONAL BLOOD TRANSFUSION SERVICE
                                                                                                                                                                                                CLINICAL SERVICES




                                                                                                                     EXAMPLE OF A TRANSFUSION COMPATIBILITY REPORT
                     COMPATIBLE WITH                                                     SURNAME              MACDONALD            HOSPITAL No. 100198E                                 COMMENTS:
                                                                                                                                                                                                                 T
                                                                                                                                                                                                         N SB TS / xR ep Fm 98( 1 )




                  DONOR
                  PACK           G101 602 597 229 N
                  No.                                      Ensure that you sign          FIRST NAME(S)
                                                                                                               MORAG               BLOOD GROUP.      O Positive


                  SURNAME
                                     MACDONALD             the compatibility report      DOB     11/07/1956                        ANTIBODY SCREEN


                                                           form to say you have          HOSP. Any Hospital WARD
                                                                                                                 4C                                                                                  1803905
                  FORENAME                                                                                                                                                              Lab Ref .


                                       MORAG               checked the blood                                                       COMPONENTS
                                                                                                               Forrest                             21/05/03 11:00                                     000082898
                  HOSPITAL                                 component against the         CONSULTANT                                REQUESTED FOR                                        Issue No.

                  No.                 100198E
                                                                                                   DONATION No .       GRO UP    COMPO NENT        EXP IRY DATE        I.D. CHECK                   I .D. CHECK                         DATE       TIME


                                                           patient’s wristband                     /   PO OL No.                                          /   TIME   (S ig n at ur e)               (Sig natur e)




                  ADDRESS                                                                1    G101 602 597 229 N         O POS       OAS           19/06/03               R GNurs e             J Doctor                              2 1/05 / 3
                                                                                                                                                                                                                                              0    1 1. 10
                                                                                                                                                                                                                                                      am

                                    25 HILL STREET
                                    TOWN CENTRE                                          2

                                                                                              G101 603 703 984N          O POS        OAS           21/06/03

                  D.O.B.                                                                 3



                                     11/07/1956
                                                                                         4

                           ABO        Rh          WARD
                                       POS        ITU      Complete
                           O                                                             5




                                                NUMBER
                                                           documentation, e.g.
                  DERESERVE DATE                OF UNITS
                                                 ISSUED    ensure donor component        6




                                                  1        number is recorded on         7




                         STORE AT 4oC.             OF
                   ANY NHS TRUST                           compatibility report form
                                                  2                                      8




                                                                                         TRANSFUSION REPORT FORM                              PLEASE FILE IN CASE NOTES AFTER COMPLETION OF TRANSFUSION




                                                                                                                                                                                                                                                   ©
                                                                                             SAMPLE COLLECTED : 20/05 /03                      RECEIVED : 20/05/03                          REPORTED : 20/05/031

            (e)
Fig. 6.4 (e) Documenting the transfusion on the compatibility report form.




                                                                                       Laupacis A, Fergusson D, Henry DA et al. Cell salvage
Further reading                                                                          for minimising perioperative allogeneic blood
                                                                                         transfusion. Cochrane Database of Systematic
Website
                                                                                         Reviews 2003; 3.
www.transfusionguidelines.org.uk. Public site giving access                            EBM reviews. Perioperative recombinant human
 to UK Handbook of Transfusion Medicine, UK standards                                    erythropoietin in hip replacement. ACP Journal Club
 for blood components, links to BCSH Guidelines,                                         1993; 119: 46.
 Cochrane library, etc.                                                                NHS Centre for Reviews and Dissemination. A meta-
                                                                                         analysis of the effectiveness of cell salvage to minimize
                                                                                         perioperative allogeneic blood transfusion in cardiac and
Systematic reviews                                                                       orthopedic surgery (provisional record). Database of
                                                                                         Abstracts of Reviews of Effectiveness 2003, issue 3.
Hill SR, Carless PA, Henry DA et al. Transfusion thresholds
                                                                                       NHS Centre for Reviews and Dissemination. The efficacy of
  and other strategies for guiding allogeneic red blood cell
                                                                                         technologies to minimise peri-operative allogeneic
  transfusion. Cochrane Database of Systematic Reviews
                                                                                         transfusion (structured abstract). Database of Abstracts
  2003; 3.
                                                                                         of Reviews of Effectiveness 2003, issue 3.
Henry DA, Carless PA, Moxey AJ et al. Pre-operative
  autologous donation for minimising perioperative
  allogeneic blood transfusion. Cochrane Database of
                                                                                       Original papers and reviews
  Systematic Reviews 2003; 3.
Henry DA, Moxey AJ, Carless PA et al. Desmopressin for                                 Beris P. The use of iron to increase red cell mass. Can J
  minimising perioperative allogeneic blood transfusion.                                 Anaesth 2003; 50 (Suppl.): S3–S9.
  Cochrane Database of Systematic Reviews 2003; 3.                                     Carson JL, Duff A, Berlin JA et al. Perioperative blood
Henry DA, Moxey AJ, Carless PA et al. Anti-fibrinolytic                                   transfusion and postoperative mortality. J Am Med Assoc
  use for minimising perioperative allogeneic blood                                      1998; 279: 199–205.
  transfusion. Cochrane Database of Systematic Reviews                                 Carson JL, Terrin ML, Barton FB et al. A pilot randomized
  2003; 3.                                                                               trial comparing symptomatic vs. haemoglobin-level-


84
                                                                                                 Effective use of blood components

  driven red blood cell transfusions following hip fracture.      transfusion trial (HATT) (abstract). Transfus Med 1998;
  Transfusion 1998; 38: 522–9.                                    8 (Suppl. 1): 36.
Carson JL, Terrin ML, Magaziner J. Anaemia and post             SAFE Study Investigators. A comparison of albumin and
  operative rehabilitation. Can J Anaesth 2003; 50                saline for fluid resuscitation in the intensive care unit.
  (Suppl.): S3–S9.                                                N Engl J Med 2004; 317: 235–40.
Cochrane Injuries Group Albumin Reviewers. Human                Sanguis Study Group. Use of blood products for elective
  albumin administration in critically ill patients:              surgery in 43 European hospitals. Transfus Med 1994; 4:
  systematic review of randomised controlled trials.              251–68.
  Br Med J 1998; 317: 235–40.                                   Schierhout G, Roberts I. Fluid resuscitation with colloid or
Hébert PC, Schweitzer I, Calder L, Blajchman M, Giulivi A.        crystalloid solutions in critically ill patients: a systematic
  Review of the clinical practice literature on allogeneic        review of randomised trials. Br Med J 1998; 31: 961–4.
  red blood cell transfusion. Can Med Assoc J 1996; 156         Serious Hazards of Transfusion Scheme (SHOT). SHOT
  (11 Suppl. 1): S9–S26.                                          Annual Report. SHOT Office, 1998.
Hébert PC, Wells G, Blajchman MA et al. A multicentre           Stainsby D, MacLennan S, Hamilton PJ. Management of
  randomized controlled clinical trial of transfusion             massive blood loss: a template guideline. Br J Anaesth
  requirements in critical care. N Engl J Med 1999; 340:          2000; 85: 487–91.
  409–17.                                                       Walsh TS, McClelland DBL. When should we transfuse
Laupacis, Fergusson. Anesth Analg 1997; 85: 1258–67.              critically ill and perioperative patients with known
McClelland DBL. Albumin: don’t confuse use with the facts         coronary artery disease (editorial)? Br J Anaesth 2003;
  (editorial). Br Med J 1998; 317: 829–30.                        90: 719–22.
McLellan SA, Walsh TS, McClelland DBL. Should we                Walsh TS, McArdle F, Maciver C, Maginnis M, McClelland
  demand fresh red blood cells for perioperative and              DBL. Age of transfused red blood cells does not affect
  critically ill patients (editorial)? Br J Anaesth 2002; 89:     indices of oxygenation after transfusion to critically ill
  537–40.                                                         patients: randomised controlled trial. Crit Care Med
Palmer JB, Maciver CR, Scott R et al. Hip fracture and            2004; 32: 364–71.




                                                                                                                              85
Chapter 7
Bleeding associated with trauma and surgery

Beverley J. Hunt




In the past, attempts at perioperative blood conser-      and mortality associated with excessive periopera-
vation have been driven by fears of transfusion-          tive bleeding.
transmitted infection, concerns about the clinical           However, there is a subset of patients in whom
efficacy and cost. The current concerns about              generalized oozing in the surgical field cannot
variant Creutzfeldt–Jakob disease (vCJD) have             be attributed to demonstrable bleeding vessels.
stimulated interest in transfusion triggers, blood        There are no adequate definitions of an ‘excessive
conservation techniques and the use of pharmaco-          bleeder’ and yet many surgeons complain when it
logical agents to obviate the need for transfusion        occurs. Some relevant factors in this respect may
(Table 7.1). The use of blood should be minimized,        include the following.
but not at the risk of increasing morbidity and           • Perioperative haemostatic changes have not
mortality in the patient from non-availability of         been extensively studied, although haemostatic
blood or under-transfusion. The use of near-              activation is thought to occur as a result of the
patient haemostatic assessment in the operating           hyperadrenergic state induced by the stress of sur-
theatre is of increasing interest and can result in       gical stimulation.
more appropriate use of blood components. Near-           • Increased fibrinolytic activity occurs during
patient haemostatic testing has developed to such         and shortly after an operation. Increased levels of
an extent that it is a prerequisite in orthotopic liver   tissue plasminogen activator (t-PA) and plasmino-
transplantation anaesthesia. Its use is common in         gen activator inhibitor (PAI)-1, with a net increase
cardiac surgery, and is being explored in other sur-      in free t-PA during and after an operation, have
gical fields.                                              been noted.
                                                          • Meninges, cerebral and prostatic tissue contain
                                                          relatively high concentrations of tissue activators
Causes of perioperative bleeding                          of plasminogen. Elimination of t-PA from the
                                                          blood is mainly regulated by the liver, with a
Excessive bleeding can be due to surgical causes          normal half-life of 3.5 min.
(i.e. suture deficiency) and/or derangement of
haemostasis. In practice, blood loss is highly vari-
able between surgeons, and there is increasing            Identifying those at risk of
interest in training surgeons in adequate surgical        excessive bleeding
haemostasis. A few minutes of the surgeon’s atten-
tion to careful haemostatic control may well save a       • A history of previous bleeding problems in
patient from returning to theatre for surgical re-        patients and/or their family, together with drug
exploration to find a bleeding point. It may also          therapy (see below for advice about withdrawing
prevent the development of haemostatic problems           aspirin and warfarin) and a full blood count, are
associated with continued bleeding, the use of            the usual prerequisites for a standard surgical
blood components and increased risk of morbidity          procedure.


86
                                                                                                                              Bleeding in trauma and surgery

Table 7.1 Methods used to reduce allogeneic blood use
perioperatively.

Accepted methods
Acceptance of lower levels of haemoglobin postoperatively                                                                               Electromagnetic
                                                                                                N                   S                   transducer
Improved surgical haemostasis
Use of pharmacological agents
Autologous transfusion (see Chapter 26)                                                                                 Pin
Other methods*
Mechanical methods of intraoperative red cell salvage (see Chapter 26)
Acute normovolaemic haemodilution                                                                                         Cup
Controlled hypotension
Preoperative erythropoietin                                                                                               0.36 mL whole
                                                                                                                          blood (clotted)
                                                                                                         4°45'
* Little evidence as yet for efficacy and safety.



• The most important determinants of the haem-                           Fig. 7.1 Operation of the thromboelastograph. See text for
orrhagic risk are the patient’s diagnosis, the pro-                      explanation.
cedure planned and whether previous surgery has
been performed.
• Prior to cardiac surgery or other major surgery
in an unwell individual, especially one who is
actively bleeding, prothrombin time (PT), acti-
vated partial thromboplastin time (APTT) and                                               a°              30 min
fibrinogen level are necessary.
                                                                                   20 mm




                                                                                                                 A30
                                                                                                    MA

• No other assays have been shown to be of value
(including preoperative bleeding time), unless
                                                                             r k
an underlying congenital bleeding disorder is
suspected.
   Non-surgical perioperative bleeding has been                                 Thrombosis                               Fibrinolysis
poorly understood and poorly managed. The
reasons for this include the following.                                  Fig. 7.2 Thromboelastograph trace. See text for
• There is failure to recognize the limitations of                       explanation.
laboratory tests.
• There are no quick and reliable laboratory
assays to investigate some components of                                 to the pot, the movement is translated into lateral
haemostasis that may be abnormal, e.g. fibrinoly-                         movements on the trace. Individual parameters
sis and platelet dysfunction. This has stimulated                        (Fig. 7.2) include the r time, the time taken for
the use of near-patient assessment such as the                           blood to clot (some equivalence to laboratory clot-
thromboelastograph (TEG) and other near-patient                          ting times), the maximal amplitude (K), and the a
haemostatic assessment devices.                                          angle, which indicates platelet–fibrin interactions.
   The principle of the TEG and its interpretation                       Fibrinolysis can be measured by the reduction in
is as follows. A blood sample is placed in a rotating                    the amplitude of the trace as the clot is lysed,
pot and a piston is suspended in this (Fig. 7.1). As                     60 min after maximal amplitude (MA).
small clots form between pot and piston, the                                The use of the TEG as a guide to intraoperative
movement of the pot is transmitted to the piston.                        transfusion has been shown to reduce postopera-
The piston is connected by a wire to a trace. Thus,                      tive transfusion requirements in high-risk surgical
as the piston is oscillated due to the clot linking it                   patients.


                                                                                                                                                          87
Chapter 7


Patients on antithrombotic medication                      regimen will allow the patient to prepare at
                                                           home and be admitted to hospital the day
Surgeons prefer their patients to have normal              before, reducing the pressure on surgical beds.
haemostasis at the time of operation. If patients       3 If a patient has antithrombin deficiency or other
are receiving aspirin, ideally this should be stopped   thrombophilic disorder, then advice from a spe-
at least 10 days prior to surgery to allow for new      cialist in thrombosis should be sought. As heparin
platelets to develop that are not affected by           acts through potentiating the effect of anti-
aspirin’s inhibition of cyclooxygenase, otherwise       thrombin, then patients with antithrombin defi-
blood loss is greater. Ideally, other antiplatelet      ciency may need antithrombin concentrates as well
drugs such as clopidogrel and non-steroidal anti-       as heparin thromboprophylaxis.
inflamamtory drugs should be stopped 2–3 days
prior to surgery. Patients receiving aspirin at the
time of surgery do have a greater blood loss.           When red cell transfusion should be given
   If patients are receiving warfarin prior to
surgery, then the following can apply.                  • Previously, anaesthetists have tended to trans-
1 If the patient can stop warfarin without the need     fuse patients when their haemoglobin concentra-
for any perioperative thromboprophylaxis, then          tion has fallen below 10 g/dL or when the
warfarin should be stopped at least 3 days              haematocrit is less than 30%. However, there is
preoperatively.                                         evidence that much lower levels of haemoglobin
2 If the patient requires perioperative thrombo-        can be tolerated without adverse effects.
prophylaxis, then the patient can be switched to        • Experimental evidence shows that in a healthy
heparin for the perioperative period. Depending         human the cardiac output does not increase until
on the underlying prothrombotic state the patient       haemoglobin falls to less than 7 g/dL. In critically
can switch to either of the following.                  ill patients, a recent randomized trial indicated
   (a) Full-dose intravenous heparin to maintain        that a threshold as low as 7 g/dL is as safe as, and
   an APTT ratio of 2–2.5. This is still the required   possibly superior to, a haemoglobin threshold of
   management for patients with artificial heart         10 g/dL, with the possible exception of those with
   valves. No low-molecular-weight heparin is           severe cardiac disease.
   licensed for this indication and thrombotic          • Moderate haemodilution is tolerated well by
   events have occurred in patients with mech-          young healthy individuals undergoing elective
   anical valves receiving low-molecular-weight         surgery, in whom there would be greater con-
   heparin. As the half-life of heparin is approxi-     cerns about the potential long-term effects of
   mately 2 h, heparin can be stopped 2 h pre-          transfusion.
   operatively and restarted as the patient’s wound     • Concern remains about moderate haemodilu-
   is being sewn up.                                    tion in those with compromised respiratory or
   (b) The modern alternative for prothrombotic         myocardial function.
   states other than artificial heart valves would be    • The ‘transfusion trigger’ should be a composite
   to switch to a low-molecular-weight heparin the      of variables specific for an individual patient.
   day after stopping warfarin. The dose used will      • The large variability in use of blood transfusion
   depend on the patient’s underlying prothrom-         between different units and surgeons in the UK
   botic state. A full treatment dose can be given in   needs to be addressed. This is one of the recom-
   those with severe prothrombotic states, and          mendations in the Department of Health’s Better
   thromboprophylaxis doses to those with lesser        Blood Transfusion initiative in the UK (see Further
   prothrombotic states. Alternatively, the latter      reading). Each hospital should have a hospital
   patient group could be given subcutaneous            transfusion committee to develop protocols that
   unfractionated heparin, but this will require        include agreed triggers for transfusion of red cells
   more frequent injections. If the patient is taught   and the management of massive blood loss
   how to administer subcutaneous injections, this      (see below and Chapter 25).


88
                                                                                                         Bleeding in trauma and surgery


Special situations                                                        of whole blood from ‘walk-in’ donors is now con-
                                                                          sidered an unacceptable and dangerous practice
Transfusion management of acute blood loss and
                                                                          due to the risks of transfusion-transmitted disease
massive blood loss
                                                                          (see also Chapter 6).
This covers situations where blood loss is profuse
for a limited period, or situations where massive
                                                                          Practical management
blood loss occurs. The latter is arbitrarily defined
as the replacement of the patient’s total blood                           • Obtain maximum venous access. One or
volume in less than 24 h. The usual presentation is                       two large-bore intravenous cannulae should be
as an emergency in accident and emergency, labour                         inserted and if possible a central line.
ward or operating theatre, but in some situations,                        • Should crystalloid or colloid be used as initial
such as liver transplantation, it can be predicted                        replacement therapy? This thorny argument has
and thus sophisticated monitoring and protocols                           continued for many years and has been fuelled by
are possible.                                                             the recent concerns about the use of albumin. If
   Management is aimed at prompt resuscitation                            crystalloids are used, then larger volumes are
(Table 7.2). It should be remembered that the most                        required.
frequent cause of death in massive transfusion is                         • Prevent tissue hypoxia by giving adequate
inadequate replacement of circulating volume and                          volume to achieve an acceptable systolic blood
red cells. Preventing tissue hypoxia by maintaining                       pressure. Initially this can be done using crys-
an adequate circulating volume of red cells is the                        talloid or colloid until red cell transfusion is avail-
most important part of resuscitation. It is impera-                       able.
tive that the prime goal is the treatment of shock;                       • Blood is required. Send a blood sample to the
achieving surgical control of bleeding and manag-                         blood transfusion laboratory for ABO group and
ing a consumptive coagulopathy can wait. Whole                            RhD group (can usually be performed within
blood is unavailable in many countries including                          5 min) and telephone the transfusion laboratory to
the UK and following the implementation of                                indicate the need for blood. If possible, wait for
universal leucodepletion of blood components it                           ABO- and RhD-compatible blood. In emergency
would not contain platelets in any case. There is no                      cases, use group O RhD-negative red cells until the
advantage in using fresh whole blood, and the use                         patient’s ABO and RhD groups are known. Switch
                                                                          to blood of the same ABO and RhD groups as the
                                                                          patient as soon as possible to avoid inappropriate
                                                                          use of group O RhD-negative red cells as their
Table 7.2 Principles of management of massive blood loss.
                                                                          supply may be limited.
Replace and maintain oxygen-carrying capacity by:                         • Also send a baseline ethylenediamine tetra-
   Maintaining blood volume                                               acetic acid (EDTA) sample for full blood count
   Optimizing PCV > 20%                                                   (FBC), and a citrate sample for coagulation
Maintain haemostasis by:                                                  screen and sample to biochemistry for urea and
   Platelet count > 50 ¥ 109/L                                            electrolytes.
   INR and APTT ratios < 1.5                                              • Administration of blood: when a very fast rate
   Fibrinogen > 1.0 g/L                                                   of transfusion is required (>50 mL/kg per h in
Avoid metabolic disturbances
                                                                          adults or >15 mL/kg per h in children), a blood
   Hypocalcaemia
                                                                          warmer should be used.
   Hyperkalaemia
                                                                          • Once the patient’s blood pressure has been
   Acid–base disturbances
   Hypothermia                                                            restored, consider surgical control of the bleeding.
Treat the cause of the blood loss                                         • Haemostasis (see also Chapter 11). An early
                                                                          coagulation screen and platelet count or TEG will
APTT, activated partial thromboplastin time; INR, international normal-   provide a guide to the use of blood components. It
ized ratio; PCV, packed cell volume.                                      is important to understand that at least 1.5 blood


                                                                                                                                   89
Chapter 7


volumes (i.e. 7–8 L in adults) must be transfused       though they decrease plasma fibronectin activity,
before the platelet count falls below 50 ¥ 109/L        but this has little clinical significance.
in an average healthy individual. There is often           Consider the use of adjunct haemostatic agents.
time to assess coagulation fully; transfusion of        Persistent bleeding will stimulate fibrinolytic activ-
blood components should be given as necessary           ity. Ideally D-dimers or a TEG trace could be used
according to the results of the screening coagula-      in this situation as a guide to fibrinolytic activity.
tion tests.                                             The use of an antifibrinolytic agent such as apro-
• Aim to (i) keep the platelet count greater than       tinin 500 000 kallikrein inhibitory units (KIU)
50 ¥ 109/L by administering platelet concentrates;      intravenously or tranexamic acid 1 g intravenously
and (ii) maintain PT and APTT ratios less than 1.5      may be beneficial.
times the control value by giving fresh frozen
plasma (FFP). Fibrinogen in the form of cryopre-
                                                        Other possible complications of blood transfusion
cipitate can be given if fibrinogen levels are dispro-
portionately low, in order to maintain fibrinogen        • Hypocalcaemia. Calcium gluconate (2 mL of
concentrations greater than 1.0 g/L.                    10% solution per unit of blood) should be given
                                                        when ionized calcium or calcium concentration
                                                        can be measured and shown to be low or there are
Coagulation problems (see also Chapter 11)
                                                        clinical signs or electrocardiographic changes.
These occur in patients with extensive bleeding         • Hyperkalaemia may occur due to the high con-
because of:                                             centration (~ 40 mmol/L) in stored blood. This is
• loss of haemostatic factors;                          usually only a problem in those with hepatic or
• consumption in clot formation;                        renal disease.
• dilution with blood components and volume             • Acid–base disturbances. Despite the presence of
expanders;                                              lactic acid in transfused blood, fluid resuscitation
• depletion of coagulation factors due to inade-        usually improves acidosis in shocked patients. Fur-
quate synthesis, although factor VIII deficiency is      thermore, transfused citrate produces an alkalosis
partially compensated for by increased synthesis        once it is metabolized.
and release as part of the stress response;             • Avoid hypothermia. Warm the patient, and the
• acidosis and hypothermia precipitate dissemin-        blood if a fast rate of transfusion is required.
ated intravascular coagulation (DIC).
   DIC may occur but cannot be predicted. Mas-
                                                        Cardiopulmonary bypass
sively transfused patients do not form a homoge-
neous group; delayed or inadequate treatment of         Cardiopulmonary bypass (CPB) has evolved into a
shock is probably the common predisposing               reliable method for total body perfusion, main-
factor, while extensive tissue damage, particularly     taining an oxygenated blood supply during the
head injuries, and pre-existing hepatic and renal       time heart surgery is performed. This is normally
failure may contribute to a deterioration in            achieved by draining venous blood under gravity
haemostasis.                                            from the right atrium into a reservoir, and then
   Volume expanders may produce other haemo-            pumping blood through an oxygenation device
static hazards, apart from dilution. Dextrans, and      back into the patient’s arterial system (Fig. 7.3).
to a lesser extent hydroxylethyl starch, have a         This procedure bypasses the heart and lungs and
fibrinoplastic effect; they accelerate the action        creates a bloodless surgical field.
of thrombin in converting fibrinogen to fibrin,              Haemorrhage is one of the most important com-
which makes clots more amenable to fibrinolysis.         plications of cardiac surgery since re-exploration
Both are adsorbed on platelet surfaces and on           for bleeding, which occurs after 2–6% of coronary
von Willebrand factor (vWF), causing decreased          artery bypass grafting procedures, has been associ-
platelet function and an acquired von Willebrand        ated with a case fatality rate as high as 22%. This
syndrome. Gelatins produce few problems, al-            is especially relevant now there are an increasing


90
                                                                                                   Bleeding in trauma and surgery




                                                                   Direction of blood flow
                                                                                                    Cannula to right atrium
                                                             Venous return from patient
                                                                          under gravity
                                                                                Cardiotomy suction lines for return
                                                                                of blood from the open chest
                                     Venous reservoir
                                      and cardiotomy
                                                filters                                              Arterial line
                                                                                                     pressure gauge
                                                                  Oxygenator

                                                                                                          Cannula
                                                                                                          to aorta


                                                                        Heat exchanger
                                                                                                      Blood returned to
                                                                                                     patient's systemic
                                                                                                    circulation bypassing
                                                                                                          the lungs
                                                          Centrifugal
                                                          pump
Fig. 7.3 Cardiopulmonary bypass
circuit.




number of patients requiring reoperation and of            owing to discharge of a granules and loss of
patients who have received anticoagulant, anti-            platelet membrane receptors such as glycoprotein
platelet or thrombolytic therapy prior to surgery          (GP)Ib and GPIIb/IIIa.
or who undergo complex surgery such as com-                • Fibrinolytic activation measured by D-dimer
bined heart and lung transplantation.                      levels has been shown to peak during CPB and
                                                           decrease at the end of CPB. There is wide variation
                                                           in fibrinolytic response to CPB. The increased
Factors associated with the bleeding diathesis
                                                           fibrinolytic activation is mainly due to an increase
of CPB
                                                           in t-PA.
• The extensive contact between blood and the              • Haemodilution is also a consequence of extra-
synthetic surfaces of the circuit causes coagulation       corporeal circulation but the fall in haematocrit,
activation, which necessitates the use of heparin.         platelet count and plasma proteins, including
Intravenous heparin is administered to the patient         coagulation factors, is about 30% and usually not
prior to CPB at a dose of 3 mg/kg, with repeated           sufficient to cause bleeding.
doses of heparin being given during CPB (approxi-             There is increasing use of minimally invasive or
mately equivalent to heparin levels of 3 U/mL).            ‘beating’ heart surgery, which avoids the use of
During CPB the activated clotting time (ACT) is            CPB. For example, using a piece of equipment
used to monitor anticoagulant therapy and is               known as an ‘Octopus’, which once placed on
maintained above 350–400 s.                                the heart will reduce movement in the area in
• Thrombocytopenia and defects in platelet                 which the surgeon wishes to operate, allows for
function are proportional to the duration of CPB,          surgery to proceed without stopping the heart
and probably related to the level of hypothermia.          beating. The haemostatic changes of these pro-
In addition, abnormalities of platelet function            cedures are less severe than those associated with
include a reduced response to aggregation stimuli          CPB.


                                                                                                                              91
Chapter 7


                                                             Some regions, especially prostatic and pelvic
Practical management of bleeding after
                                                          tissues, are rich in plasminogen activator, excessive
cardiac surgery
                                                          liberation of which may occur during the
• Check blood loss from all the chest drains. If one      following.
is filling at a greater rate than others, then this sug-   • Pelvic and prostatic surgery, especially for car-
gests a surgical cause, so discuss with the surgeon.      cinoma of the prostate.
• Obtain a history of preoperative drugs. The use         • Extensive surgery.
of aspirin and non-steroidal anti-inflammatory             • CPB.
drugs is associated with increased blood loss.            • Liver transplantation. Extremely high levels of t-
• If there is a steady blood loss in all the chest        PA occur during the anhepatic phase of orthotopic
drains of more than 300 mL/h, arrange for further         liver transplantation, probably as a result of in-
blood to be made available by calling the blood           creased endothelial release and decreased hepatic
transfusion laboratory. An FBC and clotting screen        clearance.
could be requested. While awaiting the results of         • Iatrogenic fibrinolytic bleeding can occur
these laboratory assays, an ACT may be per-               through the use of exogenous fibrinolytic activa-
formed to check that heparin has been adequately          tors such as streptokinase or urokinase in the man-
reversed with protamine. If heparin has not been          agement of thrombosis.
adequately reversed, then give the appropriate               Useful investigations include the following.
dose of protamine.                                        • A global test of fibrinolytic activity such as the
• Usually after cardiac surgery there is a throm-         TEG should ideally be available.
bocytopenia and platelet function defect, so the          • Levels of fibrin degradation products (D-dimers)
bleeding time will be prolonged and therefore of          are greatly increased.
little value in differentiating the cause of bleeding.    • PT, APTT and thrombin time are mildly pro-
A TEG trace, if available, may help.                      longed due to fibrinogenolysis.
• Discuss rationale for the use of platelet concen-       • Factors V and VIII may be normal or moder-
trates and if necessary order an adult therapeutic        ately reduced, in contrast to findings in DIC.
dose of platelets. While waiting for this to arrive, if   • Often it is difficult to exclude DIC, especially as
the patient has not received an antifibrinolytic           the most useful fibrinolytic assays are complex,
agent perioperatively consider using aprotinin            time-consuming and not widely available.
500 000 KIU.
• If bleeding continues at a rate of more than
                                                          Practical management
300 mL/h despite correction of any pre-existing
haemostatic defects and adequate haemostatic              Treatment with an antifibrinolytic agent should be
therapy, i.e. platelet transfusion given and platelet     considered if primary fibrinolytic bleeding is sus-
count greater than 100 ¥ 109/L and PT and APTT            pected. Suggested management options are:
ratios less than 1.5, with a fibrinogen level in the       • tranexamic acid up to 1 g slowly intravenously;
normal range (normally 2–4 g/L), then discuss re-         and
exploration of the wound with the surgeon.                • aprotinin 500 000 KIU as an intravenous bolus.


Hyperfibrinolysis
                                                          Pharmacological agents to
Bleeding may occur if there is excessive generation       reduce bleeding
of plasmin secondary to the release of tissue and
urokinase plasminogen activators. Plasmin is a            These have been used in two ways, either to
non-specific proteolytic enzyme and will split             prevent excessive bleeding or to treat established
peptides with arginyl-lysyl amino acid sequences.         bleeding. The majority have been used in patients
These include fibrinogen, factors V and VIII, and          having cardiac surgery with CPB. The agents used
the first component of complement.                         can be broadly classified into four groups: anti-


92
                                                                                        Bleeding in trauma and surgery


fibrinolytics, desmopressin, haemostatic sealants         should be given. Consideration should be given to
and recombinant factor VIIa.                             the future need of this drug, e.g. if a patient
                                                         requires repeat cardiac surgery but in the long term
                                                         requires a cardiac transplant, the surgeon may
Antifibrolytics                                           wish to reserve the use of aprotinin for the trans-
                                                         plant operation.
Aprotinin
                                                         • The risks of using a prothrombotic agent periop-
• High-dose aprotinin in cardiac surgery                 eratively have not been defined, especially those of
(2 000 000 KIU to the patient, 2 000 000 KIU into        increased postoperative thromboembolic disease
the CPB circuit and 50 000 KIU/h during CPB)             and particularly in relation to graft patency after
reduces postoperative drainage loss by 81%, and          coronary artery bypass grafting. Until these risks
total haemoglobin loss by 89%. It also has benefit        are defined the use of aprotinin to prevent blood
in vascular surgery and liver transplantation.           loss should be limited to its licensed indication, i.e.
Shorter operating times were also seen. This may         the prevention of blood loss in high-risk cardiac
result from the striking reduction of oozing that is     surgery.
normally seen; the operative fields remain ‘bone          • Aprotinin can also be used in established
dry’.                                                    fibrinolytic bleeding. An intravenous dose of
• Aprotinin is a basic polypeptide extracted from        500 000 KIU is a good antiplasmin dose.
bovine lung. It is able to inhibit certain serine pro-
teases by binding to their active site. In low con-
                                                         Lysine analogues
centrations, aprotinin is a powerful inhibitor of
plasmin: its molar potency in vitro is 100 and 1000      The lysine analogues, e-aminocaproic acid and
times that of tranexamic acid and e-aminocaproic         tranexamic acid, are competitive inhibitors of
acid. Its main mechanism of action is through            plasmin binding to fibrin. A continuous infusion of
an antiplasmin effect. In high doses, it also in-        tranexamic acid perioperatively in open cardiac
hibits kallikrein. The currently licensed high-dose      surgery reduces bleeding significantly by about
regimen was designed to achieve blood levels that        one-third, although it is not as effective as apro-
inhibit kallikrein (about 200 KIU/mL). Kallikrein        tinin. The dose given is 10 mg/kg over 20 min pre-
is formed during the activation of coagulation by        operatively followed by 1 mg/kg for 10 h. Both can
CPB and has a central role in the activation of the      be given to treat established fibrinolysis. The rec-
inflammatory response.                                    ommended dose for tranexamic acid is up to 1 g by
• Aprotinin has no effect on the fall in platelet        slow intravenous infusion.
count, but may have a minor effect on preserving
platelet function by preserving platelet membrane
                                                         Desmopressin
receptors, possibly by inhibiting plasmin-mediated
degradation.                                             Desmopressin acetate (DDAVP) is a synthetic
• Aprotinin, by inhibiting kallikrein, will prolong      vasopressin analogue that is relatively devoid of
in vitro tests of the intrinsic system and the ACT,      vasoconstrictor activity. It increases the plasma
because kallikrein normally operates a positive          concentrations and activity of vWF, probably by
feedback on the generation of factor XII. In order       inducing the release of vWF from Weibel–Palade
to allow for adequate levels of heparin, the ACT         bodies in the endothelium. Plasma levels of vWF
timer should be run greater than 750 s. The activa-      increase from two to five times from the baseline
tor in the ACT has traditionally been celite.            within an hour. It also improves platelet function.
Recently, kaolin has been used instead in some           It thus leads to shortening of the bleeding time in
ACT tubes, for it is less affected by aprotinin and      patients with von Willebrand’s disease, platelet
thus ACTs can be monitored in the normal way.            function defects and uraemia.
• Since aprotinin is a bovine protein and thus can          Trials of DDAVP 0.3 mg/kg given after CPB to
provoke an immunological reaction, a test dose           reduce bleeding had variable results; overall it was


                                                                                                                  93
Chapter 7


not beneficial. It may well be useful in patients        consistent with the time frame of normal wound
with platelet function defects preoperatively, but      healing.
this is not proven as yet. However, DDAVP may
yet have a place in reducing bleeding, for it may
                                                        Recombinant activated factor VII
be useful in reducing blood loss in patients
with functional platelet disorders, notably those       Recombinant activated factor VIIa (rVIIa) was ini-
patients who have received aspirin preoperatively.      tially used to treat haemophiliacs with inhibitors.
Adverse effects include flushing and an antidiuretic     The mechanism of action of factor VIIa in this
effect.                                                 setting is not fully understood but it does bypass
                                                        the need for factor VIII and IX by generating
                                                        thrombin and thus fibrin via direct activation of
Haemostatic sealants
                                                        factor X. It is unclear whether tissue factor is nec-
Haemostatic sealants mimic the final part of the         essary for this.
coagulation cascade.                                       The use of rVIIa has been explored ‘off licence’
• Fibrin sealants provide a source of thrombin          for uncontrolled bleeding in a number of clinical
and fibrinogen that when mixed together in the           scenarios. A group in Israel first described it as
presence of calcium form a clot. They can be            being efficacious in trauma patients who con-
administered by a ‘gun’ which produces mixing of        tinued to bleed despite conventional component
the reagents. Fibrin glue is best suited to secure      therapy. Subsequently, it has been used to reverse
haemostasis in patches and suture lines, for in situ-   over-anticoagulation with warfarin, uncontrolled
ations where there are high blood flow rates then it     bleeding in hepatic dysfunction, orthotopic liver
is in danger of being washed off before it has ‘set’.   transplantation, cardiac surgery and in patients
A systematic review has shown that they do reduce       with thrombocytopenia and platelet dysfunction.
allogeneic blood use and reduce bleeding but            There is a paucity of double-blind randomized
generally the trials were small and of poor             trials to assess efficacy and safety in these multiple
methodological quality.                                 potential applications, and a number of such trials
• The initial source of thrombin was of bovine          are currently in progress. The major concern about
origin, which led to the development of a bleeding      safety is the theoretical risk of thrombosis: rVIIa
diathesis postoperatively. This is due to the for-      will bind to exposed tissue factor and initiate local
mation of antibodies to bovine thrombin, which          thrombosis. Thus in patients with atheroma, tissue
cross-react with human factor V, leading to             factor in plaques may be exposed to blood at the
acquired factor V deficiency in the recipients.          time of plaque rupture, while in DIC tissue factor
Currently human thrombin is used in most fibrin          may be exposed on monocytes. Accumulating
sealants.                                               safety data suggest that the risk of thrombosis with
• Methods of preparing autologous fibrin glue            rVIIa is low, but this may relate to its low use in
have been developed. They are currently made by         those with atheromatous disease.
units with large transfusion centres and com-              A randomized double-blind trial explored the
mercial companies also exist to manufacture             use of rVIIa in preventing perioperative bleeding in
components.                                             36 patients undergoing retropubic prostatectomy
• Other haemostatic sealants, such as Floseal           randomized to receive a perioperative dose of
(Baxter), use bovine thrombin and gelatin. These        20 or 40 mg/kg rVIIa or placebo. Median blood
are reconstituted and mixed together. The mixture       loss was significantly reduced in those receiving
is applied to the tissue surface, the gelatin expands   40 mg/kg and none of this group required transfu-
to physically restrict the flow of blood and then        sion. There were no adverse events in the study
thrombin converts endogenous fibrinogen to               group. This study suggests that rVIIa requires
fibrin. The structural integrity of the gelatin–fibrin    further study to assess its efficacy and safety in pre-
matrix enables it to remain in place at the tissue      venting perioperative bleeding and reducing the
surface. The clot is resorbed within 6–8 weeks,         use of allogeneic blood.


94
                                                                                           Bleeding in trauma and surgery


                                                        girls and women of reproductive age, unless there
Organization of transfusion for patients
                                                        is life-threatening bleeding and group O RhD-
with trauma and for major accidents
                                                        negative blood is not available. Group O RhD-
(see also Chapter 6)
                                                        positive blood can be given to males with
                                                        unknown blood groups.
In a major accident, large numbers of people may
be injured within a short space of time. The rescue     • Blood components such as FFP and platelet con-
and management of patients requires a coordin-          centrates need to be available for those who are
ated approach from the rescue services and the          receiving massive transfusion.
hospital designated for admission of the casualties.    • Dealing with requests to donate blood: follow-
   A ‘major accident procedure’ is a necessity          ing a major accident, there may be a number of
within every hospital. It should be tested periodi-     telephone calls from the public, offering to donate
cally by holding a ‘major accident exercise’.           blood. These potential donors should be given the
The following must be incorporated into the             telephone number of the Blood Service National
procedure.                                              Call Centre so that they can attend one of the
• The telephone numbers of those who ‘need to           routine blood donor clinics.
know’ should be held by the hospital switchboard
and called.
• Suspend the issue of blood for non-emergency          Conclusions
cases.
• Increase the stocks of blood components to a          There have been some exciting advances in moni-
predefined level by arranging deliveries from the        toring and managing bleeding in surgical and
nearest transfusion centre and maintain the             trauma patients in the last few years. Despite this,
necessary stocks of blood and blood products            many more adequately powered, prospective
throughout the emergency. The blood transfusion         studies are required to investigate the utility and
laboratory must have a telephone line independent       safety of these approaches in different surgical
of the hospital switchboard because the main hos-       settings.
pital switchboard may be inundated with tele-
phone calls.
• The risk of errors in the identification of patients   Further reading
and blood samples can be high in an emergency.
Special care must be taken in the identification         Carless PA, Anthony DM, Henry DA. Systematic review of
of casualties, and in labelling blood samples. In         the use of fibrin sealant to minimize perioperative
accident and emergency every attempt to maintain          allogeneic blood transfusion. Br J Surg 2000; 89:
good clinical practice should be made. The                695–705.
minimum patient identification details for the           Carson JL, Poses RM, Spence RK, Bonavita G. Severity of
request form and blood sample are the hospital            anaemia and operative mortality and morbidity. Lancet
                                                          1996; 348: 1055–60.
number of the patient (or accident and emergency
                                                        Clark RAF. Fibrin sealant in wound repair: a systematic
or major incident number) and their gender.               survey of the literature. Exp Opin Invest Drugs 2000; 9:
• A telephone call from accident and emergency to         2371–91.
the blood transfusion laboratory to inform them of      Cochrane Injuries Group Albumin Reviewers. Human
estimates of potential future blood requirements is       albumin administration in critically ill patients:
essential.                                                systematic review of randomised controlled trials. Br
• To avoid errors, the practice of issuing blood in       Med J 1998; 317: 235–40. [This article received an
                                                          enormous critical response, and the reader is referred to
a major disaster should not be changed from the
                                                          one of the reviewers of the original article whose
routine practice of providing blood for urgent            response is typical of the criticism: Soni N. Human
requests.                                                 albumin administration in critically ill patients. Validity
• When the recipient’s blood group is not known,          of review methods must be assessed. Br Med J 1998; 317:
group O RhD-negative blood should be given to             883–4.]


                                                                                                                     95
Chapter 7

Friederich PW, Henny CP Messelink EJ et al. The effect of    Oz MC, Delos M, Cosgrove DM et al. Controlled clinical
  recombinant activated factor VII on perioperative blood      trial of a novel hemostatic agent in cardiac surgery. Ann
  loss in patients undergoing retropubic prostatectomy: a      Thorac Surg 2000; 69: 137–82.
  double-blind placebo-controlled randomised trial. Lancet   Porte RJ, Leebeek. Pharmacological strategies to decrease
  2003; 361: 201–5.                                            transfusion requirement in patients undergoing surgery.
Ghorashian S, Hunt BJ. Off license use of recombinant          Drugs 2002; 62: 2193–211.
  activated factor VII. Blood Rev 2004; 18: 245–59.          Sanguis Study Group. Use of blood products for elective
Hébert PC, Wells G, Blajchman MA et al. A multicenter,         surgery in 43 European hospitals. Transfus Med 1994; 4:
  randomized, controlled clinical trial of transfusion         251–68.
  requirements in critical care. N Engl J Med 1999; 340:     Segal H, Hunt B. Aprotinin: pharmacological reduction of
  409–17.                                                      perioperative bleeding. Lancet 2000; 355: 1289–90.
Horrow JC, Hlavecek J, Strong MD et al. Prophylactic         Shore-Lesserson L, Manspeizer HE, DePerio M, Francis S,
  tranexamic acid decreases bleeding after cardiac             Vela-Cantos F, Ergin MA. Thromboelastography-guided
  operations. J Thorac Cardiovasc Surg 1990; 99: 70–4.         transfusion algorithm reduces transfusion in complex
Martinowicz U, Kenet G, Segal E et al. Recombinant             cardiac surgery. Anesth Analg 1999; 88: 312–19.
  activated factor VII for adjunctive haemorrhage control
  in adults. J Trauma 2001; 51: 431–9.




96
Chapter 8
Prenatal and childhood transfusions

Irene Roberts




Obstetrics                                              • Identify RhD-negative women who require anti-
                                                        D prophylaxis (around 16% women are RhD
One of the most important aspects of obstetric          negative).
transfusion medicine is the prevention, recogni-        • Ensure swift provision of compatible blood for
tion and treatment of haemolytic disease of the         obstetric emergencies.
newborn (HDN), which causes at least 50 neona-          • Identify the fetus requiring treatment in utero or
tal deaths per year in the UK. This is considered in    in the neonatal period.
detail in this chapter. Other topics covered include:   • Identify additional red cell alloantibodies.
• aspects of maternal platelet and white cell disor-    • Identify new red cell antibodies induced by
ders relevant to transfusion;                           intrauterine transfusion.
• maternal haemorrhagic disorders, including
major obstetric haemorrhage; and
                                                        Red cell serology at the booking visit
• transfusion requirements during pregnancy of
patients with major haemoglobinopathies.                At the booking visit, which should take place
                                                        before week 16 of pregnancy, all women should
                                                        have their ABO and RhD group determined and
Antenatal red cell antibody testing
                                                        should be screened for red cell alloantibodies. If
The three factors essential in the pathogenesis of      red cell antibodies are detected at the booking visit
HDN are:                                                and/or if there is a history of HDN, the antibodies
• maternal red cell alloantibodies that cross the       should be identified, quantified and monitored as
placenta;                                               outlined below. It is particularly important to
• fetal red blood cells that express antigens against   monitor women with anti-D, anti-c and anti-K,
which the antibodies are directed; and                  since these antibodies may be associated with
• antibodies which are able to mediate red cell         severe HDN. If no red cell alloantibodies are
destruction.                                            detected at booking, all pregnant women should
Clinically relevant alloantibodies are almost           be retested at 28–36 weeks of gestation. Further
always IgG and are reactive at 37°C. Women              testing of women without detectable antibodies is
develop these antibodies as a result of previous        unnecessary since immunization later in pregnancy
transfusions, previous pregnancies or both. Identi-     is unlikely to result in antibody levels sufficient to
fication of such antibodies is the main goal of ante-    cause HDN requiring treatment.
natal screening.
                                                        Partial/weak D
Objectives of red cell antibody testing
                                                        Du (weak D) individuals are regarded as RhD posi-
in pregnancy
                                                        tive and do not form immune anti-D. Individuals
• Identify the pregnancy at risk of fetal or neona-     with known partial D status, e.g. DVI, are more
tal HDN.                                                likely to make anti-D; therefore it is important that


                                                                                                          97
Chapter 8


reagents for RhD grouping do not detect DIV             proportion of women who are RhD negative
(so that these individuals group as RhD negative).      (16%). Most anti-D antibodies are IgG1 or IgG1
                                                        plus IgG3. The presence of IgG3 alone, which has
                                                        100 times the destructive ability of IgG1, is uncom-
ABO antibodies
                                                        mon and rarely associated with HDN in utero, but
There is no need to test for ABO immune anti-           can cause severe postnatal manifestations of HDN.
bodies in antenatal samples as their presence is not       Anti-c is found most commonly in women with
predictive of HDN and such antibodies very rarely       the R1R1 genotype (CDe/CDe), which occurs in
cause significant haemolysis in utero.                   20% of pregnant women. Such women also have
                                                        the propensity to make anti-E. HDN due to anti-E
                                                        is both less common and less severe. However,
Samples at delivery
                                                        anti-E and anti-c in combination cause more severe
At the time of delivery a maternal and a cord blood     HDN than either antibody alone. Note that in
sample should be collected from all pregnancies in      such cases only the anti-E is detectable in eluates
RhD-negative women. Women with no previously            from cord blood red cells.
detected anti-D should have prophylactic anti-D            Anti-K1 is the most common red cell alloanti-
administered if the infant is RhD positive. A           body outside the ABO and Rh system. K1 is the
Kleihauer test should also be carried out on all such   principal antigen of the Kell blood group system
women to assess the requirement for additional          and is highly immunogenic; 5% of K1-negative
anti-D. A direct antiglobulin test (DAT) should be      individuals will produce anti-K1 if transfused with
performed on the cord blood: a positive DAT is a        K1-positive blood. K1 has around twice the
good predictor of HDN. It is important to note that     potency of c and E and 20 times the potency of Fya.
in women who have had prophylactic anti-D               Anti-K1 often causes severe HDN; the haemolytic
during pregnancy the anti-D remains detectable in       anaemia is compounded by suppression of erythro-
serum for up to 12 weeks and may cause confusing        poiesis due to anti-K1 inhibiting the growth of ery-
serological results in the mother and a positive        throid progenitor cells. Anti-K titres can be an
DAT in the baby in the absence of HDN.                  unreliable predictor of the severity of HDN. There-
  In the case of women with other clinically sig-       fore it is important to identify the fetuses at risk of
nificant red cell alloantibodies (see below), a DAT      HDN by determining the fetal Kell genotype in all
should be carried out on cord blood; if the DAT is      mothers with anti-K1 whose partners are heterozy-
positive, a red cell eluate may help identify the red   gous for K1 (since only 50% of such fetuses will be
cell antibody. Infants born to mothers with clini-      K1 positive). Moderate to severe HDN may also be
cally significant antibodies should be monitored         caused by anti-K2 (anti-cellano) and anti-Kpa.
for 48–72 h for the presence of haemolysis.                A number of other red cell alloantibodies have
                                                        also been reported to cause HDN of variable sever-
                                                        ity, e.g. anti-U. These initially present with a posi-
Clinically relevant red cell alloantibodies
                                                        tive indirect antiglobulin test (IAT) in maternal
The main antibodies implicated in HDN include           serum; therefore all women with a positive IAT
the following.                                          should have further investigation to try to identify
• Rh group: D, c, C, e, E, Ce and Cw.                   any clinically relevant red cell alloantibodies.
• Kell group: K1, K2 and Kpa.
• Duffy group: Fya.
                                                        Red cell alloantibodies not implicated in HDN
• Kidd group: Jka.
The antibodies most commonly implicated in severe       These include the following:
to moderate HDN are anti-D, anti-c and anti-Kell.       • anti-Lea and anti-Leb;
   Anti-D is the commonest cause of HDN                 • anti-Lua;
(approximately 40% of cases in the UK). This is         • anti-P;
because anti-D is highly immunogenic and the high       • anti-N;


98
                                                                                  Prenatal and childhood transfusions


• anti-Xga;                                             • Fetal K typing by chorionic villous sampling
• anti-Gerbish.                                         (CVS), amniocentesis or fetal blood sampling
                                                        should be performed where the father is heterozy-
                                                        gous for K1.
Management of pregnant women with red
                                                        • Fetal growth, fetal anaemia and the presence of
cell alloantibodies
                                                        hydrops should be monitored by serial ultrasound
Anti-D                                                  and Doppler and anaemia confirmed by fetal
                                                        blood sampling as indicated.
• Women with anti-D should have their anti-D
                                                        • Amniocentesis is not a good indicator of the
titres monitored monthly until 28 weeks of gesta-
                                                        severity of fetal anaemia since anaemia due to anti-
tion and then every 2 weeks.
                                                        K results from a combination of haemolysis and
• All samples should be checked in parallel with
                                                        red cell hypoplasia.
the previous sample.
                                                        • All women with anti-K should be referred to a
• An increase in anti-D by 50% or more com-
                                                        specialist fetal medicine unit early in pregnancy.
pared with the previous sample is a significant
increase irrespective of gestation.
   It is important to note that these are guidelines.   Other red cell alloantibodies
Titres of anti-D do not always correlate closely        • If the antibody is likely to cause problems with
with the development of HDN. Therefore all              provision of blood to cover an obstetric emer-
women with affected pregnancies should be               gency, it is important to inform the obstetrician in
referred early to specialist fetal medicine units for   charge of the case and the transfusion laboratory
fetal assessment by ultrasound, amniocentesis or        in the hospital and efforts should be made to
fetal blood sampling as indicated (for management       ensure that appropriate blood products can be
of the fetus and neonate, see pp. 107–9).               supplied.
                                                        • Any babies born to mothers with an IAT-
Anti-c                                                  reacting antibody must be assessed at birth for evi-
                                                        dence of HDN.
• Women with anti-c should have their anti-c
titres monitored monthly until 28 weeks of gesta-
                                                        Blood transfusion support for mother and baby
tion and then every 2 weeks.
• All samples should be checked in parallel with        Mother
the previous sample.
                                                        • Red cell components of the same ABO and RhD
• An increase in anti-c by 50% or more compared
                                                        group must be selected.
with the previous sample is a significant increase
                                                        • Group O blood may be used, provided it is
irrespective of gestation.
                                                        plasma depleted and does not contain high-titre
• Anti-c titres of greater than 10 IU/mL are asso-
                                                        agglutinins.
ciated with a moderate risk of HDN and may
                                                        • Note that in pregnancy, immunization following
require intrauterine transfusion (IUT).
                                                        a transfusion is most likely to occur in the third
• All women with anti-c should be referred to a
                                                        trimester.
specialist fetal medicine unit early in pregnancy.
                                                        • Samples used for pretransfusion testing should
                                                        ideally be taken immediately before transfusion
Anti-Kell                                               and must never be more than 7–10 days old (Table
                                                        8.1).
• Women with anti-K1 should have their anti-K
titre monitored monthly until 28 weeks of gesta-
tion and then every 2 weeks.                            Fetus and neonate: crossmatching and
• Anti-K titres may not accurately reflect the           general considerations
degree of fetal anaemia.
                                                        Management of the fetus and neonate at risk of


                                                                                                                 99
Chapter 8

Table 8.1 Pretransfusion testing of maternal samples.              10 Note that alloantibody formation is rare in the
                                                                   fetus and neonate and is usually associated with
Timing of last transfusion       Timing of pretransfusion sample   massive transfusion or with the use of fresh or
                                                                   whole blood.
3–14 days before                 24 h before transfusion
                                                                   11 Gamma-irradiation of cellular blood compon-
14–28 days before                72 h before transfusion
28 days to 3 months              1 week before transfusion
                                                                   ents to reduce the risk of transfusion-associated
                                                                   graft-versus-host disease (TA-GVHD) is recom-
                                                                   mended for:
                                                                      (a) all IUT;
HDN is discussed in detail below. The general                         (b) all transfusions to neonates previously
principles of the blood products used are summa-                      transfused in utero;
rized here.                                                           (c) exchange transfusions as long as gamma-
 1 Prior to the first transfusion, samples should be                   irradiation would not result in a delay in
obtained from the mother for ABO, RhD grouping                        transfusion;
and antibody screening and from the fetus/neonate                     (d) all transfusions from a family member;
for ABO, RhD and DAT (plus an antibody screen                         (e) all neonates with known inherited
if no maternal sample is available).                                  immune deficiencies (e.g. severe combined
 2 In the fetus/neonate the ABO group is deter-                       immunodeficiency).
mined on the cells only (as reverse grouping can                      These precautions are due to the immaturity of
detect passive maternal antibodies).                               the fetal and neonatal immune system which may
  3 Red cells which are ABO compatible with                        lead to a reduced ability to reject transfused allo-
maternal and neonatal plasma, RhD negative (or                     geneic lymphocytes, immune tolerance and the
RhD identical with neonate) should be used. (NB                    persistence of donor lymphocytes for up to 6–8
If exchange or ‘top-up’ transfusion is required                    weeks after exchange transfusion.
for HDN due to ABO incompatibility, group O red
cells with low titre anti-A and B or group O red
                                                                   HDN: guidelines for prevention
cells suspended in AB plasma should be used.)
 4 Group O blood is acceptable; units with high-                   The introduction of anti-D prophylaxis for
titre anti-A/anti-B must be excluded.                              recently delivered RhD-negative women in the UK
 5 If the mother’s blood group is unknown, blood                   in 1969 led to a reduction in new immunizations
for the fetus/neonate should be crossmatched                       against anti-D from 17% of pregnancies to 1.5%.
against the baby’s serum.                                          Every year in the UK 80 000 RhD-negative women
  6 If no atypical antibodies are present in the                   have a RhD-positive infant and despite national
maternal (or infant) sample, and if the DAT of the                 guidelines sensitization still occurs, largely due to
infant is negative, crossmatching is not necessary                 non-compliance with the guidelines. A dose of
for the first 4 months of postnatal life.                           anti-D of 125 IU (25 mg) suppresses immunization
 7 If the antibody screen or DAT is positive, full                 by 1 mL of RhD-positive red cells (i.e. 2 mL of
serological investigation and compatibility testing                whole blood). (Note that in the UK the dose of
are necessary.                                                     anti-D is given in IU, whereas in other countries
 8 An electronic crossmatch is not advisable unless                it is expressed in milligrams.) While anti-D
an appropriate algorithm has been created, as                      is extremely effective as prophylaxis, it cannot
ABO-identical adult blood transfused to an infant                  reverse immunization once it has occurred and has
with maternal anti-A or anti-B may haemolyse                       no effect on the development of non-D antibodies.
even if the pretransfusion DAT is negative, due to
stronger ABO antigen expression on adult cells.
                                                                   Indications for anti-D immunoglobulin
  9 Red cells (and platelets if given) should be
                                                                   (Table 8.2)
cytomegalovirus (CMV) negative and leucocyte
depleted.                                                          Anti-D should be given to all RhD-negative


100
                                                                                               Prenatal and childhood transfusions


                                                                     worthwhile up to 10 days after the event. In threat-
women without anti-D antibodies after the follow-
                                                                     ened abortion continuing to term, it is important
ing sensitizing events:
                                                                     to repeat the dose every 6 weeks (or use higher
• abortion (see below);
                                                                     doses; see Table 8.2) since low antibody levels can
• CVS;
                                                                     augment the antibody response.
• ectopic pregnancy;
                                                                       The dose of anti-D is as follows.
• amniocentesis;
                                                                     • Sensitizing event before 26 weeks of gestation:
• external cephalic version;
                                                                     250 IU (50 mg) i.m.
• abdominal trauma;
                                                                     • Threatened abortion continuing to term: 500 IU
• antepartum haemorrhage;
                                                                     (100 mg) every 6 weeks or 1250 IU (250 mg) every
• premature labour;
                                                                     10 weeks until term.
• pre-eclampsia; and
                                                                     • It is now recommended in the UK that routine
• intrauterine death (associated with chronic
                                                                     antenatal prophylaxis is provided in RhD-negative
fetomaternal haemorrhage).
                                                                     women without anti-D in the absence of a sensitiz-
   Anti-D should be administered following all
                                                                     ing event: 500 IU (100 mg) at 28 weeks and 34
abortions after 12 weeks, both spontaneous and
                                                                     weeks (no antibody screen is necessary before the
induced, and following abortion at any gestation
                                                                     dose at 34 weeks or at delivery).
following surgical or medical treatment, including
the use of abortifacients. Anti-D should also be
administered in cases of threatened abortion if                      Dose and schedule of administration of anti-D in
there is any bleeding after 12 weeks of gestation.                   the postnatal period
Current UK guidelines also recommend the admin-
                                                                     Therapeutic anti-D immunoglobulin to prevent
istration of anti-D immunoglobulin as antenatal
                                                                     the development of immune anti-D should be
prophylaxis since fetomaternal bleeding can
                                                                     given within 72 h of delivery; however, anti-D may
happen at any gestation.
                                                                     still be worthwhile up to 10 days after the event.
                                                                     The dose of anti-D is as follows.
Dose and schedule of administration of anti-D                        • 500 IU (100 mg) is the standard dose to cover a
during the antenatal period                                          fetomaternal bleed of 4 mL or less.
                                                                     • A Kleihauer test should always be performed to
Therapeutic anti-D immunoglobulin to prevent
                                                                     detect larger bleeds (bleeds >4 mL occur in 0.8%
the development of immune anti-D after sensitiz-
                                                                     and of >15 mL in 0.3% of deliveries) so that an
ing events should be given within 72 h of the sensi-
                                                                     additional dose of anti-D (125 IU/mL blood loss)
tizing event; however, anti-D may still be
                                                                     can be given.
                                                                     • The standard dose of anti-D in the USA and
                                                                     some European countries is higher (1500 IU).
Table 8.2 Antenatal and postnatal prophylaxis with anti-D.
                                                                        It takes 48 h following an intramuscular dose of
                                                                     anti-D to reach a good level and 72 h for clearance
Indications for anti-D         Dose and schedule of administration   of sensitized red cells. If clearance of the RhD-
                                                                     positive cells is not complete, further anti-D must
Sensitizing event <26 weeks’   250 IU (50 mg) i.m.                   be given until RhD-positive cells can no longer be
   gestation                                                         detected in maternal blood.
Threatened abortion            500 IU every 6 weeks until term or
   continuing to term            1250 IU every 10 weeks until term
RhD-negative women             500 IU at 28 weeks and 34 weeks       Kleihauer test
   without anti-D or a
                                                                     This simple and inexpensive test is used to detect
   sensitizing event
                                                                     whether there has been a fetomaternal haemor-
Standard postnatal             500 IU (1500 IU in USA and some
   prophylaxis                   European countries)
                                                                     rhage and the size of that haemorrhage. The prin-
                                                                     ciple and method for the test is as follows.


                                                                                                                             101
Chapter 8


• Fetal haemoglobin (HbF)-containing fetal red           Anti-D immunoglobulin not indicated
cells resist acid elution and therefore stain dark
                                                         Anti-D immunoglobulin is not indicated in the fol-
pink in comparison with HbA-containing cells,
                                                         lowing circumstances:
which appear as unstained ‘ghost’ cells (Plate 8.1,
                                                         • patients who are already sensitized;
shown in colour between pp. 304 and 305).
                                                         • those classified as weak D (e.g. Du);
• To quantitate fetomaternal haemorrhage, the
                                                         • if the infant is RhD negative;
numbers of pink-staining HbF-containing cells in
                                                         • for women not capable of child-bearing (follow-
each single low-power field are counted; using this
                                                         ing transfusion of RhD-positive blood);
method a count of 200 HbF-containing cells or less
                                                         • for complete abortions before 12 weeks of gesta-
in 50 low-power fields is equivalent to a fetomater-
                                                         tion if there has been no surgical treatment.
nal haemorrhage of 4 mL or less.
• Samples of maternal blood for the Kleihauer test
must be taken within 2 h of administration of anti-      Preparation of anti-D immunoglobulin
D to avoid a falsely low estimate of the size of the
                                                         Anti-D is a polyclonal antibody prepared by
fetomaternal bleed.
                                                         plasmapheresis of hyperimmunized donors, 95%
• Maternal hereditary persistence of fetal haemo-
                                                         of whom are women who have been sensitized
globin may cause a false-positive Kleihauer test
                                                         during pregnancy. Anti-D is now prepared using
due to maternal HbF-containing red cells.
                                                         US donor plasma because of concerns about trans-
                                                         mission of variant Creutzfeldt–Jakob disease.
Large fetomaternal bleeds
Larger bleeds (>4 mL) may be measured using flow          Platelet and white cell disorders in pregnant women
cytometry as well as the Kleihauer test. Larger
                                                         Differential diagnosis of thrombocytopenia
fetomaternal bleeds are associated with:
                                                         in pregnancy
• amniocentesis;
• abdominal trauma;                                      The most common causes of maternal thrombo-
• antepartum haemorrhage;                                cytopenia are the following.
• stillbirth;                                            • Gestational.
• twin pregnancy;                                        • Pregnancy induced: pre-eclampsia, eclampsia,
• manual removal of the placenta.                        HELLP (haemolysis with elevation of liver
For any fetomaternal haemorrhage >4 mL an                enzymes and low platelets) syndrome.
appropriate supplementary dose of anti-D must be         • Immune: immune thrombocytopenia (ITP) and
given immediately AND a repeat test for fetal cells      systemic lupus erythematosus (SLE).
and free anti-D should be carried out on the             • Virus associated, e.g. human immunodeficiency
mother 48 hours after the initial anti-D injection.      virus (HIV).
A further appropriate dose of IgG anti-D should be          Less common causes of maternal thrombocyto-
given to the mother:                                     penia include:
• if fetal cells are no longer present but there is no   • antiphospholipid syndrome;
residual free anti-D detectable (to make sure there      • thrombotic thrombocytopenic purpura;
is sufficient anti-D to eliminate small numbers of        • disseminated intravascular coagulation (DIC);
fetal cells below the limits of detection);              • type IIb von Willebrand’s disease;
• if fetal cells are still present but there is no       • congenital bone marrow failure (e.g. Fanconi’s
detectable anti-D.                                       anaemia);
Note that if fetal cells are still present after 48      • heparin-induced thrombocytopenia;
hours but there is still detectable anti-D, a repeat     • folate/B12 deficiency;
test for fetal cells and free anti-D should be carried   • myelodysplasia/acute leukaemia.
out after a further 48 hours to determine whether           It may be difficult to distinguish between gesta-
more anti-D IgG should be given to the mother.           tional, pregnancy-induced and immune thrombo-


102
                                                                                 Prenatal and childhood transfusions


cytopenia in pregnancy. ITP is more likely if the      moderate not severe. Fulminant pre-eclampsia pre-
platelet count was subnormal prior to or during the    cipitating early delivery may be associated with
first trimester of pregnancy. Further investigation     DIC and require treatment with platelet transfu-
depends on careful evaluation of the blood film and     sion and fresh frozen plasma (FFP) (with or
marrow smear, which may reveal characteristic          without cryoprecipitate). Thrombocytopenia in
changes (e.g. acute leukaemia). The disorders of       HELLP syndrome is more often severe and platelet
particular relevance to transfusion medicine are       transfusion may be indicated, particularly at deliv-
ITP, HELLP and type IIb von Willebrand’s disease.      ery, which is usually by urgent Caesarean section,
                                                       and postpartum.
Management of maternal ITP
ITP usually presents in an otherwise well mother       Type IIb von Willebrand’s disease
with or without a previous history of ITP or, less
                                                       Type IIb von Willebrand’s disease (vWD) is charac-
commonly, SLE. In those without a previous
                                                       terized by:
history, the diagnosis may be difficult and platelet
                                                       • low factor VIII and von Willebrand factor
antibody studies are of limited value since they
                                                       (vWF);
may be positive even in the absence of ITP.
                                                       • thrombocytopenia due to platelet activation/
   The management of the mother with active
                                                       consumption;
ITP and who is thrombocytopenic should be as con-
                                                       • reduced high-molecular-weight vWF multimers;
servative as possible. The most common approach
                                                       • the need for prophylactic treatment at delivery
to therapy is with intravenous immunoglobulin
                                                       with vWF or factor VIII.
(0.3–0.5 mg/kg daily) for 3–5 days. However, pred-
nisolone (1 mg/kg) can also be used. The indications
for treatment of maternal ITP are:                     Leukaemia in pregnancy
• platelets less than 20 ¥ 109/L in the first, second
                                                       When chemotherapy is administered during an
or early third trimester;
                                                       ongoing pregnancy, both the mother and fetus may
• aim to have platelets above 80 ¥ 109/L in the late
                                                       develop pancytopenia. Platelet transfusion for the
third trimester;
                                                       mother should be given according to the usual
• avoid epidural or spinal anaesthesia if the
                                                       guidelines, aiming to have a platelet count greater
platelet count is less than 80 ¥ 109/L;
                                                       than 80 ¥ 109/L at delivery and for the first few
• platelet transfusion is very rarely indicated;
                                                       days postpartum. The fetus should be monitored
• splenectomy should be postponed until after
                                                       and fetal blood sampling with or without
delivery if possible;
                                                       blood/platelet transfusion of irradiated blood
• fetal blood sampling and elective Caesarean
                                                       products may be indicated on rare occasions.
section for maternal ITP are unnecessary since sig-
nificant fetal thrombocytopenia is uncommon
(12%) and intracranial haemorrhage is even less        Maternal haemorrhagic problems
common (1%).
                                                       Guidelines for the investigation and management
   The fetal platelet count cannot be predicted
                                                       of haemorrhagic disorders in pregnancy have
from maternal platelet counts nor from platelet
                                                       been published by the British Committee for Stan-
serology. The most important factor predicting the
                                                       dards in Haematology (BCSH) Haemostasis and
presence and severity of fetal thrombocytopenia is
                                                       Thrombosis Task Force (1994) and are summa-
a history of maternal ITP prior to pregnancy: in
                                                       rized here.
this higher-risk group, 10–30% of babies will have
significant thrombocytopenia (<50 ¥ 109/L).
                                                       Inherited disorders
Pre-eclampsia and HELLP syndrome
                                                       The most common inherited coagulation disorder
In pre-eclampsia, thrombocytopenia is usually          is vWD. The prevalence of vWD is around 1% but


                                                                                                               103
Chapter 8


many are only mildly affected. There are three main     develop HELLP syndrome with associated hepatic
types of vWD: type I is the commonest (75%); type       failure may also develop severe DIC with low fib-
IIa occurs in 10%; type IIb occurs in 7% and may be     rinogen and extremely low antithrombin levels.
accompanied by severe thrombocytopenia; and             Maternal mortality approaches 30%; fetal mortal-
type III is the most severe. Most women with vWD        ity is around 50%.
have increased factor VIII and vWF during preg-
nancy and do not bleed excessively.                     Major obstetric haemorrhage protocol
   Bleeding in pregnant women with vWD, when it
                                                        Severe bleeding is an obstetric emergency. It is
does occur, usually causes problems:
                                                        essential to establish a major obstetric emergency
• during invasive procedures in the first trimester
                                                        haemorrhage protocol in any hospital where preg-
(e.g. CVS);
                                                        nant women are likely to deliver (see also Chapters
• after birth, particularly after surgical delivery
                                                        6 and 7). This protocol must be agreed by haema-
and/or perineal damage.
                                                        tologists, including the haematology and transfu-
   Management of vWD in pregnancy can be sum-
                                                        sion laboratory staff, obstetricians and midwives,
marized as follows.
                                                        and those responsible for transporting urgent spec-
• For first-trimester procedures factor VIIIC activ-
                                                        imens and blood products.
ity should be raised to 50 U/dL.
                                                           The most important points include the
• At delivery, operative procedures should be
                                                        following.
avoided except for obstetric indications and
                                                        • If the blood group is known and there are no
trauma should be minimized.
                                                        atypical antibodies: group-compatible red cells (6
• Type I vWD: for vaginal delivery no blood
                                                        units) should be issued.
product support is necessary if factor VIIIC is
                                                        • If there are atypical antibodies: phenotyped red
greater than 40 U/dL; if less than 40 U/dL or if Cae-
                                                        cells should be issued whenever possible.
sarean section is planned, factor VIIIC should be
                                                        • If the blood group is unknown: group O RhD-
given to raise the level to at least 50 U/dL.
                                                        negative blood (6 units) should be issued.
• Type II and type III vWD: prophylactic factor
                                                        • Samples should be obtained from the patient as
VIIIC should be started at the onset of labour in all
                                                        soon as possible for blood group, full blood count
type III patients, most type IIa patients and some
                                                        and coagulation screen.
type IIb patients; the aim should be to raise the
                                                        • Once results are available further units of red
level of factor VIIIC to more than 40 U/dL; prod-
                                                        cells, FFP, cryoprecipitate and/or platelets should
ucts with high levels of large vWF multimers
                                                        be issued as indicated.
should be used (e.g. 8Y, intermediate purity factor
                                                        • An antibody screen and retrospective cross-
VIII concentrate containing vWF); for Caesarean
                                                        match should be performed on the units issued as
section, factor VIIIC should be raised to more than
                                                        soon as time allows.
50 U/dL.

                                                        Management of the major haemoglobinopathies
Acquired disorders
                                                        during pregnancy
The most common acquired disorder is DIC. This
                                                        Sickle cell disease
occurs secondary to:
• severe eclampsia/pre-eclampsia;                       The frequency of maternal complications related
• intrauterine death;                                   to sickle cell disease, particularly vaso-occlusive
• placental abruption;                                  crises, is not particularly increased during preg-
• amniotic fluid embolism;                               nancy. However, pregnant sickle cell disease
• hydatidiform mole (chronic DIC).                      patients do have an increased risk of:
  Haemorrhage may be severe. Treatment is as for        • placental insufficiency causing intrauterine
DIC in non-pregnant patients except that the use        growth restriction;
of heparin is not recommended. Women who                • eclampsia/pre-eclampsia;


104
                                                                                         Prenatal and childhood transfusions


• preterm delivery;                                  • Desferrioxamine is discontinued during preg-
• stillbirth and neonatal death;                     nancy because of the potential for nephrotoxicity,
• urinary tract infection (SC disease);              hepatotoxicity and ear/eye damage.
• pulmonary thromboembolism (particularly SC
disease).
                                                     Fetal and neonatal transfusion
Indications for red cell transfusion in pregnant
                                                     Causes of fetal and neonatal anaemia
women with sickle cell disease
• Exchange transfusion should be carried out         The principal causes of fetal and neonatal anaemia
prior to planned operative delivery.                 are shown in Table 8.3. The commonest causes of
• Exchange transfusion should be continued in        fetal anaemia in the UK are parvovirus infection,
any patient already on a regular exchange pro-       HDN and twin-to-twin transfusion and the com-
gramme (e.g. for prevention of recurrent stroke).    monest causes of neonatal anaemia are iatrogenic
• Exchange transfusion should be considered          blood loss and anaemia of prematurity.
for severe prolonged vaso-occlusive crises during
pregnancy.
                                                     Management of neonatal anaemia (HDN is discussed
• ‘Top-up’ transfusion should be considered
                                                     in the next section)
for severe anaemia if symptomatic.
• Regular prophylactic transfusion therapy           The only available treatment for neonatal anaemia
during pregnancy does not reduce pregnancy-          is red cell transfusion. Prevention or amelioration
related complications or improve outcome.
• Red cells should be phenotyped (for Rh, Kell,      Table 8.3 Principal causes of fetal and neonatal anaemia.
Duffy, Kidd and MNS) to reduce the risk of alloim-
munization, and red cells from sickle trait donors   Impaired red cell production
                                                     Diamond–Blackfan anaemia
should not be used.
                                                     Congenital infection (e.g. parvovirus, CMV)*
                                                     Congenital dyserythropoietic anaemia
Thalassaemia major and intermedia                    Pearson’s syndrome

Both maternal and fetal morbidity are higher in      Haemolytic anaemias
pregnant women with thalassaemia major and           Alloimmune: haemolytic disease of the newborn (Rh,ABO, Kell, other)*
                                                     Autoimmune (e.g. maternal autoimmune haemolysis)
intermedia. In the mother, the degree of anaemia,
                                                     Red cell membrane disorders (e.g. hereditary spherocytosis)
the cardiovascular changes associated with preg-
                                                     Red cell enzyme deficiencies (e.g. pyruvate kinase deficiency)
nancy and pre-existing cardiac damage may aggra-     Some haemoglobinopathies (e.g. a-thalassaemia major*, HbH disease)
vate the multiorgan damage secondary to iron         Infection (e.g. bacterial, syphilis, malaria, CMV, Toxoplasma, herpes
overload. For the fetus, most reports suggest an        simplex
increase in intrauterine growth restriction and
                                                     Anaemia due to haemorrhage
preterm delivery. The rate of Caesarean section is
                                                     Occult haemorrhage before or around birth (e.g. twin-to-twin*,
increased, possibly because of cephalopelvic dis-       fetomaternal)
proportion. Management is complex, requiring         Internal haemorrhage (e.g. intracranial, cephalhaematoma)
close liaison between haematologists and             Iatrogenic: due to frequent blood sampling
obstetricians.
                                                     Anaemia of prematurity
                                                     Due to impaired red cell production, impaired erythropoietin
Management guidelines for pregnant women with
                                                     production and reduced red cell lifespan
thalassaemia major or intermedia                     Haemoglobin nadir usually 6.5–9 g/dL
• Haemoglobin should be maintained above
10–12 g/dL to optimize fetal outcome.                * Commonly present in the fetus; other causes may present during fetal
• Transfusion requirements usually increase          life but neonatal presentation is more common.
during pregnancy.                                    CMV, cytomegalovirus.


                                                                                                                       105
Chapter 8


of anaemia sufficient to reduce red cell transfusion                 reduce the need for red cell transfusion and/or
requirements may be achieved by using recom-                        donor exposure in neonates:
binant erythropoietin, although this is not useful                  • development and implementation of local trans-
in the acute setting due to the 2-week delay in                     fusion guidelines for each neonatal unit;
increasing haemoglobin using erythropoietin.                        • minimizing iatrogenic blood letting;
                                                                    • prevention and treatment of haematinic defi-
                                                                    ciencies (iron and folic acid);
Indications for ‘top-up’ transfusion
                                                                    • use of dedicated satellite packs (Paedipacks);
Guidelines for ‘top-up’ transfusion have been                       • improved antimicrobial screening;
devised by committees in a number of countries,                     • judicious use of erythropoietin (see below);
including the UK, Canada and the USA. Since                         • autologous cord blood transfusion: delayed
there is almost no objective evidence, these guide-                 clamping of the cord at delivery.
lines are based on clinical experience and represent
consensus views. Table 8.4 summarizes the indica-
                                                                    Neonatal ‘top-up’ transfusions:
tions for ‘top-up’ transfusions used in many UK
                                                                    product specification
neonatal intensive care units.
                                                                    • Small volume ‘top-up’ transfusions can be given
                                                                    without further testing provided that there are no
Hazards of transfusion in neonates
                                                                    atypical maternal antibodies in maternal/infant
Transfusion of neonates is becoming safer, espe-                    serum and the infant’s DAT is negative.
cially with the more widespread use of satellite                    • Red cells should be 35 days old or less (if in
packs. The most important hazards of transfusion                    SAG-M or similar additive system) or 28 days old
in neonates are:                                                    or less (if in CPD).
• infection (bacterial or viral);                                   • Paedipacks (aliquotted donations from a single
• hypocalcaemia (more common in neonates than                       unit) should be used wherever possible for
in infants or children);                                            repeated transfusions in order to minimize donor
• volume overload;                                                  exposure.
• citrate toxicity;                                                 • Recommended haematocrit for red cells for
• rebound hypoglycaemia (high glucose levels                        neonatal ‘top-up’ transfusion is 0.5–0.7.
from blood additives);                                              • Volume of a neonatal ‘top-up’ transfusion is
• thrombocytopenia (after exchange transfusion);                    usually 10–20 mL/kg.
• TA-GVHD (if non-irradiated products given to
those at risk; see above).
                                                                    T-antigen activation
                                                                    Severe haemolytic transfusion reactions are occa-
Strategies to minimize transfusion risk
                                                                    sionally seen in neonates or young children trans-
in neonates
                                                                    fused with adult blood or FFP containing anti-T
The following strategies have been shown to                         antibodies. This may be due to exposure or ‘activa-
                                                                    tion’ of the T antigen on neonatal red cells, usually
Table 8.4 Indications for neonatal ‘top-up’ transfusions.           as a result of infection with clostridia, streptococci
                                                                    or pneumococci and/or in association with necro-
Clinical situation                          Transfuse at            tizing enterocolitis. Up to 25% of infants with
                                                                    necrotizing enterocolitis have T-antigen activation
Anaemia in the first 24 h                    Hb < 12 g/dL
                                                                    but so do many healthy neonates and haemolysis is
Neonate receiving mechanical ventilation    Hb < 12 g/dL
                                                                    extremely rare. Therefore, although there remains
Acute blood loss                            10% blood volume lost
                                                                    some controversy, the majority of centres world-
Oxygen dependency (not ventilated)          Hb < 8–10 g/dL
Late anaemia, stable patient (off oxygen)   Hb 7 g/dL               wide feel that no special provision for neonates
                                                                    with necrotizing enterocolitis is necessary and


106
                                                                                                                 Prenatal and childhood transfusions


neither screen neonates for T activation nor
donors for high-titre anti-T.                                                                   1.0
                                                                                                0.8
                                                                                                0.6
                                                                                                0.5
Role of erythropoietin in reducing neonatal red                                                 0.4
cell transfusion                                                                                0.3




                                                            O.D. 450 nm of liquor bilirubin
                                                                                               0.25
There have been numerous clinical trials of ery-                                               0.02                    Zone 1
thropoietin for prevention or amelioration of                                                  0.16
neonatal anaemia, particularly anaemia of prema-                                               0.14                    Zone 2
                                                                                               0.12
turity, since endogenous erythropoietin produc-                                                0.08
tion is low in preterm babies for the first 6–8 weeks                                           0.06                    Zone 3
of life. These trials show that erythropoietin                                                 0.05
                                                                                               0.04
(250 units/kg daily three times per week for the
                                                                                               0.03
first 6 weeks of life) can reduce red cell transfu-                                            0.025
sions in well preterm babies but has a negligible                                             0.002
                                                                                              0.016
effect on transfusion requirements of sick preterm
                                                                                              0.012
babies, particularly those of less than 26 weeks’                                             0.010
gestation at birth. In practice this means that                                                   20   25          30           35           40
erythropoietin has a limited role in neonates as it                                                         Gestation (weeks)
works best in those that need it least.
   Therefore most neonatal units no longer use ery-       Fig. 8.1 Whitfield’s (1970) curved action line is
thropoietin routinely. The situations in which ery-       superimposed on Liley zones of severity of fetal haemolysis
                                                          based on estimations of amniotic fluid bilirubin
thropoietin can be useful are:
                                                          concentrations. This gives guidelines for appropriate
• in neonates whose parents refuse permission to          management related to gestation, i.e. intrauterine
use blood products;                                       transfusion or, if the fetus is viable, delivery and exchange
• to prevent ‘late anaemia’ in babies with HDN.           transfusion. (Adapted from Hann et al. 1991.)



Management of HDN in the fetus                            blood sampling carries a 1–2% fetal loss rate and
                                                          may cause fetomaternal haemorrhage with further
Fetal monitoring of at-risk pregnancies
                                                          sensitization).
The aims are to prevent hydrops developing in             • IUT if anaemia is severe and delivery is not pos-
utero and to time delivery so that the baby has the       sible due to extreme prematurity.
best chance of survival. Fetal monitoring includes
the following.
                                                          Intrauterine transfusion
• Regular ultrasound scans for fetal growth,
hepatosplenomegaly and/or hydrops.                        The aims of IUT are:
• Amniocentesis to measure amniotic fluid biliru-          • to prevent or treat fetal hydrops before the fetus
bin as an indirect measure of fetal haemolysis            can be delivered;
(not reliable before 25 weeks and after 34 weeks of       • to enable the pregnancy to advance to a gesta-
gestation); the bilirubin is plotted on a graph of        tional age that will ensure survival of the neonate
Liley zones modified by Whitfield (Fig. 8.1) in             (in practice, up to 36–37 weeks) with as few inva-
order to predict the severity of HDN and plan             sive procedures as possible (because of the risk of
management.                                               fetal loss).
• Fetal blood sampling if severe HDN before 24               These are achieved by starting the transfusion
weeks’ gestation is suspected, if there is a rapid rise   programme as late as safely possible but before
in maternal antibody, or if there has been a previ-       hydrops develops and by maximizing the intervals
ous intrauterine death due to HDN (note that fetal        between transfusions by transfusing as large a


                                                                                                                                               107
Chapter 8


volume of red cells as is considered safe. Transfu-     anti-c, in which case RhD-positive, c-negative
sions may be intravascular, intraperitoneal or          blood should be used). K blood is recommended to
intracardiac. All transfusions are carried out using    reduce additional maternal alloimmunization risks.
ultrasound guidance (Fig. 8.2). During transfusion      • An IAT crossmatch compatible with maternal
the point of the needle and fetal heart should be       serum and negative for the relevant antigen(s)
watched closely for signs of needle displacement,       determined by maternal antibody status should be
cardiac tamponade and bradycardia. The fetal loss       carried out.
rate associated with IUT is around 5%. IUT is gen-      • Red cells for IUT should always be irradiated
erally indicated when the haematocrit falls to          because of the risk of TA-GVHD.
below 0.25 between 18 and 26 weeks of gestation         • Red cells for IUT should be warmed to 37°C
or to less than 0.3 after 26 weeks of gestation. The    immediately prior to transfusion and transfused at
aim of the transfusion is to raise the haematocrit to   a rate of 5–10 mL/min.
0.45 and repeat transfusion is often necessary after
2–3 weeks.
                                                        Management of HDN in the neonate
                                                        The severity of HDN varies considerably, from a
Intrauterine transfusion: product specification
                                                        hydropic infant with gross hepatosplenomegaly
• Plasma-reduced red cells with a haematocrit of        who needs immediate exchange transfusion to
0.75 or greater should be used.                         mild jaundice with or without anaemia. The fol-
• The red cells should be 5 days old or less, in CPD    lowing samples should be collected at delivery
anticoagulant and sickle screen negative.               from all suspected cases:
• The red cells should be group O (low-titre            • ABO and Rh group;
haemolysin) or ABO identical with the fetus (if         • DAT;
known) and RhD negative (unless HDN is due to           • serum bilirubin;
                                                        • full blood count, reticulocyte count and blood
                                                        film.
                                                           Affected babies should be monitored by checking
                              Donor blood
                                                        their bilirubin and haemoglobin every 3–4 h.
                                                        A rising bilirubin level may require treatment
                                                        with exchange transfusion and/or phototherapy
                                                        depending upon gestational age, postnatal age and
                                                        birth weight (action charts are available for guid-
                                                        ance). Phototherapy should be given from birth to
                                                        all Rh-alloimmunised infants with haemolysis as the
                  3-way tap                             bilirubin can rise steeply after birth and this expec-
                                                        tant approach will prevent the need for exchange
                                                        transfusion in some infants. ‘Late’ anaemia presents
                                                        at a few weeks of age in some babies with milder
                                                        haemolytic disease who do not require exchange
                                                        transfusion and in babies who have had earlier
                                                        exchange transfusion; ‘top-up’ transfusion may be
                                                        required. The blood film shows evidence of ongoing
                                                        haemolysis and the anaemia is aggravated by the
                                                        normal postnatal suppression of erythropoiesis.


                                                        Exchange transfusion in neonates with HDN
Fig. 8.2 Intrauterine transfusion. (From Alter 1989.)   Exchange transfusion is used to treat severe


108
                                                                                  Prenatal and childhood transfusions


anaemia at birth, particularly in the presence of      • Jaundice may be severe but anaemia is usually
heart failure, and severe hyperbilirubinaemia. The     mild.
aim is to remove both antibody-coated red cells        • The blood film shows very large numbers of
and excess bilirubin. Exchange transfusion is          spherocytes with little or no increase in nucleated
a specialist procedure and should be undertaken        red cells.
only by experienced staff. Double-volume               • The DAT is usually, but not always, positive.
exchange (160–200 mL/kg) gives the best reduc-         • Severe HDN requiring exchange transfusion
tion in bilirubin (50%) and removes 90% of the         occurs in only 1 in 3000 births.
infant’s circulating RhD-positive cells. The pH of     • If an exchange transfusion is required, this
whole blood or plasma-reduced red cells used in        should be with group O red cells, with low-titre
exchange transfusion is around 7.0, which does         anti-A and B or with group O red cells suspended
not cause acidosis in the infant.                      in AB plasma.


Indications for exchange transfusion                   Neonatal thrombocytopenia
• Cord haemoglobin less than 8 g/dL.                   Thrombocytopenia occurs in 1–4% of neonates. It
• Cord bilirubin greater than 100 mmol/L.              is much more common in sick preterm infants,
• Rapidly rising bilirubin.                            40–50% of whom will develop thrombocytopenia
                                                       in the first 4 weeks of life. Causes of neonatal
                                                       thrombocytopenia are shown in Table 8.5. The
Exchange transfusion in the neonate:
                                                       most common cause presenting in the first few
product specification
                                                       days of life is that associated with intrauterine
• Plasma-reduced red cells with a haematocrit of       growth restriction or maternal hypertension;
0.5–0.6 should be used, as packed cells may have a     however the most important cause of severe
haematocrit up to 0.75 and cause a very high post-     thrombocytopenia (platelets < 50 ¥ 109/L) at birth
exchange haematocrit.                                  is neonatal alloimmune thrombocytopenia
• The red cells should be less than 5 days old, col-   (NAITP).
lected into CPD anticoagulant and sickle screen
negative.
                                                       Investigation of neonatal thrombocytopenia
• The most recent BCSH guidelines (2003) state
that red cells for neonatal exchange transfusion       In most cases the following tests will identify the
should be gamma-irradiated (and transfused             diagnosis.
within 24 h of irradiation); gamma-irradiation is      • Full blood count and film: the combination of
essential in the case of neonates who have previ-      mild neutropenia and large numbers of nucleated
ously received IUT and in all other cases is advis-    red cells together with moderate thrombocyto-
able unless to do so would lead to clinically          penia suggest that the cause is intrauterine growth
relevant delay.                                        restriction or maternal hypertension. The presence
• Red cells for exchange should be warmed to           of neutrophil left shift and toxic granulation with
37°C immediately prior to transfusion.                 more severe thrombocytopenia suggests that the
                                                       cause is bacterial infection with or without DIC.
                                                       • Congenital infection screen: the most common
Special features of HDN due to ABO antibodies
                                                       congenital infection associated with neonatal
• ABO haemolytic disease occurs only in offspring      thrombocytopenia is CMV.
of women of blood group O and is confined to the        • Screening for NAITP (see below) should be
1% of such women that have high-titre IgG              carried out on any case of severe thrombocyto-
antibodies.                                            penia (platelets < 50 ¥ 109/L) presenting in the first
• Haemolysis due to anti-A is more common (1 in        week of life unless there is very clear evidence of
150 births) than anti-B.                               acute infection.


                                                                                                                109
Chapter 8

Table 8.5 Causes of neonatal thrombocytopenia.                            nial haemorrhage; this occurs in 10% of cases with
                                                                          long-term neurodevelopmental sequelae in 20% of
Early onset (<72 h after birth)                                           survivors.
Placental insufficiency (pre-eclampsia, IUGR, diabetes)*
                                                                          • The diagnosis of NAITP is made by demonstrat-
Neonatal alloimmune thrombocytopenia*
                                                                          ing platelet antigen incompatibility between
Birth asphyxia
Perinatal infection (group B Streptococcus, Escherichia coli, Listeria)
                                                                          mother and baby serologically or by polymerase
Congenital infection (CMV, Toxoplasma, rubella)                           chain reaction (PCR) and is carried out in refer-
Maternal autoimmune (ITP, SLE)                                            ence transfusion laboratories (see Chapter 5 for
Severe rhesus HDN                                                         methods).
Thrombosis (renal vein, aortic)
Aneuploidy (trisomy-21, -18, -13)                                         Management of NAITP
Congenital/inherited (TAR,Wiskott–Aldrich)                                • In all suspected cases the platelet count must be
Late onset (>72 h after birth)                                            monitored for at least 72 h after birth as it may
Bacterial and fungal sepsis*                                              continue to fall during this time.
Necrotizing enterocolitis*                                                • Severely thrombocytopenic babies (platelets
Congenital infection (CMV, Toxoplasma, rubella)                           <30 ¥ 109/L) should be transfused with HPA-
Maternal autoimmune (ITP, SLE)                                            compatible platelets (available ‘off the shelf’ from
Congenital/inherited (TAR,Wiskott–Aldrich)                                transfusion centres).
                                                                          • Babies with an intracranial haemorrhage in
* The most common causes.                                                 association with NAITP should have their platelet
CMV, cytomegalovirus; HDN, haemolytic disease of the newborn; ITP,
                                                                          count maintained above 50 ¥ 109/L with HPA-
idiopathic thrombocytopenic purpura; IUGR, intrauterine growth restric-
                                                                          compatible platelets.
tion; SLE, systemic lupus erythematosus; TAR, thrombocytopenia with
absent radii.
                                                                          • If there is ongoing severe thrombocytopenia
                                                                          and/or haemorrhage despite HPA-compatible
                                                                          platelets, intravenous IgG (total dose 2 g/kg over
                                                                          2–5 days) is often useful until spontaneous recov-
Neonatal alloimmune thrombocytopenia
                                                                          ery occurs 1–6 weeks after birth.
• NAITP is analogous to HDN: maternal alloanti-                           • All babies with severe thrombocytopenia
bodies to antigens present on fetal platelets cause                       due to NAITP should have a cranial ultrasound to
immune destruction of platelets in utero.                                 look for evidence of intracranial haemorrhage
• The five principal human platelet antigens                               (Fig. 8.3).
(HPA1–5) show biallelic autosomal inheritance.
• Alloantibodies to HPA-1a, HPA-5b and HPA-3a                             Management of pregnancies at risk for NAITP (see
account for almost all cases of NAITP, the com-                           also Chapter 5)
monest being anti-HPA-1a (80–90% of cases of                              • Prenatal management of NAITP remains con-
NAITP).                                                                   troversial and all pregnancies should be monitored
• NAITP affects around 1 in 1000 pregnancies                              in a specialist fetal medicine centre with experience
and occurs in the first pregnancy in almost 50% of                         of NAITP.
cases.                                                                    • The principal options are an invasive approach
• Thrombocytopenia         is   frequently     severe                     using fetal blood sampling plus fetal transfusion
(platelets < 30 ¥ 109/L) and may present prenatally                       with HPA-compatible platelets if thrombocytope-
(as early as 20 weeks’ gestation) or at birth.                            nia is detected or a non-invasive approach relying
• The ability of an HPA-1a-negative woman                                 on non-invasive monitoring by fetal ultrasound
to form anti-HPA-1a is controlled by the HLA                              and maternal intravenous IgG therapy; each
DRB3*0101 allele: HLA DRB3*0101-positive                                  approach has evidence to support it.
women are 140 times more likely to make anti-                             • Paternal HPA genotyping is helpful since all
HPA-1a than HLA DRB3*0101-negative women.                                 fetuses fathered by men homozygous for HPA-1a
• The main clinical problem in NAITP is intracra-                         (HPA-1a/1a) will be HPA-1a positive and there-


110
                                                                                  Prenatal and childhood transfusions




Fig. 8.3 MRI studies: inversion
recovery sequence (IR 1800/600/33)
showing subacute haematoma (black
arrow) and chronic haematoma (open
arrow). (From de Vries et al. 1988.)



fore at high risk of developing severe thrombocy-      monitored daily for 2–3 days if below 200 ¥ 109/L
topenia, whereas only 50% of fetuses will be           at birth.
at risk if the father is heterozygous for HPA-1a       • If the baby is well, treatment is unnecessary
(HPA-1a/1b).                                           unless the platelet count falls below 20 ¥ 109/L.
• There is no clear correlation between the titre of   • If the baby has severe thrombocytopenia
maternal anti-HPA antibodies and the severity          (platelets <20 ¥ 109/L), treatment with intravenous
of fetal thrombocytopenia and/or the development       IgG (0.4–1 g/kg per day, total dose 2–4 g/kg) is
of intracranial haemorrhage.                           usually effective.
                                                       • Cranial ultrasound to look for intracranial
                                                       haemorrhage should be performed in all babies
Neonatal thrombocytopenia due to maternal ITP
                                                       with      severe    thrombocytopenia        (platelets
• Around 10% of infants of mothers with ITP            <20 ¥ 109/L).
or SLE develop neonatal thrombocytopenia sec-          • Platelet transfusion is reserved for life-threaten-
ondary to transplacental passage of maternal           ing haemorrhage and should be given in conjunc-
platelet autoantibodies.                               tion with intravenous IgG.
• The thrombocytopenia is usually mild and
intracranial haemorrhage occurs in less than 1%
                                                       Indications for platelet transfusion in neonates
of at-risk babies.
• The platelet count of babies born to mothers         Published guidelines for neonatal platelet transfu-
with ITP or SLE should be checked at birth and         sion acknowledge the lack of evidence on which


                                                                                                                111
Chapter 8


to base recommendations and aim for a safe                                   depending on postnatal age: less than 2.0 ¥ 109/L
approach. Suggested guidelines based on clinical                             at birth and less than 1.0 ¥ 109/L from 1 week of
experience are shown in Table 8.6. There is some                             age. The most common causes are neutropenia sec-
evidence to suggest that prophylactic platelet                               ondary to intrauterine growth restriction or mater-
transfusions are not required for healthy neonates                           nal hypertension and neutropenia secondary to
until the platelet count falls to 20–30 ¥ 109/L.                             severe sepsis. The presence of neutrophil left shift
However, a higher trigger level (<50 ¥ 109/L)                                and toxic granulation in a neutropenic neonate
should be used for babies with the greatest risk                             suggests acute bacterial infection.
of haemorrhage, especially extremely low birth
weight neonates (<1000 g) in the first week of life.
                                                                             Alloimmune neonatal neutropenia (see also
                                                                             Chapter 5)
Neonatal platelet transfusion:
                                                                             • This is analogous to HDN: there is maternal
product specification
                                                                             sensitization to fetal neutrophil antigens during
• ABO identical or compatible.                                               pregnancy.
• RhD identical or compatible.                                               • The most common implicated antibodies are
• HPA compatible in infants with NAITP.                                      anti-NA1 and anti-NA2.
• Produced by standard techniques without                                    • The estimated incidence of neonatal alloimmune
further concentration.                                                       neutropenia is 3% of live births but most cases are
• Irradiated if appropriate.                                                 mild and asymptomatic and the diagnosis may be
• Volume transfused usually 10–20 mL/kg.                                     missed.
                                                                             • Infants with severe neonatal alloimmune neu-
                                                                             tropenia develop severe cutaneous, respiratory or
Neonatal neutropenia
                                                                             urinary tract infection.
Normal neutrophil levels vary with postnatal age,                            • Treatment is with antibiotics and, if necessary,
falling in healthy babies from around 5–10 ¥ 109/L                           granulocyte colony-stimulating factor.
at birth to 2–6 ¥ 109/L by the end of the first week
of life. Neutropenia is therefore variably defined
                                                                             Granulocyte transfusions in neonates
                                                                             There is no good evidence for benefit of granulo-
Table 8.6 Guidelines for platelet transfusion in neonatal
                                                                             cyte transfusions for treatment of neonatal infec-
thrombocytopenia.
                                                                             tion. Both granulocyte colony-stimulating factor
Platelet count < 30 ¥ 109/L in otherwise well infants, including NAITP, if   and granulocyte–macrophage colony-stimulating
no evidence of bleeding and no family history of intracranial                factor can be used to increase the neutrophil count
haemorrhage                                                                  in neutropenic neonates but there is no clear evi-
                                                                             dence that this improves outcome.
Platelet count < 50 ¥ 109/L in infants with:
   Clinical instability
   Concurrent coagulopathy
   Birth weight < 1000 g and age < 1 week                                    Coagulation problems in the newborn
   Previous major bleeding (e.g. GMH–IVH)
                                                                             Causes of haemorrhage in the newborn
   Current minor bleeding (e.g. petechiae)
   Planned surgery or exchange transfusion                                   In well infants the most common causes of bleed-
   Platelet count falling and likely to fall below 30 ¥ 109/L                ing are:
   NAITP if previous affected sib with intracranial haemorrhage              • vitamin K deficiency (haemorrhagic disease of
Platelet count < 100 ¥ 109/L in infants with major bleeding                  the newborn);
                                                                             • inherited disorders, particularly haemophilias;
GMH–IVH, Germinal matrix–intraventricular haemorrhage; NAITP,                • NAITP.
neonatal alloimmune thrombocytopenia.                                          In sick infants the most common causes are:


112
                                                                                   Prenatal and childhood transfusions


• DIC, secondary to perinatal asphyxia, necrotiz-        and fibrinogen; in severe deficiency the APTT may
ing enterocolitis or, less commonly, sepsis;             also be prolonged.
• liver disease.                                         • Treatment of VKDB depends on the severity
                                                         of the bleeding. Mild cases should be given vitamin
                                                         K (1 mg) intravenously or subcutaneously as this
Vitamin K deficiency
                                                         increases the levels of active vitamin K-dependent
Vitamin K deficiency remains a clinical problem           coagulation factors within a few hours; where
largely because of the controversy in recent years       there is significant bleeding, FFP may be given in
surrounding the possible tumorigenic effects of          addition to vitamin K.
intramuscular vitamin K administered to newborn
infants to prevent haemorrhagic disease of the
                                                         Vitamin K prophylaxis
newborn. Vitamin K is necessary for the post-
translational carboxylation of factors II, VII, IX       Guidelines for the prevention of early VKDB
and X and of the natural anticoagulants protein C        recommend a single intramuscular injection of
and protein S. Levels of vitamin K and of all these      vitamin K at birth together with antenatal admin-
factors are physiologically low at birth. This physi-    istration of oral vitamin K to the mother during the
ological deficiency can be exacerbated by breast-         last 4 weeks of pregnancy. For classical and late
feeding, prematurity and liver disease, resulting in     VKDB there are several options because although
haemorrhagic disease of the newborn, often now           intramuscular vitamin K at birth prevents classical
referred to as vitamin K-dependent bleeding              and late VKDB, some studies have suggested a link
(VKDB). There are three patterns of VKDB: early,         between intramuscular vitamin K at birth and later
classical and late.                                      childhood malignancies. Although other studies
• Early VKDB presents in the first 24 h of life           have not confirmed the link with malignancy, the
usually with severe haemorrhage, including gas-          controversy is unlikely to be resolved unequivo-
trointestinal bleeding and intracranial haemor-          cally in the short term. There is no link between
rhage. It is caused by severe vitamin K deficiency in     oral vitamin K and malignancy. Both the American
utero, usually as a result of maternal medication        Academy of Pediatrics and the Royal College of
that interferes with vitamin K, e.g. anticonvulsants     Paediatrics and Child Health recommend vitamin
(phenobarbital,      phenytoin),      antituberculous    K supplementation at birth. In healthy babies the
therapy and oral anticoagulants.                         choice of which route of administration is left to
• Classical VKDB presents at 2–7 days old in             parents, who have to balance a possible risk of
babies who have not received prophylactic vitamin        leukaemia [odds ratio between 1.6 (CI 0.89–1.25)
K at birth. The risk is increased in breast-fed babies   and 1.16 (CI 0.97–1.39)] with intramuscular
and in those with poor oral intake. The incidence in     vitamin K against the slightly higher risk of VKDB
babies not receiving vitamin K supplementation is        (2.7 per 100 000) in infants given three doses of
0.25–1.7%. Classical VKDB can be prevented by a          1 mg vitamin K orally at birth, 1 week and 1 month
single intramuscular dose of vitamin K at birth.         of age.
• Late VKDB occurs 2–8 weeks after birth. It
usually presents with sudden intracranial haemor-
                                                         Use of FFP, cryoprecipitate and human albumin
rhage in an otherwise well, breast-fed term baby or
                                                         solution in neonates
in babies with liver disease. Late VKDB in healthy
breast-fed babies can be prevented either by a single    Guidelines for the use of FFP, cryopreciptate and
intramuscular dose of vitamin K or by repeated oral      albumin in neonates have been published by
doses of vitamin K over the first 6 weeks of life;        national committees in a number of countries. The
babies with chronic liver disease or malabsorption       guidelines aim to minimize their risks in the
require prolonged vitamin K supplementation.             newborn by using pathogen-inactivated products
• Diagnosis of VKDB is based on clotting studies,        and by recommending their use for a small number
which show a prolonged PT with normal platelets          of clinical indications. The only indications for


                                                                                                                 113
Chapter 8


FFP in neonates recommended in the recent BCSH          fusion, there are several groups who are frequently
guidelines and supported by evidence are DIC,           transfused, including those with inherited
VKDB and inherited deficiencies of coagulation           transfusion-dependent disorders, such as thalas-
factors.                                                saemia major, and those undergoing intensive
   Prophylactic FFP administered to preterm             chemotherapy for haematological malignancies.
neonates at birth does not prevent intraventricular     For many of these patients, including those with
haemorrhage or improve outcome at 2 years of            thalassaemia major and sickle cell disease, bone
life. Similarly, FFP is not superior to other colloid   marrow transplantation (BMT) is a possible future
or crystalloid solutions as a volume replacement        treatment. Therefore all such children for whom
solution in standard neonatal practice and there is     BMT is a possible option should receive CMV-neg-
no evidence to support its use to ‘correct’ the         ative blood components. All children on regular
results of abnormal coagulation screens.                transfusions should be vaccinated against hepatitis
                                                        B as early as possible. Those on chronic transfu-
                                                        sion therapy, particularly those with haemoglo-
Product specifications
                                                        binopathies but also those with congenital
• The current BCSH guidelines state that FFP for        dyserythropoietic anaemia, aplastic anaemia and
transfusion to neonates should be group AB (since       other bone marrow failure syndromes, should
this contains neither anti-A nor anti-B) or the same    have an extended red cell phenotype (see below)
ABO blood group as the neonate.                         performed prior to, or as soon as possible after,
• The volume transfused is usually 10–20 mL/kg,         commencing regular transfusions.
with the larger dose given if possible in order            The formula for calculating red cell transfusion
to limit donor exposure where repeated dosing is        volume in children is as follows:
likely.
                                                        Desired Hb (g/dL) – actual Hb (g/dL) ¥ weight
• FFP may be standard or pathogen inactivated. In
                                                        (kg) ¥ 3
England at the present time the Department of
Health has indicated that single-unit methylene         where ‘3’ represents 3 mL of red cells, which
blue-treated FFP should be used for neonates and        has been calculated to raise the haemoglobin by
for children born after 1 January 1996. FFP for         1 g/dL. The normal rate of red cell transfusion is
this group will be sourced from plasma from             around 5 mL/kg per h.
the USA from early 2004. Standard FFP for other
patients is prepared from leucocyte-depleted
whole blood donations from UK donors. It carries        Transfusion support for children with
a small residual risk of transmitting transfusion-      haemoglobinopathies
transmissible viruses (with the exception of CMV).
                                                        Thalassaemia major
Coagulation factor levels are 20–25% lower in
pathogen-inactivated FFP than untreated FFP.            By definition all patients with thalassaemia major
   Human albumin solution (HAS) is associated           are transfusion dependent. Transfusion therapy
with excess mortality in adults receiving intensive     is determined by the degree of anaemia and evi-
care but data about the risks of HAS in neonates        dence of failure to thrive. Most children start
are not available. Current studies suggest that         transfusion when their haemoglobin drops below
there is no good indication for the use of HAS in       6 g/dL.
standard neonatal practice.                                Current BCSH and Thalassaemia International
                                                        Federation guidelines recommend the following:
                                                        • maintain an average haemoglobin of 12 g/dL;
Transfusion in children                                 • maintain a pretransfusion haemoglobin of
                                                        9–10 g/dL;
General points
                                                        • transfusion should prevent marrow hyperplasia,
Although most children never require blood trans-       skeletal changes and organomegaly;


114
                                                                                              Prenatal and childhood transfusions


• extended red cell phenotyping should be carried        Table 8.7 Indications for transfusion in sickle cell disease.
out before starting transfusions (for Rh and Kell
antigens);                                               ‘Top-up’
                                                         Splenic sequestration*
• red cell requirements should be adjusted to
                                                         Hepatic sequestration*
accommodate growth;
                                                         Aplastic crises*
• splenectomy may be considered if hyper-
splenism develops and causes a sustained increase        Exchange transfusion
in red cell requirements;                                Chest syndrome*
• iron chelation therapy should be considered            Stroke*
                                                         Priapism
after 10 transfusions and started once ferritin is
                                                         Mesenteric syndrome
greater than 1000 ng/mL (if possible starting after
the age of 2 years because of desferrioxamine            Hypertransfusion
toxicity);                                               Stroke (to prevent recurrence)*
• since BMT is the only cure, families should be         Renal failure (to prevent/delay deterioration)
                                                         Chronic sickle lung disease
offered HLA typing of siblings as possible bone
marrow donors.                                           Surgery
                                                         Selected patients preoperatively (e.g. joint replacement)

Sickle cell disease                                      * Proven value.
Red cell transfusion in children with sickle cell
disease should not be routine but reserved for spe-      Indications for hypertransfusion in sickle
cific indications (Table 8.7). Extended red cell          cell disease
phenotyping before the first transfusion is very          • To prevent recurrence of stroke (i.e. secondary
important because up to 50% of patients other-           prevention of stroke).
wise develop red cell alloimmunization and may be        • To prevent the development of stroke in children
very difficult to crossmatch. The majority of anti-       with sickle cell disease with Doppler and/or
bodies are in the Rhesus or Kell system and may be       magnetic resonance evidence of cerebrovascular
transient and very difficult to detect, leading to a      infarction/haemorrhage in the absence of clinical
risk of delayed transfusion reactions.                   evidence of stroke (i.e. primary prevention of
                                                         stroke).
Indications for ‘top-up’ transfusion in sickle           • To delay or prevent deterioration in end-organ
cell disease                                             failure (e.g. chronic sickle lung).
Indications include splenic or hepatic sequestra-        The aims are to maintain the percentage of HbS
tion, and aplastic crisis. The aim is to raise the       below 25% and the haemoglobin between 10 and
haemoglobin to the child’s normal steady state (the      14.5 g/dL. After 3 years a less intensive regimen
haemoglobin should never be raised acutely to            maintaining HbS at 50% or less may be sufficient
>10 g/dL, since this is likely to cause an increase in   for stroke prevention.
blood viscosity).
                                                         Indications for preoperative transfusion in sickle
Indications for exchange transfusion in sickle           cell disease
cell disease                                             The BCSH guidelines are based on observational
• Acute chest syndrome.                                  studies and one large randomized controlled study
• Mesenteric (abdominal) syndrome.                       as there are no other available data. These guide-
• Stroke.                                                lines state that:
• Selected patients preoperatively.                      • ‘top-up’ transfusion (haemoglobin 8–10 g/dL) is
• Priapism (occasionally).                               as effective as exchange transfusion and may be
The aim is to reduce sickling and increase oxygen        safer;
carriage without an increase in viscosity.               • minor and straightforward procedures (e.g. ton-


                                                                                                                            115
Chapter 8


sillectomy, possibly cholecystectomy) can be safely
                                                          Gamma-irradiated blood components should be
undertaken without transfusion in most patients;
                                                          given to the following children
• exchange transfusion should be performed
preoperatively for major procedures, e.g. hip/knee        • For 2 weeks before allogeneic haemopoietic
replacement, organ transplantation, eye surgery,          stem cell transplant (SCT) and during conditioning
and considered for major abdominal surgery.               for all types of SCT until at least 6 months post-
                                                          SCT or until all immunosuppressive agents have
Practical aspects of transfusion in sickle cell disease   been discontinued, whichever is later.
• Extended red cell phenotyping (for Rh, K, Fy, Jk        • For 2 weeks before autologous SCT irradiation
and MNS) should be carried out; this should be            and during conditioning until 3 months post-SCT
done before the first transfusion and may be use-          (6 months if total body irradiation given).
fully arranged at outpatient clinic follow-up             • For SCT in children with severe combined
during the first year of life.                             immunodeficiency, irradiation should continue for
• In particular patients should be typed for U.           at least a year following SCT or until normal
• The R0 blood group (cDe/cDe) is common in               immune function has been achieved.
patients of African or Caribbean origin: all R0           • For 7 days prior to harvesting of autologous
patients should receive C-negative, E-negative            bone marrow or peripheral blood stem cells.
blood (i.e. rr or R0).                                    • For children with Hodgkin’s disease during
• The use of sickle trait-positive blood should           treatment and thereafter (susceptibility to transfu-
be avoided by testing for HbS in blood centres or         sion-associated GVHD is now considered to be
hospitals.                                                lifelong).
• During exchange transfusion in the acute situa-         • During treatment with fludarabine and for
tion a total exchange of 1.5–2 times their blood          at least 2 years or until full recovery of cellular
volume is required to achieve an HbS level of 20%         immune function.
or less; this may take two to three procedures if         • Where blood products from relatives are being
carried out manually. Automated exchange using a          used.
cell separator allows the exchange to be completed
as a single procedure. The volume of packed cells
                                                          Transfusions for children undergoing blood group-
(in mL) for each exchange is determined by: weight
                                                          mismatched BMT
(kg) ¥ 30.
• Normal saline (not FFP or albumin) should be            • Major incompatibility arises when the recipient
used as volume replacement at the beginning of the        has antibodies to the donor cells (e.g. patient
exchange prior to starting venesection to avoid           group O, donor group A).
lowering the circulating blood volume.                    • Minor incompatibility arises when the donor
                                                          has antibodies to recipient cells (e.g. patient group
                                                          A, donor group A).
Leukaemia, chemotherapy and BMT
                                                          • Current BCSH and European Blood and Bone
All children being treated with high-dose                 Marrow Transplantation Group guidelines recom-
chemotherapy/radiotherapy or those with aplastic          mend that in ABO-incompatible SCT group O red
anaemia may at some time be candidates for future         cells should be given (irrespective of the ABO
BMT. While components leucocyte depleted to less          group) until ABO antibodies to the donor ABO
than 5 ¥ 106/unit are widely considered to be             type are undetectable and the DAT is negative;
CMV-safe, not all BMT centres agree.                      thereafter red cells of the donor group are given
  Gamma-irradiation of blood components is not            (high-titre anti-A/anti-B donor units must be
necessary for most children receiving chemother-          excluded).
apy for leukaemia or solid tumours, although there        • RhD-negative red cells are given if the patient is
are several important situations where irradiation        RhD negative and/or the donor is RhD negative.
of blood products (25 Gy) is necessary.                   • After an ABO-incompatible SCT, platelets of the


116
                                                                                              Prenatal and childhood transfusions


recipient’s ABO group should be given until there                 Granulocyte transfusion in children undergoing
is conversion to the donor ABO group and ABO                      chemotherapy or BMT
antibodies to the donor ABO group are unde-
                                                                  • There is no evidence to support the use of pro-
tectable; thereafter platelets of the donor group
                                                                  phylactic granulocyte transfusions.
should be given.
                                                                  • Empirical data from some studies support their
• After an ABO-incompatible SCT, FFP of the
                                                                  use where there is severe bacterial or fungal infec-
recipient’s ABO group should be given. If there
                                                                  tion in neutropenic children, including SCT, but
is both a major and minor mismatch, group AB
                                                                  they increase the risk of platelet refractoriness.
should be given.
                                                                  • Granulocytes for transfusion should be ABO
                                                                  and RhD compatible.
Platelet transfusion in children undergoing                       • Granulocytes for all recipients should always be
chemotherapy or BMT                                               irradiated.
• Indications for platelet transfusion in children
are consensus based; those developed by the BCSH                  Haemopoietic stem cell donors
are shown in Table 8.8; in general, in non-infected
                                                                  • Children who act as bone marrow donors for
well children a platelet count of 5–10 ¥ 109/L can
                                                                  their sibling(s) usually require blood transfusion to
be used as a transfusion trigger but higher thresh-
                                                                  cover blood lost during the procedure; allogeneic
olds are used for children who are sick and/or
                                                                  blood transfused to the donor during the bone
bleeding.
                                                                  marrow harvest should always be irradiated and
• Platelets should be ABO compatible where pos-
                                                                  CMV negative unless both the patient and donor
sible because of the risk of haemolysis (see above
                                                                  are known to be CMV positive.
for ABO-incompatible SCT patients).
                                                                  • In older children (>25 kg and more than 8 years
• Platelets should be RhD compatible and RhD-
                                                                  old), autologous blood donation should be consid-
negative girls must receive RhD-negative platelets
                                                                  ered around 2 weeks prior to marrow/peripheral
because of the risk of sensitization by contaminat-
                                                                  blood stem cell donation.
ing red cells.
                                                                  • For autologous donation children should have
• A transfusion of 10–20 mL/kg is given to chil-
                                                                  no unstable cardiovascular or pulmonary prob-
dren under 15 kg and an apheresis unit for children
                                                                  lems and a haemoglobin of more than 11 g/dL. The
over 15 kg.
                                                                  maximum collected at each donation should
                                                                  be 12% of the estimated blood volume and the
                                                                  amount of citrate anticoagulant in the pack should
Table 8.8 Indications for platelet transfusion in children        be adjusted to maintain the appropriate ratio of
with thrombocytopenia.                                            blood to anticoagulant.

Platelet count < 10 ¥ 109/L

Platelet count < 20 ¥ 109/L and one or more of the following:     Further reading
   Severe mucositis
   DIC                                                            Alagappan A, Shattuck KE, Malloy MH. Impact of
   Anticoagulant therapy                                            transfusion guidelines on neonatal transfusions. J
   Platelets likely to fall < 10 ¥ 109/L before next evaluation     Perinatol 1998; 18: 92–7.
   Risk of bleeding due to local tumour infiltration               Alcock GS, Liley H. Immunoglobulin infusion for
                                                                    isoimmune haemolytic jaundice in neonates. Cochrane
Platelet count 20–40 ¥ 109/L and one or more of the following:
                                                                    Database Syst Rev 2002; 3: CD003313.
   DIC in association with induction therapy for leukaemia
                                                                  Alter BP. Methods in Haematology: Prenatal Haematology.
   Extreme hyperleucocytosis
                                                                    Edinburgh, Churchill Livingstone, 1989.
   Prior to lumbar puncture or central venous line insertion      BCSH Blood Transfusion and Haematology Task Forces.
                                                                    The estimation of fetomaternal haemorrhage. Transfus
DIC, disseminated intravascular coagulation.                        Med 1999; 9: 87–92.


                                                                                                                            117
Chapter 8

Boralessa H, Modi N, Cockburn H et al. RBC T activation      Murray NA, Howarth LJ, McMcloy M, Letsky EA, Roberts
  and hemolysis in a neonatal intensive care population:       IAG. Platelet transfusion in the management of severe
  implications for transfusion practice. Transfusion 2002;     thrombocytopenia in neonatal intensive care unit (NICU)
  42: 1428–34.                                                 patients. Transfus Med 2002; 12: 35–41.
British Committee for Standards in Haematology               National Institute for Clinical Excellence (NICE).
  Haemostasis and Thrombosis Task Force. The                   Guidelines on the use of routine antenatal anti-D
  investigation and management of neonatal haemostasis         prophylaxis for RhD-negative women. Technology
  and thrombosis. Br J Haematol 2002; 119: 295–309.            Appraisal Guidance No 41. www.nice.org.uk/pdf/
British Committee for Standards in Haematology                 prophylaxisFinalguidance.pdf. 2002.
  Transfusion Task Force. Transfusion guidelines for         Paul DA, Leef KH, Locke RG, Stefano JL. Transfusion
  neonates and older children. www.bcshguidelines.com/         volume in infants with very low birthweight: a
British Committee for Standards in Haematology. Blood          randomized trial of 10 versus 20 ml/kg. J Pediatr
  Transfusion Task force. Guidelines for pre-transfusion       Hematol Oncol 2002; 24: 43–6.
  compatibility procedures in blood transfusion              Puckett RM, Offringa M. Prophylactic vitamin K for
  laboratories. Transfusion Medicine 1996; 6: 273–283.         vitamin K deficiency bleeding in neonates. Cochrane
Bruce M, Chapman JF, duguid J, Kelsey P, Knowles S,            Database Syst Rev 2000; 4: CD002776.
  Murphy M, Williamson L Addendum for guidelines for         Ramasethu J, Luban NLC. Red blood cell transfusion in the
  blood grouping and red cell antibody testing during          newborn. Semin Neonatol 1999; 4: 5–16.
  pregnancy. BCSH Transfusion Task Force. Transfus Med       Roberts IAG, Murray NA. Thrombocytopenia in the
  1999; 9: 99.                                                 newborn. Curr Opin Pediatr 2003; 15: 17–23.
Bussel JB. Alloimmune thrombocytopenia in the fetus          Stockman JA, de Alarcon PA. Overview of the state of the
  and newborn. Semin Thromb Hemost 2001; 27: 245–              art of Rh disease: history, current clinical management,
  52.                                                          and recent progress. J Pediatr Hematol Oncol 2001; 23:
de Vries LS, Connell J, Bydder GM et al. Recurrent             385–93.
  intracranial haemorrhages in utero in an infant with       Sutor AH, von Kries R, Cornelissen EA, McNinch AW,
  alloimmune thrombocytopenia. Case report. Br J Obstet        Andrew M. Vitamin K deficiency bleeding (VKDB) in
  Gynaecol 1988; 95: 299–302.                                  infancy. ISTH Pediatric/Perinatal Subcommittee.
Gottstein R, Cooke RWI. Systematic review of intravenous       International Society on Thrombosis and Haemostasis.
  immunoglobulin in haemolytic disease of the newborn.         Thromb Haemost 1999; 81: 456–61.
  Arch Dis Child 2003; 88: F6–F10.                           Thompson J. Haemolytic disease of the newborn: the new
Grant SR, Kilby MD, Meer L, Weaver JB, Gabra GS,               NICE guidelines. J Fam Health Care 2002; 12: 133–6.
  Whittle MJ. The outcome of pregnancy in Kell               Vamvakas EC, Strauss RG. Meta-analysis of controlled
  alloimmunisation. Br J Obstet Gynaecol 2000; 107:            clinical trials studying the efficacy of rHuEPO in reducing
  481–5.                                                       blood transfusions in the anemia of prematurity.
Hann IM, Gibson BES, Letsky EA. Haemolytic disease of          Transfusion 2001; 41: 406–15.
  the newborn. In: Fetal and Neonatal Haematology.           Waldron P, de Alarcon P. ABO hemolytic disease of the
  London: Baillière Tindall, 1991; 106.                        newborn: a unique constellation of findings in siblings
Heegaard ED, Brown KE. Human parvovirus B19. Clin              and review of protective mechanisms in the
  Microbiol Rev 2002; 15: 485–505.                             fetal–maternal system. Am J Perinatol 1999; 16: 391–8.
Kadir RA. Women and inherited bleeding disorders:            Wee LY, Fisk NM. The twin–twin transfusion syndrome.
  pregnancy. Semin Hematol 1999; 36: 28–35.                    Semin Neonatol 2002; 7: 187–202.
Kelton JG. Idiopathic thrombocytopenic purpura               Wong W, Fok TF, Lee CH et al. Randomised controlled
  complicating pregnancy. Blood Rev 2002; 16: 43–6.            trial: comparison of colloid or crystalloid for partial
Kulkarni R, Lusher J. Perinatal management of newborns         exchange transfusion for treatment of neonatal
  with haemophilia. Br J Haematol 2001; 112: 264–74.           polycythaemia. Arch Dis Child 1997; 77: F115–F118.
Lee D, Contreras M, Robson SC, Rodeck CH, Whittle MJ.        Zipursky A. Prevention of vitamin K deficiency bleeding in
  Recommendations for the use of anti-D immunoglobulin         newborns. Br J Haematol 1999; 104: 430–7.
  for Rh prophylaxis. British Blood Transfusion Society      Zuppa AA, Maragliano G, Scapillati ME et al.
  and the Royal College of Obstetricians and                   Recombinant erythropoietin in the prevention of late
  Gynaecologists. Transfus Med 1999; 9: 93–7.                  anaemia in intrauterine transfused neonates with
Meyer MP, Sharma E, Carsons M. Recombinant                     Rh-haemolytic disease. Fetal Diagn Ther 1999; 14:
  erythropoietin and blood transfusion in selected preterm     270–465.
  infants. Arch Dis Child 2003; 88: F41–F45.




118
Chapter 9
Haematological disease

Michael F. Murphy and Simon J. Stanworth




Patients with haematological diseases are major         dence indicating a risk of ‘under-transfusing’
users of blood products. Over 15% of all red cell       certain groups of patients, for example those with
units are transfused to patients with haematologi-      coexisting cardiac disease.
cal disease, mostly to patients with malignant dis-
orders. The requirement for blood transfusions in
                                                        Red cell transfusions
this group are related to both the underlying con-
dition itself and the myelosuppressive/myeloabla-       The ready availability of red cell concentrates
tive effects of specific treatments used.                means that anaemia in haematology patients can
   This chapter considers the following topics:         be easily relieved. There are some specific consider-
• the indications for red cell, platelet and granulo-   ations in the management of anaemia.
cyte transfusions in haematology patients;              • Its cause should be established, and treatment
• the approaches to the management and preven-          other than blood transfusion should be used where
tion of complications associated with transfusions      appropriate, for example patients with iron
in haematology patients, including the use of           deficiency or megaloblastic or autoimmune
special types of blood components.                      haemolytic anaemias. Anaemia of malignancy may
                                                        be due to a number of causes including the effects
                                                        of marrow infiltration or therapy and ‘inhibitory’
Indications for transfusions in patients with           cytokine-mediated influences leading to the sec-
haematological diseases                                 ondary anaemias (of chronic disorders), or low
                                                        erythropoietin.
Haematological diseases requiring transfusion           • There is no universal ‘trigger’ for red cell trans-
support cover a whole spectrum of clinical disor-       fusions in haematology patients, i.e. a given level
ders: fetal, neonatal and paediatric practice           of haemoglobin at which red cell transfusion is
(Chapter 8), haemophilia (Chapter 11), haemoglo-        appropriate for all patients. Clinical judgement
binopathies, immune disorders, and bone marrow          balancing factors such as quality-of-life indices
failure syndromes, in addition to haematological        plays an important role in the decision to transfuse
malignancies.                                           red cells or not.
   The haemopoietic system has a dramatic capac-        • The clinical use of recombinant erythropoietin
ity for increasing the production of mature blood       might be considered in some situations, e.g.
cells, but this capability varies between different     delayed erythroid engraftment after allogeneic
diseases. The scenario of anaemia related to            bone marrow/peripheral blood progenitor cell
marrow ablation following chemotherapy is very          transplantation, the treatment of anaemia in
different to anaemia in an individual with a well-      patients with myeloma or myelodysplasia, and in
compensated chronic haemolytic process. It is also      the management of Jehovah’s Witnesses with
acknowledged that while much of the current             haematological disorders. Evidence supports an
impetus in transfusion practice is aimed at reduc-      association between increases in haemoglobin con-
ing inappropriate transfusions, there is now evi-       centration, reduced red cell transfusion require-


                                                                                                         119
Chapter 9


ments and improvement in quality-of-life indices        elderly patients are symptomatic even at haemo-
with erythropoietin therapy, although the findings       globin levels above 10 g/dL.
concerning quality-of-life measures are more diffi-
cult to compare between studies. Uncertainties
                                                        Haemoglobinopathies (in adults)
also remain about the factors predicting respon-
siveness, since a number of individuals fail to show    Similar principles guide red cell transfusion
adequate responses to erythropoietin. Overall           support for adult patients with transfusion-
cost-effectiveness studies have not documented a        dependent thalassaemia as for children (see
major cost–benefit for erythropoietin, but this          Chapter 8). Pretransfusion haemoglobin concen-
balance may alter in the light of changes to the        trations should be 9–10 g/dL, and iron-chelation
supply and costs of donor blood and a better            therapy will be required in all patients (see below).
understanding of the potentially beneficial effects      All patients on long-term transfusion programmes
of erythropoietin on tumour response and overall        should be vaccinated against hepatitis B.
survival.                                                  Transfusions of red cells in sickle cell disease are
                                                        used to correct anaemia or as part of the treatment
                                                        of vaso-occlusive or other complications of severe
Patients receiving intensive
                                                        sickling. Factors other than the number or percent-
myelosuppressive/myeloablative treatment
                                                        age of sickle cells may affect the risk of vaso-
There are specific considerations relating to the use    occlusion, e.g. baseline severity of anaemia,
of red cell transfusions in patients receiving inten-   infection.
sive myelosuppressive/myeloablative treatment,             Transfusion support for sickle disease may be
including the need to provide a ‘reserve’ in case of    classified by the clinical indication (e.g. aplastic
severe infection or haemorrhage, and the con-           crisis, acute chest syndrome, stroke, priapism,
venience of having a standard policy for red cell       before surgery), the predominant pathophysiologi-
transfusion in the setting of an acute haematology      cal reason (anaemia or sickling or iron overload),
service, even if this may result in some patients       or by the chosen transfusion protocol (simple
being over-transfused.                                  or additive transfusion, exchange transfusion or
   The level of haemoglobin used as the ‘trigger’       hypertransfusion). Exchange transfusions may be
for transfusion varies from centre to centre, but is    carried out manually at the bedside or by apheresis
usually in the range 8–10 g/dL. There are no def-       using a cell separator (with the possible attendant
inite data to support the use of a higher level,        problems relating to obtaining good venous
although studies in animal models of thrombocy-         access). Careful attention to fluid balance is
topenia and in uraemic patients suggest that cor-       required, and saline should be given prior to vene-
rection of anaemia also results in correction of        section at the start of an exchange procedure to
prolonged bleeding times.                               prevent falls in blood volume. The rationale
                                                        for differing transfusion requirements as part of
                                                        the management of these clinical complications
Red cell transfusions and chronic anaemias
                                                        of sickle cell disease does not relate solely to
In patients with chronic anaemia requiring regular      the desired post-transfusion reduction in HbS-
transfusions, red cell transfusions should be used      containing cells, and the exact mechanism by
to maintain the haemoglobin level just above the        which exchange transfusion, for example, is bene-
lowest level not associated with symptoms of            ficial in acute chest syndrome is not fully defined.
anaemia. There is considerable variation in this           Transfusion support may be indicated in rela-
level depending on the patient’s age, level of activ-   tion to surgery, because anaesthesia and surgery
ity and coexisting medical problems, such as car-       may increase the risk of sickling complications.
diovascular and respiratory disease; for example,       Transfusion support may not be required prior
some young patients are asymptomatic with a             to minor surgical procedures, e.g. tonsillectomy.
haemoglobin level below 8 g/dL, while some              Transfusions when indicated are given either as a


120
                                                                                            Haematological disease


‘top-up’ or as an exchange. A large randomized           per 100 000 per year. They are characterized by the
trial noted that an aggressive transfusion regimen       production of antibodies directed against high-
did not reduce the number of postoperative               frequency red cell antigens, and often exhibit
vaso-occlusive complications compared with top-          reactivity against donor red cells. The degree of
up transfusions to a haemoglobin concentration of        haemolysis depends on a number of factors,
10 g/dL, and had the disadvantage that red cell          including the characteristics of the bound antibody
alloimmunization was twice as common. One of             (e.g. class, quantity, specificity, thermal ampli-
the major concerns about transfusions in sickle cell     tude), the target antigen (e.g. density, expression),
disease is red cell alloimmunization, which may be       and other host-related genetic factors (e.g. markers
as high as 20–35% in both adults and children.           of macrophage activity). The antibody class in turn
Providing phenotyped donor red cells matched             will affect the degree of classical complement acti-
specifically for individual patients (for Rh and Kell     vation (IgM) or binding to splenic and other tissue
antigens as a minimum) reduces this risk, but it is      macrophages via Fc receptors (IgG1 and IgG3
a major logistic exercise to ensure availability of      antibodies). AIHA is divided into ‘warm’ and
appropriate units of donor red cells. The R0 group       ‘cold’ types, depending on whether the antibody
is more common in Afro-Caribbean patients and            attaches better to red cells at body temperature
such individuals should receive C-negative E-            (37°C) or at lower temperatures.
negative blood (which should also be sickle cell            In warm antibody AIHA, IgG antibodies pre-
trait negative). Providing matched blood may be          dominate and the direct antiglobulin test is posi-
unnecessary for ‘non-responder’ individuals, who         tive with IgG alone (20%), IgG and complement
rarely make red cell antibodies, but it is difficult to   (67%), or complement only (13%); the red cell
identify these individuals.                              autoantibodies usually have Rh specificity. In cold
   Hypertransfusion programmes are used to               AIHA, the antibodies are usually IgM. They easily
prevent recurrence of stroke, and may also have a        elute off red cells, leaving complement, which is
role in preventing deterioration in end-organ            detected as C3d.
failure, e.g. renal, lung. The results of the Stroke        The cause of warm antibody AIHA remains
Prevention Trial in Sickle Cell Anaemia (STOP)           unknown in more than 30% of cases, but may be
have indicated a role for regular transfusions in the    associated with lymphoid malignancies or diseases
primary prevention of stroke, but uncertainty            such as rheumatoid arthritis and systemic lupus
remains about the duration of transfusion support.       erythematosus or drugs. Therapy of warm anti-
                                                         body AIHA depends on the severity of the
                                                         haemolysis. Treatment is usually required once
Immune blood disorders
                                                         symptomatic anaemia develops. Steroids are the
In immune haemolytic anemia, antibodies bind to          first-line treatment (e.g. prednisolone in doses of
red blood cell surface antigens and initiate destruc-    1 mg/kg daily) and are effective in inducing a remis-
tion via the complement system and/or the                sion in about 80% of patients. Steroids reduce both
macrophage system. Immune haemolytic anemia              production of the red cell autoantibody and
may be alloimmune, autoimmune or drug induced.           destruction of antibody-coated cells. Splenectomy
   Alloimmune haemolytic anaemia occurs in               may be necessary if there is no response to steroids
haemolytic disease of the newborn (see Chapter 8),       or if remission is not maintained when the dose of
haemolytic transfusion reactions and after allo-         prednisolone is reduced. Other immunosuppres-
geneic bone marrow, renal, liver or cardiac trans-       sive drugs, such as azathioprine and cyclophos-
plantation when donor lymphocytes transferred            phamide, may be effective in patients who fail to
in the allograft (‘passenger lymphocytes’) may           respond to steroids and splenectomy.
produce red cell antibodies against the recipient           Blood transfusion may be required if there is ful-
and cause haemolytic anaemia (see Chapter 13).           minant haemolytic anaemia or severe anaemia not
   Autoimmune haemolytic anaemias (AIHAs) are            responding to steroids or other therapy. The pres-
uncommon, with estimates of the incidence at 1–3         ence of red cell autoantibodies on the patient’s red


                                                                                                             121
Chapter 9


cells and in the plasma can cause problems in the        and serum at 4°C and then warming the mixture
identification of compatible blood. It is important       to 37°C (Donath–Landsteiner test). Haemolysis is
to exclude the presence of red cell alloantibodies,      self-limiting, but supportive transfusions may be
and autoabsorption of autoantibodies in the              necessary. P-negative blood should be considered if
plasma using enzyme treatment of the patient’s           there is no sustained response to transfusion of
red cells may be necessary to permit the investiga-      P-positive crossmatch compatible blood.
tion of the plasma for alloantibodies (see Chapter          The issue of whether it is necessary to use an in-
25).                                                     line blood warmer when transfusing patients with
   Cold antibody AIHA is usually due to IgM anti-        cold antibody AIHA is controversial. It is logical to
bodies. Normally, low titres of IgM cold agglu-          keep the patient warm, and common practice
tinins reacting at 4°C are present in plasma and are     to use a blood warmer if the patient has florid
harmless. At low temperatures these antibodies           haemolytic anaemia. There is also debate about
can attach to red cells and cause their agglutin-        the need to use washed or leucocyte-depleted
ation in the cold peripheries of the body. In            blood when transfusing patients with AIHA; it
addition, activation of complement may cause             seems reasonable to use leucocyte-depleted blood
intravascular haemolysis when the cells return           to avoid febrile reactions that may result in delay
to the higher temperatures in the core of the            in completing the transfusion.
body. After certain infections, e.g. Mycoplasma,
cytomegalovirus (CMV), Epstein–Barr virus
                                                         Platelet transfusions
(EBV), there is increased synthesis of polyclonal
cold agglutinins producing a mild to moderate            In general, platelet transfusions are indicated for
transient haemolysis.                                    the prevention and treatment of haemorrhage in
   Chronic cold haemagglutinin disease usually           patients with thrombocytopenia or platelet func-
occurs in the elderly, with a gradual onset of           tion defects. The cause of the thrombocytopenia
haemolytic anaemia owing to the production of            should be established before platelet transfusions
monoclonal IgM cold agglutinins, usually with            are used because they are not always appropriate
anti-I specificity. After exposure to cold the patient    treatment for thrombocytopenic patients, and in
develops an acrocyanosis similar to Raynaud’s as a       some instances are contraindicated, for example
result of red cell autoagglutination. The underly-       in thrombotic thrombocytopenic purpura,
ing cause should be treated, if possible, and            haemolytic–uraemic syndrome, and heparin-
patients should avoid exposure to cold. Treatment        induced thrombocytopenia.
with steroids, alkylating agents and splenectomy
is usually ineffective. Regular blood transfusion
                                                         Bone marrow failure
is occasionally required to prevent symptoms of
anaemia.                                                 Therapeutic platelet transfusions are established as
   Paroxysmal cold haemoglobinuria is a rare con-        effective treatment for patients who are bleeding.
dition more commonly associated with childhood           The issue of the benefit of prophylactic platelet
infections, such as measles, mumps and chicken-          transfusions for the prevention of haemorrhage in
pox, but was originally described in association         chronically thrombocytopenic patients with bone
with syphilis. Intravascular haemolysis is associ-       marrow failure remains controversial. There have
ated with polyclonal IgG complement-fixing anti-          been no recent randomized trials comparing the
bodies. These antibodies are biphasic, reacting          frequencies of bleeding events and patient survival
with red cells in the cold in the peripheral circula-    in patients receiving either prophylactic or thera-
tion, with lysis occurring due to complement acti-       peutic platelet transfusions. A strategy of transfus-
vation when the cells return to the central              ing platelets only for therapeutic indications in the
circulation. The antibodies have specificity for the      context of clinical bleeding is appropriate for some
P red cell antigen. The lytic reaction is demon-         patients with chronic persisting thrombocytopenia
strated in vitro by incubating the patient’s red cells   due to bone marrow failure syndromes.


122
                                                                                            Haematological disease


  Prophylaxis for invasive procedures depends on         thrombocytopenia frequently occur after cardiac
the type of procedure.                                   bypass surgery, but prophylactic platelet transfu-
• No increase in platelet count required: bone           sions are not indicated.
marrow aspiration and biopsy.                            • Platelet transfusions should be reserved for
• Platelet count should be raised to 50 ¥ 109/L:         patients with bleeding not due to surgically cor-
lumbar puncture, epidural anaesthesia, insertion         rectable causes.
of intravascular lines, transbronchial and liver
biopsy, and laparotomy.
                                                         Granulocyte transfusions
• Platelet count should be raised to more than
100 ¥ 109/L: surgery in critical sites such as the       Severe persisting neutropenia is the principal limit-
brain or the eyes.                                       ing factor in the use of intensive treatment of
                                                         patients with haematological malignancies. It may
                                                         last for 2 weeks or more after chemotherapy
Immune thrombocytopenias
                                                         or bone marrow/peripheral blood progenitor cell
• Autoimmune         thrombocytopenias:       platelet   transplantation, and during this period the patient
transfusions should only be used in patients with        is at risk of life-threatening bacterial and fungal
major haemorrhage.                                       infections. The use of haemopoietic growth
• Post-transfusion purpura: platelet transfusions        factors, such as granulocyte colony-stimulating
are usually ineffective in raising the platelet count,   factor (G-CSF), may reduce the duration and
but may be needed in large doses to control severe       severity of severe neutropenia, but they are only
bleeding in the acute phase (see Chapter 17).            effective if the patient has sufficient numbers of
• Neonatal       alloimmune       thrombocytopenia:      haemopoietic precursors. Moreover, the time to
human platelet antigen (HPA)-matched platelet            response may be several days. Supportive treat-
concentrates are the most appropriate treatment          ment with granulocyte transfusions is a logical
for this condition (see Chapter 8).                      approach, although a number of factors have
                                                         limited its application:
                                                         • difficulties in the collection of neutrophils,
Massive blood transfusion
                                                         which are present in low numbers in normal indi-
• Clinically significant dilutional thrombocyto-          viduals and which are difficult to separate from red
penia only occurs with the transfusion of more           cells because of their similar densities (commer-
than 1.5 times the blood volume of the recipient.        cially available long-chain starch solutions now
• The platelet count should be maintained above          facilitate this separation);
50 ¥ 109/L in patients receiving transfusions for        • the short half-life of neutrophils after transfu-
massive acute blood loss (see Chapter 7).                sion, coupled with short storage times and nega-
                                                         tive effects on function of prolonged storage;
                                                         • the frequent occurrence of adverse effects such
Disseminated intravascular coagulation
                                                         as febrile reactions, including occasional severe
• In acute disseminated intravascular coagulation        pulmonary reactions and human leucocyte antigen
(DIC), where there is bleeding associated with           (HLA)       alloimmunization     causing     platelet
severe thrombocytopenia, platelet transfusions           refractoriness.
should be given in addition to coagulation factor           Various methods have been used in the past to
replacement (see Chapter 11).                            increase the number of neutrophils collected,
• In chronic DIC, or in the absence of bleeding,         including obtaining granulocytes from patients
platelet transfusions are not indicated.                 with chronic myeloid leukaemia, treating donors
                                                         with steroids, and using hydroxyethyl starch to
                                                         promote sedimentation of red cells. However, a
Cardiopulmonary bypass surgery
                                                         number of clinical trials of granulocyte transfu-
• Platelet function defects and some degree of           sions in the 1970s and 1980s suggested they had


                                                                                                             123
Chapter 9


limited efficacy in adults, and interest in their        • ventilated patients;
usage declined. Some centres continued to use           • patients with known HLA alloimmunization.
granulocyte transfusions for small children and
neonates because concentrates collected from
adult donors produced a relatively much greater
                                                        Approach to complications associated with
dose per recipient weight, and sometimes appeared
                                                        blood transfusion in haematology patients
to be clinically effective.
   There has recently been a resurgence of interest     Transfusion-transmitted CMV infection
in granulocyte transfusions because of the
                                                        Clinical features and risk factors
accumulating evidence that G-CSFs can be safely
administered to normal individuals. Much larger         CMV infection may cause significant morbidity
doses of granulocytes can then be collected from        and mortality in immunocompromised patients,
donors using regimens including G-CSF adminis-          mainly due to pneumonia. Patients who have never
tered 12–16 h prior to apheresis, together with         been exposed to CMV are at risk for primary infec-
oral steroids such as dexamethasone to further          tion transmitted by blood components prepared
improve the yields. Further evidence of the safety      from blood donors who have previously had CMV
of this approach for donors, and the efficacy of         infection and still carry the virus.
granulocyte transfusions collected in this way, are        Patients who have been previously exposed to
required before granulocyte transfusion therapy         CMV and are CMV seropositive are at risk of reac-
becomes accepted in the care of patients with           tivation of CMV during a period of immunosup-
severe neutropenia and fungal infection, in con-        pression. The extent to which CMV-seropositive
junction with other potential approaches such as        patients are at risk from reinfection with different
improved diagnostic strategies and organism-            strains of CMV remains unknown, but this risk is
targeted antimicrobials. Trials to evaluate evidence    generally considered to be low. The patients at
of survival benefit following granulocyte transfu-       risk of transfusion-transmitted CMV infection are
sions are clearly needed, but their design is compli-   shown in Table 9.1, and the generally accepted
cated by several issues including the numbers of        indications for the use of CMV-seronegative blood
patients required to power a trial and the method-      components are shown in Table 9.2.
ological difficulties related to incorporating blind-
ing in such studies.
                                                        Prevention
   High-dose granulocyte transfusions collected
using donors treated with G-CSFs might therefore        The use of CMV-seronegative blood components
be considered as indicated in patients of any age       has been shown to reduce the incidence of CMV
with severe neutropenia due to bone marrow              infection in groups at risk for transfusion-
failure under the following circumstances:              transmitted CMV infection to 1–3%. This incom-
• proven bacterial or fungal infection unrespon-        plete prevention may be due to:
sive to antimicrobial therapy, or probable bacterial    • occasional failure to detect low-level CMV
or fungal infection unresponsive to appropriate         antibodies;
blind antimicrobial therapy;                            • loss of antibodies in previously infected blood
• neutrophil recovery not expected for 5–               donors;
7 days;                                                 • transfusion of blood components prepared from
• children and lighter adults might be expected to      recently infected donors.
show better incremental responses to granulocyte           CMV is transmitted by leucocytes, and a
transfusions.                                           number of studies have found that leucocyte deple-
   Granulocyte transfusions might be considered         tion of blood components is as effective as the use
inappropriate for:                                      of CMV-seronegative blood components in the
• patients with haematological disease resistant to     prevention of transfusion-transmitted CMV infec-
treatment;                                              tion in neonates, patients undergoing remission


124
                                                                                                            Haematological disease

Table 9.1 Patients at risk for transfusion-transmitted                 blood components for patients at risk of
cytomegalovirus (CMV) infection.                                       transfusion-transmitted CMV infection when
                                                                       CMV-seronegative blood components are not
Risk well established
                                                                       available. Further information about the effective-
CMV-seronegative recipients of allogeneic bone marrow/peripheral
                                                                       ness of leucocyte depletion of blood components in
   blood progenitor cell transplants from CMV-seronegative donors
CMV-seronegative pregnant women
                                                                       the prevention of transfusion-transmitted CMV
Premature infants (<1.2 kg) born to CMV-seronegative women             infection in different patient groups is required
CMV-seronegative patients with HIV infection                           before CMV-seronegative blood components can
                                                                       be discontinued. A recent consensus conference in
Risk less well established
                                                                       Canada recommended that where universal leuco-
CMV-seronegative patients receiving autologous bone
   marrow/peripheral blood progenitor cell transplants
                                                                       cyte depletion had been implemented, both leuco-
CMV-seronegative patients who are potential recipients of allogeneic   cyte-depleted and CMV-seronegative blood should
   or autologous bone marrow/peripheral blood progenitor cell          be used for CMV-seronegative pregnant women,
   transplants                                                         intrauterine transfusions, and CMV-seronegative
CMV-seronegative patients receiving solid organ (kidney, heart, lung   allogeneic haemopoietic cell transplant recipients.
   liver) transplants from CMV-seronegative donors

Risk not established
CMV-seronegative recipients of allogeneic bone marrow/peripheral       Transfusion-associated graft-versus-host disease
   blood progenitor cell transplants from CMV-seropositive donors      Pathogenesis and clincal features
CMV-seropositive recipients of bone marrow/peripheral blood
   progenitor cell transplants                                         Transfusion-associated graft-versus-host disease
CMV-seropositive recipients of solid organ transplants                 (TA-GVHD) is a rare but serious complication
                                                                       of blood transfusion. As discussed in Chapter 18,
                                                                       there is engraftment and proliferation of donor T
                                                                       lymphocytes, and interaction with recipient cells
Table 9.2 Indications for the use of cytomegalovirus
(CMV)-seronegative blood components.
                                                                       expressing HLA antigens causing cellular damage
                                                                       particularly to the skin, gastrointestinal tract, liver
Transfusions in pregnancy                                              and spleen, and the bone marrow. Clinical mani-
Intrauterine transfusions                                              festations usually occur 1–2 weeks after blood
Transfusions to neonates and to infants in the first year of life       transfusion, and early features include fever, macu-
Transfusions to the following groups of CMV-seronegative patients      lopapular skin rash, diarrhoea and hepatitis.
   After allogeneic bone marrow/peripheral blood progenitor cell       At-risk transfused haematology patients are those
      transplants where the donor is also CMV seronegative             who are undergoing transplantation, have
   After autologous bone marrow/peripheral blood progenitor cell       Hodgkin’s disease, or have received therapy with
      transplants
                                                                       certain drugs, e.g. purine analogues.
   Potential recipients of allogeneic bone marrow/peripheral blood
      progenitor cell transplants
   Patients with HIV infection                                         Prevention
                                                                       The dose of donor lymphocytes sufficient to cause
                                                                       TA-GVHD is unknown, but may be lower than is
                                                                       achievable by current techniques for leucocyte
induction therapy for acute leukaemia and after                        depletion of blood components, since there are
bone marrow transplantation (the only prospec-                         case reports of TA-GVHD following transfusions
tive randomized trial was conducted in transplant                      of leucocyte-depleted blood. Gamma-irradiation
recipients using bedside filtration, which cannot be                    to remove the proliferative capability of donor
adequately quality controlled). These data suggest                     lymphocytes remains the method of choice to
that leucocyte-depleted blood components can be                        prevent TA-GVHD (see Chapter 18). The cur-
accepted as a substitute for CMV-seronegative                          rently recommended indications for the use of


                                                                                                                             125
Chapter 9

Table 9.3 Indications for gamma-irradiation of blood                        about the indications for special blood compon-
components in haematology patients.                                         ents, and the importance of receiving the correct
                                                                            type of blood component;
Indications
                                                                            • requests for blood components to include the
Acute leukaemia: only for HLA-matched platelets or donations from
                                                                            patient’s diagnosis and any requirement for special
   first- or second-degree relatives
Allogeneic bone marrow/peripheral blood progenitor cell
                                                                            blood components;
   transplantation: from the time of initiation of conditioning therapy     • storing of individual patient’s requirements
   and continuing while the patient remains on GVHD prophylaxis             for special blood components in the blood bank
   (usually 6 months) or until lymphocytes are greater than 1 ¥ 109/L. It   computer;
   may be necessary to irradiate blood components for SCID patients         • the prescription for blood components should
   for up to 2 years, and for patients with chronic GVHD if there is        include any requirement for special blood com-
   evidence of immunosuppression                                            ponents, enabling the ward staff to check that the
Donors of allogeneic bone marrow: to prevent TA-GVHD mediated by            blood component to be transfused complies with
   lymphocytes in donor blood transfused before or during the harvest       these requirements;
Autologous bone marrow/peripheral blood progenitor cell
                                                                            • providing patients with cards indicating their
   transplantation: during and 7 days before the harvest of
                                                                            special blood requirements, particularly for those
   haemopoietic cells, and then from the initiation of conditioning
   therapy until 3 months post-transplant (6 months if total body
                                                                            patients receiving shared care between two hos-
   irradiation is used)                                                     pitals and those with a long-term requirement
Hodgkin’s disease                                                           for gamma-irradiated blood, e.g. patients with
Patients treated with purine analogues                                      Hodgkin’s disease.
Non-indications
Aplastic anaemia (even if treated with antilymphocyte globulin)             HLA alloimmunization and refractoriness to
Non-Hodgkin’s lymphoma (although this may be reviewed following             platelet transfusions
  some recent reports of TA-GVHD in patients with B-cell non-
  Hodgkin’s lymphoma)                                                       Platelet refractoriness is the repeated failure to
HIV infection                                                               obtain satisfactory responses to platelet transfu-
                                                                            sions, and occurs in more than 50% of patients
SCID, severe combined immunodeficiency; TA-GVHD, transfusion-asso-           receiving multiple transfusions.
ciated graft-versus-host disease.                                              Various methods are used to assess response to
                                                                            platelet transfusions. If the patient is bleeding, the
gamma-irrradiated blood for                           haematology           clinical response is an important indication of the
patients are shown in Table 9.3.                                            effectiveness of the transfusion. The response to a
                                                                            prophylactic platelet transfusion is assessed by
                                                                            measuring the increase in platelet count after the
How to ensure that patients receive the correct
                                                                            transfusion. Various formulas have been used to
‘special’ blood?
                                                                            correct for the variation in response dependent on
An important issue for haematology departments                              the patient’s size and the number of platelets trans-
and hospital blood banks is how to ensure that                              fused; these include platelet recovery and corrected
patients receive special blood components (e.g.                             count increment. However, in practice, a (non-
CMV-seronegative, gamma-irradiated) when they                               sustained) increase in the patient’s platelet count of
are indicated and that standard blood components                            less than 5 ¥ 109/L at 20–24 h after the transfusion
are not transfused as this may have devastating                             can be used as a simple measure of a poor
consequences.                                                               response.
  Each hospital needs to establish its own proce-
dures so that patients receive the correct special
                                                                            Causes
blood components, where they are indicated.
These should include the following:                                         Many causes of platelet refractoriness have been
• education of ward medical and nursing staff                               described, and they can be subdivided into


126
                                                                                                                    Haematological disease


immune mechanisms, most importantly HLA                           likely, an attempt should be made to correct the
alloimmunization, and non-immune mechanisms                       clinical factors responsible, where possible, and
involving platelet consumption (Table 9.4).                       platelet transfusions from random donors should
Platelet consumption is the most frequent mech-                   be continued. If poor response to random donor
anism of platelet refractoriness, usually associated              platelet transfusions persists, the patient should be
with sepsis. However, immune-mediated platelet                    tested for HLA antibodies.
destruction remains an important cause of platelet                3 If non-immune platelet consumption appears to
refractoriness; HLA antibodies are the commonest                  be unlikely, an immune mechanism should be sus-
immune cause, and the other immune causes are                     pected, and the patient’s serum should be tested for
rare.                                                             HLA antibodies. If HLA antibodies are present,
   The precise mechanism of HLA alloimmuniza-                     the specificity of the antibodies should be deter-
tion remains uncertain, but primary HLA alloim-                   mined as this may help in the selection of HLA-
munization appears to be initiated by intact cells                compatible donors. However, HLA antibodies
expressing both HLA class I and class II antigens                 stimulated by repeated transfusions are often
such as lymphocytes and antigen-presenting cells.
Platelets only express HLA class I antigens, and
leucocyte-depleted blood components will not                      Table 9.4 Causes of platelet refractoriness.
cause primary HLA alloimunization. However,
secondary HLA alloimmunization does not re-                       Immune
quire the presence of HLA class II antigens, and                  Platelet alloantibodies
may occur in patients who have been pregnant or                      HLA
previously transfused with non-leucocyte-depleted                    HPA
blood components.                                                    ABO
                                                                  Other antibodies
                                                                     Platelet autoantibodies
Investigation and management                                         Drug-dependent platelet antibodies
                                                                  Immune complexes
If platelet refractoriness occurs, the following
algorithm can be used for investigation and man-                  Non-immune
agement (Fig. 9.1).                                               Infection and its treatment, especially amphotericin B
                                                                  Splenomegaly
1 A clinical assessment should be made for clinical
                                                                  Disseminated intravascular coagulation
factors likely to be associated with non-immune
                                                                  Fever
platelet consumption.                                             Bleeding
2 If non-immune platelet consumption appears



                                                           Poor response to random donor platelets
                                          (e.g. 24-h platelet increment <5 x 109/L with two or more consecutive transfusions)
                                                                          Clincal evaluation
                                                          For presence of infection, DIC, splenomegaly


                                           Positive clinical factors                           Negativeclinical factors


                                         Continue with random platelets          HLA antibodies present          No HLA antibodies
                                         Responses to platelet transfusions
Fig. 9.1 Algorithm for the               should improve when clinical
                                                                                    Use HLA-compatible      1 Consider ABO incompatibility
investigation and management of          factors resolve                            platelet transfusions   2 Test for non-cytotoxic HLA
patients with platelet refractoriness.                                                                         antibodies and HPA antibodies
                                                                                                            3 Ifall negative, consider causes
DIC, disseminated intravascular                                                                                such as drug-dependent
coagulation.                                                                                                   and platelet autoantibodies




                                                                                                                                       127
Chapter 9


‘multispecific’, and it is not possible to determine                                        incompatible transfusions have been used for
their specificity.                                                                          previous transfusions.
4 Platelet transfusions from HLA-matched                                                   (b) HPA antibodies, which usually occur in
donors (matched for the HLA-A, -B antigens of the                                          combination with HLA antibodies, but some-
patient) should be used for patients with apparent                                         times occur in isolation.
immune refractoriness, and the response to further                                         (c) Drug-dependent platelet antibodies, which
transfusions should be observed carefully. Figure                                          may be underestimated as a cause for platelet
9.2 shows improved responses to HLA-matched                                                refractoriness.
platelet transfusions in a patient with platelet
refractoriness due to HLA alloimmunization.
                                                                                         Alloimmunization to red cell antigens
If responses to HLA-matched transfusions are
not improved, the reason should be sought, and                                           Incidence
platelet crossmatching of the patient’s serum
                                                                                         Alloimmunization to red cell antigens is another
against the lymphocytes and platelets of one of the
                                                                                         important consequence of repeated transfusions in
HLA-matched donors may be helpful in determin-
                                                                                         haematology patients. The incidence of red cell
ing the cause, and the selection of compatible
                                                                                         alloimmunization in adult haematology patients is
donors for future transfusions.
                                                                                         in the range of 10–15% and is similar to other
5 If there are no factors for non-immune platelet
                                                                                         groups of multitransfused patients, e.g. patients
consumption and HLA antibodies are not
                                                                                         with renal failure. However, a higher proportion of
detected, consideration should be given to less fre-
                                                                                         children requiring long-term transfusion support
quent causes of immune platelet refractoriness.
                                                                                         develop red cell alloimmunization. In sickle cell
   (a) High-titre ABO antibodies in the recipient.
                                                                                         disease, the incidence is in the range of 20–30%.
   This is an unusual cause of platelet refractor-
                                                                                         The implications of these observations include the
   iness, and can be excluded by switching to
                                                                                         following.
   ABO-compatible platelet transfusions, if ABO-
                                                                                         • Patients with sickle cell disease should be pheno-
                                                                                         typed for Rh, Kell, Fy, Jk and MNS antigens before
                                                                                         the first transfusion, and patients with thalas-
                                                                                         saemia and other children requiring chronic trans-
                                  100       Random donors      HLA-matched donors
                                                                                         fusion support should be phenotyped for Rh and
                                                                                         Kell antigens.
  (20 h after platelet transfusions)




                                       80                                                • Blood for transfusion to children requiring long-
        % platelet recovery




                                                                                         term transfusion support, including patients with
                                       60                                                haemoglobinopathies, should be matched for Rh
                                                                                         and Kell antigens to prevent alloimmunization.
                                       40                                                • Phenotyping and antigen matching to prevent
                                                                                         red cell alloimmunization is not required for other
                                       20                                                groups of patients requiring repeated transfusions.


                                       0                                                 Timing of sample collection for
                                        0         10               20               30   compatibility testing
                                                            Days
                                                                                         In patients with haematological disorders receiv-
Fig. 9.2 Responses to platelet transfusions in a female                                  ing repeated transfusions, an important issue is the
patient with acute myeloblastic leukaemia undergoing                                     timing of blood sample collection in relation to the
remission induction therapy. There were poor responses to                                previous transfusion.
the initial platelet transfusions, and the patient was found                             • Where the patient is receiving very frequent
to have HLA antibodies. There were improved responses to
                                                                                         transfusion, e.g. daily, it is only necessary to
platelet transfusions from HLA-matched donors.
                                                                                         request a new sample every 72 h.

128
                                                                                                                Haematological disease


• Where the previous transfusion was 3–14 days                              blood components are given in Fig. 9.3, and can be
earlier, the sample should ideally be taken within                          briefly summarized as follows.
24 h of the start of the transfusion, although some                         • Major ABO mismatch: use red cells of patient’s
laboratories stretch this to 48 h for patients who                          ABO type until recipient ABO antibodies are unde-
have been repeatedly transfused without develop-                            tectable and the direct antiglobulin test is negative,
ing antibodies.                                                             and platelets and plasma from donors of
• Where the previous transfusion was 14–28 days                             recipient’s ABO type.
earlier, the sample should be taken within 72 h of                          • Minor ABO mismatch: use red cells of donor
the start of the transfusion.                                               ABO type throughout, and plasma and platelets of
• Where the previous transfusion was more than                              recipient type until recipient-type red cells are no
28 days ago, the sample should be taken within 1                            longer detectable.
week of the planned transfusion.                                            • Major and minor ABO mismatch: use group O
                                                                            red cells until recipient ABO antibodies are unde-
                                                                            tectable, and then switch to donor-type red cells.
ABO-incompatible bone marrow/peripheral blood
                                                                            Use group AB plasma and platelets until recipient-
progenitor cell transplants
                                                                            type red cells are undetectable.
ABO-incompatible bone marrow/peripheral blood                                  RhD-incompatible transplants can also cause
progenitor cell transplants present particular prob-                        difficulties. It is recommended that RhD-negative
lems (Table 9.5). The transplant may provide a                              blood components should be used for RhD-
new A and/or B antigen from the donor (major                                positive recipients with RhD-negative donors.
mismatch) or a new A and/or B antibody (minor                               However, no cases of immunization have been
mismatch). Recommendations for the use of donor                             reported when RhD-negative recipients have
                                                                            received RhD-positive transplants, and RhD-
                                                                            positive blood components may be used.
Table 9.5 Problems associated with ABO-incompatible
bone marrow/peripheral blood progenitor transplants.
                                                                            Iron overload
Major ABO incompatibility (e.g. recipient O, donor A)
                                                                            A major adverse consequence of repeated red cell
Failure of engraftment: risk not increased in ABO-incompatible
                                                                            transfusions over a long period is iron overload.
   transplants
Acute haemolysis at time of reinfusion: avoided by processing donor
                                                                            The body has no mechanism for excreting excess
   bone marrow/peripheral blood progenitor cells                            iron, and regular transfusion inevitably leads
Haemolysis of donor-type red cells: avoid by using red cells of recipient   to accummulation of iron. Each unit of blood
   type in the early post-transplant period                                 contains about 200–250 mg of iron, and clinical
Delayed erythropoiesis: may be due to persistence of anti-A in the          symptoms and signs of iron overload do not gener-
   recipient, but minimize transfusion of anti-A by using platelets and     ally occur until iron stores are in the range of
   plasma from group A donors                                               20–30 g, i.e. about 100 units of blood transfused.
Delayed haemolysis due to persistence of recipient anti-A: only switch         The clinical picture of iron overload resembles
   to donor red cells when recipient anti-A undetectable and direct         that of idiopathic haemochromatosis, and the
   antiglobulin test undetectable
                                                                            organs mainly affected include:
Minor ABO incompatibility (e.g. recipient A, donor O)                       • skin (causing pigmentation);
Graft-versus-host disease: risk not increased in ABO-incompatible           • endocrine glands (causing diabetes mellitus,
  transplants                                                               hypogonadism, poor growth, hypothyroidism);
Acute haemolysis at time of reinfusion: avoid by removing donor             • liver (causing cirrhosis);
  plasma if the donor anti-A titre is high                                  • heart (causing heart failure).
Delayed haemolysis of recipient cells due to anti-A produced by donor
                                                                               The haematology patient group at greatest risk
  lymphocytes (passenger lymphocyte syndrome): maximum
                                                                            for iron overload is thalassaemia major, but
  haemolysis usually occurs between days 9 and 16 post-transplant,
  and occasionally there is severe intravascular haemolysis
                                                                            patients with other inherited haematological dis-
                                                                            eases such as sickle cell disease or with acquired


                                                                                                                                 129
Chapter 9



       Recipient group              Group O          Group AB          Donor group

   Major ABO incompatibility
              Red cells

       Plasma/platelets


   Minor ABO incompatibility
              Red cells
       Plasma/platelets


   Major and minor ABO incompatibility
              Red cells
       Plasma/platelets

                                1         2                  3                 4

             1 Begin pretransplant chemotherapy                                              Fig. 9.3 Recommendations for ABO
             2 Bone marrow transplant                                                        type of blood components in ABO-
             3 ABO antibodies to donor RBC not detected. Direct antiglobulin test negative   incompatible bone marrow/peripheral
                                                                                             blood progenitor cell transplants.
             4 RBC of recipient group no longer detected
                                                                                             (From Warkentin 1983 with
                                                                                             permission.)


haematological disorders such as myelodysplasia                             Vitamin C (200 mg adults, 100 mg/day children
may also require long-term red cell transfusions                         or orange juice) is given to increase the availability
and are at risk.                                                         and hence excretion of chelatable iron, starting 2
   Iron chelation therapy is the only way to remove                      weeks after iron chelation has begun. New systems
excess iron in haematology patients and prevent                          of delivering desferrioxamine are being developed
heart failure, which is the major cause of death in                      because of the recognized problems of compliance,
transfusional iron overload. Venesection, as used                        including the lightweight silent balloon infusor
in inherited haemochromatosis, is inappropriate                          which provides continuous pressure without the
for most patients, but has been occasionally used                        need for a battery or other mechanical device.
for some sickle cell patients with iron overload.                        Administration of intravenous desferrioxamine
Only a small proportion of the body iron is avail-                       only at the time of blood transfusion is an ineffec-
able for chelation. Parenteral desferrioxamine is                        tive approach to iron chelation, but may be
established for promoting negative iron balance,                         indicated as a continuous infusion (e.g. through
reversing cardiac toxicity and prolonging life                           Port-a-Cath) for patients with consistently high
expectancy.      Subcutaneous        desferrioxamine                     levels of iron overload. It should be recalled that
(30–50 mg/kg) is typically administered as an infu-                      sickle cell (and to a degree) thalassaemia patients
sion using a syringe driver pump over 8–12 h (or                         requiring Port-a-Cath have a prothrombotic
longer if tolerated to sustain the chelation process)                    predisposition.
at least five times a week, usually overnight.                               The decision when to initiate iron chelation
However, the efficiency of currently used chelation                       therapy is not always easy, particularly in elderly
regimens is generally very low, and most of                              patients with acquired disorders such as myelodys-
the administered subcutaneous desferrioxamine is                         plasia where the patient’s life expectancy from
excreted without binding to iron atoms.                                  their underlying condition may be less than the


130
                                                                                                    Haematological disease


time it would take to develop clinically significant      Further reading
iron overload. The serum ferritin level is the sim-
plest way of monitoring iron overload, but assay         British Committee for Standards in Haematology.
of iron stores using liver biopsies is a more reliable     Guidelines on gamma irradiation of blood components
measure. Newer imaging techniques may provide              for the prevention of graft-versus-host disease. Transfus
                                                           Med 1996; 6: 261–71.
this information non-invasively in the future. Iron
                                                         British Committee for Standards in Haematology.
chelation therapy should aim to maintain the liver
                                                           Guidelines on the clinical use of red cell transfusions. Br J
iron concentration at about 3–7 mg/g liver dry             Haematol 2001; 113: 24–31.
weight, or in practice a serum ferritin level of         British Committee for Standards in Haematology.
1000–1500 mg/L.                                            Guidelines for platelet transfusions. Br J Haematol 2003;
   Desferrioxamine may cause adverse effects such          122: 10–23.
as local swelling and irritation at the sites of         British Committee for Standards in Haematology.
                                                           Transfusion guidelines for neonates and small children.
infusion. Excessive doses of desferrioxamine may
                                                           Br J Haematol 2004; 124: 433–53.
cause:
                                                         Dale DC, Conrad Liles W, Price TH. Renewed interest in
• disturbances of vision and hearing, and there-           granulocyte transfusion therapy. Br J Haematol 1997;
fore regular (yearly) audiograms and retinal exam-         98: 497–501.
ination are recommended;                                 Laupacis A, Brown J, Costello B et al. Prevention of
• bone deformities and growth retardation in               posttransfusion CMV in the era of universal WBC
children.                                                  reduction: a consensus statement. Transfusion 2001; 41:
                                                           560–9.
Long-term effects of desferrioxamine can largely
                                                         Novotny VMJ. Prevention and management of platelet
be avoided by adjusting the dose to take account of
                                                           transfusion refractoriness. Vox Sang 1999; 76: 1–13.
the degree of iron overload, ensuring that the mean      Oliveri NF, Brittenham GM. Iron-chelating therapy and the
daily dose does not exceed 40–50 mg/kg (until              treatment of thalassaemia. Blood 1997; 89: 739–61.
growth is completed), or by calculating a therapeu-      Pamphilon DH, Rider JR, Barbara JAJ, Williamson LM.
tic index of the mean daily dose of desferrioxamine        Prevention of transfusion-transmitted cytomegalovirus
(mg/kg) divided by the serum ferritin (mg/L) every         infection. Transfus Med 1999; 9: 115–23.
                                                         Petz LD. Hemolysis associated with transplantation.
6 months and keeping this ratio below 0.025.
                                                           Transfusion 1998; 38: 224–8.
   Difficulties of compliance with infusion of des-
                                                         Porter JB. Practical management of iron overload. Br J
ferrioxamine for practical and psychological               Haematol 2001; 115: 239–52.
reasons remain major issues for many patients and        Samol J, Littlewood TJ. The efficacy of rHuEPO in cancer-
have stimulated an ongoing search for alternatives;        related anaemia. Br J Haematol 2003; 121: 3–11.
the ideal would be an effective and safe iron chela-     Warkentin PI. Transfusion of patients undergoing bone
tor that can be taken orally.                              marrow transplantation. Hum Pathol 1983; 14: 261–6.




                                                                                                                     131
Chapter 10
Transfusion strategies in organ transplant patients

Derwood H. Pamphilon




Transplant patients frequently require blood com-      sion was greatest when there was more HLA
ponent transfusions. These may be given preopera-      mismatch.
tively or perioperatively. Blood transfusions are an      More recently it has been appreciated that
important consideration for transplant recipients      this ‘transfusion effect’ is less apparent when
because:                                               cyclosporin is used after grafting for immunosup-
• they may sensitize potential transplant recipi-      pression. It has been argued that pretransplant
ents to histocompatibility antigens (HLA), result-     transfusions may sensitize a proportion of patients
ing in an increased risk of graft rejection;           and that the benefit ascribed to transfusions could
• induce modulation of immune responses and            then be due to identification of non-responders
promote graft acceptance;                              and high responders. The latter would either be
• cause the acquisition of viruses such as hepatitis   excluded or transplanted with a kidney from a
C (HCV) and cytomegalovirus (CMV).                     crossmatch-negative donor. In 150 patients treated
   Blood component order schedules for ortho-          with post-transplant cyclosporin, graft survival
topic liver transplantation (OLT) place large          was not related to a history of blood transfusion
demands on transfusion services, while in kidney       but the presence of lymphocytotoxic antibodies
transplants perioperative blood transfusions are       was a major risk factor for graft failure. In a large
often not required. In some types of transplant        study of 4015 children receiving either live donor
surgery, autologous predeposit, as well as intra-      or cadaveric transplants reported by the North
operative blood salvage and the use of pharmaco-       American Paediatric Renal Transplant Co-
logical agents to minimize the use of allogeneic       operative Study, it was shown that patients who
products, may be considered.                           received between one and five transfusions had a
                                                       reduced risk of graft failure.
                                                          Blood transfusion may reduce the rate of graft
Renal transplantation                                  failure by downregulating the immune response.
                                                       This could be due to:
Pretransplant period
                                                       • generation of suppressor cells;
Patients with renal disease are frequently anaemic     • reduction in the number of cytotoxic T lympho-
and receive transfusions preoperatively. In the case   cytes reactive with donor-type HLA; or
of transplant from cadaveric donors, third-party       • formation of anti-idiotype antibodies.
blood transfusions are given. In addition, patients    There is debate as to whether the impact of pre-
who receive live donor transplants from siblings       transplant transfusion is greater if there is HLA
or other relatives may be given transfusions from      haplotype sharing between transfused blood and
the potential donor (see below). These are known       the patient. It has been shown that the frequency of
as donor-specific transfusions (DST). In 1973 it        anti-donor cytotoxic T lymphocytes was markedly
was reported that graft survival was better in         reduced in patients who received transfusions
transfused patients irrespective of matching at        where one HLA haplotype or at least one HLA-B
HLA-A, -B or -DR, although the benefit of transfu-      and -DR was shared.


132
                                                                                             Organ transplantation


   DST is thought to induce a state of immuno-          Viral infections
logical tolerance between donor and recipient,
                                                        Cytomegalovirus
perhaps as a result of persistence of donor cells
after transfusion (see below). In DST, between 8        CMV is of importance after renal transplantation
and 13% of patients are sensitized after transfu-       and can be acquired from:
sion, fewer if concurrent treatment with immuno-        • the donor kidney if seropositive;
suppressive drugs is given. Graft and patient           • CMV-positive blood transfusions;
survival of 90% at 5 years following DST has been       • reactivation of the patient’s CMV.
reported where combinations of immunosuppres-           A review of 1145 patients showed active infection
sive drugs were given after transplantation. In         in 85% of those with, and 53% of those without,
patients who became sensitized to third-party           CMV antibodies at the time of transplant, suggest-
DST, the antibody can be removed and the patient        ing that latent virus is reactivated during the trans-
‘desensitized’ by plasma exchange procedures            plant process. Reactivation itself does not appear
or immunoadsorption. Although these patients            to be detrimental to graft survival. If CMV-
may have a lower overall rate of durable renal          seronegative blood is given to CMV-seronegative
engraftment, desensitization at least allows trans-     donor–recipient pairs, then no CMV infection
plants to be done in patients who would otherwise       results. Primary infection transferred with the
be excluded as lymphocytotoxic crossmatch               donor kidney is of greater severity than infection
positive.                                               associated with reactivation. In one report, 26 of
   Recently, it has been shown that transfusion of      74 patients who received CMV-seronegative blood
donor bone marrow cells to patients who receive         and a kidney from a donor of unknown CMV
kidney and other organ transplants is associated        serostatus developed primary infection, 20 of 26
with a lower incidence of acute cellular graft rejec-   were symptomatic, and three died.
tion and lowered reactivity against donor cells in         CMV infection can be prevented in CMV-
the mixed lymphocyte reaction. This also suggests       seronegative donor–recipients by:
that transfusion of donor cells before transplanta-     • provision of CMV-seronegative blood compon-
tion may induce a state of specific tolerance. This      ents; or
could be partly due to the persistence of donor         • leucocyte depletion to less than 5 ¥ 106 leuco-
cells, which may be detected by sensitive molecular     cytes/transfused component.
analysis of chimeric status.                            Either approach is satisfactory, although the use of
   Recombinant human erythropoietin (EPO) is            leucocyte-depleted blood components will reduce
now used extensively in the preoperative period in      the rate of HLA alloimmunization as well. More-
renal transplant recipients. It reduces the degree of   over, in several European countries universal leu-
anaemia and therefore the chance of sensitization       cocyte depletion of blood components is now
induced by random blood transfusions.                   standard practice.
   It is generally recommended that all renal trans-       In ‘at-risk’ transplants, where either the patient
plant patients who require transfusion, despite the     or donor is CMV seropositive, administration of
use of EPO, should receive leucocyte-depleted           ganciclovir or valaciclovir for 3 months is effec-
blood components before transplantation to avoid        tive in preventing CMV disease. Intravenous
HLA alloimmunization. In the UK, this presents          immunoglobulin from CMV hyperimmune globu-
no difficulties as all blood components have             lin (CMVIg) may reduce the chance of CMV infec-
been routinely leucocyte depleted since 1999 (see       tion but its role is controversial.
Chapter 23). However, one or more non-
leucocyte-depleted transfusions may be given
                                                        Hepatitis viruses
deliberately      to   induce     tolerance   before
transplantation.                                        HCV, hepatitis B (HBV) and hepatitis G (HGV,
                                                        also called GBV-C) may be acquired from blood
                                                        transfusion. An increased incidence of post-


                                                                                                             133
Chapter 10


transplant liver disease has been reported in HCV-       Viral infections
seropositive patients but this does not appear to
                                                         Cytomegalovirus
influence either the graft or patient survival.
                                                         Liver transplant recipients who are CMV seroneg-
                                                         ative may acquire infection via a seropositive graft
Perioperative period
                                                         or via blood transfusions. The former route seems
The use of EPO has had a significant impact on            to be of greater importance. Recipients of CMV-
preoperative anaemia and at our institution blood        seropositive grafts are more likely to have:
is not routinely crossmatched for the transplant         • CMV pneumonia;
procedure unless the haemoglobin is below 9 g/dL.        • CMV hepatitis;
As transfusions are infrequently required during         • invasive fungal infections;
surgery, the use of autologous predeposit, intraop-      • reduced survival.
erative blood salvage or pharmacological agents to          The incidence of CMV pneumonia has been
reduce blood loss is not appropriate.                    shown to correlate with both the total number of
                                                         units of blood and the number of CMV-positive
                                                         units transfused perioperatively. CMV infection is
Orthotopic liver transplantation                         also associated with an increased chance of graft
                                                         rejection. CMV infection may also occur because
Pretransplant period
                                                         of reactivation in CMV-positive recipients. It
Precise HLA matching between donor and recipi-           appears that seroconversion to human herpes virus
ent does not influence the outcome in liver               (HHV)-6 is a marker for CMV disease.
transplantation. However, if the lymphocytotoxic            CMV infection may be prevented in seronega-
crossmatch against donor cells is positive (possibly     tive donor–recipient pairs by transfusion of
as a result of previous transfusion), it is associated   leucocyte-depleted or CMV-seronegative blood
with:                                                    components and this seems to be a sensible precau-
• poorer graft survival;                                 tion. In patients at risk of CMV infection, use of
• higher retransplant rate;                              CMVIg has been shown to reduce severe CMV-
• reduced 1-year overall survival.                       associated disease and improve long-term survival.
   The presence of lymphocytotoxic antibodies            In addition, the use of prophylactic ganciclovir in
is not necessarily associated with an increased          at-risk patients significantly reduces the incidence
chance of a positive lymphocytotoxic crossmatch          of CMV disease.
and patients who receive transfusions more than
90 days preoperatively have a reduced incidence of
                                                         Hepatitis viruses
severe or recurrent rejection episodes. This is not
reflected in an improvement in allograft or overall       • HCV: a number of patients with HCV infection
patient survival. However, there is considerable         develop severe liver disease and undergo OLT. The
interest in induction of donor tolerance pretrans-       overall outcome in this group of patients is excel-
plant and it is now appreciated that the persistence     lent, with survival as high as 75% at 5 years after
of transfused leucocytes (donor microchimerism)          transplantation. Surprisingly, grafts from HCV-
may have a clinical role in suppressing host             seropositive donors do not result in decreased
immune responses. Efforts have been made to              survival when compared with grafts from HCV-
augment this with preoperative or perioperative          seronegative donors and there is no difference in
donor bone marrow infusion. This can be done             the rate of graft rejection.
without inducing clinical graft-versus-host disease      • HGV is also acquired by blood transfusion. It is
in many types of whole organ transplant                  present in a large number of patients after trans-
recipients.                                              plantation compared with before transplantation.
                                                         It appears not to be associated with significant



134
                                                                                                       Organ transplantation


liver disease or to impact the outcome of              FFP 10 units, cryoprecipitate 10 units and platelets
transplantation.                                       12 adult therapeutic doses. Usage of blood compo-
                                                       nents is summarized in Table 10.1.
                                                          FFP treated with solvent–detergent to inactivate
Perioperative period
                                                       viruses is as effective as standard FFP in OLT.
Blood compatibility                                       Intraoperative blood salvaging may significantly
                                                       reduce the demand for allogeneic blood compon-
• It is important in OLT to ensure that there is
                                                       ents. Massive blood loss during OLT is signifi-
ABO compatibility between donor and recipient.
                                                       cantly associated with previous upper abdominal
Mismatching for ABO results in an increase in
                                                       surgery. It results in poorer graft function in the
hyperacute rejection, vascular thrombosis and
                                                       immediate postoperative period, an increased
biliary injury. It may be appropriate in some
                                                       incidence of infection, gastrointestinal and intra-
instances to use ABO-mismatched OLT in patients
                                                       abdominal complications, rejection and lower
with fulminant liver failure, where a matched liver
                                                       overall survival.
is unavailable.
• Where there is ABO mismatch, viable lympho-
cytes in the graft may induce clinically significant    Pharmacological agents
immune-mediated haemolysis in recipient red
                                                       These are discussed in more detail in Chapter 7.
cells. Usually this occurs where the donor is group
                                                       • Aprotinin: both standard and high doses have
O and the patient is group A.
                                                       been shown to inhibit fibrinolysis, thereby reduc-
• Because of preceding blood transfusions OLT
                                                       ing the requirement for red cells, FFP, cryoprecipi-
patients have a significant incidence of red cell
                                                       tate and platelet concentrates.
alloantibody formation (6.3% in one series) and
                                                       • Tranexamic acid (an antifibrinolytic drug) in
this adds to the difficulty of providing large inven-
                                                       high but not low dose has been shown to reduce
tories of compatible red cells.
                                                       intraoperative blood loss. Low-dose tranexamic
                                                       acid does inhibit fibrinolysis but is not sufficient to
Blood use                                              influence transfusion requirements.
                                                       • e-Aminocaproic acid: in one report where this
OLT patients usually have disordered coagulation
                                                       was given to patients there was no significant
before transplantation due to their underlying liver
                                                       difference in blood loss and blood requirement
disease and associated portal hypertension. Fre-
                                                       compared with those patients who did not receive
quent findings are prolonged prothrombin and
                                                       it.
activated partial thromboplastin times and throm-
bocytopenia. There is activation of fibrinolysis
during surgery, which causes increased blood loss.
In the first 100 patients transplanted at the Mayo
Clinic the average number of units of red cells,       Table 10.1 Transfusion support in liver transplantation.
fresh frozen plasma (FFP), cryoprecipitate and
platelets transfused was over 10. In addition, a       Report       Red cells      FFP        Cryoprecipitate      Platelets
mean of 5.6 units of blood were salvaged intraop-
                                                       1            12.9           13.8       17.4                 16.9*
eratively.
                                                       2            24.5           38.7       12.2                 26.2†
   Some centres prepare modified whole blood by
                                                       3            25.0           24.0        9.0                 20.0‡
separating cryoprecipitate and platelets and
returning the plasma to the red cells. The aim of      Figures given are mean number of units transfused.
this is to reduce donor exposure. In one centre the    * Apheresis platelet units.
unit standing order for components in low-risk         †
                                                         Random donor platelet units.
adult and paediatric OLT was red cells 10 units,       ‡ Type of platelets not stated.




                                                                                                                       135
Chapter 10


Cardiac transplantation                                   There is also evidence that reperfusion injury
                                                        may be reduced by the use of leucocyte-depleted
Pretransplant period
                                                        blood components.
There is some evidence that pretransplant transfu-
sions may induce tolerance and improve the
                                                        Perioperative period
outcome of transplantation. This is poorer if there
is a positive lymphocytotoxic crossmatch or dis-        Blood losses and blood order schedules
parity at HLA-DR, but not HLA-A or -B.
                                                        The requirement for blood components is much
• Administration of pretransplant transfusions
                                                        lower than in OLT. Typical requirements are
where HLA-DR is shared results in better overall
                                                        shown in Table 10.2. Heart–lung patients require
outcome and reduced vascular graft rejection.
                                                        more transfusions as shown.
• DST has, in certain strain combinations in
animals, been shown to downregulate immune
responses and improve graft acceptance.                 Pharmacological agents
• A detailed analysis in human cardiac allograft-
                                                        Aprotinin has been shown to reduce bleeding and
ing showed that the mixed lymphocyte culture
                                                        blood component requirement after both primary
response to donor-type cells was significantly
                                                        and reoperative cardiac surgery (see also Chapter
reduced at 2 years after transplantation compared
                                                        7).
with pretransplant testing. Responses to third-
party lymphocytes were still intact.
   As in renal and liver transplantation (see above),   Autologous blood
preoperative or perioperative infusion of donor
                                                        It has been shown that the haemodynamic status in
bone marrow statistically reduces the chance of
                                                        patients with cardiac or pulmonary disease who
acute cellular rejection when compared with
                                                        were candidates for cardiac transplants was com-
control patients, and responses in mixed lympho-
                                                        parable with controls following autologous blood
cyte culture are reduced. Finally, where donor
                                                        donation. This may therefore be an appropriate
HLA antibodies could be effectively removed by
                                                        strategy to reduce the requirement for allogeneic
plasma exchange before transplantation, survival
                                                        blood products during surgery. Blood collection
at 1 year was 87% compared with 25% of patients
                                                        could be augmented by the use of EPO, although
in whom plasma exchange was ineffective in
                                                        this is expensive. For a more detailed discussion of
reducing donor antibodies.
                                                        autologous transfusion strategies see Chapter 26.
                                                           Platelet concentrates may be collected immedi-
                                                        ately preoperatively in the anaesthetic room, using
Viral infection
                                                        an apheresis machine, and infused during surgery.
Cytomegalovirus                                         There is evidence that this will reduce the require-
                                                        ment for allogeneic platelets and may also impact
CMV infection adversely affects the outcome of
cardiac allografting. In 91 of 301 patients who
developed CMV infection at one centre over an 8-        Table 10.2 Transfusion support in cardiac transplantation.
year period there was an increase in:
• graft rejection;                                      Report              Red cells             FFP        Platelets*
• development of atherosclerosis resulting in
                                                        1                   3.9                   3.6        4.6
decreased survival.
                                                        2                   5.0                   4.0        None
  CMV infection may, as indicated above, be pre-        3†                  8.0                   4.0        5.0
vented by provision of:
• CMV-seronegative blood components;                    Figures given are mean number of units transfused.
• leucocyte-depleted blood components contain-          * Type of platelet component not stated.
ing less than 5 ¥ 106 leucocytes/transfusion.           †
                                                          Heart–lung transplants.


136
                                                                                                       Organ transplantation


on transfusion requirements since the platelets                 transplantation: review of the literature. Clin Infect Dis
themselves are fresh.                                           1996; 22: 537–49.
                                                              Koneru B, Harrison D, Rizwan M et al. Blood transfusions
                                                                in liver recipients: a conundrum or a clear benefit in the
                                                                cyclosporin/tacrolimus era? Transplantation 1997; 15:
Further reading                                                 1587–90.
                                                              McCarthy JF, Cook DJ, Massad MG et al. Vascular
Blajchman MA, Singal DP. The role of red blood cell             rejection post heart transplantation is associated with
  antigens, histocompatibility antigens, and blood              positive flow cytometric cross-matching Eur J
  transfusions on renal allograft survival. Transfus Med        Cardiothorac Surg 1998; 14: 197–200.
  Rev 1998; 3: 171–9.                                         Opelz G, Senger DPS, Mickey MR, Terasaki PI. The effect
Chavers B, Sullivan EK, Tejani A, Harmon WE. Pre-               of a blood transfusion on subsequent kidney
  transplant blood transfusion and renal allograft outcome:     transplantation. Transplant Proc 1973; 5: 253–9.
  a report of the North American Paediatric Renal             Palomo Sanchez JC, Jimenez C, Moreno Gonzalez E et al.
  Transplant Cooperative Study. Pediatr Transplant 1997;        Effects of intraoperative blood transfusion on
  1: 22–8.                                                      postoperative complications and survival after orthotopic
Deeg HJ, Sayers MH. Transfusion support in transplant           liver transplantation. Hepatogastroenterology 1998; 45:
  patients. In: Pamphilon DH, ed. Modern Transfusion            1026–33.
  Medicine. Boca Raton, FL: CRC Press, 1995: 177–92.          Salgar SK, Shapiro R, Dodson F et al. Infusion of donor
Falagas ME, Snydman DR, Ruthazer R et al.                       leucocytes to induce tolerance in organ allograft
  Cytomegalovirus immune globulin (CMVIG)                       recipients. J Leuk Biol 1999; 66: 310–14.
  prophylaxis is associated with increased survival after     Scudamore CH, Randall TE, Jewesson PJ et al. Aprotinin
  orthotopic liver transplantation. The Boston Center for       reduces the need for blood products during liver
  Liver Transplantation CMVIG Study Group. Clin                 transplantation. Am J Surg 1995; 169: 546–9.
  Transpl 1997; 11: 432–7.                                    van Twuyver E, Mooijaart RJD, ten Berge IJM et al.
Gratton MR, Moreno-Cabral CE, Starnes VA, Oyer PF,              Pretransplantation blood transfusion revisited. N Engl J
  Stinson EB, Shumway ME. Cytomegalovirus and its               Med 1991; 325: 1210–13.
  association with cardiac allograft rejection and            Wallington TB. Cytomegalovirus and transfusion. In: Cash
  atherosclerosis. J Am Med Assoc 1998; 261: 3561–6.            JD, ed. Progress in Transfusion Medicine. London:
Kanj SA, Sharara AI, Clavien P-A, Hamilton, JD.                 Churchill Livingstone, 1987: 26–45.
  Cytomegalovirus infection following liver




                                                                                                                       137
Chapter 11
Inherited and acquired coagulation disorders
Joanne E. Joseph and Samuel J. Machin




Blood components have long been used to treat         tenase. Factor Xa binds to factor Va (again
both inherited and acquired disorders of coagula-     activated by thrombin) which, with calcium and
tion. Attempts are continually being made to          phospholipid, rapidly converts prothrombin to
produce as ‘safe’ a product as possible, while pro-   thrombin.
viding specific concentrates of the highest purity.       The initial TF–VIIa complex is quickly inhibited
Advances in recombinant DNA technology have           by the TF pathway inhibitor; however, by this
expanded the types of products available for clini-   time, the thrombin that has already been produced
cal use.                                              activates factor XI as well as factors V and VIII,
   With so many types of product now available, it    therefore augmenting the formation of factor Xa
can be difficult deciding which is the most appro-     and ultimately the production of more thrombin.
priate to use. This chapter deals with the most       Factor XI can also be activated by factor XIIa,
common disorders of coagulation (both inherited       formed from the high-molecular-weight kinino-
and acquired) as well as detailing the most appro-    gen–prekallikrein complex on endothelial cells;
priate therapies for each condition. The use          however, this contribution to physiological
of platelet concentrates will not be specifically      haemostasis is minimal. The ultimate function of
addressed in this chapter (see Chapter 9).            thrombin is to cleave fibrinogen to fibrin and acti-
                                                      vate factor XIII that results in the cross-linked
                                                      stable clot.
Normal haemostasis                                       Fibrinolysis is also part of the normal haemosta-
                                                      tic response. Circulating plasminogen is activated
Haemostasis is a complex process involving the        to form the serine protease plasmin, which digests
interaction of many components: blood vessels,        cross-linked fibrin to form D-dimers and other
platelets, coagulation factors, coagulation factor    fibrinogen fragments.
inhibitors and fibrinolytic enzymes.
   The procoagulant cascade (Fig. 11.1) is acti-
vated when tissue factor (TF) expressed on            Investigation of abnormal haemostasis
damaged or stimulated cells (vascular cells or
monocytes) comes in contact with circulating          A careful clinical history and physical examination
factor VII and VIIa (which accounts for approxi-      should be undertaken in order to differentiate
mately 1–2% of circulating plasma factor VII).        between bleeding caused by a local factor and that
This TF–factor VIIa complex activates limited         due to an underlying haemostatic defect. Contin-
quantities of factors IX and X. Newly generated       ued oozing from venepuncture and injection sites
factor IXa forms a complex with factor VIIIa (acti-   or from wound drains suggests the possibility of
vated by traces of thrombin generated slowly by       generalized haemostatic failure.
factor Xa) in the presence of calcium and mem-           Initially, some simple ‘screening’ laboratory
brane phospholipid. This complex subsequently         tests that are easy to perform and which give quick
also activates factor X to Xa, and is known as        reliable results should be undertaken (Table 11.1).


138
                                                                                                                                      Coagulation disorders


                                                                                                                        IX

                                                                                                                             XIa
                                                                  TF

                                                                                       Ca2+                             IXa
                                                     VII/VIIa
                                                                                                                     PL, Ca2+,
                                                                 TF/VIIa                                               VIIIa
                                                     Ca2+


                                                    X                                         Xa                                            X


                                                                                  PL, Ca2+         Va




                                                                        Prothrombin
                                                                                                          Thrombin


Fig. 11.1 The procoagulant pathway.
PL, phospholipid; TF, tissue factor.                                                         Fibrinogen                            Fibrin



Table 11.1 Simple laboratory haemostasis screening tests.                         In high-dependency units, the availability of
                                                                                near-patient testing devices to rapidly assess coag-
Coagulation                                                                     ulation, e.g. prothrombin time (PT) and activated
Prothrombin time (PT)
                                                                                partial thromboplastin time (APTT), and overall
Activated partial thromboplastin time (APTT)
                                                                                global haemostasis screening (thromboelas-
International normalized ratio (INR): only in patients receiving oral
                                                                                togram) allows rapid treatment decisions to be
   anticoagulation
Thrombin time (TT)                                                              made without sending a citrated sample to the
Fibrinogen assay                                                                laboratory.
                                                                                  It is important to note that in some disorders,
Platelets
                                                                                such as mild forms of haemophilia or von Wille-
Platelet count
                                                                                brand disease (vWD), ‘screening’ tests such as
Blood film inspection
Platelet function (using PFA-100, which measures in vitro ‘high shear’
                                                                                APTT may not be overly prolonged, and hence if a
   bleeding time)                                                               bleeding disorder is strongly suspected from the
                                                                                patient’s history and clinical picture, specific factor
Fibrinolysis
                                                                                assays and/or immunological tests should be per-
D-dimers
                                                                                formed regardless of the ‘screening’ test result.
Euglobulin clot lysis time

Global haemostasis
Thromboelastogram                                                               Transfusion support for patients with
                                                                                acquired haemostatic defects
                                                                                Disseminated intravascular coagulation
If one or more of these tests suggests an abnormal-
ity, then further specialized investigations (such as                           Disseminated intravascular coagulation (DIC) is a
specific coagulation factor assays) should be per-                               disorder resulting from inappropriate and exces-
formed in order to define precisely the defect and                               sive activation of the haemostatic system that can
its severity.                                                                   be manifested by both thrombotic and haemor-


                                                                                                                                                      139
Chapter 11


rhagic pathology. DIC may be acute (uncompen-            Table 11.2 Main causes of disseminated intravascular
sated), with decreased levels of haemostatic com-        coagulation.
ponents, or chronic (compensated), with normal
                                                         Infection
or sometimes elevated levels of coagulation
                                                         Septicaemia (~60% of all cases)
factors.
                                                         Viraemia
   The main triggering mechanism for DIC is the
exposure of blood to a source of TF that initiates       Malignancy
coagulation. This can occur as a result of the           Leukaemia (especially acute promyelocytic)
                                                         Metastatic carcinomas
following:
• synthesis of TF on the surface of endothelial          Obstetric disorders
cells or monocytes stimulated by endotoxins and          Septic abortion
cytokines as a result of sepsis;                         Abruptio placentae
• release or exposure of TF as a result of direct        Eclampsia
tissue injury (as in placental abruption, cerebral       Amniotic fluid embolism
                                                         Placenta praevia
trauma) or from malignant cells;
• snake venoms may cause DIC as a result of              Shock
direct activation of coagulation factors such as         Extensive surgical trauma
factor X or prothrombin.                                 Burns
   The final consequence of coagulation activation        Heat stroke
is thrombin generation and fibrin formation,              Liver disease
which may result in microthrombus formation              Cirrhosis
(e.g. gangrene of fingers or toes, renal failure). Fol-   Acute hepatic necrosis
lowing intravascular thrombosis, secondary acti-         Transplantation
vation of the fibrinolytic pathway occurs with            Tissue rejection
subsequent lysis of fibrin and the formation of
                                                         Extracorporeal circulation
cross-linked complexes such as D-dimers, which
                                                         Cardiac bypass surgery
can be detected by a number of assays. Raised
levels of these fibrin degradation products further       Extensive intravascular haemolysis
add to the bleeding diathesis as they inhibit the        ABO-incompatible transfusion
action of thrombin and also inhibit platelet func-       Certain snake bites
tion by binding to the platelet membrane.
   Due to ongoing activation of the coagulation
cascade, hepatic synthesis of coagulation factors is
unable to fully compensate for their consumption,        • reduction of fibrinogen levels, increased levels of
and so there is a reduction in levels of all coagula-    D-dimers;
tion factors, but particularly factors V, VIII and       • thrombocytopenia;
XIII and fibrinogen. The bone marrow is unable to         • anaemia, fragmented red cells, raised reticulo-
maintain a normal platelet count, and thrombocy-         cyte count.
topenia eventuates. This combination of coagula-            The most important aspect of management is
tion factor deficiency, thrombocytopenia and the          removal or alleviation of the triggering event or
inhibitory actions of raised fibrin degradation           underlying cause, as well as treatment of any asso-
products causes the generalized and continued            ciated infection, hypovolaemia, etc. Obstetric
bleeding tendency characteristic of DIC. These           emergencies should be attended to immediately.
events are summarized in Fig. 11.2. The main             In the presence of widespread bleeding, specific
causes of DIC are listed in Table 11.2.                  replacement therapy should be given, which
   Laboratory abnormalities seen in DIC include:         includes the following.
• prolonged thrombin time (TT), variably pro-            • Fresh frozen plasma (FFP): almost all procoagu-
longed PT and APTT;                                      lant factors and inhibitors are contained within


140
                                                                                                 Coagulation disorders


                                                                        Trigger factors




                                          Activation of                  Vessel wall                Platelet
                                       coagulation cascade                damage                   activation




                                                                        Fibrin–platelet             End-organ
                                                                          thrombosis                 damage



                                                                                                              Lysis
                                                                                                              and
                                                                                                             repair




                                         Coagulation factor                                       Fibrinolysis
                                                                       Thrombocytopenia
                                            deficiency                                             activation




Fig. 11.2 Pathogenesis of acute
disseminated intravascular
                                                                     Generalized bleeding            FDPs
coagulation. FDPs, fibrin degradation                                      tendency                 generated
products.


FFP, and approximately 4–5 units should be                       Specific clotting factor inhibitor concentrates
rapidly infused.                                              (including antithrombin and activated protein C)
• Cryoprecipitate: contains fibrinogen in a ‘con-              may have a role in the management of certain
centrated’ form and 5–10 units should be infused              groups of patients (e.g. those who do not respond
with the initial FFP.                                         to simple replacement therapy or who have over-
• Platelet concentrates: approximately one to two             whelming sepsis or meningococcaemia).
complete adult doses to be given.
• Following initial replacement therapy, labora-
                                                              Liver disease
tory tests should be repeated and any further treat-
ment guided by both the clinical and laboratory               All coagulation factors, except factor VIII and von
response.                                                     Willebrand factor (vWF), and protease inhibitors
   Heparin anticoagulation may also be useful in              are synthesized by hepatocytes. The liver also
situations where initial replacement therapy has              serves to remove activated intermediates of coagu-
failed to control excessive bleeding or when DIC              lation from the bloodstream. In liver disease,
is complicated by microvascular thrombosis or                 coagulopathy may result from a number of mech-
large-vessel thrombosis. Low-dose continuous                  anisms: reduced synthesis of coagulation factors;
intravenous therapy (500–1000 IU/h) is one sug-               cholestasis and subsequent malabsorption result-
gested regimen.                                               ing in vitamin K deficiency; and acquired ‘dysfib-


                                                                                                                  141
Chapter 11


rinogenaemia’. The platelet count is often reduced       Table 11.3 Conditions associated with increased risk of
due to hypersplenism.                                    bleeding during anticoagulation.
   Laboratory abnormalities seen in liver disease
                                                         Age (possible)
include the following.
                                                         Uncontrolled hypertension
• Prolonged PT and APTT.
                                                         Alcoholism
• Prolonged TT: this may result from low fibrino-         Liver disease
gen concentration or dysfibrinogenaemia. A pro-           Poor drug or clinic visit compliance
longed reptilase time despite a normal fibrinogen         Active major bleeding
concentration implies dysfibrinogenaemia.                 Previous intracranial bleeding
• Elevated D-dimers.                                     Potential bleeding lesion (e.g. aneurysm, internal ulcer)
   It is important to note that abnormal coagula-        Thrombocytopenia
tion tests are not always associated with bleeding       Platelet dysfunction (e.g. use of aspirin)
and, in such cases, patients do not require replace-
ment therapy. However if there is active bleeding,
then replacement of clotting factors with FFP and
platelet transfusions to maintain a platelet count          If the patient is bleeding, then the anticoagulant
above 50 ¥ 109/L should be instituted. Vitamin K         effect should be reversed. Since the action of
in doses of 10–20 mg may produce some improve-           vitamin K is not maximal for at least 24 h, addi-
ment in the coagulation abnormalities. Prothrom-         tional measures are required.
bin complex concentrates (which contain factors          • FFP (10–15 mL/kg body weight) will immedi-
II, IX and X) may sometimes be used in severe            ately supply the necessary coagulation factors.
cases, but if so, with great caution, as they may        However, there are some potential problems with
precipitate DIC.                                         this type of therapy. Very large amounts of plasma
                                                         (1–2 L) may need to be infused in order to correct
                                                         the coagulopathy; and even though the INR may
Complications of anticoagulant and                       correct into the normal range, this is misleading
thrombolytic drugs                                       since it is not sensitive to factor IX, the concentra-
                                                         tion of which is only minimally increased by treat-
Oral anticoagulants
                                                         ment with FFP.
Coumarin and phenindione derivatives act by              • As a result, alternative therapies with clotting
blocking the g-carboxylation of glutamic acid            factor concentrates have been used in some
residues of vitamin K-dependent coagulation              patients. These include a combination of pro-
factors, resulting in decreased biological activity of   thrombin complex concentrate (PCC), which con-
factors II, VII, IX and X, as well as proteins C and     tains variable amounts of factors II, IX and X,
S. The international normalized ratio (INR) moni-        together with specific factor VII concentrates; or
tors their effect on the haemostatic system. Some        Prothromplex T (Immuno, Vienna), which con-
clinical situations may be associated with an            tains factors II, VII, IX and X. The dose of concen-
increased risk of bleeding during anticoagulation        trate used is calculated at approximately 50 IU
and these are listed in Table 11.3.                      factor IX per kilogram body weight. These concen-
   Management of excessive anticoagulation               trates are able to correct the defect caused by oral
depends on the INR and whether there is minor or         anticoagulants; however, they carry the potential
major bleeding. In the absence of haemorrhage,           risk of inducing thromboembolism as they often
warfarin should be stopped for a few days and            contain activated coagulation components. There-
recommenced when the INR falls into the desired          fore if using these products, caution should be
range. Small doses of vitamin K (1–2.5 mg) may be        exercised especially in high-risk groups.
given intravenously/orally if the INR is greater         • Haemorrhage occurring in a warfarinized
than 5.0, as there is a significantly greater risk of     patient with an INR in the therapeutic range
serious haemorrhage at this level.                       should be managed as above; however, additional


142
                                                                                               Coagulation disorders


investigations to exclude any underlying local            also thought that plasma from uraemic patients
lesions should also be performed.                         contains an inhibitor that interferes with normal
                                                          vWF–platelet interaction.
                                                             Dialysis is useful in reversing the haemostatic
Thrombolytic agents
                                                          defects in uraemia, although this may not correct
These agents generally cause a state of systemic          them entirely. Anaemia, particularly when packed
lysis. However, the degree to which this is affected      cell volume (PCV) is below 20%, should be cor-
varies according to the particular drug used. Strep-      rected by either blood transfusion or erythropoi-
tokinase has a greater effect on the laboratory           etin as this improves platelet function and shortens
markers of systemic lysis than does tissue plas-          bleeding time. Infusions of 1-deamino-8-D-
minogen activator; however, this does not appear          arginine vasopressin (DDAVP) (0.4 mg/kg) have
to correlate with the incidence of bleeding.              been used successfully to provide short-term cor-
   Laboratory tests such as the TT and fibrinogen          rection of the bleeding time and decreased symp-
levels will detect the presence of a systemic lytic       toms of bleeding.
state, although they do not predict the likelihood
of haemorrhage, and nowadays most protocols
                                                          Massive transfusion
use fixed-dose schedules.
   Haemorrhage complicating these agents is most          This is generally defined as blood loss requiring
commonly local (e.g. at the site of catheterization       the replacement of the patient’s total blood volume
in the groin), although intracranial or gastroin-         in less than 24 h, and may be associated with coag-
testinal bleeding may occur. Measures such as             ulation abnormalities. Thrombocytopenia can
pressure packs will often control local bleeding;         occur reasonably quickly and usually results from
more serious bleeding usually necessitates discon-        dilution, but increased consumption of platelets
tinuing thrombolysis. Most agents have a short            may also occur. The use of plasma-reduced red
half-life (minutes) and so the fibrinolytic state          cell concentrates can result in significant dilution
will reverse within a few hours of drug cessation.        of coagulation factors, although generally the
The exception to this is acylated plasminogen–            haemostatic concentration of the coagulation
streptokinase activator complex (APSAC),                  factors is well maintained and products such as
which has a half-life of 90 min. In the case of           FFP should not be given prophylactically.
life-threatening haemorrhage, infusions of cryo-             In order to prevent the indiscriminate use of
precipitate or FFP can be given to reverse the            component therapy, patients receiving massive
hypocoagulable state. Antifibrinolytic drugs such          transfusions should have routine tests of
as e-aminocaproic acid may or may not provide             haemostasis performed early in order to define
some additional benefit.                                   precise abnormalities.
                                                             Microvascular bleeding and general oozing
                                                          from wounds or venepuncture sites is particularly
Uraemia
                                                          likely when the platelet count falls below
Bleeding is a relatively common complication of           50 ¥ 109/L and platelet concentrates should be
renal failure: the major cause is that of platelet dys-   infused in order to control any microvascular
function as well as a defect in platelet–vessel wall      bleeding.
interaction. Deficiencies of coagulation factors are          If laboratory tests show that a coagulopathy is
not a common feature, unless there is complicating        present, or if clinical judgement is such that treat-
liver disease or DIC.                                     ment cannot wait for results of tests, then FFP at a
   Many qualitative platelet defects can be demon-        dose of 10–15 mL/kg will replace both fibrinogen
strated in vitro, including impaired aggregation          and other critical clotting factors. If hypofibrino-
in response to agonists as well as storage pool           genaemia is present or suspected, then cryoprecipi-
defects. However, these abnormalities do not              tate should also be given. The need for ongoing
appear to correlate well with clinical bleeding. It is    haemostatic treatment should be guided by the


                                                                                                               143
Chapter 11


patient’s clinical response and results of repeated     Table 11.4 Clinical manifestations in haemophilia A.
laboratory tests.
   In recent years, recombinant factor VIIa (Novo-      Factor VIII level (% normal)   Clinical manifestation
Seven) has been used for patients with uncontrol-
                                                        <1% (severe disease)           Usual age of onset <1 year
lable life-threatening haemorrhage. This product
                                                                                       Spontaneous bleeding common
was originally developed for use in haemophilia                                           (haemarthrosis, muscle haematoma,
patients with inhibitors to factor VIII or IX;                                            haematuria)
however, a number of clinical trials for indications                                   Bleeding post surgery and dental
other than haemophilia are currently in progress.                                         extraction
Due to its expense, its use is generally limited to                                    Post-traumatic bleeding
‘rescue’ therapy for massively transfused patients                                     Crippling joint deformity if inadequate
with persistent bleeding despite appropriate blood                                        treatment
component transfusion, haemostatic and pharma-          1–5% (moderate disease)        Usual age of onset <2 years
cological measures and surgical intervention.                                          Occasional spontaneous bleeding
Experience based solely on case reports indicates a                                    Bleeding post surgery and dental
haemostatic effect of recombinant factor VIIa in                                          extraction
doses of 60–120 mg/kg; its relatively short half-life                                  Post-traumatic bleeding
means that a repeat dose may need to be given           6–40 % (mild disease)          Usual age of onset >2 years
every 2–3 h to decrease bleeding significantly. The                                     Bleeding post surgery and dental
decision to use this product should generally be                                          extraction
made in consultation with a haematologist.                                             Post-traumatic bleeding



Transfusion support for patients with
                                                        Management
inherited haemostatic defects
                                                        The mainstay of treatment is to raise the concen-
Haemophilia A
                                                        tration of factor VIII sufficiently to arrest spon-
This sex-linked recessive disorder results in           taneous and traumatic bleeds or to cover surgery.
absent/low levels of factor VIII, although it may       There are a number of products currently available
also arise from a spontaneous mutation in up to         which can be used to treat this condition,
one-third cases.                                        including:
   Clinical features vary according to the factor       • recombinant factor VIII preparations;
VIII level and patients can be classified into mild,     • plasma-derived factor VIII concentrates (which
moderate or severe according to their factor VIII       may vary in degree of purity);
coagulant activity (Table 11.4). The severity and       • DDAVP (for mild disease only, baseline factor
type of bleeding is related to the absolute level       VIII above 15%).
of factor VIII. The minimal effective level for            The choice of which product to use is generally
haemostasis is generally about 25–30%.                  determined by cost and availability. Recombinant
                                                        products are the treatment of choice if available
                                                        and affordable, because they eliminate the risk
Investigations
                                                        of transmission of human and animal infectious
Laboratory abnormalities seen in haemophilia A          agents. They are generally recommended as the
include:                                                product of choice for prophylaxis to prevent spon-
• prolonged APTT;                                       taneous joint bleeding for children with severe
• reduction of factor VIII coagulant activity; and      haemophilia, as well as replacement therapy
• normal vWF activity (it is important to measure       for previously untreated patients. Factor VIII
vWF activity in order to exclude vWD, which will        inhibitors may develop in up to one-third of
also give low factor VIII levels).                      patients receiving recombinant products.


144
                                                                                                 Coagulation disorders


   Plasma-derived clotting factor concentrates are       • prolonged APTT;
currently considered to be ‘safe’ in terms of human      • reduction of factor IX coagulant activity.
immunodeficiency virus (HIV) and hepatitis
viruses due to effective donor screening and spe-
                                                         Management
cific viral ‘killing’ procedures, and are used when
recombinant products are unavailable.                    The main types of products currently used for
   DDAVP (0.3 mg/kg) given intravenously, subcu-         treatment include:
taneously or intranasally can be used to control         • intermediate-purity factor IX complex concen-
bleeding in mild haemophiliacs. Hyponatraemia            trates (PCC);
and water intoxication are adverse effects of this       • high-purity factor IX concentrates;
drug, and hence it is not recommended for children       • recombinant factor IX products.
under 2 years of age. It is also thought to have            The choice of factor IX replacement product has
thrombogenic potential and may need to be                been influenced by the fact that PCC contains sig-
avoided in the elderly or those with known vascu-        nificant amounts of activated coagulation factors
lar disease.                                             II, VII and X, and can cause thromboembolism
   Patients with moderate/severe haemophilia will        and DIC in some patients. Highly purified factor
require treatment with recombinant or plasma-            IX concentrates (which contain very little or no
derived factor VIII concentrates for bleeding, prior     other coagulation factors) and recombinant factor
to invasive procedures, surgery, etc. It is known        IX (unique in containing no animal or human pro-
that a dose of factor VIII of 1 unit/kg will result in   teins) are also available. Although more costly,
an increase in plasma factor VIII level by 2%. The       they are less thrombogenic than PCC and are the
level of factor VIII concentrate required to achieve     products of choice for prophylactic regimens,
adequate haemostasis will depend on the type of          surgery and those patients at high risk of venous
bleeding, but can be calculated according to the         thrombosis/DIC.
formula:                                                    The dosage of factor IX required can be calcu-
                                                         lated according to the formula:
Units of factor VIII required =
weight (kg) ¥ desired level (%) ¥ 0.5                    Units of factor IX required = weight (kg) ¥ desired
                                                         level (%) ¥ 1.0
  The plasma half-life of factor VIII is 8–12 h and
thus repeated doses at 12-h intervals are usually          The plasma half-life of factor IX is 18–30 h, and
needed. Alternatively, a continuous infusion of          therefore if repeated doses are needed they should
factor VIII can be given. For major soft tissue          be given every 12–24 h or by continuous infusion.
bleeds, levels above 50% are generally sufficient;
however, for major surgery, a preoperative level
                                                         Treatment of patients with inhibitors
of 100% is necessary and thereafter levels of
50–100% are sufficient for adequate wound                 Patients with haemophilia can develop inhibitor
healing.                                                 antibodies to factor VIII or IX, sometimes making
                                                         their treatment quite difficult. Inhibitors are most
                                                         common in patients with severe haemophilia A.
Haemophilia B
This sex-linked recessive disorder results in a defi-
                                                         Haemophilia A
ciency of factor IX. The clinical features are identi-
cal to those of haemophilia A.                           • If the inhibitor is of low titre (i.e. <10 Bethesda
                                                         units), then bleeding episodes can be treated with
                                                         higher than normal doses of human factor VIII or
Investigations
                                                         porcine factor VIII (based on the species specificity
Laboratory abnormalities seen in haemophilia B           of factor VIII inhibitors).
include:                                                 • If the inhibitor is of high titre (i.e. >10 Bethesda


                                                                                                                 145
Chapter 11


units), human factor VIII is ineffective to control     Table 11.5 Variants of von Willebrand disease.
bleeding, and management strategies include use
of porcine factor VIII, recombinant factor VIIa,        Type 1
                                                        Autosomal dominant inheritance
FEIBA or PCC. For major haemorrhage, recom-
                                                        Partial quantitative deficiency of von Willebrand factor (vWF)
binant factor VIIa (70–90 mg/kg) is generally re-
                                                        Normal vWF multimers
commended as first-line therapy (if available).          Mild bleeding disorder that decreases during pregnancy, elderly
Eradication of inhibitors with ‘immune tolerance
induction’ using factor VIII concentrates alone or      Type 2
together with immunosuppressives is considered          Autosomal dominant inheritance
                                                        Qualitative deficiency of vWF
the best long-term treatment option for these
                                                        Numerous subtypes
patients.
                                                        Abnormal vWF multimers
                                                        Generally mild bleeding disorder
Haemophilia B                                           Type 3
                                                        Autosomal recessive inheritance
• Immune tolerance using factor IX concentrates
                                                        Severe quantitative deficiency of vWF
can be attempted, otherwise PCC or recombinant
                                                        Severe haemophilia-like bleeding disorder
factor VIIa can be used for bleeding.


von Willebrand disease
                                                        treatment of choice and a dose of 0.3 mg/kg is
This is the most common of the inherited bleeding       usually given. It is important to first test an individ-
disorders and is due to a quantitative and/or quali-    ual patient’s response to DDAVP prior to using it
tative defect in the vWF protein. vWF has two           to ‘cover’ procedures.
main functions: firstly, it promotes the adhesion of     • For patients with types 2 and 3 disease, vWF
platelets to the subendothelium by binding to the       ‘replacement therapy’ is generally required. Either
platelet receptor glycoprotein Ib; secondly it pro-     a factor VIII concentrate rich in vWF or a purified
tects factor VIII:c from proteolytic degradation by     vWF concentrate is the treatment of choice. One
forming a non-covalent association.                     problem with factor VIII concentrates is that they
   vWD is a heterogeneous group of disorders and        rarely contain the highest molecular weight multi-
is classified into three different types (Table 11.5).   mers of vWF, which are very important for ade-
Depending on the type of vWD, some patients may         quate haemostasis. Similarly, the very low content
be asymptomatic whereas others will have                of factor VIII in purified vWF concentrates may
haemophilia-like bleeding.                              make it necessary (in the event of acute bleeding or
   Laboratory abnormalities seen in vWD include:        emergency surgery) to infuse vWD patients with a
• prolonged PFA-100 closure time (an in vitro           single first dose of purified factor VIII concentrate
‘high shear’ bleeding time device);                     to ensure immediate correction of the low factor
• reduction of vWF antigen;                             VIII levels.
• reduction of vWF ristocetin cofactor activity;           In the past, cryoprecipitate was used to treat
• reduction of factor VIII coagulant activity           patients with vWD; however, it is now unaccept-
(which can cause prolonged APTT);                       able to use such untreated plasma derivatives when
• abnormal vWF multimers in some subtypes.              there are ‘safer’ alternatives available.
   The goal of therapy in patients with vWD is to
correct the dual defect of haemostasis, i.e. the
                                                        Other inherited disorders
abnormal platelet adhesion and the abnormal
coagulation due to low fctor VIII levels. It is         Hereditary deficiencies of other coagulation
important to distinguish between the various types      factors are generally rare. Factor XI deficiency is
of vWD as treatment will differ.                        particularly common among Ashkenazi Jews
• For patients with type 1 disease, DDAVP is the        and is transmitted as an autosomal recessive trait.


146
                                                                                           Coagulation disorders


There is a poor correlation between factor XI          emergency when the patient’s blood group is not
levels and bleeding tendency, which usually pre-       known, group AB plasma may be safely given.
sents following surgery or dental procedures. If       RhD-compatible plasma should be given to
available, plasma-derived factor XI concentrates       women of child-bearing age since FFP contains
should be given to treat bleeding, otherwise FFP       small amounts of red cell stroma, which can boost
will suffice.                                           the response to RhD in previously immunized
   Deficiencies of factors II, V, VII, X and XIII and   patients.
fibrinogen can all be treated with FFP; however, if        Single-unit FFP is not a virally inactivated
there are more specific therapies available then        product. Other alternatives include quarantined
they should be used. Currently, there are specific      FFP, methylene blue-treated single unit, or
factor concentrates for factors VII, X and XIII        solvent–detergent (SD)-treated pooled plasma.
and fibrinogen, although these may not always be        All these products have been shown to be clinically
available. Cryoprecipitate can be used for fibrino-     effective, although SD plasma lacks the high-
gen deficiency/dysfibrinogenaemias; PCC can be           molecular-weight forms of vWF. In the UK,
given to patients with factor II or X deficiency        virally inactivated FFP from bovine spongiform
(although thromboembolic risks should be consid-       encephalopathy (BSE)-free countries is recom-
ered).                                                 mended by the Department of Health for children
   Patients with deficiencies of ‘contact factors’      born after 1 January 1996; the viral inactivation
(factor XII, prekallikrein, and high-molecular-        will be carried out by methylene blue treatment
weight kininogen) do not bleed excessively and do      method. Virus-inactivated FFP sourced from
not require any treatment.                             non-UK untransfused male donors is the safest
                                                       product for the UK to avoid the risks of
                                                       transfusion-related acute lung injury (see Chapter
Appropriate and inappropriate use of FFP,              14) and variant Creutzfeldt–Jakob disease (see
cryoprecipitate and coagulation                        Chapter 20). There are difficulties in establish-
factor concentrates                                    ing specific criteria to determine which patient
                                                       groups should receive the safest available FFP.
Fresh frozen plama
                                                       Although extending the use of virus-inactivated
FFP is obtained from either single donations or        FFP sourced from non-UK donors to all recipients
plasmapheresis collections and is prepared by          remains under consideration, the main constraint
freezing plasma to a temperature of –30°C or less      is cost.
within 6 h of collection in order to preserve the         While the use of FFP has become widespread, it
activity of coagulation factors. Each unit contains    may be transfused inappropriately. There are a
all of the coagulation factors and has a volume of     limited number of ‘definite’ indications for the use
approximately 200 mL. When ready for use it            of FFP and these are described below.
must be thawed at 37°C and — ideally it should be
infused within 2 h of thawing. It can be stored at
                                                       ‘Definite’ indications
4°C for up to 24 h as long as factor VIII replace-
ment is not required.                                  Immediate reversal of overdosing with
   The dose of FFP required will depend on the         oral anticoagulant
clinical indication; however, an initial dose of       In the event of serious or life-threatening bleeding
10–15 mL/kg is usually given. As this is an empiri-    requiring immediate reversal of the anticoagulant
cal dose, laboratory tests should be used to           effect, PCC is regarded as the treatment of choice.
monitor its efficacy, and results of these tests as     PCC does not usually contain factor VII, and thus
well as the patient’s clinical response should guide   a separate factor VII concentrate should be given
any further dosing requirements.                       (if available). The recommended dose is 50 IU
   ABO-compatible FFP should be used, although         factor IX activity per kilogram body weight. PCC
compatibility testing is not required. In case of an   is potentially thrombogenic and this should be


                                                                                                           147
Chapter 11


borne in mind if used. FFP is an alternative if PCC    opment of venous thromboembolism in patients
is not available, using a dose of 10–15 mL/kg.         with TTP.
Further doses of PCC or FFP should be given
according to laboratory results and patient’s clini-   Replacement of single factor deficiencies
cal state.                                             As more specific factor concentrates become
                                                       increasingly available, FFP should only be used as
Vitamin K deficiency                                    replacement when specific/combined factor con-
Conditions that impair vitamin K absorption (e.g.      centrates are unavailable. Currently there are no
biliary tract obstruction) as well as haemorrhagic     specific factor concentrates available for deficien-
disease of the newborn can result in a coagulopa-      cies of factors II (prothrombin) and V.
thy similar to that seen with warfarin overdosage.
Any serious or life-threatening bleeding should be     Inherited deficiencies of inhibitors of coagulation
treated in the same manner as described above.         Previously, FFP has been used as a source of
                                                       antithrombin, protein C and protein S for patients
Acute DIC                                              with inherited deficiencies of these inhibitors who
In acute DIC, activation of the coagulation and fib-    may be receiving heparin therapy for spontaneous
rinolytic systems results in depletion of platelets    thrombosis or who are undergoing surgery. Now
and coagulation factors (especially factors V and      that specific concentrates are being manufactured
VIII and fibrinogen). Treatment is always aimed at      (antithrombin and protein C), FFP should only be
removing the underlying cause. In cases of haem-       used when these are not available.
orrhage associated with laboratory abnormalities,
replacement therapy with FFP, cryoprecipitate and
                                                       Other indications
platelet concentrates is indicated. If there is no
bleeding, then replacement therapy should not be       There are several other circumstances where FFP
given in an attempt to correct the coagulopathy.       may be used appropriately to treat bleeding in the
                                                       presence of abnormal coagulation.
Thrombotic thrombocytopenic purpura
FFP in conjunction with plasma exchange has been       Liver disease
the treatment of choice for patients with throm-       Coagulation abnormalities occur quite frequently
botic thrombocytopenic purpura (TTP). Large            in patients with severe liver disease; however,
daily doses are needed, usually in the order of 3 L.   bleeding is often precipitated by an event such as
In some series, the supernatant portion formed         surgery or liver biopsy, and is rarely attributable to
during the preparation of cryoprecipitate (cryo-       the haemostatic defect alone. If there is bleeding
supernatant) has been shown to be more effective       (or a very strong possibility that bleeding will
than standard FFP when used as the replacement         occur), then FFP is indicated. Large volumes are
fluid. Cryosupernatant plasma is depleted in factor     often required to control the bleeding/correct the
VIII and fibrinogen; but whereas the factor VIII        defect, and this can be problematic in patients who
concentration may only be about 0.11 IU/mL,            may already have an expanded plasma volume.
proportionately less fibrinogen may be removed,         Complete normalization of a prolonged PT is often
leaving up to 70% remaining. All forms of FFP          not possible, and the use of PCC may be consid-
contain the metalloproteinase enzyme, which is         ered. However, one must be aware of the potential
deficient or inhibited in TTP and is responsible for    risks of inducing thrombosis or DIC in these
degrading the ultra-large multimers of vWF that        patients, particularly since they already suffer
cause the excessive platelet activation and con-       from impaired clearance of activated clotting
sumption in this condition. Cryosupernatant is         factors and reduced levels of antithrombin. Since
deficient in vWF multimers but contains metallo-        thrombocytopenia and platelet function defects
proteinase. The reduced activity of protein S in       are also a feature of hepatic disease, platelet con-
SD-treated FFP has been associated with the devel-     centrates may also need to be given.


148
                                                                                               Coagulation disorders


Massive transfusion
                                                          Cryoprecipitate
This is defined as the replacement of a patient’s
total blood volume within 24 h. Coagulation               Cryoprecipitate is prepared from plasma that is
abnormalities seen in this setting are more closely       frozen quickly and then thawed slowly at 1–6°C,
related to the clinical condition necessitating the       leaving behind a small amount of precipitated
transfusion rather than the volume of blood trans-        protein. The cryoprecipitate protein is then resus-
fused. Prophylactic ‘replacement’ regimens with           pended in a small volume of residual supernatant
FFP are not indicated. Instead, treatment with            plasma (usually 9–16 mL). It contains factor VIII,
both FFP and cryoprecipitate should be guided by          fibrinogen, vWF, factor XIII and fibronectin in
the patient’s clinical status as well as the results of   higher concentrations than are found in plasma.
laboratory tests (if available). Thrombocytopenia            The main use for cryoprecipitate is as a source of
is a frequent occurrence and should be treated with       fibrinogen and a single unit usually contains
platelet concentrates if necessary (usually when          200–250 mg fibrinogen. It can be used appropri-
the platelet count falls below 50 ¥ 109/L). More          ately in cases of hypofibrinogenaemia such as
recently, recombinant factor VIIa (NovoSeven) has         DIC, reversal of fibrinolytic agents and advanced
been used for massively transfused patients with          liver disease. Plasma levels of fibrinogen above
persistent bleeding despite appropriate blood com-        1.0 g/L are generally considered as adequate for
ponent transfusion, haemostatic and pharmaco-             haemostasis. When using cryoprecipitate, it is
logical measures, and surgical intervention.              usual to thaw and pool approximately 10–30
                                                          donor units for infusion; however, the dose will
Cardiopulmonary bypass                                    vary according to the clinical condition and the
Haemostatic disturbances during cardiopulmon-             patient’s fibrinogen level. An empirical rule is to
ary bypass are usually due to platelet dysfunction.       give one bag of cryoprecipitate for every 5 kg of
If there is persistent bleeding (despite adequate         body weight.
platelet transfusion) and a coagulopathy other               In the past, cryoprecipitate was used to treat
than that caused by heparin has been demon-               vWD and haemophilia A; however, with the
strated, then FFP should be used.                         advent of drugs such as DDAVP and the avail-
                                                          ability of specific factor concentrates, it should
Special paediatric conditions                             no longer be used for the treatment of these
FFP can be used in the treatment of neonatal DIC,         disorders.
and is also sometimes used in newborn/premature              Cryoprecipitate has more recently been used for
infants with severe sepsis, although FFP should be        the production of fibrin surgical adhesive (‘glue’),
avoided in neonates with T-antigen activation (see        which can be used in various surgical procedures
Chapter 8). Special paediatric packs of FFP that          and can be prepared from autologous donors.
contain smaller volumes than standard FFP should
be used.
                                                          Coagulation factor concentrates
                                                          Both specific and non-specific coagulation factor
Inappropriate use
                                                          concentrates are prepared from plasma using a
The use of FFP is inappropriate in the following          number of different techniques in an attempt to
conditions:                                               produce a ‘purified’ product that has undergone
• hypovolaemia;                                           viral inactivation.
• routine plasma exchange;                                   Specific factor concentrates for many of the
• part of a ‘predetermined’ replacement protocol;         coagulation factors have now been developed and
• treatment of immunodeficiency or protein-                are in clinical use as replacement therapy for inher-
losing states.                                            ited deficiencies. Their use has been discussed pre-
                                                          viously in the treatment of inherited haemostatic
                                                          defects.


                                                                                                               149
Chapter 11


   Non-specific factor concentrates such as PCC          Further reading
generally contain factors II, IX and X together
with variable amounts of factor VII. They are gen-      Bolton-Maggs PHB, Pasi KJ. Haemophilias A and B. Lancet
erally used in conditions associated with deficien-        2003; 361: 1801–9.
cies of one or more of these factors (e.g. treatment    British Committee for Standards in Haematology.
                                                          Guidelines for the use of fresh frozen plasma. Transfus
of overdosage with warfarin); however, since they
                                                          Med 1992; 2: 57–63 (revision in preparation, see
often contain ‘activated’ forms of coagulation
                                                          www.bcshguidelines.org).
factors, they have thrombogenic potential, which        Davis S. Use of factor VIIa in uncontrollable haemorrhage.
can limit their use.                                      A position statement of the NSW Therapeutic
                                                          Assessment Group Inc., October 2002 (see
                                                          www.nswtag.org.au).
Summary                                                 Machin SJ. Acquired coagulation, non-immune platelet
                                                          disorders and vascular purpuras. In: Hoffbrand AV,
                                                          Lewis SM, Tuddenham EGD, eds. Postgraduate
Coagulation disturbances are relatively common
                                                          Haematology. Oxford: Butterworth-Heinemann, 1999:
and it is important to know when to treat as well as      636–52.
the most appropriate therapy. Simple laboratory         Makris M, Watson HG. The management of coumarin-
tests will often give an indication of the problem        induced over-anticoagulation Br J Haematol 2001; 114:
and more specific tests will indicate the exact diag-      271–80.
nosis.                                                  Makris M, Greaves M, Phillips WS et al. Emergency oral
                                                          anticoagulant reversal: the relative efficacy of infusions of
   With the widespread availability of plasma and
                                                          fresh frozen plasma and clotting factor concentrates on
factor concentrates, it is important for individual
                                                          correction of the coagulopathy. Thromb Haemost 1997;
hospital centres to set up guidelines in order to         77: 477–80.
minimize wastage and inappropriate use of these         Practice guidelines for blood component therapy. A report
products. Cost is another important issue that            by the American Society of Anesthesiologists Task Force
needs to be addressed when drafting guidelines.           on Blood Component Therapy. Anesthesiology 1996; 84:
   Wherever possible, specific factor concentrates         732–47.
                                                        Smith OP, Hann IM, Machin SJ. The use of factor
should be used to treat coagulation factor deficien-
                                                          concentrates in the management of hemophilia A and
cies in preference to plasma.
                                                          other inherited coagulation disorders. In: Pamphilon D,
   Although advances in viral inactivation proce-         ed. Modern Transfusion Medicine. Boca Raton, FL: CRC
dures have resulted in the development of factor          Press, 1995: 165–76.
concentrates that are ‘safe’, it is important to        Wallington TB. Transfusion of plasma and its products. In:
remember that there are potential risks associated        Pamphilon D, ed. Modern Transfusion Medicine. Boca
with the use of these products and that not all inac-     Raton, FL: CRC Press, 1995: 139–64.
tivation procedures inactivate all known viruses.
Recombinant technology allows the provision of
safe and pure treatment, but does not effect cure of
the underlying condition.




150
Chapter 12
Uses of intravenous immunoglobulin

David J. Unsworth and Tim B. Wallington




The potential list of clinical indications for intra-   tend not to be associated with recurrent bacterial
venous immunoglobulin (IVIG) is expanding               infection, and do not merit IVIG treatment.
rapidly. Evidence justifying the use of IVIG in            Deficiency may be due to a primary/inherited
many clinical situations (e.g. systemic lupus ery-      defect where IgG deficiency is an irreversible
thematosus, IgA nephropathy, chronic fatigue            feature. The commonest primary immunodefi-
syndrome, asthma, multiple sclerosis and others) is     ciency which features IgG deficiency and suscepti-
contentious and based on poorly designed, small         bility to bacterial infection is common variable
or uncontrolled non-blinded clinical studies. There     immunodeficiency (CVID). Sometimes there is
are also concerns that demand will outstrip supply,     an obvious associated T-cell defect. CVID is an
since production depends on the availability of         umbrella term, probably encompassing several dif-
safe human plasma, from which normal IgG is             ferent lymphoid defects.
fractionated.                                              Alternatively, IgG deficiency may arise secon-
   Hepatitis C outbreaks associated with IVIG in        darily to a reversible condition. For example,
the recent past remind us that (as with cellular        marrow-toxic drugs, and other drugs including
blood products) transmission of infection is a          gold, sulphasalazine and certain anticonvulsants,
major potential adverse effect. This is not surpris-    can affect immunoglobulin synthesis. Immuno-
ing given that IVIG production depends on               globulin deficiency may also be a feature of
pooling of plasma immunoglobulin from several           lymphoproliferative disorders such as myeloma
thousand blood donors. One gram of IVIG is esti-        and chronic lymphocytic leukaemia (CLL). Recov-
mated to contain at least 7 million different anti-     ery may occur, for example once a particular drug
body specificities. One simple and expedient way         is withdrawn, but it may take several years.
of reducing the risk of viral transmission and other    Licensed uses include primary immunodeficien-
adverse effects is to restrict use of IVIG to those     cies, recent bone marrow transplantation,
clinical situations where use is of proven value.       myeloma or CLL, where documentated low IgG is
This chapter is limited to those diseases for which     associated with increased frequency and severity
the published evidence supporting efficacy is scien-     of bacterial infection.
tifically compelling (Table 12.1).                          Most cases of IgG deficiency present with global
                                                        or ‘pan-hypogammaglobulinaemia’. It is the per-
                                                        sistently low IgG levels rather than deficiency in
Clinical indications for IVIG                           IgA or IgM which, over a period of years, leaves
                                                        patients particularly vulnerable to recurrent bac-
Antibody-deficient patients
                                                        terial (and some viral) infections. Haemophilus
The only cases that merit consideration for treat-      influenzae and Streptococcus pneumoniae are fre-
ment are those with documented IgG deficiency            quently grown from sputum of IgG-deficient cases.
and a clear associated increased vulnerability to       Untreated, the majority will develop bronchiecta-
infection. Cases of isolated IgA or IgM deficiency       sis. In children, frequent bilateral ear infections



                                                                                                       151
Chapter 12

Table 12.1 Clinical indications for IVIG.                              IgG deficiency is used according to body weight,
                                                                       at a dose of 0.2–0.4 g/kg per month. Infused IgG
Prophylaxis against bacterial infection                                is consumed or catabolized, with a half-life of
Primary immunodeficiency states with IgG deficiency, e.g. Bruton’s
                                                                       arround 20 days, so that repeat infusions every
   hypogammaglobulinaemia and SCID
                                                                       2–4 weeks are required. Treatment will be lifelong
Some secondary immunodeficiency states (see text, includes paediatric
   HIV, CLL, myeloma)
                                                                       or until the associated underlying condition
Post bone marrow transplantation                                       responsible for IgG deficiency is successfully
                                                                       treated.
Immunomodulation of autoimmune conditions                                 Commercially available IVIG preparations are
Idiopathic thrombocytopenia purpura and certain other autoantibody-
                                                                       largely (>95%) composed of IgG. Repeat intra-
   mediated cytopenias, including post-transfusion purpura* and
                                                                       venous infusions will keep trough levels of plasma
   neonatal cytopenias
Kawasaki vasculitis*
                                                                       IgG above the lower limit of normal (>6 g/L) and at
Guillain–Barré syndrome* and some cases of chronic inflammatory         a level sufficient to reduce vulnerability to infec-
   demyelinating polyneuropathy                                        tion, usually around 8 g/L in our practice. The
Dermatomyositis                                                        exact dose and intervals between infusions can be
Myasthenia gravis                                                      determined by serial measurement of trough levels
                                                                       and appropriate adjustments to the treatment pro-
* Recommended first-line treatment without need to first trial pred-     tocol. It is important to remember that individual
nisolone/other treatments.                                             cases differ in their requirements, and perhaps the
CLL, chronic lymphocytic leukaemia; HIV, human immunodeficiency         most important criterion is the minimum dose
virus; SCID, severe combined immunodeficiency.
                                                                       which achieves clear clinical benefit. This may
                                                                       equate to a trough level considerably higher (or
with need for grommets is typical. Permanent                           lower) than 8 g/L. In cases of deficiency secondary
hearing impairment is a risk. Failure to thrive and                    to reversible causes (e.g. drug-induced marrow
weight loss (cachexia in adults) are seen after a                      dysfunction), the requirement may persist for
long history of recurrent infection. Parasitic infec-                  many months or years but should be regularly
tions are also seen. Giardia lamblia and certain                       reassessed and infusions stopped if endogenous
other gastrointestinal infections may lead to diar-                    B-lymphocyte function recovers. IgG deficiency
rhoea and even malabsorption. Patients also fre-                       related to protein leakage syndromes, such as
quently complain of a tendency to a productive                         nephrotic syndrome, should only exceptionally be
cough and perennial nasal blockage secondary to                        treated with IVIG, as the infused IVIG is likely to
bacterial rhinitis.                                                    be wasted.
   In some rarer cases of adult-onset antibody defi-
ciency, there may be an associated thymoma,
                                                                       Immunomodulation of autoimmune disease
which can precede or postdate the diagnosis of IgG
deficiency. In some IgG-deficient patients, lym-                         Serendipitous observations in a case of hypogam-
phadenopathy and/or hepatosplenomegaly is seen                         maglobulinaemia that happened to be associated
as a complication of IgG deficiency. Histology may                      with idiopathic thrombocytopenic pupura (ITP)
show widespread granulomatous inflammation.                             led to the realization that IVIG can have
   The prognosis is much improved if IgG is                            immunomodulatory effects. Within 24 h of IVIG
replaced passively by parenteral infusion. Replace-                    infusion in ITP, platelet numbers typically rise. The
ment can be either intravenously or by subcuta-                        degree of response varies from patient to patient
neous infusion. Both are effective in reducing                         but, in the majority, a beneficial elevation of the
infection rates. Experience is greatest with IVIG                      platelet number is seen. This can be short-lived
infusions repeated every 2–4 weeks, rather than                        (days to weeks) but is often sufficient to prevent or
the subcutaneous alternative, which is required                        reverse serious bleeding. The popular explanation
weekly to maintain adequate plasma IgG levels.                         for this effect is that infused IgG directly blocks the
   IVIG as microbiological prophylaxis in cases of                     antiplatelet autoantibody. The possible modes of


152
                                                                                                     Intravenous immunoglobulin


action of IVIG as an immunomodulator are dis-                         disease and GBS. Reputable clinical trials in GBS,
cussed below.                                                         comparing plasma exchange with IVIG, clearly
   The commonly used dose schedule is 0.4 g/kg for                    show that both treatment modalities provide
five successive days, mainly because this is effective                 impressive and comparable therapeutic effects (see
in the prototype condition — ITP. This represents                     Chapter 29). The financial costs of these two treat-
high-dose treatment when compared with the                            ments are similar but IVIG is easier to organize,
lower monthly doses used in cases of immunodefi-                       especially out of hours and remote from specialist
ciency. Many haematologists now use a single dose                     regional centres. IVIG has now replaced plasma
of 1 g/kg over 24 h, and this is as effective as the 5-               exchange as the favoured treatment in GBS. As a
day treatment in most cases. The main problem is                      generalization, conditions that respond to plasma
that beneficial effects in autoimmune disease are                      exchange tend also to respond to IVIG; this
short-lived and repeat courses are often required.                    includes myasthenia gravis. Chronic inflammatory
Also, many of the adverse effects occur more fre-                     demyelinating polyneuropathy is essentially a
quently with higher doses (Table 12.2). Little                        relapsing/remitting GBS-type illness. Patients may
research has been directed at determining                             respond to IVIG or plasma exchange and some-
minimum effective dose schedules.                                     times one treatment modality works best in a par-
   There are a number of autoimmune diseases                          ticular patient. A simplistic general overview
where high-dose IVIG therapy is widely accepted,                      would suggest that plasma exchange removes
although it is regarded as first-line therapy only in                  autoantibodies or other pathogenetic plasma
a minority (see Table 12.1). Guillain–Barré syn-                      factors, while IVIG blocks the same factors.
drome (GBS) (see Chapter 29), Kawasaki vasculi-
tis and post-transfusion purpura (see Chapter 17)
                                                                      IVIG in transplantation
are the major examples of where first-line use of
IVIG is generally agreed. Although the evidence                       IVIG is predictably useful as bacterial prophylaxis,
is compelling, not all preparations are licensed for                  and is used for that reason after bone marrow
all these indications, in all countries. Licensed                     transplantation. Studies in this context have also
immunomodulatory uses include ITP, Kawasaki                           shown reduced risk of cytomegalovirus infection
                                                                      and beneficial effect on graft-versus-host disease.
Table 12.2 Adverse effects of IVIG.                                   IVIG has also been beneficial before renal allograft
                                                                      transplantation, employing IVIG to block high
                                   Treatment                          titres of anti-HLA antibody capable of causing
                                                                      hyperacute antibody-mediated rejection.
Common
Hypersensitivity reactions         Slow down infusion or stop
   (urticarial rashes, wheeze,        antihistamine ± steroid as      Risks and adverse effects
   other)                             treatment or prophylaxis
Fevers/chills in septic patients   Ideally treat with antibiotics     Viral transmission
                                      and defer IVIG
                                                                      Safety concerns now dictate that all plasma dona-
Uncommon                                                              tions processed for IVIG production are tested for
Transmission of viral agents       May be ‘brand’ specific             human immunodeficiency virus (HIV)-1 and HIV-
Renal tubular damage*              Consider switching to              2 and hepatitis B and C antibodies. Although this
                                     different brand/product
                                                                      helps by identifying potentially infected donors, it
Aseptic meningitis*
                                                                      is disadvantageous because removal of antiviral
Cerebrovascular accident
Transmission of autoantibody       Stop infusion, report to
                                                                      (neutralizing) antibody at the same time makes
   in IVIG (haemolytic                manufacturer. Check other       viral transmission (from a donor before serocon-
   anaemia, other)                    patients receiving same batch   version for example) more likely. The hepatitis C
                                                                      outbreak in 1994 in the USA seemed to arise for
* Seen with high-dose IVIG.                                           exactly that reason, with 100 IVIG recipients


                                                                                                                          153
Chapter 12


wordwide developing clinically significant hepati-                             extracting IgG from plasma, inactivates HIV and
tis C transmission. Polymerase chain reaction                                 markedly reduces hepatitis B load.
(PCR) testing of plasma pools for hepatitis C and
other agents has therefore become standard
                                                                              Prion transmission
practice.
   Different virucidal strategies are additionally                            Outbreaks of variant Creutzfeldt–Jakob disease
used by the manufacturers to provide further con-                             (vCJD) in the UK in the 1990s led to concerns that
fidence (Table 12.3). The Food and Drug Adminis-                               the prion responsible might be transmissible by
tration in the USA requires manufacturers to                                  blood or blood products (see Chapter 20). Con-
employ model viruses to demonstrate effectiveness                             cerns were such that use of UK plasma in manufac-
of their viricidal strategies in vitro. Nonetheless,                          ture was banned, and UK producers purchased
vigilance is still required as aplastic anaemia due to                        North American plasma instead. Recently,
transmitted parvovirus B19 infection has been                                 however, occasional cases of bovine spongiform
reported. This virus is unaffected by solvent–deter-                          encephalopathy have been identified in the USA.
gent treatment, and can partially resist heat treat-                          Follow-up of patients in the UK who received
ment. Despite use of HIV-contaminated plasma in                               blood from donors who subsequently died of
the early 1980s, HIV has never been transmitted                               vCJD has provided evidence that transmission
by IVIG. Cold ethanol fractionation, the basis of                             may occur by red cell transfusion. Fractionation

                                                                                                     Table 12.3 Virus precautions in
Commercial preparations:              1          2         3       4             5             6     different commercial preparations.

Antibody tests on each donation
HAV                                   N          N         —       Y             —             Y
HBV                                   Y          Y         Y       Y             Y             Y
HCV                                   Y          Y         Y       Y             Y             Y
HIV                                   Y          Y         Y       Y             Y             Y
ALT/AST                               Y          Y         Y       Y             Y             Y
Other                                 —          —         —       Syphilis      HIV p24       —

PCR tests on donor minipools
HCV                                   Y          Y         Y       Y             —             —
HIV                                   Y          N         N       N             —             —
Other                                 HBV        HAV       —       —             —             —

Virus-inactivating steps
Cold/ethanol                          Y          Y         Y       Y             Y             Y
Solvent–detergent                     Y          —         —       Y             Y             —
Ion-exchange chromatography           Y          —         —       —             —             —
Incubation at low pH                  Y          —         —       Y             —             —
pH4/pepsin                            —          Y         —       —             —             Y
Pasteurization                        —          —         Y       —             —             —
PEG precipitation                     —          —         Y       —             —             —
DEAE-Sephadex chromatography          Y          —         Y       —             —             —

Plasma source
USA                                   Y          Y         Y       Y             Y             Y
EC (non-UK)                           —          —         —       Y             Y             Y

ALT/AST, alanine aminotransferase/aspartate aminotransferase; HAV, hepatitis A virus; HBV, hepati-
tis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus.




154
                                                                                        Intravenous immunoglobulin


models, using prion-spiked plasma, suggest that         documented pre-existing renal dysfunction and is
prion if present will be removed by most routine        therefore a potential pitfall of considering IVIG
IVIG manufacturing processes.                           treatment in myeloma patients.


Hypersensitivity reactions                              Aseptic meningitis
Cutaneous itching or wheezing occurring during          This is also seen generally with high-dose IVIG and
or soon after infusion occurs in up to 5% of            often in cases with a history of migraine. Patients
patients, although these reactions tend to be mild.     report very severe meningitic symptoms, typically
Slow infusion rates (0.01 mL/kg per min or less)        48 h after infusion. Cerebrospinal fluid analysis
tend to minimize such reactions. Severe reactions       shows high protein and white cells (neutrophils
might be expected in IgA-deficient individuals,          and lymphocytes). Switching to a different IVIG
because they are at risk of generating anti-IgA anti-   preparation often fails to resolve the problem.
bodies against the low levels of contaminating IgA      Very slow infusion rates sometimes help.
found within certain IVIG preparations. Fortu-
nately, however, the problem is very rarely seen in
                                                        Hyperviscosity/cerebrovascular accident
practice and not all experts agree that routine pre-
treatment screening for IgA deficiency is worth-         This problem is fortunately rare. It is reduced by
while. Where severe IgA deficiency does exist, it        slow infusion and the risk increases with higher
seems prudent to select an IVIG product with a          doses. Caution is sensible in arteriopathic patients.
very low (<0.005 mg/mL) content. Where an ana-          Deep vein thrombosis has also been reported.
phylactoid reaction (see Chapter 15) occurs in
association with IVIG administration the infusions
                                                        IVIG-containing ‘autoantibodies’ or haemolysins
should be stopped and, after appropriate resuscita-
tion, investigations undertaken to check for anti-      Uveitis due to antineutrophil cytoplasmic antibody
IgA antibodies.                                         and haemolysis due to high-titre ABO antibodies
   Septic patients who have been given IVIG may         have been the subject of case reports. ABO cross-
well develop feelings of severe malaise, with           matching of IVIG preparations against the
shivers and chills. This problem is well recognized     patient’s red cells is not however required in
in antibody-deficient patients. Treatment should, if     routine practice.
possible, be deferred until antibiotics have reduced
the bacterial load in these circumstances. Similar
symptoms can arise if complement-activating IgG         Possible immunomodulatory mechanisms
aggregates in IVIG preparations are infused, even       of action
in the absence of sepsis. This arises, for example,
when lyophilized IVIG preparations are reconsti-        The popular theories relating to mode of action of
tuted inefficiently. Both these types of reaction are    IVIG in suppressing certain autoimmune diseases
thought to involve immune complex formation             are summarized in Table 12.4. Certain hypotheses
with complement activation.                             are more suited to explaining the beneficial effect
                                                        in one disease, while being less plausible in
                                                        another. For example, while IgG Fc receptor block-
Renal dysfunction
                                                        ade is a good explanation in ITP, it does not ade-
High-dose treatments may cause acute oliguric           quately explain the mode of action in myasthenia
renal failure because sugars used to stabilize the      gravis, where the autoantibody targets the acetyl-
IgG present a high solute load, which damages the       choline receptors at nerve endings. An anti-idio-
proximal renal tubule. The defect is not always         type block is the more likely explanation in
reversible. It is most likely to develop in cases of    myasthenia gravis. Of interest, in ITP for example,



                                                                                                             155
Chapter 12

Table 12.4 Immunomodulation and IVIG: possible modes
of action.                                             Therapeutic benefit
                                                       Different products are assumed to show compara-
Fc receptor blockade
                                                       ble clinical efficacy, though trials have not been
IgG anti-idiotype effect
                                                       carried out. There is no evidence to the contrary.
Interference in immune complex clearance
Direct effect on lymphoid cells (T, B or NK)
Cytokine block                                         Levels of contaminating IgA
Enhanced catabolism of autologous IgG
Block of complement activation                         No product is entirely IgA free, but some products
                                                       have very low levels indeed (<0.005 mg/mL) and
                                                       are therefore preferred for truly IgA-deficient
                                                       patients.
infusion of anti-D can be effective, supporting the
hypothesis of Fc blockade in ITP.
  Although IVIG is predominantly composed of           General basic measures prior to IVIG use
IgG, it also contains contaminating factors that
might play a crucial immunomodulatary role.            • Baseline renal and liver biochemistry and a full
These incude soluble HLA molecules, soluble            blood count.
cytokine receptors capable of specific cytokine         • Storing serum and whole ethylenediamine tetra-
block, soluble CD4 molecules and others.               acetic acid (EDTA) samples before treatment for
  One key observation is that in diseases with an      future baseline comparison may be useful in
immunomodulatory action beyond dispute, the            patients receiving lifelong repeated IVIG (i.e.
benefit is seen typically within hours or a few days.   primary immunodeficiency states) to provide an
Direct and/or indirect effects of IVIG on lympho-      audit trail in case of suspected viral/prion
cye function and particularly T-cell function are      transmission.
suspected but ill understood. T lymphocytes do         • In long-term treatment, monitor liver function
carry IgG Fc receptors. One possibility is that        to check for the possibility of viral hepatitis
cytokine production is blocked or switched to          transmission.
favour an anti-inflammatory effect.                     • Monitor the underlying disease itself (platelet
                                                       counts in ITP, and blood counts to detect recovery
                                                       of marrow function in patients after treatment
Effects of different manufacturing methods             with cytotoxics), and regularly reassess whether
                                                       continued IVIG treatment is indicated.
Viral inactivation
All production methods are based on Cohn
fractionation (see Table 12.3). This inactivates       Cost considerations
HIV and markedly reduces the titre of viable
hepatitis B. Some products have good safety and        Three basic considerations will ensure that
efficacy records extending back many years,             patients are treated in the most cost-effective
including those using a pH4 pepsin step, which         manner.
seems to be fortuitously virus inactivating.           • Restrict treatment to those illnesses where the
Storage in low pH solution is also probably virus      treatment is of proven benefit (established by con-
inactivating.                                          trolled clinical trial).
   Newer products, however, include more pre-          • Alternative, more conventional (and effective)
meditated specific antiviral steps such as pasteur-     treatments (prednisolone in ITP) should be tried
ization or a solvent–detergent step. The latter        prior to IVIG whenever possible.
method inactivates enveloped viruses (including        • The dose (mg/kg), route, formulation, frequency
hepatitis C) only.                                     and duration of treatment should be the minimum


156
                                                                                                Intravenous immunoglobulin


that is clinically effective (based partly on pub-          Knezevic-Maramica I, Kruskall MS. Intravenous immune
lished trial data). Other factors such as adverse             globulins: an update for clinicians. Transfusion 2003; 43:
                                                              1460–80.
effects and compliance also need to be considered.
                                                            Mouthon L, Kaveri SV, Spalter SH et al. Mechanisms of
Whenever possible, in the context of long-term
                                                              action of intravenous immune globulin in immune
treatment, home therapy or hospital day-case                  mediated diseases. Clin Exp Immunol 1996; 104 (Suppl.
treatment is the most cost-effective.                         1): 3–10.
                                                            Sharief MK, Ingram DA, Swash M, Thompson EJ.
                                                              Intravenous immunoglobulin reduces circulating pro-
Further reading                                               inflammatory cytokines in Guillain–Barré syndrome.
                                                              Neurology 1999; 52: 1833–8.
                                                            Spellberg B. Mechanism of intravenous immune globulin
Duhem C, Dicto MA, Ries F. Side-effects of intravenous
                                                              therapy. N Engl J Med 1999; 341: 57–8.
 immune globulins. Clin Exp Immunol 1994; 97 (Suppl.
                                                            Yap PL. The viral safety of intravenous immune globulin.
 1): 79–83.
                                                              Clin Exp Immunol 1996; 104 (Suppl. 1): 35–43.
Dwyer JM. Manipulating the immune system with immune
 globulin (review). N Engl J Med 1992; 326(2): 107–16.
Haskin JA, Warner DJ, Blank DU. Acute renal failure after
 large doses of intravenous immune globulin. Ann
 Pharmacother 1999; 33: 800–3.




                                                                                                                     157
Part 3


Complications of transfusion
Chapter 13
Haemolytic transfusion reactions

Sue Knowles and Geoff Poole




A haemolytic transfusion reaction (HTR) is the        • class and subclass (in the case of IgG) of the
occurrence of lysis or accelerated clearance of red   antibody;
cells in a transfusion recipient. With few excep-     • blood group specificity of the antibody;
tions, these reactions are caused by immunological    • thermal range of the antibody;
incompatibility between the blood donor and the       • number, density and spatial arrangement of the
recipient. HTRs can be classified with respect to      red cell antigen sites;
either the time of their occurrence following the     • ability of the antibody to activate complement;
transfusion or the predominant site of red cell       • concentration of antibody in the plasma;
destruction:                                          • amount of red cells transfused.
• acute HTRs (AHTRs) occur during or within
24 h of the transfusion;
                                                      Characteristics of the antibody and antigen
• delayed HTRs (DHTRs) typically occur 5–7
days following the transfusion;                       The characteristics of the antibody (such as
• haemolysis can be predominantly intravascular,      immunoglobulin class, specificity and thermal
when it is characterized by gross haemoglobin-        range) and of the antigen sites against which anti-
aemia and haemoglobinuria, or predominantly           body activity is directed (such as site density and
extravascular, when the only feature may be a fall    special arrangement) are interrelated. Antibodies
in haemoglobin;                                       of a certain specificity, from different individuals,
• in general, intravascular haemolysis is seen in     are often found only within a particular
AHTRs and extravascular haemolysis in DHTRs.          immunoglobulin class and have similar thermal
                                                      characteristics. Red cells of a certain blood group
                                                      phenotype, from different individuals, tend to be
Pathophysiology of HTRs                               relatively homogeneous regarding the attributes of
                                                      the relevant antigen. It is for this reason that a
There are three phases involved (Fig. 13.1):          knowledge of the specificity of an antibody can be
• antibody binding to red cell antigens, which may    highly informative in predicting its clinical signifi-
involve complement activation;                        cance. Three examples illustrate this.
• these opsonized red cells interacting with and      • Anti-A, anti-B and anti-A,B antibodies are regu-
activating phagocytes; and                            larly present in moderate to high titre in the plasma
• production of inflammatory mediators.                of group O persons. These antibodies are often
                                                      both IgM and IgG, having a broad thermal range
                                                      up to 37°C, and are often strongly complement
Antigen–antibody interactions
                                                      binding. The A and B antigens are often present in
Where an immunological incompatibility is             large site numbers (e.g. up to 1.2 ¥ 106 A1 antigen
responsible, the course of the reaction depends       sites per cell) and are strongly immunogenic (pro-
upon:                                                 voking an immune response in an individual



                                                                                                       161
Chapter 13



                                               Red cells coated
                                                with antibody




                     No complement                                                     Complement
      Phase 1




                         bound                                                           bound




                                                         Activation to                                           Activation to
                                                           C3 only                                                   MAC




                FcgR binding to                   FcgR and CR1/CR3
                monocytes, macrophages            binding to monocytes,
                and NK cells                      macrophages and NK cells
      Phase 2




                Extravascular destruction         Extravascular destruction                                    Intravascular
                  phagocytosis                      phagocytosis                                               destruction—lysis
                  fragmentation                     fragmentation
                  lysis (ADCC)                      lysis (ADCC)



                                                                                     C3a            C3a, C5a
      Phase 3




                                                                  Cytokine release


Fig. 13.1 Pathophysiology of haemolytic transfusion reaction. ADCC, antibody-dependent cell-mediated cytotoxicity; MAC,
membrane attack complex; NK, natural killer.




lacking the antigen). Anti-A, anti-B and/or anti-AB                • Anti-Lua antibodies may be produced following
are frequently implicated in AHTRs.                                the immunization of a Lu(a–) person, or may be
• Anti-Jka antibodies may be produced following                    ‘naturally occurring’. They are usually IgM (but
immunization of a Jk(a–) person. They are usually                  often have IgA and IgG components), are only
IgG (but may also have an IgM component), are                      sometimes reactive at 37°C and are not usually
active at 37°C and may be complement binding. In                   complement binding. The Lua antigens show vari-
Jk(a+b–) persons, there are about 1.4 ¥ 104 Jka                    able distribution on the red cells of an individual,
antigen sites per cell. Jka antigens are not particu-              and are poorly immunogenic. The antibody may
larly immunogenic. However, the antibody is                        not be detected in pretransfusion testing, because
sometimes difficult to detect in pretransfusion                     of the fact that screening cells usually do not
testing (because of the low titre of antibody); con-               possess the Lua antigen and because antibody
sequently Jk(a+) blood may be inadvertently trans-                 levels fall after immunization. Anti-Lua antibodies
fused to patients with pre-existing anti-Jka. These                have not been implicated in AHTRs and only
antibodies are frequently implicated in DHTRs.                     rarely in (mild) DHTRs.


162
                                                                                                 Haemolytic transfusion reactions


Complement activation                                  Cytokines
Antibody-mediated intravascular haemolysis is          Cytokines are generated during an HTR as a
caused by sequential binding of complement com-        consequence of both anaphylatoxin generation
ponents (C1–C9). IgM alloantibodies are more           (C3a, C5a) and monocyte FcgRI interaction with
efficient activators of C1 than IgG, since the          red cell-bound IgG. Some biological actions of
latter must be sufficiently close together on the red   cytokines implicated in HTRs are given in Table
cell surface to be bridged by C1q in order to acti-    13.1.
vate complement. Activation to the C5 stage leads         ABO incompatibility stimulates the release of
to release of C5a into the plasma and assembly         high levels of tumour necrosis factor (TNF)-a into
of the remaining components of the membrane            the plasma, within 2 h, followed by interleukin
attack complex on the red cell surface, leading to     (IL)-8 and monocyte chemotactic protein (MCP)-
lysis.                                                 1. In IgG-mediated haemolysis, TNF-a is pro-
   Extravascular haemolysis is caused by non-          duced at a lower level together with IL-1b and
complement-binding IgG antibodies or those             IL-6. IL-8 production follows a similar time course
which bind sublytic amounts of complement. IgG         to that in ABO incompatibility.
subclasses differ in their ability to bind comple-        IgG-mediated haemolysis, as opposed to ABO
ment, with the following order of reactivity:          incompatibility, also results in the production of
IgG3 > IgG1 > IgG2 > IgG4.
   Activation of the C3 stage leads to C3b and
                                                       Table 13.1 Cytokines implicated in haemolytic transfusion
iC3b deposition on red cells, promoting binding to
                                                       reactions.
two complement receptors, CR1 and CR3, which
are both expressed on macrophages and mono-            Terminology         Biological activity
cytes, and to the release of C3a into the plasma.
Hence, C3b and iC3b augment macrophage-                Proinflammatory cytokines
mediated clearance of IgG-coated cells, and anti-      TNF, IL-1         Fever
bodies binding sublytic amounts of complement                            Hypotension, shock, death
(e.g. Duffy and Kidd antibodies) often cause more                        Mobilization of leucocytes from marrow
rapid red cell clearance and more marked symp-                           Activation of T and B cells
                                                                         Induction of cytokines (IL-1, IL-6, IL-8,TNF-a, MCP)
toms than non-complement-binding antibodies
                                                                         Induction of adhesion molecules
(e.g. Rh antibodies).
                                                       IL-6              Fever
   C3a and C5a are anaphylatoxins with potent
                                                                         Acute-phase protein response
proinflammatory effects, including oxygen radical                         B-cell antibody production
production, granule enzyme release from mast                             T-cell activation
cells and granulocytes, nitric oxide production and
                                                       Chemokines
cytokine production.
                                                       IL-8                Chemotaxis of neutrophils
                                                                           Chemotaxis of lymphocytes
Fc receptor interactions                                                   Neutrophil activation
                                                                           Basophil histamine release
IgG alloantibodies bound to red cell antigens inter-   MCP-1               Chemotaxis of monocytes
act with phagocytes through Fc receptors. The                              Induction of respiratory burst
affinity of Fc receptors for IgG subclasses varies,                         Induction of adhesion molecules
with most efficient binding to IgG1 and IgG3.                               Induction of IL-1
After attachment to phagocytes, the red cells are      Anti-inflammatory cytokines
either engulfed or lysed external to the monocyte      IL-1ra            Competitive inhibition of IL-1 type I and II receptors
membrane by lysosomal enzymes excreted by the
monocyte, i.e. antibody-dependent cell-mediated        IL, interleukin; IL-1ra, IL-1 receptor antagonist; MCP, monocyte chemo-
cytotoxicity.                                          tactic protein;TNF, tumour necrosis factor.


                                                                                                                            163
Chapter 13


the IL-1 receptor antagonist (IL-1ra). The relative                 (SHOT) confidential reporting scheme has shown
balance of IL-1 and IL-1ra may also, at least in                    that in ABO-incompatible transfusions, 20% of
part, account for some of the clinical differences                  errors occur in prescription, sampling and request,
between     intravascular    and     extravascular                  29% in the transfusion laboratory and 48% in col-
haemolysis.                                                         lection and administration. The reports have also
                                                                    highlighted that multiple errors contribute to
                                                                    ‘wrong blood’ incidents. Examples of reported
Antibody specificities associated with HTRs                          errors from several series are given in Table 13.3.
                                                                       Estimates of ABO-incompatible transfusions
These are given, together with the site of red cell                 vary and may be underestimates, since some may
destruction, in Table 13.2.                                         be unrecognized or not reported, but two recent
                                                                    surveys have found a frequency of approximately
                                                                    1 in 30 000 transfusions. Not all ABO-incompati-
Acute haemolytic transfusion reactions                              ble transfusions cause morbidity and mortality;
                                                                    mortality is dependent upon the amount of incom-
Aetiology and incidence
                                                                    patible red cells transfused, and is reported to be
These reactions arise as a result of existing anti-                 25% in recipients receiving 1–2 units of blood and
bodies, in either the recipient or donor plasma,                    reaches 44% with more than 2 units. However, as
which are directed against red cell antigens of the                 little as 30 mL group A cells given to a group O
other party. The majority of AHTRs are due to the                   recipient can be fatal.
transfusion of ABO-incompatible transfusions,                          Less frequently, Kell, Kidd and Duffy antibodies
predominantly red cells, but can also be due to the                 can be responsible and the acute reaction is due to
administration of plasma containing high titres of                  a failure to detect, or take account of, the red cell
ABO haemolysins.
   ABO-incompatible transfusions are the result of
the ‘wrong’ blood being given to the ‘wrong’                        Table 13.3 Errors resulting in ‘wrong blood’ incidents.
patient because of clerical or administrative errors,
occurring at any stage during the transfusion                       Prescription, sampling and request
process. The Serious Hazards of Transfusion                         Failure to identify correct recipient at sampling
                                                                    Correct patient identity at sampling but incorrectly labelled sample
Table 13.2 Antibody specificities associated with haemolytic         Selection of incompatible products in an emergency
transfusion reactions.
                                                                    Transfusion laboratory
                                                                    Took a correctly identified sample and aliquoted it into an improperly
Blood group          Intravascular       Extravascular
                                                                       labelled test tube for testing
system               haemolysis          haemolysis
                                                                    Took a wrongly identified sample through testing
                                                                    Tested the correct sample but misinterpreted the results
ABO, H               A, B, H
                                                                    Tested the correct sample but recorded the results on the wrong record
Rh                                       All
                                                                    Correctly tested the sample but labelled the wrong unit of blood as
Kell                 K                   K, k, Kpa, Kpb, Jsa, Jsb
                                                                       compatible for the patient
Kidd                 Jka                 Jka, Jkb, Jk3
                                                                    Incorrect serological reasoning, e.g. O-positive FFP to non-O-positive
Duffy                                    Fya, Fyb
                                                                       recipient
MNS                                      M, S, s, U
Lutheran                                 Lub                        Collection of unit
Lewis                Lea                                            Failure to check recipient identity with unit identity
Cartwright                               Yta                        Bedside administration error
Vel                  Vel                 Vel                        Recipient identity checked through case notes or prescription chart, and
Colton                                   Coa, Cob                      not wristband
Dombrock                                 Doa, Dob                   Wristband absent or incorrect




164
                                                                                           Haemolytic transfusion reactions


alloantibody in either the antibody screen or                  • hypotension, tachycardia or both;
crossmatch.                                                    • agitation, distress and confusion, particularly in
   Details of the incompatibilities resulting in               the elderly;
deaths reported to the Food and Drug Administra-               • nausea or vomiting;
tion (FDA) in the USA between 1976 and 1985 are                • dyspnoea;
provided in Table 13.4. In the UK, with voluntary              • flushing; and
reporting to the SHOT scheme, there have been                  • haemoglobinuria.
five deaths definitely attributable to an ABO-                      In anaesthetized patients, the only signs may be
incompatible transfusion, with two further proba-              uncontrollable hypotension or excessive bleeding
ble deaths and eight possible deaths attributable to           from the operative site, as a result of disseminated
the transfusion, between 1996 and 2002. Over the               intravascular coagulation (DIC). These symptoms
same period there have been 41 cases of major                  and signs can also be features of a reaction to bac-
morbidity due to an ABO-incompatible transfu-                  terial contamination of the unit.
sion and five others attributable to Kidd and Duffy
antibodies.
                                                               Complications
                                                               Renal failure develops in up to 36% of patients as
Symptoms and signs
                                                               a result of acute tubular necrosis induced by both
These may become apparent after receiving as little            hypotension and DIC. Thrombus formation in
as 20 mL of ABO-incompatible red cells. Initial                renal arterioles may also cause cortical infarcts.
clinical presentations include the following:                     DIC develops in up to 10% of patients. TNF-a
• fever, chills or both;                                       can induce tissue factor expression by endothelial
• pain at the infusion site, or localized to the loins,        cells and together with IL-1 can reduce the
abdomen, chest or head (the aetiology is unclear,              endothelial expression of thrombomodulin.
but may be related to rapid complement activation              Thromboplastic material is also liberated from
at the site of infusion, and the generation of                 leucocytes during the course of complement
bradykinin following complement activation);                   activation.


Table 13.4 Fatal acute haemolytic transfusion reactions
reported to the FDA between 1976 and 1985.                     Immediate management of suspected AHTR
                                                               Actions for nursing staff
Incompatibility                             Number of deaths
                                                               In the presence of a fever greater than 38°C, and/or
O recipient and A red cells                   80               any symptoms or signs mentioned above, the
O recipient and B/AB red cells                26               nursing staff should:
B recipient and A/AB red cells                12               1 stop the transfusion, leaving the giving set
A recipient and B red cells                    6               attached;
O plasma to A/AB recipient                     6
                                                               2 use a new giving set and keep the intravenous
B plasma to AB recipient                       1
                                                               infusion running with normal saline;
Total ABO incompatibilities                 131                3 call a member of the medical staff;
anti-K                                         5               4 check that the patient identity as provided on
anti-E + K + P1                                1               the wristband corresponds with that given on the
anti-Jkb                                       1               label on the blood pack and on the compatibility
anti-Jka + Jkb + Jk3                           1               form;
anti-Fya                                       1               5 save any urine the patient passes; and
Total non-ABO incompatibilities                9
                                                               6 monitor the pulse, blood pressure and tempera-
                                                               ture at 15-min intervals.



                                                                                                                      165
Chapter 13


                                                                           than the infusion site for the investigations listed in
Actions for medical staff
                                                                           Table 13.6.
The immediate actions depend upon the present-
ing symptoms and signs, and are summarized in
                                                                           Other reactions characterized by haemolysis
Table 13.5.
                                                                           In patients with autoimmune haemolytic anaemia,
                                                                           transfusion may exacerbate the haemolysis and be
Investigation of suspected AHTR
                                                                           associated with haemoglobinuria. Donor units of
Blood samples should be taken from a site other                            red cells may also be haemolysed as a result of:

Table 13.5 Immediate medical management of an acute transfusion reaction.

Symptoms/signs                       Likely diagnosis                            Actions

Isolated fever or fever and          Febrile non-haemolytic transfusion          Paracetamol 1 g orally, continue transfusion slowly, observation
   shivering, stable observations,     reaction (FNHTR)                             of pulse, blood presure and temperature every 15 min for 1 h,
   correct unit given                                                               then hourly. If no improvement call haematology medical staff
Fever with pruritus, urticaria       Allergic transfusion reaction               Chlorpheniramine 10 mg i.v. and other actions as for suspected
                                                                                    FNHTR
Any other symptoms/signs,            Assume to be an acute haemolytic            Discontinue transfusion, nornal saline to maintain urine output
  hypotension, or incorrect unit       transfusion reaction in first instance        >1 mL/kg per h. Full and continuous monitoring of vital signs.
                                                                                    Call haematology medical and transfusion laboratory staff
                                                                                    immediately for further advice/action. Send discontinued unit
                                                                                    of blood with attached giving set and other empty packs, after
                                                                                    clamping securely, to transfusion laboratory



Table 13.6 Laboratory investigation of suspected acute haemolytic transfusion reaction.

Blood test                              Rationale/findings

Full blood count                        Baseline parameters, red cell agglutinates on film
Plasma/urinary haemoglobin,             Evidence of intravascular haemolysis
   haptoglobin, bilirubin
Blood group                             Comparison of post-transfusion and retested pretransfusion samples, to detect ABO error not apparent at
                                           bedside. Unexpected ABO antibodies after transfusion may result from transfused incompatible plasma.
                                           The donor ABO group should be confirmed
Direct antiglobin test                  Positive in majority, pretransfusion sample should be tested for comparison. May be negative if all
                                           incompatible cells destroyed
Compatibility testing                   An IAT antibody screen and IAT crossmatch using the pretransfusion and post-transfusion sample provide
                                           evidence for the presence of alloantibody. Elution of antibody from post-transfusion red cells may aid
                                           identification of antibody, or confirm specificities identified in serum in cases of non-ABO
                                           incompatibility. Red cell phenotype should also be performed on recipient pretransfusion sample and
                                           unit in cases of non-ABO incompatibility in order to confirm absence in patient and presence in unit of
                                           corresponding antigen
Urea/creatinine and electrolytes        Baseline renal function
Coagulation screen                      Detection of incipient disseminated intravascular coagulation
Blood cultures                          In the event of septic reaction caused by bacterial contamination of unit, which may be suspected from
                                           inspection of pack for lysis, altered colour or clots

IAT, indirect antiglobulin test.


166
                                                                                  Haemolytic transfusion reactions


•   bacterial contamination;                         unique patient identifiers throughout the hospital
•   excessive warming;                               and automation within the laboratory. Access to
•   erroneous freezing;                              previous transfusion records containing historical
•   addition of drugs or intravenous fluids;          ABO groups should be available at all times.
•   trauma from extracorporeal devices; or              In the future, it is desirable that computerized
•   red cell enzyme deficiency.                       systems are used to verify at the bedside the
                                                     matches between the patient and the sample
                                                     taken for compatibility testing, and at the time of
Management of a confirmed AHTR
                                                     transfusion between the patient and the unit of
1 Maintain adequate renal perfusion by:              blood.
   (a) fluid challenges;
   (b) frusemide infusion (250 mg over 4 h); and
                                                     Prevention of non-ABO AHTRs
   (c) if necessary, inotropic support.
2 Transfer to a high-dependency area where con-      In the case of recurrently transfused patients, due
tinuous monitoring can take place.                   attention should be paid to the interval between
3 Repeat coagulation and biochemistry screens        sampling and transfusion in order to optimize the
every 2–4 h.                                         detection of newly developing antibodies. For
4 If urinary output cannot be maintained at          patients transfused within the previous 72 h, the
1 mL/kg per h, seek expert renal advice.             following pretransfusion sample should not be
5 Haemofiltration or dialysis may be required for     taken more than 24 h before the next transfusion.
the acute tubular necrosis.                          Pretransfusion samples should also only be kept
6 In the event of the development of DIC, blood      for 3 days, if the patient has been transfused within
component therapy may be required.                   the previous 14 days.
                                                        In the presence of multiple red cell alloanti-
                                                     bodies, and when it is not feasible to obtain com-
Prevention of AHTRs                                  patible red cells in an emergency, intravenous
                                                     immunoglobulin (1 g/kg daily for 3 days) with
Prevention of ‘wrong blood’ incidents
                                                     or without steroids (hydrocortisone 100 mg 6-
Prevention of the multiplicity of errors which can   hourly) has been used with anecdotal reports of
contribute to the transfusion of ABO-incompatible    preventing a haemolytic episode.
red cells must depend upon the creation of an
effective quality system for the entire process,
which will involve:                                  Delayed haemolytic transfusion reactions
• adherence to national guidelines and standards;
                                                     Aetiology and incidence
• local procedures which are agreed, documented
and validated;                                       With few exceptions, DHTRs are due to secondary
• training and retraining of key staff;              immune responses following re-exposure to a
• regular error analysis and review;                 given red cell antigen. The recipient has been pri-
• reporting to local risk management committee;      marily sensitized to the antigen in pregnancy or as
• reporting to national haemovigilance schemes to    a result of a previous blood transfusion and a few
contribute to the understanding of the extent and    days after a subsequent transfusion there is a rapid
underlying causes.                                   increase in the antibody concentration, resulting in
These aspects are specifically covered in Chapter     the destruction of red cells.
25.                                                  • The antibodies most commonly implicated and
   Since the majority of errors leading to an ABO-   reported to SHOT between 1996 and 2002 were
incompatible transfusion are due to misidentifica-    those from the Kidd blood group system, followed
tion of the patient or patient’s sample, due         by those from the Rh, Duffy and Kell systems. One
attention must be paid to the comprehensive use of   analysis showed that in approximately 10% of


                                                                                                             167
Chapter 13


reported cases, more than one alloantibody was
                                                        Management
found in the serum.
• Frequently, there are no clinical signs of red cell   The majority of DHTRs require no treatment
destruction, but subsequent patient investigations      because red cell destruction occurs gradually as
reveal a positive direct antiglobulin test (DAT) and    antibody synthesis increases. However, particu-
the emergence of a red cell antibody. This situation    larly in a bleeding patient, haemolysis will con-
has been termed a delayed serological transfusion       tribute to the development of life-threatening
reaction (DSTR).                                        anaemia and urgent investigations are required to
• Kidd and Duffy antibodies are more likely to          ensure the timely provision of antigen-negative
cause symptoms and be associated with a DHTR            units.
rather than a DSTR.                                        Expert medical advice may be required for treat-
• Estimates of the frequency of DHTR and DSTR           ment of the hypotension and renal failure. When
vary, but in a series reported from the Mayo Clinic     accompanied by circulatory instability and renal
the frequency of DHTR was 1 in 5405 units and of        insufficiency, a red cell exchange transfusion with
DSTR was 1 in 2990 units, giving a combined fre-        antigen-negative units can curtail the haemolytic
quency of 1 in 1900 units transfused.                   process. Future transfusions of red cells should
• DHTRs are in themselves rarely fatal, although        also be negative for the antigen in question.
in association with the underlying disease can lead
to mortality.
                                                        Investigation of suspected DHTR
• Of transfusion fatalities reported to the FDA
between 1976 and 1985, 10% were due to DHTR;            • The peripheral blood film is likely to show
in 75% of cases, more than one alloantibody was         spherocytosis.
present in the serum, and the same proportion           • Other evidence of haemolysis, namely hyper-
involved non-Rh antibodies.                             bilirubinaemia, reduced serum haptoglobin,
• Six deaths reported to SHOT between 1996 and          haemoglobinaemia,          haemoglobinuria        and
2002 have been due to DHTRs. Tragically, in some        haemosiderinuria, is useful to confirm the nature
instances, there were delays in diagnosis, investiga-   of the reaction and to monitor progress.
tion and provision of compatible units which led        • The DAT usually becomes positive within a few
to marked anaemia and contributed to mortality.         days of the transfusion until the incompatible cells
                                                        have been eliminated.
                                                        • Further serological testing on pretransfusion
Signs and symptoms
                                                        and post-transfusion samples should be under-
These usually appear within 5–10 days following         taken in accordance with the schedule provided for
the transfusion, but intervals as short as 24 h and     AHTR.
as late as 21 days have been recorded. The exact        • The antibody may not be initially apparent in
onset may be difficult to define since haemolysis         the post-transfusion serum but can be eluted from
can be initially insidious and may only be appreci-     the red cells. If the red cell eluate is inconclusive,
ated from results of post-transfusion samples. The      then a repeat sample should be taken after 7–10
commonest features are:                                 days, to allow for an increase in antibody titre.
• fever;                                                However, additional, more sensitive techniques
• fall in haemoglobin concentration; and                may have to be employed to detect the antibody
• jaundice and haemoglobinuria.                         and it is advisable to seek the help of a reference
Hypotension and renal failure are uncommon (6%          laboratory.
of cases).                                              • Since a significant proportion of cases have more
   In the postoperative period in particular, the       than one alloantibody in the serum, it is important
diagnosis may be overlooked and the symptoms            that the panels used for antibody identification
and signs incorrectly attributed to continuing          have sufficient cells of appropriate phenotypes to
haemorrhage or sepsis.                                  exclude additional specificities.


168
                                                                                      Haemolytic transfusion reactions


                                                         only 10–15% develop clinically significant
Prevention
                                                         haemolysis.
Access to previous transfusion records may dis-            Haemolysis in minor ABO incompatibility is
close the presence of antibodies undetectable at the     short-lived and exchange transfusion is rarely
time of crossmatching, and all patients should be        required. Plasma-containing components should
questioned regarding previous transfusions and           be of the recipient type, and red cells group O.
pregnancies. Patients found to have developed a            It has been suggested that the use of peripheral
clinically significant red cell alloantibody should       blood stem cells may increase the risk of significant
be provided with an antibody card. When the care         haemolysis since the number of lymphocytes
of patients requiring transfusion support is shared      infused with the graft is increased, and three
between hospitals, there must be adequate com-           deaths due to AHTR have been reported between
munication between laboratories and clinical             1997 and 1999 in minor ABO-incompatible trans-
teams.                                                   plants. Several cases due to anti-D have been
   Laboratories should ensure that their antibody        described, and antibody production has persisted
screen is effective in detecting weak red cell alloan-   for up to 1 year.
tibodies and that screening cells are taken from
homozygotes where the corresponding antibodies
                                                         Solid organ transplants
show dosage.
                                                         In ABO-unmatched organs, the frequency of
                                                         occurrence of donor-derived antibodies and
Delayed haemolysis following                             haemolysis increases with the lymphoid content of
organ transplantation (passenger                         the graft, from kidney to liver to heart–lung trans-
lymphocyte syndrome)                                     plants. The figures for haemolysis are 9%, 29%
                                                         and 70%, respectively. The frequency of haemoly-
Donor-derived B lymphocytes within the trans-            sis increases with an O donor and A recipient. The
planted organ may mount an anamnestic response           ABO antibodies, which appear 7–10 days after
against the recipient’s red cell antigens. Donor-        transplant, last for approximately 1 month.
derived antibodies are usually directed against          Haemolysis is usually mild, although several cases
antigens within the ABO and Rh systems. Haemol-          of renal failure and one death have been reported.
ysis occurs 7–10 days after transplantation, with        It can be prevented by switching to group O cells,
an unpredictable and abrupt onset.                       either at the end of surgery or postoperatively if the
   In minor ABO-incompatible transplants (O              DAT becomes positive.
donors and recipients of other groups), pre-                Rh antibodies have been described following
transplant isohaemagglutinin titres do not               kidney, liver and heart–lung transplants. They can
appear to predict the incidence or severity of           cause haemolysis for up to 6 months, which can be
haemolysis. In both haemopoietic stem cell and           sufficiently severe to merit therapy.
solid organ transplants, the haemolytic syndrome
is almost exclusively associated with the use of
cyclosporin and tacrolimus. The ex vivo removal          Haemolytic transfusion reactions in sickle
of T cells has a similar enhancing effect on             cell disease
the function of transplanted donor memory B
lymphocytes.                                             The frequency of alloimmunization in sickle cell
                                                         anaemia is dependent upon the nature and success
                                                         of the extended red cell antigen matching policy
Haemopoietic stem cell transplants (see also
                                                         employed. Approximately 40% of patients who
Chapter 9)
                                                         are alloimmunized have experienced or will expe-
Most patients transplanted with minor ABO-               rience a DHTR.
incompatible marrow develop a positive DAT but              Although DHTRs are characteristically mild in


                                                                                                                 169
Chapter 13


other groups of recipients, they can be responsible    • The clinical presentations are diverse and they
for major morbidity in sickle cell disease. The term   can be unrecognized or misdiagnosed.
‘sickle cell haemolytic transfusion reaction syn-      • Most fatal AHTRs are due to the transfusion of
drome’ has been suggested to capture some of the       ABO-incompatible red cells.
distinctive features which can be seen to accom-       • The transfusion of ABO-incompatible red cells
pany a reaction. These features are as follows.        is the result of an error occurring at any stage in the
• Symptoms suggestive of a sickle cell pain crisis     transfusion process.
develop or are intensified during the HTR.              • Devising and successfully implementing mea-
• Marked reticulocytopenia (for the patient).          sures to overcome these preventable and fatal
• Development of a more severe anaemia after           errors are a priority and challenge for those
transfusion than was present before: this may be       involved.
due to the suppression of erythropoiesis as a result
of the transfusion, although hyperhaemolysis of
autologous red cells (bystander immune haemo-          Further reading
lysis) has been suggested.
• Subsequent transfusions may further exacerbate       Beauregard P, Blajchman MA. Haemolytic and pseudo-
the anaemia and it may become fatal.                     haemolytic transfusion reactions: an overview of the
• Patients often have multiple red blood cell            haemolytic transfusion reactions and the clinical
alloantibodies and may also have autoantibodies,         conditions that mimic them. Transfus Med Rev 1994; 8:
which makes it difficult or impossible to find com-        184–99.
                                                       Davenport RD. Haemolytic transfusion reactions. In:
patible units of red blood cells. However, in other
                                                         Popovsky MA, ed. Transfusion Reactions. Bethesda, MA:
patients no alloantibodies are identified.                AABB Press, 1996: 1–44.
• Serological studies may not provide an explana-      Linden JV, Kaplan HS. Transfusion errors: causes and
tion for the HTR: even red cells which are pheno-        effects. Transfus Med Rev 1994; 8: 169–83.
typically matched with multiple patient antigens       Mollison PL, Engelfriet CP, Contreras M. Haemolytic
may be haemolysed.                                       transfusion reactions. In: Blood Transfusion in Clinical
• Withholding further transfusion and cortico-           Medicine. Oxford: Blackwell Science, 1997: 358–89.
                                                       Petz LD, Calhoun L, Shulman IA, Johnson C, Herron RM.
steroids (hydrocortisone 100 mg 6-hourly) and
                                                         The sickle cell haemolytic transfusion reaction syndrome.
intravenous immunoglobulin (1 g/kg daily) has            Transfusion 1997; 37: 382–92.
been beneficial in some cases.                          Ramsey G. Red cell antibodies arising from solid organ
• It is recommended that patients with sickle cell       transplants. Transfusion 1991; 31: 76–86.
disease are phenotyped prior to transfusion and        Sazama K. Reports of 355 transfusion-associated deaths:
that blood is selected for Rh and K.                     1976 through 1985. Transfusion 1990; 30: 583–90.
                                                       Serious Hazards of Transfusion. Annual Report
                                                         2002–2003. Manchester: SHOT Office, 2004
                                                         (www.shotuk.org).
Summary                                                Vamvakas EC, Pineda AA, Reisner R, Santrach PJ, Moore
                                                         SB. The differentiation of delayed haemolytic and
• HTRs are the commonest cause of immediate              delayed serologic transfusion reactions: incidence and
morbidity and mortality following a transfusion.         predictors of haemolysis. Transfusion 1995; 35: 26–32.




170
Chapter 14
Febrile reactions and transfusion-related
acute lung injury

Michael F. Murphy and Sheila MacLennan



A common adverse reaction to a blood transfusion         of patients receiving standard non-leucocyte-
is the development of fever, which can occur after       depleted blood components.
transfusion of any type of blood component. The
development of fever without haemolysis, a febrile
                                                         Clinical features
non-haemolytic transfusion reaction (FNHTR), is
relatively innocuous in itself, but it is important to   FNHTRs associated with red cell transfusions
differentiate it from more serious complications of      occur in patients who have had previous pregnan-
transfusion, particularly acute haemolytic reac-         cies and/or transfusions; FNHTRs associated with
tions and transfusion-transmitted bacterial infec-       platelet transfusions may occur in patients who
tion. FNHTRs are associated with the presence of         have not had previous pregnancies and/or
leucocytes in the blood components, and it is likely     transfusions.
that the incidence will be significantly reduced fol-        Typical clinical manifestations are:
lowing the implementation of universal leucocyte         • flushing;
depletion of blood components in the UK and              • fever;
other countries.                                         • tachycardia; and
   Transfusion-related acute lung injury (TRALI)         • sometimes rigors.
is a severe pulmonary reaction associated with           Symptoms usually occur about 30 min to 2 h after
the transfusion of blood components containing           starting a red cell transfusion, and even earlier
donor plasma. Although TRALI occurs infre-               after a platelet transfusion. In the mildest reac-
quently, it is one of the commonest causes of death      tions, patients are febrile but asymptomatic. The
associated with blood transfusion.                       temperature usually settles 2–12 h after discon-
                                                         tinuation of the transfusion.
                                                            FNHTRs are considered to be relatively innocu-
Febrile non-haemolytic                                   ous as they are transient and do not lead to more
transfusion reactions                                    serious clinical effects, although they can be
                                                         uncomfortable and distressing for patients.
Definition
                                                            Many variables influence the severity of clinical
FNHTRs are febrile episodes where there is a tem-        symptoms, including:
perature rise of 1°C or more during or soon after a      • the number of leucocytes (red cell transfusions)
transfusion, and where there is no obvious cause         or quantity of cytokines (platelet transfusions)
such as a haemolytic transfusion reaction.               transfused;
                                                         • the speed of the transfusion; and
                                                         • recipient factors such as the titre of antileuco-
Incidence
                                                         cyte antibodies.
FNHTRs have been reported to occur with an inci-
dence as high as 6.8% after red cell and 37.5%
after platelet transfusions in prospective studies


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Chapter 14


                                                                                           of clinical staff of a transfusion-transmitted
Differential diagnosis
                                                                                           bacterial infection.
Fever is the commonest early manifestation of                                                Fever due to coincidental infection and other
haemolytic transfusion reactions. Later effects                                            causes unrelated to blood transfusion should also
include back pain, headache, shortness of breath,                                          be considered in the differential diagnosis of fever
haemoglobinuria, shock and bleeding due to                                                 occurring during a transfusion.
disseminated intravascular coagulation (DIC).
Concern that fever may be the first indication of a
                                                                                           Pathogenesis
haemolytic transfusion reaction is the reason why
it is usual practice to discontinue transfusions                                           The pathogenesis of FNHTRs following red cell
associated with fever.                                                                     and platelet transfusions is different.
   There has been considerable debate about how
to manage patients developing fever during a
                                                                                           Red cell transfusions
transfusion.
• Should the blood component be returned to the                                            The importance of leucocytes in causing FNHTR
blood bank?                                                                                associated with red cell transfusion was demon-
• What investigations should be carried out to                                             strated many years ago (Fig. 14.1). The rise in
exclude a haemolytic reaction?                                                             temperature is directly related to the number of
• Should the transfusion of the implicated blood                                           leucocytes transfused, and leucocyte antibodies are
component be recommenced if the investigations                                             detectable in most patients. In one early study, the
prove negative?                                                                            reactions were prevented in all patients with a
   Transfusion-transmitted bacterial infection is an                                       history of recurrent FNHTRs by reducing the
important cause of fever during transfusion (see                                           number of leucocytes to 0.25 ¥ 109/unit, which is
Chapter 16). Not all bacterially contaminated                                              approximately 10% of the number of leucocytes in
blood components cause symptoms but, if they                                               a non-leucocyte-depleted red cell concentrate.
occur, fever and chills are common, starting during                                           FNHTRs had been assumed to be due to the
or shortly after a transfusion. Subsequent symp-                                           reaction between recipient HLA or granulocyte-
toms may include nausea, vomiting, diarrhoea,                                              specific antibodies with donor leucocytes, and the
shock, respiratory symptoms and bleeding due to                                            subsequent release of ‘pyrogens’, principally inter-
DIC; such symptoms should