Recent Advances in Haematology - 1

Recent Advances in
Haematology
Recent Advances in
Haematology
Editors
VP Choudhry
MD FIAP FIMSA FIACM FISHTM
Professor and Head
Department of Haematology
All India Institute of Medical Sciences
New Delhi-110029, India
Honorary Consultant, Armed Forces Medical Services
Former Director
Indira Gandhi Institute of Child Health, Kabul, Afghanistan
Former President
Indian Society of Haematology and Transfusion Medicine
Former President
Paediatric Haematology Oncology, Indian Academy of Paediatrics
Founder Secretary and Former President
Delhi Society of Haematology
Medical Advisor
Federation of Indian Thalassemics
National Thalassaemia Welfare Society
Editor, Indian Journal of Paediatrics
Thalassemia Care and Control in the New Millennium
Thalassaemia Perspectives
Renu Saxena
MD FIMSA
Professor, Department of Haematology
Coordinator, ISHTM-AIIMS EQAP Programme
Ex-Editor, Delhi Society of Haematology, Newsletter
HP Pati
MD
Additional Professor, Department of Haematology
Senior Specialist, Armed Forces Hospital, Kuwait
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Recent Advances in Haematology
© 2004 VP Choudhry, Renu Saxena, HP Pati
All rights reserved. No part of this publication should be reproduced, stored in a
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First Edition: 2004
ISBN 81-8061-252-X
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Printed at Replika Press Pvt Ltd., 310 EPIP, HSIDC, Kundli, Sonipat (Haryana)
Dedicated
to
Our respected Teachers and Parents
for their constant inspiration
and
Our patients for whose welfare
we have been striving to offer them the best care
Contributors
A Chaturvedi H Subramanya
Haematology Section Haematology Section
Department of Pathology Department of Pathology
Armed Forces Medical College Armed Forces Medical College
Pune, Maharashtra Pune, Maharashtra
AK Karak MD PhD HP Pati MD
Associate Professor of Pathology Additional Professor
All India Institute of Medical Department of Haematology
Sciences AIIMS, New Delhi
New Delhi
Jawed Fareed PhD
Amish Vora DM
Loyola University of Chicago
Department of Medical Oncology
Maywood, IL 60153
Institute Rotary Cancer Hospital
All India Institute of Medical Joseph Loscalzo MD PhD
Sciences Whitaker Cardiovascular Institute
New Delhi Evans Department of Medicine
Barbara Voetsch MD PhD Boston University School of
Whitaker Cardiovascular Institute Medicine
Evans Department of Medicine Boston
Boston University School of
M Kannan MSc
Medicine
Boston Department of Haematology
All India Institute of Medical
Debra A Hoppensteadt PhD Sciences
Loyola University of Chicago New Delhi
Maywood
IL 60153 Madhu Choudhry
Department of Haematology
DK Mishra AIIMS, New Delhi
Haematology Section
Department of Pathology Maitreyee Bhattacharyya MD
Armed Forces Medical College Department of Haematology
Pune, Maharashtra AIIMS, New Delhi
GS Chopra Manisha Bhutani DM

Head, Department of Pathology Department of Medical Oncology
and Laboratory Medicine Institute Rotary Cancer Hospital
Army Hospital (R and R) All India Institute of Medical
Delhi Cantt Sciences, New Delhi
viii Recent Advances in Haematology
Manoranjan Mahapatra MD Rajive Kumar MD

Assistant Professor Additional Professor of
Department of Haematology Haematology and Head,
AIIMS, New Delhi Laboratory Oncology Unit, IRCH
AIIMS, New Delhi
MB Agarwal MD MNAMS
Haematologist and Haemato- Ramesh Kumar Arya
Oncologist, Bombay Hospital MBBS MD PhD
Institute of Medical Sciences Al-Adan Hospital, Ministry of
Mumbai Health, Kuwait
Mona Anand MD Renu Saxena MD

Senior Research Associate Department of Haematology
Laboratory Oncology Unit All India Institute of Medical
IRCH, AIIMS, Sciences, New Delhi
New Delhi
S Tyagi MD
MR Lokeshwar Assistant Professor
Consultant Pediatrician and Department of Haematology
Pediatric Hematologist-Oncologist AIIMS, New Delhi
and Head of Dept. of Pediatrics
S Varma MD
P.D. Hinduja National Hospital
Professor and Head
and Medical Research Center,
Department of Internal Medicine
Mahim, Mumbai PGIMER, Chandigarh
Lilavati Hospital and Medical
Research Center Sarfraz Ahmad PhD
Bandra Reclamation Loyola University of Chicago
Mumbai Maywood, IL 60153
Center for Hemostasis and
Nitin Shah Thrombosis, Florida Hospital
Part-time Consultant Cancer Institute, Orlando,
PD Hinduja National Hospital and FL 32804, USA
Medical Research Center
Mahim, Mumbai Sumitra Dash MD
Professor of Haematology
R Saxena MD Postgraduate Institute of Medical
Professor of Haematology Education and Research,
Department of Haematology Chandigarh
Sciences Tanu Singhal
New Delhi Clinical Associate, PD Hinduja
National Hospital and Medical
Rajat Kumar Research Center, Mahim, Mumbai
MD (Med) DNB (Med) FRCP (London)
FRCP (Edin) Taqdees Sheikh PhD
Ex. Army Hospital Research and Loyola University of Chicago
Referral, New Delhi Maywood, IL 60153
Contributors ix
Velu Nair MD Vinod Kochupillai MBBS FRCP

Hematologist, Army Hospital Department of Medical Oncology
(R andR), Delhi Cantt Institute Rotary Cancer Hospital
Vijaylaxmi Ray MD
Sciences
Ex-Prof and Head, Department of
New Delhi
Transfusion Medicine, Sanjay
Gandhi Post Graduate Institute of VP Choudhry
Medical Sciences, Lucknow, UP
MD FIAP FIMSA FIACM FISHTM
Harprit Singh MD Professor and Head
Senior Resident, Department of Department of Haematology
Transfusion Medicine, Sanjay All India Institute of Medical
Gandhi Post Graduate Institute of Sciences
Medical Sciences, Lucknow, UP New Delhi
Preface
Knowledge in the field of Haematology continues to expand and

accumulate with astonishing pace. Molecular haematology has added
new facets and has taken the diagnosis of haematological diseases to
the molecular level. It has enabled to provide the antenatal diagnosis
for a variety of inherited disorders. Initiation of community screening
programme along with antenatal diagnosis has made it feasible to
initiate the control programme. Several countries have initiated the
programme and controlled the birth of haematological diseases such
as thalassaemia, sickle cell anaemia, haemophilia, etc. The pharmaceuti-
cal industry has kept pace with these developments. They have
developed molecules which act at molecular levels and have offered
complete cure for diseases such as chronic myeloid leukaemia, acute
promyelocytic leukaemia, etc. Other molecules have offered newer
approaches for management of cancers and other several conditions. It
has been unfortunate that the benefits of these developments have
remained in the developing countries.
In the developed countries like ours the facilities for diagnosis and
management have been limited. Haematology in our country has
developed only in metro cities where the Departments of Haematology
have been developed. Degree courses of Doctorate in Medicine, in
Haematology have been initiated in some of these institutions.
Therefore, the book on Advances in Haematology will come long way in
providing the current state-of-art on major topics of greater interest. In
addition it will impart the knowledge to doctors practicing in the field
of Haematology, Medicine, Paediatrics, Pathology as topics of greater
relevance for the developing countries. In addition it will further help
in expanding the Haematology in our country.
We acknowledge all the authors who have made special efforts to
include the Indian data on prevalence of haematological disorder,
clinical spectrum and the outcome of various therapeutic modalities
alongwith the current state-of-art of management with their results.
We would like to acknowledge the support of Mr. Jitendar P. Vij
Chairman and Managing Director of Jaypee Brothers Medical
Publishers as well as the assistance of editorial and production staff to
bring out this edition.
VP Choudhry
Renu Saxena
HP Pati
Contents
1. Blood Substitutes ............................................................................. 1

Ramesh Kumar Arya
2. Stem Cell Transplantation ............................................................ 22
Rajat Kumar
3. Apheresis ......................................................................................... 56
Col Velu Nair
4. Blood Component Therapy: In Paediatric Practice ................. 70
VP Choudhry
5. Haematopoietic Stem Cells .......................................................... 84
HP Pati
6. Biological Therapy of Cancers .................................................... 93
M Mahapatra, HP Pati, VP Choudhry
7. Management of AML ...................................................................112
M Mahapatra, VP Choudhry, R Saxena, HP Pati
8. Paroxysmal Nocturnal Hemoglobinuria:
Pathogenesis and Molecular Biology ...................................... 135
S Varma
9. Inherited Bone Marrow Failure Syndrome ........................... 147
VP Choudhry, Maitreyee Bhattacharyya
10. Prevention and Control of HIV:
Among Blood Products and IV Drug Users ............................ 165
Madhu Choudhry, VP Choudhry
11. Transfusion Medicine: Audit ..................................................... 183
Vijaylaxmi Ray, Harprit Singh
12. Multiple Myeloma: Some Specific Aspects ............................ 195
Mona Anand, Rajive Kumar
13. Minimal Residual Disease (MRD) In Leukaemias ............... 219
DK Mishra, A Chaturvedi, H Subramanya, GS Chopra
14. Diagnostic Dilemmas in Lymphoproliferative
Disorders ....................................................................................... 239
Sumitra Dash
15. Immunohistochemistry on Marrow
Trephine Biopsy ........................................................................... 249
AK Karak
16. Carrier Detection in Haemophilia A ........................................ 262
R Saxena, RPH Ahmed
17. Genetic Determinants of Atherothrombotic Disease ........... 275
Barbara Voetsch, Joseph Loscalzo
xiv Recent Advances in Haematology
18. Anticoagulation Therapy ........................................................... 289

MB Agarwal
19. Laboratory and Clinical Diagnosis of
Heparin-Induced Thrombocytopenia and
Its Therapeutic Options .............................................................. 303
Sarfraz Ahmad, Renu Saxena, Debra A Hoppensteadt,
Taqdees Sheikh, Jawed Fareed
20. Neonatal Thrombosis .................................................................. 321
Renu Saxena, M Kannan
21. Anaemia in the Newborn ........................................................... 333
MR Lokeshwar, Tanu Singhal, Nitin Shah
22. Thalassaemia Intermedia Syndrome ....................................... 358
S Tyagi, VP Choudhry
23. Cytogenetics of Leukemias and Myelodysplastic
Syndrome ...................................................................................... 372
S Tyagi
24. Adult ALL ...................................................................................... 388
Manisha Bhutani, Amish Vora, Vinod Kochupillai
Index ................................................................................................ 401

1
Blood Substitutes
Ramesh Kumar Arya
Key Words
Transfusion • Transfusion alternatives • Red cell
substitutes • Platelet substitutes
INTRODUCTION
The term “Blood Substitutes” was first used for plasma expanders and
later for blood components. Presently, it is applied to ex vivo produced
therapeutic materials with potential to replace or reduce transfusion
requirement of blood and blood components.1 Such materials that on
therapeutic administration can perform function of the blood
component, which is meant to be replaced, are referred to as “real blood
substitutes”. Those that help to reduce its transfusion requirement by
enhancing in vivo production of the desired component, such as
therapeutic erythropoietin, G-CSF, thrombopoietin, interleukins,
DDAVP, etc. are called “virtual blood substitutes”.2 The latter also
includes management guidelines that help to rationalize and thus
reduce the use of blood component in a given situation.1 In future, in
vitro activated stem cells and gene therapy may become major players
in this field.3
REAL BLOOD SUBSTITUTES

Among the real blood substitutes, major development has occurred
for two components: (a) red blood cell substitutes, and (b) platelet
substitutes.
Need for Development of Blood Substitutes

The following arguments appear convincing:
1. With rapid advances in surgical sophistication and increasingly
aggressive protocols of cancer management in the recent past, the
transfusion requirements of both red cells and platelets have
outgrown human resource. Transfusion alternatives and blood
2 Recent Advances in Haematology
substitutes are necessary to narrow the gap between demand and

supply.4
2. Risk of transmissible infection such as HIV, HVB, CMV, malaria,
and syphilis, Variant Creutzfeldt Jacob Disease, etc. with blood
transfusion has occupied front line attention. 5 Although,
mandatory regulations for pre-transfusion screening of allogenic
blood have now reduced the panic, yet no component can be
declared 100 per cent safe. Moreover, there is no guarantee that
some new infectious agent will not emerge.6 Blood substitutes
which can be subjected to vigorous in vitro sterilization can
alleviate both the risks and costs in this regard.
3. Sudden transfusion requirements triggered by massive blood loss
in accidents, natural disasters, conflicts and war cannot be entirely
met with allogenic blood transfusion. Off-the-shelf available blood
substitutes can be life saving.7
4. Immunogenic risks of allogenic transfusion like incompatibility
reactions; graft-versus-host reaction, allergic and febrile reactions
and transfusion related lung injury, though presently much
reduced, couldn’t be completely ignored. Success to enzymatically
scrub or cloak red cell membranes of blood group antigens is still
elusive.8 The cost of donor blood collection, transport, storage in
cold, compatibility testing, processing for leucocyte depletion,
irradiation and other manipulations is enormous. Blood
substitutes that are devoid of immune identity markers and thus
universally acceptable help to cut across all these problems.
5. Limited shelf life of allogenic components resulting in wastage of
beyond-the-expiry-date unused packs are a serious economic
handicap. Blood substitutes on the other hand, show promise of
much longer shelf life, even for several years when packed in
lyophilized state.
6. It is also important to realize that some people on account of their
religious belief (Jehovah’s witnesses) are prohibited from taking
blood from other persons. 1 Blood substitutes are the only
alternatives.
Efforts toward development of a desired blood substitute have ,
therefore, been directed to produce a product that (a) is available as
non-toxic, stable, pre-packed material that can be easily stored and
transported, has long shelf life and can be easily reconstituted for
immediate administration (b) should withstand vigorous sterilization
against viruses and bacteria (c) is completely non-immunogenic and
universally acceptable without compatibility testing (d) is cost effective
for mass pharmaceutical production and above all (e) should be bio-
effective with efficacious performance of the functions of the blood
component that it is meant to substitute.
Blood Substitutes 3
DEVELOPMENT OF RED CELL SUBSTITUTES

Although efforts have been made to develop blood substitutes for all
the cellular elements, yet it is the red blood cell substitutes that have
attracted most of the researches and resources. By far the largest
transfusion requirement pertains to red blood cells all over the world.
According to the estimates of pharmaceutical industry, nearly 12-20
billion dollar market exists for a red cell substitute.
As a result of massive research and competition in pharmaceutical
industry, three categories of red cell substitutes have emerged:
• Cell-free haemoglobin solutions
• Encapsulated haemoglobins
• Perfluorochemical emulsions.
Cell-Free Haemoglobin Solutions

The most important function of red blood cells is to carry oxygen and
carbon dioxide by virtue of its haemoglobin content. Therefore, in search
of an alternative to red blood cell transfusion, RBC free haemoglobin
solution has rightly attracted the attention. However, the following
major problems were encountered in its use and have demanded
solutions:
1. When outside the RBC, the normal tetrameric molecular
configuration of haemoglobin molecule undergoes rapid
fragmentation into dimeric form, which is imbued with the
following untoward effects:
i. Haemoglobin dimers are easily filtered through renal glomeruli
and precipitated in proximal convoluted tubules causing
serious renal damage.1,9,10 Acute renal failure as hallmark of
intravascular haemolysis is well known.
ii. Dimeric fragments have increased ability to transmigrate
endothelial cell barriers and act as endothelial cell toxin. Iron
moiety of haemoglobin molecule provides excellent nidus for
increased free oxygen radicals.9
iii. Dimeric fragments rapidly bind nitric oxide and thus result in
significantly increased vasoconstriction causing hypertension
and oesophageal spasm. Scavenging of nitric oxide also wipes
away its protective action on endothelial cells from the
damaging effect of platelet and white cell binding.11
2. Outside the red cell milieu, free haemoglobin looses its natural
buffering environment of 2,3-diphosphoglyceraldehyde (2,3-
DPG). This adversely affects the binding and release of oxygen
by the free haemoglobin molecule. Normally, in the oxygen
dissociation curve that is depicted by sigmoid pattern, 50 per cent
oxygen saturation (P 50) of RBC enclosed haemoglobin is at
approximately 26 torr. Once removed outside the erythrocyte, free
haemoglobin has much greater affinity for oxygen with the oxygen
dissociation curve moving considerably to the left with P50 at
around 15-16 torr,12 meaning thereby, that free haemoglobin does
not release oxygen as easily as when it is inside the RBC. When
free haemoglobin solution is utilized for exchange transfusion in
animals, the mixed venous oxygen saturation that reflects tissue
oxygenation may drop drastically, though haemoglobin itself is
well saturated.12
3. Intra-erythrocytic environment provides for the important
reductive enzymes and mechanisms which combat the very
oxidative nature of oxyhaemoglobin.
4. RBCs contain catalase, superoxide dismutase and other enzymes
that are significantly important in counteracting tissue injury
caused by free oxygen radicals.10 It is well known that ischaemia
consequent to lack of tissue oxygen supply (such as in haemor-
rhagic shock, stroke and other causes of inadequate circulation)
leads to production of hypoxanthin. When the ischaemic tissue is
re-perfused with oxygen, xanthin oxidase converts hypoxanthin
to superoxide that results in formation of free oxygen radicals.
Red cell enzymes, quite importantly, help to prevent this.
Superoxide dismutase converts superoxide to hydrogen peroxide
and catalase breaks up hydrogen peroxide to water and oxygen.
Ex-RBC free haemoglobin devoid of these red cell enzymes is,
therefore, incompetent to prevent “re-perfusion tissue injury”.13
The above mentioned problems in using cell-free haemoglobin as
red blood cell substitute have engaged researchers for many years. To
overcome these difficulties, three strategies have emerged leading to
development of the following products
1. Modified haemoglobins
• Polymerized (cross-linked) haemoglobins (or Polyhaemo-
globins)
i. Inter-molecularly cross-linked haemoglobins
ii. Intra-molecularly cross-linked haemoglobins
• Conjugated haemoglobins
2. Recombinant haemoglobins
3. Encapsulated haemoglobins (artificial RBC)
4. Perfluorochemical emulsions.
CROSS-LINKED HAEMOGLOBINS (MODIFIED HAEMOGLOBINS)

Taking advantage of the existence of many amino-groups (rich in lysin
residues) on the surface of haemoglobin molecule, it has been possible
to cross-link several haemoglobin molecules (inter-molecular cross-
linkage) to create haemoglobin molecule of larger size and higher
Blood Substitutes 5
molecular weight. Such polymerized haemoglobins (polyhaemo-

globins) show two important advantages:
i. These do not break into dimeric fragments in circulation and are
thus devoid of renal toxicity and endothelial cell damage. Nitric
oxide scavenging is also eliminated which obviates the risk of
hypertension and oesophageal spasm.1
ii. Change in surface configuration of polymerized haemoglobins
renders them unrecognizable by the host immune system. Thus
these polyhaemoglobins are non-immunogenic and universally
acceptable for transfusion without cross-matching.14
Inter-Molecularly Cross-Linked Haemoglobins

First attempts for inter-molecular cross-linking were made with
bifunctional “diacid” reagent-diaspirin, 15 but later replaced by
gluteraldehyde.16
Northfields Lab’s “polyHaeme” and Biopure’s “Haemopure” utilized
gluteraldehyde for cross-linking human and bovine source haemo-
globin respectively. Addition of 2,3-DPG analogue–pyridoxal phosphate
to cross-linked haemoglobin improved their P50.17 Other cross-linkers
are being developed, some of which have the dual function of cross-
linking and acting as 2,3-DPG analogues.17 One such approach presently
in clinical trials involves the use of a dialdehyde prepared from
oxidizing a sugar molecule to form open ring raffinose. Hemosol’s
“Hemolink” utilizes this approach. “O-raffinose” polymerized haemo-
globin shows good P50 without addition of 2,3-DPG.18
Intra-Molecularly Cross-Linked Haemoglobins

Cross-linkers mentioned above have been used for both inter-molecular
and intra-molecular polymerization. Specifically, intra-molecular cross-
linking of 2-beta subunits of haemoglobin molecule is brought about
with a bifunctional agent, 2-nor-formopyridoxal 5-phosphate which is
also a 2,3-DPG analogue.17 Another 2,3-DPG-pocket modifier, bis (3,5-
dibromosalicyl) fumerate has been successfully used for intra-molecular
cross-linkage of 2-alpha subunits of haemoglobin molecule. Intra-
molecular cross-linking has been successful in preventing dimer
formation and shifts the oxygen dissociation curve to the right, thus
improving P50 to facilitate oxygen release. Many other bi-functional
2,3-DPG-pocket modifiers are also in the pipeline.10
Conjugated Haemoglobins
Conjugated haemoglobins are obtained by cross-linking soluble
haemoglobin polymers to other large inert molecule. Apart from the
usual advantages of cross-linked polyhaemoglobins, conjugated
haemoglobins show improved circulation time (T/2 bio-availability)
after infusion.4
Apex biochemical’s and Ajinomoto of Japan have experimented with

conjugation of polyoxyethelene to human pyridoxilated polyhaemo-
globin17 and Enzon Inc. has used polyethylene glycol to conjugate bovine
haemoglobin.
RECOMBINANT HAEMOGLOBINS
With advances in bioengineering technology, recombinant haemoglobin
has been successfully produced in E.coli. 19 Somatogen who have
pioneered this technology claim that their recombinant haemoglobin
“Optro” in which fusion of 2-alpha subunits has also been achieved,
retains its tetrameric structure without breaking up into dimers, thus
eliminating the renal toxicity and reducing the other toxic effects caused
by dimeric fragments.20,21 Further modifications have resulted in
achieving improved P 50 and a second generation tailor-made
recombinant haemoglobin has been created in which the receptor site
for nitric oxide has also been blocked.22
Usage and Limitations of Modified Haemoglobins

Since modified haemoglobins produced by cross-link polymerization,
conjugation and recombinant technology are resistant to fragmentation
into dimers, renal toxicity and nitric oxide scavenging has been
considerably controlled and eliminated. Additional linkage with 2,3-
DPG pocket modifiers to cross-linked haemoglobins has improved their
P50 for oxygen delivery. Careful filtration to completely remove all cell
membrane fragments and stromal elements has almost completely
eliminated the dangers of complement activation and endothelial cell
micro-vascular toxicity. The risk of re-perfusion tissue injury that had
remained unsolved with first generation modified haemoglobins has
also been diminished by successful addition of superoxide dismutase
and catalase enzymes to cross-linked haemoglobins.23,24
All modified haemoglobins can be subjected to rigorous sterilization
against transmissible infections. These can be conveniently stored and
transported. Devoid of immunogenicity, the modified haemoglobins
can be used as universal therapeutic agents without risks of
incompatibility. These can be easily administered by paramedicals, like
any other pharmaceutical intravenous fluid.
Therefore as oxygen carriers, modified haemoglobins have come a
long way to be accepted for human use. Clinical trials (see update
below) for some of these products are in phase III, with applications
already with Regulatory State Health Authorities for approval.
However, a serious limitation that has, so far, defined all efforts is
their short bio-availability (25-30 hours) after infusion.25 Compared with
RBC-enclosed haemoglobin that circulates for nearly 3 months,
bioavailability of modified haemoglobins for 30 +/– hours is only
meager. Therefore, it is suitable only for short-term or emergency blood
Blood Substitutes 7
replacement or as bridge transfusion until compatible allogenic blood

becomes available. Some of these conditions, in which modified
haemoglobins have found effective application include cardio-
pulmonary bypass surgery, haemorrhagic shock, and emergency
resuscitation in natural disasters, accidents and conflicts/war
casualties.7,25 Medical uses include cardioplegia, balloon angioplasty,
thrombosis and embolism to improve distal tissue oxygenation where
RBC cannot reach. In addition, modified haemoglobins have been found
to be beneficial in normo-volumic exchange transfusion and in auto-
transfusion.4
Clinical Trials Update of Modified Haemoglobins

Several clinical trials with modified haemoglobins as red cell substitutes
have been conducted. The results reported until August 2001 have been
reviewed by Chang26 and are summarized here.
i. Northfield Laboratories, in 1998 reported the results of their
prospective randomized trial comparing therapeutic benefit of
gluteraldehyde cross-linked human haemoglobin “Polyhaeme”
with allogenic red cell transfusion in the treatment of haemorrhagic
shock.27 In 1999, they reported a randomized trial of polyhaeme
in acute trauma and in emergency surgery. Polyhaeme was shown
to maintain satisfactory total haemoglobin concentration and
helped to reduce the need for donor blood transfusion to the extent
of nearly fifty per cent. Now well-into Phase III clinical trial, they
have reported no side-effects after having infused 10,000 ml of
their product. FDA approval is awaited. (http://
www.northfieldlabs.com/polyhaeme.htm).
ii. Biopure reported in 1999 that their gluteraldehyde cross-linked
bovine polyhaemoglobin “haemopure” has been infused in large
amounts and repeatedly. It has shown no side effects with infusion
amounts up to 10,000 ml in human subjects. In a single blind multi-
centric study in the year 2000, involving 72 patients requiring
aortic surgery, their product HBOC-201 could help to reduce
allogenic blood transfusion requirements significantly.28 It has
been accorded FDA approval for routine use in veterinary
medicine for treatment of canine anaemia.29 For human use,
approval has been given in South Africa selectively for surgical
patients with acute anaemia. In June 2000, Biopure have reported
having safely and successfully used their bovine polyhaemoglobin
in a patient with auto-immune haemolytic anaemia.30 Recently,
(August 2001) they have described findings about its safety and
efficacy in pivotal phase III clinical trials (http://
www.biopure.com)
iii. Hemosol’s o-raffinose cross-linked polyhaemoglobin “hemolink” has
completed phase III clinical trials in coronary artery bypass
surgery (CABG) in Canada and UK and is awaiting regulatory

approval for its routine use. Their recent analysis has confirmed
that it was safe in cardiac surgery patients and that no clinically
significant side effects were observed.31 Hemosol have also
completed their phase II clinical trials for using “hemolink” in
orthopaedic surgery and acute anaemia.26
iv. Enzon have reported on their phase II clinical trials with
polyethyleneglycol (PEG) conjugated bovine haemoglobin for
sensitizing tumours to chemotherapy in cancer patients.25
v. Apex Bioscience (Ajinomoto) is currently involved in clinical trials
using polyethylene human pyridoxolated (PHP) conjugated
haemoglobin for scavenging nitric oxide in septic shock and a
few other applications.18
vi. Baxter had produced intra-molecularly alpha-cross-linked human
haemoglobin “Hemassist” by using bis- (dibromosalicyl) fumerate
and were vigorously persuing FDA approval in 1998, when they
suddenly discontinued due to safety concerns. Later, joined by
Somatogen they have now been involved in developing recombi-
nant haemoglobin, with high P50. They have also succeeded in
creating a second generation recombinant haemoglobin in which
the receptor for nitric oxide has been successfully blocked, thereby,
counteracting vaso-constrictive effects when infused into
animals.22
ENCAPSULATED HAEMOGLOBINS (ARTIFICIAL RBC)

First attempt to encapsulate free haemoglobin with artificial membranes
mimicking RBC was reported in 1957.32 The membrane enclosed
haemoglobin exists in tetrameric state and exhibits oxygen dissociation
properties similar to natural RBC-contained haemoglobin. It remains
protected from breakdown into dimeric fragments.32 Also, 2,3-DPG
could later be added into the membrane enclosure, in addition to other
enzymes like carbonic anhydrase and catalase. The encapsulated
catalase could be successfully used for antioxidant effect against
hydrogen peroxide in experimental animals.33 These contents could be
membrane enclosed in 1-5 μm diameter spherules mimicking red blood
cells. But the major problem encountered was their rapid removal from
circulation by reticulo-endothelial cells allowing circulation time of only
a few hours. An observation that removal of sialic acid from the
enclosing membrane material further enhanced their disappearance
from circulation proved to be of significant value. Preparations of
artificial cells with modification of surface properties including addition
of sialic acid rich polysaccharides and negative surface charge has
helped to improve circulation time. Yet, it was not enough for practical
application. Further attempts to enhance circulation time saw the
development of smaller 0.2 μm diameter lipid membrane enclosed
Blood Substitutes 9
spherules. Several workers have since carried out extensive research

and have developed bilayer lipid membrane material including
addition of sialic acid analogues and appropriate modification of surface
charge.34 This has resulted in improving the circulation time of such
artificial RBC to more than 24 hours.34
Extensive further work is on-going. For example:
i. Rapid progress has been made to solve the problem of methaemo-
globin by using artificial reductive systems and by addition of
methaemoglobin reductase enzyme to the haemoglobin containing
lipid spherules.33
ii. Studies for interaction with human complement by using in vitro
screening methods have almost been completed.26
iii. Lipid human haemoglobin vesicles have successfully been used
in treatment of massive haemorrhagic shock in experimental
animals.35
iv. No adverse changes have been observed in histology of brain,
heart, kidneys and lungs in experimental animals, even on
repeated infusions.35
v. Large scale commercial production appears quite feasible and it
is likely that clinical testing will be carried out soon.34
Along with development of bilayer lipid membrane material, efforts
have also succeeded to develop biocompatible and biodegradable
polymer membrane material to prepare liposome enclosed haemo-
globin. The biodegradable polymers like polyactide and polyglycolide
provide greater strength and more porosity to the enclosing membranes,
thus making the spherules permeable for glucose entry from outside
plasma and for exit of deleterious metabolic products from inside the
microspherules.36 Lately, by using nano-technology, it has now been
possible to create nano-capsules of 80-200 nanometres in diameter with
biodegradable membrane material to function as artificial RBC.37
Further, it has been possible to increase the content of haemoglobin in
these nano-capsules. Superoxide dismutase and catalase can also be
added in addition to methaemoglobin reductase system with promising
results of conversion of methaemoglobin to haemoglobin in in vitro
studies.37 Such nano-capsules have been successfully infused into
animals to the extent of one-third of their blood volume without any
deleterious effect.37 Attempts to increase their circulation time are in
active pursuit.
PERFLUOROCHEMICALS
Perflurochemicals (PFC) are fluorinated (hallogenated) hydrocarbons.
The very concept of their use as red cell substitutes is based on the fact
that these chemicals have exceptionally high solubility for nonpolar gasses,
and are one of the most stable compounds.38 Another unique property
of these compounds relevant to their oxygen carrying ability is that
they dissolve and release oxygen (and also carbon dioxide) along simple
partial pressure gradient for diffusion.39
Physico-Chemical Properties of Perfluorochemicals39,40

To fully understand the importance of perfluorochemicals as red blood cell
substitutes it is important to describe their relevant physico-chemical and bio-
effective properties. This is followed by an account of their development as
oxygen carrying blood substitutes for human use.
a. PFCs are hydrocarbons that are produced by substituting single
hydrogen of each carbon atom of the benzene ring with fluoride
radical in the selected hydrocarbon base.
b. PFCs are extremely stable compounds in which non-polar gasses
(such as respiratory gasses) are highly soluble. Although much of
the research on these compounds has been aimed at oxygen
transport, but these can also carry other gasses in solution, such
as nitrogen, carbon dioxide, xenon, argon, hydrogen and helium.
c. Oxygen solubility in PFC is dependent on its equilibrium with
atmospheric partial pressure of oxygen. In fact, this is the single
important determinant factor for the amount of oxygen dissolved.
Gasses move in and out of this liquid against partial pressure
gradient for simple diffusion. Unlike the haemoglobin molecule,
oxygen is not chemically bound to PFC. Instead it is simply held
in solution. In pure form, PFC can carry 40-60 volumes per cent
oxygen at 100 per cent partial pressure of oxygen at sea level.
This reflects into PFC capacity of carrying 21 volumes per cent of
oxygen in blood when it is in equilibrium with 100 per cent oxygen
in the inspired air. However, if the gas pressure is increased,
theoretically there is no limit to gas solubility. This fact may assume
unique application in undersea medicine.
d. Pure PFC exists as liquid oil. Because of this, the following two
points are important for its biological use.
i. Being an oil, it is completely immiscible with blood. This means
that PFC cannot be administered into the bloodstream in pure
form. Therefore a stable non-toxic emulsion must be created
to take advantage of its immense oxygen carrying capacity.
When emulsions are made, a significant amount of dilution
takes place that limits its oxygen transport ability depending
upon the proportion of PFC that can be driven into the
emulsion
ii. Depending upon the volatility of the particular hydrocarbon
chain, pure PFC may have tendency to vaporize at body
temperature. Therefore, for biological use, it is important to
select the parent hydrocarbon chain that is not volatile at 37°C.
At the same time, it should not have such a high boiling point
Blood Substitutes 11
that it does not vaporize at all and thus create problems of

clearance from the body.
Physico-Chemical Properties of PFC in Relevance to Bio-

Application as Blood Substitute38-42
a. Since it is necessary to emulsify pure PFC, search for suitable
emulsifiers has been important. The earlier used emulsifying
agent-Pluronic F68 used in “Flusol-DA-20 per cent” (see et seq)
was found to activate complement and was hepatotoxic. Although
further investigations have led to safer emulsifiers, but search is
on going.
b. A significantly important physical property of PFC is that the
amount of oxygen dissolved in this liquid is chiefly determined
by the atmospheric pressure of oxygen with which it is in
equilibrium. Based upon this property, the following considera-
tions are important in relevance to its bio-use as oxygen carrier.
i. To increase the oxygen carrying capacity of PFC, it is important
that the patients breathe a high inspired oxygen concentration.
Therefore, the patients in haemorrhagic shock and those with
poor lung perfusion may not be able to make full use of oxygen
carrying capacity of PFC. On the other hand, however, in
circumstances such as cardiopulmonary bypass, the membrane
oxygenator might well provide the perfect way to utilize the
enhanced oxygen carrying transport capabilities of PFC.
ii. Since oxygen in PFC is not chemically bound and its release is
determined simply on flow across a diffusion pressure
gradient, all the oxygen dissolved in PFC is potentially
available for metabolic use. Similarly, other respiratory gasses
such as nitrogen and carbon dioxide from tissues to the lungs
can be completely removed from circulation in one pass, if there
is no partial pressure of these gasses in the lungs.
iii. After being cleared from circulation by reticulo-endothelial
cells, pure PFC is transiently lodged in the liver, but without
any hepato-toxicity or release of hepatic enzymes. From the
liver, it is slowly carried back to the lungs where it vaporizes
and is exhaled as colourless and odourless vapors. There is no
hepatic metabolism or renal clearance. A small proportion
(nearly 10%) may also be transpired through the skin.
DEVELOPMENT OF PERFLUOROCHEMICALS FOR

THERAPEUTIC USE
The first generation perfluorochemical was developed by Green Cross
Corporation, Japan. Their product “Fluosol-DA-20 per cent” was a 20 per
cent w/v emulsion of two chains of fluorocarbons (7 parts of
perfluorodecalin and 3 parts of perfluorotripropylamine). It was

emulsified in Pluronic F68, into which only 10 per cent of pure PFC
could be driven, the rest of 90 per cent being emulsifier.43 Because of
the low concentration of pure PFC in “Fluosol-DA-20 per cent”
emulsion, its oxygen dissolving capacity was limited. It could not carry
more than 1 volume per cent of oxygen. Moreover, the emulsifying
agent (Pluronic F68) also showed considerable hepatotoxicity, inhibition
of leucocytes and complement activation38 The total administrable dose
of “Fluosol-DA-20 per cent” was, therefore, limited to less than 500 ml.
Moreover, limitations of “Fluosol-DA-20 per cent” also included the
need for stem emulsion to be stored in frozen state and the need to
thaw and reconstitute before use; the whole exercise is taking about an
hour.38
Because of these reasons, its general use as oxygen carrying blood
substitute could not be supported. However, it was found effective in
patients undergoing balloon angioplasty in as much as it could supply
oxygen efficiently to the tissue beyond the inflated balloon catheter.44
Patients who received “Fluosol-DA-20 per cent” had fewer myocardial
infarctions and had much better outcome than those who did not receive
this infusion.45 FDA approval for “Fluosol-DA-20 per cent” was, thus,
accorded for its use in this limited indication. The other first generation
perfluorochemicals, Emulsion II and Perflubron (from China and Russia
respectively), and Oxypherol (from Green Cross) were developed but
could not be promoted.
The second generation perfluorocarbons such as “Oxygent” of Alliance
Pharmaceuticals (58 w/v perfluorodecyl bromide) with egg yolk lecithin
as surfactant46 and “Oxyfluor” of Hemogen (76% w/v perfluorodi-
chloroctane) with sunflower triglycerides and egg yolk lecithin as
emulsifier have shown greater promise. The higher concentrations of
pure PFC that can be driven into these emulsions, have made it possible
for these products to carry significantly larger amounts of oxygen,
provided the patient breathes 100 per cent oxygen in inspired air.47
With change in emulsifier (from Pluronic F68 to egg yolk lecithin),
hepato-toxicity and complement activation have nearly been eliminated
when used in controlled doses. These also showed improved stability
characteristics, hence longer shelf life and better bio-compatible and
excretion properties.
In phase II clinical trials, “oxygent” when used in the dose of
0.9 g/Kg has been able to help avoid the need of up to 2 units of blood
in surgical patients. The present emphasis is, therefore, to study the
use of PFC in surgical patients with the objective of reducing the need
for some amount of blood during surgery.48 Moreover, with the help of
PFC infusion, surgery could be initiated at lower haematocrit, thereby
reducing intra-operative blood loss.45 Its use in autologous transfusion
has also been appreciated.48
Among the other applications of second generation PFC, the

following are significant:
i. The use of PFC in lower dose levels is logical in patients with
thrombosis, atherosclerosis and other causes of vascular
obstruction, wherein the small particle size of emulsion has the
advantage of finding its way past the area of vascular occlusion.39
This together with increased oxygen pressure in the inspired air
may help to alleviate the ischaemic damage in the affected tissue
beyond the occluded blood vessel.
ii. PFC enhances oxygen solubility in plasma by 25-100 folds. Oxygen
cannot normally be carried in plasma since plasma is a very polar
fluid. With PFC present, any oxygen molecule that is released by
haemoglobin can travel down the column of PFC and follow a
diffusion gradient rapidly to its target mitochondria.39
iii. Presently, air embolism is a serious problem in several surgical
situations, particularly in neurosurgery. The almost universal
problem of neuro- psychiatric dysfunction after cardio-pulmonary
bypass is believed largely to be due to micro-air-embolism. Near
elimination of air embolism has been achieved in a number of
animal studies in cardio-pulmonary bypass surgery with addition
of PFC to the bypass prime.39,49
iv. Several small animal studies have shown PFC to be excellent in
treatment of decompression sickness. It is, though, not clear
whether direct bubble absorption occurs through enhanced
nitrogen solubility in PFC (nitrogen is 1000-10000 times more
soluble in PFC) or actually unloading and quick perfusion of
oxygen is responsible.41
v. PFC has also been investigated for its use as adjunct to
chemotherapy and radiotherapy for solid tumours. Like free
haemoglobin preparations, it increases the oxygen content in the
center of the tumour and thereby enhances its susceptibility to
chemotherapy and radiotherapy.42
vi. By virtue of the small size of emulsion particles, PFC is also carried
in lymphatics and, therefore, significant tissue oxygenation can
occur through trickle flow. This should be an important aspect of
its use in acute angina, stroke and transient ischaemic attacks.40
vii. The pure perfluorocarbons (without emulsifier) are being
investigated for use as (i) respiratory tract infiltrates through liquid
ventilation medium for both adult and paediatric respiratory
distress syndrome39,46,50 (ii) as priming fluid for cardiopulmonary
bypass machines39 (iii) as perfusate for isolated organs39,51 and
(iv) in surgical tools in ophthalmology.52
viii. Radio-opaque and acoustic impedance properties of some of the
perfluorochemicals (Perflubron) have been utilized for contrast
enhancement for X-rays in gastro-enterology and bronchoscopy

and as target specific contrast agent in imaging technology.53
Thus the promise of perfluorochemicals beyond their use as blood substitutes,
in several other applications is also encouraging and exciting.
The greatest merit of perfluorochemicals is that these are synthetic
agents which are accessible for mass production by chemical means
without dependence upon donor blood or other biological sources.
Simply, being chemical compounds, these can be subjected to intense
sterilization, can be stored and transported without cold, are non-
immunogenic; thus universally acceptable and can be safely infused
like other pharmaceutical fluids.
However, significant restrictions for use of PFC today, concern
i. limitation of administrable dose to restrict hepato-toxicity and
complement activation
ii. retention in reticuloendothelial tissues and delayed clearance
iii. need for the patient to breathe 100 per cent oxygen in inspired air.
PLATELET SUBSTITUTES
Need to Develop Platelet Substitutes
Like in case of red blood cell substitutes, it is important to consider the
reasons that have directed the efforts and research for developing
alternatives to platelet transfusions. These can be summarized as
follows:
i. Demand on platelet transfusion has been mounting rapidly not
only for its use in non-immune thrombocytopenia and platelet
functional disorders, now diagnosed more often, but more so
because, availability of platelet transfusion has allowed develop-
ment of increasingly intense cancer chemotherapy protocols.
Platelet transfusions have also made extracorporeal bypass
surgery a safer procedure. Therefore, there is universal and ever
increasing gap between demand and availability of platelet supply.
ii. Shelf life of stored platelets is too short (5 days) that restricts its
easy availability off-the-shelf and makes wastage difficult to
control.
iii. Since platelet concentrates are stored at room temperature, the
risk of introducing infection into stored platelets is not unrealistic.
iv. Donor collection of platelets through apheresis requires special
settings that are not within the means of routine health care
establishments. Maintenance of donor panels and procedural
management for donor apheresis for platelet collection has
constantly expanded the costs.
v. Development of anti-platelet antibodies to major histocompati-
bility (MHC)-class-I antigens and platelet antigens consequent to
repeated platelet transfusions is an important event that keeps on
making ever increasing demands on platelet supply.
As mentioned earlier, development efforts have been directed to

produce a product that (a) should be available as non-toxic, stable, pre-
packed material that can be easily stored and transported, has long
shelf life and can be easily reconstituted for immediate administration
(b) should withstand vigorous sterilization against viruses and bacteria
(c) should be completely non-immunogenic and universally acceptable
(d) should be cost effective for mass pharmaceutical production and
above all (e) should be bio-effective with efficacious haemostatic
performance.
Development of Platelet Substitues

Although this chapter concerns the “real blood substitutes”, yet it is
not out of order to mention that serious efforts have also been made
for helping to reduce demands for platelet transfusion with
implementation of rational guidelines for its use. And, research in
photodynamic treatment of platelets in additive solutions with
psoralens54 and ultraviolet irradiation has also helped to eliminate
transfusion transmitted infections, febrile reactions and transfusion
associated graft versus host rejection, thereby reducing wastage1,4 and
controlling avoidable increased demands.
REAL PLATELET SUBSTITUTES

The following three products have shown good promise
• Lyophilized platelets
• Infusible platelet membranes
• Fibrinogen coated micro-spheres (artificial platelets)
Lyophilized Platelets
In search for alternatives to fresh platelets for transfusion, three
modifications, that could be derived from pooled out-dated platelets
were developed and tried. These included:
i. Thawable frozen platelets
ii. Sonicated platelets
iii. Freeze-dried or lyophilized platelets
Of these only the lyophilized platelets showed promise in being as
effective as stored fresh platelets in tests of haemostasis in vitro and to
provide effective haemostasis to thrombocytopenic animals in vivo.55,56
Additionally, these could stand intense sterilization, important to
alleviate the risk of transmissible infections.
However, lyophilized platelets still remain immunogenic and can
stimulate antibody response almost similar to stored fresh platelets.
Clinical trials also remain to be completed.
Infusible Platelet Membranes

Platelet membranes that can be infused as platelet substitute are derived
by fragmentation of both fresh as well as out-dated platelets, in the
process of preparing freeze-dried preparations. These products have

been shown to promote haemostasis in animal studies and in phase II
clinical trials.57
Infusible platelet membranes can be subjected to intense sterilization
procedures and are, therefore, safer in respect of donor transmissible
infections. There is considerably reduced expression of major histo-
compatibility (MHC) class I antigens and therefore these are less
immunogenic.57
In addition, since infusible platelet membranes can be prepared from
out-dated platelets, these can be more easily accessible for mass
procurement .
Fibrinogen Coated Microspheres

It has been possible to create microspheres of human albumin coated
with human fibrinogen in an approach to produce synthetic platelet
substitute (synthocytes).58 In both animal and human studies, such
microspheres have been found to show remarkable haemostatic
qualities.58 However, their ability to promote platelet plug formation
involves interaction with recipient’s own platelets, though this can occur
quite effectively even when the host platelet count is considerably low.59
No immediate toxicity has been noticed in rodents and primates,
but studies about their safety and overall efficiency in human subjects
are still underway.
This product can be intensely sterilized for safety against transmis-
sible infections. Since, fibrinogen coated microspheres are independent
of platelet source and are devoid of platelet or MHC class-I antigens,
these are entirely non-immunogenic in terms of production of anti-
bodies to platelet antigens.
Alternative forms of synthetic platelet substitutes involving
liposomes and other carriers containing platelet receptors have also
been evolved.60 These still remain in pre-clinical trial stages.
Efficiency of all the three above mentioned platelet substitutes, that
is lyophilized platelets, infusible platelet membranes and fibrinogen
coated microspheres still remains to be studied in prophylaxis of
bleeding in severely thrombocytopenic patients. More immediate
application of these products especially fibrinogen coated microspheres
may be in improving haemostasis where the patient’s own platelet count
is moderately reduced. Their application as adjunct to therapy where
patients have become refractory to platelet transfusions through allo-
immunization is also of considerable value.
SUMMARY
The search for “blood substitutes” as alternatives to donor derived
blood component transfusion was chiefly driven by safety concerns in
terms of donor transmissible viral infections and the ever increasing
gap between demand and supply. Ready-to-use availability of

efficacious, stable, non-toxic, safe, and pharmaceutically producible
products have been the goals of intense research and development.
Convenient storage, easy transport, longer shelf life and more
importantly universal acceptability without need for cross matching
have been the development goals.
Massive efforts and resources have been expended on red cell
substitutes. Three strategies have been persued. (a) modified (cross-
linked, conjugated and recombinant) cell-free haemoglobin (b)
encapsulated haemoglobin (c) perfluorochemical (inert respiratory gas-
dissolving) emulsions. All have come a long way. There were initial
concerns about ex-RBC free haemoglobin in circulation in respect to
renal toxicity, vasoactivity (due to nitric oxide scavenging), inadequate
oxygen dissociation (due to lack of 2,3-DPG), methaemoglobin
reduction and free oxygen radical toxicity (due to absence of reduction
systems) and hydrogen peroxide accumulation (due to lack of catalase
and superoxide dismutase enzymes). Similarly, there were concerns
about perfluorochemicals in respect of endotoxic toxicity and
complement activation. Most of these problems have now been
resolved. Nano-technology based haemoglobin containing nano-
capsules (artificial RBC) to which most of the native RBC enzymes can
be added appear immensely promising. However, very short bio-
availability of all these red cell substitutes remains a serious limiting
factor. Presently, their use is thus largely restricted to emergency
resuscitation in post-traumatic/haemorrhagic shock, peri-surgical
haemodilution, acute surgical anaemia, cardio-pulmonary bypass
surgery, cardioplegia and balloon angioplasty in as much as that
requirement of donor blood transfusion can be considerably reduced
and that these substitutes act as bridge transfusion, until safe allogenic
blood is available. Some of these products have already completed
Phase III clinical trials and await regulatory approval for marketing.
Platelet substitutes include (a) lyophilized platelets (b) infusible
platelet membranes and (c) fibrinogen coated micro-spheres (artificial
platelets). Though these products largely meet the required quality
characteristics and are shown to be efficacious in animal settings, yet
their functionality depends upon the availability of at least a small
number of functional host platelets. Some of these products are in phase
II clinical trials, some in pre-clinical stage of development.
Apart from the above-mentioned blood substitutes to be used as
replacement alternatives, several pharmaceutical products and
biomaterials (like haemopoietic growth factors) have been developed,
which promote in vivo production of blood cells. These along with
rigorous implementation of regulatory guidelines to rationalize the use
of blood components render considerable help in avoiding blood
transfusion.
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in adult patients with acute respiratory distress syndrome. JAMA 1996;275:383-
89.
51. Geywe RP, Monrose RG, Taylor K. Survival of rats totally perfused with
fluorocarbon-detergent preparation. In Norman Jc, Folkman J, Haridson WG,
Rudolf LE and Veith FJ. Organ Perfusion and Preservation. Appleton Century
Crofts, New York 1968;85-96.
52. Peyman GA, Schulman JA, Sullivan B. Perfluorocarbon liquids in
Ophthalmology. Surv Ophthalmol 1995;39:375-95.
53. Mattrey RF. The potential role of perflurochemicals (PFCs) in diagnostic
imaging . Artificial Cells, Blood Substitutes and Immobilization Biotechnology-
An International Journal 1994;22:295-13.
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concentrates:Current research perspectives. Transfusion. Med Review
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to evaluate the in vivo hemostatic properties of platelets and platelet substitutes.
Transfusion Med Review 1997;12:95-105.
56. Lee DH, Blajchman MA. Novel platelet products and substitutes. Transfusion
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Transfusion 1998;38:997-98.
2
Stem Cell Transplantation
Rajat Kumar
Key Words
Autologous stem cell transplantation • Bone Marrow
Transplantation (BMT) • Stem cell transplantation
(SCT) • Cord blood transplant (CBT) • Allogeneic
BMT
INTRODUCTION
Bone marrow transplantation (BMT) or stem cell transplantation (SCT)
is a life-saving procedure for a number of malignant and non-malignant
life-threatening diseases.1,2 The procedure itself has many technical
variations according to the primary disease, age of the patient, facilities
available and experience of the centre. In India the procedure is being
carried out in many centres and more than 1000 transplants have been
performed till now. The majority of patients who have undergone BMT
will lead productive lives.
Allogeneic stem marrow transplantation involves the transplantation
of haematopoietic stem cells derived from bone marrow of an HLA
identical donor, ideally an HLA identical sibling, into the patient. In
autologous transplants, the patient’s own stem cells can be harvested
and then transplanted back in what is known as autologous transplant.3
Haematopoietic stem cells can also be collected from the peripheral
blood4, cord blood5 or foetal liver.
INDICATIONS FOR SCT

The indications for haematopoietic stem cell transplantation can be
conveniently divided into two groups (Table 2.1):
a. Malignant disorders-like leukaemias, lymphomas, multiple
myeloma and solid tumours like breast cancer, testicular cancer.
In all these indications, the cure or palliation is by the high-doses
of chemotherapy or radiation therapy, while the transplant serves
to rescue the patient from the myelotoxic effects of the anti-cancer
Stem Cell Transplantation 23
Table 2.1: Indications for stem cell transplantation

Malignant Disorders Non-Malignant Disorders
Chronic myeloid leukaemia Thalassaemia
Acute myeloid leukaemia Aplastic anaemia
Acute lymphoblastic leukaemia Fanconi’s anaemia
Multiple myeloma Combined immune deficiencies
Chronic lymphocytic leukaemia Inborn errors of metabolism
Myelodysplastic syndromes Autoimmune diseases
Hodgkin’s disease
Non-Hodgkin’s lymphoma
Neuroblastoma
Glioma
Soft tissue sarcoma
Germinal tumours
Breast cancer
Ewing’s sarcome
Ovarian cancer
Lung cancer
Other solid tumours
therapy. In allogeneic type of transplants, there is an additional

immunological advantage of graft versus cancer effect, which
contributes to the cure.
b. Non-malignant diseases-like aplastic anaemia, thalassaemia,
Gaucher’s disease, etc. In these conditions, the abnormal marrow
is deliberately destroyed and replaced by the healthy donor
marrow. In this setting, autologous transplantation cannot be
effective for obvious reasons. The use of autologous stem cell
transplantation to treat autoimmune diseases is a recent
development and is considered experimental. It has been used in
rheumatoid arthritis, juvenile chronic arthritis, systemic lupus
erythematosus, and systemic sclerosis and initial responses in most
patients were good to excellent.6
ALLOGENEIC SCT
Donor Requirement
For an allogeneic BMT, an HLA identical sibling is the ideal donor. A
sibling who is identical in the HLA-A, B, DR loci is considered HLA
identical. In spite of HLA identity, there is always variation in the minor
histocompatibility loci. These antigenic differences lead to graft rejection
or graft versus host disease unless immunosuppression is used. It is
also possible to have a successful transplant using a partially matched
sibling as a donor, or an unrelated HLA identical donor, but the
complications of graft versus host disease and graft rejection increase.7
Most centres in India are not conducting any unrelated BMTs. Unlike
most other organ transplants, in bone marrow transplantation, ABO
compatibility is not essential.8 After successful BMT, the blood group
of the recipient will also change.
Stem Cell Source

The three sources of stem cells used in haematopoietic stem cell
transplantation are the bone marrow, peripheral blood and cord blood.2
The three sources differ in the stem cell content, composition and state
of activation of immune cells. In the earliest days of bone marrow
transplantation, bone marrow was collected to achieve a dose of 1 to 3
x 10 8/kg nucleated cells which was considered as adequate for
engraftment.9
It is well-known that the peripheral blood contains a small per cent
of stem cells, approximately 0.1 per cent. This number can be increased
by administration of colony stimulating factors, like G-CSF and GM-
CSF.10 For allogeneic donors, administration of colony stimulating
factors, like G-CSF and GM-CSF for 4 to 5 days results in high circulating
stem cells which can be collected by a cell separator. The procedure
requires venous access and takes two to four hours. The donor need
not be admitted, does not require anaesthesia and is spared the pain of
marrow aspiration.11 With refinements in techniques for collection of
stem cells from the peripheral and discovery that CD34 antigen was a
marker for stem cells, it has become possible to manipulate the dose of
stem cells and lymphocytes for transplantation.
Quantitatively, peripheral blood represents the richest and cord blood
the poorest stem cell source. Peripheral blood contains more
lymphocytes than the other two sources. The most rapid engraftment
is observed with peripheral blood transplants and the slowest with
cost blood transplants. The risk of developing graft versus host disease
(GVHD) also varies with the source of stem cells. Peripheral blood
transplants show a possible increase in acute and a difinite increase in
chronic GVHD compared with bone marrow, while cord blood
transplants have a lower risk of GVHD.9 Immune reconstitution may
be better with PBSCT as there are more lymphocytes in the graft as
compared to marrow.12
Placental blood, which is routinely discarded in clinical practice, is
potentially a vast supply of allogeneic foetal haematopoietic stem cells.
An estimated 2000 patients have undergone CBT thus far.13 With sibling
donors who matched the recipients’ HLA types completely or partially,
transplants of placental haematopoietic cells in children have engrafted
and produced normal blood cells without exposing the recipients to a
high-risk of graft-versus host disease. It has also been shown that HLA-
mismatched placental blood from unrelated donors is an alternative
source of stem cells for haematopoietic reconstitution in children.14

Studies now suggest that successful long-term engraftment of placental
haematopoietic stem cells is possible in adults who receive relatively
small amounts of placental blood.15
In this article, bone marrow transplantation and differences with
the other sources of stem cells have been reviewed. Comparison of dif-
ferent cell doses achieved with the three sources is shown in Table 2.2.
ENGRAFTMENT AFTER BMT vs PBSCT

Engraftment after allogeneic HLA identical sibling BMT or PBSCT
occurs in more than 99 per cent of cases receiving adequate cell dose.
Graft failure is a risk when there is HLA disparity between the donor
and recipient, inadequate immunosuppression of the recipient, low cell
dose of the stem cells and in patients of aplastic anaemia. A number of
randomized studies have shown that engraftment after PBSCT occurs
faster than after BMT. A comparison of haematopoietic recovery after
BMT and PBSCT is shown in Table 2.3.
G-CSF accelerates haematopoietic recovery after BMT and PBSCT
while methotrexate used for GVHD prophylaxis delays engraftment.
Table 2.2: Stem cell dose in allogeneic transplants

Median Dose of Cells (Range)
BM PB CB Ref
TNC × 108/kg 3.1 (1.6-4.5) 7.0 (2.6-14) – BM vs PB16
8
TNC × 10 /kg – – 0.2 (0.1-0.63) Adult CBT17
CD 34 × 106/kg 2.4 (0.1-14.3) 6.4 (0.7-32.0) 0.12 (0.02-1.7) BM vs PB17,18
CD3 × 108/kg 0.3 (0.01-1.6) 3.7 (1.2-30.8) 0.05 (0.009-0.09) BM vs PB17,18
TNC= total nucleated cell.
Table 2.3: Haematological recovery comparing BMT vs PBSCT

Days to absolute neutrophil Days to Platelet
count > 0.5 × 109/L count > 20 × 109/L
Ref Stem Number Median Range Number Median Range
cell Of Pts Of Pts
source
19. BM 91 21 13-36 91 19 7-71
PB 81 16 11-29 84 13 5-41
20. BM 19 23 18-32 16 18 12-41
PB 18 17.5 11-28 19 11 7-16
21. BM 30 23 18-32 30 21 12-36
PB 28 17 11-29 28 13 8-90
18. BM 113 23 13-68 114 22 0-100
PB 106 19 12-35 96 16 0-100
Occurrence of acute GVHD, a higher grade of genetic disparity between

the donor and recipient, T-cell depletion of the stem cells and infections
contribute to delayed engraftment.
Conditioning Procedure
The standard preparatory regimens given prior to BMT are myelo-
ablative.22 Patients receive extremely high doses of chemotherapy or
radiotherapy or both. The aim of conditioning is threefold:
a. Eradication of malignant cells or, in cases of genetic disorders it is
eradication of the abnormal clone of cells,
b. Suppression of the immune system of the host (recipient) so that
the allograft is not rejected, and
c. Clearing a “physical space” to allow adequate growth of the donor
stem cells. The conditioning, which is myeloablative, is also toxic
to various organs like the liver, lungs, kidneys, gastrointestinal
tract and reproductive system.
Technical Aspects of Allogeneic Transplantation

The actual SCT is technically not complicated. The donor’s marrow is
harvested by repeated aspiration from the posterior and anterior iliac
crests, under general or spinal anaesthesia. The marrow is collected in
a bag with anticoagulant. Bone marrow is transfused through the veins
and the donor marrow cells home into the recipient’s marrow space
and start engrafting. Engraftment is considered established when the
peripheral neutrophil count reaches 500/cu mm on 3 successive days.
SUPPORTIVE CARE OF THE PATIENT

Protective Isolation
After transplantation of the marrow, it takes about two to three weeks
before engraftment occurs, that is the time when the stem cells start
producing adequate number of neutrophils, platelets and erythrocytes.
During this period very intensive support is required. Most centres
keep the patients in protective isolation. An analysis from the
International Bone Marrow Transplant Registry (IBMTR) of 5065
patients undergoing allogeneic transplant, indicated an early survival
benefit for leukaemia patients treated in laminar air flow (LAF) and/
or high efficiency particulate air (HEPA) filtered units in contrast to
“conventional” isolation in single rooms. The benefit was more for
transplants from donors other than HLA-identical siblings, where there
were fewer fungal infections. The report mentioned that HEPA/LAF
isolation could be an indirect indicator of unidentified aspects of
supportive care.23
In contrast, in a study of 288 patients with myelodysplasia and
leukaemia who underwent allogeneic transplantation without
protective isolation, the transplant-related mortality at 100 days was

only 1 per cent for patients having early leukaemia receiving matched
sibling transplants. For higher risk patients the mortality was 21 per
cent. These figures compare favourably with the IBMTR data, and
suggest that in some environments it may be safe to perform SCT
without confining patients continuously in hospital.24 Similar results
have been reported from other centres without increase in morbidity
or mortality.25 The most likely benefit of isolation of patients undergoing
BMT would be its effect on the acquisition of airborne pathogens such
as Aspergillus, as invasive infections from such organisms carry a high
mortality.26 In autologous SCT, a number of studies have shown the
safety and efficacy of performing these procedures in the outpatient
approach.27,28
Venous Access
The transplant process typically involves the use of a long-term, silastic,
multilumen, flexible catheter for chemotherapy administration, infusion
of stem cells and supportive care management including frequent blood
sampling, intravenous antibiotics, blood components and parenteral
nutrition.29,30
Blood Component Support

After conditioning therapy, patients require multiple red cell and
platelet transfusions during the 2 to 4 weeks period of pancytopenia,
till engraftment occurs. Patients are profoundly immunosuppressed
and at risk of developing transfusion associated-graft versus host
disease (TA-GVHD) after receiving cellular blood products.31 This is
mediated by viable lymphocytes in the blood product, which recognise
HLA differences in the host, and mount a graft versus host disease
with a mortality rate of 84 per cent. To prevent this, all cellular blood
products should be irradiated prior to transfusion, to inactivate the
donor lymphocytes.32 As BMT can be performed even with ABO
incompatibility, haemolysis may occur during infusion of ABO-
incompatible stem cells, or later as a result of the production by donor
lymphocytes of isoagglutinins directed against recipient ABO-antigens.
With proper precautions this is not a major problem.8 Major ABO
incompatibility occurs when patients have antibodies directed against
donor red blood cell antigens; minor incompatibility occurs when donor
plasma contains antibodies against the patients’ red cells. For some
patients bi-directional ABO incompatibility may be present.8 For major
ABO incompatible bone marrow transplantation, the bone marrow
should be depleted of red cells by centrifugation or sedimentation. In
peripheral blood stem cell transplantation, the volume of red cells in
the leucopheresis product is too small to be of significance. For minor
ABO-incompatible marrow, plasma depletion needs to be done if the

donor isoagglutinin titres are raised (IgG and/or IgM > 1:128).8
The ABO type of blood components transfused after SCT is defined
by the blood groups of the donor and recipient. For major or bi-
directional ABO incompatible transplants, blood group O red cells are
used. For AB group recipients with A or B donors, the donor type red
cells can be infused. This is continued till the recipient isoagglutinins
directed against the donor ABO type are undetectable for 2 consecutive
weeks. After this donor type red cells can be infused till required. For
platelet infusion the preferred ABO type is that of the donor for major
ABO incompatibility and of recipient for minor ABO incompatibility.
Haematopoietic Growth Factors

Haematopoietic colony stimulating factors (CSF) like G-CSF and GM-
CSF are often administered to patients after infusion of stem cells in
order to reduce the duration of neutropenia.33 Although G-CSF has
been shown to accelerate the time to neutrophil engraftment, the exact
timing of its administration is still not well established. In a recent study,
241 patients with breast cancer were studied with one group getting
G-CSF 5 mcg/kg starting on day of stem cell infusion, another group
getting the G-CSF five days later and a third group not getting any G-
CSF. There was a definite difference in neutrophil recovery in patients
receiving G-CSF, but no difference in the two groups getting G-CSF at
different timings. The study concluded that G-CSF should be started
on day +5 in this setting.34
Toxicity Related to Conditioning

The conventional myeloablative therapy given before infusion of bone
marrow causes organ toxicity, in addition to myelotoxicity.
Veno-occlusive Disease (VOD) of the Liver

VOD of the liver is a well-known complication in BMT patients,
responsible for significant morbidity and mortality. The incidence varies
from 10 to 60 per cent.35 This is diagnosed by the presence of at least
two of the following features within 30 days of marrow infusion:
a. Jaundice
b. Haepatomegaly and right upper quadrant pain
c. Ascites or unexplained weight pain.36 Treatment is largely sup-
portive with maintenance of intravascular volume, treatment of
secondary hyperaldosteronism and management of hepatic
encephalopathy.
Multiorgan failure (MOF) is common in severe VOD. Many different
approaches to prophylaxis have been studied. These include ursode-
oxycholic acid, heparin, ATIII concentrates, but none have been of
definite benefit. Prophylaxis with low molecular weight heparin has

shown promise in a recent trial from Israel. 37 The treatment of
established VOD is unsatisfactory. In a study of 42 patients with VOD,
tissue plasminogen activator (tPA) was given along with heparin for
severe VOD. Response was seen in 29 per cent, but 24 per cent
developed severe secondary bleeding secondary to treatment. The
authors concluded that tPA/heparin should not be given to patients
with MOF and treatment should be given early in the course of disease
or not at all.38 Recently a number of trials have evaluated Defibrotide
(DF). DF, a single stranded polydeoxyribonucleotide, is an adenosine
receptor agonist which has anti-thrombotic, anti-ischaemic and
thrombolytic properties, especially on microvasculature. DF does not
produce significant systemic anti-coagulant effects. Studies have
suggested that DF improves survival in patients with severe VOD and
MOF with minimal toxicity. A phase II randomized study has been
carried out in patients with severe VOD to determine the effective dose
comparing either a dose of 25 mg/kg/d or 40 mg/kg/d. Results showed
a promising complete response rate of 51 per cent, and day +100 survival
of 46 per cent, and favourable tolerability profile.39
Haemorrhagic Cystitis
This is characterized by the presence of haematuria, dysuria, and
urinary frequency in a patient with sterile urine. A common cause is
the use of high dose cyclophosphamide in the conditioning, and this
complication is reduced by use of mesna as prophylaxis.40
Seizures
Drug induced seizures can occur after high-dose busulphan, and in
most regimens an antiepileptic is administered prophylactically along
with the drug.
Pulmonary Complications
During the early transplant period pulmonary complications are a major
cause of morbidity and mortality. The non-infectious complications
include diffuse alveolar haemorrhage, ARDS, idiopathic interstitial
pneumonitis, or non-cardiogenic pulmonary odema.41,42
Skin and Mucosal Changes

Alopecia, nail changes and oral mucositis are common after con-
ditioning. Oral mucositis can result in painful ulcers making parenteral
nutrition necessary. Intestinal mucosal toxicity with nausea, vomiting
and diarrhoea are also seen frequently.
FAILURE OF ENGRAFTMENT
Failure to engraft after haematopoietic stem cell transplantation (graft
dysfunction) or to sustain engraftment (graft rejection) is a formidable
complication due to many possible factors. These include inadequate
stem cell numbers, infections, graft-versus-host disease and
immunological mediated processes. Bone marrow graft may get rejected
by functional host lymphocytes which survive the conditioning
regimen. In early years the immunosuppression used was inadequate,
with a higher rate of graft rejection. In an update of the Seattle
experience, 333 patients with severe aplastic anaemia transplanted
between 1970 to 1996 were reviewed.43 The rate of rejection was 35 per
cent in the period of 1970 to 1976, it decreased to 12 per cent in the
period 1977 to 1981 (p < 0.001) and was < 9 per cent in the period 1982
to 1996. Fortunately, this complication is now uncommon. Multiple
treatment alternatives have been explored including haematopoietic
growth factors, additional infusions of stem cells alone, with augmented
immunosuppression or with additional cytotoxic therapy. 44 The
incidence is higher in unrelated donor BMT and whenever there is
presence of any HLA mismatch. Depletion of the marrow of T-cells
also increases graft rejection.45
GRAFT VERSUS HOST DISEASE

In allogeneic BMT patients, a unique complication occurs: Graft versus
Host disease (GVHD).46 There are two types of GVHD, acute and
chronic.
Acute GVHD
Acute GVHD is an immunologic event which involves activation and
clonal expansion of the donor’s effector T-cells in response to the
recipient’s disparate histocompatibility antigens, and leads to injury
of the target organs: skin, gut, and liver.47 This occurs within the first 3
months after transplant. The severity can be graded according to the
extent of skin involvement, degree of hyperbilirubinaemia and severity
of diarrhoea. Comparison of Ac GVHD after BMT and PBSCT is shown
in Table 2.4. Despite the best prophylaxis, the incidence of Ac GVHD
varies from 30-60 per cent after haematopoietic SCT from an HLA
identical sibling donor and the incidence increases with a mismatched
or unrelated donor transplantation.48 Initial treatment consists of
intensifying the dose of corticosteroids. In 30 to 60 per cent of patients
steroid resistant acute GVHD develops, necessitating secondary
intervention. Anti-thymocyte globulin (ATG) is commonly used as first-
line therapy in this setting. In a study of 58 patients with steroid-resistant
acute GVHD, an initial improvement was noted in 30 per cent of
patients. Skin disease was more likely to improve with ATG (79%), while
Table 2.4: Acute GVHD (grades II to IV)

PB BM
Reference No of Patients with Ac % No of Pts with Ac %
GVHD gr II-IV/Total GVHD gr II-IV/Total
No of Patients No of Patients
19. 52/81 64 52/91 57
20. 10/20 50 9/19 47
21. 6/30 20 3/30 10
18. 51/117 44 47/107 44
Range 20-64 10-57
progression of gut and liver acute GVHD was observed in 40 per cent
and 66 per cent of patients respectively. Despite initial improvement,
52 patients (90%) died at a median of 40 days after ATG therapy from
progressive acute GVHD and/or infection (74%), ARDS (15%) or relapse
(11%).48
In unrelated BMT acute GVHD is a limiting factor. T-cell depletion
is one way of reducing GVHD in this setting. In a randomised study, T-
cell depletion/cyclosporine (TCD) was compared with methotrexate/
cyclosporine (M/C) as GVHD prophylaxis. The incidence of neutrophil
engraftment (day 42) and platelet engraftment (1 year) was similar for
recipients of the two types of prophylaxis. There was reduced the risk
of severe acute GVHD and regimen related toxicity with T-cell
depletion. Overall, incidence of grade 3 to 4 acute GVHD at 100 days
with TCD versus M/C was 0.15[CI, 0.11-0.20] vs 0.27 [CI, 0.21-0.34].34
However, it was associated with a greater risk of relapse in recipients
of chronic myeloid leukaemia in chronic phase.49
Chronic GVHD
This develops later than 100 days after transplant and often follows
acute GVHD but may also develop de novo. The mortality varies from
20 to 40 per cent.50 Comparison of chronic GVHD after BMT and PBSCT
is given in Table 2.5. Chronic GVHD is one of the most common
Table 2.5: Extensive chronic GVHD

PB BM
Reference No of patients with % No of patientss with %
extensive chronic extensive chronic
GVHD/Total GVHD/Total
No of patients No of patients
19. 37/81 46 32/91 35
20. 6/20 30 5/19 26
21. 4/30 13 2/30 6
18. 40 30
problems affecting long term survivors of allogeneic SCT.51 Up-to 60

per cent of patients surviving more than 4 months after allogeneic
transplantation develop chronic GVHD. The pathophysiology of
chronic GVHD is poorly understood. The main effect of chronic GVHD
is on immune function and infections account for the majority of deaths
in these patients. Patients have an increase in peripheral auto-reactive
T lymphocytes which act with interferon gamma to produce the
increased collagen seen histopathologically in chronic GVHD. Chronic
GVHD can be classified as per the type of onset, clinical manifestations,
or extent of disease.
a. Based on onset: Progressive-chronic GVHD evolves directly from
Ac GVHD and this has the worst prognosis; Quiescent-this follows
a period of recovery from Ac GVHD and has intermediate
prognosis; De novo- chronic GVHD develops with no prior Ac
GVHD and has a relatively good prognosis.
b. Based on clinical manifestations: Lichenoid or sclerodermatous
based on skin manifestations.
c. Based on extent of disease: Limited disease- this constitutes
localized skin involvement, with or without hepatic dysfunction,
and does not require any treatment; extensive disease- these
patients have generalized skin involvement or limited skin
involvement in association with eye involvement, oral involve-
ment, hepatic dysfunction with abnormal liver histology or
involvement of any other target organ and require treatment.
A more recent grading system has been reported which divides
patients into risk categories according to the clinical characteristics at
diagnosis.52 Three variables were found to be risk factors for shortened
survival by multivariate analysis: extensive skin GVHD involving >
50 per cent of the body surface area; platelet count of < 100,000/cu
mm; and progressive type onset. This model was validated using data
from 1108 patients from the IBMTR, Fred Hutchinson Cancer Research
Centre, University of Nebraska and University of Minnesota.51 The
median time of diagnosis of chronic GVHD is day 201 after HLA-
identical sibling transplant, day 159 after mismatched related
transplant, and day 133 after an unrelated donor transplant.53 The
clinical manifestations of major organs are summarized in Table 2.6.
The therapies for chronic GVHD are immunosuppressive and need
to be continued for a long-time, hence the diagnosis should be
confirmed before initiating therapy. Infections are the leading cause of
death among patients with chronic GVHD and they should receive
prophylaxis for PCP and encapsulated bacteria. Endocarditis
prophylaxis should be given prior to any dental or invasive procedure.
Vaccinations should be delayed till one-year after completion of
treatment of GVHD, otherwise patients will not be able to mount a
response.
Table 2.6: Manifestations of chronic GVHD

Organ Clinical Manifestation and Intervention
Investigation
Skin Erythematous popular rash Moisturise
(lichenoid) or thickened, tight, (petroleum jelly),
fragile skin (sclerodermatous). topical steroids for
Biopsy local areas.
Sweat glands Destruction leading to hyperthermia Avoid excessive heat
Eyes Dryness, photophobia, progression to Preservative free
corneal abrasion. Schirmer’s test. tears and ointment.
Mouth Dry, sensitivity to spicy food, lichen Avoid foods which
planus lesions in cheeks and tongue, are irritants. Dental
erythema and painful ulcerations. care. Topical steroid
Require viral and fungal cultures. rinses followed by
antifungal agents.
Respiratory tract Bronchiolitis obliterans can manifest Therapy is investi-
with dyspnoea, wheezing cough, gational
obstruction on pulmonary function
tests and a normal CT scan. Chronic
sinopulmonary symptoms or
infection can occur.
Liver Cholestasis. Liver biopsy is needed No specific therapy
to confirm liver involvement. is superior.
Immune system Profound immunodeficiency, PCP prophylaxis till
functional asplenia. High-risk of 6 months after no
pneumococcal sepsis, PCP, invasive GVHD and pneumo-
fungal infections. For 6 months after coccal prophylaxis
GVHD has resolved, ll patients lifelong.
should be assumed as severely
immunocompromised and asplenic.
The most widely used therapy is alternating day cyclosporine and

prednisolone. In high-risk patients treated with prednisolone alone,
the 5-year survival was only 26 per cent while with alternating day
cyclosporine and prednisolone the 5-year survival exceeded 50 per
cent.54 Patients are treated with daily prednisolone 1mg/kg per day
and daily cyclosporine at 10 mg/kg per day divided in two doses. After
two weeks, if the disease has not progressed, the steroids are tapered
by 25 per cent per week to 1mg/kg prednisolone on alternate days.
After this tapering of steroids, cyclosporine is reduced by 25 per cent
per week till to alternate day dosing such that the patient takes
cyclosporine 10 mg/kg per day in two divided doses, every alternate
day. If the disease has completely resolved after 9 months, patients are
slowly weaned off both medications, with dose reductions every two
weeks. Those with incomplete response are kept on therapy for another
3 months. If response is still inadequate, salvage regimes are required.55
There is no standard approach for patients refractory to the initial

therapy. The use of tacrolimus (FK506) is steroid resistant patients has
been reported by Tzakis et al. In their series of 17 patients with extensive
chronic GVHD, 6 showed response.56 Tacrolimus has been combined
with mycophenolate mofetil (MMF) and in 26 patients treated with
this combination, there was a response rate of almost 50 per cent.57
Etretinate, a synthetic retinoid, has been used to treat patients with
sclerodermatous and fascial chronic GVHD. In a report of 27 patients,
20 showed some improvement in skin and range of movements.58 Other
drugs which are being evaluated in various trials, usually in combi-
nation with other immunosuppressive drugs, are clofazimine, hydroxy-
chloroquin, thalidomide, Pentostatin, Rapamycin and PUVA.51
INFECTIONS
During and following haematopoietic SCT infections are expected due
to different types of immunodeficiencies, which vary from one type of
transplant to another. There are three phases for opportunistic infections
following SCT:
a. pre-engraftment,
b. early post engraftment, and
c. late post-engraftment. The duration of each phase varies according
to the type of transplant. The frequency and type of infections
can vary enormously and an overview is shown in Table 2.7.59
Pre-engraftment Phase
This phase starts early after transplant and lasts until engraftment (first
2 to 4 weeks). During this period patients develop neutropenic fever
which is commonly bacterial and, if neutropenia is prolonged, the risk
of fungal infections increases. Viral reactivation, typically herpes
simplex virus (HSV), is common. The fastest neutrophil engraftment
occurs with autologous transplant, followed by HLA identical sibling
SCT, with maximum delay with matched unrelated SCT. In each group
PBSCT allows faster engraftment than BMT. Thus, the types of infections
vary.
Early Post-engraftment
This phase ranges from the time of neutrophil engraftment till day 100
and occurs in both autologous and allogeneic transplants. It is
characterized by impaired cell mediated and humoral immunity. This
phase is prolonged if GVHD develops as both the GVHD as well as its
treatment lead to further immune deficiencies. During this phase viral
and fungal infections are common, especially in allogeneic SCT. The
most important viruses causing infections after SCT are CMV, HSV,
and VZ.60
Table 2.7: Pattern of opportunistic infections after stem cell transplantation

Period of Risk Autologous SCT Allogeneic HLA Allogeneic HLA
identical sibling identical sibling
SCT without SCT with GVHD
GVHD
Pre-engraftment
Gram-positive bacteria +++ +++ +++
Gram-negative bacteria + + +
Candida ++ ++ ++
Aspergillus + + +
HSV +++ +++ +++
Early post-engraftment
Gram-positive bacteria +++ ++ ++
Gram-negative bacteria – – +
Candida – + ++
Aspergillus – + ++
CMV + + ++
HSV + + +
Late post-engraftment
Gram-positive bacteria – – ++
Gram-negative bacteria - – +
Candida – – +
Aspergillus – – +
CMV +/– + +++
VZV ++ ++ +++
The number + and-indicate the relative risk of infection.

CMV = cytomegalovirus, HSV= herpes simplex virus, VZV=varicella zoster virus
SCT = stem cell transplantation
GVHD=graft versus host disease
Late Post-engraftment
This phase ranges from day 100 till normal immunity is regained.
Patients with allogeneic transplant are at the greatest risk. Those who
develop chronic GVHD have prolonged impairment of T-cell
dysfunction. Patients are prone to bacterial infections mainly caused
by encapsulated bacteria (S pneumoniae, H influenzae, N meningitides),
fungi (candida and Aspergillus) and viruses (CMV).61
Graft Versus Tumour Effect and Donor Lymphocyte Infections

Tumour Relapse
A successful BMT does not always mean that the primary disease is
cured. A certain number of patients will relapse from the original
malignancy, as the tumour cells survive the chemo/radiotherapy and

graft versus tumour effect.
Graft Versus Tumour Effect

In 1956, Barnes et al suggested the existence of a graft-versus-tumour
(GVT) effect when they observed eradication of leukaemia in irradiated
mice receiving allogeneic but not syngeneic marrow transplants.62
Similar effects have been shown in humans.63 A number of studies have
shown that relapse rates were least in patients who develop acute or
chronic GVHD, higher in those with no clinically evident GVHD, and
highest in recipients of allogeneic marrow depleted of T-cells and in
recipients of syngeneic marrow.64 A major component in the success of
allogeneic SCT can be ascribed to the anti-leukaemic effect exerted by
the donor T-cells.65
Donor Lymphocyte Infusions (DLI)

Further evidence for the existence of a GVT effect has come from the
use of donor lymphocyte infusions (DLI) to treat patients who have
relapsed after allogeneic transplant. In 1990, Kold et al reported the
first evidence that adoptive transfer of lymphocytes from the original
marrow donor could restore remission in patients with CML who
relapsed post-SCT.66 CML is exquisitely sensitive to immune recog-
nition, as the rate of response to DLI as assessed by most studies varied
from 60 to 73 per cent.67,68 The only major factor predictive of response
is the disease stage at the time of DLI. Patients whose disease is detected
only at the molecular or cytogenetic level fare better than those with
haematologic relapse, and among haematologic relapses, patients in
chronic phase respond better than those with advanced disease.
Following the success in CML, DLI has also been carried out in relapses
of acute leukaemia, but it is far less effective than in CML.65 European
and American multicentre studies have reported a low response rate
in acute leukaemia of both lymphoid (0 to 18%) and myeloid(15 to
29%) origin.67,68 It appears that ALL is less responsive than AML to
DLI. A possible role for DLI in the treatment of multiple myeloma
relapsing after allogeneic SCT has been suggested. In 13 patients with
relapsed multiple myeloma, 8 (61%) responded, although only 4 of the
responders achieved complete remission.69 There is inadequate data
of the role of DLI in lymphomas and solid tumours, partly because the
role of allogeneic SCT in these conditions is not fully established.
Apart from an antitumour effect, DLI can also be used for non-
malignant complications of allogeneic SCT. The cell mediated immunity
restored by this procedure is effective in reversing defective bone
marrow engraftment due to graft rejection and in displacing residual
host haematopoiesis after allografts for nonmalignant haematologic
diseases.65
DLI is associated with the complications of acute or chronic GVHD,

with significant morbidity and mortality. GVHD also correlates with
response. In the North American study the incidence of grade 2 or
higher acute GVHD was 60 per cent,67 while it was 41 per cent in the
European experience.68
NON-MYELOABLATIVE TRANSPLANTS
Rationale for Nonmyeloablative SCT
Conventional allogeneic SCT using marrow ablative doses of
chemoradiotherapy is accompanied by considerable toxicity. In most
studies the non-relapse mortality ranges from 10 per cent in good risk
patients to 20 per cent or more in patients in relapse undergoing SCT.
These rates are higher in older patients and in those receiving stem
cells from unrelated or mismatched donors.63 This has restricted the
use of conventional SCT to patients younger than 50 to 55 years. Many
of the haematalogic malignancies affect patients who are older, but they
are ineligible for allogeneic SCT due to the toxicity of the procedure.
The use of DLI to reinduce remissions in patients who have relapsed
after an allogeneic SCT, has demonstrated the effectiveness of the graft
versus tumour effect of donor lymphocytes.
This has led to the development of different less intensive preparative
regimens for the older and medically debilitated patients. The aim of
this approach is to allow engraftment of allogeneic progenitor cells to
allow graft-versus-tumour effect, and to reduce the toxicity with a
milder preparative regimen. This would also result in reduced release
of inflammatory cytokines, and therefore, diminish GVHD. A number
of different regimens have been reported for nonmyeloablative
transplantation. Some of these are shown in Table 2.8. There is
insufficient information yet available to make firm conclusions about
the antitumour effects of nonmyeloablative SCT. In general, complete
responses have been more frequently seen in patients with less tumour
burden and in patients with slower-growing malignancies such as CML,
CLL, multiple myeloma and the indolent lymphoma. The results to
date in patients with overt progressive acute leukaemia are generally
discouraging with few long-term cures. Results of some trials are shown
in Table 2.9. Until randomized trails are conducted comparing
myeloablative with nonmyeloablative regimens, patients who are
otherwise eligible for myeloablative SCT should not be treated with
nonmyeloablative transplantation.75
CORD BLOOD STEM CELL TRANSPLANTATION

The first successful cord blood transplant (CBT) was performed in a
child with Fanconi anaemia in 1988 by Eliane Gluckman.76 Cord blood
stem cells have distinctive proliferative advantages which include an
Table 2.8: Approaches to nonmyeloablative transplantation

Approach Reference
Low-dose total body irradiation (TBI)-based regimens 70.
Chemotherapy based regimens
Melphalan + Fludarabine 71.
Busulphan + Fludarabine+ Antithymocyte globulin (ATG) 72.
Cyclophosphamide + fludarabine 73.
Cyclophosphamide + ATG + thymic external beam radiation 74.
Table 2.9: Results of different non-myeloablative approaches

Refe- No. Diag- Condition- Postgraft Acute Chr Outcome
ren- of nosis ing Immuno- GVHD GVHD (No. or %
ces pati- Suppress- Gr II-IV of Patients)
ents ion
71. 86 HM F+M T+MTX 34 21 2-yr OS: 28%
2-CDA +M 2-yr DFS: 23%
72. 26 HM/GD F+B+ATG CSA 10 9 OS: 22,
2 after DFS: 21
DLI Median 240
days
73. 15 HM/ST F + CY CSA 10, 4 OS:8
1after 121-409 days
DLI posttrans-
plant
74. 21 HM CY+ATG CSA 12, NA OS: 11
± TI 6 after DFS:7
DLI Median 445
days.
OS = overall survival; DFS = disease free survival; HM = haematological malignancies;

GD = genetic diseases; ST = solid tumours; F = fludarabine; M = melphalan; B =
busulphan; ATG = antithymocyte globulin; CY = cyclophosphamide; TI = thymic
irradiation; T= tacrolimus; CSA = cyclosporine A; MTX = methotrexate; DLI = donor
lymphocyte infusion; 2-CDA = 2-chlorodeoxyadenosine.
enriched proportion of immature stem cells, higher clonogenic growth

advantage, increased cell cycle rate, autocrine growth factors production
and increased telomere length. 77 The small number and relative
immaturity of native T-cells of cord blood lymphocytes is expected to
reduce the risk and severity of graft versus host disease (GVHD).
Nevertheless, HLA matching is needed for a successful transplant.
Clinical Aspects of CBT

Cord blood transplants were initially done utilizing placental blood
collected from a sibling and although delays in myeloid engraftment
were noted, the probability of event-free survival was 72 per cent after
a median follow-up of 1.6 years.78 In 25 consecutive unrelated cord

blood transplants, of which 24 were partially HLA-mismatched,
engraftment in occurred in 23/25 patients with an overall 100-day
survival of 64 per cent.79 In most cases unrelated cord blood has been
used because a related bone marrow donor is not available. The overall
survival is nevertheless better in related versus unrelated cord blood
transplants. In a series of 143 patients reported from Europe, for
recipients of cord blood from related donors the one-year survival was
63 per cent, while for unrelated transplants it was only 29 per cent.5 A
recent study in paediatric patients showed that the outcomes of
unrelated umbilical cord blood transplantation with 0 to 3 HLA
mismatches, compared with HLA matched unrelated bone marrow
transplantation, were similar.14 The majority of patients were 1 or 2
HLA-antigen mis–matched, and these results cannot be generalised to
recipients of 3 antigen-mismatched grafts.
Engraftment Factors for CBT

In contrast to bone marrow and peripheral stem cell transplantation
where engraftment usually occurs in about two weeks time, the time
taken for engraftment is much longer in cord blood transplantation.
The median time taken to reach an absolute neutrophil count of 500/
cu mm was 30 days (range: 8-56) and to reach a platelet count of 20,000/
cu mm was 56 days (range 9-180) in the European series of cord blood
transplantation.5 The dose of nucleated cell dose available in a cord
blood unit is limited, and the dose infused in recipients is about 1 log
less than in bone marrow or peripheral blood transplants. There is
concern about engraftment on account of this lower dose. The critical
factors for engraftment are the number of cells infused and HLA
compatibility: increasing the number of cells infused decreased the
influence of HLA mismatches while the number of cells necessary for
engraftment was less in HLA identical transplants.80 The major factor
in engraftment is the number of nucleated cord blood cells infused per
kilogram of the recipient’s weight, and in a recent report 160 of 562
patients receiving cord blood transplants failed to achieve myeloid
engraftment.81 If a higher the dose of nucleated cells is infused,
engraftment is faster and the risks of graft failure are less.
Related Donor CBT

An analysis by Rocha et al compared the outcomes of HLA-identical
sibling BMT with HLA-identical CBT.82 This study compared 113
recipients of CBT with 2,052 BMT recipients transplanted between 1990
and 1997. The median time to neutrophil recovery was 26 days for CBT
versus 18 days for BMT. The cumulative incidences of grade II to IV
acute GVHD were 0.14 (95%CI, 0.08-0.22) in CBT versus 0.24 (95%CI,
0.22-0.26) in BMT recipients (p=0.02). These findings suggest that HLA-

identical sibling donor CBT is a valid alternative to BMT in the
paediatric setting.
Unrelated CBT in Children

Clinical series of unrelated CBT in children have shown haematopoietic
recovery and sustained engraftment in the vast majority with lower
than anticipated risk of acute GVHD.81
Comparison of Unrelated CBT and BMT

At the University of Minnesota, a matched pair analysis was made of
unrelated transplants, comparing paediatric recipients of 0-3 HLA
mismatched cord blood with recipients of HLA-A, B, DRB1 matched
bone marrow. The study showed that despite increased HLA disparity,
probabilities of engraftment, GVHD, and survival after 1 to 2 antigen
mismatched unrelated CBT was comparable to HLA matched BMT.14
For unrelated donor haematopoietic SCT, a simultaneous search for
both bone marrow and cord blood donors should be performed. The
selection of the unrelated donor cord blood versus bone marrow should
be based on the urgency of the transplant, the availability of 6/6
matched bone marrow donors, the cell dose of the cord blood units,
and the degree of HLA disparity of the cord blood unit. A 0 to 2 HLA
mismatched unrelated donor cord blood unit is routinely selected over
a 1 HLA mismatched unrelated bone marrow donor, provided the cell
dose of the cord blood unit is greater than 1.5 × 107nucleated cells/
kg.13
CORD BLOOD BANKING

A number of cord blood banks have been established throughout the
World. Cord blood is collected from the placenta after delivery, which
may be vaginal or caesarian, and the routine clinical management of
the delivery is not altered because cord blood is being collected. After
cleaning the cord, the umbilical vein is cannulated with the needle of a
standard blood collecting bag, and the placental blood is drained into
the collection bag and subsequently processed and cryopreserved.83
The New York Blood Centre is the largest and most active. In Europe
cord blood banking has been developed in several countries including
Germany, France, UK, Italy, Belgium, The Netherlands and Spain. These
facilities are expensive, and therefore to recover the cost, one unit of
cord blood, which is released, costs around $ 15,000.00.84
The advantages and disadvantages of cord blood transplantation
for allogeneic transplantation are shown in Table 2.10.
Table 2.10: Advantages and disadvantages of cord blood cells for

haematopoietic stem cell transplantation
Advantages Disadvantages
No risk to mother or newborn. Delayed engraftment.
Low viral contamination with Limited number of stem cells collected.
CMV and Epstein-Barr virus. Not feasible to collect additional stem
cells for DLI.
Low incidence of acute and chronic Potential risk of genetic disease
graft versus host disease. transmission.
Less HLA restriction.
Cord blood banks can provide.
HLA matched stem cells on demand.
Frozen cord blood is conveniently
shipped and thawed.
In donor registries there are donor
losses due to advancing age, disease,
or geographical relocation. This does
not occur with cord blood banks.
The concept that telomerase activity
is age dependent, suggests that cord
blood stem cells may provide longer
functional activity compared to adult
donor stem cells.
AUTOLOGOUS STEM CELL TRANSPLANTATION

Autologous bone marrow or peripheral blood stem cell transplantation
is a procedure similar to allogeneic BMT, with the difference that the
patient’s own stem cells are used for engraftment. The concept of
offering autologous stem cell transplant is to permit administration of
very high doses of chemoradiotherapy, which would otherwise be fatal
due to myelotoxicity. By first collecting the patient’s marrow or stem
cells prior to this chemotherapy, it is used to ‘rescue’ the patient from
the myelotoxicity. The procedure is mainly indicated for malignancies
which are chemo/radiosensitive like leukaemias, lymphomas, multiple
myeloma, Ewing’s sarcoma and gonadal tumours.2 Peripheral blood
stem cell transplantations (PBSCT) have virtually replaced bone marrow
for autologous stem cell transplantation. The advantage of autologous
transplant over allogeneic transplant is that there is no graft versus
host disease, and once engraftment occurs then graft rejection is unlikely.
Thus, there is a significant decrease in the complication rate as compared
to allo-BMT. However, there is a higher risk of tumour relapse as
compared to allogeneic BMT.
EUROPEAN GROUP MARROW TRANSPLANTATION (EBMT)

DATA
A recent report from EBMT reveals the changes, reflects current status,
and provides medium-term projections of haematopoietic stem cell
transplantation development in Europe.2 Data on 132 963 patients,
44 165 with allogeneic haematopoietic SCT (33%) and 88 798 with an
autologous haematopoietic SCT transplant (67%), collected
prospectively from 619 centres by the European Group for Blood and
Marrow Transplantation (EBMT) in 35 European countries between
1990 (4234 haematopoietic SCTs) and 2000 (19 136 haematopoietic SCTs)
illustrate utilization of haematopoietic SCT. These transplants increased
in all European countries and for all indications. There were major
differences depending on disease indication and donor type. Projections
on medium-term development for each disease based on a weighted
sensitivity analysis predict an ongoing increase in allogeneic
haematopoietic SCT except for chronic myeloid leukaemia. This is
because of novel therapies such as the bcr/abl tyrosine kinase inhibitor
imatinib mesylate for chronic myeloid leukaemia. For breast cancer a
lot of hopes were placed in haematopoietic SCT, but frustration about
the results have halted its use. The rapid increase in autologous SCT
for breast cancer in the early 1990s, with a peak between 1997 and 1998
was followed by a rapid decline.
Stem cell source varied over time and was dependent on donor type.
In 1990, almost all haematopoietic SCT were bone marrow derived. This
has changed within the decade. Of the 19 136 SCT in 2000, only
3555 (19%) were still bone marrow derived; 15 581 (81%) were from
peripheral blood stem cells or were combined bone marrow and
peripheral blood stem cell transplants. There are differences in stem
cell source for autologous and allogeneic haematopoietic SCT. Of the
12732 autologous haematopoietic SCTs, only 566 (4%) used bone
marrow and 12 166 (96%) used peripheral blood stem cells. Of the
6404 allogeneic haematopoietic SCTs, 2989 (47%) were bone marrow
derived and 3415 (53%) were peripheral blood stem cell transplants.
Peripheral blood was used in 57 per cent of HLA-identical sibling donor
transplants, in 81 per cent of transplants from other family members,
in 76 per cent of twin donors, and in 39 per cent of unrelated donors.
INTERNATIONAL BONE MARROW TRANSPLANT REGISTRY

(IBMTR)/ AND AUTOLOGOUS BLOOD AND MARROW
TRANSPLANT REGISTRY (ABMTR)
Since 1972, the International Bone Marrow Transplant Registry (IBMTR)
has collected and analysed outcome data from blood and marrow
transplant centres worldwide. In 1991, the Autologous Blood and
Marrow Transplant Registry (ABMTR) began collecting outcome data
on autotransplants from centres in North and South America. Currently,

492 centres participate in the IBMTR/ABMTR. The trends in use and
outcome of allogeneic and autologous haematopoietic stem cell
transplants prepared by IBMTR/ABMTR is described below.85 It is
estimated that 15,000 allogeneic and over 25,000 autologous transplants
were carried out in 2000.
Most allogeneic transplants have been bone marrow transplants, but
in 1998 to 2000 there was a steady increase in use of peripheral blood
stem cells, especially in older recipients. Over 95 per cent of autotrans-
plants in adults and 80 per cent in children and adolescents use
haematopoietic progenitor cells collected from blood. The remainder
use bone marrow alone or in combination with cells collected from
blood. There was a steady increase in the number of non-myeloablative
transplants being carried out between 1997 and 2001.
In North America the most common indications for allo- and
autotransplants differ. The most common indications for allotransplants
are acute and chronic leukaemias, myelodysplasia (MDS), and non-
malignant diseases (aplastic anaemia, immune deficiencies, inherited
metabolic disorders). Autotransplants are generally used for non-
Hodgkin’s lymphoma (NHL), MM, Hodgkin’s lymphoma, and solid
tumours. In 2000, NHL and MM were the most common indications
for transplant in North America, accounting for over one-third of all
transplants. Most allotransplants are from HLA-identical sibling donors.
However, only about 30 per cent of transplant candidates have such a
donor. Transplants from unrelated donors now account for approxi-
mately 25 per cent of allogeneic transplants.
The 100-day mortality rates are often used as a gauge of transplant
related mortality (TRM). Among HLA-identical sibling transplants
carried out 1999 to 2000 and reported to the IBMTR, 100-day mortality
rates ranged from about 10 per cent for patients with acute leukaemia
in first remission to almost 40 per cent for those with advanced
leukaemia. The 100-day mortality rates after transplants for aplastic
anaemia and immune diseases ranged between 10 per cent and 15 per
cent. Recurrence or progression of the primary disease is responsible
for over 30 per cent of all deaths following HLA-identical sibling
transplants, with GVHD and infection each responsible for approxi-
mately 20 per cent of deaths.
TRM is higher for recipients of unrelated donor transplants. The 100-
day mortality ranged from about 20 per cent for patients with acute
leukaemia in first remission to over 50 per cent for those with advanced
acute lymphocytic leukaemia (ALL) and chronic myeloid leukaemia
(CML). The 100-day mortality rates after transplants for MDS, aplastic
anaemia and immune diseases ranged between 15 and 30 per cent.
Recurrence or progression of the primary disease and infections were
the most common causes of mortality in this group. Early mortality is

generally lower following auto-than allotransplants. Among patients
receiving autotransplants in 1999–2000, those transplanted for NHL or
Hodgkin’s lymphoma, MM or acute leukaemia in remission had 100-
day mortality of < 10 per cent, while patients treated in relapse had
higher early mortality. Recurrent disease continues to account for the
overwhelming majority of deaths in autotransplant recipients.
Increasing age is associated with increased 1-year TRM after allografts.
TRM remains a significant problem, being higher than 30 per cent for
patients over 50 years of age.
DISEASE SPECIFIC RESULTS

CML
Allotransplants are an effective treatment for CML. Among 5,816
recipients of HLA-identical sibling transplants carried out for CML in
chronic phase between 1994 and 1999, reported to the IBMTR, 3-year
probabilities of survival were 69 ± 2 per cent for 2,876 transplants carried
out within 1 year of diagnosis and 57 ± 3 per cent for 1,391 patients
transplanted > 1 year after diagnosis. Unrelated donor transplants can
cure CML but are associated with higher risks of GVHD and TRM.
Additionally, unrelated donor transplants are often delayed because
of the time required to identify a donor and reluctance to risk the higher
TRM. Delaying transplantation may adversely affect outcome. For
patients receiving unrelated transplants for CML in chronic phase, the
3-year probability of survival was 54 ± 5 per cent for 613 patients
transplanted within the first year of diagnosis, and 46 ± 3 per cent for
936 patients transplanted beyond the first year from diagnosis of CML.
Among 5,126 recipients of allogeneic transplants for acute myeloid
leukaemia (AML) carried out between 1994 and 1999, reported to the
IBMTR, 3-year probabilities of survival for recipients of HLA-identical
sibling transplants were 60 ± 2 per cent for 3,298 patients in first
remission, and 44 ± 4 per cent for 837 patients in second or subsequent
remission. Survival was generally worse in 991 patients receiving
transplants from unrelated donors. The 3-year probabilities of overall
survival for recipients of unrelated donor transplants in first or second
vs subsequent remission were and 40 ± 5 and 37 ± 5 per cent,
respectively.
Among patients receiving autotransplants for AML between 1994
and 1999, reported to the ABMTR, the 3-year probability of survival
was 59 ± 8 per cent for 209 patients (< 20 years of age) and 50 ± 4 per
cent for 991 patients (> 20 years of age) transplanted in first remission.
Patients transplanted in relapse or persistent disease did poorly, with
3-year probabilities of survival of 23 ± 18 per cent for 29 patients < 20
years of age and 24 ± 7 per cent for 244 patients > 20 years of age.
ALL
Most patients with ALL are cured with conventional chemotherapy.
Consequently, bone marrow transplants are reserved for patients failing
conventional therapy, i.e. in relapse or second or subsequent remission,
or, patients in first remission with prognostic factors predicting a high-
risk of failure with conventional therapy. The most frequent indications
for transplantation in first remission are older age, high leukocyte count
at diagnosis, Philadelphia and other chromosome abnormalities and
difficulty obtaining a first remission. Among 2,820 recipients of HLA-
identical sibling transplants between 1994 and 1999, reported to the
IBMTR, 3-year probabilities of survival were 61 ± 4 per cent for 561
recipients and 48 ± 4 per cent for 909 recipients age > 20 years of age in
first remission, and 47 ± 6 per cent for 962 recipients < 20 years of age
and 30 ± 5 per cent for 388 recipients > 20 years of age transplanted in
second or subsequent remission. Although associated with higher TRM,
unrelated donor transplants may be considered for patients with ALL
unlikely to be cured by chemotherapy alone. Among 280 patients < 20
years of age and 223 patients > 20 years of age who received unrelated
donor transplants for ALL in first remission reported to the IBMTR, 3-
year probabilities of survival were 50 ± 3 per cent and 40 ± 8 per cent
respectively; among 805 recipients < 20 years of age and 215 recipients
> 20 years of age who received their transplant in second or subsequent
remission, 3-year probabilities of survival were 39 ± 4 per cent and 28
± 7 per cent respectively.
Among 416 recipients of autotransplants for ALL between 1994 and
1999, reported to the ABMTR, 3-year probabilities of survival were 44
± 9 per cent for 187 transplants carried out in first remission, 36 ± 9 per
cent for 168 transplants carried out in second or subsequent remission,
and 12 ± 9 per cent for 61 transplants carried out in relapse.
CLL
In CLL transplants have primarily been carried out for poor prognosis
patients. In 316 recipients of HLA-identical sibling transplants for CLL
between 1994 and 1999, the 3-year probability of survival was 47 ± 7
per cent. The experience with autologous transplantation for CLL is
more limited. Among 164 recipients of autotransplants for CLL and
reported to the ABMTR, the 3-year probability of survival was 84 ± 9
per cent.
MDS
Allogeneic bone marrow transplantation can cure some patients with
myelodysplastic syndromes. Among 1,095 recipients of HLA-identical
sibling transplants between 1994 and 1999, reported to the IBMTR, 3-
year probabilities of survival were 73 ± 15 per cent for 48 recipients
< 20 years of age and 49 ± 7 per cent for 255 recipients > 20 years of age
with refractory anaemia (RA) or refractory anaemia with ringed
sideroblasts (RARS). Among 97 recipients < 20 years of age and 695
recipients > 20 years of age with refractory anaemia with excess blasts
(RAEB), refractory anaemia with excess blasts in transformation (RAEB-
T), or chronic myelomonocytic leukaemia (CMML), the 3-year
probabilities of survival were 52 ± 12 and 37 ± 4 per cent, respectively.
Among 440 recipients of unrelated donor transplants reported to the
IBMTR, the 3-year probabilities of survival were 37 ± 11 per cent for 33
recipients < 20 years of age and 25 ± 7 per cent for 82 recipients > 20
years of age with RA/RARS. Among 95 recipients < 20 years of age
and 230 recipients > 20 years of age with RAEB/RAEB-T/CMML the
3-year probabilities of survival were 38 ± 22 per cent and 25 ± 7 per
cent respectively.
Aplastic Anaemia
Allogeneic transplantation is the treatment of choice for young patients
with aplastic anaemia who have an HLA-identical sibling. Three-year
probabilities of survival after 1,689 HLA-identical sibling transplants
between 1994 and 1999, and reported to the IBMTR, were 76 ± 3 per
cent for 844 patients < 20 years of age and 67 ± 3 per cent for 845 older
patients. Results were not as good in 358 recipients of unrelated donor
transplants: 53 ± 6 per cent in 244 patients < 20 years of age and 32 ± 10
per cent in 114 older patients.
Multiple Myeloma
In multiple myeloma (MM) haematopoietic stem cell transplantation
is now considered standard therapy. Survival rates are better for patients
transplanted early, compared to those transplanted more than 18
months from diagnosis. For 3,277 recipients of autotransplants who
were transplanted < 18 months from diagnosis, the 3-year probability
of survival was 59 ± 2 per cent, compared to 48 ± 4 per cent in 1,038
recipients who were transplanted > 18 months from diagnosis. The 3-
year survival rates were better for recipients of HLA-identical sibling
transplants: 47 ± 4 per cent for 642 patients transplanted within 18
months from diagnosis compared to 38 ± 7 per cent for 258 patients
transplanted > 18 months from diagnosis.
Hodgkin’s Disease
Most patients with Hodgkin’s disease are cured with conventional
chemotherapy. However, for the 20 to 30 per cent failing conventional
therapy, autotransplants are effective salvage therapy. Among 3,356
autotransplants between 1994 and 1999, reported to the ABMTR, 3-year
probabilities of survival were 81 ± 8 per cent for 184 transplants in first

remission, 76 ± 4 per cent for 734 transplants in second or subsequent
remission, 63 ± 3 per cent for 1,806 transplants in relapse, and 55 ± 5 per
cent for 632 patients with persistent disease.
Non-Hodgkin’s Lymphoma (NHL)

NHL is the most common indication for haematopoietic stem cell
transplantation. Among 1,698 patients receiving autotransplants for
follicular lymphoma between 1994 and 1999, 3-year probabilities of
survival were 81 ± 8 per cent for 150 patients in first remission, 71 ± 6
per cent for 296 in second remission, 66 ± 4 per cent for 894 in relapse,
and 63 ± 6 per cent for 358 never achieving remission with standard
chemotherapy. Relapse is less frequent but TRM is higher with HLA-
identical sibling transplants. Among 403 patients with follicular
lymphoma the 3-year probability of survival was approximately 60
per cent regardless of remission status pre-transplant.
Diffuse Large Cell Lymphoma

Among 3,676 patients receiving autotransplants for diffuse large cell
lymphoma, 3-year probabilities of survival were 68 ± 6 per cent for 362
patients in first remission, 53 ± 5 per cent for 657 in second remission,
42 ± 3 per cent for 1,746 in relapse and 49 ± 4 per cent for 911 patients
never achieving remission with conventional chemotherapy. Most
failures after autotransplants for NHL are due to relapse. Higher TRM
offsets the lower relapse rate seen with HLA-identical sibling
transplants for these lymphomas. The 3-year survival rates among 326
patients transplanted between 1994 and 1999 from HLA-identical
siblings for diffuse large cell lymphoma were 46 ± 23 per cent in 25
patients in first remission, 32 ± 9 per cent for 177 patients in relapse
and 24 ± 9 per cent for 124 patients with persistent disease.
Indian Experience
In India the first BMT was performed in Tata Memorial Hospital (TMH),
Mumbai in March 1983. At TMH, more than 200 transplants have been
done till now and at present about 30 SCTs are being performed every
year (personal communication-Dr TK Saikia). TMH has a large
experience in transplantation in chronic myeloid leukaemia.86
The next centre to start BMT/SCT was Christian Medical College,
Vellore where the first allogeneic BMT was done in 1986. Till June 2002,
335 allogeneic and 395 autologous SCTs have been done (personal
communication-Dr A Srivasatava). At present about 5 transplants are
being done every month. CMC Vellore has a large experience in
performing allogeneic SCT for Thalassaemia major.87 Their disease free
survival rates of around 90 per cent for Class II and 50 per cent for
Class III patients compare favourably with the results of the largest
centre in the world at Pesaro, Italy.
AIIMS, New Delhi is the third largest centre in the country with
about 145 transplants done till date (personal communication-Dr Lalit
Kumar). They have a large series of SCTs performed in multiple
myeloma.88 A new transplant centre is being constructed at AIIMS.
Apollo Hospitals Chennai is another large centre where stem cell
transplantation was started in May 1995. Till January 2003, 117 SCTs
have been performed. Of these, 45 are autologous, 72 allogeneic, 3 are
cord blood transplants and one is a mini-allotransplant (personal
communication-Dr T Raja). The majority have been PBSCTs and about
16 to 18 transplants are being done every year.
At Army Hospital Research and Referral, New Delhi, SCTs were
started in January 1998 and allogeneic transplants form the majority of
transplants performed.89 Till January 2003, 40 transplants have been
performed.
Other centres which have performed more than 10 transplants till
date include Cancer Institute (WIA) Adyar Chennai, SGPGI Lucknow,
Inlaks Hospital Pune, Command Hospital (Southern Command) Pune.
Smaller numbers have been performed in Rajiv Gandhi Cancer Institute
Delhi, and other hospitals in Mumbai, Kolkata, and Bangalore. In the
next few years the number of transplants performed in India will
increase sharply.
FUTURE PROSPECTS OF SCT

Gene Therapy
Haematopoietic stem cells are attractive targets for gene therapy
because of their capacity for self-renewal and the wide systemic
distribution of their progeny. Collecting a patient’s autologous stem
cells, correcting the genetic defect, and reinfusing the modified cells is
a likely mode of delivering gene therapy. Despite this promise, clinical
success has been limited by poor rates of gene transfer, poor engraftment
of modified cells, and poor levels of gene expression.90 Recent improve-
ments in retroviral transduction protocols have resulted in the first
successful amelioration of a human haematologic disease — a form of
severe combined immunodeficiency — by haematopoietic stem cell
gene transfer.91
Plasticity of Stem Cells

In the last few years, the discovery that adult tissue specific cells, such
as haematopoietic stem cells, have the ability to “transdifferentiate”
into other tissues has generated much excitement among cell biologists
and transplant clinicians.92 Experimental studies suggest that stem cells

derived from the bone marrow can differentiate into tissue such as
endothelial cells, liver, muscle, and neurones. This property, termed as
plasticity, allows adult stem cells to switch to make other specialised
sets of cells appropriate to their new location.93 This has therapeutic
potential for many liver, heart and neurodegenerative diseases. Adult
bone marrow contains haematopoietic stem cells and mesenchymal
stem cells. Adult mesenchymal stem cells can be expanded in vitro and
stimulated to form either bone, cartilage, tendon, muscle or fat cells
and this makes them attractive for tissue engineering and gene therapy
strategies.94
Autologous cell transplantation for the treatment of damaged myo-
cardium after myocardial infarction is becoming an increasingly
promising strategy. Strategies to regenerate myocardium with stem cells
either extract stem cells from the bone marrow and inject these cells
into the damaged area or they attempt to increase the efficiency of the
natural reparative process by increasing the mobilization of bone
marrow-derived stem cells after myocardial infarction.95 In a clinical
phase I trial, bone marrow derived stem cells were transplanted into
the infarcted myocardium in 10 patients who were undergoing coronary
artery bypass grafting (CABG). There was improvement in local
perfusion in 5 patients. The study showed that transplantation of
autologous stem cells into the myocardium can be safely performed,
but controlled studies are required to determine the true efficacy.96 In
spite of these encouraging reports, before stem cells can be used
therapeutically in patients with different organ dysfuntion, the pro-
perties of such cells must be characterised and the factors responsible
for their regulation defined and functionally proved.
CONCLUSION
Stem cell transplantation is curative in many potentially fatal conditions.
The procedure is expensive as a lot of resources in the form of supportive
therapy are required. Complications such as veno-occlusive disease of
the liver, acute and chronic GVHD, and infectious conditions remain
major obstacles for the success of allogeneic haematopoietic stem cell
transplantation. Despite progress, GVHD and disease recurrence remain
a challenge. In future, the promise of gene therapy and plasticity of
stem cells is likely to expand the application of stem cell transplantation
to many different clinical conditions.
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3
Apheresis
Col Velu Nair
Key Words
Apheresis • Plateletpheresis • Plasmapheresis
• Therapeutic apheresis • Leucocytapheresis
Aphaeresis (or haemapheresis) is the collection of blood from a donor

or patient followed by separation and removal of a cellular component
(s) and/ or plasma and return of the remaining blood components to
the donor or patient. Cytapheresis is the procedure by which cellular
elements of the blood, platelets (platelet apheresis or thrombocyta-
pheresis), leucocytes (leucocytapheresis), lymphocytes (lymphocyta-
pheresis ) or red cells (erythrocytapheresis), are selectively removed
from the blood. Plasmapheresis is the selective removal of plasma. It is
used to collect upto 600ml of plasma from healthy donor (without any
fluid replacement) or to exchange plasma from a patient to remove a
constituent (antibody, immune complexes, paraproteins, lipoproteins
and toxins) which is causing harm to the body. Here several litres of
plasma is removed and replaced by colloid and crystalloid solutions.
It can also be used to replace or replete a deficient plasma constituent.
Adverse effects with current techniques are infrequent and usually mild.
As a procedure it is not curative and does not change the natural course
of the disease, however, it can be life-saving in many situations and
buys time to effect more definitive therapy.
INTRODUCTION
The term apheresis is derived from a Greek verb, meaning “ to take
away or withdraw”. It involves removal of one blood component with
return of the remaining components to the donor. Like phlebotomy,
originally it was used to treat patients but later on became more
important for collection of blood components for transfusion.1 The term
apheresis is interchangeably used with haemapheresis. Flieg in 1910
was the first to experiment with plasmapheresis as a method of
removing substances from the blood. He demonstrated in rabbits first
Apheresis 57
and then in a human subject with uraemia wherein 600 ml of blood

was removed on three occasions and patients own red cells were washed
and suspended in saline and reinfused back to the patient in the process
removing plasma along with the uraemic toxins. This resulted in
substantial clinical improvement. It was not until 1960 that the technique
was applied in the treatment of human disease. With the development
of plastic bags, manual apheresis became a possibility, with the selective
removal of the desired product, usually plasma. This method has been
used primarily for the collection of plasma for fractionation. The
development of mechanical devices, called cell separators, has changed
the approach to apheresis and has resulted in the application of this
methodology to the collection of single donor products such as plasma
or platelets, as well as the treatment of certain diseases. In the USA the
annual figures for some apheresis procedures are.2
• Plateletpheresis > 6 million/year
• Plasmapheresis > 10 million/year
• Therapeutic apheresis >100,000 /year
Plateletpheresis using cell separators results in the collection of single
donor platelets which is now a days extensively used to combat severe
thrombocytopenia which results post high dose chemotherapy and
following conditioning for BMT. Apheresis is more extensively used to
harvest platelets and plasma from healthy donors than as a therapeutic
modality now a days. In the latter situation, it is referred to as a
“therapeutic apheresis” which could involve removal of blood cells
(therapeutic cytapheresis) or plasma (therapeutic plasma exchange).
In this article the main emphasis will be on various aspects of
therapeutic apheresis.
PRINCIPLE AND MECHANISM OF ACTION OF APHERESIS

(TABLE 3.1)
A. Physical removal of a circulating blood component, either cells
(cytapheresis) or some plasma solute (Plasmapheresis).
B. Most often the circulating substance directly responsible for the
disease is targeted for removal. However, it can also mobilize cells
and plasma components from tissue depots. Lymphocytes can be
mobilized from the spleen and lymph nodes in chronic
lymphocytic leukaemia (CLL) and low-density lipoproteins (LDL)
from tissue stores in patients with familial hypercholesterolaemia.
C. Most commonly available cell separators remove platelets and
lymphocytes very efficiently. However, granulocytes and
monocytes can not be cleanly separated from other cells using
standard centrifugal apheresis equipments.
D. Reinfusion of a deficient plasma factor as in thrombotic
thrombocytopenic purpura (TTP).
Table 3.1: Role of Plasmapheresis

S.No Disease/syndrome Factor to be removed
1. Cryoglobulinemia Cryoglobulins
2. Anti GBM disease Anti GBM antibody
3. Acute demyelinating polyneuropathy Antimyelin antibody
(Guillain-Barre)
4. Hyperviscosity syndrome IgM
5. Microangiopathic thrombocyotopenia Anti-endothelial cell antigen
(TTP/HUS) von Willebrand’s factor
multimers
6. Homozygous familial LDL cholesterol
hypercholesterolaemia
7. Myasthenia gravis crisis ACh-receptor antibody
8. Overdose with certain drugs e.g. Digitalis
9. Poisoning involving protein bound Amanita phalloides
toxins (herbicides, mushrooms)
10. Coagulation factor inhibitors Factor VIII inhibitor
11. Hemolytic disease of newborn IgM
E. Enhances splenic clearance of immune complexes in certain

autoimmune disorders.3
THERAPEUTIC CYTAPHARESIS
The common indication for therapeutic cytapheresis are listed in Table
3.2.
Platelet Pheresis
Therapeutic plateletpheresis (TP) is valuable for the acute management
of patients with symptomatic thrombocythaemia where a rapid
reduction in platelet count is required. It is also useful in those who
cannot tolerate drug therapy (hydroxyurea or anagrelide).4,5 TP helps
reverse clinical manifestations of myocardial or cerebral ischaemia,
Table 3.2: Common Indications for Therapeutic Cytapheresis

Red cell exchange
Sickle cell disease
Acute complications
Prophylaxis for recurrent stroke
Frequent severe pain crises
Hyperparasitemia (malaria, babesiosis)
Leukapheresis
Leukaemia with hyperleucocytosis syndrome
Cutaneous T-Cell lymphoma (Photopheresis)
Peripheral blood stem cell collection
Plateletpheresis
Symptomatic thrombocytosis
Apheresis 59
pulmonary embolism and gastrointestinal bleeding. Platelet counts can

be lowered by 50 per cent with each procedure of TP. In pregnancy
with thrombocythaemia, TP is the preferred treatment as drug therapy
is contraindicated and rapid lowering of platelet counts helps prevent
placental infarction and fetal death.6
Leucocytapheresis
This procedure is used most commonly to deplete malignant leucocytes
in both acute and chronic leukaemias to prevent or treat the leucostatic
syndrome wherein pulmonary and/or cerebral dysfunction are likely
to develop once the total leucocyte count (TLC) is above 200,000/cumm
in acute myeloid leukaemia (AML) and above 300,000/cumm in chronic
myeloid leukaemia (CML). However, it may occur in AML even when
TLC is 75,000/cumm. Hence, treatment in such situation needs to be
individualized. Many centres resort to leucocytapheresis once TLC is
greater than 100,000/cumm in AML cases, as this is considered a risk
factor for early death.7 In acute lymphoblastic leukaemia (ALL) cases
with high counts, leucocytapheresis was associated with lower
incidence of electrolyte abnormalities. Chronic leucocytapheresis is
resorted to, in cases of CML patients with pregnancy where drugs could
have a damaging effect on the fetus.
In the leukaemic phase of cutaneous T cell lymphoma (CTCL) called
sezary syndrome, repeated leucocytapheresis has helped in reducing
the circulating malignant cells also called the sezary cells. This results
in either resolution or improvement of the skin lesions.8,9 Photopheresis
where leucocytes are removed by apheresis then exposed to ultraviolet
A light in presence of 8-methoxypsoralen, then reinfused to the patient
has helped in bringing about sustained remissions in CTCL.10 The
mechanism of action is believed to be photochemical damage of DNA,
immunomodulating the malignant cell which in turn stimulates host
antitumour activity.11
Though the extent to which white cells should be lowered is not
known with certainty, in practice the endeavour is to lower the TLC by
30 to 50 per cent which can be achieved usually by processing at least
two patient blood volumes.
Erythrocytapheresis
Red cells apheresis has been used with great benefit in sickle cell disease
(SCD) during severe crises such as strokes, chest syndrome, cholestasis
and priapism.12,13,14 The goal of exchanging the patients sickle red cells
for red cells containing haemoglobin A is to interrupt the vicious cycle
of stasis, sickling and hypoxia. The proportion of haemoglobin A
required to achieve this goal is not known with certainty but most
exchanges aim for post transfusion levels of 70 per cent or more of
haemoglobin A. This procedure has also been used prophylactically in

pregnancy with SCD and prior to general anaesthesia in these patients,
though these are not yet established indications for red cell apheresis
in SCD patients.15 Red cell exchange is being used to lower parasite
loads in severe falciparum malaria16 and babesiosis.17
BLOOD COMPONENT EXCHANGE

Exchange of one plasma volume will lower the intravascular
concentration of a substance by about 65 per cent, and a second such
exchange lowers it by only 23 per cent demonstrating, thus that the
early portion of a plasma exchange is more efficient than later ones.
When IgG antibodies have to be removed then a series of single plasma
volume exchanges separated by intervals adequate to allow
reequilibration between intra and extravascular spaces is resorted to,
as these antibodies have a significant extravascular reservoir. On the
contrary since IgM is mainly intravascular, even a single plasma
exchange significantly lowers IgM levels. During such plasma
exchanges, replacement fluid is in form of 5 per cent albumin or plasma.
The former is preferred as there is no risk of viral transmission or
urticarial reactions and blood group is not a limiting factor. There is a
depletion of coagulation factors with transient prolongation of
prothrombin and partial thromboplastin time following plasma
exchange. All coagulation proteins return to normal levels within 6-24
hours except fibrinogen.18,19 IgG and IgM take several weeks to recover
their levels after a TPE. Replacement by plasma is indicated when TPE
is done in a patient with bleeding diathesis or in TTP where a plasma
constituent is deficient.
Therapeutic Plasma Exchange (TPE)

The term plasmapheresis is used to describe the removal of plasma
without fluid replacement. This includes the collection of plasma from
normal donors. A therapeutic plasma exchange (TPE), on the other
hand, is the removal of a large proportion of patient plasma that is
replaced with crystalloid or colloid fluids. These terms are often used
interchangeably. TPE is a rapid, safe and easy procedure wherein two
plasma volumes can be exchanged within 3 hours and levels of
intravascular solutes such as antibodies, monoclonal proteins, immune
complexes and protein bound toxins can be lowered by 50 to 65 per
cent with each exchange.The theoretical efficiency of TPE is shown in
Table 3.3. The therapeutic goals of plasma exchange are to remove the
offending plasma components such as monoclonal proteins and
cryoglobulins, immune complexes, lipoproteins, or toxins responsible
for physical or metabolic problems. This may include removal of
Apheresis 61
Table 3.3: Plasma Exchange: Theoretical Efficiency

Number of Plasma Volumes Exchanged % of Original Plasma Remaining
0.5 60
1.0 35
1.5 20
2.0 12
autoantibodies, as in myasthenia gravis and alloantibodies in post-

transfusion purpura or in patients with factor VIII inhibitors. The
existence and pathogenetic role of antibodies or immune complexes is
presumed in several situations often treated with apheresis, in
neurologic disorders such as Guillain-Barre syndrome, polyneuropathy,
and various nephritides. In thrombotic thrombocytopenic purpura,
plasma exchange with cryo-poor plasma or FFP replacement or
transfusion may be life saving. In hyperviscosity syndrome, TPE is
particularly effective as IgM is largely distributed in the intravascular
compartment. Even exchange of as little as 500-1000 ml of plasma by
manual bag techniques brings about a reduction in viscosity, which
relieves both haemorrhagic, and ischaemic symptoms. However, larger
volume exchanges will result in greater relief from the symptoms due
to hyperviscosity. This is explained by the fact that the relationship
between paraprotein concentration and viscosity is nonlinear. Certain
recent indications for TPE are conditions like familial hypercholestero-
lemia and acute renal failure secondary to multiple myeloma. In the
former chronic TPE can reduce total plasma LDL levels and lead to
regression of atherosclerotic changes in blood vessels and promotes
resorption of xanthomas and atheromas.20 In patients with familial
hypercholestrolemia, on line selective extraction of lipoproteins is
carried out by chemical and immunological means. In Refsums disease,
removal of phytanic acid by TPE helps prevent or reverse the neuro-
logical manifestations.21
Early institution of TPE maximises its benefit as seen in therapy of
anti-GBM disease and Gullain-Barre syndrome. TPE is also useful in
many diseases mediated by circulating antibodies targeting host tissue
antigens as enumerated in Table 3.4. TPE has been successfull in cold
agglutinin induced autoimmune hemolytic anaemia (AIHA) where
once the antibody titres fall the degree of haemolysis is reduced. In
warm antibody (IgG) AIHA, the role of TPE is limited to, refractory
cases, as an adjunct to therapy with IVIG and /or pulse cychophospha-
amide. In Haemophiliacs development of both auto and alloantibodies
is a major therapeutic challenge especially when these patients have to
undergo surgery and adequate factor VIII levels are not achieved due
to presence of coagulation factor inhibitors. TPE has an important role
Table 3.4: Examples of specific antibodies in diseases treated with plasma

exchange
Antibody specificity Disease
Autoantibodies
1. Motor endplate acetylcholine receptor Myasthenia gravis
2. Nerve ending (calcium channel active Lambert-Eaton myasthenic
zone) syndrome
3. Peripheral nerve myelin Guillain-Barre syndrome, chronic
inflammatory demyelinating
polyneuropathy
4. Red cell I/i Cold agglutinin disease
5. Factor VIII Acquired haemophilia
6. A3 chain of type IV collagen Goodpasture syndrome
Alloantibodies
1. HPA-1a or other platelet antigen Posttransfusion purpura
2. Anti-A, anti-B ABO incompatible transplant
3. Anti-D Hydrops fetalis
4. Factor VIII Haemophilia A inhibitors
in removing these antibodies, which act as coagulation factor inhibitors.

Lowering of antibody titres allows the replaced coagulation factors to
circulate, though only temporarily.
In ABO-incompatible marrow transplants the alloantibodies to red
blood cells can be removed from the patient by TPE and the donor
incompatible red cells can also be removed from the graft by apheresis.22
Maternal IgG antibodies to the fetus in Rh negative pregnant women
who are sensitised can lead to destruction of fetal red cells. TPE can
help remove these antibodies.23
TPE is an established form of treatment in neurological diseases such
as Gullain-Barre syndrome (GBS), chronic inflammatory demyelinating
polyneuropthy (CIDP), myasthenia gravis (MG), and lambert-eaton
syndrome (LES). Antimyelin antibodies in GBS and CIDP and auto-
antibodies to the acetylcholine receptor and nerve endings in MG and
LES respectively lead to tissue damage in these diseases. These
antibodies are effectively removed by TPE and help in amelioration of
the disease.24,25,26
In renal and rheumatic diseases, TPE has improved the outlook,
especially in Goodpasture syndrome where autoantibodies are
deposited in the basement membrane in the lung and renal tissue
leading to lung haemorrhage and glomerulonephritis. In this condition
early institution of TPE and cyclophosphamide is the treatment of
choice.27 In SLE with nephritis, controlled studies have not shown any
advantage by adding TPE to cyclophosphamide.28 TPE has also been
beneficial in rapidly progressive glomerulonephritis with dialysis
Apheresis 63
dependant renal failure.29 However, no benefit has been noted with

TPE in renal transplant rejections.30,31
Thrombotic thrombocytopenic purpura (TTP) responds to simple plasma
infusion and better still to TPE.32 This has been explained because of a
two fold action in TPE. Firstly, possible removal of an IgM autoantibody
which acts as a specific protease inhibitor and secondly by replacing a
deficient factor, most probably a specific protease which limits the size
of the circulating von Willebrand factor multimers.32,33,34,35
Guidelines for clinical practice have been developed by the American Society
for Apheresis (ASFA). The disorders for which TPE has been used are
divided into four categories: category I, TPE is standard and acceptable,
category II, TPE is generally accepted; category III, there is insufficient
evidence to establish the efficacy of TPE; category IV, a lack of efficacy
has been demonstrated through controlled trails. Some of the
indications for TPE (categories I and II) are listed in Table 3.5.
Erythrocytapheresis
This has been described in 3-C.
CELLULAR THERAPY
Cell separators are used in the cell processing laboratory for removal
of T lymphocytes, for selecting progenitor cells from autologous bone
marrow harvests and peripheral blood stem cell (PBSC) collection
products. These progenitor cells are cryopreserved and later used for
reconstitution of bone marrow following ablative chemotherapy.36
There is a promising role for use of progenitor cells and other mono-
nuclear cells in the field of gene therapy.37,38 Immunotherapy with
lymphocyte concentrates has found a place in treatment of leukaemias
and solid tumours.39,40
REPLACEMENT FLUID FOR PLASMA EXCHANGE

The main goal of the replacement fluid is:
a. To maintain intravascular volume and to replace a deficient factor
as in TTP. Here fresh frozen plasma (FFP) or cryoprecipitate-poor
plasma replaces the von Willebrand factor cleaving protease.
b. Restoration of important plasma proteins.
c. Maintenance of colloid oncotic pressure and electrolyte balance.
d. Preservation of trace elements.
e. In patients having or prone to bleeding diathesis secondary to
deficiency of coagulation factors, replacement of these factors is
paramount while undertaking TPE.
For less than 500 ml of plasma removal as is the case, in most cell
collection and cell depletion procedures no replacement fluid beyond
the anticoagulant and saline priming solution is warranted.
Table 3.5: Guidelines for therapeutic haemapheresis by

American Society of Apheresis
Efficacy demonstrated as primary therapy or first line adjunct to other initial therapy
(category I)
1. Hyperviscosity (macroglobulinemia, myeloma)
2. Thrombotic thrombocytopenic purpura
3. Post transfusion purpura
4. Cryoglobulinemia
5. Eaton-Lambert myasthenic syndrome
6. Guillain-Barre syndrome
7. Myasthenia gravis
8. Chronic inflammatory demyelinating polyneuropathy
9. Goodpasture syndrome
10. Refsum disease
11. ABO-incompatible marrow transplant (red cell removal from the marrow)
12. HIV related polyneuropathy
13. Cutaneous T cell lymphoma (photoapheresis)
14. Leukaemia with hyperleucocytosis syndrome
15. Peripheral blood stem cell collection for transplantation
16. Familial hypercholesterolemia
17. Sickle cell anaemia (select indications)
Sufficient evidence to suggest efficacy but only on an adjunctive basis to support more
definitive therapies (category II)
1. Systemic lupus erythematous
2. Haemolytic uraemic syndrome
3. Rapidly progressive glomerulonephritis
4. Autoimmune thrombocytopenia
5. Myeloma and paraproteinemias
6. Coagulation factor inhibitors
7. Renal allograft rejection
8. Certain drug overdose, toxins
9. Pemphigus vulgaris
10. Hyperparasitemia (erythrocytalpheresis in malaria)
11. Cutaneous T- cell leukaemia (cytoreduction)
12. Raynaud’s disease
13. Cold agglutinin hemolytic anaemia
Inconclusive evidence for efficacy (Category III)
1. Hemolytic disease of newborn
2. Multiple sclerosis
3. Progressive systemic sclerosis
4. Pure red cell aplasia
5. Thyrotoxicosis
6. Transfusion refractoriness due to alloantibodies (red cells, platelets, HLA)
7. Life threatening haemolytic transfusion reactions
Lack of efficacy in controlled trials (Category IV)
1. AIDS (for symptoms of immunodeficiency)
2. Aplastic anaemia
3. Polymyositis
4. Psoriasis
5. Leukaemia without hyperleucocytosis syndrome
6. Chronic ITP
Apheresis 65
Removal of larger volumes of plasma in TPE necessitates replacement

with 5 per cent albumin or FFP and crystalloid solutions. The replace-
ment fluids which are used in apheresis or summarized in Table 3.6.
Table 3.6: Comparison of replacement of fluids

Replacement Solution Advantages Disadvantages
Crystalloids Low cost Large volumes required
Hypoallergenic Hypo-oncotic
No hepatitis risk No coagulation factors
No immunoglobulins
Albumin Iso-oncotic High cost

No contaminating No coagulation factors
“inflammatory mediators” No immunoglobulins
No hepatitis risk
Plasma Protein Fraction Cheaper than albumin Possible induction of

allergic and hypotensive
reactions
Fresh Frozen Plasma Maintains normal levels Hepatitis risk

of: -
• Immunoglobulins Citrate load
• Fibrinogen ABO incompatibility
• Complement Allergic reactions
• Antithrombin III Sensitization
Table 3.7: Complications of Plasma pheresis

S.No. Related to vascular access
1. Haematoma
2. Pneumothorax
3. Retroeperitonead bleeding
S.No. Related to procedure
1. Hypotension from externalization of blood due to extracorporeal
circulation; due to decreased intravascular volume.
2. Bleeding from reduction in plasma levels of coagulation factors
3. Edema due to decreased intravascular oncotic pressure
4. Loss of cellular elements (Platelets)
S.No. Related to anticoagulant used
1. Bleeding specially with heparin (rarely used)
2. Hypocalcaemic symptoms, specially with citrate (commonly used)
• Arrhythmia
• Hypotension
• Numbness and tingling of extremities
• Metabolic alkalosis from citrate
66
Table 3.8: Comparison of cell seperators

Sl Manufacturer/ Principle of Operating protocols Year of Cost
No. Model No. operation Introduction
1. Baxter CS 3000 and CS Continuous bag Plasma Platelets, PBSC, Introduced in 1979
3000 Plus (USA) centrifugation and Plasma Exchange
2. Baxter Amicus (USA) Continuous bag centri- Plasma Platelets and Introduced in 1995 Approx US $ 50000.00
fugation PBSC only (Rs.25 Lakh)
3. Cobe 2997 (USA) Continuous Tubular belt Plasma, Platelets, PBSC Introduced in 1980
centrifugation and Plasma Exchange
4. Cobe/Gambro Continuous Tubular belt Plasma Platelets, PBSC, Introduced in 1987 Approx US $ 48000.00
Spectra/LRA (USA) centrifugation and Plasma Exchange
5. Cobe Trima Accel (USA) Continuous belt centri- Platelets, PBSC and Introduced in 1996 Approx US $ 50000.00
fugation Packed Cell Collection
Recent Advances in Haematology
6. Fresenious AS 104 Continuous belt centri- Plasma, Platelets, PBSC Introduced in 1991
(Germany) fugation and Plasma Exchange
7. Fresenious AS 204 Continuous Belt centri- Plasma, Platelets, PBSC Introduced in 1995
(Germany) fugation and Plasma Exchange
8. Dedico Vivacel (Italy) Intermittent Bowl Centri- Plasma, Platelets Semi automa-
fugation ted PBSC and Plasma Exchange
9. Haemonetics MCS-3p Intermittent Bowl Centri- Platelets (LDP), Totally auto- Introduced in 1991
(USA) fugation mated PBSC, Bone Marrow
Processing, Packed RBC
10. Haemonetics MCS+ Intermittent Bowl Centri- Platelets (LDP), Totally auto- Introduced in 1995 Approx US $ 45000.00
(USA) fugation mated PBSC, Bone Marrow (Rs.22 lakhs)
Processing, Packed RBC
+ Double Packed RBC
Apheresis 67
Table 3.9: Comparison on plasma seperators

Sl Manufacturer/ Principle Operating Year of Cost
No. Model No. of operation protocols Introduction
1. Baxter Auto C Continuous Source Plasma 1991 Approx US
Filtration $ 20000.00
2. Asahi, Japan Continuous Source 1992 Approx US
Filtration Plasma $ 24000.00
3. Haemonetics Centrifugation Platelet Rich 1986
PCS Plasma and
Source Plasma
4. Haemonetics Centrifugation Platelet Rich 1994 Approx US
PCS 2 Plasma and Platelet $ 20000.00
Poor Plasma
COMPLICATIONS
Apheresis is a safe procedure and the current generation of cell
separators is very reliable and is equipped with sensitive alarm systems
to alert the operator of any potential problems. Most reported deaths
have been due to cardiac and respiratory causes in patients critically ill
prior to apheresis. Estimated mortality is 3 in 10,000 procedures. The
frequency of complications is dependent on the experience of the
operator and the nature of the patient population under treatment.41
The most common adverse effect of apheresis is symptomatic hypocal-
caemia due to infusion of calcium chelating citrate ions in the anticoagu-
lant. Hyperventilation exacerbates these symptoms. Calcium gluconate
(10 %) injection given intravenously can promptly correct this problem.
The most severe allergic complication occurs when plasma is used as
the replacement fluid and this risk increases with repeated exposures.41
Hypotension occurs in 1 to 2 per cent cases. The list of complications is
enumerated in Table 3.7. The various types of cell separators and plasma
separators available in the market are enumerated in Table 3.8 and 3.9
respectively.
REFERENCES
1. Abel JJ, Rowntree LG, Turner BB: Plasma removal with return of corpuscles
(Plasmapheresis). J Pharmacoi Exp Ther 1914;5:625.
2. Rock G, Sutton DMC: Apheresis: man versus machine. Transfusion 1997;37:993.
3. Lockwood SM, Worlledge S, Nicholas A et al: Reversal of impaired splenic
function in patients with nephritis or vaculitis by plasma exchange. N Engl J
Med 1979;300:524.
4. Pineda AA. Brzica SM. Tasweek HF: Continuous–and semi-continuous flow
blood centrifugation systems: Therapeutic applications. With Plasma-,
platelet-, lympha-, and eosinapheresis. Transfusion 1977;17:407.
5. Taft EG: Apheresis in platelet disorders. Plasma Ther 1982;2:181.

6. Mercer B, Drouin J, Jolly E, d Anjou G: Primary thrombocythemia in pregnancy:
A report of two cases. Am J obstet Gynecol 1988;159:27.
7. Ventura GJ, Hester JP, Smith TL, Keating MJ: Acute myeloblastic leukaemia
with hyperleukocytosis: Risk factor for early mortality in induction. Am J
Hematol 27:1988.
8. Edelson R, Factor M, Andrews A: Successful management of the Sezary
syndrome. N Engl J Med 1974;291:93.
9. Bongiovanni MB, Katz RS, Tomaszewski JE: Cytapheresis in a patient with
Sezary syndrome. Transfusion 1981;21:332.
10. Lim HW, Edelson RL: Photopheresis for the treatment of cutaneous T-cell
lymphoma. Hematol Oncol Clin North Am 1995;9:1117.
11. Marjs DI, Rockman SP, Oziemski MA, Fox RM: Mechanisms of lymphocyto-
toxicity inducted by extracorporeal photochemistry for cutaneous T CELL
lymphoma. J Clin Invest 86:2080, 190.
12. Miller ST, Jensen D, Sreedhar PR: Less intensive long-term transfusion therapy
for sickle cell anaemia and cerebrovascular accident J Pediatr 1992;120:54.
13. Vichinsky EP, Syles LA Colangelo LH: Acute chest syndrome in sickle cell
disease: clinical presentation and course. Blood 1997;89:1787.
14. Hamre MR, Harmon EP, Kirkpatrick DV: Priapism as a complication of sickle
cell disease . J Urol 1991;145:1.
15. Koshy M, Weiner SJ, Miller ST: Surgery and anesthesia in sickle cell disease.
Blood 1995;86:3676.
16. Kramer SL, Cakmpbell CC, Moncreif RE: Fulminant Plasmodium faclciparum
infection treated with exchange blood transfusion. JAMA 1983;249:44.
17. Cahill KM, Benach JL, Reich LM: Red cell exchange:Treatment of babesiosis
in a splenectomized patient . Transfusion 1981;21:193.
18. Keller AJ,Chirnside A, Urbaniak SJ: Coagulation abnormalities produced by
plasma exchange on the cell separator with special reference to fibrinogen
and platelet levels. Br J Haematol 1979;42:593.
19. Simon TL: Coagulation disorders with plasma exchange. Plasma Ther Transfus
Technol 1982;3:147.
20. Ginsberg HN: Update on the treatment of hypercholesterolemia, with a focus
on HMG-CoA reductase inhibitors apd combination regimens Clin Cardiol
1995;18:307.
21. Gibberd FB: Plasma exchange for Refsums disease. Transfus Sci 1993;14:23.
Brain HG, Sensenbrenner LL, Wright SK et al: Bone marrow transplantation
with major ABO incompatibility using erythrocyte depletion of marrow prior
to infusion. Blood 1982;60:420.
22. Bensinger WL, Baker DA, Buckner CD: In vitro and in vivo removal of anti-A
erythrocyte antibody by adsorption to a synthetic immunoadsorbent.
Transfusion 1981;21:335.
23. Rock G, Lafrebuere I, Chan L, McCombic N: Plasma exchange in the treatment
of hemolytic disease of the newborn. Transfusion 1981;21:546.
24. French Corporative Group on Plasma Exchangeand Gullain-Barre Syndrome:
Efficiency of Plasma exchange in Guillain-Barre syndrome:role of replacement
fluids. Ann Neurol 1987;22:753.
25. Connolly AM, Pestronk A, Trottler JL: High-titre selective anti-betatubulin
antibodies in chronic demyelinating polyneuropathy. Neurology 1993;43:557.
Apheresis 69
26. Seybold ME: Plasmapheresis in myasthenia gravis. Ann NY Acad Sci

1987;505:84.
27. Kalluri R, Gunwas S, Reeders ST: Goodpasture syndrome:Localization of the
epitope for the autoantibodies to the carboxy terminal region of the alpha
3(IV) chain of basement membrane collagen. J Biol Chem 199;266:24018
28. Lewis EJ, Hunsicker LG, Lan s-P: A controlled trial of Plasmapheresis therapy
in severe lupus nephritis. N Engl J Med 1992;326:1373.
29. Pusey CD, Ress AJ, Evans DJ: Plasma exchange in focal necrotizing
glomerulonephritis without anti-GBM antibodies. Kidney Int 1991;40:757.
30. Kirubakaran MG, Disney APS, Norman J: A controlled trial of plasmapheresis
in the treatment of renal allograft rejection. Transplantation 1981;32:164.
31. Blake P, Sutton D, Cardella C: Plasma exchange in acute renal transplant
rejection Prog. Clin Biol Res 1990;31:698.
32. Rock GA, Shumak KH, Buskard NA: Comparison of plasma exchange with
plasma infusion in the treatment of thrombotic thrombocytopenia purpura.
N Engl J Med 1991;325:93.
33. Furlan M, Robles R, Solenthaler M, Lammle B: Acquired deficiency of von
Willebrand factor-cleaving protease in a patient with thrombotic thrombo-
cytopenic purpura Blood 1998;91:2839.
34. Furlan M, Robles R, Galbusera M: von Willebrand factor-cleaving protease in
thrombotic thrombocytopenia purpura and the hemolytic uremic syndrome.
N Engl J Med 1998;339:1578.
35. Tsai H-M Lian EC-Y: Antibodies to von Willebrand factor-cleaving protease
in acute thrombotic thrombocytopenic purpura . N Engl J Med 1998;339:1585.
36. Leitman SF, Read EJ: Haematopoietic progenitor cells. Sem Haematol
1996;33:41.
37. Cassel A, Cottle-Fox M, Doren S, Dunbar C: Retroviral-mediated gene transfer
into CD34-enriched human peripheral blood stem cells. Exp Hematol
1993;21:585.
38. Brenner MK: Emerging applications of gene transfer in the haematopoietic
cancers, J Pediatr Hematol Oncol 1997;19:1.
39. Kelin HG, Leitman SF: Adoptive immunotherapy in the treatment of malignant
disease. Transfusion 1989;29:170.
40. Rosenberg SA: The immunotherapy and gene therapy of cancer.J Clin Oncol
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41. Sutton DMC, Nair RC, Rock G and the Canadian Pheresis Study Group:
Complications of plasma exchange. Transfusion 1989;29:124.
4
Blood Component Therapy:
In Paediatric Practice
VP Choudhry
Key Words
Red blood cells • Platelet transfusion • Factor concen-
trate replacement • Thalassaemia • Sickle cell
disease • Haemophilia
INTRODUCTION
Transfusion of blood and its component requires careful consideration
in children because of continuous anatomical and physiological
developments. A correct diagnosis is essential for proper choice and
use of various components. The weight and age of the patient
determines the transfusion requirements and risks of complications.
Potential problems often arise because of the small blood volume and
difficulty in administration of larger amount of blood products if the
concentrates are not available. Premature infants and neonates under
the age of four months present unique problems in paediatric
transfusion therapy. Premature infants are often over transfused.1 Better
understanding of the pathophysiological changes and advances in the
management of critically ill newborns has necessitated changes in the
blood bank policies and preparation of smaller units. The adminis-
tration of blood components have been altered to suit their special
needs. Appropriate paediatric transfusion practice requires the
understanding of neonatal physiology.
A full-term neonate has an average blood volume of 85 ml/kg and a
premature infant has about 100-110 ml/kg of the body weight. The
blood volume reduces after birth as a result of transudation of the
plasma. A newborn cannot compensate for volume depletion or over
transfusion as effectively as children because of limited cardiovascular
adaptive capabilities. Normal haemoglobin (Hb) level in the neonates
varies between 16 to 18 g/dL. Neonatal haemoglobin is not able to
Blood Component Therapy: In Paediatric Practice 71
deliver oxygen to tissues as effectively as an adult haemoglobin. The

bone marrow shows poor response to anaemia, primarily due to low
erythropoietin level and poor reticulocyte response.
Metabolic problems may arise as a result of immature kidneys which
have poor capability of excreting potassium and acid and/or calcium
loads. Similarly, acidosis or hypocalcaemia may occur because of
immature liver which cannot metabolise citrate efficiently.
BLOOD COMPONENTS
The various blood components used for paediatric transfusion are
shown in Table 4.1.
Table 4.1: Blood components

1. Cellular components
i. Red blood cells
ii. Platelets
iii. Granulocytes
2. Plasma components
i. Fresh frozen plasma
ii. Cryoprecipitate
iii. Factor concentrates
Components also can be leucodepleted, irradiated or saline
washed as per specific requirements.
RED BLOOD CELLS (RBC)

There are marked differences in transfusion practices in children and
adults:
1. Children require relatively small quantities of blood and blood
products, usually 10-15 ml/kg of body weight and thus require
only fraction of a unit.
2. Repeated blood sampling, even of small quantities, can lead to
volume depletion in a neonate. Sometimes red cell transfusions
are needed following repeated blood sampling for laboratory tests
and blood-gas analysis.1 The sick infant requires frequent blood
sampling for various tests and therefore needs red cells, plasma
and platelet products. 1 It is generally recommended that
micromethods should be used for various investigations such as
biochemical parameters, blood-gas analysis, blood cultures etc.
for neonates and infants.
3. Since neonates have maternal antibodies, in addition to their own,
till the age of 3-6 months, therefore, it is preferable that red cells
intended for transfusion should be crossmatched with the
mother’s serum, as well.
Currently the knowledge of haematopoiesis during neonatal and
perinatal period and response to RBC (i.e., packed red cells) transfusion
is limited. The tissue needs for oxygen cannot be measured directly

and presence of high foetal haemoglobin cannot release the oxygen to
tissues as freely as in adults. Therefore, the indications for red cell
transfusions are not well defined. 1,2 However, packed red cell
transfusion in presence of cardiorespiratory failure secondary to
anaemia is unquestioned. But the role of packed red cell transfusion in
situations such as poor weight gain, respiratory difficulties, apnoea,
patent ductus arteriosus or maintenance of a predetermined blood
haemoglobin concentration etc. remains controversial.
The concept of ‘fresh’ red cells needs to be clarified. Platelets and
factor VIII are lost within 48 hours of storage at 4°C. There is an increase
in potassium and ammonia along with a reduction in pH after 5-7 days
of storage. Blood less than 5 days old is often used for large volume
transfusions, e.g. exchange transfusion in neonates. Blood less than
5 days old ensures that the plasma electrolyte concentration within
tolerable limits for infants and is now considered as fresh blood.
Indications for Red Cell Transfusions

The indications of red cell transfusion are given in Table 4.2. Paediat-
ricians can modify them according to the general condition of the patient
and their hospital transfusion practices.
Table 4.2: Guidelines for red blood cell transfusions

Neonates
i. Physiological anaemia of infancy/anaemia of prematurity
ii. Haemoglobin (Hb) < 10 g/dL, if neonate is symptomatic
iii. Hb < 13.0 g/dL, in cardiopulmonary disease
iv. Hb < 10.0 g/dL, in cases of failure to thrive
v. Replacement of iatrogenic blood loss
vi. Situations requiring large volume transfusions, e.g. exchange
transfusion
Infants and older children
i. Hb < 7 g/dL, or in presence of symptomatic anaemia
ii. Blood loss due to haemorrhage
PHYSIOLOGICAL ANAEMIA OF INFANCY

The anaemia, resulting from the decrease in red cell mass due to
physiological factors during the first eight weeks of life is termed as
“physiological anaemia of infancy.” Haemoglobin values in-term infant
fall to about 11 ± 2 g/dL by 8 weeks of age.1 The erythropoietin (EPO)
response to anaemia is relatively diminished and the fall in haemoglobin
is variable, during the neonatal period with lowest value being
9 ± 2 g/dL.1 Clinical trials with recombinant human erythropoietin
(rhEPO) for the physiological anaemia of infancy have been encoura-
ging.3
Hb Less than 10 g/dl, If Symptomatic

In neonates, particularly with respiratory distress, red cell transfusions
should correct systemic hypoxia, improve peripheral oxygen delivery
and assure adequate oxygen perfusion. The adequacy of transfusion is
assessed by haemoglobin level or haematocrit.
Hb<13.0 g/dl, In Cardiopulmonary Disease

Neonates requiring oxygen and ventilator support for longer period
should be transfused red cells. It will provide satisfactory oxygen
delivery during the period of illness associated with diminished
pulmonary functions. It has been recommended that haemoglobin
should be maintained above 13.0 g/dl. This level of haemoglobin
ensures optimal oxygen delivery in severe cardiac diseases with
cyanosis or congestive heart failure while in patients with patent ductus
arteriosus the rise in haemoglobin levels following red cell transfusion
has been found to be useful as it increases pulmonary vascular resistance
which enhances the ductal closure and alleviates high output failure.
Red cell transfusion should be given slowly and should be monitored
closely as transfusion may transiently increase blood volume leading
to cardiac failure.
Hb < 10 g/dl In Infants with Failure to Thrive

Anaemia is one of the several possible causes for growth failure,
especially in presence of other signs of distress (e.g., tachycardia,
respiratory difficulty, less vigorous cry and activity and poor sucking).
RBC transfusion is recommended in infants having anaemia with
growth failure, if Hb level is less than 10 g/dl in the absence of other
causes for poor weight gain.
REPLACEMENT OF IATROGENIC BLOOD LOSS

Red blood cell transfusion for iatrogenic blood loss accounts for about
90 per cent of transfusions during the neonatal period.4 Repeated blood
sampling may be necessary for laboratory monitoring in the intensive
care unit (ICU). Therefore, it is recommended that only minimal
necessary investigations should be undertaken. Secondly, these
investigations should be done by micromethods to prevent anaemia. It
is generally advised that red cells should be replaced on volume to
volume basis.
INFANTS AND OLDER CHILDREN

Symptomatic Children with Hb Less than 7 g/dl
Causes of severe anaemia could be secondary to diminished erythro-
poiesis in conditions such as leukaemia, aplastic anaemia, following
chemotherapy, infiltration by tumour cells, myelofibrosis or due to
ineffective erythropoiesis secondary to various haemolytic anaemias,

congenital dyserythropoietic anaemia, haemoglobinopathies etc. The
usual practice is to raise Hb above 10 g/dl. Red cell transfusions may
be required in asymptomatic infants with anaemias, e.g. radiotherapy,
which requires adequate Hb for optimal effectiveness and for children
undergoing major surgery.
Blood Loss Due to Haemorrhage

Blood loss usually occurs during surgical procedures and more in
presence of thrombocytopenia or following accidents. The aim of
transfusion is to replace the red cell mass and to maintain the oxygen
carrying capacity with adequate tissue perfusion. In massive transfusion
(> 1 blood volume replacement in 24 hours) whole blood may be given
as it also provides plasma for maintaining oncotic pressure.
TRANSFUSION OF THE RBC PRODUCT

Packed red cells (hematocrit 70-80%) in a dose of 10-15 ml/kg body
weight is the product of choice for neonates. Clinical and laboratory
evidence suggest that RBCs preserved till 42 days in extended storage
media (AS-1 or AS-3) are safe for neonatal transfusions.5,6
Donor exposure should be limited by collecting RBCs in multiple
bags (e.g., triple bag or pentapack) and splitting the bag into individual
small volume units, which retain the expiry date of the original red cell
unit. Alternatively, the required amount of red cells can be transferred
to a transfer bag using a sterile connecting device under all aseptic
measures to ensure sterility.
The age of blood does not matter for small volume top-up trans-
fusions. Hyperkalaemia is not a common complication because of
higher amounts of plasma potassium in stored RBCs except when large
volume transfusions are given.7 Storage of blood often results in
depletion of 2, 3-DPG (2,3-diphosphoglycerate) and the resultant high
oxygen affinity is a theoretical disadvantage of aged blood, but does
not constitute any significant problem in clinical practice. The 2,3-DPG
rapidly regenerates following transfusion. 2,3-DPG is greater than 70
per cent during the first 5 days of storage, therefore it is preferable that
blood collected within 5 days should be used for exchange and other
massive transfusions.
For other infants and older children the dose for transfusion is 15
ml/kg body weight, irrespective of the age. It results in rise of Hb by 2
to 2.5 g/dl. Smaller repeat doses (5 ml/kg) spread over 24 hours may
be given to children with severe anaemia. Children may be given 15
ml/kg of body weight. Very slow transfusion with constant monitoring
along with use of frusemide may be undertaken in infants with severe
anaemia. Otherwise, a partial exchange should be done to avoid volume
overload.
PLATELET TRANSFUSION
Platelet concentrates may be either random donor platelet (RDP) units
or single donor platelet (SDP) units. RDP units are prepared from one
unit of whole blood and contain 5.5 × 1010 platelets in 40-60 ml of plasma.
SDP units are harvested from a donor connected to the cell separator
(apheresis) and contain 3 × 1011 platelets. Both are stored at +22°C on a
platelet agitator and have a short shelf life of 3-5 days depending on
the quality of the bag.
The normal platelet count in infants and children is similar to adults
with a range of 150-450 × 109/L. General guidelines for platelet therapy
are given in Table 4.3. The primary indication for platelet transfusion
is thrombocytopenia with active bleeding. Thrombocytopenia may
result from decreased platelet production, increased platelet destruction
or following massive blood transfusion. There is no absolute threshold
or critical value below which bleeding always occurs. However, the
risk of bleeding is high when platelet count is below 10 × 109/L and
very high when platelet count is below 5 × 109/L. In actively bleeding
patients or those undergoing major surgical procedures the platelets
should be transfused if the platelet count is below 50 × 109/L. Platelet
counts should be maintained above 20 × 109/L in those who have
thrombocytopenia following bone marrow failure or undergoing minor
surgical procedures. Consensus conference statement (1998) 8
recommended that platelet count should be maintained above 10 ×
109/L for stable thrombocytopenic patients and 20 × 109/L for those
with fever, infection and related conditions with prophylactic platelet
support. In neonates, the Consensus suggests that a higher threshold
should be maintained as there is considerable danger of haemorrhage.
However, in developing countries, it is more practical to treat clinical
bleeding rather than platelet count in view of poor availability of platelet
concentrates, high cost and limited facilities.
Table 4.3: Guidelines for platelet transfusion

1. Platelet count < 50 × 109/L in premature infants
2. Platelet count < 10 × 109/L with
i. active bleeding or
ii. major invasive or surgical procedure
3. Platelet count < 20 × 109/L with
i. marrow failure or
ii. minor surgical procedure
4. Cardiovascular bypass surgery
5. Excessive haemorrhage, regardless of the platelet count
6. Immune thrombocytopenia
7. Neonatal thrombocytopenia
i. Neonatal alloimmune thrombocytopenia
ii. Secondary to maternal ITP
Platelet transfusions may also be required in neonatal immune

thrombocytopenia (secondary to maternal idiopathic thrombocytopenic
purpura (ITP) or neonatal alloimmune thrombocytopenia)
In thrombocytopenia secondary to maternal ITP the platelet
antibodies have broad reactivity against all platelets. Transfusion of
platelets from all donors including the mother and family members
have uniformly short survival. It should be given only in life-threatening
situations. Intravenous immunoglobulin is an effective therapy for both
the categories of immune thrombocytopenia. In neonatal alloimmune
thrombocytopenia, maternal platelets can be used as they are the most
readily available source of compatible platelets. Platelets can be washed
to remove incompatible plasma and irradiated to avoid graft-versus-
host disease (GVHD). If PLA-1 negative platelets are necessary, SDP
should be preferred.
Platelets should be rapidly infused over 30 to 40 minutes. One RDP
unit/5 kg body weight is sufficient to stop haemorrhage. Platelets have
a lifespan of 7 days but are probably haemostatically effective for only
the first 4 or 5 days. About two-third remain in the circulation while
the rest comprise a freely exchangeable splenic pool. Determination of
a 1 hour post-transfusion corrected count increment (CCI) has been
suggested to assess the response to platelet therapy. However, the
cessation of haemorrhage is the way to determine the effectiveness of
platelet therapy. If the adequate platelet therapy does not arrest the
bleeding in the absence of infection and other causes such as dissemi-
nated intravascular coagulopathy, platelet refractoryness should the
considered. In these cases one should investigate for platelet antibodies.
Special platelet products like HLA-matched platelets, should be
considered to control the bleeding episodes in presence of platelet
alloantibodies.
Random donor platelet (RDP units) are usually given irrespective of
ABO and Rh-blood group if the RDP is not visibly contaminated with
red cells. It has been observed that platelet survival is better when ABO
compatible platelets are transfused. Heal et al9,10 found a higher median
CCI with ABO identical platelet transfusions and lowest with platelet
incompatible transfusion, i.e. when recipient has antibodies to donor
ABO antigens.
Transfusion of ABO incompatible plasma is more dangerous in the
paediatric recipients, especially in the neonates, than in adults due to
their smaller blood volume. If necessary the plasma volume of platelets
(RDP) can be reduced. Volume reduction is also necessary to prevent
volume overload.
There is a possibility of recipient sensitization to Rh-(D) antigen in
Rh-(D) negative patients as small numbers of donor erythrocytes are
always present in platelet products. Although the ability of the host to
respond to immunogenic stimuli may be compromised by disease or

by therapy, but it is essential to administer the anti-D immunization to
Rh-negative individuals.
GRANULOCYTE TRANSFUSIONS
The quantitative and qualitative abnormalities in myelopoiesis and
neutrophils in the newborn have suggested a possible role for granulo-
cyte transfusions in addition to microbial therapy for treatment of over-
whelming sepsis. Neonates are more susceptible to severe bacterial
infections than older individuals.
Optimal use of granulocyte transfusion for neonates is under
investigation and is yet to be established beyond doubt. The only
granulocyte preparations whose efficacy is documented in the clinical
trials are those harvested by leukopheresis. The average cells per
leukopheresis unit is 2 × 1010, nearly two times of daily production in
neonates and about 10 per cent of the daily granulocyte production in
adults. Stauss11 has analysed the data from seven reports of granulocyte
transfusions. He has concluded that the precise role of granulocyte
transfusion for neonatal sepsis is unclear. Some patients of neonatal
sepsis respond to antibiotics alone but have high mortality.
The dose of granulocytes transfused should be at least 1-2 × 109/kg
body weight given once in 24 hours or more frequently as required.
The volume of infusion is usually 10-15 ml/kg. The granulocyte product
should be red cell compatible and preferably irradiated if the infant is
premature or immunodeficient. Since granulocyte function deteriorates
with storage, they should be transfused within a few hours of collection.
FRESH FROZEN PLASMA (FFP)

FFP is the fluid portion of the blood which is separated and frozen at
lower than -30°C within 6-8 hours of collection. In general FFP is used
to replace coagulation factors in infants with acquired or congenital
coagulation factor deficiencies (Table 4.4).
Table 4.4: Recommendation for transfusion of FFP

1. Vitamin K dependent coagulopathy in haemorrhagic disease of newborn (HDN)
with significant haemorrhage
2. Replacement of isolated factor deficiencies, when specific component therapy
is not available (factor VIII, factor IX, fibrinogen deficiency, factor X, V, II
deficiency etc.)
3. Replacement therapy in antithrombin III, protein C or S deficiency
4. DIC
5. Reversal of haemostatic disorders following massive transfusion
6. Therapeutic plasma exchange in thrombotic thrombocytopenic purpura (TTP)
7. Coagulopathy secondary to L-asparaginase therapy
Since maternal clotting factors do not cross the placenta, hereditary

deficiencies of clotting factors may become apparent at birth.
Haemorrhagic disease of the newborn, caused by deficiency of vitamin
K dependent coagulation factors (factor II, VII, and IX and X) may result
in significant cutaneous, gastrointestinal or CNS haemorrhage at 2-4
days of age. Now this disease is rare as prophylactic intramuscular
vitamin K is routinely given at birth. Vitamin K deficiency may also
occur in paediatric patients in the setting of malnutrition and /or
alteration of the gut microflora following broad spectrum antibiotic
therapy or interference of vitamin K metabolism by certain newer
cephalosporins.
Coagulopathy is also a feature of disseminated intravascular
coagulation (DIC) or impaired hepatocellular function. Management
of DIC includes treatment of the underlying cause and transfusion of
FFP and platelets till the process of DIC is controlled.12 Haemorrhage
prior to invasive procedures in patients with impaired hepatocellular
function (e.g., hepatitis or hepatotoxicity due to chemotherapy) is also
managed with FFP. In thrombotic thrombocytopenic purpura (TTP),
the first line therapy is plasma exchange with large volume FFP infusion,
or cryo-poor plasma. FFP is more effective as it is able to provide a
labile platelet aggregation inhibitor.13 FFP also reduces the incidence
of coagulopathy associated with L-asparaginase therapy in acute
lymphoblastic leukaemia (ALL).
Each unit of FFP contains 1 unit of activity/ml of therapeutically
useful clotting factors. FFP is administered usually in the dose of 10
ml/kg body weight over 1-2 hours and repeated every 30 minutes as
necessary to control the haemorrhage. The dose can be repeated every
12 or 24 hours depending on the clinical situation.
FFP should not be used for the sole purpose of treating hypovolaemia
as FFP carries the risk of transfusion transmitted infections. FFP should
definitely be preferred for infants who require both plasma volume
expansion and clotting factor replacement, as occurs in septic shock or
nercotizing enterocolitis (NEC).
CRYOPRECIPITATE
It is prepared by thawing FFP at 4°C. The cryoprecipitate provides a
concentrated form of fibronogen, vWF and F VIII as compared to FFP
(approximately a five-fold increase in factor VIII concentration). A
commonly used dose is 20 ml/kg.
Cryoprecipitate is useful for acquired hypofibrinogenaemic states
besides for management of bleeding episodes in factor VIII deficiency,
von Willebrand’s disease (Table 4.5).14
Table 4.5: Recommendations for use of cryoprecipitate

1. Haemophilia A
2. von Willebrand disease
3. Hypofibrinogenaemia or dysfibrinogenaemia
4. DIC
5. Preparation of fibrin glue (fibrin sealant)
MODIFIED COMPONENTS
Gamma-Irradiated Blood Products
Transfusion associated graft-versus-host disease (TA-GVHD) is due to
the immune response mounted by the donor T-lymphocytes against
host tissues. Gamma-irradiation in doses of 25 to 30 Gy prevents the
GVHD by inactivating these T cells.15 Irradiated blood products are
also indicated in severely immunodeficient foetus requiring intrauterine
transfusions and children getting transfusion from first degree relatives.
Though occasional instances of TA-GVHD have been reported in the
neonates. There is no definitive data to suggest the need for irradiated
blood to be given to healthy term infants.16 Deleterious effects of
irradiation have been noted on recovery of red cells and on extracellular
potassium which increases with the storage. Therefore, it is preferable
to irradiate blood products just prior to dispensing.
Besides whole blood, the blood components also can cause TA-
GVHD. The usual practice is to irradiate (with a minimum of 25 Gy)
these blood components used for intrauterine transfusions or for
neonatal patients with birth weight < 1200 grams9 or whenever the
donor and recipient are first-degree relatives. Components that have
been frozen (deglycerolized RBCs, fresh frozen plasma, and
cryoprecipitate) or blood products such as albumin, plasma protein
fraction, and gammaglobulin need not be irradiated.
USE OF SPECIAL FILTERS AND LEUCOCYTE DEPLETED

BLOOD
Currently available third generation leucodepletion filters achieve a
3 log depletion, i.e. they remove 99.9 per cent of the leucocytes.
Leucocyte depletion diminishes the chance of alloimmunization to WBC
antigens, decreases the risk of CMV transmission, and possibly also
reduces the effects of immunomodulation. Several studies have failed
to confirm the postulated high-risk of transfusion-transmitted CMV in
the neonates who received CMV seropositive blood and blood products.
These neonates do not have an increased mortality or morbidity.17
The genuine benefits of WBC reduction in transfusion are limited.
In view of the technical difficulties and high costs of filters the routine
use of leucocyte depletion for all transfusions is not justified.
SALINE-WASHED RED BLOOD CELLS AND PLATELETS

Washing of RBCs removes about 90 per cent of the leucocytes and 99
per cent of plasma proteins and electrolytes. Saline-washed red cells
are recommended in patients who are hypersensitive to plasma to
prevent urticarial reactions in IgA deficient patients to avoid
anaphylaxis, in multitransfused patients, e.g. thalassaemia, to prevent
nonhaemolytic febrile transfusion reactions and in babies with
paroxysmal nocturnal haemoglobinuria to remove the complements.
Saline-washed platelet reduces the plasma content by 95 per cent
without any significant platelet loss. Saline washed platelets are of great
benefit in patients with severe allergic reactions. Similarly, washed
platelets of the mother are recommended in neonates with alloimmune
thrombocytopenia.
TRANSFUSION IN DISEASE STATES REQUIRING MULTIPLE

TRANSFUSIONS
Thalassaemia
The aim of management in thalassaemia is to maintain the Hb level
above 10 gm/dL. Packed red cell transfusions may be required initially
at short intervals to achieve the required haemoglobin levels. Subse-
quently packed red cell transfusions are given at 2-3 weekly
intervals.18,19
Generally, RBC units that have been stored for fewer than 7-10 days
are advocated for 2 reasons: (a) Higher 2, 3 DPG levels and (b) better
red cells survival in these units compared to units stored for 35-42 days.
However, both these reasons are not valid as red cell stored for relatively
short periods do not increase the post-transfusion red cells survival or
decrease the transfusion requirements.
Thalassaemics may develop allergic and/or febrile non-haemolytic
trasnfusion reactions following multiple transfusions. Leucocyte filters
are recommended from the beginning with transfusion to prevent the
development of these side effects. However, since the cost of filters is
high and the fact that all patients do not develop these reactions,
therefore, practical strategy for a developing country is to use the filters
when children develop these reactions. Alternatively, saline washed
red cells, which have negligible plasma proteins and less than 10 per
cent of the leukocytes have been recommended.
Sickle Cell Disease (SCD)

Red cell transfusions are given to patients with SCD with the objectives
to (a) correct anaemia which increases the oxygen carrying capacity in
the anaemic patients, (b) replace the abnormal HbS by normal HbA,
and (c) to reduce the viscosity of blood which in turn reduces the
incidence of sequestration in blood records. Reduction of sickle
haemoglobin level to about 40 per cent in the blood alleviates the pain,
prevents local hypoxia and prevents pooling of blood in the spleen
and at other sites.
The red cells can be given as a simple transfusion or exchange
transfusion depending upon the severity of disease. In a simple
transfusion, red cells (10 ml/kg) are given at 12 hourly intervals till
haemoglobin level attains normal range. Subsequently, blood may be
given at 3-4 weekly interval to ensure that the proportion of sickle
haemoglobin in the blood is kept below 40 per cent. Exchange
transfusion may be undertaken in infants during acute crisis.
Haemolytic Disease of the Newborn (HDN)

In HDN, the red cells of the foetus are coated with IgG alloantibodies
of maternal origin resulting in accelerated destruction foetal red cell at
birth. Exchange transfusion is performed in severe haemolytic disease
of the newborn (HDN) with the objectives of:
1. Reducing the load of accumulated bilirubin and of antibody
molecules by removal of the plasma from the newborn.
2. Reducing the antibody/coated cells prevents the haemolysis, there
by reduces bilirubin production;
3. Provides additional albumin to bind residual bilirubin;
4. Restoring the haemoglobin level.
In severe HDN, in spite of a successful exchange, bilirubin may
reaccumulate in plasma due to continued haemolysis or redistribution
of bilirubin from other compartments. Exchange transfusion can be
repeated if required.
Haemophilia
The availability of factor concentrates has drastically reduced the use
of cryoprecipitate as the source of coagulation factors.14 The factor
concentrate are much safer as they are virus inactivated. Recombinant
factor concentrates are now available. However, in developing
countries, due to limited availability of factor concentrates and its high
cost , fresh frozen plasma (FFP) is being used in majority of cases.
The dose of factor VIII for haemophilia A is calculated as follows:
0.5 units of factor VIII/kg body weight raises the plasma levels by 1
per cent. Factor VIII has a short half-life of 8-12 hours. Therefore, factor
VIII is administered at 12 hourly. The indication of factor replacements
for various situations has been reviewed recently.20 Currently few
centres are using factor replacement prophylactically to ensure near
normal life and to prevent development of arthropathy.
In haemophilia B, the dose of factor IX is 1 unit/kg body weight to
raise the factor IX level by 1 per cent.
Oncology Patients
Patients especially with leukaemia and other malignancies present with
anaemia. Chemotherapy may also lead to anaemia and thrombo-
cytopenia. These children need repeated red cell and platelet
transfusions for several weeks or months during and after chemo-
therapy. These cases need to be monitored periodically. Leucodepleted
blood products are advisable if the bone marrow transplant is being
considered for treatment of cancer to reduce the risk of alloimmu-
nization and CMV disease. Irradiated blood products are recommended
to prevent TA-GVHD.
REFERENCES
1. Strauss RG: Transfusion therapy in neonates. Am J Dis Child 1991;45:904-11.
2. Strauss RG, Sacher RA, Blazina JF et al: Commentary on the small volume red
cell transfusion for neonatal patients. Transfusion 1990;30:565-70.
3. Messer J, Haddad J, Donate I et al: Early treatment of premature infants with
recombinant human erythropoietin. Pedaitrics 1993:92;519-23.
4. Choudhry VP, Krishnamurti L. Hemato-oncologic emergencies in principles
of paediatric and neonatal emergencies (Ed). Sachdev HPS, Puri RK, Bagga
A, Choudhry P. Publication of Ind. Paediatrics. Jaypee Publishers 1994;201-
220.
5. Strauss RG, Burmeister L, James T, Miller J: A randomized trial of fresh versus
stored RBCs for neonatal transfusion (abstract): Transfusion
1994;34(suppl):66S.
6. Strauss RG, Burmeister LF, Johnson K: AS-1 red cells for neonatal transfusions:
A randomized trial assessing donor exposure and safety. Transfusion
1998;38:873-78.
7. Voak D, Cann R, Finney RD: Guidelines for administration of blodo products:
Transfusion of infants and neonates. British Committee for Standards in
Haematology Blood Transfusion Task Force. Transfusion Medicine 1994;4:63-
69.
8. Consensus Conference on Platelet Transfusion. Br J Cancer 1998;78:290-91.
9. Heal J, Rowe J, McMican A: The role of ABO matching in platelet transfusion.
Eur J Haematol 1993;50:110-17.
10. Heal JM, Blurnberg N, Masel D: An evaluation of crossmatching. HLA and
ABO matching for platelet transfusions to refractory patients. Blood 1987;70:23-
30.
11. Strauss RG: Current issues in neonatal transfusions. Vax Sang 1986:51:1-9.
12. Choudhry VP, Sharma LM, Kashyap R. Disseminated intravascular
coagulation in Recent Advances in Paediatrics. Ed Gupte S. Jaypee Publications
2001;11:75-86.
13. Rock GA, Shumak KH, Buskard NA: Comparison of plasma exchange and
plasma infusion in the treatment of thrombotic thrombocytopenic purpura.
N Engl J Med 1991;325:393-97.
14. Kashyap R and Choudhry VP. Management of hemophilia in developing
countries. Ind Pediatr 2001;68:151-57.
15. Rosen NR, Weidner JG, Bolot HD, Rosen DS: Prevention of transfusion
associated graft-versus-host disease: Selection of an adequate dose of gamma-
irradiation. Transfuson 1993;33:125-27.
16. Strauss RG: Practical issues in neonatal transfusion practices. AM J Clin Pathol
1997;107(1):557-83.
17. Preiksaitis JK: Indications for use of cytomegalovirus seronegative blood
products. Trans Med Rev 1991;5:1-17.
18. Marous RE, Wonke B, Bantock HM: A prospective trial of young red cells in
48 patiens with transfusion dependent thalassemia. Br J Hematol 1985;60:153-
59.
19. Choudhry VP, Ahlawat S, Pati HP. Current management protocol in
thalassemia in current management protocols in paediatric practice. Ed
Kukreja S Publishers CBS 1995;178-86.
20. Kashyap R, Choudhry VP. Inhibitors in hemophilia in Recent Advances in
Paediatrics Ed. Gupte S. Jaypee Publications 2001;11:314-23.
5
Haematopoietic Stem Cells
HP Pati
Key Words
Stem cell • Progenitor cell • Peripheral blood stem
cell • Haematopoietic stem cell • Stem cell markers
• Haematopoiesis • Cytokines • Stem cell plasticity
INTRODUCTION
Haematopoiesis is the process by which primitive stem cells proliferate
and differentiate to produce mature blood cells. These cells continually
replenish the supply of the essential blood cells, from oxygen-carrying
erythrocytes and blood-clotting platelets to the infection-fighting
lymphocytes and myeloid cells. Haematopoietic stem cells also have
the uncanny ability to replicate themselves and retain their pluripotent
potential. It is driven by highly coordinated patterns of gene expression
under the influence of growth factors and the micro-environment.
There is a recognizable and predominant cells component, called
the haematopoietic cells precursors. They constitute the major marrow
compartment cells with easily recognizable cells of different cell line
which give rise to mature cells and released to blood circulation. The
smaller, unrecognizable cell component can be divided into stem cell
and progenitor (lineage committed) cell components. Approximately
1 in 10 5 bone marrow cells are haematopoietic stem cells (HSC) and 1
in 200 cells is a progenitor cell. Rest of the marrow cell component is
the precursor cells. The stem cells are able to initiate and sustain
haematopoiesis throughout adult life till death.
HAEMATOPOIETIC STEM CELLS (HSCs)

Dorsal aorta/gonads/mesonephros (AGM region) is considered site
of the early human embryonic definitive HSC formations, which express
CD34 and leukocyte common antigen CD45 and expression of
haematopoietic transcription factors Runx-1, c-myb and SCL.1
Haematopoietic Stem Cells 85
STOCHASTIC MODEL
The stem cell renewal versus differentiation towards a particular lineage
and its regulation is not yet very well understood. In stochastic model
of haematopoiesis, a stem cell randomly differentiates along a particular
way.2 But the role of cytokines, cytokine receptors and the genetic
program is not fully understood.
Cell sorting studies with density gradient centrifugation, labelling
with antibodies, lectins, immuno-magnetic bead selection 3 and
bioassays have allowed stem cells to be arranged into a hierarchy based
on their capacity for self-renewal (repopulation with identical daughter
stem cells) and for generating their mature progeny (differentiation/
maturation). The most ancestral stem cells are multi-potential and are
detectable by long-term in vivo repopulation tests. Many conventional
assays fail to measure long-term repopulating ability and maximal
differentiating ability, which are the most important characteristics of
the primitive HSC. In vivo assay of human stem cells include measuring
human marrow repopulating cells (MRC) in non-obese diabetic /severe
combined immunodeficient (NOD/SCID) mouse.4 Long-term culture
initiating cell (LTC-IC) test recognizes somewhat more mature stem
cells, which are able to sustain haematopoiesis in suspension cultures
usually in the presence of a stromal cell under layer.5 LTC-IC cells are
less than 1 per cent of the CD 34 positive cells present in marrow. A
more mature subset of stem cells in semisolid agar culture is able to
form blast cells (Bl-CFC, blast-colony forming cells).6
Cells that do not normally proliferate or that proliferate rarely are
suspended at a stage called G0. A protein called p21 has been identified
that appears in some cells to play a role in controlling whether the cell
remains at G0 or it enters the cell cycle to proliferate, but its role in stem
cells had been unknown.7 p21 helps in protecting the haematopoietic
system from a variety of toxic effects. Studies in quiescent stem cells
from human bone marrow have shown high levels of p21 in them.
STEM CELL MARKERS

Stem cells are CD 34 antigen +, thy1 (CD 90) +, and lack in HLA-DR8 as
well as the antigens present on more lineage–restricted progenitors like
CD 38, CD33, CD 20, CD 14 (Lin-). They express growth factor receptors
like c-kit (CD 117), CD 123 and CD 135. Possibly even CD 34 –ve stem
cells also do exist which co-express c-kit and ostocalcin. In a recent
study, it was shown that the human CD34+ population could act as a
reservoir for generation of CD34- cells. Human CD34+/CD38- cells
could generate CD45+/CD34- progeny in a long-term xenograft model,
and that those CD45+/CD34- cells could regenerate CD34+ progeny
following secondary transplantation.9 A CD 34+ cell is apparently
dynamic in their expression pattern and related to the activation state

of the stem cells themselves.4 The comparative phenotypic markers in
human and murine stem cells are given in Table 5.1.
PROGENITOR CELLS
There are both multipotent and unipotent progenitor cells and could
be assayed by their ability to form colonies in vitro on semisolid medium,
stimulated by appropriate growth factor(s).
They are often defined as colony forming cells (CFC) for their capacity
to produce in vitro colonies (granulocytes, monocytes, mast cells,
megakaryocytes, dendritic cells, natural killer cells, lymphocytes). They
differ from stem cells in that they are mostly in cell cycle and have little
capacity for self-renewal. Mitotic activity in these cells is much more
than the stem cells. A progenitor cell to be unipotent involves acquisition
of some specific growth factor receptors and loss of some others. These
cells are CD 34+, HLA-DR+ and Lin+.
Some examples of progenitors are:
1. Multipotent colony forming cell-mixed (Mix-CFC/CFU-GEMM)-
produce granulocytic cells, erythroid cells except lymphoid cells.
2. Bust forming unit-erythroid (BFU-E)-produce colony forming
Unit-Erythroid (CFU-E).
3. Granulocyte-monocyte colony forming cell (GM-CFC)-give rise
to granulocyte-colony forming cell (G-CFC) and monocyte colony
forming cell (M-CFC).
4. Megakaryocyte-colony forming cell (Meg-CFC)–produce
megakaryocytes.
5. Eosinophil colony forming cell (Eo-CFC)-produce eosinophils.
6. Basophil colony forming cell (Ba-CFC)-produce basophils.
7. There are also B-cell, T-cell, NK-cell and dendritic cell progenitors
arising from lymphoid stem cells.
Table 5.1: Phenotype characteristics of murine and human HSCs

Murine Human
CD34-/low CD 34 +
thy1+ thy1+ (CD 90)
Rhodamine dull Rhodamine dull
CD 38+ HLA-DR -
SCA1+ Lin-
Lin- CD14-, 20-, 33-,38-/low, CD77-
c-kit + c-kit +/low( CD 117)
CD 123+
CD 135+
? CD 34-ve stem cell(and CD 90,117,123,135+)
None of the markers are unique to any stem cell function
YOLK SAC HAEMATOPOIESIS

During embryogenesis, haematopoiesis starts in extra-embryonic yolk
sac, fetal liver and bone marrow. Yolk sac haematopoiesis include only
erythropoiesis that is established in yolk sac by day 18 of gestation
from mesoderm and BFU-E, CFU-E are present at 4th week of
gestation.10 The growth factors inducing mesoderm formation such as
transforming growth factor-b (TGF b), fibroblast growth factor and bone
morphogenetic protein-4 (BMP-4) are likely to have role in the onset of
early haematopoiesis. Yolk sac erythroblasts have some different
characteristics, i.e. they differentiate inside blood vessel (not in extra-
vascular space), have higher sensitivity to erythropoietin (EPO), remain
nucleated throughout their life and released as such into circulation,
larger in size similar to megaloblasts and have shorter life span
compared to fetal and adult erythroblasts. After 7th week of gestation,
haematopoietic progenitors are no longer detectable in yolk sac.
Removal of yolk sac in earlier studies have prevented haematopoiesis
within the embryo proper, suggesting that HSC possibly migrate from
this to fetal liver, bone marrow or other organs through blood circulation
or by direct tissue migration.
Though culture studies show that the yolk sac cells can yield to adult
erythroid cells in culture, but actual maturation to adult cells don’t
occur in situ. Possibly the environment in which haematopoietic
progenitors reside alter the differentiation process as well.
HEPATIC HAEMATOPOIESIS
In contrast to yolk sac, all haemopoietic cell lines including
lymphopoisis occur in liver. BFU-E appears in fetal liver by 5th week
of gestation 10 and is the major site of haematopoiesis after 12th week
and continues till 24th week and red cells entering circulation are non-
nucleated. Platelets appear in circulation by 8-9th week of gestation. B-
lymphocytes with surface IgM are present in liver and lymphocytes
appear in circulation by 9th week of gestation. Primitive fetal liver stem
cells that give rise to both myeloid and NK cell progenitors are found
in CD34+, CD 38-, HLA DR+ subset.11
BONE MARROW HAEMATOPOIESIS

Haematopoietic cells appear in marrow by 12th week of gestation and
become major site of haemopoiesis by 24th week. The active marrow
site in first 2-3 yrs after birth are in all bones throughout their length
and by early adulthood is confined to epiphyses of long bones and the
sternum, ribs, vertebrae, and pelvis.
The microenvironment cells, termed stromal cells in marrow
elaborate important mediators and provide the solid support upon
which haematopoiesis occur.12 Stromal cells appear quite diverse.
Homing is the process of migration of HSCs to the bone marrow micro-

environment. But whether it is an active process or mere capturing of
circulating HSCs at specific sites of the micro-environment is not clear.
Homing is regulated by the interaction of integrins and extracellular
matrix and chemokines.13,14
GROWTH FACTORS and CYTOKINES

There are more than 25 growth factors produced from fibroblasts,
macrophases, T-cells, marrow stromal cells, endothelial cells. The
common ones are Erythropoietin, G-CSF, GM-CSF, IL-3 (multi-CSF),
M-CSF and Thrombopoietin. Many synergise with other cytokines only.
ex. Kit-ligand (stem cell factor, SCF). IL-1 modulates production of other
cytokines. Marrow stromal cells produce a large number of growth
factors ie. IL-1, IL-6, IL-11, SCF, M-CSF, G-CSF, GM-CSF. Probably actual
contact between stromal cells with haematopoietic stem cell is needed
and the stimuli are transmitted from the stromal cell membrane to stem
cells. Extracellular matrix (ECM) molecules present on the membrane
of stromal cells are known to bind IL-3 and GM-CSF. Therefore, either
the growth factors produced by stromal cells or sequestrated from
circulation are available to stem cells through cell-cell or cell-matrix
interaction.
Growth factors exert their effect at the level of stem cell and
progenitor cell levels. Stem cells have several types of GF receptors
than committed cells, which have only specific receptor.
MOBILIZATION OF HAEMATOPOIETIC PROGENITOR CELLS

Now it is an established procedure to mobilize stem cells by giving
single cytokines like G-CSF or GM-CSF or IL-3 + G-CSF, IL3+GM-CSF;
but still the exact mechanism of normal homing and mobilization is
far from clear. However, it appears to be multi-factorial with
involvement of integrins, cytokines and SDF-1.
Adhesion Molecules
Circulating peripheral blood stem cells (PBSCs) and BM cells express
adhesion molecules (integrins, selectins, selectin ligands), but of
different quantity, i.e. b1 integrin VLA-4 (very late acting antigen) and
b2 integrin LFA-1 (leucocyte function antigen) are expressed low on
circulating PBSC compared to BM cells, suggesting that down-
modulation of adhesion molecules may have role in their release from
marrow.
Marrow stromal and endothelial cells express VCAM-1 (vascular cell
adhesion molecule) and ICAM-1 (intercellular adhesion molecule).
Antibody to VLA-4 has been shown to mobilize progenitors by
disrupting the adhesion and antibody to VLA-4 and VCAM inhibit

homing of stem cells in BM.
Cytokines
Cytokines act as adhesion molecules when they are membrane-bound,
i.e. Receptor for SCF (c-kit) is expressed on marrow stromal and
endothelial cells. The circulating PBSC and progenitor cells have low
c-kit, which would facilitate their emigration into circulation from
marrow.
SDF-1 (Stromal-Cell Derived Factor)

It is a chemo-tactic factor for trans-endothelial migration of marrow
progenitors and has been shown to have definite roll in stem cell
mobilization and homing. But many questions need to be yet answered.
But, how is this regulated? Reduced production or Down-modulation
of SDF-1 receptors on progenitor cells.
There is a difference between bone marrow CD34+ cells and it from
the PBSCs. Mobilized CD34 +ve PBPC have less c-kit, CD71, more CD38,
and HLA-DR.They are mostly (99%) not in cell cycle (S-phase)
compared to BM CD34+ve cells (30% cells in S-phase). The significance
of theses difference is not known.
PLASTICITY of HSCs
Morphological and in vitro culture studies have supported the existence
of a bipotential haematopoietic-vascular precursor: the Haemangio-
blast.15 Both of them share common antigens CD34, Flk-1, SCL, GATA-
2 and LMO2. But in other studies, the endothelial cells are found to be
precursor to haematopoietic cells,16,17 suggesting specialized endothelial
cells in the dorsal aorta and umbilical arteries as the source of HSCs.
Bone marrow cells enriched for HSC have the potential to give rise
to muscle.18,19 In addition, bone marrow stromal cells can differentiate
into astrocytes 20 and bone marrow is also a source of primitive
mesenchymal cells.21 Studies with rodents indicate that brain cells can
turn into blood.22 Two recent studies on human patients23,24 have
reported that hepatocytes were derived from bone marrow cells and
they are derived from HSC.25 The self-renewing adult mice haemato-
poietic stem cells have functional haemangioblast activity, that is, they
can clonally differentiate into all haematopoietic cell lineages as well
as endothelial cells that revascularize adult retina.26 It would suggest
that adults maintain a reservoir of haematopoietic stem cells that can
enter the circulation to reach organs in need of regeneration. Adult
stem cell plasticity is also shown in study in which enriched bone
marrow populations have been used to restore myocardial function in
infarct models in irradiated mouse and donor derived myocardial and

vascular cells were detected.27,28
It suggests that marrow HSC can produce variety of cells and also
marrow HSC are present in many other tissues. Though some studies
suggest conversion of muscle stem cells to haemopoietic cells,29 but
more likely that presence of migratory HSCs within muscle.30 In similar
way, other tissue specific stem cells can reside in marrow cell population.
CONCLUSION
Still a lot of study needs to be done to understand stem cell in its totality.
i.e., identification of new lineage-specific transcriptional factors or cell
surface receptors, and their mechanisms of action. Little is known about
the control of many of the known haematopoietic genes at the
transcriptional level, including those for structural proteins, enzymes
and receptors. Trans-acting factors that control the developmental
pattern of globin gene expression have been postulated on the basis of
experimental data but their identities remain unknown. Silencers have
been identified for embryonic and fetal globin genes but the mechanism
of their action is unknown.
A more complete understanding of the normal control of haemato-
poietic cell differentiation and the abnormal processes leading to
pathogenesis will increase our understanding of haematologic diseases
and will aid the development of rational therapies.
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Dystrophin expression in the mdx mouse restored by stem cell transplantation.
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Hepatocytes from non-hepatic adult stem cells. Nature 2000;406:257.
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Purified Haematopoietic Stem Cells can differentiate into Hepatocytes in vivo.
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6
Biological Therapy of Cancers
M Mahapatra, HP Pati,
VP Choudhry
Key Words
Interferons • Biological agents • Vaccines, inter-
leukins • Growth factors
INTRODUCTION
Cancer is a major cause of morbidity and mortality throughout the
world. At present, about 50 per cent of patients with cancer can be
cured with treatment modalities such as chemotherapy, radiotherapy
and surgery.1 Chemotherapy alone contributes to cure in only 17 per
cent of patients.2 Until recently, cytotoxic agents remained the main
pharmacotherapeutic strategy against human cancers. The application
of modern molecular genetics had led to a greater understanding of
underlying mechanisms of neoplasm and new agents like monoclonal
antibodies (mAbs), interferons (IFNs), interleukins (ILs) have been
developed to inhibit the genes or function of genes with aim to arrest
the proliferation of neoplastic cells. These new therapeutic modalities
have been termed as biological therapy or biological response modifiers
(BRMs) in cancer.3 The primary aim of all these modalities is passive or
active immunization, however some of these agents have antiproli-
ferative, anti-angiogenic and cytotoxic activity. The advantages of the
biological therapy over chemotherapy are shown in Table 6.1.
The history of biological therapy can be traced back to 1893, when,
William B Coley observed that cancer patients improved or underwent
complete remission if they developed postoperative infection.4 Coley
gathered several pathogens and injected them into a few cancer patients.
He observed that patient underwent complete remission if the patient
survived the feverish infection period and concluded that fever and
infection upregulated or recharged the immune system to recognize
and destroy the invading tumour cells by some unknown mechanisms.
Coley’s ‘cocktail’ was branded as quackery but now scientists recognize
him as ‘Father of biological response modifiers’.5 After promising results
Table 6.1: Advantages of newer forms of therapy7

Property Chemotherapeutic agents Biologic agents
Mechanism of action Less specific Specific target
Antitumour effect Regression Stabilization +/– regression
Duration of response Short, retreatment may Response is over longer
be necessary period, retreatment may not
be necessary
Route of administration IV, oral Oral > SC > IV
Toxicity Usually high Usually mild, as they
induce normal biologic
response
Monotherapy Possible with high Yes, for maintenance
single-agent activity therapy
Combination therapy Frequently used Needs investgation
Effective dose Near maximum tolerated Below MTD
dose (MTD)
Variability of response High Low
in animal studies, many large clinical trials were conducted to stimulate

the immune system by using the bacteria Bacillus Calmette-Guérin
(BCG) and Corynebacterium parvum.6 The results of these trials were
discouraging; thus immunotherapy as a possible modality for cancer
treatment lost momentum. However, the better understanding of cycle
control pathways, apoptosis and recent advances have prompted a
renewed interest in BRMs and today biological therapy has emerged
as an important area in cancer research and treatment.
MOLECULAR PATHWAYS OF ACTION OF BIOLOGIC AGENTS7

Several molecular pathways are being investigated to determine the
most effective and safe biological therapy for different cancer. These
pathways include:
a. Signal transduction pathway,
b. Cell-cycle control pathway,
c. Apoptosis pathway,
d. Angiogenesis-metastasis pathway.
Signal Transduction Pathway

This relates to the various methods of selectively inhibiting intracellular
signal transduction. Among various intracellular signal transductions,
the promising agents are tyrosine kinase inhibition, farnesyltransferase
inhibition, and vaccines against epitopes expressed by malignant cells.
Tyrosine kinases trigger a cascade of events when phosphorylation
stimulates additional enzymes or when it prompts protein to change
Biological Therapy of Cancers 95
their locations. Tyrosine phosphorylation is an early event in a complex

signaling system. Many receptors are actually tyrosine kinases, so
signals are quickly transmitted from outside the cell into the nucleus
after ligand binding at the cell surface. Kinases use adenosine tripho-
sphate as a source of phosphate, but if an inhibitor binds to the enzyme
instead of adenosine triphosphate, the kinase cannot phosphorylate
proteins and the signaling stops, like an “off” switch.7 Thus, the complex
signaling system does not function which inhibits the cell proliferation
or impairs the cellular function.
Cell-cycle Control Pathway

Cyclin-dependent kinases (cdks) regulate the transition from one cell-
cycle phase to the next by phosphorylating key structural and
regulatory substrate molecules.8 This activity is regulated by the orderly
appearance of cyclins and endogenous inhibitors of cdk activity (p16
[inhibits cdk 4 or 6]; and p27 [inhibit cdk 1,2,4, or 6]).
The expression of cyclin D and the emergence of cdk 4 or 6 activity
are particularly important because of their critical regulation of the
phosphorylation of tumour suppressor gene product (TSGP).
Phosphorylation of TSGP results in a loss of affinity for the transcription
factors. These transcription factors govern the transcription of many
genes necessary for progression through the S phase. It is also important
to mention that cdks can serve an architectural role, functioning as a
scaffold for assemblies of molecules important in nuclear function.8
Apoptosis Pathway
Apoptosis may be defined as multiple converging pathways resulting
from a variety of initiating events and culminating in a common,
irreversible execution phase in which proteases and nucleases digest
the doomed cell. There are a variety of pathways that may become
activated as a result of oncogenic transformation to counteract the
signals leading to apoptosis. The p53 tumour suppressor and the Bcl-2
protein family, as well as other intracellular transdution pathways play
a role in deciding whether a cell will live or die and therefore are
potential therapeutic targets.9
Angiogenesis-metastasis Pathway
The formation of new blood vessels and the permeability of blood
vessels are regulated by a family of vascular endothelial growth factors
(VEGFs) and receptors. The neovascularization response is mediated
by VEGF, a growth factor that is induced by hypoxia, as well as several
pathways. Immature tumour vessels are particularly vulnerable to
VEGF withdrawal.
The angiopoietins are other growth factor families specific to vascular

endothelial cells and work together with VEGF in all aspects of vascular
biology. In contrast, thrombospondin-1, angiostatin, endostatin,
vasostatin, and interferon alpha (alpha 2a and alpha 2b), among others,
are endogenous inhibitors of angiogenesis. They inhibit endothelial
proliferation and migration. Thus, the local balance between molecules
that stimulate or inhibit the process determines the extent of
angiogenesis.10 The development of inhibitors will inhibit tumour
angiogenesis which can be targeted in several ways.
1. Selective inhibition of VEGF: A humanized anti-VEGF monoclonal
antibody is undergoing clinical development as a potential therapy
for several solid tumours. SU5416 is a highly selective inhibitor
of the FLK-1/KDR/VEGFR-2 tyrosine activity and ZD4190 is
another potent inhibitor of VEGF RTK, which interfere with VEGF
signaling in vascular endothelial cells.11 The antibody AA98
inhibits the proliferation and sprouting of vascular endothelial
cells, and it also mediates complement-dependent-cytotoxicity
along with antibody-dependent-cellular cytotoxicity on
proliferating endothelial cells. Selective peptides that bind
receptors expressed on the vasculature of the target tissues are
being developed. In hormone-dependent tumour, tamoxifen
significantly inhibits angiogenesis and metastasis.12
2. Targeting endothelial cell function and migration: Vascular endothelial
cadherin (VE-cadherin) links endothelial cells and is important
for the assembly of vascular structures. Antibodies to VE-cadherin
inhibit experimental angiogenesis. Moreover, metallo-proteinases
play a role in matrix degradation and tumour invasion by new
vessels.13 Metalloproteinase inhibitors are currently undergoing
clinical trials alone and in combination with conventional therapy.
3. Coagulation-initiating proteins: Antibodies linked to a genetically
engineered form of coagulation-initiating protein promote
thrombotic occlusion of the tumour vasculature. Combretastatin
A4 (CA4) induces a rapid and prolonged reduction in tumour
blood flow with induction of necrosis in a number of transplanted
and spontaneous tumours.14 Combretastatin A4 phosphate CA4P-
a prodrug) is different from angiogenic inhibitors currently in
development, because it attacks pre-existing tumour vasculature
rather than preventing the formation of new vessels, changing
the shape and permeability of endothelial cells, specially actively
proliferating cell.
CLASSIFICATION7
A classification of the various biologic agents used in cancer therapy
based on their mechanism of action is depicted in Table 6.2.
Table 6.2: Classification of biologic agents based on mechanism of action7

Mechanism of action Biologic agent
A. Directly acting upon the 1. Monoclonal antibodies
cell-cycle/apoptosis 2. Interferon
3. Interleukin
B. Acting upon cell receptor/ 1. Hormones
micro-environment
C. Indirectly depriving nutrition 1. Anti-angiogenic agents
and growth of the cells 2. Coagulating factors
D. Enhancing immunologic 1. Tumour accine
processes and rejection of 2. Processed lymphocytes
tumour cells
E Immunomodulators, i.e. growth 1. GM-CSF
factors that change the way the body 2. G-CSF
responds to cancer itself or its treatment 3. Erythropoietin
F. Inhibit formation of oncogenic 1. Gene therapy
protein and thereby cell growth
INTERFERONS
Interferons are small proteins that inhibit viral replication and promote
cellular (T-cell) immune response. Their exact mechanism of action is
not well-known, however, they have direct antiproliferative activity
because they inhibit ornithine decarboxylase production, resulting in
slowing of cell cycle. They also inhibit new blood vessel formation and
certain oncogenes.15 Interferons were used for treatment in late 1970s
when biotechnological advances enabled their mass production. There
are three major types of IFNs (alpha, beta, and gamma), each with
similar yet distinctive capabilities for altering biological response. Both
IFN-alpha and IFN-beta are type-1 IFNs, i.e. acid-stable proteins that
act on the same receptor on target cells. IFNs-gamma, a type-2 IFN, is
acid-labile and acts on a separate receptor on target cell. IFN-alpha is
produced from leucocytes while IFN-beta is produced from fibroblasts
and epithelial cells and IFN-gamma has been produced from activated
T lymphocytes.
IFN-alpha was the first BRM approved by the FDA in 1986 for the
treatment of hepatitis C. It was approved for use in hairy cell leukaemia,
chronic myelogeneous leukaemia and AIDS-associated Kaposi’s
sarcoma,2,17 and has therapeutic effectiveness in cutaneous T-cell
lymphoma, melanoma and multiple myeloma.18,19 The recommended
dosage varies according to the condition, ranging from 3 million units
thrice weekly to 36 million units daily. IFNs can be administered by
the intravenous (bolus or infusion), intramuscular, subcutaneous or
intrathecal route. They can also be given intranasally and recently, the
oral use of IFN-alpha has been recommended.20 IFNs have variable on
side effects depending upon the frequency and intensity of the dose
and formulation of IFN. The side effects are common during a patient’s
first exposure to IFN, but usually decrease in intensity with continued
therapy.21 Now peglated IFN have been produced which needs to
administered once a week and have minimal side effects.
IFN-beta binds to the same receptor as IFN-α and has an immuno-
modulatory action. It enhances the natural killer (NK) activity, induces
class I MHC expression and inhibits the mixed lymphocyte reaction.
IFN-β has cytostatic action and does not have cytotoxic effect. However,
IFN-β has potent antiproliferative effect for colorectal and melanoma
cell lines.22 IFN-β can induce tumour cell differentiation and alter
oncogenes activity. In renal cell cancer, 20-30 per cent response rates
have been observed with IFN-β alone or in combination with IL-2.23,24
IFN-gamma has broader range of immunological activities than other
interferons. It has antiproliferative and antiviral activity and is a potent
activator of macrophage anti-tumour activity and releases tumour
necrosis factor (TNF). It also promotes differentiation of the tumour
cells and upregulates the receptors for TNF, lymphotoxin and
interleukin-1 (IL-1). IFN-gamma plays an important role in inflam-
mation and immunity.25 Phase I and II trials have shown that IFN-
gamma is effective in renal cell carcinoma, ovarian cell carcinoma,
malignant mesothelioma. It has been evaluated in CML and response
rate IFN-gamma alone has varied between 38-64 per cent without
reversal of the Philadelphia chromosome positivity. The combination
of IFN-alpha and IFN-gamma has not improved the survivals.26
INTERLEUKINS
These are cytokines that occur naturally in the body and have been
produced in the laboratory. They mediate their antiproliferative and
anti-tumour effects on cell surface receptors in relevant target cells.
Many ILs have been identified and numbered in the order of their
discovery (IL-1 through IL-23).27
IL-2 has been the most widely studied in cancer treatment. The
naturally produced IL-2 is a glycoprotein, which is secreted by activated
helper T lymphocytes. It has a mitogenic effect on T and B lymphocytes,
NK cells and mononuclear phagocytes and plays a key role in the
development of antigen specific and antigen non-specific immune
response. It also affects the haemopoiesis by stimulating the T lympho-
cyte to produce other cytokines and the overall haematopoietic effect
depends upon the balance between the production of growth inhibiting
and stimulating cytokines. The proposed mechanisms of IL-2’s anti-
tumour activity include: (1) IL-2 induced activation of immune effector
cells such as lymphokine activated killer (LAK) cells, NK or T-cells

that kill the tumour cells,( 2) Secretion of secondary cytokines such as
IL-1, IFN-gamma or tumour necrosis factor (TNF), (3) Enhanced
antibody dependant cell cytotoxicity, and (4) An indirect effect on
tumour vasculature leading to tumour necrosis.28
IL-2 therapy in patients with metastatic or unresectable renal cell
cacinoma has shown response rate of 25 per cent, with 5 per cent of
patients achieving complete remission. Similarly, 13 per cent of patients
with melanoma achieve objective response with IL-2 alone.29 Clinical
trials in patients with metastatic malignant melanoma have resulted
in response rate of 35 to 83 per cent and extended response duration of
more than 23 months.30 IL-2 has been approved for the treatment of
advanced metastatic renal cell carcinoma and malignant melanoma.16
Table 6.4 gives the response rates and survival in patients receiving
high doses of IL-2 alone. Role of IL-2, either alone or in combination
with other agents is being evaluated in colorectal, ovarian and small
cell lung cancer.31 The cure rates with IL-2 alone or in combination
with other cytokines in patients with NHL have been minimal.32 In
acute leukaemia, IL-2 therapy has shown benefit in patients with early
relapse and produced complete remission in some patients.33 It also
prolongs the duration of remission after bone marrow transplantation
in haematological malignancies.34 The other interleukins currently
under evaluation are IL-3, IL-4, IL-12, IL-15 and IL-18.35,36
The half-life of IL-2 in human is short; therefore, most clinical
schedules have used continuous infusion, multiple intermittent dosing.
Liposome encapsulated IL-2 and a conjugation of IL-2 with
polyethylene glycol to extend the half-life have been developed to
enhance its delivery to immune cells in tumours. 37,38 The most
important anti-tumour activity has been demonstrated with most
intensive dosing schedules: continuous intravenous infusion for 5 days
on alternate weeks for 2 cycles, or intravenous bolus doses every 8
hours daily for 5 days on alternate weeks.
The adverse effects of IL-2 therapy are listed in Table 6.3. The severest
of these result from the ability of IL-2 to increase capillary permeability.
This may cause hypotension, ascites and pulmonary oedema. Chills,
fever and flu-like symptoms occur within a few hours after the
administration of IL-2. Liver dysfunction is common during therapy
but resolves once treatment is stopped. Anaemia, thrombocytopenia
and skin rashes are more likely with higher and cumulative doses.39
HAEMATOPOIETIC GROWTH FACTORS

Granulocyte and granulocyte-macrophage colony stimulating factors.
Colony stimulating factors (CSF) are a diverse group of glycoproteins
regulating the growth and proliferation of haemopoietic stem and
progenitor cells. Granulocyte-colony stimulating factor (G-CSF) is
produced by mononuclear cells, fibroblasts and endothelial cells in

response to IL-1, TNF and bacteria. It stimulates neutrophil colony
formation by progenitor cells that have been commited to myeloid
differentiation. Granulocyte-macrophage colony stimulating factor
(GM-CSF) is produced by activated T lymphocytes, B lymphocytes,
endothelial cells, fibroblast and macrophages in response to specific
activating agents and acts on intermediate progenitor levels and
supports the proliferation of neutrophil, eosinophil and monocytes. In
addition, GM-CSF enhances the function of neutrophil, monocytes and
macrophages.40 Studies have shown that CSFs have the potential to:
1. protect or restore bone marrow function in combination with
chemotherapy or radiotherapy, thus permitting the patient to
tolerate higher and more effective doses;41
2. stimulate immune system components, thus enhancing anti-
tumour activity;42 and
3. help treat fever, infections and bleeding that may occur in patients
who have received chemotherapy.43
CSFs have been produced in sufficient quantities for clinical use by
recombinant DNA technology. Some have been approved for use and
others are in various stages of clinical trials.44-46 G-CSF and GM-CSF
are approved by the FDA for the management of neutropenic fever
secondary to cytotoxic chemotherapy. Both are administered as
intravenous bolus, continuous infusion or subcutaneously by daily
injection. Their serum half-lives are 2-7 hours after IV or SC adminis-
tration. In the treatment of chemotherapy induced neutropenia, G-CSF
5 mg/kg/day, or GM-CSF 250 mg/m2/day, is usually started within
24-72 hours after completing chemotherapy. According to the 1997
guidelines issued by American Society of Clinical Oncology (ASCO),
this therapy should continue until the absolute neutrophil count is more
than 10,000-cells/ml.47 However, the current clinical practice is to stop
CSF therapy once the neutrophil count has reached a range 2500-5000
cells/ml. GM-CSF may be preferred as it stimulated granulocyte and
macrophage system. Stimulation of macrophage system may have
better immunogenic potential. G-CSF therapy is associated with
minimal toxicity, though GM-CSF produces more systemic toxicities.48
(Table 6.3). The bone pain associated with CSF therapy is possibly due
to expansion of cells and increased blood flow in medullary space; blood
levels of alkaline phosphatase and aminotransferases may also be
increased.49
Other Growth Factors

Erythropoietin (EPO) is a 30 to 34 KD protein with 166 amino acid and
is produced by the kidneys. Decreased red cell production results in
tissue hypoxia, which stimulates erythropoietin production by the renal
interstitial cells. EPO acts on the receptors on the erythroid colony
Table 6.3: Important adverse effects of various biological respone modifiers1

Agent Adverse effects
Monoclonal antibodies Allergic reactions, dyspnoea, wheezing, fever,
chills, headache, rash, tachycardia, nausea,
vomiting
Interferons Flu-like syndrome, fever, chills, myalgias,
decreased blood counts, nausea, anorexia,
confusion, seizures, acute renal failure, alopecia
Interleukins Hypotension, ascites, pulmonary oedema, flu-like
syndrome, fever chills, skin rash, liver dys-
function, anaemia, thrombocytopenia
Colony stimulating factor Bone pain (mainly in lower back, sternum and
pelvis), fatigue, fever, muscle ache, rash (more
with GM-CSF)
forming units (CFU) and enhances erythropoiesis. Trials with recombi-

nant human EPO in doses of 100-150 units/kg thrice weekly have shown
to increase the mean haematocrit and haemoglobin level in patients
undergoing cancer chemotherapy.50 It is also effective in treating
anaemia in patients with bone marrow involvement with multiple
myeloma and lymphoma. Patients of multiple myeloma with renal
failure respond to EPO with a rise Hb level within 4-10 weeks of starting
treatment. Patients with low or appropriate levels of serum
erythropoietin show good response to recombinant human EPO therapy
when compared with those with increased levels.50
Macrophage colony stimulating factor (M-CSF) stimulates survival,
proliferation and differentiation of mononuclear macrophages. It
enhances the function of monocytes and macrophages and increases
their capacity for intracellular killing of fungal and bacterial micro-
organisms.51 IL-11 induces megakaryocytic proliferation and is FDA-
approved for use in patients with a reduced platelet count. Opervekin
is the recombinant form of IL-11 and is approved for clinical use.52 The
half-life of IL-11 is 7-8 hours when injected subcutaneously and may
limit the amounts of platelet transfusion required. Recombinant
thrombopoietin is also being investigated but there have been reports
of patients developing neutralizing antibodies to it.53
MONOCLONAL ANTIBODIES
Monoclonal antibodies (mAbs) have several advantages and offer hope
in the future of cancer management. The purity and specificity of mAbs
allow for decreased toxicity, reduced cross-reactivity to normal tissues
and uniform coupling to isotopes, drugs and toxins, and ability to
manufacture in large quantities make them ideal for anticancer therapy.
The use of mAbs involves the development of specific antibodies

directed against antigens located on the surface of tumour cells. Samples
of the patient’s tumour cells are processed to develop specific antibodies
against the tumour-associated antigens. Monoclonal antibodies are
developed against tumour cells unique antigens which are different
from the antigens expressed by normal cells. The antibodies thus
developed can be used either alone to kill cancer cells or as carriers of
other substances used for therapeutic or diagnostic purposes. The naked
mAbs bind to tumour cells and cause lysis while the conjugated mAbs
deliver cytotoxic agents or radioactive substances to the tumour cells.54
A variety of antigens that are ideal targets for monoclonal antibody
therapy include: (a) oncofetal antigens (b) differentiation antigens, e.g.
carcinoembryonic antigens (c) cell surface receptors such as platelet
derived growth factor (PDGF) receptor, epidermal growth factor
receptors55,56 (EGFR). Antibodies have been raised to several epitopes
on the epidermal growth factor receptors (EGFR) including HER-2. The
monoclonal antibodies to EGFR compete with ligand binding resulting
in a decrease in receptor activity. This in turn leads to reduce cell
proliferation and cell cycle arrest.57,58
Monoclonal antibodies can also be coupled to an unrelated protein,
enzymes, toxins, cytotoxic drugs and cytokines for giving novel binding
specificity and greater therapeutic benefit.59 Immunotoxin conjugates
involve mAbs conjugated to various toxins. These toxins could be of
plant (ricin), bacterial (Pseudomonas exotoxin) or fungal origin and
are effective cytotoxic agents.60 Antibodies coupled to ricin have yielded
response rates of 40-50 per cent in leukaemias and lymphomas. AR-
209, a monoclonal antibody against HER-2 is undergoing phase-I
clinical trials in carcinoma breast, prostatic, ovarian and small-cell lung
cancer. 60,61 Drug conjugates, which involve linking of a chemo-
therapeutic agent such as doxorubicin or vincristine to the antibody,
are also being investigated. One such mAb is BR96, which binds
strongly Lewis-y, a carbohydrate antigen found in large quantities on
a number of tumours, including carcinomas of colon, breast, pancreas,
and lung.54 The conjugate of BR96 and doxorubicin, once bound to the
tumour cell, internalizes to produce a selective cytotoxic effect. Various
isotopes of high radioactivity have been coupled with antibodies
develop to radioimmunotherapy agents for treatment of cancers.
Although these conjugates have yielded response rates as high as 70
per cent in human lymphomas, they have been less effective in other
solid tumours, mainly because the radioactivity needed to achieve the
desired therapeutic effect cannot be used as it is toxic to bone marrow.
Work is in progress to develop radioconjugates that could deliver a
higher radiation to tumour sites while sparing the healthy organs.62
FDA has approved many humanized mAbs and many others are in
various stages of clinical trials. They have been found to be effective
for the treatment of haematological malignancies such as acute and

chronic leukaemias, lymphomas, melanomas, colorectal cancer and
neuroblastomas. Table 6.4 gives the efficacy of some of the FDA-
approved mAbs in treatment of various cancers.
Trastuzumab, a recombinant DNA-derived, humanized mAb, binds
to the extracellular domain of membrane-anchored HER-2 with
nanomolar affinity, downregulating the kinases signaling of this
receptor to reduced cell proliferation.63 There is also an unexplained
increase in cell sensitivity to TNF-α,64 and cisplatin,65 paclitaxel and
doxorubicin.66 Trastuzumab is approved for the treatment of metastatic
breast cancer in patients whose tumour expresses HER-2.67 Rituximab
is a chimeric murine-human mAb that binds to the CD20 molecules on
B lymphocytes and is approved for the therapy of non-Hodgkin’s B
cell lymphoma.68 The CD33 antigen is expressed on blast cells in 80-90
per cent of acute myeloid leukaemia (AML) cases but is not expressed
on pluripotent haematopoietic stem cells or on non-haematological cells.
Gemtuzumab is a recombinant humanized antiCD33 mAb that delivers
the potent cytotoxin calicheamicin into cells.69
Despite these uses, mAbs have multiple limitations7 (Table 6.5).
Antibodies are commonly raised in mice by antigenic stimulation. These
mAbs, when administered to human beings, often stimulate the produc-
tion of antimouse-antibodies (HAMA), which form immune complexes
Table 6.4: Clinical efficacy of FDA approved biological response modifiers1
Agent Indication Response Survival rate
rate
Monoclonal antibodies
Trastuzumab Breast cancer 50%* Median of 25.1months84
Rituximab Non-Hodgkin’s 72% 77% progression-free
Lymphoma (NHL) at 1 year85
Gemtuzumab Acute myeloid 30% 31% at 1 year;
Leukaemia (AML) median 5.9 months69
Interferon-alpha Chronic myeloid
Leukaemia (CML) 38% 79% at 5 years86
Hairy cell 83% disease- free
Leukaemia (HCL) 88.9% at 31 months87
Multiple myeloma 6.6%• 7.9 months over standard
chemotherapy88
Interleukin-2 Renal cell 23% 9% at 5 years89
carcinoma
malignant 15% 6% at 3 years90
melanoma
* 50 per cent response rate seen with trastuzumab and standard chemotherapy
compared to 32 per cent response rate with chemotherapy alone.
• 6.6 per cent increase in response rate compared to chemotherapy alone.
Table 6.5: Limitations to the clinical application of monoclonal antibodies7

• The vast majority of mAbs are not tumour-specific
• Adverse effects of mAbs on normal tissues (cytokines related symptoms as mAbs
bind to blood cells and are cleared by the reticuloendothelial system) or toxic
effects due to the cytotoxic substance attached to mAbs
• Heterogeneity of the distribution of antigens on tumour cells
• Emergence of cells that do not express antigens and consequently are resistant to
any specific mAb.
that are neutralized and cleared rapidly by the liver, thus deactivating
the conjugate before its therapeutic effect is achieved. In addition, mAbs
may lack specificity for tumour antigens. When tumour cell antigens
are sufficiently different from those on normal cells, antibodies interact
with antigens on both normal and cancer cells, leading to the destruction
of normal cells.70 Another problem associated with antibody conjugates
relates to the linkers employed to connect the antibody to the drug or
toxins, reducing the potency of the antibody, drug or both. The side
effects of mAbs include dyspnoea, wheezing, fever, chill, headache,
rash, nausea, vomiting and tachycardia (Table 6.3).
ANTICANCER VACCINES1
Recent advances in tumour immunology have helped to develop a
variety of novel and specific vaccine approaches. Activated T and B
cells which evolve into memory cells and develop antibodies in large
quantities to destroy some antigen if enters the body again. The various
strategies include vaccines based on tumour cells, carbohydrates,
peptides and heat shock proteins. Recombinant bacteria and viruses
are used to deliver antigens or the DNAs coding for them.71
Tumour Cell Vaccines

The immunogenicity of tumour cells is enhanced by physical or genetic
manipulations such as gamma-irradiation, viral oncolysates or the
insertion of genes encoding cytokines or costimulatory molecules,
e.g.IL-2 and TNF-alpha.72 The major advantage is that it allows
immunization against all tumour proteins, and obviates the need for
identification of individual tumour antigens. It is highly effective in
inducing protective immunity and thus preventing recurrence, but its
efficacy in the treatment of established tumours is very limited. Most
current clinical trials using tumour cell vaccines have been limited to
patients with malignant melanoma.73
Carbohydrate Vaccines
Antibody responses against carbohydrate antigens on the surface of
encapsulated organisms such as Neisseria meningitidis, Streptococcus
pneumoniae and Haemophilus influenzae can provide protective immunity,

as similar antigens on human cancers are also potential target immune
recognition.74 Early clinical trials are in progress in patients with
melanoma and carcinoma prostate.
Peptide Vaccines
These permit specific targeting of the immune response against one or
two unique antigens, thereby preventing the autoimmune cross-
reactivity. Native peptides are less immunogenic, so synthetic peptides
are made by amino acid deletion or substitutions to enhance the
immunogenicity. Clinical trials are in progress in patients with
melanoma and carcinomas of the cervix, pancreas, breast and
ovaries.75,76 Heat-shock protein-peptide vaccination circumvents the
need to specifically identify immunogenic determinants by immunizing
against the entire repertoire of antigens expressed by a tumour. This
approach is being evaluated in several animal studies and clinical trials
as an adjuvant therapy in carcinoma pancreas.77
Plasmid DNA
DNA-based vaccination is accomplished either by direct inoculation
of plasmid DNA encoding the antigen of interest or by the use of a
hand-held helium-powered ‘gene gun’ which can deliver DNA-coated
gold particles directly.78 These vaccines are safe, easily purified and
permit immunization against a known antigenic determinant without
inducing a host immune response to the vaccinating vector.
Recombinant Bacteria and Viruses

Many viruses elicit very-specific, strong and lifelong immunity. Vaccinia
virus, fowlpox virus and adenovirus have been identified which have
number of sites within their genome where large amounts of foreign
DNA can be stably inserted without affecting the viral replication or
packaging.79 Several viral vaccines have been developed and are in
clinical trials of patients with cancers of the colon, prostate and cervix,
and melanoma.80
There are several obstacles for development of different vaccines.
The vector should must be stable in vivo with easy route of
administration. Most vaccines are delivered by the intramuscular,
intravenous or subcutaneous routes or by scarification.81 Independent
host factors such as immunodeficiency state of individual because of
underlying cancer or by prior chemotherapy, radiation therapy, etc.
affect the generation of the immune response to the vaccine. The choice
of adjuvant is another important factor for successful immunization.
Although cytokines such as IL-2, TNF-alpha and IL-12 remain attractive
agents for co-administration with vaccines, non-cytokine adjuvants
exhibiting non-specific immunostimulatory methods have been used

more frequently.82 These agents include BCG, Corynebacterium parvum,
alum, saponins and oil emulsions and others.
The immunotherapeutic response needed for regression of an
established tumour is likely to require repetitive rather than single
immunization. Heterologous boosting strategies in which different
vectors are used to prime and then boost may result in a greater
cytotoxic response and enhanced immunotherapeutic effect. The poor
results observed with homologous boosting may be due to the induction
of a strong antibody response to the vaccinating vector.83
CONCLUSION
The success of biological therapy has been established in some
malignancies (melanoma, breast cancer, certain leukaemias, Kaposi’s
sarcoma and renal cell carcinoma). The results of biological therapy
are not very encouraging as yet. The results of BRMs in other tumours
has remained elusive. High cost is a major prohibitive factor for the
widespread use of these agents. A major obstacle in developing
immunotherapy against cancer is that cancer cells arise from healthy
host cells and the antigens expressed by them are similar. Success of
BRMs depends largely on the identification selective cancer antigens.
The next few years will yield results from a number of clinical trials on
biological therapy. Development of these newer strategies, will come a
long way in improving the long-term event free survival and the cancer
will no longer remain as incurable.
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7
Management of AML
M Mahapatra, VP Choudhry,
R Saxena, HP Pati
Key Words
Cytogenetics in AML • Induction therapy • R elapsed
and refractory AML • AML in elderly
INTRODUCTION
Acute myeloid leukaemia (AML) remains a formidable disease for
internists, haematologists and patients. Like most other malignant
diseases, newer treatment strategies for AML have evolved as a
consequence of advances in the understanding of both molecular and
cytogenetic pathogenesis.1 This research has led to new treatments that
have significantly improved the survival rate. Before 1970, the 5-year
survival rate was less than 15 per cent, whereas the present survival
rate for all myeloid leukaemias is about 40 per cent. Different treatment
modalities and newer drugs in different combinations are being
evaluated in an effort to improve the event free survival further,
particularly among older adults, in whom 5-year survival rates are less
than 10 per cent.2
In this review, we focus on AML primarily from the clinician’s point
of view, summarizing some of the latest treatment approaches,
including targeted chemotherapeutic agents, newer biologic therapies
and stem cell transplantation. In addition, the current developments
in molecular/cytogenetic aspects are being reviewed in brief. Relapsed
and refractory AML and AML in older patients have been reviewed
along with the future directions.
CYTOGENETICS IN AML
Historically, the treatment of AML has involved the administration of
intensive antileukaemic chemotherapy. This empiric approach to
treatment is undergoing changes, due to improvements made in the
identification of characteristic chromosome abnormalities that are
Management of AML 113
associated with the various subtypes of AML. In conjunction with the

recently completed Human Genome Project, more target genes are being
localized, leading to the recognition of leukemogenic pathways
potentially amenable to target-specific therapy.
The clonal nature of AML has been recognized for years, with 80 to
100 per cent of newly diagnosed patients demonstrating a chromosomal
abnormality.3 It is likely that most cases of AML are the result of genetic
mutations that occur in haematopoietic progenitor cells. It also seems
that the majority of these mutations are acquired during an individual’s
lifetime.
A recent retrospective analysis of over 1600 patients confirmed what
clinicians had already recognized: 5-year survival is directly related to
cytogenetics at presentation.4 Patients with normal cytogenetics or with
favourable cytogenetic abnormalities, such as t(8;21), t(15;17), and
t(16;16), have better prognoses. Patients with deletions in the long arm
of chromosome 7 and 5, deletions or inversions of chromosome 3, t(6;9),
and the Philadelphia chromosome t(9;22), as well as abnormalities of
chromosome 11q23, have relatively poor prognoses.
In AML, genes located at translocation breakpoints are typically
responsible for cellular transcription and transduction of growth signals.
Most translocations in de novo AML result in a fusion of the affected
genes, with production of hybrid proteins. When aberrant fusions occur,
the normal function of transcription factors (i.e., the regulation of
cellular differentiation) becomes disordered or blocked. The (15;17)
translocation found in most APL illustrates this mechanism.
The promyelocytic leukaemia (PML) gene on chromosome 15 is fused
to the retinoic acid receptor (RAR) alpha gene on chromosome 17,
leading to production of a nonfunctional RAR that does not respond to
normal physiologic levels of circulating retinoic acid. Cell transcription
and differentiation is thus arrested by the inability of the RAR to activate
normal target genes. The PML gene seems to function as a histone
deacetylator, which also serves to suppress gene transcription and
cellular maturation. The administration of all trans-retinoic acid (ATRA)
allows the histone deacetylation complex to dissociate from the RAR-
PML fusion protein, enabling the RAR to function appropriately, which,
in turn, leads to cellular differentiation. In rare APL variants, such as
t(11;17), administration of ATRA enables removal of the histone
deacetylase complex from RARalpha, but does not dislodge it from the
fusion protein. Consequently, gene transcription remains suppressed.
Another well-characterized gene rearrangement in AML involves
the transcription factor complex core-binding factor (CBF). CBF is
composed of an alpha subunit that directly contacts DNA and a CBF-
beta subunit that facilitates binding of AML1 to DNA. Three genes
(AML1, AML2, and AML3) encode the alpha subunit. It is thought that
translocations involving the CBF result in the loss of normal CBF

properties, converting CBF from transcriptional activator to inhibitor,
thus leading to suppression of transcription of several target genes,
including genes for interleukin-3 (IL-3), myeloperoxidase, granulocyte-
macrophage colony-stimulating factor (GM-CSF), and the T-cell
receptor beta.5
The (8;21) translocation, seen in approximately 46 per cent of AML-
M2 patients, results in the production of a fusion protein known as
ETO/AML, of which the ETO portion from chromosome 8 is involved
in histone deacetylation.6 This, in turn, serves to block activation of
AML1 gene expression, resulting in lack of IL-3 and GM-CSF receptor
activation and loss of cellular differentiation/maturation. Inv (16),
found in AML-M4 with eosinophilia, is associated with fusion of CBF-
beta to the smooth muscle myosin heavy chain (MYH11) gene. This
CBF-beta-MYH11 chimeric protein directly represses AML1-mediated
transcriptional activity by forming inactive complexes in the cellular
cytoplasm.7
Treatment-related AML, especially following use of topoisomerase
II inhibitors, seems to involve translocations at locus 11q23 (the MLL
gene). A site within the MLL breakpoint cluster region has been
identified as being prone to double-stranded DNA breaks as a result of
exposure to topoisomerase II inhibition.8 To date, at least 38 different
translocations have been identified that involve the MLL gene, but the
mechanisms of action of these translocations are not well understood.
It is apparent, from the work done in APL, that further characteriza-
tion of the common molecular pathways shared by many of the
chromosomal translocations found in AML will lead to more specific
treatments like ATRA. And, like APL, it is hoped that the response rates
and survival statistics in other myeloid leukaemias will improve
significantly in near future.
CHEMOTHERAPY FOR NEWLY DIAGNOSED AML

In general, treatment for AML consists of an induction phase, followed
by a postremission or consolidation phase. The necessity for
consolidation treatment was demonstrated in a randomized study
conducted by the Eastern Co-operative Oncology Group (ECOG) in
which patients who were treated with induction therapy alone
experienced a higher rate of relapse following a shorter period of
remission. 9 Subsequent clinical investigation demonstrated that
consolidation is essential, whether treatment is given immediately
following remission or delayed for several months.10
From the mid-1960s to the present day, combinations of cytotoxic
chemotherapeutic agents have been used to treat AML. Initially,
cytarabine and thioguanine were employed in both induction and
consolidation therapy, achieving an approximately 50 per cent remission

rate with modest improvements in survival. In the 1970s, the
daunorubicin, was added to the preceding combination and then to
cytarabine alone, producing long-term survival in about 15 per cent of
those patients achieving complete remission.11
A retrospective review by ECOG examined analysis in more than
1400 newly diagnosed AML who were treated with induction
daunorubicin and cytarabine in 5 clinical trials conducted between 1976
and 1994.12 A complete remission rate of 62 per cent was attained, but
76 per cent of these patients relapsed or died. Overall survival at 5
years was 15 per cent, ranging between 9 and 33 per cent for patients
less than 55 years old, and between 6 and 15 per cent for patients 55
years old and older. Both disease-free and overall survival was better
in younger patients, when more intensive postremission treatments
were employed.
Thus, the “standard” chemotherapy regimen of daunorubicin and
cytarabine for AML induction has been in use for almost 30 years.
Dosing schedules were “standardized” in studies performed by the
Cancer and Leukaemia Group B (CALGB) in the 1980s, in which 3 days
of daunorubicin and 7 days of cytarabine were shown to be superior to
2 + 5 days, while 10 days of cytarabine therapy afforded no advantage
over 7 days.13,14 The dose of daunorubicin was established at 45 mg/
m2 for patients less than 60 years of age and was shown to be less toxic
than an equivalent dose of doxorubicin. 15 Dosing studies with
continuous infusion cytarabine demonstrated that 100 mg/m2 was as
effective as 200 mg/m2.16
Since the advent of daunorubicin-cytarabine induction, other clinical
studies have substituted potentially less cardiotoxic agents (e.g.,
aclarubicin, mitoxantrone, and the synthetically manufactured
idarubicin) for daunorubicin, with mixed results. Response rates,
especially among younger patients, were superior with idarubicin,
compared with response rates to daunorubicin in 3 of 4 prospective
randomized trials. 17-19 More myelosuppression occurred in the
idarubicin treatment arms, raising the question of whether any improve-
ment in outcome might be secondary to a higher biologic dose of
idarubicin. No randomized clinical trial has attempted to clarify this
point. Despite higher complete response rates in patients treated with
idarubicin, no overall survival advantage has been shown over dauno-
rubicin.
Both mitoxantrone and aclarubicin have shown similar efficacy to
daunorubicin among younger patients, with less extramedullary
toxicity.20,21 Amsacrine, a DNA intercalator, showed higher complete
response rates, earlier achievement of remission, and higher overall
survival rates when compared with daunorubicin.22 Nevertheless, the
3 + 7 anthracycline/cytarabine regimen using idarubicin ( 12-13 mg/

m2 daily) is still considered to be a standard therapy for the majority of
newly diagnosed younger patients with AML, although daunorubicin
at 60 mg/m 2 or 70 mg/m 2 daily may be equivalent. Complete
remissions are achieved in approximately 50 to 75 per cent of patients
below 60 years. While the CALGB data showed no difference between
200 mg/m2 vs 100 mg/m2 doses of continuous infusion cytarabine,
while several clinical trials investigated the use of higher doses of
cytarabine. One such trial employed a dose of 500 mg/m2 of cytarabine,
but also increased the dose of daunorubicin to 60 mg/m2.23 Results
were no different from those seen in patients treated with 200 mg/m2
of cytarabine.
The use of high-dose cytarabine for induction therapy has produced
mixed results. Two large prospective randomized studies showed
improvement in disease-free survival with high-dose treatment,
compared with standard induction, but no change in response rates
were observed when compared with regular dose cytarabine. A trial
conducted by the Australian Leukaemia Study Group (ALSG) 24
compared high-dose cytarabine (3 g/m2 every 12 hours every other
day for 8 days) plus daunorubicin and etoposide vs induction with
etoposide. A Southwest Oncology Group (SWOG) trial25 compared
high-dose cytarabine (2 g/m 2 every 12 hours for 6 days) plus
daunorubicin (45 mg/m 2) vs standard daunorubicin/cytarabine.
Neither study showed a higher complete remission rate, but both
studies reported higher disease-free survival among patients who
received high-dose cytarabine. Overall survival among all patients did
not differ between two treatment arms. Toxicity was considerably
greater among patients treated with high-dose cytarabine, with higher
rates of haematologic and nonhaematologic complications.
In contrast, a German study employed a double induction regimen
(with standard-dose cytarabine, daunorubicin, and thioguanine in both
arms, followed by the same regimen in one arm vs high-dose cytarabine
plus mitoxantrone in the other arm) and observed no improvement in
either disease-free or overall survival between those two treatment
arms.26 Potential benefits were seen in younger patients, with more
favourable cytogenetic characteristics when treated with high-dose
cytarabine.
A previous ALSG trial concluded that the addition of etoposide to
standard daunorubicin/cytarabine induction therapy did not improve
the complete remission rates or overall survival, although a significantly
longer disease-free survival was observed. 27 However, survival
appeared to be prolonged in the subset of younger patients (aged less
than 55 years), and, in a recent update, overall survival was found to
be significantly longer at 5 and 10 years (25% and 25%, respectively) in
the etoposide treated group vs 5- and 10-year survival rates (17% and
14%, respectively) among patients receiving daunorubicin/standard
cytarabine.28
The role of etoposide in AML therapy was further evaluated in a
large randomized trial (AML10) conducted by the Medical Research
Council (MRC) of the United Kingdom.29 More than 1800 patients, 55
years of age and younger, were randomized to receive either 7 + 3
thioguanine or 7 + 3 etoposide (100 mg/m2 daily for 5 days). Treatment-
related mortality, as well as complete remissions rates and disease-free
and overall survival rates, were equivalent in both arms. There were
no additional benefits by addition of etoposide.
INDUCTION THERAPY FOR

ACUTE PROMYELOCYTIC LEUKAEMIA (APL)
APL has been defined previously by virtue of its pathognomonic
chromosomal abnormality t(15;17), its PML-RAR alpha fusion protein,
which results in abnormal RAR expression as well as histone
deacetylation, and its association with a severe coagulopathy, primary
disseminated intravascular coagulopathy (DIC). It is also known that
ATRA can induce remission in 70 to 90 per cent of patients with t(15;17)-
positive APL.30 Coagulopathies appear to resolve quickly with ATRA
administration, compared with conventional chemotherapy. Patients
must be monitored for development of hyperleukocytosis and onset of
respiratory distress, which is characteristic of retinoic acid syndrome.
Fenaux and associates of the European APL group recently updated
their APL91 trial at 73 months, following last patient entry.31 The original
trial randomized 101 chemotherapy-naive patients with newly
diagnosed APL to either ATRA plus daunorubicin and cytarabine or
daunorubicin and cytarabine only, followed by 2 cycles of consolidation
therapy. Kaplan-Meier estimates of extended disease-free survival and
relapse at 4 years were 63 and 31 per cent, respectively, compared with
78 and 17 per cent, respectively, in the daunorubicin/cytarabine only
group. Overall survival at 4 years was 76 per cent in the ATRA group
and 49 per cent in the chemotherapy group (P = .03). In the chemo-
therapy group, 23 of 25 patients who relapsed were subsequently treated
with ATRA. By Kaplan-Meier estimate, 60 per cent remained in
remission at 30 months. All 10 patients who relapsed in the ATRA group
were retreated with ATRA, and all of them attained a second complete
remission. This study confirmed the role of ATRA in first-line treatment
for APL, as well as in relapse.
A prospective randomized trial sponsored by the North American
Intergroup assigned patients to receive induction ATRA alone vs
chemotherapy.32 Patients who achieved complete remission were then
given another course of the same induction regimen, followed by a
second consolidation of high-dose cytarabine and daunorubicin. At the

completion of consolidation, patients were randomized to either obser-
vation or 1 year of ATRA maintenance. While no difference in complete
remission rates were observed, both disease-free and overall survival
rates were significantly superior in each arm that included ATRA.
ATRA was given for 5 days before standard anthracycline-based
induction chemotherapy and compared with concurrent ATRA plus
chemotherapy in an MRC-sponsored trial. 33 The investigators
hypothesized that initial administration of ATRA might reduce the
complications of coagulopathy that are often seen in APL. In the
concurrent therapy arm, a higher complete response rate was observed
(87% vs 69%, P = .001). It was also noted that the early death rate was
lower (12% vs 23%, P = .02). Overall survival at 3 years was superior in
the concurrent therapy arm (74% vs 53%, P = .003).
From the preceding studies, it can be argued that standard therapy
for APL should consist of concurrent ATRA plus chemotherapy. A study
by the PETHAEMA (Program for the Study and Treatment of
Haematologic Malignancies) co-operative group in Spain suggested
that cytarabine might no longer be needed in induction or consoli-
dation.34 After induction therapy there is consensus that two cycles of
consolidation therapy are essential. However, it remains unclear
whether maintenance therapy with ATRA is required, although both
the North American Intergroup Trial and the European APL group
seemed to suggest that patients with APL may benefit from maintenance
ATRA, with or without low-dose chemotherapy—particularly those
who are at high-risk for recurrent disease.33,35
NEWER INDUCTION CHEMOTHERAPY APPROACHES IN AML

As discussed above, histone acetylation plays a significant role in
cellular transcription and leukaemogenesis. Histone acetylation allows
for greater exposure of DNA to transcription factors by enabling DNA/
histone contacts to loosen or relax, thus promoting gene expression
and cellular differentiation. Conversely, histone deacetylation is
expected to inhibit gene expression and arrest cellular differentiation.
The fusion genes PML-RAR and AML-1/ETO have already been
described as promoting histone deacetylation. Thus, histone acetylation
promoters and histone deacetylation (HDAC) inhibitors are logical
agents for clinical study in AML.
Several HDAC inhibitors are currently under clinical investigation.
These include depsipeptide, sodium butyrate, and retinoic acid. Phase
1 trials showed minimal histone deacetylation activity in vitro with the
use of maximally tolerated doses of sodium phenylbutyrate, precluding
further single-agent studies. In one effort to overcome this obstacle,
retinoic acid and sodium phenylbutyrate were combined. As a result,
significant cellular differentiation was achieved, prompting further

clinical investigation.36 A case report from Memorial Sloan-Kettering
Cancer Centre described a case of APL who failed to respond to ATRA,
high-dose cytarabine, arsenic trioxide, and allogeneic transplantation
achieved complete remission at molecular level following ATRA and
sodium phenylbutyrate therapy for more than 6 months before eventual
relapse.37
Angiogenesis has been much discussed in recent years and seems to
play a role in leukaemogenesis. Overexpression of fibroblast growth
factor and vascular endothelial growth factor has been demonstrated
in lymphoid and myeloid leukaemia, respectively.38,39 There are a
number of potential targets for angiogenic inhibition, including
suppression of angiogenic factor release, binding of the free circulating
factor, receptor blockade, or direct interference with endothelial cell
function. Clinical trials with thalidomide and the tyrosine kinase
inhibitors (SU5416 and SU6668) are currently being evaluated.
Inspired by the success of ATRA, investigators have looked at other
retinoid compounds for use in leukaemia. Fenretinide (N-[4-
hydroxyphenyl] retinamide), first developed as a chemopreventive
agent,40 was shown to induce apoptosis in malignant cells whether or
not these cells possessed an RAR.41 Phase 1 testing demonstrated
acceptable toxicity.42
Protein kinase C (PKC) activation has been linked with the develop-
ment of nucleoside analogue resistance. Bryostatin, a PKC inhibitor,
has been studied in a number of solid and haematologic malignancies
and is currently being testing in combination with cytarabine, fludara-
bine, and 2-chlorodeoxyadenosine, as well as in combination with
ATRA. UCN-01 (7-hydroxystaurosporine), a selective, but non-specific,
kinase inhibitor with good activity against PKC, has been shown to
reverse cytarabine resistance in leukaemic cell lines.43 Moreover, both
bryostatin and UCN-01 have been shown to promote apoptosis via
phosphorylation of Bcl-2. Other strategies for overcoming drug resis-
tance are being tested, including reversal of the multidrug resistance
protein gene with quinine and cyclosporine.
POSTREMISSION CHEMOTHERAPY
It is presumed that most patients still have residual disease after the
completion of induction chemotherapy. Treatment options include
consolidation therapy with intensive cytarabine-based chemotherapy
regimens, high-dose chemotherapy or chemoradiotherapy followed by
autologous bone marrow stem cell support, or high-dose marrow-
ablative therapy followed by allogeneic bone marrow transplantation
(alloBMT).
Most nontransplant consolidation chemotherapy regimens

incorporate cytarabine. While the mortality rate associated with such
treatments varied from 10 to 20 per cent, disease-free survival rates
ranged from 20 to 50 per cent in several published studies.44-47 One of
these trials, a large study conducted by the CALGB, randomized 596
patients who achieved complete remission following standard 7 + 3
induction therapy, to receive 1 of 3 cytarabine-based consolidation
regimens for 4 courses.46 This was followed by 4 monthly cycles of
cytarabine (100 mg/m2 given by subcutaneous injection every 12 hours
for 5 days) with daunorubicin (45 mg/m2 on day 1). The first regimen
consisted of cytarabine (100 mg/m2 daily for 5 days) by continuous
infusion. The second regimen administered 400 mg/m2 of cytarabine
daily for 5 days by continuous infusion. The third regimen consisted of
3 g/m2 of cytarabine, given over 3 hours every 12 hours on days 1,3,
and 5.
With a median follow-up of 52 months, disease-free survival was
markedly different among the 3 treatment groups, with a 44 per cent
probability of remaining disease free in the high-dose cytarabine arm,
compared with 24 and 29 per cent, respectively, in the 100 mg/m2 and
200 mg/m2 arms. The authors concluded that there was a dose-response
effect for cytarabine in patients up to 60 years old for consolidation of
AML.
Subset analysis indicated that high-dose cytarabine was particularly
beneficial for patients with favourable prognosis cytogenetics, whereas
among patients with intermediate risk cytogenetics, the benefits of high-
vs intermediate-dose cytarabine were obscured. Those patients with
poor risk cytogenetics did not fare better in any treatment arm. Neither
the effect of maintenance therapy nor the number of consolidation cycles
on treatment outcome were independently analysable and remain
important questions that must be answered in the context of pros-
pective, randomized, clinical trials.
An ECOG trial published almost a decade ago is of greater inter-
pretive value in this regard.48 Following uniform induction therapy,
those patients younger than 41 years who had a histocompatible sibling
underwent alloBMT. The other patients were randomized to receive
either 2 years of continuous maintenance therapy with cytarabine and
thioguanine (83 patients) vs a single course of high-dose cytarabine, 3
g/m2 every 12 hours for 12 doses followed by amsacrine 100 mg/m2
daily for 3 days (87 patients). At 4 years, a 27 per cent relapse-free
survival was observed among patients younger than 60 years who were
treated with high-dose cytarabine plus amsacrine, compared with a 16
per cent relapse-free survival rate among those patients receiving
maintenance chemotherapy (P = .07). Mortality for those patients older
than 60 years of age who were treated with high-dose chemotherapy
was unacceptably high (57%). The 4-year event-free and overall survival
rates were similar in both alloBMT and consolidation chemotherapy;
however, both were clearly superior to maintenance therapy.
AML IN ELDERLY
Since the median age at presentation for AML is 65 years, most patients
have the unfavourable prognostic risk group by virtue of age alone.
Moreover, older adults tend to have presence of other conditions that
make treatment even more difficult by the very nature of the cytotoxic
drugs. Clearance of such cytotoxic drugs may also be impaired by
hepatic or renal dysfunction. Previous SWOG data have disclosed that
multidrug resistance overexpression was observed in more than 70 per
cent of de novo AML patients over the age of 55 years and is highly
predictive for failure to achieve complete remission.49 There is a greater
incidence of AML arising from prior myelodysplasia, which, in turn, is
accompanied by less favourable cytogenetic anomalies. All of these
factors contribute to poor prognosis.
The European Organisation for the Research and Treatment of Cancer
(EORTC), in their AML9 trial, observed median survival of 9-month
and a 5-year survival rate of 8 per cent in patients older than 60 years.50
A 5-year survival rate of less than 5 per cent in patients aged over 60
years was reported during the 4th International Workshop on
Chromosomes and Leukaemia.51 It is likely that the real survival rate
may be even worse as large number of patients are not included because
of the presence of various exclusion criteria, selection bias, and other
complicating factors.
The ideal induction regimen for older patients has not yet been
defined. While cytarabine/anthracycline-containing induction
regimens produce complete remission rates of approximately 70 per
cent in patients under the age of 60 years, those same regimens result
in complete remission rates of 45 to 50 per cent in patients older than
60 years.46,52 In younger patients, as discussed previously, disease-free
as well as overall survival advantages have been demonstrated in trials
that have incorporated idarubicin or mitoxantrone with higher doses
of cytarabine. On the other hand, these same trials show no advantage
within older populations, when compared with standard 7 + 3 therapy.
An ECOG trial (E3993) that compared daunorubicin vs mitoxantrone
vs idarubicin showed no clinical advantage with either mitoxantrone
or idarubicin.53
Few studies have focussed specifically on older adults in terms of
the best postremission consolidation therapy, but high-dose cytarabine,
with or without mitoxantrone, has not been found to confer clear
benefit.46,54 In the previously cited CALGB trial,46 which randomized
patients to 3 consolidation arms, only 29 per cent of patients over the
age of 60 years were able to receive all 4 courses of high-dose cytarabine,

compared with a 60 per cent completion rate among younger patients.
This has led some investigators to use low-dose chemotherapy in both
induction and consolidation phases (the focus of several small trials,
not all of them prospectively randomized), while others have opted to
use no therapy at all.
The “no therapy” approach was demonstrated in an EORTC trial in
which patients, median age 71 years, were randomized to receive
standard therapy at time of diagnosis vs no therapy until clinical
deterioration was apparent.55 There were profound differences between
treatment arms, with 58 per cent of those treated upfront achieving
complete remission, while no patients in the delayed treatment group
achieved remission. This translated into substantially different survival
rates at 2 years (17% vs 0%, respectively).
Another multi-institutional European trial randomized elderly
patients to receive either intensive induction chemotherapy or low-
dose cytarabine induction therapy.56 The results demonstrated a clear
advantage for those patients receiving the more intensive regimen, with
a complete remission rate of 52 per cent, compared with a 32 per cent
remission rate in the low-dose cytarabine arm. A higher overall survival
rate was noted in the more chemointensive treatment arm (12.8 vs 8.8
months). This improvement in response and survival was, however,
associated with a 31 per cent induction mortality rate vs 10 per cent in
the low-dose cytarabine arm, as well as an increased need for trans-
fusion support.
As is the case in younger patients, the number of postremission
chemotherapy cycles remains undefined for older patients.
Once again, the question of prolonged maintenance therapy has been
raised; because standard or dose-intensive consolidation regimens are
not well tolerated by older patients. A study by the leukaemia co-
operative group of the EORTC examined disease-free survival among
elderly patients who were randomized to low-dose cytarabine
maintenance therapy (10 mg/m2 subcutaneously every 12 hours for 12
days every 6 weeks) or observation, after completion of induction
therapy with either mitoxantrone/cytarabine or daunorubicin/
cytarabine. While no difference in overall survival was noted, a 5-year
disease-free survival advantage of 13 vs 7 per cent (P = .006) was
reported in the low-dose cytarabine-treated cohort, compared with
those patients who were merely observed.
Clearly, newer treatment strategies for management of AML in the
elderly are essential. Given the higher incidence of multidrug resistance
gene expression (particularly the gp170 multidrug resistance protein)
in this population, it is logical to develop therapies that target this entity.
A recent SWOG trial showed positive results targeting gp170 multidrug
resistance with cyclosporin.57 Clinical studies with valspodar (PSC833),

a nonimmunosuppressive multidrug resistance inhibitor, have either
not shown improvement in outcome or were stopped early because of
toxicity.
Non-myelosuppressive immunotherapeutic approaches include
immunostimulatory agents, such as IL-2, monoclonal antibody therapy,
and vaccine therapy. IL-2 can instigate a graft vs leukaemia effect via
activation of T and natural killer cells and has demonstrated the efficacy
and feasibility in clinical trials.58,59 A phase 3 CALGB study is ongoing
in patients aged 60 years or more who remain in remission following
induction and consolidation therapy for AML. Patients are randomized
to either observation or a 90-day course of subcutaneous low-dose
IL-2.
Vaccine therapies, using murine leukaemic cells that have been
transfected with adenovirus vectors that express immunologically
detectable antigens, have already been developed. These therapies have
demonstrated an ability to protect vaccinated subjects against
inoculations of wild-type leukaemia cells.60 Mice with active leukaemia
have gone into remission following inoculation with these vaccines.
Other forms of vaccine manipulation have stimulated AML cells to
express dendritic cell antigens or have fused AML cells to autologous
dendritic cells in order to make them amenable to intrinsic immune
responses.61
POSTREMISSION TRANSPLANTATION
As the dose-intense postremission chemotherapy confers a survival
advantage in younger patients, it appeared logical that the dose
escalation followed by either autologous stem cell transplantation or
allogenic bone marrow transplantation (alloBMT) should improve the
survival. Subsequently, it was well established that alloBMT from a
human lymphocyte antigen-matched sibling can cure 50 to 60 per cent
of recipients. 62-64 However, the benefits of BMT must be weighed
against the risks of immunosuppression or mortality (20% to 40%)
secondary to graft-vs-host disease (GVHD), veno-occlusive disease of
the liver, interstitial pneumonitis, or graft failure. Currently most centres
feel that allogeneic transplants may be undertaken for patients younger
than 55 years following first remission.
Autologous bone marrow transplantation (ABMT), permits the
administration of dose-intensive chemotherapy, but, unlike alloBMT,
does not confer the potentially favourable graft-vs-leukaemia effect.
Additionally, ABMT may be associated with reinfusion of occult
residual leukaemia cells. Peripheral blood haematopoietic progenitor
cells were successfully harvested and later reinfused with more rapid
recovery of haematopoiesis than autologous BMT.65 With improvements
in “in vivo” purging, the results of autografting with peripheral blood

stem cell transplantation (PBSCT) were simillar to ABMT.66,67 Both
PBSCT and ABMT produce disease-free survival rates ranging between
35 and 50 per cent in AML patients after first remission. Treatment-
related mortality is lower in PBSCT and ABMT (10% to 20%). Several
controversial issues persist in PBSCT and ABMT, including optimal
timing for ABMT, the necessity or degree of consolidation therapy prior
to ABMT, and the role of graft purging with various chemotherapeutic
or immunologic agents.
A prospective trial from the City of Hope Cancer Centre studied the
issues of consolidation therapy and graft purging in 60 patients with
AML who were in first remission.68 Study participants were treated
with 1 course of high-dose cytarabine before marrow collection. A total
of 44 patients proceeded on to ABMT, following fractionated total body
irradiation, as well as myeloablative etoposide/cyclophosphamide. At
2 years, the intent to treat disease-free survival was 49 per cent,
comparable with survival rates achieved with alloBMT and regimens
requiring autologous purging.
Combined European and Italian data comparing alloBMT vs ABMT
vs high-dose consolidation cytarabine chemotherapy after first
remission have demonstrated equivalent survival in both transplant
arms, whereas comparable survival is achieved in the high-dose
chemotherapy arm when relapsed patients underwent ABMT.69 A
similarly designed US collaborative trial also found no difference in
disease-free survival between alloBMT, ABMT, or dose-intensive
chemotherapy with high-dose cytarabine.70 Two paediatric studies have
shown no advantage to purged ABMT overdose-intense consolidation
chemotherapy regimens.71-72 No superior response was demonstrated
when unpurged ABMT grafts were compared with dose-intensive
consolidation chemotherapy in an adult AML cohort.73 Contradictory
results observed in these studies may be secondary to variations in the
types of AML that predict for outcome. This has been shown, to some
degree, in a retrospective cytogenetic analysis of 999 patients treated
with alloBMT or ABMT following first remission.74 Those patients with
poor prognostic cytogenetics did not fare as well following alloBMT,
compared with their normal or favourable karyotypic counterparts.
Decision regarding transplantation should be based from case to case.
The short-and long-term toxic effects of transplantation must be
weighed carefully against the risk of relapse. Conversely, most clinicians
would support alloBMT following first remission in patients with poor
prognostic risk factors.
RELAPSED AND REFRACTORY AML

Majority of non-APL AML patients treated with chemotherapy alone,
tend to relapse. Several factors responsible for relapse include (a) patient
age (b) cytogenetic findings, and (c) most important factor is duration
of first remission.75 Those patients whose remission has lasted for 2
years or more will achieve a second remission in 50 to 60 per cent of
cases, when treated with same regimen. If the first remission lasted 12-
14 months, the patient has a 40 per cent chance of attaining a second
remission. In patients whose remissions lasted less than 1 year or who
failed to achieve a first remission (primary refractory disease), complete
remission was achieved in 10 to 20 per cent cases when treated with
the same regimen. Long-term survival at 3 years varied from 20 to 25
per cent in patients with longer first remission as compared to no
survival in patients with shorter-duration remission.76 Therefore,
treatment decisions must be based, in large part, upon an individual’s
potential for obtaining and maintaining a remission. Those patients
who are at greater risk for failure should not be offered standard
therapies as a matter of course.
Whether high-dose cytarabine should be used as investigational
therapy is an important consideration. Estey and colleagues 77,78
suggested that high-dose cytarabine is better able to induce second
remissions following short-, intermediate-, and long-term first remission
relapses when compared with a variety of investigational therapies. In
the group that had a first remission lasting less than 12 months, high-
dose cytarabine was no better than investigation strategies for extending
survival, even though higher initial rates of complete remission were
achieved. These results were attributed to (a) short second remission
durations and (b) higher mortality rates associated with dose-intensive
chemotherapy. Therefore, the data does not support the use of
conventional high-dose cytarabine regimens in such cases. Similarly,
the data does support the use of high-dose cytarabine in patients whose
first remissions lasted for more than 1 year.
Ideally, patients with short duration of first remissions, barring other
complicating factors, should be considered as candidates for alloBMT
and ABMT and these patients can achieve approximately 30 per cent
long-term survival rates.79,80 Petersen and colleagues81 and Buckner
and colleagues82 have shown that alloBMT and ABMT can be offered
at time of relapse without benefit of prior chemotherapy with similar
survival, irrespective of whether chemotherapy preceded transplan-
tation.
Patients younger than 55 years with primary refractory disease,
alloBMT seems to be superior to ABMT, as demonstrated in data from
the City of Hope Cancer Centre and the International Bone Marrow
Transplant Registry.83,84 For patients who relapse at 1 year and less than
2 years after first remission, following standard or high-dose cytarabine
chemotherapy, alloBMT is still the preferred therapy. Comparing
alloBMT with chemotherapy in first relapse, the International Bone
Marrow Transplant Registry identified a leukaemia-free survival

advantage (41 vs 17%, respectively) among transplanted patients who
were younger than 30 years old with at least 1 year or more of initial
complete remission.85 A second group of patients, older than 30 years
with less than 1 year of initial complete remission, also achieved a higher
3-year leukaemia-free survival compared with chemotherapy (18 vs
7%, respectively). 86 Similar results were noted in patients who
underwent ABMT.
Patients with APL who relapse following treatment with ATRA and
an anthracycline-based chemotherapy regimen may be retreated with
ATRA, as long as resistance has not been demonstrated. For those
patients who fail to respond to a retinoid and chemotherapy, arsenic
trioxide (ATO) has emerged as the treatment of choice. Chinese
investigators reported patients treated with ATO between 1986 and
1998.87 A total of 104 patients received ATO-67 who relapsed after ATRA
treatment, 10 who relapsed following standard chemotherapy, 17 who
relapsed after prior ATO treatment, and 10 patients who relapsed post-
ABMT/alloBMT treatment. Half of the patients received only ATO
monotherapy, while the other half of the patients were given both ATO
and serial chemotherapy. Complete remission for the ATO arm was 55
per cent, and the median survival of complete responders was 4.8 years.
A total of 22 patients (43%) eventually relapsed. For the ATO plus
chemotherapy group, the relapse rate was lower (31%), but the median
complete remission duration was longer (7 years).
Many other studies on the use of ATO have been conducted in China,
and the findings were confirmed in a US multicentre study.88 Among
40 APL patients experiencing a first or second relapse who were treated
with daily infusions of ATO, up to a maximum of 60 doses, 34 patients
(85%) achieved complete remission, 31 (91%) of whom had post-treat-
ment cytogenetic tests that were negative for t(15;17). Among the
patients who were assessible by reverse transcriptase polymerase chain
reaction (RT-PCR), 86% converted from positive to negative for the
promyelocytic leukaemia/RA. Ralpha transcript following one course
of ATO. The most common side effect was electrocardiographic QT
prolongation, which was seen in 63 per cent of patients.
As the high complete response rate seen in first-line APL with the
use of ATRA, it is doubtful that ATO will become as initial therapy.
However, ATO remains a highly successful salvage alternative, one that
is well tolerated and has an acceptable level of toxicity. In developing
country, ATO may be used as first-line of therapy for those patients
who cannot afford ATRA.
MONOCLONAL ANTIBODY THERAPY IN AML

One of the more innovative approaches to treatment of malignancy
within the last decade has been the development of monoclonal
antibody (MAb) therapy. By targeting features unique to malignant

cells, these treatments conceptually allow for eradication of malignant
clones while sparing normal tissue. The first MAb developed was
rituximab, an unconjugated anti-CD20 Mab. It was approved by Food
and Drug Administration (FDA) in 1997 for the treatment of B-cell non-
Hodgkin’s lymphoma. In 1998, trastuzumab gained FDA approval for
the treatment of Her2/neu receptor-positive metastatic breast cancer.
Since then, MAbs, including the unconjugated anti-CD52 MAb
alemtuzumab, the anti-CD33 immunoconjugate MAb gemtuzumab,
and the anti-CD20 radioimmunoconjugate 90Y-ibritumomab tiuxetan,
have become available for the treatment of leukaemia and lymphoma.
Early in haematopoiesis, it is believed that a pluripotent stem cell
gives rise to committed precursor cells that are responsible for
production of granulocytes, monocytes, erythrocytes, and platelets.
Both stem cells and precursor cells express the CD34 antigen. In contrast,
the CD33 antigen is expressed in committed myeloid precursor cells,
but not in haematopoietic stem cells.89 The CD33 antigen is also
expressed by leukaemic blasts in at least 90 per cent of patients with
AML.90 The notion of employing “naked” unconjugated monoclonal
antibody therapy against AML does not seem logical as the effectiveness
of unconjugated antibody therapy depends upon the patient’s relatively
intact immune system to mediate antibody-dependent cellular cyto-
toxicity. AML results from dysregulation of the immune response. Acute
myeloid leukaemia seems to be an ideal target for delivery of mono-
clonal antibody therapy because leukaemia cells are generally well
vascularized, thereby allowing antibody access to all neoplastic cells.
Gemtuzumab ozogamicin (Mylotarg; Wyeth-Ayerst Laboratories;
Madison, New Jersey) is composed of an anti-CD33 IgG4 antibody,
complexed to calicheamicin, an anti-tumour antibiotic that generates
double-stranded DNA breaks, resulting in cellular death. 91
Gemtuzumab ozogamicin has been approved by the FDA for relapsed/
refractory CD33-positive AML in patients aged 60 years or older who
are not considered candidates for other types of cytotoxic chemotherapy.
Phase 1 and phase II trials revealed promising results, however, further
studies are required for proper evaluation.92,93
The CD45 antigen is expressed on all leucocytes and leucocyte
precursors and is found in more than 90 per cent of AML patients, as
well as in most all patients. Unlike the CD33 antigen, CD45 does not
internalize after antibody binding.94 Phase 1 testing of the radio-
immunoconjugate131I BC8 (Murine anti-CD45), in combination with
total body irradiation and cyclophosphamide as a pretransplant
marrow-ablative regimen among 34 patients, defined the maximum
tolerated dose of antibody-delivered radiation. The liver was
determined to be the dose-limiting organ at a maximum tolerated dose
of 10.5 gray (Gy). An average of 24 Gy to marrow and 50 Gy to spleen

could be delivered at the maximum tolerated liver dose. 95 This
radioimmunoconjugate is now in ongoing phase 2 testing. Radio-
labelled131I anti-CD45 antibody has also been given to patients with
AML in first remission, again as a preparative regimen with cyclophos-
phamide and busulfan for ABMT. 96 Whether the addition of the
radioimmunoconjugate confers a benefit to high-dose busulfan and
cyclophosphamide or whether straight busulfan/cyclophosphamide
followed by PBSCT alone is just as efficacious, remains to be determined
in randomized phase 3 testing.
CONCLUSIONS
The treatment of AML is advancing rapidly, and, like therapy in other
malignant states, is favouring treatment strategies “tailored” to specific
leukaemia subtypes. As more becomes known about the genetic and
molecular characteristics of leukaemia cells, and the pathways of
leukaemogenesis are further elucidated, it is hoped that future therapies
will be directed specifically toward the least toxic clonal malignant cells.
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8
Paroxysmal Nocturnal
Haemoglobinuria: Pathogenesis
and Molecular Biology
S Varma
Key Words
PNH • GPI • Complements • PIG-A gene
INTRODUCTION
Paroxysmal nocturnal haemoglobinuria (PNH) is an uncommon,
acquired, clonal haematopoietic stem cell disorder resulting in chronic
haemolytic anaemia classically associated with recurrent haemoglobi-
nuria that may also show features of bone marrow failure (cytopenias)
and vascular thrombosis.1,2 However, many patients may present with
chronic anaemia or other manifestations without any definite history
of paroxysmal haemoglobinuria, though evidence for intravascular
haemolysis in the form of haemosiderinuria may be evident. Though
an uncommon disorder with an estimated prevalence of 1-10 cases/
million population, it has been extensively studied and many of the
mysteries relating to its pathogenesis have been unravelled. Whereas
convincing explanation for the thrombotic episodes is still lacking,
intravascular haemolysis has been related to increased sensitivity of
the RBCs to the haemolytic action of activated complement.3 This
increased sensitivity was shown to result from the inability of
erythrocytes to inactivate the complement on their surface due to the
absence of specific cell surface proteins on the RBCs. Since several
proteins were found to be deficient, an abnormality to anchor these
proteins to the cell surface that occurred due to the absence of glycosyl
phosphatidyl inositol (GPI) was demonstrated4 (Fig. 8.1). This, in the
last decade, was finally linked to mutation in X chromosome (PIG-A
gene).5 Some details related to the present state of the knowledge are
reviewed in the subsequent text.
Increased sensitivity to
Haemolysis
complement mediated lysis
Absence of complement regulatory

membrane proteins
PIG-A
gene defect Impaired/ absent GPI anchor
Fig. 8.1: Pathogenesis of haemolysis in paroxysmal nocturnal haemoglobinuria
COMPLEMENT SENSITIVITY
Haemolysis in PNH is related to the sensitivity of the RBCs to the effect
of complement activation that may occur through the alternate or the
classical pathway.2,3 Depending upon the degree of sensitivity to the
effects of complement, the PNH cells have been divided into three
types.5 PNH I cells are those without any increase in sensitivity, PNH
II cells are 3-5 times (moderately) sensitive whereas PNH III cells are
15-20 times (exquisitely) sensitive to complement mediated lysis. The
proportion of cells of different types is variable and is responsible for
the phenotypic expression of the disease.
THROMBOSIS IN PNH
As opposed to intravascular haemolysis, the pathogenesis of thrombosis
is poorly understood. It is hypothesised that urokinase plasminogen
activator receptor (uPAR) which is GPI linked protein is deficient in
leucocytes of PNH patients.6 It might lead to impaired fibrinolysis. The
other hypotheses relate to activation of the coagulation cascade6 or
hyperactivity of the platelets by the activated complement pathway
products.6 Though undefined so far the possible mechanism may be
due to a combination of factors or primarily related to the platelet
abnormality.
GPI LINKED PROTEINS

PNH blood cells are deficient in a number of proteins that are linked to
the cell surface by a glycosyl phosphatidyl inositol (GPI) molecule.
There are several such proteins that require GPI anchor and belong to
different groups such as enzymes, receptors, complement regulators,
adhesion molecules, blood group antigens, etc. Over 100 such proteins
have been identified to date in mammalian cells.7 The first deficient
enzyme described in PNH was alkaline phosphatase, which was
Paroxysmal Nocturnal Haemoglobinuria 137
followed by the description of the deficiency of acetyl choline-esterase.

Though the degree of deficiency of the later enzyme correlated with
the complement sensitivity, no causal relationship could be established
between the two. First protein linked to the control of the complement
activity on the cell surface found deficient was decay accelerating factor
(DAF, CD 55). Though this is known to inhibit the formation of C3
convertases and their stability on the cell surface, it was observed that
its deficiency increased the sensitivity to complement only marginally.
Subsequently another protein that inhibited the membrane attack
complex, termed membrane inhibitor of reactive lysis (MIRL, CD 59)
was described. CD 59 hinders the insertion of C9 polymers (MAC/
membrane attack complexes) into the RBC membrane and thus prevents
complement-mediated lysis.6 Its congenital absence has been associated
with PNH like syndrome.7 Degree of CD 59 deficiency (complete or
partial) is related to the phenotype of PNH. When the deficiency is
complete, it gives rise to PNH III cells whereas partial deficiency is
associated with PNH II cells and Type I cells have normal expression.
Abnormalities of GPI anchored proteins can be demonstrated on
the haematopoietic stem cells as well as on other peripheral bloods
cells.7 However, blood cells other than erythrocytes deficient in CD 59
are not lysed despite in vitro demonstration of increased sensitivity to
complement compared to normal controls. This phenomenon is related
to the expression of an additional complement regulatory protein called
membrane co-factor protein (MCP, CD 46) on these cells8 and its absence
on the human erythrocytes. Additionally, substantially more MACs are
needed to lyse a leukocyte than an erythrocyte.
GPI ANCHOR
The structure and biosynthesis of the GPI anchor have been highly
conserved among the animal species. The GPI anchor consists of a
molecule of phosphatidylinositol (PI), a glycan core formed by a
molecule of N-acetyl glucosamine with three molecules of mannose
and a molecule of ethanolamine.13 PI end is attached to the lipid bilayer
of the cell membrane and proteins are attached to the ethonalamine
end through their carboxy end (Fig. 8.2). Biosynthesis of the GPI anchor
takes place in the endoplasmic reticulum. N-acetyl glucosamine is
transferred to the inositol moiety on phosphatidyl inositol molecule
from UDP-N-glucosamine under the influence of enzyme N-gluco-
samine transferase. This is followed by deacylation of N-glucosamine
and then the addition of three mannose and an ethanolamine to
complete the glycan core.6
The first step is complex and has been shown to be controlled by
three gene products that have been designated as class A, C and H in
murine cell lines. Somatic cell fusion complementational studies
DAF(CD55)
DAF (CD55)
MIRL (CD
MIRL (CD59)
59)
B11
B
B Mutant PIGAA
Mutant PIG
A
A
AA
PNH cells
Normal Haematopoietic PNH cells
Cells
Normal Hematopoietic Cells
Fig. 8. 2: Graphic representation of the molecular basis of PNH defect. Alphabet ‘A’
denotes transmembrane protein attached to RBC membrane (parallel black lines).
B denotes GPI anchored proteins that are absent in the PNH cells (B1). Unlike
transmembrane proteins, GPI anchor lacks transmembrane and cytosolic domains.
GPI anchor is a glycolipid composed of phosphatidylinositol (filled hexagon), N-
glucosamine (filled circle), three mannose moieties (unfilled circles) and
ethanolamine (straight line) to which protein is attached. Acyl/ alkyl glycerol of the
lipid anchors the structure in the lipid bilayer of the cell membrane. A somatic
mutation in the PIG-A gene results in failure of transfer N-acetyl glucosamine to
inositol from UDP-N-acetylglucosamine resulting in absence of GPI anchored
proteins in haematopoietic stem cells. (Adapted from Parker CJ Stem Cells
1996;14:396-411)
between murine cell lines and cultured cell lines or granulocytes from
PNH patients have demonstrated class-A complementational defect in
PNH.15 The gene for this defect was located on X chromosome and has
been termed PIG-A gene. It has been subsequently found that patients
with PNH have mutations in this gene and c-DNA from normal gene
can complement the cell lines from the PNH patients.
PIG-A GENE
Mutations in the PIG-A gene have been found in patients with PNH. It
has been mapped to the short arm of X chromosome (Xp22.1). It consists
of 6 exons and spans over 17 kb c-DNA, has 1455 base pairs and encodes
for a putative 60 kD protein having 484 amino acids.5 This protein is
located on the endoplasmic reticulum with amino terminus on the
cytosolic side and has α 1-6 N-acetyl glucosamine transferase activity.
By the end of year 2000, approximately 174 mutations had been
described in this gene5. Some of these result in complete functional
inactivation of the gene product due to large deletions, frame shifts or
non-sense mutations and may relate to PNH III defect. Other mutations
that resulted in mis-sense mutations or small in-frame deletions are
possibly related to the partial deficiency of the GPI anchor (PNH II
cells). The two types may frequently co-exist in the same patient that
indicates presence of more than one PNH clones. Mutations in the PIG-
A gene are located in all the exons with no definite hot spots. Most mis-
sense mutations are located on exon 2. These mutations are acquired
and are not familial.
CLONALITY OF PNH
The fact that mutations in PIG-A gene and deficiency of GPI anchored
proteins are demonstrable in more than one cell line in patients with
PNH is indicative of the mutation arising in the haematopoietic stem
cell. It was also borne out by demonstration of different G-6-PD isotypes
in PNH cells in double heterozygote female patients.6 Similarly, two
different populations of BFU-E, one sensitive and the other resistant to
the complement mediated lysis, were demonstrated in the blood of
patients with PNH. Finding identical mutation in different type of blood
cells of the same patient is the ultimate proof of the clonal origin of the
disorder.
DEVELOPMENT OF PNH
Demonstration of the GPI anchor abnormality and its relationship to
somatic mutations in the PIG-A gene provides an explanation for the
clinical features related to intravascular haemolysis. However, reasons
for expansion of this clone at the expense of normal haematopoietic
cells are far from clear. Hypothetically it could occur if the defect offered
a growth advantage to the PNH clone or alternatively if this clone could
escape a pathologic process that impairs the growth of normal
phenotype. However, no growth advantage over normal cells has been
demonstrated by experimental data and clinical observations also do
not support this contention. Spontaneous remissions with persistence
of small number of PNH cells are known to occur in this disease.
Similarly, small number of PNH cells, recognisable by flowcytometry
may be present in some patients or in normal adults without any clinical
manifestation. Culture assays of haematopoietic cells have also failed
to show any growth advantage.6 Though a decrease in apoptosis was
reported in one study, it has not been reproduced.
The second hypothesis relates to the existence of an extrinsic factor
that might result in expansion of the PNH clone. An association of PNH
with aplastic anaemia (AA) is well documented9 and it is possible that
the clone expands if bone marrow failure occurs due to any reason.
However, PNH clone does not occur universally in all AA patients and
appears to correlate with immunologically mediated bone marrow
failure only. It has been suggested that absence of GPI linked proteins
might allow PNH cells to escape immune injury, whereas the normal
clone might be affected. Recent data on lymphoma patients treated
with CAMPATH 1 H (which recognises GPI linked CD 52), demons-

trating appearance of GPI deficient cells on treatment and their
disappearance after its withdrawal support this hypothesis. It is possible
that there are other conditional events that help in expansion of the
PNH clone.
CLINICAL FEATURES
PNH is a rare disorder with an estimated annual incidence of 1 to 10
cases per million persons.10 It has been reported at all ages but is
diagnosed more commonly in fourth or fifth decade with a median
age of 35 years. Reports suggest that its incidence may be higher in
South East Asia and Far East.11 In two published case series from AIIMS
Delhi12 and Hyderabad,13 the patients were young adults with a male
preponderance. Our unpublished data shows slightly higher incidence
in females (Table 8.1).
Clinical presentations of PNH are complex and at times the diagnosis
is delayed considerably. Three main clinical syndromes with which
these patients present are related to: (i) chronic haemolysis with or
without acute exacerbations, (ii) tendency to thrombosis and (iii)
cytopenias of varying severity. Clinical picture depends upon the
presence or absence and the severity of each of these in a given patient.
Recognising the clinical heterogeneity, different designations based
upon the clinical presentation, blood findings and the size of the PNH
clone have been proposed (Table 8.2).
Haemolysis
Classic presentation with early morning haemoglobinuria is reported
initially by less than 25 per cent patients.14 Most patients would present
with chronic anaemia, at times have minimal jaundice and palpable
spleen. Persistent haemosiderinuria may result in iron deficiency.
Table 8.1: PNH Indian experience

AIIMS Hyderabad PGIMER
n 16 11 27
Age 17-62 16-42 16-43
M:F 16:0 9:2 12:15
Hb 2.5-9.6 4.0-7.9 1.7-9.9
Retics 0.6-10 2-30 0.5-30
TLC 1.2-10.0 2.9-9.0 0.8-9.2
Plat ↓ in12 variable 23-226
Hypo BM 2 0 7
Thrombosis ?1 4 0
Table 8.2: Clinical heterogeneity in patients with PNH clone and proposed
designations
Predominant Blood findings Size of Designation
clinical feature PNH clone
Haemolysis ± thrombosis Anaemia, little or Large Florid PNH
no other cytopenia
Haemolysis ± thrombosis Anaemia; mild to Large PNH,
moderate other hypoplastic
cytopenia/s
Purpura and/or infection Moderate to severe Large AA/PNH
pancytopenia
Purpura and/or infection Severe pancytopenia Small AA with PNH
clone
Thrombosis Normal or moderate Small Mini-PNH
cytopenia/s
(Tremmi G, Karadimitris A, Luzzatto L. Paroxysmal nocturnal hemoglobinuria:
learning about PNH cells from patients and from mice. Haematology. 1998;1:12-
20)
Haemolysis and consequently the severity of symptoms may be

aggravated by infections. During the phase of acute intravascular
haemolysis, the patient may also complain of abdominal and back pain.
The degree of haemolysis is directly proportional to the size of the PNH
clone, severity of PNH defect (PNH III versus PNH II cells) and the
degree to which the complement is activated.6
Thrombosis
Tendency to venous thrombosis has been widely documented in
patients with PNH. The reported incidence has been much higher in
American-European patients2,15 compared to those from South East and
Far East Asian regions.16 Even in India, the incidence of thrombotic
complications has been low (Table 8.1). The patients may present with
intra-abdominal thrombosis resulting in Budd-Chiari syndrome or
mesenteric venous thrombosis, cerebral venous thrombosis or that of
veins of the limbs, skin or other sites. Vascular thrombosis is important
cause of morbidity and mortality in these patients.15
Cytopenias
Many patients with PNH have varying degree of granulocytopenia,
thrombocytopenia and relative reticulocytopenia. These indicate
deficient haematopoiesis.6 At times it may be hypoproliferative and
some patients of PNH may progress to aplastic anaemia. Such patients
may have clinical course complicated by haemorrhage or infections.
Appearance of PNH clone in patients with de-novo aplastic anaemia
has been demonstrated by several observers. We demonstrated PNH
clone by flowcytometery in 31 per cent patients of aplastic anaemia on
follow up as opposed to 8.9 per cent at diagnosis.17 However, Hams

Acidified Serum Test was positive in only 3 patients.17 It is possible
that bone marrow failure favours the expansion of PNH clone.
DIAGNOSIS
Laboratory investigations would reveal anaemia with or without other
cytopenias, features of intravascular haemolysis. Bone marrow is
hypercellular to varying degree of hypocellular marrow. Conventional
laboratory tests used are demonstration of haemosiderinuria (as a result
of chronic intravascular haemolysis) and Hams Acidified Serum Lysis
Test (HAST) or sucrose lysis test. The latter two are related to the
increased sensitivity of PNH cells to complement at acidic pH or
haemolysis as a result of increased absorption of complement compo-
nents by the red cells in the presence of low ionic strength solutions.
With the availability of flowcytometeric techniques the PNH clone
can be demonstrated by showing deficiency of GPI anchored proteins
on the blood cells.18 Deficiency of DAF (CD55) and/or MIRL (CD59) is
indicative of PNH clone. Absence of uPAR (urokinase like plasminogen
receptor) or s-uPAR (soluble uPAR) on the granulocytes has recently
been shown to be equally sensitive. A rapid test using gelcard to detect
GPI anchor deficient cells is also available. Finally PCR based techniques
can be used to detect the mutations in the PIG-A gene.
MANAGEMENT
Approach to managing a patient with PNH is largely empirical and is
directed towards symptomatic management, attempt to control
haemolysis, treat thrombotic episodes and managing bone marrow
failure by immunosuppression or stem cell transplantation.
Replacement Therapy
Supplemental folic acid and iron is needed in most patients. At times
iron supplementation might result in haemoglobinuria due to the
outpouring of reticulocytes necessitating the use of prednisolone or
RBC transfusion.19 Patients with anaemia can be transfused if indicated.
Since haemolysis of complement sensitive cells can occur with blood
transfusion, especially with whole blood, it is preferable to give packed
red blood cells.
Steroids
Prednisolone has been found to be useful in controlling or suppressing
haemolysis. In chronic haemolysis, it may be used as an alternate day
therapy in 0.3 to 0.5 mg/kg dose whereas in acute haemolysis higher
daily dose may be used. A dose administered in early evening can
prevent a nocturnal episode. It is unlikely to have any effect on bone

marrow failure. Androgenic hormones and attenuated androgens like
Danazol have been used for the treatment of anaemia in PNH and have
been shown to be effective in some patients.19
Management of thrombotic episodes Venous thrombosis is a major
cause of morbidity and mortality. Acute thrombosis is treated with
anticoagulation. Fibrinolysis using tPA or other agents has been shown
to be useful in patients with recent onset thrombosis.20 Anticoagulation
should be initiated with heparin and followed with warfarin. Role of
prophylactic anticoagulation has not been established. Antiplatelet
agents may be required in some patients who continue to have thrombo-
sis despite anticoagulation.21
Management of Cytopenias
If an HLA identical sibling is available, allogeneic haematopoietic stem
cell transplant would be the treatment of choice for a young patient.
Transplant from unrelated donors has been associated with unaccepta-
bly high complication rates and remains experimental at present.21
Immunosuppression with ATG or ALG along with cyclosporine is the
alternative for other patients.6,19 Patients presenting primarily with
hypoplastic marrows tend to have better response compared to those
with haemolysis as the predominant presentation.22 Anecdotal reports
of beneficial effects of growth factors like G-CSF, GM-CSF and high
dose erythropoietin administration are available in literature.5,23 A
controlled trial would be needed to clarify the role for these factors in
the management of PNH.5
Future Strategies
Strategies based upon gene therapy to correct the gene defect by using
stem cells transfected with PIG-A gene,21 transfer of GPI anchor
proteins,24 use of anti-complement drugs or development of inhibitors
of effector pathways may prove useful in future.
PROGNOSIS IN PNH
PNH is a chronic disease with a variable clinical course depending
upon the severity of the presenting illness, associated complications
and progression to secondary leukaemia or marrow failure. However,
most patients have prolonged survival and spontaneous remission may
be noted in an occasional patient. Many patients may live 20 years or
beyond. In a retrospective analysis of 220 patients, Socie et al reported
a median survival of 14.6 years and survival estimate of 78 per cent at
5 years, 66 per cent at 10 and 58 per cent at 15 years from the time of
diagnosis.15 Same investigators identified thrombosis, evolution to
pancytopenia, evolution to myelodysplastic syndrome or acute

leukaemia and age > 55 years at the onset as the indicators of poor
prognosis (Table 8.3).
Table 8.3: Poor prognostic factors in PNH

Factors RR CI
Thrombosis 10.2 6-17
Evolution to
pancytopenia 5.5 2.8-11
MDS/AL 19.1 7.3-50
Age > 55 4 2.4-6.9
Need for additional treatment 2.1 1.3-3.6
Thrombocytopenia 2.2 1.3-3.8
Lancet 1996,348:560
CONCLUSIONS
Despite the rarity of the disease, PNH has been extensively studied
and the molecular basis has been clearly defined. It is an acquired,
clonal, somatic disorder that presents with intravascular haemolysis,
cytopenias and thromboses. IV haemolysis is due to increased
complement sensitivity that occurs as a result of partial or complete
absence of GPI anchored protein (CD59, MIRL). Pathogenesis of
thrombosis is unclear, though platelet activation and impaired
fibrinolysis may be responsible. GPI anchored proteins are absent
because of mutation(s) in the PIG-A gene that result in blocking of
transfer of N-acetyl glucosamine from UDP-N-acetyl glucosamine to
PI moiety due to lack of α 1-6 N-acetyl glucosamine transferase activity.
Expansion of PNH clone occurs probably due to a second event that
helps in cellular selection. Although great progress has been made in
understanding the molecular basis of the disease, several aspects of its
biological behaviour need further elucidation. The disease demonstrates
clinical heterogeneity with prolonged survivals at one hand and
occurrence of serious life-threatening complications on the other.
Treatment strategies need to be individualised for the moment and in
selected individuals with appropriate donor, stem cell transplant is
curative. Gene therapy, anti-complement drugs and transfer of GPI
anchor proteins need to be explored as future treatments.
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9. Nagarajan S, Brodsky RA, Young NS, Medof ME: Genetic defects underlying
paroxysmal nocturnal haemoglobinuria that arises out of aplastic anaemia.
Blood 1995;86:4656-61.
10. Luzzatto L, Bessler M, Rosoli B. Somatic mutations in paroxysmal nocturnal
haemoglobinuria-a blessing in disguise? Cell 1997;88:104.
11. Issargrisil S. Epidemiology of aplastic anaemia in Thailand. Thai aplastic
anaemia group. Int J Hematol 1999;70:137-40.
12. Saxena R, Malhotra OP, Saraya AK. A clinico-hematological profile of
paroxysmal nocturnal haemoglobinuria. JAPI 1991;39:741-43.
13. Koduri PR, Gowrishankar S. Paroxysmal nocturnal haemoglobinuria in
Indians. Acta Hematol 1992;88:126-28.
14. Nishimoura T, Murakami Y, Kinoshita T. Paroxysmal nocturnal haemoglobin-
uria: an acquired genetic disease. Am J Hematol 1999;62:175-82.
15. Socie G, Mary JY, de-Gramont A, Rio B, Leporrier M, Rose C et al. Paroxysmal
nocturnal haemoglobinuria: long term follow-up and prognostic factors.
Lancet 1996;348:573-77.
16. Kruatrachue M, Wasi P, Na-Nakorn S. Paroxysmal nocturnal haemoglobinuria
in Thailand with special reference to aplastic anaemia. Br J Hematol
1978:39:267-76.
17. Varma N, Varma S, Vohra H, Malik K, Garewal G. Flowcytometric detection
of PNH defect and response to therapy in aplastic anaemia patients. Methods
in Cell Science 2002;24:77-08.
18. Ward MS. The use of flowcytometery in the diagnosis and monitoring of
malignant hematological disorders. Pathology 1999;31:381-92.
19. Rosse WF. Paroxysmal nocturnal haemoglobinuria as a molecular disease.
Medicine 1997;76:63-93.
20. Hauser AC, Brichta A, Pabinger-Fasching I, Jager U. Fibrinolytic therapy with
rt-PA in a patient with paroxysmal nocturnal haemoglobinuria and Budd-
Chiari syndrome. Ann Hematol. 2003;82:299-302.
21. Meyers G, Parker CJ. Management issues in paroxysmal nocturnal
haemoglobinuria. Int J Hematol. 2003;77:125-32.
22. Packman CH. Pathogenesis and management of paroxysmal nocturnal
haemoglobinuria. Blood Reviews 1998;12:1-11.
23. Fujimi A, Matsunaga T, Kogawa K, Ohnuma T, Takahira N, Abe T et al. A

patient with paroxysmal nocturnal haemoglobinuria in whom granulocyte
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62.
24. Soland EM, Maciejewski JP, Dunn D, Moss J, Brewer B, Kirby M et al. Correction
of the PNH defect by GPI anchored protein transfer. Blood 1998;92:4439-45.
9
Inherited Bone Marrow
Failure Syndrome
VP Choudhry,
Maitreyee Bhattacharyya
Key Words
Marrow failure • Chromosomal breakage • Bone
marrow transplantation • Gene therapy
INTRODUCTION
Genetic disorders characterised by bone marrow failure resulting in
decreased production of one or more haematopoietic lineages are in
frequent states. These states were considered as constitutional, as most
of these disorders were associated with congenital abnormalities. With
the better understanding and identification of various genetic
mutations, now these disorders have been classified as inherited
marrow failure syndrome. Now, it is believed that genetic mutations
interfere with haematopoiesis and cause the marrow failure, although
the specific molecular basis has not been identified for some of these
disorders. Acquired factors may also be operative and may interact
with the putative genetic mutations to evolve into overt disease with
varying clinical expression. Pathophysiological factors such as viruses,
drugs, chemicals or toxins might provide the ‘second hit’ and result in
progression of the disease.
Several studies from different countries have shown that these
syndromes comprise 30-35 per cent of cases of paediatric marrow failure
and among these Fanconi’s anaemia represents about 2/3rd of cases.1
The various types of inherited bone marrow failure syndromes along
with the inheritance pattern are given in Table 9.1.
Fanconi’s Anaemia (FA)

It was first described by Fanconi in 1927 in three brothers who had
pancytopenia and physical abnormalities at birth. Detailed variable
Table 9.1: Inherited marrow failure syndromes

Syndromes Genetics Lineage involved
Fanconi’s Anaemia AR Pancytopenia
Monocytopenia-bi or
pancytopenia
Diamond-Blackfan Anaemia AR, AD Anaemia
Dyskeratosis Congenita XLR, AR, AD Monocytopenia to
Pancytopenia
Kostmann’s syndrome AR Neutropenia
Shwachman-Diamond syndrome AR Neutropenia bicytopenia
Pancytopenia
Thrombocytopenia with absent radii AR Thrombocytopenia
Amegakaryocytic thrombocytopenia XLR, AR Thrombocytopenia
Pancytopenia
* AR-Autosomal Recessive, AD-Autosomal Dominant, XLR-X-Linked Recessive.
clinical picture of approximately 1000 cases over the ensuing 70 years

has been recently reviewed by Alter BP.2 The true incidence of Fanconi’s
anaemia is difficult to ascertain, because of its wide age range at
presentation.3 The mean age at diagnosis is 7.9 years in males and 9
years in females with median age of 6.5 and 5 years, respectively. Its
diagnosis has been made from birth to 32 years in males and from birth
to 48 years in females. Patients with associated congenital anomalies
are generally diagnosed 4 years earlier as compared to patients without
any birth defects.
Inheritance and Environment

Fanconi’s anaemia is an autosomal recessive disorder involving all races
and ethnic groups. There is considerable heterogeneity within families
as evident from a family in which one brother had typical Fanconi’s
anaemia while another had paroxysmal nocturnal haemoglobinuria and
their cousin died of leukaemia.3 The brother with PNH was diagnosed
to have Fanconi’s anaemia after 30 years by diepoxy butane (DEB)
induced chromosomal breakage studies and developed cancer at the
age of 66 years. Fanconi’s anaemia gene, in different external and
internal environments often results in different phenotypes. Develop-
ment of aplastic anaemia in Fanconi’s anaemia (homozygotes) has been
reported following viral illness, hepatitis and chloramphenicol therapy
suggesting that the environment plays a major role in modifying the
course of the disease.
Inherited Bone Marrow Failure Syndrome 149
Genetics and Pathogenesis

Eight different genetic groups have been described by complemen-
tation, FA-A through FA-H. The genes for three of these groups FANCA,
FANCC and FANCG have been cloned.4,5 Mutations in FANCA
accounts for 65 per cent of Fanconi’s anaemia patients whereas FANCC,
FANCG mutations account for another 15 and 5 per cent of mutation
respectively.6 The relationship among birth defects, haematopoietic
failure, malignancies and chromosome breakage continues to be elusive.
Clastogenic agents causing chromosomal breakage include radiation
and bifunctional cross-linkers such as Mitomycin C (MMC), nitrogen
mustard and Diepoxybutane (DEB). It has been proposed that FA cells
are damaged by free oxygen radicals.7 Red cell levels of superoxide
dismutase (SOD) were low. Culture of lymphocytes in the presence of
increased oxygen resulted in an increase in number of spontaneous
breaks in FA cells and not in normal cells. The haematopoietic defect is
at the level of pluripotent stem cells. Colonies from bone marrow CFU-
GM, CFU-E and BFU-E as well as from blood BFU-E were all decreased
in patients with aplastic FA.8
Fanconi’s cells are defective in DNA repair. Cellular features of FA
are hypersensitivity to DNA cross-linking agents and accelerated
telomere shortening.9 Fanconi’s anaemia cells have an impairment of
RAD-51 dependent homologous recombination pathway for DNA
repair, which results in chromosomal instability and extreme sensitivity
to DNA cross-linking agents.10
Clinical Presentation
Fanconi’s anaemia is characterised by short stature, abnormal thumbs,
microcephaly, café au lait as well as hypopigmented spots and a
characteristic facial appearance including a broad nasal base, epicanthic
folds and micrognathia. The skin is involved most frequently, followed
by poor growth, anomalies of the upper limbs, male hypogonadism
and microcephaly. The number and size of the pigmentary changes
increases with age. Café au lait spots are actually more common than
hyperpigmentation. Upper limb anomalies involve thumbs most
frequently with absence, hypoplasia, supernumerary, bifid or
triphalangeal thumb. Radii are often absent or hypoplastic. In Fanconi’s
anaemia if radii are affected, thumb is always abnormal, while in
thrombocytopenia absent radii (TAR) syndrome radii are absent and
thumb is always present. In male patients underdevelopment of the
genitalia is most frequently followed by undescended testes. Most
common abnormalities of the eyes include microphthalmia and stra-
bismus while deafness and structural abnormalities are associated with
ear defects. Renal malformation include ectopic, pelvic or horse shoe
kidneys. Low birth weight and failure to thrive is other common
manifestation. Less common defects include congenital heart disease,

gastrointestinal anomalies and a variety of others.
Diagnosis
Patients have anaemia alone or may be associated with bi or pan-
cytopenia. Pancytopenia often occurs with advanced age. Differential
counts are generally within the normal range. Fanconi’s anaemia is
diagnosed by demonstrating increased breaks, gaps, rearrangements,
exchange and reduplication in metaphase preparation of cultured
peripheral blood lymphocytes. Breakage of chromosomes dramatically
increases on exposure to clastogenic agents such as (DEB), nitrogen
mustard and mitomycin C. Patients with Fanconi’s anaemia often do
not show spontaneous breaks but are positive for DEB. Fanconi’s
anaemia homozygotes have a mean of 8.96 breaks (range 1.3 to 23.9)
per cell following culture of peripheral blood lymphocytes with DEB
compared to mean of 0.06 (range 0 to 0.36) in normals as per
International Fanconi’s Anaemia Registry.11
Fanconi’s anaemia can also be diagnosed using flow cytometry to
demonstrate that Fanconi’s anaemia cells treated with alkylating agents
fail to divide but undergo DNA replication and accumulate in the G2
phase of the cell cycle, where they are detected because of increased
amount of DNA per cell.12 Fanconi’s anaemia patients are currently
identified by the characteristic chromosomal response to clastogenic
stress even in the absence of abnormal physical examinations and
normal haematologic picture.
Complications
A major feature of the Fanconi’s anaemia phenotype is the propensity
to develop cancer. The karyotype data, the defects in DNA repair and
the cellular damage that occur in Fanconi’s anaemia translate into an
enormous predisposition for malignancy. Over 80 patients (of 1000
published cases) have developed leukaemia, 30 developed liver
tumours and 47 had other cancers giving an overall incidence of
malignant transformation of about 20 per cent.
Seventeen patients of Fanconi’s anaemia with leukaemia never had
any preceding history of anaemia when they presented with
leukaemia.14 Acute myeloid leukaemia often evolves in these cases.
Over 40 patients developed into cancer other than leukaemia and
liver tumours. The majority of the tumours were gastrointestinal.13 Next
most common malignancy were gynaecological. Most of the cancers
are squamous cell carcinoma while hepatic tumours were hepatocellular
carcinoma followed by hepatomas and adenomas.13
Development of malignancy following Fanconi’s anaemia needs to
be treated differently. The myeloid and erythroid stem cells in Fanconi’s
anaemia have increased sensitivity to chemotherapy and irradiation.
Therapy
Initial therapy of Fanconi’s anaemia was limited to supportive
treatment, comprising of repeated blood transfusion, and treatment of
infections. Earlier, over 80 per cent of patients died within 2 years of
onset of aplastic anaemia and virtually all within 4 years.15 However,
with the current therapy, projected median age of survival is nearly 30
years for patients as observed over the last 10 years.16
Treatment with androgen was first initiated by Shahidi and
Diamond.17 The overall response rate is approximately 50 per cent,
although most patients relapse following withdrawal of therapy. In
children, androgens are used along with steroid to prevent early
epiphyseal closure. Oxymethalone is preferred and is recommended
in dose of 2-3 mg/kg/day. If response occurs, the androgen should be
tapered slowly but should not be discontinued. Patients treated with
androgen should be carefully monitored with liver function tests and
ultrasonography.
Role of G-CSF has been investigated in 12 Fanconi’s anaemia patients
with neutropenia. Eight of the 10 patients who completed 40 weeks of
G-CSF treatment showed increases in the percentage of marrow and
peripheral blood CD34+ cells.18
Bone Marrow Transplantation (BMT)

Currently BMT offers the potential for cure of cases with Fanconi’s
anaemia and for prevention for its progression to leukaemia. However,
BMT will not prevent the development of other malignancies.
Conditioning regimen for BMT may accelerate the appearance of
secondary malignancies. BMT is indicated when a patient fails to
respond adequately to androgen and/or cytokine therapy necessitating
repeated transfusions, the presence of a persistent clonal cytogenetic
abnormality in marrow cells such as monosomy 7 and overt
transformation to MDS/AML . Initial results of BMT using unmodified
conditioning regimen of high-dose cyclophosphamide were discoura-
ging with only 47 per cent survival.13 However, recent protocols using
low dose cyclophosphamide (5mg/kg/d × 4 d) with 500 cGy of
thoracoabdominal irradiation has shown 75 per cent survival rate.19
The International Bone Marrow Transplant Registry (IBMT) has recently
reviewed the results of bone marrow transplantation in 228 patients of
Fanconi’s anaemia at 42 centres between 1978 to 1992.20 A number of
different conditioning regimens and graft versus host disease
prophylaxis were used by various centres. Multivariate analysis
revealed that younger age, use of antithymocyte globulin in the
conditioning regimen, use of cyclosporin for GVHD prophylaxis and
conditioning with limited field irradiation and low dose cyclophos-
phamide were significantly associated with decreased post-transplant
mortality. Results of transplantation for patients lacking fully

compatible related sibling donor are less satisfactory. The IBMTR study
reported a 29 per cent two years survival in alternative donor
transplants including mismatched sibling, matched and mismatched
parent donors, other related donor as well as 15 HLA matched unrelated
donors. Small series using HLA matched unrelated donors have been
reported more recently from Minnesota. Seven patients underwent
unrelated donor transplant, conditioning was done with cyclosphos-
phamide 40 mg/kg and 400-450 cGy total body irradiation. Five patients
were evaluable with three survivors at > 9 months. Major issues
included were GVHD and engraftment.21
The most exciting transplant possibility is the use of umbilical cord
blood. Seventeen patients of Fanconi’s anaemia have received unrelated
cord blood transplants in the United States and of which 10 are alive.22
Gene Replacement
Gene replacement therapy for Fanconi’s anaemia is an important
alternative for cure as the gene for several of the complementation
groups have now been identified and cloned. Retroviral vectors have
been developed that allow the expression of Fanconi’s anaemia proteins
in the human haematopoietic progenitor cells, and in vitro studies have
confirmed the presence of both retroviral sequences and expressed
protein in transducted cells. Evidence for long-term correction of
Fanconi’s anaemia cells in vivo has been elusive. However, recently Liu
JM et al were able to demonstrate the corrected stem cells in peripheral
blood for only brief period and only at low levels.23
DIAMOND BLACKFAN ANAEMIA (DBA)

DBA is a rare congenital hypoplastic anaemia which presents early in
infancy. Retrospective studies in the UK and the Netherlands are
consistent with an incidence of 4-5 per million live births.24 It is evident
that it is inherited as dominant and recessive in 12-25 per cent of cases.25
The disease is characterized by moderate to severe anaemia, occasional
neutropenia and/or thrombocytosis and a normocellular bone marrow
with erythroid hypoplasia.
Current diagnostic criteria of DBA include (a) Normochromic usually
macrocytic but occasionally normocytic anaemia presenting in 90 per
cent cases in first 12 months of life. (b) profound reticulocytopenia (c)
normocellular bone marrow with selective, marked deficiency of red
cell precursors (d) increased levels of erythropoietin (e) normal or
slightly decreased white cell count and (f) normal or often increased
platelet count.
Inheritance
More than 500 cases have been described. Occurrence of sporadic cases
are frequent (about 75%).16 It is evident that familial cases have both
dominant and recessive patterns of inheritance, thereby suggesting that
different molecular defects can result in the DBA phenotype. In 20
reported families with dominant inheritance, male and female patients
were equally represented. In 32 recessive families males are more often
affected than females suggesting X linked inheritance in some families.26

A patient with a X:19 chromosome translocation provided the first clue
to localization of DBA gene.27 Majority of familial cases suggested X-
linked inheritance. Linkage analysis of 13 multiplex families was
consistent with localization in chromosome 19q 13.2 in all the families
which included both dominant and recessive pattern of inheritance.
The X chromosome translocation break point was cloned and showed
disruption of a ribosomal protein gene RPS 19. Analysis of 40 cases
showed that 10 patients had missense, nonsense, splice site or frameshift
mutation.28 The discovery that mutation in a ribosomal protein gene
causes DBA is of considerable interest. How mutation in such a protein
can lead to specific defect in erythropoiesis and the other characteristic
features of DBA remains unclear. DBA like FA is genetically hetero-
genous. Besides mutation in RPS 19, which accounts for 25 per cent of
both sporadic and familial cases, there is evidence for the involvement
of at least two other genes.
Despite the effort of many investigators and recent identification of
a gene mutation in about 25 per cent of cases, the pathophysiology of
DBA remains obscure. In vitro studies of erythropoiesis revealed severe
or moderate deficiency of BFU-E and CFU-E.29 Subsequent studies
suggested heterogeneity of the disease reflected by the colony forming
cell assays by three different patterns of growths a) normal BFU-E; b)
less than 70 per cent BFU-E with a reduction of CFU-granulocyte
macrophage (GM) as well as c) less than 5 per cent of BFU-E with
reduced CFU-GM.30 A circulating inhibitor to erythropoiesis was
suggested but could not be confirmed.31
Cellular immune process suggested by Hoffman 32 was not
confirmed.33 A defect of the haematopoietic microenvironment was
suggested by Ershler et al.34 Currently it is believed that in DBA there
is defect of erythroid progenitor cells as bone marrow transplantation
offers cure.
In a review of 80 children, pallor was most common with median age
at diagnosis being between 2-4 months. 24 Congenital anomalies
occurred in approximately 25-30 per cent cases which included thumb

malformations (sublaxation, supernumerary, bifid, triphalangeal, flat
thenar eminence with or without absent radial pulse), craniofacial
anomalies (cleft or high arched palate, hypertelorism with flat nasal
bridge, strabismus, ptosis, cataracts), urogenital anomalies or multiple
abnormalities.
Therapy
Corticosteroids are considered as the first line of therapy. Steroid should
be started initially in a dose of 2mg/kg/day. Reticulocytes usually
appear within 1-2 weeks and when Hb reaches 10 gm/dl, its dose should
be gradually tapered. Approximately 60 per cent of patients are steroid
dependent, while 30 to 40 per cent fail to respond. Extremely high
dosages of I.V. methylprednisolone can induce a steroid independent
remission in DBA who fail to respond to conventional dosage of
prednisolone.
Patients refractory to steroid therapy can be managed by regular
tansfusion. The goal of transfusion therapy is to maintain adequate
growth, development and activity while maintaining haemoglobin
concentration between 8-9 gm/dl. The major complication of chronic
red blood cell transfusion therapy is the development of progressive
iron overload requiring chelation with desferioxamine. Spontaneous
remission may occur in 15 to 20 per cent cases of DBA.
Treatment with haematopoietic growth factors has been used in a
number of patients. Neimeyer et al observed no reticulocytes or
haemoglobin response in nine patients treated with rEPO, dosage as
high as 2000U/kg/day. However, in a recent trial with IL3, six out of
37 patients showed significant response.26
Allogenic BMT provides viable therapeutic option for patients with
resistant DBA who have a compatible sibling donor. Till date BMT have
been performed in heavily transfused patients who had sustained
significant end organ damage due to iron overload or transfusion
transmitted infections. Despite these reservations, there were 25 long-
term survivors among 30 patients who underwent either matched
allogenic BMT (28) or cord blood transplantation (2).26
DYSKERATOSIS CONGENITA (DC)

Dyskeratosis congenita is an inherited disease characterized by the triad
of abnormal skin pigmentation, nail dystrophy and mucosal
leukoplakia. A variety of non-cutaneous (dental, gastrointestinal,
genitourinary, neurological ophthalmic, pulmonary and skeletal)
abnormalities have been reported (Table 9.2).36 Bone marrow failure is
the principal cause of mortality with an additional predisposition to
malignancy and fatal pulmonary complications. X-linked recessive,
autosomal dominant and autosomal recessive forms of the disease have

been recognized.
Table 9.2: Frequencies of abnormalities in dyskeratosis congenita
Characteristics X-linked(%) AR(%) AD(%)
M M F M F
Skin pigmentation 94 88 80 58 67
Nail dystrophy 92 100 68 50 63
Leukoplakia 71 75 52 33 20
Eye abnormalities 52 75 36 8 7
Teeth 21 50 28 8 7
Developmental delay 18 38 24 0 0
Skeletal abnormalities 18 25 28 8 7
Hyperhidrosis 13 13 8 8 7
Short stature 13 38 28 0 7
Urinary tract abnormalities 11 0 12 0 7
Hair loss 10 50 32 25 27
Gastrointestinal 10 63 16 0 7
Gonads 5 13 12 0 0
Bone marrow failure 47 38 64 17 7
Malignancies 12 13 12 8 0
AR=Autosomal recessive, AD=Autosomal dominant
Dyskeratotis Congenita Registry (DCR) was established in 1995 at

the Hammersmith Hospital (London) in which 92 DC families
comprising of 148 patients from 20 different countries have been
recruited till November 1999.

The majority of patients with DC recruited on the DCR were male
suggesting that X-linked recessive form of DC represents the majority
(> 80%) of cases. Through linkage analysis in one large family DKCI
gene responsible for X-linked DC was mapped to Xq 28.37
DKCI mutations have been identified in approximately 40 per cent
of DCR families. Majority are missense mutations.38 The precise function
of DKCI in the human cell and how mutations in this gene lead to
clinical phenotypes including bone marrow failure is currently
unknown. The corresponding protein of DKCI gene has been identified
and it is possible that deficiency of this protein has maximum impact
on tissues with high turnover like skin and bone marrow which are
affected in DC.
Haemopoietic progenitor studies have shown reduced number of
all progenitors which decreased further with advancing age. It is
believed that the haemopoietic system appears to undergo ‘premature

ageing’ with a reduction in the proliferative potential of clonogenic
progenitors.39
Clastogenic stress studies using bleomycin, diepoxybutane,
mitomycin C have not shown any differences between DC and normal
lymphocytes.40 However, fibroblast have shown unbalanced chromo-
somal rearrangements in the absence of any clastogenic agents.41 Blood
and bone marrow metaphases from some patients show numerous
unbalanced chromosomal rearrangements in the absence of any
clastogenic agents.42 The demonstration of chromosomal instability
suggest that cells of tissues with high turnover, such as BM, skin and
gastrointestinal tract may accumulate progressive DNA damage which
could explain the association of bone marrow failure with the epithelial
abnormalities seen in these patients.
Women who are obligate carriers of DC showed complete skewing
of X-chromosome inactivation pattern (XCIP) and in addition, women
predicted to be carriers on the basis of genetic analysis also had skewed
XCIP.43 The presence of the extremely skewed pattern of X inactivation
in peripheral blood cells suggests that cells expressing the defective
gene have growth survival disadvantages over those expressing in
normal allele.
Data from the DC registry revealed that males are predominantly
affected 127 of 148 (86%). Among 16 families of the 92 DC families
there was one or more affected female. These 16 families probably
represent autosomal form of the disease.
Somatic abnormalities were not present at birth but developed at a
variable rate at later age. The mucocutaneous features (skin pigmen-
tation, nail dystrophy and leukoplakia) usually appeared between 5-
10 years. Reticulated mottled hyperpigmentation involves the face,
neck, shoulder and trunk. Longitudinal ridges develop in the nail plates.
Leukoplakia appears later in life, most common site involved is the
oral mucosa but it may involve anal and genital mucosa also. Excessive
watering from eyes (epiphora) and blephiritis is common due to
blockage of lacrimal duct. Other abnormalities found in these patients
include sparse hair, premature greying of hair, urinary tract abnor-
malities like stenosis, phimosis, penile leukoplakia. The gastrointestinal
abnormalities include oesophageal stenosis, webs and diverticula.
Haematological abnormalities, bone marrow failure resulting in
peripheral cytopenia is most consistent. Nearly 90 per cent of patients
had a peripheral cytopenia affecting one or more lineages while 80 per
cent had cytopenia of two or more lineages. Patients who developed
pancytopenia, 80 per cent of them had developed before 20 years of
age.
Pulmonary complications were observed in 20.3 per cent of patients

evident by reduced diffusion capacity and/or restrictive defect. Post-
mortem studies revealed pulmonary fibrosis and abnormalities in the
pulmonary microvasculature.41 The development of the pulmonary
abnormalities may in part explain the high incidence of early and late
fatal pulmonary complications after BMT.
Analysis of DCR showed that 67 per cent of patients died either of
bone marrow failure or complication of its treatment. Only 9 per cent
died of sudden pulmonary complications. Among patients who under-
went BMT, 9 per cent died of fatal pulmonary complications, six percent
died from malignancy and 9 per cent from unrelated causes.44 Malig-
nancies developed in 13 (8.8%) out of 148 patients. These included four
cases of myelodysplasia (2 with RA and 2 RAEB). There was also one
case of Hodgkin’s lymphoma and eight cases of carcinoma.45
Therapy
Treatment of DC remains unsatisfactory. Androgens (oxymetholone)
may produce transient improvement in bone marrow function in some
patients. Transient successful response to granulocyte-macrophage
colony stimulating factor (GM-CSF), granulocyte colony stimulating
factor (G-CSF) and (erythropoietin) have also been reported.46 The main
treatment of marrow failure is however allogenic bone marrow
transplantation (BMT).47 Unfortunately because of early and late fatal
pulmonary and vascular complications, the results of allogenic BMT
are unsatisfactory.
SHAWCHMAN DIAMOND SYNDROME (SDS)

In 1964, Shawchman first described the syndrome of pancreatic
insufficiency and bone marrow dysfunction. It is a rare autosomal
recessive disorder but is second most common cause after cystic fibrosis
for pancreatic insufficiency in children.

Precise pathogenic defects responsible for haematologic problem is not
known. Abnormal bone marrow stroma is present in many cases. Fas
expression on haematopoietic progenitor cells was significantly higher
in these patients than in normal controls, implying that exaggerated
sensitivity following Fas ligand binding with sequential activation of
caspases may occur and could be an important clue regarding
pathophysiologic mechanism of bone marrow failure and myelo-
dysplastic changes.48
This disease commonly affects boys, male to female ratio is 1.8:1 and
males are more likely to develop acute leukaemia following a variable
period of pancytopenia. Disease manifests in early childhood with

diarrhoea, malabsorption, steatorrhoea and failure to thrive. These
children are often malnourished and have short stature (> 60% cases).
In 15 per cent of cases mental retardation was found. Other common
physical findings include protuberant abdomen and icthyotic skin rash.
Neutropenia leads to bacterial infection of skin, upper and lower
respiratory tract, middle ear and oropharynx. Skeletal abnormalities
like metaphyseal chondrodysplasia of the hips occurs in a majority of
cases. Other joints which are affected include knee, shoulder, wrist and
ankle metaphyses. The thoracic cage is elongated with a coat hanger
configuration of the ribs in approximately 50 per cent of cases.
Recently a prospective study showed that these group of patients
also suffered from immune dysfunction involving B, T and NK cells49
which may explain higher frequency of infections in these cases.
Elevated liver enzymes and/or hepatomegaly are frequent in younger
patients and tend to improve with age.50 In some cases myocardial
fibrosis was observed and all these patients died of cardiac failure.51
Neutropenia is most common, often from neonatal period, occurring
in 88-100 per cent of patients (intermittent in 66% and constant in the
remainder). Defective neutrophil mobility was observed in 13 patients
tested. Anaemia with decreased reticulocytes was recorded in 80 per
cent cases. Peripheral cytopenia does not correlate with bone marrow
cellularity. Bone marrow cellularity varied from hypoplasia to normal
and increased cellularity.
Leukaemic transformation occurred between 12 to 25 per cent of
cases.52 Myelodyspoiesis often precedes the development of acute
myeloid leukaemia. Cytogenetic abnormalities was reported in 25 SDS
patients, chromosome seven was most commonly involved.
Therapy
Treatment of pancreatic insufficiency is oral pancreatic enzyme
replacement. Red blood cell and platelet transfusions are required for
correction of anaemia and thrombocytopenia. Regular blood transfusion
are essential to maintain haemoglobin. Granulocyte colony stimulating
factors given for profound neutropenia has been effective.53 These
patients need to be treated promptly with appropriate antibiotics to
control infection. At present haematopoietic stem cell transplantation
provides the only curative option.
KOSTMANN SYNDROME (KS)

Kostmann in 1956 was first to describe an autosomal recessive disorder
with severe neutropenia. In 1994 severe chronic neutropenia
international registry (SCNIR) was established in which 383 patients
had registered till December 2000.54

Cytogenetic studies at diagnosis are often normal but may change
during the course of the disease. Cytogenetic abnormalities such as
monosomy 7 is the most frequent aberration. Patients may go on to
develop myelodysplastic syndrome or acute myeloid leukaemia.55
Kostmann’s syndrome is an autosomal recessive disorder that
primarily affects the neutrophils. The underlying genetic defect of KS
has not been fully identified. Genetic screening for mutations in patients
of KS has shown that neutrophil elastase mutations were often present.56
G-CSF receptor mutation affecting the cytoplasmic domain are present
in most patients who developed leukaemia57 suggesting an important
role of this mutation in leukaemogenesis.
The G-CSF receptor mutations have not been detected at birth
indicating that these mutations are not responsible for the neutropenia.
G-CSF receptor analysis cannot be used for diagnostic purposes for
the underlying disease but might be helpful for screening for the risk
of leukaemia.
These patients present with recurrent infections involving pulmonary
and gastrointestinal system. Absolute neutrophil count (ANC) is usually
within a range of 0-0.2 × 109/L. These patients may have mild anaemia,
thrombocytosis and increased blood monocytes and eosinophils. Bone
marrow shows ‘maturation arrest’ of neutrophil precursors at the level
of promyelocytes/myelocytes. Promyelocytes number is slightly
increased and often reveal morphological abnormalities.
Therapy
These patients need to be treated with appropriate antibiotics early in
presence of infections. More than 95 per cent of 383 patients registered
in SCNIR responded to G-CSF treatment with an increase in ANC to
>1.0 × 109/L. G-CSF is started at a dose of 5 µg/kg/d, escalated to 10
µg/kg/d and then increased by 10 µg/kg/d every 2 weeks if ANC
<1.0 × 109/L.
Non-responders are defined by patients who do not respond even
at a dose more than 120 µg/kg/d. In some of these patients, a combi-
nation of G-CSF with stem cell factor (SCF) led to further increase in
ANC. The potential allergic side effects of SCF have limited its use. For
patients who do not respond to G-CSF either alone or in combination
with SCF, bone marrow transplantation is the only option for treatment.
AMEGAKARYOCYTIC THROMBOCYTOPENIA
Congenital amegakaryocytic thromboctypenia (CAT) is a rare marrow
failure syndrome characterized by the absence or decreased numbers
of megakaryocytes in bone marrow. Two subtypes have been described.
i. Thrombocytopenia with absent radii (TAR)16 characterized by

spontaneous improvement after the first year of life.
ii. Congenital amegakaryocytic thrombocytopenia without
associated radius malformation. There are X linked and autosomal
recessive patterns of inheritance.58
Thrombocytopenia develops in the first year of life and is associated
with physical abnormalities in 40 per cent of patients. Most frequent
physical findings are neurologic and cardiac anomalies. Most frequent
anomalies are neurologic and cardiac and at a mean age of 3.5 years,
half of the patients develop aplastic anaemia. The risk or incidence of
malignant conversion is difficult to determine because of the rarity of
the disease and paucity of published data.
These children present with easy bruisibility and bleeding manifesta-
tion. Skin and mucosal bleeds are common. Children with severe
thrombocytopenia may develop severe and life threatening bleeds.
Therapy
Mucosal and serious bleeding episodes can be managed with
appropriate platelet support. Antifibrinolytic agents may be able to
control bleeding in mild episodes.
Clinical trials with IL3, GM-CSF was initiated for 5 patients with
CAT. IL3 but not GM-CSF resulted in improved platelet count. Bone
marrow transplantation is the only curative option for these children.
CONGENITAL THROMBOCYTOPENIA WITH RADIO-ULNAR

SYNOSTOSIS—A NEW SYNDROME
Thompson et al60 reported 3 cases from two different families with
thrombocytopenia and proximal radio- ulnar synostosis. There was no
history of consanguinity and in a family, two children and father had
radio-ulnar synostosis. All the three cases developed bleeding
manifestations in the neonatal period. Peripheral blood showed
thrombocytopenia, bone marrow was cellular with either absent or
marked paucity of megakaryocytes. Other physical abnormalities found
in these patients were clinodactyly and dislocation of hip joint. One
patient progressed to pancytopenia. None of them showed increased
chromosomal breakage with clastogenic stress. One of them had
sensorineural deafness. These three cases underwent bone marrow
transplantation and two are alive without any haematological
abnormalities. Dakol et al in 1989 reported proximal radio-ulnar synos-
tosis in two unrelated kindreds. In one family radio-ulnar synostosis
was present over four generations, only two affected individuals
suffered from haematological abnormalities (thrombocytopenia in one
and aplastic anaemia in other). In the second family 10 individuals

over two generations had proximal fusion of radius and ulna and two
developed aplastic anaemia.60 The pattern of inheritance and long-term
prognosis is not well known.
KEY POINTS
1. Fanconi’s anaemia diagnosed by chromosomal breakage in
patients who may have normal physical examination and may
not have haematological disease.
2. DKC1 gene responsible for X-linked dyskeratosis congenita.
3. In Shwachman Diamond Syndrome, cytopenia does not always
correlate with marrow cellularity.
4. G-CSF mutations are not responsible for neutropenia in Kostmann
Syndrome.
ACKNOWLEDGEMENT
Our heartiest thanks to Mr. Harinder Kumar for typing this article.
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10
Prevention and Control of HIV:
Among Blood Products
and IV Drug Users
Madhu Choudhry, VP Choudhry
Key Words
HIV-1 • HIV-2 • HIV prevalence in thalassemics
• Transfusion transmitted infections
INTRODUCTION
Acquired Immunodeficiency Syndrome (AIDS) presently, is a growing,
worldwide fatal pandemic.1 It has become one of the biggest global
challenges in the history of public health. Today, its modes of trans-
mission, the social and economic conditions, which facilitate its spread,
are well understood. Despite all the knowledge, risk behaviours and
risk environments persist and the Human Immunodeficiency Virus
(HIV), cause of AIDS continues to spread like a wild fire among indivi-
duals, crossing geographic borders throughout the world.
ETIOLOGY
Human Immunodeficiency Virus (HIV), the etiologic agent of AIDS,
belongs to Genus Lentivirus in the Family Retroviridae. These
lentiviruses are grouped into two types HIV-1 and HIV-2 on the basis
of serologic properties and sequence analysis of molecularly cloned
viral genomes.2 The HIV-1 is closely related to Simian Immuno-
deficiency Virus (SIV) isolated from chimpanzees in 1990. HIV-2 is more
closely related phylogenetically to the SIV found in sooty mangabey.
Both HIV-I and HIV-2 differ in geographical distribution, biological
and molecular characteristics and extent of transmissibility.
THE AGENT
HIV is 120-130 nm icosahedral, enveloped RNA virus. The envelope
consists of a lipid bilayer with uniformly arranged 72 spikes or knobs
of glycoproteins, gp 120 and gp 41. Glycoprotein, gp 120 protrudes out

on the surface of the virus and gp 41 is embedded in the lipid matrix.
Inside, there is a protein core surrounding two copies of RNA. HIV–I
and HIV–2 genomes have similar genomic structure and subtype
specific genes have been identified to differentiate the two. Both HIV-1
and HIV-2 genomes, have genes that encode the structural proteins of
the virus namely, “gag”-group antigen which encodes for capsid proteins
including p-24 antigen; “pol”-polymerase which encodes for reverse
transcriptase (RNA dependent DNA polymerase), a protease and an
endonuclease (integrase) and “env”-envelope which encodes for the two
major envelop glycoproteins gp 120 and gp 41.
In addition to these characteristic retroviral genes, the HIV genomes
encode regulatory proteins, namely “rev”-regulatory of virion protein,
“tat”-transactivator and “nef” negative regulatory factor. Besides, there
are accessory proteins that help in the maturation and release of the virus
particles.3 These are “vif” and “vpr” which are found in both HIV-1 and
HIV-2. There is “vpu” which is HIV-1 specific and “vpx” which is HIV-
2 specific and this forms the basis of differentiation between both the
types.
There is a high degree of genetic variation because of extremely high
replication rate in the infected individuals and this variation is not
evenly distributed throughout the genome. The differences are seen
particularly in the “env” region.4 On the basis of molecular studies HIV-
1 has been divided into three groups, group M (major), group O (outlier)
and group N (New).5,6,7 The M group is the predominant group and is
distributed all over the world. It is subdivided into 10 subtypes or clades
designated as A to H, J and K and they differ from each other by 30 per
cent in their “env” coding sequences and by 14 per cent in their “gag”
coding sequences. The epidemiological studies have revealed the
geographic distribution of these subtypes as given in Table 10.1.
Table 10.1: Distribution of HIV-I group M

Subtype Geographical area
Subtype A Western Africa
Subtype B Europe, North America, Japan, Australia
Subtype C South Africa and India
Subtype D Central and East Africa
Subtype E Thailand and Central African Republic
Subtype F Romania and Brazil
Subtype G,H,J,K Africa, Russia, Taiwan and Sweden
Latest survey shows that subtype C constitutes 56 per cent of all

circulating subtypes of HIV-I group M viruses in the world.8 The “O”
group is a relatively rare form, is more divergent from other subtypes
and is found in Cameroon, Gabon and France. “N” group has been
Prevention and Control of HIV 167
reported from Cameroon. Similarly, HIV-2 isolates have been classified

into 5 subtypes (A through E).9 HIV-2 is generally prevalent in West
Africa, Caribbean and South America. However, isolated cases have
been reported from India and other parts of the world.10 It has been
observed that the genomic variation occurs both, within a single infected
individual and also as the virus spreads through the host populations.
This genetic variation allows rapid antigenic diversification and this is
a cause of concern as it accounts for drug resistance, evasion from
immune response and problems in vaccine development.11
EPIDEMIOLOGY
According to estimates from WHO and UNAIDS12 there were 40 million
people around the world living with HIV at the end of 2001. During
2001 it has been estimated that there were 5 million new HIV infections
and 3 million deaths due to HIV/AIDS. It has also been estimated that
about 14 thousand new HIV infections occurred every day in the year
2001. More than 95 per cent of these were in developing countries, 2000
were in children under 15 years of age and about 12000 were in persons
aged 15 to 49 years of whom almost 50 per cent were women.
The epidemic is now spreading rapidly in Asia, where new infections
are increasing faster than anywhere else in the world. AIDS first
appeared in South East Asia in the 1980s. By 1997, over 65,000 cases
had developed AIDS and an estimated 3.75 million HIV-infected
persons were reported.13 By the end of 2001, WHO and UNAIDS
estimated that approximately 6.1 million people living with HIV/AIDS
and more than 135,000 AIDS cases have been reported in South East
Asia.
India has the second highest number of estimated HIV infected
people. The significant levels of HIV/AIDS in India suggest that the
country is now at an advance stage of epidemic. Epidemiological
analysis at the end of year 2000 shows that.14
1. Estimated number of HIV infected persons are 3.86 millions.
2. Predominant mode of transmission of infection in AIDS patients
is through heterosexual contact (80.86%) followed by blood
transfusion and blood product infusion (5.52%), IDUs (5.30%) and
perinatal transmission 0.72 per cent and others 7.60 per cent.
3. Males account for 77 per cent of AIDS cases and females 23 per
cent (a ratio of 3:1).
4. Estimated aggregate costs of HIV/AIDS by the year 2000 were
$11 million (5% of India’s GDP).
TRANSMISSION
Since AIDS was first identified in homosexuals, it was originally termed
as a “gay” disease being transmitted from one man to another by sexual
contact. Now it is clear that HIV can be transmitted by following ways:
1. Intimate sexual contact with an HIV infected individual

• Homosexual
• Heterosexual
• Oral
2. Exposure through infected blood
• Blood Transfusion
• Organ transplantation
• Infected needles and syringes
• Sharing of drug equipment
3. Mother to fetus or newborn (vertical transmission)
• In utero
• During labour or delivery
• Breast feeding
HIV is not transmitted by casual contact, shaking hands, hugging,
sharing utensils, sharing food or objects handled by people with HIV/
AIDS, sharing toilet seats or by mosquito bites or donating blood.
Individuals who are at higher risk of HIV infection are given in Table
10.2.
PATHOGENESIS
HIV crosses the epithelial barrier through a process known as
transcytosis. It is believed that after crossing the epithelium, HIV is
picked up by the antigen presenting cells which are primarily dendritic
cells. This is, however, debated and sufficient evidence is there which
suggests that possibly HIV directly infects CD4 T lymphocytes in
mucosal associated lymphoid tissue (MALT), establishes a fulminant
local infection within a few days, and then spreads quickly throughout
the body. Recent data suggests that viral reservoirs are established early
during mucosal infection. HIV binds with two surface proteins to infect
a cell, CD4 and CXCR4 (formerly known as fusin), or CCR-5. CD-4
Table 10.2: Individuals at higher risk of HIV infection

1. Homosexual or bisexual men
2. Intravenous drug users
3. Individuals with sexually transmitted diseases
4. Persons with hemophilia or other coagulation disorders
5. Recipients of transfusion of blood, blood components, or tissues
6. Children born to mother with HIV infection
7. Heterosexual men and women who have:
— Sex with an IV drug user
— Sex with multiple partners
— Sex with a person with hemophilia
— Sex with a person born in an endemic area
— Sex with a transfusion recipient
— Sex witth an HIV infected individual
molecules are present on T helper lymphocytes, monocytes and

neurons. HIV virus may be either T tropic or M tropic. T tropic viruses
bind to CD4 and CXCR4 and are transmitted via blood and blood
products. These viruses are syncytia inducing (SI) and infect T cells. In
contrast, M tropic HIV viruses bind to CD4 and CCR5 and these are
transmitted via sexual contact. They are not syncytia inducing (NSI)
viruses and they infect macrophages and T cells. Generally speaking,
SI strains tend to appear late in the disease shortly before stage IV
disease develops. Primary infection is usually with the NSI phenotype,
however, about 50 per cent of AIDS patients never develop SI viruses
and high viral load develops without conversion to SI phenotype.
HIV binds to CD4 molecule via viral envelope glycoprotein gp120
and then binds to CxCR4 or CCR5. This is followed by entry of viral
RNA into the cell cytoplasm. With the help of viral reverse transcriptase,
complementary DNA is produced which is then transported into the
cell nucleus and it gets integrated into the host cell DNA. The integrated
proviral DNA now commands for virus production. The extent of viral
production is dependent on the expression of HIV regulatory and
accessory genes. It also possibly effects programmed cell death in
noninfected cells.
TRANSFUSION OF BLOOD AND BLOOD PRODUCTS

Blood is vital and it is the most essential life saving substance. It can be
obtained only from human beings, as there is no artificial substitute.
Millions of lives are saved through blood transfusions. The major aim
of blood transfusion is to make transfusion safe and beneficial.
Transmission of HIV through blood/blood products happens to be
the most efficient way of acquiring infections. Most transfusions are
given to people who are injured in accidents, children with severe
anemia, patients with haematological disorders, surgical and cancer
patients and women who loose large quantities of blood as a compli-
cation of pregnancy. Patients requiring multiple blood transfusions or
blood products are at a higher risk. It has been observed that the
prevalence of HIV infection increases with the increase in number of
blood transfusions and transfusion of factor concentrates as seen in
hemophilics and thalassemics (Table 10.3 and 10.4). However, there is
no risk of HIV transmission in haemophilics if factor concentrates
derived from DNA recombinant technology are used.
Although blood contaminated with HIV was transfused as early as
1979, it was only in 1982 that the first transfusion related case of AIDS
was identified. In 1985, HIV screening became available, and blood
supply was considered as safe. During this 6 year period, from 1979 to
1985, thousands of people contracted HIV through blood transfusions.
Over 5300 transfusion related AIDS cases had been identified by 1993.
Table 10.3: Transmission of HIV in Indian haemophilics

Percentage reactivity
Authors Place Pt(n) HIV
Sengupta, et a15 Calcutta 37 24.3
De M, et al16 Calcutta 20 4.4
Singh, et al17 Delhi 124 12.1
Chandra C18 Calcutta 105 3.8
Table 10.4: Transmission of HIV in multitransfused Indian thalassemic children

Study Place No. HIV
Kumar et al 9419 Manipur 406 8.9
Chopra 9820 Mumbai 50 18.0
Chowdhary 9821 Lucknow 39 2.6
Sharma 9922 Jammu 58 6.25
Chandra 9923 Calcutta 75 0.0
Choudhry 200024 Delhi 199 0.0
HIV transmission has become rare since donors are being routinely
screened for both HIV antibody and antigen in the developed countries.
With the consistent support of National AIDS Control Organization
and routine screening of donated blood the risk of transmission through
blood has decreased (Table 10.5). However, these figures suggest that
blood in our country is still not very safe.
It is important to remember that even today inspite of present
screening methods in our country, there is an appreciable risk of
transfusion transmitted infections. Whenever, any patient requires
blood transfusion or blood products the clinician should weigh the
risk versus benefit. Single unit of blood transfusion should be avoided.
Almost 80 per cent of the world’s population lives in developing
countries, and the people in the developing countries are supported
by only 20 per cent of the world’s blood supply. In developed countries,
blood donations are mainly from voluntary unpaid donors, whereas
in developing countries 80 per cent of blood comes from paid or
replacement donors. Prevalence of various viral infections is higher in
the general community which increases the risk of transfusion
Table 10.5: Prevalence of AIDS through blood transfusion and IV drug users
NACO publications N Blood (%) IVDU (%)
December 9625 3161 7.7 8.2
March 9826 5204 7.05 7.3
June 9927 7012 7.79 6.68
March 200028 11251 5.50 5.20
February 200229 33794 3.17 3.23
transmitted infections (TTI) in developing countries. WHO has warned

that inadequate availability of safe blood, the attraction of cash
payments for blood donations and the inappropriate use of blood and
blood products are putting people at higher risk of TTI.30
It has been estimated that between 5-10 per cent of all HIV infections
worldwide have been acquired through transfusion of contaminated
blood and blood products. Globally, each year unsafe transfusion and
injection practices cause an estimated 8-16 million cases with hepatitis
B infections, 2.3-4.7 million with hepatitis C infections and 80,000-
160,000 HIV infections.30
Current estimated risk of being infected with a unit of screened blood
are 1/1,000,000 for Hepatitis A, 1/30,000-1/250,000 for Hepatitis B,
1/30,000-1/150,000 for Hepatitis C, 1/200,000-1/2,000,000 for HIV.
Viruses such as CMV and EB virus also pose a potential risk following
transfusion of blood or blood components.31
Now people are quite aware of the adverse outcome of the
transfusions and TTI. Supply of safe blood has world over generated a
lot of public concern and every one is looking towards the government
commitment for implementation of various measures to ensure safe
blood. WHO is working with national authorities to promote safe blood
and to reduce the spread of HIV and other TTI in all countries, by
advocating and assisting in the development of policies, infrastructures
and the training of personnel.
ISSUES/CHALLENGES RELATED TO TRANSFUSION

TRANSMITTED HIV
Safe Donors and Unsafe Blood Donations
Paid donors serve as a major source of blood in developing countries.
They come from poor sector of society, are undernourished, are not
very healthy and are more likely to suffer from multiple infections.
Some of these donors are also drug addicts who sell their blood to buy
drugs for injections. The poverty forces them to use unsterilized syringes
and needles and sharing of these equipment which further increases
the risk of contracting HIV.
Good donor selection is very important for which trained staff is
required. People need to be educated and motivated so that they are
able to take correct decisions while donating blood-the most important
aspect being self exclude or self defer. Self exclusion implies that they
of their own exclude themselves from donation if they are aware of the
fact that their blood is likely to be unsafe because of their behavioural
practices. Self deferral is to postpone the blood donation in presence of
any infection or any other temporary reason for it.
Education, motivation, recruitment and retaining of low risk donors
is very necessary and this itself is a big challenge. One needs to have a
well trained staff and a well funded blood donor recruitment unit which
will be totally dedicated to their job. It will be their responsibility to
produce educative material for blood donors, to have campaigns at
appropriate places for motivation and education, to have donor registry,
to have set procedures for donor selection, donor deferral and donor
notification, retention of donors, counseling, etc.
Screening of Blood
Health is mostly a neglected sector and donors are not properly screened
due to lack of planning and funding in developing countries. A well
planned and totally organized blood transfusion service is not available
everywhere. Blood transfusion services are generally available in urban
areas and even in these areas there are shortcomings in form of adequate
supply of test kits, quality of the kits and well qualified staff. In case of
HIV there are several types of tests based on different technologies.
The test selected for screening of donated blood should preferably be
combined for HIV-1 and II and should be highly sensitive. Such a test
will rarely produce false negative results which is essential for safe
blood. Problem however, comes when a person donates blood in the
window period. Donors during window period can be tested by HIV
p24 antigen tests. But one has to keep in mind that these tests are not
cost effective in most of the blood banks and have not been recom-
mended for developing countries.
Inappropriate Use of Blood

Transfusions are many times given when they are not really required.
Minimizing unnecessary transfusions reduces the risk of transmitting
HIV and other TTI. Unnecessary transfusions lead to shortage of blood
which again calls for active participation of professional donors for
blood supply. This is another major challenge for which medical
community needs to be trained to avoid unnecessary and inappropriate
blood transfusions. Use of blood substitutes for volume replacement,
and use of blood components needs to be encouraged in place of whole
blood.
Donor Notification
The units of donated blood which are reactive for HIV or give indeter-
minate test results, are considered as probably infected and are
discarded. If donors are to be notified, the test should first be confirmed
and thereafter the donor should be counseled or he/she can be referred
to a Voluntary Counseling and Testing Centre (VCTC). If these people
are not informed and counseled, they remain unaware of their status
and will continue donating here and there. This amounts to huge
national wastage in terms of test reagents, man power, disposables,
blood bags, etc. Donor get a false sense of satisfaction that he has
donated the blood which has been accepted and he is free of HIV
infection. Even though he may not be having other risk behaviours,
but likelihood of his passing the infection to the spouse is high and
subsequently vertical transmission is a cause of concern.
Inappropriate Testing
Some countries import blood products and test them for HIV and other
infectious agents. However, this is not required and is not advisable
too. The reason being that the plasma/serum used for preparation of
products is supposedly screened before processing. Secondly, the HIV
antibody tests are designed for screening serum or plasma and not the
final products for, e.g. Immunoglobulins, albumin, and other plasma
derivatives. These assays may give non specific false positive results.32
Inactivation of Viruses in Blood Components31

There has been substantial progress in inactivating viruses in plasma
products. Safe and efficient inactivation procedures have been
developed and applied successfully for coagulation factor concentrates,
immunoglobulins, lymphokines and other growth factors. An
important scientific and technical challenge is to achieve similar safety
procedures for cellular blood products such as platelet concentrates
and packed red cells.
Attempts made to develop inactivation procedures to destroy the
infectivity of viruses for blood components have been unsuccessful so
far. This has been due to the inherent lability of membranes and sensiti-
vity of cellular proteins. Viral inactivation is often accompanied by loss
of cellular function. Consequently whole blood and cellular components
including red cells, platelets and leucocyte continue to carry a risk of
virus transmission. Therefore, the prevention of transfusion-transmitted
diseases remains a major challenge of transfusion medicine research.
There have been a number of approaches to inactivate or remove
viruses from blood and blood components. Removal of leukocytes by
filtration was shown to reduce CMV transmission to susceptibible
patients; however, elimination of HIV-infected cells by this method was
incomplete, as filtration could not remove cells infected with viruses.
The use of UV or ionizing irradiation is simple, however, the dose of
gamma irradiation used to inactivate lymphocytes is not sufficiently
virucidal, and the virucidal doses are generally cell destructive. UV
irradiation was found to inactivate HIV under conditions generally
favourable to platelets. It was found that suboptimal doses for viral
inactivation induce virus activation and/or viral replication. Ozone
has been shown to inactivate HIV but virucidal doses often cause
haemolysis. Good results have been obtained with photodynamically

active dyes. The precise biologic mechanisms of in vitro photochemical
inactivation of pathogens are not well understood. In order to minimize
the damage to platelets caused by irradiation, the process must be
conducted in absence of oxygen or in the presence of agents that remove
damaging reactive intermediate compounds. This is currently being
evaluated in clinical trials.
INTRAVENOUS DRUG USER

Intravenous drug use causing HIV infection accounts for a significant
proportion of the total cases of HIV world over. The number of cases
have been increasing at an enormous rate everywhere-US, Europe, Asia
and elsewhere. Since the use of drugs is illegal, it is not easy to reach
these high risk people and is also difficult to make accurate assessment
of the extent of HIV in them. The association of HIV in IVDU was
considered when identical opportunistic infections were recognized
both in homosexuals with AIDS and in IVDU who had no history of
male to male sex.33 Later, similar opportunistic infections were observed
in non drug using female sexual partners of male IVDU.34 These obser-
vations suggested that the causative agent was initially acquired by
the male partner through parenteral drug use and then transmitted
sexually. The mode of transmission of HIV among IVDU could be
determined after isolation of HIV from blood contaminated needle,
syringes and injection paraphenalia.35
HIV was most probably introduced in the IVDU risk group in New
York, North America in 1976. Cross sectional serosurveys on HIV
prevalence conducted on sera collected from New York city IVDU
entering drug treatment in 1978 showed 9 per cent of specimens were
already seropositive.36 HIV has spread at an alarming rate since then,
it rose to 26 per cent in 1979, 38 per cent in 1980 and 50 per cent during
1981-1983, reaching a plateau thereafter. Similar patterns of rapid spread
were seen in localized areas in Europe. In Edinburgh, Scottland,
seroprevalence rose rapidly up to 51 per cent in early 1980s, with most
seroconversions occurring within 2 year period.37 In Milan, Italy,
seroprevalence increased from 5 per cent to almost 50 per cent in IVDU
in less than 3 years and reached to 62 per cent in early 1987.38 Situations
have been similar amongst IVDU in Asia. In Bangkok, Thailand
seroprevalence among IVDU was estimated at 1 per cent in late 1987,
but rose to 43 per cent by Dec. 1988.39 In Manipur, India, estimated
seroprevalence among 15000 IVDU rose from 9 to 50 per cent over 6
months from 1989 to 1990.40
In 1996 an overall national seroprevalence was estimated in US and
despite various variables it was projected by Holmberg that prevalence
rate of HIV infection was 14 per cent and incidence of 1.5 per cent
among IVDU during the study period.41
At the start of IV injection, blood enters the needle and syringe and
these get infected if used by person who is HIV positive. The reuse of
blood contaminated needle or syringe serves a source of HIV
transmission as the virus enters the blood stream directly. In addition,
sharing of drug equipment by I.V. drug users serves as a means of
spreading HIV. Infected blood can be introduced into drug solution by
using blood contaminated syringes to prepare drugs, reusing of water,
reusing of bottle caps, spoons, or other container used to dissolve drugs
in water and to heat drug solutions. Repeated use of small pieces of
cotton to filter particles which could block the needles can serve as a
source of spread of HIV infection. Rag pickers may collect the used
syringes, which are repacked and sold as sterile syringes and there is a
strong likelihood that these syringes may spread HIV infection.
Recycling of these syringes is quite common in developing countries.
RISK FACTORS FOR HIV INFECTIONS IN IVDU

Transmission of HIV in IVDU occurs primarily through HIV infected
blood contamination of injection paraphernalia which is reused by an
uninfected IVDU. The frequency of needle sharing, a number of partners
with whom the paraphernalia are shared, the probablity of partners
being HIV infected, are important risk factors towards acquisition of
HIV. Behaviours which increase the liklihood, frequency and magnitude
of exposure to infected blood and other factors such as low income,
male gender, antisocial personality etc. are responsible for increased
risk of incfection.37,43 It has been seen in clinical studies that anti social
personality disorder may be prevalent in 40 per cent of IVDU as
compared with 3 per cent of general population, and it is associated
with more frequent infections, with more frequent needle sharing with
greater number of sex partners and greater use of alcohol and cocaine.42
Using a mathematical model based on seroprevalence rates in New
Haven, and the prevalence of HIV in syringes collected in the local
needle exchange program, Kaplan and Heimer estimated that the
average risk of infection from an HIV infected apparatus was 0.0067
per injection episode i.e. 1:150 approximately.43 Risk factors among
female IVDU include frequent trading of sex for drugs or money and
having a male sexual partner who is also an IVDU.44 Homosexual and
bisexual male IVDU are at high risk for HIV infection because of sexual
transmission as well as high prevalence of HIV in their needle sharing
peer group.
Biological differences in susceptibility to acquiring HIV have not as
yet been demonstrated to explain differences in respect of race, gender,
ethnicity, etc. Some recent reports strongly associate deletion of a
nucleotide with beta chemokine receptor 5 (CCR5) in individuals

remaining uninfected despite frequent sexual exposure to HIV.45 This
allele was found in 8 per cent of white gay men enrolled in the
Multicenter AIDS Cohort Study, at Chicago but not in participants of
African or Asian descent.
ISSUES AND CHALLENGES RELATED WITH DRUG USE

There are various issues related to intravenous drug use. The high
degree of risk the IVDU carry to contract HIV infection and the lethality
of HIV infection is a cause of concern. Although HIV infection is a
medical problem but social and ethical implications are of still greater
importance. Conducting research activities in form of controlled trials
is not possible because of non-cooperation of these individuals. Besides,
studies require establishment of prevalence and incidence in a given
community and recurrent representative samples of that population,
which is not easy. Other problems are in form of limitation of funding
for health promotion programs, negative attitude of public towards
IVDU and lack of political commitment etc. The most reliable way to
eliminate the risk of transmission is by totally abstaining from injection
and by practicing safe sex but this is not easy to achieve.
De-addiction is very important but is all the more difficult as the
effective therapy for de-addiction is not well established. It has been
seen that a small percentage of the addicts who use cocaine and
amphetamine, can be treated by drug free techniques, e.g. counseling,
group support and modifying their social structure.46 Treatment in the
form of methadone is available for opiate users and almost 50 per cent
reduction in needle sharing and lower mortality has been demonstrated
in some clinical trials.47-50 Those who continued with drug injections,
possible causes could be traced to inadequate methadone doses, lack
of stable home life, job and income.51 Throughout the world it has been
observed that there is a strong imbalance between demand for effective
treatment for drug dependency and availability of supportive care.
As the cessation of IVDU behaviour is difficult, therefore, the disinfec-
tion of contaminated injection paraphernalia plays an important role
in prevention of spread of HIV infection. However, studies have failed
to demonstrate the protective effect of bleach as the IVDU do not give
required contact time to the injection paraphernalia for achieving
disinfection.52 IVDU need to be educated and encouraged for proper
use of bleach in proper concentration and for enough time for proper
disinfection in the absence of sterile equipments.
The introduction of needle exchange programmes is one of the most
effective means of preventive intervention to reduce HIV transmission
among IVDU. In this programme one for one exchange of used syringes
and needles is undertaken for sterile equipment and results are viewed
in context of changes in injection and sexual behaviour, rate of drug

use, prevalence of contaminated syringes in the community, hepatitis
B seroprevalence, HIV seroprevalence, etc. These studies demonstrated
that there has been reduced frequency of needle sharing and sharing
partners.53-56
Another important benefit of needle exchange programmes is that
one gets an opportunity to contact and educate the IVDUs who are
otherwise inaccessible for any preventive interventions. During this
contact, they can be addressed regarding safe sex and counseling. It
has been reported that there is reduction of sexual risk behaviour
associated with safe sex interventions in collaboration with the needle
exchange programmes. The incidence of IVDU-related hepatitis B
infection also declined signicantly with the implementation of syringe
exchange programme.57
CONCLUSIONS
Various studies depict that there is a high prevalence of TTI in our
country. Reports are also there which suggest that multitransfused
patients e.g. patients of leukaemia, haemophilia, thalassemia etc. are
at a high risk for HIV infection. The risk of TTI increases with the
number of transfusions or factor concentrates administered to these
patients. Presently, prevention of HIV infection and AIDS is dependent
upon deferral of blood donations by persons at increased risk of HIV
infection, testing of donated blood for HIV antibodies and treatment
of clotting factor concentrates. Routine counseling and HIV testing is
not done for blood transfusion recipients. Efficient blood transfusion
services are not existing as of today in our country. The infrastructure
is poor and problems relating to manpower, procurement of equipment
and its subsequent maintenance is a perpetual problem at most centres.
Quality assessment programme for blood screening is lacking. System
of post transfusion auditing is non existent.
Sharing of needles and injecting equipment among injection drug
users is also serving as a fuel for rapid spread of HIV in this group.
These high risk populations are difficult to reach for preventive
interventions such as safe needle exchange and education programmes.
Treatment of these persons infected with HIV poses a big challenge,
the worst being stigmatization and discrimination by the society. The
drugs for treatment are not easily available. Though the cost of drugs
has been markedly slashed but still, the management of these cases
involves exorbitant expenditure by the family as the drugs have to be
taken for a prolonged period of time and regular monitoring has to be
done by expensive tests namely CD4 counts and viral load assays.
Facilities for monitoring of antiretrovirals are not available everywhere
and the techniques are not very well standardized. The side effects of
drugs are many and generally the patients do not tolerate the drugs
for longer periods. There is uncertainty regarding the cure rates.
Combination of these factors leads to poor compliance of treatment
among these patients.
There are several reports that persons with transfusion transmitted
HIV and injection drug users have passed on the infection to their sexual
partners.58 Women are at higher risk of getting infected with HIV,
because of gender inequality, cultural and social customs, poor access
to knowledge, information and education and for biological reasons.
HIV infection among women, who are not sex workers, is increasing in
India and worldwide and the most likely mode of transmission is
through unprotected sex with their husband. The high infection rate
among the non sex workers has been confirmed in studies from Indian
National AIDS Research Institute, Johns Hopkins’ University and
National Institute of Allergy and Infectious diseases.59 As per joint report
from UNAIDS/WHO Dec 2001, there have been 12000 persons with
new infections a day in the year 2001 and out of these 50 per cent were
women.12
The increased prevalence of HIV infection among women has far
reaching implications as the neonate can get HIV infection through
perinatal transmission. Surveillance studies show that in the high
prevalence states of Maharashtra, Tamil Nadu, Karnataka, Andhra
Pradesh, Manipur and Nagaland, HIV prevalence in antenatal women
is more than 1 per cent. The reported risk of mother to child transmission
is between 13-60 per cent. According to Kumar et al the vertical
transmission rate in India is 48 per cent, whereas study conducted by
Arcon in Mumbai, Dongaonkar et al reported mother to child trans-
mission rate of 36 per cent.60
Health education and behaviour modification are the only ways of
interrupting transmission. Cultural taboos that surround sexual
behaviour, general talks about sex, negotiating safe sexual practices
etc pose a significant challenge. It is, therefore, important to have
information and education programmes for all men and women,
including adolescents. It is very important that sex education be given
to adolescents before they become sexually active. It implies that
education must begin in early teenage years and studies demonstrate
that sex education is the most effective way of postponing sexual
activity.61
We are confronted today with a very serious public health problem
where multiple partners such as medical faternity, paramedical people,
school and college teachers, NGOs, religious leaders, media, scientists
and politicians need to play their respective role for prevention and
control of HIV infection. A strong will on the part of the government is
required to create policies and to exercise leadership for campaigns
and development of resources. There have to be policies formulated to
support an enabling environment for preventive interventions, to fight

against stigma and discrimination and to ensure access for management
of patients infected with HIV. Further, research is essential for
development of newer safe drugs, vaccines and for community research
to determine suitable methodology for care and control of HIV infection
through blood and intravenous drug use..
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11
Transfusion Medicine: Audit
Vijaylaxmi Ray, Harprit Singh
Key Words
Transfusion audit • Audit criteria • Audit types • Audit
schedule • Audit area • Hospital transfusion
committee • Haemovigilance • Audit methodology
DEFINITION
The term audit means a formal examination or review of any procedure
or outcomes with a view to rectify the shortcomings. Initially audit
was used for settlement of accounts but now this has relevance in any
sector of quality management. In fact, auditing has gained more promi-
nence in recent years in the health care management system. In blood
transfusion services (BTS) audit could be applied as a methodical and
defined review of practices and policies to ensure safe and appropriate
transfusions. It means analysis and evaluation of processes involved
in the production of blood components at manufacturer level (Blood
centre, that deals with procurement and distribution of blood
components), also analysis at consumer level (clinician) for the most
beneficial outcome. So that auditing involves at BTS compliance with
good manufacturing practices (GMP) and in clinical practices. Mallett
et al.1 have reinforced role of transfusion audit and practice guidelines.
AIMS AND OBJECTIVES OF AUDIT

Transfusion audits have gained considerable importance due to rapid
advances in transfusion services, acceptability of national blood policy
and stringency of licensing authority, encroachment of cost benefit and
cost effective analysis. Thus, the objective of audit is to improve the
transfusion services and to reduce errors or accidents in a scientific
manner. Therefore, auditing associates with GMP along with
modification of physician’s behaviour towards patient’s transfusion
needs good clinical practices (GCP) in a cost-effective way.
SCHEDULE
Routine Audit Schedule
Routine audit schedule is based on the needs of BTS and can be
performed monthly, quarterly, half-yearly or annually. But whatsoever
frequency is decided a regular follow-up is a must to improve the
outcome.
Emergent
Emergent audit is performed whenever some error comes into notice
and immediate steps are to be taken for the corrections by formal or
informal communication with concerned staff. These audits are
performed in day to day practices and require active error and incidence
reporting.
METHODOLOGY OF PERFORMING AUDITS

Audit Criteria
Audit performance requires criteria, defined as predetermined,
measurable elements, chosen to assess whether practices conform to
the stated indications.2 The indications are based on standard guidelines
and may be modified to the gradual understanding of local need of
transfusion services and of clinical practices. Therefore, criteria should
take into consideration operational strategies of transfusion services.
The audit criteria are defined in number of ways.
Explicit Criteria
Explicit criteria are easily measurable and clearly defined parameters,
e.g. haemoglobin levels of patient for red cell transfusion.
Implicit Criteria
Implicit criteria means that criteria are not easily measurable and
involve individual judgment, e.g. clinical assessment for red cell
transfusion.
An appropriate and safe transfusion includes both manufacturing
practices and clinical practices, thus audits performed in transfusion
services can be divided into compliance with current GMP and
compliance with GCP (Box 11.1).
TYPES OF AUDIT FOR COMPLIANCE WITH CURRENT GMP

The GMP audits are of several types depending on the level of blood
centre and component production involved. The audits can be carried
out by an external agency involved only in such activities or internal
by the staff of the organization.
Transfusion Medicine: Audit 185
Box 11.1: Types of Audits

For compliance with good manufacturing practices (GMP)
• Self auditing
• Internal audit
• External audit
• Vendor audit
For compliance with good clinical practices
• Prospective
• Retrospective
Self-inspection Audit
Self-inspection audit is one in which, the staff responsible for current
GMP within specified area carries out the procedure, e.g. staff involved
in component production ensures strict adherence to standard operative
procedures (SOP).
Internal Audit
Internal audit is carried out within blood centre by staff that does not
bear direct responsibility to the area being audited. Such audits can
involve hospital or transfusion service policy and may be influenced
by the guidelines of hospital transfusion committee. All parts of
transfusion services are covered in such audits and form a baseline for
the future practices.
External Audit
External audit is carried out by trained auditors outside the
organization. This can be mandatory or voluntary. Drug inspectors for
issuing/renewal of blood banking licenses carryout a mandatory one.
A failure in such audit may lead to grave consequences like delicensing
of the premises. While a voluntary external audit is performed on
request by the transfusion services in question. Such types of audits
are usually restricted to some particular area of transfusion services.
The example of such an audit is one carried out by International
Standard Organization (ISO) and certifications given, international
haemovigilance, that involves issues related to transfusion safety,
reviews errors and serious sequelae in transfusing blood components,
external quality assessment of blood component usage by College of
American Pathologist (Q-probes of CAP) audit usage and wastage of
blood components.
Vendor Audits
Vendor audits are carried out by the organization to ensure safe and
reliable supply of proper reagents or raw material to the organization,
e.g. purchase of reagents of kits and reagents, blood collection containers

by certified suppliers.
AREAS OF BTS FOR AUDIT

Auditing essentially comprises of all areas of BTS and can be divided
into following sections. All personals involved are assigned duties and
follow-up of protocols are involved. Audit reports the overall func-
tioning of transfusion services, hence focusing on critical areas is crucial.
The auditors have special interest in following areas (Box 11.2).
Donor Recruitment and Blood Collection Policies

As blood donors provide a raw material for transfusion services, donor
selection has been vital issue in this aspect of transfusion services. In
fact, such audits also stress on voluntary recruitment and abolishing
paid or professional donors. Proper cleansing and use of local antiseptics
before phlebotomy, setting up of time limits for component preparation
are to name a few guidelines that has led to present day component
preparation practices. Donor reactions to plateletpheresis has been
reviewed at national level by Makar et al.3 and have concluded that the
procedure is relatively safe with incidence of severe citrate toxicity
comparable to severe fainting reactions after whole blood donations.
Component Preparation
As per GMP and standard operating procedure (SOP) is required. In
India minimum standards have been laid down under Drug and
Cosmetics Act, 1940. European countries have carried out universal
leucodepletion (LD), i.e. 3 to 5 log reduction of the leucocytes of all
cellular blood components manufactured. However, certain negative
impacts of this policy are seen that includes inevitable loss of therapeutic
constituents and increased cost considerably without expected benefit.4
Cost-effective analysis have shown multicomponent donation as a cost-
effective and dose directed method.5
Box 11.2: Critical areas for audits in blood transfusion services.

• Donor recruitment and blood collection policies
• Component preparation
• Infection screening
• Release of blood components for transfusion and
disposal of biohazardous byproducts
• Immunohaematological follow-up of post-transfusion
complication
• Blood group discrepancy work-up
• GMP, QA and SOP and validation
• Research methodology, evaluation and policy issues
Infection Screening
For endemic transfusion transmitted infection is required. The national
requirements and policies are available for HIV, HBV, HCV, malaria
and syphilis. Active reporting of incidences of such infection not only
gives snapshot of donor selection in transfusion services but also
provide regional scenario of such infections. However, challenges of
recently discovered infections are to be looked into. In October 2002,
FDA, USA has issued a guidance document that provides recommenda-
tions for the assessment of donor suitability and blood and blood
product safety in cases of known or suspected West Nile virus infection.6
At present there has been other new agents such as GB virus-C/hepatitis
G virus (GBV-C/HGV) for hepatitis, putatively hepatitis related TT
virus (TTV), possibly recently announced SEN-V for hepatitis,7 human
herpes virus 8 for Kaposi’s sarcoma (HHV-8/KSAV), new variant (vCJD)
for CJD are to name a few, that are under close scrutiny as they have
been shown to be transmitted by blood but risk factor and patient
implications are still to be fully analyzed. Retrovirus epidemiology
donor study (REDS) in conjunction with CDC, funded by National,
Heart, Lung and Blood Institute (NHBLI), is collecting a repository of
linked donors and recipient specimens that is known as REDS allogeneic
donor and recipient repository (RADAR) that aims at quantifying the
transfusion risk with reasonable level of certainty.8,9 Various countries
such as Canada have a look back policy over transfusion-transmitted
infection (TTI) to prove the worthiness of curtailing such infections.10
Release of Blood Components for Transfusion and

Disposal of Biohazardous Byproducts
Hospital transfusion committee formulates standard guidelines and
audit indications and maximum surgical blood ordering schedule
(MSBOS) to minimize wastage as well as safe disposal of biohazardous
material are taken into consideration. Patient’s consent is required prior
to transfusions of blood products in European and North American
countries. As treating physicians give first hand information of
advantages and disadvantages of blood transfusion as well as
alternative to transfusion, Rock et al. have analyzed current concepts
in transfusion practices among physicians of various fields recently.11
They have shown less than half of treating physicians have basic
knowledge of blood transfusions and some sort of corrective action is
required in this regard.
Immunohaematological Follow-up of
Post-transfusion Complication
Immunohaematological follow-up of post-transfusion complication is
vital not only from medicolegal implication but also for corrective actions
to reduce such incidences. Serious hazards of transfusion (SHOT) under
haemovigilance is step in this direction.12 It reported more than half of

cases as wrong blood to patient (54.2%) episodes including ABO
incompatible. Clerical errors still plays as major cause of serious
transfusion reactions. Transfusion transmitted infections account for a
very small proportion of reported post-transfusion complications (3%).
Blood Group Discrepancy Work-up

Blood group discrepancy work-up in routine service and this requires
working knowledge of blood group systems. Blood centre can send
the samples along with clinical details to reference laboratory for further
work. Blood grouping and crossmatching policies defined on the basis
of higher incidence antigen antibody cannot be underestimated. The
Bombay phenotype (Oh) prevalence in Maharashtara in India is an
evidence of this fact. In Mumbai, Maharashtara blood donors and
patients are regularly screened for Oh phenotype. On the other hand,
universal screening of A2 phenotype is no more recommended at
various centres.13
GMP, QA and SOP and Validation

GMP, QA and SOP and validation is key to best component production.
With more and more of experience in the field of component production,
there is improvement of GMP, e.g. use of additive solutions for red cell
and platelets to increase the yield of fresh frozen plasma (FFP) and
reduce the risk of transfusion reaction.14 With advances in screening
methods of infections and use of nucleic acid amplification test (NAT)
has decreased the risk of infectious agents to minimal level.15 In order
to improve the quality of components, now 90 to 100 per cent of
components tested should meet the standards when compared to
previous limits of 75 per cent of the tested ones.16,17 Validation of new
procedures and equipments at manufacturer is required to comply with
GMP as well as minimum requirements by FDA/DCA.
Research Methodology, Evaluation and Policy Issues

Research methodology, evaluation and policy issues are one of the most
difficult areas for auditing, as no well set predefined criteria is always
available. Moreover ethical issues and confidentiality of patients as well
as long-term implication of such trials pose a major challenge in this
issue. However, an interim audit during research is recommended so
as to justify the utility, efficacy and safety of such projects at predefined
intervals.
TYPES OF AUDIT FOR COMPLIANCE WITH CURRENT GMP

The compliance with good clinical practice (GCP) audits are performed
prospectively, concurrently or retrospectively based on the indication
for transfusion and preservation of these requisitions are of utmost

importance. The guidelines for the number of requisitions for auditing
have been laid down by Joint Commission on Accreditation of Health
Organizations (JCAHO).18,19 Universal review, i.e. review of all requests
is no more required. For high volume blood components, studies can
be performed on 5 per cent of cases or 30 of total cases whichever is
larger. Universal auditing is done in case of components with fewer
numbers or when baseline for practices have not been fully established,
e.g. granulocyte transfusions.
Hospital Transfusion Committee

Hospital transfusion committee plays a central role in establishment
and continuous monitoring of safe and effective transfusion therapy.
This committee comprises of head of transfusion services, director of
the hospital and other key personals involved in blood transfusions,
e.g. surgeons, anaesthesiologist, obstetrics and gynaecologists and
whosoever uses a large volume of blood components. This committee
should take an appropriate step to improve the services as well as the
clinical practices. A recent study by Torella et al.20 have reiterated the
usefulness of hospital transfusion committee to change transfusion
practices in patients undergoing coronary artery bypass grafts
(CABG’s), Transurethral resection of prostate (TURP) and hip
replacements. Thus, in hospitals, this committee is among the powerful
tools for appropriate utilization of blood products (Box 11.3).
Prospective Audits
Prospective audits can be performed by blood centre staff on the basis
of physician’s requisitions. Such audit may need informal communi-
cation among physicians and transfusion experts. Audits based on
Box 11.3: Role of Hospital Transfusion Committee34

• Establish broad policies for blood transfusion therapy.
• Develop audit criteria for transfusion practices.
• Assess blood and blood component use for ways to improve patient care.
• Review and analyze statistical reports of the transfusion services.
• Audit transfusion reactions; post-transfusion infections and other adverse
events affecting all inpatients and outpatients.
• Reaudit previously identified problem areas to evaluate improvement.
• Promote continuing education in transfusion practices for the hospital staff.
• Assist the blood suppliers in blood procurement efforts.
• Assess adequacy and safety of the blood supply.
• Ensure that written policies and procedures are reviewed annually and
conform to established standards.
• Report to the committee responsibilities for overall quality assessment activities
and recommend corrective actions when indicated.
physician blood requisition can be used as an educational tool to

understand the transfusion needs in particular diagnoses or
management. This form of audits is usually not feasible under urgent
situations where laboratory reports may not be available or justified
for example, in case of acute bleeding where haemoglobin reports may
not be available or may not reflect the true picture of patient status.
Such requisitions are based on individual’s experiences and may not
comply with established guidelines.
The prospective audits based on explicit criteria, e.g. laboratory
parameters are more objective. These are more formal when, physician
in charge of the patient has to fulfill necessary predefined criteria. Audit
of such cases have shown decline in inappropriate transfusions of
platelet concentrates by 56 per cent at the time when patient load
increased by 38 per cent in Walter Reed Army Medical Centre.21 Thus,
a transfusion based on predecided criteria is more beneficial and is
gradually becoming an acceptable policy. These audits are carried out
by transfusion personals and are concurrent with indications written
in blood requisitions. If justification is not clear, physicians should be
contacted or be discussed in transfusion committee. Meanwhile issuing
component on such request should be based on whether request is
routine or urgent. The analysis is done on the following day of trans-
fusion of components as described by William Beaumont Hospital.22
Whenever, an inappropriate transfusion is considered, physician in-
charge of the patient is contacted personally.
Since the first inception of process in mid 1980’s there has been lot of
reports of the beneficial effect of auditing and education of physicians
by this method, that is the use of explicit criteria and discussion with
physician, has been used for red cells, platelets and FFP issue. Use of
the post-transfusion criteria has been modified by American association
of blood bank (AABB) in 1987 that incorporated use of both explicit
and implicit criteria.23 AABB recommends that medical technologist
screen the component request on basis of predefined criteria and
prepared algorithm. In case of inappropriate transfusion observed by
the medical technologist, the case is reported to resident doctor who
reviews the transfusion and patient’s requirements and judges the need
of transfusion and the matter sorted out. If irregularities are still there,
the cases are reported to blood bank director and formal communication
is established for justification. But keeping the patients safety
uppermost urgent demands can be issued and matter can be discussed
later. However, routine component issue can be withheld till situation
is clearer. It is seen that immediate communication for such demands
has more impact than delaying the same. Though these methods do
not prevent inappropriate transfusion in all cases, continuing medical
education (CME) does decrease number of inappropriate transfusions
in the long-run. It is the responsibility of auditing committee to have

consensus on such preset criteria and can be modified based on
transfusion requirements of particular transfusion services. This form
of audit poses a problem when manpower in the field is limited as
happens in our part of the world.
Retrospective Audits
Retrospective audits are based on evaluation of requisitions already
available. Such auditing is helpful whenever no clear-cut guidelines
are available or auditing is in initial stages of establishments. A
retrospective audit is helpful in comparing trends and changing patterns
of transfusion practices over a period. Indication, dosage as well as
patient’s follow-up can be evaluated. Retrospective audits are one of
most common audits performed in transfusion practice. 24 A
comprehensive approach of retrospective audits, analyzing problematic
area, corrective action taken and reanalysis of cases in prospective
manner has been a standard manner of auditing blood component
transfusions in number of situations. In 1997, retrospective analysis of
FFP utilization was carried out at Karachi, Pakistan. High incidence of
inappropriate transfusion was observed and corrective action in form
of educating anaesthetists and surgeons was carried out. A prospective
audit was carried out 2 years later and great improvement was
observed. Thus, the study showed the relevance of retrospective audit
and prospective audit for improving transfusion practices.25 There are
a few papers from developing countries too, which shows that a large
amount of plasma is inappropriately transfused by prospective
audit.25,26
Use of manual or computer in auditing is based on convenience. In
case of reviewing low volume audits, manual data analysis can be used.
However, for high volume audits computer database can be faster and
reproducible.
Corrective Action
Corrective action taken is undoubtedly essential for improving the
observed irregularities. Such corrective actions should be taken with
positive approach by concerned personals. CME is most important tool
to create established practice. The effectiveness of CME was observed
in 1990’s when it showed decline in the transfusions of red cell and
FFP transfusions in spite of increasing workload in hospitals. But
increasing platelet transfusions during the same period was of concern.
A review of American Society of Clinical Oncologist (ASCO) on
prophylactic transfusions concluded that therapeutic platelet
transfusions should be practiced rather than the prophylactic approach
as practiced presently.27
Better utilization of blood components by improving inventory

control has been forwarded by ‘Blood stocks management scheme’
(BSMS) established in April 2001 in U.K.28 The scheme collects red cell
stock and wastage data with the objective of understanding the blood
supply chain, including the levels of blood held in blood centres and
hospitals to optimize hospital and National blood services (NBS) centre
stock management. The wastage of red cell in the NBS as a percentage
of issue was less than 1 per cent with a downward trend at one-year
interim report.28 Under this scheme total of 2217418 red cell units were
issued to 310 hospitals (68% to district general hospital, 29% to teaching
hospitals and 3% to private hospitals).28
HAEMOVIGILANCE
Haemovigilance consists of detection, gathering and analysis of
information regarding untoward and unexpected effect of blood
transfusion. 17 A voluntary, anonymous reporting scheme for
haemovigilance in U.K. is being carried out in 1996 onwards. At present
more than 90 per cent of hospitals are participating in serious hazards
of transfusion (SHOT) scheme under haemovigilance system. According
to the latest report, the major increase is in wrong component
transfusion (from 48% of total to 67% of total) 29 rather than
immunological adverse effects or transfusion transmitted infections in
the last five years. The acceptance of haemovigilance is rising rapidly
across globe on voluntary or mandatory basis.30 Therefore, CME, i.e.
educating treating physicians has been prime target with the help of
various methods latest of this being e-learning program.31 Guidelines
by JCAHO for blood utilization were published in 1994 were a milestone
for improving blood utilization.32 Herbert reviewed transfusion of red
cells in intensive care unit (ICU) patients.33 They have stressed the need
for restrictive transfusion satisfying patient’s need rather than
physician’s thinking in critically ill-patients. It has been demonstrated
in TRICC that trigger of 7 gm/dl is as good as 9 gm/dl. Restrictive
strategy is superior, as it decreased transfusion by 54 per cent with a
better clinical outcome. Therefore, adoption of restrictive protocol, that
is transfusion trigger of 7 gm/dL, in most ICU patients has been recom-
mended.32 However, in the same study restrictive use of blood products
in patients of acute coronary artery disease has not been recommended
due to lack of evidence in this regard. Thus, it can be inferred that
same guidelines cannot be applied to all cases but patients individuali-
zed needs are to be considered and given priority.
CONCLUSION
Audit in BTS aims to improve the transfusion services and to reduce
errors or accidents in scientific manner. The uniform transfusion criteria
and compliance with regulatory guidelines, GMP and are required to

achieve the goals of safe and effective transfusions. Thorough and
regular audit and corrective actions will ultimately improve the blood
services.
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hazards of transfusion (SHOT): the U.K. approach to haemovigilance. Vox
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13. Chaudhary RK, Shukla JS, Ray VL. A2 and A2B subgrouping: is it necessary
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21. Simpson MB. Prospective concurrent audits and medical consultation for
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22. Shanberge JN, Quattrociocchi-Longe T. Analysis of fresh frozen plasma
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12
Multiple Myeloma:
Some Specific Aspects
Mona Anand, Rajive Kumar
Key Words
Cell of origin of myeloma • Cytogenetic and molecular
abnormalities • Growth factors in myeloma • Cell
adhesion molecules • Angiogenesis in myeloma,
prognosis
INTRODUCTION
Multiple myeloma (MM) is characterised by accumulation of malignant
plasma cells in bone marrow (BM) accompanied nearly always by
monoclonal immunoglobulins in serum and/or urine, and often,
suppression of normal immunoglobulin levels. Morbidity and mortality
are primarily related to skeletal, haematological and renal complications
of the disease.
The following topics that have been touched upon, deal with those
aspects of multiple myeloma that fall outside the scope of routine
diagnostic tests for the disease.
a. Cell of origin of myeloma
b. Cytogenetic and molecular abnormalities.
c. Growth factors in myeloma
d. Cell adhesion molecules
e. Angiogenesis in myeloma
f. Human herpes virus-8
g. Diagnostic criteria, classification and prognosis.
CELL OF ORIGIN IN MYELOMA

Studies of immunoglobulin (Ig) gene rearrangement are as germane to
multiple myeloma, monoclonal gammopathy of undetermined
significance (MGUS), or other B-lymphoproliferative disorders as they
are to a study of normal B-cell ontogeny. They shed light on the precise
stage of B-cell development at which the malignant clone emerges. For

the same reason, such studies can also be used to find out if progression
of the disease involves a new B-cell clone.1
It is useful to understand the background that underlies gene
rearrangement studies in myeloma.
NORMAL B-CELL ONTOGENY AND GENERATION OF

ANTIBODY DIVERSITY
B-lymphocytes derive from haematopoietic stem cells by a complex set
of molecular events that are aimed at producing immunocompetent
lymphoid cells capable of handling a large variety of antigens.
Differentiation of haematopoietic stem cells into B-cells occurs first in
the foetal liver and later in adult life, chiefly in the bone marrow.
The key events in the B-cell development are centred on genetic
changes that create a diverse array of surface immunoglobulin mole-
cules that act as antigen receptors. These crucial events take place in
pro(genitor)–B-cells which are the earliest committed B-lineage cells,
and the pre(cursor)-B-cells, the cells which develop from the pro-B-
cells.
Experiments on L chain genes in the late 1970’s2,3 established the
fundamental correctness of the hypothesis of Dryer and Bennet about
what the key genetic changes might be, that gave rise to antibody
diversity. Dryer and Bennet hypothesized that there was a single C-
region gene encoded in the germline, separate from not one, but multiple
V region genes. In the course of B-cell development, one of the V region
genes comes to lie with the C-region gene. This completed (C+V) gene
is then expressed by the cell. Mechanisms that increase the diversity in
the V region genes thus leave the single C-region gene at a distant locus
untouched. This idea of gene rearrangement occurring independently
in each lymphocyte was revolutionary in that it violated the then
accepted dogma that DNA is the same in all cells of an organism.
The genes for heavy chains and kappa and lambda light chains lie on
3 different chromosomes. As cloning and sequencing of light and heavy
chain DNA has shown, each of these genes instead of being encoded
by a single continuous DNA sequence, is coded for, by sets of gene
segments. In the germ line DNA, each of these multigene families
contains several coding sequences or coding genetic segments separated
by non-coding region.
The variable region of the kappa and lambda light chains are encoded
by the V-and J-gene segments. The variable region of the heavy chain
(VH) is encoded by V, D and J gene segments. The number of these
segments in the heavy and light chains has been worked out in man
and mouse. In humans there are over 50 potentially functional VH genes
that are grouped into structurally related families, VH1 to VH7.
Multiple Myeloma: Some Specific Aspects 197
During the development of B-cells, germ line heavy and light chain
gene segments, each, somatically recombine or rearrange so as to form
a continuous functional gene. Rearrangement in heavy chain precedes
that in light chains. The heavy chain gene segments undergo a VDJ
recombination while the light chain genes undergo a VJ recombination
to produce functional genes. After the initial rearrangment is complete,
the entire gene, including the exons and introns, is transcribed. The
primary RNA transcripts thus formed, undergo splicing to remove the
introns and produce an mRNA for translation into heavy and light chain
polypeptides. These undergo assembly to form a complete immuno-
globulin molecule.
In the ontogeny of B-cells that take place in the bone marrow, the
pro-B-cells undergo a heavy chain DH to JH gene rearrangement. This
is followed by a VH to D HJ H rearrangement. Productive VHDHJ H
rearrangement defines the next stage of development, i.e the pre-B-
cell. A pre-B-cell has surface mu chain paired with a surrogate light
chain. Light chain gene rearrangement, VL to JL, now occurs and surface
IgM appears. The cell is now designated immature B-cell. Change in
RNA processing then causes IgD also to appear on the cell membrane.
A cell with both surface IgM and IgD is designated mature B-cell.
Development in the bone marrow, an antigen independent process,
ends here.
The mature B-cell leaves the bone marrow and enters peripheral
lymphoid organs where the subsequent steps in B-cell development-
activation, proliferation and differentiation occur in response to antigen.
In the first day after immunization, antigen-activated B-cells start to
proliferate in the follicles of the peripheral lymphoid tissue. Germinal
centres develop as a result of clonal expansion of the B-cell blasts. In
this proliferative phase affinity maturation and class switching occur
and memory B-cells and plasma cells are formed. Affinity maturation
is a result of hypermutation mechanism that gets under way once B-
cells start proliferating. From the standpoint of studies in myeloma and
other B-cell neoplasms it is important to understand the hypermutation
process well.
Hypermutation introduces point mutations into the rearranged Ig
variable region genes and their adjacent flanking regions at a frequency
of about one nucleotide exchange per cell division. This occurs when
the germinal centre is established, i.e. well into the primary response.
Because of the hypermutation, the B-cell antibody repertoire gets
diversified. However, random mutations inserted into the V region will
only rarely lead to increased specificity of the antigen. Yet it is known
that the affinity of the antibody rises 10-fold in 2 weeks. Analysis of
hybridoma cell lines has shown that this affinity maturation is due to
selection of high affinity variants that arise from the hypermutations,4
such that only those B-cells which have come to acquire high affinity
receptors are expanded.5 Selection in germinal centre takes place in the
light zone among the non-dividing centrocyte population. Follicular
dendritic cells of the germinal centre appear to play a crucial role in
this through antigen presented on their surface. This is a major factor
that decides whether a centrocyte is negatively selected and dies or
experiences positive selection and emerges from the germinal centre.
Normal somatically mutated IgM positive B-cells in the germinal centre
either enter blood as memory cells6 or undergo isotype switch and
differentiation to plasma cells.7 Circulating memory IgM+ memory B-
cells may move to the bone marrow.8
Very elegant studies shed light on the approach that has been used
to establish, first, that hypermutation occurs in the germinal centre,
and second, that gene sequencing studies can show a group of cells to
be of common descent.9
As the study involved picking up individual cells for study from the
germinal centre and outside it, it was important to be able to pick up
from among the many, just those cells which were responding to the
antigen used for immunization. Hapten 4-hyroxy-3-nitrophenylacetyl
(NP) conjugated with chicken gammaglobulin as carrier was chosen as
the immunogen, because the initial response to this hapten is dominated
by a particular heavy chain gene rearrangement and the use of a lambda
light chain. Therefore, antibodies raised against the idiotype of this
particular antibody, could easily be employed immunocytochemically
to distinguish responding B-cells present in thin sections of spleens taken
from animals immunized with NP. These cells from germinal centres
and outside the germinal centre were microdissected, and the particular
VH gene (mouse VH 186.2) were amplified, cloned and sequenced. While
the sequences of Ig genes obtained from B-cells in germinal centres
had many mutations, those from activated B-cells from non-germinal
centre foci had very few. More importantly, from the perspective of
this chapter, when the mutated sequences of the B-cells picked up from
the germinal centre were compared, it was clear that many had
sequences sufficiently similar that indicated they were related by
common descent from the same precursor cell. It was thus possible to
build a genealogic tree showing the progenitor-progeny relationship
among the cells.
The entire process of Ig gene rearrangement is discussed in detail in
several standard immunology texts, which should be consulted.
IMMUNOGENETIC STUDIES
(V GENE ANALYSIS) IN B-CELL TUMOURS
Since a pro-B-cell in the course of its evolution to a plasma cell or
memory cell undergoes several sequential changes in its Ig genes,
genetic analysis of the Ig genes has been carried out in a number of
B-cell neoplasms as well. These immunogenetic studies entail, chiefly,

amplification, cloning and sequencing of H-chain genes. Interpretation
of the results of such an analysis in anyone disease is made possible by
comparison with what happens in normal immune response and also
with data from other B-neoplasms.
As discussed above, a VH-DH-DH transcript is generated by a process
of genetic recombination. The particular D-segment chosen in this
process and the imprecision of D-J joining introduces heterogeneity
and uniqueness in amino acid sequence of the third complementarity
determining region (CDR). The CDR3 sequence is, therefore, a clonal
signature for a B-cell.10 For the same reason the CDR3 sequence acts
also as a clonal marker for B-cell neoplasms. Hypermutations occurring
in germinal centres introduce further changes on this background. This
means that the heterogeneity in tumour cell clones resulting from hyper-
mutation will have, as a common feature, the same signature CDR3,
signifying origin from the same clone. Thus, in neoplastic B-cells, the
CDR3 is not only a useful marker for detection of a tumour clone, but
also for tracking its fate. The place to look for the changes introduced
by hypermutation is especially the nucleotide sequences of the three
CDR’s. As stated earlier, the sequence diversification that occurs in the
hypermutation process is a random event, but the limited amount of
antigen in the germinal centre selects only those cells which have high
affinity Ig on the surface. As contact with antigen is made via CDR1,
CDR2 and CDR3 regions of the surface Ig, the imprint of antigen selec-
tion is naturally seen as a clustering of nucleotide changes, representing
replaced amino acids, in these regions.
From the above considerations it is clear that the uniqueness of the
nucleotide sequences that result from recombination and the changes
that take place in the differentiation process in B-cell ontogeny, serve
not only as clonal markers for tracking B-cell clones, but also for working
out the point reached in the differentiation process and hence the
maturational stage by B-cell tumours. This kind of study of various B-
cell tumours and other pathological states also shed light on the differen-
ces, if any, in the usage of different groups of VH genes in different B-
cell tumours. In addition, one is able to decipher heterogeneity,
signifying presence of more than one type of clone, in seemingly homo-
geneous population of cells of a particular tumour, as exemplified by
chronic lymphocyte leukaemia (qv, below). Similarly, questions such
as emergence of a new clone in the course of treatment of one of the B-
neoplasms can be answered by this approach.
MATURATIONAL STAGES OF B-CELL TUMOUR

(BY V GENES ANALYSIS)
Study of B-cell neoplasm in this manner has allowed their classification
into 3 categories based on their maturational stage.10-12
a. Those neoplasm, which like native B-cells, have unmutated V

genes showing that the cell of origin does not enter the germinal
centre. A subset of chronic lymphocytic leukaemia is an example.
Another group of CLL cases with a better prognosis and typified
by the presence of chromosome 13 abnormality, shows mutations
indicative of a different maturational state.13
b. Tumours, such as follicular cell lymphoma, when V genes are
mutated and in addition, show heterogeneous sequences. The
heterogeneity is intraclonal as seen by the fact that the
heterogeneous clonal sequences share the same CDR3.
Heterogeneity of mutated V genes implies that the mutation of V
genes continue to take place evenafter the transformation has
occurred. This shows that the tumour not only arises from the
germinal centre but also continues to reside in this milieu.
c. Tumours with mutated V genes, where, in contrast to follicular
lymphoma, the mutated V genes have homogeneous sequences,
i.e. show no intraclonal heterogeneity. This means that the cell of
origin is post-follicular, i.e., has traversed the germinal centre but
has not stayed there and is thus no longer exposed to the mutator.
This situation is exemplified by multiple myeloma, which for this
reason would be considered to arise from a more mature B-cell.
VH GENE ANALYSIS IN MYELOMA

Cloning and sequencing of VH gene in multiple myeloma shows that
the clonal gene rearrangement that occurs in myelomas are
characterised by somatic hypermutation, which serves as evidence of
antigen selection and also class switch.14-17 There is an absence of
intraclonal variation, i.e. there is a lack of additional Ig gene diversity
between malignant clones in patients. This means that the final
oncogenic event in multiple myeloma occurs after somatic
hypermutation and class switches are complete, i.e. in a terminally
differentiated post-germinal centre B-cell, such as memory B-cell,
plasmablast or plasma cell. Class switch of course accounts for the fact
that myeloma can be of different isotypic classes/subclasses.
Elucidation of the particular VH genes that are used in myeloma is
informative and is best understood in the context of knowledge of what
is normal and what happens in other diseases. As mentioned earlier,
the V H genes are categorized into 7 families based on sequence
homology. The sizes of the families vary. For example, whereas VH1
and VH6 have only one gene each, VH3 has 22.18 As might be expected,
the VH gene usage in the normal human B-cell antibody repertoire is
proportional to the number of functional germ line genes within each
VH families.19,20 Some asymmetry in usage of individual VH gene has
however been noted. For example, the gene V H 4.2 (V H 4.34) is
overrepresented in the normal B-cell repertoire.19,21-24 Similarly, VH 4.21

(VH 4.34) is used for encoding IgM auto-antibodies of I/i specificity
against red cells in cold agglutinin disease.25,26 It is also overrepresented
in CLL (4-22% cases), acute lymphoblastic leukaemia (9-33% cases) and
in diffuse large cell lymphoma (as high as 65% cases).27-30 In the contrast
to this and for reasons not entirely known31,32 use of VH 4.34 in myeloma
is exceptional. As in the case of the normal B-cell , in myeloma too,
there is only a minor bias in usage compared to the normal expressed
repertoire. The genes VH 3 to 30 and VH 3 to 23 are overrepresented and
VH 1 to 69 and VH 3 to 9 appear unique to the disease.31 Thus, the VH
gene usage in myeloma is unique compared with that in malignant
and non-malignant B-cell populations and unlike other B-cell
neoplasms, VH 4.21 (VH 4.34) does not appear to have a role in develop-
ment of myeloma.
VH Gene Study in Myeloma Progression

Absence of intraclonal variation seen in myeloma at presentation
persists unchanged even in the course of disease progression, showing
that disease progression does not involve a new B-cell clone.1
IgM+ B-cells Clonally Related to Myeloma Cells

Absence of intraclonal variation in myeloma is taken to mean that
neoplastic event has occurred at a post-follicular stage. While this may
be true of the bulk of the tumour cell population consisting of plasma
cells, the finding in myeloma, of bone marrow IgM+ B-cells that are
clonally related to the malignant plasma cells, i.e. have identical VH
sequence as the isotype switched plasma cells, has not been adequately
explained.33-36 If one recalls that normal somatically mutated IgM+ B-
cells, after emerging from the germinal centre, either escape into the
blood as memory cells or undergo isotype switch and differentiation to
plasma cells, it is natural to ask if the IgM+ B-cells that are showing
clonal relationship to the myeloma cells are the cells where the neoplastic
event first occurs, followed by its further differentiation, through isotype
switching, to the malignant plasma cells. This would mean that in
myeloma there is a less mature B-cell that could constitute the growth
fraction of the tumour thereby feeding the plasma cell compartment.
The work of Van Ness’s group argues in favour of this view.34 It has
been suggested that the transition to a malignant plasma cell from such
a B-cell entails a second genetic change.37
V Genes in MGUS and its Transition to Myeloma

There have been, understandably, few studies of V genes in MGUS38
and fewer still of MGUS transforming to myeloma.39 Three types of
findings have been seen in MGUS:
a. No intraclonal heterogeneity within the tumour cell population

b. Intraclonal heterogeneity within the tumour population
c. Presence of IgM+ precursor cell with mutational heterogeneity.38
As in the case of follicular cell lymphoma and unlike that in myeloma,
the heterogeneous group shows that the tumour cell in MGUS,
continues to be exposed to the mutator after transformation. It further
suggests that the tumour cells instead of replicating in the bone marrow,
reach there from the germinal centre. This is consistent with the lower
plasma cell labelling index in MGUS as compared to myeloma.40
The MGUS cases with intraclonal homogeneity suggest that the
transformation or the crucial genetic event in this group of MGUS
patients affects a post-follicular cell. Alternatively, absence of intraclonal
heterogeneity may be due to differences in the relative growth rates of
members of the clone with the dominant clone causing self-cloning, a
possibility that needs investigating MGUS patients from a very early
stage of the disease.32
The finding in one patient of IgM+ precursor cell with the same
mutational heterogeneity as the tumour cell, suggests that like in
myeloma, it is still able to undergo isotype switching.
V gene status in transition from MGUS to myeloma has been studied
in 2 patients,39 one with intraclonal heterogeneity and the other with
intraclonal homogeneous sequence. In both, the emergent myeloma
clone was homogeneous. In the former, however, the intraclonally
homogeneous myeloma sequences co-existed with residual hetero-
geneous sequences from the MGUS.
CYTOGENETIC AND MOLECULAR CHANGES

The molecular pathogenesis of myeloma involves chiefly chromosomal
translocations; proto-oncogene mutations and rarely inactivation of
tumour suppressor genes.41 In addition, patients may show hyper-
diploidy (60-65% patients) pseudodiploidy (10-20% patients) and
hypodiploidy (10-30% patients).42
Chromosomal translocations involving chromosome 14 (IgH locus)
occur in approximately one-third cases and are important. The partner
in these translocations varies and has included in different cases,
chromosome 1, 4, 6, 11 and 16.43
Study of the (1;14) translocation has allowed cloning of 2 putative
genes at 1q: MUM2 and –3. Involvement of chromosome 4 affects
fibroblast growth factor receptor–3 (FGFR3) gene; chromosome 6,
insulin-response factor gene IRF4; chromosome 11, cyclin D1; and
chromosome 16, v-maf.44,45
The t(4;14) (p16.3;q32.3) translocation is seen uniquely in 10 to 25 per
cent patients of multiple myeloma and in human myeloma cell lines.46,47
It is the first example of an immunogloblin heavy chain (IgH)
translocation that simultaneously dysregulates two potential oncogenes

on two chromosome derivatives: FGFR-3 on der(14) and MMSET on
der(4).48,49 Wild-type FGFR3 (fibroblast growth factor receptor 3), a
weakly transforming molecule in murine haemopoietic models acquires
kinase-activating mutations50,51 that are strongly transforming in the
NIH3T3 cells and in the haemopoietic model. The FGFR’s are tyrosine
kinase receptors that bind 9 related fibroblast growth factors.52 Normal
haemopoietic cells for all practical purposes do not express FGFR3. Its
expression is limited, in multiple myeloma, to cases with 4p16
breaks.53,54 The MMSET (multiple myeloma set domain) gene codes
for a protein that shares a number of domains with nuclear proteins
involved in chromatin remodeling. Most notable among the domains
are the PHD fingers and the SET, characteristic of the trithorax group
of proteins in Drosophila and of the hrx gene involved in translocations
in human acute leukaemia. Translocation leads to deregulation of
MMSET.
The t(6;14) (p25;q32) translocation brings IgH in apposition to the
mum-1 gene (multiple myeloma-1, also known as the IRF4 for interferon
regulatory family). The IRF-4 encodes a member of the interferon
regulatory factor family which play a role in the control of B-cell
proliferation and differentiation.55 The resulting overexpression of the
IRF-4 gene may contribute to the oncogenesis.
The t(11;14) translocation. Breaks at 11q13 are seen in a fourth of
myeloma cases.56 The overexpression of cyclin D1 in myeloma differs
from that in mantle cell lymphoma. Mantle cell lymphoma shows
rearrangement of BCL-1 (B-cell leukaemia-1/cyclin D1) regions and in
approximately one-third cases, the breakpoints in chromosome 11
region q13 are clustered. Multiple myeloma shows breakpoints that
are scattered throughout the 11q13 region in the cyclin D1 genes.
Clustering is not seen. The presence of t(11;14) translocation in myeloma
is associated with lymphoplasmacytic morphology, a more aggressive
clinical course and a poorer prognosis.57,58 It has been proposed that a
common feature of all plasma cell neoplasms is the ultimate up-
regulation of the cyclin D pathway.59,60 This may occur as a direct
consequence of a t(11;14)(q13;q32) translocation up-regulating cyclin
D1 or because of upregulation of the cyclin D2 and cyclin D3 genes, the
latter directly as a consequence of chromosomal translocations or as
downstream effect of the other IgH translocations. Using gene
expressing profiling, up-regulation of at least one cyclin-D gene was
detected in almost all cases of plasma cell neoplasms.59,60
Recent work has shown that underlying genetic aberrations can help
classify myeloma into unique biologic subgroups of the disease.61,62
Data has been presented to show that the 2 major subgroups of the
disease are patients with recurrent IgH translocations (primary IgH
translocations) and patients with hyperdiploid karyotype and a low

prevalence of IgH translocations. In addition, there is an intermediate
group of patients with one of several non-recurrent secondary IgH
translocations who can have a variable ploidy.63 This unifying model
still needs validation. It does however provide a framework on which
to base future investigations and ultimately, a step toward targeted
therapeutics.
The t(14;16) translocation. This translocation causes deregulated
expression of c-maf proto-oncogene.64 The gene c-maf is a member of a
large family of basic zipper transcription factors, including jun, fos,
and nfil-6, many of which form heterodimers with one another. This
family of transcription factors takes part in many cellular processes
including proliferation, differentiation and responsiveness to 1L-665-67
whose role in myeloma growth is discussed below.
Less than 5 per cent of myeloma cases show mutations of p53 gene.
With disease progression this increases to over one-third.68 Interstitial
deletions at the site of the retinoblastoma (RB) gene on the chromosome
13 occur in approximately 40 per cent cases and carry an unfavourable
prognosis.
Early in its natural history multiple myeloma is IL-6 dependent, but
this property is lost later in the course of the disease and in cell lines.
This suggests that members of the signaling pathway may be
constitutively active in disease progression and points to a search for
mutations in them. The IL-6 signal is sent via two independent
pathways. The first of these is the RAS/MAP (reticular-activating
system/mitogen activated protein) kinase pathway and the second,
the Jak/STAT (Janus kinase/signal transducers and activations of
transcription) pathway.43 Mutations of Jak/STAT pathway have not
been described but those of the RAS/MAP kinase pathways do exist.
The frequency of RAS mutation increases with disease progression,
being the maximum in plasma cell leukaemia, followed by myeloma
and rarely, if ever in MGUS.69
GROWTH FACTORS IN MYELOMA

Interleukin-6
Myeloma cells require for in vitro growth, stromal interaction and
specific growth factors, some secreted by the tumour cells themselves.
The discovery of IL-6 played a major role in understanding the growth
factor requirements of myeloma cells. IL-6 was discovered as a T-cell
derived factor that caused differentiation of B-cell to immunoglobulin
secreting cells. The importance of IL-6 as a growth factor in myeloma
was shown in studies demonstrating secretion of IL-6 as well as
expression of IL-6 receptors (IL-6R) by freshly isolated human myeloma
cells.70,71
IL-6 appears to be a major autocrine, and more importantly, paracrine

growth factor for myeloma cells.67 The paracrine stimulus comes from
the bone marrow stromal cells. Bone marrow stromal cells provide a
microenvironment for normal haematopoiesis by direct cell contact and
by secretion of cytokines, including IL-6. In multiple myeloma, the
stromal cells, not part of malignant population, mediate the paracrine
stimulation of cell growth and suppression of apoptosis of malignant
plasma cells. The importance of stromal cell support was shown with
the demonstration that long-term bone marrow cultures from myeloma
patients maintained and supported division of monoclonal plasma cells
for over 6 weeks,72 and stromal cells from patients with multiple
myeloma produce higher levels of IL-6 than those from normal
individuals.73 Myeloma cells produce very low levels of IL-6 but are
able to induce stromal cell to upregulate their production of IL-6.74
IL-6 prevents apoptosis of myeloma cells induced by serum depri-
vation or other means.75,76 Retinoic acid induces apoptosis in myeloma
cell lines by down regulating the IL-6 receptor (IL-6R) expression.77
IL-6 also, by serving as a powerful osteoclast-activating factor,
contributes to the bony disease and hypercalcaemia seen in the disease78
and its possible role in renal failure of myeloma has been shown by the
demonstration of the characteristic histopathologic changes in the
kidney of IL-6 transgenic mice.79
Elevated serum IL-6 predicts a poor prognosis and active disease.
The same has been show to hold for soluble IL-6R.80,81
IL-6 affects cell cycle kinetics and supports myeloma cell proliferation
by its action on retinoblastoma protein and p21, another cell cycle
regulatory protein.82,83
Interleukin 6 acts in unison with interleukin-1 (IL-1), transforming
growth factor-beta (TGFB) and vascular endothelial growth factor
(VEGF) to maintain malignant plasma cell clone. Vascular endothelial
growth factor and other plasma cell products induce vascularization
of the tumour through angiogenesis.
IL-6 receptor belongs to the category of Type-I cytokine receptor.
The receptor functions in conjunction with a second molecule, gp130,
which is responsible for signal transduction.84,85
Inhibiting IL-6 pathway as a means of therapy has been tried by
administering anti-IL-6 antibodies.86
Interleukin-1 beta
Interleukin-1 beta (IL-1B), a bone resorption factor is produced by
myeloma cells.87 Cells from patients with lytic bone lesions are more
likely to have increased IL-1B transcripts compared to those without.
IL-1B can also induce the stromal cells to produce IL-6 and thus can
serve as a mediator of paracrine induced myeloma cell growth.
Tumour Necrosis Factor-alpha

Tumour necrosis factor-alpha protects myeloma cells from IL-6
deprived apoptosis.87 Serum levels of TNF-alpha are often increased in
patients with MGUS and elevated levels of TNF-alpha may antedate
transformaton to myeloma.88 TNF-alpha is an important bone resorbing
cytokine and has been found in increased amounts in the supernatants
from unfractionated myeloma bone marrow.89 The effects of TNF-alpha
and several other cytokines, including IL-1 are mediated through NF-
kB (nuclear factor kappa B). The central role of NF-kB in bone resorption
is shown by the fact that NF-kB knockout mice suffer from
osteopetrosis.90 Another biochemical pathway intimately associated
with bone resorption through NF-kB is that of RANK and RANKL.
RANK is a receptor for activation of NF-KB and a member of the
TNF receptor family. RANK is expressed on the surface of osteoclasts.
The ligand RANKL that activates RANK is not a soluble molecule but
is present on osteoblasts and stromal cells. This interaction forms the
basis of osteoclastogenesis, a process that has been known to involve
direct interaction of osteoblasts or stromal cells with osteoclasts.91 TNF-
alpha stimulates osteoblasts to increase expression of RANKL and also
differentiation of osteoclast.92 Myeloma cells also express RANKL and
therefore can themselves stimulate osteoclast formation in the myeloma
bone marrow environment.
Knowledge of the above mechanism is important in the context of
the action of osteoprotegerin (OPG), a decoy soluble receptor, that binds
RANKL and thus prevents the binding of the ligand to RANK.91 Quite
expectedly, animals that lack OPG show osteoporosis93 and this delicate
balance between soluble OPG and RANKL determines the quantum of
bone loss. This observation has understandably led to studies in which
in murine model, OPG prevented and reversed hypercalcaemia of
malignancy94 blocked cancer induced bone destruction and bone pain.95
These promising preclinical studies have spurred clinical trials in
myeloma and breast cancer patients with bone metastases. Since
myeloma cells express RANKL it is obvious that blockage of the
RANKL-RANK interaction may not only inhibit osteoclast stimulation,
but suppress the tumour cells themselves.
ADHESION MOLECULES IN MULTIPLE MYELOMA

CD56 (NCAM, neural-cell adhesion molecule), a mediator of
haemotypic adhesion is overexpressed on myeloma cells.96 Patients with
CD56 disease have an aggressive course. There is an inverse relationship
between the expression of CD56 and the extent of peripheral blood
involvement and patients with extramedullary disease do not express
CD56.97
All plasma cells express ICAM-1 (intercellular adhesion molecule-1;

CD54) but those that coexpress LFA-1 (CD18), a leucocyte cell surface
glycoprotein, spontaneously aggregate because of the interaction
between these homotypic adhesion molecules.98
CD44 (HCAM) is expressed by all plasma cells but alternative splicing
can cause formation of variant isoforms such as the 9v domain, known
to be associated with poor prognosis.99
CD138 (syndecan-1) is expressed on plasma cells and plays a role in
their adhesion to collagen. Myeloma cells express CD38 more strongly.
VLA5 (CD49e), like CD138, is adhesion molecule that binds to
fibronectin and may play a role in the homing of plasma cells to BM.
Clinical response after induction therapy has been correlated with the
percentage of VLA-5 negative plasma cells present in the BM.100
ANGIOGENESIS
There is a significantly increased microvessel density in active myeloma
compared with non-active myeloma, MGUS and normal subjects. A
low degree of angiogenesis is a favourable prognostic factor.101
Thalidomide may mediate some of its beneficial effect in myeloma
through inhibition of angiogenesis. Factors which mediate increased
angiogenes include vascular endothelial growth factor and basic
fibroblast growth factor.102
HUMAN HERPES VIRUS—8

Kaposi sarcoma-associated herpes virus (KSHV), also known as HHV-
8, is a member of the gamma herpes virus family. It has been shown to
be associated with human disease like Kaposi sarcoma, systemic
Castleman’s disease, primary effusion lymphoma (PEL). What links
multiple myeloma to Kaposi sarcoma is the presence of a biologically
active homologue to human IL-6, termed vIL-6, in the KSHV genome.103
KSHV DNA and vIL-6 RNA transcripts have been shown to be
presented in bone marrow dendritic cells of multiple myeloma
patients.104 This concept has received support from studies is patients
who were given chemotherapy to mobilize stem cells.105 Other groups
have however failed to corroborate the association between multiple
myeloma and KSHV.106
MULTIPLE MYELOMA: DIAGNOSTIC CRITERIA,

CLASSIFICATION AND PROGNOSIS
These topics are adequately covered in major haematology texts. Three
aspects are presented below to supplement what is given in the major
textbooks:
1. International Myeloma Working Group’s recommendation on the
diagnosis of plasma cell dyscrasias, and
2. Notes on the practical aspects of diagnosis,

3. Prognostic factors.
IMWG Recommendations for Clinical Definitions

In order to bring uniformity to the diagnostic criteria and prognostic
categories of plasma cell neoplasms, Kyle’s group has brought out an
update, now published in the British Journal of Haematology, on behalf
of the International Myeloma Working Group (IMWG).107 The following
notes63 are provided only to supplement this update.
The disease categories recommended by the IMWG include
asymptomatic (smouldering) myeloma, symptomatic myeloma, non-
secretory myeloma, solitary plasmacytoma of bone, extramedullary
plasmacytoma, multiple plasmacytomas (+/–recurrent) and plasma cell
leukaemia. In formulating the defining characteristics of the various
disease categories, the IMWG has used simple, easy-to-use criteria based
on easily available laboratory tests, and has not tried to covered every
possible diagnostic situation. One of the key aspects of disease categori-
zation is that it is based on the presence or absence of end-organ damage
(related organ or tissue impairment-ROTI) as it was felt that it was this
feature, more than anything else that distinguishes multiple myeloma
from MGUS and smouldering or asymptomatic myeloma. Four features
define ROTI. These are hypercalcaemia, renal insufficiency, anaemia,
and bone lesions (CRAB). In addition, other features also count, e.g.,
symptomatic hyperviscosity, amyloidosis, and recurrent bacterial
infection. It is also recognized by the group that evidence of myeloma
ROTI frequently may not be clear-cut and recognition of transition from
asymptomatic myeloma to multiple myeloma requiring treatment
(symptomatic myeloma) may require multi-disciplinary critical
assessment.
The project to formulate a prognostic classification has been divided
into 2 phases. The first phase has used commonly available clinical and
laboratory variables for prognostic classification of patients. The second
phase will incorporate and validate the use of novel prognostic markers
such as molecular changes, gene expression arrays and cytogenetics.
In the first part of the project, through a study of more than 11,000
patients over a period of 21 years, the investigators sought to identify 2
or 3 variables that would best predict survival and identify patients
most at risk. It was found that the disease could be classified, based on
beta 2 microglobulin and serum albumin into 3 stages that could predict
survival (Table 12.1). Prior to this also there have been several attempts
to synthesize clinical and laboratory parameters into a prognostic
classification or staging system. Of these, the staging system of Durie-
Salmon is the most popular. It is based on a combination of factors that
correlate with the myeloma cell mass. This clinical system is however
thought to be unreliable in many instances and has significant
Table 12.1: Prognostic classification of myeloma (IMWG)

Criteria Definition Stage Median survival
(months)
Low B2M and B2M mg/L<3.5 and albumin > 3.5g/dL 1 61
normal albumin
High B2M B2Mmg/L >5.5 3 44
All others B2M mg/L <3.5 and albumin<3.5g/dl or 2 29
B2M 3.5-5.5
B2M=beta 2 microglobulin
shortcomings, especially in the designation of bone lesions.108 In a study

109
of over 150 patients in which seven staging systems were evaluated
for their ability to stratify the patients into good-risk and poor-risk
groups with significant differences, it was found that some differences
in survival were significant and others were not. The British Medical
Research Council staging system110 was the best. More importantly,
none of the staging system was clearly superior to certain single risk
factors, especially anaemia (Hb> or < 8.5 g/dl) and renal impairment
(creatinine < or > 177 u mol/L). Another study that evaluated some
staging systems along with serum beta-2 microglobulin and the rate of
bone resorption found serum beta-2 microglobulin and serum albumin
to be better than any staging system.111 The IMWG staging in fact is
based on these 2 parameters.
It is felt by the International Myeloma Working Group that their
staging system may be an improvement over the Durie-Salmon staging
classification on the following grounds:
a. It segregates patients into 3 equal-sized cohorts
b. It is simple to use
c. It is easily reproducible
d. It is easy to remember
e. It predicts survival for patients treated with conventional or high-
dose therapy for all geographical regions and for all age groups.
NOTES ON PRACTICAL ASPECTS OF ELECTROPHORESIS

Agarose should be used for HRE of serum or urine, bands quantified
by densitometry and monoclonal nature of any suspected M band
confirmed by immunofixation (IMWG). We feel that from the
perspective of users in India, the choice of the densitometer is crucial.
Densitometers made by several reputed firms are excellent but suffer
from the disadvantage that they accept only pre-poured gels made by
the same manufacturer. We recommend purchase of densitometers that
accept home-poured gels of a wide range of sizes. Not only are these
densitometers cheaper, but as laboratory-poured gels are used, there is
no dependence on costly and recurring imports of pre-poured gels.
Urine Electrophoresis
In the absence of albuminuria, dipstick testing may be negative even
when monoclonal light chain is present. Urine electrophoresis and
immunoelectrophoresis are the recommended methods for detection
of Bence Jones proteins.
As the concentration of urinary M-protein is generally too low to be
detected on HRE, urine sample must be concentrated prior to use. This
can be done using a commercial concentrator or by dialysis. The urine
sample may be an aliquot from a 24-hour urine collection or the morning
first sample, preferably the former.
INTERPRETATION OF ELECTROPHORESIS PATTERN

The following points help:
1. Paraproteins appear as discrete additional bands anywhere within
the separation but most often in the beta or gamma region. The
width and density of any band corresponds respectively to the
width and height of the peak that it produces on densitometry.
2. Monoclonal IgD, IgE and free heavy chains may result in small
diffuse bands which can easily be missed.
3. The normal bands of the beta region, transferring and complement
C3, can mask serum paraproteins, particularly small bands of
monoclonal IgA. Densitometry can help in picking up suspicious
bands of this kind by showing the concentration of the normal
band to be outside the normal range. The importance of estab-
lishing the normal range in Indian subjects therefore is obvious.
4. Other proteins may give rise to bands that can be mistaken for
paraproteins. These include, among others, fibrinogen, haemoglo-
bin-haptoglobin complex, haemoglobin and C-reactive proteins.
5. A band in urine may be due to free light chains, or to a serum
protein that may have leaked into the urine.
6. Monoclonal free light chains may be found in addition to serum
paraprotein in myeloma patients.
7. The serum and urine paraproteins from an individual patient can
show different relative mobilities.
PROGNOSTIC FACTORS IN MYELOMA

This has been partially discussed above and the following account
summarizes the main points pertaining to single risk factors.
Plasmablastic Morphology
Greipp’s classification of myeloma into mature, intermediate, immature
and plasmablastic morphology has shown that the plasmablastic
morphology is associated a higher incidence of renal insufficiency,
higher plasma cell indices, a higher serum IL-6 receptor levels, more
ras mutations, more aggressive disease and a shorter survival.112
Serum Beta-2 Microglobulin

In patients with myeloma with normal renal function, rising seurm beta-
2 microglobulin predicts progression, clinical stage of Durie and Salmon
and response to therapy.113
S-Phase Estimation
The proportion of plasma cells in the S-phase of the cell cycle is
important, > 3 per cent signifying poor survival as an independent
prognostic factor.114
Tumour Cell Karyotype

Among newly diagnosed myeloma patients treated with standard-dose
chemotherapy as well as in those receiving stem cell transplantation,
presence of cytogenetic abnormalities has prognostic value.115,116 In the
latter study, abnormalities involving chromosome 11 and 13 were the
most unfavourable. A Mayo clinic study found significant correlation
between the presence of cytogenetic abnormalities and a high plasma
cell labelling index.117 In general, cytogenetic studies are not widely
performed because only 20 per cent patients have abnormalities
detectable on conventional karyotype analysis.
Cell Surface Phenotype

Certain immuno-phenotypic features have been suggested as having a
prognostic value. While presence of CD20+ plasma cells is associated
with a more aggressive course, the value of CD10 positivity in myeloma
is less clear.118
Serum Cytokine/receptor Levels

Serum IL-2 levels are higher in myeloma than in normal controls and
higher IL-2 levels have been reported to be associated with a prolonged
acturial survival.119 Serum IL-6 has been reported to correlate with
advanced disease and decreased survival,120 an elevelated serum IL-6
receptor level is an independent predicator of poor outcome in
myeloma.121
Lactate Dehydrogenase
Because increased serum LDH is seen in only a small proportion of
patients (5-11%), the prognostic value of LDH in myeloma is limited.122
Circulating Plasma Cells and Peripheral Blood Labelling Index

Circulating myeloma cells are more easily detected by flow cytometry
and their presence in large numbers is associated with poor prognosis.123
The peripheral blood plasma cell labelling index is an additional
prognostic factor. Circulating plasma cells are predictive for patients
with smoldering multiple myeloma who are likely to rapidly evolve to

active disease.124
ACKNOWLEDGEMENT
The authors thank Mr Pradeep Kotnala for the typing of the manuscript.
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13
Minimal Residual Disease (MRD)
In Leukaemias
DK Mishra, A Chaturvedi,
H Subramanya, GS Chopra
Key Words
Leukaemia • minimal residual disease
(MRD) • morphology • cytogenetics • immuno-
phenotype • molecular remission • disease free
survival • leukaemia free survival • southern
blotting • RT-PCR • FISH
INTRODUCTION
Leukaemic disorders are comprised of biologically and clinically
heterogeneous subsets. The pathologic mechanisms of these
malignancies are now better understood with application of advance-
ments in the field of immunology and genetics. The detection of chromo-
somal translocations by cytogenetics, followed by identification of genes
involved, their breakpoints and novel gene products have shed light
onto the biologic mechanisms of tumour growth. This in turn has
provided the ground for development of very sensitive diagnostic
molecular techniques to detect tumour cells in very small numbers
(minimal residual disease), which were hitherto missed on conventional
microscopy.1,2 Therapeutic modalities have consequently been modified
and improved.
Though a high percentage of patients with leukaemia achieve
complete clinical remission after initial treatment, a large majority of
these patients will finally relapse. The conceptual solution to this puzzle
is strongly considered to be due to surviving leukaemic cells which
depending on the underlying malignant disease and therapeutic
treatment may proliferate, remain quiescent or vanish. The aim therefore
is to predict impending relapse in subsets of patients with specific clonal
abnormalities.3
Minimal residual disease (MRD) has variously been defined as:

• A stage in leukaemia treatment when the patient is in remission,
symptoms of the disease are absent but small numbers of
leukaemic cells still remain in the body.
• When in complete clinical remission; the absence of malignant
disease detectable by clinical imaging, standard histologic or
cytologic means and when malignant cells can only be found by
very sensitive immunologic or molecular techniques.
• Lowest level of disease detectable in patients in complete clinical
remission, by the most sensitive methods available.4
Detection of MRD has been attempted using morphologic and
immunologic techniques; cytogenetic and molecular visualization (in
situ hybridization) studies; by RFLP combined with southern blotting
and in vitro amplification of specific DNA and RNA sequences (PCR
based studies).
Broadly speaking, MRD evaluation serves to identify and quantify
occult leukaemic cells and then using this information to determine
prognosis and modify therapeutic protocols with the long-term
intention to individualize and improve treatment outcome. Measure-
ment of MRD is seen as the key to gauge risk of relapse. More specific
questions that MRD analysis seeks to answer are: quantification of
leukaemic cell load at various stages of disease and treatment; thereby
attempting to classify responders and their risk groups, thus
determining their need for adjuvant therapy and to evaluate newer
therapeutic modalities. It has also been used to determine quality of
grafts, the efficacy of procedures used to remove malignant cells from
autografts and to determine its clinical significance if any.
Though, answers to many of the above problems have been found,
challenging questions on optimum strategies in determining MRD with
regards to techniques, timing of follow-up samples and the ideal
samples etc. are being put forward. The key is to know whether it is
possible to identify patients whose prognosis is different from what is
expected from the use of statistical risk factors. Will it be possible to
design tailor made therapies, avoiding over-treatment of patients whose
disease does not warrant it or under-treatment of poor responders who
require more aggressive treatment.
To understand the concept of minimal residual disease, the
management protocols in leukaemias have to be better appreciated.
Leukaemia therapy can be divided into two phases, i.e. induction and
post-induction/post-remission therapy. The initial goal of therapy is
induction of a complete remission, usually requiring a period of bone
marrow aplasia.
The therapeutic approach in acute myeloid leukaemia (AML) is
chemotherapy alone (Acute promyelocytic leukaemia/non-acute
Minimal Residual Disease (MRD) in Leukaemias 221
promyelocytic leukaemia), bone marrow transplant or immuno-

therapy. Of these, chemotherapy is given in phases. With the induction
therapy, 60 to 85 per cent of individuals show a complete remission.
Post-remission therapy follows this induction phase and could either
be intensification, consolidation or maintenance phases. The National
Cancer Institute (NCI), defines complete remission (CR) in AML (except
acute promyelocytic leukaemia), by normalization of neutrophil counts
to at least 1.5 × 10 9 /L and platelet counts of greater than 100 × 10 9/L,
a marrow aspirate and biopsy that demonstrates at least 20 per cent
cellularity with less than 5 per cent blasts, no Auer rods and absence of
extra medullary leukaemia. These parameters should be sustained for
at least a period of four weeks or until initiation of intensification
therapy if earlier than four weeks. A partial remission is defined as
bone marrow blasts of 5 to 25 per cent.
In acute lymphoblastic leukaemia (ALL), treatment is divided into
three phases i.e. induction, intensification and maintenance. The NCI
remission criteria for ALL are, a normo-cellular bone marrow with less
than 5 per cent blasts, no lymphoblasts in the PBS, absolute neutrophil
count of > 500/mm3, platelets >100,000/mm3 and the absence of extra-
medullary disease. In addition, attainment of CNS remission is specific
to ALL.
In spite of all the therapeutic advances, long-term leukaemia free
survival has not been achieved. A major reason in the past has been
imprecise MRD monitoring methods. Thus, new understanding of
minimal residual disease, its quantification and relationship to relapse
is helping individualize therapy and offset the risk of recurrence,
thereby improving prognosis. Most current schemes for classification
of risk rely primarily on clinical features, but it is not impractical to
hope that with the recent advances in understanding of leukaemia
biology and its inherent promise to lead to more rigorously proven
and understood classifications, effective management protocols would
be established in the not so distant future.
DIAGNOSTIC TOOLS IN MRD EVALUATION: TECHNICAL

ASPECTS
Depending on the understanding of tumour specific biology, different
investigative modalities have been found useful in evaluating MRD
for each leukaemic subset.
Detection of MRD by morphological and Immunological

Technique
i. The sensitivity or detection limit of morphologic (cytologic,
cytochemical and histologic) assessment is 5 per cent blast cells
(detection limits 10–1 to 10–2). Though it is a standard procedure,
it is limited by its low sensitivity.4 Following chemotherapy the

bone marrow is heterogeneous in blast cell distribution and often
a hypoplastic bone marrow further compounds the pitfalls of
morphologic assessment.
ii. a. By standard immuno-phenotypic analysis, 1 to 5 per cent
leukaemic cells can be detected.
b. By using fluorescence activated cell sorter (FACS), it detects
specific combinations of cell surface proteins that may
characterize the original blast population and uses this aberrant
combination as an immunologic “footprint” in follow-up
studies. Using combinations of monoclonal antibodies and
multicolour flow cytometry,5 it is possible to increase sensitivity
to 0.1 per cent and further up to 0.01 per cent if immuno-
phenotyping is done on cytospin preparations. But, unfortu-
nately all immunologic techniques are hampered by a lack of
adequate number of tumour specific antigens and antibodies.4
Conventional Cytogenetics and Molecular Techniques

Standard karyotypic analysis (G-banding) has shown chromosomal
abnormalities in 50 to 75 per cent of haematological malignancies.6
Sensitivity is low (5 per cent), with a detection limit of 10–1 to 10–2,
which is similar to a simple morphologic assessment. This disadvantage
is further compounded by the fact that, at least 2 to 5 per cent cells are
required to have cytogenetic abnormalities and a minimum of 20 to 50
good quality metaphases have to be scored. This requires both technical
expertise and time.
Fluorescence in situ hybridization (FISH) uses specific probe (Fig.
13.1, Plate 1) that detects the presence or absence of particular
chromosomes (centromeric probes) or abnormal sequences (cosmid
probes) using chromosome or aberration specific DNA probes. It has a
sensitivity of 1 to 5 per cent which is similar to that of routine cyto-
genetics, but one advantage of this technique is that dividing cells are
not required. The examination can be performed on interphase nuclei,
and a large number of cells can be analyzed in a short period of time.
But then, though it can be carried out on interphase nuclei (Fig. 13.2,
Plate 1), there is significant false positivity and the technique is labour
intensive. Double colour FISH (D-FISH) on interphase nuclei and hyper-
metaphase FISH seems to be about 10 times more sensitive. I-FISH
(Interphase FISH) analysis in most cases is sufficiently sensitive and a
highly reliable way of monitoring the outcome of therapy.7
Recently a new multiparametric cell scanning system (Duet) has been
introduced in the field of MRD detection, which automatically scans a
large number of cells and performs combined analysis of morphology
and FISH on the same cell.8
Cell Culture Methods

Modification of growth conditions to preferentially allow leukaemic
cell growth and inhibit normal progenitor cells; have been used to
develop colony assays and detect MRD by employing PCR on `plucked’
colonies. This technique is limited by the difficulty in maintaining
culture conditions and also in standardization of the cell culture
methods to determine cut off levels of residual disease.9
Molecular Targets
Analytical techniques in leukaemia search for mainly two types of
targets at the molecular level:
a. Abnormal genes and/or transcripts (Molecular equivalents of
chromosomal alterations):
A few leukaemias are associated with constant non-random
chromosomal translocations and the consequent new tumour
specific fusion genes and m-RNA transcripts. An increasing
number of MRD studies have utilized techniques, which identify
and quantify these novel genes and their products.8 Though these
genomic alterations occur over large regions, they usually generate
fusion m-RNA which apart from small variations are constant for
specific leukaemias making them amenable to detection by
molecular means, provided a first reverse transcriptase step is
added to the procedure (RT-PCR).
b. Clonality Markers: (Immunoglobulin/TCR gene rearrangements):
During the ontogeny of B and T lymphocytes, whether normal or
malignant, Ig and TCR genes are rearranged. The coding instruc-
tions for the antibody or TCR variable region are generated
according to a common mechanism; three (V, D, and J for Ig heavy
chains and TCR) or two (V and J for Ig light chains) genes are
joined together to form first a DJ junction, to which a V gene is
secondarily fused to give rise to a final VDJ mature Ig or TCR
gene. During this process, some nucleotides are randomly deleted
from the joining ends of the V,D and J genes while others are added
by the terminal deoxy-ribonucleotidyltransferase giving rise to a
V N D N J region, which can be regarded as a fingerprint of a
particular clone.
Since most B and T cell malignancies originate from immune cells,
which have already undergone this physiologic gene rearrangement
during differentiation, all cells belonging to a malignant clone carry
the same gene rearrangement as a unique clonal marker.
Based on the above principles two primary tools have been used for
detecting leukaemic cells:
DNA Restriction Fragment Length Polymorphism (RFLP)

Analysis Combined with Southern Blot Hybridization
It uses DNA labelled probes to look for chromosomal aberrations and
clonal markers. However, at least 1 per cent of cells under investigation
should be clonal cells. It is a laborious, time consuming technique
hampered by a low sensitivity of 1 per cent and restricted by the number
of tests that can be carried out simultaneously.
In Vitro Amplification of Specific DNA and RNA Sequences

Amongst the comparatively more recent tools, PCR techniques (Fig.
13.3) have dramatically changed the manner in which nucleic acids are
studied and analyzed.10 It has emerged as the most promising approach
to date for MRD detection and analysis. The inherent problems of low
sensitivity, labour intensiveness and technique specific difficulties
associated with the previously described methods: like inadequacy of
tumour markers (immuno-phenotyping), have been offset by the newer
generation of PCR analytical tools. It is well-established now that quanti-
tative MRD data have a higher predictive value than qualitative ones.11
In the past, quantitative PCR techniques were not used as often,
because they were thought to be semi-quantitative or because they are
very laborious with a high-risk of false positive results due to conta-
mination or carry-over of previously amplified DNA. These quanti-
tative techniques have been:
PCR Techniques
Multiplex PCR Nested PCR
1 2 3 4 5 B B K BV 1 2 3 4 5 B M5B M
- BCR
– b3a2
- b3a2 – b2a2
- b2a2
BCR-ABL competitive PCR # 1 BCR-ABL competitive PCR # 2
1 2 4 1 2 4
104 103 102 106 105 104 103 102 10 104 103.5 103 106105.5105 3 10 0
6 × 103 2 × 105 < 10
Fig. 13.3: Different RT-PCR techniques in the detection and quantification of

BCR-ABL fusion transcripts in CML and ALL
• Limiting dilution assays combined with a single or two step PCR.12

• Competitive PCR techniques—based on the observation that the
amount of added standard increases as the native signal
decreases.13
• Non-competitive PCR techniques—which use quantification of two
products synthesized in parallel under conditions when the native
signal is not altered by the standard.4
It is now, with the introduction of real time quantitative PCR
techniques, which is based on a kinetic cycle-by-cycle quantification
using target gene specific fluorogenic probes, that the field of MRD
analysis has taken a quantum leap (Fig. 13.4, Plate 2). This technique
uses an integrated system for thermal cycling, real time fluorescence
detection in closed tubes and subsequent computerized analysis (Light
Cycler of Roche Diagnostics and Taqman of Applied Biosystems).4,14
The sensitivity of most PCR assays carried out with genomic DNA
is one positive cell in 105-6 normal cells and it can reach even up-to one
in 107 normal cells if stochastic analyses are included.15 The sensitivity
of the PCR technique is one of the most important factors while
evaluating MRD in serial samples of single patients as well as large
groups of patients in clinical studies, aiming at a comparative analysis
with results of other trials. But there are many technical limitations
that could lead to false negative and false positive results. For a standard
PCR analysis, the total amount of mRNA used for c-DNA synthesis
and final PCR is important for sensitivity. For PCR combined with
stochastic analysis up-to 1 to 10 μg DNA can be tested, and in this
setting a 2 step PCR is recommended.
TUMOUR SPECIFIC CHROMOSOMAL ABERRATIONS (FUSION

GENES)4,16 MYELOID LINEAGE
Acute Myeloblastic Leukaemia (AML)
t (8;21) (q22; q22) AML1-ETO RT-PCR
inv (16) (p13; q22)/t(16;16) CBF β-MYH11 RT-PCR
t (15; 17) (q22; q12) PML-RAR• RT-PCR
t(9;11) (p21-22; q23) MLL-AF9 RT-PCR
Chronic Myeloid Leukaemia (CML)

t (9;22) (q 34.1; q 11.2) BCR-ABL RT-PCR
LYMPHOID LINEAGE
Acute Lymphoid Leukaemia
• B Lineage:
t (9; 22) (q34.1;q11.2) BCR-ABL RT-PCR
t (1;19) (p23;p13) E2A-PBX1 RT-PCR
t (17; 19) (q23;P13) E2A-HFL RT-PCR

t (8;14) (q24;q32) MYC-IgH DNA -PCR
t (4;11) (q21;q23) MLL-AF4 RT-PCR
• T Lineage
t (11;14)(p13;q11) RHOM2 -TCRδ DNA -PCR
t (1;14)(p34;11) TAL1-TCRα DNA -PCR
t (10 ;14)(p34;11) HOX11-TCRα DNA -PCR
CLONAL REARRANGEMENTS
• B cell leukaemia IgH-CDR 3 DNA-PCR
rearrangement
• T cell leukaemia TCR-rearrangement DNA-PCR
MRD IN LEUKAEMIA: CLINICAL APPLICATIONS

Acute Myeloblastic Leukaemia
AML is a complex and heterogeneous disease in all respects of cytologic
features, stages of differentiation, antigen expression, chromosome
abnormalities, gene expression, growth regulation and response to
treatment. Current classification systems and treatment regimens are
inadequate, as they do not represent their biologic heterogeneity.
Among the prognostic markers poor cytogenetics is taken to be the
presence of t (9; 11)-MLL/AF9, t (11; 19)-MLL/ENL, chromosome 5, 7,
8 abnormalities and complex karyotypes. Good cytogenetics on the
other hand, is the presence of t (8; 21)-AML1/ETO and inv (16)-CBF β/
MYH 11. As discussed below, these chromosomal aberrations have been
used in MRD analysis using various methodologies.
Management of AML practically starts by first differentiating non -
APL and APL subgroups. Non -APL is treated with cytarabine along
with an anthracycline derivative with or without another agent
(etoposide) included in the regime. The effectiveness of therapy has
been demonstrated by a number of studies. In a study of forty-five
patients (median age< 60 yrs) a complete remission was seen in 80 per
cent individuals.17 Compared to this, APL therapy is primarily with
all-trans retinoic acid (ATRA) but, there is a subgroup which has shown
decreased ATRA sensitivity. In a study of eighty-five previously
untreated APL patients published by Fenaux et al all showed a complete
remission (CR) with ATRA.18 As, paradoxically, no molecular remission
is seen on ATRA therapy, a combination of ATRA and chemotherapy is
generally given. But, evenafter complete remission has been achieved,
due to persistence of submicroscopic residual leukaemia a final cure
rate of zero per cent is usually seen if this minimal residual disease is
not treated.19 Therefore, post-remission therapy after ATRA has been
essential to eliminate MRD and achieve complete cure.20
MRD Monitoring in AML ( Non -APL)

Various techniques have been used in MRD detection and follow-up in
a number of settings in AML. The role of conventional cytogenetics is
well proven. In a study of 71 cases of AML in morphologic remission,
20 of whom had abnormal metaphases identical to those at diagnosis,
all the twenty individuals relapsed within a period of 78 weeks, thereby
proving a positive karyotype to be a reliable predictor of relapse; but,
25 of the remaining 51 negatives also relapsed implying that, a failure
to detect abnormal clones does not guarantee a durable remission.21 In
situ hybridization and FACS have also been used in MRD evaluation.
In one case study of a patient of AML-M7, the disappearance of
aneuploid peaks coincided with attainment of morphologic remission.22
Apart from these studies, the bulk of MRD analysis in AML has focused
on the following genetic abnormalities:
i. t (8; 21) (q22 ; q22.3) A reciprocal translocation which leads to the
formation of AML1-ETO fusion gene and transcript; has been
found in 10 per cent AML patients, especially of the M2 subtype.
Serial analysis by competitive or real time quantitative PCR in
this setting seems to be useful in identifying patients at risk for
relapse, since increases in transcripts (molecular relapse) can be
found 2 to 4 months before clinical relapse, providing an opportu-
nity for early therapeutic interventions to prevent haematological
relapse.23
ii. inv (16) (p13;q22) and t (16; 16) (p13; q22).These are associated with
acute myelomonocytic leukaemia with increased eosinophils
(AML-M4Eo). The latter results in the formation of CBFβ-MYH
11 (core binding factor β gene-smooth muscle myosin heavy chain)
fusion gene and transcript. Quantification of minimal residual
disease (MRD) by competitive CBF β/MYH11 reverse-transcrip-
tase polymerase chain reaction (RT-PCR) in the bone marrow (BM)
cells during post-remission therapy with high-dose cytarabine
(HiDAC) or after BM transplantation with a matched unrelated-
donor marrow (MUD-BMT) has been carried out. Molecular moni-
toring by conventional two-step RT-PCR has yielded comparable
results only when multiple assays per time point were performed,
while single-assay RT-PCR gave misleading results. Competitive
RT-PCR has been found to be a valuable tool for molecular
monitoring during post-remission chemotherapy, as well as after
BMT in this setting.24
Negative PCR results obtained 8 months after a complete elimination
have indicated a low-risk of relapse. A complete elimination of CBF β-
MYH 11 mRNA positive cells have been observed after allogeneic
BMT.25
MRD Monitoring in Acute Promyelocytic Leukaemia (APL/M 3)

APL constitutes 5 to 10 per cent of AMLs. t(15; 17)-PML/ RARα is the
defining chromosomal abnormality. MRD monitoring in APL has been
carried out using FISH and RT-PCR for PML/ RAR a. t(15;17)
(q22;q21) with PML-RAR a transcripts and variant translocations t (5;
17) (q32;q31) (NPM-RAR a) and t (11;17) (q 13;q21) (NuMA-RAR α)
benefit from a combination therapy of ATRA and chemotherapy. On
the other hand, patients with t (11; 17) (q23; q21) (PZLF-RAR α) are
ATRA resistant.4
The majority of patients treated with chemotherapy and ATRA will
become PCR negative by applying conventional PCR assays with a
sensitivity of 10–4.26 The persistence of PML-RAR α transcripts is highly
predictive of relapse, especially at the end of induction therapy. Delayed
disappearance of t(15;17) associated transcripts is an independent
prognostic factor in APL (27). In a study 11/13 PCR positive patients at
4 months relapsed; whereas, 22 PCR negatives at 4 months (Disease
free survival for 3 months to 5 years) and 9 APLs on 4 to 12 years long-
term remission (Chemotherapy plus BMT) remained PCR negative.28
Also in a setting of autologous BMT, monitoring suggests a prolonged
clinical and molecular remission with transplantation of a PCR negative
bone marrow, but PCR positivity before transplant is associated with a
high-risk of relapse. Presence of PCR detectable transcripts 3 months
after BMT seems to indicate a high-risk of relapse.
Acute Lymphoblastic Leukaemia (ALL)

The population based surveillance epidemiology and end result (SEER)
data show 69 per cent 5-year survival of ALLs in the 0 to 14 year age
group, for the years 1978 to 1986,29 which increased to 79 per cent for
the period 1986 to 1992.30 Thus, a ten per cent decrease in relapse was
seen. One of the major reasons for this decline has been an improvement
in management protocols, including post-induction intensification
according to the risk group category of the patient. With the present
management protocols, cure rates of 75 per cent in childhood ALLs
and 25 to 40 per cent in unselected adult ALLs are the norm. The princi-
pal barrier to long-term leukaemia free survival (LFS) has been relapse.
Therapy in ALL begins with the phase of induction which is for a
period of 3 to 4 weeks with various protocols (CCG, UK-ALLVIII DFCI,
NCI, EORTC, St Judes, BFM etc.) using Vincristine (V), Prednisolone
(P), Anthracycline, L-Asparaginase, , Cyclophosphamide , Methotrexate
(Mtx) Arabinoside-Cytosine (Ara-C), and CNS radiotherapy in different
combinations. This is followed by post-induction intensification lasting
for 3 to 6 months, with combinations of 6-Mercaptopurine (6-MP),
Prednisolone, L-Asparaginase, Methotrexate and Ara-C. Finally,
maintenance therapy is instituted over 2 to 3 years with Mtx, 6-MP,
Vincristine and Prednisolone.
The classic prognostic markers for ALL are age, sex, race, WBC count,
haemoglobin, platelets, extramedullary disease (nodes, liver, spleen,
mediastinal mass), FAB morphology (L1, L2, L3), immunophenotype
(Early Pre-B, B-ALL, T-ALL) and cytogenetics: hyperdiploidy , 6q-versus
t ( 9;22 ), t (8;14 ),t ( 4;11). The time to remission after start of treatment
is an important and independent prognostic indicator, based on the
‘Goldie-Coldman’ hypothesis, which implies that failure to decrease
leukaemic mass fast permits leukaemic clones to arise.31 In a study,
post-induction MRD levels were compared to the relapse rates and the
outcome groups were categorized into bad, intermediate and good. 8/
9 patients with high ( >10–3), 12/26 with intermediate (10–3 to 10–5) and
29/117 patients having low (<2 × 10–5) levels of MRD had a relapse.32 A
day 5 to 14 bone marrow or blood examinations in children and a day
28 evaluation in adults showing clearance of lymphoblasts from the
same is also associated with longer remission and fewer relapses.33
Another major prognostic factor in MRD is CNS Leukaemia/CNS
Relapse which is seen in 3 per cent childhood ALLs at diagnosis and
connotes a poor risk group. In both children and adults it is associated
with a lower rate of remission induction, a higher risk of relapse and
shorter survival. Its severity is further defined by criteria given by the
NCI: CNS 1-No blasts, CNS 2-<5 WBCs/ml with blasts, CNS 3->5
WBCs/ ml with blasts or cranial nerve involvement. A single Tdt
positive cell in CSF is taken as positive for CNS involvement.34
Treatment remains the single most important prognostic factor, with
the National Cancer Institute34 subclassifying ALLs into low/standard
risk and high-risk groups, the former being treated by a three or four
drug combination therapy and the latter with four to seven drug
intensive chemotherapy. The success of management protocols based
on this approach especially in high-risk ALL, has weakened the power
of many other adverse prognostic factors.
MRD analysis in ALL has been done by application of FISH using
chromosome specific repetitive centromeric probes, 35 combined
multiparametric systems (DUET),8 gene rearrangement studies, RT-
PCR36 and even cell culture colony assays.9
When acute lymphoblastic leukaemia is diagnosed in a patient, the
total number of leukaemia cells is approximated to be 1012 to 1013. A
majority of patients reach complete remission (CR) after about 4 weeks
of chemotherapy.11 One in four patients have residual disease at clinical
remission, which falls to 17 per cent at week 14th of consolidation
therapy, then to 5 per cent at week 32nd and zero at week 120th when
therapy is complete.37
Differences in survival for MRD positive versus negative patients
have been found to be statistically significant at all time points. In
general, presence of MRD at higher level and persistence of MRD at all
time points (0-2, 3-5, 6-9, 10-24 months) have a poorer outcome and
increased risk of relapse regardless of other prognostic features.38 High
levels of MRD at week 14th of consolidation therapy and residual
leukaemia at 6 months of maintenance therapy are ominous signs.
Patients with no residual leukaemia at the end of remission induction
therapy have a 90 per cent chance of survival without relapse.37 Hemato-
logical relapse in patients with acute leukaemia (>5% blasts) could
theoretically still mean a leukaemic burden at 1010 leukaemic cells.4
MRD analyses is also applied in risk categorization. A child classified
as low risk on clinical parameters at the end of remission induction
therapy would be reclassified as higher-risk if MRD positivity is found.
And if residual disease persists after the third month of consolidation
therapy with other high-risk features, alternative therapies like bone
marrow transplantation are then considered.37
MRD studies in ALL have primarily been divided into three broad
categories:
i. In the patients with standard risk B -lineage ALL (characterized
by absence of high-risk chromosomal aberrations viz. t (9; 22),
t(1; 19) or t (4; 11)) the detection of MRD is carried out using IgH/
TCR gene rearrangement studies. IgH-CDR3 leukaemia specific
clonal rearrangements have primarily been used for this purpose.39
TCR-γ, β, δ genes have also been used to follow-up T-ALL
malignant clones. These gene rearrangements have also been
found in B-ALL (50-70%) cases.4 The sensitivity of PCR assays
with clone specific probes is low (ranging from 1 leukaemic cell
in 103 to 1 in 106 cells). To be detectable by PCR, the sample must
contain at least 5 to 10 per cent clonal cells. Therefore, whenever
possible, detection of MRD in ALL is carried out by following at
least two marker genes to avoid false-negative results.40
In 12 to 26 per cent of T-ALL, SIL-TAL 1 fusion gene on chromo-
some 1 has a clone specific sequence, representing an ideal target
for MRD analysis.41
ii. Ph1 chromosome or BCR-ABL mRNA transcripts have been found
in 3 to 5 per cent of paediatric patients with ALL and in 12 to 43
per cent of adults. Ph1 positive ALLs have a very poor prognosis
with conventional chemotherapy having a very poor success rate,
and thus autologous and allogeneic BMT have been used.
Allogeneic BMT has been introduced as first line therapy after
induction.42 The median time-interval from the first positive PCR
assay (molecular relapse) to clinical relapse is about 100 days43
and during this window period other therapeutic interventions
like donor lymphocyte infusions have been successfully used.44
MRD has also been evaluated in patients started on Glivec (STI-
571) in Ph positive ALLs.45
iii. t (1; 19) (q 23; p13.3) detected cytogenetically in 5 per cent ALL
(mainly pre-B ALL) with chimeric transcript of E2A (Chr.19) and
PBX1 (Chr.1) genes, is associated with a poor prognosis though
PCR analyses for MRD have shown that a substantial proportion
of children achieve a molecular remission, and persistently
negative PCR assays indicate a low-risk of relapse, when the
patient is treated with intensive chemotherapy.46 Also ALLs
associated with t (17; 19) (q22; p13.3) with an E2A/HLF fusion
gene, on follow-up for MRD using RT-PCR have shown a very
poor prognosis.47
Chronic Myelogenous Leukaemia (CML)

Ninety per cent of CML patients show a Philadelphia chromosome
translocation t(9; 22) (q 34.1; q11.21) which results from a reciprocal
translocation of long arms of chromosome 9 and 22 resulting in a new
fusion gene consisting of 5’-BCR (22q11) and 3’-ABL (9q34) sequences.9
This has been used in MRD studies in CML.48,49 Commonly, in children,
a gene fusion e13a2 (b3a2) and in adults e14a2 (b2a2), with the
consequent protein products named p210/p230/p190 are formed. These
aberrations that are looked for in MRD analysis using conventional
cytogenetics, FISH and RT-PCR techniques. In a further 5 per cent, a
variant Ph translocation involving 9, 22 and one other chromosome
occurs. In a small number of patients the karyotype is normal although
in a half of these the BCR-ABL fusion can be detected submicro-
scopically and at the molecular level. The remaining cases do not have
this fusion gene signifying a separate molecular basis,14 characterised
as atypical CML.
During monitoring of patients of CML the following variables are
evaluated:
i. Haematologic response which is defined by normalization of
peripheral counts, no immature cells and no systemic symptoms
and absence of splenomegaly.
ii. Cytogenetic response which is subclassified as complete
cytogenetic response= Ph+ 0 per cent, partial cytogenetic
response=Ph+ 1-34 per cent, and minor cytogenetic response=Ph+
35 to 90 per cent, and
iii. Molecular response which does not have an agreed set of criteria,
but RT-PCR negativity with an assay with 10–5 to 10–6 sensitivity
is taken to be a positive response.
Relapse at the molecular level also does not have a precise definition,
but a BCR-ABL/ABL ratio of > 0.05 per cent, with one or more confir-
matory analyses has been taken to be indicative of a relapse.
In patients treated with interferon-α (IFN-α) or allogeneic BMT, CML
monitoring has been performed by repeated examination of bone
marrow morphology and karyotyping, with their attendant problem

of poor sensitivity.48 Cytogenetic studies on peripheral blood samples
of patients in haematologic remission have proven to be difficult. Also,
leukaemic cells from individuals who are IFN-α and Imatinib mesylate
(STI-571) treated have either low cell counts or fail to grow well in
culture. The only advantage offered is in the detection of additional
karyotypic abnormalities.
The use of FISH techniques to detect BCR-ABL fusion gene (Fig.
13.2) in Ph positive leukaemic cells increased the sensitivity of
cytogenetic studies to a great extent. A fused red green colour in D-
FISH studies indicates a BCR-ABL fusion gene.50 However, random
co-localization of red and green signals could also occur in normal cells
and could give a high false positive rate of 10 to 15 per cent. A high
false negative rate could also be expected depending on the probes
used and the precise location of ABL breakpoint.51 These problems have
been overcome by probe systems currently in use, which reduce false
positivity to around 0.2 per cent. Also because 100 to 500 cells are scored,
peripheral blood can be examined avoiding the need for a bone marrow
aspirate. It is likely to play an important role in monitoring CML but is
technically demanding and requires care in a number of areas.
Molecular techniques of southern blot, western blot and RT-PCR have
been used but the former two techniques have largely been rendered
obsolete.
In the majority of cases the t (9; 22) translocation juxtaposes exons
e13 (previously b2) or e14 (previously b3) adjacent to exon 2 and ABL
gene yielding BCR-ABL fusion transcripts referred to as e13a2 or e14a2
(previously b2a2 and b3a2) respectively which are then analyzed by
RT-PCR.7 Peripheral blood expression of BCR-ABL by RT-PCR is nearly
equal to that of bone marrow and thus eliminates need for routine BM
samples.
A sensitivity of 10–5 to 10–6 is achievable by nested PCR.14 Qualitative
MRD analysis provides little clinical information; therefore quantitative
and semi-quantitative RT-PCR assays have come into vogue. Newer
introductions have been competitive PCR and now real time quanti-
tative PCR (RQ-PCR), which is in the market as two systems Taq-man
(Fig. 13.4, Plate 2) and fluorescence resonance energy transfer (FRET).14
In a setting of Allogeneic SCT quantitative PCR is now regarded as
the method of choice for MRD analysis, whereas with IFN-• therapy
cytogenetic analysis is still the standard method. FISH is increasingly
replacing conventional cytogenetics. XY-FISH probes can be used to
detect donor-recipient cells in sex mismatched allogeneic stem cell
transplants to assess complete donor chimerism (Fig. 13.5, Plate 2). RT-
PCR has also been used extensively (Fig. 13.4). Quantitative PCR has
been invaluable in-patients on IFN-α treatment,52 because virtually all
who have attained complete cytogenetic remissions are persistently
RT-PCR positive. Cytogenetic monitoring in these individuals is often

impractical as IFN-α treatment can lead to a hypoplastic fibrotic
marrow. In individuals where Imatinib mesylate (Glivec) is being tried
the clinical relevance of cytogenetic analysis and other laboratory tests,
as reliable ‘surrogate’ markers for survival are still to be determined.14
The need for sequential cytogenetic and molecular analysis in the
management of patients with CML and for the evaluation of minimal
residual disease in patients on therapy has been emphasized.53 Serial
RQ-PCR analyses post-BMT is recommended to distinguish low versus
high-risk for relapse.54 Also, a BCR-ABL/ABL ratio<0.045 per cent has
been seen to be associated with a considerable decrease in risk of relapse,
therefore with time an individual can be weaned off IFN-• therapy
without risk of relapse.55
Chronic Lymphocytic Leukaemia (CLL)

Conventional cytogenetic studies have been problematic in CLL because
neoplastic cells divide infrequently. However, I-FISH studies now
permit detection of chromosomal anomalies with prognostic signi-
ficance in CLL.56
With the use of BMT in CLL and emergence of purine analogues,
which are capable of inducing complete response in a substantial
proportion of patients, MRD studies have become more relevant.56
Studies using IgH PCR and IgH clonogenic probes have shown
conflicting results when attainment of molecular CR was assessed.57
CONCLUSION
In practice, it is ‘Impossible’ for every single neoplastic cell to be
eliminated, therefore MRD detection and subsequent treatment aims
to decrease disease load to levels where risk of relapse is least. The
efficacy of minimal residual disease analysis in optimal treatment of
leukaemia patients has been rigorously proven by statistically sound
studies. There is no doubt that in the future, therapeutic measures in
leukaemia management will be closely interlinked to close monitoring
of leukaemic cell load in the patients.
Determination of MRD is an evolving field in which the technology
and understanding of the results are continually being refined.
Morphologic examination, cytogenetics, fluorescence-activated cell-
sorting (FACS) analysis, and now polymerase chain reaction (PCR) all
detect MRD, albeit with differing sensitivities. However, there are still
a number of unanswered questions regarding the modalities and means
of attaining the optimal approach to MRD analysis with regards to the
specific leukaemic subsets. It is possibly in these areas that current
research would have to be directed. Utilizing these modalities in a
clinically fruitful manner is what the study of minimal residual disease
promises to achieve.
KEY POINTS
1. MRD is the lowest level of detectable disease by a given technique.
2. Conventional BM morphologic assessment has the lowest
sensitivity (10–1 to 10–2).
3. Cytogenetic abnormality detected if any; is both diagnostic and
prognostic: t(9;22), t(15;17), t(8;21), t(8;14) etc.
4. FISH cytogenetics is superior to conventional cytogenetics.
5. Multi-parametric flow cytometry is useful for initial characteriza-
tion and subsequent follow-up.
6. Immunoglobulin(Ig)/TCR gene rearrangement studies are
excellent molecular techniques for clonality.
7. Detection of fusion transcripts by RT-PCR (BCR-ABL, PML-RARA,
AML1-ETO) can be routinely practiced.
8. If facilities are available, employ multiple techniques to monitor
the residual disease.
9. Real time PCR technology can quantify residual disease at the
molecular level.
10. Optimal approach to MRD analysis and therapeutic measures in
leukaemia management should be closely inter-linked.
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14
Diagnostic Dilemmas in
Lymphoproliferative Disorders
Sumitra Dash
Key Words
Lymphoproliferative disorders • Haematogenes
• Biphenotypic leukaemia • Extramedullary LPD
INTRODUCTION
Lymphoproliferative disorders usually pertain to lymphocytic
malignancies. These can originate from cells that are at a stage prior to
T-and B-lymphocyte differentiation from a primitive stem cell, or from
cells at stages of maturation after stem cell differentiation. Variability
in expression of a lymphopoietic stem cell disorder may result in the
spectrum of lymphocytic diseases. Dilemmas and mistakes in diagnosis
may arise (Table 14.1), albeit infrequently in modern times with the
Table 14.1: Common Pitfalls in the diagnosis of paediatric leukaemia

Infections Viral including HIV
Bacterial in infants
Leishmaniasis
Immune diseases ITP
Juvenile rheumatoid arthritis
Macrophage disorders Osteopetrosis
Haemophagocytic lymphohistocytosis
Congenital bone marrow diseases Pure red cell aplasia

Congenital neutropenia
Bone marrow failure Acquired aplastic anaemia
Solid tumours Neuroblastoma
Rhabdomyosarcoma
Medulloblastoma
Primitive neuroectodermal tumour
Nutritional deficiency Megaloblastic anaemia
availability of better gadgets. A misdiagnosis however subjects patients

to the risk of cytotoxic therapy. Choosing the wrong treatment for
patients who have acute leukaemia significantly reduces their chance
of cure. Both errors are disastrous for the patient.
The international lymphoma study group have proposed a new
classification termed Revised European American Lymphoma (REAL)
classification,1 which was published in 1994 and has since become
widely accepted in lymphoma diagnostic practice. This classification
scheme makes use of the pathologic, immuno-phenotypic, genetic and
clinical features of a given lymphocyte tumour to delineate them into
separate disease entities. At the end of 1999 WHO came out with a new
lymphoma classification.2 This embodied the principles of the REAL
classification. Some lymphomas considered as provisional entities in
the REAL system are now accepted as definite entities within the WHO
classification.
RISK FACTORS FOR DIAGNOSTIC PROBLEMS

Undue Haste in Starting Treatment
Before sufficient good quality diagnostic material is obtained to enable
morphologic, immuno-phenotypic and cytogenetic diagnosis. A panic
situation usually prevails when acute leukaemia is suspected. However,
usually these cases need prompt treatment for symptoms of bone
marrow failure rather than immediate cytotoxic treatment. There should
never be hesitation in repeating bone marrow aspiration to obtain
adequate material. In those cases where treatment is urgent as due to
high leucocyte count, diagnosis is usually obvious.
Inappropriate Reliance on Modern Technology

Inappropriate reliance on modern technology, use of wide panel of
monoclonal antibodies in the classification of leukaemias has lead to
confusion over the diagnosis of biphenotypic leukaemia, as many ALL
cases may show myeloid antigen expression and true biphenotypic
leukaemias are rare and extremely rare in children. In children a high
proportion of lymphocytes having early B-cell phenotype, being CD
19, CD 10 and TdT positive may be present in the blood and bone
marrow. A young child with enlargement of lymph nodes, liver and
spleen, a blood count, which is normal, or lymphocytosis may have a
bone marrow full of reactive lymphoid cells with phenotype of
immature B-cells and raise concern about possible malignancy. A lymph
node biopsy in these cases will be consistent with reaction to infection.
Bone marrow lymphocytosis may be interpreted as showing relapse in
children with ALL. Interpretation of immuno-phenotyping also may
create problem in diagnosis of acute leukaemias in infants, specially
Diagnostic Dilemmas in Lymphoproliferative Disorders 241
the early B-cell lineage ones, which may be positive for CD 15 but have
MLL gene rearrangement.
Leukaemia or Bone Marrow Failure

About 1 to 2 per cent cases of childhood ALL and a smaller proportion
in adults are preceded by a period of apparent bone marrow failure,
subsequent haematological remissions within weeks and later
development of acute leukaemia, usually within 3 to 9 months.3 In
contrast to acquired aplastic anaemia,these cases usually have
neutropenia more pronounced than thrombocytopenia. Bone marrow
though shows hypocellularity, there may be increase in reticulin laying
down. Diagnostic confusion can be avoided in these cases by:
• Administering prompt supportive care
• Careful follow-up
• Repeating the bone marrow aspirate in 4 to 6 weeks if patient
remains pancytopenic.
• Cytogenetic analysis may be helpful but it is often difficult to
obtain sufficient material for diagnosis.
• No specific treatment till confident about aplasia and later, the
development of acute leukaemia.
• Prognosis is as good as ALL if no inappropriate treatment such as
steroids or immunosuppression is given to these cases.
Bone marrow in both acquired aplastic anaemia and congenital bone
marrow disorders may contain a high proportion of early B
lymphocytes. This might lead to misdiagnosis of congenital
neutropenia, Diamond Blackfan anaemia and transient erythro-
blastopenia of childhood. Here again careful consideration of the clinical
picture and a period of observation should obviate mistakes in
diagnosis.
OTHER PRIMARY BONE MARROW DISORDERS

Pancytopenia, fever and increasing hepatosplenomegaly are features
of haemophagocytic lymphohistiocytosis, a rare condition characterized
by infiltration of the spleen, liver, bone marrow and CNS with histiocytic
cells.4 The hallmark of this disease is finding of haemophagocytosis in
the bone marrow or other tissues, but a number of associated diagnostic
features of macrophage activation syndrome may be present. Repeated
bone marrow aspiration and or liver biopsy may be needed to confirm
the diagnosis.
A leucoerythroblastic blood film with blast cells in blood may cause
confusion with acute leukaemia and is a feature of many disorders
including severe infection, bone marrow infiltration with solid tumours
and thalassaemia major. All of which are associated with hepatospleno-
megaly. Bone marrow examination will differentiate in most cases.
Clinical picture of ITP may superficially resemble acute leukaemia.

Blood counts are usually normal apart from reduced platelets. Bone
marrow examination is necessary in these cases.
A severe megaloblastic anaemia may stimulate acute leukaemia due
to peripheral pancytopenia and some organomegaly. Serum levels of
vitamin B12 and folate along with bone marrow examination will resolve
the diagnosis.
SYSTEMIC DISEASE AND INFECTION

Bone pains, joint pains and swelling are common presenting features
of ALL in children. There may also be lytic lesions in skeletal radiograph.
Periosteal and metaphyseal changes may be present in ALL. In these
cases with low leucocyte count, diagnosis of leukaemia may not be
apparent on blood film. 5 Previously many such cases use to be
diagnosed as juvenile rheumatoid arthritis. These cases usually respond
to steroid therapy and relapse as leukaemia. Bone marrow examination
is a must before giving steroid treatment.
A large number of infections may mimic acute leukaemia, parti-
cularly in young infants. These include acute bacterial infection, CMV,
parvovirus, rubella and toxoplasmosis. HIV infection may also mimic
acute leukaemia in infancy and in older patients is associated with
lymphomas and a variety of other malignancies6. Epstein-Barr virus
(EBV) infection is well recognized as superficially resembling leukaemia
but in practice appropriate investigations exclude it.
Usually, visceral leishmaniasis is associated with fever, progressive
pancytopenia and an enlarging liver and spleen and may cause
confusion with acute leukaemia.7
LEUKAEMIA OR OTHER MALIGNANCY

The most common solid tumour involving bone marrow in paediatric
age group is neuroblastoma. The clinical picture of bone pain and
anaemia is similar to that of acute leukaemia. Although abnormal cells
are rarely seen in the blood film, replacement of the bone marrow by
clumps and sheets of malignant cells may be present. Immuno-pheno-
typing and immunocytochemistry may be helpful in distinguishing
solid tumours from leukaemia blasts. Other disseminated cancers which
may mimic leukaemia include soft tissue sarcoma, germ-cell tumours,
medulloblastomas. There can be bone marrow involvement without
any obvious primary lesion. Diagnosis of solid tumour should be
considered in all cases with large atypical blasts with doubtful
haemopoietic origin. Immuno-phenotyping and immunocytochemistry
may be helpful to distinguish solid tumours from leukaemic blasts.
The diagnosis of neuroblastoma is usually confirmed by finding raised
catecholamines and a primary mass in the abdomen or posterior thorax.
Cytogenetics is helpful specally to differentiate primary neuroecto-

dermal tumour t(11;22) and alveolar rhabdomyosarcoma t(1;13)(2;13).
WHICH TYPE OF LEUKAEMIA

To give appropriate first line treatment it is necessary not only to
distinguish AML from ALL but also the subtypes. Identifying the 1 to 2
per cent cases of ALL with Smlg +ve B-cells who are highly curable
with short-term high-dose chemotherapy, as used in B-cell NHL.8
Cytogenetic and immuno-phenotyping, is necessary and would
determine the approach to treatment (Table 14.2).
In most cases morphological difficulties arise in distinguishing L2
ALL and undifferentiated M0-AML or M5-AML and in recognition of
M6 and M7 AML (from ALL). The distinctions are facilitated by the
use of appropriate cytochemistry and a panel of monoclonal antibodies.
With such measures virtually all acute leukaemias can be classified. It
is probable that most cases of acute undifferentiated leukaemia (AUL)
in children are probably cases of ALL.
ABERRANT ANTIGEN EXPRESSION AND

BIPHENOTYPIC LEUKAEMIA
Treatment strategy become confusing in patients with acute leukaemias
co-expressing both lymphoid and myeloid antigens. These are variously
described as mixed lineage, hybrid or biphenotypic leukaemias. It has
become clear that aberrant antigen expression is a feature of both ALL
and AML and attempt have been made to develop more stringent
criteria, for the diagnosis of true biphenotypic leukaemia. One proposed
scoring system (Table 14.3) has been put forward by Matutes et al (1997).9
It is clear from Table 14.3, that most emphasis has been given to the
presence of highly specific antibodies, for example, the expression of
both CD 13 and CD 33 does not render ALL a biphenotypic leukaemia.
Table 14.2: Diagnostic panel of antibiodies as recommended by the British

Society for standards in haematology (1994)
Primary Panel
B-Lymphoid CD19, Cd22, CD10, CD79a
T-Lymphoid CD7, cyCD3, CD2
Myeloid CD13, CD33, anti-MPO
Non-Lineage related TdT
Supplementary Panel
B-Lymphoid cyµ, Smlg
T-Lymphoid CD5, CD1, CD4, CD8
Myeloid CD41/61, antiglycophorin.
Table 14.3: Suggested criteria for diagnosis of biphenotypic leukaemia

Score Myeloid B-Lineage T-Lineage
2 MPO cy CD22 cy CD3
CD79a
1 CD13 CD10 CD2
CD33 CD19 CD5
0.5 CD 11b/11c TdT TdT
CD14 CD7
CD15
Leukaemia is classified biphenotypic if 2 or more lineages score > 2 points.
Myeloid associated antigen expression in particular CD 15 and CD 65

is frequently seen in acute leukaemia of infancy associated with MLL
gene rearrangements.10 Such infants with ALL are now usually treated
on protocols which include elements active in AML in an attempt to
improve their prognosis.
In the MRC-UK ALL XI trial, 17 per cent of children had ALL with
either CD13 or CD 33 positively. A number of studies have now
confirmed that aberrant antigen expression which does not fulfill the
criteria for mixed lineage leukaemia , has no prognostic significance,
contrary to what was thought before to give poor prognosis. One should
remember that immuno-phenotype is generally not an independent
prognostic variable with current treatment regimes for high-risk ALL.
Similarly, the expression of lymphoid associated antigens is of no prog-
nostic significance in AML cases.11
BONE MARROW INFILTRATION IN LYMPHOMAS

Bone marrow infiltration is frequent in lymphomas. The pattern of
infiltration is important in the differential diagnosis of lympho-
proliferative disorders (LPD) and also has prognostic significance.
Immunohistochemical techniques performed on sections of paraffin
embedded tissue are useful in establishing or confirming the nature of
lymphoid infiltrates. Molecular genetic analysis using PCR to detect
Ig heavy chain (IGH) gene rearrangements and T-cell receptor (TCR)
gene rearrangements can be useful in confirming clonality in difficult
cases. IGH rearrangements will detect around 57 to 80 per cent of cases,
with infiltration by B-cell lymphoma in trephine biopsy sections.
There may be discordance between the type of lymphoma seen in
the marrow and that present in the lymph node, or other tissues. The
frequency of such discordance varies from 6 to 40 per cent. Also in
about 6 to 21 per cent cases there is discordance as to grade of
lymphoma.12 One common discordance is follicular lymphoma in the
lymph node and lymphoplasmacytic lymphoma in the marrow. This

may represent differentiation of tumour in the marrow.
Lymphomatous infiltration of the bone marrow needs to be
distinguished from infiltration by reactive lymphocytes. Both pattern
of infiltration and the cytological characteristics have to be taken into
account.
• Interstitial infiltration can occur in both
• Para-trabecular infiltration and packed marrow is usually present
in neoplastic state.
• Nodular infiltrates with well-defined margins and a poly-
morphous population of immunoblasts, macrophages and plasma
cells, are usually reactive.
• Nodular infiltrates with ill-defined margins, often extending
around fat cells, with relatively homogeneous cellular composition
are usually neoplastic.
• Immuno-phenotyping and clonality demonstration by IgH or TCR
gene rearrangements may be helpful.
SOME DISTINGUISHING POINTS BETWEEN

LEUKAEMIA AND LYMPHOMA
• Majority cases of precursor B-LPD present as ALL. Only 10 to 15
per cent present as lymphoma.
• T-lineage LPDs usually present as lymphoma.
• In precursor B-ALL, bone marrow is markedly hypercellular and
heavily infiltrated with leukaemia.
• When there is infiltration, it cannot be distinguished from ALL.
• In precursor B-cell lymphomas, bone marrow is often normal. All
initial stages of bone marrow infiltration is patchy with intervening
areas of surviving haemopoietic tissue and fat.
• By convention, cases with fewer than 25 per cent or 30 per cent of
bone marrow lymphoblasts are categorized as lymphoblastic
lymphoma and cases with heavier infiltration as ALL.
DIAGNOSTIC PITFALLS DURING MANAGEMENT

OF ACUTE LEUKAEMIAS
Problems in Diagnosis of Repalse
Problems arise in interpretation of bone marrows done during or
sometime after therapy for ALL. Increased numbers of immature
lymphoid cells resembling lymphoblasts of L1 morphology are
sometimes seen in children. These are termed haematogens and could
be interpretated as relapse. Although they may be positive for CD34,
CD10 and TdT, but expression of TdT is usually stronger and CD 10
and CD 19 is weak in the haematogens. The reverse occurs in ALL
blasts. Following antileukaemic therapy, most patients enter
morphologic or haematologic complete remission. Sometimes there is

lymphocytic rebound. It is however ideal to base treatment decisions
on molecular measurement of minimal residual disease.13 Table 14.4
enlists the methods of detecting minimal residual leukaemia in marrow
or blood. It is extremely important that such techniques are rigorously
standardized and that treatment decision based on minimal residual
disease are instigated as part of rigid prospective studies with adequate
technical supervision rather than performed or an adhoc basis.
Usually intensive intrathecal chemotherapy and cranial or
craniospinal irradiation is given to children with cranial nerve infil-
tration or a CSF pleocytosis of more than 5 cells/mm3, with recognizable
blasts at diagnosis, or the time of relapse. Children with lower cell counts
are treated as ‘clear’ CSF and receive no additional treatment. However,
the interpretation of cytospins of CSF is more difficult than widely
admitted. So, it has now been suggested that the presence of equivocal
CNS findings or bloody CSF at diagnosis may be associated with worse
prognosis and will benefit from additional intrathecal treatment.14
Another difficult area is the interpretation of CSF findings in children
who are undergoing regular intrathecal chemotherapy when a low
number of blasts are found in CSF. Despite the report that children
with these findings do not have an adverse prognosis,15 it has been
found that recurrent low-grade pleocytosis may be a prelude to CNS
relapse, sometimes with focal rather than generalized disease. In these
Table 14.4: Methods for detecting minimal residual leukaemia in marrow or blood
Technique Approx. Comments
Sensitivity
(cells)
Morphologic or <5-10 Variable sensitivity dependent
cytochemical on unique features
Cytogenetics 5-10 Cells must undergo mitosis
Immuno-phenotyping 1-5 Greater sensitivity if unique
leukaemia-specific profile
defined by multicolour analysis
is used.
FISH 2-5 Applicable only if appropriate
probe available, must exceed
background levels of false
positivity
PCR <0.01 Applicable only if appropriate
probe available, may be utilised
for gene rearrangement studies
and assessment of specific
translocations
cases CSF should be repeated after 5 to 6 weeks without giving more

intrathecal therapy, to avoid masking the diagnosis. Immuno-pheno-
typing cytogenetics or FISH may prove useful if sufficient material-
available.16
Extramedullary Tumours
Extramedullary tumours of lymphoproliferative disorders are often
misdiagnosed. Their morphologic appearance is variable and can be
confused with undifferentiated non-haematopoietic malignant
tumours. Extramedullary infiltrates of immature T or B-cells are
diagnosed as to lymphomatous or leukaemic origin by arbitrary criteria.
ALL is diagnosed when lymphoblasts are > 25 per cent of the marrow
elements. Infiltrates in lymph node, spleen, liver and mediastinum are
common manifestations of ALL. CNS and testes infiltrates usually occur
in relapse in ALL. In general, T-cell lymphoblastic infiltrates predomi-
nant in extramedullary sites. Paraffin immunoperoxidase detection of
cyCD 3, CD 43, and CD 45 is consistent in T-cell lymphoblastic infiltrates
with variable expression of TdT and CD 34. However, detection of CD
45 and CD 20 is frequently not detected in B-cell lymphoblastic
infiltrates. In these cases CD 79a may be useful. TdT and CD 34 expres-
sion may or may not be present in them. The actual paraffin immuno-
peroxidase profile of extramedullary tumours lack specificity, thus a
comprehensive immunoperoxidase panel should be used if cell sus-
pension or electron microscopy is not available.
CONCLUSION
A variety of conditions may mimic acute lymphoblastic leukaemia and
other lymphoproliferative disorders. The spectrum is wider particularly
in the young child. Clinical examination and morphological review of
well-stained blood, marrow films and appropriate biopsy material
remain the essential tools for diagnosis of lymphoproliferative disorder.
Immuno-phenotyping, cytogenetics and molecular genetics allow a
rational approach to diagnosis and management, but are not substitutes
for traditional morphology. Most problems in diagnosis arise from
undue haste and failure to recognize that these tools are our servants
and not our masters.
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2. Harris NL, Jafle ES, Diebold J, Flandrin G, Muller Hermelink HK, Vardman J
et al. The World Health Organization classification of neoplastic diseases of
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2000;36:69-86.
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JJ. Terminal deoxynucleotidyltransferase (TdT) positive cells in cerebrospinal
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Blood 1989;74:416-22.
15
Immunohistochemistry on
Marrow Trephine Biopsy
AK Karak
Key Words
Immunohistochemistry • Bone marrow trephine
biopsy • Immunodiagnosis • Immuno-phenotyping
An accurate diagnostic and prognostic assessment of any haematologic

disorder is ideally achieved by means of an integrative approach of all
available data on peripheral blood and bone marrow cytomorphology,
histomorphologic features of marrow trephine biopsy, immuno-
phenotypic information on the cellular components involved in the
disease process, cytogenetic and molecular genetic analysis and
presenting clinical features. However, the necessity for adoption of such
an all-exhaustive investigative approach does not arise in every case
and it is important to be aware of the strengths and limitations of each
of the aforementioned investigative procedures in order to make most
efficient utilization of these. For example, detailed cytomorphologic
features could only be evaluated in Romanowsky-stained preparations
of peripheral blood smear, marrow aspirate or imprint preparation;
whereas, histologic features of marrow trephine biopsy brings out best
the topographical relationship of various components of a complex
cellular infiltrate; and cytogenetic or molecular genetic analysis is the
preferred mode for revealing the subvisual and subcellular anomaly
of a disease process.
The present article examines bone marrow trephine biopsy (BMTB)
as a candidate for providing diagnostically useful immunophenotypic
information. BMTB is a rich of source material for establishment of
diagnosis, staging, monitoring of therapeutic response and assessment
of residual disease status in diverse haematologic disorders. Sometimes,
in case of “dry aspirate” or “dry tap”, it is the only material available
for examination. Immunohistochemical (IHC) studies on BMTB can be
performed using cryostat (frozen undecalcified), plastic-embedded or
paraffin-embedded tissue sections. The cryostat sections have the all-

important advantage over either plastic or paraffin-embedded sections
in that there is complete preservation of all surface and cytoplasmic
antigens. However, the technique, although successfully employed by
some investigators,1,2 never found wide acceptance, mainly because of
suboptimal morphology. It is difficult to section frozen tissue with dense
bony element and considerable architectural distortion results from
compression or tearing of the specimen. Thus, sections of one-cell
thickness are almost impossible to obtain, leading to considerable
interpretative difficulty of IHC results. Plastic-embedded sections allow
for the best possible morphologic details, but it also causes difficulties
for IHC studies because of the slow and irregular penetration of reagents
in the plastic matrix. Water-soluble plastics such as glycol-methacrylate,
on the other hand, bind to amino-terminal portions of proteins during
polymerization and thus mask the antigenic epitopes. Nevertheless,
application of IHC on plastic-embedded sections is possible adopting
a suitably modified method.3 High cost and technical difficulties are
major limiting factors restricting the routine use of plastic-embedded
sections. Paraffin embedding is thus the most widely used routine
method worldwide for processing of BMTB. The rest of the article is in
concern with the application of IHC on routinely processed, formalin
fixed, decalcified, paraffin-embedded marrow trephine biopsy sections.
Successful application of a wide range of immunohistochemical
investigations on routinely processed marrow trephines became
possible because of two crucial factors:
a. Development and commercial availability of a number of lineage-
associated and differentiation stage-specific antibodies capable of
recognizing formalin-resistant epitopes (‘paraffin-reactive
antibodies’) and
b. Introduction of efficient antigen-retrieval procedures4,5 capable
of unmasking antigenic sites. However, method of decalcification
is a crucial determinant for successful application of IHC on BMBT.
While hydrochloric acid based decalcification procedures cause
irreversible loss of most of the useful antigens, the antigenic
epitopes are however well-preserved in EDTA/electrolytically
decalcified marrow biopsy.
MERITS OF DIAGNOSTIC IMMUNOHISTOCHEMISTRY ON

PARAFFIN-EMBEDDED BONE MARROW TREPHINE BIOPSY
The technique of immunohistochemical phenotyping on routinely
processed BMTB is particularly attractive because it is inherently
endowed with several distinct advantages over other methods, namely:
i. Well preserved morphologic details compared to cryostat sections.
ii. Direct visualization of positive staining of exact cell types and
their topographic relationship to histologic microarchitecture.
Immunohistochemistry on Marrow Trephine Biopsy 251
iii. Easy transport of stable diagnostic material (paraffin-embedded

tissue block) to distant referral centre.
iv. Permits study of archieval material.
v. Generates relatively permanent stained preparation allowing
storage for subsequent review and comparison.
vi. Much more cost-effective compared to flow cytometry. There is
very little capital expenditure for any new equipment.
vii. Easily adaptable by routine laboratory conversant with
immunohistochemical investigations on formalin fixed soft tissue
specimens.
SCOPE OF IMMUNOHISTOCHEMICAL INVESTIGATION ON

PARAFFIN-EMBEDDED BONE MARROW TREPHINE BIOPSY
FOR IMPROVEMENT OF QUALITY OF DIAGNOSIS
Results of work done by a number of investigators have now clearly
established that a wide spectrum of useful antibodies can be used on
routine paraffin sections with high degree of reliance for improving
the quality of diagnosis on BMTB. A list (not exhaustive) of these
antibodies is presented in Table 15.1.
Table 15.1: Immunohistochemical markers suitable for paraffin sections of

marrow trephine biopsy.
Markers Reactivity
bcl-2 (100/D5) Neoplastic germinal centres in follicular
lymphomas express high levels of the onco-
protein whereas the normal or hyperplastic
germinal centres are negative
CD1a (NA1/34) Langerhans cells, immature T-cells
CD3 (PS1) Pan T
CD5 (4c7) Pan T, B-cell subset
CD8 (C8/144B) Cytotoxic/suppressor T
CD10 (56C6) Precursor B, follicle centre cells
CD 15 (LeuM1) Reed-Sternberg cells, granulocytes
CD20 (L26) Pan B
CD21 (1F8) Follicular dendritic cells
CD23 (1B12) B-cell subsets, follicular dendritic cells
CD30 (Ber-H2) Reed-Sternberg cells, anaplastic large cell
lymphoma cells, activated lymphoid cells
CD34 (QBEnd10) Immature lymphoid or myeloid cells or stem
cells, endothelial cells
CD43 (DF-T1) Pan T, myeloid cells
CD45 (PD7/26+2B11) Leucocyte common antigen
CD45RA (4KB5) Pan B
CD45RO (UCHL1) Pan T, a few granulocytic, monocytic and B-
cells
contd...
contd...
Markers Reactivity
CD56 (123C3.D5) NK cells, cytotoxic T-cell subsets
CD57 (HNK1) T-cells, NK cell subsets
CD61 (Y2/51) Megakaryocytes
CD68 (KP1) Myelocytes, monocytes/histiocytes
CD68 (PGM1) Monocytes/histiocytes
CD79a (JCB117) Pan B
CD99 (HO36-1.1) Ewing’s sarcoma, lymphoblasts
CD138 (MI15) Plasma cells
Chromogranin A (DAK-A3) Neuroendocrine cells
Cyclin-D1 (DCS-6) In the context of lymphoid tumours, expres-
sion of this protein is highly selective for
mantle cell lymphoma
Cytokeratin (MNF116) Epithelial cells
DBA.44 (DBA.44) Hairy cells, B-cell subset
E-cadherin (HECD-1) Strong membrane expression in normal
erythroid precursors (cells of erythroleukae-
mia lack membrane expression)
Epithelial Membrane Antigen Epithelial cells, plasma cells, anaplastic large
(E29) cell lymphoma cells
Factor VIII-related antigen (F8/86) Megakaryocytes, endothelial cells
Fascin (55K-2) Reed-Sternberg cells and variants except L
and H type, dendritic cells, endothelial cells
Glycophorin A (JC159) Erythroblasts, erythrocytes
Immunoglobulins G, M, A, Kappa, Lambda
Ki-67 (MIB-1) Cells in active phases of the cell cycle (G1, S,
G2, M)
Lysozyme (Polyclonal) Granulocytes, monocytes/histiocytes
Myeloperoxidase (Polyclonal) Granulocytes, monocytes
Synaptophysin (Polyclonal) Neuroendocrine cells
TdT (Polyclonal) Immature lymphoid cells, lymphoblastic
lymphoma/leukaemia, rarely undifferen-
tiated acute myeloid leukaemia
Tryptase (AA1) Mast cells
A large volume of work exists in the literature on usefulness of

various panels of antibodies for improving the diagnostic accuracy of
various lymphohematopoietic disorders in the context of paraffin-
embedded BMTB. A detailed presentation of all the results is outside
the scope of the present chapter. Briefly, the significant impact of IHC
as reported in the literature may be summarized as follows:
i. Diagnosis and phenotyping of acute leukaemia with feasibility of
further subtyping of both ALL and AML.6-10
ii. Diagnosis and subtyping of chronic lymphoproliferative disorders

particularly low-grade peripheral B-cell neoplasms.11-15
iii. Differential diagnosis of reactive lymphoid infiltrate versus non-
Hodgkin’s lymphoma.16-19
iv. Sensitive detection of marrow involvement in non-Hodgkin’s
lymphomas for accurate staging.20-24
v. Diagnosis of plasma cell dyscrasia including recognition of
residual disease.25-27
vi. Diagnosis and prognostically useful histologic stratification of
primary myelodysplastic syndromes.28-31
vii. Recognition of chronic/accelerated/blastic phases of chronic
myeloid leukaemia.32
viii. Differential diagnosis of metastatic deposit versus lympho-
haematopoietic neoplasm.33
Thus, the diagnostic utility of the technique of IHC on routinely
processed BMTB is now fairly well established. However, the studies
conducted so far have focussed attention within the narrow confines
of either solving a defined diagnostic dilemma or to investigate the
fidelity of immunostaining behaviour of one or more antibodies in the
context of marrow trephine biopsies. We have recently conducted a
retrospective study34 with a significant difference in the design. Instead
of working on any particular defined area of diagnostic difficulty, our
aim was to test the usefulness of the technique for improving the quality
of H and E-based diagnosis in the context of real-life situation of a
tertiary health care set-up wherein marrow trephines are generated as
part of diverse clinical situations in various disciplines of medicine,
consequent upon which there is wide scope of diagnostic possibilities
to be entertained before reaching any conclusion. Out of a total of 513
consecutively available BMTBs during a period of three months, review
of histologic features of H and E stained preparations revealed the
necessity of IHC marker study in 183 (35.7%) cases. IHC was conducted
in 171 of these cases with adequate tissue in paraffin block, following
the guiding principle of discretionary choice of various sets of antibody
combinations from a repertoire panel of 15 antibodies, on the basis of
‘minimum yet efficient’ strategy, for various classes of H and E-based
histologic diagnosis in need of qualitative improvement. Our results
revealed that IHC has crucial contribution (“significant impact”) for
improvement of quality of diagnosis in 40.9 per cent cases and useful
contribution (“worthwhile impact”) for providing objective reconfirma-
tion of diagnosis, basic immuno-phenotypic information and certain
prognostically relevant information in 53.8 per cent cases. In 5.3 per
cent cases, IHC failed (“null impact”) to improve diagnostic status.
Number of antibodies used in the study for various classes of H and E-
based diagnosis ranged from a minimum of two (for example,
myeloperoxidase and glycophorin-A for objective confirmation of

erythroid versus myeloid precursors) to a maximum of eight (CD34,
CD3, CD79a, myeloperoxidase, CD68-KP1, CD68-PGM1, glycophorin-
A and CD61) in cases of acute leukaemia. Thus, a total of 669 IHC studies
were required for 171 biopsies: an average of 3.9 immunostaining for
each biopsy deserving immunohistochemical investigation. Finally, the
stand-alone diagnostic efficiency of IHC on BMTB, in absence of
information on marrow aspirate/imprint preparation was found to be
93.0 per cent. This is the maximum diagnostic potential the technique
could offer, as observed in our study. There is no comparable data on
this topic in existing literature. A few examples of cases with significant
diagnostic impact of IHC are presented in.
GUIDELINES ON USE AND INTERPRETATION OF IMMUNO-

HISTOCHEMISTRY ON PARAFFIN-EMBEDDED BONE MARROW
TREPHINE BIOPSY
Following points need strong emphasis:
1. A diligent study of the conventional morphologic features of a
well-stained, thin, H and E preparation of EDTA-decalcified
marrow trephine biopsy remains the cornerstone for accurate
diagnosis. IHC is only auxiliary means either to subtly refine or
help sub-categorization of the basic morphologic diagnosis. Under
no circumstances, immunohistochemical or any other specialized
investigation including molecular biology is capable of
compensating the deficiency on morphologic observations. The
outcome in such an event is always disastrous. It is unfortunate
that this point cannot be overemphasized and there is a tendency
to take refuge in special studies to cover morphologic interpre-
tative deficiency. It is also critically important to remember that
the best diagnostic results are obtained only when the morphologic
and special studies are interpreted in the context of the clinical
features of the patient.
2. The only means for determination of B-cell clonality in tissue
sections is by assessment of relative proportions of κ and λ light
chain reactive cells. It is important to be aware of the fact that
although the antibodies to κ and λ light chains react well in fixed,
decalcified sections, the reactivity is usually restricted to cells that
contain intracytoplasmic immunoglobulin such as plasma cells.
The technique of paraffin section immunohistochemistry is
generally not sufficiently sensitive to determine the clonality in
lymphoproliferative disorders characterized by the presence of
only surface immunoglobulin; and except for plasma cell
dyscrasias it is uncommon for the cells of a B-lineage malignancy
to exhibit sufficient cytoplasmic immunoglobulin to be detected.
Thus, the histologic pattern of the process and the morphology of

the individual cells in the bone marrow sections must be used in
conjunction with the antibody studies to distinguish a benign from
a malignant process. The monoclonality of plasma cell population,
however, could be readily determined by calculating light-chain
ratio wich is number of cells reactive for the predominant light-
chain divided by the number of cells reactive for the minority
light-chain. A light-chain ratio of 4 or more is considered to confer
monoclonality.25
3. Unlike flow cytometric immunodiagnosis of haematological
disorders, there are no standard well-accepted antibody-protocols
for diagnostic immunohistochemistry on BMTB. Although steady
progress is being made, the discipline is yet to reach that stage. A
judicious and careful selection of antibody combinations based
on the comprehensive data in the existing literature is possible,
but best results are obtained only on the basis of first-hand
experience with various antibodies in one’s own set-up. For
example, PGM1 epitope of CD68 was reported in the literature to
be reactive in a significant proportion of M4 and M5 FAB subtypes
of acute myeloid leukaemia. However, results in our laboratory
reveals that although PGM1 has very high specificity, the sensi-
tivity of the antibody is too low to be useful for immuno-identifica-
tion of monocytic differentiation in cases of acute leukaemia.
4. It is prudent to defer inclusion in the diagnostic panel of an
antibody whose novel and apparently promising reactivity pattern
has been only recently reported, till such time the findings are
either authenticated by other independent investigators or a first-
hand experience could be obtained in one’s own set-up. For
example, we encounter cases of severe megaloblastic anaemia,
which closely mimic acute leukaemia in H and E morphology.
Glycophorin-A positive immunostaining of the cells help us to
correctly identify these cases in the light of marrow aspirate/
imprint findings and presenting clinical features. Immunohisto-
chemistry alone is however incapable of confidently disting-
uishing these cases from M6 AML. A recent report35 documents
that cells of erythroleukaemia lack membrane expression of E-
cadherin, in contrast to strong membrane expression of the antigen
by normal erythroid precursors. If megaloblasts also show a
reactivity pattern akin to the normal precursors, then it might be
useful in the context of diagnostic difficulty between megaloblastic
anaemia and M6 AML. However, there is currently no information
available on this aspect. Therefore, we have recently procured E-
cadherin in order to validate the findings reported in the literature
and also to extend the study to cases of megaloblastic anaemia in
our laboratory, before recruiting antibody against E-cadherin to

cautions diagnostic use.
5. Obtaining a technically sound good immunostained preparation
is not the end of the story. Accurate diagnostic interpretation of an
immunohistochemical preparation is an expertise by itself and
needs to be learnt only by practical experience. Immunostain inter-
pretation belies the common simplistic expectation of an “all or
none” formula for either a positive or negative staining pattern in
favour of or against a particular diagnosis. Briefly, the prerequi-
site knowledge and guiding principles for acquiring an accurate
interpretative skill for IHC preparations may be outlined as follows:
i. A comprehensive knowledge of the spectrum of normal
histologic features in BMTB including the normal immuno-
histochemical staining profile of various cell components of
the marrow is indispensable. The topic has been excellently
reviewed by Brown and Gatter.36 Few additional articles deal
with the immunohistochemical reference ranges for B and T
lymphocytes in bone marrow biopsy paraffin sections.16,37
ii. Distinction between a significant (diagnostically relevant) and
non-significant (diagnostically irrelevant) positive staining
pattern for each antibody is very important. For example, only
nuclear positivity of cyclin-D1 immunostaining is relevant for
diagnosis of mantle cell lymphoma and cytoplasmic staining,
however intense, needs to be considered negative. Similarly,
positive nuclear immunostaining of S-100 protein in a
suspected case of Langerhans cell histiocytosis will establish
the diagnosis but absence of nuclear positivity, despite
cytoplasmic positivity, would have to be considered as negative
staining pattern.
iii. Positive immunostaining for a lineage-associated marker
complemented with negative staining for markers of other
lineages, is reliable. Results of staining for any single lineage-
marker should not be interpreted in isolation.
iv. Assessment of in-built positive or negative controls is necessary
in order to establish the authenticity of the staining characteris-
tics of the cell population of interest. For example, a cyclin-D1
immunostained preparation in a suspected case of mantle cell
lymphoma will be considered truly negative (thereby rejecting
the suspected diagnosis) if the in-built positive control of a
few stromal and endothelial cells do show a positive nuclear
staining. Otherwise, a technical deficiency should be suspected
and the staining results are to be considered diagnostically non-
contributory.
v. A thorough knowledge and awareness of various staining
artifacts are necessary for accurate interpretation of
immunohistochemical preparations. Examples are edge-

artifact, “dot” artifact of cytoplasmic staining, nonspecific
staining due to retraction and crushing artifact etc.
vi. Morphologic features are the solid anchor for diagnosis.
Therefore, immunostaining pattern that is contradictory and
unforeseen in the morphological context needs thorough
scrutiny. Possibility of ‘antibody mix-up’ at technical level must
be ruled out by repeating the immunostains.
Methodological Considerations
Published literature on the topic does provide the guidelines. However,
it is important to appreciate that it is obligatory for each laboratory to
optimize the technique and to determine the reactivity patterns of
various antibodies on the specimens processed in that laboratory. It is
crucial to understand that the differences in fixatives and decalcification
procedures may lead to different results from those published in the
literature or specified by the manufacturer. Trephine biopsies fixed in
adequate amount of neutral buffered formalin for overnight, followed
by gentle decalcification in 10 per cent EDTA solution in distilled water
for 36 to 48 hours, would retain most of the diagnostically useful
antigens for immunohistochemical detection. Decalcification using
mineral acid not only renders the biopsy material unsuitable for
application of any immunohistochemical procedure but also results in
unacceptably poor preservation of morphologic features in H and E
stained preparation. For most of the antigens, application of suitable
antigen-retrieval pretreatment is a requirement for successful
immunostaining of EDTA-decalcified marrow trephines. Also, use of a
sensitive avidin-biotin based visualization system in which a
biotinylated secondary antibody reacts with several peroxidase-
conjugated streptavidin molecules are crucial for immunodetection of
antigens, some of which are often present in low concentrations.
Diaminobenzidine remains the choice of chromogenic substrates. The
optimal dilution, pretreatment requirements, clone and source of 27
antibodies successfully used in our laboratory for IHC on paraffin-
embedded BMTB are given in Table 15.2.
KEY POINTS FOR CLINICAL PRACTICE

1. Bone marrow trephine biopsy is a rich source material for
establishment of diagnosis in diverse haematologic disorders.
Sometimes it is the only available material for examination as in
cases of ‘dry tap’.
2. Availability of a wide spectrum of paraffin-reactive antibodies and
efficient techniques of antigen-retrieval have now made successful
Table 15.2: Optimal dilution, pretreatment requirement, source and clone of 27

antibodies successfully used in our laboratory for immunohistochemistry on
marrow trephine biopsy.
Sr. Antibody Dilution Pretreatment Clone Source
No.
1. CD3 1:200 MW Citrate* Rabbit Polyclonal Dako
2. CD5 1:100 MW EDTA** 4C7 Dako
3. CD10 1:50 MW EDTA 56C6 Neomarkers
4. CD15 1:150 MW Citrate MMA Neomarkers
5. CD20 1:150 MW Citrate L26 Dako
6. CD23 1:100 MW EDTA 1B12 Neomarkers
7. CD30 1:150 MW Citrate 30C02 Neomarkers
8. CD34 1:300 MW Citrate QBend10 Dako
9. CD45 1:200 MW Citrate 2B11 + PD7/26 Dako
10. CD45RO 1:150 MW Citrate UCHL1 Dako
11. CD61 1:45 Pronase Y2/51 Dako
12. CD68(KP1) 1:700 None KP1 Dako
13. CD68 (PG-M1) 1:200 MW Citrate PG-M1 Dako
14. CD79a 1:150 MW Citrate JCB117 Dako
15. CD138 1:200 MW Citrate MI15 Dako
16. DBA.44 1:50 MW Citrate DBA.44 Dako
17. Kappa 1:200,000 MW Citrate Rabbit Polyclonal Dako
18. Lambda 1:200,000 MW Citrate Rabbit Polyclonal Dako
19. Cyclin D1 1:500 MW EDTA DCS-6 Dako
20. MPO 1:5,000 None Rabbit Polyclonal Dako
21. Glycophorin A 1:400 MW Citrate JC159 Dako
22. Fascin 1:200 MW Citrate 55K-2 Dako
23. Pan-CK 1:100 MW Citrate MNF116 Dako
24. EMA 1:100 MW Citrate E29 Dako
25. Chromogranin 1:50 MW Citrate DAK-A3 Dako
26. Synaptophysin 1:50 MW Citrate Rabbit Polyclonal Dako
27. Ki-67 1:200 MW Citrate MIB-1 Dako
* MW Citrate: Microwaving in 10 mmol/L citrate buffer pH 6.0.

** MW EDTA: Microwaving in 1 mmol/L EDTA buffer pH 8.0.
application of IHC on EDTA-decalcified paraffin sections of

marrow trephines feasible, leading to substantial value-addition
for diagnostic potential of BMTB. It has been estimated that
judicious use of IHC on deserving cases is capable of providing
accurate diagnosis in up to 93 per cent cases, in absence of informa-
tion on marrow aspirate/imprint preparations.
3. A significant impact of IHC for improvement of quality of diag-
nosis on BMTB is observed in: diagnosis and phenotyping of acute
leukaemia, diagnosis and subtyping of low-grade peripheral B-

cell neoplasms, more accurate staging of lymphomas by sensitive
detection of marrow involvement, differential diagnosis of reactive
lymphoid infiltrate from non-Hodgkin’s lymphoma, diagnosis of
plasma cell dyscrasias including recognition of residual disease,
prognostically useful histologic stratification of primary myelo-
dysplastic syndromes, diagnosis and characterization of metastatic
tumours.
4. The outstanding merits of the technique of immunohistochemical
phenotyping on paraffin sections of BMTB are:
i. ability to directly visualize the positive staining of exact cell
types and their topographic relationship to histologic micro-
architecture;
ii. feasibility of easy transport of stable diagnostic material
(paraffin-embedded tissue block) to distant referral centre.
Besides, the technique also permits study of archival material,
is cost-effective and easily adaptable by routine laboratory
conversant with immunohistochemical investigations on
routine soft tissue specimens.
REFERENCES
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immunohistological analysis of undecalcified human bone marrow trephine
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2. Falini B, Martelli MF, Tarallo F, Moir DJ, Cordell JL, Gatter KC et al:
Immunohistological analysis of human bone marrow trephine biopsies using
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chemistry in the diagnosis of haematopoietic and lymphoid neoplasms. Clin
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6. Arber DA, Jenkins KA: Paraffin section immunophenotyping of acute
leukaemias in bone marrow specimens. Am J Clin Pathol 1996;106:462-68.
7. Chuang SS, Li CY: Useful panel of antibodies for the classification of acute
leukaemia by immunohistochemical methods in bone marrow trephine biopsy
specimens. Am J Clin Pathol 1997;107:410-18.
8. Toth B, Wehrmann M, Kaiserling E, Horny HP: Immuno-phenotyping of acute
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Immunohistochemistry can be used to subtype acute myeloid leukaemia in
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cytometry. Am J Clin Pathol 2000;113:814-22.
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13. Chen CC, Raikow RB, Sonmez-Alpan E, Swerdlow SH: Classification of small
B-cell lymphoid neoplasms using a paraffin section immunohistochemical
panel. Appl Immunohistochem Mol Morphol 2000;8:1-11.
14. Pezzella F, Munson PJ, Miller KD, Goldstone AH, Gatter KC: The diagnosis of
low-grade peripheral B-cell neoplasms in bone marrow trephines. Br J
Haematol. 2000;108:369-76.
15. Kremer M, Dirnhofer S, Nickl A, Hoefler H, Quintanilla-Martinez L, Fend F:
p27(Kip1) immunostaining for the differential diagnosis of small b-cell
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16. Horny HP, Wehrmann M, Griesser H, Tiemann M, Bultmann B, Kaiserling E:
Investigation of bone marrow lymphocyte subsets in normal, reactive, and
neoplastic states using paraffin-embedded biopsy specimens. Am J Clin Pathol
1993;99:142-49.
17. Krober SM, Horny HP, Greschniok A, Kaiserling E: Reactive and neoplastic
lymphocytes in human bone marrow: morphological, immunohistological,
and molecular biological investigations on biopsy specimens. J Clin Pathol
1999;52:521-26.
18. Thiele J, Zirbes TK, Kvasnicka HM, Fischer R: Focal lymphoid aggregates
(nodules) in bone marrow biopsies: differentiation between benign hyperplasia
and malignant lymphoma—a practical guideline. J Clin Pathol 1999;52:294-
300.
19. West RB, Warnke RA, Natkunam Y: The usefulness of immunohistochemistry
in the diagnosis of follicular lymphoma in bone marrow biopsy specimens.
Am J Clin Pathol 2002;117:636-43.
20. Chetty R, Echezarreta G, Comley M, Gatter K: Immunohistochemistry in
apparently normal bone marrow trephine specimens from patients with nodal
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involvement in anaplastic large cell lymphoma. Immunohistochemical
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1995;103:82-89.
22. Labouyrie E, Marit G, Vial JP, Lacombe F, Fialon P, Bernard P et al:
Intrasinusoidal bone marrow involvement by splenic lymphoma with villous
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20.
23. Skinnider BF, Connors JM, Gascoyne RD: Bone marrow involvement in T-
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24. Pittaluga S, Tierens A, Dodoo YL, Delabie J, De Wolf-Peeters C: How reliable

is histologic examination of bone marrow trephine biopsy specimens for the
staging of non-Hodgkin lymphoma? A study of hairy cell leukaemia and
mantle cell lymphoma involvement of the bone marrow trephine specimen
by histologic, immunohistochemical, and polymerase chain reaction
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25. Peterson LC, Brown BA, Crosson JT, Mladenovic J: Application of the immuno-
peroxidase technic to bone marrow trephine biopsies in the classification of
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1990;94:742-46.
27. Thiry A, Delvenne P, Fontaine MA, Boniver J: Comparison of bone marrow
sections, smears and immunohistological staining for immunoglobulin light
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Histopathology 1993;22:423-28.
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prognostic significance of immunohistochemical assessment of bone marrow
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29. Horny HP, Wehrmann M, Schlicker HU, Eichstaedt A, Clemens MR, Kaiserling
E: QBEND10 for the diagnosis of myelodysplastic syndromes in routinely
processed bone marrow biopsy specimens. J Clin Pathol 1995;48:291-94.
30. Oriani A, Annaloro C, Soligo D, Pozzoli E, Cortelezzi A, Lambertenghi Deliliers
G: Bone marrow histology and CD34 immunostaining in the prognostic
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64.
31. Baur AS, Meuge-Moraw C, Schmidt PM, Parlier V, Jotterand M, Delacretaz F:
CD34/QBEND10 immunostaining in bone marrow biopsies: an additional
parameter for the diagnosis and classification of myelodysplastic syndromes.
Eur J Haematol 2000;64:71-79.
32. Orazi A, Neiman RS, Cualing H, Heerema NA, John K: CD34 immunostaining
of bone marrow biopsy specimens is a reliable way to classify the phases of
chronic myeloid leukaemia. Am J Clin Pathol 1994;101:426-28.
33. Chu PG, Chang KL, Arber DA, Weiss LM: Practical applications of
immunohistochemistry in haematolymphoid neoplasms. Ann Diagn Pathol
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biopsies’ submitted in November 2002, to the Faculty of All India Institute of
Medical Sciences, New Delhi in partial fulfillment of the requirements for the
degree of Doctor of Medicine (Pathology).
35. Acs G, LiVolsi VA: Loss of membrane expression of E-cadherin in leukaemic
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36. Brown DC, Gatter KC: The bone marrow trephine biopsy: a review of normal
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chemical reference ranges for B lymphocytes in bone marrow biopsy paraffin
sections. Am J Clin Pathol 1995;104:517-23.
16
Carrier Detection in
Haemophilia A
R Saxena, RPH Ahmed
Key Words
Haemophilia • Carrier Detection • Intron 22 inversion
• Linkage analysis
Introduction
Haemophilia A is the most common hereditary form of severe bleeding
disorder in humans, affecting one in 5000 male newborns. The
phenotype is due to the deficiency or absence of coagulation factor
VIII:C caused by deleterious mutations in the factor VIII (FVIII) gene.
Affected individuals develop a variable degree of hemorrhage
predominantly into joints and muscles with the severity and frequency
of bleeding symptoms correlating well with the factor VIII activity in
blood plasma. About 50 per cent of patients exhibit a severe phenotype
while 10 per cent show a moderate and 40 per cent a mild expression
of the disease.1 The replacement of human factor VIII derived from
plasma or from recombinant sources is the treatment of choice in this
disorder. The major complication of replaced therapy is the develop-
ment of antibodies against exogenous factor VIII, rendering therapy
ineffective.
The FVIII gene maps to the distal end of the long arm of the X-
chromosome at Xq28 and spans 186 kb of genomic DNA (Fig. 16.1). It
consists of 26 exons2,3 encoding a mature protein of 2332 amino acids.
One large exon, exon 14, covers about 40 per cent of the coding
sequence and encodes the B domain, which is not essential for FVIII
activity.4 Intron 22 contains two more genes, designated F8A and F8B,
with so far unknown functions.5,6 The management of bleeding
manifestations, including life-threatening bleeds, consists of
replacement by the factor VIII, which may be in the form of plasma
derived FVIII, recombinant FVIII, fresh frozen plasma or cryopreci-
pitate. Since these are expensive and poorly available in India they
Carrier Detection in Haemophilia A 263
Fig. 16.1: Schematic representation of the chromosomal localisation and structure

of the Factor VIII gene, located about 1000kb from the Xq telomere (Xqter)
have a limited role in management Prevalence of hemophilia in India

is 51,000 (6/10,000). Prenatal termination of a hemophilic child
diagnosed in utero, offers a good strategy for control of hemophilia. A
prerequisite for prenatal diagnosis of hemophilia is identification of
carrier women, who would be the potential candidates for prenatal
diagnosis. Mutation analysis in the factor VIII gene is valuable with
regard to both genetic counselling and medical care of affected families.
An important issue in genetic counselling is the determination of the
risk of recurrence, in particular given the high frequency of de novo
mutations.7 This is complicated by the high mortality from blood-borne
viruses in previous years which has often led to a situation where an
index patient is not available for genetic diagnosis.8,9 In these instances,
it is necessary to directly genotype potential carriers without prior
knowledge of the mutation, a situation in which the test sensitivity is
of critical importance. For patient care, knowledge of the genotype is
helpful as it facilitates the prediction of risk of anti-FVIII antibody
development.10 In the future, this knowledge will become a precondi-
tion for entering gene therapy programs.11
Methods for Carrier Detection

Pedigree Analysis
A family history is extremely important in assisting diagnosis of carrier
state. The disease has an X-linked recessive inheritance. Thus, the
female relatives may be the carriers of disease while the males may
suffer. There is 50 per cent chance of the sister of a hemophilic to be a
carrier if the mother is a carrier. Based on inheritance patterns, several

types of carriers can be identified. Obligate carrier is a woman who is a
mother of 2 hemophilic children or is a daughter of a hemophilic father
is a sister of a hemophilic male and has a hemophilic child or has
hemophilia in her son and in her sister’s son. Probable carriers are the
sisters of a hemophilic male. Possible carrier is a mother who has one
hemophilic child with no family history of hemophilia. By Pedigree
analysis, one can suspect carrier ship in mother in 40 per cent cases.
FVIII:C/VWF Ag Ratio
Due to one defective X chromosome in a carrier woman, it is logical
that the FVIIIC levels would be approximately 50 per cent of normal.
However, since these levels are affected by several physiological
conditions like pregnancy, etc. and the FVIII Ag levels unaffected, it is
preferable to measure ratio of FVIIIC/ VWFAg which should ordinarily
be 1. In carrier of hemophilia, it may be reduced. Due to extreme
lionization, low levels of FVIIIC may not always be found. Its sensiti-
vity is approximately 50-55 per cent for carrier detection.
Linkage Analysis
Since FVIII gene is a large gene (186 Kb) and direct mutation analysis
is labor intensive and not practical, linkage analysis is widely used.
This is based on the principle that there is a tendency of genes to be
inherited together as a result of their location on the same chromosome
and in close proximity. Several polymorphisms, which are allelic
variations, (changes in DNA affecting the restriction patterns of
restriction enzymes), are present in and near the FVIII gene and can
be used in linkage analysis of hemophilia A . Dinucleotide repeats in
introns 13 and 2212,13 are generally the first choice of markers as they
have the highest rate of heterozygosity. A wide variety of methods
can be used for their analysis, the method selected will depend upon
laboratory circumstances. The simplest method involves PCR
amplification followed by electrophoresis on a native polyacrylamide
gel, and detection by ethidium bromide staining. Silver staining
obviates the need for darkroom facilities (Fig. 16.2).
Alternatively, one primer can be end labelled with 32P, and the
labelled PCR products detected following electrophoresis on a
denaturing polyacrylamide gel. This method is tedious, but effective
at allele discrimination. For fluorescent detection, one primer is end
fluorescent labelled, product is detected following electrophoresis on
a gel in an automated sequencer used for genotyping.14 This method
has the advantage of automation, but may be expensive.
Fig. 16.2: Schaematic diagram of technique for Bcl 1 restriction site
Dimorphisms—PCR Analysis
Commonly used dimorphisms in the FVIII gene which can be analysed
by PCR include those detected by digestion with Bcl I in intron 18,15
Hind III in intron 1916, and G/A in intron 7 , which can be detected
using an introduced Alw NI site.17 Of these, Bcl I is the most widely
used. Hind III is in strong linkage disequilibrium with Bcl I, so only
one of these two markers should be analysed (Fig. 16.3).
Fig. 16.3: Carrier detection in a Haemophilia a patient’s family (with positive

family history) using B cl 1linkage analysis
The fragment amplified for Hind III dimorphism analysis also

contains a constant Hind III restriction enzyme site. Dimorphisoms
may be intragenic like BCl 1, Hind III, Xba 1, MSp1 or extragenic like
Bgl 1, St 14. In addition, several sequence alteration have been detected
in intron 13 and 22 i.e. (CA)n tandem repeats in intron 13 and (GT)n
(AG)n repeats in intron 22 which can be used for linkage analysis. All
analysis of the dinucleotide repeats suffers to an extent from stutter
bands which occur due to polymerase slippage during PCR amplifica-
tion. These additional bands can complicate allele size identification.
Since there are several ethnic variations, the choice of marker to be
used depends on the allelic and heterozygosity frequency (Figs 16.4
and 16.5) of a particular marker in the population and its proximity to
the gene of interest, to prevent recombination.
Techniques like restriction fragment length polymorphisms may
be used for restrictions enzyme polymorphisms and specific primers
to do PCR for intron 13/22 can be used for detection of tandem repeats
(Fig. 16.6).
Based on inheritance patterns, it is well known that two STR loci
are located in the FVIII gene, that is, (CA)n in intron 13 and (GT)n(AG)n
in intron 22. The allele number of the former is at least 10, and its
heterozygosity rate is about 91 per cent.12 It was reported that the allele
number of STR within intron 22 is at least 6 and the heterozygosity
rate is 33 per cent.13 The rate in the Chinese population is slightly
Intragenic
Intron 18 Bcl 1 poly.
Intron 19 Hind III Poly.
Intron 22 Xba 1, MSP 1 Poly.
Intron 22 (GT)n (AG)n repeats
Intron 13 (CA)n repeats
Extragenic Bgl 1 Poly.

St 14 Poly.
Fig. 16.4: FVII markers commonly used in linkage analysis
Caucasion Japanese Chinese Indian Saxena et al

(2002)
Bcl 1 0.27 (–) 0.30 (–) 0.21 (–) 0.57 (–) 0.42 (–)
0.73 (+) 0.70 (+) 0.79 (+) 0.43 (+) 0.58 (+)
Xba 1 0.44 (–) 0.41 (–) 0.42 (–) 0.17 (–) 0.46 (–)
0.56 (+) 0.54 (+) 0.58 (+) 0.83 (+) 0.54 (+)
Fig. 16.5: Informativity of Bcl 1 and Xba 1

Fig. 16.6: Schematic diagram of technique for (CA) repeats
lower.18 These discrepancies among published reports may be due to

the different genetic background and the existence of sampling bias.
Usually, linkage analysis of genetic polymorphisms can only be applied
if the family is not a sporadic one, and an affected male and related
family members are available for analysis. Furthermore, the female
carrier must be heterozygous for the marker analyzed and the
polymorphism analysis may always possibly have crossover events.
As a result of all these limitations, it should be used in combination
with direct diagnostic methods. Using all of the 5 markers, the total
informative rate is about 94.7 per cent, implying effective genetic in
familial haemophilia A.counselling should be provided to the HA
families (Fig. 16.7).
Fig. 16.7: Heterozygosity frequency in female relatives (n=45) of haemophilia A.

Patients at department of Haematology AIIMS
However, the diagnostic rate for the intragenic markers is relatively

low, decreasing their value for diagnosis. In addition, there are families
which are uninformative for all these markers, so it is necessary to
find more informative intragenic loci. These are based on PCR which
is more rapid, convenient and cost-effective than the original methods
and avoids radioactive contamination. The St14 locus is a multi-allelic
VNTR locus tightly linked to the FVIII gene.19 In normal Chinese
females, the heterozygosity rate is approximately 60 per cent.20
However, this locus is an extragenic one with a recombination rate of
2 per cent, which should not be neglected.
The results of carrier detections at Dept. of Haematology, AIIMS,
using BCl, Xba and CA repeats in intron 13 are given in Figures 16.8
and 16.9.
One major limitation of linkage analysis for carrier detection arises
when the haemophilic child has died and no living issue is present. In
such cases, inversion 22 defect and direct mutation analysis are a good
alternative.
Gene Rearrangements (Intron 22 Inversion)

Gross gene rearrangements reported consist almost entirely of a unique
inversion elaborated quite recently, yet now known to be responsible
for more than 40 per cent of all cases of severe haemophilia A. The
genetic defect in 40 to 50 per cent of patients with severe haemophilia
A are represented by intrachromosomal inversions involving intron
22 of the FVIII gene.21 With this molecular defect, we can easily trace
Positive family history (14)
Sisters (16) Mothers (14)
Probable carrier
Traced defective X
chromosome (11)
Carriers Non-carriers
(8) (8)
Potential candidate
for prenatal Δ
Fig. 16.8: Carrier detection in 30 haemophilia A families using B cl 1, Xba 1 and

intron 13 polymorphism
Negative history (16)
Female subjects (25)
Sister (9) Mother (16)
Non-carriers (3) Probable carrier (6) Possible carrier (16)

FVIII assay FVIII assay
UF (3) Carriers (3) Carriers (3) UF (3)
Inv 22 Identified X 10 severe

chromosome haemophilic M
UF: Uninformative Daug. carrier Inv 22

prenatal diagnosis
Fig. 16.9: Carrier detection in 30 haemophilia A families using B cl 1, Xba 1 and

intron 13 polymorphism
the female relatives in an HA family. This defect is identified in 45-50

per cent of severe haemophilics. During intensive attempts to define
the causative mutations in a population of severe patients by PCR
amplification of all 26 exons, mutations were found in only about 50
per cent of cases21: in the rest, all the exonic sequences appeared
normal. However, on RT-PCR of FVIII mRNA from these cases it was
found that no amplification was possible between exons 22 and 23.22,23
It is now known that in all these patients there is a large inversion and
translocation of exons 1-22 (together with introns) away from exons
23-26, the mechanism of which is homologous recombination between
the F8A and one of the extragenic F8A copies 400Kb 5' to the FVIII
gene.23,24 Fig. 16.10 shows how a simple crossover event during the
meiotic division of spermatogenesis can lead to fragmentation of the
gene with subsequent severe disease. Indeed, family studies show that
the origin of such inversions is almost exclusively a gamete supplied
by a normal male.25 A recent study1 determined that of the two
common types of inversion, the distal F8A copy is responsible for 35
per cent of severe haemophilia A cases, while crossover with the
proximal copy results in a further 7 per cent of cases. The inversion
Fig. 16.10: Schaematic representation of the mechanism of one type of the common
inversion of the factor VIII gene due to intrachromosomal crossing-over between
the homologous sequences a1 and a3. The partial inversion includes exons 1-22
of the gene
may be detected by southern blotting or PCR. Detecting the inversion

by Southern blot is an expensive, time-consuming, labor-intensive
method necessitating the use of radioisotope. Liu et al developed a
PCR assay that combines overlapping PCR with long-distance PCR to
make the genetic diagnosis of inversions.26 After optimization of reac-
tion conditions, the LD-PCR analysis can be performed successfully
within a day. Intron 22 inversion may be detected by PCR or southern
blot in the haemophilic child and in the carrier mother.
Schaematic of the PCR assay: At the top, the locations of four primers
(P.Q. A and B) are represented by arrows. The upper box represents
Int 22h1, and the dashed lines indicate the flanking sequences. The
lower box represents Int 22h2 and Int22h3, and the wavy lines indicate
the flanking sequence. Deleterious inversions can occur by
recombination between Int 22h1 and either Int 22h2 or Int22h3 (dotted
lines). The PCR amplifies overlapping and multiplex segments from
genomic DNA with four primers PQ.AB, PB and AQ. Two primer P
and Q are specific to the flanking sequences of Int22h1, located at 1212
bp before and at + 1334 bp after the homolog two primers, A and B are
specific to the flanking sequence of Int 22h2 and Int 22h3, located at -
167 bp before and at + 118 bp after the homologs. P and Q anneal at
different distance form A and B to differentiate PQ (12 kb), AB (10 kb)
and PB+AQ is generated in males with the inversion and both PQ
and PB+AQ are present in the carriers. AB is always produced and
serves as a positive control, because at least one copy of Int 22h2 and
Int 22/3 remains intact. S-PCR was performed with each primer at 0.2
PM. The relative copy numbers of segments PQ, AB and PB+AQ before
amplification are indicated at the bottom, although some individuals

have one or more extra copies of Int22h3 located on the X chromosome.
In cases where all the above are non informative, direct mutation
detection may be looked for by techniques like single stranded confor-
mation polymorphism (SSCP), chemical mismatch cleavage (CNC),
denaturing gradient gel electrophoresis (DGGE) or denaturing high
performance liquid chromatography (dHPLC).
DHPLC
Knowledge of the genotype is helpful as it facilitates the prediction of
risk of anti-FVIII antibody development.10 In the future, this knowledge
will become a precondition for entering gene therapy programs.11 In
recent years, the haeterogeneity and size of the FVIII gene have
hampered mutation testing in haemophilia A patients. In 1991, the
first systematic analysis of the complete coding sequence of the FVIII
gene was performed by applying denaturing gradient gel electro-
phoresis (DGGE) after PCR amplification of individual exons.20
Notably, causative mutations were found in about 90 per cent of non-
severe haemophilia A,20 whereas molecular defects could be identified
in only 60 per cent of the more severely affected patients.27 This
discrepancy was resolved in 1993 when Naylor, et al28 and Lakich et
al29 discovered a prevalent intron 22 inversion. Detection of carrier by
any of the above would enable not only genetic counselling but also
help in prenatal diagnosis of haemophilia in these subjects.
dHPLC offers a rapid and sensitive technology that is amenable to
automation and thus is well suited for routine applications specifically
for large genes such as FVIII. Sequencing the complete coding region
of the factor VIII gene in those patients where the current protocol is
not able to identify the mutation can be used to further optimize
dHPLC sensitivity. Reanalyzing newly identified mutations by dHPLC
will then lead to the establishment of additional parameters. Following
this strategy the protocol can be systematically improved until maximal
sensitivity is achieved. This possibility of continuous optimization of
the dHPLC protocol is, in our opinion, a major advantage of dHPLC
technology; however, it may become necessary to develop a multi-
tier strategy to maintain an efficient system when the number of
conditions, and consequently the number of injections, increases.
Under such a strategy, patients would be screened first.
It is, thus, concluded that carrier detection in Haemophilia can be
successful in >90 per cent of female relatives of Haemophilia. A using
linkage analysis and inversion intron 22 detection. In the rest, dHPLC
offers a good alternative. The approach to carrier detection is given in
Fig. 16.11.
Positive F/H Negative F/H
(Bcl 1, Xba 1, intron 13) (Bcl 1, Xba 1, intron 13)
UF UF I
Inv. 22 Inv 22
I
Pro/Pos. Carriers Non Carriers
UF
Non Obl. FVIII mother/Sister

Carriers Carriers
Carriers UF
CVS
UF: Uninformative CVS Cord blood FVIII

I: Informative
Fig. 16.11: Approach for carrier detection in female relatives of haemophilia A
REFERENCES
1. Antonarakis SE, Rossiter JP, Young M, Horst J, de Moerloosw P, Sommer SS.
Factor VIII gene inversions in severe haemophilia A: results of an international
consortium study. Blood 1995;86:2006-12.
2. Toole JJ, Knopf JL, Wozney JM, Sulzman LA, Buecker JL, Pittman DD.
Molecular cloning of a cDNA encoding human antihaemophiliac factor. Nature
1984;312:342.
3. Vehar GA, Keyt B, Eaton D, Rodriguez H, O’Brien DP, Rotblat F. Structure of
human factor VIII. Nature 1984;312:337-42.
4. Pittman DD, Alderman EM, Tomkinson KN, Wang JH, Giles AR, Kaufman
RJ. Biochemical, immunological, and in vivo functional characterization of
the B-domain deleted factor VIII. Blood 1993;81:2925-35.
5. Levinson B, Kenwrick S, Lakich D, Hammonds G, Gitschier J. A transcribed
gene in an intron of the human factor VIII gene. Genomics 1990;7:1-11.
6. Levinson B, Kenwrick S, Gamel P, Fisher K, Gitschier J. Evidence for a third
transcript from the human factor VIII gene. Genomics 1992;14:585-9.
7. Strauss HS. The perpetuation of haemophilia by mutation. Pediatrics
1967;39:186-93.
8. Erfle V, Hehlmann R, Mellert W, Kruger G, Seifried E, Heimpel H. Prevalence
of antibodies to HTLV-III in AIDS risk groups in West Germany. Cancer Res
1985;45(Suppl):4627-9.
9. Peake I. Genetic services available for counselling and prenatal diagnosis of
haemophilia. Haemophilia 1998;4(Suppl):24-5.
10. Schwaab R, Brackmann H-H, Meyer C, Seehafer J, Kirchgesser M, Haack A.

Haemophilia A: mutation type determines risk of inhibitor formation. Thromb
Haemost 1995;74:1402-6.
11. Kaufman RJ. Advances toward gene therapy for haemophilia at the millenium.
Hum Gene Ther 1999;10:2091-107.
12. Lalloz MRA. Haemophilia A diagnosis by analysis of a hypervariable
dinucleotide repeat within the human factor VIII gene. Lancet 1991;338:207-
11.
13. Lalloz MRA. Haemophilia A diagnosis by simultaneous analysis of two
variable dinucleotide tandem repeats within the factor VIII gene Br J Haematol
1994;86:804-09.
14. Noble JS. Automated genotyping in diagnosis In: Methods in molecular
medicine: Molecular diagnosis of genetic diseases. Elles R. ed Humana Press
Inc Totowa, NJ (1996).
15. Kogan SC. An improved method for prenatal diagnosis of genetic diseases by
analysis of amplified DNA sequences-Application to haemophilia A. New
England Journal of Medicine 1987;317:985-90.
16. Graham JB. The utility of a HindIII polymorphism of factor VIII examined by
rapid DNA analysis. British Journal of Haematology 1990;76:75-79.
17. Kogan S, Gitschier J. Mutations and a polymorphism in the FVIII gene
discovered by denaturing gradient gel electrophoresis. Proceedings of the
National Academy of Sciences USA 1990;87: 2092-2096. m repeats within the
factor VIII gene. Br J Haematol 1994;86:804-809.
18. Yip B, Chan V, Chan Tk: Intragenic dinucleotide repeats in factor VIII gene for
the diapgnosis of haemophilia A. Br J haematol 1994;88(4):889-91.
19. Richards B, Heilig R, Oberle I. Rapid PCR analysis of the St14 (DXS52) VNTR.
Nucl Acids Res 1991;19(8):1944.
20. WZ Chen, DB Zhou, YJ Wu. The polymorphic distribution of DXS52 VNTR
locus. Chin J Hematol 1998;19(4):209-10.
21. Higuchi M, Antonarakis SE, Kasch L, Oldenburg J, Economou-Petersen E,
Olek K. Molecular characterization of mild-to-moderate haemophilia A:
detection of the mutation in 25 of 29 patients by denaturing gradient gel
electrophoresis. Proc Natl Acad Sci USA 1991;88:8307-11.
22. Naylor JA, Green PM, Rizza CR, Giannelli F. Factor VIII gene explains all
cases of haemophilia A. Lancet 1992;340:1066-67.
23. Naylor JA, Brinke A, Hassock S, Green PM, Giannelli F. Characteristic mRNA
abnormality found in half the patients with severe haemophilia A is due to
large DNA inversions. Hum. Mol. Genet. 1993;2:1773-78.
24. Lakich D, Kazazian HH Jr, Antonarakis SE, Gitschier J. Inversions disrupting
the factor VIII gene are a common cause of severe haemophilia A. Nat. Genet.
1993;5:236-41.
25. Rossiter JP, Young M, Kimberland ML, Hutter P, Ketterling RP, Gitschier J et
al. Factor VIII gene inversions causing severe haemophilia A originate almost
exclusively in male germ cells. Hum. Mol. Genet. 1994;3:1035-39.
26. Liu Q, Nozari G, Sommer SS. Single-tube polymerase chain reaction for rapid
diagnosis of the inversion mutation in haemophilia A . Blood 1998;92(4):1458-
59.
27. Higuchi M, Kazazian HH, Kasch L, Warren TC, McGinnis MJ, Phillips JA.
Molecularcharacterization of severe haemophilia A suggests that about half
the mutations are not within the coding regions and splice junctions of the
factor VIII gene. Proc Natl Acad Sci USA 1991;88:7405-9.
28. Naylor JA, Green PM, Pizza CR, Gianelli F. Analysis of factor VIII mRNA
reveals defects in every one of 28 haemophilia A patients. Hum Mol Genet
1993;2:11-7.
29. Lakich D, Kazazian Jr X, Antonarakis SE, Gitschier J. Inversions disrupting
the factor VIII gene as a common cause of severe haemophilia A. Nature Genet
1993;5:236-41.
17
Genetic Determinants of
Atherothrombotic Disease
Barbara Voetsch, Joseph Loscalzo
Key Words
Arterial thrombosis • Pathogenesis • Platelet
receptors • Antioxidants • Glutathione peroxidase
INTRODUCTION
Many of the currently established risk factors for atherothrombosis are
environmental in nature and not alone sufficient to explain completely
the variations in incidence and risk of developing atherothrombotic
disease. Numerous studies indicate that there is a significant genetic
component underlying the occurrence of atherothrombosis, and a range
of specific genes contributing to disease risk have been defined. We
now recognize that the pathogenesis of atherothrombotic disease
involves multiple genetic and environmental factors related to
atherosclerosis and thrombosis, as well as their interaction. In this
review, we discuss the role of the most relevant genetic markers
associated with atherothrombotic disease, and describe their functional
implications as well as association with disease risk.
COAGULATION FACTORS
Fibrinogen
Among the components of the coagulation system, elevated fibrinogen
has been most consistently associated with atherothrombotic disorders.
Its independent association with myocardial infarction (MI), ischaemic
stroke (IS), and peripheral vascular disease (PVD) has been shown in
prospective studies of both healthy subjects and patients with pre-
existing vascular disease. The relative risk of vascular disease
determined in several studies is –2.0 for the highest versus lowest
quartiles of fibrinogen, independent of other risk factors.1,2 Several
mechanisms explain the association of increased fibrinogen with arterial
disease, including increased fibrin formation, plasma viscosity, platelet

aggregation, and vascular endothelial and smooth muscle cell
proliferation. Fibrinogen is an acute phase reactant and levels are subject
to much biologic variation; the acute-phase response arising from viral
infection and smoking in particular is strongly implicated in the
development of cardiovascular disease. Alternatively, it has been
postulated that elevated fibrinogen may reflect the inflammation
associated with atherosclerosis rather than being a causal risk factor.3
In addition, several studies indicate that plasma fibrinogen levels
strongly correlate with other cardiovascular risk factors, including age,
diabetes mellitus, and smoking.
Fibrinogen is composed of three pairs of polypeptide chains, α, β,
and γ, encoded by three separate genes clustered on the long arm of
chromosome 4. Multiple polymorphic sites have been identified in these
genes; yet, as the synthesis of the fibrinogen β-chain is rate-limiting in
vitro, most studies have focused on this gene. The main β-chain variants
include the Bcl1, Arg448Lys, -148C/T, and-455G/A polymorphisms,
which are in strong linkage disequilibrium with each other. The-455G/
an substitution lies adjacent to promoter response elements, in
particular an IL-6 response element, and has been most extensively
studied. The-455A allele is present in approximately 20 per cent of the
population and correlates with fibrinogen levels that are 10 per cent
higher than in individuals with the GG genotype. Nevertheless, the
relationship between the-455G/A variant and risk of atherothrombotic
disease is controversial, with some case-control studies indicating an
association and other large studies reporting no association.4 Similarly,
the association of the Bcl1 polymorphism and vascular disease remains
unclear.
In addition to the β-chain polymorphisms, a polymorphism in the
α-chain that codes for a Thr312Ala substitution has been described that
lies near a factor XIII crosslinking site and may influence clot stability.
Although this variant has been found to have a gene-dose-related
influence on post-stroke mortality rates in one study,5 it seems to be a
more relevant determinant of venous thrombosis.
Factor VII
Owing to its role in the initiation of coagulation, there has been
considerable interest in the influence of factor VII on atherothrombotic
disease. Several prospective studies have examined the association of
factor VII coagulant activity (VIIc) and cardiovascular disease, and
yielded discrepant results. Although initial studies described a strong
positive association between factor VIIc and coronary artery disease
(CAD), later reports failed to confirm this finding, possibly due to the
adjustment for other cardiovascular risk factors. 4 Similarly, to
Genetic Determinants of Atherothrombotic Disease 277
fibrinogen, plasma factor VII levels are affected by various environ-

mental factors, including body mass index, triglyceride levels, and age.
In addition, five polymorphisms in the factor VII gene have been
described, of which the most commonly studied are the strongly linked
Arg353Gln substitution and the hypervariable region 4 (HVR4)
polymorphism that involves 37bp repeats in intron 7. In vitro expression
studies suggest that the Arg353-encoding allele and the longest of the
three HVR4 alleles (termed H7) produce higher levels of factor VII. In
clinical studies, however, the effect of these factor VII genotypes on
factor VII plasma levels and factor VIIc was not as clear, and the relative
influence of each polymorphic site on factor VII levels remains to be
fully clarified.
Few studies have provided evidence of an association between these
polymorphisms and arterial disease. The most representative is an
Italian case-control study of young adults with familial myocardial
infarction; patients with the Gln353Gln or HVR4 H7H7 genotypes had
a significant decrease in risk of myocardial infarction (OR, 0.08 and
0.22, respectively), in addition to lower levels of both factor VII antigen
and factor VIIc than those with the Arg353Arg or HVR4 H6H6
genotypes. 6 In contrast, most other studies failed to detect any
association with the risk of MI or IS.2,4 Thus, the evidence to date
suggests that if there is any effect of factor VII genotypes on athero-
thrombotic disease, it is very small and only detectable in highly
selected populations.
Factor V/Prothrombin
Numerous studies have examined the role of the factor V G1691A (factor
V Leiden) and the prothrombin G20210A polymorphisms in
atherothrombotic disease, and most have yielded negative results, even
among patients who suffered a vascular event at a young age.1,7 The
studies that have shown an association of these polymorphisms with
CAD, MI, or IS have either been performed in highly selected popula-
tions or in children,8 or have considered interactions with environmental
risk factors. Rosendaal and colleagues reported an increased risk of
nonfatal MI among young women who carried factor V Leiden, with
an OR of 2.4.9 The mutation had little effect on nonsmokers, while it
led to a significant increase in risk among smokers (OR=3.6), resulting
in a 32-fold higher risk of MI in smokers who also carried factor V
Leiden compared to noncarriers who did not smoke. Furthermore,
carriers of the prothrombin 20210A allele had a four-fold increase in
risk of MI that was again increased over 40-fold in smokers.10 In a
combined analysis of factor V Leiden and the prothrombin 20210A allele
in this population, the effect of major coronary risk factors was enhanced
four-to six-fold by the presence of one of these inherited prothrombotic

risk factors. These interactions were confirmed in two case-control
studies of men who had an increased risk of MI associated with factor
V Leiden and the prothrombin G20210A variant that was most
pronounced in the presence of other cardiovascular risk factors. 11,12
Factor XIII
Factor XIII forms covalent bonds between the γ-chains of adjacent fibrin
monomers, thereby stabilizing the fibrin clot. Its role in atherothro-
mbotic disease has not been extensively studied. Factor XIII is a tetramer
consisting of two catalytic subunits (A subunit) and two nonenzymatic
subunits (B subunit). Several polymorphisms have been identified in
the A subunit, including one that codes for a valine-to-leucine
substitution at postition 34 (Val34Leu), only three amino acid residues
from the thrombin cleavage site.13 The less common Leu34 allele has
been associated with increased factor XIII activation by thrombin and
increased crosslinking activity. Paradoxically, recent studies have found
an association between the Leu34 allele and a decreased risk of MI, IS,
and venous thrombosis. 2,4 The mechanism for this protective effect
remains to be understood but may reflect alternate activities of factor
XIII, such as the promotion of angiogenesis (Aida Inbal, personal com-
munication).
PLATELET RECEPTORS
GP IIb/IIIa:
Surface membrane glycoproteins (GP) are essential for adhesion of
platelets to exposed subendothelial extracellular matrix and for platelet-
platelet interactions. Glycoprotein IIb/IIIa (known also as integrin
αIIbβ3) is the primary platelet surface receptor for fibrinogen, and also
binds von Willebrand factor (vWF) and several other adhesion ligands.
In the GP IIIa gene, a common T-to-C polymorphism in exon 2 leads to
a substitution of proline for leucine at amino acid 33 (Leu33Pro),
resulting in a conformational change in the amino-terminal disulfide
loop of GP IIIa relative to the fibrinogen binding site.14 This poly-
morphism is found in approximately 15 per cent of Caucasians; the
more frequent allele is known as PLA1 (HPA-1a), and the 33Pro allele as
PLA2 (HPA-1b). The homozygous A2/A2 form is well known to be
associated with posttransfusional purpura and neonatal alloimune
thrombocytopenia, conditions in which allo-antibodies are formed
against the A1 allele. In 1996, Weiss and colleagues first published an
association of PLA2 with the risk of acute coronary thrombosis, which
was strongest in patients under the age of 60 years (relative risk of
6.2).15 Several subsequent studies analyzed the role of the PL A2 allele

in CAD and IS, of which some confirmed an association, but most did
not.1,4 Possible mechanistic associations that have been postulated
between the PLA2 polymorphism and thrombosis include increased
sensitivity to platelet aggregation by several agonists and altered
sensitivity to aspirin.
GP Ia/IIa and Ib/IX/V

Glycoprotein Ia/IIa (integrin α2β1) is the major platelet collagen receptor
and is responsible for adhesion to the exposed vessel wall; the GP Ib/
IX/V complex is the main receptor for vWF. Polymorphisms in the
genes of these GP’s are emerging as possible candidates for an
association with atherothrombotic disease. These include a length
polymorphism of the GP Ibα subunit with a variable number of tandem
repeats of 39bp and a linked C-to-T polymorphism at nucleotide 3550
that leads to a Thr145Met substitution.16 A small number of reports
have inconsistently correlated these genetic variations with CAD and
IS, particularly in younger patients. A silent exonic C-to-T nucleotide
substitution at position 807 in the gene coding for the GP Ia/IIa α2
peptide has been found to increase receptor density and may be
associated with MI, again in younger patients.17 Finally, a second
polymorphism in the α 2 peptide gene, A1648G, which causes a
Glu505Lys substitution, is another recent candidate for an association
with vascular disease, although a functional correlation has yet to be
identified.
FIBRINOLYTIC SYSTEM
Tissue-type plasminogen activator (tPA) is the main endothelial-derived
activator of the fibrinolytic system; the major inhibitor of tPA is
plasminogen activator inhibitor 1 (PAI-1). Elevated levels of PAI-1 have
been associated with vascular risk4 and atherosclerotic progres-
sion18with relative consistency, although adjustment for potentially
confounding vascular risk factors, mainly diabetes mellitus,
hypertriglyceridemia, and obesity, may result in loss of statistical
significance for the association. In addition, increased levels of tPA have
also been identified as a risk marker. PAI-1 exists in the circulation in
great excess over tPA to permit local clot lysis without systemic bleeding,
and most tPA is inactive and complexed with PAI-1. In consequence,
tPA and PAI-1 levels are moderately positively correlated (r=0.65), which
can complicate the interpretation of fibrinolytic assays and explain this
apparently paradoxical association of elevated tPA levels with
cardiovascular disease.1
The 4G allele of a 4G/5G insertion/deletion polymorphism located
at position-675 of the promoter is the most frequently studied of the
PAI-1 polymorphisms and has been correlated with higher levels of

gene transcription and elevated PAI-1 plasma levels. This promoter
site exhibits genotype-specific responses to tryglicerides, with the
highest levels of PAI-1 occurring in carriers of the 4G/4G genotype
who are also hypertrygliceridemic. A triglyceride-responsive region
has been identified adjacent to the 4G/5G site that explains this
interaction.19 While several case-control studies have demonstrated an
increased risk of MI, CAD, and IS in carriers of the 4G allele, these
findings have not been confirmed in several larger studies. A meta-
analysis of nine studies that included approximately 1,500 cases and
2,100 controls yielded an overall very slightly increased risk of MI
associated with the 4G allele (OR=1.23, 95 per cent CI: 1.04-1.45); this
risk was, however, confined to subgroups of high-risk populations.6 In
addition to the promoter 4G/5G polymorphism, a CA(n) dinucleotide
repeat in intron 3 and a 3’ HindIII site have been identified, for which
no role in atherothrombotic disease has yet been established.
Of the several nucleotide sequence changes that have been identified
in the tPA gene, the most studied in atherothrombotic disease is a Alu
insertion/deletion in intron 8. An association between the number of
Alu repeats and thrombosis has been suggested; however, a clear
functional effect of the polymorphism remains to be characterized.
HYPERHOMOCYSTEINEMIA
Homocysteine is a sulfur-containing amino acid that is formed as an
intermediary compound during methionine metabolism, an essential
amino acid derived from dietary protein. It is well established that
hyperhomocysteinemia is an independent risk factor for athero-
thrombosis. Experimental evidence suggests that the deleterious effects
of homocysteine result mainly from endothelial dysfunction, followed
by platelet activation and thrombus formation. Although severe
hyperhomocysteinemia is rare, abundant epidemiologic evidence has
demonstrated that even mild hyperhomocysteinemia, which occurs in
approximately 5 to 7 per cent of the general population, is an indepen-
dent risk factor for atherosclerosis in the coronary, cerebral, and
peripheral vasculature.20
Homocystinuria and severe hyperhomocysteinemia are caused by
rare inborn errors of metabolism, the most common of which is
cystathionine β-synthase deficiency, resulting in marked elevations of
plasma and urine homocysteine concentrations. Mild-to-moderate
elevations in homocysteine levels can be caused by homozygosity of a
common C677T point mutation in the coding region of the methyle-
netetrahydrofolate reductase (MTHFR) gene, a key enzyme involved
in the metabolism of homocysteine, which leads to the substitution of
valine by alanine in a potential folate binding site. In addition, nutri-
tional deficiencies in the vitamin cofactors required for homocysteine
metabolism (folate, vitamin B 12 , and vitamin B 6 ) may lead to

hyperhomocysteinemia. An inverse correlation between homocysteine
and serum levels of vitamins B6, B12, and folate has been clearly docu-
mented.21 Patients with mild hyperhomocysteinemia have none of the
clinical signs of homocystinuria, and are typically asymptomatic until
the third or fourth decade of life when premature CAD can develop, as
well as recurrent arterial and venous thrombosis.
Although the MTHFR 677TT genotype has been identified as the
most common genetic cause of mild to moderate hyperhomo-
cysteinemia, its association with atherothrombotic disease remains
controversial. Studies in certain populations have shown an over three-
fold increase in risk of CAD and IS associated with homozygosity for
MTHFR-T, while others found no association.22,23 Brattstrom and
colleagues 24 recently reviewed twelve case-control studies, and
determined that individuals homozygous for the MTHFR 677T allele
generally have increased fasting homocysteine levels, -2 to 4 mmol/L
higher than in heterozygous or normal individuals. Nevertheless, eight
of these studies did not report an increased risk of cardiovascular
disease associated with the homozygous MTHFR 677TT genotype, and
the authors concluded that the MTHFR polymorphism is, at most, a
modest risk factor for atherothrombotic disease. A possible explanation
for these results stems from recent studies indicating that plasma
homocysteine concentrations in MTHFR 677T homozygotes are
dependent upon folate levels; homocysteine levels are elevated only in
the setting of low folate status. Thus, folate supplementation can
confound the interpretation of genotype association studies, and may
be a means of preventing atherothrombotic events in these patients.
ENDOTHELIAL NITRIC OXIDE SYNTHASE

Nitric oxide (NO), a product of the normal endothelium, regulates
vascular tone and has antithrombotic effects, mainly by inhibiting the
adhesion, activation, and aggregation of platelets. In addition, NO limits
vascular smooth muscle cell proliferation and leukocyte adhesion to
the endothelium, and NO-dependent endothelial dysfunction is now
accepted as a key initial step in atherogenesis. Nitric oxide is produced
in the vasculature by the constitutive endothelial isoform of the nitric
oxide synthases (NOS), as a by-product of the conversion of arginine
to citrulline. The endothelial NOS (eNOS) gene is located on chromo-
some 7q35-36 and comprises 26 exons.
Numerous polymorphic sites have been identified in the eNOS gene,
most of which are intronic. Two distinct variable nucleotide tandem
repeats (VNTR) in introns 4 and 13 have been examined in association
with vascular disease. The intron 4 VNTR is characterized by the
presence of either four (minor allele) or five (major allele) copies of a
27-base pair repeat. A mild but significant reduction in plasma levels

of NOx had been observed in homozygotes of the minor allele.25 A few
Japanese studies have found an association of the minor allele with
MI; however, this association was not confirmed in other populations.
In intron 13, between 17 to 44 copies of a CA repeat have been described,
and the presence of a minimum of 38 repeats has been associated with
an independent 2.2-fold increase in risk of CAD in one study.26
Three strongly linked single base pair changes, T(-786)C, A(-922)G,
and T(-1468)A, have been identified in the promoter region of the eNOS
gene and associated with coronary spasm in the Japanese population.27
The authors determined that the substitution at position-786 resulted
in a significant reduction in eNOS gene promoter activity, as assessed
by luciferase reporter gene assays, while the other two polymorphisms
had no effect. Japanese carriers of the-786CC genotype have also been
shown to have reduced cerebral flood flow, yet this effect was only
observed among smokers. Recently, two Italian studies found an
association of the-786C allele with angiographically defined CAD, as
well as with endothelial dysfunction among hypertensive individuals
as measured forearm flow-mediated dilation; 28,29 however, numerous
other studies have failed to confirm these observations.
A G-to-T base pair change at position 894 in exon 7 predicts a
Glu298Asp substitution, which is the only polymorphism that alters
the primary structure of the protein.30 This amino acid change influences
enzyme stability, the 298Asp isoform being degraded more rapidly than
its Glu counterpart. Numerous studies have attempted to find a role
for this polymorphism in atherothrombotic disease and, similarly to
findings with the promoter-786 polymorphism, most positive studies
have been carried out in the Japanese population, where an
approximately two-fold increase in risk of MI and hypertension has
been shown in carriers of 298Asp isoform.
ANTIOXIDANT ENZYMES
Paraoxonase
Serum paraoxonase (PON1) is a calcium-dependent esterase
synthesized by the liver and bound exclusively to high-density
lipoprotein (HDL) in plasma. This enzyme has been extensively studied
in the field of toxicology owing to its ability to detoxify organophos-
phate insecticides and nerve gases; its physiological role, however,
remains unclear. Recently, PON1 has been implicated in the patho-
genesis of atherosclerosis and cardiovascular disease. It has been shown
to preserve HDL function and to protect low-density lipoprotein (LDL)
from oxidative modification by hydrolyzing lipid peroxides, thus
exerting antioxidant and antiatherogenic effects.31 In addition, PON1
protects against the induction of monocyte-endothelial interactions in
the artery wall by metabolizing biologically active lipids in oxidized

LDL. Both peroxidation of LDL and the secondary inflammatory
responses are key steps in the initiation of atherogenesis. Thus, PON1
is now thought to be responsible, at least in part, for the cardioprotective
properties of HDL. PON1-deficient mice are more susceptible to diet-
induced atherosclerosis than their wild-type littermates, and HDL
isolated from these animals is unable to prevent LDL oxidation in vitro.32
In addition, clinical reports have demonstrated that PON1 activity is
reduced in patients with acute MI and conditions associated with
accelerated atherogenesis, such as familial hypercholesterolemia and
diabetes mellitus.31
There is a 10- to 40-fold interindividual variability in serum PON1
activity as measured by rates of hydrolysis of the exogenous substrate
paraoxon. Two common polymorphisms in the coding region of the
PON1 gene, which lead to a Gln-to-Arg substitution at position 192
and a Leu-to-Met substitution at position 55, independently influence
PON1 activity.33 In addition, five new polymorphic sites in the promoter
have been described recently: C(-107)T, G(-126)C, G(-160)A, G(-824)A,
and G(-907)C.34 Reporter gene constructs and genotyping studies in
healthy populations established that in particular the C(-107)T and
G(-824)A substitutions have a strong impact on gene expression and
serum concentrations of the enzyme. Several case-control studies have
investigated the association between the PON1 coding region
polymorphisms and CAD, yielding conflicting results. While some
reports have shown an increased susceptibility to CAD, carotid intima-
media thickness, and IS among carriers of the 192R allele, with risk
estimates ranging between 1.7 and 8.8, others have reported a lack of
association.35,36Homozygosity for the PON1 55L allele has also been
demonstrated to increase the risk of CAD independently, yet results of
several later studies failed to confirm this association. With respect to
the promoter polymorphisms, two studies have analyzed the
prevalence of the C(-107)T substitution in CAD and found that the low
expressor genotype (-107TT) is associated with a moderately increased
risk in selected populations.37,38 We have investigated the role of the
promoter polymorphisms in a group of young patients with IS and
found that, although the -107T allele had a modest influence on the
risk of AIS when analyzed individually, its interaction with the
paraoxonase 192RR genotype led to a 17-fold increase in risk compared
to individuals carrying neither variant (Voetsch and colleagues, unpub-
lished data).
Plasma Glutathione Peroxidase

Plasma glutathione peroxidase (GPx-3) is a major antioxidant enzyme
in plasma, and as part of the GPx family scavenges reactive oxygen
species (ROS) produced during normal metabolism or after oxidative

insult, thereby maintaining the bioavailability of NO. Deficiencies of
the cellular and plasma isoforms of GPx have been associated with
CAD in clinical studies.39 Recently, Freedman and colleagues studied
the plasma of two brothers with a history of idiopathic childhood stroke
and provided evidence for a new prothrombotic mechanism associated
with AIS.40 The childrens’ plasma impaired the antiplatelet effects of
NO, and this effect was found to be a consequence of a reduction in
GPx-3 activity. These investigators proposed that a deficiency of GPx-
3 reduces bioavailable NO owing to the impaired metabolism of ROS,
and increases the risk of thrombosis. Interestingly, the reduction in GPx-
3 activity was present also in the unaffected mother. In a second study
including seven families with childhood stroke, Kenet and colleagues
confirmed the familial distribution of these observations, suggesting a
heritable trait. 41
In an attempt to identify the molecular basis of this defect, we recently
studied the entire GPx-3 gene of young IS patients and healthy age-
and gender-matched controls by single-stranded conformational
polymorphism analysis and sequencing of fragments with electro-
phoretic shifts. We identified four novel polymorphisms in the promoter
of the GPx-3 gene which were strongly linked:-622 A→T,-688 T→C,
-703 A→C, and-68 A→T (Voetsch and colleagues, unpublished data).
Carriers of the haplotype combining nucleotides-622T,-688C, and-703C
had a two-fold increase in risk of AIS as compared to non-carriers
(OR=2.09, 95 per cent CI, 1.12 to 3.92). These risk estimates remained
unchanged after adjustment for inherited prothrombotic and
conventional vascular risk factors. In addition, in individuals
simultaneously exposed to vascular risk factors that enhance oxidative
stress, such as smoking and hypertension, the risk associated with the
GPx-3 polymorphisms was amplified over four-fold. Expression studies
are currently underway in our laboratory to analyze the effect of the
GPx-3 polymorphisms on enzyme activity levels.
CONCLUSION
The past decade has been marked by a rapidly expanding literature
attempting to characterize the genetic basis of atherothrombotic disease.
Numerous polymorphisms have been identified; however, as described
in this review and summarized in Table 1, the relationship between
most polymorphisms and disease is highly controversial. A striking
point in several studies is the inconsistency in relating genotype,
intermediate phenotype, and clinical outcome. Although early reports
in the literature revealed positive associations, numerous negative
studies have followed, and the initial hope that inherited risk factors
might contribute significantly to the development of atherothrombotic
disease has largely been unconfirmed. Therefore, there is currently no

evidence that screening for any of the above haemostatic markers either
within the general population or among subgroups, such as individuals
who develop atherothrombotic disease at a young age, would have
any prognostic or therapeutic consequence.1
Genetic association studies need to be interpreted taking several
aspects of into consideration. First, the study design and characteristics
of patients and controls vary greatly among different reports. Different
clinical endpoints chosen by different investigators, such as CAD, MI,
carotid stenosis, stroke, and PVD render their comparison difficult, as
the pathophysiology of these disorders may have subtle, yet relevant,
specific differences. As an example, among reports on cerebrovascular
disease, some authors include patients with transient ischaemic attacks,
while others only study patients with an acute neurological deficit
lasting more than 24 hours or if a brain imaging study shows an
ischaemic lesion appropriate to the symptoms; some investigators
analyze carotid intima-media thickness, while others only enroll cases
with severe carotid stenosis. Secondly, many of these polymorphisms
or mutations have low prevalences in the general population. As a
result, large sample sizes are required to yield statistical power to
demonstrate an effect. Thirdly, the prevalence of genetic polymorphisms
may vary greatly within and between racial groups, as has been clearly
described for factor V Leiden, the prothrombin variant, the MTHFR
C677T polymorphisms, and the paraoxonase coding region poly-
morphisms. In the case of ethnically heterogeneous populations, false-
positive associations may arise if the frequency of the genetic marker
varies because of population admixture rather than as a consequence
of a true association with the disease phenotype. In addition, the genetic
marker of interest may not be directly involved in disease susceptibility,
but rather be in linkage disequilibrium with the actual causative
mutation located in the same gene or another gene nearby.
Finally, there is compelling evidence that atherothrombosis is a
complex disorder and multifactorial in nature. Its pathogenesis involves
multiple genetic and environmental factors related to atherosclerosis
and thrombosis, as well as their interaction (i.e., gene-gene and gene-
environment interactions). After the identification of inherited
deficiencies of the coagulation inhibitors, antithrombin III, protein C,
and protein S, and of the associated high risk of venous thrombo-
embolism, it was believed that atherothrombosis most likely would
have a similar molecular basis and also be a single gene disorder;
however, most carriers of these genetic markers never experience
vascular events, demonstrating the importance of other genetic and
acquired risk factors in the onset of disease. For several polymorphisms,
the contribution to risk is only detectable in association with other
environmental risk factors. Factor V Leiden and the prothrombin variant
have been found to influence the risk of atherothrombosis only in a

subgroup of female smokers. Elevated plasma homocysteine
concentrations in patients homozygous for the MTHFR 677TT genotype
have been shown to have a multiplicative effect on risk among cigarette
smokers and patients with hypertension. The demonstration that
individuals who are homozygous for this mutation appear to have
elevated plasma homocysteine levels only in the setting of folic acid
depletion further indicates the importance of environmental influences
on the genetic risk of vascular disease. Several studies have
demonstrated that the risk of disease is exponentiated if the genetic
marker occurs simultaneously with an environmental risk factor,
attesting(???) to an interaction between these factors. A more novel
concept is that of gene-gene interactions; these may occur between
different genes and lead to multiple deficits(?) in their gene products,
or be due to the presence of multiple polymorphisms within one gene.
Some studies have shown evidence that individual polymorphisms
within a gene may not correlate with disease risk, while the analysis of
haplotypes clearly demonstrates an influence on risk. The unique
interaction of multiple polymorphisms within a haplotype may affect
the biologic phenotype and outcome, while individual polymorphisms
may not play a relevant role in the determination of disease. In the
future, studies will most likely need to focus on these gene-gene and
gene-environment interactions to unravel the complex pathophysiology
that underlies the development of atherothrombosis.
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18
Anticoagulation Therapy
MB Agarwal
Key Words
Anticoagulants • Antithrombotics • Heparin • LMW
heparin • Pentasaccharide • Thrombin inhibitors
• Warfarin
INTRODUCTION
Recent advances in anticoagulation therapy have made significant in-
roads in the prophylaxis and treatment of thromboembolic disorders.
Heparin and warfarin have remained the mainstay of anticoagulation
for many decades. Clinical indications for anticoagulation therapy,
which often involves elderly patients, have been on rise. Safe and
effective anticoagulation for long periods has been difficult due to
narrow therapeutic index of these two compounds. This has lead to
development of multiple new anticoagulants.1 This became possible
because of our better understanding of coagulation, coagulation
proteins, their interactions with cell membrane, their substrate binding
sites etc. The goal has been to develop more efficacious, safer, and easier
to use anticoagulants. Orally bioavailable drugs for the long-term treat-
ment of thrombotic disorders, particularly those that do not require
monitoring, are under aggressive development. Local delivery of
anticoagulants or genes modulating anticoagulant control at sites of
increased thrombogenecity, is a promising treatment modality. At the
same time, improved patient management with existing drugs, i.e.
heparin and warfarin by instituting anticoagulation clinics, promoting
patient-self-monitoring and improving efforts to educate patients and
health care providers about the use of these compounds are equally
important.
An ideal anticoagulant drug should have following features:
• Effectiveness (preferably at the site of pathologic thrombus for-
mation).
• Safety and total lack of serious toxicities
• A wide therapeutic window
• No need for monitoring

• Rapid onset of action
• Oral bio-availability (at least for long-term use)
• Mechanism of action independent of vit-K metabolic pathway and
cytochrome P-450 system
• Lack of binding to plasma proteins
• Easy reversibility (an available antidote)
• Safety during pregnancy
• Low cost
• A short half-life for those used in acute setting of thrombosis and
a long half-life for those used for prophylaxis
Although no such drug exists, availability of many drugs fulfilling
most of these criteria would soon expand the therapeutic options for
physicians who must balance the dangers of clotting against the risk of
excessive haemorrhage in patients who often have multiple medical
problems. Success of such agents, however, would be determined by
their cost and by the value they offer to the treatment regimen in terms
of efficacy, safety, and ease of use.
Table 18.1 enlists the anticoagulants approved for use by the FDA in
US/Europe or Australia (as in 2003). 1 Table 18.2 enlists newer
anticoagulants on the horizon1 (Fig. 18.1) gives the sites of action of
newer anticoagulants.2
Table 18.1: Approved anticoagulants as in year 20031

Generic name Trade name Mechanism of Route of
anticoagulation administration
Warfarin Coumadin and Vit-K antagonism Oral
others
Heparin Various agents Thrombin and I.V., S.C.
(unfractionated) Factor-Xa inhibitor
LMW-heparin Clexane Thrombin and I.V., S.C.
Fraxiparine Factor-Xa inhibitor
Fragmin
Others
Danaparoid Orgaran Thrombin and I.V.
Factor-Xa inhibitor
Argatroban Argatroban Direct thrombin I.V.
inhibitor
Lepirudin Refludan Direct thrombin I.V.
inhibitor
Bivalirudin Angiomax Direct thrombin I.V.
inhibitor
Fondaparinux Arixtra Xa inhibitor S.C.
Activated protein-C Xigris Va and VIIIa I.V.
inhibitor
Anticoagulation Therapy 291
Table 18.2: Newer anticoagulants in the pipeline1

Mechanism of action Examples
Inhibitors of coagulation initiation Inhibitors of tissue factor expression
Anti-tissue factor antibodies
Tissue factor pathway inhibitor (TFPI)
Recombinant nematode anticoagulant
peptide c2
(rNAPc2)
Inhibitor of VIIa: TF
Inhibitors of coagulation amplification Factor-Xa inhibitors:
Fondaparinux (Arixtra)
Danaparoid (Orgaran)
Factor-IXa inhibitors
Thrombin inhibitors Melagatran
Ximelagatran
Enhancers of natural anticoagulant Activated protein-C (Xigris)
system Soluble thrombomodulin
Others Oral heparin coupled to carrier
molecule
LMW derivatives-Vasoflux
Fig. 18.1: Steps in blood coagulation and sites of action of new anticoagulants2
We will now discuss some issues with the following old and new
drugs:
a. Unfractionated heparin
b. LMW heparin
c. Vitamin-K antagonists
d. Heparinoid (Danaparoid)
e. Direct thrombin inhibitors (Hirudin, Lapirudin, Bivalirudin,
Argatroban, Ximelagatran)
f. Pentasaccharides (Fondaparinux-Arixtra)
g. Activated protein-C (Xigris)
h. Oral Heparins
i. Heparin derivatives (Vasoflux)
j. Thrombolytic therapy (Plasmin)
Unfractionated Heparin
Unfractionated heparin (UFH) has been the mainstay of anticoagulant
treatment for acute thromboembolic disorders for many decades. It is
a crude product, derived from the intestine of pigs. It has many
limitations.3
• An unpredictable anticoagulant response due to the binding to
various acute-phase plasma proteins, proteins released from
activated platelets (platelet factor-4) and endothelial cells.
• The need to monitor it’s effect.
• Limitations of present monitoring system.
• Lack of wide availability of anti-factor-Xa assay.
• Dependence on antithrombin (antithrombin-III) for anticoagulant
action.
• Inability to bind to and inactivate cell-membrane-bound and clot-
bound thrombin and platelet-bound factor-Xa.
• Potential for development of heparin-induced-thrombocytopenia
with or without thrombosis (HIT and HIT-T).
• Osteoporotic effect with long-term application.
• Dose-dependent clearance and lack of linear dose-response curve
• Lack of oral bio-availability.
However, there are certain advantages of unfractionated heparin and
hence even today, there are few indications for using UFH as shown in
Table 18.3.
Low Molecular Weight Heparins3

LMW heparins are fragments of standard UFH produced by controlled
enzymatic or chemical depolymerisation methods. UFH has a molecular
weight of up to 30,000 daltons while LMW heparin has a mean
molecular weight of 5000 daltons. LMW heparins are today widely
used as parenteral anticoagulants for the prevention and treatment of
thromboembolic diseases.
Table 18.3: Advantages and indications of UFH over LMW-heparin31

Advantages Indications
Short half-life Renal failure
Easy reversibility Intensive care unit
Extra-renal metabolism Operating room
Their advantages include:

• A predictable anticoagulant response
• Better bio-availability
• Long half-life
• Safety without laboratory monitoring
• Use by subcutaneous route
• Less osteoporosis
• Decreased risk of inducing HIT and HIT-T
LMWHs accelerate antithrombin-mediated inactivation of factor-Xa.
They are relatively less effective on thrombin. Table 18.4 gives
characteristics of few commonly available LMW heparins, including
anti-factor-Xa to anti-factor-IIa ratio.3 There are very few studies directly
comparing different LMWHs. None of them demonstrate a clinically
meaningful therapeutic superiority of one over another. Patients
receiving prophylactic or therapeutic doses of LMWHs do not require
therapeutic monitoring. The dose is weight adjusted. However, when
needed, it has to be anti-factor-Xa activity assay. The optimum time to
perform this is 4 hours after a SC injection of LMWH.
There are, however, certain issues.4 These include, their use in patients
with:
• Renal insufficiency
• Pregnancy
• Malignancy with Trousseau’s syndrome
• Morbid obesity
• Paediatric age group
• Regional anaesthesia (epidural and spinal)
• Intensive care units
• Surgery
Table 18.4: LMW heparins31

Preparation Market names Manufacturer Anti-Xa / Anti-IIa ratio
Enoxaparin Clexane Aventis 3.8
Nadroparin Fraxiparine Sanofi Synthelabo 3.6
Reviparin Clivarine — 3.0
Dalteparin Fragmin Pharmacia and Upjohn 2.7
Tinzaparin Logiparin Bristol Meyer Squibb 1.9
Ardeparin Normiflo — 1.9
During their use in conditions mentioned above, therapeutic

monitoring may be essential. LMWHs should be avoided in patients
with HIT and they should be used with great caution in patients needing
neuraxial anaesthesia. In addition, for developing world, cost is an issue.
Vitamin-K Antagonists5,6
While several parenteral anticoagulant drugs are available, for decades,
the vitamin-K antagonists have been the only oral anticoagulants. Two
classes of them are in the market, the coumarins (e.g. warfarin,
acenocoumarol etc.) and the indandiones (e.g. anisindione, phenin-
dione, fluindione etc.). Rising number of clinical indications for anti-
coagulation, which often involve elderly patients, has lead to increased
problems with long-term management.
This group of drugs has several disadvantages, e.g.:
• A narrow therapeutic window
• Large inter-individual dosing differences
• Interactions with dietary vitamin-K
• Need for monitoring using INR
• Interactions with a prodigious number of other medications due
to their dependence on the cytochrome P-450 system
• The potential for serious and even fatal bleeding in patient’s
receiving therapeutic doses.
• Recurrences of thromboembolism in spite of therapeutic INRs
• The need for thorough patient education and compliance
Recently, patients have been identified who are exquisitely sensitive
to warfarin due to mutation in the cytochrome P-450 CYP2C9 enzyme
which increases the half-life of warfarin dramatically. Such individuals
can develop life-threatening bleeding. The applicability and cost-
effectiveness of large-scale screening are unknown, but genetic testing
may prove useful, particularly for high risk patients. Patients with the
mutations would benefit from using warfarin derivatives like
Acenocoumarol, which are less influenced by CYP2C9.
Until a newer oral anticoagulant (vide-infra) becomes available,
warfarin will remain the mainstay of outpatient long-term anticoagu-
lation. Identifying high risk patients by clinical characteristics and
perhaps genetic testing, vigilant maintenance of a therapeutic INR
(through anticoagulation clinics or home-monitoring) and prompt
treatment of asymptomatic increase in INR as well as bleeding
complications are important considerations. If careful attention is paid
to these issues, oral anticoagulation safety can be greatly improved.
Heparinoid (Danaparoid)7
Danaparoid is a heparinoid, composed of low molecular weight non-
heparin glycosaminoglycans, primarily heparin sulfate (84%). It is
administered subcutaneously twice a day. It is effective and relatively

safe in patients with HIT (heparin-induced thrombocytopenia).
Direct Thrombin Inhibitors7

Indirect thrombin inhibitors include heparins-UFH as well as LMWH
and their active Pentasaccharide moiety (Fondaparinux-Arixtra). While,
they all are very efficient inhibitors of thrombin in solution, they do
not affect surface-bound thrombin including fibrin clot bound thrombin.
Direct thrombin inhibitors directly interact with thrombin without
an intermediate such as antithrombin. They block thrombin’s catalytic
activity on a wide range of substrates. There were two major forces
leading to development of direct thrombin inhibitors. These included
(a) their ability to inhibit thrombin bound to fibrin clots and (b) their
relative safety in cases of heparin induced thrombocytopenia with or
without thrombosis. Table 18.5 gives details of various agents useful in
patients with heparin-induced thrombocytopenia (HIT).8-10
There are five direct thrombin inhibitors:
• Hirudin
• Lepirudin
• Bivalirudin
• Argatroban
• Melagatran/Ximelagatran
Table 18.5: Newer anticoagulants for pts with HIT2

Variables Lepirudin Argatroban Danaparoid Bivalirudin
(Refludan) (Novastan) (Orgaran) (Angiomax)
Action Direct thrombin Direct thrombin Factor-Xa and Direct
inhibitor inhibitor thrombin thrombin
(ATIII inhibitor
dependent)
Half-life 1.5 hr 40 min 19 hr 25 min
Route I.V. or S.C. I.V. I.V. or S.C. I.V.
Elimination Renal Hepatic Renal Renal
Monitoring aPTT, ECT aPTT Anti-factor-Xa ACT or aPTT

assay
INR Prolonged Prolonged No effect Prolonged
Indication HIT HIT VTE PCI

prophylaxis
ATIII: Antithrombin-III, ECT: Ecarin clotting time, ACT: Activated clotting time,
HIT: Heparin induced thrombocytopenia, PCI: Percutaneous coronary intervention
Hirudin is a leech protein. It’s anticoagulant properties are known

since the late-1800, but its clinical development is recent.10 Its biologic
function is to maintain the blood liquid after a bite so that the leech can
continue to drink. The best source of medicinal leech was a lake in
central Hungary and hence it was inaccessible during the cold war.
This delayed it’s clinical development. Today, besides Hirudin, its
modified form, i.e. Bivalirudin and recombinant form, i.e. Lepirudin
are already in clinical use. Argatroban, however, is synthetic Arginine-
based molecule while Melagatran is a dipeptide with low molecular
weight. Melagatran has low oral bio-availability but Ximelagatran is
an inactive pro-drug which can be taken by mouth and is converted to
the active Melagatran.
Direct thrombin inhibitors are likely to play a significant role in the
management of thrombotic disorders. Their advantage over heparin
includes independence from antithrombin and their ability to inhibit
surface-bound thrombin. Ximelagatran has the advantage of being
orally effective with efficacy similar to warfarin but greater convenience
due to simpler dosing and lack of a monitoring requirement.
Pentasaccharides
Arixtra (Fondaparinux Sodium)11-19
Arixtra is the first of a new class of selective factor-Xa inhibitor. It is a
pentasaccharide produced entirely by chemical synthesis.12-14
Arixtra binds with high affinity to antithrombin (AT) III and
specifically catalyses the inactivation of factor-Xa, interrupting the
coagulation cascade at the point where the intrinsic and extrinsic
coagulation paths merge, (Fig. 18.2, Plate 3).15 Inactivation of factor-Xa
prevents thrombin formation and the activation of factors V, VIII, XIII
and protein C. The binding of Arixtra to AT-III induces a conformational
change in the AT molecule, potentiating AT-mediated inhibition of
factor-Xa by 300-fold.12-14
Comparative analysis of Arixtra v/s LMWH is shown in Table 18.6.
Arixtra is 100 per cent bio-available when administered subcutaneously.
It has a terminal half-life of 17 hours and therefore once daily dosing is
possible. It has no binding affinity for S. proteins. Hence, the potential
for drug-drug interactions is limited. Importantly, it does not interact
with aspirin, warfarin and piroxicam. Peak plasma levels are reached
by 2 hours and hence it can be given post-operatively for DVT prophy-
laxis. There is very little intra- and inter-subject variability and therefore
routine monitoring and dose adjustments are unnecessary. Arixtra is
not metabolised and is eliminated unchanged in urine.12-14
The recommended dose for DVT prophylaxis is 2.5 mg sub-
cutaneously once a day, starting 6 hours post-operatively. It has an
excellent therapeutic index.12-14
Table 18.6: Fondaparinux-arixtra v/s LMW heparin11

Variable Fondaparinux LMWH
Mol. Wt (Dalton) 1,728 2,000-10,000
Source Chemical synthesis Porcine mucosa
Dispersity Homogeneous Heterogeneous
A single chemical entity Mixture of polysaccharides
Target Single target, i.e. Multi-targeted agent
Factor-Xa
Pharmacological Activity expressed in Activity expressed as IU
effect microgram or micromoles
HIT/HIT-T Never Occurs
No binding to PF4 Binds to PF4
Platelets No effect on function Significant effect
and aggregation
Bio-availability 100% 85-95%
(SC route)
Half-life 17-hours (OD) 4-hours (often BD)
PREVENTION OF VTE: CLINICAL DATA

The high prevalence of VTE in orthopaedic surgery demands critical
appraisal of the therapies available. Data indicates that Arixtra reduces
the incidence of VTE by over 50 per cent when compared to LMWH
prophylaxis, with enoxaparin in patients undergoing major orthopaedic
surgery (i.e. hip replacement, hip fracture and knee replacement)
without increasing clinically relevant bleeding. Four phase-III studies—
Ephesus,16 Pentathlon 200016 , Penthifra17 and Pentamaks18—formed
part of a world-wide VTE prevention programme. There was a risk
reduction in favour of Arixtra of 55.2 per cent (95% CI, 45.8-63.1). Safety
analysis showed that the incidence of clinically significant bleeding
leading to death, re-operation or bleeding occurring in a critical organ
did not differ between the two groups.16-18 The Penthifra Plus study,11
the first to evaluate 4 weeks’ prophylaxis following hip fracture surgery,
reported a 96 per cent risk reduction in favour of Arixtra. The number
of patients with symptomatic events indicative of DVT and PE was
reduced from 2.7 to 0.3 per cent. There were no clinically relevant
differences in bleeding events between Arixtra and placebo.
TREATMENT OF VTE19
In two clinical trials, Arixtra has been used in the treatment of DVT
and PE (MATISSE study).19 At a dose of 7.5 mg once daily Arixtra has
been found to be effective and well-tolerated.19
Clinical trial data show that Arixtra fulfils the two main criteria
required of a new prophylactic agent for VTE; greater efficacy and safety.
Compared with enoxaparin, significantly greater reductions in the risk

of VTE are reported after prophylaxis with Arixtra without any increase
in the risk of clinically significant bleeding. Its ease of administration,
as a once daily dose without the need for dose adjustments or laboratory
monitoring, suggest that patient compliance is likely to be high. All of
these factors make Arixtra a promising new agent in the prevention
and treatment of VTE.
Arixtra is currently being evaluated for its efficacy in the management
of VTE in medical patients and patients undergoing general surgery,
as well as patients with coronary artery disease. Clinical trial results
are awaited with interest.
Activated Protein-C (XIGRIS)20

Activated protein-C (APC) has multiple biological property. Besides
anticoagulant action, it has anti-inflammatory, profibrinolytic and anti-
apoptotic properties. Together, these explain its role in reducing
mortality in patients with sepsis and associated organ dysfunction.
Based on PROWESS trial and many animal studies, it has received US-
FDA approval for adult patients having severe sepsis. It is also useful
in reducing the volume of cerebral infarction after a stroke. Potential
utility of APC for severe sepsis, thrombosis and stroke appears to be
highly promising.
Oral Heparins21-23
Because of their large size and anionic structure, heparin is not reliably
absorbed when given orally. Their coupling to a carrier such as the
aminoacid compound or deoxycholic acid, enhances intestinal
absorption and bio-availability. SNAC and DOCA heparins are two
such examples. However, except for oral administration, all the other
disadvantages of heparin therapy (vide-supra) remain. In future, it is
possible that the synthetic compound Fondaparinux (the Penta-
saccharide) may be linked to a carrier protein leading to an orally
effective molecule which is safer and equally effective.
Heparin Derivatives24
Vasoflux is a LMW heparin, i.e. chemically modified to reduce its affinity
for antithrombin. It is administered intravenously and it inhibits factor-
IX/IXa activity. It has little anti-factor-Xa activity.
Thrombolytic Therapy25
Plasminogen activators (PA) which include streptokinase, urokinase,
tissue plasminogen activator, alteplase-a recombinant form of wild-
type TPA molecule and plasmin are the mainstay of thrombolytic
therapy used for myocardial infarction, stroke, peripheral arterial
occlusion and even VTE. The current therapeutic agents have reached
a plateau of maximum potency. However, a significant improvement
in safety, especially regarding the avoidance of intracranial
haemorrhage, is still an unfulfilled goal. Regional infusion of plasmin
offers a distinct safety from this angle.
NEW DELIVERY SYSTEMS26,27

In the future, prevention and treatment of thrombosis would be the
targeted manipulation of the local vascular milieu. Local intervention
would minimise systemic bleeding complications. Down regulation of
tissue factor or delivery of tissue factor inhibitors are such examples.
Topical placement of stents or grafts with virally transduced confluent
endothelial cells secreting anticoagulant is another example. Gene
therapy can also be used.
IMPROVED PATIENT MANAGEMENT2,28,29

Better anticoagulation control and higher patient satisfaction can be
achieved by:
• Establishing coordinated anticoagulation clinics
• Self-monitoring of anticoagulation by patients2 (Table 18.7 and
Figure 18.3a Plate 3, and b, c Plate 4 and d Plate 5).
• Patient education
• Health care provider education
It has been shown that these activities have lead to2 :
Table 18.7: Capillary whole blood (point-of-care) PT instruments2

Instrument Clot detection Home use
methodology approval
CoaguCheck Plus Clot initiation : Thromboplastin
CoaguCheck Pro Clot detection : Cessation of blood
flow through capillary channel
CoaguCheck Pro/DM
CoaguCheck Clot initiation : Thromboplastin Yes
Thrombolytic Clot detection : Cessation of
movement of iron particles
Assessment system
ProTime monitor Clot initiation : Thromboplastin Yes
Hemochron Jr Clot detection : Cessation of blood
flow through capillary channel
GEM PCL
Avocet-PT-Pro Clot initiation : Thromboplastin Yes
AvoSure-PT-Pro Clot detection : Thrombin generation
detected by fluorescent thrombinpro
• Improved anticoagulation control

• Reduced bleeding
• Reduced thromboembolic events
• Reduced emergency department visits
• Reduced hospitalisation
• Reduced health care costs
Similar goal can also be achieved through2 :
• Patient self-management
• Point-of-care instruments (Table 18.7 and Figure 18.3a Plate 3, and
b, c Plate 4 and d Plate 5)2
• Self-monitoring of INR
• Guideline-based management of anticoagulant dosing
Unfortunately, although widely available in some countries, such
instruments are not yet available in India.
In conclusion, although, we have emphasized the newer agents, the
older ones, i.e. UFH and warfarin are unlikely to be abandoned. Rather,
the novel drugs should expand the therapeutic options for us and we
should now balance the dangers of clotting against the risk of haemor-
rhage.30 As stated earlier cost, safety and ease of use (monitoring) would
remain the critical issues.30
KEY POINTS FOR THE CLINICAL PRACTICE

• Heparin—both unfractionated and low molecular weight together
with vitamin-K antagonists, i.e. Warfarin form and remain the
main stay of anticoagulation even today.
• Role of unfractionated heparin has now got restricted to patients
with renal failure, in intensive care units and in operating rooms.
Unfractionated heparin in these situations is preferred due to its
extra renal metabolism, short half-life and easy reversibility.
• Newer anticoagulants which have wider therapeutic index and
which often require little laboratory monitoring are likely to
replace the present anticoagulants because of their safety, easier
handling and greater efficacy.
• Direct thrombin inhibitors, i.e. lepirudin, bivalirudin, argatroban
and ximalagatran are promising agents especially in patients with
heparin-induced thrombocytopenia.
• Pentasaccharide (Fondaparinux-Arixtra) is likely to be marketed
soon. It is a synthetic selective factor-Xa inhibitor which may be
preferred over LMW heparin for DVT prophylaxis as once a day
subcutaneous drug.
• Activated protein-C (Xigris), besides an anticoagulant, is an
important anti-inflammatory, profibrinolytic and anti-apoptotic
agent which reduces mortality in patients with sepsis and
associated organ dysfunction.
• Improved patient management by establishing coordinated

anticoagulation clinics and self-monitoring by patients using
point-of-care instruments and better patient education would
achieve improved care with lesser complications and cost.
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Haematol 2002;39:145-57.
2. Ansell JE, Weitz JI, Comeroto AJ. Advances in therapy and management of
antithrombotic drugs for venous thromboembolism. Haematology 2000:266-
84.
3. Weitz JI. Low molecular weight heparins. N Engl J Med 1997;337:688-98.
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6. Levine MN, Raskob G, Landefeld S, Kearon C. Hemorrhagic complications of
anticoagulant treatment. Chest 2001;119:108S-121S.
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2002;39:187-96.
8. Fitzgerald D, Murphy N. Argatroban: a synthetic thrombin inhibitor of low
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disease. Circulation 1999;100:2049-53.
10. Stringer KA, Lindenfeld J. Hirudins: antithrombin anticoagulants. Ann
Pharmacother. 1992;26:1535-40.
11. Turpie AGG. Pentasaccharides. Semin Haematol 2002;39:158-71.
12. Walenga JM, Jeske WP, Bara L. Biochemical and pharmacologic rationale for
the development of a synthetic heparin pentasaccharide. Thromb Res
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13. Herbert JM, Petitou M, Lormeau JC. SR 90107 A/Org 31540, a novel anti-
factor Xa antithrombotic agent. Cardiovasc Drug Rev 1997;15:1-26.
14. Turpie AGG, Gallus AS, Hock JA. A synthetic pentasaccharide for the
prevention of deep vein thrombosis after total hip replacement. N Engl J Med
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15. Bauer KA. Fondaparinux sodium: a selective inhibitor of factor Xa. Am J
Health-Sys Pharm 2001;58(Supl.2):S14-7.
16. Turpie AGG. Overview of the clinical results of pentasaccharide in major
orthopedic surgery. Acta Haematol 2001;86:59-62.
17. Eriksson BI, Bauer KA, Lassen MR. Fondaparinux compared with enoxaparin
for the prevention of venous thromboembolism after hip-fracture surgery. N
Engl J Med 2001;345:1298-1304.
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for the prevention of venous thromboembolism after elective major knee
surgery. N Engl J Med 2001;345:1305-10.
19. Rembrandt Investigators. Treatment of proximal deep vein thrombosis with a
novel synthetic compound (SR90107A/ORG31540) with pure anti-factor Xa
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20. Joyce D, Yan B, Basson HR. Disseminated intravascular coagulation in severe

sepsis patients treated with recombinant human activated protein C, a
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(Abstract).
21. Gonze MD, Salartash K, Sternberg WC. Orally administered unfractionated
heparin with carrier agent is therapeutic for deep venous thrombosis.
Circulation 2000;101:2658-61.
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Res. 2000;17:1259-64.
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24. Peters RJ, Spickler W, Theroux P. Randomized comparison of a novel
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International Trial for Acute Myocardial Infarction Lysis). Am Heart J. 2001.
142;237-43.
25. Marder VJ, Stewart D. Towards safer thrombolytic therapy. Semin Hematol
2002;39:206-16.
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major bleeding complications in older patients receiving warfarin. A
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Semin Hematol 2002;39:172-78.
19
Laboratory and Clinical Diagnosis of
Heparin-Induced Thrombocytopenia
and Its Therapeutic Options
Sarfraz Ahmad, Renu Saxena,
Debra A Hoppensteadt,
Taqdees Sheikh, Jawed Fareed
Key Words
Heparin-induced thrombocytopenia • Pathophysiology
• Anti-heparin-PF4 antibodies • Clinical presentation
• Diagnosis • Therapy
INTRODUCTION
Of the known side effects of heparin therapy, thrombocytopenia is
without a doubt the most frequent and dangerous effect. There are two
types of heparin-induced thrombocytopenia (HIT). HIT I is characteri-
zed by a transitory and asymptomatic reduction in the platelet count,
rarely below 100 × 109/L, that resolves spontaneously and does not
require removal of the drug. The origin of HIT I is not yet completely
known but is thought to be related to a phenomenon of heparin-induced
platelet clumping.1 No immunologic components are involved in HIT
I and pathologic manifestations are insignificant.
HIT II has an immunologic predisposition2 and is characterized by
a significant reduction in platelets (>30%) generally after the fifth day
of therapy. In the case of previous exposure to heparin, thrombo-
cytopenia may appear earlier.3 The thrombocytopenia usually resolves
5-15 days after heparin has been removed, but in some cases it may
take months. 4 The pathophysiologic manifestations of HIT II are
complex and involve thrombosis at arterial, venous and microangio-
pathic sites. Other systemic manifestations result in myocardial
infarction, thrombotic stroke and massive occlusive disease.
EPIDEMIOLOGY
The true incidence of HIT II is not well-defined because reported studies
are mostly retrospective and differ regarding the characteristics of the
patients considered, type of heparin administered, dosage, route of
administration, duration of therapy, definition of thrombocytopenia,
and laboratory tests employed for diagnostic confirmation.5 With
reference to prospective studies in which the diagnosis was clinically
based, there are important differences in the definition of thrombo-
cytopenia. In some studies, the threshold value is 150 × 109/L,6 while
in others, it is 100 × 10 9/L. 7 Other investigators instead use the
percentage fall as their reference.8 A relationship between the incidence
of HIT II (defined only on a clinical basis), dosage and type of
unfractionated heparin used emerged from the study of Warkentin.9
The incidence was about 5 per cent for therapeutic dosages of bovine
heparin, and 1 per cent for porcine heparin, while it was less than 1 per
cent with prophylactic dosages of porcine heparin. In this series, the
incidence of secondary thrombotic complications was about 20 per cent.
In a later review of prospective clinical trials, the incidence of HIT II
varied from 1 to 30 per cent in patients treated with high dosages of
intravenous (I.V.) heparin, while it was less than 2 per cent in patients
administered low dosages of subcutaneous (s.c.) heparin.5
Schmitt and Adelman reviewed 23 randomized or cohort prospective
studies for a total of 2,160 patients in order to evaluate the impact of
the various methodological characteristics, such as HIT II definition,
frequency with which platelet count was verified, and diagnostic
criteria. This analysis confirmed that the incidence of HIT is
overestimated in studies that do not include a “repeatedly abnormal
platelet count. The cumulative incidence of HIT II in studies that
employed “a frequently lowered platelet count was 2.9 per cent for
bovine heparin, and 1 per cent for porcine heparin, 1.7 per cent for I.V.
administration and 0 per cent for s.c. administration. Even if this trend
does not reach statistical significance, it speaks in favour of porcine
heparin and s.c. administration of low dosages.
Our retrospective study disclosed a higher incidence.10 Independent
of the route of administration, 6 per cent of the patients had a clinical
score suggestive of HIT II, with a 30 per cent incidence of thrombotic
complications and a 30 per cent mortality rate, in-line with literature
reports.11 However, using more selective clinical criteria, the percent
incidence lowered to 3 per cent and the diagnosis was confirmed by
the presence of anti-heparin-platelet factor 4 (anti-H-PF4) antibodies
only in a fraction of patients.10
On the other hand, Kappers-Klunne et al.12 reported a particularly
low (0.3%) HIT incidence in 558 cardiologic and neurologic patients
treated with I.V. administration of heparin. In this study, both functional
Laboratory and Clinical Diagnosis of Heparin-Induced Thrombocytopenia 305
and immunologic tests were used for laboratory confirmation of the

clinical diagnosis. Anecdotal reports13 describe HIT II induced by low
molecular weight heparin (LMWH), but one clinical study indicated
that its use is associated with a lesser incidence of thrombocytopenic
and thrombotic complications than heparin.14
Heparin induced thrombocytopenia is also observed in patients who
are administered drugs intravenously and heparin flush is used for
the patency of the intravenous lines. Reports on the incidence of HIT
in patients with biomedical devices coated with heparin and
intravascular stents have recently become available and warrant a
registry.
The incidence of HIT in children and elderly is not well investigated.
A recent report from our institution described the absence of AHPF4
antibodies primarily due to immuno-compromised status of this group.
Also no information in the elderly and the effect of ethnic origin is
available. Immunomodulation by disease and drug and its impact on
HIT is not studied.
A recent double-blind randomized study compared s.c. administered
heparin with LMWH in 655 patients undergoing orthopedic surgery.14
The clinical diagnosis of HIT II was confirmed by means of the14
C-serotonin release assay (SRA). HIT II was documented in 2.7 per cent
of the patients treated with s.c. heparin, and in none of the patients
receiving LMWH (p=0.0018). Thrombotic complications were also more
frequent in the former (88.9%) vs. the latter (17.8%) group (p<0.001). In
a sub-group of patients, independent of the presence of HIT II, more
heparin-treated patients than LMWH-treated patients had a positive
functional test (7.8 vs. 2.2%, p=0.02). Thrombotic episodes, however,
were more frequent in the patients who developed HIT II than in those
with only a positive functional test. Thus, the frequency of laboratory
confirmed HIT II seems to be about 2 per cent in patients receiving
heparin while it is much lower in those who receive LMWHs.
PATHOPHYSIOLOGY
HIT II has an immunologic aetiology.15 The immunologic basis of HIT
II was first advocated by Rhodes et al.16 who showed that the IgG
fraction from the serum of patients with HIT caused in vitro platelet
aggregation in the presence of therapeutic concentrations of heparin.
It was also reported that immunoglobulin-heparin complexes formed
only in the presence of platelets.17 The pro-aggregating effect of heparin
depends on the degree of sulfation and the molecular weight,18 and is
mediated by the release of substances from the platelet α-granules.19
Several platelet proteins/chemokines were proposed as the putative
receptors of heparin-dependent antibodies,20 and platelet factor 4 (PF4)
was identified as the main co-factor.21 The antibody is not exclusively
specific for the heparin-PF4 (H-PF4) complex, but also for complexes
made up of PF4 and other glycosaminoglycans (GAGs), based on the
degree of sulfation and the length of the polysaccharide.22
The heparin and PF4 ratio appears to be critical for the constitution
of the multimolecular antigenic complex, with an optimal heparin :
PF4 ratio ranging from 1:4 to 6.23 The most accredited pathogenic model
at present is the following: at therapeutic concentrations ranging from
0.1 to 1.0 U/ml, heparin displaces PF4 from endothelial heparin sulfate,
or releases it directly from the platelets; numerous PF4 molecules bind
to a heparin molecule, and the complex becomes immunogenic; the
immune complexes made up of anti-H-PF4 antibodies (mainly IgG)
activate the platelets, and provoke an immune-mediated endothelial
lesion,24 with thrombocytopenia and/or thrombosis. The IgG anti-H-
PF4 immune complex activates the platelets through the bond with
the FcγRIIa (CD32) receptor,25 whose platelet surface expression ranges
from 700 to 4,000 binding sites, and is further increased by the immune
complex bond.26,27 Indeed, platelet activation is blocked by both the
monoclonal antibody (IV 3), specific for the FcγRIIa receptor,28 and the
F(ab’) 2 fractions from patients with HIT II. 29 The Arg/His poly-
morphism at position 131 of the FcγRIIa receptor influences platelet
reactivity to the immune complexes, in particular, the His/His
phenotype is more reactive to the IgG2 isotype. Nonetheless, while some
studies have demonstrated a greater prevalence of HIT II and
thrombotic complications in subjects with the His/His phenotype,30
others have not confirmed these findings.31 Other data are consistent
with the hypothesis that H-PF4 complexes bind directly to platelets
and these complexes are the target for the F(ab’)2 fraction of the
antibody.32
The mechanism by which the anti-H-PF4 antibodies cause
thrombosis is not clear at this time. In general, IgG2 isotype anti-heparin
antibodies are not particularly more frequent than the other sub-classes
in patients with HIT II,33 and IgM and IgA, which are not able to bind
to FcγRIIa receptors are also present in significant percentages in these
patients.34 This suggests that the mechanism of platelet activation may
occur independent of the FcγRIIa receptor for IgG. Moreover, the
antibody isotype tends to modify in relation to the duration of the
treatment. The antibodies are still detectable in patient serum for about
4-6 weeks, and cases of antibody persistence for longer periods of time
have been described. Although the thrombotic complications in HIT
syndrome are well-described, only limited data have become available
on the inflammatory components in this disease.
We have proposed a functional heterogeneity of anti-H-PF4 anti-
bodies based on the fact that heparin is a heterogeneous mixture of
sulfated mucopolysaccharides with molecular, structural, and physical
heterogeneity. Thus, heparins likely form multiple complexes with PF4,

and depending upon the nature of this interaction, the allosteric
modifications in PF4 leading to a neoantigen formation may also vary.
To characterize the anti-H-PF4 antibody in terms of functional
heterogeneity, we obtained IgG fractions from HIT II patients’ serum
utilizing ammonium sulfate precipitation (ASP) and H-PF4-Sepharose
4B affinity chromatography methods.35 With affinity purification, two
major components, Peaks I and II, with high anti-H-PF4 antibody titers
were eluted [purity was established by sodium dodecylsulfate-
polyacrylamide gel electrophoresis (SDS-PAGE)]. Table 19.1 sum-
marizes the platelet activation responses measured by SRA. While Peak
I did not induce serotonin release from platelets in a heparin-dependent
manner, Peak II and the IgGs obtained with the ASP method, exhibited
a strong and concentration-dependent activation in the presence or
absence of heparin (as well as LMWHs). This suggests the generation
of ‘super-active’ HIT antibodies capable of activating platelets without
heparin. The anti-H-PF4 antibody titers of Peak I, Peak II and the ASP-
IgG as measured by HPIA-ELISA (Stago, Asnieres, France) were similar.
To rule out the possible presence of a heparin contamination in the
above IgG preparations (and thus to confirm antibody activity
independent of heparin), heparinase digestion, the use of an ion-
exchange resin (Heparsorb®), and dialysis against 1x phosphate-
buffered saline (PBS) were performed on the HIT sera, Peak II, and
IgG-ASP. None of these treatments resulted in a significant decrease in
the activation of platelets. These observations underscore the complex
pathophysiology of HIT syndrome and suggest that there may be a
HIT antibody active in a non-heparin dependent manner.
Since it is believed that the pathophysiology of HIT II involves the
activation of platelets, endothelium and leukocytes, it would be
Table 19.1: Platelet activation in the presence of heparin (0.1 U/ml) and HIT
immunoglobulin preparations
% Serotonin Release
Sample Range Mean ± S.D.
HIT Negative Serum 0-5 3.2 ± 1.1
HIT Positive Serum 60-92 76 ± 6.2
Ig Peak I* 2-8 4.1 ± 3.2
Ig Peak II* 70-95 79 ± 3.6
IgG-ASP# 35-92 63 ± 9.7
* Isolated from patients (n=3) clinically diagnosed with HIT II; protein content
adjusted to 5 μg/ml
# Isolated from patients (n=3) clinically diagnosed with HIT II; protein content
adjusted to 50 μg/ml (n=6 platelet donors per sample)

expected that the associated activation products, including soluble

selectins and/or cellular adhesion molecules, would be upregulated
in the circulating blood of patients with HIT. Our studies showed that
selectin levels were markedly elevated in HIT II patients (Table 19.2).
Treatment of these patients with the direct thrombin inhibitor
argatroban (Novastan®; Texas Biotechnology Corp., Houston, TX) was
associated with some decrease in the level of selectins at 24 and 72
hours post-treatment. A similar decrease of soluble P-selectin levels
was also noted when the HIT II patients were treated with other direct
thrombin agents (e.g., hirudin and hirulog) as alternate anticoagula-
tion.36 Since thrombin plays an important role in the activation of
platelets resulting in microparticle formation, the selectin level down-
regulation may be related to the inhibition of thrombin generation. In
addition, we have reported an increase in the adhesion molecules ICAM
and VCAM in HIT patients.36 These adhesion molecules may also play
a role in the pathophysiology of HIT.
Additional uncertainties regarding the above pathogenic scheme also
emerged from an experimental model in the mouse, in which the
formation of autoantibodies against the H-PF4 complex produced
thrombocytopenia, but not thrombosis. From a pathogenic point of view,
it is likely that a state of platelet and endothelial pre-activation, and
probably other currently still unidentified features of the patient,
contribute to the thrombotic phenomena.37
Inflammation is an important component in the mediation of HIT
pathophysiology. Recent studies on CRP, IL-6, TNFα and other
mediators have provided additional data on the pathogenesis of this
syndrome. It is therefore important to consider anti-inflammatory drugs
in the management of HIT syndrome.
Table 19.2: Soluble P-, E-, and L-selectin levels in patients from the ARG 911
clinical trial of HIT
24 h post- 72 h post-
Marker Normal Pre-treatment treatment treatment
sP-Selectin
(ng/ml) 27 ± 8 98 ± 10 78 ± 7 69 ±6
sE-Selectin
(ng/ml) 63 ± 12 101 ± 15 95 ± 14 80 ± 11
sL-Selectin
(ng/ml) 1,245 ± 140 1,690 ± 40 1,525 ± 165 1,406 ± 149
Citrated blood plasma samples were collected at pre-treatment and after 24 and
72 h of argatroban anticoagulation (2-10 μg/kg/min infusion adjusted to a
therapeutic aPTT of 60-100 sec). The selectins were measured using ELISA-based
assays (R and D Systems)
CLINICAL PRESENTATION
In HIT II, the onset of thrombocytopenia appears to be independent of
the type of heparin, dosage, and route of administration. The entity of
thrombocytopenia usually varies from 50-100 × 109/L, but severe cases
are frequent.38 There is no gender predominance; although elderly
patients undergoing post-surgical prophylaxis or treatment for deep
vein thrombosis (DVT),39 in particular orthopedic and cardiovascular
surgery, seem to be at higher risk. In more than 60 per cent of the cases,
there are other concomitant prothrombotic factors such as diabetes,
neoplasm, cardiac insufficiency, systemic lupus erythematosus,
anti-phospholipid syndrome, infection and trauma. Besides thrombo-
cytopenia, cutaneous allergic manifestations and skin necrosis may be
present.
Despite the thrombocytopenia, haemorrhagic events are not frequent,
while the major clinical complication is thrombosis. Both arterial and
venous thrombosis can complicate the course of HIT II. The thrombotic
event is a worsening of the thrombosis which necessitates the initial
heparin treatment, or a new thromboembolic complication.40 The
thrombotic complications may appear even in the absence of
thrombocytopenia.41 Arterial thrombosis was the first reported event
to be associated with HIT.42 Nonetheless, today arterial and venous
thrombotic complications are considered to be equivalent.43 Arterial
thrombosis seems to be more frequent in patients with cardiovascular
disease and venous complications are more often found in patients
undergoing post-surgical prophylaxis. The most common arterial
complications are thromboses of the large vessels with gangrene and
limb amputation, stroke, myocardial infarction, and cardiac thrombosis.
Venous complications are DVT, pulmonary embolism, thrombosis of
the cerebral venous sinus, and closure of arterial-venous fistula in
dialyzed patients, disseminated intravascular coagulation and
haemorrhagic adrenal necrosis have been documented occasionally.
DIAGNOSIS
It is commonly held that HIT II is still under-diagnosed. During heparin
therapy, platelet counts must be checked regularly, at least twice weekly,
especially in patients receiving treatment for more than 4 days, or those
who show resistance to heparin or treatment-related to skin
manifestations (Table 19.3). Once thrombocytopenia is confirmed, the
diagnosis of HIT II should be formulated on the basis of clinical criteria
and the in vitro demonstration of heparin-dependent anti-platelet
antibodies. Nonetheless, the diagnosis is still only clinically based
(associated negative lab results) in more than 20 per cent of the cases.43
To evaluate the clinical probability of HIT II, various score systems
have been proposed44 based on the entity of thrombocytopenia, recovery
Table 19.3: Diagnosis of HIT

• Platelet counts twice weekly, especially in patients with:
– Heparin treatment > 4 days
– Heparin resistance
– Related skin manifestations
• Clinical score system:76,77
– Thrombocytopenia
– Recovery following heparin suppression
– Thrombosis
– Cutaneous complications
– Exclusion of other causes
• Laboratory confirmation:
– SRA
– Aggregation
– ELISA-antibody titer
following drug suspension, onset of thrombotic and cutaneous

complications, and the exclusion of other causes of thrombocytopenia.
A score less than 3 suggests a diagnosis of HIT II improbable, from 4-6
it is possible, and greater than 6 would be highly probable.
Among the functional laboratory tests, the SRA is the reference
procedure. This test is based on the capacity of heparin-dependent HIT
antibodies to induce the release of 14C-serotonin from platelets. The
serum from a patient suspected of having HIT II is incubated, in the
presence of therapeutic heparin concentrations (0.1-1.0 U/ml), with
washed donor platelets labeled with 14 C-serotonin. If
heparin-dependent antibodies are present, platelets are activated and
the labeled serotonin is released. In the presence of high heparin
concentrations (10-100 U/ml) the release is inhibited. This method has
several disadvantages in that it requires the use of radioactive material.
The result is highly dependent on the characteristics/reactivity of the
donor platelets45,46 and the procedure is time-consuming. Clearly, a test
that is able to provide results within a few hours with higher sensitivity
could be more useful from a clinical point of view.
The platelet aggregation test, which utilizes a similar principle as
the SRA, is able to furnish quicker results. This test measures platelet
aggregation induced by the HIT patient serum in the presence of
therapeutic concentrations of heparin. While this method is widely used
due to its relative simplicity, the results vary considerably more than
those reported for SRA in relation to the different heparin concentrations
and donor platelet variability.5,7,32 The overall sensitivity of this test is
less than that of the SRA.46,47
The heparin-induced platelet aggregation test (HIPA) on micro-
ELISA plates demonstrates greater reliability and correlation with the
SRA than the platelet aggregation test.48 This test is based on the visual
evaluation of the aggregation of washed platelets from different donors

in the presence of heparin utilizing a magnetically shaken microplate.
There are no reported studies on the use of this test except by one lab.
Regardless of the functional method used to detect the HIT anti-
bodies, the selection of the donor platelets is crucial. The use of washed
platelets vs. platelet-rich plasma improves the test sensitivity because
there could be a platelet over-reactivity in response to heparin not due
to the presence of HIT antibodies. Under most optimal conditions, the
sensitivity of the aggregation and the SRA methods can reach 88 and
94 per cent, respectively.49 Typical response, however, is 50-60 per cent
sensitivity of these assays.
Recently, other functional tests have been suggested, such as the
bioluminescent adenosine nucleotide release assay,50 or the binding of
annexin V to platelet membrane anionic phospholipids utilizing flow
cytometry.51 However, as these tests also employ donor platelets, they
too would be affected by platelet variability. Other flow cytometric
assays have also been developed using small volumes of patient
platelets or whole blood. This approach could provide a major
advantage overall other tests described if sensitivity and specificity to
HIT are proven. Another recently described test employs a solid-phase
adherence method to demonstrate the presence of heparin-dependent
antibody.
Following the demonstration of antibodies against the H-PF4
complex in the serum of patients with HIT II, the ELISA technique for
the detection of these antibodies was introduced. In this technique, a
well-defined ratio of heparin and PF4 complexes is bound to microtiter
plates by means of a covalent or electrostatic bond. The patient serum
is appropriately diluted and incubated with the complex. The presence
of HIT antibodies is detected with a secondary antibody conjugated
with peroxidase or alkaline phosphatase.
The ELISA method shows a good correlation with the SRA procedure,
but comparison with the aggregation method is less reliable. The ELISA
method is characterized by a greater sensitivity than the functional
tests and a greater reproducibility because it does not employ donor
platelets. Moreover, this procedure is technically easier to perform and
may reveal IgM and IgA isotype antibodies as well as IgG. Nonetheless,
the ELISA method has demonstrated questionable specificity in that
anti-H-PF4 antibodies are detected in heparin-treated patients who did
not present thrombocytopenia and in most patients undergoing heart
surgery. More importantly, this test was negative in patients with HIT
confirmed clinically or by positive functional tests.
It is possible that the complete development of HIT II only occurs
with high antibody titers, after persistent exposure to heparin, or that
antigens different from the H-PF4 complex are involved in its
pathogenesis. Caution is advised in interpreting the ELISA assay data
as assays from different manufacturers have different sensitivities and

specificities. However, the presence of H-PF4-dependent IgG seems to
be related to the clinical probability of HIT II, as judged by the high
score values in patients with antibodies against the H-PF4 complex.
In general, aside from the varying sensitivity of the methods and
the lack of standardization, ELISA tests have proven to be predictive
of the diagnosis of HIT II or of the thrombotic risk. Therefore, especially
in the absence of a highly suggestive clinical picture, it appears appro-
priate to support the clinical diagnosis with a functional test as well as
the measurement of anti-H-PF4 antibody titer by ELISA test.
Improved ELISA methods based on the detection of IgG subtype
antibodies will be more useful in the diagnosis of Heparin Induced
Thrombocytopenia. Binary immunoassay with heparin platelet factor
4 complex along with the anti-IgG II heparin platelet factor 4 will be
useful.
A recent consensus publication has provided the recommendation
of the College of American Pathologists Expert Group Conference XVI
on the diagnosis of HIT. Several issues remained unresolved and the
need for a more reliable diagnostic test is identified.
THERAPY
The best therapeutic strategy for patients with HIT II is not yet clearly
established but reasonable guidelines have a wide consensus (Table
19.4). If HIT II is clinically probable, heparin therapy must be immedi-
ately discontinued even in the absence of a confirmatory laboratory
test. Platelet transfusion is contraindicated because it may worsen the
thrombotic picture. Anticoagulant therapy with vitamin K antagonists
should be initiated 3 or 5 days after heparin suspension when platelet
Table 19.4: Therapeutic management of patients with HIT
• Primary
– Discontinue heparin
– Plasmapheresis
– Immunoglobulin
– Vitamin K antagonist (3-5 days after heparin with increasing
platelets)
– Heparinoid if negative lab test
– Antiplatelet agents (GPIIb/IIIa inhibitors)
– Anti-thrombin agents
• Secondary
– Prostaglandin
– Ancrod
– Thrombolytics
– Venacava filter
– Thrombectomy
– Immune suppression
counts are increasing, but preferably before resolution of the thrombo-

cytopenia in order to avoid potential worsening of the thrombotic
picture. However, heparin suspension alone and substitution with
dicumaroids do not prevent the onset of severe thrombotic compli-
cations in nearly 50 per cent of the patients. On the basis of these
disappointing results, new approaches have been proposed that
included the use of LMWH, heparinoids, anticoagulating agents such
as ancrod, prostaglandin (Iloprost®), antiplatelet drugs, and thrombin
inhibitors (argatroban and hirudin).
Among these various drugs, LMWHs, heparinoids, ancrod,
argatroban and hirudin have been used in a significant number of
patients. A small clinical trial was conducted with ancrod, a viper-
derived venom with anticoagulant action which showed efficacy. Other
reports describe the use of plasmapheresis to remove immune
complexes or high doses of immunoglobulin alone or associated with
LMWH and a heparinoid.52 In addition to the alternative anticoagulants,
also indicated for use in patients with HIT II are thrombolytic agents,
the insertion of filters in the inferior vein cava, and in the case of arterial
thrombosis with limb ischaemia, embolectomy or thrombectomy.
LMWHs or the heparin pentasaccharide could theoretically represent
a therapeutic option for the HIT II. The rationale resides in the fact that
with a decrease in the molecular weight and degree of sulfation, the
interaction with PF4 diminishes.53 However, the cross-reactivity with
heparin-induced antibodies in vitro ranges from 60 to 100 per cent.54
The general opinion, therefore, is to not administer LMWHs unless the
absence of cross-reactivity has been demonstrated by an in vitro test.
Nonetheless, some reports describe cases in which the use of LMWH
was efficacious in controlling HIT even though cross-reactivity with
heparin had been evidenced. On the other hand, no in vitro study has
ever demonstrated a positive response with the ultra-low molecular
weight pentasaccharide. Future clinical studies of pentasaccharide in
patients with HIT are needed to prove its efficacy.
In a study from our group, two synthetic heparin pentasaccharides
(SR90107A/Org31540 and SanOrg34006) which are in clinical
development for the prophylaxis of post-surgical DVT, were tested in
comparison to heparin and a LMWH (enoxaparin) for their relative
platelet activation potential in HIT assays. Sera from HIT II patients (n
= 30), validated for heparin-dependent aggregation responses, and
antibodies purified by H-PF4-Sepharose column separation were used
to study the effects of the four drugs using platelet aggregation. At
comparable concentrations, heparin and enoxaparin consistently
produced platelet activation (Table 19.5), whereas both pentasaccharides
failed to produce a response at concentrations up to 100 U/ml
(–50 μM). Similarly, in the SRA and flow cytometric assays, both heparin
Table 19.5: HIT sera-mediated platelet activation determined by

functional assays
% Platelet % Serotonin % P-selectin % Microparticle
Agents Aggregation Release Expression Formation
Heparin (U/ml)
Saline 12 ± 3 68 ± 8 5±2 4.1 ± 3
0.1 54 ± 10 76 ± 6.2 50 ± 7 24 ± 17
100 15 ± 4 5±3 10 ± 2.5 8 ± 3.5
Enoxaparin (U/ml)
0.1 45 ± 6 80 ± 9 45 ± 8 30 ± 9
100 11 ± 3.9 49 ± 7 12 ± 4 9±3
Pentasaccharide (U/ml)
1.0 16 ± 3 7 ±2 6±2 4±1
10 18 ± 2 8±4 5 ±2 10 ± 3
100 19 ± 4 7±3 7±3 5±2
Data represent mean percent (± SEM) responses of responses to 25 HIT patient

sera.
and enoxaparin produced positive responses, whereas the two

pentasaccharides consistently failed to produce any effect.
We have further shown that in patients from a clinical trial sub-study
where pentasaccharide was administered for the prophylaxis of DVT
after hip surgery, no anti-H-PF4 antibody was detected during the
treatment period (n = 10). However, in a comparable study with
enoxaparin, 6 out of 20 patients without clinical thrombocytopenia
showed a positive anti-H-PF4 antibody titer. These observations suggest
that the pentasaccharides with highly selective anti-Xa activity are
devoid of generating anti-H-PF4 antibody, do not produce HIT
responses in the presence of antibody.
At present, the use of a low molecular weight heparinoid, and direct
thrombin inhibitors is considered the most safe and efficacious
therapeutic approach for HIT. The major reported experiences concern
danaparoid sodium (Org10172, danaparoid, Lomoparan®; Organon,
Oss, The Netherlands), a mixture containing heparin sulfate (85%),
dermatan sulfate (10%), and chondroitin sulfate (5%), whose
cross-reactivity with heparin in vitro is less than 10 per cent. More than
600 patients with HIT II have been successfully treated with this drug,
with a remarkable reduction in mortality due to thrombotic
complications and in overall mortality in patients treated early,
compared to others.55 However, cases of failure of treatment or of
danaparoid-induced fatal thrombotic thrombocytopenia56 have also
been reported. In particular, treatment with danaparoid resolved the
thrombocytopenia in 91 per cent of the cases and significantly reduced
mortality due to thrombotic complications of HIT from 28 to 5 per cent,
but not total mortality which was 20 per cent. Danaparoid has been
approved for the treatment of HIT II in New Zealand, Denmark,
Luxembourg, Belgium and Portugal.
Direct thrombin inhibitors are also indicated for the treatment of
HIT II syndrome. The first large scale clinical trial of HIT patients treated
with a thrombin inhibitor used argatroban, a thrombin inhibitor based
on the L-arginine structure. A number of patients with HIT II associated
thrombosis requiring angioplasty were also successfully treated with
argatroban. Hirudin, another thrombin inhibitor, was evaluated for use
in the treatment of HIT II mostly in Germany, where it was found to be
efficacious. Recent studies have shown a differential therapeutic index
of different thrombin inhibitors in the management of Heparin Induced
Thrombocytopenia from the review of clinical trials and post-marketing
surveillance studies clearly show that Argatroban is a better
anticoagulant for this syndrome. This is due to the multiple sites of
action of this agent. Furthermore, a recent alert from European
medicines equivalence agency (EMEA) regarding the anaphylactic
responses upon re-exposure with hirudin have raised concerns over
the safety of this drug. Additional studies on safety issues on all of the
anti-thrombin agents are needed at this time.
Another possibility, especially for patients with severe thrombotic
complications refractory to thrombin inhibitor treatment alone is the
use of anti-platelet agents. It has recently been demonstrated by several
in vitro studies that antagonists of GPIIb/IIIa, are able to inhibit platelet
aggregation induced by the serum of patients with HIT II. The inhibition
by GPIIb/IIIa inhibitors was more effective than inhibition of platelet
activation by aspirin. The clinical usefulness of this treatment, combined
with low dose anti-thrombin agents has shown beneficial results.
We have recently shown that glycoprotein (GP) IIb/IIIa receptor
inhibitors, such as Aggrastat®, Integrilin®, and other synthetic GPIIb/
IIIa receptor inhibitors in development (e.g., SR121566A; Sanofi,
Toulouse, France), inhibit HIT serum-induced platelet aggregation. Our
studies revealed that HIT antibodies, in the presence of low heparin
concentrations (0.1-1.0 U/ml), showed strong platelet aggregation and
14
C-serotonin release responses. GPIIb/IIIa inhibitors produced a strong
inhibition of the HIT antibody mediated platelet aggregation responses
(IC50 in the range 40-60 nM). In addition, the platelet aggregation and
14
C-serotonin release responses induced by these antibodies were shown
to be independent of heparin; however, at >10 U/ml heparin, an
inhibition of these responses was noted. A weaker inhibition of these
‘super-active’ (heparin independent) HIT antibodies (IC50 in the range
400-300 nM) was observed.
Currently, several newer antithrombotic drugs are being developed.
These include synthetic anti-Xa inhibitors with both parenteral and
oral bio-availability, tissue factor inhibitors, thrombin activatable

fibrinolytic inhibitor modulators, PAI-1 inhibitors and heparinase. These
agents may have a value in the overall management of HIT. However,
with the advances in technology heparin manufacturing processes can
be improved and structure can be modified to reduce its ability to bind
with PF4 to trigger antibody generation.
Recently we proposed that the prevalence of HIT antibodies in
patients treated with immunosuppressive agents (such as cyclosporine)
would be lower than in non-treated patients. Cyclosporine is used to
suppress the immune system of the transplant recipient to prevent the
production of antibodies against the foreign major histocompatibility
(MHC) factor of the donor organ thus reducing the incidence of
rejection.
We tested the anti-H-PF4 antibody levels in cardiac surgery patients
(n = 48, who received heparin) at baseline (pre-surgery), 5-7 and 14
days post-surgery using an ELISA test and compared the values to anti-
H-PF4 antibody titers in a group of cardiac transplant patients (n =
30).57 The samples showing high antibody titer were tested for the
presence of functional HIT antibody using the SRA. At 1 week, 23 per
cent of the cardiac surgery patients had positive antibody titers with
6.3 per cent of these positive by SRA (Table 19.6). In contrast, only 10
per cent of transplant patients exhibited positive antibody titers and
none of these were SRA positive. Both groups showed diminished
antibody titers and SRA responses at 14 days. In an additional study,
two groups of patients with rheumatoid arthritis (n = 9) and anti-
phospholipid syndrome (n = 21) were treated with heparin and
immunosuppressive therapy. None of these patients exhibited a positive
anti-H-PF4 antibody titer.
These observations suggest that patients treated with immunosup-
pressive agents have a decreased generation of anti-H-PF4 antibodies.
The clinical implication of this finding is that patients at high risk may
be prophylactically treated with mild immunosuppression prior to
heparinization to minimize the risk of HIT II. Clinical investigations
are needed to validate this hypothesis.
Table 19.6: HIT assay results in cardiac surgery and transplant patients
receiving cyclosporine
Positive Antibody Titer* SRA Positive
Group Number of Patients (% Prevalence) (% Prevalence)
Cardiac Surgery 48 11 (22.9%) 3 (6.3%)
Cardiac Transplant 30 3 (10.0%) 0 (0%)
* Diagnostic Stago HPIA-ELISA.

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20
Neonatal Thrombosis
Renu Saxena, M Kannan
Key Words
Thrombosis • Neonates • Lab Investigation and
Management
INTRODUCTION
Neonatal thrombosis is a serious event that can cause mortality or result
in severe morbidity and disability.1 Neonates and infants, less than one
year of age-account for the largest proportion of thrombotic events seen
in the paediatric population.2 These events, however, remain relatively
uncommon and often occur in sick-term and pre-term infants: the most
important risk factor for the development of thrombosis during the
neonatal period is the presence of an indwelling central line and conse-
quently the vessels involved tend to be those most frequently used for
catheterization. Other documented risk factors for the development of
neonatal thrombosis include asphyxia, septicemia, dehydration,
maternal diabetes and cardiac disease. Spontaneous, noncatheter related
thrombotic events are uncommon in the neonatal period and most
frequently involve the renal vein. The majority of cases of renal vein
thrombosis present during the first few days of life and in approximately
a quarter of these the thrombosis is bilateral with a smaller proportion
also developing extension into the inferior vena cava. Moreover, the
coagulation and the fibrinolytic systems of infants are different from
those of adults. Normally, the plasminogen, when activated, is
converted to plasmin, which lyses the fibrin clot. Newborn infants have
dysfunctional and decreased concentration of plasma plasminogen and
tissue plasminogen activators. They also have increased concentration
of tissue plasminogen activator inhibitors.3 In addition, severe transient
deficiencies of antithrombin III, protein C and protein S in sick newborn
infants increase the risk of vascular thrombosis.4,5
Pathophysiology
Thrombosis in the neonatal period and in childhood is mainly arterial.
This is puzzling since infancy is often accompanied by a hypocoagulable
state. Normal levels of clotting factors are usually achieved within six
to twelve months. However, it has to be remembered that vascular
lesions play an important role in neonatal thrombosis. Atrial thrombosis
has been described in a variety of conditions with or without atrial
fibrillation; it has also been documented in defects of clotting inhibitors.
The causative role of congenital deficiencies of anticoagulants in
neonatal thrombosis is clear only in the homozygous/double hetero-
zygous deficiencies of Protein C and Protein S which result in neonatal
purpura fulminans.
Protein C
Protein C is a Vitamin K dependent protein and levels are physio-
logically reduced in the newborn. Homozygous protein C deficiency
is usually easily diagnosed in the neonate with levels often undetectable
at presentation. In contrast, the wide range of protein C levels seen in
the normal neonate can make heterozygous protein C deficiency
difficult to diagnose and assays may have to be repeated after six
months to confirm a deficiency. Assays of other vitamin K dependent
coagulation proteins for comparison can be of help in the diagnosis, as
can the measurement of parental levels of protein C.
Protein S
Total protein S levels in the neonate are low when compared with adult
ranges but the protein is present almost totally in its free form. Free
protein S levels, however, are low when compared with adult values
and increase to the adult range by 4 months of age, total protein S
increasing similarly in the first 10 months of life. As with protein C,
homozygous protein S deficiency is usually associated with low or
undetectable levels of protein S in this age group and again, the
heterozygous state can be difficult to diagnose definitively until a later
age. Antithrombin levels are reduced in the term neonate and more so
in the premature infant: they will remain reduced at least for the first 3
months of life.
Resistance to Activated Protein C

The wide variations in neonatal factor VIII levels make this an
inappropriate test in this age group and the Factor V Leiden genotype
should be looked for directly.
Factor V Leiden and Prothrombin 20210A

Each of these single point mutations is associated with an increased
risk of venous thrombosis in adults and children and is identified using
Neonatal Thrombosis 323
PCR techniques.6,7 Approximately 4 per cent and 2 per cent respectively

of Caucasians are heterozygous for these gene defects. Their causative
role in neonatal thrombosis is unknown but they may have a contri-
butory role in the pathogenesis of thrombosis in this particular age
group.
Abnormalities of the Fibrinolytic System

Plasminogen levels at birth are approximately 50 per cent of adult values
but homozygous plasminogen deficiency does not appear to be
associated with thrombosis8 and it would therefore not seem justified
to include this investigation in thrombophilia testing.
Other Investigations of Thrombophilia

Babies of women with systemic lupus erythematosus and/or
antiphospholipid syndromes may infrequently develop thrombosis in
the presence of associated autoantibodies (lupus anticoagulant,
anticardiolipin antibodies). Neonatal thrombotic events have
occasionally been reported in association with maternal systemic lupus
erythematosus due to the transplacental passage of antiphospholipid
antibodies.9 This also may occur secondary to DIC. Testing thrombotic
neonates for these disorders in the absence of a maternal history is not
justified because of the rarity of such findings. Associations of neonatal
thrombosis and other thrombophilic defects such as the methylene-
tetrahydrofolate reductase (MTHFR) C677T genotype and increased
serum levels of lipoprotein have recently been reported.10 It is becoming
increasingly recognized that there is a high prevalence of other
thrombophilic defects in children who develop venous thrombosis,10-
12
though the causative nature of these defects and their influence on
the natural history of the thrombotic event still remain uncertain. It is
not possible, however, to make the assumption that the aetiology of
thrombosis in children and adults is similar or that the response to
treatment and long-term complications of thrombosis are the same in
these two age groups.13 Acquired deficiencies of protein C and protein
S, particularly in sick pre-term infants, may also increase the risk of
thrombosis.14
DISSEMINATED INTRAVASCULAR COAGULATION (DIC)

DIC always occurs as a secondary event to another disease entity. The
incidence is particularly high during the neonatal period, especially in
preterm infants. The clinical spectrum of neonatal DIC varies greatly
from apparently asymptomatic cases of low grade compensated DIC
to fulminant DIC characterised by bleeding and thrombosis.
Intrapulmonary and intraventricular haemorrhage may be exacerbated
in preterm infants by thrombocytopenia and an uncompensated
coagulopathy. Disorders associated with neonatal Disseminated

Intravascular Coagulation include Fetal/neonatal disorders and
Maternal/Obstetric Disorders.
Clinical Feature of Neonatal Thrombosis

Thrombosis of Congenital thrombophilia aetiology should be
considered in any child with a clinically significant thrombosis, parti-
cularly a spontaneous thrombotic event, unanticipated or extensive
venous thrombosis, ischaemic skin lesions or purpura fulminans and
particularly with a positive family history of neonatal purpura
fulminans. Asymptomatic neonates should not be investigated unless
there is a significant medical history such as previous neonatal purpura
fulminans or thrombosis. Parents need counseling before any investiga-
tions are performed either on themselves or their children so that the
consequences of testing asymptomatic individuals can be fully
explained.
Protein C and S Deficiencies

Onset is usually within the first few days of life and can occur within
hours of birth. The microcirculation is characteristically affected first
with the development of purpura fulminans associated with laboratory
evidence of DIC. Cerebral and renal vein thromboses are common and
can be presenting features. Ocular manifestations are also characteristic
and it is likely that cerebral and ophthalmic thrombosis often occur as
intrauterine events. Cases of associated entities with neonatal gangrene
include prematurity, congenital syphilis, asphyxia, maternal diabetes,
polycythemia, dehydration, protein C deficiency and emboli from
closing patent ductus arteriosus.15-20 Although peripheral gangrene is
not seen after most difficult and traumatic deliveries, oligohydramnios
or ‘dry’ labor and birth trauma were noted as causes of gangrene.21
Less well appreciated is the rare problem of limb gangrene presenting
at birth or shortly following birth in the absence of any obvious
precipitating cause. Turnpenny et al reported that the gangrene in the
neonate appeared to predominate in the upper limbs, male cases
predominate over female and in the majority of reports no aetiologic
factor was identified.22 Doppler ultrasonography is the most useful
and most popular method of diagnosis of neonatal thrombosis,
however, its diagnostic accuracy remains uncertain. Angiography is
the best method for confirming the diagnosis.23,24
LABORATORY INVESTIGATION OF NEONATAL HAEMOSTASIS,

THROMBOSIS AND FIBRINOLYSIS
Laboratory investigation should identify neonates with inherited or
acquired disorders of coagulation. The physiological deficiencies of
procoagulant and anticoagulant proteins can cause difficulties in

distingwishing the pathological alterations from the physiological ones.
Moreover the correct interpretation of such investigations is dependant
on an awareness of the ‘normal’ levels of these proteins in the neonate
and how these are influenced by gestational age, postnatal age and by
external factors such as sepsis or vitamin K deficiency. In particular,
the diagnosis of mild congenital factor deficiencies will require repeat
testing later in infancy for confirmation of the diagnosis. The gestation
dependent differences between neonates and adults together with the
evolving state of the newborn’s coagulation system in the early weeks
and months of life necessitate sequential reference ranges which reflect
the effects of gestational and postnatal age. Since screening tests and
factor assays are influenced by a number of variables including test
methodology, reagents and instrumentation, laboratories should
wherever possible establish their own normal ranges for neonates of
different gestational ages.25
INVESTIGATION OF THE NEONATE

Sampling
Sample Collection
Present day automated coagulometers have reduced the need for
laboratories to establish manual microtechniques for coagulation
screening. One ml of blood is sufficient to perform a coagulation
screening and an additional one ml will be necessary for subsequent
procoagulant or anticoagulant assays. Blood sampling from a neonate
should avoid contamination with intravenous fluids, particularly
heparin. Neonatal units should have established blood discard
procedures to minimise the risk of contamination. Platelet clumping is
common in such samples, resulting in spurious thrombocytopenia. All
neonatal samples should be inspected for fibrin strands and platelet
clumps to help interpretation of results.
Sample Anticoagulant
Ideally the volume of anticoagulant in the sample tube should be based
on the volume of plasma and not on the total volume of blood taken. It
should, therefore, be reduced in proportion to the increased neonatal
haematocrit in order to avoid dilution of coagulation factors and this is
particularly pertinent for neonates with very high haematocrits.26
Laboratories and neonatal units usually take a more pragmatic
approach to this problem and accept a degree of ‘artefactual’ prolonga-
tion of the coagulation times. Blood is therefore usually taken into 3.2
per cent buffered sodium citrate, one part citrate to nine parts blood.
Laboratory Investigation
Main laboratory findings in the diagnosis of hypercoagulable states,
include:
• Short global tests (PTT)
• Decreased levels of inhibitors (AT III)
• Increased resistance to activated protein C
• Defective fibrinolysis (basal and after stimuli)
• Increased levels of clotting factors (fibrinogen, factor VII, factor
VIII, etc.)
• Increased and/or hyperactive platelets
• Increased whole blood and/or plasma viscosity
• Antiphospholipid antibodies.
As far as the vein walls are concerned, laboratory investigation is
less complicated but equally important. Careful echodoppler and/or
compression ultrasonography of the veins of the extremities, CT and/
or MRI of the thorax and/or abdomen for the venae cavae, and
phlebography may be needed.
LABORATORY EVALUATION OF THE FIBRINOLYTIC SYSTEM

Global Tests
Whole blood clot lysis time, Euglobulin lysis time, Fibrinolytic activity
on fibrin plates and Thromboelastogram (TEG)
Tissue Type Plasminogen Activator (TPA) and Urokinase

a. Functional assay: chromogenic methods
b. Antigen assay: ELISA methods
Plasminogen Activator Inhibitor (PAI)

a. Functional assay: chromogenic method
b. Antigen assay: ELISA method
Plasminogen and Antiplasmin

a. Functional assay: chromogenic method
b. Antigen assay: electroimmunoassay, radial diffusion, crossed
immunoelectrophoretic assay, lasernephelometer.
Fibrinogen/fibrin Degradation Products and D-dimer

a. Latex agglutination
b. ELISA
Molecular Biology Analysis

a. FV Leiden
b. P20210
c. MTHFR
Levels of the vitamin K dependent coagulation proteins (factor II,

VII, IX, X) and the naturally occurring inhibitors protein C and protein
S are physiologically low at birth and these proteins are functionally
inactive in the absence of vitamin K.
MANAGEMENT OF NEONATAL THROMBOSIS

The management of arterial and venous thromboembolic events in the
neonatal period remains controversial and it is generally acknowledged
that there is an urgent need for large multicentre studies on which to
base recommendations. Treatment options include supportive care only,
anticoagulant therapy with heparin or low molecular weight heparin
(LMWH), thrombolytic therapy and surgery. Warfarin is not indicated
in the neonatal period because of the difficulties in establishing
consistent levels of anticoagulation.
Supportive Therapy
In the absence of controlled studies indicating the efficacy of more
aggressive therapy, supportive care alone may be appropriate manage-
ment for clinically silent thrombosis, which will, therefore, include the
majority of small, asymptomatic catheter related events.27 Regular
objective monitoring should be performed to detect extension of the
original thrombus and in the case of catheter related events, catheter
removal is recommended.
Anticoagulant Therapy
In the presence of more extensive, clinically significant thrombosis,
particularly where there is evidence of organ or limb dysfunction,
consideration should be given to the use of anticoagulant therapy.
Unfractionated heparin remains the most frequently used anticoagulant,
although there is increasing experience with LMWH in this age group.
The use of heparin in the neonatal period is complicated by the
physiological immaturity of the haemostatic system, with reduced
levels of antithrombin, resulting in relative heparin resistance.28
HEPARINISATION OF NEONATES AND DOSE ADJUSTMENT

LOADING DOSE OF HEPARIN
Heparin is given in the loading dose of 75 IU/kg body weight, then
continuous infusion: 28 IU/kg/hour. Monitor APTT level prior to and
after administration of heparin, to avoid over heparinization. In the
absence of a validated therapeutic range for the use of heparin in
neonates, the APTT should be prolonged to a therapeutic range
corresponding to an anti-Xa level. Also, standard anti Xa assays will
tend to underestimate heparin unless the neonatal antithrombin
deficiency is fully corrected in the test system. Nomograms are available

for dose adjustment,29 and can be modified by individual laboratories
in order to achieve their own therapeutic APTT range. Thrombocyto-
penia is common in the sick neonate and every attempt should be made
to maintain a platelet count above 50 × 109/l during heparin therapy.
The optimal duration of anticoagulation remains undefined but short
term therapy (e.g. 10-14 days) is commonly used, with objective
radiological monitoring performed both during and after completion
of anticoagulant therapy. Low molecular weight heparin is becoming
increasingly used in the treatment of neonatal thrombosis. Dose finding
studies have been published and indicate that as with standard heparin
dose requirements in the neonate are higher than in older children.30,31
For example, the recommended dose of enoxaparin is 1.5 mg/kg/dose
administered subcutaneously twice per day which should result in a
therapeutic anti- Xa level of between 0.5 to 1.0 units/ml by chromogenic
assay at 4 hours post-dose.
Thrombolytic Therapy
Thrombolytic therapy should be considered in the presence of extensive
thrombosis with organ dysfunction or where limb viability is
threatened. Such therapy should not be used within 10 days of surgery
or in the presence of pre-existing bleeding problems. Streptokinase,
urokinase and tissue plasminogen activator (tPA) have all been used
in neonates but overall experience is relatively limited and results
conflicting.32 As with heparin the response to thrombolytic agents in
the neonate is significantly different from that seen in older children,
reflecting physiologically reduced levels of plasminogen. In vitro studies
have demonstrated that tPA is more effective than streptokinase and
similar to urokinase at lysing thrombi in plasmas with decreased con-
centrations of plasminogen. Tissue plasminogen activator (tPA) and
urokinase are therefore the preferred agents for thrombolytic therapy
in neonates.
Prophylactic Anticoagulation
Heparin prophylaxis is recommended for neonates with indwelling
umbilical artery catheters and those undergoing cardiac catheterisation.
Umbilical artery patency can be prolonged by the use of low dose
heparin (35 U/hr) by continuous infusion. Thrombotic complications
associated with cardiac catheterisation can be reduced by the
administration of a bolus of heparin (100-150 units/kg) as the femoral
artery is catheterised.33
Management of Protein C Deficiency

The most important aspect of management in the acute phase is the
immediate and adequate replacement of the deficient inhibitor. Protein
deficient neonates should receive protein C concentrate as a starting

dose of 40 IU/kg, subsequent dosage being based on protein C recovery
data. If concentrate is not immediately available, FFP 10-20 mls/kg
should be used as a temporary measure. During the early stages of
replacement therapy when DIC is ongoing the half life of the infused
protein C may be as short as 2 to 3 hours, necessitating frequent dosing.
This usually improves to about 10 hours once the DIC is controlled,
enabling a single daily treatment to be given.34 Protein C levels should
remain in excess of 0.25 U/ml to prevent further thrombosis.35
There is no currently available protein S concentrate and FFP is,
therefore, used for replacement therapy (10-20 ml/kg every 8 to 12
hours). Long term management remains controversial but oral
anticoagulants with or without concomitant replacement therapy are
used. It is difficult to warfarinise infants until their vitamin K dependent
factors have physiologically increased and affected babies should
preferably receive replacement therapy. Older children receiving
warfarin seem to require an INR at the upper end of the therapeutic
range (3 to 4.5) to prevent recurrent skin necrosis and, therefore, require
careful monitoring. Where possible, dosing should be individualised
to identify the minimum dose required to remain symptom free. In the
event of recurrent problems during warfarin therapy, temporary or
longer term reintroduction of protein C replacement may be required.
Similar problems have been reported in the management of
homozygous protein S deficiency where again intermittent replacement
with FFP may be required in addition to oral anticoagulants.36 Low
molecular weight heparin may be a useful therapeutic option in the
long term treatment of homozygous protein C deficiency in which
plasma levels remain detectable but its place in the management of
cases with undetectable levels has not been determined as yet.37
Other Homozygous Defects

Homozygous Antithrombin deficiency has been recorded very
infrequently.38 Long-term anticoagulation has been successfully used
in this disorder. Homozygosity for the Factor V Leiden mutation rarely
presents in childhood and a significant number of adults with this
mutation also remain symptom- free throughout their lives, the same
being true for individuals homozygous for the Prothrombin G20210A
mutation.
Management of DIC
The most important aspect of management is reversal of the underlying
disease process. Acidosis should be corrected, tissue perfusion
maintained and the neonate well oxygenated. Beyond this, there are
no clear guidelines on the optimal management of neonatal DIC and a
virtual absence of recent randomized controlled trials addressing the

available treatment options. Blood product replacement is indicated
for the treatment of clinical bleeding in the presence of laboratory
confirmation of DIC.
CONCLUSIONS
It is our impression that no clear distinction can be made between
venous and arterial thrombophilia. Laboratory evaluation is common
to the two conditions since all aspects of blood coagulation, platelet
function, fibrinolytic system and rheological parameters should be
carefully evaluated in every patient with a family history of thrombosis
or with a past history of thrombosis. Arterial wall changes should also
be taken into due account. A thrombophilic state is a prothrombotic
state, namely a condition predisposing to thrombosis, and therefore
should be carefully considered as such. The thrombotic event, venous
or arterial, may merely be the result of additional acquired conditions
which act as a trigger. The role of these triggering factors is widely
recognized but few, if any, systematic studies have been carried out.
For example, given a thrombophilic state, regardless of the type and
nature, arterial thrombosis may manifest itself only if vascular lesions
are present. It is possible that arterial thrombosis is a more severe
manifestation of a thrombophilic state than venous thrombosis. This
appears to be sustained, for example, by the observation that the
homozygous protein C and protein S deficiency is associated with severe
arterial thrombosis, whereas heterozygous patients present mainly
venous thrombosis. Furthermore, arterial thrombosis may be less
frequent than venous thrombosis but it is still present in venous and
arterial thrombophilia.
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thromboembolic complications (VTE) in children: First analyses of the
Canadian Registry of VTE. Blood 1994;83:1251-57.
3. Symnasky MR, Fox HA. Umbilical vessel catheterization: Indications,
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prothrombin gene is associated with elevated plasma prothrombin levels and
an increase in venous thrombosis. Blood 1996; 88: 3698-3703.
8. Mingers AM, Philapitsch A, Schwarz HP, Zeitler P, Kreth HW. Poly-
morphonuclear elastase in patients with homozygous type I plasminogen
deficiency and ligneous conjunctivitis Seminars in Thrombosis and
Haemostasis 1998;24:605-12.
9. Tabbutt S, Griswold WR, Ogino MT. Multiple thromboses in a premature infant
associated with maternal phospholipid antibody syndrome. J Perinatol
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12. Clagett GP, Ledesma DF, Commentary on Lawson JA. In situ vein grafts require
more secondary procedures than reversed grafts after infrainguinal bypass
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13. Walker ID, Greaves M, Preston FE. Guidelines: Investigation and management
of heritable thrombophilia. Br J Haematol 2001;114:512-28.
14. Manco-Johnson MJ, Abshire TC, Jacobson LJ, Marlar RA. Severe neonatal
protein C deficiency: prevalence and thrombotic risk. J Pediatr. 1991;119:793-
98.
15. Payne RM, Martin TC, Bower RJ, Canter CE: Management and follow-up of
arterial thrombosis in the neonatal period. J Pediatr 1989;114:853-58.
16. Jaiyesimi F, Effiong CE, Lawson EAL: Congenital gangrene of an extremity in
a Nigerian neonate. Clin Pediatr 1976;15:283-85.
17. Corrigan JI: Neonatal thrombosis and the thrombolytic system: Pathophysilogy
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18. Idowu J, Hertzler JH, Philipart AI: Occlusion of abdominal aorta in the
newborn. J Pediatr Surg 1986;21:971-72.
19. Nazer H, Rajab AA, Qaryouti S, Zubrow A, Razi N, Askin SR et al: Neonatal
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20. Stravosky M, Iellin A, Spirer Z: Acute ischemia of the limb in a newborn treated
successfully by thrombectomy. Am J Surg 1975;129:337-40.
21. Johnson D, Rosen JM, Khoury M, Stevenson D. Infarction of the upper limb
associated with oligohydramnios and intrauterine compression. J Hand Surg
1988;13A:420-22.
22. Turnpenny PD, Stahl S, Bowers D, Bingham P: Peripheral ischaemia and
gangrene presenting at birth. Eeur J Pediatr 1992;151:550-54.
23. Schmidt B, Andrew M. Report of scientific and standardization subcomittee
on neonatal haemostasis. Diagnosis and treatment of neonatal thrombosis.
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24. Andrew M, Paes B, Johnston M. Development of the human coagulation
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Antithrombotic properties of heparin in a neonatal piglet model of thrombin
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WE, Homozygous protein S deficiency: 7 year follow up. Thrombosis and
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21
Anaemia in the Newborn
MR Lokeshwar, Tanu Singhal,
Nitin Shah
Key Words
Haematologic parameters in newborn • Foeto-
maternal haemorrhage • Foeto-foetal haemorrhage
• Congenital defects of red cells
INTRODUCTION
Haematology of newborn, particularly anaemia in neonatal period
remains cardinal concern even today, not only because of unique blood
picture during this period, normal variation in haematological
parameters, but also in no other period of life anaemia is known to
occur due to such varied causes, as occurs in first few weeks of life.
Neonatal period is the most dynamic period in human life, as during
this period there occur profound alterations and adjustments, especially
during transit of foetus from dependent, hypoxic, intrauterine life-to
totally independent extrauterine existence. Although the foetus is
nourished and protected by the mother during this period, he/she may
suffer adverse effects related to maternal malnutrition, illnesses,
infections, drug ingestions etc. The proximity of two circulatory systems
(mother and the baby) also permit the free passage of formed blood
elements between mother and foetus as seen in foeto-maternal haemor-
rhage leading to anaemia and sensitization to RBC antigens and
materno-foetal haemorrhage leading to hyperviscocity syndrome.
Erythrocytic system undergoes serial adaptation to meet progressively
changing demand to oxygen in embryo, foetus and neonates. Numerous
physiological changes occur in succession and rapidity to adapt new
pattern of life. This leads to rapid change in normal haematological
parameters from foetal period to immediately after birth and through-
out neonatal period even hours, days and weeks after birth. A switch
finally occurs, mainly after delivery wherein foetal haemoglobin is
replaced by adult haemoglobin, because breathing neonate starts to
extract oxygen from the lungs rather than from its mother’s blood.
Hence, interpretation of laboratory findings and institution of
appropriate therapy requires understanding of maturational process
and normal physiological variations that takes place during this
period.1-4
Normal Values of Haematological Parameters in the Newborn

Many factors influence what is considered as normal haematological
parameters in newborn period. Various workers have reported range
of normal values during newborn period. Table 21.1 A and B shows
haematological parameters in the full-term normal infants studied at
LTMG Hospital, Bombay3
Table 21.1: Normal haematological values during neonatal period

of the term infant3
A: Haemoglobin, haematocrit and RBC count
Hb Haematocrit RBC
(gm%) (%) (million/mm3)
Cord blood 16.2 ± 3.6 46.66 ± 5.1 4.9 ± 1.2
12-18 hours 18.79 ± 2.8 49 ± 4.8 5.3 ± 0.8
72 hours 17.38 ± 3.0 46.9 ± 5.3 5.2 ± 0.6
7 days 17.0 ± 2.4 45.0 ± 4.0 5.0 ± 1.1
15 days 16.36 ± 2.2 43.4 ± 4.1 5.01 ± 0.9
28 days 14.17 ± 2.4 42.1 ± 3.8 4.7 ± 1.0
B: MCV, MCH, MCHC and normoblast count

MCV MCH MCHC Normoblasts
(fl) (Pg) (%) (cells/mm3)
Cord Blood 113.04 ± 5.3 34.33 ± 1.4 33.9 ± 0.8 600 ± 186
12-18 hrs 108.96 ± 5 35.1 ± 1.9 34.4 ± 0.6 283 ± 122
72 hrs 98.54 ± 2.9 35.82 ± 0.8 34.9 ± 0.5 36 ± 48
7 days 96.0 ± 3.4 34.0 ± 1.0 34.6 ± 0.8 –
15 days 95.5 ± 4.0 33.2 ± 9.0 34.54 ± 0.5 –
28 days 96.1 ± 3.2 31.6 ± 0.93 34.2 ± 0.7 –
2 per cent of neonates in this study has Hb value less than 13 gm per cent in
the cord blood.3
Various authors have reported values for the normal mean

haemoglobin (Hb) concentration of cord blood ranging from 15.7 gm
per cent to 17.9, gm/dl (Table 21.2). Approximately 95 per cent of all
values fall between 13.7 gm/dl to 20.1 per cent gm/dl1,3,5-12
Naiman and Oskin,1 Mollison et al,5 Sisson,27 Dalal and Lokeshwar3
and others6,8,9,11 have suggested that 13.5 gm/dl be considered lowest
normal value. Hb value for umbilical artery blood tend to be about 0.5
gm/dl higher than sample obtained from umbilical vein.14 At the end
Anaemia in the Newborn 335
Table 21.2: Normal cord blood haemoglobin by various authors

Mean Hb(gm/dl) Range
Mollison (1951)5 16.6 –
Dochain et al (1952)6 17.9 14.4 – 21.6
Walker et al (1953)7 16.5 –
Marks et al (1955)8 16.9 12.3 – 22
Guest et al (1957)9 17.1 13.6-25
Sturgeon (1956)10 15.7 –
Dalal and Lokeshwar3 16.2 13.2-22
Rama Rao11 – –
of 1st week of life Hb concentration is as high as it was in the cord

blood, though during first several hours after birth there is increase in
Hb concentration. Hb increases by 17-20 per cent of the initial value at
the first 24 hours of life but then falls slightly during next 24 hours.15
Table 21.3 gives normal haematologic values during first two weeks of
life in-term infants.1
Table 21.3: Normal haematologic values during the first two weeks of
life in-term infant1
Value Cord blood Day 1 Day 3 Day 7 Day 14
Hb gm/dl 16.8 18.4 17.8 17.0 16.8
Haemaocrit (%) 53.0 58.0 55.0 54.0 52.0
Red cells (mm3) 5.25 5.8 5.6 5.2 5.1
MCV (fl) 107 108 99.0 98.0 96.0
MCH (Pg) 34 35 33 32.5 31.5
MCHC (g/dl) 31.7 32.5 33 33 33
Reticulocytes (%) 3-7 3-7 1-3 0-1 0-1
Nucleated RBCs 500 200 0-5 0 0
Platelets 290 192 213 248 252
X10 3/mm3)
HAEMATOCRIT
Reports of normal values of haematocrit ranges from mean of 51.3 per
cent to 56 per cent.1,9,16,17 Just as Hb value, haematocrit value also shows
increase during the first few hours of life and reaches the original value
of cord blood by one week. Gatti et al18 reported capillary haematocrit
on first day of life to be 62.9 ± 3.2 per cent reaching 56.6 ± 2.6 by day 7,
and 53.7 ± 2.5 per cent by day 10, whereas Guest and Brown19 recorded
mean cord blood haematocrit of 52.3 and 58.2 per cent on first day,
54.31 per cent on 3rd day and 54.9 per cent on 7th day. Mean capillary
haematocrit value is two-percentage point higher than the mean venous
haematocrit values1 at 1 week of age.
RED CELL COUNT AND RED CELL INDICES

Red cell count also shows a great variability at the time of birth and
ranges from 4.6 to 5.2 million/cumm.1,3,9,8,20 Our study of 100 newborn
babies2,3 showed red blood cell count in cord blood 4.9 ± 1.2 reaching
5.3 ± 0.8 million after 12-18 hours and 5 ± 1.1 at 7th day and stabilizing
at some level thereafter throughout the neonatal period.
Other studies also have reported similar changes in red blood cell
count.1,8,9,21 Newborn red cells are generally much larger than adults
and average diameter of RBC is 8.5 to 9.3 μ at birth reaching the adult
value of 7.5 μ around 6 months of age.21,22,25 Relative macrocytosis is
observed in the newborn period and MCV at birth ranges from 104 to
118 fl. compared to normal adult value of 82-92 fl. Mean corpuscular
volume rapidly decrease in first week of life and at the age of 2 months,
cell size is comparable to those in adult cells.7,23-26
MCV Values less than 92 fl. should strongly suggest alpha-
Thalassaemia trait or iron deficiency. MCV is higher in preterm infants-
115 ± 5 fl.1,26 and decline to mean value of 95 ± 5 by 7th weeks of life.27
MCH is also increased in newborn period, values ranging from 33.5
to 41.4 Pg. as compared to adult values of 27-31 Pg.1,3 However, MCHC
in the newborn period is quite similar to that in adult ranging from 30-
35 per cent in newborn and 30-36 per cent in adults.1,3
RETICULOCYTE COUNT AND NUCLEATED RBC

Average retic count at birth ranges from 1.6 to 6.2 per cent, (majority
studies reported 4.1 to 6.3%) with an approximate count of 3,00,000
reticulocyte/cumm.1,3,23,28-30 Term infants in other words have an
average 7.3 nucleated RBC/100 WBC with normal range of 0-2430 at
birth. Infants born prematurely have higher retic counts, with values
ranging between 6-16 per cent in infants born between 30th and 36th
weeks of gestation. This increased retic count in first 2-3 days of life
reflects very active erythropoiesis during newborn period and value
drops to about 1 per cent by 7th day.1 Reticulocyte count increases
slightly during first 2-3 days after birth and abruptly drops 1 per cent
by 7th day of life.1,19,29-32
Persistent reticulocytosis in cord blood suggests (a) blood loss, (b)
hypoxia or (c) haemolytic process.1
The term infant has approximately 500 nucleated RBCs/cmm at birth
(0.1% of red cell population) which drops to 50 per cent by 12 hours
and to 20-30 nucleated RBC/cmm by 48 hours. It is unusual to see
nucleated RBCs in the peripheral smear of term infant after the age of 4 days.1,20
In other words the term infants has 7.3 nucleated red cells per 100
leucocytes at birth (ranging from 0-24). In premature infants the average
figure in 21 nucleated RBC/100 WBC.31 In premature babies nucleated
red cells may vary 1000 to 1500/mm3 at birth1 and decreases rapidly
during first week of life. However, occasional nucleated RBCs may be

seen in the peripheral blood smear of 7 days old infant. Increased number
of nucleated RBCs are seen after haemorrhage,2 hypoxia,3 haemolytic disease
of newborn,4 Down’s syndrome5 and congenital anomalies.1,32
FACTORS AFFECTING NORMAL HAEMATOLOGICAL VALUES IN

NEWBORN
Various variables influence the interpretation of so called normal values
of Hb, HCT, RBC indices, reticulocyte count at the time of birth and
during early weeks of life. These include:
1. Site of sampling
2. Time of sampling
3. Treatment of umbilical vessels at the time of delivery
4. Position of neonate after delivery
5. Gestational age of the infant
6. Foeto-maternal and materno-foetal transfusion.
Site of Sampling
Capillary sampling collected by skin prick from heel or the toe has a 5-
10 per cent higher haemoglobin concentration than simultaneously
collected venous sample.1 Oettinger and Mills32 reported this difference
around during 1st hour of life 3.6 to 8 gm per cent. However, in some
instances the capillary haemoglobin-venous Hb different may exceed
5-10 gm per cent.32-35,38 Stasis of the blood in the peripheral vessel
because of sluggish circulation and resultant transudation of plasma is
believed to be the cause of higher capillary haemoglobin as compared
to venous haemoglobin.1
Capillary/venous haematocrit ratio is greater than 1 in virtually all
infants. A ratio higher than 1.2 is observed in premature infants (before
30 weeks of gestation) infants with acidosis (pH less than 7.2) and
hypotension.35 Thus capillary Hb and haematocrit are falsely elevated
in sick infants with altered microcirculation. However, an accurate
determination of Hb concentration is most important in the clinical
management. Capillary/venous HCT ratio gradually decreases with
increasing gestational age. Infant born between 26 and 36 weeks of
gestation have average ratio of 1.21, infant born between 31-32 weeks
1.12, those born between 33-35 weeks 1.16 and between 36-41 weeks,
1.12.35 Infant with birth weight less than 1500 gms have mean venous
capillary difference of approximately 5 per cent at 12 weeks of age.36
Diagnosis of anaemia can be missed by evaluating capillary blood Hb.
Moe et al38 (1967) in their study of 54 infants with erythroblastosis
foetalis 25 out of 41 infants found to anaemic whereas only 14 could be
considered as anaemic according to the value obtained from capillary
sample.36
Difference in haemoglobin concentration between venous and

capillary blood can be minimised by first warming the extremities
before skin prick and obtaining good spontaneous blood flow,
discarding the first few drops before obtaining the sample.35,37 Hence,
capillary values should not be compared to previously obtained cord
venous blood values when one is looking for changes in Hb concen-
tration during first week of life and venous blood should be obtained
for this purpose. The selection of the vein is unimportant as blood from
different sites of vein gives similar results.17,34
Time of Sampling
During the first few hours after birth, an increase in haemoglobin
concentration takes place (as great as 2.5-6 gm/dl)1,39 which is due to
a) placental transfusion that occurs during the time of delivery and b)
readjustment of the blood volume after birth resulting in increased red
cell count, haematocrit and Hb concentration.1,34,38,39 Magnitude of
increase depends on the amount of placental transfusion.34
Treatment of Umbilical Vessels

At birth, the blood volume of the infant may be increased by as much
as 61 per cent by allowing complete emptying of placental vessels before
cord is clamped.39 Placental vessels contain 75-125 cc of blood i.e.
1/3rd to 1/4th foetal blood volume.43 The amount of blood received
by the neonate depends upon time of clamping of umbilical cord. Within
15 seconds of birth about a quarter and at the end of 1 minute, about
half of placental transfusion takes place.12 Placental transfusion occurs
more rapidly in women who receive ergometrin derivatives at the onset
of 3rd stage of labour.1,3,39-43 During first hour of birth, plasma leaves
the circulation. Greater the placental transfusion, greater is the plasma
loss. On the third day of life, there are only small differences in total
blood volumes regardless of method of cord clamping.44 In the group
with late cord clamping, at 24 hours of age the red cell mass was approxi-
mately 32 per cent greater and haematocrit was 15 percent higher.44,45
Hb per cent difference can range from 2-4 gm per cent between early
clamping and delayed clamping with haematocrite difference of 2-16
per cent by various authors42,43,48,49 during first week of life, by 6 weeks
and the difference are no longer apparent.43,46-49
Position of the Neonate After Delivery

As umbilical arteries generally constrict shortly after birth, no blood
flows from the infant to mother. However, as the umbilical vein remains
dilated, it permits the blood flow in the direction of gravity. Infants
held below the level of placenta, as it happens during normal delivery,
continue to gain blood from placenta, whereas infants held above the
placenta which is often seen during caesarian delivery, infant may bleed
into mother, thus leading to anaemia in the neonate.39,44 In infants
delivered at-term with caesarian section, maximal placental transfusion
is achieved in 40 seconds after birth.43 Delay of 3 minutes in cord
clamping after caesarian section has been associated with sings of
respiratory and metabolic acidosis indicating that earlier clamping may
be preferable.43 Infants born to caesarian section it is advisable to keep
baby at least 20 cm below the placenta for approximately 30 sec before
clamping the cord in order to ensure a partial placental transfusion.
However, when infant suspected to have blood group incompatibility,
cord should be clamped promptly in order to minimize the transfer of
sensitized cells. During normal deliveries umbilical cord should be
clamped within 30 seconds after birth, provided infant is not elevated
above the level of uterus.1 Delayed clamping tends to have higher values
of Hb during first week as there is greater placental transfusion and
greater plasma loss. Infants with delayed cord clamping had an average
red cell mass of 49 ml/kg at 72 hours as compared to 31 ml/kg in
infants with immediate cord clamping.40,45 The infant with delayed
clamping tend to have higher Hb value during first week of life than
those whose clamping was delayed. By 6 weeks, however differences
are no longer apparent.49 However, in infants in whom cord ligation
was delayed, decreased incidence of respiratory distress syndrome have
been reported and hence delayed cord clamping is indicated for
premature infants. However, there have been reports of circulatory
overload and congnitive cardiac failure in the setting of delayed
clamping (symptomatic neonatal plathora). Premature infants with
increased red cell mass, as consequence of delayed cord clamping, have
been found to have higher serum bilirubin level.47,48 However, when
there is obvious evidence of foeto-placental or maternal bleeding before
or during the birth and infant appears pale and in shock, cord clamping
should be delayed, if resuscitation can be given simultaneously.
CRITERIA FOR ANAEMIA

The definition of anaemia in newborn is made difficult because of
several important factors that influence normal blood picture in the
newborn infants such as site of sampling, time of sampling, treatment
to the umbilical vessels at the time of delivery, gestational age of the
infants, time of clamping the umbilical cord and possibility of foeto-
metarnal or materno-foetal transfusion etc. Oski et al1 have suggested
a Hb less than 14 gm per cent in capillary blood whereas Mollison5 and
Sisson27 have suggested less than 13 gm/dl from venous blood in the
first 2 weeks of life as evidence for anaemia.3,25 In our study of 100
newborn babies, only 2 per cent of neonates had Hb level less than 13
gm per cent in cord blood.3
AETIOLOGY OF ANAEMIA
Nutritional anaemia (unlike in older children) is not a common cause
of anaemia during neonatal period even when mother is severely
anaemic, as foetus is an effective parasite in the mother. Anaemia in
the newborn often accompanies and is complicated by conditions like
asphyxia, shock, jaundice which make the situation even worse.
Anaemia may be caused by a widespectrum of diseases and could be
due to (1) haemorrhage (2) haemolysis, (3) failure of red cell production.
Haemorrhage
It has been reported that 25 per cent of all infants admitted to neonatal
intensive care and 5-10 per cent of all severe neonatal anaemias are
due to haemorrhage and haemorrhage is reported in 1 per cent of all
newborn nursery admissions.50,51 Haemorrhage leading to anaemia
could be due to (1) occult haemorrhage-haemorrhages associated with
malformation of placenta, umbilical cord, obstetric accident-foeto-
maternal haemorrhage or foeto-foetal haemorrhage, (2) internal
haemorrhages and (3) haemorrhage after birth.
Malformation of Placenta or Cord or Obstetric Accidents

Severe foetal haemorrhage may accompany various placental anomalies
like placenta praevia, abruptio placenta, accidental incision of placenta
during the caesarian section. It is reported that 10 per cent of all infants
born following placenta praevia and 4 per cent of infants born following
abruptio placenta present with severe anaemia.52,53 Other causes include
rupture of umbilical cord, or of aneurysm of cord or aberrant vessels
and due to velamentous insertion of the cord.51,54,55 Approximately 1
per cent of all pregnancies have velamentous insertion of the cord. This
abnormality is more common in multiple pregnancies and incidence
of haemorrhage is seen between 1 and 2 per cent.55 Unattended preci-
pitous delivery may lead to rupture of normal umbilical cord. When
cord ruptures tear generally occurs in foetal third and bleeding is
immediate and profuse.56 Severe bleeding may result in still birth; may
manifest with severe respiratory distress and asphyxia. The following
table differentiates pallor and asphyxia in the newborn.
In asphyxiated neonate, pulse is slow, respiration may be absent and
irregular, bradycardia is often associated. Cyanosis improves with
oxygen and assisted ventilation whereas a neonate with severe blood
loss has tachycardia, rapid shallow breathing and minimal cyanosis
with no improvement of colour after oxygenation. Venous pressure on
introduction of catheter through the umbilical vein is found to be low.
In acute haemorrhage, the Hb concentration is initially often normal
and if infant is in shock due to severe bleed, the capillary haemoglobin
may be misleadingly high owing to peripheral stasis and hence Hb
estimation performed on venous blood is mandatory. However, if Hb

is initially high, Hb estimation should be repeated and followed closely
during next 12 to 24 hours of life to rule out haemorrhage. Careful
examination of placenta and the cord helps in diagnosis.
FOETAL HAEMORRHAGE
Foetal haemorrhage in maternal circulation. The passage of foetal
erythrocytes in maternal circulation occurs commonly during
pregnancy. In 50 per cent of pregnancies some foetal cells are passed in
maternal circulation at some times during gestation or during birth
process.57,58 In about 8 to 10 per cent of pregnancies and trasplacental
blood loss ranges from 0.5 cc to 40 ml of blood and in about 1 per cent
of cases the loss may be even greater as much as 100 ml.59 The nucleated
foetal erythrocytes can be detected in maternal circulation as early as 4
to 8 gestational age.59 Foetal haemorrhage may also occur in substances
of placenta or may result in retro-placental haemorrhage.60,61 More
common type of foeto-maternal haemorrhage occurs when infant is
held above placenta during delivery particularly after caesarian section
before cord is clamped. Infant also may be deprived of placental blood
when born with tight nuchal umbilical cord. The pressure on cord
obstruct umbilical vein before it obstructs umbilical artery and hence
infant is deprived of placental blood via umbilical vein while infants
blood continue to return to placenta by umbilical artery.62 This may
lead to severe bleeding resulting in stillbirth or child may be born with
severe pallor or irregular gasping respiration. When blood volume is
reduced by 10 per cent, the newborn develops abnormalities in both
central and peripheral circulatory response.
Causes of Foeto-maternal Haemorrhage

1. Diagnostic amniocentesis : 10 to 32 per cent.63,64
2. Traumatic injury to mother during pregnancy, secondary to
vehicular accidents, fall, abdominal trauma.
3. Use of intravenous oxytocin.
4. External cephalic version.
5. Manual removal of placenta.
6. Application of fundal pressure during 2nd stage of labour.
7. Maternal toxaemia.
8. Erythroblastosis foetalis.
9. Placental chorio-angioma and chorio-carcinoma seen in 1 per cent
of placenta.65,66
Foeto-maternal or placental haemorrhage may present as:
Acute Haemorrhage
This may result in stillbirth or child may be born with severe pallor or
irregular gasping circulation or shock may be present. Tachycardia is
Table 21.4: Differential diagnosis of pallor in the newborn51

Asphyxia Acute Severe Blood Loss Haemolytic Disease
1. Respiratory findings: 1. Rapid shallow 1. Hepatosplenomegaly,
retractions, cyanosis respirations jaundice CCF may be
improves with O2 Minimized cyanosis asociated
No improvement after O2
2. Moribund appearance 2. Decrease in venous and 2. Positive Coomb’s test
Normal venous arterial pressure Normal venous pres-
pressure Low CVP sure
3. Bradycardia 3. Tachycardia 3. Tachycardia ±
4. Acyanotic
initially normal Hb
4. Stable haemoglobin 5. Drop in haemoglobin 4. Anaemia
Capillary Hb
misleading.
often associated. However, pallor should be differentiated from other

causes of respiratory distress as shown in Table 21.4. Mother may
present with shaking chills as consequences of transfusion reaction
particularly when there is blood group incompatibility. Occasionally
the haemolytic transfusion reaction in mother may lead to acute renal
failure.1,69
Chronic Foeto-maternal Haemorrhage

If haemorrhage occurs repeatedly during course of pregnancy and has
been prolonged, anaemia in foetus occurs slowly with the result foetus
tries to compensate and gets the opportunity to adjust haemodynami-
cally and infant when born may present only pallor, unexplained
anaemia and mild hepatosplenomegaly. Chronic blood loss may lead
to iron deficiency anaemia and haemoglobin value may range from 4
to 6 gm/dl and newborn may be stable with cardio-respiratory compen-
sation. Though complete obstrestical history gives clue to diagnosis,
history of vaginal spotting during last trimester or prior to delivery,
placenta previa, abruptio placenta, non-elective caesarian section and
difficult labour with possibility of cord compression should be noted
in history.67,68
DIAGNOSIS AND LABORATORY INVESTIGATIONS

Degree of anaemia depends upon whether acute or chronic.
Haemoglobin value as low as 3 to 4 gm/dl have been recorded.
Following acute haemorrhage Hb may not drop first 24 to 48 hours.
High index of suspicion in an anaemic neonate without jaundice and
with negative direct Coomb’s test will help in suspecting diagnosis of
acute haemorrhage. If haemorrhage has been acute and shock is present,
Hb value may not reflect the magnitude of blood loss. Several hours
may elapse before profound anaemia is documented. If the neonate is

in shock, Hb determination should be performed on venous blood as
capillary Hb may be misleadingly high. Even if Hb is initially normal,
neonate should be repeatedly followed up closely during next 12 to 24
hours and falling Hb may be noticed after some time due to
haemodilution that accompanies. Examination of placenta and cord
should be performed before it is thrown to ascertain the site of blood
loss. Jaundice is absent and bilirubin level not increased.
In acute haemorrhage peripheral smear examination may show
normochromic, normocytic RBCs, whereas hypochromic, microcytic
anaemia seen in chronic blood loss. Negative Coomb’s test, increased
retic count and increased number of nucleated RBCs are seen in both
acute and chronic haemorrhages. In chronic haemorrhage the serum
iron values are decreased and bone marrow aspiration if done may
show no stainable iron. However, bone marrow aspiration is not
indicated for diagnosis.
Diagnosis of foeto-maternal haemorrhage is confirmed by demons-
trating the presence of foetal blood in maternal circulation by Kleihauer
Betke’s test, differential haema-gglutination, mixed agglutination or
fluorescent antibody technique. All these techniques are very sensitive
and are capable of detecting as little as 0.1 cc of foetal blood in maternal
circulation. In the presence of maternal Thalassaemia minor, sickle cell
anaemia, or hereditary persistence of foetal haemoglobinopathy,
Kleihauer Betke’s test may be false positive and is not useful and
differential haemagglutination test should be employed. If ABO blood
group incompatibility is associated, diagnosis can be missed as infant’s
A or B cells are rapidly cleared from maternal circulation by maternal
anti-A and anti-B antibodies. The amount of blood lost in a foeto-
maternal haemorrhage, can be calculated by the following formula:
Foetal RBC × 2400
cc of foetal blood = ——————————
Maternal RBCs
or 1 foetal RBC in 1000 maternal RBCs indicates 2 cc of foeto-maternal
haemorrhage.
FOETO-FOETAL HAEMORRHAGE
(TWIN TO TWIN TRANSFUSION)
Simultaneous occurrence of anaemia in one of the twins and poly-
cythemia in other should always arouse a suspicion of twin to twin
transfusion. The donor twin is pale and evidence of listlessness and
shock may be present. Besides, the donor twin is usually smaller than
the recipient. The recipient twin is plethoric and has associated
polyhydramnios and polycythemia. Hydramnios in one placenta and

oligohydramnios of other as well as marked difference in the size of
the placenta is usually seen. 70 per cent of monozygous twin preg-
nancies are monochorionic72 and the incidence of twin-twin transfusion
is estimated to be 15-30 per cent of all monochorionic twins.70,71
Anaemic donor infant may develop congestive heart failure whereas
the plethoric twin may manifest symptoms and signs of the
hyperviscosity syndrome, disseminated ventravascualr coagulation and
respiratory distress, venous thrombosis and hyperbilirubinemia. When
twin to twin transfusion is suspected, detailed placental examination
should be carried out.
Twin to twin transfusion should be suspected when the Hb difference
greater than 5 gm/dl exists between identical twins in contrast to 3.3
gm/dl in cord blood Hb in dizygotic twins1,74 and the weight difference
between the 2 exceeds 20 per cent of weight of the larger twin.73 Anaemic
donor baby show significant reticulocytosis. Hb value of recipient twin
may vary from 20-30 g per cent with a haematocrit as high as 82 per
cent75 and many recipient twins develop hyperbilirubinemia needing
light therapy and exchange transfusion.
INTERNAL HAEMORRHAGE
Anaemia that appears in the first 24-72 hours after birth and not
associated with jaundice is commonly due to internal haemorrhage
and associated negative Coomb’s test. However, when internal
haemorrhage takes place in newborn it may not be recognized till shock
has occurred.
Haemorrhage in the postnatal period may be either obvious or occult.
The commonest causes of obvious bleeding in the newborn are slipped
ligature of the cord, haemorrhagic disease of newborn, or the bleeding
disorders in the neonate. Haemorrhage also may occur into the adrenal
gland, kidney, spleen, retroperitoneal area particularly following breach
delivery. Various other causes that lead to internal haemorrhage are
traumatic deliveries resulting in subdural, subarachenoid haemorrhage,
cephalhaematoma, blood loss in subaponeurotic area of the scalp.
Subapneurotic haemorrhage usually extends throughout the soft tissue
of the scalp and covers entire calveria and this blood loss can lead to
exanguination and death. Boggy swelling of the head extending from
frontal region to nape of the neck may be present and may be associated
with the swelling of the eyelids and Hb may drop as low as 2.2 g/dl at
48 hours of age and infant may be in shock. It can be estimated that for
each centimeter of increase in head circumference above that expected,
38 ml loss of blood and may also develop hyperbilirubinemia.76
IMPACT OF BLOOD SAMPLING ON

HAEMOGLOBIN LEVEL OF NEWBORN INFANTS
Cumulative blood loss through sampling for blood monitoring of small
infants are often surprisingly high. Blanchette and Alvin Zupersky in
their study of 52 premature infants studied during first 6 weeks of life
reported mean blood loss through sampling 22.9 ± 10 ml of packed
cells.69 46 per cent of infants studied had cumulative losses that
exceeded their circulating red cell masses at birth. They also noted 10
per cent of blood loss during sampling for laboratory monitoring is
“hidden” and represents blood on cotton swabs, in the dead space of
syringe or tubing of the butterfly needle.
High index of suspicion particularly following traumatic delivery
in infant found to be anaemic during first few days is key to diagnosis.
Clinical picture including sudden collapse, cyanosis, limpness, irregular
respiration, presence of flank mass accompanied by bluish discoloura-
tion of overlying skin should lead to suspicion of adrenal haemorrhage.
Rupture of the liver with resultant anaemia is more often found at
autopsy (1.2-5.6 per cent) and haemorrhage may be subcapsular or free
blood may be present in the peritoneal cavity. An infant ruptured liver
generally normal first 24-48 hours and suddenly goes into shock, as
haematoma ruptures the capsule causing haemoperitonem. Upper
abdomen may appear distended. Mass in the right hypochondrium
may be palpable. Presence of the free fluid can be demonstrated by
clinical examination as well as a ultrasound evaluation. Jaundice may
appear due to break down of RBCs from these entrapped haemorrhages.
Prompt blood transfusion and surgical repair of the laceration helps
though in general prognosis is poor. Splenic rupture should be kept in
mind in large babies with pallor, abdominal distension, scrotal swelling
and radiographic evidence of peritoneal effusion following difficult
delivery or in babies with erythroblastosis foetalis. Umbilical venous
pressure is decreased rather than increased.
IATROGENIC ANAEMIAS
With the advent in the neonatal care and intensive care units, frequent
monitoring of critically ill neonates has become a rule. Therefore
anaemia appearing during first week of life, may be caused by frequent
blood sampling. It should be remembered removal of 8-10 cc of blood
from 1500 g. neonate constitutes a loss of 8 per cent of blood volume
which is equivalent to about 400 cc of blood from an adult. Various
studies have shown blood withdrawal for investigations during first
weeks of hospitalization can range from 5-45 per cent of total blood
volume or 50.5 ml/kg per 28 days period.
ANAEMIA DUE TO INCREASED RBC DESTRUCTION

The normal life span of RBC is 120 days. However, red cell life survival
in-term infants may be 60-80 days and newborn 36-32 weeks gestation
only 20-30 days.1
HAEMOLYTIC ANAEMIAS
Anaemia as a consequence of haemolytic process is common in the
newborn period. Since destruction of 1 g/dl of haemoglobin results in
production of 35 mg of bilirubin, haemolytic anaemia in the newborn
is always associated with significant hyper-bilirubinemia and often may
need light therapy or exchange transfusion. Haemolytic disease of
newborn may be caused by isoimmunization, congenital or acquired
defects of RBCs.
Haemolytic disease of newborn should be suspected when there is:
1. A rapid fall of haemoglobin concentration in the absence of
evidence of haemorrhage
2. Evidence of increased red cell production i.e. reticulocytosis and
normoblasts in peripheral smear.
3. Jaundice during first 24-48 hours of life.
4. Abnormal erythrocyte morphology,
5. Haemoglobinuria
6. Positive Coomb’s test
ISOIMMUNIZATION
Haemolytic disease of newborn as a consequence of isoimmunization
is caused by passage of foetal red cells into the maternal circulation
where they stimulate the production of antibodies. These antibodies of
IgG class return to foetal circulation, attach to the antigenic site on the
foetal red cells leading to haemolysis of these cells. Isoimmunization
can be caused by ABO, Rh, or minor blood group incompatibility. As
few as 0.05 to 0.1 cc cells are sufficient to produce isoimmunization.
While in utero, excess of bilirubin produced by haemolysis is removed
by the placenta and hence at the time of birth, child may not be
jaundiced. Jaundice usually appears in the first 24-48 hours of life as
neonatal immature liver is not in a position to conjugate excessive
bilirubin load. This may be accompanied by anaemia and hepatospleno-
megaly. Laboratory investigations may reveal blood group incompati-
bility and may show reticulocytosis with increase in the number of
nucleated RBCs. A direct Coomb’s test usually positive in Rh-incompati-
bility and may be weakly positive in ABO incompatibility. Bilirubin
levels are usually raised. In ABO incompatibility increased number of
spherocytes may be seen on peripheral blood smear examination. Levels
of IgG, anti-A, or anti-B in the mothers of babies with ABO haemolytic
disease are significantly higher than in the mother whose infants do
not have the disease. ABO incompatibility is common and occurs in

approximately in 20 per cent of all pregnancies. Haemolytic disease
due to minor blood group incompatibility should be suspected when
Coomb’s test is positive and there is no evidence of major blood group
incompatibility in mother and child. The principal antibodies found
are-anti-E, anti-C, anti-Kell, Duffy, Kidd, Fu, etc.
With advent of Rh-prevention programme and routine use of Rh-
anti-D immunoglobulin, the problem of Rh-haemolytic disease has
become less pronounced. The direct Coomb’s test is positive and
peripheral blood smear shows polychromasia, increased number of
erythroblasts. Measures available to predict severity of foetal haemolytic
diseases are maternal antibody titre, prior history of haemolytic disease,
amniotic fluid spectrometry to assess foetal bilirubin level. Additionally
amniotic fluid PCR testing may detect Rh or Kell-antigen states of the
foetus.
Management of severely affected foetus consist of early delivery with
or without foetal transfusions depending upon gestation of foetus.
Management of neonate depends on degree of haemolysis and level of
indirect hyperbilirubinemia. Hyperbilirubinemia must be aggressively
treated to prevent kernicterus with phototherapy, exchange transfusion.
Whenever required top-up transfusion with packed red blood cells may
be given for symptomatic anaemia. All Rh-negative mothers with Rh
positive foetus Rh-immunoglobulin given at 28-30 weeks of gestations
and 72 hours after delivery and after spontaneous or therapeutic
abortion.
Anaemia may be associated with bacterial and viral infections in
the newborn period and is often associated with jaundice (both
conjugated and unconjugated fraction) and hepatosplenomegaly.
Intrauterine, viral infections like congenital CMV, toxoplasmosis,
congenital syphilis, malaise, herpes, all may be associated with
haemolytic anaemia in the neonatal period. Presence of autoimmune
haemolytic anaemia in mother may result in haemolytic anaemia in
the newborn infant.
CONGENITAL DEFECTS OF RED CELLS

These abnormalities includes conditions associated with defects of red
cell metabolism, red cell membrane, and haemoglobin synthesis.
Common defects of red cell metabolism include G6PD deficiency-less
common disorders is pyruvate kinase deficiency. G6PD prevents
oxidative damage to the red blood cells. However, with G6PD enzyme
deficiency, oxidation of membrane protein leading to precipitation of
denatured haemoglobin (Heinz bodies) thus shortening the RBC life
span. Because of the diminished capacity of neonatal RBCs to deal with
oxidative stress, as a result of lower glutothione paroxidase, catalases
as well as relative deficiency of Vit. E, newborn infants with G6PD

deficiency are at a greater risk of developing haemolytic anaemia than
are adults. This particularly is the case in more severe types affecting
Asians and Mediterranean group. Hyperbilirubinemia in G6PD
deficient males has been reported is newborns from Greece, Italy,
Singapore, Taiwan, India and China. There is a tendency for jaundice
to occur more frequently in particular families and communities,
indicating that the genetic and environmental factors may influence
the incidence of the disease. In India, G6PD deficiency is most
commonly seen in Parsi, Bhanushali, Sindhi and Punjabi Khoja
communities. In this group of patients, jaundice may be severe leading
to kernicterus. Jaundice that occurs, appears to be due to accentuation
of physiologic jaundice of newborn, although jaundice may appear in
some during first 24 hours. In some instances abnormal RBC
morphology with evidence of Heinz bodies, intravascular haemolysis
may be associated. Normal G6PD levels during acute haemolysis may
not rule out G6PD deficiency as younger RBCs contain high level of
enzymes. Hence, the test may have to be repeated after 6 weeks. The
other common red cell enzyme deficiency leading to haemolytic
anaemia in newborn during first week of life is pyruvate kinase
deficiency. This disorder is generally characterised by evidence of
haemolysis with increased retic count, few or no spherocytes on the
peripheral smear, no blood group incompatibility and a negative
Coomb’s test.
Abnormalities of Red Cell Membrane

In approximately 50 per cent of patients with hereditary spherocytosis,
a history of neonatal jaundice can be obtained and may be of a sufficient
magnitude so as to need photo-therapy and exchange transfusion, and
may lead to kernicterus if left untreated. A family history of chronic
anaemia, splenectomy, cholecystectomy, pain in abdomen, unhealed
ulcers may be present. Physical examination of parents may reveal mild
to moderate splenomegaly. Examination of peripheral blood reveals
characteristic micro-spherocytes and the osmotic fragility of
erythrocytes may be increased. Spherocytes however may be seen in
the newborn period in other conditions like ABO incompatibility,
septicaemia, red cell enzyme deficiency, etc.
The Thalassaemia Syndrome

Alpha Thalassaemia Syndrome
Of the 4 varieties of alpha Thalassaemia the homozygous alpha
Thalassaemia which results from the absence of all four genetic loci for
alpha chain synthesis, presents in the neonatal period. In the absence
of alpha chains, cord blood contains Bart’s haemoglobin (Gamma 4)

and Haemoglobin H (Beta 4). Most affected infants are stillborn,
although some may live for a few hours after birth. These infants are
hydropic at birth and thus are similar in appearance to neonates with
severe erythroblastosis due to Rh-incompatibility.
Gamma Thalassaemia Syndrome

The production of gamma polypeptides is regulated by four genes.
The complete absence of gamma chains is incompatible with foetal
life.Intermediate reduction of gamma-polypeptide synthesis may
produce a mild to moderate neonatal anaemia characterised by a
reduced percentage of foetal haemoglobin. This type of anaemia
resolves when significant beta chain synthesis begins.
Other unusual causes of neonatal anaemia include congenital
dyserythropoietic anaemia, leukaemia, microangiopathic, haemolytic
anaemia, DIC etc.
Anaemia due to under Production

Impaired red cell production is an unusual cause of anaemia in the
newborn period. Congenital pure red cell anaemia (Diamond Blackfan
syndrome) is an uncommon disorder in which red cell precursors in
the bone marrow are markedly reduced or virtually absent while white
blood cell and platelet production remains normal. It may be associated
with increased incidence of prematurity, abnormalities of pregnancies
and physical abnormalities. Mothers of affected children may have
increased incidence of miscarriages and abortion. Physical abnormali-
ties associated are triphalangeal or duplicated thumb, cleft palate, ocular
defects like epicanthal folds, hypertelorism, ptosis, short or webbed
neck, congenital heart diseases and short stature. Laboratory
abnormalities include macrocytic anaemia, absent reticulocyte count,
elevated HbF, within normal values of leucocytes and platelets.
Diagnosis is confirmed by demonstrating a virtual absence of erythroid
precursors in the bone marrow. Erythroid-myeloid ratio ranges from
1:6 to 1:240.
Pancytopenia, accompanied by reticulocytopenia, leukaepenia and
thrombocytopenia may be seen in severe septicaemia, congenital
infections like rubella, other TORCH group of infections. Transplacental
transmission of parvo virus B19 occurs in one third of acutely infected
pregnant women, causing hypoplastic anaemia, when severe may lead
to intrauterine death and when survive intrauterine infection, may be
born with hydrops foetalis and rarely transcobalamine II deficiency.
Fanconi’s anaemia usually does not manifest in the newborn period
and often presents with anaemia around the age of 5-8 years. Anaemia
in the newborn period could be due to marble bone disease/

osteoporosis, haemolysis as well as for non-production and anaemia
with jaundice has been observed during this period. The disease is
associated with hydrocephalus, hepatosplenomegaly and marked
increase in the density of the bone particularly of long bones, ribs and
base of the skull. Other causes include transcobalamine II deficiency,
refractory sederoblastic anaemia.81,82 However, associated congenital
anomalies should make the consultant to follow the child regularly.
Table 21.5 shows etiology of anaemia in the newborn period as seen in
the haematology clinic at L.T.M.G.Hospital, Sion.
Table 21.5: Aetiology of anaemia in the newborn period (study done at L.T.M.
Medical College and L.T.M.G.Hospital, Bombay)
Etiology of Anaemia 1979-80 (%) 1990-91 (%)
Haemolytic
Rh-incompatibility 66 10
ABO incompatibility 13 15
Hydrops foetalis – 2.5
Minor blood group incompatibility 4 –
G6PD deficiency 2 1.25
Pyruvate kinase deficiency – 1.25
Sepsis 16 20
DIC 4 10
Malaria – 1.25
TORCH – 2.5
Syphilis 12 1.25
Spherocytosis – 2.5
Haemorrhage
Haemorrhagic disease of newborn 4 3.75
Extrinsic blood loss (iatrogenic) 6 3.75
Intracranial haemorrhage 2 7.5
Subgaleal haemorrhage – 1.25
Congenital afibrinogenemia – 1.25
Foeto-foetal haemorrhage – 5.0
Foeto-maternal haemorrhage 2 5.0
Hypoplastic
Transient erythroblastopenia of childhood – 1.25
TAR Syndrome - 1.25
TREATMENT
1. The treatment of a neonate with anaemia due to blood loss
depends on the degree of hypovolemia or anaemia and whether
the blood loss has been acute or chronic. A diagnostic approach to
anaemia in newborn is shown in Table 21.6 and Fig. 21.1. Baby
born pale at birth should be differentiated from an asphyxiated
baby as shown in Table 21.6.
2. Pale babies usually will have tachycardia with minimal or no

cyanosis, decreased CVP and a rapid fall in haemoglobin with
circulatory collapse. The following guidelines may be employed
in managing such cases.
i. Clear the airway, administer oxygen and intubate if necessary.
ii. Insert the catheter in the umbilical vein and measure CVP and
obtain blood specimen for investigation. If CVP is very low,
administer 20 cc/kg of available solution-in order of preference
of ‘O’ –ve blood, plasma, 5 per cent albumin and isotonic saline.
Table 21.6: Diagnostic approach to anaemia in newborn

Anaemia in newborn Diagnosis
Age at which anaemia is first noted Haemorrhage or haemolysis
at the time of birth and 1st week of life
First two days Internal or external haemorrhage
After 48 hours with jaundice Haemolytic disease
H/o anaemia in male siblings G6PD deficiency
H/o attacks of jaundice in other Spherocytosis
members of the family
Obstetric History-
H/o amniocentesis/external podalic Foeto-maternal haemorrhage
version
Abruptio placenta, Placenta previa, OR Foeto-placental haemorrhage
Caesarian section
Bleeding P.V. in mother Blood Loss
Traumatic delivery/Breech delivery Occult haemorrhage
Twin delivery Foeto-foetal transfusion
Premature delivery/drug ingestion Haemorrhagic disease of newborn
in mother (Dilantin, Aspirin) and drug induced.
No Vit. K. given to neonate
Improper ligation of the cord Slipped ligature
Healthy breast fed newborn with G.I. Haemorrhagic disease of
bleeds newborn.
Healthy baby bleeding after circumcision Haemophilia syndrome
Mother Rh –ve Rh-incompatibility
Blood group incompatibility in mother Haemolytic disease of newborn.
and child (Rh,ABO,minor)
Sick newborn, icterus, signs of sepsis. D.I.C.
Anaemia + Hepatosplenomegaly + Intrauterine infection /
Purpura ± jaundice Sepsis with DIC/Cong.leukaemia
Anaemia + Jaundice + Haemolytic disease of newborn
Mild hepatosplenomegaly /Sepsis
Associated with congenital malformation like Diamond Blackfan syndrome
triphalangial /Bifid thumb Fanconi’s anaemia
APPROACH TO NEONATAL ANEMIA

Hb./HCT, PS examination, Direct Coomb’s test, Red cell indices,
Retic count
N/Increased Decreased
Coomb’s Test (DCT) Diamond Blackfan syndrome

Transcobalamine II def.
Sideroblastic anaemia,
Marrow infiltration,
+ve or –ve
Perform bone marrow
weakly +ve
Isoimmunization MCV
MCV
Rh/ABO/Minor
Maternal autoimmune
haemolytic anaemia
Low MCV MCV N/High

PS examination
Hypochromic, micro- Morphologic abnormalities No specific abnormalities

cytic chronic haemor- Spherocytosis Normocytic, normochromic
rhage Elliptocytosis
Foeto-maternal, Stomatocytosis
Foeto-foetal and Heinz Bodies-G6PD Jaundice
Thalassaemia triat Fragmented cells-DIC
Hb electrophoresis
Kleihaur Betke’s Test No jaundice Jaundice present
on mother’s smear
Acute blood loss Splenomegaly
Absent Present
G6PD and other enzyme def. TORCH infection

Septicemia, laeukemia
Osteopatrosis,
Galactocaemia
Spherocytosis
Fig. 21.1: An alogrithm suggesting approach to case of neonatal anaemia
iii. If whole blood is not administered initially or venous pressure

does not return to normal, then repeat 10-20 cc/kg of whole
blood.
iv. After resuscitation, investigate the child to determine the cause
of blood loss, examine the cord and placenta, mother’s blood
smear for the evidence of foeto-maternal haemorrhage.
An anaemic infant with evidence of congestive cardiac failure may
be given partial exchange transfusion or packed cell transfusion
and should be treated with diuretics like frusemide in the dose of
1 mg/kg.
3. Neonates who are anaemic at birth without signs and symptoms
of failure due to chronic blood loss with no distress, do not require
blood transfusion.Elemental iron in the dose of 2 mg/kg body
weight daily for three months is required to replenish iron stores
and return the haemoglobin to normal.
4. Iron therapy is also needed for the infant who also receives blood
transfusion for symptoms at acute distress, because the trans-
fusions are not sufficient to totally replace the iron lost due to
haemorrhage.
Calculations of Dosage of Blood for Simple Transfusion
A haemoglobin of 15 mg/dl corresponds to red cell mass of 30
ml/kg. This implies that a transfusion of 2 ml RBC/kg will increase
the haemoglobin concentration by 1gm/dl. Whole blood
(haematocrit of 33%) contain 2 ml RBCs per 6 ml of whole blood.
Thus the transfusion of 3 ml packed RBCs/kg or 6 ml of whole
blood/kg will increase haemoglobin concentration by 1 gm/dl.
5. The treatment of haemolytic anaemia consists mainly of manage-
ment of hyperbilirubinemia which includes phototherapy and
exchange transfusion. For other types of anaemia, blood trans-
fusion or packed cell transfusion may be given if anaemia is
significant. 6 cc of blood or 3 cc of packed cell/kg body weight
raises the haemoglobin by 1 g/dl.
6. Diamond Blackfan syndrome can be managed by corticosteroids
and blood transfusions while anaemia of transcobalamin II
deficiency requires weekly intramuscular injections of 100 micro-
grams of vitamin B12.
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22
Thalassaemia Intermedia Syndrome
S Tyagi, VP Choudhry
Key Words
Thalassaemia intermedia • Mutation • Infections
• Chelation • Hypersplenism
INTRODUCTION
Thalassaemia intermedia is a state in which patients have mild to
moderate anaemia and are either transfusion independent similar to
Thalassaemia trait or require minimal red cell transfusion. In view of
clinical heterogeneity, varied haematological spectrum and genetic
inheritance in alpha or beta chains alone or in combination with other
haemoglobinopathies, it has become a subject of controversies regarding
its diagnosis and management. Apart from reduction in globin chain
synthesis genetic interaction of various Thalassaemia genes like co-
inheritance of alpha Thalassaemia or compound heterozygosity for beta
and delta beta Thalassaemia or coinheritance of structural haemoglobin
variant seems to be responsible for its diverse clinical spectrum. The
genetic determinants which enhance the gamma chain production are
known to ameliorate its severity. The various controversial issues along
with pathophysiological changes, iron overload, chelation therapy, etc.
are being reviewed.
PATHOPHYSIOLOGY
The basic defect in Thalassaemia is a partial or total absence of synthesis
of either alpha or beta globin subunit leading to an imbalance between
alpha and beta globin chain ratio. The precipitation of excess unbound
alpha or beta chain causes destruction of red cells both intramedullary
as well as in the peripheral blood with consequent development of
variable degree of anaemia. In addition, splenic entrapment of red cells
which have alpha or beta chain deposition also leads to marked
erythropoietic drive with expansion of bone marrow. These manifesta-
tions lead to abnormally high metabolic activity, nutritional deficiency
Thalassaemia Intermedia Syndrome 359
and muscle wasting. Further, red cells containing high HbF in children
with beta Thalassaemia have less alpha chain and therefore, have a
survival benefit over the cells containing subnormal amount of
haemoglobin A.1 However, high levels of HbF are associated with low
P50 volume and 2,3-DPG levels. These patients are unable to
compensate for their anaemia by shifting O2 dissociation curve to the
right as HbF interact with 2,3 DPG much less readily than HbA.
Increased serum erythropoietin levels leading to increase in
erythropoietic activity in the presence of anaemia and subsequently
erythroid hyperplasia is probably responsible for increased iron
absorption from GI tract. Repeated blood transfusion further add iron
to already overloaded tissues in thalassemic patients and lead to liver
damage and cirrhosis and other complications of iron overload. Apart
from liver, other commonly involved organs are heart and endocrine
organs affecting the growth and development in thalassemic children.
As the imbalance between α and β chains in Thalassaemia intermedia
syndrome (TIS) is less severe than children with Thalassaemia major,
patients with TIS present later in life compared to beta Thalassaemia
major, therefore, progression and severity of symptoms depends upon
the haemoglobin levels maintained by these children besides nutritional
deficiency or infections which may increase the severity of anaemia or
result in transient severe anaemia. Adaptation to these haematological
changes makes patients able to maintain their haemoglobin at a level
which is compatible with survival in steady state until stress situation
like infections, hypersplenism or nutritional deficiency of vitamin B12
and/or folic acid occur which may cause severe anaemia and other
related symptoms.
The genetic mechanisms responsible for production of TIS are still
not well understood. However, several factors like inheritance of
relatively milder beta alleles, coinheritance of alpha Thalassaemia and
genetic determinants that increase the gamma chain production have
been implicated and considered to ameliorate the severity resulting in
a diverse clinical spectrum of TIS (Table 22.1). All these factors result in
less globin chain imbalance Although the red cells are hypochromic
but are relatively more efficient.
On the other hand coinheritance of triplicated α globin gene
arrangement in beta Thalassaemia trait on one chromosome (ααα/αα)
may cause severe clinical phenotype.2,3 Severity in these patients vary
with the individual capacity with which excess unbound alpha chains
are removed by proteolytic mechanism.3
It is now well known that increased production of fetal haemoglobin
or hereditary persistence of fetal haemoglobin can effectively
compensate for reduced beta chain production4 but the underlying
factors which determine the increased production of HbF are still not
clear. Whether it is a basic molecular defect or certain racial groups
Table 22.1: Genetic mechanism responsible for phenotype of beta thalassemia

intermedia
1. Mild deficit in β globin production
• Homozygous silent or mild β+ thalassemia
• Compound heterozygosity for severe β° or β+ and mild or silent β+
thalassemia
2. Reduced globin imbalance due to coinheritance of α and β thalassemia
• Homozygous or compound heterozygous severe β° or β+ thalassemia
with deletional or nondeletional α gene mutation.
3. Severe β thalassemia with increased γ chain synthesis
• Homozygous or compound heterozygous β° or β+ thalassemia with
heterocellular HPFH.
4. Deletion forms for delta beta thalassemia and HPFH
• Homozygous δβ° or (Ayδβ°) thalassemia
• Compound heterozygous for β° or β+ and δβ° or (Aγδβ°) thalassemia
5. Compound heterozygosity of β or δβ thalassemia and β chain structural variants
• Hbs S/E/D
6. Miscellaneous
• β thalassemia trait associated with ααα or αααα gene arrangements.
• Dominant β thalassemia
• Highly unstable β globin chain variants.
who have a tendency to synthesize gamma chain more efficiently in

response to anaemia and ineffective erythropoiesis.5
Apart from this, certain acquired factors like severe infections,
hypersplenism or folate deficiency are important and known to
influence the phenotype resulting into a severe clinical phenotype.
Compound heterozygous state for delta beta Thalassaemia and beta
Thalassaemia has also been found to be associated with a clinical picture
of Thalassaemia intermedia. Inheritance of β Thalassaemia mutation
along with structural haemoglobin variants such as HbE, sickle cell or
HbD may also manifest as TIS.
DIAGNOSIS
Clinical Features
The clinical manifestations of TIS are extremely variable. Approximately
10 per cent patients present early in life with relatively severe anaemia
while others (≅60%) remains asymptomatic or present later in life.6
Unlike Thalassaemia major, where patients survival is dependent on
regular blood transfusion, patients with TIS are able to maintain a
haemoglobin level between 6-8 g/dl with minimal or no red blood cell
transfusion. Hence, presentation of patients with TIS is late and
generally after 2 years of age. The major symptoms in early childhood
are anaemia and mild jaundice with variable degree of splenomegaly.
These patients fails to thrive and develop growth retardation. Majority
of patients have bone changes in the form of maxillary prominence,

frontal bossing or depressed nasal bridge and termed as having
haemolytic facies. The bony changes are extremely variable and range
from almost none to severe skeletal deformity as seen in patients with
Thalassaemia major. The clinical course is generally one of well compen-
sated anaemia. The definite cut off for haemoglobin levels at
presentation or steady state haemoglobin levels continues to be the
subject of controversy to differentiate if from Thalassaemia major as
well from Thalassaemia minor. Therefore, misdiagnosis may occur. Well
compensated anaemia in TIS, if associated with exacerbating factors
like hypersplenism, infections or nutritional deficiency may lead to
misdiagnosis of beta Thalassaemia major. Similarly, poor socioeconomic
conditions, inadequate health facilities, lack of awareness for
investigation facilities etc. may have a bearing on the age at initial
presentation.
BETA THALASSAEMIA IN ASSOCIATION WITH STRUCTURAL

HAEMOGLOBIN VARIANTS
Interaction of structural haemoglobin variants with beta Thalassaemia
alleles are complex clinical conditions. Clinical course resembles to TIS.
It is a widespread public health problem in different parts of the world.
Common structural haemoglobin variants are HbS, C, E and HbD. Their
interaction with beta Thalassaemia result in HbS-beta Thalassaemia
HbE-beta Thalassaemia and HbD-beta Thalassaemia which are quite
frequent in different population group7 in India.
Sickle Cell-beta Thalassaemia

Sickle cell-beta Thalassaemia is a remarkably heterogenous disorder,
the clinical course may range from one extreme in which the condition
is Indistinguisable from severe sickle cell anaemia to a disorder which
is completely symptomless and which may be detected incidentally
during a family study or population screening. These patients present
with mild chronic anaemia. The clinical phenotype is dependent upon
the (a) type of beta Thalassaemia gene inherited, (b) amount and sickling
properties of HbS which in turn is dependent upon mutations for HbS
and (c) relative amount of HbA and HbF. The milder clinical phenotype
is due to inheritance of milder beta thalassemic allele which is associated
with higher amount of HbA generally in the range of 20-30 per cent7
and also higher levels of HbF which has an inhibiting effect over sickling
process. The milder subtype of HbS-beta Thalassaemia is found in
African countries and the severe variant is seen in Mediterranean region
which is associated with lower levels of HbA (0-15%). These patients
have a clinical presentation similar to sickle cell anaemia except slightly
higher propensity to have splenomegaly and foetal losses.8 However,
splenic atrophy occurs later as compared to sickle cell anaemia.9 Other

complications like sickling crisis, aplastic crisis, leg ulcers and bone
and joint disease also occurs with almost equal frequency in both
disorders but are often milder in patients having inheritance for β
Thalassaemia also.
The differentiation can be made on the basis of high HbA2 levels in
patient or a positive family history of beta Thalassaemia trait.
Laboratory evaluation reveals variable degree of anaemia and
reticulocytosis. Red cell indices like MCV and MCH are reduced. HbF
levels are variable in different population groups depending upon the
inheritance pattern of hereditary persistence of fetal haemoglobin
(HPFH) or xmn polymorphism which are responsible for higher levels
of HbF. Variants with high HbF values are found in Orissa and are
associated with milder phenotype.10
HbE-beta Thalassaemia
HbE-beta Thalassaemia is common public health problem and
widespread in southeast Asia, parts of India, Pakistan, Bangladesh and
Sri Lanka. The gene frequency of HbE is approximately 27-50 per cent
in eastern states of India.11 The heterozygous state for HbE is completely
asymptomatic. The homozygous state is very similar to beta
Thalassaemia trait with mild anaemia and microcytosis. However,
interaction between HbE gene with beta Thalassaemia alleles is
associated with relatively severe clinical course.
The Clinical phenotype of HbE-beta Thalassaemia is variable and
range from mild anaemia to moderate disorder. However, patients
generally present at an advanced age. Growth retardation, infections,
Hypersplenism, aplastic crisis, iron overloading and cardiopulmonary
dysfunctions secondary to pulmonary thromboembolism or abnormal
platelet aggregation are common complications. Investigations reveal
moderate to severe degree of anaemia with haemoglobin being in the
range of 5-7 g/dl. Bilirubin levels are increased. HbF shows a wide
variation from 5-85 per cent. Prognosis varies with severity of illness.
The usual cause of death is infection. Therapy is primarily supportive.
HbD-beta Thalassaemia
HbD-beta Thalassaemia frequently found in the state of Punjab and
hence the name HbD Punjab is given. The patients present with mild
anaemia and occasional enlargement of spleen. The haemoglobin varies
between 8-12 g/dl with red cell indices similar to beta Thalassaemia.
The diagnosis is based on the presence of HbD on electrophoresis, which
is present in the range of 82-95 per cent. The rest is contributed by HbA
(0-7%) and HbF (1-7%). HbD Punjab is also known as HbD Los Angeles.
HbC-beta Thalassaemia
HbC is not prevalent in India. It is commonly found in Africa and Medi-
terranean regions. This disorder has a wide heterogeneity depending
upon the type of beta Thalassaemia alleles. The levels of HbA are
dependent on type of beta Thalassaemia alleles and range from 0 to 20
per cent. HbF varies between 2-5 per cent. HbC constitute a major part
of total haemoglobin generally in the range of 65-80 per cent.
HbO Arab-beta Thalassaemia

HbO Arab-beta Thalassaemia is found in Afro-American Arabs,
Romanies and Egyptians. Patients present with mild to moderate
anaemia with HbF range between 6-8 g/dl. The RBC morphology is
predominantly microcytic. Splenomegaly is a common feature.
Interaction of Beta Thalassaemia with Unstable Haemoglobin

Interactions of unstable haemoglobins like HbSaki, Hb Duarte, Hb
Leiden and Hb Mississippi with beta Thalassaemia have also been
described. There is marked variation in clinical phenotype. The relative
high oxygen affinity, severe globin chain imbalance and oxidant damage
are the important mechanism responsible for the clinical course.
HAEMATOLOGICAL FEATURES
Patients with TIS have variable degree of anaemia with haemoglobin
levels range between 6-8 g/dl. The children present at a later age.
Symptoms of other associated disorders are present with variable
severity. These children have growth retardation as a result of chronic
anaemia. There is a generalized osteoporosis and long bones are
susceptible to fractures. Haemolytic and skeletal abnormalities and
organomegaly occur secondary to excessive erythropoiesis. The red
cell changes are indistinguishable from that of beta Thalassaemia major.
Variable presence of nucleated red cells may be associated with elevated
reticulocyte count. The red cell indices like MCV and MCH are generally
low. Bone marrow examination shows erythroid hyperplasia.
Other haemoglobin like HbF and HbA2 also show marked variability
due to genetic heterogeneity. In Thalassaemia intermedia no single
pattern of HbF is typical. HbF may range from 10-90 per cent12 but can
be as low as 5 per cent13 or as high as 99 per cent.14 A recent transfusion
can alter the levels of HbF. Patients who are heterozygous for beta
Thalassaemia and presenting as Thalassaemia intermedia may have
Hb A2 levels significantly elevated. The red cell survival studies have
shown moderate to considerable reduction in red cell survival time
ranging from 10-16 days. 15 Most of the time the diagnosis of
Thalassaemia intermedia is based only on clinical features as no specific
diagnostic tests are available for TIS. A steady state haemoglobin level,
transfusion requirement, mild growth retardation and skeletal changes

are the main differentiating features between beta Thalassaemia major
and TIS. If a patient present after two year of age, should be followed-
up for any fall in general activity, haemoglobin levels, growth velocity,
increase in spleen size or development of skeletal deformity. If the
progression of disease is mild and child does not require blood
transfusion, a close observation and assessment over a period of time
is required before categorizing them into TIS.
Other causes of anaemia secondary to concomitant folate and/or
vitamin B12 deficiency, hypersplenism, red cell aplasia secondary to
parvovirus infection and aplastic or sequestration crisis should always
be excluded before considering a diagnosis of beta Thalassaemia major
or TIS.
COMPLICATIONS
The development of complications in TIS depend upon the degree of
imbalance between alpha and beta globin chain and consequent red
cell destruction, severity of ineffective erythropoiesis, iron overload
and various precipitating factors like infections, hypersplenism etc.
Iron accumulation occur at a slower rate in TIS as compared to
Thalassaemia major and manifestations of iron overload develop at a
relatively higher age.16,17 However, it is not always possible to correlate
high iron levels with age or haemoglobin levels.18 Other factors which
influence are diet, erythropoiesis status and requirements of blood
transfusions. These patients need to be monitored with serum ferritin
levels and other parameters for assessing iron overload. These children
should be managed with chelating agents such as desferrioxamine and
deferiprone etc.
Endocrine Dysfunctions
Diabetes mellitus, hypopituitarism, hypogonadism are the common
endocrine dysfunctions seen in patients with TIS.19,20,21 Abnormality
in other endocrine organs like thyroid or adrenal is less frequent.
Endocrine dysfunctions are generally less common in TIS as compared
to inadequately chelated transfusion dependent Thalassaemia major
as the iron overload is not very high and secondly the complication
appears at higher age.22 Patients with TIS need to be investigated for
various endocrine functions periodically for their early detection and
appropriate management.
Skeletal Deformity and Bone and Joint Disease

Skeletal deformity secondary to massive expansion of bone marrow
and production of extramedulary haematopoietic tumours are very
much similar to Thalassaemia major in some of the patients of TIS.23
Other skeletal abnormalities like bone and joints pains, radiological

changes like widening of medullary spaces, thinning of cortical bone,
microfractures osteopenia and osteomalacia are also seen in TIS.20,24,25
Sometimes they may be severe enough to cause the disability especially
in the older patients. The only way for prevention of skeletal abnormali-
ties is to initiate blood transfusion at an early age.
Hypersplenism
Progressive enlargement of spleen aggravates the anaemia by reducing
red cell survival and also by haemodilutional effect.26 However, the
pathophysiology of anaemia in Thalassaemia is complex. Spleen being
a site of extramedullary erythropoiesis attribute towards increasing
metabolic demands in the situation of ineffective erythropoiesis, thereby,
suppressing the growth and development in these children. Splenec-
tomy may be of help in reducing the requirement of blood transfusion
and correction of anaemia, thrombocytopenia and neutropenia. Hyper-
splenism may be avoided by instituting an early and regular transfusion
therapy in children with Thalassaemia major.27 However, no such study
is available in TIS. Thus maintaining optimum level of haemoglobin
by any means is essential to avoid development of hypersplenism and
may really cut down the need of splenectomy and its associated
complications.
Iron Overload
Mild to severe iron overload is frequently seen in patients with TIS
which generally becomes evident after second and third decades of
life. Increased iron absorption is the chief mechanism of iron loading
and leading to haemosiderosis in transfusion independent or minimally
transfused patients with TIS.18 Iron overloading is associated with
cardiac failure, endocrine dysfunctions, diabetes mellitus, hypopi-
uitarism and hypogonadism.16,28,29 Therefore, patients with high serum
ferritin levels need to be diagnosed early and managed appropriately
with chelating agents to prevent iron overload and its complications.
Pregnancy
Patients with TIS have normal puberty and it is expected that they
would have normal sexual development and pregnancy as well.
However, patients who are inadequately transfused either due to social
or economic constraints and maintain a low haemoglobin level (< 7 g/
dl), may have 30-50 per cent reproductive wastage.30 On the other hand,
patients who were well transfused and maintained a haemoglobin level
of more than 10 g/dl had full term normal delivery in 50-85 per cent
cases.30,31,32
Thromboembolic Disease
Thromboembolic phenomenon in TIS have been observed uncommonly.
Chronic hypercoagulable state in TIS may be associated with increased
platelet activation secondary to procoagulant effect of RBC membrane33
similar to Thalassaemia major and this does not have any association
with age. An approximate incidence of thromboembolic phenomenon
in TIS has been observed to be 9.61 per cent.34 However, no particular
risk factor has been found to be associated except a possible association
of chronic liver disease or pulmonary hypertension.34,35 It is also not
clear whether, increased thrombosis in TIS is related to splenectomy.36
Patients with hypercoagulable state may present with pain depending
upon the site of thrombus, fever and local swelling and doppler
ultrasound and/or CT scan would demonstrate the venous or arterial
thrombus and extent of obstruction.
Infections
Patients with TIS are quite susceptible to develop infections by hepatitis
B and C, parvovirus and yersinia enterocolitica, especially those who
receive multiple blood transfusion for severe anaemia. IL-8 levels are
increased secondary to infection leading to the development of hyper-
active macrophages which may precipitate chronic haemolysis.37
Other complications like leg ulcers hyperuricaemia, gout20 Gall
stones 15 and folic acid deficiency 6 have also been reported in
Thalassaemia intermedia.
THERAPEUTIC OPTIONS
The conventional treatment in TIS remains controversial. Earlier therapy
has been limited to transfusion and chelation only. Recently novel
therapeutic modalities have been developed based on pathophysiology
and molecular pathology of the disease and have been found to be
associated with a better response and outcome.38
Red Cell Transfusion

The objective of transfusion regimen in beta Thalassaemia is to correct
anaemia and to suppress endogenous erythropoiesis. As yet there is
no consensus regarding the transfusion regimen in TIS. Presently it is
advisable not to transfuse a child on the basis of haemoglobin levels
alone and apart from steady state haemoglobin levels (6-8 g/dl), other
parameters like the clinical assessment of child activity, growth and
development, spleen size, progression of skeletal changes and other
complications related to TIS should be considered before initiating
transfusion therapy. Decision of transfusion therapy should be based
upon the individual needs rather than haemogobin levels.
A regular blood transfusion regimen similar to Thalassaemia major

should be started if there is any decline in growth velocity or early
skeletal deformity or anaemia hampering the child’s activity. However,
in presence of crisis following infections, hypersplenism or in cases
where Hb falls below 6 g/dl, blood transfusion therapy becomes
necessary. In cases with normal growth and development and in absence
of early bony changes or hypersplenism patients should be kept under
regular follow-up. Medullary width (> 0.5 cm) in the midpoint of second
left metacarpal regardless of age or haemoglobin levels may have been
used as an independent criteria for initiation of transfusion treatment
in patient with TIS.39
In the absence of an established guidelines it is felt that multicentre
study is required to define the minimal baseline haemoglobin level
which is just sufficient to correct anaemia and suppressing erythro-
poiesis along with prevention of iron overload. Further the risks and
benefits of transfusion therapy should be analysed on individual basis.
Iron Chelation Therapy

Iron overload in patients with TIS appear to be much lower than in
transfusion dependent beta Thalassaemia major patients. Increased
erythropoiesis is mainly responsible for abnormally high iron
absorption and subsequent development of haemosiderosis in non-
transfused or minimally transfused patients with TIS29 and this tends
to increase progressively with age of the patients.
Iron chelation in patients with TIS is indicated generally after 5 years
of age. The body iron should be assessed periodically in all patients of
TIS at an interval of 3-4 months, if serum ferritin levels are in excess of
1000 ng/l, iron chelation therapy should be instituted40 using either
oral iron chelator deferiprone or by subcutaneous infusion of
desferrioxamine. In patients with very high ferritin levels or in presence
of complications of iron overload it may be desirable to initiate combina-
tion therapy (daily deferiprone along with desferrioxamine twice a
week) to chelate iron more effectively.
The therapy may be tailored according to individual needs of each
patient and should be established by close monitoring of serum ferritin
levels. Spleen probably has some role in regulation of iron metabolism
as serum ferritin is found to be higher in splenectomized patients
compared to non-splenectomized patients.18
Other Pharmacological Agents

Recently several drugs like hydroxyurea, arginine butyrate, 5
azacytadine, myeleran and recombinant erythropoietin have been used
for treatment in patients with TIS. These drugs have the potential of
improving the general status, haemoglobin levels and reduce the need
of transfusion. Hydroxyurea acts by enhancing the synthesis of HbF

using excess alpha chains and reduces the imbalance between alpha
and beta chains, thereby ameliorates the severity of disease. At present
hydroxyurea is being used at several centres effectively. It activates
gamma globin gene when used in doses of 10-20 mg/kg/day and has
low toxicity.
Apart from its role in gamma globin biosynthesis, it also appear to
have a role in increased synthesis of beta globin as evident from increase
in total haemoglobin level with a relative reduction in percentage of
HbF.41 Hydroxyurea can be used alone or in combination with sodium
phenylbutyrate or other experimental drugs. 42,43 Patients with
Thalassaemia intermedia can also be treated effectively using
recombinant human erythropoietin.44 Currently studies are in progress
where hydroxyurea along with erythropoietin is being used to evaluate
its efficacy.
Splenectomy
In severe form of beta Thalassaemia splenectomy has been a part of
therapeutic regime over several years. Splenectomy has also been
recommended for past few years in patients of Thalassaemia
intermedia. However, the data is insufficient regarding the optimum
haemoglobin levels and spleen size before splenectomy which could
determine the efficacy and risks of splenectomy. It was postulated that
splenectomy may improve steady state haemoglobin levels and reduce
the transfusion requirement.6 Decision regarding the splenectomy
should made either on clinical or laboratory evaluation or both.
Laboratory assessment includes red cell survival studies, site of RBC
destruction, red cell pool in spleen, extent of splenic erythropoiesis and
evidence for bi or pancytopenia. However, these studies are time
consuming, expensive and can be carried out only at reference
laboratory. Size of spleen cannot be a sole criteria for performing
splenectomy. Splenectomy in patients of Thalassaemia intermedia is
recommended if hypersplenism is associated with > 50 per cent increase
in annual requirement of blood transfusion or physical discomfort due
to massive splenomegaly or development of pancytopenia and presence
of bleeding symptoms during the course of the disease. Moreover, fall
in steady state haemoglobin level or reduction in the rate of growth
velocity with or without associated anaemia and early skeletal changes
have to be considered before performing splenectomy in Thalassaemia
intermedia patients.38 Splenectomy is a safe procedure but post-
operative complications are several, and includes infections, thrombo-
embolism, large liver syndrome and iron overload.18 Severe and
overwhelming infections due to streptococcus pneumoniae, haemo-
philus influenzae and neisseria meningitis are frequent and seen
especially in young children who are less than 6 years of age. Children
should be given H influenzae, pneumococcal vaccination at least 4-6
weeks prior to splenectomy.
Removal of spleen reduces the primary immune response to
encapsulated organisms. Therefore, the general practice is to defer the
splenectomy till 5 years of age. However, the age of the child is not an
absolute contraindication and potential benefits of splenectomy must
be balanced against postoperative risks of infections. The postoperative
period should be observed carefully for any change in steady state
haemoglobin levels, growth velocity and disappearance of symptoms
of anaemia. These patients should be given prophylactic penicillin
preferably life long or at least till 20 years of age to prevent post
splenectomy sepsis.
ANTICOAGULANT THERAPY
Major thrombotic episodes, particularly postsplenectomy should be
treated initially with heparin followed by long-term oral anticoagulant
therapy such as warfarin to maintain an INR between 1.5-3.
OTHER SUPPORTIVE CARE

1. All patients with intermediate Thalassaemia should be put on
regular folate supplementation.
2. Drinking strong tea or coffee after major meals has been reported
to reduce the quantum of iron overload.
3. Food rich in iron or which enhances the absorption of iron like
citrus fruits should be avoided.
4. Patients should be encouraged to take milk and diet rich in
calcium.
5. Cholecystectomy may be performed in patients who develops
manifestations of cholengitis, cholestasis, and cholilithiasis in the
form of recurrent abdominal pain and swelling.
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in Thalassaemia major. Br J Hematol. 1974;28:77.
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Thalassaemia. Blood. 1997;89:739.
28. Banherman RM, Keusch G, Kreimer-Birnbaum M, Vance VFK, Vaughan S.
Thalassaemia intermedia with iron overlaod, cardiac failure, diabetes ellitus,
hypopituitarism and porphyrinuria. Am J Med. 1967;42:476.
29. Calada A. Iron overload in a non-transfused patient with Thalassaemia
intermedia. Scand J Haematol. Feb. 1982;28(2):169-74.
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hematological profile of thalasemia intermedia patients. Int J Hum Gen. 2003
(In Press).
32. Scordis N, Christou S, Koliou M, Pavlides N, Angastinatis M. Fertility in
females patients with Thalassaemia. J Paediatric. Endocrinol Metabol
1998;11:935-45.
33. Ruf A, Pick M, Deutsch V. In vivo platelet activation correlates with red cell
anionic phospholipid exposure in patients with B Thalassaemia major Br. J.
Haematol 1997;98:51.
34. Borgna-pignatti C, Bugolotto S, De Stafano P. Survival and disease
complications in Thalassaemia major. Ann N Y Acad Sci. 1998;50-222.
35. Taher A, Abou Mourad Y, Abchee A, Zalouaa P, Shamseddine A. Pulmonary
thromboembolism in beta Thalassaemia intermedia: Are we aware of this
complication. Haemolgobin 2002;26(2):107-12.
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splenectomy for Paediatric Haematologic disease. J Peadiatr Surg. 1993;28:1109.
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Thalassaemia intermedia. Haematological 1995;80:431.
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Minerva Ginecol, Jan-Feb; 2000;52(1-2):29-31.
23
Cytogenetics of Leukaemias and
Myelodysplastic Syndromes
S Tyagi
Key Words
Cytogenetics • Molecular • Acute leukaemia
• Myelodysplastic syndromes • Chronic myeloid
leukaemia
INTRODUCTION
The discovery of Philadelphia chromosome in 1960 by Nowell and
Hungerford1 has made a large impact in the understanding of the
leukaemogenesis of chronic myeloid leukaemia (CML). However, later
in 1970 the discovery of banding techniques by Caspersson and others2,3
made it possible to identify the individual chromosome or part of
chromosome. An international system for human chromosome
nomenclature (ISCN) has4 allows a precise description and identifica-
tion of chromosomal rearrangements which are responsible for
molecular abnormalities present in various leukaemias and lymphomas.
Several large studies on chromosomes in leukaemias 5-7 have
correlated the cytogenetic abnormalities with clinical features and
morphological FAB subtypes of acute myeloid leukaemia (AML).8 This
helps in identification of AML subgroups with distinct clinical
behaviour and response to therapy. Epidemiological and aetiological
factors were also found to be correlated with genetic changes.
Correlation between chromosomal translocations with immuno-
logical subtypes of acute lymphoblastic Leukaemias (ALLs) both in
children and adults helped in defining the various prognostic groups
and this is required for the institution of targeted therapy.
With the advancement of molecular genetic technology, the
mechanism of the genetic control of normal haematopoiesis, gene
rearrangements, disregulated cell proliferations and consequently the
development of cancer has been studied extensively.9 The formation of
Cytogenetics of Leukaemias and Myelodysplastic Syndromes 373
novel fusion protein as a result of chromosomal translocation is the

commonest pathway for leukaemogenesis, e.g. BCR-ABL fusion protein
which is known to cause CML by increasing tyrosine kinase activity.
However, other mechanism for gene mutation like loss of tumour
suppressor genes or transcriptional upregulation of gene also exist.
Chromosomal analysis can be performed by standard cytogenetics
using banding techniques, by means of fluorescence in situ hybridiza-
tion (FISH) and southern blot analysis using specific probes for
particular chromosomal abnormalities especially for the translocations
or by more sensitive and advanced molecular techniques like multiplex
polymerase chain reaction (PCR).
These methods can also be used for monitoring the course of the
disease and for detecting relapse as well as the remission status at the
molecular level.
Cytogenetic analysis of malignant diseases is based on the study of
the tumour cells themselves. In leukaemia, the specimen is usually
obtained by bone marrow aspiration. Rarely, bone marrow biopsy can
be processed. Peripheral blood can be cultured in patients with high
total leucocyte count (TLC >10,000/μl) or blast count of more than 10
per cent. Similarly specimen containing tumour mass or involved
lymph nodes or effusion fluid may also be processed for the analysis
of chromosomes in lymphomas. Cytogenetic studies are possible only
for those specimens which contain viable dividing cells. Therefore,
sample should immediately be transported to the laboratory for analysis
to get adequate results.
Chromosomal abnormalities are described according to ISCN.
Structural rearrangements (e.g. translocations, deletions, inversions)
or gain of the same chromosome in at least two cells or loss of the same
chromosome in at least three cells is required to document the presence
of abnormal clone. However, one cell with normal karyotype indicates
the presence of normal cell line.
Different Approaches for Carrying out Chromosomal Analysis

Chromosomal analysis can be carried out using different approaches:
1. Karyotype analysis using banding techniques: Both numerical as well
as structural abnormalities can be identified in proliferating cell clones.
However, it has limited sensitivity due to limited resolution of banding
and presence of submicroscopic deletions and requires highly skilled
personnel. The technique depends on the quality of leukaemic chromo-
somes and the mitotic index which can be enhanced by adding growth
factors and/or synchronization techniques thereby more subtle
abnormalities can be reliably diagnosed. These modifications are
specific for the type of leukaemia like myeloid or lymphoid. The sample
should also be provided with adequate clinical history regarding age
of patient, type of leukaemia and recent therapy, if any.
2. Molecular cytogenetics: Single stranded DNA can be hybridized

specifically with complementary sequences and this property is used
in in situ hybridization techniques. DNA probes with known specificity
are commercially available which hybridize with DNA sequences of
chromosome in the interphase cells or metaphases and can be visualized
using fluorochrome. This is termed as FISH. FISH bridges the gap
between conventional cytogenetics and advanced molecular DNA
techniques like PCR. FISH has the intermediate sensitivity and can be
used on interphase cells using larger probes and, therefore, is an ideal
technique for follow-up studies of patients with leukaemia as well for
the detection of minimal residual disease. The main advantage of this
technique is that a large population of cells could be screened using
interphase cells. Chromosome painting techniques also allow the
detection of complex karyotype due to multichromosomal rearrange-
ments which could be easily missed by standard cytogenetic techniques.
Complex karyotypes are generally seen in myelodysplastic syndromes
(MDS) and secondary leukaemia.
Now a new technique called comparative genomic hybridization
(CGH) has come up where DNA from normal and leukaemic cells can
be labeled with different fluorochromes (e.g. red and green). They are
mixed and then applied to normal chromosomes. If there is no numeric
aberration of DNA, the chromosome is yellow and if chromosome
rearrangement is present the affected region will be green or red
depending on the colours used to label the different DNAs.
Recently techniques like spectral karyotyping (SKY) or multicolour
FISH (M-FISH) are also coming where using single or multiple fluors,
the involved chromosome is identified. The disadvantage here is that
the specific region of chromosome is not identified. Therefore,
additional analysis with specific DNA probes is required to identify
the abnormal karyotype.
A precise cytogenetic and genetic description of leukaemia is
becoming increasingly important in instituting the proposed gene
therapy in future.
CYTOGENETIC ABNORMALITIES IN LEUKAEMIA

Chromosomal abnormalities in leukaemia are clonal. The defect is
present either in the pleuripotent stem cells or committed progenitors
and thereafter consistently seen in the differentiated cells. Although
standard cytogenetic techniques are highly sensitive (10-2). Non-
detection could be secondary to poor quality of sample, inadequate
transport, technical insufficiencies or presence of subtle aberrations
which are not visible by standard banding techniques. However, if done
properly chromosomal aberrations are identified in more than 90 per
cent of cases. Common lesions are usually reciprocal translocations,
insertions or inversions seen in children and young adults with

leukaemia. Other defects are deletions, isochromosomes or rings seen
in MDS or secondary leukaemias.
Numeric changes (gain or loss of whole chromosome) are frequent
non-random changes usually found in pre-malignant conditions and
secondary leukaemia. They lack specificity, e.g. trisomy 8, may be seen
in several malignant and pre-malignant conditions like AML, MDS or
myeloproliferative disorder.
Gene amplification like homogenous staining regions (HSR) or
double minutes (dm) are commonly seen in tissue malignancies but
rarely seen in haematological malignancies.
The frequency of various specific non-random chromosome aberra-
tions associated with different types of leukaemias is variable ranging
from less than 1 per cent to more than 90 per cent. Numeric aberrations
and deletion like +8, +12, -5 or 5q-, -7 or 7q-, 9q-, 20q- have a frequency
approximately 40 per cent and are seen in secondary leukaemias or in
leukaemia following MDS. They are often associated with complex
karyotypic abnormalities and carry an unfavourable prognosis with
short survival. The frequency of rare balanced translocations like t(1;22),
t(6;9), t(3;21) and trisomy 4 or trisomy 11 is less than 1 per cent.
THE COMMON CHROMOSOMAL ABERRATIONS IN AML

1. t(8;21)(q22;q22): This was the first translocation identified in human
cancer in 1972.10 It is most frequently observed specific chromosomal
translocation involving AML gene on chromosome 21 and ETO gene
on chromosome 8 in children and young adults with AML with a
frequency of 20 and 10 per cent respectively. It is strongly associated
with FAB subtype AML-M2 which has a characteristic morphology with
prominence of auer rods and strong myeloperoxidase positivity.9 More
than 90 per cent AMLs with t(8;21) defect were found to have FAB-M2
morphology while as many as 30-40 per cent AML cases with FAB-M2
morphology have t(8;21) abnormality. The characteristic immuno-
phenotype in these cases is CD13, CD33, CD19, CD34 and CD56
positivity. Therefore combination of FAB-M2 morphology with this
immunophenotype pattern strongly suggests the presence of t(8;21).
However, t(8;21) can also be found in few cases with FAB-M4
morphology, MDS and as a secondary change in blast crisis of Ph+
CML.5,9 Although t(8;21) usually predicts good prognosis in young
adults with high remission rate and longer median survival, the survival
time is shorter compared to that of AML cases with inv(16) and t(15;17).
The disease with t(8;21) usually relapses frequently but the recurrence
remain responsive to aggressive chemotherapy.11 The results obtained
with chemotherapy alone are very good and comparable to allogenic
BMT. Therefore detection of this molecular genetic lesion at diagnosis
may be helpful in deciding the treatment plans and really cut down
the transplant associated morbidity and mortality. However, paediatric
AML with t(8;21) have much less favourable prognosis.12,13
2. Inv(16) (p13;q22) and t(16;16) (p13; q22): Inversion 16 inv (16) is a
subtle chromosome abnormalities seen in approximately 8-12 per cent
cases of all AMLs 9 and in at least 25 per cent cases of acute
myelomonocytic leukaemia (AML M4E0) which has been characterized
morphologically by prominence of >40 per cent eosinophils and their
precursors with abnormal basophilic granules in the bone marrow
which are positive for periodic acid-schiff (PAS) and chloroacetate
esterase. The percentage of these abnormal eosinophils has been
reported to be as low as 5 per cent in some studies.14 Patients with inv
(16) or t(16;16) or del(16) have a very high remission rate (>95%).
However, relapses are also seen. This chromosomal abnormality may
also be seen in FAB AML-M2 and M5 subtypes. Inv(16) or t(16;16) results
in an unusual chimaric fusion. The inversion break point at 16q22 occurs
near the end of the coding region of CBFB which encodes one subunit
of a novel heterodimeric AML1/CBFB transcription factor. Fusion of
aminoterminus of the CBFB transcription factor to the carboxyl
terminus of the cytoplasmic smooth muscle myosin heavy-chain gene
(MYH II)12,15 produces a fusion protein which can be detected by RT-
PCR assay. How this protein contributes to leukaemiogenesis is still
not known clearly. Possibly it disrupts the function of AML1/CBFB
transcription factor. Other types of fusion transcripts have also been
reported. The frequency of CBFB/ MYH II fusion is >90 per cent in
AML cases with inv(16) or t(16;16). The detection of inv(16) by standard
cytogenetic technique is quite difficult. Approximately, 10 per cent cases
with AML M4 morphology, lacking eosinophilic abnormalities have
detectable CBFB/MYH II transcripts by RT-PCR. Leukaemias with
inv(16) have various similarities with t(8;21). Both involves the gene
encoding the core binding protein, a heterodimeric transcription factor
required for haematopoiesis. Secondly, both have distinct specific
morphological FAB subtypes and overall prognosis is good in both
conditions.
3. t(15;17)(q22;q21): Approximately 12-15 per cent of all AML cases in
young adults are acute promyelocytic leukaemia (APML) which is
classified into FAB (AML M3) subtype based on typical morphology of
blasts and clinical features. Presence of t(15;17) seen in cases of AML
M3 results from fusion of a large part of the retinoic acid receptor alpha
(RARA) gene on chromosome 17 to promyelocytic leukaemia (PML)
gene on chromosome 15. The PML gene function as a tumour suppressor
gene while RARA has differentiation promoting and growth
suppressing activities. The aberrant PML/RARA protein acts as a
dominant negative inhibitor of both PML and RARA gene and therefore,
shows an acquired higher growth rate of myeloid precursor cells

consequent to diminished apoptotic cell death. This aberrant protein is
very sensitive to all trans retinoic acid (ATRA). Therefore, the newer
therapeutic approaches based on high doses of retinoic acid are
currently being used for treatment of APML cases. Patients with APML
are generally young adults who develops bleeding diathesis with
dissiminated intravascular coagulation like picture which require an
early and specific induction therapy with ATRA to reduce the mortality
associated with it. Patients who survive this critical phase respond well
to chemotherapy. Therefore it is essential to detect t(15;17) to determine
which patient will be benefited by ATRA induction therapy. Recently a
monoclonal antibody CD56 directed against PML/RARA fusion protein
has been developed which has a diagnostic specificity for APML. This
can be demonstrated by immunocytochemical method to allow a rapid
diagnosis of APML. t(11;17)(q23;q21) is a variant in APML and results
from fusion of a zinc finger gene called PLZF on chromosome 11 to
RARA gene on chromosome 17.2,9 Morphologically it is different from
classic APML in the sense that nucleus is more regular and abundant
cytoplasm with coarse fine granules. APML with t(11;17) are resistant
to ATRA therapy. Another variant is t(5;17) (NPM/RARA).
4. t(11q23): This chromosomal abnormality is very frequently seen and
can be found both in AMLs as well as in ALLs. t(9;11)(p22;q23),
t(6;11)(q27;q23) and t(11;19)(q23;p13) are common in (FAB M5) and
represent 5-6 per cent of all AMLs whereas approximately 7-10 per cent
cases of ALLs show chromosomal aberrations like t(11;19) (q22;p13)
and t(4;11)(q21;q23).16,17 t(11q23) is highly prevalent in MDS and patients
with secondary leukaemia especially secondary to drugs that target
topoisomerase II activity. 18 t(11q23) translocation carries a poor
prognosis in all age groups and phenotypes of leukaemia.16,19 Patient
generally presents with aggressive clinical behaviour and are less
responsive to intensive chemotherapy. The survival rate is low (3-5%).20
These characteristic features of t(11q23) suggest the probability of a
defect that is present in pluripotent progenitor cells which could
differentiate into either lymphoid or myeloid cell.
The t(11q23) occur in upto 70-80 per cent cases of infants leukaemia
irrespective of the phenotype.9,20 It has been shown that mixed lineage
leukaemia (MLL) gene is involved in about 95 per cent cases of 11q23
translocations in both AML and ALL. 12 However, MLL gene is
invariably involved in all cases of infant leukaemia. Although the MLL
fusion gene and proteins are poorly understood, it appears that
duplication of MLL gene or its fusion with a partner gene forms a fusion
protein which disregulates the homeobox (HOX) gene expression,
leading to leukaemogenesis.
5. Monosomy 5,7, 5q-, 7q-: Complex or multiple chromosomal

abnormalities seen in elderly patients with AML, therapy related AML
or MDS and are associated with poor prognosis. Recent cytogenetic
studies revealed that multiple distinct regions are involved. Two regions
7q22 and 7q32-34 and four different regions like 5q11, 5q13-21, 5q31
and 5q33-35 are involved in 7q- and 5q- chromosomal abnormalities
respectively. Since -5/5q- and -7/7q- abnormalities are common in
elderly patients. It is proposed that perhaps the cumulative environment
exposure gives rise to genomeic instability which is responsible for these
rearrangements and deletions and subsequently the development of
leukaemia.
CYTOGENETIC ABNORMALITIES IN ALL

Initially patients with ALL were classified into relevant risk groups
based only on clinical features like age and white blood cell counts at
diagnosis and response to treatment, etc. 21 Later on, with the
improvement in cytogenetic techniques and development of molecular
methods, a number of structural and chromosomal changes have been
detected and incorporated into the existing risk classification system.
However, cytogenetic studies are technically more difficult to perform
in ALL than for AML because metaphase chromosomes are short, fuzzy
and poorly spread. Techniques like FISH, RT-PCR made it possible to
detect the exact molecular defects and thus the low and high risk groups
of patients with ALL are defined.
PLOIDY SYSTEM
The ploidy system is very informative and used from the time when
banding techniques were not developed properly and resolution was
poor. There are five classes.
1. High hyperdiploidy: When chromosome number range from 50-
60 (average no. 55) with DNA index more than 1.16. The prognosis
is excellent with well tolerated drug regimen. This is seen in
approximately 25-30 per cent of children with ALL22 but rare in
adults (<5%). Commonly non-random gain of chromosomes 21,
X, 18, 14, 4, 6, 10 and 17 are seen. However, presence of additional
structural abnormalities has a negative prognostic impact in this
group.
2. Low hyperdiploidy: When chromosome number range from 47-
50 (DNA index >1.00 but <1.16). It is found in 8-15 per cent of
childhood ALL cases.23 Trisomy 21 is most frequent numeric
change seen in this group followed by trisomy 8 and 10. The
frequency of associated structural abnormalities is very high
(~75%). The overall prognosis is less favourable than high hyper-
diploidy group.
3. Hypodiploidy: When the chromosome number is less than 46.

The frequency is 7-8 per cent. The prognosis is generally unfavour-
able. A subtype of this group termed as near haploidy is a rare
entity where chromosome number are less than 30. Near haploid
leukaemia is associated with poor prognosis.
4. Pseudodiploid: This group is characterized by diploid number
of chromosomes accompanied by structural chromosomal abnor-
malities. It carries a worst prognosis and found in 40-50 per cent
patients of leukaemia.
5. The diploid Karyotype: Seen in 9-30 per cent of cases of leukaemia.
It is seen in approximately 30 per cent of cases of T-ALL.
STRUCTURAL CHANGES IN ALL

a. t(8;14)(q24;q32): This translocation is seen in B cell ALL (FAB-L2)
and Burkitt lymphoma indicating that both are different
manifestations of the same disease. This group of patients present
with organ involvement at diagnosis. The prognosis is poor.
However, with intensive chemotherapy the event free survival is
approximately 80 per cent. Other variant translocations seen in
B-cell ALL and Burkitt lymphoma are t(2;8)(p12;q24) and
t(8;22)(q24;q11). The basic functional abnormality is juxtaposition
of MYC oncogene (8q24) to immunoglobulin gene regulatory
element on chromosome 14,2 or 22 resulting in an upregulation
of the expression of MYC oncogene.24
b. t(1;19)(q23;p13): It is reciprocal translocation and seen approxi-
mately in 5-6 per cent cases of childhood ALL. It shows a strong
association with Pre-B ALL (25-30% of Pre-B ALL). This
rearrangement can occur in both forms—in balanced form (25%)
as well as an unbalanced form (75%). Better outcome was observed
in patients who had balanced t(1;19) as compared to patients with
unbalanced form.25
t(1;19) produces fusion of two genes E2A on chromosome 19 and
PBX1 on chromosome 1. The E2A/PBX1 fusion proteins activate
the gene expression of adverse target genes in either Pre-B cells or
their progenitors that could directly contribute to malignant cell
transformation.
c. t(12;21)(p12;q22): The most common cytogenetic anomaly found
in children with B-ALL is t(12;21). It is difficult to detect by
conventional cytogenetics alone as morphology of the involved
chromosomes remained intact due to similar size and banding
pattern of both the chromosomes 12 and 21. However, it can be
detected by other sensitive molecular techniques like FISH or PCR.
It is seen in 15-30 per cent of childhood B-ALL and 3-4 per cent of
adult ALL cases.26,27 There is a fusion of TEL gene at chromosome
12 with AML1 gene at chromosome 21 to form a fusion protein
TEL/AML1 which causes malignant transformation in B cells due

to loss of function of normal TEL allele. Patients with this
translocation fall in to favourable prognostic group. In one series
patients with t(12;21) were found to have 91 per cent five year
event free survival, compared to 65 per cent for patients without
this rearrangement.27 Patients are generally young (1-10 yrs) and
are positive for B-lineage immunophenotype (CD10+, CD19+).
d. t(9;22)(q34;q11): Philadelphia chromosome or t(9;22) is present in
approximately 30 per cent of adult ALL cases.26 It is rare in children
with ALL with a low frequency of 3-5 per cent. These patients
generally have poor outcome with high relapse rate. The long term
survival is a rare phenomenon. Patients with t(9;22) are associated
with higher white blood cells (WBC) and blast count at diagnosis
and have FAB L2 morphology. They generally express myeloid
markers (CD13 and CD33) along with lymphoid markers.
However, studies revealed good outcome in patients with ALL
who presented with low WBC count at diagnosis (<25 × 109/L) or
had a good initial response to steroid or chemotherapy.28,29 The
treatment of choice in these children is allogenic bone marrow
transplantation.
e. 11q23 in ALL: Translocation involving 11q23 is seen in 5-7 per
cent of ALL cases. The most common amongst these is
t(4;11)(q21;q23) and t(11;19)(q23;p13). Translocation involving
11q23 can also be found in cases with AML in equal proportion.
f. T-cell ALL: The common recurring translocations in T-cell ALL
are t(1;14)(p21;q11), t(11;14)(p13;q11), t(10;11)(q24;q11) and
t(7;9)(q34-35;q34). Of these t(11;14)(p13;q11) is commonest (3-7%)
followed by t(10;14) (3%). T-cell specific chromosome abnormali-
ties have also been observed in lymphomas of T-cell origin. In
addition patients with T-cell ALL also found to have certain
deletions like 6q-, 9p-, 5q-, 7q-, 11q-, 12p- and 13q-. The frequency
of these deletions range from 2.5-10 per cent and they are
associated with variable outcome in patients with T-cell ALL.
Detection of deletional defects by conventional cytogenetics is
difficult in T-cell ALL. But they can be detected easily by southern
blotting or PCR technique.
The prognosis is usually unfavourable in children with T-cell ALL
as compared to those with B-cell ALL. However, 65-75 per cent long
term survival have been observed with intensive chemotherapy.30 In
adults the survival is slightly lower (~ 40%) than in children. The
recurring chromosomal translocations have not been found to be helpful
in formulating therapy for patients with T-cell ALL as it is in B-cell
ALL. Also the other clinical and laboratory features like age, sex and
WBC counts were only partially useful in assessing the risk groups for
the patients with T-cell ALL.
CYTOGENETIC ABNORMALITIES IN
MYELODYSPLASTIC SYNDROME
MDS is a heterogenous group characterized by refractory cytopenia. It
includes refractory anaemia (RA), refractory anaemia with ring
sideroblasts (RARS), refractory anaemia with excess blasts (RAEB),
RAEB in transformation (RAEB-t) and chronic myelomonocytic
leukaemia (CMML). Cytogenetic abnormalities are very frequent in
MDS patients and correlates well with the severity of MDS. However,
specific recurring chromosomal aberrations often seen in AML cases
are not observed in MDS. The overall incidence of chromosomal
aberrations is approximately 30-50 per cent in de novo MDS and 70-80
per cent in secondary MDS.2,31 The frequency is low (25-30%) in low
risk MDS like RA and RARS. High risk groups like RAEB and RAEB-t
which have a higher tendency to transform into AML shows a higher
percentage of cases (60-70%) with clonal abnormalities (Table 23.1).4,5,32
There is no close correlation with specific clinical or morphological
subtypes of MDS except 5q- syndrome which is associated with good
prognosis, predominates in RA and seen in older women, with low
blasts counts and normal or increased platelet counts. Others are 13q
and 20q which are associated with ring sideroblasts and prominent
dyserythropoiesis respectively. Monosomy 7 and complex abnormali-
ties are associated with poor prognosis and they generally predominate
in RAEB and RAEB t. Some of the clonal changes are seen in both AML
as well as in MDS, e.g. +8,-5, 5q-, -7, 7q- and 20q-. Some chromosomal
rearrangements are common in AML and myeloproliferative diseases
but not found in MDS, these include t(9;22), t(15;17).33,34 Patients with
MDS generally have single or multiple chromosome abnormalities at
diagnosis. However, new chromosomal aberrations may develop
during the course of the disease. Sometimes unrelated clonal
abnormalities may be seen in MDS cases (5%). Which is higher than
those observed in AML cases (<1%).
Cytogenetic abnormalities in MDS are helpful in predicting the
survival and progression to AML. Patients who have complex
cytogenetic abnormalities have a shorter survival and a higher incidence
of progression to AML than those who have a normal karyotype or
Table 23.1: Cytogenetic abnormalities according to FAB classification of MDS

Percentage of abnormal karyotype
Abnormality % abnormal Del5q -7/del 7q +8 Der/del Der/del
Karyotype 11q 12p
RA 20-36 70 <5 10 <5 0
RARS 15-20 20 <1 20 20 0
RAFB(t) 45-60 40 25 20 20 5
CMML 25-30 <1 20 15-20 <2 10
single chromosomal abnormality4,5,32 like -y alone, 5q-or 20q.

The international MDS risk analysis workshop32 has studied 816
patients and defined cytogenetic abnormalities, percentage of bone
marrow blasts, number of cytopenias as major variables and
incorporated these parameters into international scoring system for
evaluating survival in MDS patients and their progression to AML cases.
Based on the presence of cytogenetic abnormality three risk groups
were defined—good, intermediate and poor. The median survival of
patients within these 3 groups were 3.8, 2.4 and 0.8 years respectively.
The time for 25 per cent of these patients to undergo evolution into
AML were 5.6, 1.6 and 0.9 years.
Cytogenetic abnormality like -7, 7q- or +8 can also be used to
differentiate hypocellular MDS from aplastic anaemia. Complex
cytogenetic abnormalities involving at least three chromosomes occur
in about 15 per cent of de novo MDS and in 50 per cent of patients with
secondary MDS. Involvement of chromosomes 7 and 5 are found in 60
per cent and 40 per cent of those cases respectively (Table 23.2).
Certain chromosomal aberrations are specific with the age in MDS,
e.g. Monosomy 7 as a single abnormality is the most frequent
rearrangement seen in 25 per cent cases of pediatric MDS while only 7
per cent patients of MDS, older than 50 years, showed isolated -7
abnormality.31 Secondary MDS have isolated -7 abnormality, in 50 per
cent of cases, 5q- in about 25 per cent of patients and complex
cytogenetic abnormalities are seen in approximately 50 per cent of cases.
Although 5q- is associated with a good prognosis but when present in
secondary MDS it rarely occurs in isolation and carry a poor prognosis.
Balanced translocations are rare in secondary MDS, as compared to
unbalanced translocations, which lead to partial monosamy or trisomy
and they often involve chromosome 5, 7 and 17.
SPECIFIC ENTITIES BASED ON

CYTOGENETIC FINDINGS IN MDS
-5q /5q- syndrome: Del 5q is the most common cytogenetic abnormality
in MDS and is seen in 10 per cent of cases of MDS. However, the
frequency is higher in therapy related MDS. A typical 5q- syndrome is
seen in elderly woman, in 70-75 per cent cases of RA, and have an
association with macrocytic anaemia, erythoblastopenia, hypolobulated
megakaryocytes and thrombocytosis. These cases rarely progress to
AML. 5q- abnormality observed in RAEB or RAEB-t is usually
associated with complex karyotype often with abnormalities of
chromosome 7.4 -5 or 5q- abnormality can also be seen in all FAB
subtypes of AML with a highest frequency (∼ 60%) being in AML -M6.
-7/7q: This is the second most frequent chromosomal abnormality
seen in MDS/AML. In AML it is seen in all subtypes with highest
Table 23.2: Cytogenetic abnormalities in MDS

Chromosomal Approximate incidence (%)
Abnormalities De novo MDS Secondary MDS
Partial chromosomal deletion
Del 5q 20 20
Del 20 3-4 <1
Del 7q 1-2 10
Del 11 q 2-3 <1
Del 12 p 1-2 3-4
Del 13 q <1
Chromosomal loss
Monosomy 7 10-15 50
Loss of Y chromosomes 3-4 10
Monosmy 3 5-7
Chromosomome gain
Trisomy 8 11-15 10
Trisomy 11 3 1
Trisomy 21 2 1
Translocations
T(3;3)(q21;126) 1-2 3
T(1;7) (p11;p11) <1 4-5
T(5;17) (p11;p11) 1-2 4-5
T(7;17) (p11;p11) 1-2 2-3
T(5;7) (p11;p11) <1 2
Others
Iso (17q) <1 3-4
Inv (3) <1 3
Complex karyotype
(< 3 chromosomal abnormality 15-20 50
frequency (45%) observed in AML M6. Monosomy 7 is also found in

preleukaemic conditions like fanconi’s anaemia (25-40%), severe
congenital neutropenia (75%), neurofibromatosis type I (25%) and
familial monosomy 7 syndrome (100%).10,30 Monosomy 7 syndrome is
generally seen in male children, younger than 5 years of age, having
splenomegaly, thrombocytopenia, leukocytosis and monocytosis, and
have a poor prognosis. Juvenile Myelomonocytic leukaemia (JMML)
is a close differential diagnosis.
Although there is a spectrum of diseases associated with -5/del 5q
and -7/del 7q, triliniage dysplasia is a common morphological finding.
Abnormalities of chromosome 5, 7 or both are associated with a poor
response to therapy and poor survival.10
The pathogentic role of -7 in MDS is not clearly known. It is seen in
wide variety of preleukaemic conditions in children once they evolve
to MDS or AML. Also in some cases of MDS the occurrence of -7
abnormality has been found to be developed during the course of the

disease. These findings suggest that -7 could serve as a cytogenetic
markers in AML that developed secondary to constitutional genetic
instability or toxic gene damage.35
In MDS karyotypic study at diagnosis is important for predicting
the survival and progression of disease and a repeat karyotypic study
during follow-up can add further prognostic information.
CHRONIC MYELOPROLIFERATIVE DISORDERS

Chronic Myeloid Leukaemia (CML)
The first chromosomal abnormality detected in a patient with CML
was philadelphia (Ph) chromosome t(9;22). The defect was a balanced
reciprocal translocation between chromosome 9 and chromosome 22.
The t(9;22) also represent the first rearrangement which was
demonstrated to have a fusion protein (BCR/ABL). About 92 per cent
patients with CML have the typical Ph chromosme t(9;22) positivity,
however, certain group of patients with CML lack the Ph chromosome.
Some of these patients contain a DNA rearrangement indentical to
t(9;22) and can only be detected by molecular techniques. For these
cases the term Ph negative CML is used. Some of these cases resemble
to the myelodysplastic syndromes (RAEB or CMML). There is a new
variant of CML called atypical CML. In variant CML cases, there is
three way translocation involving chromosome 9,22 and a third
chromosome. Therefore t(9;22) and subsequent BCR/ABL fusion
protein is considered to be characteristic of all CML cases. The detection
of t(9;22) can be done by conventional cytogenetics. However, BCR/
ABL fusion gene can only be detected by sensitive techniques like, FISH,
southern blot analysis or PCR reactions. These techniques are also useful
in monitoring the therapy and detecting the minimal residual disease.
More than 80 per cent patients of CML who develop blast crisis, show
a new cytogenetic abnormalities in addition to Ph chromosome and
this is a grave prognostic sign.36,37 Common additional abnormalities
could be gain of chromosome 8 or 10 or an iso (17q). The less commonly
seen abnormalities include –7, –17, +17, +21, or t (3; 21). No correlation
has been observed with myeloid or lymphoid blasts crisis, with the
exception of inv (16) in myelomonocytic (M4) blast crisis. The t(9;22)
can also be seen in ALL but it involve a different breakpoint in BCR
whereas lymphoid blast crisis in CML is a multilinage disease and
always involve a break in the bcr of the BCR gene and has a good
prognosis compared to Ph positive ALL.
Polycythaemia Vera (PV)

Cytogenetic abnormalities are rare (14%) in untreated PV cases but a
higher frequency (39%) has been observed in cases who have been
treated with cytotoxic agents or who have progressed to acute

leukaemia (85%).36 The most common chromosomal abnormalities seen
in PV are gain of chromosome 8 or 9, del 20q or duplication of 1q. In
patients who have progressed to leukaemia after using the oral
alkylating agents, –7 or del 5q are the common chromosomal
abnormalities.
Essential Thrombocythaemia
Chromosomal abnormalities are rarely (5%) seen and no recurring
abnormalities has been found in these patients. However, in myeloid
metaplasia and myelofibrosis clonal abnormalities are seen in
approximately 35 per cent of patients. Of which most common are +8.
-7 or 7q-, 11q-, 13q- and 20q-.37
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Leukaemia. 1988;2:403.
12. Yasuhide Hayashi: The molecular genetics of recurring chromosome
abnormalities in acute myeloid leukaemia. Semin Haematol. 2000;37:368-80.
13. Mrozek K, Heinonen K, Dela chapelle A. Clinical significance of cytogenetics
in acute myeloid leukaemia. Semin oncol. 1997;24:17-31.
14. Le Beau MM, Larson RA, Bitter MA. Association of an inversion of
chromosome. 16 with abnormal marrow eosinophils in acute myelononocytic
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15. Liu P, Tarle SA, Hajra A. Fusion between transcription factor CBF B/PEBP2p
and a myosin heavy chain in acute myeloid leukaemia. Science 1993;261:1041-
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16. Huret JL, Brizard A, Slater R. Cytogenetic heterogeneity in t(11;19) acute
leukaemia: clinical hematological and cytogenetic analysis of 48 patients—
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17. Hagemeijer A, Van Dongen JJM, Slater RM. Characterization of blast cells in
acute leukaemia with translocation (4;11) Report of 8 additional cases and of
one case with a variant translocation. Leukaemia. 1987;1:24.
18. Pedersen B, Jergaard Jp, Philip P. Balanced translocations involving
chromosome bands 11q23 and 21q22 are highly characteristic of myelo-
dysplasia and leukaemia following therapy with cytostatic agents targeting
at DNA —topoisomerase II. Blood. 1991;78:1147.
19. Pui C-H, Frankel LS, Carroll AJ. Clinical characteristics and treatment out-
come of childhood acute lymphblastic leukaemia with the t(4;11)(q21;q23): A
collaborative study of 40 cases. Blood. 1991;77:440.
20. Chen D-S, Sorensen PH, Domer PH. Molecular rearrangements on
chromosome 11q 23 predominate in infant acute lymphoblastic leukaemia
and are associated with specific biologic variables and poor outcome. Blood
1993;81:2386.
21. Cortes JE, Kantarjian HM: Acute lymphoblastic leukaemia. Cancer.
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22. Climent JM. Molecular cytogenetic of childhood hematological malignancies
(Review). Leukaemia. 1997;11:1999-21.
23. Raimondi SC, Pui C-H, Hancock ML. Heterogeneity of hyper diploid (51-67)
childhood acute lymphoblastic leukaemia. Leukaemia.1996;10:213-24.
24. Croce CM; Nowell PC. Molecular basis of Human B cell neoplasia. Blood.
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25. Secker-Walker LM, Berger R. Fenaux P. Prognostic significance of the balance
t(1:9) and unbalanced der (19) t (1;19) translocation in acute lymphoblastic
leukaemia. Leukaemia. 1992;6:353.
26. Faterl S, Kantarjain HM, Talpaz M, Estrov Z. Clinical significance of cytogentic
abnormalities in adult acute lymphoblastic leukaemia. Blood 1998;91:2995-
4019.
27. Rubnitz JE, Downing JR, Pui C-H. TEL gene rearrangment in acute
lymphblastic leukaemia: A new genetic marker with prognostic significance.
J Clin oncol. 1997;15:1150-57.
28. Schrappe M, Arico M, Harbott J. Philadelphia chormosome positive (Ph+)
Childhood Acute Lymphoblastic Leukaemia Good initial Steroid Response
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30. Uckun FM, sensel MG. Biology and treatment of childhood T-lineage acute
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Hematol. 1996;33(2):127-38.
32. Grenberg P, Coxc, Le Beau MM. International scoring system for evaluating
prognosis in myelodys plastic syndromes. Blood 1997;89:2079-88.
33. Morel P, Hebber M, Lai JL. Cytogenetic analysis has strong prognostic value
in de novo myelodysplastic syndrome and can be incorporated in a new
scoring system: A report on 408 cases. Leukaemia 1993;7:1315-23.
34. Toyama K, Yohyashiki K, Yoshida Y. Clinical implication of chromosomal
abnormalities in 401 patients in MDS: A multicentric study in Japan. Leukaemia
1993;7:499-508.
35. Luna-Fineman S, Shannon K, Lange BJ. Childhood monosomy 7: Epidemio-
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36. Rowley JD, Testa JR. Chromosome abnormalities in malignant hematologic
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37. Demory JL, Dupriez B, Fenaux P. Cytogenetic studies and their prognostic
significance in agnogenic myeloid metaplasia a report on 47 cases. Blood
1988;72:855-59.
24
Adult ALL
Manisha Bhutani, Amish Vora,
Vinod Kochupillai
Key Words
Acute lymphoblastic leukaemia • Adults • Induction
therapy • Cytogenetics
INTRODUCTION
Based on several reports, it is widely accepted that more than one-
third and perhaps as many as 40 per cent of unselected adults with
ALL are cured by modern chemotherapeutic regimens. However, this
may be overstatement in view of the fact that the results of treatment
in adult (but not childhood) ALL are conventionally reported as disease-
free survival, thereby considering only those patients who enter CR,
excluding 20 to 40 per cent of individuals who fail to achieve CR.
Further, more if other biases of selection, inclusion, withdrawal, follow-
up, reporting and quoting of published results are eliminated, it is
reasonable to conclude that about 20 per cent of adults with ALL,
compared to about 70 per cent of children with same disease, have
been cured by modern chemotherapeutic regimens.1
By better biologic characterization of ALL, in terms of immuno-
phenotype and cytogenetic and molecular genetic aberrations, it is now
clear that the results are not uniformly inferior in adults, but vary by
disease subtype. Importantly, specific therapies directed at relatively
homogenous subgroups of ALL are now emerging and have improved
the results for those subtypes. This review provides a treatment option
overview and summarizes recent advances in the biology of adult ALL
and their application to the design of more effective treatment for this
disease.
INCIDENCE
In children, ALL accounts for 25 per cent of all cancers and 80 per cent
of all leukaemia cases and represents the most common malignancy,
Adult ALL 389
both in industrial as well as developing nations. Among adults (usually

defined as 15 years of age and older), the age-adjusted incidence,
approximately 2 persons per 100,000 in the United States annually,
amounts to about one-third of that in children.2
The incidence of ALL in developing countries, in general, tends to
be lower than in the USA or Europe, although there is country-to-
country variation. The reasons for relatively lower incidence in
‘emerging’ compared to ‘developed’ world are not entirely clear;
probably the differences in the genetics of different ethnic groups and
differences in exposure of various populations to carcinogens in the
environment explains this pattern.
TREATMENT OF ADULT ALL

Although the application of therapeutic strategies proven effective for
childhood ALL has improved results in adults with ALL, it is clear that
older age is an unfavourable prognostic feature even when using similar
therapies. As disease in adults is distinct from that seen in children,
substantial improvement in results in adults will likely require
innovative therapeutic strategies, rather than attempts to apply strate-
gies effective in most children to adults.
Induction Therapy
The traditional goal of induction therapy in ALL is to reduce the number
of leukaemic cells to below that detectable by conventional methods.
The success of induction therapy, as measured by the ability to quickly
achieve CR, is an important prognostic factor in adult ALL patients.
Randomized and non-randomized studies show that the addition of
anthracyclines to backbone of vincristine and prednisone improve the
CR rates in most adult studies from 50 to 60 per cent to greater than 70
per cent and improve duration of remission.3,4
Attempts to further intensify induction treatment by addition of L-
asparaginase and/or cyclophosphamide in randomized trials, have not
been shown to improve the survival.5,6 Various new approaches are
currently being explored to improve CR rates including:
• High-dose Ara-C given up-front or secondary to conventional
chemotherapy; two recent studies reported CR rates of 85 and 93
per cent.7,8 However, the impact of a potentially improved
remission quality on LFS, need to be confirmed.
• Dose-intensive anthracyclines; Todeschini et al reported a trial with
application of 270 mg/m2 daunorubicin as three 3-day cycles
during induction that resulted in a CR rate of 93 per cent in a
patient cohort with a median age of 34 years.9
• Pegylated-asparaginase (PEG-A), a new preparation of Eschericia
coli A, is synthesized by conjugation to polyethylene-glycol,
leading to significantly longer half-life of 5.7 days compared with

1.2 days for native E. coli A and 0.65 days for Erwinia A.10 Thus,
one application of PEG-A may be sufficient to maintain sufficient
asparaginase activity for at least 14 days. Preliminary results with
the use of PEG-A (2000 U/m2) in adult ALL show high CR rate of
93 per cent albeit with high incidence of toxicity.11 Further studies
are exploring reduced doses of PEG-A (1000 U/m2).
• Dexamethasone has been shown to exert many fold higher
antileukaemic activity compared with prednisolone in in vitro
trials.12 Dexamethasone also has a longer half-life in the cerebro-
spinal fluid than prednisolone. Thus, dexamethasone has been
approved during induction in some adolescent and adult ALL
trials, currently underway.
• Replacement by doxorubicin analogue (Epirubicin) did not
enhance the remission rate and survival, although it was found
equally efficacious.13
Post-remission Therapy
In patients after successful induction therapy, 108 to 109 residual tumour
cells can generally be detected with molecular techniques. The goal of
post-induction therapy, once remission is achieved, is to eradicate all
malignant cells, i.e. those that are undetected by conventional
techniques.
The drugs used during consolidation phase are cell cycle specific,
given in a manner of intensive-rotational cycles. A retrospective analysis
from large cohorts of adult ALL patients show, in general, a superior
outcome among trials implementing intensive multi-drug consolidation
therapy.14 Unfortunately, the results of randomized trials in adult ALL
are inconclusive, with some,15 but not others,16 showing improvement
in LFS with early/late intensification.
High-dose chemotherapy, mainly MTX or Ara-C, has been used to
overcome drug resistance and to achieve therapeutic drug levels in the
cerebrospinal fluid. A high CR rate (88%) and LFS rate (45% at 9 years)
were reported with a regimen that included conventional induction
and intensive rotational consolidation/maintenance chemotherapy
over a total treatment period of 3 years.17
Maintenance Therapy
Maintenance therapy, consisting of 6-mercaptopurine and MTX, with
additional vincristine or more intensive cycles, is usually administered
for total treatment duration of 2 to 3 years. Earlier attempts to omit
maintenance therapy after intensive induction and consolidation clearly
led to inferior results unless patients were transplanted.18
Adult ALL 391
CNS Prophylaxis
Prophylactic treatment of the CNS, based on the premise that the CNS
is a sanctuary for leukaemic cells that are protected from cytotoxic
concentration of drugs by the blood brain barrier, is an integral part of
virtually all current adult ALL treatment protocols. It typically consists
of cranial irradiation plus intrathecal methotrexate. Recent trials have
reported effective CNS prophylaxis with high-dose systemic therapy
with either MTX and/or Ara-C. The overall superiority of anyone
prophylactic therapy over another has not been established.
Stem Cell Transplantation

Transplantation is clearly the most effective way of eliminating
leukaemia in most patients. Although much of the data is inconclusive,
most studies have achieved better results with allogeneic trans-
plantation than with auto transplantation. The timing of stem cell
transplantation in the treatment of adult ALL is controversial.
Despite the use of modern chemotherapy, more than half of adult
patients with ALL relapse, most often within the first two years. In
general, once relapse has occurred, allogeneic SCT produces results
better than salvage chemotherapy and is appropriate in patients less
than 55 years of age with a histocompatible sibling donor. Twenty per
cent to 30 per cent of adults undergoing allogeneic SCT from HLA-
identical sibling donors in second CR are long-term leukaemia-free
survivors.19 It is unclear whether results in second remission are better
than those in early relapse.
Transplantation in first remission is controversial, but is clearly the
best option in some subgroups. Patients who are Ph’-positive have the
worst prognosis with few cures despite a substantial rate of achieving
complete remission. The IBMTR reported LFS of 38 per cent in 33 Ph-
positive ALL patients who underwent allogeneic SCT in first CR.20
Individuals with t(4;11) have a similarly dismal prognosis with
chemotherapy alone but can be cured by allogeneic transplantation
and, therefore should undergo transplantation in first CR if an HLA-
identical donor is available. Patients with these translocations who have
no sibling donors should be considered for transplants from matched
unrelated donors early in the course of their disease. Other risk factors
such as older age, high WBC count, and slow response to induction
therapy are not clear indications for transplantation because these
appear to adversely affect transplant outcome as well.
Patients who do not achieve a CR within 6 to 8 weeks have an
exceedingly poor long-term prognosis. Because cure is unlikely with
second-line therapy, allogeneic SCT early in the course of disease in
patients failing induction therapy is appropriate if HLA-compatible
donors are available. Roughly 20 per cent of patients with primary
induction failure are reported to achieve sustained leukaemia-free

survival with allogeneic SCT.
Newer Approaches
STI 571, a selective inhibitor of the Abl tyrosine kinase, has been used
in phase II trial to treat patients with relapsed/refractory Ph’-positive
patients. It produced a complete remission in 29 per cent of the
patients.21
REASONS FOR POOR OUTCOME IN ADULTS

Compared to children, adults with ALL have lower CR rates and
substantially poorer rates of long-term survival. The differing prognosis
in adults and children have been attributed to differences in disease
biology. Besides biological factors, there are also differences in treatment
tolerance that distinguish these two age groups.
DISEASE BIOLOGY AND PROGNOSIS

B vs T lineage ALL
With the wider availability of facilities for flowcytometry it is possible
to classify ALL, into B- and T- cell lineage, based upon the expression
of specific pattern of cell surface markers, i.e. immunophenotype. It is
now well recognized that each subtype has distinct biologic, clinical
and prognostic features.
Within B-lineage ALL, precursor B-ALL, comprising of early pre-B
or common ALL (c-ALL) (52%) and pre-B-ALL (10%), is the most
common subgroup. Classically well known adverse prognostic factors,
like high WBC count >30,000/μl, cytogenetic aberration t(9; 22), late
achievement of CR (> 4 weeks) and age (> 35 yr, > 50yr) are applied to
this subgroup to divide it into standard- and high-risk. Patients with
only favourable prognostic features are defined as standard-risk
patients with a survival of 40 to 50 per cent. Patients with one or more
adverse prognostic features are high-risk patients with a survival of 10
to 30 per cent.22 The high incidence of Ph’ positive ALL (>40%) within
c-ALL partly explains the poorer results in adults (LFS 30%) compared
to children (LFS 80%).14
Pro-B ALL (incidence, 11%) is characterized by high WBC count, co-
expression of myeloid antigens (50%), and t(4;11) (50%). Previously
associated with poor outcome, the results have improved to 40 to 50
per cent with the use of high dose Ara-C based chemotherapy or
allogeneic BMT.23 Similarly in mature B-ALL (incidence, 4%), the
outcome has significantly improved from < 10 per cent to greater than
50 per cent, with the use of recent short intensive regimens containing
fractionated moderate dose cyclophosphamide, high dose Ara-C and
MTX.24
Adult ALL 393
T-lineage ALL consists of pre-T- (6%) and mature-T- (18%) subtypes.

It is characterized by male predominance (73%), younger age (75% <
35 years), WBC count greater than 30,000/μl (62%), mediastinal tumour
(60%) and CNS involvement (15%), and a higher rate of CNS relapse.
Within T-lineage ALL WBC count > 100,000/μl is an adverse prognostic
factor.14 The T cell phenotype used to be associated with a worse
outcome, however, when treated with inclusion of cyclophosphamide
and cytarabine pulses, the results have improved. In a large series
covering several studies of the CALGB, the LFS in T-ALL after 3 years
was clearly superior (62%) to that of B-lineage ALL (42%).25
Cytogenetics
With improvement in spreading and banding techniques, most studies
report chromosomal changes in 60 to 85 per cent of ALL cases. The
Third International workshop on Chromosomes in Leukaemia (TIWCL)
found the majority of cytogenetic changes in cases of B-lineage ALL,
with only 39 per cent occurring in T-ALL.26
Most studies on karyotypic abnormality and their clinical significance
have been performed in childhood ALL. Adult ALL may show non-
random chromosomal abnormalities similar to those found in childhood
ALL, but their distribution is different.
Hyperdiploidy has been associated with good outcome in adult ALL
in some27 but not all studies.28 TIWCL showed higher CR duration and
median survival time in both children and adults with hyperdiploidy,
children still survived longer than adults for modal numbers > 50 or
47-50.26 The less favourable prognosis in adult ALL with hyperdiploid
karyotype, compared to children, may be explained by the higher preva-
lence of associated unfavourable structural changes.
Of the clonal structural changes, the translocations are the most
common (30%) abnormalities. The incidence of specific chromosomal
translocations associated with poor prognoses, especially Ph’
chromosome, is higher in adults than children. Although fewer than 5
per cent of childhood ALL cases are positive for Ph’ chromosome, the
frequency of positivity increases steadily with age, and approximately
30 per cent of adults with ALL have Ph’- positive disease.29
Recently three study groups published the results of cytogenetic
analyses in large patient cohorts with a total of 1049 patients.27,30,31 All
trials confirmed the poor prognosis of t(9; 22) and t(4; 11), detected in
23 and 4 per cent of adults, and reported the prognostic impact of further
clonal aberration grouped to favourable, intermediate, and poor risk.
The favourable group consisted of 12 p aberrations, t (10; 14) and high
hyperdiploid karyotype, whereas hypodiploid karyotype was
identified as poor prognostic feature. Patients with normal karyotype
had an intermediate prognosis in all three trials.
The CALGB suggested stratification into three prognostic subgroups:

poor [including t(9; 22), t(4; 11), -7, +8], normal diploid, and
miscellaneous (all other structural aberrations]. The leukaemia free
survival rates were 11, 38 and 52 per cent, respectively.30 These findings
indicate that cytogenetic findings may add reasonable information for
risk stratification, although the number of ALL cases analyzed is still
low and the treatment regimens applied vary greatly.
Molecular Genetics
The molecular detection of BCR-ABL and ALL-AF4 fusion genes related
to the translocations t (9; 22) and t (4; 11), respectively, is part of standard
diagnosis at major centres. Their presence identifies additional 10 to 30
per cent patients with these poor-risk aberrations not detected on
conventional cytogenetics. The detection of other molecular aberrations
has, however, not been reported for larger adult ALL patient cohorts.
Candidate genes for possible prognostic relevance are: TEL-AML1 and
E2A- PBX1 fusion genes, associated with cryptic translocations t (12;
21) and t (1; 19) respectively, which are associated with favourable
prognosis with the former and poor prognosis with the latter in children
and infants.
Minimal Residual Disease

In adult ALL, the prognostic value of minimal residual disease (MRD)
is still under evaluation but the few data available so far confirm the
findings obtained in childhood ALL, although MRD levels are generally
higher and predictive time-points for prognosis could be later in adults.
At the end of induction, with high level of MRD (>10-3) the risk of
relapse was 89 per cent, whereas with low MRD (<10-3) it was 46 per
cent.32 The incidence of MRD positive results decreased constantly in
patients with continuous CR (40% at month 1 to 0% at months 10 to12),
whereas patients with eventual relapse showed consistently high rates
of positive MRD results (76% at months 1 to 6). At early time points
(months 1 to 3), no correlation between MRD status and relapse risk
was found. At later time points, the prognostic value of MRD status
increased significantly.
German group has started a prospective study of MRD-based
treatment decision after 1 year of chemotherapy. Patients will be risk
stratified based upon the MRD results. In low-risk MRD patients, treat-
ment will be stopped after one year and maintenance will be omitted.
TREATMENT TOLERANCE
There are differences in metabolism of chemotherapeutic agents related
to age. Individuals vary in their processing of antimetabolites such as
Adult ALL 395
methotrexate (MTX) and 6-mercaptopurine. For instance, poly-

glutamated forms of MTX are retained within the cell longer than the
parent drug and increased formation in vitro correlates with better
prognosis in pediatric B-lineage leukaemias. In adults, B-lineage blasts
with ALL accumulate significantly lower levels of MTX polyglutamates
compared to children with same disease.33 This may contribute to
shorter remission duration in adults because MTX is commonly
administered drug in post-induction therapy.
Another potential factor in the poorer response to chemotherapy in
adults may be related to expression of the multidrug resistance (MDR
1)-associated membrane protein (p170). MDR 1 functions as an
adenosine triphosphate-dependent efflux pump for many compounds
including a variety of chemotherapeutic agents. Expression of MDR 1
at diagnosis had no effect on probability of entering CR for pediatric
patients, but in adults, the CR rate was significantly lower in MDR-
positive cases (56%) compared with MDR-negative cases (93%). MDR-
positive patients were significantly more likely to relapse in both age
groups (100% of adults and 73% of children).34 Adults, but not children,
commonly express MDR 1 at time of relapse. Development of drugs
that can be used clinically to overcome MDR 1-mediated resistance
would permit tailored therapy based on expression of MDR 1.
Additional reasons for poorer outcome in older patients include
greater hematologic toxicity leading to treatment delays and a higher
incidence of life-threatening infections, and non-hematologic toxicity,
e.g. hepatic and cardiotoxicity resulting in higher morbidity and
mortality. It has been observed that adults who receive less than
specified treatment fare substantially worse than those receiving full
treatment.
INDIAN SCENARIO
Exact incidence of ALL from India is difficult to obtain because
leukaemias are classified as myeloid- and lymphoid without any
distinction between acute- and chronic-, in the results of survey
conducted by population based cancer registries. The frequency as per
hospital-based series varies from 9-39 per cent of all leukaemias, highest
being in Kerala.13
Patients often present with features reflecting more advanced disease,
like hepatosplenomegaly (80%), lymphadenopathy (79%) and total
leukocyte count (>50,000) (26%).35 Reported incidence of T- ALL is
higher (21-44%) in India versus the West (11-25%), whereas that of c-
ALL is lower (21-68% vs 60-80%).13 The relatively high incidence of T-
ALL may either be related to low socio-economic status or to high
proportion of lymphoblastic lymphoma cases with bone marrow
involvement and peripheral spill. At present, however, c-ALL has begun
to increase in children and adults, but T-ALL incidence remains high.

In Mumbai, an earlier study reported frequencies of c-ALL and T-ALL
as 31 and 33 per cent,36 respectively, whereas a later study showed
corresponding figures to be 68.6 and 20.7 per cent respectively,35 indica-
ting an increasing trend of c-ALL.
Preliminary research on the molecular characteristics of ALL in India
suggests that chromosomal translocations associated with a poorer
prognosis in western series are more frequent, and those associated
with a good prognosis, less frequent. For instance, T-cell receptor (TCR)
gene rearrangement is demonstrated in 58 per cent vs 15 to 29 per cent
and Ph chromosome in 24 per cent vs 2-5 per cent in children in India
compared to the West.37,38 On the other hand, good prognostic factors
like hyperdiploidy and TEL/AML 1 translocation occur less
frequently.39,40
The MCP 841 protocol designed in collaboration with the National
Cancer Institute (US) was conceived with the idea to determine whether
a more intense chemotherapy program than previously used would
give improved results in developing nations. The results using this
protocol are available from four major centres in India and are consistent
with approximate overall and disease-free survival at 5 years of 50 and
60 per cent, respectively, for patients aged < 25 years.13 These figures
represent a significant improvement in survival rates compared to
earlier reports using various other protocols indicating the effectiveness
of MCP 841. The results, however, seem inferior to those reported from
developed countries.
Besides different disease biology, there are other reasons to explain
poorer results in our patients. There is generally delay in seeking access
to specialized hospitals and institutes for treatment and then in getting
admission due to limited bed availability. Moreover many patients even
when they reach hospitals, family’s inability to pay for the treatment
due to socioeconomic factors or the lack of health insurance often
impacts upon the type of therapy that is ultimately provided to the
patient. Co-morbidities such as hepatitis, malaria and malnourishment
are much more common, which may affect the patients’ ability to
tolerate treatment. Continuation of treatment and follow-up is often
difficult, again because of socioeconomic factors.
The paucity of research in India is a loss not only to patients here,
but also to patients with ALL worldwide. The vast panoply of environ-
ments, lifestyles, and ethnic differences, provides a spectrum of
opportunities, which, if taken advantage of, would lead to much more
rapid increase in our understanding of the disease and in our ability to
improve results.
Adult ALL 397
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18. Dekker AW, van’t Veer MB, Sizoo W. Intensive postremission chemotherapy
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29. Faderl S, Kantarjian HM, Talpaz M, Estrov Z. Clinical significance of
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30. Wetzler M, Dodge RK, Mzorek K. Prospective karyotype analysis in adult
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31. Secker-Walker LM, Prentice HG, Durrant J. Cytogenetics adds independent
prognostic information in adults with acute lymphoblastic leukaemia on MRC
trial UKALL XA. Br J Haematol. 1997;96:601-10.
32. Brisco MJ, Hughes E, Neoh SH. Relationship between minimal residual disease
and outcome in adult acute lymphoblastic leukaemia. Blood 1996;87:5251-56.
33. Goker E, Lin JT, Trippett T. Decreased polyglutamation of methotrexate in
acute lymphoblastic leukaemia blasts in adults compared to children with
this disease. Leukaemia 1993;7:1000.
34. Goasguen JE, Dossot JM, Fardel O. Expression of the multidrug resistance-
associated P-glycoprotein (p-170) in 59 cases of de novo acute lymphoblastic
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of prognostic factors using a single treatment regimen. Ann Oncol. 1999;10:167-
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36. Kamat DM, Gopal R, Advani SH. Pattern of subtypes of acute lymphoblastic
leukaemia in India. Leuk Res. 1985;9:927-34.
37. Sazawal S, Bhatia K, Gurbuxani S. Pattern of immmunoglobulin and T-cell
receptor gene rearrangements in childhood acute lymphoblastic leukaemia
in India. Leuk Res. 2000;24:575-82.
38. Gurbuxani S, Lacoste JM, Raina V. Detection of BCR-ABL transcripts in acute
lymphoblastic leukaemia in Indian patients. Leuk Res. 1998;22:77-80.
39. Amare P, Gladstone B, Varghese C. Clinical significance of cytogenetic findings
at diagnosis and in remission in childhood and adult acute lymphoblastic
leukaemia. Experience from India. Cancer Genet Cytogenet. 1999;110:44-53.
40. Inamdar N, Kumar SA, Banavali SD. Comparative incidence of the
rearrangement of TEL/AML1 and ALL1 genes in pediatric precursor B
lymphoblastic leukaemias in India. Int J Oncol. 1998;13:1319-22.
Index
A treatment of 389, 394

CNS prophylaxis 391
Abnormalities of red cell
disease biology and
membrane 348
prognosis 392
Acquired immunodeficiency
induction therapy 389
syndrome 165 maintenance therapy 390
challenges related to post-remission therapy 390
transfusion 171 Aetiology of anaemia 340
donor notification 172 ALL 45
inactivation of viruses in blood Amegakaryocytic thrombocyto-
components 173 penia 159
inappropriate testing 173 clinical presentation 160
inappropriate use of blood 172 therapy 160
safe donors and unsafe blood Angiogenesis 207
donations 171 Angiopoietins 96
screening of blood 172 Anticancer vaccines
challenges related with drug carbohydrate 104
use 176 peptide 105
epidemiology 167 tumour cell 104
etiology 165 Anticoagulant therapy 327, 369
Anticoagulation therapy 289
intravenous drug user 174
Antioxidant enzymes 282
pathogenesis 168
Aphaeresis 56
risk factors for HIV infections 175
complications 67
transfusion of blood and blood principle and mechanism of
products 169 action 57
transmission 167 Aplastic anaemia 46
Activated protein-C 298, 322 Apoptosis 95
Acute lymphoblastic leukaemia 228 Arixtra 296
Acute myeloblastic leukaemia 226 Audit
Acute myeloid leukaemia 112 aims and objectives 183
AML in elderly 121 areas of BTS 186
chemotherapy for newly diagnosed methodology 184
AML 114 schedule 184
cytogenetics 112 types compliance with current
induction therapy 117 GMP 184
newer induction chemotherapy external 185
approaches 118 internal 185
postremission chemotherapy self-inspection 185
vendor 185
119
types of compliance with current
Acute promyelocytic leukaemia 117
GCP 188
Adhesion molecules 88
prospective 189
Adult ALL retrospective 191
incidence 388 Autologous stem cell transplan-
Indian scenario 395 tation 41
B C
Beta thalassaemia 361 Cancer
Biologic agents 94 biological therapy 93
classification 96 Carrier detection 262, 268
molecular pathways 94 Cell separators 66
Biphenotypic leukaemia 243 Cellular therapy 63
Blood 79 Chromosomal aberrations in AML 375
Blood collection policies 186 Chromosomal analysis 373
Chronic myelogenous leukaemia 231
Blood component exchange 60
Chronic myeloid leukaemia 384
Blood components 71
CLL 45
Blood group discrepancy 188
CML 44
Blood sampling 345 Coagulation factors 275
Blood substitutes 1, 16 factor V 277
cell-free haemoglobin 3 factor VII 276
cross-linked haemoglobins 4 Coagulation-initiating proteins 96
conjugated 5 Congenital defects of red cells 347
inter-molecularly cross-linked 5 Congenital thrombocytopenia 160
development of red cell 3 Cord blood banking 40
encapsulated haemoglobins 8 Cord blood stem cell transplantation
need for development 1 37
perfluorochemicals 9 clinical aspects 38
other applications 13 engraftment factors 39
physico-chemical properties related donor 39
10, 11 unrelated CBT in children 40
therapeutic use 11 Corrective action 191
platelet substitutes 14 Criteria for anaemia 339
Cryoprecipitate 78
development 15
Cyclin-dependent kinases 95
fibrinogen coated microspheres
Cytopheresis 58
16
Cytogenetic abnormalities in
infusible 15 leukaemia 374, 378
lyophilized 15 Cytogenetic findings in MDS 382
need to develop 14 Cytogenetics 393
real 15 Cytokines 88, 89
recombinant haemoglobins 6
clinical trials update 7 D
usage and limitations 6
DHPLC 271
Bone marrow infiltration in
Diamond blackfan anaemia 152
lymphomas 244
inheritance 153
Bone marrow transplantation 22, 151
genetics and pathogenesis 153
Bone marrow trephine biopsy 249 clinical presentation 153
guidelines on use and interpre therapy 154
tation of immunohisto- Diffuse large cell lymphoma 47
chemistry 254 Dimorphisms 265
merits of diagnostic immunohisto- Disseminated intravascular coagu-
chemistry 250 lation (DIC) 323
methodological considerations 257 DNA restriction fragment length
scope of immunohistochemical polymorphism 224
investigation 251 Donor lymphocyte infections 35
Index 403
Donor lymphocyte infusions 36 H

Dyskeratosis congenita 154
Haematocrit 335
clinical presentation 156
Haematology of newborn 333
therapy 157 factors affecting 337
position of the neonate after
E delivery 338
site of sampling 337
Electrophoresis 209, 210
time of sampling 338
Endocrine dysfunctions 364
Endothelial nitric oxide synthase 281 treatment of umbilical vessels
Erythrocytopheresis 59, 63 338
Erythropoietin 100 normal values of haematological
European group marrow transplan- parameters 334
tation 42 Haematopoiesis 84
Extramedullary tumours 247 bone marrow 87
hepatic 87
F yolk sac 87
Factor XIII 278 Haematopoietic growth factors 99
Fanconi’s anaemia 147 Haematopoietic progenitor cells 88
clinical presentation 149 Haematopoietic SCT infections 34
complications 150 Haematopoietic stem cells 84
diagnosis 150 platicity 89
genetics and pathogenesis 149 stochastic model 85
inheritance and environment 148 Haemolytic anaemias 346
therapy Haemolytic disease of the newborn 81
bone marrow transplantation Haemophilia 81
151 Haemorrhagic cystitis 29
gene replacement 152 Haemovigilance 192
Fibrinogen 275 HbD-beta thalassaemia 362
Fibrinolysis 324 complications 364
Fibrinolytic system 279, 323, 326
haematological features 363
Foetal haemorrhage 341
HbE-beta thalassaemia 362
Foeto-foetal haemorrhage 343
Heparin derivatives 298
Foeto-maternal haemorrhage 341, 342
Fresh frozen plasma 77 Heparin-induced thrombocytopenia
303
G clinical presentation 309
diagnosis 309
Gamma-irradiated blood products 79
pathophysiology 305
Gene rearrangements 268
Gene replacement 152 therapy 312
Gene therapy 48 Heparinoid (danaparoid) 294
Genetic disorders 147 Hodgkin’s disease 46
GP 278 Hospital transfusion committee 189
GPI anchor 137 Human herpes virus 207
GPI linked proteins 136 Human immunodeficiency virus 165
Graft versus host disease Hyperhomocysteinemia 280
acute 30 Hypermutation 197
chronic 31 adhesion molecules 206
manifestations 33 classification and prognosis 207
Granulocyte 77 IMWG recommendations for
Growth factors 88 clinical definitions 208
cytogenetic and molecular changes L

202 Leiden and prothrombin 20210 A 322
diagnostic criteria 207 Leucocyte depleted 79
gene analysis 200 Leucocytopheresis 59
progression 201 Leukaemia and lymphoma 245
transition 201 Leukaemic disorders 219
growth factors 204 Low molecular weight heparins 292
interleukin-1 beta 205 Lymphoproliferative disorders 239
interleukin-6 204 risk factors for diagnostic problems
tumour necrosis factor-alpha 240
206 inappropriate reliance on
prognostic classification 209 modern technology 240
prognostic factors 210 leukaemia of bone marrow
cell surface phenotype 211 failure 241
circulating plasma cells 211 leukaemia or other malignancy
lactate dehydrogenase 211 242
peripheral blood labelling index primary bone marrow disorders
211 241
plasmablastic morphology 210 systemic disease and infection
S-phase estimation 211 242
serum beta-2 microglobulin 211 undue haste in starting
serum cytokine/receptor levels treatment 240
211
tumour cell karyotype 211 M
Hypersplenism 365
MDS 45
Minimal residual disease 220, 394
I diagnostic tools in MRD evaluation
Iatrogenic anaemias 345 221
Iatrogenic blood loss 73 cell culture methods 223
Infection screening 187 conventional cytogenetics and
Inherited marrow failure syndromes molecular techniques 222
148 molecular targets 223
Interferons 97 morphological and immuno-
Interleukins 98 logical technique 221
Internal haemorrhage 344 tumour specific chromosomal
International bone marrow transplant aberrations 225
registry 42 Molecular cytogenetics 374
Investigation of the neonate 325 Molecular genetics 394
Iron chelation therapy 367 Monoclonal antibodies 101
Iron overload 365 Multiple myeloma
Isoimmunization 346 cell of origin 195
normal B-cell ontogeny and
K generation of antibody
Karyotype analysis 373 diversity 196
Kostmann syndrome 158 immunogenetic studies 198
clinical presentation 159 maturational stages 199
genetics and pathogenesis 159 Multiple myeloma 46
therapy 159 Myelodysplastic syndrome 381
Index 405
N Post-transfusion complication 187

Neonatal haemostasis 324 Postremission transplantation 123
Neonatal thrombosis 321, 324 Pregnancy 365
Progenitor cells 86
management 327
pathophysiology 322 Prophylactic anticoagulation 328
Newer anticoagulants 291 Prothrombin 277
Proximal radio-ulnar synostosis 160
Non-Hodgkin’s lymphoma 47
Non-myeloablative transplants 37
Nucleated RBC 336 R
Recombinant bacteria and viruses 105
O Red blood cells 71
transfusions 72
Oncology patients 82
RBC product 74
Oral heparins 298
Red cell count 336
Red cell indices 336
P Red cell transfusion 366
Paraoxonase 282 Replacement fluid 63, 65
Paroxysmal nocturnal haemoglobi- Reticulocyte count 336
nuria 135
clinical features 140 S
cytopenias 141
Saline-washed red blood cells 80
haemolysis 140
Seizures 29
thrombosis 141
Shawchman diamond syndrome
clonality 139
clinical presentation 157
complement sensitivity 136
development 139
therapy 158
diagnosis 142
Sickle cell disease 80
management
Sickle cell-beta thalassaemia 361
replacement therapy 142
Signal transduction 94
steroids 142
Skeletal deformity 364
prognosis 143 Southern blot hybridization 224
thrombosis 136 Special filters 79
Pedigree analysis 263 Splenectomy 368
Pentasaccharides 296 Stem cell markers 85
Perfluorochemicals 9 Stem cell transplantation 22, 391
Philadelphia chromosome 372 allogeneic 23
Physiological anaemia stem cell source 24
infancy 72 engraftment 25
older children 73 conditioning procedure 26
PIG-A gene 138 technical aspects 26
Plasma glutathione peroxidase 283 indications 22, 23
Plasmapheresis 58, 60, 65 infections 34
Plasma separators 67 supportive care of the patient 26
Plasmid DNA 105 blood component support 27
Plasticity of stem cells 48 failure 30
Platelet 75 haematopoietic growth factors
Platelet pheresis 58 28
Platelet receptors 278 haemorrhagic cystitis 29
Ploidy system 378 protective isolation 26
Polycythaemia vera 384 pulmonary complications 29
seizures 29 Thrombolytic therapy 298, 328

skin and mucosal changes 29 Thrombophilia 323
toxicity 28 Thrombosis 324
veno-occlusive disease (VOD) of Thrombotic thrombocytopenic
the liver 28 purpura 63
venous access 27 Transfusion of the RBC product 74
Stromal-cell derived factor 89 cryoprecipitate 78
Structural haemoglobin variants 361 fresh frozen plasma 77
Supportive therapy 327 granulocyte 77
platelet 75
T Translocation 203
Thalassaemia 80 Tumour relapse 35
Thalassaemia intermedia syndrome Twin to twin transfusion 343
358
diagnosis 360 U
pathophysiology 358 Unfractionated heparin 292
Thalassaemia syndrome 348 Urine electrophoresis 210
Therapeutic haemapheresis 64
Therapeutic plasma exchange 60
Thrombin inhibitors 295
V
Thrombocythaemia 385 Veno-occlusive disease (VOD) of the
Thromboembolic disease liver 28
therapeutic options 366 Vitamin-K antagonists 294

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Recent Advances in Haematology - 1

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Recent Advances in

GS Chopra Manisha Bhutani DM

Manoranjan Mahapatra MD Rajive Kumar MD

Mona Anand MD Renu Saxena MD

Velu Nair MD Vinod Kochupillai MBBS FRCP

Knowledge in the field of Haematology continues to expand and

1. Blood Substitutes ............................................................................. 1

18. Anticoagulation Therapy ........................................................... 289

Index ................................................................................................ 401

REAL BLOOD SUBSTITUTES

Need for Development of Blood Substitutes

substitutes are necessary to narrow the gap between demand and

DEVELOPMENT OF RED CELL SUBSTITUTES

Cell-Free Haemoglobin Solutions

CROSS-LINKED HAEMOGLOBINS (MODIFIED HAEMOGLOBINS)

molecular weight. Such polymerized haemoglobins (polyhaemo-

Inter-Molecularly Cross-Linked Haemoglobins

Intra-Molecularly Cross-Linked Haemoglobins

Apex biochemical’s and Ajinomoto of Japan have experimented with

Usage and Limitations of Modified Haemoglobins

replacement or as bridge transfusion until compatible allogenic blood

Clinical Trials Update of Modified Haemoglobins

surgery (CABG) in Canada and UK and is awaiting regulatory

ENCAPSULATED HAEMOGLOBINS (ARTIFICIAL RBC)

spherules. Several workers have since carried out extensive research

Physico-Chemical Properties of Perfluorochemicals39,40

that it does not vaporize at all and thus create problems of

Physico-Chemical Properties of PFC in Relevance to Bio-

DEVELOPMENT OF PERFLUOROCHEMICALS FOR

perfluorodecalin and 3 parts of perfluorotripropylamine). It was

Among the other applications of second generation PFC, the

enhancement for X-rays in gastro-enterology and bronchoscopy

As mentioned earlier, development efforts have been directed to

Development of Platelet Substitues

REAL PLATELET SUBSTITUTES

Infusible Platelet Membranes

process of preparing freeze-dried preparations. These products have

Fibrinogen Coated Microspheres

gap between demand and supply. Ready-to-use availability of

38. Lowe KC. Perfluorochemical respiratory gas carriers. Applications in medicine

INDICATIONS FOR SCT

Table 2.1: Indications for stem cell transplantation

therapy. In allogeneic type of transplants, there is an additional

Stem Cell Source

source of stem cells for haematopoietic reconstitution in children.14

ENGRAFTMENT AFTER BMT vs PBSCT

Table 2.2: Stem cell dose in allogeneic transplants

TNC= total nucleated cell.

Table 2.3: Haematological recovery comparing BMT vs PBSCT

Occurrence of acute GVHD, a higher grade of genetic disparity between

Technical Aspects of Allogeneic Transplantation

SUPPORTIVE CARE OF THE PATIENT

protective isolation, the transplant-related mortality at 100 days was

Blood Component Support

ABO-incompatible marrow, plasma depletion needs to be done if the

Haematopoietic Growth Factors

Toxicity Related to Conditioning

Veno-occlusive Disease (VOD) of the Liver

definite benefit. Prophylaxis with low molecular weight heparin has

Skin and Mucosal Changes

GRAFT VERSUS HOST DISEASE

Table 2.4: Acute GVHD (grades II to IV)

Table 2.5: Extensive chronic GVHD