Professional Documents
Culture Documents
Associate Editors
BL Bhardwaj
MD MAMS FCCP FIMSA FIACP FNCCP FGSI FIACM
Professor
Department of Medicine
Government Medical College
Patiala, Punjab, India
Kusum K Thakur
MD PGDMCH PGDHHM MISBTI MIMA MAATM
Lecturer
Department of Transfusion Medicine
Government Medical College
Patiala, Punjab, India
Secretary, ISBTI (Punjab Chapter)
Rajni Bassi
MD MISBTI MIMA
Lecturer
Department of Transfusion Medicine
Government Medical College
Patiala, Punjab, India
Assistant Editor
Harnoor Singh Bhardwaj MBBS
Junior Resident
Government Medical College
Patiala, Punjab, India
Forewords
KK Talwar
TR Raina
Neelam Marwaha
An ISBTI Publication
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Transfusion Update
ISBN: 978-93-5152-598-1
Printed at
Dedicated to
My loving parents
Mrs Surjit Saini and Er BK Saini
and my teachers
Contributors
A Surekha Devi MD Anupam Verma MD PDCC Bimal Kumar Agrawal MD
Consultant and Head Additional Professor Professor and Head
Department of Transfusion Medicine Department of Transfusion Medicine Department of Medicine
Global Hospitals Sanjay Gandhi Postgraduate Institute MM Institute of Medical Sciences
Hyderabad, Andhra Pradesh, India of Medical Sciences (SGPGIMS) and Research
surekhadevi@gmail.com Lucknow, Uttar Pradesh, India Ambala, Haryana, India
aver2211@gmail.com aver@sgpgi.ac.in bkagrawal2001@yahoo.com
Aastha Miranpuri MD
Rochester, New York, USA BK Rana
Aradhna Sharma MD PhD
aasthamiranpuri@gmail.com Joint Director
Medical Officer
Department of Transfusion Medicine National Accreditation Board for Hospitals and
Achhar Singh MD Healthcare Providers (NABH) Quality Council
Assistant Professor Government Medical College
Patiala, Punjab, India of India
Department of Pulmonary Medicine New Delhi, India
Gian Sagar Medical College asharma130798@gmail.com
bkrana@nabh.co
Banur, Patiala, Punjab, India
kusum_gmcbb@yahoo.com Arun Bansal MS MCh (Neurosurgery) BL Bhardwaj
Neurosurgeon
MD MAMS FCCP FIMSA FIACP FNCCP FGSI FIACM
Ajata Shatru Kapoor MS MCh (Plastic Surgery) Amar Hospital
Professor
Assistant Professor Patiala, Punjab, India
Department of Medicine
Department of General Surgery drarunneuro@gmail.com
Government Medical College
MM Institute of Medical Sciences and
Patiala, Punjab, India
Research, Ambala, Haryana, India Arvind Chahal MBBS bachan_bhardwaj@yahoo.co.in
kapoorajatashatru@yahoo.com Junior Resident
Department of Medicine Devinder Singh Sandhu
Ajay Bahl MD DM
Pt BD Sharma Postgraduate Institute MD (Medicine) DM (Medical Oncology)
Consultant
of Medical Sciences Consultant
Department of Cardiology
Rohtak, Haryana, India Oncologist and Hematologist
Postgraduate Institute of Medical Education
med.pgims@hry.nic.in Sandhu Cancer Center
and Research (PGIMER)
Chandigarh, India Ludhiana, Punjab, India
drajaybahl@hotmail.com Aseem K Tiwari MD drsandhu@sandhucancercentre.com
Associate Director
Ajit C Gorakshakar PhD Department of Transfusion Medicine Dharam Paul MS
Scientist E (Deputy Director) Medanta: The Medicity Hospital Civil Surgeon (Retd)
National Institute of Immunohematology Gurgaon, Haryana, India Khanna, Punjab, India
Indian Council of Medical Research aseem.tiwari@medanta.org partola@rediffmail.com
KEM Hospital Campus, Parel
Mumbai, Maharashtra, India Ashish Jain MD Dinesh Ahluwalia MD
ajit5678@yahoo.com Director
Assistant Professor
Dinesh Path Lab
Department of Transfusion Medicine
Alpna Thakur MD Patiala, Punjab, India
Postgraduate Institute of Medical Education
Assistant Professor and Research (PGIMER) drdineshahluwalia@gmail.com
Department of Dermatology Chandigarh, India
Hind Institute of Medical Sciences DP Singh MS ISS (Cleveland) FICLS
ashishjain16@gmail.com
Lucknow, Uttar Pradesh, India Professor (Surgery)
alpna.30@gmail.com Government Medical College
Ashok Sharma MD Patiala, Punjab, India
Amit Aggarwal MD Professor and Head dpsingh.7@gmail.com
Consultant Department of Medicine
Fortis Escorts Heart Institute Indra Gandhi Medical College Gagandeep Kaur MD
New Delhi, India Shimla, Himachal Pradesh, India Assistant Professor
agrawalamit@gmail.com drashoksharmamd@me.com Department of Transfusion Medicine
Government Medical College and Hospital
Anupam Sachdeva MD Bharat Singh MD Chandigarh, India
Director Consultant Pathologist and Head Regional kaurgagandeep2701@gmail.com
Pediatric Hematology-Oncology and Bone Blood Transfusion Centre
Marrow Transplantation Guru Teg Bahadur Hospital and University Hamed Bashir MD DM
Institute for Child Health College of Medical Sciences Consultant Cardiology
Sir Ganga Ram Hospital, New Delhi, India New Delhi, India Max Healthcare Institute
anupamace@yahoo.co.in bsdirsbtc@yahoo.co.in Saket, New Delhi, India
viii Transfusion Update
Hari Krishan Dhawan MD Former Chairman Manjit Kaur Mohi MD FICOG FIMSA
Assistant Professor Board of Governors Professor and Head
Department of Transfusion Medicine Medical Council of India Department of Obstetrics and Gynaecology
Postgraduate Institute of Medical Education Former President Government Medical College
and Research (PGIMER) National Academy of Medical Sciences Patiala, Punjab, India
Chandigarh, India Former Director manjitmohi@yahoo.co.in
hkdpgimer@gmail.com Postgraduate Institute of Medical Education
and Research (PGIMER), Chandigarh, India
Manoj A Kahar MD PhD (Pursuing)
Harnoor Singh Bhardwaj MBBS Former Professor and Head
Blood Bank
Junior Resident Department of Pathology
Department of Cardiology
Government Medical College Government Medical College
All India Institute of Medical Sciences (AIIMS)
Patiala, Punjab, India Surat, Gujarat, India
New Delhi, India
harnoorbhardwaj@yahoo.co.in manoj_kahar@yahoo.com
kktalwar@hotmail.com
Harprit Singh MD Manuj Wadhwa MS MCh Ortho (UK)
Kshitija Mittal MD
National Programme Officer Ranawat Joint Replacement Fellow (USA)
Department of Transfusion Medicine
DBTS, Directorate of AIDS Control Director and Head
Government Medical College and Hospital
New Delhi, India Max Elite Institute of Orthopaedics
Chandigarh, India
harprit_1@hotmail.com and Joint Replacement
drkmittal@gmail.com
Mohali, Chandigarh, India
Hemchandra Pandey MD Kulbir Kaur MD manuj.wadhwa@gmail.com
Additional Professor Director Principal
Department of Transfusion Medicine Meenu Bajpai MD
Professor (Pathology and Transfusion
Sanjay Gandhi Postgraduate Institute of Associate Professor
Medicine)
Medical Sciences (SGPGIMS) Transfusion Medicine
Punjab Institute of Medical Sciences
Lucknow, Uttar Pradesh, India Institute of Liver and Biliary Sciences
Jalandhar, Punjab, India
New Delhi, India
Hitish Narang MD drkulbirkaur@yahoo.co.in meenubajpai@hotmail.com
Senior Consultant
Kunal R Patel DNB (Orth) D Orth meenubajpaii@gmail.com
Transfusion Medicine
Consultant
Satguru Pratap Singh (SPS) Apollo Hospital Mina Sidhu MD
MAX Hospital
Ludhiana, Punjab, India Assistant Professor
Mohali, Chandigarh, India
hitishnarang@gmail.com Department of Transfusion Medicine
drkunalpatel@gmail.com
HS Sandhu MD Government Medical College
Kusum K Thakur Jammu and Kashmir, India
Professor and Head
MD PGDMCH PGDHHM MISBTI MIMA MAATM minathapa@gmail.com
Department of Medicine
Lecturer
Government Medical College Mini Bhatnagar Sud MD
Department of Transfusion Medicine
Patiala, Punjab, India Associate Professor
Government Medical College
harendrasandhu@yahoo.co.in Patiala, Punjab, India Department of Medicine
Secretary MM Institute of Medical Sciences and
Ishwar Chouhan MBBS
Indian Society of Blood Transfusion and Research, Ambala, Punjab, India
Junior Resident
Department of Medicine Immunohaematology (ISBTI) Mohanvir Kaur MD
Government Medical College (Punjab Chapter) Assistant Professor
Patiala, Punjab, India kusum_gmcbb@yahoo.com Department of Pathology
i.chouhan05@gmail.com M Joseph John MD DM Government Medical College
Associate Professor Patiala, Punjab, India
Jitendra Kumar Pehalajani MBBS
Clinical Haematology, Haemato-oncology mohanvirkaur@gmail.com
Junior Resident
Department of Medicine and Bone Marrow (Stem Cell) Transplant Unit Neelam Marwaha MD FAMS FISHTM
Pt BD Sharma Postgraduate Institute of Christian Medical College Professor and Head
Medical Sciences Ludhiana, Punjab, India Department of Transfusion Medicine
Rohtak, Haryana, India mjosephjohn@gmail.com Postgraduate Institute of Medical Education
jitnanhe@gmail.com M Joshua Daniel Jeyakumar MD and Research, Chandigarh, India
Associate Professor and Technical Expert neelam2918@yahoo.com
Kanchan Bhardwaj
MD MAMS FIAC FIMSA MISBTI MIAPM MISHTM MIMA CDC-CMA1 NACO AMS Project Nidhi Bhatnagar MD
Professor and Head Vinayaka Missions Medical College and AMP Lead Assessor (NABH Blood Banks)
Department of Transfusion Medicine Hospital Associate Professor
Government Medical College Salem, Tamil Nadu, India Department of Transfusion Medicine
Patiala, Punjab, India drjoshuadaniel@gmail.com BJ Medical College
drkanchan_bhardwaj@yahoo.com drjoshuadaniel@yahoo.com Ahmedabad, Gujarat, India
Manisha Shrivastava MD bhatnagarnidhi@ymail.com
KK Talwar
MD DM FAMS FNA FACC FIMSA FIACS (Canada) DSc (hc) Associate Professor and Head Nidhi Mehta MD
Advisor Department of Transfusion Medicine (Secretary-Indian chapter–AATM)
Government of Punjab Bhopal Memorial Hospital and Research Consultant Transfusion Medicine
Health and Medical Education Centre Kokilaben Dhirubhai Ambani Hospital
Member Bhopal, Madhya Pradesh, India Mumbai, Maharashtra, India
Punjab Governance Reforms Commission manishasdr@gmail.com drnidhimehta@yahoo.co.in
Contributors ix
Foreword
It is a great pleasure to write a foreword for Transfusion Update. This is a result of the concentrated efforts
of the authors and the editorial team in bringing out a book containing the latest practices in transfusion
medicine. While covering a majority of the important and challenging topics in the field of transfusion, the
authors and editors have made sincere efforts to bring forth the practical points concerning patient care
from the transfusion medicine. The challenging problems seen in the practice of transfusion medicine are
dealt with in a simplified manner.
Transfusion Update is indispensable for transfusion medicine specialists and clinicians. I am sure
that postgraduate students will also find this book containing 81 chapters with pearls of immense value.
The book is having four sections consisting of clinical hemotherapy, cellular therapies, blood safety with pathogen reduction
technology and miscellaneous. To sum-up, the book is a practical and reference manual for clinicians on issues related to
transfusion medicine.
As this transfusion medicine update is being released on the occasion of the 39th Annual National Conference of the Indian
Society of Blood Transfusion and Immunohaematology (ISBTI) scheduled to be held at Government Medical College, Patiala,
Punjab, India, I congratulate Dr Kanchan Bhardwaj, Editor-in-Chief, for taking great pains in bringing out this volume of over
300 pages for the benefit of dedicated and busy clinicians of our country. I hope the TRANSCON 2014 and the book will add
to everyone’s knowledge and help everyone to face the challenges of transfusion practice in the years ahead. Dr BL Bhardwaj,
Dr Kusum K Thakur and Dr Rajni Bassi deserve special congratulations for successfully completing this important project.
(KK Talwar)
TR Raina MD (Path)
Former Professor and Head
Department of Transfusion Medicine
Government Medical College
Jammu, Jammu and Kashmir
Secretary General
Indian Society of Blood Transfusion and Immunohaematology (ISBTI)
Panchkula, Haryana, India
Foreword
Transfusion medicine is a continually involving field. It has a wide spectrum of activities involving
the community for purposes of donor motivation, recruitment and retention, complex laboratory
technologies for blood component preparation and blood testing, regulatory aspects of blood transfusion
services and most importantly clinical transfusion practices, monoclonal antibodies for therapeutics and
cellular therapies.
Transfusion Update being released on the occasion of TRANSCON 2014 has been diligently compiled
by the Organizing Committee, under the initiative of Dr Kanchan Bhardwaj, Organizing Secretary of the
Conference. The contents fit in well with the theme—Optimizing Transfusion Therapy. Best practices,
recent advances and challenges in clinical transfusion in the fields of medicine, surgery, oncology, obstetrics and neonatology
have been included in the scientific program. Articles on scope of apheresis technology, special transfusion support in
hemoglobin disorders and hemophilia have also been contributed by experts.
A major section has been devoted to cellular therapies—the most challenging and controversial areas in regenerative
medicine. The role of platelets as growth and repair promoters, current status of gene therapy, blood grouping and cord blood
transplantation have also been included in the contents. The section on blood safety brings into focus the regulatory, ethical
and legal issues in transfusion medicine. It also highlights the hazards of blood transfusion–hemovigilance and its reporting
mechanism in the country. State-of-the-art technology, platforms for immunohematology, transfusion transmitted viral
infections and bacterial contamination of blood components have also been contributed by the experts. The contents also
included quality assurance principles and practices in transfusion services.
All the contributors deserve appreciation for the efforts, they have made in preparing manuscripts and I wish to put on
record my sincere praise and appreciation for the Organizing Committee for compiling updated scientific knowledge in the
field of transfusion medicine. Transfusion Update would definitely benefit all the readers and help in improving transfusion
practices.
Editor-in-Chief
Contents
59. Natural Killer Cells in Tumor and • Direct Methods of Bacterial Detection 238
Transplantation 211 • Direct Rapid Detection Methods 239
Sunil K Arora, Rajendra Kumar • Pathogen Reduction 239
• Origin and Distribution of Natural Killer Cells 211 66. Pathogen Reduction Technology 242
• Mechanism of Cytotoxicity 211 Kanchan Bhardwaj
• Role of Natural Killer Cells in Transplant • Pathogen Inactivation in Platelet
Immunology 212 Concentrates 242
• Natural Killer Cells and Cancer Surveillance 212 • Platelet Concentrates with Amotosalen
60. Potential and Hazards of Gene Therapy 214 Photochemical Treatment 242
Ajit C Gorakshakar • Riboflavin and Ultraviolet Light Treated
Platelets 243
• Historical Aspect 214
• Methylene Blue Treated Platelets 243
• Gene Therapy in Transfusion Medicine 214
• Pathogen Inactivation in Fresh Frozen Plasma
• Prerequisite for Gene Therapy 215
Preparation 243
• Gene Delivery Systems 215 • Potential System for Pathogen Inactivation
• Vector Design 215 in Red Cell Concentrates 243
• Viral Delivery Systems 215
• Nonviral Delivery Systems 215 67. Discarding of Blood and Blood Components 245
• Regulatory Issues 216 Bharat Singh, Preeti Dewakar
• Recommendations 251
Section 3: Blood Safety 68. Adverse Transfusion Reactions 252
Manoj A Kahar
61. Leucoreduction 221 • Classification 252
Kanchan Bhardwaj • Acute Adverse Transfusion Reaction
• Approximate Leucocytes in Various Red Blood with Immune Causes 252
Cell Preparations 221 • Investigations to be Done for Immediate
• Clinical Indications for Leucoreduction 221 Hemolytic Transfusion Reaction 254
• Methods of Leucodepletion 222 • Acute Adverse Transfusion Reaction
• Adverse Effects of Filtration and with Nonimmune Causes 256
Leucodepletion 224 • Delayed Adverse Transfusion Reaction
• Universal Leucodepletion versus Selective with Immune Causes 257
Leucodepletion 224 • Delayed Adverse Transfusion Reaction
• Leucodepletion—Indian Perspective 225 without Immune Causes 258
62. Improving Blood Donor Screening by Nucleic 69. Hemovigilance and its Launch in India 260
Acid Technology 226 Neelam Marwaha
Rekha Hans • Hemovigilance Systems 260
• Need for Nucleic Acid Technology 226 • Outcome of Hemovigilance around the World 260
• Nucleic Acid Technology 226 70. Advances in Serological Assays for Screening
• Blood Donor Screening 226 Blood Donors for Infectious Agents 264
• Experiences of Different Countries 227 Paramjit Kaur
63. ID-NAT Versus MP-NAT 230
Rajesh Kumar Section 4: Miscellaneous
64. Occult Hbv Infection: A Risk for Transfusion 232
Sheetal Malhotra 71. The Evidence-based Transfusion Medicine 269
Kulbir Kaur
65. Current Status of Bacterial Detection in • Evidence-based Red Blood Cells Utilization
Blood Components 236 Guidelines 270
Mina Sidhu • Evidence-based Platelet Utilization Guidelines 270
• Clinical Relevance 237 • Evidence-based Fresh Frozen Plasma Utilization
• Methods to Reduce Risk of Post-transfusion Guidelines 271
Sepsis 237 • Evidence-based Cryoprecipitate Utilization
• Methods to Detect Bacterial Contamination Guidelines 271
in Blood Component 237 • Limitations of Evidence-based Transfusion
• Indirect Rapid Bacterial Detection Tests 237 Medicine 271
xxvi Transfusion Update
72. Blood Bank Accreditation 273 77. Molecular Genotyping and its Applications
BK Rana to Transfusion Medicine 292
• Types of Blood Banks/Centres 273 Swati Kulkarni
• Scope of Accreditation 273 • Solving ABO Blood Group Discrepancies 292
73. National Plasma Policy for Access to Plasma • Identification of Weaker and Partial Variants
Derived Proteins for Clinical/Therapeutic Use 278 of RhD Gene 293
Harprit Singh, Shobini Rajan, Shanoo Mishra, • RhD Zygosity Testing 293
Sunil D Khaparde • Identification of DEL Phenotypes 293
• Objectives of the Policy 278 • Prenatal Diagnosis of RhD in Fetus 293
• Antigen Profiling in DAT Positive Cases 294
74. Ethical Issues in Transfusion Medicine 281 • Differentiation between Alloantibody and
Kusum K Thakur, Achhar Singh Autoantibody 294
• International Society of Blood Transfusion Code • Quality Control in Reagent Red Cells 294
of Ethics 281 • Screening for Rare Blood Group Donors 294
• A Code of Ethics for Blood Donation and
Transfusion 281 78. Rare Blood Group Registry 297
• Ethical Issues Related to Donors 282 Swati Kulkarni
• Ethical Issues Related to Patients 283 • Defining Rare Blood 297
• Ethical Issues Related to Blood • International Rare Donor Program 297
Establishments 283 • National Status 298
• Future Prospectives 283 • Need for National Registry 298
75. Gel Based Technology: Past, Present and Future 285 • Identification of Rare Donors 299
Nidhi Bhatnagar • Methods for Typing Blood Group Types 299
• Principle of Gel Technology 285 • Future Directions 299
• Practical Applications of Gel Technology 286 79. New Versus Old Blood 301
• Advantages of Gel Technology 286 Rimpreet Singh Walia
• Past, Present and Future 287
80. Importance of Safe Blood Administration
76. Automated Blood Grouping and Antibody Practices 305
Screening 288 Sharad Jain
Ashish Jain
• Automation in Serologic Testing 288 81. Strategies to Reduce Unnecessary
• Solid-phase Immunoassays 288 Transfusions 308
• Erythrocyte-magnetized Technology 289 Hemchandra Pandey, Rajendra K Chaudhary
• Validation of Automated Systems 291
• Edge of Automation 291 Index 311
Section
1
Clinical
Hemotherapy
Transfusion Practice in Medicine
Transfusion in Bone
Marrow Failure Syndromes
Table 1: Comparison between rcc and whole blood Table 2: Recommended coagulation parameters for common
RCC Whole blood procedures
Table 3: Expected platelet increment • CMV negative, immunosuppressed and HIV positive
patients are at high risk for CMV infection.
Weight 1 Unit 4 Units 6 Units
• Bacterial testing in platelet concentrates has significantly
(kg) 1.0 × 1011 4.0 × 1011 6.0 × 1011
decreased this risk from platelet transfusion.
23 kg 22,000 88,000 132,000
45 kg 11,000 45,000 66,000 Transfusion Reactions
68 kg 7,400 30,000 44,000
A transfusion should be stopped immediately whenever a
91 kg 5,500 22,000 33,000 transfusion reaction is suspected.
• Hemolytic transfusion reaction: Fatal hemolytic
transfusion occurs once in 60,000 transfusions. It usually
Indications of Granulocyte Transfusions9 occurs after transfusion of incompatible blood component.
Fever, hypotension, nausea, vomiting, tachycardia,
• Granulocyte therapy may warrant consideration in
dyspnea, chest or back pain and hemoglobinuria are various
severely neutropenia patients with bacterial infections
signs and symptoms. May cause renal failure and DIC.
that are unresponsive to typical antimicrobial therapy.
• Delayed hemolytic transfusion reaction: These reactions
• A brief trial of granulocyte infusions may be warranted in
occurs in previously sensitized patient and can cause
patients with self-limited neutropenia and documented
symptomatic or asymptomatic hemolysis several days
fungal infection who are refractory to standard antifungal
after transfusion.
therapy.
• Febrile transfusion reaction: These reactions are
• Although prophylactic granulocyte transfusion may
due to sensitization to antigens on cell components
decrease the risk of septicemia, the increased incidence of
particularly leukocytes. Leukocyte depletion and removal
adverse effects observed with this therapy may outweigh
of plasma may be helpful. Rarely may be due to bacterial
the beneficial effects.
contamination.
• Transfusion related acute lung injury: These reactions
Doses and Target Level occur when donor plasma contains antibodies against the
• The recommended dose is 1 × 109 granulocytes/kg of body patient’s HLA or leukocyte specific antigen. Symptoms
weight. include dyspnea, hypotension and fever usually 30
• Granulocytes should be administered on a daily basis until minutes to 6 hrs after transfusion. Ventilatory support may
the patient’s endogenous neutrophil count rises to 0.5 × be required.
109/L or until the infection clears. • Urticarial and allergic type reactions: Most commonly
due to allergies due to specific proteins in donor’s plasma.
If severe washed RBC’s should be used. IgA deficiency may
Synthetic oxygen carriers be thought in severe reactions.
Various oxygen carrier compound available including the
following: Conclusion
• Perfluorochemicals: These are fluorinated hydrocarbons,
Stored in frozen state Fluosol-DA has been found to Early diagnosis of the etiology and specific therapy is
decrease the size of myocardial and cerebral infarctions in imperative in bone marrow failure syndromes. Appropriate
animals. and timely transfusion can reduce mortality as well as
• Chemically modified hemoglobin solutions: They morbidity of the patients, thus the judicious use of the blood
are used to increase oxygen transport in place of viable product is needed to minimize the side effect. It is essential
red cells. They have been used to supply oxygen in to use blood component therapy and not the whole blood so
experimental animals. that not only there is no waste of other blood component but
also there are less complications and transfusion reactions.
Rajesh B Sawant
2
Indications for Transfusion • If the case is complex and the transfusion services cannot
readily perform the appropriate investigations or provide
Red blood cell transfusion is a significant component of the appropriate blood, then the transfusion medicine-MD
supportive care for all patients with Autoimmune hemolytic should consult a hematologist.
anemia (AIHA). Although many patients will present with mild
to moderate anemia that does not require urgent transfusion,
Technical
an occasional patient presents with life-threatening anemia,
and the need for red blood cell transfusions is emergent. • Investigations should be expedited to obtain crossmatch
Patients with auto-antibodies requiring transfusion may compatible blood as quickly as possible.
pose difficulties in compatibility testing, and may be at • Perform appropriate technical procedures.
risk for hemolyzing transfused blood and/or developing • If the transfusion service is unable to perform these
alloantibodies. investigations, send samples immediately to a reference
Careful communication between the clinician and the regional laboratory for investigation.
transfusion service is required, since transfusion may be • Consult with seniors as indicated in technical procedures,
required before the serologic evaluation is completed. Even or as necessary based on technologist’s skills, additional
after thorough serologic evaluation, the optimal blood for information on the clinical situation, or laboratory
transfusion may be serologically incompatible. The clinician examination results.
must understand that in some cases, serologically incompatible
blood is safe for transfusion and can be expected to have in vivo Pretransfusion Examinations
survival comparable to the patient’s own red cells. Reluctance
to transfuse these patients because of serologic incompatibility The following examinations need to be done on pre-
or an incomplete work-up can be devastating. Patients with transfusion samples:
severe anemia may appear to be hemodynamically stable, • Preliminary antibody investigation to determine whether
but these patients have life-threatening anemia and should the auto-antibody demonstrates any specificity.
be transfused immediately regardless of the status of the • Other examinations to identify underlying alloantibodies.
serologic evaluation or compatibility. Such as prewarm, autoadsorption, differential alloadsorp
tion.
• Full red blood cell phenotype may be required in some
Responsibilities of cases.
Transfusion Medicine • Red cells provided for transfusion should not express
clinically significant antigens which the patient lacks, or
Clinical should be negative for clinically significant antigens, if the
patient phenotype is unknown.
• Advise the clinician(s) of the duration of delay in obtaining
compatible blood or discuss transfer of the patient to a
larger center. Selection of Blood for Transfusion
• Be familiar with the policies, processes, and procedures to Transfusion management is complicated for patients with
release “least incompatible” blood. AIHA, due to their serologic complexities. Although patients
8 Section 1 Clinical Hemotherapy
with AIHA can present with an ABO discrepancy, ABO hematocrit of greater than 30% or a hemoglobin level greater
typing can usually be performed following removal of IgG than 10 g/dL is reached. Once the treatment goal is achieved,
autoantibody in warm AIHA or warm washing red cells to the dose of prednisone is reduced to 20–30 mg/d within
remove IgM autoantibody in cold agglutinin syndrome. In a few weeks. Thereafter, the prednisone dose is tapered
an emergency or if results are not clear-cut, Group O donor slowly (by 2.5–5 mg/d per month) under careful monitoring
red cells can be used. Rh typing can also be difficult if the of hemoglobin and reticulocyte counts. An alternate-day
patient’s cells are heavily coated with autoantibody. Low regimen (reducing the dose gradually to nil on alternate days)
protein, monoclonal reagents are available for Rh typing in may reduce the side effects of steroids. If the patient is still in
the setting of immunoglobulin-coated red cells. remission after 3–4 months at a dose of 5 mg of prednisone/
The most pressing problem in a patient with previous day, an attempt to withdraw steroids is made. If a patient is
pregnancy or transfusions is detecting and identifying refractory to the initial corticosteroid treatment, a diagnostic
alloantibody, which may be hidden or masked by the re-evaluation with regard to a possible underlying disease
autoantibody. The exclusion of underlying clinically should be made. Patients with malignant tumors, benign
significant allo-antibodies is time consuming, and the clinical ovarian teratomas, or with warm IgM antibodies are often
situation may not allow for completion of these studies steroid-refractory.
before transfusion is needed. In the untransfused patient,
autologous adsorption studies are required to investigate Autoimmune Hemolytic Anemia
the presence of underlying alloantibody. If the patient has
been transfused in the preceding 3 months, more complex,
Controversies Regarding Transfusing
differential allogeneic adsorption studies are required.
In the un-transfused patient, determination of the red Crossmatch-Incompatible Blood
cell phenotype is invaluable. Techniques and procedures In patients with autoimmune hemolytic anemia (AIHA),
are available to dissociate autoantibody so that phenotyping antibodies are present, which react with both the patient’s
can be performed. Once a red cell phenotype has been own red blood cells and nearly all donor RBCs. Therefore,
determined, this phenotype can guide the exclusion of allo- serologically compatible blood is rarely available for
antibodies by indicating which antigen specificities are transfusion. The inability to provide crossmatch-compatible
at risk of eliciting an alloantibody. Shirey et al (2002) has blood has led to concerns that transfusion of these patients
shown that when available, phenotypically matched red could precipitate acute hemolysis with potentially severe
cells are an efficient method for providing safe red cells for consequences (hemoglobinemia, hemoglobinuria, acute renal
transfusion in this setting. Additionally, patients with AIHA failure, disseminated intravascular coagulation and death).
are at risk for requiring chronic transfusion therapy; knowing These concerns are based on a few published reports
the red cell phenotype of the patient is helpful in identifying of post-transfusion hemolysis in such cases. Because of
alloantibodies if they develop over time. the limited amount of this data and anecdotal experience
suggesting a lack of severe adverse events after transfusion,
Patient Management the indications for and advisability of transfusion in these
patients remain controversial issues.
• In a stable patient, hemoglobin levels as low as 60 g/L may Concern regarding the precipitation of acute hemolytic
be tolerated in an attempt to avoid exposing the patient to transfusion reactions leading to morbidity and mortality after
blood transfusion, and the attendant risk of alloantibody transfusion of red blood cells in patients with autoimmune
development. hemolytic anemia stems from a few early reports of fatalities
• In a symptomatic patient, blood transfusion should not be in these patients. While a temporal relationship between
withheld and can be initiated under a steroid cover. transfusion and hemoglobinemia/hemoglobinuria has been
• Red cells for transfusion should be selected that are seen in a handful of cases, a careful analysis of these reports
the “least incompatible”, now preferably termed “Best does not support a cause and effect relationship between
Matched” with the patient’s plasma/serum to minimize transfusion and death. The available safety and efficacy
the risk of alloantibody formation. data for transfusion in patients with autoimmune hemolytic
anemia, while somewhat limited, suggests that blood
Role of Steroids in Treatment of AIHA transfusion is well-tolerated and efficacious.
The mainstay of treatment of newly diagnosed primary
warm antibody autoimmune hemolytic anemia (WAIHA) is Suggested reading
glucocorticoids (steroids). According to accepted recommen 1. Kohan et al. Vox Sanguinis. 1994;67(2):195-8.
dations, start treatment immediately with an initial dose 2. Mauro et al. Blood. 2000; 95(9):2786-92.
of 1 mg/kg/d prednisone orally or methylprednisolone 3. Narvios et al. Curr Issues Trans Med. 2001;9(3):1-5.
intravenously. This initial dose is administered until a 4. Shirey et al. Transfusion. 2002;42(11):1435-41.
3
Sugar Free Antibodies: A
New Treatment for Idiopathic
Thrombocytopenic Purpura and
Autoimmune Hemolytic Anemia
HS Sandhu, Ishwar Chouhan
Introduction In their study, Collin et al. first showed that EndoS, at low
concentration, efficiently hydrolyzes the glycan on IgG in the
Autoimmune destruction of self-tissue is the consequential complex environment of human blood in vitro. They then went
result of a convergence of several factors, both genetic and on to show that the hydrolysis could be achieved efficiently in
environmental, that effectively dislocates the immune system’s the circulation of a live animal by injecting rabbits with EndoS
ability to tolerate self-antigens but simultaneously retains its and monitoring total IgG. Low concentrations of EndoS were
focus on those perceived as foreign. Although many of these effective without perturbing the total IgG concentration or
autoimmune targeted self-antigens are known, immune system disrupting other serum glycoproteins. Furthermore, the IgG
triggers can be more elusive. Nevertheless, once autoimmunity glycan hydrolysis effect was observed even in the face of a
has been initiated, immune complexes (IC) play as a significant host antienzyme response. To investigate whether the glycan
driving force in maintaining chronic inflammation and modification might have utility in modifying IgG activity,
subsequently pathology in many of these conditions. ICs are they chose a mouse model of a human autoimmune disease,
antibody/antigen assemblies, usually IgG antibody subtype, immune thrombocytopenic purpura (ITP), in which platelets
sometimes found systemically but primarily at the site of are targeted by autoantibodies. In the model, polyclonal
autoimmune self-antigen recognition.1 Sugar free antibodies antiplatelet IgG is injected into the mice to cause disease.
are deglycosylated IgG having a dominant suppressive effect Collin et al. showed that pretreatment of the antiplatelet
on inflammation and points to a unique class of therapeutic IgG with EndoS or treatment of the mice with EndoS after
immunoglobulins for the treatment of autoimmunity. initiation of disease prevented lethal thrombocytopenia in
The enzyme EndoS from Streptococcus pyogenes is an the majority of animals. Most impressively, even if disease
immunomodulatory molecule hydrolyzing the conserved was allowed to proceed to symptoms, as might be the case
glycans in the effector part of immunoglobulin G (IgG). for humans presenting with ITP, EndoS treatment was able to
EndoS is remarkably specific for IgG, and hydrolysis having provide considerable benefit.3
profound effects on IgG effector functions. EndoS pre-
treatment of IgG, or direct administration to animals with How does EndoS Exercise a Protective
experimental antibody-mediated autoimmune diseases,
inhibits development of disease or cures animals from Function?
established disease. The properties of EndoS make it a unique One plausible mechanism is that “activating” signals from
experimental tool and an attractive alternative to current C1q, Fc_Rs, and MBL binding are all reduced or abolished,
therapies of conditions involving pathogenic antibodies.2 whereas the interaction with the “inhibitory” Fc_RIIB
receptor is preserved. This mechanism may reflect the
Sugar Free Antibodies in Immune natural immunosuppressive role of this enzyme. S. pyogenes
has evolved several mechanisms to evade and diminish the
thrombocytopenic purpura
adaptive immuneresponse, one of these appears to effectively
Collin et al.3 adopt a novel experimental approach to this switch off the effector functions of IgG.
challenge: enzymatic removal of carbohydrates from IgG Fc
in vivo. The enzyme chosen, EndoS, is isolated from a common Sugar Free Antibodies in Autoimmune
human pathogen Streptococcus pyogenes and specifically
hydrolyzes the Fc glycans between the two core GlcNAc hemolytic anemia
residues. This modification of Fc abolishes the binding of The presence and pathogenicity of autoantibodies directed
IgG to Fc receptors and reduces complement activation. against red blood cells (RBCs) have been extensively
10 Section 1 Clinical Hemotherapy
investigated and implicated in a number of autoimmune EndoS Inhibits Phagocytosis and Adherence of
diseases, such as autoimmune hemolytic anemia (AIHA)
Sensitized RBCs by Monocytes
and systemic lupus erythematosus.4 The RBC surface
contains many epitopes recognizable by alloantibodies The effect(s) of EndoS on phagocytosis of anti-D sensitized
and autoantibodies, including more than 300 blood group RBCs by monocytes was analyzed using monocyte monolayer
antigens located on glycoproteins and glycolipids but also assay. Monolayers of monocytes were cultured on slides,
nonpolymorphic/nonblood group protein structures.5 The and RBCs presensitized with anti-D were added to the
most common targets of pathogenic RBC autoantibodies monolayers. The degree of phagocytosis was determined
are epitopes on the Rh protein, encoded by 2 homologous using light microscopy and expressed as the number of
genes on chromosome 1, RHD and RHCE.6 More than half monocytes with adherent/phagocytosed RBCs by analyzing
of all autoantibody specificities in patients with AIHA of 100 monocytes. As expected, the phagocytosis of anti-D–
the common so-called warm type include immunoglobulin sensitized RBCs was significantly greater than nonsensitized
G (IgG) antibodies against these Rh epitopes, which are RBCs and was efficiently eliminated by pretreatment of anti-D
consequently often used to model AIHA experimentally. with EndoS (p < 0.001). This clearly shows that the treatment
Human antibodies against RhD (anti-D) or rabbit of anti-D with EndoS before sensitization of RBCs abrogates
antibodies against human RBC (anti-RBC) can result in the phagocytosis-enhancing activity of anti-D and decreases
accelerated phagocytosis and removal of RBC from the the accumulation of intracellular hemoglobin in monocytes.
circulation by monocytes/macrophages in the spleen and
liver.7,8 Fcγ receptor (FcγR)-mediated erythrophagocytosis, EndoS Inhibits Oxygen Metabolite Production by
in addition to other IgG effector functions, such as activated
complement, oxidative burst, and cytokine production by
Monocytes Induced by Sensitized RBCs
FcγR expressing effectors cells, play an essential role in In an attempt to further elucidate the effects of EndoS on
shortened RBC survival.9-13 The ability of normal human monocyte-driven cell cytotoxicity to sensitized RBCs, we
monocytes to bind and phagocyte IgG sensitized RBCs via measured the release of oxygen metabolites from monocytes
FcγR and mediate immune RBC destruction is highly related using a chemiluminescence test. RBCs were opsonized with
to the glycosylation status of the Fc domain on the anti-RBC immune plasma with high titers of anti-D from 3 persons.
antibodies.14-19 Monocyte responses to sensitized RBCs were compared with
A study was undertaken to establish the effects of EndoS their response to unsensitized cells and expressed as a ratio
on antibody-mediated destruction of RBCs with a future (opsonic index). RBCs incubated with immune plasma gave
therapeutic application in mind. Here it was shown, using positive results in chemiluminescence test with opsonic index
monocytes from blood and the THP-1 monocytic cell 2.5. In contrast, monocyte responses to RBCs incubated with
line, that pretreatment of anti-RBC with EndoS abrogates immune plasma pretreated with EndoS were significantly
erythrophagocytosis and subsequent activation of monocytes. decreased (plasma 1, p < 0.07; plasma 2, p < 0.02; plasma 3,
The complement-dependent hemolytic effects of anti-RBC p < 0.015) and in some cases even below the control plasma.
added to whole human blood were greatly decreased by These results suggest that treatment of immune plasma with
pretreatment of this antibody with EndoS. Finally and most EndoS abrogates in vitro metabolic oxidative responses by
importantly, EndoS showed inhibitory effects on a model of monocytes.
AIHA in mice. These results suggest that anti-RBC antibodies
can be rendered less pathogenic by treatment with EndoS, EndoS Inhibits IL-8 Secretion from Monocytes
which may be regarded as a possible future inhibitor of
antibodymediated destruction of RBCs and AIHA. Induced by Sensitized RBCs
It has been reported that IL-8, besides being produced
Mechanism of Action by monocytes in response to lipopolysaccharide, tumor
necrosis factor-α, or IL-1, is implicated as an indicator
Treatment of Anti-D with EndoS does not of RBC incompatibility and mediator of the pathologic
events in hemolysis in, for example, hemolytic transfusion
Affect the Binding of Anti-D to RBCs reactions.21-23 It has been shown that monocytes exposed to
EndoS inhibits binding of IgG to soluble and monocyte- sensitized RBC’s release IL-8, which was measured by ELISA
bound FcγR by hydrolyzing the N-linked glycan on the heavy method, as expected a significant rise in levels of IL-8 were
chain of IgG.20 In a study conducted by Allhorn M, the results observed. Noticeably, RBCs sensitized with anti-D pretreated
showed that the treatment of anti-D with EndoS did not affect with EndoS showed significantly decreased secretion
the antigen recognition by this antibody and the amount of of IL-8 in THP-1 cells (p < 0.001) and bloodmonocytes
RBC-bound IgG was not influenced by EndoS treatment. (p < 0.003), which was comparable with the secretion of IL-8
Chapter 3 Sugar Free Antibodies: A New Treatment for Idiopathic Thrombocytopenic Purpura... 11
by THP-1 cells and monocytes because of stimulation with of C1q and C3 deposits in mice injected with complement-
nonsensitized RBCs (p < 0.001 and p < 0.002, respectively. activating 34-3C IgG2a, no significant differences were
These results demonstrate that EndoS does not have any visible compared with mice without EndoS treatment. Data
direct cytotoxic effects on monocytes. indicate that EndoS partially inhibits development of anemia
mediated by anti-RBC reactive antibodies in an IgG subclass-
EndoS Inhibits Hemolysis of Sensitized RBCs and dependent manner in mice.
Platelets 50% patients will not survive. DIC with sepsis has a significantly
higher rate of death than DIC associated with trauma.16
Platelet transfusion may be considered in patients with DIC
and severe thrombocytopenia, in particular, in patients with
bleeding or in patients at risk for bleeding (e.g. in the early References
postoperative phase or if an invasive procedure is planned). 1. Vincent JL, De Backer D. Does disseminated intravascular
The threshold for transfusing platelets varies. Platelet coagulation lead to multiple organ failure? Crit Care Clin.
replacement is usually required in non-bleeding patients 2005;21(3):469-77. [Medline].
if platelet counts drop below 20 × 109/L. In some instances, 2. Levi M, Ten Cate H. Disseminated intravascular coagulation.
N Engl J Med. 1999;341(8):586-92. [Medline].
platelet transfusion is necessary at higher platelet counts,
3. [Guideline] Taylor FB Jr, Toh CH, Hoots WK, Wada H,
particularly if indicated by clinical and laboratory findings.10 Levi M. Towards definition, clinical and laboratory criteria, and
In actively bleeding patients, platelet levels from 20 × 109/L to a scoring system for disseminated intravascular coagulation.
50 × 109/L are grounds for platelet transfusion (1 or 2 U/kg/ Thromb Haemost. 2001;86(5):1327-30. [Medline].
day). 4. Levi M, de Jonge E, van der Poll T. New treatment strategies
for disseminated intravascular coagulation based on current
understanding of the pathophysiology. Ann Med. 2004;36(1):
Coagulation Factors 41-9. [Medline].
Cryoprecipitate and coagulation factor concentrates should 5. Matsuda T. Clinical aspects of DIC–disseminated intravascular
not routinely be used as replacement therapy in DIC, because coagulation. Pol J Pharmacol. 1996;48(1):73-5. [Medline].
6. Franchini M, Lippi G, Manzato F. Recent acquisitions in the
they lack several specific factors (e.g. factor V). Additionally,
pathophysiology, diagnosis and treatment of disseminated
worsening of the coagulopathy via the presence of small intravascular coagulation. Thromb J. 2006;4:4. [Medline]. [Full
amounts of activated factors is a theoretical risk. Specific Text].
deficiencies in coagulation factors, such as fibrinogen, can 7. Levi M. “Disseminated Intravascular Coagulation”. Critical
be corrected by administration of cryoprecipitate or purified Care Medicine. 2007;35(9):2191-5.
fibrinogen concentrate in conjunction with fresh frozen 8. Hematology: Basic Principles and Practice, 6th edn. Elsevier
plasma administration. Saunders. 2012. ISBN 1437729282.
The consumption-induced deficiency of coagulation 9. Levi M, Toh CH, et al. “Guidelines for the diagnosis and
factors can be partially rectified by administering large management of disseminated intravascular coagulation”.
British Journal of Haematology. 2009;145(5):24-33.
quantities of fresh frozen plasma (FFP), particularly in
10. Levi M, Opal SM. Coagulation abnormalities in critically ill
patients with an international normalized ratio (INR) higher patients. Crit Care. 2006;10(4):222. [Medline]. [Full Text].
than 2.0, a 2-fold or greater prolongation of the aPTT, or a 11. Wada H, Asakura H, Okamoto K, Iba T, Uchiyama T, Kawasugi
fibrinogen level below 100 mg/dL.11 The suggested starting K, et al. Expert consensus for the treatment of disseminated
dose is 15 mg/kg.12 intravascular coagulation in Japan. Thromb Res. 2010;125(1):
Repeated measurement of global clotting tests (e.g. aPTT 6-11. [Medline].
and PT) might be useful for monitoring the coagulation 12. Levi M, Toh CH, Thachil J, Watson HG. Guidelines for the
defect. In case of a (relative) vitamin K deficiency in the diagnosis and management of disseminated intravascular
coagulation. British Committee for Standards in Haematology.
face of consumption, administration of vitamin K may be
Br J Haematol. 2009;145(1):24-33. [Medline].
required.5,13,14 13. Levi M. Disseminated intravascular coagulation: What's new?
FFP: 10–20 mL/kg of body weight will increase factor levels Crit Care Clin. 2005;21(3):449-67. [Medline].
by 20–30%. 14. Levi M. The coagulant response in sepsis. Clin Chest Med.
Frequency of transfusion depends on the half-life of the 2008;29(4):627-42, viii. [Medline].
deficient factor(s). 15. Franchini M, Manzato F, Salvagno GL, et al. Potential role
of recombinant activated factor VII for the treatment of
severe bleeding associated with disseminated intravascular
Prognosis coagulation: a systematic review. Blood Coagul Fibrinolysis.
Prognosis varies depending on the underlying disorder and 2007;18(7): 589.
16. Becker, Joseph U, Charles RW. Disseminated intravascular
the extent of intravascular thrombosis. The prognosis for DIC,
coagulation at eMedicine, 10 September 2009.
regardless of underlying cause, is often grim: between 10 and
Transfusion Practice in Oncology
Table 1: Comparison of risks and benefits of erythropoiesis-stimulating agents versus red blood cell transfusion for chemotherapy-related
anemia in patients with solid tumors
Erythropoiesis stimulating agents Red blood cell transfusion
Risks Thrombotic events Transfusion reactions
Potentially decreased survival Circulatory overload
Viral infection
Iron overload
Development of multiple alloantibodies
Benefits Gradual improvement in hemoglobin/hematocrit Rapid improvement in hemoglobin/hematocrit
Gradual clinical improvement, avoidance of RBC transfusions in Rapid clinical improvement
some patients, net reduction in transfusion requirements
16 Section 1 Clinical Hemotherapy
• ESAs: Epoetin and darbepoetin: Clinical trials have • The use of ESA should be discontinued after eight weeks if
established that epoetin and darbepoetin are effective the Hb has not increased by more than 1 to 2 g/dL or there
in raising hemoglobin levels and decreasing transfusion is no diminution in the need for RBC transfusion.6,7
requirements in a substantial number of patients with • For all patients treated with ESAs, supplemental iron be
chemotherapy-induced anemia. Both ESAs appear to be given to maintain a transferrin saturation ≥20% and a
equivalent with regard to efficacy and safety.6,7 serum ferritin ≥100 ng/mL.
Flow chart 1 Thrombocytopenia: Diagnostic algorithm starting with complete blood count
Abbreviations: CAMT, congenital amegakaryocytic thrombocytopenia: CBC, complete blood count; DITP, drug-induced immune
thrombocytopenia; FTLS, familial thrombocytopenis-leukemia syndrome; HIT, heparin-induced thrombocytopenia; HIV, human immunodefici
ency virus; HCV, hepatitis C virus; ITP, immune thrombocytopenia; TAR, thrombocytopenia-absent radius syndrome; VCF, velocardiofacial
syndrome, WAS, Wiskott-Aldrich syndrome.
18 Section 1 Clinical Hemotherapy
There are two types of drug-induced thrombocytopenia:10 for life-threatening bleeding (ideally transfused following
1. By direct myelosuppressive effect (e.g. chemotherapy- intravenous immunoglobulin to “protect” the platelets).
induced thrombocytopenia). If the cause of thrombocytopenia is unknown and there
2. By immune-mediated destruction of platelets due to are no contraindications, such as infections, corticosteroids
an idiosyncratic drug reaction (drug induced immune may be used to increase the platelet count.
thrombocytopenia [DITP]).
NEUTROPENIA
Complete Blood Count (CBC)
Background
Isolated thrombocytopenia is typically associated with
immune-mediated disorders11 and inherited disorders Neutropenia, usually defined as an absolute neutrophil
(e.g. Bernard-Soulier, TAR syndrome), but is uncommon in count (ANC) below 1.5 × 109/L (1500/mm3), encompasses
malignant processes involving bone marrow. a wide range of diagnoses, from normal variants to life-
Thrombocytopenia associated with anemia and leuco threatening, acquired and congenital disorders. The
penia (pancytopenia) can be caused by bone marrow sup functional consequences depend largely, but not exclusively,
pression by various medications (commonly chemotherapy, on the severity of neutropenia: ANC of 1.0–1.5 × 109/L does
rarely antihypertensive medications and antibiotics e.g. not impair host defense, but may warrant investigation of the
chloramphenicol); viral infections (HIV); bacterial infections underlying cause; ANC of 0.5–1.0 × 109/L may slightly increase
(e.g. leishmaniasis); severe folate and B12 deficiency; the risk of infections, but only if other arms of the immune
paroxysmal nocturnal hemoglobinuria; systemic lupus system are impaired; ANC of 0.2–0.5 × 109/L is associated with
erythematosus (SLE); inherited disorders (e.g. dyskeratosis an increased risk of infections in most patients. ANC of 0.2 ×
congenita, Fanconi anemia); malignancy (metastatic disease, 109/L or less carries a risk of severe, life-threatening infections
leukemia, lymphoma with bone marrow involvement, multiple with susceptibility to opportunistic organisms.12,13
myeloma, rarely solid tumors) or bone marrow failure (e.g.
aplastic anemia, myelodysplastic syndrome). Classification of Neutropenia
Neutropenia can be described as transient (or “acute”)
Additional Investigations (Table 3) or chronic (or “persistent”) (Table 4); extrinsic
or intrinsic; by descriptive names (e.g. neonatal isoimmune
• Elevated blood lactate dehydrogenase (LDH) and indirect
neutropenia of infancy, cyclic neutropenia, severe congenital
bilirubin, combined with low haptoglobin level and a
neutropenia) and as syndromes (e.g. Kostmann, Shwachman-
positive direct antiglobulin test (direct Coombs test).
Diamond, and Barth syndromes).14,15
• Elevated LDH combined with renal function impairment
may indicate TTP or hemolytic uremic syndrome (HUS).
Table 3: Causes of transient (acute) neutropenia (other than
• Blood coagulation tests disturbance: Prolonged prothrom- chemotherapeutic agents)
bin time (PT), low fibrinogen and elevated D-dimers are
Infection
typical for DIC.
• Elevated liver enzymes with or without elevated bilirubin, Viral Cytomegalovirus, Epstein-Barr virus, HIV,
LDH and alkaline phosphatase should lead to the investi influenza, parvovirus
gation of hepatic disease (viral hepatitis, drug induced Bacterial Brucella, paratyphoid, tuberculosis,
or toxic hepatitis), cirrhosis, and viral infection such as tularemia, typhoid; Anaplasma
cytomegalovirus (usually only liver enzymes and LDH are phagocytophilum and other rickettsia
elevated). Protozoan Plasmodium vivax, Plasmodium falciparum
• Serological tests for viruses, blood cultures, anti-platelet Drugs
antibodies, bone marrow biopsy and many other
Anticonvulsant Carbamazepine, valproate
diagnostic tests can be performed at the discretion of the
Antimicrobial Sulfonamides, penicillins, trimethoprim/
physician according to the presenting signs and course of
sulfamethoxazole
the disease.
Antipsychotic Clozapine, olanzapine, phenothiazines
Antirheumatic Gold, levamisole, penicillamine
Treatment of Thrombocytopenia
Antithyroid Methimazole, propylthiouracil
In a case of severe bleeding, if the etiology of thrombocytopenia Other Aminopyrine, deferiprone, rituximab,
is unknown, but not thought to be immunologic, platelet levamisole-adulterated cocaine
transfusion can be used to provide an immediate platelet
Immune
increase. In contrast, if the underlying cause is immune,
Neonatal isoimmune
the effect from platelet transfusion may be minimal and
at best very short-lived, and it should be reserved only Autoimmune
Chapter 5 Transfusion Practice in Oncology 19
Sudhir Varma
6
Introduction Among high-risk CAD patients with anemia8 or severe
bleeding,4 blood transfusion was required in >20% and was
Coronary care has taken a quantum leap after the advent of associated with increased in-hospital mortality (adjusted
angioplasty. Current pharmacoinvasive approach during odds ratio: 2.02, p < 0.001)8 and increased 1-year mortality
angioplasty permits use of a number of antithrombotic drugs. (relative risk: 2.03, p = 0.028).4
Major bleeding after percutaneous coronary intervention Collectively, these studies demonstrate a robust
(PCI) is associated with increased mortality.1–6 The nature association among major bleeding, blood transfusion, and
of this relationship and its implications still remain unclear. increased mortality in patients undergoing PCI.4,8 They
Is bleeding a surrogate for high-risk patients, because those suggest decreased survival among patients with acute
patients are at increased risk for bleeding complications too? coronary syndromes who experience major bleeding6 or
Bleeding may be an important, but until now overlooked, require blood transfusion.6,11
cause of excess mortality following PCI. There is now robust It is important to emphasize, however, that none of these
data that implicates direct and indirect effects of bleeding on studies can establish a causal connection between bleeding
subsequent adverse events.7,8 Growing suspicion also has events (or blood transfusion) and increased risk of death
focused on potentially harmful effects of red blood cell (RBC) after PCI.2,6 Although the relationship between bleeding and
transfusion.1,4,5,8 mortality is described in several studies as an independent
The scope of the triad of bleeding, blood transfusion and one, many of the correlates of bleeding (such as advanced age
morality is beyond academic debates and no longer limited to and renal failure) are themselves associated with mortality. As
discussions. Major bleeding is dependent not only on patient a result, resolving the question of association versus causality
characteristics5 but also to a large extent on choice of vascular has been a tremendous challenge, but emerging mechanistic
access strategy (radial vs. femoral),9,10 and to a lesser extent on data have begun to warm up the debate.
the choice of an antithrombotic regimen.11 Earlier neglected,
inclusion of bleeding as a part of the primary end point in
some recent PCI trials has highlighted it as a major issue, Why Bleeding and Excess Morality
drawing equivalence between this complication and other POST Percutaneous coronary
major adverse events (such as periprocedural myocardial intervention?
infarction) in the evaluation of novel therapeutic strategies in
coronary interventions. The biological impact of bleeding after PCI is likely to be
Data from more than 90,000 patients are now multifaceted. The location (intracranial) or torrential
available, derived from a combination of unselected1,5–8 nature of the hemorrhage (gastrointestinal, retroperitoneal)
and randomized trial populations.2–4 Although bleeding may result in death, regardless of any previous cardiac
definitions and patient characteristics varied, a consistent procedure. However, other consequences of bleeding
finding of increased mortality among patients who experience may have specific detrimental effects in patients who have
major bleeding has emerged. Blood transfusion risk is there recently undergone coronary intervention and may offer an
beyond usual concerns. Emerging evidence suggests that explanation for the association between femoral bleeding
transfusion in PCI settings may be associated with risks over complications and mortality.14-16
and above usual concerns regarding microbial transmission Interactions between activated platelets and the
and acute antigen-antibody reactions.12,13 A number of clotting cascade produce a rapid hemostatic response at the
studies have examined the impact of RBC transfusion on site of vascular injury. Systemic amplification of this localized
long-term outcomes after PCI and found a deleterious effect. response is prevented by a number of mechanisms, including
Chapter 6 Transfusion Practice in Coronary Interventions 21
control of the activated procoagulants and platelets by that it takes 48 hour from the time of donation to complete
antithrombotic pathways.12 These antithrombotic pathways the required testing and preparation of a unit of RBCs for
are predominantly active within vascular endothelial cells, transfusion. Hypoxic vasodilation by banked RBCs correlated
but little is known about the function of these pathways strongly with the amount of SNO-hemoglobin, underscoring
in patients with cardiovascular disease. Deficiency of the physiologic significance of the findings.20
such antithrombotic protective mechanisms (in tandem Prothrombotic effects are noted with blood transfusions.
with systemic endothelial dysfunction) could lead to a The transfusion of blood products has been associated with
hypercoagulable state induced by bleeding, which would an acute platelet release of CD40 ligand.21 Transfusion also is
pose obvious risks to the patient who had recently undergone associated with increased exposure to another procoagulant
PCI. protein, plasminogen activator inhibitor (PAI)-1. There is a
Furthermore, experimental data suggest that increased 3- to 6-fold increase in PAI-1 content of packed RBCs stored
synthesis and release of erythropoietin in response to anemia for greater than 35 days,22 and serum levels of PAI-1 increase
caused by bleeding might sustain a systemic prothrombotic after allogeneic blood transfusion.23 Adenosine diphosphate,
state beyond the acute phase, by causing platelet activation a well-recognized mediator of platelet activation, might
and inducing plasminogen activator inhibitor-1 (a also participate in transfusion related vascular thrombosis.
procoagulant cytokine).3 Treatment with erythropoietin Adenosine diphosphate is released from activated platelets
has been associated with increased risk of thrombosis in and damaged endothelial cells and is also released by
critical care patients.23 Finally, aspirin, clopidogrel, or other erythrocytes in stored blood.24 Finally, free NO acts not only
antithrombotic medication may have to be discontinued as a potent vasodilator but also as an inhibitor of platelet
after a major bleeding event, thus increasing the risk for stent activation.21 Deficiencies of NO transport and release by
thrombosis, a complication that frequently is fatal.14 stored RBCs may therefore not only impact adversely on
The significance of these issues in everyday clinical practice regional blood flow in zones of hypoxia but also on risk for
is difficult to quantify, but recent data have confirmed an thrombosis at sites of endothelial dysfunction.
increased risk of acute ischemic events after major bleeding Other adverse effects of blood transfusion. Trans
suggesting that the hypothetical concerns may be justified. fusion related immunomodulation (TRIM) is an immuno
suppressive, or in some cases proinflammatory, effect
Can Blood Transfusion in of blood transfusion that may contribute to adverse
outcomes among recipients of blood.21 The specific effects
Percutaneous coronary intervention
of this phenomenon on plaque biology, thrombosis, and
Patients be Harmful ? microvascular function are as yet undefined, but given the
Impairment oxygen delivery to the tissues is an important inflammatory nature of atherosclerosis, the potential for such
issue. Hemoglobin level elevation can occur post blood interplay is of obvious concern. Finally, severe hemolytic
transfusion but paradoxically whereas transfusion increases reactions and the consequences of large-volume transfusion
oxygen delivery, but measures of tissue oxygenation either (such as coagulopathy and electrolyte disturbance) also may
decrease or do not change.16 The reason for this paradox increase mortality associated with blood transfusion.
(greater oxygen delivery but no increase in tissue use) is Duration of storage of transfused blood. Many of the
unclear.18 Stored RBCs are low in 2, 3-diphosphoglyceric aforementioned concerns ultimately appear to hinge upon
acid (2, 3-DPG); therefore, the hemoglobin has high oxygen the duration that blood is kept in storage before use. The
affinity (i.e. will tend not to release oxygen to the tissues). Food and Drug Administration USA allows packed red cells
Traditionally, this finding been regarded as the pre-eminent to be stored for up to 42 days, allowing blood centers the
mechanism underlying reduced tissue extraction of oxygen flexibility to manage this scarce resource but also influencing
from the circulation after blood transfusion.17 However, 2, the quality of blood that is transfused. Whether clinical
3-DPG levels partially recover within several hours after outcomes are adversely affected by longer storage, however,
transfusion, and the results of experimental studies now is still uncertain. No data are available currently regarding
indicate that the physiologic impact of 2, 3-DPG depletion the impact of storage duration on outcomes for patients who
may have been overstated.18 Recent data suggest that other require transfusion after PCI. There are, however, reasonable
structural and biochemical changes that occur in RBCs grounds for concern that administration of blood that has
during storage might have a greater impact on their function been stored for longer than 14 days also may be associated
in vivo.15,28,29 with worse outcomes for PCI patients, given the similarities
Recent data indicate that this biochemical function is in clinical characteristics with cardiac surgery patients.
significantly disrupted by storage of RBCs, with a rapid Implications for clinical practice. The concerns
decrease in S-nitrosothiol hemoglobin (SNO-hemoglobin) outlined herein should not lead clinicians to withhold blood
concentrations to 30% of initial levels observed within transfusion when it is clearly indicated (i.e. severe anemia
1 day of storage (>20% within 1 week).19 It is important to note causing symptoms or other evidence of ischemia, especially
22 Section 1 Clinical Hemotherapy
for patients who exhibit signs of oxygen-supply dependency). The situation is likely to be even more complex among
Efforts to reduce the impact of bleeding complications on the patients undergoing PCI, because factors such as clinical
PCI population should rather focus on: (1) the identification presentation and completeness of revascularization will
of measures to decrease the incidence of bleeding probably alter the risk/benefit ratio for transfusion.
complications without increasing risk for ischemic events; Baseline anemia is associated with increased need
and (2) the development of strategies for more targeted and for blood transfusion and greater mortality after PCI.27
safer use of blood transfusion. Aggressive strategies to increase hemoglobin levels with
A prognostic risk score for major bleeding in patients the use of iron repletion, erythropoietin, or other disease-
undergoing PCI via the femoral approach has been specific measures would probably reduce the need for
published, allowing the identification of high-risk patients transfusion by providing a greater window of tolerance for
before the procedure.25 Seven variables were proposed based bleeding. It is reasonable to assume that such an approach
on an analysis of bleeding events from the REPLACE-2 trial: could also improve tolerance of myocardial ischemia during
age ≥ 55 years, female sex, estimated glomerular filtration long, complex cases. Although the value of postponing
rate ≤ 60 mL/min/1.73 m2, pre-existing anemia, use of low- elective PCI to treat anemia has not yet been tested, given
molecular Weight heparin within 48 hour before PCI, use of the frequency of this finding in routine clinical practice
glycoprotein IIb/IIIa inhibitors, and the use of the intra-aortic such a study would clearly be of tremendous interest. There
balloon pump2 Weighting for each was assigned by the use of are ongoing efforts to improve the quality of transfused
different integer scores, with a total score of >10 associated blood through refined harvest and storage techniques. For
with a major bleeding risk of >5%. Although this risk scoring example, decreased deformability of stored RBCs may be
system remains to be validated in real-world cohorts, such a ameliorated by correction of intracellular pH and restoration
tool may help inform decisions regarding initial management of adenosine triphosphate levels.28 The immune response
strategy (for patients with borderline indications for PCI) and to TRIM is attenuated by leukodepletion of blood products
enable the interventionalist to tailor the strategic approach before transfusion, suggesting that either a component or
according to bleeding risk for those who do proceed with byproduct of donor leukocytes elicits the recipient response.29
intervention. The age of RBCs at the time of collection might also affect the
The single most effective way for the operator to reduce impact of storage time on RBCs. In normal circumstances,
major bleeding is to use radial rather than femoral access.10 erythrocytes have a lifespan of approximately 120 days.
A systematic review of randomized trials revealed an odds It would be expected therefore that a unit of blood would
ratio of 0.20 (95% confidence interval: 0.09 to 0.42; p < 0.0001) contain a proportion of cells that are approaching the end
for access-site complications after radial rather than femoral of their lifespan and may be more prone to storage induced
PCI.14 Furthermore, in a retrospective study of almost 39,000 changes than younger cells. With a growing understanding
procedures, transradial PCI was associated with one-half of storage-related hazards and the development of assays to
the number of bleeding complications, markedly reduced measure these, it appears likely that quality criteria for RBCs
transfusion requirements, and lower adjusted 30-day and will expand in the future to include functional biochemical
1-year mortality (p > 0.001).9 Selection bias could certainly properties in addition to current regulations based on hemo
account for the finding of decreased mortality, as it seems lysis and hemoglobin mass. On the basis of current data the
likely that the most complex cases requiring large devices use of arbitrary cutoffs (such as a hemoglobin of >8 g/dL)
and hemodynamic support would have been performed to trigger transfusion after PCI should be avoided in most
from the femoral route. However, it is possible that decreased circumstances. Risks and potential benefits of transfusion
bleeding complications (and transfusion requirements) also should be weighed on clinical grounds.25,29 Among patients
could have contributed, at least in part, to the finding of lower with low hemoglobin who exhibit evidence of ischemia
mortality among patients treated via the radial artery. despite successful revascularization, there is clear potential
Refinements in the use of blood transfusion. In addition for blood transfusion to have a net beneficial effect.27 When
to reducing the incidence of major bleeding complications, there is clearly no evidence of ongoing ischemia, and therefore
refinements in the use of blood transfusion may lead to minimal potential gain, adverse effects of transfusion may be
better outcomes for patients undergoing PCI. Appropriate more likely to predominate (an exception to this may be the
indications for transfusion after PCI have not yet been asymptomatic patient who is considered at very high-risk
defined, specifically with regard to the hemoglobin level for rebleeding from a noncompressible site, where a greater
above which the risks of transfusion might outweigh any reserve of red cell mass may be desirable).
benefit. Among patients with critical illness, a randomized
trial has demonstrated that those treated with the use of a Conclusion
restrictive transfusion policy had better outcomes (when
compared with a more liberal transfusion policy),26 although There is a strong association of major bleeding, blood
older patients with cardiac disease these findings might not transfusion, and the risk of death after PCI. It remains to be
be applicable. proven, however, that strategies to reduce bleeding and/or
Chapter 6 Transfusion Practice in Coronary Interventions 23
blood transfusion will consistently lower all cause mortality. 13. Taylor JE, Henderson IS, Stewart WK, et al. Erythropoietin and
The development of risk algorithm for bleeding following PCI spontaneous platelet aggregation in haemodialysis patients.
can be useful strategy for preventing such events. Despite Lancet. 1991;338:1361-2.
14. McFadden EP, Stabile E, Regar E, et al. Late thrombosis in drug-
frequent use of blood transfusion in PCI patients, vexing
eluting coronary stents after discontinuation of antiplatelet
issues like thresholds for blood transfusion and blood storage therapy. Lancet. 2004;364:1519-21.
remain and need to be addressed to. 15. Greenburg AG. A physiologic basis for red blood cell transfusion
decisions. Am J Surg. 1995;170:44S-8S.
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801. 25. Nikolsky E, Mehran R, Dangas G, et al. Development and
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9. Chase AJ, Fretz EB, Warburton WP, et al. The association Requirements in Critical Care Investigators, Canadian Critical
of arterial access site at angioplasty with transfusion and Care Trials Group. A multicenter, randomized, controlled
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11. Lincoff AM, Kleiman NS, Kereiakes DJ, et al. Long-term efficacy 29. Vamvakas EC. Meta-analysis of randomized controlled trials
of bivalirudin and provisional glycoprotein IIb/IIIa blockade investigating the risk of postoperative infection in association
vs heparin and planned glycoprotein IIb/IIIa blockade with whiteblood cell-containing allogeneic blood transfusion:
during percutaneous coronary revascularization: REPLACE-2 the effects of the type of transfused red blood cell product and
randomized trial. JAMA 2004;292:696-703. surgical setting. Transfus Med Rev. 2002;16:304-14.
12. Lane DA, Philippou H, Huntington JA. Directing thrombin.
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Neurological ICU: Role of Red
Blood Cell Transfusion
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Conclusion JO Jr. Low hematocrit levels increase intracranial pressure in an
animal model of cryogenic brain injury [published corrections
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brain injury patients who are not actively bleeding is Trauma. 2009;66(3):720-6.
warranted. Although RBCT may augment brain oxygenation 11. Claudia S Robertson, H Julia Hannay, José-Miguel Yamal,
in compromised tissue, it may also result in a paradoxical et al. Effect of Erythropoietin and Transfusion Threshold
decrease in perfusion. This variable effect may be the result on Neurological Recovery After Traumatic Brain Injury A
of prolonged blood storage. It is possible that patients with Randomized Clinical Trial. JAMA. 2014;312(1):36-47.
impaired baseline perfusion benefit from RBCT more 12. Monisha A Kumar. Red Blood Cell Transfusion in the
Neurological ICU. Neurotherapeutics. 2012;9(1):56-64.
than those without. Transfusion may be more effective in
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randomized controlled trials are needed to better define the in neurocritical care. Crit Care. 2009;13:R89. [PMC free article]
[PubMed]
role of anemia, optimal hemoglobin threshold, and utility
16. Nishiyama T, Hanaoka K. Hemolysis in stored red blood cell
of RBCT and are of paramount importance to improve the concentrates: Modulation by haptoglobin or ulinastatin, a
management of patients in the neurological ICU. protease inhibitor. Crit Care Med. 2001;29:1979-82. [PubMed]
17. Zallen G, Moore EE, Ciesla DJ, Brown M, Biffl WL, Silliman CC.
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28 Section 1 Clinical Hemotherapy
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Chapter 7 Neurological ICU: Role of Red Blood Cell Transfusion 29
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Blood Transfusion Alternatives
Vitamin K depletion usually causes prolongation of PT increase the yield of peripheral blood stem cells (PBSCs) from
out of proportion to APTT because factor VII which has the peripheral blood stem cell donors. Side effect is bone pain.
shortest half life out of vitamin K dependant factors has
Thrombopoietin Its clinical trials are in progress:
little effect on APTT. Deficiency of vitamin K dependant
coagulation factors is best managed by treating the underlying
disease and by giving parenteral vitamin K, provided liver Recombinant Human Antihemophilic
function is adequate. Factor (Rh-Factor VIII)
It offers a good and safe alternative to human derived factor
Recombinant Hematopoietic VIII concentrate as it carries no risk of transmission of
Growth Factors hepatitis or AIDS.
Recombinant hematopoietic growth factors are the synthetic
forms of naturally occurring molecules capable of stimulating Red Cell Substitutes
endogenous hematopoiesis. Although blood transfusion is effective, it is not without
risks. Allogenic blood transfusion may cause fatal hemolytic
Recombinant Human Erythropoietin reactions, transmit blood-borne infectious agents and
(R-HU EPO) compromise overall immune function. It is therefore highly
desirable to have an artificial oxygen carrying fluid that is
Erythropoietin (EPO) is produced by the kidney. It stimulates readily available, free of infectious agents and can be used
red cell production by interacting with erythroid progenitor independent of the recipient blood type.
cells via specific receptor site on erythroid colony forming
units (CFU-E). The purification of erythropoietin and the Constitution of an Ideal Blood Substitute
development of recombinant human erythropoietin have
proved effective in correcting various chronic anemias,1 e.g. Blood substitutes or synthetic blood are currently labeled as
• Anemia of chronic renal failure “Oxygen carriers”. This is because they are unable to mimic
• Anemia secondary to zidovudine (AZT) therapy in HIV many of the other functions of blood; they do not contain
patients cells, antibodies, or coagulation factors. Their main function
• Anemia secondary to chronic disorders, e.g., connective is to replace lost blood volume and oxygen carrying capacity.
tissue disorder The ideal blood substitute could be defined by its increased
• To facilitate autologous blood collection within predeposit availability as compared to donated blood; oxygen carrying
program. capacity, equaling or surpassing that of biological blood;
Erythropoietin therapy has reduced considerably the need volume expansion; universal compatibility: elimination of
of blood transfusion especially in patients of chronic renal cross matching; pathogen free: elimination of blood contained
failure. Erythropoietin dose is 50–100 IU/Kg body weight infections; minimal side effects; survivability over a wider
three times a week either in I/V or S/C (more effective). range of storage temperatures; long shelf life and cost efficient.
Erythropoietin may have substantial role in reducing the Partial blood substitutes not involved in oxygenation are
need for homologous blood transfusion by increasing the platelet substitutes. They cover a different realm than oxygen
red cell production prior to surgery. Contraindication of carriers and are created for mainly one purpose: coagulation.
erythropoietin is uncontrolled hypertension. All the patients They are considered mainly with the hopes of eliminating
on erythropoietin therapy should have a hemoglobin level risks of bacterial contamination and the short shelf life.
measured at least once a week until suitable level is achieved The blood oxygen carriers are divided into two categories:
and periodically thereafter. 1. Hemoglobin based oxygen carriers (HBOCs)
2. Perfluorocarbon emulsions (PFCEs).
Other Blood Cell Growth Factors
Hemoglobin Based Oxygen Carriers
Recombinant Granulocyte-colony stimulating factor (G-CSF) (HBOCs)
and Granulocyte macrophage-colony stimulating factor
(GM-CSF) stimulate endogenous granulocyte production. • Stroma free hemoglobin and cross linked hemoglobin
These are used for the treatment of chemotherapy induced • Microencapsulated hemoglobin.
neutropenia as it decreases the duration of neutropenia and
also increases the tolerance to cytotoxic drugs. GM-CSF has Stroma Free Hemoglobin and Crosslinked
been approved for use in patients undergoing autologous
Hemoglobin
marrow transplantation, allogenic marrow transplantation or
in patients having bone marrow suppression due to anti-viral Stroma free hemoglobin is prepared from outdated red blood
agents. These growth factors are also used to stimulate and cells. In the solution hemoglobin dissociates into dimers and
32 Section 1 Clinical Hemotherapy
monomers.21 This hemoglobin is modified by pyridoxalation • Immunological response—Immunological response to
which reduces its oxygen affinity and subsequent cross linking the foreign protein remains a challenge.
with various biocompatible polymers increases its molecular • Short half-life—Current HBOc products have chemically
size so that it is not filtered by renal glomeruli. As yet there is or genetically cross-linked Hb chains resulting in larger
no report of the use of modified hemoglobin in human. molecules that are not readily filtered by the glomerulus,
thus possessing a greatly increased half-life.
Microencapsulated Hemoglobin • Increased oxygen affinity—Hb in the plasma has a much
higher affinity for O2 than it does within a red blood
The stroma free hemoglobin can be enclosed in artificial cell. The increased affinity for O2 is due to lack of 2,3
membranes, made readily from phospholipids (synthetic diphosphoglycerate (DPG) in the plasma. Consequently,
membranes).2 Hemoglobin in these artificial cells is no the HbO2 dissociation curve shifts to the left, making such
longer exposed to extracellular environment. The synthetic a high affinity Hb not an ideal oxygen delivery substance.
membrane is more stable than biological RBC membrane. However, chemically cross-linking the Hb structure has
There are no blood group antigens and artificial cells do not the net effect of decreasing O2 affinity and optimizing
react with blood group antibodies. Although these cells have intracellular oxygen delivery.3
proved effective in carrying oxygen in rats but they have very • Vasoactive properties—Vasoactive properties are one of
short stay in circulation after intravenous injection. This the major challenges facing the development of HBOCs.
hemoglobin does hold promise as an emergency resuscitative
fluid. Perfluorochemical Compounds
Table 1 shows the comparison of HBOCs to transfused red
blood cells.1 Perfluorochemicals (PFCs) are large organic compounds
in which all the hydrogen atoms have been replaced by
fluorine atoms. The high solubility of gases is a major reason
Benefits for the current biological use. For the use in blood substitute
The general benefits of HBOCs over transfused red blood preparations, liquid PFCs are preferred. Oxygen transported
cells are: No prior planning is needed, faster and better by a perfluorocarbon is carried in a solution. It can dissolve
O2 distribution, ready to use, no wastage, no equipment 40–70% oxygen per unit volume, almost 3 times the oxygen
is needed, long shelf life, no refrigeration, universally capacity of blood. They contain no antigens,4,5 do not carry
compatible, no clerical errors, immediately offloads oxygen, carbon monoxide and that is why could provide oxygen to
no 2,3-DPG, can be used by Jehovah’s witnesses. carbon monoxide victim until the patient is able to replace
the abnormal red cells.
PFCs, have a shelf life of 2 years and minimizes the risk
Challenges
of infection or immunologic reaction from a transfusion. In
The challenges associated with the development of HBOC are: addition, PFCs dissolve oxygen (unlike hemoglobin, which
• Availability—Development of HBOCs faces supply binds the molecule), which allow them to load/unload
challenge. While production of human Hb by recombinant oxygen 2 times faster than hemoglobin and extract >90% of
DNA could be a possible solution.2 the transported oxygen.
Table 1: Comparison between hemoglobin based oxygen carriers and red blood cells
Infused hemoglobin based oxygen carriers Transfused red cells
Onset of action Immediate 2,3 DPG dependent
Oxygen affinity Red cell 2,3 DPG not required for oxygen release Red cell 2,3 DPG required for oxygen release
Oxygen transport Red cells plus plasma Red cells only
Risk of disease transmission Sterile pharmaceutical; no leukocyte exposure Risk minimized by improved donor selection;
leukocyte depletion
Storage Room temperature; no loss of efficacy Refrigeration required; progressive loss of efficacy
Shelf life 36 months 42 days
Compatibility Universal Type-specific
Preparation Ready to use Requires typing and cross-matching
Viscosity Low High
Duration of action Maximum of 3 days Estimated 60–90 days
Chapter 8 Blood Transfusion Alternatives 33
Another potential use relates to the PFCs small particle size, Using inhibitors of cytoskeleton actin assembly prevents
which enables the suspension to penetrate occluded vessels in the disc-to-sphere-shape change seen in chilled platelets.
situations such as cerebral ischemia or myocardial infarction. Thrombosol has been reported to give greater retention of
PFCs have now been licensed for use in percutaneous in vitro function. Antifreeze glycoproteins are found in the
transluminal coronary angioplasty (PTCA). Perfluorocarbon circulation of polar fish. The use of these glycoproteins in cold
must be emulsified before administrating intravenously. storage of platelets has been shown to inhibit platelet shape
Since emulsified PFC particles have little osmotic and change and cold-induced activation, but no in vivo findings
oncotic pressure properties, electrolytes and plasma volume have been reported.
expanders must be added to provide these needs.
In contrast, both the PFC and oncotic agent (hydroxyethyl Photochemically Treated Platelets
starch) are essential when the substitute is given to replace
Photochemical treatment of liquid-stored platelets with
blood lost by hemorrhage.
psoralen and long-wavelength ultraviolet radiation has been
explored as a means to inactivate bacteria, protozoa it acts by
Disadvantages disrupting their DNA. The photochemically treated platelets
• PFCs do not preferentially extract oxygen from the air as have normal hemostatic function, but have slightly in vivo
haemoglobin does, so the oxygen level in a perfluorocarbon recovery and survival.
solution equilibrates with oxygen level in the atmosphere.
Perfluorocarbon therapy therefore requires the concurrent Lyophilized Platelets
administration of 60–100% oxygen.4,5 A high oxygen Lyophilization platelets are prepared using 1.8% paraformal-
environment can render the patient vulnerable to oxygen dehyde and freeze-dried with 5% albumin. Rehydrated plate-
toxicity, especially when the concentration of oxygen lets have a similar appearance to fresh platelets and have
in inspired gas is above 10% for more than 24 hours. most of the same surface proteins.6
Disadvantage is of oxygen toxicity.
• Second disadvantage of PFCs emulsion is their instability,
Platelet-derived Microparticles
resulting in the need for frozen stage.
• A third disadvantage of perfluorocarbon is its retention in Platelet microparticles are microvesicles of platelet mem
the liver and spleen. branes which are formed spontaneously during storage. The
• The most extensively studied fluorocarbon is fluosol-DA microparticles have similar hemostatic properties as intact
developed by Japanese. Fluosol contains two types of platelets. They can be produced from outdated platelets.
perfluorocarbons in a mixture of balanced electrolytes,
lecithin and hydroxyethyl starch (HES). Culture-derived Platelets
In 1995, the process of growing platelets in the laboratory
Platelet Substitute from megakaryocyte progenitors was described. The resulting
platelets appeared to be functionally and morphologically
Platelet-derived Products similar to fresh donated platelets in vitro.
Ashok Sharma
9
Historically, blood was transfused as whole blood without these factors degrade rapidly during storage. The platelets
further processing. Whole blood is obtained from human undergo conformational changes even with short-term
donors by venesection and is collected into a sterile, dis refrigerated storage that causes rapid removal of transfused
posable plastic pack, which contains an anticoagulant- platelets by the reticuloendothelial system and thus are
preservative solution. This solution usually contains citrate, unlikely to provide much improvement in hemostasis.
phosphate, dextrose and adenine. There are variations in the Around 92 million blood donations are collected annually
volume collected and in the type of anticoagulant used in from all types of blood donors. The average annual collection
different countries. Blood collected in an anticoagulant can be per blood center is 30000 in high-income countries, 7500 in
stored and transfused to a patient in an unmodified state. This middle-income countries and 3700 in low-income countries.
is known as ‘whole blood’ transfusion. However, blood can be Only 31% of the blood collected in low-income countries is
used more effectively if it is processed into components, such separated into blood components. The capacity to provide
as red cell concentrates, platelet concentrates, plasma and patients with the different blood components when they
cryoprecipitate. In this way, it can meet the needs of more require, is thus still limited in these countries.
than one patient. Some blood banks have replaced this with
platelets collected by plateletpheresis because whole blood Indications of Whole Blood
platelets, sometimes called “random” platelets, must be
pooled from multiple donors to get enough for a therapeutic
Transfusion
dose.
The appropriate use of blood and blood products means Acute Hemorrhage
the transfusion of safe blood products only to treat a condition Whole blood can be transfused to replace red cells in acute
leading to significant morbidity or mortality that cannot be bleeding when there is also a need to correct hypovolemia. As
prevented or managed effectively by other means. Blood whole blood transfusion is limited to acutely hemorrhaging
transfusion can be a life-saving intervention. However, like individuals, dosing should be based on the patient’s clinical
all treatments, it may result in acute or delayed complications condition, estimated blood loss, and other measures being
and carries the risk of transfusion-transmissible infections, used to maintain hemodynamic stability. Such as seen
including HIV, hepatitis viruses, syphilis, malaria and Chagas with major trauma, requiring massive transfusion and
disease. rapid correction of anemia, coagulopathy, acidosis, and
Blood is an expensive, scarce resource. Unnecessary hypothermia. Studies supporting this approach include
transfusions may cause a shortage of blood/components military trauma where they are able to transfuse very fresh
for patients in real need. Intravascular transfusion of whole (<24 hours old) whole blood which is not currently routinely
blood has long been a well-recognized life saver during and available in civilian institutions.
after major surgery and where there has been massive loss of
blood in an accident or in childbirth. Whole blood provides
Autologous Transfusion
red cells, stable clotting factors, and volume in each unit
that make it potentially beneficial in rapidly hemorrhaging Whole blood is no longer commonly available or used in most
patients. Each unit (~450–500 mL) provides the equivalent of of the United States. The most common use of whole blood
one unit of RBCs and one unit of plasma. However, the plasma in the United States is currently autologous donations for
may be deficient in labile clotting factors (V and VIII) since elective surgery.
36 Section 1 Clinical Hemotherapy
Management of Preoperative
Anemia in Patients Undergoing
Elective Surgery
Manisha Shrivastava
10
Preoperative anemia is a very common finding in elective cardiorespiratory function tolerate, at least briefly, a
surgical patients and remains undiagnosed till the patients hemoblobin concentration of 5 g/dL. Serum lactate and
undergo preoperative diagnostic examination. Preoperative oxygen consumption do not increase, and mild cognitive
anemia is associated with excess morbidity and mortality. impairment is reversible. However, this low level of
Correcting anemia by blood transfusion was a long standing hemoglobin may not be benign to elderly patients with
modality for such patients, however, understanding of cardiopulmonary disease. Many questions remain regarding
the complications associated with allogenic transfusions when we should transfuse red cells for acute anemia.3
such as infection, renal injury, and acute lung injury has
lead to adaptations of different strategies other than blood Procedures that Usually Do versus Do Not
transfusion. The data suggesting that morbidity and mortality
amongst patients receiving blood before or during surgery
Require Transfusion
is higher has also brought about reduction in the use of Surgical procedures that usually require transfusion include
allogenic blood to manage preoperative anemia. coronary artery bypass graft, major vascular surgery, hip
replacement, major spine surgery with instrumentation,
Prevalence Depends on Many Factors hepatic resections, radical prostatectomy, and selected
neurologic procedures such as resection of arteriovenous
The reported prevalence of anemia in surgical patients varies malformation. Surgical procedures that usually do not require
widely from 5–76% and depends on the patient’s disease and transfusion include cervical spine fusion, intervertebral
co-morbidities, the surgical procedure and associated blood discectomy, mastectomy, hysterectomy, reduction mammo
loss, and the definition of anemia used. The prevalence of plasty, cholecystectomy, tonsillectomy, transurethral resec
preoperative anemia increases with patient age and is higher tion of the prostate, and vaginal and cesarean deliveries.4
in women than in men.1 Pathophysiology of preoperative, perioperative and post
operative anemia.
Minimum Acceptable Hemoglobin Perioperative anemia is a common clinical entity. It is
Concentration usually due to combination of various mechanisms, including:
pre-existing anemia prior to surgery; anemia due to impaired
The National Institute of Health Perioperative Consensus erythropoiesis, including alterations of metabolism of iron and
Conference concluded that “otherwise healthy patients erythropoietin (EPO); anemia due to increased destruction of
with hemoglobin values of 10 g/dL or greater rarely require red blood cells (RBCs); and anemia due to iatrogenic causes.
preoperative or perioperative transfusion, whereas those Postoperatively, anemia resembles anemia of chronic disease
with acute anemia with resulting hemoglobin values of less and is probably related to the effects of inflammatory mediators
than 7 will frequently require red cell transfusions.” Whether released during and after surgery on the production and
patients with hemoglobin values between 7 and 10 g/dL survival of RBCs. Pro-inflammatory cytokines, such as tumor
should receive transfusions depends on the patient’s clinical necrosis factor, impair erythropoietin-dependent signalling
status.2 and iron homeostasis. Iatrogenic causes, notably excessive
The minimum acceptable level of hemoglobin varies phlebotomies, remain a major cause of perioperative anemia.
for patients because individual persons vary in their ability With increasing emphasis on restrictive blood transfusion
to tolerate and compensate for anemia. For example, at strategies, understanding these mechanisms is important for
rest, normovolemic young healthy persons with normal the clinician.5
38 Section 1 Clinical Hemotherapy
Significance of Preoperative Anemia the World Health Organization criteria (Grade 2C). The
group also recommended that further laboratory testing to
Preoperative anemia is associated with adverse outcomes evaluate anemia for nutritional deficiencies, chronic renal
after cardiac surgery. In a study outcomes after non-cardiac insufficiency, and/or chronic inflammatory disease (Grade
surgery were studied with an aim to assess the effect of 1C). It was recommended that nutritional deficiencies be
preoperative anemia on 30-day postoperative morbidity and treated (Grade 1C). The suggestion that erythropoiesis-
mortality in patients undergoing major non-cardiac surgery. stimulating agents be used for anemic patients in whom
The study concluded preoperative anemia, even to a mild nutritional deficiencies have been ruled out, corrected,
degree, is independently associated with an increased risk of or both (Grade 2A) and anemia should be viewed as a
30-day morbidity and mortality in patients undergoing major serious and treatable medical condition, rather than simply
non-cardiac surgery.6 abnormal laboratory value. The authors concluded that the
implementation of anemia management in the elective
Recommendations for Detection, orthopedic surgery setting will improve patient outcomes.
Evaluation and Management of Since the data suggested that the mortality and length of
stay are worsened with liberal use of transfusion, medical
Preoperative Anemia in Orthopedic alternatives to transfusion include iron supplementation
Surgery [Network for Advancement and erythropoiesis-stimulating agents (ESAs) could be
of Transfusion Alternatives (NATA)] used to reduce the need for perioperative blood transfusion
compared with placebo, they are associated with an increased
A standardized approach for the detection, evaluation, risk of thrombotic events in surgical patients.7
and management of anemia before surgery is of utmost
importance in the hospital settings of developing countries.
A multidisciplinary panel of physicians was convened by Management of Preoperative Anemia
the Network for Advancement of Transfusion Alternatives The indication to treat preoperative anemia depends on the
(NATA) with the aim of developing practice guidelines for severity of the anemia, as well as the expected surgical blood
the detection, evaluation, and management of preoperative loss. For instance, if a patient has currently a hemoglobin
anemia in elective orthopedic surgery. A systematic literature level of 9 g/dL and is expected to lose more than 500–1000
review and critical evaluation of the evidence was performed, mL of blood, there is an increased likelihood of dropping the
and recommendations were formulated according to the hemoglobin to critical values (<7 g/dL) where transfusion is
method proposed by the grades of recommendation assess indicated. In this group of patients, the hemoglobin should be
ment, development and evaluation (GRADE) working optimized, raising it by 2–3 g/dL preoperatively, to compensate
group. It was recommended that elective surgical patients for the expected postoperative drop and attempt to prevent
have a hemoglobin (Hb) level determination 28 days before the critical transfusion threshold.8,9 There is a definite link
the scheduled surgical procedure if possible (Grade 1C). between preoperative anemia in patients undergoing surgery
It was also suggested that the patient’s target Hb before and perioperative needs of blood and the management plan
elective surgery be within the normal range, according to of elective and emergency cases has been depicted Table 1.
Table 1: Management: blood delivery to operating room beginning with perceived need
Elective cases Emergency cases
Consider autologous and directed donor blood Consider whether uncrossmatched blood
is indicated
Request blood bank to coordinate autologous, directed, and random Request to blood bank with patient’s blood specimen and
blood needed. Draw blood specimen from patient for blood bank, indicate urgency
before the day of surgery, if possible
Transport blood to operating room on the day of surgery Facilitate transport logistics if urgent, e.g. send person to blood
bank to pick up blood
Check blood units and patient identity by two persons before Check blood units and patient identity by two persons before
transfusion transfusion
Transfuse autologous before directed and directed before random units. Monitor for adverse reactions
Monitor for adverse reactions
Return unused units to blood bank inventory Return unused units to blood bank inventory
Chapter 10 Management of Preoperative Anemia in Patients Undergoing Elective Surgery 39
Blood management is a patient-centered standard of hour to reduce blood utilization and improve and prevent the
care whereby strategies and techniques are used to reduce, adverse outcomes; the program has achieved some reduction
eliminate or optimize blood transfusions to improve patient in blood utilization in its first 7 months. Preoperative anemia
outcomes. It ensures safe, efficient use of the many resources is associated with adverse outcomes after cardiac surgery but
involved in the complex process of blood component therapy. outcomes after non-cardiac surgery are not well established.
Its objectives are the translation of evidence into clinical
practice to minimize blood utilization, ensure consistency References
among different physicians' practice behavior and
implement preoperative treatment focused on the etiology 1. Kumar A. Perioperative management of anemia: limits of
of anemia as a “best practice” for elective surgical patients. blood transfusion and alternatives to it. Cleve Clin J Med.
2009;76(Suppl 4):S112-8.
Perioperative blood management is poised at the confluence
2. Consensus conference: Perioperative red cell transfusion.
of patient safety, resource utilization and system integration, National Institutes of Health. Conn Med. 1988;52(10):593-6.
with consequent improvement in the overall quality of care. 3. Weiskopf RB, Viele MK, Feiner J, Kelley S, Lieberman J, Noorani
Blood transfusion to correct anemia is currently considered a M, et al. Human cardiovascular and metabolic response to
marker of poor outcomes in medicine for multiple reasons. In acute, severe isovolemic anemia. JAMA. 1998;279(3):217-21.
the current state of a decreasing donor pool, arbitrary use of 4. NIH Expert Panel on Autologous Blood: Transfusion alert: Use
blood impedes effective resource allocation. It is associated of autologous blood transfusion. 1995;335:703-13.
with serious complications (e.g. transfusion-related acute 5. Singh S, Gudzenko V, Fink MP. Pathophysiology of perioperative
anaemia. Best Pract Res Clin Anaesthesiol. 2012;26(4):431-9.
lung injury, immune-modulation, transfusion-associated
6. Musallam KM, Tamim HM, Richards T, et al. Preoperative
circulatory overload), and there is substantial cost associated anaemia and postoperative outcomes in non-cardiac surgery:
with the blood banking process. In addition, recent studies a retrospective cohort study. Lancet. 2011;378:1396-407.
have shown that a blood-restrictive approach is associated 7. Goodnough LT, Shander A, Spivak JL, et al. Detection,
with improved outcomes in hip surgery patients.10 evaluation, and management of anemia in the elective surgical
patient. Anesth Analg. 2005;101:1858-61.
8. Boucher BA, Hannon TJ. Blood management: a primer for
Conclusion clinicians. Pharmacotherapy. 2007;27(10):1394-411.
9. Patel M, Auron M, Benitez-Santana SM, et al. Preoperative
Anemia is a potent risk factor for mortality and morbidity
blood management—paving the path to blood less surgery.
in surgical patients, and its management has begun to shift J Hosp Med. 2011;6(Suppl 2):S124.
away from allogenic blood transfusion in recent years. A 10. Carson JL, Terrin ML, Noveck H, et al. Liberal or restrictive
blood management program aimed at reducing allogenic transfusion in high-risk patients after hip surgery. N Engl J
blood exposure for greater patient safety, hand in gloves with Med. 2011;365:2453-62.
the national anemia prevention program is the need of the
Blood Conservation Strategies
DP Singh
11
INTRODUCTION
Blood conservation is a collaborative, advanced medical
approach to patient care that reduces the need for donor
blood transfusions or blood components. Blood conservation
is important in surgery because of risk associated with blood
transfusion and due to shortage of blood supply. Potential
health benefits include faster recovery reduced hospital
stays, reduced risk of infections and reduction in donor blood
transfusions.1
METHODS
With the arrival of minimal invasive surgery, robotic surgery
and new vessel sealing instruments it is possible to perform
bloodless surgery. Blood conservation measures include2–4:
• Perioperative Figure 1 Harmonic scalpel
• Intraoperative
• Postoperative.
Blood conservation strategies start from perioperative
period which include:
• Anemia correction
• Identify and manage bleeding risk
• Manage anticoagulation carefully
• Procedure planning and timing surgery
• Autologous blood donation.
Intraoperative methods:
• Meticulous hemostasis
• Blood sparing surgical techniques
• Permissive hypotension
• Autologous blood options
• Hemostatic agents.
There are various devices available for obtaining meticu-
lous hemostasis which include:
• Harmonic scalpel (Fig. 1)
Figure 2 Ligasure
• Ligasure (Fig. 2)
Chapter 11 Blood Conservation Strategies 41
• CUSA—used in neurosurgery, liver surgery (Fig. 3) and robotic surgical techniques it is necessary to come
• Robotic arms which provide 7 degrees of freedom (Fig. 4) together and share our experience with each other as team
for welfare of our patients and humanity so that they can get
Postoperative care includes:
maximum benefit.
• Avoiding secondary hemorrhage
• Control of infection
• Maintain normothermia. REFERENCES
1. LD Petz., et al’s Clinical Practice of Transfusion Medicine, 3rd
edn.
CONCLUSION 2. Haemostasis and Thrombosis Task Force (for the British
Committee for Standards in Haematology). Guidelines on oral
Optimal patient blood management is the key to success to
anticoagulation: third edition. British J Haem. 1998;101:374-87.
conserve this scarce, expensive and potentially infectious 3. Guidelines for Autologous Transfusion part II: peri-operative
resource. With our current understanding of pathophysiology haemodilution and cell salvage. Br J Anaes. 1997;6:768-71.
of blood transfusion, cell savers, autologous blood transfusion 4. Page C, Retter A and Wyncoll D Blood conservation devices in
and that we had learned from damage control resuscitation in critical care: a narrative review. Annals of Intensive Care. 2013;
warfare and with the invention of modern minimal invasive 3:14.
Thromboelastography
Anupam Verma
12
To assess the viscoelastic properties of blood there are R value is time from the beginning of the trace until
currently various coagulation monitoring devices in the amplitude of 2 mm is reached. This denotes the time taken
market namely Thromboelastograph (TEG) (Hemoscope from the placement of the blood sample in the cuvette to
Corporation, USA), ROTEM (Pentapharm GmbH, Germany), the initial fibrin formation. It represents a deficiency in
and Sonoclot analyzer Sienco Inc., USA. These coagulation coagulation factors, effects of endogenous heparin, and/or
analyzers provide graphical representations of the dynamics hemodilution. The treatment with FFP or no treatment would
of fibrin polymerization in citrated or noncitrated whole depend on the clinical picture. Conversely, a shortening
blood, platelet-rich or platelet-poor plasma, with or without in the R value (<2 min) would indicate a hypercoagulable
activation. picture and treatment with an anticoagulant of choice may
The paradigm shift in our understanding about classical prove beneficial.; K is the time from the end of R until a fixed
coagulation cascade occurred due to introduction of a cell- level of clot strength is reached, i.e. amplitude of the trace
based model which emphasizes the importance of tissue factor is 20 mm. A low value may indicate the need for fibrinogen
as the initiator of the coagulation cascade and the pivotal role or cryoprecipitate administration, and a high value may
of platelets for intact hemostasis. This new understanding indicate hypercoagulability for which the patient may benefit
explains the poor correlation between traditional tests of from anticoagulant therapy; α-angle (clot formation rate)— It
coagulation and clinical bleeding and has generated renewed is the angle of the trace from the horizontal at a point on the
interest in viscoelastic tests for diagnosing derangement in trace until amplitude is 20 mm. It measures the rate of fibrin
hemostasis and to guide transfusion therapy. build up and cross linking and to some extent platelets also
TEG assesses the viscoelastic properties of blood samples contribute to α angle. Both K and α angle measure speed of
under low shear conditions. It measures the clot’s physical clot formation or clot kinetics; MA (maximum amplitude)—It
property by using a stationary cylindrical cup that holds the is a measure of maximum strength of the clot and assesses the
blood sample and oscillates through an angle of 4°45 with function of the platelets and to some extent fibrinogen also;
each rotation cycle lasting 10 sec. The TEG tracing shows A low value can result from hypofibrinogenemia, decreased
complete dynamics of clot formation and lysis over time platelet function, or quantity. Transfusing platelets may
(Fig. 1). The main TEG parameters are defined as under. improve hemostatic condition. An MA >69 is an evidence
of a prothrombotic state, and an anticoagulant of choice
may be clinically indicated. CI (coagulation index). The
patient’s overall coagulation profile is calculated from the R,
K, MA and angle. The qualitative analysis thus obtained from
TEG can facilitate administration of right blood product or
anticoagulant in correct amount. The TEG assay can also be
used for preoperative risk stratification based on individual
hemostatic conditions.
Besides standard TEG, Platelet Mapping which is an
extension of TEG technology, in addition to providing
information on clot formation and lysis of whole blood
samples, it quantifies the contribution of fibrin, adenosine
diphosphate (ADP) receptor and thromboxane A2 (Tx A2)
Figure 1 TEG on citrated blood sample in a patient of DIC receptor in clot strength.1 The TEG Platelet Mapping assay
Chapter 12 Thromboelastography 43
enables relating the percent platelet inhibition to the Variations in hematocrit are known to affect the results
individual’s maximum uninhibited platelet function. This with increased and decreased hematocrit leading to TEG
information allows monitoring of effectiveness of anti- picture that indicate hypocoagulable and hypercoagulable
platelet agents, aspirin and clopidogrel, via inhibition of states, respectively. Thromboelastographic results need
TxA2 and ADP receptors. This investigative tool is important to be carefully interpreted in presence of severe anemia,
where we need to monitor the effects of antiplatelet drugs thrombocytopenia and hemodilution as these conditions can
especially in patients from cardiology, cardiac surgery, variably affect the test results.5 TEG has still not been adopted
Neurology, Interventional radiology. The existence of a high in mainstream clinical practice due to poor standardization
level of individual variability in platelet responsiveness to and validation and artifacts it can introduce if not performed
antiplatelet therapy and on-treatment platelet reactivity have correctly as it is not fully automated. Bubbles can appear in
been confirmed in the majority of clinical studies examining the cup if pipetting technique is not good which can affect the
antiplatelet therapy efficacy in patients undergoing test result.
percutaneous coronary intervention. Platelet mapping can As this technology diversifies with different reagents and
also depict whether a delay in surgery is necessary or, may applications, it is important to understand these differences
possibly proceed earlier than anticipated. when interpreting any data generated. Overall, TEG is a
Various studies suggest that TEG could be a complimentary powerful tool, but its implementation requires thought and
test to current standard coagulation assays and in some planning to insure that the test is performed correctly and
cases, a superior method altogether. In theory, TEG has results are interpreted cautiously taking into consideration
many of the properties of an ideal coagulation test. However, the clinical picture and results of other laboratory tests.
there are issues with this technology as well. First, TEG was
designed for use with fresh whole blood samples, but use of References
fresh whole blood requires initiation of the test within 4–6
minutes of sample collection which is not always possible. 1. Agarwal S, Coakley M, Reddy K, Riddell A, Mallett S.
Thus citrated blood samples were used for laboratory based Quantifying the effect of antiplatelet therapy: A comparison
of the platelet function analyzer (PFA-100) and modified
testing. However, both systematic differences and artifacts
thromboelastography (mTEG) with light transmission platelet
in TEG tracings have been described when comparing fresh aggregometry. Anesthesiology. 2006;105:676-83.
whole blood to citrated blood samples.2,3 Differences in 2. Camenzind V, Bombeli T, Seifert B, et al. Citrate storage affects
patient populations, use of clot activators, and other factors thromboelastograph analysis. Anesthesiology. 2000;92:1242-9.
make the studies difficult to directly compare. Beyond 3. Johansson PI, Bochsen L, Andersen S, Viuff D. Investigation
choice of sample types and issues of timing between sample of the effect of kaolin and tissue factor-activated citrated
collection and testing, TEG and other viscoelastic tests are whole blood, on clot forming variables, as evaluated by
subject to unique pre-analytical and analytical artifacts thromboelastography. Transfusion. 2008;48:2377-83.
4. Scarpelini S, Rhind SG, Nascimento B, et al. Normal range
that can significantly affect the results. The manufacturer’s
values for thromboelastography in healthy adult volunteers.
manual provides normal reference values for most but not all Braz J Med Biol Res. 2009;42:1210-17.
different techniques. It is recommended that one should have 5. Bolliger D, Seeberger MD, Tanaka KA. Principles and practice
local reference values for TEG which is not very practical.4 of thromboelastography in clinical coagulation management
Besides it cannot detect the disorders of primary hemostasis and transfusion practice. Transfusion Medicine Reviews. 2012;
and not sensitive to mild coagulation factor deficiencies. 26:1-13.
Transfusion Support
in Cardiac Surgery
Rajendra K Chaudhary
13
Cardiac surgery, above all other major surgical procedures, when the rates of all-cause mortality at 30 days and severity
continues to place the largest demand on the available blood of organ dysfunction when a restrictive strategy of red-cell
supply. Despite major advances in perioperative blood transfusion, i.e. hemoglobin concentration between 7 and
conservation, transfusion rates in cardiac surgery remain 9 g/dL, was compared with a liberal strategy, i.e. hemoglobin
high with large variations among individual centers. Blood concentration at 10–12 g/dL. The restrictive strategy of RBC
transfusion is commonly used in cardiac surgical patients transfusion was at least as effective as and possibly superior
and accounts for about 10–20% of blood transfused. It is to the liberal transfusion strategy. The restrictive strategy also
noted that approximately 10–20% of patients consume 80% of appeared to be safe in critically ill patients with cardiovascular
total blood transfused among cardiac surgery patients. disease, with the possible exception of patients with acute
myocardial infarction and unstable angina.
Transfusion variability in Another randomized study in 428 elective, primary
coronary artery bypass grafting (CABG) patients found that
Cardiac Surgery there were no differences in clinical outcomes, including
In spite of published guidelines there are tremendous morbidity and mortality, between two groups of patients
variations in transfusion practices among physicians and assigned to receive RBC transfusion if the postoperative
institutions. The incidence of blood transfusion among hemoglobin was <8 g/dL or <9 g/dL, respectively. A
patients undergoing cardiac surgery has been reported to vary number of database studies in cardiac surgery reported that
between 27 and 92%. The observed variability in transfusion decreased perioperative hematocrit values is associated with
practices may be reduced by altering blood use in this high decreased survival. In a study in 6,980 patients undergoing
usage group. These wide differences in transfusion rates CABG, the lowest hematocrit during cardiopulmonary
may be explained by a variety of reasons including different bypass (CPB) was significantly associated with increased risk
patient populations, differences in procedure-related factors, of in-hospital mortality, intra- or postoperative placement
traditions, and norms. Therefore, individual centers should of an intra-aortic balloon pump, and return to CPB after
pay attention to their current transfusion practice. attempted separation.
No “optimal” hematocrit or hemoglobin concentration
Potential benefits of red blood cell is established in cardiac surgery. The classical “10/30” rule,
suggesting that transfusion should be given to maintain a
transfusion in cardiac surgery hemoglobin concentration of 10 g/dL and a hematocrit of
Allogeneic blood transfusion may be given to treat life- 30%, was abandoned several years ago. Today, much lower
threatening hemorrhage during surgery, but most often hemoglobin and hematocrit values are accepted. However,
transfusion of blood components is given to increase there are no clinically available methods for measuring
the oxygen-carrying capacity of the blood or to improve regional tissue oxygenation to identify when there is a need
hemostasis. There is, however, a lack of clear evidence for RBC transfusion. Observational studies among CABG
regarding the benefit of RBC transfusion when it comes patients, who refused blood transfusions for religious
to enhancing the oxygen-carrying capacity of blood. reasons, suggest that survival without transfusion is possible
Only a few randomized trials were carried out to identify at low hemoglobin concentrations.
the potential benefits of RBC transfusion. Randomized, During the past three decades, traditional transfusion
controlled study in 838 critically ill, normovolemic patients triggers were constantly challenged. Despite published
in an intensive care unit (ICU) produced equivalent results blood transfusion guidelines and reviews on transfusion
Chapter 13 Transfusion Support in Cardiac Surgery 45
Predictors of allogeneic
red blood cell transfusion
during Cardiac Surgery
Knowledge about predictors of bleeding and transfusion
is a prerequisite for preoperative risk stratification and
use of rational blood conservation techniques. Not all
patients are at the same risk for transfusion requirement.
Some patient variables can be used to predict the risk for
perioperative transfusion. Need for packed red blood cell
(PRBC) transfusion in isolated, first-time CABG patients
can be predicted preoperatively. The strongest predictors
are use of CPB, hematocrit ≤ 40%, body weight ≤ 70 kg, and
serum creatinine > 100 μmol/L. Female gender, older age,
higher Euro score, and number of distal anastomoses are
also significant predictors. Ability to identify patients at
risk of PRBC transfusion would save blood bank efforts and Figure 1 Mechanism of bleeding during cardiopulmonary bypass
resources and allow the employment of a targeted blood
conservation policy in CABG patients.
Recently, an externally validated transfusion risk under an important and delicate balance between anticoagulation
standing scoring tool (TRUST) for assessing this risk in adult during CPB and proper hemostasis after CPB. These
patients undergoing cardiac surgery has been proposed patients are at risk for excessive perioperative blood loss
(Table 1). often requiring transfusion of blood products. In the past,
clinicians administered blood products empirically due to
long turn around times of laboratory-based coagulation tests.
Mechanism of Bleeding in Patients
This practice exposes patients to inappropriate blood therapy
Undergoing Cardio Pulmonary Bypass resulting in increased morbidity and mortality and hospital
Bleeding during cardio pulmonary bypass is due to various costs. Today, point-of-care (POC) devices are available
factors as shown in the Figure 1. providing rapid bedside monitoring to aid the clinician in
directing appropriate targeted therapy. The majority of POC
devices used today can perform multiple coagulation tests.
Point-of-Care (POC) Tests in Cardiac Surgery The use of transfusion algorithms in conjunction with POC
The hemostatic management of patients undergoing cardiac testing has been shown to reduce both transfusion require
surgery with cardiopulmonary bypass (CPB) necessitates ments and blood loss in cardiac surgery.
Table 2: Postoperative morbidity associated with allogeneic red blood cell transfusion in cardiac surgery
Studies Number of patients Outcome associated with transfusion
Michalopoulos et al. 1998. Eur J Surgery 2,615 Severe sepsis
Leal-Noval et al. Chest. 2001;119:1461 738 Nosocomial pneumonia
Chelemer et al. Ann Thorac Surg. 2002;73:138 533 Respiratory and surgical site infection
Koch et al. Crit Care Med. 2006;34:1608 11,963 Renal failure, prolonged ventilatory support, serious infection,
neurologic events
Rogers et al. Am Heart J. 2006;152:1028 8,518 Infection, pulmonary dysfunction
Scott et al. Ann Card Anaesth. 2008;11:15 1,746 Increased time to extubation, stay in the ICU, and postoperative
length of stay
Koch et al. Ann Thorac Surg. 2009;88:1410 16,847 Pulmonary morbidity
Chapter 13 Transfusion Support in Cardiac Surgery 47
A predonation program obviously does decrease the need for Typically the loss and transfusion rate is less than total hip
allogenic blood in primary THA. However, there are still some arthroplasty but a 40–50% rate is not uncommon. In a study
serious concerns: of 477 patients only the preoperative Hb was a predictor of
• Autotransfusion is not completely safe. Clinical errors and transfusion risk. If > 13.5 g/dL there was only a 2.8% risk. This
infection can still occur. actually could produce a lower complication rate but is not
• There is a high level of wastage. About half the units are not generally accepted as good practise.9
used. Hemodilution and intraoperative salvage are not
• Preoperative anemia is more likely with predonation. The appropriate for most primary TKAs as many are performed
erythropoietic response to phlebotomy is unexpectedly under tourniquet and the blood loss and transfusion rate is
rather poor. not particularly high in most cases.
• Predonation is neither cheap nor easy to arrange.
• The use of hypotensive epidural anesthesia has been Bilateral Total Knee Arthroplasty
shown to reduce the blood loss significantly and is used in
many orthopedic centers routinely.6 The blood loss is significantly higher than that seen in
• Acute hemodilution is safe and effective but probably not unilateral cases leading to an increased transfusion rate. In
required for the majority of primary THAs. It requires a patients with no predonation this rises from 18 to 57%. This
high level of skill and is time consuming. necessitates the application of one or more techniques to
• There is some debate concerning the effect of different avoid allogenic blood. There was a low rate of complications
implants. 25 matched pairs of THAs, half uncemented and good clinical outcome in all groups at a mean follow-
and half hybrid or cemented, were compared. There up of four years (1 to 7.2). The group staged at a one-week
was no significant difference noted in the blood loss and interval had the least blood loss (p = 0.004).10
transfusion rate.7 In a comparative study of tranexamic acid (TEA) against
• Intraoperative salvage is costly in time and staffing and control showed significant reduction in blood loss. The total
rarely warranted in primary THA. Postoperative reinfusion postoperative drain output was found to be lower in patients
drains can be used but most blood loss is intraoperative who received TEA as compared to the control group (275 mL
and the trend is now to avoid the routine use of drains.8 vs 810 mL) and this relation was also found to be statistically
significant (p = 0.000). The sample size was large enough for
this conclusion to be made (power 1.00 in all). This implied a
Revision Hip Replacement
decrease in total blood loss in patients who were administered
Templating and rehearsal of the procedure and familiarity TEA during bilateral total knee arthroplasty (TKA). As such
with the instruments and implants are vital. Revision surgery the amount of allogenic blood transfusion (BT) requirements
is accounting for an increasing percentage of hip arthro were also reduced in these patients as compared to the
plasty. The implants inserted in the 70s and 80s are now control group (0.80 units vs 3.17 units).11
reaching 15–20 year follow-up and the failure rate is rising Patients with osteoarthritis of the knees were injected
significantly. Revision surgery is much less predictable than with 5 mL (25 mg/mL) TEA and 5 mL analgesic containing
primary surgery and preoperative planning is more difficult. epinephrine (3 μg/mL) solution were injected at several
At times the surgery is no worse than a primary procedure, points into the posterior capsule before installation of the
but more likely there will be problems with dense scar prosthesis. The femoral medullar canal was closed with
tissue, large areas of exposed bone, osteolysis and fractures autograft bone and then sealed compressively with cement.
and a greatly increased operative time. All this adds up Before the tourniquet was released, 10 mL TEA solution and
to increased blood loss, typically 50–100% greater than a 10 mL analgesic containing epinephrine were injected at
primary procedure. Typically 3 units of blood are transfused. several points into the periosteum, synovium, joint capsule,
Breakthrough allogenic transfusion with a predonation tendons, and deep fascia tissue (injection of analgesic
program is highest with revision hip surgery at 21% in one containing epinephrine into subcutaneous fat and dermis
series.1 was avoided). The residual nail holes in the bone and the
uncovered bone section were covered with bone wax. TEA
solution (20 mL) was injected into the articular cavity after
Primary Total Knee Arthroplasty wound closure. The drainage tube was clamped for 4 hours,
In 100 consecutive primary total knee arthroplasties at Guys then opened. Intraoperative blood loss, drainage volume, total
Hospital in 1998, the measured blood loss ranged from postoperative blood loss, and number of patients requiring
50 to 2,590 mL with an average of 780 mL. When calculated allogenic blood transfusion were significantly reduced.12 Cell
(based upon pre- and postoperative Hb and the transfusion saver techniques for blood salvage is an important component
given) this average was closer to 1,300 mL. The average loss is reducing blood loss. OrthoPAT system is an important tool
in males was much higher than in females 980 mL vs 650 mL. in autoblood transfusion intraoperatively.13
50 Section 1 Clinical Hemotherapy
7. Tric ME, Walker RH, DLima DD Morris BA, Colwell CW,
REFERENCES Jr. Blood Loss and Transfusion Rate in Noncemented and
1. Bierbaum MD, et al. An Analysis of Blood Management in Cemented/Hybrid Total Hip Arthoplasty. Is there a difference?
Patients Having a Total Hip or Knee Arthroplasty. J Bone Joint A comparison of 25 matched pairs. Orthopedics. 1999;22:S141.
Surg. 1999;81a:2-10. 8. Chandrataya A, et al. To drain or not drain: literature versus
2. de Andrade JR, Jove M, Landon G, Frei D, Guilfoyle M, Young practice. JR Coll Surg Edinb. 1998;43:404-6.
DC. Baseline hamoglobin as a predictor of risk of transfusion 9. Lotke, Garino JP, et al. Autologous blood after total knee
and response to epoetin alfa in orthopedic surgery patient. Am arthoplasty: Reduction in non-surgical complications.
J Orthop. 1996;25:533-42. Presented at Am Acad Orthop Surg. 1997.p.265.
3. Faris PM, Ritter MA, Abels RI. The American Erythropoetin 10. Forster MC, Bauze AJ, Bailie AG, Falworth MS, Oakeshott
Study Group. The effects of recombinant human erythropoietin RD. A retrospective comparative study of bilateral total knee
on perioperative transfusion requirements in patients having replacement staged at a one-week interval. JBJS (Br) 2006;88-
a major orthopedic operation. J Bone Joint Surg. 1996;78-A: B(8):1006-10.
62-72. 11. Dhillon MS, Bali K, Prabhakar S. Tranexamic acid for control of
4. Nuttall GA, Santrach PJ, Oliver WC Jr, Horlocker TT, Shaugh blood loss in bilateral total knee replacement in a single stage.
nessy WJ, Cabanela ME, et al. The predictors of red cell Indian Journal of Orthopedics. 2011;45(2):148-52.
transfusions in total hip arthroplasties. Transfusion. 1996;36: 12. Zhaohui Liu, Wanshou Guo, Qidong Zhang, Guangduo Zhu.
144-9. Topical hemostatic procedures control blood loss in
5. Walker RW, et al. Blood loss during primary total hip arthro bilateral cemented single-stage total knee arthroplasty.
plasty: use of preoperative measurements to predict the need Journal of Orthopedic Science. 2014.
for transfusion. Ann R Coll Surg Engl. 1997;79:438-40. 13. Kyle C, Sinclair BS, Henry D, Clarke MD, Brie N, Noble BS.
6. Sharrock NE, Minoe R, Urquhart B, Salvati EA. The effect of two Blood management in total knee arthroplasty: a comparison of
levels of hypotension on intraoperative blood loss during total techniques. Orthopedics. 2009;32(1):19.
hip arthroplasty performed under lumbar epidural anesthesia.
Anesthesia Analgesia. 1993;76:580-4.
Restricting Transfusion
Requirements in Major Orthopedic
Surgery and its Alternatives
Ravisha Bhardwaj, RS Boparai
15
Introduction a biological product, allogenic blood transfusion will never
be risk-free: (i) screening methods for the detection of new
In major orthopedic surgical procedures, perioperative infectious threats will only be implemented once a significant
blood loss and blunted postoperative erythropoiesis, due to proportion of the transfused population has already been
surgery-induced inflammation, may lead to postoperative infected; (ii) “clerical mistakes” and administration of
anemia in almost 90% of patients. Allogenic blood transfusion “wrong blood” are still too frequent (1/15,000–1/20,000); and
is the most frequently used measure for treating acute intra- (iii) the risk of complications such as graft-versus-host
and postoperative anemia, especially in patients with low disease, metabolic disorders, bacterial contamination,
preoperative hemoglobin levels and/or undergoing non- transfusion-related acute lung injury (TRALI), transfusion-
elective surgery, as it may increase the patients’ hemoglobin associated circulatory overload (TACO), and transfusion-
levels though transitorily, quickly and effectively.1 related immunomodulation (TRIM) still persists.4
The rationale behind allogenic blood transfusion is to In order to reduce variability in transfusion practice,
restore oxygen delivery and provide a reserve, should further both in the proportions of patients receiving allogenic
bleeding occur. It is generally assumed that, in the event of blood transfusion and in the volume of blood administered
tissue hypoxia, the benefits on survival conferred by the per transfused patient, scientific societies have developed
allogenic transfusion in certain patients clearly outweigh evidence-based guidelines and recommendations on the
the risks. This goal can only be achieved if dual indicators for indications of allogenic blood transfusion. The final objective
blood transfusion (level of oxygen carriers and evidence of of these guidelines is a more rational and “restrictive” use of
the oxygen tissue debt of anemic origin) are used. However, allogenic blood transfusion in patients for whom pharma
in everyday clinical practice, reliable indicators of oxygen cological options are not available or cannot be implemented
deficit of anemic origin (mixed venous blood saturation, (e.g. acute severe anemia).1 Accordingly, in making a
partial tissue oxygen pressure, etc.) are often not available2 transfusion decision in euvolemic, nonbleeding patients:
and, consequently, decisions on transfusion are usually • The risk of anemia and the risks and benefits of red
made just on the basis of the patient’s hemoglobin level cell transfusion should be carefully balanced for each
and/or symptoms. Thus, many allogenic blood transfusions individual patient;
are unnecessarily given to patients with only relatively low • The so-called “liberal” transfusion protocols (pre-
hemoglobin levels, just expecting that transfusion of red transfusion hemoglobin concentration >9–10 g/dL)
blood cells will increase oxygen transport, thus alleviating should be generally avoided;
tissue hypoxia and improving outcome. Evidence based • Should allogenic blood transfusion be deemed necessary,
medicine shows that allogenic blood transfusion produces a single unit transfusions are desirable; and
variable increment in brain tissue oxygenation, as assessed • Patients should be reassessed between transfusions to
by direct measurement of partial tissue oxygen pressure, in determine the remaining transfusion needs.
patients with neurological trauma and documented oxygen In this regard, the transfusion requirements in critical
deficit.3 care (TRICC) trial demonstrated that the introduction of a
As a result of improved screening for detecting trans- restrictive transfusion protocol with transfusion trigger of
fusion transmitted infections and better product conserva- Hb <7 g/dL, reduced the rate of allogenic blood transfusions
tion methods, allogenic blood transfusion is considered a by 33% and the allogenic blood transfusion index by 3 units
safe treatment option in developed countries. However, as per patient compared to a liberal transfusion protocol with
52 Section 1 Clinical Hemotherapy
transfusion trigger of Hb <10 g/dL. There were no differences can be lowered with the use of special frames or supports thus
in mortality rates between groups, not even for the subgroup preventing congestion of the epidural veins.
of patients with significant cardiac disease, but the restrictive
protocol resulted in a lower mortality rate among patients
who were younger (<55 years) or less critically ill (APACHE
Choice of Anesthesia
score <20).5 It has long been recognized that spinal anesthesia for total hip
However, blood transfusion conceals a number of well- replacement surgery can have significant advantages. More
recognized risks and complications and blood products have recently the beneficial effect of hypotension on intraoperative
become more expensive because of their specific preparation blood loss during total hip surgery under epidural anesthesia
procedure. Even small changes to routine procedures can has been reported. The surgical blood loss was reduced from
lead to enormous benefits for patients, physicians, hospitals 700 mL to approximately 250 mL.7
and society in general.
Orthopedic surgery can be divided into the fields of elective
orthopedics and trauma. The elective aspects, particularly Perioperative Use of Pharmacological Agents
joint replacement and spinal surgery, are well suited to the There is only limited experience in the use of these agents
many techniques available to help reduce the use of allogenic in orthopedic surgery. It is suggested that the use of
blood. Trauma by definition is acute, unpredictable and a Desmopressin with Harrington rod spinal fusion surgery can
much bigger challenge. reduce blood loss by 32.5%. No benefit was noted with total
hip surgery however. Aprotinin has been reported to reduce
Techniques aPPLICAble to blood loss in total hip arthroplasty.
orthopedic surgery6
In USA and many European countries predonation of blood Perioperative Erythropoietin
for autologous transfusion is perhaps the most widely utilized
Many studies have confirmed the safety and efficiency
method. Lowering the transfusion trigger is the simplest way
of recombinant human erythropoietin. Used alone in
to reduce allogenic transfusion. Too often blood is given to
the perioperative period or as an adjunct to maximize a
patients on an empirical basis. At times patients undergoing
predonation program, red blood cell production is increased
total knee arthroplasty with a tourniquet inflated were
and allogenic transfusions are reduced.
transfused even before any blood was lost. It is essential to
educate the relevant staff members who may order this trans
fusion. This may be the anesthetist, the junior surgical staff Preoperative Optimization of Medical Status
or, not infrequently, the surgeon himself who is unwilling
to change his practice despite overwhelming evidence to The majority of patients undergoing orthopedic surgery
the contrary. Secondly we can make the choice of surgical have a number of comorbid conditions, which need to be
procedure, which reduces blood loss like the use of closed addressed preoperatively. Probably half are hypertensive.
techniques for nailing long bone fractures. Similarly, the Many are taking aspirin, nonsteroidal anti-inflammatory
use of external fixators for unstable pelvic fractures not only agents or anticoagulants. Coagulopathies should be recog
reduces blood loss but at times can be life saving. Thirdly, nized and treated.
preoperative planning and rehearsal like surgeon being
familiar with the patient, the procedure, the instruments and Predonation of Autologous Blood
the implants can save valuable time and reduce blood loss.
Modification of surgical techniques is other very simple and It has been the standard of care in many orthopedic units
cost-effective way of reducing the use of allogenic blood. across the world for a number of years. Although generally
Adherence to good surgical principles like taking more time effective there are increasing concerns related to cost factors,
to identify and control bleeding vessels before the surgery the increase in preoperative anemia and the large number of
proceeds. Many procedures have their own potential wasted units.
problems. Perforating branches of profunda femoris artery
are often injured when approaching the shaft of the femur. Hemodilution Techniques
Branches of the circumflex vessels are at risk with a posterior
approach to the hip. The superior lateral geniculate artery As yet there is only limited information available in the
may be cut during knee surgery. A few extra seconds to locate orthopedic setting. There are probably indications for its use
and avoid or control these arteries can make a big difference when blood loss is expected to be moderately high and the
to overall blood loss. Appropriate positioning particularly patient has no coronary artery, renal, hepatic or pulmonary
helpful in spinal surgery where the intra-abdominal pressure disease.
Chapter 15 Restricting Transfusion Requirements in Major Orthopedic Surgery and its Alternatives 53
Sandeep Kundra
16
Introduction not compromise subcutaneous tissue oxygen tension due
to compensation with increasing blood flow. However,
Neurosurgical procedures involve surgery on head and neck Weiskopf, et al.2,3 showed that cognitive abilities are
or spine. Brain is a highly vascular organ and is supplied by compromised by acute hemodilution below 6 g/dL. Thus,
internal carotid arteries and vertebral arteries which form an the application of end point for transfusion at Hb of 6 g/
anastomosis named, the ‘circle of Willis’ at the base of brain. dL, in the context of injured brain is fraught with risks. The
Overall, brain receives roughly 15–20% of cardiac output. possibility of worsening brain injury has been demonstrated
Brain is a metabolically highly active organ, with a high even at hemoglobin levels above the commonly accepted
requirement for oxygen. threshold of 7.0 g/dL.4 Decisions about when to transfuse
The result of these things is two fold: neurosurgical patients must further weigh the putative
1. Any injury or surgery on brain leads to greater bleeding as benefits of increasing oxygen carrying capacity and delivery
compared to other body organs. against our increasing understanding of the serious risks
2. Blood loss which results in decreased oxygen carrying associated with the transfusion of blood products. These risks
capacity of brain may cause irreversible brain injury. include infection, hemolysis, transfusion-related acute lung
Among the neurosurgical techniques, surgeries on injury, alloimmunization, and immunosuppression,5 as well
cranium accounts for greater blood loss as compared to as concerns about whether or not transfused red cell products
spine. Among the cranial surgeries, meningiomas being more effectively augment oxygen delivery.6,7
vascular, tend to bleed more. Neurosurgery differs from other surgical specialties on
The purpose of this presentation is to highlight some of
three main accounts:
the recent work and issues surrounding the factors, which
1. Being highly vascular structures, surgery on nervous tissue
must be considered when making a decision to transfuse
entails significant blood loss which needs to be replaced.
a neurosurgical patient. The focus is on the perioperative
2. Patients can bleed to death in a very short time if accidental
period. As with all treatment decisions, transfusion decisions
injury to venous sinuses or arterial vasculature of brain
must be taken in the context of the individual patient’s co-
occurs.
morbidities and the clinical scenario, such as ongoing
3. Many patients need critical care, wherein, most critical
bleeding. Although the issues discussed center primarily
care specialists tend to keep the Hb around 10 g/dL.
on the use of blood components and derived products, the
contributions of other equally important factors such as So, most neurosurgeons and neuroanesthetists tend
surgical hemostasis, temperature, pH, and serum calcium to transfuse blood at much higher Hb levels of usually 8 or
must be acknowledged lest we neglect to consider them 9 g/dL.
during our clinical decision-making. The need for blood product transfusion in neurosurgery
can broadly be divided into three categories:
Threshold for transfusion in 1. For acute blood loss.
2. For correcting chronic anemia prior to taking up for
Neurosurgery is uncertain surgery.
The threshold hemoglobin at which transfusion should be 3. Correcting coagulation defects.
performed in neurosurgical patients is very uncertain. In To highlight clinical relevance, information is presented
healthy population, Hopf et al.1 demonstrated that acute according to presentations and procedures common to
hemodilution down to 5 g/dL in healthy volunteers does neuroanesthetic practice.
56 Section 1 Clinical Hemotherapy
Cerebrovascular Surgery would be desirable under this circumstance. Again, the exact
hemoglobin value below which one should be concerned is
The incidence of intraoperative transfusion for cerebro uncertain. A subset analysis of head injured patients from the
vascular procedures such as aneurysm clipping and carotid transfusion requirements in critical care trial15 did not detect
endarterectomy has been described as relatively low, with a difference between populations maintained at hemoglobin
some published reports of intraoperative transfusion rates of 7.0–9.0 g/dL versus 10.0–12.0 g/dL,16 though it may have
well under 10%.8 When the preoperative and postoperative been somewhat underpowered. A number of studies have
phases are taken into account, transfusion rates are higher. demonstrated an apparent improvement in cerebral well
Ruptured intracranial aneurysms represent a unique being after transfusion based on available neurophysiologic
circumstance as classically treatment involves treatment with monitors17,18 whereas other studies looking at clinical
induced hypertension and hypervolemia and hemodilution. outcome have demonstrated a lack of improvement with
The rationale for this treatment is to strike a balance between transfusion.19,20 Taken together, the appropriate course
reduced blood viscosity and preserved oxygen carrying of action becomes even less evident. Transfusion of
capacity. In spite of this theory, hemodilution as a strategy to blood components has an additional role in TBI due to
improve outcome in symptomatic vasospasm has been called the association of coagulopathy with brain injury, and its
into question.9,10 There is evidence that higher hemoglobin
association with poor outcome.21-23 Brain parenchyma is a
levels in patients with subarachnoid hemorrhage (SAH) is
rich source of tissue factor, which activates coagulation when
associated with improved outcome.11 Although there seems
released. By this mechanism, disseminated intravascular
to be reasonable evidence to suggest that higher hemoglobin
coagulation (DIC) may be induced. It has been suggested
levels are good, it is not clear what hemoglobin level is
that this clotting activation may in some cases be matched by
optimal. A recent study has identified a cut-off of 9 g/dL, or
fibrinolysis, resulting in a net consumption of clotting factors,
hematocrit of 0.27, below which an increased incidence of
but possibly avoiding the microembolic phenomenon
cerebral hypoxia is observed via invasive monitors of brain
of DIC. Owing to the complex, heterogeneous, and likely
tissue oxygenation.12 To further complicate matters, it is
multifactorial nature of the coagulopathy associated with
unclear as to whether or not it is useful to achieve or maintain
TBI, it is not surprising that guidelines for treatment of
higher hemoglobin levels through transfusion. It has been
coagulopathy caused by brain injury do not exist. Depending
asserted that transfusion in the setting of SAH in particular,
on the mechanism contributing to the problem, blood
may be associated with increased risk for vasospasm (at least
products such as plasma, platelets, and cryoprecipitate likely
radiographically, if not clinically)13 and poor outcome. So
have a role in replacement of depleted coagulation substrate.
most neurointensivists tend to target a hemotocrit of 27–30%,
The association of TBI with the multiply injured poly-
albeit the evidence to support or refute this practice is lacking.
trauma patient also merits mention. Brain injury is often not
the only source of bleeding and coagulopathy, as a significant
Intracerebral Hemorrhage proportion of brain injured patients may present with other
Intracerebral hemorrhage (ICH) is most commonly due injuries. Recent literature regarding massive transfusion in
to hypertension. Sometimes, patients on anticoagulant trauma has focused on the role of increased ratios of plasma
therapy may also develop ICH. Surgery is performed when and platelets to red blood cells.24 In the context of closed head
ICH is causing mass effect resulting in increased intracranial injury, avoidance of coagulopathy or its rapid and adequate
pressure (ICP). Transfusion depends on the amount of blood reversal is arguably even more important.
loss and the pre-existing Hb with most neuroanesthetists
targeting a Hb of 9 g/dL. The most significant recent Tumor Surgery
development in the treatment of intracerebral hemorrhage
(ICH) has been the result of the phase III FAST trial, As with traumatic brain injury, resection of brain tumors
investigating the role of activated factor VII in treatment.14 has been associated with a clinically significant incidence
Although hematoma growth was reduced, there was no of DIC.25,26 The development of coagulopathy in the context
demonstrated benefit for survival or functional outcome. of tumor resection is again associated with poor outcome.
One issue regarding transfusion, which is particular to tumor
surgery, is the role it may play in tumor progression.27 It is
Traumatic Brain Injury speculated that the immune-modulatory effects of trans-
Traumatic brain injury (TBI) represents a complex and fusion impair the body’s innate abilities to suppress
heterogeneous group of patients in which mechanisms tumor growth and spread. The exact significance of these
that normally help to ensure an adequate supply of blood observations as applied to brain metastases or primary
and oxygen to brain parenchyma, such as autoregulation, neurological tumors remains to be established. For now, it
are variably impaired. It may seem logical that higher is at the minimum yet another reason why we must strive to
hemoglobin levels and greater oxygen carrying capacity avoid unnecessary transfusion.
Chapter 16 Transfusion Support in Neurosurgery 57
Interventional Neuroradiologic Procedures clear, although there are animal data to suggest that PCCs
may be more effective.37
(Embolization, Acute Stroke)
Advances in the field of interventional radiology continue to Blood conservation strategies
have an important impact on procedures which pose a risk
for perioperative hemorrhage. Preoperative embolization of Given many of the conflicting advantages and disadvantages
tumors and arteriovenous malformations (AVMs) can greatly of blood transfusion in the neurosurgical patient, perhaps
attenuate blood loss at the time of surgery, and reduce the an aggressive implementation of perioperative blood
requirement for transfusion. Advances in the field naturally conservation strategies is the answer. If transfusion counter
lead to more questions. What International Normalized acts the benefit of increased hemoglobin levels in the peri
Ratio (INR) or platelet count is appropriate for a patient operative setting, one of the main interventions with which
undergoing embolization of an AVM or treatment for acute we are left is the scrupulous avoidance of activities which
stroke? Patients presenting with coagulopathy enrolled in contribute to anemia. The goal of any blood conservation
the MERCI and multi-MERCI trials underwent interventional strategy is to reduce or eliminate the exposure to allogeneic
radiology procedures for cerebral artery revascularization blood. Autologous predonation, erythropoietic support,
without pre-procedure correction of their coagulation status, acute normovolemic hemodilution, intraoperative cell
and did not demonstrate any higher rate of symptomatic salvage, induced hypotension, and the use of pharmacological
intracranial hemorrhage.28 A hemoglobin target that is most agents in addition to meticulous surgical hemostasis have
appropriate for a patient undergoing an intervention for acute all been associated with reductions in allogeneic blood
embolic stroke is unknown. Most of the work to date suggests use, yet the cost effectiveness of these strategies has been
that extremes of hematocrit are poor prognostic indicators questioned.38,39 Many of these techniques have been adopted
in acute stroke. The study by Allport et al.29 showed reduced into use in neurosurgery; however, few have been studied in
reperfusion and tissue survival with increasing hematocrit this patient population. Limited evidence appears to show
in a patient population with median hematocrit levels of that intraoperative cell salvage and perioperative autologous
39–42%. Hemodilution for the purpose of reduced viscosity transfusion is well-tolerated and offers a modest reduction
and improved flow through narrowed vessels has been shown in allogeneic blood transfusion rates during intracranial
to lack efficacy as well.30 surgery.40 The antifibrinolytics epsilon-aminocaproic acid
and tranexamic acid have been studied in the setting of
preventing rebleeding of aneurysmal acute subarachnoid
Anticoagulated Patient hemorrhage. Both agents have shown considerable reduction
Patients treated with vitamin K antagonists such as warfarin in rebleeding rates; however, high dose and prolonged use
need special care when they present for emergency neuro (>72 hours) in this population is associated with increased
surgical procedures. Whether it be intracerebral hemorrhage, risk of vasospasm and hydrocephalus negating any overall
trauma, or subarachnoid hemorrhage, these patients require benefit. These conclusions were drawn in a time when early
rapid reversal of their anticoagulation. Supplementation surgical aneurysm clipping was not recommended. Both
with vitamin K, although appropriate, does not act as epsilon aminocaproic acid and tranexamic acid have been
quickly as one might like in these situations. Replenishment re-evaluated in view of changes in surgical management of
of vitamin K dependent clotting factors with plasma has aneurysmal subarachnoid hemorrhage and have shown
long been viewed as appropriate in these patients, but positive results in reducing early rebleeding rates yet sparing
may not provide reversal in the time frame desired,31,32 the complications found in earlier work.41,42
and alternatives have been advocated.33,34 Prothrombin
complex concentrates (PCCs) provide more rapid reversal Conclusion
of vitamin K antagonists, are quick to prepare, are virally
inactivated, and do not require the volume load of plasma. It is known that in the context of neurosurgery, higher
There is, however, no evidence for improved outcomes hemoglobin levels are associated with better outcomes in
in patients treated with PCC versus plasma. The exact general. Unfortunately, maintaining these levels through
components of different PCC products vary by manufacturer, transfusion of banked, allogeneic red blood cells appears to
and different products may not be equally effective in be associated with worse outcome. As neuro-anesthetists,
restoring coagulation,35 so the clinician must be familiar we can contribute by minimizing unnecessary losses,
with the specific product stocked at one’s own institution. such as increased bleeding from venous congestion, and
Activated factor VII has also been used to rapidly reverse avoiding superfluous blood draws, although the latter issue
coagulopathy associated with vitamin K antagonists and is perhaps more applicable over the longer term in the ICU
intracranial bleeding.36 The decision as to whether one environment. Notable questions for the future include
should choose activated factor VII or PCCs is not entirely the exact role of transfusion sparing practices such as cell
58 Section 1 Clinical Hemotherapy
salvage or acute normovolemic hemodilution, and whether 8. Couture DE, Ellegala DB, Dumont AS, et al. Blood use in
or not they may be associated with improved neurological cerebrovascular neurosurgery. Stroke. 2002;33:994-7.
outcomes as compared with allogeneic transfusion. The role 9. Treggiari MM, Deem S. Which H is the most important in
of strategies to address anemia preoperatively prior to elective triple-H therapy for cerebral vasospasm? Curr Opin Crit Care.
2009;15:83-6.
neurosurgical procedures also remains to be addressed with
10. Ekelund A, Reinstrup P, Ryding E, et al. Effects of iso- and
regard to outcome. hypervolemic hemodilution on regional cerebral blood
A great deal of focus has been directed toward monitoring flow and oxygen delivery for patients with vasospasm after
modalities such as: aneurysmal subarachnoid hemorrhage. Acta Neurochir.
• Brain tissue oxygen tension 2002;144:703-12; discussion 712-713.
• Near infrared spectroscopy 11. Naidech AM, Jovanovic B, Wartenberg KE, et al. Higher
• Jugular bulb catheter sampling, to name just a few. hemoglobin is associated with improved outcome after
Indications from these monitors of inadequate regional subarachnoid hemorrhage. Crit Care Med. 2007;35:2383-9.
or global oxygenation have been used to advocate for 12. Oddo M, Milby A, Chen I, et al. Hemoglobin concentration and
transfusion, with a resultant improvement in the monitored cerebral metabolism in patients with aneurysmal subarachnoid
hemorrhage. Stroke. 2009;40:1275-81.
parameter. Increased use of these monitors may aid us in
13. Smith MJ, Le Roux PD, Elliott JP, et al. Blood transfusion
balancing cerebral requirement of blood thus optimizing
and increased risk for vasospasm and poor outcome after
transfusion. Once issues such as reduced perfusion pressure, subarachnoid hemorrhage. J Neurosurg. 2004;101:1-7.
vasospasm, pyrexia, and seizures have been addressed 14. Mayer SA, Brun NC, Begtrup K, et al. Efficacy and safety
without a satisfactory improvement in monitored parameters, of recombinant activated factor VII for acute intracerebral
one might reasonably consider transfusion. hemorrhage. N Engl J Med. 2008;358:2127-37.
As for the exact number to target, we feel that the avail- 15. He´ bert PC, Wells G, Blajchman MA, et al. A multicenter,
able evidence currently supports a hemoglobin level of randomized, controlled clinical trial of transfusion
8.0–9.0 g/dL. In the context of ongoing bleeding in the requirements in critical care. Transfusion Requirements
operating room in particular, targeting a lower number in Critical Care Investigators, Canadian Critical Care Trials
(such as 7.0 g/dL) may result in brief periods of even more Group. N Engl J Med. 1999;340:409-17.
16. McIntyre LA, Fergusson DA, Hutchison JS, et al. Effect of a
profound anemia, which appear to be clearly linked with
liberal versus restrictive transfusion strategy on mortality in
inadequate brain perfusion. It cannot be stressed strongly patients with moderate to severe head injury. Neurocrit Care.
enough, however, that these targets may need to be modified 2006;5:4-9.
or revised in the context of significant comorbidities such as 17. Leal-Noval SR, Rinco´ n-Ferrari MD, Marin-Niebla A, et al.
coronary artery disease or hypoxemia. We must continue to ransfusion of erythrocyte concentrates produces a variable
examine these issues to identify which patient groups in the increment on cerebral oxygenation in patients with severe
heterogeneous neurosurgical population will benefit from traumatic brain injury: a preliminary study. Intensive Care
the interventions that we might offer. Med. 2006;32:1733-40.
18. Zygun DA, Nortje J, Hutchinson PJ, et al. The effect of red blood
cell transfusion on cerebral oxygenation and metabolism after
References severe traumatic brain injury. Crit Care Med. 2009;37:1074-8.
19. George ME, Skarda DE, Watts CR, et al. Aggressive red blood
1. Hopf HW, Viele M, Watson JJ, et al. Subcutaneous perfusion and
oxygen during acute severe isovolemic hemodilution in healthy cell transfusion: no association with improved outcomes for
volunteers. Arch Surg (Chicago, Ill: 1960). 2000;135:1443–9. victims of isolated traumatic brain injury. Neurocrit Care.
2. Weiskopf RB, Kramer JH, Viele M, et al. Acute severe isovolemic 2008;8:337-43.
anemia impairs cognitive function and memory in humans. 20. Salim A, Hadjizacharia P, DuBose J, et al. Role of anemia in
Anesthesiology. 2000;92:1646-52. traumatic brain injury. J Am Coll Surg. 2008;207:398-406.
3. Weiskopf RB, Feiner J, Hopf HW, et al. Oxygen reverses deficits 21. Saggar V, Mittal RS. Hemostatic abnormalities in patients with
of cognitive function and memory and increased heart rate closed head injuries and their role in predicting early mortality.
induced by acute severe isovolemic anemia. Anesthesiology. J Neurotrauma. 2009;51(3):567-71.
2002;96:871-7. 22. Talving P, Benfield R, Hadjizacharia P, et al. Coagulopathy in
4. Hare GMT, Tsui AKY, McLaren AT, et al. Anemia and cerebral severe traumatic brain injury: a prospective study. J Trauma.
outcomes: many questions, fewer answers. Anesth Analg. 2009;66:55-61.
2008;107:1356-70. 23. Pasternak JJ, Hertzfeldt DN, Stanger SR, et al. Disseminated
5. Hendrickson JE, Hillyer CD. Noninfectious serious hazards of intravascular coagulation after craniotomy. J Neurosurg
transfusion. Anesth Analg. 2009;108:759-69. Anesthesiol. 2008;20:15-20.
6. Weiskopf RB, Feiner J, Hopf H, et al. Fresh blood and aged 24. Holcomb JB, Wade CE, Michalek JE, et al. Increased plasma
stored blood are equally efficacious in immediately reversing and platelet to red blood cell ratios improves outcome in
anemia-induced brain oxygenation deficits in humans. 466 massively transfused civilian trauma patients. Ann Surg.
Anesthesiology. 2006;104:911-20. 2008;248:447-58.
7. Tinmouth A, Fergusson D, Yee IC, He´ bert PC. Clinical 25. Brecknell JE, McLean CA, Hirano H, et al. Dissemi-nated
consequences of red cell storage in the critically ill. Transfusion. intravascular coagulation complicating resection of a
2006;46:2014-27. malignant meningioma. Br J Neurosurg. 2006;20:239-41.
Chapter 16 Transfusion Support in Neurosurgery 59
26. Eom KS, Kim JM, Kim TY. Disseminated intravascular coagula 35. Holland L, Warkentin TE, Refaai M, et al. Suboptimal effect of
tion in a patient undergoing removal of metastatic brain tumor. a three-factor prothrombin complex concentrate (Profilnine-
J Korean Neurosurg Soc. 2008;44:341-4. SD) in correcting supratherapeutic international normalized
27. Atzil S, Arad M, Glasner A, et al. Blood transfusion promotes ratio due to warfarin overdose. Transfusion. 2009;49:1171-7.
cancer progression: a critical role for aged erythrocytes. 36. Ilyas C, Beyer GM, Dutton RP, et al. Recombinant factor VIIa
Anesthesiology. 2008;109:989-97. for warfarin associated intracranial bleeding. J Clin Anesth.
28. Nogueira RG, Smith WS. Safety and efficacy of endovascular 2008;20:276-9.
thrombectomy in patients with abnormal hemostasis: 37. Dickneite G. Prothrombin complex concentrate versus
pooled analysis of the MERCI and multi MERCI trials. Stroke. recombinant factor VIIa for reversal of coumarin anticoagula
2009;40:516-22. tion. Thromb Res. 2007;119:643-51.
29. Allport LE, Parsons MW, Butcher KS, et al. Elevated hematocrit 38. Goodnough LT, Brecher ME, Kanter MH, et al. Transfusion
is associated with reduced reperfusion and tissue survival in medicine. Second of two parts: blood conservation. N Engl J
acute stroke. Neurology. 2005;65:1382-7. Med. 1999;340:525-33.
30. Asplund K. Hemodilution for acute ischemic stroke. Cochrane 39. Goodnough LT, Monk TG. Blood conservation in patients
Database of Systematic Reviews 2002, Issue 4. Art. No.: undergoing noncardiac surgery. Curr Opin Anaesthesiol.
CD000103. 2000;13:365-70.
31. Aiyagari V, Testai FD. Correction of coagulopathy in warfarin 40. Cataldi S, Bruder N, Dufour H, et al. Intraoperative autologous
associated cerebral hemorrhage. Curr Opin Crit Care. blood transfusion in intracranial surgery. Neurosurgery.
2009;15:87-92. 1997;40:765-71.
32. Lee SB, Manno EM, Layton KF, et al. Progression of warfarin 41. Chwajol M, Starke RM, Kim GH, et al. Antifibrinolytic therapy
associated intracerebral hemorrhage after INR normalization to prevent early rebleeding after subarachnoid hemorrhage.
with FFP. Neurology. 2006;67:1272-4. Neurocrit Care. 2008;8:418-26.
33. Ansell J, Hirsh J, Hylek E, et al. Pharmacology and management 42. Komotar RJ, Zacharia BE, Mocco J, et al. Controversies in the
of the vitamin K antagonists: American College of Chest surgical treatment of ruptured intracranial aneurysms: the
Physicians Evidence-Based Clinical Practice Guidelines (8th First Annual J. Lawrence Pool Memorial Research Symposium:
Edition). Chest. 2008;133:160S-98S. controversies in the management of cerebral aneurysms.
34. Levy JH, Tanaka KA, Dietrich W. Perioperative hemostatic Neurosurgery. 2008;62:396-407.
management of patients treated with vitamin K antagonists.
Anesthesiology. 2008; 109:918-926.
Maternal, Fetal and Neonatal Transfusion Practice
Intrauterine Transfusion The blood for possible transfusion must be typed and cross-
matched against the mother’s serum to help rule out any other
Intrauterine transfusion (IUT) is a process where blood is possible hemolytic antibodies. Additionally, the unit of red
transfused to the fetus, in order to provide a better chance blood cells should be cytomegalovirus-negative, irradiated,
of survival from life threatening conditions associated with less than 5 days old, and with a hematocrit level between 75
severe anemia, including Rh isoimmunization. The incidence and 80%. This can require several hours. The large variety
of Rh negativity in India is about 1–5% and the rate of Rh of equipment, such as transfusion tubing, blood warmer,
sensitization to be approximately 0.79% of live births. heparinized syringes, and an accurate machine to obtain a
Intrauterine infusion of red blood cells (RBCs) into rapid hematocrit value, must be prepared and calibrated.
the fetus is one of the most successful means of in utero The fetus should be monitored with continuous fetal
therapy. This has contributed to survival of several severely monitoring for the ensuing 4 hours (both during the procedure
anemic fetuses. Although universal use of Rh (D) immune and immediately after because decelerations of the fetal heart
globulin has dramatically reduced the need for intrauterine rate are common and must be managed cautiously).
transfusions, the procedure continues to be an essential
modality for treatment of severe fetal anemia from a variety suggested reading
of causes. IUT is indicated for fetal anemia resulting from
1. Basu S, Kaur R, Kaur G, Jain S. Severe hemolytic disease of
varied causes like blood group isoimmunization, parvovirus
newborn due to non Rh D Antibody. Indian J Pediatr. 2011.
B19 infection, fetomaternal hemorrhage, twin anemia- 2. Basu S, Kaur R, Kaur G. Hemolytic disease of the fetus and
polycythemia syndrome in complicated monochorionic twin newborn: Current trends and perspectives. Asian J Transfus
pregnancies, hemoglobinopathies, dyserythropoiesis, red- Sci. 2001;5:3-7.
cell enzymopathy like G6PD deficiency. 3. Furukawa K, Nakajima T, Kogure T. Example of a women
Intrauterine transfusion of RBCs is indicated to prevent with multiple intrauterine death due to anti M who delivered
fetal death from severe anemia. The procedure is generally a live child after plasmapheresis. Exp Clin Immunogenet.
limited to fetuses between 18 and 34 weeks of gestation 1993;10:167-7.
4. Guidelines for blood grouping and antibody screening in the
because of technical limitations before 18 weeks and
antenatal and perinatal setting. Australian and New Zealand
excessive fetal risk compared with delivery after 34 weeks. The Society of Blood Transfusion Ltd. 3rd edn. 2007.
introduction of measures to both predict the severity of fetal 5. Guidelines for blood grouping and red cell antibody testing
disease and treat it has greatly reduced perinatal mortality during pregnancy. Transfus Med. 1996;6:71-4.
rates and augmented survival rates. 6. Jeon H, Calhoun B, Pothiawala M, Herschel M, Baron BW.
Should titers rise to 1:16 or higher, fetal assessment is Significant ABO haemolytic disease of the newborn in a
indicated via noninvasive methods. The only definitive group B infant with a group A2 mother. Immunohematology.
means of diagnosing fetal anemia and acidosis is via fetal 2000;16:105-8.
blood sampling (FBS), also known as cordocentesis or 7. Kennedy MS. Perinatal Issues in Transfusion Practice In
American Association of Blood Banks Technical Manual. 16th
percutaneous umbilical blood sampling (PUBS),
64 Section 1 Clinical Hemotherapy
edn. American Association of Blood Banks, Bethesda: 2008. p. 14. Thakral B, Agrawal SK, Dhawan HK, Saluja K, Dutta S, Marwaha
633. N. First report from India of haemolytic disease of the newborn
8. Marwaha N, Dhawan HK, Thakral B, Kaur R, Basu S, Parmar by anti c and anti E in Rh (D) positive mothers. Haematology.
V. Severe ABO hemolytic disease of the newborn with a 2007;12:377-80.
positive direct antiglobulin test. Indian J Pathol Microbiol. 15. Tondon R, Kataria R, Chaudhry R. Anti - M: Report of two cases
2009;52:292. and review of literature. Asian J Transfus Sci. 2008;2:81-3.
9. Moise KJ Jr. Non anti-D antibodies in red cell alloimmunisa 16. Toy PT, Reid ME, Papenfus L, Yeap HH, Black D. Prevalence
tion. Eur J Obstet Gynecol Reprod Biol. 2000;92:75-81. of ABO maternal-infant incompatibility in Asians, Blacks,
10. Mollison PL, Engelfret CP, Contreras M. Hemolytic disease Hispanics and Caucasians. Vox Sang. 1988;54:181-3.
of the fetus and the newborn. In: Mollison PL, Engelfret CP, 17. Voak D, Mitcheli R, Bowell P, Letsky E, de Silva M, Whittle M.
Contreras M, eds. Blood transfusion in clinical medicine, 11th Guidelines for Blood grouping and red cell antibody testing
edn. Oxford Blackwell Science; 2005. p. 508. during pregnancy. Transfus Med. 1996;6:71-4.
11. Pepperell RJ, Barrie JU, Fliegner JR. Significance of red cell 18. Walker RH. Relevance in the selection of serologic tests for the
irregular antibodies in the obstetric patient. Med J Aust. obstetric patient. In: Garratty G, ed. Hemolytic disease of the
1997;2:453-6. newborn. Arlington, VA: American Association of Blood Banks;
12. Pollock JM, Bowman JM. Anti-Rh (D) subclasses and severity of 1984. p. 173.
Rh haemolytic disease of the newborn. Vox Sang. 1990;59:176-9. 19. Weinstein L. Irregular antibodies causing haemolytic disease
13. Prasad MR, Krugh D, Rossi KQ, O'Shaughnessy RW. Anti D in Rh of newborn: a continuing problem. Clin Obstet Gynaecol.
positive pregnancies. Am J Obstet Gynecol. 2006;195:1158-62. 1982;25:321-32.
Antenatal Screening of
Thalassemia
Reena Das
19
Hemoglobin (Hb) is a fascinating molecule which binds fetuses after appropriate genetic counseling, being much
oxygen in the lungs and delivers it in a precise and orderly cheaper, is a cost effective measure to control TM.4
manner to the tissues. Human Hb consist of four heme groups Since most marriages in India are arranged by the parents,
and four globin chains (2 α and 2 globin chains). Normal marriages are prevalent within the same castes and this leads
human adults have predominantly (98%) adult Hb (ΗbΑ; to higher frequency of thalassemia in certain communities.
α2β2) and small (<2%) amounts of HbA2 (α2δ2,) and fetal Hb Preventive strategies include a) Population screening at either
(HbF; α2γ2) of <1%.1 Reduced synthesis of one or the other premarital or preconception stage, b) genetic counseling and
type of globin chains results in decreased hemoglobinization c) prenatal diagnosis. Earlier screening for βTT was carried
of the erythroid cells and an excess of the non-affected chains out in schools and colleges for determining the prevalence
which continue being synthesized normally, but cannot be of βTT in the general population. However, following up
effectively removed and become harmful for the cell. This with genetic counseling does not yield productive result
group is known under the general term “thalassemia”, further since many years may lapse between conveying the report
subdivided in alpha– and beta- form, according to the chain and planning the birth of their child. Presently a consensus
whose synthesis is reduced. Defects in the structure of globin has evolved that screening the antenatal women during the
chains are called Hb variants or hemoglobinopathies.2 first antenatal visit is the most cost effective measure of
Due to the autosomal recessive pattern of inheritance of screening the population. All women who are found to be
beta thalassemias, both the parents of an affected child with carriers are advised to get their spouse screened. This strategy
thalassemia major (TM) are obligate carriers of one defective has been found to be most effective as the concern to have a
beta globin chain and are known as β thalassemia trait (β normal healthy baby is a universal phenomenon. The other
TT). Individuals with βTT are apparently normal and being group where targeted screening yields good return is ‘cascade
unaware that they are carriers, they constitute the reservoir or extended family screening’ in family members who have
of the disease. The diagnostic hallmark to identify a βTT an index case of thalassemia major or severe thalassemia
is by quantitation of HbA2. Patients with a clinical severity intermedia. This subgroup is also initiated to the process of
intermediate between βTT and TM are called thalassemia screening and the frequency of finding βTT’s is much higher
intermedia (TI) and they show marked clinical and molecular than the general population screening.5
heterogeneity. Patients with TM present in infancy with We are indeed fortunate that a screening strategy exists
progressive pallor and hepatosplenomegaly and require by way of a simple blood test to identify individuals who are
frequent three weekly intervals of blood transfusion to carriers of a grave genetic disorder such as beta thalassemia.
sustain a reasonable quality of life. If not treated with regular A through hematologic evaluation including complete blood
blood transfusions patients with TM die within their first few count (CBC) using automated blood cell counters, which are
years. Also children who receive regular blood transfusions readily available, can be used as a basic screening strategy.
develop marked secondary iron overload and require regular Almost universally all individuals who are βTTs show
chelation therapy to reduce the deposition of siderotic iron. hypochromic microcytosis (usually MCV will be <80 fl and
The cost of treatment of a child with TM is approximately Rs. MCH <27 pg) with relative erythrocytosis (increased RBC
1.5 lakh/year. Lifelong treatment is expensive and causes counts) and this is used to screen the population or specific
immense financial strain to the patient and the family. Bone target groups such as antenatal women. Since the availability
Marrow transplant from a HLA matched sibling is the only of cation exchange high performance liquid chromatography
cure and costs Rs 7–10 lakhs.3 Therefore, screening for βTT (HPLC) from BioRad, this has become the screening method
and antenatal diagnosis and selective termination of affected to analyze the HPLC pattern due to the speed, convenience
66 Section 1 Clinical Hemotherapy
as well as accuracy of the results. Quantitation of HbA2 of ≥4% problems encountered are occasional non-amplification
is the diagnostic hallmark to identify a βTT. Any individual of primers. Most centers are performing VNTRs to exclude
identified to be a βTT should be advised to get a screening maternal contamination. Though our country has the
done of the partner before conception of a child. In the event expertise and technical knowhow and many more centers
of finding any abnormal peak on the HPLC, a hemoglobin throughout the country are required to tackle the burden
electrophoresis should be carried out before reporting the of the disease. Offering prenatal diagnostic services is the
result of HPLC. This is due to the fact that many abnormal most effective way of controlling thalassemia. The problems
peaks can be found in the same location such as Hb D Iran and encountered in our country include, a vast population, no
Hb E overlap in the region of HbA2 and Hb electrophoresis national screening program, unawareness amongst the
is required to differentiate the two conditions. Though the medical fraternity and shortage of trained centers and staff.
regional prevalence’s of the abnormal hemoglobins are Many patients present to the obstetricians and pediatricians
segregated, the population migration as well as inter caste late in their pregnancy to work up for the antenatal testing.
marriages is blurring such differences.6 DNA analysis for (Unpublished data) A few subjects are ‘silent’ carriers who
specific beta gene mutations is commonly carried out by pose to be diagnostic difficulties in screening programs and
either ARMS-PCR or by Reverse Dot Blot analysis. Many of it is important to keep this fact in mind during the process of
the specific abnormal hemoglobin analysis are carried out by genetic counseling.8
specific PCR-RFLP such as Hb D Punjab, Hb E, Hb S and Hb
O Arab.7 References
More than 2200 children with TM are on a regular blood
transfusion program in the Punjab region and unaccounted 1. Higgs DR, Thein SL, Wood WG. Human haemoglobin. In:
for number of affected children with TM have died due to the Weatherall DJ, Clegg B, eds. The thalassaemia syndromes. 4th
edn. Oxford, England: Blackwell Science, 2001.
complications of the disease. The department of Hematology
2. Weatherall DJ, Clegg JB, editors. The thalassemia syndromes.
at PGIMER, Chandigarh has been providing diagnostic 4th edn. Oxford: Blackwell Science, 2001.
support to the patients of the region since 1993 and holistic 3. Verma IC, Bijarnia S. The burden of genetic disorders in India
management of children with TM is provided by the Hemato- and a framework for community control. Community Genetics.
oncology unit, Pediatrics department. Since the last five years, 2002;5:192-6.
with the help of the Obstetrics and Gynecology department 4. Garewal G, Das R. Spectrum of β thalassemia mutations
routine complete screening (comprising of automated blood in north India. International Journal of Human Genetics.
cell counts and HPLC for Hb subtyptes) is being provided 2003;3:217-9.
5. Garewal G, Das R, Kaur J, Marwaha R K, Gupta I. Establishment
to all antenatal women referred to the department. Prenatal
of Prenatal Diagnosis for β-Thalassemia: A step towards its
diagnosis has been carried out on over 700 pregnancies in the Control in a Developing Country. Annals of Human Biology.
region. Many transfusion centers such as those in PGIMER, 2005;32(2):138-44.
Chandigarh; GMCH, Sector 32, Chandigarh; GMCH, Patiala 6. Madan N, Sharma S, Sood SK, Colah R, Bhatia HM. Frequency
and GMCH, Amritsar are among the many centers in Punjab of b-thalassemia and other hemoglobinopathies in India.
who are committed to provide safe blood to the patients with Indian Journal of Human Genetics. 2010;16:16-25.
TM and TI. Due to the marked health burden to our limited 7. Colah RB, Gorakshakar AC, Nadkarni AH. Invasive
health resources and cost involved only 10% of the patients are and non-invasive approaches of prenatal diagnosis of
being optimally treated with regular blood transfusions with haemoglobinopathies: Experiences from India. Indian Journal
of Medical Research. 2011;134:552-60.
safe blood and iron chelation in India. Therefore, aggressive
8. Garewal G, Das R, Awasthi A, Ahluwalia J, Varma N. Marwaha
preventive strategies are needed to be taken to decrease the RK. The clinical significance of the spectrum of interactions of
prevalence of TM. CAP+1 (A®C), a silent beta globin gene mutation, with other
We have been involved in prenatal diagnosis of >700 beta thalassemia mutations and globin gene modifiers in
cases by using ARMS-PCR, RFLP and DNA sequencing. The North Indians. Eur J Haematol. 2007;79(5):417-21.
Neonatal Transfusion Practice
A Surekha Devi
20
Sick neonates are one of the most heavily transfused groups of for top-up transfusion may be irradiated at any time up to 14
patients in modern medicine. The transfusion requirements days after collection, and stored for 14 days from irradiation.
of the neonate are recognized as unique. Like any therapeutic Platelets transfused in utero to treat alloimmune throm
decision, neonatologists should decide to transfuse based on bocytopenia and platelet transfusions given after birth
the risks, benefits and alternatives to transfusion. Recipients to infants who have received either red cells or platelets
run the risk of both over-transfusion and under-transfusion. in utero should be irradiated.
Administration of blood components is done on a per weight All granulocytes should be irradiated for patients of any
basis. Guidelines are derived from consensus, with several of age and transfused as soon as possible after irradiation.
them demonstrating appropriateness in published studies.
Blood and blood component specifications: Indications of Irradiation of Cellular
Blood Components
General recommendations (fetuses,
neonates, infants and children) • Intrauterine transfusions (IUT)
• Exchange transfusion (ET) of red cells
• Top-up transfusions after IUT
Donors • Directed donations from a first- or second-degree relative
Components to children under 1 year of age must be prepared • Immunodeficiency
from blood donated by donors who have given at least one
previous donation within the past 2 years, which was negative Plasma and Platelet Compatibility
for all mandatory microbiological markers.
Platelets should be ABO and RhD identical with the recipient,
if not available compatible components lacking high titer
Special Needs anti-A or anti-B should be transfused to group A or B
Leukocyte depletion: All components other than granulo recipients.
cytes concentrates should be leukocyte depleted (not more Group AB FFP is recommended for transfusion in the first
than 5 × 106 leukocytes per unit) at the time of manufacture. year of life.
Cytomegalovirus (CMV): The guidelines of the UK trans
fusion services state that blood transfused in the first year of Administration
life should be CMV negative. Fetuses, infants weighing under
All components should be transfused through a standard
1.5 kg, immunodeficient patients and stem cell transplant
blood giving set with a screen filter (170–220 microns). Where
recipients are at risk of transfusion transmitted CMV. In an
small volumes are drawn into a syringe an appropriate filter
emergency, where CMV-seronegative blood components are
must be used. Microaggregrate filters are not required for
not available, transfusion of leukodepleted components is an
leukoreduced components.
acceptable.
Irradiation: Cellular blood components are irradiated to
a dose of 25 Gy. For IUT and large volume transfusion, e.g.,
Pretransfusion Testing
exchange transfusion, the component should be used within Samples from both mother and neonate should be obtained
24 hours of irradiation and within 5 days of donation. Red cells for initial ABO and RhD group determination.
68 Section 1 Clinical Hemotherapy
Intrauterine Transfusion
Transfusions of preterm infants
Intrauterine red cell transfusion is indicated to correct fetal
anemia caused by red cell alloimmunization (RhD, Rhc and • All NICUs should have written, consensus based guidelines
K), or for fetal parvovirus infection. Intrauterine platelet that underline transfusion policy. Guidelines should be
transfusions are indicated to correct fetal thrombocytopenia regularly updated in-line with the best available evidence
caused by platelet alloimmunization. Aims of IUT are and their implementation should be regularly assessed by
• To prevent or treat fetal hydrops before the fetus can be audit.
delivered • Most preterm infants with birth weight of less than 1000 g
• To enable the pregnancy to advance to a gestational age will require RBC transfusion, majority of infants with birth
that will ensure survival of the neonate weight greater than 1500 g do not require RBC transfusion.
– Starting transfusion as late and safely possible but • Minimizing donor exposure is important in reducing the
before hydrops develops risk of transfusion-transmitted infections. Infant who is
– Maximizing the intervals between transfusion by likely to require recurrent RBC transfusion should have
transfusing large volume of red cells pedipack assigned to them.
• Blood loss due to repeated phlebotomy is important cause
of anemia of prematurity requiring transfusion support in
Red Cell Preparations low birth infants.
• Be group O or ABO identical with fetus, RhD negative, • The aim of top up transfusion is to restore or maintain
K-negative. In case of hemolysis due to maternal anti-c, adequate tissue oxygen delivery without a marked increase
RhD positive, c-negative blood is given in oxygen consumption.
Chapter 20 Neonatal Transfusion Practice 69
• Erythropoietin (EPO) cannot be recommended as the re transfusions up to four postnatal months if atypical mater
cent Cochrane Database meta-analysis (2005) showed an nal antibodies present).
increased risk of retinopathy of prematurity with early • Be 35 days old or less (if in SAG-M or similar additive
EPO treatment. solution) or 28 days old or less (if in CPD).
• Have a hematocrit of 0.50–0.70.
Neonatal transfusions • Be irradiated if appropriate.
• Be infused in a volume of 10–20 mL/kg.
• Be aliquotted donations (pedipak) from a single unit dedi
Exchange Transfusion (ET)
cated to one infant.
Exchange transfusion is used to manage severe anemia
at birth, in the presence of heart failure and to treat severe Guidelines for Administration of Red Cells
hyperbilirubinemia, caused by HDN.
Aim is to remove both the antibody-coated red cells • Transfusion thresholds for red cell transfusion
and the excess bilirubin. It should be done by staff who are − Anemia in the first 24 hour—Hb 12 g/dL
experienced in the procedure. − Acute blood loss/cumulative blood loss in 1 week—10%
Single volume exchange: Exchanging the estimated BV
volume of the baby’s blood in a single volume exchange will − Neonate receiving intensive care—Hb 12 g/dL
remove 75% of red cells. − Chronic oxygen dependency—Hb 11 g/dL
Double volume exchange (160–200 mL/kg) removes 50% − Late anemia, stable patient—Hb 7 g/dL
of available intravascular bilirubin. • Surrogate markers of anemia include respiratory irregu
larity, tachycardia, poor weight gain, lethargy, poor suck
and increased blood lactate levels.
Component and Procedure Specification
• Patients with higher oxygen extraction ratio (>40%)
• Be group O or ABO compatible with maternal and neonatal
plasma, RhD negative (or RhD identical with neonate). Platelets for Neonatal Transfusions
• Be negative for any red cell antigens to which the mother
has antibodies. • Be ABO identical or compatible, Rh identical or compatible.
• Be IAT-cross-match compatible with maternal plasma. • Be HPA compatible in infants with alloimmune thrombo
• Be 5 days old or less. cytopenia.
• Be collected into CPD anticoagulant. • Be produced by standard techniques (plateletpheresis).
• Be CMV seronegative. • Be irradiated if appropriate.
• Be irradiated and transfused within 24 hours of transfus • Be infused in a volume of 10–20 mL/kg.
ion. Irradiation is essential if the infant has had a previous • Administration of platelets:
IUT and is recommended for all ETs. − Preterm or term neonate, with bleeding—50 × 109/L
• Have a hematocrit of 0.50–0.60. − Sick preterm or term neonate, not bleeding—30 ×
• To warm the blood product prior to transfusion. 109/L
• Volume transfused is 80–160 mL/kg for a term infant and − Stable preterm or term neonate, not bleeding—20 ×
100–200 mL/kg for a preterm infant. 109/L
ABO Hemolytic Disease of the Newborn (HDN) Fresh Frozen Plasma for Neonatal
• Group O blood compatible with the maternal plasma,
Transfusions
should be used for transfusion. • Be group AB, or compatible with recipient’s ABO red cell
• If an ET is required in ABO HDN, group O red cells with antigens.
low titer plasma anti-A and anti-B, or with group O red • Be infused in a volume of 10–20 mL/kg.
cells suspended in AB plasma should be used. • Virus inactivated plasma should be used for treatment of
patients with inherited coagulation deficiencies.
Red Cells for Top-up Transfusions
in Neonates Cryoprecipitate for Neonatal Transfusions
• Be ABO compatible with mother and infant, and infant’s Common indication for cryoprecipitate is to restore fibrinogen
RhD group. levels in patients with acquired hypofibrinogenemia in DIC
• Be IAT compatible with maternal plasma (if available) or and massive transfusions.
neonate’s plasma for first transfusion (and subsequent • Should be transfused only ABO-compatible cryoprecipitate.
70 Section 1 Clinical Hemotherapy
• Cryoprecipitate separated from AB plasma can also be • Whole blood contains a significant amount of relatively
given fresh plasma containing all coagulation factors other than
• Dose is 5 mL/kg which raises fibrinogen level to about FVIII and FV
0.6–1.0 g/L • Red cells in additive solution are not advised for priming
• Platelet transfusions are given to maintain platelet count
Granulocytes for Neonatal Transfusions above 100 × 109/L and FFP given to manage excessive
bleeding.
• Be ABO and RhD compatible with the recipient. • Fibrinogen level should be maintained above 0.8–1.0 g/L
• Dose is 1–2 × 109 granulocytes/kg. with cryoprecipitate 5 mL/kg.
• Irradiated to a dose of 25 Gy prior to administration. • Antithrombin infusion is given to keep the levels adequate
• Be CMV seronegative. for heparin function.
• Recommended two or more daily infusions of an appro • A normal hematocrit (of around 0.45) has been associated
priate dose. with increased risk of clotting in the circuit, which may be
reduced by lowering the hematocrit to 0.35.
Neonates are the longest living survivors of blood trans
Transfusions in necrotizing fusion and are at risk for the long-term consequences of
enterocolitis (NEC) transfusion transmitted infections. Every effort has to be
made to reduce transfusions and donor exposure in this most
• Be transfused with saline washed red cells or red cells in
vulnerable group of transfusion recipients.
SAG-M as this is relatively plasma-free saline washed red
cells or
• ET may be necessary if a patient with NEC develops he suggested reading
molysis, and T-activation is likely cause. 1. JMO’ Riordon, et al. Transfusion of blood components to
• Platelets, FFP and cryoprecipitate should be administered infants under four months: Review and Guidelines. Irish Medi
only when clearly indicated. cal Journal Supplement June 2007, Volume 100, Number 6.
2. NA Murray, IAG Roberts. Neonatal transfusion practice. Arch
Dis Child Fetal Neonatal Ed.2004; 89:F101-F107.
Transfusions in Extracorporeal 3. Neonatal transfusion recommendations at RCH, Melbourne,
Australia. Contents authorized by Webmaster. Last updated
membrane oxygenation (ECMO) 12 Dec, 2012.
This is highly specialized respiratory support, infants are 4. Strauss RG. Current issues in neonatal transfusion. Vox Sang.
anticoagulated with heparin, and require regular monitoring 1986;51(1):1-9.
5. Susan D.Roseff. Pediatric transfusion. A physcian’s Handbook.
of coagulation parameters and platelet count.
2nd edn. 2006. AABB, Bethesda, Maryland, USA.
• Following the initial ‘coating’ prime with albumin, priming 6. Transfusion guidelines for neonates and older children.
with whole blood, packed red cells or packed cells and FFP Blackwell Publishing, British Journal of Haematology. 2004;
may be indicated 124:433-53.
• Blood should be as fresh as possible not >5 days old
Transfusion Practice in Transplantation
Transfusion Practice in
Bone Marrow Transplantation
and HLA Matching
M Joseph John
21
Hematopoietic stem cell transplantation (HSCT) is today infected cells. Activated helper T-cells stimulates appropriate
widely used to treat hematologic, immune and genetic B-lymphocytes to produce an antibody.
diseases. The annual rate of allogenic bone marrow trans These antigens determine whether an organ would be
plants now exceeds 60,000 world wide and more than 1200 accepted or rejected when transplanted into another indivi
in India [as per the ISCTR data (Indian Stem Cell Transplant dual.
Registry data)]. In thalassemia, BMT now provides a cost HLA typing can be done by serological or DNA PCR based
effective alternative to transfusion/chelation therapy with methods. Of late the PCR based test is being done. Before the
long-term transfusion and iron overload free life. availability of DNA based 10 antigen typing was available, 6
Hematopoietic stem cells (HSCs) have the potential of antigen typing with serological method was being done for
indefinite self renewal, proliferation and differentiation into AB and DR. However, now with the 10 antigen typing HLA-A,
different cell lineages. Stem cells are found in the bone mar B, C, DR and DQ up to the allelic resolution can be done. This
row, cord blood and a small number in the peripheral blood. helps in choosing the most appropriate family donor or a
matched unrelated donor.
HLA Typing HSCT differs from many other solid organ transplant for
the fact that it can be done across the blood groups even for
Major histocompatibility complex (MHC) is located in the
major, minor and major-minor mismatches. This is essentially
short arm of chromosome 6. Human leukocyte antigens are
because blood group antigens are not expressed on the stem
the gene products of MHC. They are all cell surface antigens.
cells and once the engraftment occurs, the donor stem cells
Class I: HLA A, B and C. They are located farthest from the will produce the red blood cells of the donor blood group. In
centromere. Present in all nucleated cells except mature short if a B group patient is transplanted with A group, post-
trophoblast and neural tissue. transplant that person would become A group.
Class II: HLA, D-DR, DP and DQ. Located closest to the
centromere. Present on B lymphocytes, monocytes, macro Transfusion Practices
phages, dendritic cells, endothelial cells, Langerhans cells
and activated T lymphocytes. It is important to consider certain principles and transfusion
practices to minimize the complications related to trans
Class III: Serum proteins, complement, TNF, properidin.
fusion of blood components in the peri-transplant period.
They separate Class I and II in their location.
Hematopoietic stem cell transplant (HSCT) patients often
Function of HLA: HLA antigens restrict and therefore regulate require intensive blood component support. Transfusion
the immune response in a highly specific way. The function of may be complicated by transfusion transmitted infection
antigen presenting cells, is to incorporate antigen, to process (TTI)—both viral and bacterial, transfusion-associated
it, and to present it either intact or fragmented in association (TA)-GvHD, febrile non-hemolytic transfusion reactions
with self HLA so that it can be recognized by T-lymphocytes. (FNHTR) and transfusion-related acute lung injury (TRALI).
This dual recognition generates cytochemical signaling which Alloimmunization (AI) to red cell antigens may cause
results in the proliferation of cells subsets of T-lymphocytes, difficulties in selecting compatible blood while AI to the
activated cytotoxic T-lymphocytes subsequently attack all human leukocyte antigens (HLA) present on platelets may
cells expressing self HLA\foreign antigen combination of their cause refractoriness to subsequent transfusions of randomly-
surface. This is an efficient mechanism for destroying viral selected platelets.
72 Section 1 Clinical Hemotherapy
Key Points to Remember while Transfusing is currently the recommended method for TA-GVHD
prevention. Leukodepletion by current filtration technology
the Blood in a Transplant Patient (Pre or Post)
is inadequate for this purpose.
• Standard blood bank practices TA-GVHD occurs when donor lymphocytes from
• Use a leukodepletion filter transfused blood engraft in the recipient and mount an
• Irradiate the blood before transfusion immune response. There appears to be a particular risk
• Choosing the appropriate product in case of incompati when donor and recipient share a particular haplotype,
bility. as occurs within families or in populations with restricted
haplotypes. Under certain circumstances, these cells engraft
Importance of Using Leukodepleted Products and proliferate in the patient. Interaction between donor T
lymphocytes and recipient cells carrying either class I or II
Transfused leukocytes cause alloimmunization (Table 1) to HLA antigens results in cellular damage which may be natural
HLA Class 1 antigens (HLA AI) in a proportion of patients. This killer (NK) cell mediated. The risk is highest in patients who
may be manifested clinically as febrile non-hemolytic are immunosuppressed. Typically TA-GVHD occurs 7–14
transfusion reactions (FNHTR), although these may also days post transfusion with clinical features of fever, skin
be caused by antibodies to neutrophils, platelets or plasma rash, hepatitis, diarrhea and pancytopenia. Although the
proteins and cytokines which accumulate in stored blood incidence is approximately 1%, it is fatal in more than 90%
components, especially packed red blood cells. patients.
Table 2: Selecting appropriate blood products for recipients of For minor ABO mismatch use red cells of the donor type,
ABO/Rh mismatched stem cell transplants i.e. group O throughout.
Give platelets and plasma of recipient type until recipient-
Donor Recipient Red cells Platelets FFP
type red cells are no longer detected.
Major ABO A O O A A For major and minor ABO mismatch use group O red
incompatibility B O O B B cells until recipient ABO antibodies are undetectable and the
antiglobulin test is negative and then switch to donor type.
AB O O AB AB
For platelets and plasma use group AB until recipient red
AB A O,A AB AB cells are undetectable.
AB B O,B AB AB Following graft rejection, revert to recipient-type red cells
Minor ABO O A O A A and platelets.
incompatibility O B O B B
O AB O AB AB
suggested reading
A AB O,A AB AB 1. EBMT Hand book on Stem cell transplantation. 2008. Chapter
7. Transfusion policy.
B AB O,B AB AB 2. Guidelines on gamma irradiation of blood components for the
Bidirectional ABO A B O AB AB prevention of transfusion associated graft versus host disease.
incompatibility BCSH Blood transfusion task force. Transfusion medicine,
B A O AB AB
1996;6:261-71.
3. James L Gajewski, et al. A review of transfusion practice before,
until recipient ABO antibodies are undetectable and the during, and after hematopoietic progenitor cell transplantation.
antiglobulin test is negative. Give platelets and plasma from Blood. 2008;112:3036-47.
donors of the recipient’s ABO type until recipient red cells are
no longer detected.
Transfusion Practice in Renal
Transplants Recipients
Unlike the B lymphocyte mediated hyperacute rejection, During the pre-cyclosporin era, studies suggested that
this reaction is mediated through alloreactive T cells.2 non-transfused renal graft recipients were at higher risk for
Activated cytotoxic T cells infiltrate the graft and trigger an graft rejection as compared to those transfused recipients5,6
immune response. They can induce graft injury by inducing (Table 1).
apoptosis and by secreting cytotoxic proteins (perforin and Subsequently, many studies attempted to define the
granzyme B). Pre-transplant assessment for the presence or optimal dose and timing for the transfusion effect. With
absence of alloantibodies and T cell activities to HLA antigens the introduction of cyclosporin in the early 1980s, leading
reduces the risk of acute rejection. to improved renal graft and patient survival, the beneficial
role of blood transfusions and HLA matching was again
Humoral Rejection questioned.18 Meanwhile, some preliminary trials had shown
Outside of the hyperacute rejection state, humoral rejection the use of per-protocol transfusion: matched pre-transplant
can still occur although less frequently than cell mediated blood transfusion (motifs) or donor-specific transfusion
acute rejection.3 Humoral rejection is characterized by B (DST) to be beneficial. The evidence supporting the
lymphocytes injuring the allograft through immunoglobulin effects (positive, negative, neutral) of pre-transplant blood
and complement activities.1 transfusion, regardless of therapeutic or protocol transfusion,
in renal transplantation is still not well-established. It is
Chronic Rejection unclear whether the benefits, if any, of pre-transplant blood
transfusion may be due to the modulation of immune
The definition of chronic rejection is ambiguous, and is
response in which tolerance is induced.
sometimes recognized as chronic allograft nephropathy or
There are two keys questions that need to be answered for
any immunological responses that results in slow loss of graft
understanding of effect of transfusion on renal transplant.
function with histopathological processes: tubular atrophy,
Question 1. Do red blood cell transfusions prior to renal
interstitial fibrosis, and fibrous intimal thickening of arteries.4
transplant impact allograft rejection/survival and what is
the magnitude of that effect relative to other factors (e.g.
Evolution of Transfusion in Renal pregnancy, prior transplantation)?
Transplantation In summary, prior transplant may induce increase in
High-volume therapeutic use of blood transfusion was rejection and worsening graft survival or no significant
originally used in attempts to maintain ESRD patients who changes may occur but benefits are unlikely to result from
were anemic with red cell mass ranges of 20–25%. Due these outcomes. Transfusions may be related to decreasing
to concerns with transfusion-induced infections such as rejection and benefiting graft survival or no significant
hepatitis and the production of anti-HLA antibodies resulting changes may occur but it is unlikely that these outcomes
from the exposure to blood products, efforts were made would worsen as a result of transfusion (Table 1). Prior
in the 1970s to avoid the use of blood transfusions in renal pregnancy had very scant data and could not be well assessed
transplant recipients.3 (Table 2).
Table 2: Impact of prior transplant and pregnancy related to transfusion on rejection graft and patient survival
Study , year Anaylsis type Outcome Multivariate results Significant effect
Impact of previous transplant
Reed A, 1991 Poisson Rejection episodes NR, NA
N=12715 P=0.40
Sanfilippo F, 1986 Cox Irreversible graft RR 1.409, Transplant worsens
N=381116 rejection P=0.0002 rejection
Sanfilippo F, 1986 Cox Irreversible graft RR 1.884, Transplant worsens
N=381116 rejection P=0.0006 rejection
Impact of pregnancy
Reed A, 1991 Poisson Rejection episodes NR, Pregnancy benefits
N=12715 P=0.017 rejection
78 Section 1 Clinical Hemotherapy
Question 2. Is any such impact of red blood cell transfusions and B blood group antigen. These antibodies may cause
on renal transplant outcomes altered by variables such as: hyperacute/acute humoral rejection of the organ due to
• The number of transfusions, the number of units of blood, endothelial damage because A and B antigens are expressed
and/or the number of donors (Table 3). on the vascular endothelium. ABOi exists in approximately
• The use of leukocyte depleted blood (Table 4). 35% donor-recipient pairs by virtue of ABO blood group
distribution in general population. The A2 blood group
HLA and ABO Sensitization and has reduced expression of A antigen on their RBCs and
endothelium and, therefore, group A2 donors are preferred
Desensitization in Renal Transplantation over group A1 donor for group O or B recipients in kidney
Despite a marked increase in the number of living donor transplantation with very low-risk for graft rejection. Minor
allografts (LDA) performed annually, many potential incompatibility occurs where the organ donor has naturally
recipients with otherwise suitable donors are relegated to occurring ABO antibodies against the recipient.
the ever expanding deceased donor waiting list secondary
to either: Preformed human leukocyte antigen (HLA) anti Current Management/Treatment
bodies. These are acquired via pregnancy, transfusion, or
prior transplant or ABO blood group incompatibility (ABOi). Most published reports on ABOi solid organ transplantations
If not adequately removed, the presence of such antibodies suggest removal of anti-A or anti-B antibodies in conjunc
is likely to result in severe antibody mediated rejection (AMR) tion with immunosuppressive treatment with drugs such as
and early graft loss. tacrolimus and Mycophenolate mofetil (MMF); and mono
Patients with a willing but HLA- or ABO- incompatible clonal antibodies like Daclizumab and Rituximab. Rituximab
donor may wait for a deceased donor organ or receive a living is effective in B cell ablation but does not affect plasma cells.
donor organ through desensitization, kidney paired donation Other immunotherapy modalities including intravenous
(donor swap) or a combination of both. The choice between immunoglobulins (IVIG) and antithymocyte globulins (ATG)
desensitization and kidney paired donation for an individual have important roles in the transplant process.
recipient depends upon the likelihood of finding a suitable
match through kidney paired donation and the chances of Rationale for Therapeutic Apheresis
successful desensitization. The increased risk of hyperacute
There are no controlled clinical trials using TPE in ABOi solid
antibody mediated rejection (AMR) and subsequent allograft
organ transplantations. Due to past experiences of hyperacute
loss when transplanting against either preformed HLA
and acute rejection of ABOi solid organs, TPE has been used as
antibodies or ABOi has engendered a general avoidance
an adjunct therapy to reduce anti-A or anti-B antibody titres in
of this practice and started using various desensitization
the peri-transplant period, thus preventing hyperacute/acute
protocols designed to reduce the amount of pre-existing
rejection and improving graft survival. TPE has been included
antibody to a level that allows for successful engraftment
in preparatory regimens for ABOi solid organ transplantation
leading to successful transplantation across HLA and/or ABO
as an adjunct with different immunosuppression therapies
barriers.
and IVIG. In ABOi kidney transplantation, TPE is part of a
preconditioning protocol to lower antibody titre to <8/<4
ABO Desensitization prior to the transplant procedure. Apart from TPE, specific A
Major incompatibility refers to the presence of natural or B antigen immuno-adsorption columns have been used in
antibodies in the recipient against the donor’s A or/ Europe to selectively remove anti-A or anti-B antibodies.
Duration and Discontinuation/ defined but include the use of cyclosporine, tacrolimus,
mycophenolate mofetil, azathioprine, and anti-thymocyte
Number of Procedures
globulin.
The goal should be to reduce the antibody titre (IgM and IgG) Treatment of AMR includes increasing immunosuppres
to ≤ 8/4 in renal transplantation. This titre can be achieved sive medications as well as rituximab, TPE, IVIG or splenec
usually in 2–5 days, depending upon the baseline titres. The tomy. In severe cases or cases refractory to TPE+IVIG,
antibody titres may increase 3–7 days after transplantation; splenectomy and rituximab have been used. Clinical trials
therefore, daily antibody titre for the first 2 weeks post- have demonstrated improved graft survival with TPE/IVIG
transplantation is necessary. During the following 2 weeks, versus TPE alone or IVIG alone, and TPE + rituximab versus
antibody titre measurement every second day helps to TPE alone.
prevent immunologic graft events. If the antibody titre is
high (>32) with or without humoral rejection, TPE should be Rationale for Therapeutic Apheresis
performed again in the post-transplantation period. In renal
transplantation, usually three more TPEs are performed In antibody-mediated rejection DSA are generated after
postoperatively (every second or third day followed by IVIG). transplantation. These antibodies can be removed with
If the antibody titre can be maintained at <8 in post-transplant plasma exchange, double filtration plasmapheresis and
first week and 16 in second week, the risk of humoral rejection immunoadsorption. Therapeutic apheresis is always in
is decreased. combination with other immunosuppressive drugs, such as
antithymocyte-globulin, glucocorticosteroids, rituximab, and
intravenous immunoglobulin.
HLA Desensitization and AMR TPE can also be used prior to transplant to remove HLA
Patients with an elevated HLA antibody screen have difficulty antibodies. TPE (some series have used double filtration
finding an HLA compatible donor and remain on the plasmapheresis and one small series used Prosorba column)
transplantation list significantly longer than unsensitized is used in combination with immunosuppressive drugs
patients. The goal of desensitization protocols is to allow pre-transplant until cross-match is negative. TPE is usually
these individuals to be transplanted using a donor kidney continued postoperatively also and re-initiated in cases
that would otherwise not be usable due to the high likelihood where AMR occurs. The ability to obtain a negative cross-
of graft loss. match depends on the DSA titre. Using approximately 5 TPE
Allograft rejection has traditionally focused on T cell pre-operatively, will allow the titre of < 32 to become negative.
mediated process causing cellular rejection. Recently a clear
histological diagnosis of AMR can be made based on the Duration and Discontinuation/
Sixth Banff Conference on Allograft pathology in 2007. The
Number of Procedures
diagnosis is based on:
• Documentation of donor specific antibody (DSA). For AMR, some protocols use a set number of procedures,
• Histologic evidence of acute tissue injury, such as acute usually 5 or 6, daily or every alternate day. Other protocols
tubular injury, neutrophils in peritubular capillaries and/ guide number of treatments based on improvement in renal
or glomeruli, and/or capillary thrombosis, or intimal arte function and decrease in DSA titres. It is also undecided if low
ritis/fibrinoid necrosis/intramural or transmural inflam dose IVIG (100 mg/kg) should be used after every procedure
mation in arteries. or at the end of the series or not at all. For desensitization
• C4d in peritubular capillaries or immunoglobulin and protocols, TPE is performed daily or every other day per
complement in arterial fibrinoid necrotic areas by protocol until cross-match becomes negative. TPE is also
immunohistology. performed postoperatively for a minimum of 3 procedures.
AMR affects fewer than 10% of renal allografts. Recipients
at higher risk include those with previous transplant and high References
panel-reactive antibodies.
1. Gabardi S, Olyaei AJ. Solid organ transplantation, chapter 55.
In, Chisolm-Burns MA, Ed. Pharmacotherapy Principles and
Current Management/Treatment Practice, 2nd edn. McGraw-Hill, NY, NY. 2010. pp. 939-64.
2. The Organ Procurement and Transplant Network (OPTN).
New immunosuppressive drugs are continually being
Available at: http://optn.transplant.hrsa.gov/latestdata/
developed to prevent and treat acute renal allograft rejection, viewdatareports.asp. Accessed: 2010.
and to decrease antibody titres. All transplant recipients are 3. Opelz G, Sengar DP, Mickey MR, et al. Effect of blood
placed on immunosuppressive therapy but individuals with transfusions on subsequent kidney transplants. Transplant
a high likelihood of acute rejection, including those with Proc. 1973;5:253-9.
HLA antibodies and recipients of cadaveric organs, receive 4. Halloran PF. Call for revolution: a new approach to describing
more intense regimens. The optimal regimen has yet to be allograft deterioration. Am J Transplant. 2002;2:195-200.
80 Section 1 Clinical Hemotherapy
5. Opelz G, Terasaki PI. Improvement of kidney-graft survival 13. Albrechtsen D, Flatmark A, Brynger H, et al. Impact of blood
with increased numbers of blood transfusions. N Engl J Med. transfusions and HLA matching on national kidney transplant
1978;299:799-803. programs: the first Swedish-Norwegian Study of cyclosporine.
6. Fuller TC, Delmonico FL, Cosimi B, et al. Impact of blood Transplant Proc. 1988;20:257-60.
transfusion on renal transplantation. Ann Surg. 1978;187:211-8. 14. Barbari A, Stephan A, Masri MA, et al. Donor specific
PMID: 343736. transfusion in kidney transplantation: effect of different
7. Marti HP, Henschkowski J, Laux G, et al. Effect of donor- immunosuppressive protocols on graft outcome. Transplant
specific transfusions on the outcome of renal allografts in the Proc. 2001;33:2787-8.
cyclosporine era. Transpl Int. 2006;19:19-26. 15. Reed A, Pirsch J, Armbrust MJ, et al. Multivariate analysis
8. Hiesse C, Busson M, Buisson C, et al. Multicenter trial of one of donor-specific versus random transfusion protocols in
HLA-DR-matched or mismatched blood transfusion prior to haploidentical living related transplants. Transplantation.
cadaveric renal transplantation. Kidney Int. 2001;60:341-9. 1991;51:382-4.
9. Alexander JW, Light JA, Donaldson LA, et al. Evaluation of pre- 16. Sanfilippo F, Vaughn WK, LeFor WM, et al. Multivariate
and post-transplant donor-specific transfusion/cyclosporine A analysis of risk factors in cadaver donor kidney transplantation.
in non-HLA identical living donor kidney transplant recipients. Transplantation. 1986;42:28-34.
Cooperative Clinical Trials in Transplantation Research Group. 17. Corry RJ, West JC, Hunsicker LG, et al. Effect of timing of
Transplantation. 1999;68:1117-24. administration and quantity of blood transfusion on cadaver
10. Opelz G, Vanrenterghem Y, Kirste G, et al. Prospective renal transplant survival. Transplantation. 1980;30:425-8.
evaluation of pretransplant blood transfusions in cadaver 18. Melzer JS, Husing RM, Feduska NJ, et al. The beneficial effect
kidney recipients. Transplantation. 1997;63:964-7. of pretransplant blood transfusions in cyclosporine-treated
11. Sharma RK, Rai PK, Kumar A, et al. Role of preoperative donor- cadaver renal allograft recipients. Transplantation. 1987;43:
61-4.
specific transfusion and cyclosporine in haplo-identical living
19. Nube MJ, Persijn GG, van Es A, et al. Beneficial effect of HLA-A
related renal transplant recipients. Nephron. 1997;75:20-4.
and B matched pretransplant blood transfusions on primary
12. Aalten J, Bemelman FJ, van den Berg-Loonen EM, et al.
cadaveric kidney graft survival. Transplantation. 1983;35:556-
Pre-kidney-transplant blood transfusions do not improve
61.
transplantation outcome: a Dutch national study. Nephrol Dial
Transplant. 2009;24:2559-66.
Incompatible Blood Type
Kidney Transplantation
negative.These histological studies indicate that the detection ADVERSE EFFECT OF ABOi-KT
of C4d alone in ABO incompatible graft does not indicate
AMR and support a concept of accommodation in ABOi-KT. Infection
Therefore, AMR after ABOi-KT can only be diagnosed on the
basis of morphological evidence, serological evidence and The improvement in ABOi-KT graft survival rate has come
the clinical course. at the expense of increased posttransplant infection. The
Morphologically transplant glomerulopathy (TG) at 1 year infection rate in ABOi-KT is significantly higher than in
after transplantation was reported as an indicator of poor ABOc-KT (60% vs 29.8%). The rates of infection including
outcome. ABOi-KT had more severe TG than ABOc-KT without cytomegalovirus (CMV), herpes simplex virus, varicella zoster
HLA antibody at 1 year posttransplant. However, there were virus and BK virus (BKV) in ABOi-KT were also significantly
no differences in interstitial fibrosis, tubular atrophy, chronic higher than in ABOc-KT. The most common viral infection
vasculopathy and allograft function between both groups. was BKV in 25% of ABOi-KT compared to only 8.5% of ABOc-
In the absence of prior AMR, histological change at 1 year KT. However, the incidences of rejection, graft survival rate
posttransplant was mild irrespective of ABO compatibility. and function of ABOi-KT patients were compatible with these
Moreover, prior AMR in ABOi-KT was associated with TG of ABOc-KT patients. On the contrary, Genberg et al. showed
and interstitial fibrosis and not to arteriolar hyalinosis and that there was no statistical difference in overall infection
chronic vasculopathy. Consequently, ABO incompatible complications between ABOi-KT with RIT and living ABOc-KT
grafts with TG and/or interstitial fibrosis had lower GFR at (40% vs 63.3%). However, ABOi-KT patients who were treated
1 year after transplantation than those with normal histology. with RIT may have had different infection profiles. Grim
et al. retrospectively analyzed the incidence of posttransplant
infection in HLA sensitized KT or ABOi-KT treated with RIT
INCIDENCE OF ACUTE CELLULAR AND and compared to HLA sensitized KT without RIT. The acute
ANTIBODY MEDIATED REJECTION IN rejection rate in RIT treated KT was similar to KT without RIT
ABOi-KT (40% vs 33%). However, posttransplant infection rate was
48.0% RIT with KT, but only 11.1% without RIT. Kamar et al.
As previously described, the outcome of graft survival in reported that infection rate was 45.5% in KT with RIT which
ABOi-KT has been similar to ABOc-KT. However, there is an was similar to KT without RIT (53.9%). Bacterial, viral and
increased risk of AMR in ABOi-KT due to anti-A/B antibody. fungal infection were observed in 36.3%, 18.2% and 16.9% in
Protocol biopsies at 3 months post-transplant in ABOi-KT KT with RIT, against 31.6%, 34.3% and 5.32% in KT without
had a significantly higher incidence of AMR compared to RIT. Polyoma virus infection rate (64.3%) was relatively high
ABOc-KT (17.9% vs 1.1%). However, there was no significant in RIT. Moreover, infection related-deaths were significantly
difference in the rate of acute cellular rejection between ABOi- higher in RIT treated patients. This data ascertained that RIT
KT and ABOc-KT (48.4% vs 35.7%) In the acute lesion score was associated with severe infection which causes death
based on Banff classification, t2-3 and g2-3 following ABOi-KT rather than an increased risk of infection. Other report
was higher than that of ABOc-KT (t2-3: 42.9% vs 19.4%, g2-3: confirmed earlier observation showing that the incidence of
28.6% vs 6.5%). Gloor et al. described in the study of protocol posttransplant infection in RIT-treated recipients was similar
biopsies at 1 year posttransplant that there was a significant to RIT-untreated recipients (52.2% vs 40.2%). However, as in
difference in the incidence of acute rejection between ABOi- earlier studies the incidences of CMV and BKV infection in
KT and ABOc-KT without HLA antibody (50% vs 13.6%). Acute RIT-treated recipients were higher than in non RIT-treated
rejection in ABOi-KT was mainly AMR (73.3%) as compared recipients (CMV: 16.4% vs 5.7%, BKV: 13.4% vs 8.0%).
to ABOc-KT without HLA antibody (12.5%). Setoguchi et al.
also compared the histologic findings of protocol biopsies in
48 ABO incompatible and 133 compatible grafts. There was no
Malignancy
difference in clinical and subclinical rejection between ABO It is generally accepted that immunosuppression is associated
incompatible and compatible grafts (clinical: 37.5% vs 25.6%, with an increased incidence of malignancy in KT recipients
subclinical: 10.4% vs 15%). However, ABO incompatible grafts compared to the general population. However, several
had a high incidence of AMR compared to ABO compatible studies have demonstrated that ABOi-KT did not increase
grafts (27% vs 5.3%). Interestingly, rejection was detected in the risk of posttransplant malignancy compared with ABOc-
only 15.0% at 1 mo in ABOi-KT compared to 34.7% in ABOc- KT. Yamamoto et al. analyzed the risk of ABOi-KT compared
KT, but in 30.0% at 6–12 mo compared to 10.5%. Wilpert et al. to ABOc-KT retrospectively. ABOi-KT recipients were
demonstrated that the rejection rates in ABOi-KT were similar older than ABOc-KT recipients and all ABOi-KT recipients
to that in ABOc-KT. Acute cellular rejection was detected in received splenectomy, in this study despite increased age
23.2% of ABOi-KT and in 22.5% of ABOc-KT. Acute AMR was and splenectomy, there was no significant difference in the
shown in 4.7% of ABOi-KT, which was similar to ABOc-KT incidence of malignancy between ABOi-KT and ABOc-KT
(5.0%). (4.8% and 4.2%). Similarly, Hall et al. showed that 7 of 318
86 Section 1 Clinical Hemotherapy
ABOi-KT recipients experienced posttransplant cancer. The 5. Lindberg L, Johansson SM, Liu J, Grufman P, Holgersson J. Is
incidence rate ratio (IRR) of cancer in ABOi-KT was identical there a clinical need for a diagnostic test allowing detection
to that in matched control ABOc-KT (IRR: 0.99). This limited of chain type-specific anti-A and anti- B. Transfusion. 2011;
51(3):494-503.
data reassuringly indicates that ABOi-KT is not associated
6. Lynch RJ, Platt JL. Accommodation in renal transplantation:
with an increasing incidence of malignancy after KT. Thus, a unanswered questions. Current Opinion in Organ Transplan
further analysis of long-term observations in ABOi-KT after tation. 2010;15(4):481-5.
RIT is needed. 7. Moon HW, Yun YM, Hur M, Park JH, Lee HW, Chang SH, et al.
An experience of ABO-incompatible kidney transplantation
CONCLUSION using plasmapheresis and anti-CD20 monoclonal antibody.
Korean Journal of Laboratory Medicine. 2009;29(6):585-8.
Since first performed over 50 years ago, ABOi-KT has become 8. Oettl T, Halter J, Bachmann A, Guerke L, Infanti L, Oertli D,
an accepted source of KT. Reassuringly, despite lack of control et al. ABO blood group-incompatible living donor kidney
trials in ABOi-KT, more than satisfactory outcomes have been transplantation: a prospective, single-centre analysis including
serial protocol biopsies. Nephrology, Dialysis, Transplantation.
observed in adult and pediatric recipients, in many studies
2009;24(1):298-303.
equivalent to living ABOc-KT. Preconditioning treatment of 9. Szczepiorkowski ZM, Winters JL, Bandarenko N, Kim HC,
ABOi-KT, such as antibody reduction and desensitization, Linenberger ML, Marques MB, et al. Guidelines on the use
is more intensified and complicated than that of ABOc-KT. of therapeutic apheresis clinical practice--evidence-based
With current protocols, the occurrence of early graft loss approach from the Apheresis Applications Committee of the
and AMR are not completely abolished. Preconditioning American Society for Apheresis. Journal of Clinical Apheresis.
strategy in ABOi-KT has evolved over time. RIT has replaced 2010;25(3):83-1217.
splenectomy which was once thought a crucial procedure for 10. Tanabe K, Ishida H, Shimizu T, Omoto K, Shirakawa H,
ABOi-KT, although this is increasingly abandoned in favor Tokumoto T. Evaluation of two different preconditioning
regimens for ABO-incompatible living kidney donor transplan
of IAs and IVIG. Overall, ABOi-KT is more expensive than
tation. A comparison of splenectomy vs. rituximab-treated
ABOc-KT which may restrict its adoption in resource poor non-splenectomy preconditioning regimens. Contributions to
countries. We believe that a live donor ABOi-KT is a viable Nephrology. 2009;162:61-74.
alternative to waiting on deceased donor list. 11. Tobian AA, Shirey RS, King KE. ABO antibody titer monitoring
for incompatible renal transplantation. Transfusion.
Suggested reading 2011;51(3):454-7.
12. Tobian AA, Shirey RS, Montgomery RA, Cai W, Haas M, Ness
1. Crew RJ, Ratner LE. ABO-incompatible kidney transplantation: PM, et al. ABO antibody titer and risk of antibody-mediated
current practice and the decade ahead. Current Opinion in rejection in ABO-incompatible renal transplantation.
Organ Transplantation. 2010;15(4):526-30. American Journal of Transplantation. 2010;10(5):1247-53.
2. Genberg H, Kumlien G, Wennberg L, Tyden G. Isoagglutinin 13. Tobian AA, Shirey RS, Montgomery RA, Tisch D, Ness PM,
adsorption in ABO-incompatible transplantation. Transfusion King KE. Therapeutic plasma exchange reduces ABO titers to
and Apheresis Science. 2010;43(2):231-5. permit ABO-incompatible renal transplantation. Transfusion.
3. Haidinger M, Schmaldienst S, Körmöczi G, Regele H, Soleiman 2009;49(6):1248-54.
A, Schwartz D, et al. Vienna experience of ABO-incompatible 14. Valli PV, Puga YG, Fehr T, Schulz-Huotari C, Kaup N, Güngör
living-donor kidney transplantation. Wiener klinische T, et al. Changes of circulating antibody levels induced by
Wochenschrift. 2009;121(7-8):247-55. ABO antibody adsorption for ABO-incompatible kidney trans-
4. Jeon BJ, Kim IG, Seong YK, Han BH. Analysis of the results of plantation. American Journal of Transplantation. 2009;9(5):
ABO-incompatible kidney transplantation: in comparison 1072-80.
with ABO-compatible kidney transplantation. Korean Journal
of Urology. 2010;51(12):863-9.
Transfusion Practice in
Heart Transplant
A Surekha Devi
25
Liver transplantation is the treatment of choice in patients ultimate aim of being able to translate this practice to all
with acute or chronic end stage liver disease(ESLD), general surgical procedures.3
irresectable primary liver tumors, and metabolic disorders.1 Contributing factors to blood loss during liver trans
Orthotopic liver transplantation(OLT) is the replacement of plantation can be categorized based on factors related to
a diseased liver with a healthy liver in the normal anatomic preoperative and intraoperative factors.
position. The operative procedure is extensive, complex and
technically challenging with multiple vascular transections Transfusion predictors
and anastomoses.2 In addition, OLT is associated with several
hemostatic defects that contribute to risk of massive blood
loss.2 The liver is an extremely vascular organ. The associated
Preoperative Factors
coagulopathy, anemia, malnutrition and severe portal Preoperative factors associated with blood loss during OLT
hypertension have made this procedure more daunting and include liver failure, cirrhosis, cholestasis and splenomegaly.2
the use of blood products almost universal.3 Hence of all solid The liver plays a central role in hemostasis.
organ transplantations, OLT has placed the greatest demands Two scoring systems have been used to grade the severity
on clinical transfusion services.4 Although blood use steadily of ESLD and prioritize the patients on waiting list for OLT.
declined with improved surgical and anesthesiological
techniques, better graft preservation, better intraoperative Child-Turcotte-Pugh (CTP) Score
monitoring of coagulation status and pharmacologic
treatment of fibrinolysis during the last decade, OLT still CTP score is a measure of disease severity and is classified
frequently demands transfusion equal to one blood volume into CTP class A to C with a scoring system of 5–15.2
(massive transfusion).5 Because the transfusion needs during Class A: 5–6 (less severe)
OLT are unpredictable, transfusion services must remain Class B: 7–9
prepared to effectively deliver massive transfusion support. Class C: 10–15 (more severe)
Liver transplant recipients present unique challenges not
only in terms of blood supply, but requirements of specialized Model for End-stage Liver Disease (MELD)
blood components, serologic problems, and immunologic MELD a numerical scale, ranging from 6 (less ill) to 40 (gravely
effects of transfusion on both the allograft and the recipient.6 ill), used for liver transplant patients, gives a score based on
Studies have observed that increased blood requirements are how urgently the patient needs a liver transplant within the
associated with higher incidence of infections, drug overdose, next three months. Its impact on transfusion requirements
prolonged stay in ICU, serologic problems, and immunologic can be translated directly to the timing of the transplantation.
effects of transfusion on both the allograft and the recipient, With longer waiting times the liver disease progresses, as
graft rejection or graft death and patient death.1-6 However, reflected in higher Child-Pugh/MELD score and thereby
whether these differences in outcome are related to the increasing transfusion requirement.
transfusion as an independent risk factor or whether the
transfusion is a marker for a technically more difficult surgery Etiology of Liver Disease
remains unclear. Yet, bloodless liver transplantation has
been achieved in Jehovah’s witness patients.2 This subset of Patients with chronic active hepatitis have more advanced
patients has allowed us a distinctive opportunity to develop disease and require more blood products than patients with
strategies toward a transfusion-free environment, with primary biliary cirrhosis.2
90 Section 1 Clinical Hemotherapy
whereby the inferior vena cava is preserved and venovenous Postoperative Factors
bypass is avoided.2 Portosystemic shunting is done in
patients with liver failure in order to decrease preoperative Primary Nonfunction (PNF) of Graft or
complications associated with portal hypertension (e.g. Delayed Graft Function
bleeding varices, ascites, sepsis). This decreases blood
requirement and operative time.2 The experience of Failure of graft to function contributes to postopera
surgical and anesthesiologists team impacts blood loss and tive bleeding, causing coagulopathy.2 Appropriate blood
transfusion requirement. Additionally, modifications in component therapy is given to these patients.
surgical technique, including use of electrocautery, argon
beam coagulation and use of fibrin glue minimizes blood Leaks at Anastomosis
loss thereby minimizing transfusions.2 Meticulous surgical
Leaks at vascular suture lines or bleeding from the cut
technique and securing hemostasis on table remains priority
surfaces at bowel anastomoses due to technical failures result
for the surgeon, and indeed long operating times translate
in postoperative bleeding which may require reintervention
into higher transfusion requirement.
and blood transfusions.2
Blood Loss
Graft Versus Host Disease (GVHD)
Blood loss is directly proportional to the duration of surgery.
Consumption of banked blood (no. of RBC units) reflects GVHD can occur from transfer of donor-derived passenger
the degree of blood loss. Intraoperative bleeding remains a lymphocytes and manifests as hemolysis during 7–14 days
significant problem affecting the immediate outcome after after transplantation.6 This is controlled by transfusion of
transplantation of liver.14 Preoperative parameters cannot donor-specific RBCs.6
predict operative bleeding accurately and the mainstay to
prevent bleeding is a meticulous surgical technique during Sepsis
the hepatectomy and correction of coagulation abnormalities Sepsis also results in multiple blood transfusions during post-
throughout the procedure.14 operative period.
Hemodynamics Thrombocytopenia
Central venous pressure (CVP) monitoring is an important It causes bleeding which is associated with platelet consump
aspect of OLT. Patients undergo volume expansion prior tion, platelet-associated IgM and IgA antibody production,
to hepatic resection to prevent bleeding complications, sequestration, thrombin generation, viral infection and
but expansion increases CVP. Deliberate lowering of the ABO-incompatible GVHD.2 This further necessitates platelet
CVP during liver resection assists in bleeding control by transfusions.
decreasing the blood pressure gradient over which bleeding
occurs during dissection.2 Massicotte et al showed that
maintenance of a low CVP prior to the anhepatic phase,
Drugs that minimize blood loss
avoidance of plasma transfusions and restricting volume
replacement during OLT was associated with a decrease in Antifibrinolytics
RBC transfusions during OLT.15 Increased fibrinolysis is observed in some patients during
second and third phases of OLT. Various antifibrinolytic
Cell Salvage agents like aprotinin (AP), tranexamic acid (TA) and epsilon-
The use of cell salvage to collect and reinfuse shed, aminocaproic acid (EACA) have been used to counter
autologous blood is common practice in OLT, when high this accelerated fibrinolysis.2 Prophylactic inhibition of
blood loss is anticipated.2 Autologous blood transfusion hyperfibrinolysis with the biological serine protease inhibitor
reduces the risks associated with allogenic transfusions. AP or the synthetic lysine analog TA is common clinical
Cell salvage is not indicated in the presence of sepsis and practice in OLT.18 Aprotinin has been shown to significantly
hepatocellular carcinoma (HCC).2 Large volume transfusion decrease blood loss, transfusion of RBC units, FFPs, platelets
of salvaged blood can cause postoperative hypofibrinogene and cryoprecipitate and duration of surgery.19 It also has an
mia, thrombocytopenia, prolonged prothrombin and partial effect on platelet function, anti-inflammatory properties and
thromboplastin time and elevated fibrin split products.16 the need for intraoperative ionotropes.19,20 Tranexamic acid
This cell saver induced coagulopathy can be prevented by also decreases transfusion requirements in some but not all
simultaneous platelet and cryoprecipitate transfusions along studies.2
with reinfusion of salvaged blood, as salvaged blood does not
provide platelets and fibrinogen.16,17 Saline washing of red Recombinant Factor VIIa (rFVIIa)
cells increases sodium levels and decreases potassium and
calcium levels. Hence, supplementation of potassium and has been shown to
calcium is done during cell salvage. improve hemostasis during liver transplantation.5 A single
92 Section 1 Clinical Hemotherapy
dose of 80 mcg/kg rFVIIa significantly reduced transfusion Group AB patients: Can receive RBCs of any group. ‘AB’
requirements during OLT.5,21 A study was done where rFVIIa RBCs available through outdating should first be used, then
was given as a bolus just before surgery to patients with switched to group ‘A’ RBCs. After transfusing 10 units of
preoperative risk of high intraoperative blood loss, including group ‘A’ RBCs, patient can be switched to group ‘A’ plasma.
severe uncorrected coagulopathy.2 There was immediate
Group O patients: Only group ‘O’ RBCs can be used in these
correction of coagulation after administration of rFVIIa and
patients, they can receive any ABO group plasma.
these patients were adjusted to normal risk group without an
increased risk for thrombotic complications.2
Rho (D) Selection 6,7
OLT without Transfusion Rh-negative patients should be provided with D- RBCs to
avoid sensitization to D. When transfusion requirements
Literature includes cases of orthotopic liver transplantation
become excessive, D-women over child-bearing age, and
(OLT) performed without transfusion of any blood products
adult men may be switched to D+ RBCs, provided anti-D is
and OLT performed safely without additional blood products
not detected before transfusion. Effort should be made to
if blood loss is limited to 1600–3400 ml.2 Reports have
maintain D- women of child bearing age and children on
described OLT in Jehovah’s witness patients (for religious
D- blood, although the risk of D alloimmunization in OLT
reasons, Jehovah’s witnesses refuse transfusion of blood
is low perhaps due to postoperative immunosuppression.
products), who received no blood products.2 Jabbour
Such patients should be given Rh immunoglobulin (RhIg)
and colleagues continue to lead the field in performing
after administration of Rh-positive blood components. A
OLT without the use of blood products. They reported
case of severe anti-D mediated hemolysis resulting from
favorable results in 27 consecutive patients who underwent
liver transplantation in a O Rh+ man who received an
transfusion-free liver transplantation.2,3 This team used
O Rh- liver allograft is reported.23 The female O Rh- donor had
a combination of preoperative stimulation of red cell
alloantibodies against D,C, and K. This patient required two
production using recombinant human erythropoietin and
red blood cell exchanges and intermittent red cell transfusions
iron and intraoperative hemodilution, cell salvage, and
negative for D,C, and K. A normalization of hemoglobin levels
tolerance to moderate anemia.3 They reported successful use
and decrease in serum bilirubin occurred after a splenectomy
of rFVIIa in 10 patients, just prior to the incision.3 Another
on postoperative Day 321.23
team led by Olivier Detry reported successful transfusion-free
liver transplantation in 9 Jehovah’s witnesses.22
Selection of Red Cell Units in Patients with
Transfusion Support Clinically Significant Alloantibodies
Clinically, significant alloantibodies are present in 6% of liver
ABO Grouping transplant patients, due to sensitization through previous
Liver transplants must be ABO compatible, because of blood transfusions.6 To minimize the risk of hemolysis,
complement-fixing effect of ABO antibodies on endothelial these patients are managed by using antigen-negative units
cells.6 Efforts to cross the traditional ABO barrier fall for the first 5–10 units, switching to antigen unscreened
into three categories: (i) neonatal recipients (ii) A2 and/or partially matched units in the middle of the case, and
organs (iii) fully ABO-incompatible organs.7 Major ABO- then switching back to antigen-negative units for the last
incompatible liver transplants do not undergo hyperacute 5–10 units to prevent postoperative hemolysis.6 This strategy
rejection, but their short-term graft survival is significantly requires close cooperation between anesthesiologist and
reduced. These protocols utilize plasmapheresis, multiple transfusion services. An additional strategy would be to use
immunosuppressive agents, and sometimes splenectomy.7 preoperative plasmapheresis to remove clinically significant
An error in ABO compatibility for organs can be life- low-titer antibodies.7
threatening for the patient. Hence, United Network for Organ
Sharing (UNOS) instituted new requirements for additional Use of Autologous Blood
clerical verification of correct ABO types for transplant donors
Preoperative autologous blood deposit, perioperative
and recipients.7 UNOS also proposed new policies to require
isovolemic hemodilution, and reinfusion of salvaged blood
the performance of two separate ABO typings of each organ
by cell saver during surgery minimizes allogenic transfusions.7
donor and recipient.7
as cytomegalovirus (CMV) infection.6 Steps should be taken Transfusion Protocol in Minor ABO Mismatched
to prevent transmission of CMV through blood transfusions
Liver Transplants6,7
to liver transplant recipients who have not been previously
infected (i.e. CMV-seronegative recipients of seronegative Transfusing exclusively donor group red blood cells(RBCs)
donors).24 Prevention can be achieved by leukocyte reduction from the beginning of surgery:
of the cellular components, by selection of blood from CMV- • Transfusing recipient ABO group of red cells at the
seronegative donors, or both.24 beginning of surgery, but switching to donor ABO group
of red cells after early reperfusion phase up to six weeks
Leukocyte-reduced Blood Components in postoperative period. This replaces susceptible red cells
with cells that will not be hemolyzed. Plasma products are
Transfusions before, during, and after transplant surgery transfused based on recipient ABO group to reduce the risk
should be leukocyte reduced to reduce the risk of developing of hemolysis by providing soluble ABO antigen capable
HLA alloimmunization.24 If HLA antibodies develop in of neutralizing DDAb. After 6 weeks, if DAT and anti-A/
patients waiting for an organ transplant, the risk of rejecting anti-B antibodies are negative, red cells are switched over
the transplanted organ increases and the chance of finding a to recipient’s ABO group.
compatible organ decreases.24 • Transfusing recipient group RBCs, but performing
postoperative DAT and switching to donor group RBCs if
Irradiated Blood Components DAT is positive.
• Transfusing recipient group RBCs until incompatibility is
Graft versus-host disease (GVHD) is a rare complication noted in a crossmatch.
of organ transplantation and is almost always due to donor
organ. These few cases do not support a policy of routine
irradiation of cellular blood components for solid organ Transfusion Protocol During
transplant recipients.6 Liver Transplantation
Criteria for replacement of blood products are as follows:
Male only Plasma Administration of red cell units to maintain Hemoglobin
The strategy to issue plasma from male donors for transfusion (Hb) levels at 8%; FFP (1 unit/20 kg) in case of hemorrhage
is an effort to reduce the incidence of transfusion related associated with International Normalized Ratio (INR) >2.0;
acute lung injury (TRALI), as plasma components linked to apheresis platelets to maintain platelet count >50 × 103/mm3
female donors (parous women) with neutrophilic antibodies (>80 × 103/mm3 in massive transfusion) and cryoprecipitate
are responsible for the majority of probable TRALI cases.25 (1 unit/10 kg) for fibrinogen levels <100 mg/dl in case of
ongoing bleed (<120 mg/dl in massive transfusion).14,26
Hourly laboratory monitoring of hemoglobin (Hb), platelet
Minor ABO-incompatible Transplants count, PT and fibrinogen are done throughout the surgery.
When group O allografts are transplanted to nongroup-O Cell saver is used to salvage blood for autologous transfusion.
patients/nongroup-AB allografts are transplanted to group In case of massive blood loss, rapid infusion pump is used
AB patients, donor passenger lymphocytes are transferred to facilitate fluid resuscitation and transfusions are given in
with the organ at the time of transplantation and produce blood warmer. Air-warming blankets are used to maintain
alloantibodies against host antigens in the recipient causing body temperature.
delayed hemolysis.7 Clinically significant hemolysis caused In OLT, there is institutional variability in transfusion
by anti-Jk(a), produced by passenger lymphocytes transferred practice, which mandates reassessment of the rational use of
from the donor’s liver to the recipient has been reported.6 blood products.27
These donor derived antibodies(DDAb) develop 7–14 days When transfusions exceed more than one blood volume
after transplantation and is heralded by the development within 24 hours, it is considered as massive transfusion.18
of positive direct antiglobulin test (DAT).6 Serum antibody
is predominantly IgG. DDAb are short lived antibodies that Conclusion
persist for 2–3 weeks. Ramsey reported the incidence of
DDAb and hemolysis in OLT as 40% and 29%.6 Hemolysis
is usually mild and self-limiting. This is more likely in group
Multidisciplinary Approach
A recipients of group O livers who receive cyclosporin Proper patient selection, better preservation of graft, short
or tacrolimus immunosuppression.6 In most cases the CIT, meticulous surgical technique, skilled anesthesia
hemolysis associated with DDAb is mild and can be treated practice, intraoperative autotransfusion, use of antifibrino
with transfusions.6 Several cases of hemolysis-induced acute lytic drugs and rFVIIa during surgery, use of TEG, and
renal failure have been reported. In fulminant cases, red cell administration of appropriate components, optimal
exchange with donor ABO group or group O red cells, or use of available new technologies and drugs minimizes
plasma exchange, has been performed.6 transfusions during OLT. Efforts to reduce intraoperative
94 Section 1 Clinical Hemotherapy
bleeding leading to limitation of the amount of blood used 14. Mor E, Jennings L, Gonwa TA, Holman MJ, Gibbs J, Solomon
are therefore desirable not only to improve results but also to H, et al. The impact of operative bleeding on outcome in
control costs, preserve the blood pool for other emergency or transplantation of the liver. Surg Gynecol Obstet. 1993;176:219-
27.
routine surgeries and to decrease the program’s dependence
15. Luc Massicotte, Serge Lenis, Lynda Thibeault, Marie-Pascale
on blood availability. Goal should be to develop strategies Sassine, Robert F Seal, Andre Roy. Effect of low central venous
towards transfusion free environment. pressure and phlebotomy on blood product transfusion
requirements during liver transplantations. Liver Transpl.
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Institution experience of 27 consecutive liver transplants in Walter Wijns, Luc de Pauw, Jean Vandestadt et al. Comparison
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2005;3:412-7. and red blood cell transfusion requirement during the late
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Petz, Scott N Swisher, Steven Kleinman, Richard K Spence, 19. Victor W Xia, Radolph H. Steadman. Antifibrinolytics
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Medicine, 3rd edn. New York: Churchill Livingstone; 1996. pp. controversies. Liver Transpl. 2005;11:10-8.
792-802. 20. Findlay JY, Kufner RP. Aprotinin reduces vasoactive medication
5. Herman GD Hendriks, Karina Meijer, Joost Th MDE Wolf, Ids J use during adult liver transplantation. J Clin Anaesth. 2003;15:
Klompmaker, Robert J Porte, Peter Jan De Kam, et al. Reduced 19-23.
transfusion requirements by recombinant factor VIIa in 21. Lodge JP, Jonas S, Jones RM, Olausson M, Mir-Pallardo J, Soefelt
orthotopic liver transplantation: A pilot study. Transplantation. S, et al. Efficacy and safety of repeated perioperative doses of
2001;71:402-5. recombinant factor VIIa in liver transplantation. Liver Transpl.
6. Darrel J Triulzi. Transfusion support in liver transplantation. 2005;11:973-9.
Current Hematology Reports. 2004;3:444-9. 22. Oliver Detry, Arnaud De Roover, Jean Delwaide, Abdour
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Clinical Principles and Practice, 2nd edn. Bethesda, MD: 23. Fung MK, Sheikh H, Eghtesad B, Lopez-Plaza I. Severe
American Association of Blood Banks press; 2005. pp. 265-74. hemolysis resulting from D incompatibility in a case of ABO-
8. McNicol PL, Liu G, Harley ID, McCall PR, Przybylowski identical liver transplant Transfusion. 2004;44:1635-9.
GM, Bowkett J, et al. Patterns of coagulopathy during liver 24. Ted Eastlund. Tissue and Organ Transplantation and the
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9. Broering DC, Sterneck M, Rogiers X. Living donor liver (Eds). Technical Manual, 16th edn. Bethesda, MD: American
transplantation. J Hepatol. 2003;38:S119-35. Association of Blood Banks; 2008. pp. 850-2.
10. Heaton N. Small-for-size liver syndrome after auxillary and 25. Anne F Eder, Ross Herron, Annie Strupp, Beth Dy, Edward pp.
split.liver transplantation: donor selection. Liver Transpl. Notari, Linda A Chambers, et al. Transfusion-related acute lung
2003;9: S26-8. injury surveillance (2003-2005) and the potential impact of the
11. Richard S Mangus, A Joseph Tector, Avinash Agarwal, Rodrigo selective use of plasma from male donors in the American Red
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and University of Wisconsin solution (UW) in adult liver Larma, Laura Llado, Juan Figueras, et al. Intraoperative red
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12. Erick B Edwards, Richard B Freeman. Effect of cold ischemia patient outcome, prediction requirements, and measures to
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conclusions_and_recommendations.pdf.
Massive Transfusion
is recommended that covering exposed bowel with moist Use of warmed intravenous fluids is one of the simplest
towels be avoided because it can increase evaporative heat and most effective means of providing heat to the core
loss by nearly 250%.24 Dry towels or plastic bags are superior. in patients requiring massive fluid resuscitation. Current
fluid warmer technology allows large volumes of warmed
Active Core Rewarming fluids to be infused quickly at 42°C, the current standard
The hypothermic trauma patient requires active core recommended by the American Association of Blood Bank.28
rewarming which may include airway rewarming, heated Blood-warming methods include surface-contact warmers,
body cavity lavage, heated intravenous fluids, continuous counter-current warmers, and heated-saline admixture.29 In-
arteriovenous rewarming (CAVR), and extracorporeal line microwave blood-warming technology (in development)
circulatory rewarming. Humidified ventilator circuits can be has been shown to heat blood safely to 49°C and shows great
warmed to 41°C. promise for the future.30
98 Section 1 Clinical Hemotherapy
EVIDENCE DEFINITIONS
• Class I: Prospective randomized controlled trial.
• Class II: Prospective clinical study or retrospective analysis of reliable data. Includes observational, cohort, prevalence, or case control studies
• Class III: Retrospective study includes database or registry reviews, large series of case reports, expert opinion.
• Technology assessment: A technology study which does not lend itself to classification in the above-mentioned format. Devices are evaluated in terms
of their accuracy, reliability, therapeutic potential, or cost effectiveness.
LEVEL OF RECOMMENDATION DEFINITIONS
• Level 1: Convincingly justifiable based on available scientific information alone. Usually based on Class I data or strong Class II, evidence if randomized
testing is inappropriate. Conversely, low quality or contradictory Class I data may be insufficient to support a Level I recommendation.
• Leve 2: Reasonably justifiable based on available scientific evidence and strongly supported by expert opinion. Usually supported by Class II data or a
preponderance of Class III evidence.
• Level 3: Supported by available data, but scientific evidence is lacking. Generally supported by Class III data. Useful for educational purposes and in
guiding future clinical research.
In continuous arteriovenous rewarming (CAVR),31 8.5 deleterious effects of acidosis on the cardiovascular system
French femoral arterial and venous catheters are placed include decreased cardiac contractility and cardiac output,
percutaneously, an extracorporeal arteriovenous circuit vasodilation and hypotension, decreased hepatic and renal
that uses the heating mechanism of a counter-current fluid blood flow, bradycardia, and increased susceptibility to
warmer are created. This is more effective in comparison with ventricular dysrhythmias.33 Acidosis directly reduces the
traditional warming techniques.29 activity of the extrinsic and intrinsic coagulation pathways as
measured by PT and PTT and also diminishes platelet function
as measured by platelet aggregation and platelet factor III
ACIDOSIS release.24 These adverse effects are generally not seen until pH
The association between high lactate levels and increasing decreases below 7.2.33 Therapy for metabolic acidosis remains
risk of death was with metabolic acidosis as demonstrated directed toward correcting the underlying hypoperfusion.
by arterial pH, lactate, and base deficit clearance.32 The Resuscitation endpoints include normalization of arterial pH,
Chapter 26 Massive Transfusion Protocol 99
base deficit, and lactate. Bicarbonate administration should viscoelastic changes in whole blood as clotting progresses and
be deferred until the pH persists below 7.2, despite optimal fibrin strands form between the cup and pin. Clot strength is
fluid loading and inotropic support.34 decreased by hypofibrinogenemia, thrombocytopenia, low
F XIII level, or reduced thrombin generation. Rapid TEG
TRANEXAMIC ACID (coagulation is initiated by the addition of tissue factor) gives
faster results compared to kaolin TEG. ROTEM, EXTEM
Tranexamic acid is an antifibrinolytic agent that has (uses recombinant tissue factor to activate coagulation)
historically been shown to decrease the need for blood and FIBTEM (measures contribution of fibrinogen to clot
transfusions in patients undergoing elective surgery. In strength) allow differential diagnosis of thrombocytopenia
2010, a multinational randomized, double-blind placebo- and hypofibrinogenemia. Higher hemoglobin levels reduce
controlled trial (CRASH-2) showed that the tranexamic acid TEG/ROTEM signals.
group had a significantly lower all-cause mortality at 28
days.35
Application
RESUSCITATION IN PATIENTS REQUIRING Hyperfibrinolysis was identified in the most severely injured
patients, and correlated with mortality.38,39 Kashuk et al.
MASSIVE TRANSFUSION demonstrated primary fibrinolysis in 34% of patients requiring
Early control of hemorrhage is essential to limit consumptive massive transfusion using TEG.40 Early administration of
coagulopathy and thrombocytopenia, and decrease usage an anti-fibrinolytic agent (tranexamic acid) to adult trauma
of blood. Limiting the use of isotonic crystalloid will help patients resulted in reduced all-cause mortality in the
prevent dilutional coagulopathy. Limiting the use of treatment group, reduced mortality from bleeding and a
0.9% saline may prevent further acidosis. Hypotensive safe side effect profile in bleeding trauma patients.41 It is
resuscitation (systolic blood pressure 80–100 mm Hg) until recognized, however, that severely injured patients who pre
hemorrhage is controlled is generally recommended, unless sent fibrinolysis have increased mortality14,42 so the efficacy
there is concern for traumatic brain injury. Early use of of tranexamic acid in such patients is questionable. TEG/
plasma, platelets as well as RBCs is necessary in an attempt to ROTEM assays may signal the selection of patients who would
improve survival with massive transfusion. Fibrinogen levels most benefit from this intervention.
above 1.5 g/L are targeted.36 These levels can be achieved with
cryoprecipitate 50 mg/kg or fibrinogen concentrate doses of FACTOR rVIIa AND MTP
3–4 g.36 Above base deficit and lactate levels are monitored to
assess adequacy of resuscitation in restoring oxygen delivery Evidence shows benefit for rFVIIa, especially in coagulopathic
and tissue perfusion. Electrolyte abnormalities caused by trauma patients, however this remains an off-label use
dilution and transfusion should be corrected: hyperkalemia and controversial. rFVIIa is not a first-line therapy for
from large volume of banked RBCs, hypocalcemia from hemorrhage. Adequate fibrinogen levels (e.g. > 1.5–2 g/L) and
citrated anticoagulants, and Na and Cl abnormalities from platelet count (e.g. > 50–100 × 109/L) are necessary for drug
crystalloid resuscitation. Evidence is growing for use of point efficacy. Control of surgical bleeding and normalizing acid-
of care coagulation testing to guide hemostatic therapy base status and core temperature are of obvious importance.
(thromboelastography, thromboelastometry, Sonoclot)6,37 In a meta-analysis of patients with body trauma, use of rFVIIa
Standard coagulation tests such as PT, PTT, INR, platelet reduced ARDS and transfusion requirements, but had no
count, and fibrinogen usually require 30–60 minutes for effect on mortality or thromboembolism.43 Because of the
results to be available. For massive transfusion patients risk of serious adverse effects, treatment with rFVIIa must be
requiring acute interventions, results of standard coagulation individualized based on a risk-benefit analysis.
tests may not be an accurate reflection of coagulation
function. Measurement of platelet count and fibrinogen only COMPLICATIONS OF MTP
provides absolute amounts, not functional activity and may
Blood transfusion is an independent predictor of multi-organ
over estimate the levels.
failure in a retrospective and prospective study of trauma
patients with injury severity score > 15 and survival greater than
THROMBOELASTOGRAPhY (TEG) AND 24 hours.9 There is a 2–6 fold increase in SIRS, 4 fold increase
ROTATIONAL THROMBOELASTOMETRY in ICU admission and mortality in transfused patients. One
study showed that transfusion was an independent predictor
(ROTEM)
of death in non-operative blunt trauma and the risk of death
Two available viscoelastic whole-blood assays measured at increased with each additional RBC unit.44 Multiple studies
37°C give an in vitro measure of clotting. These hemostatic have linked transfusion to increased development of and
assays provide a graph of the displacement caused by mortality from ARDS. TRALI occurs with a 1:5000 incidence in
100 Section 1 Clinical Hemotherapy
the general transfusion population and is likely increased in assays may allow for goal directed therapy in coagulopathy
massive transfusion, although the extent is unknown. TRALI of trauma and massive transfusion including the use of
is likely under-diagnosed after severe injury. It is difficult to antifibrinolytics when appropriate. Single agent therapy
differentiate TRALI from transfusion associated circulatory such as rFVIIa may have a role in coagulopathic trauma
overload or other causes of lung injury in trauma. For non- patients but safety is still a concern. A restrictive transfusion
massively transfused trauma patients, plasma administration strategy should be adopted once hemorrhage is controlled to
is associated with increased complications (ARDS, multiple minimize unnecessary exposure to blood.
organ dysfunction, pneumonia, and sepsis) with no improve The MTP practice is still fraught with many unresolved
ment in survival.45 Despite the claim that high ratio MTP issues such as use of fibrinogen and/or prothrombin complex
reduces dilutional coagulation, some clotting factors do get concentrate and blunt vs penetrating trauma. Understanding
diluted by the shear volume of components and the limited the mechanism of hemorrhage is not universal and is
availability of factors in plasma may not be sufficient to different in the obstetrical population as it is in pediatric
overcome this defect. As such, the early and sometime the or cardiovascular patients. This may add to the limitation
sole use of coagulation factors and fibrinogen concentrates is of universal adoption of a single ratio driven MTP. Well
being adopted in European centers. The use of these agents designed, prospective randomized trials are required to
in the US is off label and many are not FDA approved. Firm determine optimal transfusion ratios and timing of blood
data on efficacy and safety of this practice is needed and is component administration.
slowly accruing.
REFERENCES
MTP AFTER CONTROL OF HEMORRHAGE 1. ASA Committee on Blood Management. Charles E Smith,
A restrictive approach to blood product utilization is advo Andrew M Bauer, Evan G. Pivalizza, Kenichi Tanaka,
cated once bleeding is stopped and the patient is stable.9 Leonard Boral, Aryeh Shander, Jonathan H. Waters, 2012.
file:///C:/Users/win8/Downloads/MTP%20for%20ASA%20
RBC transfusion is generally indicated for hemoglobin
Transfusion%20Committee%20Final%20(1).
< 7 g/dL. For hemoglobin above 7 g/dL, hypovolemia is 2. O'Keeffe T, Refaai M, Tchorz K, Forestner JE, Sarode R. A
treated with IV fluids. Signs and symptoms of impaired massive transfusion protocol to decrease blood component
oxygen delivery and decreased tissue perfusion are closely use and costs. Arch Surg 2008;143:686-90, discussion 690-1.
monitored. RBC transfusion is generally required for 3. Riskin DJ, Tsai TC, Riskin L, Hernandez-Boussard T, Purtill
persistent base deficit, lactic acidosis, signs of end organ M, Maggio PM, Spain DA, Brundage SI. Massive transfusion
ischemia, and low mixed venous oxygenation. protocols: the role of aggressive resuscitation versus product
ratio in mortality reduction. J of the Am Coll of Surg. 2009;
209:198-205.
MTP IN PEDIATRICS 4. Cotton BA, Gunter OL, Isbell J, Au BK, Robertson AM, Morris
JA Jr, St Jacques P, Young PP. Damage control hematology: the
Massive transfusion for pediatric trauma patients generally
impact of a trauma exsanguination protocol on survival and
follow similar principles as in adults.46 An age and weight- blood product utilization. J Trauma. 2008;64:1177-83.
based approach is required. Paterson used a MTP protocol for 5. Cotton BA, Au BK, Nunez TC, Gunter OL, Robertson AM, Young
a 5-year-old child with massive bleeding during neurosurgery PP. Predefined massive transfusion protocols are associated
(estimated loss of between five and six blood volumes over with a reduction in organ failure and post injury complications.
1.5 hours.47 The first blood volume was replaced with J Trauma 2009;66:41-8; discussion 48-9.
crystalloid (40 ml/kg), RBCs (30 ml/kg) and plasma (20 mL/ 6. Johansson PI, Stensballe, Hemostatic resuscitation for massive
kg FFP). RBCs, plasma, platelets, cryoprecipitate and rFVIIA bleeding: the paradigm of plasma and platelets—a review of
were used subsequently. The child survived. Further studies the current literature. Transfusion. 2010;50(3):701.
7. Tien H, Nascimento B Jr, Callum J, Rizoli S. An approach to
are necessary to validate use of MTP in children.
transfusion and hemorrhage in trauma: current perspectives
on restrictive transfusion strategies. Can J Surg. 2007;50(3):202-
SUMMARY 9
8. Sihler KC, Napolitano LM. Massive transfusion: new insights.
Massive hemorrhage and resuscitation can result in refractory Chest 2009;136(6):1654-67.
coagulopathy if not aggressively treated. The use of MTPs 9. Sihler KC, Napolitano LM. Complications of massive
facilitate rapid availability of components. Increased ratios transfusion. Chest, 2010;137:209-20.
of plasma and platelets to RBCs and their timely adminis 10. Johansson PI, Ostrowski SR, Secher NH. Management of major
blood loss: an update. Acta Anaesthesiol Scand. 2010;54:1039-
tration improve outcome in trauma, decrease coagulopathy
49.
and transfusion requirements. Large volumes of plasma are 11. Schuster KM, Davis KA, Lui FY, Maerz LL, Kaplan LJ. The
required to correct coagulopathy, so early administration is status of massive transfusion protocols in United States
presumably more efficacious. The approach would be diffe trauma centers: massive transfusion or massive confusion?
rent when specific factor concentrates are used.48 Viscoelastic Transfusion. 2010;50(7):1545-51.
Chapter 26 Massive Transfusion Protocol 101
12. Repine TB, et al. The use of fresh whole blood in massive human packed red blood cells. Crit Care Med. 1995;23:1243-
transfusion. J Trauma. 2006;60(6 Suppl):S59-69. 50.
13. Bolliger D, Görlinger K, Tanaka KA. Pathophysiology and 31. Gentilello LM, Cortes V, Moujaes S, et al. Continuous
treatment of coagulopathy in massive hemorrhage and arteriovenous rewarming: Experimental results and thermo
hemodilution. Anesthesiology. 2010;113(5):1205-19. dynamic model simulation of treatment for hypothermia. J
14. Brohi K, Cohen MJ, Ganter MT, Schultz MJ, Levi M, Mackersie Trauma. 1990;30:1436-49.
RC, Pittet JF. Acute coagulopathy of trauma: hypoperfusion 32. Abramson D, Scalea T, Hitchcock R, et al. Lactate clearance and
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Trauma. 2008;64(5):1211-7. 33. Wildenthal K, Mierzwaiak DS, Myers RW, et al. Effects of acute
15. Brohi K, Cohen MJ, Ganter MT, Matthay MA, Mackersie lactic acidosis on left ventricular performance. Am J Physiol.
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Ann Surg. 2007;245(5):812-8. and Principles of Therapy. Philadelphia: FA Davis; 1992:267.
16. Frith D, Brohi K. The acute coagulopathy of trauma shock: 35. CRASH-2 trial collaborators. Effect of tranexamic acid on
Clinical relevance. Surgeon 2010;8:159-63. death, vascular occlusive events, and blood transfusion in
17. Bolliger D, Szlam F, Molinaro RJ, Rahe-Meyer N, Levy JH, trauma patients with significant haemorrhage: a randomized,
Tanaka K. Finding the optimal concentration range for placebo-controlled trial. Lancet, 2010;376:23-32.
fibrinogen replacement after severe haemodilution: an in vitro 36. Rossaint R, et al. Management of bleeding following major
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18. Hiipala, et al. Hemostatic factors and replacement of major 37. Johansson PI, et al. Thromboelastography and thromboelasto
blood loss with plasma-poor red cell concentrates. Anesth metry in assessing coagulopathy in trauma. Scand J Trauma
Analg. 1995;81:360-5. Resusc Emerg Med. 2009;17:45.
19. Kashuk JL, et al. Primary fibrinolysis is integral to the 38. Levrat, et al. Evaluation of rotation thromboelastography
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protocol for the exsanguinating trauma patient. J Trauma. 9.
2010;68:1498-505. 40. Kashuk JL, et al. Postinjury coagulopathy management: Goal
21. Cotton BA, Dossett LA, Haut ER, et al. Multicenter validation directed resuscitation via POC thromboelastography. Ann
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Trauma. 2010;69 Suppl 1:S33-9. 41. Effects of tranexamic acid on death, vascular occlusive events,
22. Massive Transfusion for Coagulopathy and Hemorrhagic Shock, and blood transfusion in trauma patients with significant
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transfusion%20protocol%202012. trial. Lancet, 2010.
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Blackbourne LH. Impact of policy change on US Army combat a review of mechanisms. J Trauma. 2008;65:748-54.
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24. Gentilello LM, Jurkovich GJ. Hypothermia. In: Ivatury RR, hospital use of recombinant factor VIIa for off-label indications.
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Baltimore: Williams & Wilkens; 1996;995-1005. 44. Robinson WP, et al. Blood transfusion is an independent
25. Luna GK, Maier RV, Pavlin EG, et al. Incidence and effect predictor of increased mortality in non-operatively managed
of hypothermia in seriously injured patients. J Trauma. blunt hepatic and splenic injuries. J Trauma. 2005;58(3).
1987;27:1014-8. 45. Inaba K, et al. Impact of plasma transfusion in trauma patients
26. Frank S, Beattie C, Christopherson R, et al. Unintentional who do not require massive transfusion. J Am Coll Surg.
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27. Britt L, Dascombe W, Rodriguez A. New horizons in product use in the pediatric trauma patient. Seminars in
management of hypothermia and frostbite injury. Surg Clin Pediatric Surgery 2010;19:286-91.
North Am. 1991;71:345-70. 47. Paterson NA. Validation of a theoretically derived model for
28. American Association of Blood Banks. Technical Manual. the management of massive blood loss in pediatric patients—a
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Bethesda, MD: American Association of Blood Banks; 1998. 48. Schochl H, et al. Goal-directed coagulation management of
29. Iserson K, Huestis D. Blood warming: Current applications and major trauma patients using thromboelastometry (ROTEM®)-
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30. Pappas C, Paddock H, Goyette P, Grabowy R, Connolly R, prothrombin complex concentrate. Critical Care 2010;14:R55.
Schwaitzberg S. In-line microwave blood warming of indate
Massive Transfusion in
Obstetrical Hemorrhage
Sangeeta Pathak
27
PRINCIPLE • Loss of 50% of blood volume within 3 hours.
• Loss of blood at a rate in excess of 150 mL per minute.
The massive transfusion protocol (MTP) for obstetrical
Hemorrhage is intended for antepartum, intrapartum
Causes of Massive Blood Loss
or postpartum patients deemed candidates based on
requirement for massive blood volume replacement. This Obstetric haemorrhage may occur before or after delivery,
protocol has been prepared to meet the special needs of the but more than 80% of cases occur postpartum.
obstetrical hemorrhage patient. Worldwide, massive obstetric hemorrhage, resulting from
the failure of normal obstetrical, surgical and/or systemic
DEFINITION hemostasis, is responsible for 25% of the estimated 358,000
maternal deaths each year.2
Any blood loss occurring in the peripartum period,
revealed or concealed, that is likely to endanger life. Blood loss may be:
Physiological and hematological changes induced by • Antepartum: Hemorrhage after 24th week of gestation and
pregnancy can hide signs of hypovolemic shock and patient before delivery
can collapse suddenly. − Placenta previa, placental abruption, bleeding from
Obstetric hemorrhage remains a major cause of maternal vaginal or cervical lesions
mortality in the UK, with substandard management identified • Postpartum (primary): Within 24 hours of delivery
as a contributor in 80% of the cases.1 It is estimated that − Tone (uterine atony)
there are more than 4000 cases of severe hemorrhage each − Tissue (retained products)
year in the UK. The majority of these cases will need blood − Trauma (cervical and genital tract damage during
transfusion. delivery)
Retrospective analyses of the clinical scenarios often − Thrombin (coagulation disorder)
criticise the employment of blood transfusion as ‘too little, • Postpartum (secondary): 24 hours to 6 weeks post-delivery
too late’. Women at high risk of losing greater than 1000 mL − Uterine atony, retained products, genital tract trauma,
should be strongly advised to deliver in a setting where blood uterine inversion.
transfusion and intensive care facilities are available. Blood loss can be notoriously difficult to assess in
Blood transfusion may be a life-saving procedure but it is obstetric bleeds. Bleeding may sometimes be concealed and
not without risk. Recipients may rarely develop transfusion- the presence of amniotic fluid makes accurate estimation
transmitted infection as well as suffer immunological challenging.3
sequelae such as red cell alloimmunization. Post-partum hemorrhage (PPH) has been defined as a
The major risk of blood transfusion, however, is of a patient blood loss of 500 mL or more during puerperium and severe
receiving an ‘incorrect blood component’. PPH as a blood loss of 1000 mL or more.4
Strict adherence to correct sampling, crossmatch and
administration procedures is therefore of paramount Goals in the Management of Transfusion
importance, even in an emergency. in Severe Hemorrhage Include
Massive blood loss may be defined as: • Rapid resuscitation with crystalloids to restore and
• Loss of one blood volume within a 24-hour period. maintain the circulating blood volume to prevent tissue
(7% of lean body weight (5 liters in an adult). and organ hypoperfusion.
Chapter 27 Massive Transfusion in Obstetrical Hemorrhage 103
• Maintenance of tissue oxygenation using red blood cells pressure but they are complicated by pulmonary edema,
and exacerbation of thrombocytopenia and coagulopathy due
• Reversal or prevention of coagulopathy using appropriate to hemodilution.
blood and plasma components, i.e. platelets, fresh frozen • On the other hand, permissive hypotension, where systolic
plasma (FFP), for the provision of clotting factors and blood pressure of 80–100 mm Hg is tolerated and have
cryoprecipitate or fibrinogen concentrate as a source of shown survival benefit in some studies.
fibrinogen. • It is contraindicated in patients with traumatic head
Prevention and treatment of hypothermia, acidosis and injuries.
hypocalcemia will ensure optimal function of transfused
coagulation factors.2 Packed Red Blood Cells (PRBCs)
Obstetric conditions associated with the need for blood
transfusion may lead to morbidity and mortality if not The majority of protocols recommended six units of PRBCs
managed correctly. The increasingly important issues in blood be prepared and available and hematocrit be maintained
transfusion are adverse events associated with transfusion, minimally at 21–24%.1,6,7,9,11-13 These recommendations are
including potential infection and potential transmission consistent with a recent survey of obstetricians and practice
of prions, rising costs and the possible future problems of guidelines from the American Society of Anesthesiologists.14
availability. Unfortunately, information from randomized Ideally, the use of a single unit of PRBCs should increase
controlled trials to inform best practice is largely unavailable hematocrit by approximately 3–4% in a 70 kg patient.15
in the discipline of blood transfusion. However, the expected increase in hematocrit may be slightly
The aim of this guideline is to offer guidance about the less due to expanded blood volume during pregnancy. As
appropriate use of blood products that neither compromises noted elsewhere in this toolkit, any patient with continued
the affected woman nor exposes her to unnecessary risk. bleeding after initial measures have failed (Stage 2), should
Strategies to maximize the hemoglobin (Hb) level at delivery have two units of PRBCs released from the blood bank. If
as well as to minimize blood loss are also discussed. It is these are not readily available, consideration should be given
envisaged that this guideline will be incorporated into a for the use of uncross–matched O negative blood while the
protocol for each unit to disseminate locally. blood bank is completing the patient’s type and cross match.
Consistent with recommendations from this toolkit, for
Aim for and Maintain any patient that reaches Stage 3, a massive OB hemorrhage
• Temperature >35°C (pre-warm resuscitation fluids using pack should be prepared, which includes an additional 4
temperature controlled blood warmers and use forced air or 6 units of PRBCs as pre-arranges with the blood bank for
warming blanket). massive transfusion protocol. Good communication with the
• pH >7.2 laboratory regarding the urgency of the situation is essential.
• Base excess < -6
• Lactate < 4 mmol/L Fresh Frozen Plasma
• Ca2+ >1.1 mmol/L
Fresh frozen plasma (FFP) contains nearly all coagulation
• Platelets > 50 × 109/L
factors and can be used up to 24 hours after thawing and up
• PT/APTT <1.5 × normal
to 5 days if relabeled as “thawed plasma.” Concomitant use of
• INR ≤1.5
FFP and PRBCs is recommended during massive hemorrhage.
• Fibrinogen >1.5 g/L
Using a high ratio of PRBCs to FFP (1.5:1 or 1:1) has been
It is optimal to have a single person coordinating commu-
shown to significantly improve survival from hemorrhage after
nication with the transfusion service, laboratory staff and the
trauma.16 Similar recommendations have been established at
hematologist and/or MedSTAR.
centers with existing massive OB hemorrhage protocols with
• Phone the transfusion service and identify themselves
the goal of maintaining the INR at <1.5–1.7. 1,6,11,17,18 If diffuse
(and how to be contacted) as the clinician making the
bleeding is noted, or there is laboratory evidence of DIC and
notification.
the patient has not been crossmatched, initial requests for 4
• Provide the name, unit record (UR) number and location
units of AB-FFP are recommended. AB plasma is usually in
of the patient.
short supply and reserved for infants. However, 2–3 units of
• Inform the Transfusion Scientist “Massive Transfusion
mismatched plasma can be transfused to adults while type
Protocol needs to be activated”
and cross matching is completed. FFP usually requires 30
• Arrange appropriate care post-event, e.g. care in HDU,
minutes to thaw and will not be available immediately. Pre-
adult ICU or inter-hospital transfer.
arrangement with the blood bank for an inventory of thawed
plasma for immediate issue may be considered if plasma
Blood Component Therapy usage volume is sufficient. If the patient enters into Stage 3
• Historically, aggressive fluid resuscitation with crystalloid/ status, there should be no delays in preparation of FFP while
colloid is recommended to maintain normal blood waiting for laboratory results.
104 Section 1 Clinical Hemotherapy
Contd...
11. University of Southern California. Emergency Obstetrical Affects Mortality in Patients Receiving Massive Transfusions
Hemorrhage Protocol. Approved and in use in Department of at a Combat Support Hospital. The Journal of Trauma.
Obstetrics, Women’s and Children’s Hospital. 2007;63(4):805-13.
12. Alfirevic Z, Elbourne D, Pavord S, Bolte A, Van Geijn H, Mercier 17. Stinger H, Spinella P, Perkins J, Grathwohl K, Salinas J, Martini
F, et al. Use of recombinant activated factor VII in primary W, et al. The Ratio of Fibrinogen to Red Cells Transfused
postpartum hemorrhage: the Northern European registry Affects Survival in Casualties Receiving MassiveTransfusions
2000-2004. Obstet Gynecol. 2007;110(6):1270-8. at an Army Combat Support Hospital. The Journal of Trauma.
13. ACOG. Clinical Management Guidelines for Obstetrician- 2008;64(2):S79-S85.
Gynecologists. Obstetrics and Gynecology. 2006;108(4):1039- 18. Riskin D, et al. Reduced Mortality After Implementation of
47. a Massive Transfusion Protocol: A Single Trauma Center
14. Matot I, Einav S, Goodman S, Zeldin A, Weissman C, U E. A Experience. American College of Surgeons. 2008. pp. 13-6.
Survey of physicians' attitudes toward blood transfusion in 19. Ahonen J, Jokela R, Korttila K. An open non-randomized
patients undergoing cesarean section. American Journal of study of recombinant activated factor VII in major postpartum
Obstetrics and Gynecology. 2004;190(2):462-7. haemorrhage. Acta Anaesthesiol Scand. 2007;51(7):929-36.
15. Puget Sound Blood Center. Available at http://www.psbc.org/ 20. Bhuskute N, Kritzinger S, Dakin M. Recombinant factor
therapy/index.htm. VIIa in massive obstetric haemorrhage. Eur J Anaesthesiol.
16. Borgman M, Spinella P, Perkins J, Grathwohl K, Repine T, 2008;25(3):250-1.
Beekley A, et al. The Ratio of Blood Products Transfused
Platelet Therapy
Methodology
Patient’s clinical history and family history of bleeding are
very important. Laboratory investigations carried out in case
of platelet disorders are:
• Platelet count and peripheral blood smear
• PT, APTT, TT to rule out coagulation disorders
• Bleeding time has gradually fallen out of favor as a
clinically useful test. But a prolonged bleeding time should
be considered sufficient to perform additional tests.
• Platelet function analyzer (PFA-100)2
• Platelet aggregation studies using agonists such as ADP,
thrombin, epinephrine, ristocetin.
• Measurement of platelet ADP and ATP content and release
are useful in diagnosis of storage pool and release defects.
• Flow cytometry can be used to measure surface receptor
density in BSS, GT.
Chapter 28 Inherited and Acquired Platelet Disorders: Diagnosis and Therapy 109
• Sequestration as in hypersplenism. treat menorrhagia; rFVIIa may help arrest bleeding. BMT and
Thrombocytosis may be primary (e.g. polycythemia vera, gene therapy have been tried.
MDS) or secondary (e.g. iron deficiency, malignancy).
Uremia: Hemodialysis/peritoneal dialysis; RBC transfusion
Qualitative platelet disorders6 may be: and EPO have been observed to decrease bleeding and
• Congenital: increase platelet adhesiveness but some patients become
– Disorders of adhesion—Bernard Soulier syndrome7 hypercoagulable.
(BSS) (defect in GP Ib-IX) is a rare autosomal recessive
disorder. Giant platelets8 are seen on peripheral blood Platelet Transfusion Triggers10
smear in BSS.
In Von Willebrand disease (vWD), vWF binding to GP Prophylactic: Maintain platelet count in all patients > 10 ×
Ib-IX-V is reduced. 109/L or in stable patients > 5 × 109/L and in patients with
– Disorders of activation—storage pool deficiency coagulopathy > 20 × 109/L. In patients who are already
– Disorders of aggregation—Afibrinogenemia, Glanz bleeding or who are about to undergo a hemostatic challenge
mann’s Thrombasthenia (GT) (defect in GP IIb-IIIa) is such as a surgical procedure, attempts are made to keep the
a rare autosomal recessive entity. platelet count > 50 × 109/L. An even higher target of 100 × 109/L
– Disorder of platelet procoagulant activity – Platelet is often used for intracerebral, pulmonary and ophthalmic
Factor V Quebec hemorrhage. The role of anemia should also be considered in
• Acquired (more common): prevention or treatment of hemorrhage.
– Uremia Therapeutic dose of platelets is 1 unit of RDP/10 kg body
– Myeloproliferative disorders like polycythemia vera, weight or 1 unit of SDP in adults. Calculating the corrected
myelofibrosis and essential thrombocytosis count increment (CCI) and percent platelet recovery (PPR)
– MDS and acute leukemias are clonal disorders in which will help in determining the adequacy of response to platelet
all cell lines are abnormal transfusion.
– Dysproteinemias-paraproteins may coat platelets non-
specifically and interfere with binding
Conclusion
– Cardiopulmonary bypass is associated with platelet
dysfunction Understanding the basic roles of platelets, bone marrow,
– Antiplatelet antibodies spleen and immune system in normal conditions and specific
– Liver disease, various drugs (e.g.) aspirin, heparin. diseases will aid in determining the cause of an abnormal
platelet count. A fundamental knowledge of mechanisms
Clinical Features of Platelet Disorders involved in platelet adhesion and aggregation allows for
better understanding of platelet functional disorders.
Bleeding occurs with falling platelet counts. Sites of bleeding
may be Cutaneous (petechiae, purpura), Mucosal (epistaxis,
menorrhagia, GIT bleed) or within the central nervous system. References
In qualitative platelet disorders, count is usually normal 1. Buchanan GR. Quamtitative and Qualitative platelet disorders.
but bleeding time (BT) is prolonged due to abnormalities Clin Lab Med. 1999;19:71-86.
in function. Clinical features vary. Most patients have 2. Cines DB, Blanchette VS. Immune thrombocytopenia purpura.
mucocutaneous bleeding. N Engl J Med. 2002;346:995-1008.
3. Drews RE, Weinberger SE. Thrombocytopenic disporders in
critically ill patients. Am J Respir Crit Care Med. 2000;162:347-
Diagnosis 51.
Bedside examination, accurate patient and family history is a 4. Fausett B, Silver RM. Congenital disorders of platelet function.
key element in assessing platelet disorders. Clin Obstet Gynecol. 1999;42:390-405.
5. George JN. Platelets. Lancet. 2000;355:1531-9.
6. Lankford KV, Hillyer CD. Thrombotic thrombocytopenic
Management purpura: new insights in disease pathogenesis and therapy.
Transfus Med Revs. 2000;12:244-57.
Bernard Soulier syndrome: General and specific treatment 7. Mhawech P, Saleem A. Inherited giant platelet disorders. Am J
of bleeding, contraceptive therapy after puberty in females, Clin Pathol. 2000;113:176-90.
Platelet transfusion (preferably HLA matched) to treat 8. Rodgers GM. Overview of platelet physiology and laboratory
bleeding during surgery, Desmopressin and rFVIIa. Rarely evaluation of platelet function. Clin Obstes Gynecol. 1999;
hematopoietic stem cell transplant (HSCT). 42:349-59.
9. Shapiro AD. Platelet function disorders. Haemophilia. 2000;
Glanzmann’s thrombasthenia9: Prophylactic platelet trans 6(Suppl 1):120-7.
fusion, Fibrin sealants and antifibrinolytics like Tranexamic 10. Tullu MS, Dixit PS, Nair SB, et al. Glanzmann’s thrombasthenia.
acid to treat localized bleeding, high doses of Progesterone to Indian J Pediatr. 2001;68:563-6.
Transfusion Practice in Dengue
proteins C, S and antithrombin III were noted to decrease the higher levels for patients without additional bleeding
with increasing severity of shock, likely due to increased risk factors such as sepsis, concurrent use of antibiotics or
capillary leakage. Increased thrombomodulin, tissue factor other abnormalities of homeostasis, prolonged high fever (as
and plasmin activator inhibitor type-1 (PAI-1) were observed seen in dengue patients with severe hepatitis and secondary
with thrombomodulin levels increasing with severity of shock, bacterial infections), sepsis, vascular compromise, anatomic
and PAI-1 levels increasing with severity of bleeding. Direct aberrations, significant coagulopathy, uncorrected hemo
activation of fibrinolysis by dengue virus has been implicated, dynamic instability, DHF and severe dengue. Further as
a process that distinguishes hemorrhage in dengue from that per BCSH guidelines,9 a platelet transfusion threshold of
seen in classic disseminated intravascular coagulopathy. 5 × 109/L is identified for patients without any bleeding risk
The presence of fever is viewed as an additional risk factor factors if alloimmunization leading to platelet refractoriness
for bleeding in the presence of severe thrombocytopenia. is a concern. Annals Academy of Medicine Singapore3
Severe thrombocytopenia in dengue occurs around the tail propose that prophylactic platelet transfusion in dengue
end of the febrile period. There is no significant relationship patients may be safely withheld for platelet counts as low as
between bleeding severity and degree of thrombocytopenia.3 5 × 109/L in the absence of additional bleeding risk factors
and should be reserved for patients with major bleeding
(Grade 2 and above in WHO bleeding scale). Careful
Prophylactic Platelet Transfusions in Dengue
observation without platelet transfusion is warranted in
Quantifiable increases in platelet counts and other coagulation stable dengue patients with only minor ‘dry’ bleeding such
parameters are transient in dengue, even more so than in as petechiae, ecchymosis and minor ‘wet’ bleeding such as
other conditions where prophylactic platelet transfusions are mucosal oozing (WHO bleeding scale Grade 1). Singapore’s
administered. In DSS patients who underwent prophylactic clinical practice guideline recommends that in the presence
platelet transfusion, platelet counts, prothrombin time ratio of additional bleeding risk factors or when there is rapid
(PTR), and partial prothrombin time (PTT) returned to pre- decline in platelet count, prophylactic platelet transfusion
transfusion values in less than 5 hours, demonstrating the in pts < 20 × 109/L should be given.10 As per the guidelines
lack of clinical efficacy as an intervention which happens of the Directorate of National Vector Borne Diseases Control
to target a parameter—platelet counts—which is not the Program, Director General Of Health Services, Govt of India5
implicated culprit of major bleeding in dengue.8 guidelines for dengue treatment, platelet transfusion should
The British Committee for Standards in Hematology be given at platelet count < 10 × 109/L without additional risk
(BCSH)9 states that the threshold of 10 × 109/L is as safe as factors.
112 Section 1 Clinical Hemotherapy
Flow chart 1 Volume replacement flow chart for patients with DHF Grades I and II5
Improvement : Hematocrit falls, pulse rate and blood pressure stable, urine output rises.
No improvement : Hematocrit or pulse rate rises, pulse pressure falls below 20 mm Hg, urine output falls.
Unstable vital signs : Urine output falls, signs of shock.
Flow chart 2 Volume replacement flow chart for patients with DHF Grades III and IV 5
• Serial platelet and hematocrit determinations:—Drop in platelets and rise in hematocrit are essential for early diagnosis of DHF.
• Cases of DHF should be observed every hour for vital signs and urinary output.
Table 3: Platelet increments after one unit of random donor surgery, and concurrent amphotericin B therapy. Non-
platelet transfusion5 immune platelet consumption is the most likely cause of a
low CCI when no alloantibody can be found by the laboratory
In adult : 5–10 × 109/L in an adult of 70 Kg body weight
and the patient has been transfused for less than three weeks
In child : 20 × 109/L in child of 18 Kg body weight (unless there has been prior exposure to HLA antigens by
In infant : 75–100 × 109/L transfusion, organ transplantation or pregnancy).12
8. Lum LC, Abdel-Latif Mel-A, Goh AY, Chan PW, Lam SK.
References Preventive transfusion in Dengue shock syndrome—is it
1. Shivbalan S, Anandnathan K, Balasubramanium S, et al. necessary? J Pediatr. 2003;143:682-4.
Predictors of spontaneous bleeding in dengue. Ind Pediatr. 9. Guidelines for the use of platelet transfusions. Br J Haematol.
2004;71:33-6. 2003;122:10-23.
2. Dengue Guidelines for Diagnosis, Treatment, Prevention and 10. Health Sciences Authority–Ministry of Health Clinical
Control–World Health Organization, 2009. Available at http:// Practice Guidelines–Clinical Blood Transfusion, Singapore.
whqlibdoc.who.nt/publications/2009/9789241547871_eng. 2011;3(5):48.
pdf. Accessed July 1, 2011. 11. Schrezenmeir H, Seifried E. Buffy-coat–derived pooled platelet
3. The Rationale Use of Platelet Transfusion in Dengue Fever. concentrates and apheresis platelet concentrates: which
Transfusion Newsletter 8th edn. 2013.pp.1-4. Blood Trans product should be preferred? Vox Sang. 2010;99(1):1-15.
fusion Service of Health Bureau Government of Macao SAR, 12. Delaflor-Weiss E, Mintz PD. The evaluation and management
crystal @ssm. gov. mo. of platelet refractoriness and alloimmunization. Transfus Med
4. Chairulfatah A, Setiabudi D, Agoes R, Colebunder R. Rev. 2000;14:180-96.
Thrombocytopenia and platelet transfusion in dengue and 13. Fatal bacterial infections associated with platelet transfusions−
dengue shock syndrome. WHO Dengue Bulletin. 2003;27:141-3. United States, 2004. MMWR Morb Mortal Wkly Rep. 2005;
5. Clinical Management. In: Guidelines for Clinical Management 54:168-70.
of Dengue fever, dengue haemorrhagic fever and dengue shock 14. Epidemiological News Bulletin: Management of guidelines for
syndrome. Directorate of National Vector Borne Diseases dengue patients at Tan Tock Seng Hospital and communicable
Control Program. DGHS Ministry of Health and Family Welfare, Disease center Singapore. Platelet transfusion. 2005. p.49.
Govt of India. 2008.pp.14-24. 15. Dodd RY. Current viral risks of blood and blood products. Ann
6. Changa K, Dimatatac F, Diana LT Teo, et al. When less is Med. 2000;32:469-74.
More: Can We Abandon Prophylactic Platelet Transfu 16. Dwyre DM, Fernando LP, Holland PV. Hepatitis B, hepatitis
sion in Dengue Fever? Ann Acad Med Singapore. 2011;40: C and HIV transfusion − transmitted infections in the 21st
539-45. century. Vox Sang. 2011;100:92-8.
7. Wills BA, Oragui EE, Stephens AC, et al. Coagulation abnor 17. Chuansumrit A. Control of Bleeding in children with dengue
malities in dengue hemorrhagic Fever: serial investigations in haemorrhagic fever using recombinant activated factor VII: a
167 Vietnamese children with Dengue shock syndrome. Clin randomized, double blind, placebo-controlled study. Blood
Infect Dis. 2002;35:277-85. Coagul Fibrinolysis. 2005;16(8):549-55.
Fetal and Neonatal Alloimmune
Thrombocytopenia
Prasun Bhattacharya
30
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) with the HLA DRB 3*0101. The other antigens involved are
is the most common cause of severe thrombocytopenia in the HPA–5b, HPA-1b and HPA-15.9
fetus and otherwise healthy neonates.1 The overall incidence
of this entity in the Caucasian population has been estimated Clinical Presentation
by prospective studies to be 1 in 800 to 1 in 1000 live birth.2,3
This condition results from incompatibility between the Since its first description by Harrington in 195310, the
parents for platelet-specific antigens. diagnosis of NAIT in the first born child is usually unexpected
These antigens are inherited in the fetus in an autosomal by the family and health care providers and is frequently made
co-dominant fashion. Fetomaternal passage of platelets at birth. The infant can present with petechiae, bruising, and
results the development of the specific antibodies to the bleeding but will otherwise look well. The neonate may be
exposed fetal antigen, which is absent in the pregnant mother. asymptomatic with incidental thrombocytopenia (platelets
This syndrome may be comparable to the hemolytic disease count< 150 x 10 9/L).
of the newborn, although severe thrombocytopenia may With the development of fetal medicine, fetal alloimmune
occur during the index pregnancy.4-6 The thrombocytopenia thrombocytopenia has been recognized as the most severe
results from the maternal immunization against specific form of thrombocytopenia. By the end of the 1st trimester
platelet alloantigens paternally inherited by the fetus. These the fetal platelet count reaches the threshold value, i.e. 150 ×
allo-antibodies can cross the placental barrier as early as 14 109/L. Fetal alloimmune thrombocytopenia may be suspected
weeks of gestation.7 The maternal IgG coated fetal platelets in a case of ICH upto 80% and of them 40% occurring before
are cleared in the reticuloendothelial system resulting mild to 30th gestational weeks.11
severe thrombocytopenia. The most significant complication
of FNAIT is intracranial hemorrhage (ICH) which occurs in Laboratory Investigations
10–20% of affected pregnancies, with 75% of them occurring
before birth.8 When to suspect NAIT in the fetus:
• If fetal thrombocytopenia is incidentally discovered
• If there is a fetal ICH, hydrocephalus or ventriculomegaly
Pathogenesis of FNAIT
or cerebral cysts
Human platelet antigens (HPA) are specific proteins on • If there is a family history of NAIT if there is unexplained
platelet membrane, HPA system is used in the nomen fetal anemia
clature of platelet antigens. There are 24 platelet-specific • If there are unexplained, recurrent miscarriages after the
alloantigens, that have been recognized. They are numbered first trimester
according to the chronological order of discovery. The Detection of maternal platelet-specific antibodies has
platelet alloantigen is then further defined according to the to carried out by two techniques, indirect platelet immuno
frequency in which it appears in the population. A high- fluorescence test (PIFT) and by monoclonal antibody
frequency alloantigen is designated ‘‘a’’ and a low-frequency specific immobilization of platelet antigens (MAIPA). Platelet
alloantigen is designated ‘‘b.’’ HPA-1a is the most common typing of the parent and the newborn may be done by the
platelet alloantigen in the Caucasian population and is molecular techniques as polymerase chain reaction (PCR)
responsible for 80% of the cases of NAIT. The presence of SSP (sequence specific primer) and PCR-RFLP (restriction
antibodies to the HPA-1a antigen is also strongly associated fragment length polymorphism).
Chapter 30 Fetal and Neonatal Alloimmune Thrombocytopenia 117
Drug-induced thrombocytopenia Usually 7–10 days following introduction of a new drug. Platelets <20,000/µL, bleeding complications
Autoimmune thrombocytopenia Not associated with heparin medication
Diabetic ketoacidosis Acute thrombocytopenia with onset of illness
GP IIb/IIIa inhibitor-induced Begins within 12 hours of IIb/IIIa-inhibitor infusion, platelets <20,000/µL, bleeding complications
thrombocytopenia (important differential diagnosis: Pseudothrombocytopenia)
Post-transfusion purpura 7–14 days after transfusion in preimmunized patients (>95% women) , Platelets <20,000/ µL, bleeding
complications
former is performed by ELISA and is highly sensitive but with a optical density value above 0.4 in the PF4-PVS well that can
specificity that ranges from 50 to 90%. In this target complexes be inhibited by high-dose heparin confirms the presence
of PF4 and heparin or heparin-like molecules are immobilized of a heparin-dependent antibody in the patient’s sample.
on a solid phase. To perform the test, the patient’s serum is Although IgG antibodies are the most clinically relevant
added to pre-made complexes of PF4 and heparin or heparin- antibodies causing this syndrome,9 occasional patients with
like molecules (e.g. polyvinyl sulfate, or PVS) alone and in HIT appear to have only non-IgG (IgM or IgA) antibodies.10
the presence of high-dose (100 U/mL) heparin. Heparin- PF4 ELISA assays are available in two forms: those that detect
dependent antibody binds to the complexes and is detected but do not differentiate IgG, IgM and IgA heparin-dpendent
via enzyme-conjugated antihuman immunoglobulin. An antibodies, and those that detect only IgG.
Chapter 31 Clinicopathological Syndrome in Immune-mediated Heparin-induced Thrombocytopenia 121
Timing of platelet count fall Days 5–10, or ≤1 day if heparin >Day 10 or unclear (but fits with HIT), or ≤1 day (no recent heparin)
exposure within past 30 days ≤1 day if heparin exposure within past
30–100 days
Thrombosis or other Proven thrombosis, skin necrosis, Progressive, recurrent, or silent None
sequelae or, after heparin bolus, acute thrombosis; erythematous skin lesions
systemic reaction
Other cause for None evident Possible Definite
thrombocytopenia
Points assigned in each of four categories are totalled, and the pre‐test probability of HIT by total points is as follows: 6 to 8 = high, 4 to 5 = intermediate; 0 to 3
= low. (Adapted with permission from Warkentin et al. Hematology/the education program of the American Society of Hematology. Copyright 2003, American
Society of Hematology.11)
122 Section 1 Clinical Hemotherapy
Although exposure to heparin is usually avoided following • Warfarin should not be used until the platelet count has
the diagnosis of HIT, the antibodies do not usually persist recovered.
beyond 100 days and re-exposure to heparin after this time
does not generally lead to recurrence of HIT. Information to the Patient and
Record Keeping
Prevention
The diagnosis of HIT should be clearly recorded in the
The incidence of HIT can be reduced by: patient’s notes and marked as a serious allergy. The condition
• Limiting courses of heparin to <5 days, if possible. should be clearly explained to the patient and an information
• Using LMWH in place of unfractionated heparin for leaflet may be helpful in this respect. The patient should be
thromboprophylaxis in high‐risk postoperative patients. issued with an antibody card.
• Porcine UFH is associated with lower incidence of HIT
than bovine UFH.
• In general, monitoring the platelet count at least every
References
other day between days 4 and 14 of heparin exposure or 1. Franchini M. Heparin-induced thrombocytopenia: an update.
until heparin is discontinued. Thrombosis Journal. 2005;3:14.
• Taking care not to automatically initiate heparin if a 2. Kelton JG, Smith JW, Warkentin TE, et al. Immunoglobulin
patient is re‐admitted for a thrombotic event; the medical G from patients with heparin-induced thrombocytopenia
binds to a complex of heparin and platelet factor 4. Blood.
record must be thoroughly reviewed for use of heparin on a
1994;83:3232–9, PubMed.
previous admission (within the past 100 days). Unfortu 3. Keeling D, Davidson S, Watson H. Guideline: the management
nately, in some cases, the previous heparin exposure of heparin‐induced thrombocytopenia. Br J Haematol.
may not be recorded in the medical record (for example, 2006;133:259-69, PubMed.
heparin flushes). 4. Greinacher A. Heparin-induced thrombocytopenia. J Thromb
• Orders for use of heparin flushes and heparinized normal Haemost. 2009;7(Suppl.1):9-12.
saline must be written and signed by a physician and use 5. Kickler TS, Ness PM, Herman JH, Bell WR. Studies on
heparin flushes and heparinised normal saline must be the Pathophysiology of posttransfusion purpura. Blood.
documented in the patient’s medical record 1986;68:347-50.
6. Christie DJ, Pulkrabek S, Putnam JL, et al. Posttransfusion
purpura due to an alloantibody reactive with glycoprotein 1a/
Key points IIa (anti-HPA-5b). Blood. 1991;77:2785-9.
7. Seidenfeld AM, Owen J, Glynn MF. Post-transfusion purpura
• Heparin-induced thrombocytopenia (HIT) is an immune‐ cured by steroid therapy in a man. Can Med Assoc J.
mediated event that can have severe life‐ and limb‐ 1978;118:1285-6.
threatening complications. 8. Warkentin TE, Greinacher A. Heparin-induced throm
• Despite thrombocytopenia, bleeding is rare; rather, HIT is bocytopenia: recognition, treatment, and prevention:
strongly associated with thromboembolic complications. the Seventh ACCP Conference on Antithrombotic and
• When a diagnosis of HIT is suspected immediate cessation Thrombolytic Therapy. Chest. 2004;126:311S–37S.
of all forms of heparin, including unfractionated heparin 9. Nugent D, McMillan R, Nichol JL, Slichter SJ. Pathogenesis
of chronic immune thrombocytopenia: increased platelet
(UFH), low molecular weight heparin (LMWH) and
destruction and/or decreased platelet production. Br J
heparin flushes, is imperative. Haematol. 2009;146:585-96.
• Treatment of HIT should be initiated based on clinical 10. McMillan R. Therapy for adults with refractory chronic immune
suspicion and must never be delayed pending laboratory thrombocytopenic purpura. Ann Intern Med. 1997;126:307-14.
confirmation of HIT. 11. Warkentin TE, Aird WC, Rand JH. Platelet–endothelial
• A direct thrombin inhibitor, such as lepirudin, danaparoid interactions: sepsis, HIT, and antiphospholipid syndrome.
or argatroban, is considered the agent of choice for Hematology (Am Soc Hematol Educ Program). 2003.pp.497–
treatment of HIT. 519.
Single Donor Platelets Versus
Random Donor Platelets
Table 1: Relative merits of single donor platelets and random donor platlets
Single donor platelets Random donor platelets
Platelets > 3 × 1011 (equal to platelets obtained from 5–6 whole blood 5.5 × 1010 platelets
donations)
Plasma volume 200 ml Plasma volume 30–40 ml
Leukocytes <5.5 × 106 Leukocytes 5.5 × 108 in each unit
Obviates the need of filtration Filtration is required to reduce leukocytes
Red cells—traces Red cells—more
pH-6.0 or more pH-6.0 or more
Exposes a patient to one donor Exposes a patient to multiple donors
Less exposure to infection More exposure to infection
Low-risk to alloimmunization Relatively more risk to alloimmunization
HLA- or platelet-matched donor product can be prepared for the Not possible
patients who have become refractory to platelets
Decreased risk of bacterial contamination and easy handling More risk of bacterial contamination platelets are pooled
Sophisticated equipment required Routine donation, can be made from whole blood
Highly trained persons required Not much
Poonam Singal
33
Patients with severe thrombocytopenia are presumed to patient’s primary disease, the presence of fever or sepsis,
be at increased risk for bleeding, and consequently, it has concurrent medications, and overall coagulation status. The
been a standard practice for the past four decades to give transfusion trigger varies in different clinical settings as given
allogenic platelet transfusions to severely thrombocytopenic here.
patients as supportive care.1 Thrombocytopenia can result
from myriad causes including marrow suppression e.g. due
to drugs, irradiation, infiltration, intrinsic marrow disease
Transfusion Triggers
such as myelofibrosis, massive transfusion (dilutional
thrombocytopenia), or immune destruction (e.g. immune Prophylaxis for Surgery3
thrombocytopenic purpura, thrombotic thrombocytopenic • Bone marrow aspiration and biopsy may be performed in
purpura, neonatal alloimmune thrombocytopenia). Patients patients with severe thrombocytopenia without platelet
with congenital or acquired platelet disorders, such as support, provided that adequate surface pressure is
Bernard-Soulier syndrome or platelet storage pool deficiency, applied.
may also benefit from platelet transfusion. These individuals • For lumbar puncture, epidural anesthesia, gastroscopy
will frequently have normal platelet counts but diminished and biopsy, insertion of indwelling lines, transbronchial
platelet function that is inadequate for normal hemostasis.2 biopsy, liver biopsy, laparotomy or similar procedures, the
Platelet transfusions may be given either prophylactically platelet count should be raised to at least 50 × 109/L.
to reduce the risk of bleeding, in the absence of clinical • For operations in critical sites such as the brain or eyes, the
hemorrhage (prophylactic transfusions) or to control platelet count should be raised to 100 × 109/L.
active bleeding when present (therapeutic transfusions). It should not be assumed that the platelet count will
Platelet transfusions are not indicated in all causes of rise just because platelet transfusions are given, and a
thrombocytopenia and may indeed be contraindicated in preoperative platelet count should be checked to ensure that
certain conditions. Thus, the cause of the thrombocytopenia the above thresholds have been reached.
should be established before a decision about the use of
platelet transfusion is made. Any decision must also be based
Chronic Stable Thrombocytopenia
on an assessment of risk versus benefit. Risks associated with
platelet transfusions include alloimmunization, transmission In patients who are chronically thrombocytopenic due to
of infection, allergic reactions and transfusion-related acute chemotherapy, bone marrow transplantation or for marrow
lung injury; potential benefits include reducing morbidity conditions resulting in thrombocytopenia such as aplasia or
associated with minor hemorrhage and reducing morbidity/ myelodysplasia:
mortality resulting from major bleeding.3 While there is a • A threshold of 10 × 109/L is as safe as higher levels for
universal consensus to give platelet transfusions in the face patients without additional risk factors. Risk factors
of severe bleeding, controversy persists as to the optimal dose include sepsis, concurrent use of antibiotics or other
of platelets to use, and the appropriate platelet count level at abnormalities of hemostasis.
which to transfuse the nonbleeding patient. It is now generally • For patients without any risk factors, a threshold of
accepted that the decision to transfuse platelets or any blood 5 × 109/L may be appropriate, if there are concerns that
product should almost never be based solely on transfusion alloimmunization could lead to platelet refractoriness.
thresholds (or transfusion triggers). Patient factors should However, accurate counting of low platelet numbers may
always be considered when deciding when an individual create difficulties when trying to reduce the threshold
patient should be transfused. These factors may include the below 10 × 109/L.
Chapter 33 Prophylactic Platelet Transfusion 127
A specific threshold for transfusion may not be appropriate Glanzmann’s thrombasthenia. However, acquired causes of
for patients with chronic stable thrombocytopenia who platelet dysfunction can exacerbate bleeding in patients who
are best managed on an individual basis depending on the already have impaired hemostasis.
degree of hemorrhage. It is recommended that for prophylaxis before invasive
procedures for patients with a known or suspected platelet
Massive Transfusion and Disseminated function disorder:
Intravascular Coagulation • Withdraw drugs known to have antiplatelet activity
• Correct any underlying condition known to be associated
A platelet count of around 50 × 109/L should be maintained. with platelet dysfunction, if possible
In chronic disseminated intravascular coagulation (DIC), • Correct the hematocrit to > 0·30 I/L in patients with renal
or in the absence of bleeding, platelet transfusions should not failure
be given merely to correct a low platelet count. • Consider the use of Desmopressin DDAVP, in patients
with inherited dysfunction defects
Cardiopulmonary Bypass Surgery • Consider the use of DDAVP or cryoprecipitate in patients
with uremia
Cardiopulmonary bypass (CPB) results in the destruction • Use platelet transfusions where the above methods are not
of a significant fraction of the patient’s platelets. In addition appropriate or are ineffective.
there is evidence that the remaining platelets may have their Another important aspect of prophylactic platelet
function impaired by the bypass procedure. Further: transfusion therapy is regarding the dose of platelets to
• There is no place for prophylactic transfusion of platelets be transfused. In practice, a single dose of transfused
in patients undergoing CPB but platelets should be readily platelets generally contains approximately 3 × 1011 platelets
available in all institutions undertaking cardiac surgery. corresponding to 1 apheresis derived (AD)-platelet product
• The preoperative assessment of patients attending for or 5 to 6 pooled whole blood (WB)-platelet units. The optimal
cardiac surgery should include a thorough review of all adult dose of platelets has not been clearly established, and
medications likely to interfere with platelet function. doses are often determined based on factors unrelated to
• The decision to transfuse platelets in patients following efficacy such as cost and availability. For example, an adult’s
CPB remains a clinical decision based on the evidence body weight is not usually considered when determining
of microvascular bleeding in association with excessive the number of platelets to administer. In general, the higher
postsurgical blood loss. a patient’s post transfusion platelet increment, the longer
the interval will be before the next platelet transfusion
Bone Marrow Failure (Due to Disease, is required. Endogenously produced platelets normally
survive approximately 9–10 days in the absence of diseases
Cytotoxic Therapy or Irradiation)
that decreases platelet survival. Transfused platelets do not
Prophylactic platelet transfusions have become standard circulate this long in thrombocytopenia patients, as they are
practice for patients with bone marrow failure. The platelet more rapidly consumed, for instance, to maintain vascular
count cut off for prophylactic platelet transfusion is deter integrity. At platelet count below 100 × 109/ L, a direct
mined in part by the concept that while thrombocytopenic relationship exists between low platelet count and shorter
patients may develop petechiae and ecchymoses (dry platelet life span in the circulation.
bleeding) they will not develop fatal hemorrhagic
complications without first developing extensive mucous Large/Infrequent Versus Small/Frequent
membrane bleeding (wet bleeding). It is recommended to:
Platelet Dosing Strategies2
• Transfuse platelets at count <10 × 109/L in patients with no
or only “dry” bleeding. Several transfusion specialists advocate transfusing doses of
• Prophylactic threshold should be 20 × 109/L in patients platelets larger than the standard 6 WB platelet pool albeit
with risk factors like infection, fever or prior to minor at longer intervals between transfusions, e.g. 10–12 WB-
surgery. platelet units every 2–3 days. The rationale for this practice
is largely based on decreasing the number of individual
Platelet Function Disorders transfusion episodes. This is more convenient in outpatient
settings. As expected, it is possible to obtain a higher post
Patients with platelet function disorders rarely need platelet transfusion platelet increment by transfusing higher platelet
transfusions. Even patients with severe inherited platelet numbers. However, it is unclear whether this practice is
function disorders such as Glanzmann’s thrombasthenia more effective than standard platelet doses in reducing the
only have sporadic bleeding and may have no bleeding risk of spontaneous bleeding. Alternatively smaller doses of
for many years. Negligible or no excessive bleeding can be platelets transfused at shorter intervals (e.g. 3–4 WB-platelets
expected in patients with acquired platelet function disorders per transfusion) may reduce the total number of platelets
as the impairment in platelet function is much less than in required during a patient’s thrombocytopenic period. This
128 Section 1 Clinical Hemotherapy
strategy could decrease the overall number of platelets duration of patients thrombocytopenia will be reduced,
needed for a large patient population and decrease the further decreasing the need for transfused platelets.5
number of exposures for an individual patient. Thus, smaller Therefore, an optimal dose of platelets has not been
platelet doses may be more economical but this practice clearly defined, and transfusion practices will likely remain
has been criticized for possibly increasing the number of largely based on local preferences which will often be heavily
individual transfusions, which increases overall costs.4 influenced by costs and supply.
Another factor which favors the lowering of transfusion
trigger and low platelet dose is the role of thrombopoietin. References
Thrombopoietin has been recognized as the major hema 1. Blajchman MA, Slitcher SJ, Heddle NM, et al. New strategies
topoietic growth factor that stimulates platelet production for optimal use of platelet transfusions. Haematology Am Soc
and it is constitutively made in the liver at a constant rate. Haematol Edu Program. 2008.pp.198-204.
TPO binds to its ligand on the surface of platelets and 2. Fisk JM, Pisciotto PT, Snyder EL, et al. Platelets and Related
megakaryocytes and in the absence of megakaryocytes or Products. Blood Banking and Transfusion Medicine, 2nd edn.
platelets, TPO levels rise. It has been documented that TPO Churchill Livingstone. 2007.p.326.
is adsorbed on the surface of transfused platelets. Therefore, 3. Guidelines for the use of platelet transfusions. British Journal
reductions in the number of transfused platelets by decreasing of Haematology. 2003.pp.10-23.
4. Ackerman SJ, Klumpp TR, Guzman GI, et al. Economic
the platelet transfusion trigger and the platelet dose should
consequences of alterations in platelet transfusion dose:
further reduce the number of platelet transfusions required as analysis of a prospective randomized, double blind trial.
elevated TPO level will stimulate platelet production as soon Transfusion. 2000;40:1457-62.
as megakaryocytes appears in the marrow. With endogenous 5. Slichter SJ. Evidence based platelet transfusion guidelines.
TPO stimulated megakaryocyte platelet production the Haematology Am Soc Hematol Educ Program. 2007.pp.172-8.
Apheresis
Prashant Agarwal
34
There are several challenges in the field of transfusion medi improvement, it is possible to collect multiple doses of
cine in several countries. These are, shortage of blood supply blood element (e.g. platelets, RBCs) or combination of blood
which may be due to less motivation among the population components (RBCs and plasma; platelets and RBCs).1
about blood donation, more need of specific components
Donation criteria—The requirement of donors for MCA are
as compared to blood donation, various regulatory issues,
same as whole blood donation except there are some specific
public pressure from safety of transfusion transmitted
requirements. These are: (1) Extracorporeal volume should not
infections and cost of blood components. So, in the current
exceed 13% of total blood volume, (2) If we plan to collect one
scenario of transfusion medicine it is very necessary to
or more plateletpheresis units, previous platelet count should
advance and implement the technology which can collect
not be less than 150 × 10 9/L, (3) To obtain more than one
multiple components.1
plasma unit, donor should have a total amount of proteins over
Now-a-days the main aim of transfusion medicine are self-
6 g/dL, (4) Erythropheresis require Hb more than 12.5 g/dL for
sufficiency, good quality of blood components, blood donor
women and 13.5 g/dL for men in order to obtain a single unit.
health and satisfaction of patient at the lower cost. Aging of
For double erythropheresis requirements are: volume over
the population, more stringent donor criteria, economic
5000 mL and hemoglobin over 14 g/dL and hematocrit >42%.
and social challenges and increasing complexity of modern
medicine all are responsible for the blood shortage. These Donor selection: Due to shortage of donors it is possible to
are hardly compensated by blood collection unless multi- select a special pool of donors for different routine protocols,
component collection (MCC) strategies are simultaneously e.g.
developed. MCC is an exciting concept in modern era of • B and AB donors are better for plasma and platelets.
transfusion medicine but switching donors from conventional • O and A negative are interesting for RCC (single or double
blood collection to individual components collection which dose)
is termed as apheresis technology, is not so easy.2 • Donors with blood volume > 5000 mL and platelet count
There are several studies which states that over 95% of > 250 × 109/L can be reserved for double platelet protocols.
donors can be switched to MCC and only 15 to 20% refuse • Donors with blood volume > 4500 mL and platelet count
to enlist in MCC programme. But in reality only from 15 to > 270 × 109/L can be used for double dose of platelets, or
20% of eligible donors undergo MCC for reasons ranging from single dose of platelets and a red cell concentrates.
structure limitations, poor organization of blood drives, lack
Blood components: The yield of units should be set to provide
of information, limited number of machines, lack of qualified
maximum benefit to the patients without compromising
personnel and poor appreciation of cost involved. Another
donor’s health, e.g.
reason why MCC is underutilized is because it took years for
• Unit of platelets: 3.5 × 1011 per unit
the industries involved in apheresis to fully understand the
• Double platelets: 7 × 1011 per unit
advantage of MCC in terms of potential for expansion of their
• Unit of plasma: 230 mL
market.2
• Double plasma: 460 mL
Multi-component apheresis (MCA)—Apheresis technology • RCC: 180 mL.
is well established in modern era of transfusion medicine. It Now-a-days it is possible to collect apheresis units of
was first incepted in 1970s. It is widely used to collect platelets, platelets and red cells leucodepleted, HLA and HPA matched
plasma and red cells from allogeneic donors although donors for patients with alloantibodies and more volume of
autologous donation are also utilized. With the technology plasma for donors like IgA deficiency.3
130 Section 1 Clinical Hemotherapy
Multi-component connection has advantages like more References
frequency of donation, more donor safety (less vasovagal re
action), enhance regulatory compliance because by collecting 1. Mark A. Popovsky: Multicomponent apheresis blood collection
in the united states; current status and future direction.
double units of platelets or red cells, less inventory management
Transfusion and Apheresis Science. 2005;32:299-304.
is needed, less donor exposure by double units and collection 2. M Valbonesi, et al. Multicomponent collection as of 2005.
of product from special donors. MCA is one of the tools that Transfusion and Apheresis Science. 2005. pp. 287-97.
enables blood collectors to meet the challenges of supply and 3. Lydia Blanco. Tailored collection of multicomponent by
better quality.1 apheresis. Transfusion and Apheresis Science. 2002. pp. 123-7.
Therapeutic Plateletpheresis
Tulika Chandra
35
Introduction Cerebrovascular events most common clinical finding.
Complications not directly related to platelet count, can be
Plateletpheresis (more accurately called thrombocytapher seen with platelet counts between 500 × 109 – 5000 × 109/L.
esis or thrombapheresis, though these names are rarely Many patients are asymptomatic despite high counts.
used) is the process of collecting thrombocytes, more Thromboses more common in the elderly (> 60) or those with
commonly called platelets, a component of blood involved in cardiovascular risk factors, including previous episodes.
blood clotting. The term specifically refers to the method of Therapeutic plateletpheresis achieves rapid reduction
collecting the platelets, which is performed by a device used in of platelet count, and is usual ly reserved for patients
blood donation that separates the platelets and returns other with acute serious thrombotic or hemorrhagic events, or
portions of the blood to the donor. Platelet transfusion can high risk patients with very high platelet counts (> 1000
be a life-saving procedure in preventing or treating serious × 109/L) (ASFA/AABB Category I Indication). Follow-
complications from bleeding and hemorrhage in patients up procedures can help maintain the lower count
who have disorders manifesting as thrombocytopenia (low until pharmacologic agents start to take effect. Pharmaco
platelet count) or platelet dysfunction. This process may therapy (hydroxyurea, anagrelide) eventually required to
also be used therapeutically to treat disorders resulting in lower platelet count.
extra ordinarily high platelet counts such as essential throm
bocytosis. Practical Considerations of TP
• Can be performed with any centrifugal apheresis
Therapeutic plateletpheresis instrument.
This is occasionally used in patients with myeloproliferative • In emergent cases, peripheral venous access can be used
disorders and symptomatic thrombocytosis until chemo instead of central venous catheter (and avoid the attendant
therapy and/or antiplatelet drug therapy takes effect. risks of catheter placement).
• Instrument configuration, anticoagulation, etc. same as
in a platelet donation, however, a higher flow rate in the
Thrombocytosis and the Rationale for removal line may be desirable.
Therapeutic Plateletpheresis (TP) • Large volume of cells must be removed. Available data
suggest that 30–50% of circulating platelets can be
Thrombocytosis may occur in 3 settings:
removed after processing ~ 1.2 blood volumes over a wide
1. Reactive thrombocytosis in response to splenectomy, Fe
range of platelet counts. STAT intraprocedural count may
deficiency, acute hemorrhage, chronic inflammation, and
be helpful.
malignancy.
• No consensus on the target platelet count after procedure.
2. In myeloproliferative disorders such as CML or essential
thrombycythemia.
Sample Therapeutic Plateletpheresis Orders
3. Rare inherited forms.
Symptoms are only seen in patients with myeloproliferative When ordering plateletphersis, following orders should be
disorders, including both thrombosis and hemorrhage. considered:
• Hemorrhage: Due to abnormal platelet function • Procedure target:
• Thrombosis maybe arterial or venous. – Volume: 2 × TBV (total blood volume).
132 Section 1 Clinical Hemotherapy
• Frequency of procedure: – Use blood warmer.
– Daily until platelet count < 100 × 109/L. – Normal saline: 1–2 L to prime, rinse-back and infuse
• Anticoagulation: during treatment.
– ACD-A Therapeutic plateletpheresis is generally reserved for
• Medication orders: patients with myeloproliferative disorders and hemorrhage or
– Ca gluconate to prevent citrate reaction symptoms. thrombosis associated with an increase in circulating platelets.
– Ca gluconate 10%: Normal saline IV piggyback PRN Many centers consider using plateletpheresis when the
citrate reaction. patient’s peripheral platelet count is greater than 1000 × 109/L,
– Ca gluconate 10%: Normal saline during treatment. although no consistent relation between the level of platelet
• Central venous access care: elevation and the occurrence of symptoms has been found, and
– Flush each lumen of catheter with 10 mL NS followed no generally accepted assay of platelet dysfunction predicts
by Heparin + NS mL to equal internal volume of each which patients are at risk. A single cytapheresis procedure can
lumen. lower the platelet count by 30 to 50%. Plateletpheresis can have
– Flush each lumen of catheter with 10 mL of NS followed dramatic effects for selected patients such as those with evolving
by ACD-A to equal the internal volume of each lumen. digital gangrene. Attempts to maintain thrombocythemic
• laboratory orders: patients at normal platelet counts by cytapheresis alone have
– Preprocedure: CBC with platelet count. not been successful; more practical long-term chemotherapy
– Postprocedure: CBC with platelet count. should be instituted concurrently.
• Standing orders:
– Two CaCO3 tablets (e.g. Tums) PO/PRN every 30 minutes
for citrate symptoms. suggested reading
– Notify consultant doctor for BP < 90/50 mm Hg or 1. Joint United Kingdom (UK) Blood Transfusion and Tissue
> 180/100 mm Hg and for pulse rate < 50 minutes or Transplantation Services Professional Advisory Committee.
> 150 minutes 2014.
– NS IV: 250 mL × 2 and/or 250 mL 5% albumin for 2. Therapeutic plateletpheresis in thombocythemia. Transfusion.
hypotension (BP systolic < 90 mm Hg) PRN. 1979;19(2):147-52.
Therapeutic Plasma Exchange
Aseem K Tiwari
36
Apheresis is the process in which whole blood is withdrawn • Continued production of the substance or mobilization
from a person’s circulation, a component such as plasma is from tissues into the intravascular space will result in less
separated out and retained, and the remainder is returned to apparent reduction despite an equal or even greater total
the patient usually with some replacement fluid. amount removed.
Apheresis may be performed in a healthy blood donor to For a substance that is strictly intravascular, 36.8% of the
harvest a blood component like platelets, red cells, plasma or initial concentration will remain after a single blood volume
even hematopoietic stem cells or apheresis may be performed exchange by TPE. However, if the substance is also present
in a patient as a therapy and is called therapeutic apheresis. in the extravascular space with a total volume of distribution
Therapeutic apheresis is a branch of transfusion medicine equal to twice the blood volume, for example, 60.6% will
encompassing the treatment of diseases through removal of remain after processing a single blood volume. Generally
blood components or specific blood substances. 1–1.5 volume exchange is performed every alternate day
The goal of therapeutic apheresis is to remove a pathologic for most of the disease conditions. However, the exchange
element from the blood. The element may be a plasma volume and frequency may both change depending on the
protein such as an autoantibody as in myasthenia gravis, red disease to be treated.
cells as in sickle cell crisis, leukocytes as in hyperleukocytosis It is very crucial to understand as to which are the right
accompanying acute leukemia, or platelets as in marked indications for TPE. American Society for Apheresis (ASFA)
thrombocytosis. Therapeutic apheresis is sometimes classified has done a very good classification of indications for TPE.
as therapeutic cytapheresis and therapeutic plasmapheresis. Although there are many case reports of successful treatment
Therapeutic cytapheresis is the removal of cellular blood of a large variety of disease and conditions by apheresis,
elements. If red cells are removed and replaced with donor there have been few high-quality trials for justifying TPE in
red blood cells (RBCs), it is called red cell exchange. Thera various indications. ASFA has published a categorization of
peutic plasmaphereis is the separation and removal of plasma indications for apheresis based on the best available evidence
from whole blood with replacement by a colloid solution Table 1.
(typically albumin or plasma) or a combination of crystalloid/ We would like to share our experience in TPE in last few
colloid solution. It is also called therapeutic plasma exchange years. Ideally, physicians ordering therapeutic apheresis
(TPE). So the term therapeutic plasmapheresis and thera procedures on their patients for various indications should
peutic plasma exchange (TPE) is used synonymously. TPE is be aware of the “right” indications. However, actually many
also the most common therapeutic apheresis modality. The practicing physicians commonly overuse, under-use, and
effectiveness of TPE depends on concentration of element in misuse this therapeutic intervention. Transfusion medicine
blood, volume of blood processed and equilibrium between specialists who perform this therapy on patients may steer
blood and the extra-vascular volume of distribution. The the treating physicians in right direction through continuing
principles of TPE are as follows: medical education (CME) which gives them an opportunity
• Apheresis is most efficient at the beginning of the to keep abreast with the latest “right” criteria of ordering
procedure and least efficient at the end. therapeutic apheresis.
• Exchange of a single blood volume will eliminate We at department of transfusion medicine in Gurgaon
approximately two-thirds of a substance (if it does not reviewed, collected, and interpreted the effect of formal CME
move from extravascular sites to the intravascular space). interventions (conferences, clinical rounds, meetings and
134 Section 1 Clinical Hemotherapy
Nidhi Mehta
37
Apheresis is a general term describing a procedure in which Therapeutic Cytapheresis
blood is removed from the body; a particular component of
blood is separated, and rest blood is then returned to body. It is most often used to remove defective RBCs and substitute
Therapeutic apheresis includes plasma exchange and normal ones in patients with sickle cell anemia who have
cytapheresis, which are generally tolerated by healthy donors. the following conditions: acute chest syndrome, stroke,
However, many minor and a few major risks exist. pregnancy, or frequent, severe sickle cell crises. RBC
exchange achieves Hb S levels of < 30% without the risk of
increased viscosity that can occur because of increased Hct
Plasma exchange (PE) with simple transfusion.
It is a procedure in which the plasma is separated from the It may also be used to reduce severe thrombocytosis
blood, discarded in total, and replaced with a substitution or leukocytosis (cytoreduction) in acute leukemia or in
fluid such as albumin or with donated plasma from a healthy accelerated or blast crisis phase of chronic myelogenous
person. This is generally performed to remove toxins or leukemia when there is risk of hemorrhage, thrombosis, or
autoantibodies that have accumulated in the plasma. pulmonary or cerebral complications of extreme leukocytosis
(leukostasis).
Cytapheresis
Erythrocytapheresis
Therapeutic cytapheresis removes cellular components from
blood, returning plasma. A donor procedure in which the equivalent of 1 or 2 units of
The Apheresis Applications Committee of the American RBCs are removed to produce RBCs for transfusion.
Society for Apheresis periodically evaluates potential Therapeutic erythrocytapheresis (TEA) has been used in
indications for apheresis and categorizes them from I to IV different diseases such as polycythemia vera (PV), secondary
based on the available medical literature. The following are erythrocytosis or hemochromatosis as a process of the
some of the indications, and their categorization, from the less cumbersome but more expensive phlebotomy. TEA is
society’s 2013 guidelines. preferred in emergency conditions such as thrombocytosis
or in conditions such as porphyria cutanea tarda (PCT) or
Category I: Disorders for which apheresis is accepted as first-
erythropoietic porphyria when plasma exchange (PEX) is
line therapy, either as a primary standalone treatment or in
often combined with TEA to reduce extracellular levels of
conjunction with other modes of treatment.
uroporphyrin which contribute to plasma hyper viscosity.
Category II: Disorders for which apheresis is accepted as
second-line therapy, either as a standalone treatment or in Leukocytapheresis
conjunction with other modes of treatment.
A procedure in which the WBCs are separated from the blood.
Category III: Optimum role of apheresis therapy is not
The cells may be discarded, as when used to decrease WBC
established. Decision-making should be individualized.
count in acute leukemia, or used for transfusion, as in the case
Category IV: Disorders in which published evidence of granulocyte collection or the collection of hematopoietic
demonstrates or suggests apheresis to be ineffective or progenitor cells.
harmful. IRB approval is desirable if apheresis treatment is Therapeutic WBC removal (leukapheresis) can remove
undertaken in these circumstances. kilograms of buffy coat in a few procedures, and it often
136 Section 1 Clinical Hemotherapy
relieves leukostasis. However, the reduction in WBC count • For intraerythrocytic parasite infections
itself may be mild and only temporary. • Cerebral malaria
• ABO incompatible transfusions
Plateletapheresis • Prevention of Rh immunization after RhD+ transfusions
into RhD recipients
A donor procedure in which platelets are removed to produce
• CO intoxication
a platelet product for transfusion is called plateletpheresis.
• Aniline poisoning
• AIHA
Thrombocytapheresis • Hb-Cheverly
A therapeutic procedure in which platelets are removed and • Hereditary hemochromatosis.
discarded from a thrombocythemic patient. Cytapheresis is Therapeutic erythrocytapheresis (TE) has become a new
effective in thrombocytosis because platelets are not replaced therapeutic modality, which potentially offers a more efficient
as rapidly as WBCs. One or 2 procedures may reduce platelet method to remove iron overload with fewer procedures.
counts to safe levels. Red cell exchange using a cell separator causes rapid
Other uses of cytapheresis include collection of peripheral removal of parasites from the circulation of patients with
blood stem cells for autologous or allogeneic bone marrow high parasite load with renal, pulmonary and cerebral
reconstitution (an alternative to bone marrow transplantation) complications. It offers a rapid approach to treat acute, severe
and collection of lymphocytes for use in immune modulation cases of malaria.
cancer therapy (adoptive immunotherapy).
Babesiosis Conclusion
Tick-borne, malaria-like illness caused by infection of RBCs RCE use on a larger scale will reduce the cost of the procedure.
with Babesia protozoa. Characterized by flu like syndrome, A well-trained team is necessary along with matching
headache, chills, sweats, myalgia and arthralgia. Although equipment, which will make it a clinically very effective
clinical trials are lacking, several case reports and case procedure with low percentage of side effects that are not
series suggest that, given the absence of exo-erythrocytic dangerous to the patient. Despite these wide applications,
phase of infection, RCE combined with antibiotic therapy, data concerning RCE is still limited. We are looking into
can be beneficial in severe cases of babesiosis with >5% this interesting field of therapeutic apheresis for further
parasitemia.1,4,7 randomized trials and widening of applications.
3. Cho G, Hambleton IR. Regular long-term red blood cell 12. Romanovsky A, Djogovic D, Chin D. A case of sodium chlorite
transfusions for managing chronic chest complications in toxicity managed with concurrent renal replacement therapy
sickle cell disease. The Cochrane library. Published online: and red cell exchange. J Med Toxicol. 2013;9:67-70.
8 Jan 2014. Available from: http://onlinelibrary.wiley.com/ 13. Saxena H, Saxena AP. Acute methaemoglobinemia due to
doi/10.1002/14651858.CD008360.pub3/pdf ingestion of nitrobenzene (paint solvent). Indian J Anaesth.
4. Draser E, Igbineweka N, Vasavda N, Free M, Awogbade M, 2010;54(2):160-2.
Allman M, et al. Blood transfusion usage among adults with 14. Adamski J, Hanna CA, Reddy VB, Litovsky S, Evans CA,
sickle cell disease—a single institution experience over ten Marques MB. Multiorgan failure and bone marrow necrosis in
years. Br J Hemat. 2011;152;766-70. three adults with sickle cell beta thalassemia. Am J Hematol.
5. Mahfoudhi E, Lecluse Y, Driss F, Abbes S, Flaujac C, 2012;87:621-4.
Garcon L. Red cell exchanges in sickle cell disease lead 15. Stokley S. Guideline for exchange transfusions in children and
to selective reduction of erythrocytes—derived blood adolescents with sickle cell disease. (internet) 2011 (cited 2014
microparticles. Br J Hemat. 2012;156:545-7. Jun15). Available from: https://www.nuh.nhs.uk/handlers/
6. Swerdlow PS. Red cell exchange in sickle cell disease. downloads.ashx?id=48971.
Hematology Am Soc Hematol Edu Program. 2006.pp.48-53. 16. Wilkinson K, Harris S, Gaur P, Haile A, Armour R, Teramura G,
7. Cheung ATW, Miller JW, Miguelino MG, To WJ, Lin X, Chen et al. Molecular blood typing augments serologic testing and
PC, et al. Exchange transfusion therapy and its effects on real- allows for enhanced matching of red blood cells for transfusion.
time microcirculation in pediatric sickle cell anemia patients: in patients with sickle cell disease. Transfusion 2012;52:381-8.
An intravital microscopic study. J Peditr Hematol Oncol. 17. Baron JM, Baron BW. Red cell exchange is not effective
2012;34(3):169-74. for patients with sickle cell anemia and coexisting warm
8. Udani S, Deshpande A, Kalgutkar S. Exchange transfusion for autoantibody hemolysis. Blood Transfus. 2010;8:303-6.
severe malaria: A comparison of red cell exchange with whole 18. Hackenberg LA, Staudinger T, Bojic A, Locker G, Leitner
blood exchange. IJCCM. 2003;7(2):124-7. GC, Graninger W, et al. Automated red cell exchange as an
9. Riddle MS, Jackson JL, Sanders JW, et al. Exchange transfusion adjunctive treatment for severe Plasmodium falciparum
as an adjunct therapy in severe Plasmodium falciparum malaria at the Vienna General Hospital in Austria: a retro
malaria: A meta analysis. CID. 2002;34:1192-8. spective cohort study. Malar J. 2012;11:158.
10. Cabrera LS, Arroyo MF, Gonzalez FR, et al. Erythrocytapheresis 19. Deshpande A, Kalgutkar S, Udani S. Red cell exchange using
in the management of severe falciparum malaria. J Emerg cell separator (Therapeutic eryththrocytapheresis) in two
Trauma Shock. 2010;3(2):206. children with acute severe malaria. JAPI. 2003;51:926-6.
11. Srivastava A, Chaturvedi A, Gupta SK, et al. Acute nitrobenzene 20. Watanaboonyongcharoen P, Park YA, Poisson JL, et al. Rapid
poisoning: case fatality and importance of methylene blue. Sri increase in parasitemia following red cell exchange for malaria.
Lankan Journal of Anaesthesiology. 2010;18(2):91-3. J Clin Apher. 2011;26(6):315-9.
Chronically Transfused Patients
Transfusion Practice in
Thalassemia
Gagandeep Kaur
39
Introduction Transfusion therapy
Thalassemia is considered as the most common monogenic The current management of thalassemia is based on regular
disorder worldwide with a particularly high frequency in a transfusions and effective chelation therapy. The goals of
broad belt, extending from Mediterranean region, Africa, the transfusion therapy are the correction of anemia, suppression
Middle East, the Indian subcontinent, Myanmar and South- of erythropoiesis and inhibition of gastrointestinal iron
east Asia. About 10% of total world thalassemia patients absorption, which occurs in transfused patients as a conse
belong to the Indian subcontinent, among them 3–4% are quence of increased, although ineffective erythropoiesis.
carriers. These disorders occur due to decreased or absent
synthesis of the globin chains. Alpha and beta thalassemias
are two most important forms of the disorder. Clinically the Whom to transfuse
most severe form of the disorder is classified as thalassemia
The decision to start transfusion in patients with a confirmed
major and is transfusion dependent, less severe but clinically
diagnosis of thalassemia should be based on the presence of
symptomatic disease is called thalassemia intermedia and
severe anemia (Hb < 7 g/dL on 2 occasions, more than two
the generally asymptomatic carrier state is the thalassemia
weeks apart, excluding all other contributory causes such
trait.
as infections). However, even in patients with hemoglobin
> 7 g/dL, other factors should be considered, including age at
Pathophysiology presentation of first symptoms, facial changes, poor growth,
In thalassemia major, the genetic defect results in absence evidence of bony expansion and increasing splenomegaly.
of normal globin chain synthesis with resultant excess of the When possible, the decision to start regular transfusions
other unaffected globin chains. The excess chains precipitate should not be delayed until after the third year, as the risk
in the erythroid precursors leading to intramedullary death of developing multiple red cell antibodies increases, with
and ineffective erythropoiesis.1 The damage is much more subsequent difficulty in finding suitable blood units.
with excess alpha chains hence ineffective erythropoiesis is
markedly increased in beta thalassemia major. The resultant Compatibility testing
anemia leads to increased erythropoietin production; hence
there is stimulation of erythropoiesis both medullary and The usual transfusion policy is to perform ABO and Rh(D)
extramedullary. Increase in plasma volume as a result of typing of donors and patients and subsequent compatibility
shunting through expanded bone marrow and progressive testing. However, there are many minor but clinically signi
splenomegaly aggravate the anemia. Bone marrow expansion ficant blood groups which can lead to alloimmunization
also results in characteristic deformities of the skull and face. in these patients. Once the alloantibodies develop, finding
There is increased gastrointestinal iron absorption due to compatible units may become difficult. Following steps
paradoxical hepcidin suppression from dyserythropoiesis, should be followed before starting the transfusion therapy:
which leads to progressive iron deposition in tissues and a • Extended red cell antigen typing of the patient before
variety of growth, metabolic and organ abnormalities. starting transfusion: The patient should be typed for
Chapter 39 Transfusion Practice in Thalassemia 141
common antigens of the Rh, Kell, Kidd and Duffy systems Patients with thalassemia suffer from chronic anemia and
to prevent RBC alloimmunization. hence require red blood cells. Despite increasingly sophisti
• Ethnically related donors: Homogeneity between donor cated blood additives, RBCs suffer from ex vivo storage and
and patient populations leads to a lower prevalence of undergo biochemical, structural, enzymatic, morphological
alloimmunization. Some workers emphasize on recruit and functional deterioration.4 Although many of these changes
ment of phenotypically matched donors. are reversible following transfusion, the current practice is to
• Antibody screening in the patient prior to each transfu- transfuse red cells in appropriate additive solutions for less
sion. than one week and hence as fresh as possible.5,6
• Hepatitis B vaccination.
• Human leukocyte antigen (HLA) typing of siblings Modified Packed RBCs
and patients should be done. Transfusions from 1st
degree relatives should be avoided if bone marrow
Leukoreduced
transplantation (BMT) is required in future.
• Family study for genetic counseling. To avoid transfusion reactions from antiplatelet antibodies
and antileukocyte antibodies, patients should receive
Transfusion regimen leukoreduced packed red cells. Removal of leukocytes and
platelets is obtained by filtration of whole blood. Reduction
The superiority of regular repeated transfusions, as compared to to 1 × 106 or fewer leukocytes per unit is considered. The
transfusions only for symptomatic anemia, was first recognized critical threshold for preventing adverse reactions attributed
by Orsini in France and later by Wolman and Piomelli in US, to contaminated leukocytes.7
who suggested a transfusion program aimed at monitoring a
basal Hb level sufficient to eliminate hypoxia.2 Several different
Washed Packed RBCs
regimens have been proposed over the years, but at present the
majority of centers choose to transfuse at a pretransfusion Hb In rare patients sensitized to plasma proteins, washed red
level of 9–10 g/dL, and to reach a post-transfusion level of 13– cells may be beneficial.
14 g/dL. This prevents growth impairment, organ damage and
bone deformities, allowing normal activity and quality of life, Neocytes
and is associated with relatively low rates of blood requirement
and of iron accumulation. Post-transfusion hemoglobin These are young red cells with low specific gravity and can
should not exceed 15 g/dL to prevent thromboembolism. survive in the circulation longer than older cells. They can
In untreated beta thalassemia major, the erythropoiesis be separated from donor units’ ex-vivo using cell washers
exceeds 10 times the normal leading to bony abnormalities. or using continuous flow cell separators. Usually 50% of
Gastrointestinal iron absorption increases over and above the the original red cell volume is recovered as neocytes. The
body iron losses when the erythroid activity exceeds 5 times clinical benefits of neocyte transfusions are (a) increase of
normal. There is almost complete suppression of endogenous transfusion interval, (b) reduction in packed RBC transfusion
erythropoiesis with hyper transfusion and super transfusion requirements and (c) reduction in transfused iron by 15%
regimen; however with moderate transfusion regimen the per year per patient. However, two disadvantages are: (a)
erythroid activity exceeds 2.4 times normal. This is below the considerably increased donor exposure and (b) increase cost
critical threshold of five times normal.3 of processing.
Parveen Mittal
40
INTRODUCTION transfusion dependent, whereas the disease has a later
clinical onset and is associated with longer survival
India is an ethnically diverse country with an approximate compared with thalassemia major.
population of 1.5 billion. The frequency of beta-thalassemia • Thalassemia minor: mild asymptomatic disease.8
trait has variously been reported from <1 to 17% and an
Recent advances in detection and therapeutic management
average of 3.3%.
of thalassemia has resulted in substantial improvement of
The word “thalassemia” comes from the Greek word
patients’ survival.
“thalassa” (sea) because of the high prevalence of the disease
in the countries bordering the Mediterranean Sea. Beta-
thalassemia had been traditionally prevalent in and confined ROAD MAP
to the Mediterranean basin, Middle East, North India,
Southeast Asia and the Indochina Peninsula.1-3 • Diagnosing thalassemia
It has been estimated that the prevalence of pathological • Educating and counseling parents
hemoglobinopathies in India is 1.2/1,000 live births, and with • Initiation and monitoring of treatment
approximately 27 million births per year this would suggest • Follow-up
the annual birth of 32,400 babies with a serious hemoglobin • Preventive strategy.
disorder. Within this overall disease classification a 1989 WHO
working group on guidelines for the control of hemoglobin Diagnosing Thalassemia
disorders estimated a 3.9% carrier frequency for β-thalassemia
There are only a few causes for anemia in first year of
in India, encompassing all types of β-thalassemia trait.4–6
life, thalassemia being one of the major causes among
Beta-thalassemia is caused by the reduced synthesis
them. Punjab being a belt of increased incidence of beta
of beta-globin chains, which leaves an erythrocyte excess
thalassemia, we should have a high degree of suspicion in
of unopposed alpha-chains; the resulting ineffective
diagnosing the disease. The delay in diagnosing and treating
erythropoiesis leads to chronic hemolytic anemia.7
the disease portends multisystem complications leading to
Three clinical forms of beta-thalassemia are distinguished, poor quality of life and even mortality.
depending on clinical severity: Patients with the beta thalassemia trait generally have no
1. Thalassemia major: The typical phenotype arising either unusual physical findings. In patients with beta thalassemia
from homozygous or from compound heterozygous major, the physical findings are related to severe anemia,
defects. This kind of anemia is severe, develops during the ineffective erythropoiesis, extramedullary hematopoiesis,
first year of life, and requires life-long transfusion therapy and iron overload resulting from transfusion and increased
for survival. iron absorption. The skin may show pallor from anemia and
2. Thalassemia intermedia: Accounts for up to 25% of cases jaundice from hyperbilirubinemia, and the skull and other
and encompasses a wide variety of molecular defects, bones may be deformed secondary to erythroid hyperplasia
including homozygous, compound heterozygous, and with intramedullary expansion and cortical bone thinning.
double heterozygous ones. The phenotypic spectrum Heart examination may reveal findings of cardiac failure and
extends between the clinically severe thalassemia major arrhythmia, related to either severe anemia or iron overload.
on one end and asymptomatic thalassemia carrier state Abdominal examination may reveal changes in the liver,
on the other end. Anemia in thalassemia intermedia is gallbladder, and spleen. Hepatomegaly related to significant
generally milder than that of the major form and not extramedullary hematopoiesis typically is observed.
144 Section 1 Clinical Hemotherapy
Patients who have received blood transfusions may have between 9.5 and 10.5 g/dl, thus improving his or her sense
hepatomegaly or chronic hepatitis due to iron overload. The of well-being while simultaneously suppressing enhanced
gallbladder may contain bilirubin stones formed as a result of erythropoiesis. But blood transfusion brings along a wagon
the patient’s lifelong hemolytic state. Splenomegaly typically of demerits such as–blood born infections and iron overload,
is observed as part of the extramedullary hematopoiesis which are discussion in detail later.
or as a hypertrophic response related to the extravascular
hemolysis. Iron overload may also cause endocrine Chelation Therapy
dysfunction, especially affecting the pancreas, testes, and
thyroid. Transfusion-associated viral hepatitis resulting in Chronic blood transfusion may lead to iron overload if not
cirrhosis or portal hypertension also may be seen. proper chelation is instituted. Increased amount of iron
One of the most common dilemmas faced by a may lead to multisystem involvement in form of secondary
pediatrician is to differentiate thalassemia from its common diabetes mellitus, hypothyroidism, hypoparathyroidism,
hematological mimickers, iron deficiency anemia being one hypogonadotropic gonadism, congestive heart failure and
of the most common. A major proportion of children in our cardiac arrhythmias.
country are malnourished with a resultant high incidence of
nutritional anemia. There should exist an in hospital protocol Deferoxamine
to differentiate between these similar yet different conditions.
Hematological and biochemical parameters useful in • it may be administered as a subcutaneous infusion over
differentiating these two conditions have been summed up in 10–12 hours 5–6 days a week.
the table given below. • Side effects—ototoxicity with high frequency hearing
loss, retinal changes, peripheral neuropathy and bone
Variable Beta-thalassemia Iron deficiency anemia dysplasia with truncal shortening.
Mentzer index* < 13 > 13
Red cell Normal Increased
Deferasirox
distribution width • The recommended initial oral daily dose is 20 mg/kg
Serum ferritin Normal or increased Decreased body weight. An initial daily dose of 30 mg/kg may be
Serum iron Normal to high <30
considered for patients who require reduction of elevated
body iron levels and who are also receiving more than
Total iron-binding Normal > 360 14 ml/kg/month of packed red blood cell.
capacity (TIBC)
• Side effects—abdominal pain, diarrhea, dizziness, earache
Percent transferrin 30–80 < 10 or pain in the ear, nausea, voice changes, vomiting.
saturation
Hb electrophoresis Increased HbA2, HbF Normal Folic Acid
* Mentzer index = mean corpuscular volume (MCV, in fL) divided by the red
blood cell count (RBC, in Millions per microliter) 200–500 mcg/day may be supplemented so as to tide over the
high turnover state.
MANAGEMENT
Stem Cell Transplantation
Management of beta thalassemic children has to have a multi
prong strategy. Beta-thalassemia can only be cured by allogenic hemato
poietic stem cell transplantation (HSCT). HSCT treatment
of thalassemia has substantially improved over the last two
Caretaker Counselling decades, with advancements in preventive strategies, control
Education of the care takers occupies a major cog in the of transplant-related complications, and preparative regimens.
wheel. Nature of the disease, its chronicity, treatment options
and genetic counselling should be made available for the Splenectomy
parents even at the first sitting.
It may be indicated for patients with thalassemia intermedia
who have a falling steady-state hemoglobin level and for
Blood Transfusion transfused patients with a rising transfusion requirement.
Anemia in thalassemia is due to hemolytic process existing
in the system to clear out the damaged RBCs. This leads to
myriad of clinical symptoms which brings a child to our
Complications of Thalassemia
attention. Decision for transfusion should be individualized Complications associated with beta-thalassemia, aside from
with a goal of maintaining pretransfusion hemoglobin the aforementioned anemia, are as follows:
Chapter 40 Pediatrician and β-Thalassemia 145
• Extramedullary hematopoiesis—pathogenic bone frac- from lifelong transfusions and enhanced iron absorption
tures, spinal cord compression. results in secondary iron overload.
• Growth failure—higher metabolic need, hepatospleno-
megaly interfering with nutritional intake, chelation ther-
apy, endocrinopathy. PREVENTION
• Iron overload Effective prevention programs for thalassemia have been
– Hyperglycemia demonstrated in many European countries where the
– Hypocalcemia due to hypoparathyroidism numbers of carriers of abnormal genes are high. Premarital
– Hypothyroidism screening for thalassemia should be made a standard
– Hypogonadotropic hypogonadism. practice. Most at-risk couples are identified early for prenatal
• Transfusion-associated infections. diagnosis in the first pregnancy and genetic counseling may
• Cardiac arrhythmias and failure. be offered to these individuals to have healthy offspring.
• Cholelithiasis. Screening program need to be supported by public education
and regulatory structures empowering individuals to make
FOLLOW-UP informed decisions and to ensure that people are protected
against discrimination as a result of their test results.
Transfusion requirement in every child varies and the same Thalassemia carriers may be detected mainly through the
should be individualized. The consulting pediatrician should expanded family study of cases with thalassemic diseases.
device a follow-up algorithm incorporating surveillance over Local data are still not available due to inherent difficulties
the major complications of thalassemia and its treatment so in the diagnosis of thalassemia traits. However, laboratory
as to identify the complications early and institute effective progress during the last few years has enabled us to undertake
management. mass screening for beta-thalassemia and Hb E carriers, with
The follow-up algorithm should include: accurate diagnosis using automatic high performance liquid
• Anthropometric assessment including plotting growth chromatography (HPLC).
charts In our community reliable estimates for the prevalence of
• Hemodynamic parameters thalassemia genes are not available at present, and diagnoses
• Echocardiography of the diseases and management of the patients are still far
• Effort tolerance from satisfactory. Thus, the patients and families suffer
• Nutrition miserably due to ignorance about the disease, misunder
• Viral markers standing and malpractices. To improve this situation will
• Periodical monitoring of thyroid, parathyroid and liver require proper education of health personnel, not only the
functions physicians but downstream to the auxiliaries working in the
• Glycemic status communities. Genetic counseling, at least for families already
• Status of iron overload—serum ferritin, iron studies, liver afflicted by the diseases or in those who request it, should
biopsy, T2 weighted MRI of liver and heart be provided. This requires accurate diagnoses and a good
• Monitoring for adverse effects of chelation therapy. understanding of the different disorders. While conventional
therapy involving blood transfusions and iron chelation
PROGNOSIS are intolerably expensive, prenatal diagnosis and selective
abortion are more economical and cost-effective. However,
Individuals with thalassemia minor (thalassemia trait) usually these should not be carried out indiscriminately.
have mild, asymptomatic microcytic anemia. This state does
not result in mortality or significant morbidity. The prognosis PRENATAL DIAGNOSIS
of patients with thalassemia major is highly dependent on the
patient’s adherence to long-term treatment programs, namely • Chorionic villous sampling and DNA analysis in the first
the hypertransfusion program and lifelong iron chelation. trimester.
Allogenic bone marrow transplantation may be curative. • Molecular analysis–β-thalassemia is extremely hetero
geneous with more than 200 mutations described
worldwide.9 In India, about 64 mutations have been
Morbidity and Mortality characterized by studies done at different centers10-13
The major causes of morbidity and mortality in beta- • Fetal blood analysis in the second trimester–In India,
thalassemia are anemia and iron overload. The severe second trimester diagnosis is still done as many couples
anemia resulting from this disease, if untreated, can result at risk are identified late during pregnancy. Fetal blood
in high-output cardiac failure; the intramedullary erythroid sampling is done by cordocentesis at 18–20 week
expansion may result in associated skeletal changes such as gestation and after confirming that there is no maternal
cortical bone thinning. Increased iron deposition resulting contamination in the fetal sample by fetal cell staining
146 Section 1 Clinical Hemotherapy
using the Kleihauer-Betke method, it is analysed by HPLC 5. Borgna-Pignatti C, Rugolotto S, De Stefano P, Piga A, Di
on the variant hemoglobin testing system. Gregorio F, Gamberini MR, Sabato V, Melevendi C, Cappellini
MD, Verlato G. Survival and disease complications in
thalassemia major. Ann N Y Acad Sci. 1998;850:227-31.
CONCLUSION 6. World population data sheet. Washington DC: Population
Reference Bureau; 2009.
In a resource poor setting like that of rural Punjab, pediatricians
7. Kremastinos DT, Toutouzas PK, Vyssoulis GP, Venetis CA,
can make a difference in by forming a local protocol for Avgoustakis DG. Iron overload and left ventricular performance
diagnosis and management of thalassemia. Beta- thalassemia in beta thalassemia. Acta Cardiol. 1984;39:29-40.
being a genetic disorder clusters in families. Management 8. Kremastinos DT, Tsiapras DP, Tsetsos GA, Rentoukas EI,
should be started even before the disease start to exist. The Vretou HP, Toutouzas PK. Left ventricular diastolic Doppler
current era of giant strides in the fields of medicine allows us characteristics in beta thalassemia major. Circulation.
to predict, prevent and eradicated many diseases. Let us heal 1993;88:1127-35.
the world, make it a better place, for the entire human race. 9. Globin Gene Server: http://globin.cse.psu.edu/, accessed on
October 5, 2010).
10. Colah R, Gorakshakar A, Nadkarni A, Phanasgaonkar S, Surve
REFERENCEs R, Sawant P, et al. Regional heterogeneity of beta thalassemia
mutations in the multi ethnic Indian population. Blood Cells
1. Kremastinos DT, Tsiapras DP, Kostopoulou AG, Hamodraka ES,
Mol Dis. 2009;42:241-6.
Chaidaroglou AS, Kapsali ED. NT-proBNP levels and diastolic
11. Sinha S, Black ML, Agarwal S, Colah R, Das R, Ryan K, et al.
dysfunction in beta-thalassaemia major patients. Eur J Heart
Profiling β-thalassemia mutations in India at state and regional
Fail. 2007;9:531-6.
levels: implications for genetic education, screening and
2. Modell B, Khan M, Darlison M. Survival in beta-thalassaemia
counseling programmes. Hugo J. 2009;3:51-62.
major in the UK: data from the UK Thalassaemia Register.
12. Edison ES, Shaji RV, Devi SG, Moses A, Viswabandhya A,
Lancet. 2000;355:2051-2.
Mathews V, et al. Analysis of β-globin mutations in the Indian
3. Olivieri NF, Nathan DG, MacMillan JH, Wayne AS, Liu PP,
population: presence of rare and novel mutations and region
McGee A, Martin M, Koren G, Cohen AR. Survival in medically
wise heterogeneity. Clin Genet. 2008;73:331-7.
treated patients with homozygous beta-thalassemia. N Engl J
13. Kumar R, Tamhankar PM, Panigrahi I, Dalal A, Agarwal S.
Med. 1994;331:574-8.
A novel β-globin mutation (HBB; c: 107 A>G; or codon 35 β
4. Engle MA, Erlandson M, Smith CH. Late cardiac complications
(A to G) at alpha-beta chain interfaces. Ann Hematol. 2009;88:
of chronic, refractory anemia with hemochromatosis.
1269-71).
Circulation. 1964;30:698-705.
Iron Overload in Chronically
Transfused Patients: Diagnosis
and Treatment
Praveen C Sobti, Shruti Kakkar
41
Regular blood transfusion is required for management of a hepatitis and depressed in vitamin C deficiency. Studies have
variety of hematological conditions (Table 1). Each unit of demonstrated a significantly lower risk of cardiac failure and
packed cells transfused adds 200 mg of iron to the body’s iron death if serum ferritin is kept below 2500 µg/L over a decade.
pool. Moreover, many of these patients also have increased A serum ferritin of less than 1000 µg/L, is associated with
absorption of iron from the gut due to chronic anemia. longer survival and reduced risk of cardiac complication and
There is no physiological mechanism for removing this hypogonadism.
excessive iron from the body. Without iron chelation, iron Serum ferritin is measured serially every 3–6 months
gets deposited in liver, heart and endocrine organs leading interval in patients with thalassemia major and chelation
to complications. Adequate iron chelation is the cornerstone therapy tailored accordingly. A trend in serum ferritin is
of management of these disorders. The goal of iron chelation more important than a single value while deciding upon the
has shifted from treating iron overload to prevention of iron chelation regimen.
deposition in tissues.
Liver Iron Concentration
Assessment of Iron Overload Liver iron concentration is the reference standard for
estimating the total body iron.
Regular and accurate assessment of the iron overload forms
Liver iron concentration can be measured by chemical
the basis of an effective iron chelation regimen.
determination on a liver biopsy specimen or by noninvasive
methods like superconducting quantum interference device
Serum Ferritin (SQUID) and MRI.
Serum ferritin is a readily available, inexpensive, indirect Liver biopsy: Liver biopsy is considered the gold standard
measure of body iron stores. Serum ferritin has been shown for estimating the iron overload as it is a direct measure of
to correlate with the body iron stores in thalassemia major but LIC and also gives additional information on liver histology.
underestimate the iron loading in thalassemia intermedia. Normal LIC values are up to 1.8 mg/g dry weight of the liver.
Serum ferritin can be falsely elevated in inflammation, Values of up to 7 mg/g dry weight have been seen in normal
population without any adverse effects. High liver iron
content (LIC > 15–20 mg/g dry weight) has been associated
with worsening prognosis, liver function abnormalities
Table 1: Hematological conditions which may require repeated and progression to liver fibrosis. Liver biopsy is an invasive
transfusions and iron chelation
procedure but the rate of complications in experienced hands
Congenital Acquired has been reported to be less than 0.5%. Fallacious values can
Thalassemia major Myelodysplastic syndrome be obtained due to inadequate sample size (< 2.5 cm core
Thalassemia intermedia Aplastic anemia size) or uneven distribution of iron especially in presence of
cirrhosis.
Sickle cell disease Paroxysmal nocturnal
hemoglobinuria
SQUID
Sideroblastic anemia
Liver iron content (LIC) can be measured accurately by mag
Pure red cell aplasia
netic biosusceptometry using a superconducting quantum
Fanconi’s anemia interference device (SQUID). Only 4 such machines are
148 Section 1 Clinical Hemotherapy
available worldwide, limited availability, high maintenance Table 3: Cardiac T2* and iron overload
cost and requirement for dedicated trained staff limits its
Grading Myocardial T2*(ms)
widespread utilization.
None > 20
MRI Mild 12–20
The concept of magnetic resonance imaging (MRI) assessment Moderate 8–12
of iron overload by MRI came up in 1980’s but only recently Severe <8
the advances in the MRI techniques have made it possible to
use MRI for widespread assessment of iron overload.
The underlying principle is that the organs containing Table 4: Pancreatic R2* and iron overload
iron darken more rapidly than normal tissues on a MRI. The Pancreatic R2* Iron overload
echo or observation time taken by a tissue to become twice as < 30 Hz Normal
dark is represented as T2*. The rate of darkening of a tissue is
30–100 Hz Mild
expressed as R2*. T2* is inversely related to R2* and vice versa
(T2* = 1000/R2*). MRI machines can also measure T2 and R2, 100–400 Hz Moderate
but this is technically more demanding and slow. > 400 Hz Severe
MRI values are affected by the tissue water concentration.
An increase in R2 and R2* is found in dehydrated states due
to concentration effect whereas opposite occurs in fluid
overload. MRI Assessment of Pancreatic Iron Overload
Pancreatic R2* is currently not being used in routine clinical
MRI Assessment of Liver Iron practice but gives complimentary information regarding iron
overload in addition to liver and heart estimates. Pancreatic
Liver iron content (LIC) can be calculated from R2 and
iron overload is a reflection of chronic NTBI exposure.
R2*. R2* is easy to obtain but requires a special software for
Pancreatic R2* is a better predictor of cardiac iron overload
generating iron estimates (Table 2). Both R2 and R2* can be
than liver iron as the kinetics of pancreatic iron loading
converted to biopsy equivalents using calibration curves.
and overloading are intermediate between heart and liver.
The maximum liver iron concentration that can be reliably
Correlation between R2* and pancreatic siderosis is shown
obtained from a MRI is between 30 and 40 mg/g dry weight
in Table 4. Longitudinal studies have demonstrated that
on a 1.5 Tesla machine.
a pancreatic R2* < 100 Hz rules out cardiac iron overload
(negative predictive value 95%). Some authors have used a
MRI Assessment of Cardiac Iron staged approach where patients with pancreatic R2* of < 100 Hz
Cardiac T2*or R2* can be measured using the same scanner are followed with abdominal MRI only but this approach
and software as liver R2* but is more labor intensive. It can needs to be validated. Pancreatic R2* > 100 Hz is also predictive
also give additional information regarding the chamber size of impaired glucose tolerance. Pancreatic MRI is difficult
and function at the same time. Cardiac iron content can be in older patients with thalassemia who have undergone
calculated from T2* as splenectomy due to lobular atrophy and fat stranding and can
Cardiac Fe = 45(T2*)–1.22 give inaccurate assessment of iron overload in these patients.
Cardiac T2* > 20 milliseconds is considered normal and
corresponds to a cardiac iron content of 1.6 mg/g dry weight. MRI in other Iron Overload Conditions
Cardiac T2* of < 10 ms is considered severe iron overload
MRI has been extensively studied in thalassemia major
and these patients are at high risk of developing heart failure
but is also required in other chronically transfused states
(Table 3). Studies have demonstrated that 50% of patients
like Diamond Blackfan anemia, thalassemia intermedia,
with cardiac T2* < 6 ms develop heart failure within a year.
myelodysplastic syndromes and sickle cell anemia. Cardiac
iron overload is higher in Diamond Blackfan anemia and
Table 2: Liver T* and iron overload least common in thalassemia intermedia.
Grading Hepatic T2*(ms)
None > 6.3
Iron Chelation
Mild 2.7–6.3 The primary objective of chelation therapy is to keep body
Moderate 1.4–2.7
iron within safe limits. The labile plasma iron constitutes the
chelatable iron pool as iron cannot be directly removed from
Severe < 1.4 ferritin or hemosiderin. This accounts for the slow removal of
Chapter 41 Iron Overload in Chronically Transfused Patients: Diagnosis and Treatment 149
iron once it is accumulated in the body. What constitutes the Prospective studies of changes in serum ferritin in thalassemia
safe iron levels depends upon the diagnosis and the chelation major patients have shown a dose dependent fall in serum
being used. ferritin with DFO. A mean daily dose of 42 mg/kg lead to a
decrease in serum ferritin of 364 µg/L over a year compared
When to Start Chelation to a fall of 1000 µg/L with a mean daily dose of 51 mg/kg.
A trend in serum ferritin gives information on compliance
Iron chelation should be initiated as soon as transfusions have rather than iron content of body at a given time.
caused enough iron accumulation to cause tissue damage.
Traditionally, with desferrioxamine iron chelation is started
when the following criteria are met due to fear of toxicity. Effect on Liver Iron
• Age of the child ≥ 2 years DFO is effective in controlling liver iron if administered in
• After first 10–20 transfusions adequate doses at least 5 times a week. Randomized trials
• Serum ferritin > 1000 µg/L have demonstrated that the effect of DFO on iron balance
• LIC > 7 mg/g dry weight. depends on the rate of transfusional iron loading and the
With the availability of newer oral iron chelators, how early dose given. A negative iron balance could be achieved in 75%
chelation can be initiated remains to be seen. The primary patients receiving DFO at 39–45 mg/kg/d, five days a week, if
goal should be prevention of iron loading of hepatocytes thus transfusional iron loading rate was between 0.3 and 0.5 mg/
preventing the secondary distribution to endocrine organs kg/d. With higher transfusional iron loading rates (> 0.5 mg/
and heart. kg/d), negative iron balance could be obtained in less than
50% patients at same dose, but increased to 86% at a dose of
Desferrioxamine 50 mg/kg/d.
Desferrioxamine (DFO) is the first iron chelator discovered
in 1962. This hexadentate chelator is obtained from Effect on Cardiac Iron
Strepyomyces Pilosus. Due to its high molecular weight, the
oral bioavailability is negligible. It has a short half-life of 20 Subcutaneous DFO changed the clinical progression in
min and the iron free drug is rapidly metabolized in liver. thalassemia major. Before the introduction of DFO, most
The iron is excreted in both urine and feces (Table 5). The patients of thalassemia major succumbed to iron induced
urinary iron is derived from breakdown of red blood cells cardiac failure in second decade of life. Treatment with
in macrophages whereas the fecal iron is derived from iron continuous intravenous DFO has been shown to reverse the
chelated within the liver. symptomatic cardiac failure.
Continuous intravenous DFO has also been shown to
reduce the cardiac iron overloaded in severely iron loaded
Effect on Serum Ferritin hearts (T2* < 6 ms). It lead to an average increase in T2* of
Desferrioxamine (DFO) lead decrease in serum ferritin about 3 ms/year. It would take several years for the cardiac
has been seen in clinical experience over 4 decades now. T2* to normalize at this linear rate.
Yazdi Italia
42
Sickle cell anemia is a hereditary hemoglobin disorder found frequencies of alpha thalassemia and of high levels of fetal
amongst different tribal populations of India. So far it is a hemoglobin (HbF) in those populations.
neglected rural health issue and there is no national policy for These steady state levels are maintained despite rapid
comprehensive sickle cell program. Gujarat is the first state hemolysis by expansion of the bone marrow and greater
to include Comprehensive Sickle Cell Program in the state erythropoietic turnover, manifest in the peripheral blood by
health services. an increased reticulocytes count. Hemoglobin levels fall if
When we talk about anemia, we always think about iron this erythropoietic response is limited by nutritional factors
deficiency and treatment starts with iron therapy. But in case of (iron, folate or other deficiencies), metabolic abnormalities
sickle cell anemia iron is mostly normal unless malnourished. (renal impairment), or infections (septicemia, parvovirus-
Hence all the patients, whose blood is not examined for sickle induced aplastic crisis), with accelerated hemolysis
cell tests are misdiagnosed and mistreated. (chronic hypersplenism) or red cell pooling (acute splenic
Hemoglobin S can cause RBCs to change to a sickle shape. sequestration).
The sickle cells are rigid. Rigidity and abnormal shape reduce The rate of decline is determined by the specific complica
their ability to be propelled through tiny/narrow capillaries tion and is most rapid in acute splenic sequestration (falls
and formation of entangled masses of cells in larger blood of 3-5 g/dl within hours), the aplastic crisis (1 g/dL/day),
vessels. The obstruction of the blood flow produces temporary or septicemias, and a gradual decline to a new hematologic
or permanent organ dysfunction or structural changes. equilibrium may occur in chronic hypersplenism, nutritional
The sign and symptoms of sickle cell disease is pallor, deficiencies and chronic renal failure. The symptoms
frequent jaundice, bone and body ache, enlarged spleen, are determined to some extent by the speed of decline in
retarded growth, frequent infections and dactilytis. hemoglobin levels and in chronic nutritional deficiency or
Life span of RBC in sickle cell disease is less than 30 days renal failure, levels as low as 2-4 g/dL may be surprisingly well
instead of 90 to 120 days. Anemia results from the bone tolerated.
marrow’s inability to produce enough blood cells to keep pace The role of transfusion in the management of lowered
with the rate of destruction. This is one of the major factors for hemoglobin levels in SS disease is determined by the
mild to moderate anemia in sickle cell disease patients. underlying cause and there are often other more appropriate
Homozygous sickle cell (SS) disease is associated with therapies to address the diagnosis.
a lowered hemoglobin level which is characteristic of each Sickle hemoglobin within the red cell behaves with a low
patient in the clinical steady state. This clinical steady state is a oxygen affinity4,5 releasing more oxygen in the periphery than
state at particular hemoglobin level the sickle disease patient HbA. Oxygen affinity of HbS varies widely between patients
can perform his normal duties, without much discomfort. with SS disease and is inversely related to hemoglobin level,6
This level varies widely between patients and is influenced patients with the greatest shift in their oxygen dissociation
by genetic and environmental factors. In disease of African curves tending to run lower hemoglobins, not necessarily
origin, levels are typically 6–9 g/dl but steady state values in because their hemolysis outstrips the bone marrow response
individuals may vary as widely as 5–11 g/dL.1 but because at steady state levels, oxygen delivery may be
In the sickle cell disease associated with the Asian near normal even at low hemoglobin level.
haplotype, occurring in the eastern Province of Saudi Arabia Because of this variation, hemoglobin levels cannot be
and western and central India, steady state hemoglobin levels assumed to reflect oxygen delivery and patients should be
are typically 8–11 g/dl,2,3 probably influenced by the higher treated according to symptoms rather than their hemoglobin
154 Section 1 Clinical Hemotherapy
level. Transfusions requested in patients with hemoglobin Sickle cell disease is one of the major public health
levels ≥ 8.0 g/dl, is difficult to justify outside the context of a problems in rural India where blood transfusion services are
regular chronic transfusion program. Sickle disease patients still under development, where there are maximum numbers
should not be treated like thalassemic patients and regular of sickle disease patients. Further, in India we have highest
transfusion is not indicated depending upon the hemoglobin numbers of patients of sickle cell disease in the world. Thus
content. there is an urgent need to strengthen blood bank services and
Blood transfusions can be life saving for sickle cell disease also to avoid unnecessary blood transfusions to sickle cell
patients in certain life threatening complication like disease patients in rural part of India.
• hemolytic crisis Optimal therapy in sickle cell disease requires regular
• sequestration crisis review of patients when clinically well to document the
• Aplastic anemia steady state levels and provide the baselines against which
• Acute chest syndrome to evaluate changes in hematological indices.6 Hence blood
• Stroke etc. transfusion in sickle cell anemia should not depend upon the
Further blood transfusion also has an important role in hemoglobin percentage but look at the patient’s symptoms
prevention of life threatening conditions in sickle cell disease and vital signs and decide for transfusion, i.e. Treat the
patients like stroke, postoperative acute syndrome, etc. Patient and Not the Number...........
Transfusion is not a harmless procedure but carries risks
of red cell alloimmunization, transfusion acquired infections,
and delayed transfusion reactions and in those receiving References
repeated transfusions, there are additional problems of iron
overload and of maintaining venous access. Furthermore, the 1. Hayes RJ, Beckford M, Grandison Y, Mason K, Serjeant BE,
unit cost of processing and matching blood and the delivery Serjeant GR. The haematology of steady state homozygous
of transfusion have to be considered. Even the patients of sickle cell disease. Frequency distributions, variation with age
and sex, longitudinal observations. Br J Haematol. 1985;59:369-
sickle cell disease in India are getting free blood as per the
82.
February 2014 directives of NBTC, a judicious use of blood 2. Padmos MA, Roberts GT, Sackey K, Kulozik A, Bail S, Morris JS,
is recommended. All transfusions should always be weighed Serjeant BE, Serjeant GR. Two different forms of homozygous
against the risk and benefits to the patients. sickle cell disease occur in Saudi Arabia. Br J Haematol.
It is currently impossible to state whether all transfusions 1991;79:93-8.
are clearly indicated and result in patient benefit. Alternative 3. Kar BC, Satapathy RK, Kulozik AE, Kulozik M, Sirr S, Serjeant
therapies such as splenectomy for chronic hypersplenism BE, Serjeant GR. Sickle cell disease in Orissa State, India.
have been shown to be effective and require assessment in Lancet ii: 1986. pp. 198-201.
4. Seakins M, Gibbs WN, Milner PF, Bertles JF. Erythrocyte Hb-S
India where this complication may be more common. There
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is an urgent need to document the patients’ steady state blood in sickle cell anemia. J. Clin Invest. 1973;52:422-32.
hematological indices, to record reticulocyte counts and 5. Abdu A, Gomez-Marquez, Aldrich TK. The oxygen affinity of
red cell indices and carefully assess the possible causes of sickle hemoglobin. Respir Physiol Neurobiol. 2008;161(1):92-4.
lowered hemoglobin levels. Until such data are available, a 6. Mehta V, Mistry A, Raicha B, Italia Y, Serjeant G. Transfusion
more conservative transfusion policy may benefit both the in Sickle Cell Disease: Experience from a Gujarat Centre. The
patients and the health care delivery services. Indian Journal of Pediatrics 2013.
Advanced Therapies for
Treatment of Hemophilia
Bleeding Disorders:
Lab Diagnosis
plasma should be prepared by centrifugation at 2000 gyri for of the system. So each lab should calculate its own normal
15 minutes at 4°C. range. The common causes of prolonged APTT are:
• DIC
Prothrombin Time • Liver disease
• Massive transfusion with stored blood
Prothrombin time assess the extrinsic and common pathway. • Administration of heparin or contamination of sample
Tissue factor, thromboplastin and calcium are added to with heparin
platelet poor citrated plasma. Prothrombin measures efficacy • Circulating anticoagulant
of V, VII and X on the fibrinogen concentration of plasma. • Deficiency of coagulation factors other than factor VII.
Recently recombinant thromboplastins are used and they
are replacing the thromboplastin from rabbit brain. For the A long APTT with a normal PT indicates possible deficiency
control of oral anticoagulation, a preparation is calibrated of factor VIII, IX, XI, or XII. When APPT is prolong, 50:50
against International thromboplastin, hence commercially mixture of normal and test plasma should be tested. If in this
available thromboplastin will have its ISI determined and mixed plasma APPT corrects by more than 50% of difference
clearly available. With most rabbit thromboplastin, the between the two clotting time tests, a factor deficiency is
normal range of thromboplastin is between 11 and 15 seconds indicated. Poor correction suggests an inhibitor, which may
and for recombinant thromboplastin it is between 10–12 be either one of the clotting factors or of the lupus type.
seconds. Each laboratory should establish its own normal e.g. APTT control : 30 sec.
range. For the purpose of oral anticoagulant monitoring, APTT Test : 60 sec.
international normalized ratio (INR) has been widely 50:50 mix : 40 sec.
adapted. This overcomes the reagent's variability when using This shows good correction, means there is factor deficiency.
ISI. A sensitive reagent has low ISI, around 1.0. If 50:50 mix : 52 sec.
The results are expressed in ratio to calculate INR This shows poor correction, it means there is circulating
inhibitor which includes lupus anticoagulant.
Test PT = Prothrombin ratio (PTR) APTT is used as a test for screening of lupus anticoagulant,
Control PT different conditions and reagents may be employed to
International normalized ratio (INR) optimize sensitivity. Lupus anticoagulant is present in up
INR = [PTR]ISI to 2% of plasma samples and not associated with increased
bleeding. In general the APTT is insensitive to the presence
Prolongation of PT Indicates of low molecular weight heparin (LMWH). The test may
be normal or only modestly prolonged in the phase of
• Administration of oral anticoagulant drugs therapeutic LMWH levels in the plasma. Those bleeding due
• Liver disease particularly obstructive to overdose of LMWH need assay for antifactor Xa activity.
• Vitamin K deficiency Factor XII deficiency is amongst the most common causes
• DIC of unexpected prolongation of APTT and is present in 2%
• Very rarely undiagnosed Factor VII, X, V of prothrombin of healthy subjects. Mixing studies are done to distinguish
deficiency or defect. between factor deficiency and the effect of an inhibitor.
Information Provided by TEG fixed on the top of rotating shaft, which is guided by a high
precision ball bearing system. In this the cycle time is 10 per
• The time it takes for the first fibrin strand to be formed, is minute. The exact position of axis is detected by reflection of
similar to activated clotting time, PT, APTT. light by a small mirror, which is attached to the shaft. The loss
• Gives information about the kinetics of the clot formation. of elasticity upon clotting of the sample leads to a change in the
• The strength of clot rotation of the shaft. The opto-mechanical detection method
• Quantitative and qualitative information about the hypo provides good protection against the impact of vibrations
coagulation, normocoagulation and hypercoagulation and mechanical shocks. Transportation and installation of
disorder in patients. instrument is simple.
• Reaction time (hr) measures the intrinsic pathway hence
factor VIII IX XI and XII.
Platelet Mapping
There are various parameters of TEG (r) tracing, which
measures different stages of clot development: Good correlation between Thrombelastography (TEG)
R : This is time period from initiation of test to Platelet Mapping and LTA is seen. Perioperative TEG Platelet
the initial fibrin formation. Mapping is used as routine service for patients presenting
K : This represents the dynamics of clot formation; for surgery who have failed to omit their antiplatelet
it is the time from beginning of clot formation medication. Conservative value of <30% platelet inhibition
until the amplitude of thromboelastogram is considered safe to proceed for surgery. Postponement of
reaches 20 mm. surgery was avoided in 85% patients with recent ingestion
Alpha angle : This provides information about kinetics of of antiplatelet drugs. In addition, TEG platelet mapping
fibrin build up and cross linking. enables to administer informed rather than empirical platelet
MA : Maximum amplitude, reflects strength of clot transfusions in both elective and emergency patients. Recent
which is dependent on number and function use of antiplatelet drugs by patients before operation poses a
of platelets and its interaction with fibrin. dilemma as to whether to proceed with surgery or not. TEG
MA60 : This measures the rate of amplitude 60 allows rapid quantification of an individual's response to
minutes after MA and represents the stability antiplatelet agents, TEG platelet mapping to be a very useful
of clot. perioperative tool, which has led to improved patient care,
EPL : This measures degree of lysis 30 minutes after empirical platelet transfusions and financial savings in terms
MA. (Estimated percentage lysis). of reduced surgical cancellation.
Meenu Bajpai
45
The liver is the primary site for synthesis of most procoagulant Defects in Secondary Hemostasis
and anticoagulant factors. When the liver is compromised
there is a decreased synthesis of both categories of the factors Liver is site of:
leading to a tenuous rebalance of hemostasis. These patients • Synthesis of coagulation/anticoagulation factors
have a much lower reserve and are therefore at risk of both • Metabolism of coagulation/anticoagulation factors
hemorrhage and thrombosis. Routine laboratory assays • Gamma decarboxylation of coagulation factors.
are not able to assess the risk of bleeding and therefore it is Liver disease patients may have hypocoagulability or
difficult to plan prophylactic blood component therapy. hypercoagulability.
Chronic liver disease affects primary and secondary Hypocoagulability may be due to vitamin K deficiency
hemostasis as well as the fibrinolytic system. as decreased bile production causes decreased vitamin K
absorption leading to decreased decarboxylation of Factors
II, VII, IX, X. There is an inadequate production of other
Defects in Primary Hemostasis
coagulation factors synthesized in liver along with an
There are changes in number as well as function of platelets
in patients with liver diseases. Thrombocytopenia is due to
increased platelet sequestration by the liver and spleen. There Table 1: Site of synthesis of the coagulation factors
is also a reduced production of thrombopoietin by the liver. Factor name Roman numeral Source
The half life of platelets is also reduced due to auto-antibodies
Fibrinogen I Liver
(more common in patients with HCV infection and primary
biliary cirrhosis). Prothrombin II Liver**
In addition to low platelet counts there are also defects in Tissue factor III Damaged cells
platelet function in these patients. The causes of thrombo Calcium IV Gut and bone
cytopathy are as follows.
Preaccelerin V Liver and platelets
Proconvertin VII Liver**
Intrinsic Factors
Antihemophilic VIII Platelets and endothelium
• Defective TxA2 synthesis factor
• Storage pool deficiency Christmas Factor IX Liver**
• Abnormalities of the platelet Gp Ib (proteolysis of receptors
Stuart-Prower X Liver**
by plasmin).
factor
Plasma XI Liver
Extrinsic Factors thromboplastin
• Abnormal HDL antecedent
• Reduced hematocrit Hageman Factor XII Liver
• Endothelium-derived NO and prostacyclin, two potent Fibrin-stabilizing XIII Liver
platelet inhibitors. factor
Hemostasis is maintained in these patients by com
** Dependent on vitamin K for synthesis in liver
pensatory mechanisms such as increased production of von Source: Nowale TJ, Handford GA (2009). Pathophysiology: Concepts and Appli-
Willebrand factor by the endothelium and diseased liver. cations for Health Care Professional. (3rd edn). McGraw-Hill. NY.
Chapter 45 Management of Coagulopathy in Liver Diseases 163
Flow chart 1 Secondary hemostasis in liver disease to decreased hepatocyte function associated with increased
consumption due to DIC or hyperfibrinolysis. The levels of
coagulation factors VIII and vWF are increased as they are
acute phase reactants and are synthesized by both liver and
endothelial cells. Fibrinogen is also an acute phase reactant
and may be normal or Increased. These alterations lead to a
concomitant thrombophilic predisposition (Flow chart 1).
Fibrinolysis
All factors involved in fibrinloysis are produced in the liver
exceptions being tissue plasminogen activator (tPA) and
plasminogen activator inhibitor-1 (PAI-1). Hyperfibrinolysis
is the result of of alterations in the levels of the various factors
involved. In acute liver failure there may be hypofibrinolysis
dus to elevated PAI-1 (Flow chart 2).
Conclusion
The typical liver disease patient has multiple and opposing
factors that influence hemostasis and clot formation.
This explains the apparent paradox of the prolonged global
coagulation tests and their poor prediction of bleeding risk.
increased consumption of by ongoing fibrinolysis (D dimers)
due to increased tPA levels along with low levels of plasmin
Overview of Management
inhibitor factor. Liver disease also leads to dysfibrinogenemia
due to increased sialic acid residues on fibrinogen leading to The management of coagulopathy in liver disease patients
impaired fibrin polymerization (Flow chart 1). involves the following:
Hypercoagulability is due to decreased levels of anti- • Red blood cell transfusions
coagulant factors—Protein C, Protein S, ATIII, protein Z due • Vitamin K
• Half life of 2–3 hours • Dose in liver disease patients 5 mg oral or 10 mg subcuta-
• Dosage ranges from 30 to 120 µgm/Kg neous daily over 5 consecutive days.
• Dramatic improvement in PT and arrests bleeding • Intramuscular route avoided due to risk of hematoma.
• Effect short-term, very expensive • Intravenous route has a higher risk of reactions.
• May be used prior to procedure/not for prophylaxis.
Summary
Vitamin K Hemostasis in liver disease patients is in a rebalanced state and
• Bile salts and products of pancreatic lipolysis are res laboratory values may not correctly represent the coagulation
ponsible for absorbtion of Fat soluble vitamin K. profile of the patient. Blood component support is imperative
• It is a cofactor for the post-translational gamma car in bleeding patients. In patients who are not actively bleeding
boxylation of coagulation factors II,VII, IX and X as well as unnecessary transfusions should be avoided. A summary of
protein C and S. guidelines for prophylactic component transfusions in liver
disease patients undergoing invasive procedures is given in
Table 3.
coagulation factors, thrombocytopenia and acidosis. Its with inhibitors. However, more complications/events have
activity is maintained at lower temperature when overall been reported with the off label use of rFVIIa.
hemostatic function of the body is impaired. rFVIIa is being used as a general hemostatic agent to
Optimal dose and dosing regimen is still not well defined for enhance hemostasis in patients with bleeding who may
rFVIIa. The standard modality is intermittent infusion of rFVIIa or may not have coagulation defects. However, the drug
every 2 to 6 hours. However, studies have reported continuous is expensive. The literature reports no survival benefit in
infusion to be more effective for maintaining rFVIIa levels. patients with off label indications. Thus, it should be used
Laboratory monitoring of hemostatic efficacy in patients cautiously in non approved situations.
treated with rFVIIa is complex. Standard PT and aPTT test do
not correlate well with the clinical picture. A modified version
of the Staclot VIIa-rTF (Diagnostica Stago, France) assay is Suggested reading
being used for monitoring patients on treatment with rFVIIa. 1. Dutta TK, Verma SP. Rational use of recombinant Factor VIIa
Thromboelastography has also shown promising results in in clinical practice. Indian J Hematol Blood Transfus. 2014;30:
monitoring of hemostatic effects when using rFVIIa. 85-90.
2. Midathada MV, Metha P, WanerM, Fink LM. Recombinant
Side Effects Factor VIIa in the treatment of bleeding. Am J Clin Pathol.
2004;121:124-37.
Few side effects in the form of thromboembolic events have 3. Poon MC. Use of recombinant Factor VIIa in hereditary
been reported with the use of rFVIIa in hemophilia patients bleeding disorders. Curr Opin Hematol. 2001;8:312-8.
Plasma Versus Recombinant
Clotting Factors
Rajesh C Gopal
47
Management of coagulation disorders, say hemophilia has inhibitors, the usual forms of clotting factors may not
come a very long way with the advancement of transfusion effectively prevent or stop bleeding. Children are more likely
medicine and myriad technological breakthroughs support to develop inhibitors than adults.
ing its pursuits.
The two nightmarish and relatively common coagulation
disorders viz., hemophilia A and hemophilia B have
Viral Infections/TTIs
been traditionally managed with the ever increasing By the late 1960s, scientists and manufacturers developed
armamentarium which has included but is not limited to: methods for separating factor VIII and factor IX from pooled
• ‘Fresh’ whole blood plasma, resulting in neatly packaged bottles of freeze-dried
• Fresh frozen plasma (lyophilized) factor VIII or factor IX concentrates. Each bottle
• Low or intermediate potency, cryoprecipitate had a label indicating the amount of factor VIII or factor IX it
• High potency human factor VIII, animal factor VIII contained, allowing more accurate dosing. By the early 1970s,
• Recombinant Clotting Factors: the availability of these concentrates led to home treatment,
– Recombinant human factor VIII greatly changing the lives of people with hemophilia.
– Licensed recombinant factor IX product However, there was a big price to be paid for this
– Recombinant activated factor VII. advancement. Thousands of plasma donations were
Till the 1960s, fresh frozen plasma (FFP) was the mainstay combined for preparation of one batch of plasma-derived
of treatment for hemophilia A and hemophilia B. One bag of factor VIII or factor IX concentrate. In the early 1980s, human
FFP contains only small amounts of factor VIII and factor IX, blood, plasma, and plasma-derived products were found
thus large volumes of intravenously administered FFP are to be the responsible for transmission of potentially lethal
required to effectively manage bleeding episodes. Children blood-borne viruses, including hepatitis viruses and HIV.
generally need to be hospitalized for treatment of bleeding Manufacturers of plasma-derived clotting factor concentrates
into a knee, an elbow, or any other joint. did make all efforts to kill these viruses with dry heat, solvent-
detergent treatment and pasteurization, but could not
Clotting factor concentrate succeed in inactivating all the pathogens. In the USA, by
mid-eighties most patients with hemophilia were switched
Plasma factor concentrate is made from human plasma. The to heat-treated concentrates, but many of them had already
plasma is processed to separate the clotting factors from the acquired HIV infection and that ultimately proved fatal for a
other parts of the plasma. This creates the clotting factor big percentage of them. Safety of plasma-derived products
concentrate. The concentrate is processed to kill any viruses in the hemophilia community emerged as a major concern
that might have been in the plasma. amongst all the stakeholders.
Subsequently better screening methods for blood donors
Demerits of Plasma factor were developed, improving the safety of donated plasma.
Screening of donors for the hepatitis B virus was already in
concentrates place, and in 1989 the hepatitis C virus (HCV) was isolated,
allowing HCV antibody testing of donors to begin in 1990.
Inhibitors
HIV was identified in 1984, and by 1985 a blood test for HIV
Some people develop antibodies to the injected clotting antibodies was instituted in blood and plasma collection
factors. These antibodies are called inhibitors. If one develops facilities.
Chapter 47 Plasma Versus Recombinant Clotting Factors 169
A great advancement came in the mid-1960s with musculoskeletal function in adolescents and young men with
the discovery of a method for preparing factor VIII from hemophilia.
FFP by allowing it to thaw in the cold (cryoprecipitated There is almost no risk of an infection from a recombinant
plasma). This preparation could be stored in frozen form as clotting factor. Recombinant clotting factors are made in a
“cryoprecipitate.” This allowed intravenous administration laboratory/by genetic engineering. They are not obtained
of more factor VIII in a smaller volume, allowing outpatient from blood and are made with recombinant DNA technology.
treatment for bleeds and even elective surgery in persons They are concentrated into a powder form that is then mixed
with hemophilia A. This more concentrated form of clotting with sterile water and injected.
factor VIII rapidly became the preferred treatment for acute Since their transition into the clinic, the recombinant
bleeding episodes in patients with hemophilia A. versions of both factor VIII and IX have proven to be
The risk of getting a viral infection from plasma factor remarkable facsimiles of their plasma-derived counterparts.
concentrates is very low. These products are processed to kill The broad adoption of recombinant therapy throughout the
viruses. The products are also tested to make sure they do not developed world has significantly increased the supply of
contain viruses. clotting factor concentrates and helped advance aggressive
The successful cloning of the factor VIII gene in 1984 was therapeutic interventions such as prophylaxis.
a major breakthrough, allowing production of recombinant Incidentally, the recombinant era for hemophilia began in
human factor VIII (r factor VIII). It was followed by clinical
the early 1980s with the cloning and subsequent expression
trials in humans.
of functional proteins for both factors VIII and IX. Efficient
By 1992, two pharmaceutical companies had licensed r
production of recombinant clotting factors in mammalian cell
factor VIII products for use in hemophilia A. Cloning of factor
culture systems required overcoming significant challenges
IX was first reported in 1982, and a licensed r factor IX product
due to the complex post-translational modifications that
(Bene FIX) became available for people with hemophilia B in
were integral to their pro-coagulant function. The quick
1997.
Other significant advances that have been made in recent development and commercialization of recombinant
years include treatment for patients with inhibitors (anti clotting factors was, in part, facilitated by the catastrophic
bodies that inhibit or interfere with the function of factor VIII impact of viral contamination of plasma-derived clotting
or factor IX) and prophylaxis (treatment to prevent disease). factor concentrates at the time. By 1992, two pharmaceutical
Inhibitor antibodies develop in approximately 30–35% of companies had licensed recombinant factor VIII products for
people with hemophilia A and 1–3% with hemophilia B. use in hemophilia A. Cloning of factor IX was first reported
Scientists have also developed innovative treatment in 1982, and a licensed recombinant factor IX product (Bene
products for ‘bypassing’ the inhibitor, such as recombinant FIX) became available for people with hemophilia B in 1997.
activated factor VII (r factor VIIa), which was first licensed for The major on-going challenges in hemophilia care have
use in hemophilia in 1997. been effectively addressed by recombinant DNA technology
Newer second-generation products to treat inhibitors are as follows:
actively being developed, and while much progress has been • Reduced costs of therapy
made in treating inhibitors, this complication remains the • Increased availability in the developing world
greatest problem in the management of hemophilia today. • Improved functional properties of these proteins.
With safer clotting factor replacement products, pre On June 26, 2013, the US. Food and Drug Administration
ventive (prophylactic) treatment is being widely accepted approved Rixubis [Coagulation Factor IX (Recombinant)] for
thereby resulting in larger preservation of normal joint and use in people with hemophilia B who are 16 years of age and
• 1201 consecutive open heart surgical procedures in 1169 Blood product Number of Mild reactions Serious TRALI
transfusions reactions
patients
– Adults: 921 Erythrocytes 1.552.216 1748 / 114.8* 101 / 6.5* 5
- Transfused 610 (66%) Thrombocytes 157.858 462/292* 20 / 12.7* 4
- Transfused with Octaplas: 450 Octaplas 350.751 65/18.5* 6 / 1.7* 0
– Octaplas only: 108
*n = 12
- Children (≤15 years): 279 Transfused 198 (71%)
- Transfused with Octaplas: 122
– Octaplas only: 63. Risk of Fibrinolysis
The study showed In 2003, de Jonge et al reported laboratory signs of fibrinolysis
• That Octaplas is an adequate substitute for FFP in open during OLT when using Octaplas, but no significant differences
heart surgery in blood loss. After introduction of low dose Aprotinin neither
• Only mild allergic reactions venous thromboembolism (VTE) nor fibrinolysis occurred in
• No red blood cell antibodies 120 subsequent OLT treated with Octaplas.
• Viral safety was specially analyzed in 343 patients with
adequate preoperative and 6–12 months follow-up blood Risk of Venous Thromboembolism
samples
– This analysis demonstrated no transmission of viral • In Norway where more than 3000 PE have been performed
disease caused by: HBV, HCV, HIV, HTLV, CMV or HAV with Octaplas, we consider TTP patients to be at risk
and Parvovirus. for VTE when thrombocytes normalize during PE, and
• Octaplas in liver transplantation therefore routinely take precautions with
– The period 2005-2009 was specially analyzed • LMW heparin
– 250 consecutively transplanted patients (214 adults, 36 • Elastic stockings.
children)
– No major thromboembolic events Pharmacoeconomics
– No episodes of TRALI (or other severe transfusion
Cost-effectiveness of European SD plasma
reactions)
• Because of low residual risk for recognized viral infections,
• Plasma exchange with octaplas
cost-effectiveness of pathogen reduction is low in
– Over 3000 plasma exchanges with Octaplas (10–20
industrialized countries.
patients per year since 1993).
• Cost of a quality adjusted life year is over US $ 7.5 million.
– In 2010-2011: 435 plasma exchanges were performed
– Cost-effectiveness of SD treatment combined with
on TTP/HUS patients, and in no instances was there
elimination of TRALI and significant reduction of
observed venous thromboembolism as a complication
allergic/anaphylactic adverse events (Octaplas/
of the plasma exchange.
Octaplas LG)
Experience: Octaplas is safe and effective, allergic reactions • Depending on the estimated frequency of TRALI the cost
are only rarely observed (Table 2). of a quality adjusted life year has been calculated between
US $ 8315 and US $ 40000.
Clinical experience and studies with European SD plasma
• 18 studies and retrospective analyses (4 prospective
randomized) covering all indications for European SD
Conclusion with European SD plasma
plasma, document the excellent clinical efficacy and • Over 12.5 million bags of Octaplas or licensees have been
tolerance of Octaplas. transfused to more than 4 million patients
• The studies included different kind of patients • Octaplas can be used in all indications for all patient
– Coagulopathy/DIC associated with surgical bleeding, groups including newborns
cardiac surgery, obstetric complications, intrauterine – TTP and liver transplant patients can be transfused
transfusions, liver disease or liver transplantation. safely
– Inherited coagulation disorders • There is no need for regular FFP
– Plasma exchange due to TTP or immune modulation • Special blood cell components including exchange trans-
– Neonates, children and adults. fusions, can be prepared in Octaplas
Chapter 48 Solvent Detergent Treated Plasma in Clinical Use 173
• Incidence of anaphylactic and allergic reactions including 4. Hellstern P, Solheim BG. Use of Solvent detergent treatment
serious adverse reactions, are significantly reduced in pathogen reduction of plasma. Transfus Med Hemother.
• No TRALI reactions have been documented 2011;38(1):65-70.
5. Lerner RG, Nelson J, Sorcia E, Grima K. Evaluation of solvent/
• No cases of virus disease transmission have been reported
Detergent treated plasma in patients with a prolonged
• Hospital blood banks and clinicians are satisfied with the prothrombin time, Vox Sanguinis. 2000;15(3):161-7.
safety and clinical efficacy of the standardized product 6. Riedler GF, Haycox AR, Duggan AK, Dakin HA. Cost
with a predictable effect effectiveness of solvent/detergent-treated fresh frozen plasma,
Vox Sanguinis, 2003. p. 85.
Suggested Reading 7. Sinnott P, Bodger S, Gupta A. Presence of HLA antibodies
in single donor derived FFP compared with pooled solvent
1. Edel E, Al-Ali HK. Efficacy and safety profile of solvent/ detergent treated plasma (Octaplas), European Journal of
detergent plasma in the treatment of Acute thrombocytopenic Immunogenetics. 2004;31(6):271-4.
Purpura. Transfus Med Hemother. 2010;37(1):13-9. 8. Theusinger OM, Baulig W, Seifert B, Spahn DR. Relative
2. Flamholz R, Hye-Ran Jeon, Baron JM. Study of three patients concentrations of haemostatic factors and cytokines in solvent/
with Thrombotic Thrombocytopenic Purpura exchanged with detergent treated plasma and FFP, International Scientific
solvent/detergent treated plasma: Is its decreased protein S Meeting in Anaesthesiology, 2013.
activity clinically related to their development of deep venous 9. Ulrich JH Sachs, Dorte Kauschat. White blood cell reactive
thrombosis, Journal of clinical apheresis. 2000;15(3):169-72. antibodies are undetectable in solvent/detergent plasma,
3. Giancarlo Liumbruno, Angela Lattenzio, Gina Rossetti. Recom Transfusion. 2005;45(10):1628-31.
mendations for the transfusion of plasma and platelets, Italian 10. WHO Technical Report: Guidelines on viral inactivation and
society of Transfusion Medicine and Immunohaematology. removal procedures intended to assure the viral safety of
2009;7(2):132-50. human blood plasma products, World Health Organization,
Series no-924,2004.
Cryoprecipitate
Aradhna Sharma
49
Cryoprecipitate is the common term for the cold insoluble • Factor XIII deficiency
plasma proteins that precipitate in fresh frozen plasma when • Source of Fibrin Glue used as topical hemostatic agent
it is thawed between 1°C and 6°C and then centrifuged, in surgical procedures and to remove fragmented renal
collected and refrozen. calculi.
Initially developed in the mid-1960s by Judith Pool and • Uremic coagulopathy
colleagues, cryoprecipitate revolutionized the treatment of • Reversal of thrombolytic therapy
hemophilia and von Willebrand disease (vWD), previously • Massive transfusion with bleeding.
dependent on whole blood or fresh plasma.
Common misuses
Cryoprecipitate constituents
• Fibrinogen replacement with normal fibrinogen levels and
and their stability no evidence of increased dysfunction or consumption.
The main constituents of cryoprecipitate are the high • Reversal of Warfarin therapy.
molecular-weight proteins fibrinogen, factor VIII, von- • Treatment of impaired surgical hemostasis in the absence
Willebrand factor (vWF), factor IX, and fibronectin. Remain of hypofibrinogenemia.
ing components include small amounts of immunoglobulin • Treatment of hepatic coagulopathy with multiple factor
G, IgM, and albumin. deficiencies.
Each bag has approximately.
Plasma : 10–15 mL Doses and Administration of
Factor VIII : 80–100 IU Factor VIII Concentrates
Fibrinogen : 150–250 mg Cryoprecipitate contains a negligible amount of red blood
von-Willebrand Factor : 40–70% cells and only a small amount of the isohemagglutinins anti
Fibronectin : 55 mgm A/or anti B so cryo may not be ABO identical, however, ABO
compatible cryoprecipitate is preferred. The compatibility
Factor XIII : 20–30% of the original
testing is not necessary, Rh type need not be considered when
Several studies show that fibrinogen levels remain stable using this component.
and adequate up to 24 hrs after thaw at room temperature In neonatal recipients, cryoprecipitate should be ABO
and in units kept up to 8 years in frozen storage.1 In contrast, compatible because of the smaller plasma volumes in infants.
factor VIII levels appear less stable than fibrinogen over time.2 A dose of 1 unit of cryoprecipitate per 10 kg body weight
can be expected to increase the fibrinogen concentration by
Indications approximately 50 mg/dL in the absence of heavy consumption
or bleeding. The frequency and duration of administration is
• Hemophilia A based on the underlying condition.
• von Willebrand disease Factor VIII content of cryoprecipitate varies with each
• Congenital or acquired fibrinogen deficiency donor and blood group. In blood group A, factor VIII level is
• Acquired factor VIII deficiency (e.g. DIC, massive usually found more than other groups.3
transfusion)
Chapter 49 Cryoprecipitate 175
In calculating the dose, it can be assumed that 1 IU of Factor VIII assay should be done to serve as a guide to
factor VIII/kg body weight will increase the patients factor therapy. In emergencies, the aPTT can be used as a rough
by 2% (0.02 IU/mL). Thus, if the patients base line factor VIII guide to factor VIII activity.
level is 0.01 IU/mL, a dose of 20 IU/kg body weight would be In mild or moderate hemophilia A, Desmopressin
expected to raise the level to 40% (0.4 IU /mL). (DDAVP) 0.3 ug/kg intravenous, is the treatment of choice.
The half-life of factor VIII is 10-12 hours. Antifibrinolytic agents (aminocaproic acid and tranexamic
If 20 units/kg body weight are required, a person of 70 kg acid) are useful in controlling bleeding in oral cavity. The
weight will require 1400 IU of factor VIII. recommended dose of aminocaproic acid is 75 mg/kg body
CRYO could be used to supply 1400 IU of factor VIII, but at weight every 4–6 hours, that of tranexamic acid is 25 mg/kg
80 IU per bag this would require at least 17–18 bags of cryo. body weight every 6–8 hours. Antifibrinolytic agents are given
1 day preoperative and continued for 7–10 day. The therapy of
The amount of factor VIII required can also be calculated as
choice for severe hemophilia A is Factor VIII concentrate. Factor
follows:4
VIII concentrate, 500 IU bottle, available as pharmaceutical
Weight (kg) × 70 mL/kg : Blood volume (mL) product, is the product of choice for most of the hemophiliacs.
Blood volume (mL) × (1.0- : Plasma volume (mL)
Hct) Preparation, storage and
Units of factor VIII required : (Desired factor VIII level in units/mL quality control
Initial level of factor VIII/mL) × Blood is collected in a triple CPD/CPDA blood collection
plasma volume in (mL)
bag and fresh frozen plasma is separated. Frozen plasma
for cryoprecipitate preparation may be stored for up to 12
Example months at −30oC. The cryoprecipitate may be prepared at any
time during these twelve months.
A 70 kg severe hemophiliac with hematocrit of 40% has initial
Frozen plasma is allowed to thaw in a thawing water
factor VIII level of 2 IU per dl (0.02) IU/mL, 2% activity). How
bath at 4oC or in a refrigerator overnight. Overnight thawing
many units of factor VIII concentrate should be given to raise
process results in a plasma protein precipitation rich in factor
his factor VIII level to 50 IU/dl?
VIII, in the form of white flakes of varying size.
70 kg × 70 mL/kg : 4900 mL (Blood volume) The bags are removed and centrifuged at 5000 × for five
minutes at 4oC (heavy spin). The supernatant plasma is
4900 × (1.0-0.40) : 2940 mL (Plasma volume)
separated into transfer bag by hanging the bag in the inverted
2940 × (0.50-0.02) : 1411 IU of Factor VIII required position, leaving the cryoprecipitate adhering to the walls of
bag.
This dose will increase the level to 50 IU/dl. The tubing is sealed and the cryoprecipitate bag is
The duration of treatment with factor VIII depends upon separated immediately. The cryoprecipitate is stored at −18oC,
the type and location of hemorrhage. After major surgery, the transported in a frozen state, and used within 12 months of
factor VIII level should be maintained above 40–50 IU/dl for the original date of collection.
at least 10 days. To assure the quality of cryoprecipitate, the CFR dictates
that quality of antihemophilic factor and fibrinogen should
be tested monthly on at least four representative containers of
Hemophilia A- Recommended Doses of Factor VIII
cryoprecipitate. With every quality assurance testing, the AABB
Type of bleeding Doses Frequency standard states that unpooled components of cryoprecipitate
(Factor antihemophilic factor should contain a minimum of 150 mg of
VIII IU/kg)
fibrinogen and 80 IU of coagulation factor VIII.
Acute hemarthrosis
Early 10 Seldom required Quality control of cryoprecipitate
Late 20 20 IU every 12 hours Parameter Quality control Frequency of control
Intramuscular hemorrhage 20–30 20 IU every 12 hours Volume 10–20 mL Occasionally
Life-threatening situation 50 25-30 IU every
Factor-VIII 80–120 units Occasionally
Intracranial hemorrhage 8–12 hours
Major surgery Von Willebrand factor 40–70% of the original Occasionally
Major trauma Factor-XIII 20–30% of the original Occasionally
Severe abdominal pain 20 20–25 IU every 12 hours Fibrinogen 150–250 mg Occasionally
Extraction of permanent 20 20 IU every 12 hours Fibronectin 55 mg Occasionally
teeth Often not necessary
75% units sampled and tested should have the values
Tongue or mouth laceration 20 20 IU every 12 hours indicated here.
176 Section 1 Clinical Hemotherapy
2
Cellular Therapies
Stem Cell Transplantation:
Brief Overview
Pankaj Malhotra
50
There have been recent advances in the field of stem cell from unrelated donor who has HLA match or when donor and
transplantation especially in the last two decades. This recipient differ by one HLA antigen. There is also increasing
procedure is the only curative option for some of the use of peripheral blood stem cell or progenitor cell transplant
benign as well as malignant hematological disorders. The (PBPC) for allografting. Even in autologous bone marrow
procedure should preferably be called hematopoietic stem transplant (BMT), there is an increasing use of peripheral
cell transplantation (HSCT) because the source of stem blood stem cell (progenitor cell) transplant. The use of PBSC
cells could be from bone marrow, peripheral blood or results in more rapid engraftment, shorter hospital stay and
umbilical cord. HSCT is generally performed for treatment thus decreased medical costs.
of patients with genetic disorders like thalassemia major; Before BMT/PBPC transplantation, patient’s bone
malignant disorders like acute leukemia and in conditions marrow is conditioned with high dose chemotherapy with or
of hypofunctioning of marrow like aplastic anemia. The without total body irradiation. The purpose of conditioning
procedure is called Allogeneic if donor and recipient are is to destroy the recipient marrow including the tumor
of different genetic origin and autologous if patient’s own cells if present. By destroying the cells populating the bone
marrow is transfused (transplanted) after chemotherapy and marrow, twin objectives of creating space for engraftment
or radiotherapy (Table 1). and destroying host immune effector cells to reduce rejection
For Allogeneic BMT, selection of donor is determined by of the allografted stem cells are achieved.
human leukocyte antigen (HLA) matching. HLA is composed The donor stem cells are collected by stem cell apheresis
of closely linked genes on chromosome 6. There is one in technique from peripheral blood or by multiple marrow
four chance of HLA matching between two siblings. The most aspiration from iliac crests. Mononuclear cell count or
widely used transplants are those between HLA-identical preferably CD 34 counts is calculated on these products to
siblings. However, there is now increasing use of marrow know the adequacy of collection. It is then given to recipient
by intravenous infusion (there is no surgery involved in stem
Table 1 Types of hematopoietic stem cell transplantation cell/bone marrow transplantation). The donor’s marrow cells
Source Type Donor almost go exclusively to recipient marrow and start producing
different series of normal cell lines usually within 2–4 weeks.
Bone marrow Allogeneic Sibling
Use of colony stimulating factors (G-CSF, GM-CSF) has
Voluntary donor shortened the period further down to between 1–2 weeks.
Syngeneic Twin sibling However, during this period these patients need utmost
Autologous Self care to prevent complications due to thrombocytopenia
and granulocytopenia. Life-threatening bleeding due to
PBSC* Allogeneic Sibling
thrombocytopenia is prevented by platelet transfusions.
Voluntary donor Granulocytopenia is a major risk factor for bacterial and
Syngeneic Twin sibling fungal infections in immediate post transplant period and
Autologous Self is prevented by appropriate antibiotics and antifungals. All
blood products are given to these patients after irradiation to
Umbilical cord blood Allogeneic Sibling
prevent graft versus host disease.
Unrelated Successful engraftment is signaled by rise in granulocyte
* Peripheral blood stem cell and platelets and appearance of reticulocytes of the donor’s
180 Section 2 Cellular Therapies
marrow origin in the peripheral blood film. Proof of expanding. However, it still continues to be a procedure
engraftment depends on use of cytogenetic and other genetic with fairly high morbidity and mortality. It is hoped that
markers to distinguish donor from host cells. the results shall improve following better understanding of
The complications that follow successful transplant are transplantation biology as well as better methods for control
graft versus host disease (GvHD) which can be acute or and management of various complications.
chronic infections, veno-occlusive liver disease, recurrence
of leukemia and miscellaneous (hemmorrhagic cystitis, Suggested Reading
sterility, leukoencephalopathy, cataract, etc.). Immunological
reactions due to immunocompetent cells from donor are not 1. Baron F, Storb R. Allogeneic hematopoietic cell transplantation
always harmful. In fact to a large extent, graft versus tumor as treatment for hematological malignancies: a review.
Springer Semin Immunopathol. 2004;26:71-94.
effect is thought to be responsible for cures among patients
2. Giralt S, Bishop MR. Principles and overview of allogeneic
with malignant disorders. hematopoietic stem cell transplantation. Cancer Treat Res.
The success of transplant depends upon the age of the 2009;144:1-21.
patient and the disease for which the transplant is performed. 3. Gyurkocza B, Rezvani A, Storb RF. Allogeneic hematopoietic
It is more successful in younger age group (less than 40 years) cell transplantation: the state of the art. Expert Rev Hematol:
although allogeneic BMT and PBPC are performed even 2010;3:285-99.
after this age. The overall results of BMT and PBPC are still 4. Körbling M, Freireich EJ. Twenty-five years of peripheral blood
poor in elderly age group because of decreased tolerability to stem cell transplantation. Blood. 2011;117:6411-6.
5. Vaughan W, Seshadri T, Bridges M, Keating A. The principles
conditioning regimen and increased risk of infections.
and overview of autologous hematopoietic stem cell trans
With improvement in supportive techniques and use of plantation. Cancer Treat Res. 2009;144:23-45.
effective treatment for graft versus host disease; there has 6. Wingard JR, Hsu J, Hiemenz JW. Hematopoietic stem cell trans
been an improvement in the outcome. The list of indications plantation: an overview of infection risks and epidemiology.
for hematopoietic stem cell transplantation has been Infect Dis Clin North Am. 2010;24:257-72.
Stem Cell in Regenerative
Medicine: Applications and
Cautions
RR Sharma
51
Stem cells are considered the master cells, capable of unknown. In addition, we must understand the role of
both self-renewal and multilineage differentiation. Recent immune cells that may reject donor cells or trigger other
investigations have identified them as a potentially novel immune reactions leading to failure of the therapy or even
cell therapy for regenerative medicine largely because of worse, death of the patient. Added to these biological issues
their ability to differentiate into many functional cell types. are the regulatory requirements for conducting clinical trials
It is anticipated that stem cell therapy could solve many and the complexities of cellular products. Many aspects of
medical challenges facing humanity, increase our knowledge drug development including toxicology, development of
of pathogenesis, allow us to screen for new drugs for safety potency assessment, determination of dosing and safety
and effectiveness, and treat a variety of diseases. It is highly assessment in animal models are not readily applicable to
expected that detailed investigations of stem cell biology cellular products. For example, testing in animal models is
and clinical applicability will result in revolutions in medical complicated due to the human origin of the clinical cellular
technologies. Most early work on stem cells was carried out products and xeno rejection in animals. In many cases the
with pluripotent embryonic stem (ES) cells derived from inner animal equivalent is biologically different and cannot be
cell mass of blastocyst embryo, which, however, introduced used in place of the human cellular product. Scientists have
a series of ethical problems in clinical applications. To developed mouse models of immune compromised mice
avoid such ethical issues and create histocompatibility, new that enable engraftment of the human cells. However, these
technologies have enabled tissue cells to become induced models obviously do not evaluate the immune aspects of
pluripotent stem (iPS) cells.1 One characteristic of ES cells the cellular therapy. So how can Regenerative Medicine be
and iPS cells is their ability to form teratomas, which, in turn, developed?
is a major concern for future clinical application.2,3 Moreover, As detailed in the perspective article by Daley,7 there are
it is likely that iPS cells are more tumorigenic than ES cells.4 many clinical trials in progress around the world evaluating
The teratoma-forming property of stem cells is considered cellular approaches to disease. Many of these studies
a major obstacle for biomedicine by the US Food and Drug involve testing mesenchymal stem cells (MSCs) in a range
Administration (FDA).5 of clinical indications. The early results from these trials,
along with extensive animal data, suggest limited integration
Translation of stem cell science into of the infused cells into the target tissue and concluding
regenerative medicine that the benefits observed may be due to production of
cytokines or other soluble mediators. Further it appears that
Regenerative medicine has been proposed to provide benefits obtained from these treatments may be transient
in the near future cures for debilitating diseases such as due to the lack of engraftment of the MSCs. As many of
diabetes, cardiovascular disease, and Alzheimer’s disease.6 the treatments are invasive procedures, such as catheter
In particular, the use of stem cells to repair damaged delivery of cells to the heart,8 it is unlikely that multiple
tissues has led to a number of clinical trials in wide range treatments will be feasible. A major challenge will be to
of disorders.6 However, despite the enormous potential of continue with clinical trials when the initial studies show
stem cells for regenerative medicine, there are many hurdles limited efficacy. Given the history of cellular therapies in
to overcome to develop these therapies. Although, we are bone marrow transplantation and immunotherapy9,10 it
able to isolate, culture, expand and differentiate stem cells is only through carefully designed incremental studies that
in vitro, the requirements for in vivo survival, proliferation these approaches will be optimized. Some cellular products
and differentiation into functional tissue, are essentially will be developed through commercial entities and the same
182 Section 2 Cellular Therapies
questions will become critical to the long term survival of the cell banks that could be the source of cells “off-the-shelf”. The
companies with stock holders and the stock market expecting polymorphism of histocompatibility genes and the resulting
breakthrough results in clinical trials. Another consideration variety of tissue types is far too great in human populations
relates to the multicellular composition of tissues and organs. to expect banks to be able to supply perfect tissue matches
Ischemic events will damage all the cells of the tissue or organ for all potential patients. Instead, one might envision banks
and so multicellular products will most likely be needed to of cells derived from donors with highly common genotypes
restore full capacity. This may require sequential treatments of the histocompatibility genes. This type of approach would
with an initial cellular product, such as MSCs, to repair the be greatly facilitated by cell strains with homozygosity of
tissue microenvironment, followed by a second cellular histocompatibility loci. Past approximations of the number of
product to replace the functional mature cells of the tissue. cell lines that would be needed in such a repository or master
In the absence of repair to the microenvironment, infusion of cell bank, based on modeling data from pools of kidney
functional cells may result in poor engraftment of survival. transplant patients and recipients in the United Kingdom and
Scientific advances in stem cell biology are being driven by Japan, have suggested that a bank comprised on the order of
the current intellectual ferment and excitement of the field, 10–50 cell lines might effectively provide a single HLA antigen
but when and how these advances will be translated into match (deemed a minimal requirement for acceptable solid
successful treatments remain fertile questions for debate. organ transplantation) for approximately 80% of the local
Will cell therapies remain a highly patient-focused endeavor population (Gourraud et al.,11 2012; Nakajima et al.,12 2007;
performed solely in academic medical centers, akin to bone Taylor et al.,13 2011). While encouraging, these numbers
marrow or solid organ transplantation? Or will stem cells ever suggest that some kind of dual system might well be needed
become commercial, pharmaceutical grade “off-the-shelf” in which the vast majority of individuals can benefit from
products? off the- shelf therapies, but personalized autologous cells
One might imagine a future in which medical centers offer derived via reprogramming would be needed for those with
highly customized, patient focused approaches to stem cell difficult-to-match tissue types.
treatments, perhaps utilizing the products of personalized
induced pluripotent stem cells. IPS cells have enormous Threats to clinical translation and
theoretical appeal as vehicles for combined gene repair and to the integrity of regenerative
cell replacement therapy for genetic disease Newer forms
of stem cell transplant could replicate the current status of
medicine
bone marrow transplantation, which has developed into a Translating the basic discoveries of stem cell biology into
remarkably complex infrastructure for capturing cellular and robust, effective, and safe new modalities of care will
molecular information in international registries for literally mean solving new challenges; before success, regenerative
millions of potential donors, and entails lengthy, costly, and medicine will suffer many setbacks. While translating too
risky interventions in intensive clinical care settings. Given the timidly might deprive needy patients of precious time
imperative of treating patients in need, stem cell transplants and life quality, testing cells in patients before a deeper
for genetic and acquired diseases will emerge from academic understanding of how stem cells work is risky too. We need
centers because clinician investigators will develop them to be confident that we understand the full spectrum of safety
and patients will demand them. Like gene repair (“gene concerns and can therefore avoid placing patients at undue
therapy”), cell replacement therapies will likely serve rare risk. We also need to design rigorous controlled trials where
conditions first, and pertain to small numbers of patients evidence for clinical efficacy can be defined precisely, rather
receiving highly individualized treatments, perhaps coupling than depend upon anecdote and clinical observation alone.
gene repair with autologous cell replacement approaches, for Given that patients and practitioners may carry unrealistic
example for blood diseases. Such small-scale applications expectations of clinical efficacy, there is a high likelihood
will dominate until and unless generic interventions and off- for a robust placebo effect as well as interpretive bias in
the-shelf approaches prove feasible. reporting of clinical results. We also need to be conscious of
The prospects for more widespread stem cell-based not exhausting resources that would be better spent on more
treatments depends on either solving the immune rejection practical health care needs. Premature application runs the
barrier, through advances in promoting immune tolerance risk of high-profile failure that would sully the credibility of
to allogeneic tissues, or accepting the use of immune this still-developing field.
suppression—even lifelong—to facilitate allogeneic cell With the goal of advancing clinical investigation while
therapies. Immune suppression is already standard for organ preserving rigor, promoting medical innovation while protecting
transplantation, so we know that its use to facilitate life- patients, and ensuring integrity in regenerative medicine while
sustaining cell therapy is feasible. Because cell manufacture respecting autonomy of individual practitioners and patients,
is likely to be the most costly and time-consuming aspect the International Society for Stem Cell Research (ISSCR)
limiting cell therapies, the prospects for realizing economies assembled an international group of scientists, surgeons, gene
of scale would seem to call for the establishment of master therapists, bioethicists, patient advocates, and attorneys and
Chapter 51 Stem Cell in Regenerative Medicine: Applications and Cautions 183
occurs. These pilot studies have prompted the design of large 4. Olivares EL, Ribeiro VP, Werneck de Castro JPS et al. Bone
RCTs to confirm these findings including the Regenerate- marrow stromal cells improve cardiac performance in healed
DCM study which recently began recruiting patients. This is infarcted rat hearts. Am J Physiol Heart Circ Physiol. 2004;287:
H464-H70.
the first randomized, double blind, placebo-controlled trial
5. Schuster MD, Kocher AA, Seki T, et al. Myocardial neovascular-
worldwide investigating the role of G-CSF and BMSCs to ization by bone marrow angioblasts results in cardiomyocyte
improve cardiac function in patients with DCM.34 regeneration. Am J Physiol Heart Circ Physiol. 2004;287:H525-
H32.
Concerns 6. Gnecchi M, He H, Liang OD, et al. Paracrine action accounts
for marked protection of ischemic heart by Akt-modified
So far several clinical studies exploring the safety and mesenchymal stem cells. Nat Med. 2005;11:367-8.
feasibility of stem cell therapy have been conducted. 7. Moore KA, Lemischka IR. Stem cells and their niches. Science.
These studies have used a myriad of different cell types 2006; 311:1880-85.
and preparations, each in a small number of patients with 8. Nygren JM, Jovinge S, Breitbach M, et al. Bone marrow-
derived hematopoietic cells generate cardiomyocytes at a low
different disease states. Present clinical evidence suggests
frequency through cell fusion, but not transdifferentiation. Nat
that stem cell therapy might work, though the efficacy is Med. 2004;10:494-501.
modest at best. Initial clinical studies have generated a great 9. Fujii T, Yau TM, Weisel RD, Ohno N, Mickle DAG, Shiono N,
deal of hope, we should take into account the lessons learned Ozawa T, et al. Cell transplantation to prevent heart failure: a
from the translation of therapeutic angiogenesis into clinical comparison of cell types. Ann Thorac Surg. 2003;76:2062-70.
studies, where great expectations raised by open studies have 10. Beltrami AP, Barlucchi L, Torella D, et al. Adult cardiac stem
not been confirmed by subsequent randomized trials. cells are multipotent and support myocardial regeneration.
Although studies are underway, fundamental questions Cell. 2003;114:763-6.
need to be addressed experimentally. What is the fate of the 11. Oh H, Bradfute SB, Gallardo TD, et al. Cardiac progenitor cells
from adult myocardium: homing, differentiation, and fusion
injected cells after transplantation? How long do they survive?
after infarction. Proc Natl Acad Sci USA. 2003;100:12313-8.
Do the cells incorporate, or is transient retention sufficient to 12. Kuethe F, Figulla HR, Herzau M, et al. Treatment with
promote functional effects? Genetic and transgenic markers granulocyte colony-stimulating factor for mobilization of bone
should be used to determine the lineage commitment of marrow cells in patients with acute myocardial infarction. Am
engrafted cells. Heart J. 2005;150:115.
13. Jackson KA, Majka SM, Wang H, et al. Regeneration of ischemic
cardiac muscle and vascular endothelium by adult stem cells.
Conclusion J Clin Invest. 2001;107:1395-1402.
• Stem cell therapy is an exciting novel treatment strategy for 14. Caplan AI. Mesenchymal stem cells. J Orthop Res. 1991;9:641-
the management of patients with left ventricular systolic 50.
15. Beltrami AP, Urbanek K, Kajstura J, et al. Evidence that human
dysfunction.
cardiac myocytes divide after myocardial infarction. N Eng J
• The efficacy of cell therapy in improving cardiac Med. 2001;344:1750-7.
function has been proved in animal models although the 16. Messina E, De AL, Frati G, et al. Isolation and expansion of
mechanisms of action need to be clearly defined. adult cardiac stem cells from human and murine heart. Circ
• Clinical trials of cell therapy have been performed in the Res. 2004;95:911-21.
settings of acute myocardial infarction, chronic ischemic 17. Oh H, Bradfute SB, Gallardo TD, et al. Cardiac progenitor cells
heart failure and dilated cardiomyopathy. These trials have from adult myocardium: homing, differentiation, and fusion
uniformly shown the safety of this treatment regardless of after infarction. Proc Natl Acad Sci USA. 2003;100:12313-8.
18. Menasche P, Hagege AA, Scorsin M, Pouzet B, Desnos M,
study design. Unfortunately clinical trials have shown at
Duboc D, Schwartz K, et al. Myoblast transplantation for heart
best only modest improvement in left ventricular function. failure. Lancet. 2001;357:279-80.
• The clinical efficacy of cell therapy remains to be fully 19. Assmus B, Schächinger V, Teupe C, et al. Transplantation
translated and needs far more basic research and closer of Progenitor Cells and Regeneration Enhancement in
collaboration between scientists and clinicians. Acute Myocardial Infarction (TOPCARE-AMI). Circulation.
2002;106:3009-17.
20. Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous
References bone-marrow cell transfer after myocardial infarction: the
1. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells BOOST randomised controlled clinical trial. Lancet. 2004;
regenerate infarcted myocardium. Nature. 2001;410:701-5. 364:141-8.
2. Condorelli G, Borello U, De Angelis L, et al. Cardiomyocytes 21. Meyer GP, Wollert KC, Lotz J, et al. Intracoronary bone marrow
induce endothelial cells to transdifferentiate into cardiac cell transfer after myocardial infarction: 5-year follow-up from
muscle: implications for myocardium regeneration. Proc Natl the randomized-controlled BOOST trial. Eur Heart J. 2009;
Acad Sci USA. 2001;98:10733-8. 30:2978-84.
3. Kudo M, Wang Y, Wani MA, et al. Implantation of bone marrow 22. Schächinger V, Erbs S, Elsässer A, et al. REPAIR-AMI Investi-
stem cells reduces the infarction and fibrosis in ischemic gators. Intracoronary bone marrow-derived progenitor cells in
mouse heart. J Mol Cell Cardiol. 2003;35:1113-9. acute myocardial infarction. N Eng J Med. 2006;355:1210-21.
188 Section 2 Cellular Therapies
23. Huikuri HV, Kervinen K, Niemela M, et al. Effects of 29. Assmus B, Honold J, Schachinger V, et al. Transcoronary
intracoronary injection of mononuclear bone marrow cells on transplantation of progenitor cells after myocardial infarction.
left ventricular function, arrhythmia risk profile, and restenosis N Eng J Med. 2006;355:1222-32.
after thrombolytic therapy of acute myocardial infarction. Eur 30. Fischer-Rasokat U, Assmus B, Seeger FH, et al. A pilot trial
Heart J. 2008;29:2723-32. to assess potential effects of selective intracoronary bone
24. Janssens S, Dubois C, Bogaert J, et al. Autologous bone marrow-derived progenitor cell infusion in patients with
marrow derived stem-cell transfer in patients with ST-segment nonischemic dilated cardiomyopathy: final 1-year results of the
elevation myocardial infarction: double-blind, randomised transplantation of progenitor cells and functional regeneration
controlled trial. Lancet. 2006;367:113-21. enhancement pilot trial in patients with nonischemic dilated
25. Lunde K, Solheim S, Aakhus S, et al. Intracoronary injection cardiomyopathy. Circ Heart Fail. 2009;2:417-23.
of mononuclear bone marrow cells in acute myocardial 31. Seth S, Bhargava B, Narang R, et al. The ABCD (Autologous
infarction. N Eng J Med. 2006;355:1199-209. Bone Marrow Cells in Dilated Cardiomyopathy) trial a long-
26. Hirsch A, Nijveldt R, van der Vleuten PA, et al. Intracoronary term follow-up study. J Am Coll Cardiol. 2010;55:1643-4.
infusion of mononuclear cells from bone marrow or peripheral 32. Bocchi EA, Bacal F, Guimarães G, et al. Granulocyte-colony
blood compared with standard therapy in patients afteracute stimulating factor or granulocyte-colony stimulating factor
myocardial infarction treated by primary percutaneous associated to stem cell intracoronary infusion effects in non
coronary intervention: results of the randomized controlled ischemic refractory heart failure. Int J Cardiol. 2010;138:94-7.
HEBE trial. Eur Heart J. 2011;32:1736-47. 33. Vrtovec B, Poglajen G, Lezaic L, et al. Effects of intracoronary
27. Menasche P, Alfieri O, Janssens S, et al. The Myoblast CD34+ stem cell transplantation in nonischemic dilated
Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) cardiomyopathy patients: 5-year follow-up. Circ Res. 2013;112:
trial: first randomized placebo-controlled study of myoblast 165-73.
transplantation. Circulation. 2008;117:1189-200. 34. Arnous S, Mozid A, Mathur A. The Bone Marrow Derived Adult
28. Dib N, Dinsmore J, Lababidi Z, et al. One-year follow-up of Stem Cells for Dilated Cardiomyopathy (Regenerate-DCM)
feasibility and safety of the first U.S., randomized, controlled trial: study design. Regen Med. 2011;6:525-33.
study using 3-dimensional guided catheter-based delivery of
autologous skeletal myoblasts for ischemic cardiomyopathy
(CAuSMIC study). JACC Cardiovasc Interv. 2009;2:9-16.
Stem Cell Therapy for
Diabetes Mellitus
Rajesh Rajput
53
Introduction whole-organ pancreas transplants. A pancreas transplant can
be carried out in three ways:
Diabetes is a group of diseases characterized by abnormally 1. Simultaneous kidney pancreas transplant (SPK): Both
high levels of the glucose in the bloodstream. This uncontrolled the pancreas and kidneys are transplanted together, from
hyperglycemia is responsible for most of the microvascular the same donor. This is the most common type, accounting
and macrovascular complications seen in diabetes. Broadly for nine out of 10 transplants. It is used in people who have
diabetes is classified into Type 1 diabetes (T1DM) and Type kidney disease as a result of type 1 diabetes.
2 diabetes (T2DM). T1DM typically affects children and 2. Pancreas after kidney transplant (PAK): A person first
young adults and is characterized by immune mediated receives a kidney transplant from a living or deceased
destruction of pancreatic beta cell, as a result all patients with donor. This is then followed by a pancreas transplant from
T1DM needs insulin therapy for proper management of their a deceased donor.
disease. T2DM the more common type of disease is seen in 3. Pancreas alone transplant (PTA): Only the pancreas
around 90% of adults and tends to affect older, sedentary, is transplanted. This is a treatment for patients with very
and overweight individuals with a family history of diabetes. poorly controlled type 1 diabetes who have hypoglycemic
T2DM results from combination of insulin resistance and attacks without warning, and which may threaten their
relative insulin deficiency and needs combination of diet, life.
exercise, and oral medication. But a good number of patients To prevent the body from rejecting the transplanted
with increasing duration of T2DM ultimately need insulin pancreas, patients must take powerful immunosuppressive
therapy for control of hyperglycemia. Despite the fact that drugs for their entire lives, a regimen that makes them
large number of drugs available for management of diabetes, susceptible to a host of other diseases. However, the demand
there is currently no permanent cure for diabetes and large for transplantable pancreas outweighs their availability and
number of patients need different type of complex insulin because of this attempts have been made to cure diabetes by
regimen which puts them at risk of hypoglycemia and weight injecting patients with pancreatic islet cells—the cells of the
gain. pancreas that secrete insulin and other hormones. However,
It is estimated that by year 2013, approximately 382 the requirement for steroid immunosuppressant therapy
million people worldwide are suffering from diabetes out to prevent rejection of the cells increases the metabolic
of which 90% suffers from type 2 diabetes. This number is demand on insulin-producing cells and eventually they may
expected to rise to 592 million by 2035. Statistics shows that exhaust their capacity to produce insulin. The deleterious
in year 2012 and 2013, diabetes resulted in 1.5 to 5.1 million effect of steroids is greater for islet cell transplants than for
deaths per year making it as 8th leading cause of death and whole-organ transplants. As a result, less than 8% of islet cell
each year, diabetes affects more people and causes more transplants performed before last year had been successful.
deaths than breast cancer and AIDS combined. For decades, More recently, James Shapiro and his colleagues in
diabetes researchers have been searching for ways to Edmonton, Alberta, Canada, have developed an experimental
replace the insulin-producing cells of the pancreas that are protocol for transplanting islet cells that involves using a
destroyed by a patient's own immune system. To overcome much larger amount of islet cells and a different type of
barriers associated with use of insulin therapy and provide immunosuppressant therapy that avoids use of steroids.
patients chance of permanent cure of diabetes, every year, Despite advances in immunosuppressive regimens, many
approximately 1,300 people with type 1 diabetes receive hurdles still remain in using this approach on a wide scale to
190 Section 2 Cellular Therapies
treat diabetes. First, donor tissue is not readily available and the cell types of the body. Scientists have identified specific
islet cells used in transplants are obtained from cadavers, and growth inhibitors that induce mouse embryonic stem cells
the procedure requires at least two cadavers per transplant. to adopt, in vitro, various characteristics of pancreatic islet
The islet cells must be immunologically compatible, and the cells, including insulin production, glucose-dependent
tissue must be freshly obtained—within eight hours of death. insulin release and formation of islet-like aggregates.
Because of the shortage of organ donors, these requirements Also, when transplanted into diabetic mice, these islet-
are difficult to meet and the waiting list is expected to far like clusters significantly improve diabetic symptoms.
exceed available tissue, especially if the procedure becomes The exact pathways that embryonic stem cells take to
widely accepted and available. Further, islet cell transplant become pancreatic cells are yet to be identified, however,
recipients face a lifetime of immunosuppressant therapy, and embryonic stem cells themselves present a number
which makes them susceptible to other serious infections and of scientific challenges. In general, embryonic stem cells
diseases. These challenges opened a new era of utilizing the are hard to maintain in culture in their undifferentiated
option of stem cells for treatment of diabetes. state. Because of their uncontrolled growth, embryonic
stem cells may have tumor-forming potential if not fully
Stem Cell Therapy for Diabetes differentiated before transplantation into patients. There is
also the risk of immune rejection following transplantation,
Stem cells are self-renewing, unspecialized cells that give so patients receiving the transplants would likely have to
rise to multiple specialized cell types through a process of be maintained on immune-suppressant medication.
differentiation. Theoretically, stem cells could be induced Adult stem cells, specifically autologous stem cells which
in the laboratory to become any specialized body cell type, are derived from mature somatic tissues, may avoid the issue
including pancreatic islet cells, and then transplanted of immune rejection but present distinct challenges of their
back into a patient to replace diseased or damaged tissue. own. Adult stem cells do not grow indefinitely in the lab and,
Transplantation of stem cells or stem-cell derived tissue although they can be found in a great number of tissues,
into human patients has not yet reached the clinical level, these special cells are generally rare and hard to identify.
however, and many questions remain regarding the safety In addition, adult stem cells are thought to have a more
and efficacy of such therapies. Various types of stem cells limited differentiation profile than embryonic cells, with only
with potential to treat diabetes mellitus include: the capacity to differentiate into the specialized cells of the
tissue from which they were derived (e.g. a brain stem cell
Source Type of stem cells could become a neuron but not a blood cell). Preliminary
Embryonic stem cells Blastocyst-derived embryonic stem studies in which adult stem cells of non-pancreatic origin
cells differentiated to become islet-like cells suggest that
Induced pluripotential stem cells adult stem cells may have greater plasticity, or ability
Umbilical cord stem cells Cord blood hematopoietic stem cells to develop into a greater number of cell types than previously
believed. In one case, scientists demonstrated that bone
Cord blood or cord wall mesenchymal
marrow-derived cells transplanted into lethally irradiated
stem cells
mice are able to differentiate into functional, insulin-
Embryonic-like stem cells producing islet cells in the pancreases of the recipients.
Adult stem cells Hematopoietic stem cells It is not known, however, whether these differentiated
Mesenchymal stem cells cells were the progeny of hematopoietic stem cells
(which normally do not give rise to pancreatic cells) or
Pancreatic cells Pancreatic stem cells
pancreatic stem cells that happen to reside in the bone
Reprogrammed adult pancreatic marrow. In another study adult hepatic oval cells, stem
exocrine cells cells of the liver that do not normally give rise to pancreatic
tissue, were shown to differentiate into pancreatic islet-like
In order to better gauge the therapeutic potential of clusters in vitro that are able to reverse diabetic symptoms in
stem cells, much basic research and animal model testing mice upon transplantation.
is currently being conducted. Scientists have focused on Progressive and inexorable β-cell dysfunction is the
stem cells from two sources: early-stage embryos and stem hallmark of type 2 diabetes mellitus (T2DM) and β-cell
cells from adult tissue. Embryonic and adult stem cells have regeneration using stem cell therapy may prove to be an
different characteristics and are thought to have different effective modality. Bhansali et al. in their study involved a
developmental potential. Embryonic stem cells are derived total of 10 patients (8 men) with T2DM for >5 years who failed
from the inner cell mass of blastocysts approximately five on triple oral antidiabetic drugs, were on insulin (≥ 0.7 U/
days after fertilization in vitro, and appear to have the greatest kg/day) at least for 1 year, and glutamic acid decarboxylase
therapeutic potential. These cells grow indefinitely in the lab antibody negative. Patients on stable doses of medications
and are termed pluripotent, as they can develop into any of for past 3 months were recruited. Primary end points were
Chapter 53 Stem Cell Therapy for Diabetes Mellitus 191
reduction in insulin requirement by ≥50% and improvement release insulin in this biphasic manner. Instead insulin is
in glucagon-stimulated C-peptide levels at the end of 6 months released in an all-or-nothing manner, with no fine-tuning
of autologous hematopoietic stem cell transplantation for intermediate concentrations of glucose in the blood.
(HSCT), while secondary end points were a change in weight Therefore, many researchers believe that it will be preferable
and HbA1c and lipid levels as compared to baseline. Seven to develop a system in which stem or precursor cell types
patients were responders and showed a reduction in insulin can be cultured to produce all the cells of the islet cluster
requirement by 75% as compared to baseline. Mean duration in order to generate a population of cells that will be able to
to achieve the primary objective was 48 days. Three patients coordinate the release of the appropriate amount of insulin to
were able to discontinue insulin completely, although it the physiologically relevant concentrations of glucose in the
was short-lived in one. Mean HbA1c reduction was 1% and blood.
3 of the 7 responders had HbA1c value <7%. A significant
weight loss of 5.5 kg was noted in the responders, whereas,
non responders gained 2.2 kg of weight. However, weight
Ethical Issues with use of Stem Cells
loss did not correlate with reduction in insulin requirement The main ethical concern with stem cell research is
(r = 0.68, p = 0.06). There was a significant improvement in balancing the pursuit of a potential therapy for diabetics
both fasting and glucagon-stimulated C-peptide level in with the proper respect for and protection of human life,
the group (p = 0.03) and responders (p = 0.03). HOMA-B an immediate issue when considering the destruction of
increased significantly in the whole group (p = 0.02) and living human embryos that is involved in the derivation
responders (p = 0.04) whereas, HOMA-IR did not change of embryonic stem cells. An embryo is a unique human
significantly (p = 0.74). Reduction in insulin doses correlated entity whose biological identity is determined from the
with stimulated C-peptide response at the baseline (r = 0.83, moment of fertilization; this identity is unchanging through
p = 0.047) and mononuclear cell count of infused stem cells any subsequent differentiation and growth. Natural law,
(r = 0.57, p = 0.04). No serious adverse effects were noted. or those rules for action that all can know through reason
Our observations indicate that SCT is a safe and effective and observation of the world, states that all human persons
modality of treatment to improve β-cell function in patients possess certain basic rights including the right to life and
with T2DM. However, further large-scale studies are needed physical integrity. Because these rights, or justifiable claims
to substantiate these observations. on others, flow from each human person's intrinsic dignity
Autologous HSCT had been tried across the globe by as sharers in a common human nature, all living human
various researchers for treatment of T1DM. It has been beings must be regarded as persons and treated in a morally
observed that high dose immunosuppression followed by equivalent manner. If the human embryo is a person,
HSCT shows better results among other immunotherapeutic then he or she cannot be used as a means toward any end,
treatments for the disease as patients with adequate beta however good. No projected therapeutic good that may
cell reserve achieve insulin independence. However, come from the use of embryonic stem cells can outweigh
this response is not maintained and as seen with T2DM the immediate harm (death) to the human embryo when its
reoccurrence of the disease is a major challenge with use of stem cells are extracted. Therefore, research that involves
HSCT in future to prevent pr control relapse of T1DM. the derivation of stem cells from living human embryos is
Another area of concern while utilizing stem cells for morally unacceptable, even if the projected benefits can be
treatment of diabetes is that it is not clear whether it will gotten in no other way. The creation of "surplus" embryos
be desirable to produce only beta cells—the islet cells that that one is not planning to implant, a routine part of in vitro
manufacture insulin—or whether other types of pancreatic fertilization procedures, must also be rejected.
islet cells are also necessary. Studies by Bernat Soria and Other scientifically and ethically viable avenues of stem
colleagues, for example, indicate that isolated beta cells— cell research do exist that appear to hold great promise for
those cultured in the absence of the other types of islet cells— the eventual treatment of diabetes, including the use of adult
are less responsive to changes in glucose concentration stem cells, and these should be pursued. Although much
than intact islet clusters made up of all islet cell types. basic research remains to be done, adult stem cells appear to
Islet cell clusters typically respond to higher-than-normal have great therapeutic potential and may even have an edge
concentrations of glucose by releasing insulin in two phases: over embryonic cells, in that they avoid issues of immune
a quick release of high concentrations of insulin and a slower rejection.
release of lower concentrations of insulin. In this manner the
beta cells can fine-tune their response to glucose. Extremely
high concentrations of glucose may require that more insulin Suggested Reading
be released quickly, while intermediate concentrations of 1. Anil Bhansali, Vimal Upreti, et al. Stem Cells and Development.
glucose can be handled by a balance of quickly and slowly 2009;18(10):1407-16.
released insulin. Isolated beta cells, as well as islet clusters 2. Congregation for the Doctrine of the Faith. Donum Vitae.
with lower-than-normal amounts of non-beta cells, do not Boston: St. Paul Editions, 1987;I:1-5.
192 Section 2 Cellular Therapies
3. Hori Y, et al. Growth inhibitors promote differentiation 7. Soria B, et al. Insulin secreting cells derived from emdryonic
of insulin-producing tissue from embryonic stem cells. stem cells normalize glycemia in streptozotocoin induced
Proceedings of the National Academy of Sciences. USA. 2002; diabetic mice. Diabetes. 2000;49:157-62.
99:16105-10. 8. US Department of Health and Human Services. Stem Cells:
4. Ianus A, et al. In vivo derivation of glucose-competent Scientific Progress and Future Research Directions. 2001.
pancreatic endocrine cells from bone marrow without evidence <http://www.nih.gov/news/stemcell/scireport htm>
of cell fusion. Journal of Clinical Investment. 2002;111:843-50. 9. Voltarelli TC, et al. Stem cell therapies for type 1 diabetes
5. Lee V, Stoffel M. Bone Marrow: an extra-pancreatic hideout for mellitus. Indian Journal of Experimental biology. 2011;49:395-
the elusive pancreatic stem cell? Journal of Clinical Investment. 400.
2003;111:799-801. 10. Yang L, et al. In vitro trans-differentiation of adult hepatic stem
6. Meyer J. Human embryonic stem cells and respect for life. cells into pancreatic endocrine hormone-producing cells.
Journal of Medical Ethics. 2000;26:166-70. Proceedings of the National Academy of Sciences. USA. 2002;
99:8078-83.
Stem Cell Therapy in Peripheral
Artery Disease
are not enough to compensate for a large occluded vessel. Idei et al.4 studying the long term clinical outcome of CLI
Arteriogenesis refers to growth of small collateral branches patients showed that in 25 patients who received BMMNCs
from sides of arterial wall. Intra-arterial pressure build-up the 4 year AFS WAS 48% as compared to 0% in PAD controls
within the vessel proximal to the occlusion increases the who did not get stem cells. The 4 year survival rate was 76% in
shearing forces against vessel wall which induces increment the BMMNCs group and 67% in the control group.
of diameter of pre-existing collateral arterioles. Balber5 suggested aldehyde dehydrogenase rich bright
It has been proposed that bone marrow derived cells (ALDHbr) could be used for angiogenesis in CLI
mononuclear cells are central to such regenerative and patients.
reparative processes of angiogenesis and arteriogenesis. Perin et al.6 compared 2 groups of patients of CLI one
These BMMNCs (stem cells) adhere to and invade the vessel of whom received ALDHbr with the other that received
wall. It is not clear whether these invaded cells proliferate autologous Bone Marrow derived mononuclear cells
and differentiate into endothelial cells. A growing number of (ABMMNCs) and concluded that stastically significant
studies suggest that these circulating EPCs/BMMNCs do not improvement was observed in Rutherford category at 12
engraft into blood vessel, rather they regulate vascular repair weeks (mean, 4.09+/–0.03 to 3.46+/–1.04; p==0.05) and in ABI
via paracrine mechanism by release of cytokines which result at 6 and 12 weeks.
in proliferation of resident endothelial cells. Probably these Huang P et al.7 randomized 28 patients of diabetes
BMMNCs are circulating endothelial progenitor cells. It has with critical limb ischemia and studied the effect of G-CSF
been observed that patients with risk factors like diabetes, mobilized PMNCs administered by intramuscular injection
smoking, advanced age have lower number of circulating of affected limb. Mean ABI increased from 0.50+/–0.21 to
BMMNCs. 0.6+/–0.25 (p<0.001). Out of 18 foot ulcers 14 healed in the
Stem cell therapy in PAD is intended to boost the natural transplanted patients and no amputations were required
reparative process of angio- and arteriogenesis. But it requires whereas in the control group 5 amputations were performed
large number of functional autologous precursor cells. (p=0.007 transplant vs Control).
About 250–500 mL of bone marrow aspirated and centrifuged Tateishi-Yuyama8 et al. studied 25 patients of unilateral
to prepare Buffy coat concentrate. Another method is to limb ischemia in a randomized controlled trial by injecting
collect these myelo-monocytoid cells (CD34+/FLK1+) from BMMNCs including endothelial PCs into gastrocnemius
peripheral blood following injections of GM-CSF. Stem cells muscle of ischemic limb and saline in the less ischemic limb
after being isolated is either injected intra-arterially or into and in the second group they took patients of bilateral limb
the gastrocnemius (multiple sites, 20 to 30) muscle. ischemia and randomly injected one limb with BMMNCs
and the other with PMNCs as control. In the transplant group
Measures of Outcome of Stem Cell Therapy there was improvement in ABI in the limb with BMMNCs as
compared to that with PMNCs diff 0.009 (95% CI 0.06–0.11;
Amputation free survival (AFS)—composite end point of p<0.0001, transcutaneous oxygen pressure 13(9-17); p<0.001
mortality and amputation. and pain free walking time (1.2(0.7-1.7)); p=0.025.
Improvement in Rutherford stage of limb ischemia due Iafrati MD9 et al. in a multicenter double-blind randomized
to PAD—category 0-Asymptomatic, category 1-mild claudi- control trial involving 48 patients with no option CLI who
cation, category 2-moderate claudication, category 3-severe received bone marrow concentrate injected percutaneuos
claudication, category 4-rest pain, category 5-ischemic ulcer- in the ischemic limb and controls who received dilute blood
ation of digits, category 6-extensive ulceration or gangrene. injections showed statistically insignificant trends favoring
• Ulcer healing the bone marrow recipients vs control in major amputation
• Pain free walking (400 meter walk) (17.6% vs 28.6%), improved pain (44% vs 25%) improved ABI
• Transcutaneous oxygen pressure (TrO2)-measured by (32.4% vs 7.1%).
oxymonitor, normal value is 70–90 mm Hg Watt and Gullo10 were of the opinion that MSCs have
• Significant improvement in ABI (increase in ABI =/> 0.1). tremendous potential as they are expandable ex vivo and
as such may be used to re-establish vascular supply by
Clinical Trials on stem cell therapy regeneration new blood vessels in ischemic tissues. However,
concerns remain about their ability to differentiate into
for PAD
vascular cells or cardiomyocytes. Their efficacy, safety,
Arai et al.3 divided 39 patients of atherosclerotic PAD into delivery route and dose also need to be standardized by
3 groups subjecting the first group to conventional drug further clinical trials.
therapy alone, the second to conventional drugs and BMT Wang et al.11 conducted a meta-analysis sourced from
while the third received conventional drugs and G-CSF PubMed, Cochrane Central Registry of controlled trials,
injections. After one month it was noted that there was Elsevier database and EBSCO for autologous cell therapy up
improvement in symptoms in the G-CSF and BMT groups. to October 30 2013 and found that clinical end points, i.e.
ABI and Transcutaneous oxygen pressure (trCut O2) also ABI, Tr-cutO2 pressure, pain scale and AFS were improved
improved in the same groups. following autologous stem cell therapy in patients with CLI
196 Section 2 Cellular Therapies
but the limited duration of follow-up and limited number of CLI—Critical limb ischemia.
randomized controlled trials limited their importance. G-CSF—Granulocyte colony stimulating growth factor.
iPSCs—Induced pluripotent stem cells, genes expressed in
Current status of stem cell therapy embryonic stem cell is introduced into adult cells (mature
in PAD differentiated cells), which are then dedifferentiated into
Most trials testing the efficacy and safety of stem cell therapy pluripotent stem cells.
in PAD involved a small number of patients who were MSCs—Mesenchymal stem cells, sometime called as marrow
suffering from critical limb ischemia but were not candidates stromal cells-these are multipotent cell that can differentiate
for percutaneuos or surgical therapeutic intervention. There into a variety of cells like osteoblasts, chondrocytes,
were few randomized, double blinded multicenter placebo adipocytes.
controlled trials.1,2,11 PSCs—Progenitor stem cells, early descendants of stem cells
Trials have shown that endothelial progenitor cells are that can differentiate into one or more kind of cells but cannot
vasculogenic as they provide structural framework and divide and reproduce indefinitely.
growth factors for vascular regeneration.2 Pericytes also
provide structural framework and may be used. Lack of PMNCs—Peripheral blood mono nuclear stem cells.
knowledge of the exact mechanism of action has limited our
selection of best type of cells to be used but most studies in References
the field of vascular regeneration have used ABMMNCs,
1. Crystal M Botham, William L, et al. Clinical trials of adult stem
Endothelial progenitor cells, Autologous CD34+ Cells or cell therapy for peripheral artery disease. MDCVJ/IX 2013.
Aldehyde dehydrogenase rich bright cells (ALDHbr) collected 2. Thekkeparambil Srijaya, Thamil Ramasamy, Noor Kasim.
with the help of flow cytometry from autologous bone Advancing stem cell therapy from bench to bedside; lessons
marrow or G-CSF mobilized peripheral blood cells.5,6 The from pharmaceutics for bio-pharmacy. Journal of Translational
route of administration used in these trials was intramuscular Medicine. 2014;12:243.
injections in the ischemic limb, intrarterial or percutaneous. 3. Arai M, Misao Y, Nagai H, Kawasaki M, Nagasimha K, Suzuki
The dose of stem cell and duration of follow-up was variable. K, et al. Granulocyte-colony stimulating factor. A non-invasive
Clinical end points of most trials included Ankle brachial therapy for treating atherosclerotic peripheral artery disease.
Circ J. 2006;70(9):1093-8.
index, transcutaneous oxygen tension (TcO2), duration of
4. Idei N, Soqua J, Hota T, et al. Autologous bone marrow
pain free walking and amputation free survival (AFS). Most mononuclear cell implantation reduces long-term amputation
trials concluded that stem cell therapy resulted in significant risk in patients with critical limb ischemia. A comparison of
improvement in ABI, duration of pain free walking and AFS. atherosclerotic peripheral artery disease and Burgers’ disease.
Circ. Cardiovascular Interv. 2011;4(1):15-28.
5. Balber AE. Concise review; aldehyde dehydrogenase bright
Summary stem cells and progenitor cell population from normal tissue.
A new era of medical science has begun with the advent of Characteristics activities and emerging uses in regenerative
stem cell technology in the field of therapeutics. Clinical trials medicine.
of this treatment modality in the field of peripheral artery 6. Perin EC, Silva G, Grahremanpour A, et al. A randomised
controlled study of autologous therapy bone marrow derived
disease have shown promising results in limb salvage and
aldehyde dehydrogenase bright cells in patients with CLI.
symptom relief. Though limited by small sample size, lack of Catheter Cardiovascular Interv. 2011;78(7):1060-7.
standardized procedures and paucity of placebo controlled, 7. Huang P, Li S, Han M, Xiao Z, Yang R, Han ZC. Autologous
randomized, double blinded multicenter studies; yet it transplantation of Granulocyte colony stimulating factor
may be hoped that stem cell therapy will find a place in the mobilised peripheral blood mononuclear cells improves critical
treatment of PAD in future when more refined techniques limb ischemia in diabetes. Diabetes Care. 2005;28(9):2155-60.
shall be developed and tested and proven to be effective. 8. Tateishi-Yuyama E, Matsubara H, MuroharaT, Ikeda U, Shintai
S, Masaki H, et al. Therapeutic angiogenesis for patients with
limb ischemia by autologous transplantation of bone marrow
Glossary of terms cells: A pilot study and a randomised control trial. Lancet.
2002;360(9331):427-35.
ABMT—Autologous bone marrow transplant.
9. Iafrati MD, Hallett JW, Geils G, Pearl G, Lumsden A, Peden E,
ABI—Ankle brachial index. et al. Early and lessons learnt from a multicentre, randomised,
double-blind trial of bone marrow aspirate concentrate in
AFS—Amputation free survival. critical limb ischemia. J Vasc Surg. 2011;1:54(6):1650-8.
ALDHbr—Aldehyde dehydrogenase rich bright cells, hemat- 10. Watt SM, Gullo. Angigenic properties of Mesenchymal stem/
opoietic stem cells are rich in aldehyde dehydrogenase. To stromal cells and their therapeutic potential. Br Med Bull.
isolate stem cells a fluorescent substrate is used which makes 2013;108:25-33.
the cell bright. 11. Wang ZX, Li D, Cao JX, et al. Efficacy of autologous bone
marrow mono nuclear cells therapy in patients with peripheral
BMMNCs—Bone marrow derived mono nuclear cells. artery disease. J Atherosclerosis Thromb. 2014.
Role of Platelet Rich Plasma
in Regenerative Medicine
Table 1: Growth factors identified within platelet rich plasma and their physiologic effect
Factor Target cell/ tissue Function
PD-EGF Blood vessel cells, outer skin cells Cell growth, recruitment, differentiation, skin closure cytokine
fibroblasts and many other cell types secretion
PDGF A+B Fibroblasts, smooth muscle cells, chondrocytes, Potent cell growth, recruitment, blood vessel growth, granulation,
osteoblasts, mesenchymal stem cells growth factor secretion; matrix formation with BMPs (collagen and
bone)
TGF-β1 Blood vessel tissue, outer skin cells Blood vessel (+), collagen synthesis
fibroblasts, monocytes TGF gene family includes the growth inhibition, apoptosis (cell death)
BMPs, osteoblasts-highest levels of TGF-Br differentiation, activation
IFG-I, II Bone, blood vessel, skin, other tissues Cell growth, differentiation, recruitment collagen synthesis with
fibroblasts PDGF
VEFG, ECGF Blood vessel cells Cell growth, migration, new blood vessel growth, anti-apoptosis
(anti-cell death)
bFGF Blood vessels, smooth muscle, skin Cell growth, cell migration, blood vessel growth
fibroblasts, other cell types
Abbreviations: PD-EGF, platelet-derived epidermal growth factor; PDGF, platelet-derived growth factor; BMP, bone morphogenetic protein; TFG, transforming
growth factor; IGF, insulin-like growth factor; VEGF, vascular endothelial growth factor; ECGF, endothelial cell growth factor; bFGF, basic fibroblast growth factor.
number of proteins, cytokines and other bioactive factor as (30–40 mL) from patient‘s elbow vein with a syringe to a
shown in Table 1 that initiate and regulate basic aspects of test tube which is anti-coagulated with citrate phosphate
wound healing most notably in acute surgical conditions and dextrose adenine (CPDA) with a ratio of 1 : 9 to the blood, to
in the management of chronic nonhealing wounds.6 bind ionized calcium and inhibit the clotting cascade.
It is followed by two centrifugations, one ten minutes
PLATELET RICH PLASMA THERAPY centrifugation at 2000 rpm which separates blood into three
layers; red blood cells, platelets and platelet poor plasma as
This therapy is safe and natural alternative to surgery. Platelets shown in Figure 2.
contain an abundance of growth factors and cytokines that Red cell layer is drawn from the tube. The remainder
can affect inflammation, postoperative blood loss, infection, is agitated for several seconds and will undergo a second
osteogenesis, wound, muscle tear and soft tissue healing. centrifugation at 2000 rpm for ten minutes. There will be two
They also release many bioactive proteins responsible layers, upper supernatant is platelet poor plasma (PPP) and
for attracting macrophages, mesenchymal stem cells and residual is platelet concentrates. About the three fourth of
osteoblasts that not only promote removal of degenerated
and necrotic tissue, but also enhance tissue regeneration and
healing. This therapy relieves pain by promoting long lasting
healing. PRP therapy relieves pain without the risk of surgery,
general anesthesia or hospital stays and without a prolonged
recovery. In fact most people return to their jobs right after
the procedure.3
supernatant is discarded and residual platelet concentrate • Chronic wound healing: In the United States, the
is drawn into a syringe. Another syringe with 10% calcium diabetic foot (Fig. 3J) problem accounts for 20% of all
chloride will be integrated with PRP syringe in a ratio of 6 : 1 to diabetic hospital admissions and 50% of all nontraumatic
activate the PRP for delivery to the desired site.4 Clotting leads amputations. Ulcers occur in 15% of diabetics, and 6–20%
to platelet activation, resulting in release of growth factors of all hospitalized diabetic patients have foot ulcers.8,10
from alpha granules. Approximately 70% of stored growth The topical use of the platelet rich plasma may play an
factors are released within 10 minutes of PRP activation, and important role in the initiation of the repair process
nearly 100% within one hour and rest till 8–10 days.6 PRP is of chronic wounds (Fig. 3E). Various protocols have
injected/applied to and around the point of injury/wound. documented such an improved healing of surgical wound
The procedure time takes about one to two hours, including in cardiac surgery (Figs 3C and D) and plastic surgery
preparation and recovery time. Twice weekly dressings (Fig. 3K).
done for maximum ten dressings for chronic wounds and • Cancer: PRP has a definite role in the cancer, which is
maximum up to three injections can be given for injuries probably due to their proangiogenesis stimulation and
within six months time frame, usually performed two to three regulation of new blood vessel growth through numerous
weeks apart but considerable or complete relief may be gained stimulators and inhibitors of angiogenesis, such as
after first to second injection.3,4 Before application informed endostatin and VEGF, by several pathways.8
patient’s consent is must after explaining the procedure, risk • Neurology: There is role of PRP therapy in cases of
and benefits.4 ischemic damage to a nerve due to scar tissue banding
during percutaneous procedures.8
THERAPEUTIC PLATELET PRODUCTS8 • Other applications: Platelet gel mixed with centrifuged
fat has been successfully applied for the patients affected
• Platelet rich plasma: Platelets concentrate obtained after by Parry-Romberg syndrome, i.e. progressive hemifacial
two centrifugations as described above. atrophy and volumetric deficit, which is an uncommon
• Platelet gel: Platelets are exposed to an agonist, which degenerative and poorly understood condition.8
induces activation resulting in release of intrinsic
substances that are applied to the target area where they
CONTRAINDICATIONS11
accelerate wound healing.
• Platelet leukocyte gel: Contain high concentrations of • Absolute: Platelet dysfunction syndrome, critical
platelets and leucocytes with antimicrobial properties in thrombocytopenia, hemodynamic instability, septicemia,
addition, at wound. local infection at the site of procedure and patient
unwilling to accept risks.
INDICATIONS • Relative: Consistent use of NSAIDS within 48 hours of
procedure, corticosteroid injection at treatment site
• Dermatology and esthetic surgery: Fine wrinkles around within one month, systemic use of corticosteroids within
the eyes, bags and dark circles under the eyes, neck, chest two weeks, tobacco use, recent fever or illness, cancer
and décolletage, lips, around the ears, cheeks, naso- hematopoietic or bone, hemoglobin less than 10 g/dL and
labial folds, acne and scars, back of hands and arms, hair platelet count less than 100 × 109/L.7
growth and change of color. It is safe and effective tool
in the management of mucosal skin lesions related to
the graft-versus-host disease (GVHD) involving dermis
SAFETY PROFILE
subcutaneous or oral mucosa and is applied as local Since PRP is prepared from autologous blood, there are
therapy (Figs 3A and B).8 minimal risks for disease transmission, immunogenic
• Orthopedics: Tendonitis, ligament sprains, intra- reaction or cancer as compared to standard therapies. Till
articular injuries, joint pain, arthritis, knee meniscus date, PRP has been widely used in the treatment of at least
repair, acute and/or chronic muscle strain. PRP therapy thirty diseases in more than hundred thousand patients,
is a revolutionary new treatment that relieves pain by and there have been no reports about side effects of PRP
promoting lasting healing of musculoskeletal injuries injection.5
(Figs 3G to I).3,8
• Oral and craniofacial surgeries: Maxillofacial settings
where it is considered standard treatment of bone defects,
SUMMARY
artificial endosseal implants, oral reconstructions, Studies have shown that with PRP application, there
maxillary sinus augmentation (Fig. 3F).8 is complete absence of keloid formation, hyperplasia,
• Ophthalmology: PRP has been successfully used for ocular metaplasia or dyschromia of healed lesion, comfort and pain
diseases, such as for dry eye syndrome and enhanced diminution, less edema and ecchymosis, reduced mortality
healing of corneal surface and ocular surgery (Fig. 3L).8,9 after cardiac surgery, reduced infection rate after cardiac and
200 Section 2 Cellular Therapies
A D G J
B E H K
C F I L
Figures 3A to L: Pictures showing some of the applications of PRP12-23:A and B : Dermatology and esthetic use; C and D : Cardiothoracic
surgery; E: Chronic wound healing, F: Dentistry; G to I: Orthopedics and musculoskeletal pain relief; J: Diabetic wound healing, K: Plastic surgery;
L: Cosmetic ophthalmic surgery
orthopedic surgery, reduced transfusion supply and hospital Consequently, we can say that the platelet-derived products
stay after orthopedic surgery and reduced amputation rate currently represent a valuable therapeutic modality, offering
in diabetic foot. Practically there are no or minimal ill effects opportunities for various applications in regenerative
when compared to standard therapies.8 medicine and tissue engineering.8
Umesh Sharma
56
Platelet Rich Plasma Platelet Rich plasma in Plastic Surgery
Platelets are more than just first-line responders to bleeding
injury. Every platelet is also a biochemical storehouse of Platelet Rich Plasma in Hair Transplantation
regulatory, signaling and growth-factor molecules that
participate in recovery and healing of tissue as well as The potential for using PRP to promote healing and hair
emergency response to injury. Growth-factor molecules growth after hair transplantation is centered in three
associated with platelets include: functional applications:4
• Platelet-derived growth factor (PDGF): Promotes blood 1. To preserve and enhance hair follicle viability during and
vessel growth, cell replication, skin formation. after hair transplantation.
• Transforming growth-factor-beta (TGF-b): Promotes 2. To promote and enhance tissue repair and healing after
growth of matrix between cells, bone metabolism. hair transplantation; and
• Vascular endothelial growth factor (VEGF): Promotes 3. To reinvigorate dormant hair follicles and stimulate new
blood vessel formation. hair growth.
• Epidermal growth factor (EGF): Promotes cell growth
and differentiation, blood vessel formation, collagen
formation.
To Preserve and Enhance Hair Follicle Viability
• Fibroblast growth factor-2 (FGF-2): Promotes growth of Between the time that hair follicles are removed from a donor
specialized cells and blood vessel formation; and area of the scalp and transplanted into a recipient area, they
• Insulin-like growth factor (IGF): A regulator of normal are subject to damage from several causes:
physiology in nearly every type of cell in the body. • Dehydration if the donor follicles are inadequately
moistened between the times of removal and
Rationale for use of Platelet transplantation.
Rich plasma • Oxygen and nutrient starvation due to being removed
from blood supply during the harvest-to-transplantation
The rationale for use of PRP in surgery is to artificially increase time period.
the number of platelets at a site where the storehouse of • Temperature and acid/alkaline changes in the follicle
growth factors in the platelets can be put to use in enhancing environment; and
tissue recovery, repair and health.1 Search for a way to enhance survival of transplanted
Platelet rich plasma (PRP) is also finding increasing use hair follicles and promote healing with minimal scarring
in orthopedics, dental implant surgery and other surgical after transplantation led to trials of PRP.
specialties as a wound treatment. Safe use of PRP has been A common approach to maintaining donor hair follicle
documented in neurosurgery, ophthalmology, urology, viability during the transition period is to keep them in a
cardiac surgery, thoracic surgery, maxillofacial surgery and storage solution that provides a protective environment of
cosmetic surgery.2,3 Approval of these uses by the US Food appropriate temperature, chemical balance and nutrient
and Drug Administration (FDA) is not required as long as the supply. Recent research has indicated that addition of PRP
use is procedural and part of the treatment process, and no to the storage solution improves follicle viability during
advertised claims of its efficacy are made. and after transplantation, enhances post-transplantation
Chapter 56 Role of Platelet Rich Plasma in Plastic Surgery 203
Paramjit Dhot
57
Introduction to Gold". The first cord blood transplant was carried out in a
boy with Fanconi’s Anemia by Dr Eliane Gluckman in Paris,
Stem cells are the mother cells of the human body. Stem cells in 1988, from stem cells of his sister and he is well and cured
are self-renewing and can give rise to more specialized cells of his life threatening disease.2
of human body, such as muscle cells, blood cells and brain Lifecell International is the first, largest, oldest and most
cells. They are best described in the context of normal human accredited cord blood bank in the country with over 100
development. When a sperm fertilizes an egg, the product is collection centers in India and abroad namely Bahrain,
a single cell that has the potential to form an entire organism. Dubai, Abu Dhabi and Nepal. More than 100,000 units of cord
These stem cells are being used for curing blood cancers blood are stored by Lifecell International at Chennai and dual
and diseases like thalassemia and cerebral palsy. Clinical storage facility at Manesar. Of these 30 cords blood units have
trials are in progress to use the stem cells in heart attack, been retrieved for transplantation. There were 21 cord blood
diabetes mellitus, spinal injury, osteoarthritis and nonunion transplants for Thalassemia Major, 5 for cerebral palsy and
of bones. The sources of stem cells are from the bone marrow, 4 for leukemias. The cerebral palsy transplant patients have
peripheral blood stem cells, cord blood stem cells and cord shown neurological and functional improvement. A Public
tissue stem cells. Cord Blood Bank has been launched by Lifecell, on 27 July
Cord blood transplantation has come of age. Umbilical 2014, to store 10,000 units at a cost of Rs 30 Crores.
cord blood banking efforts have increased dramatically in The advantages of cord blood are that it is easily available
the past two decades in response to increasing demands and has less graft versus host disease. In the coming decade
for alternative sources of stem cells to support patients Cord Blood stem cells will be used extensively in the clinics
requirement stem cell transplantation. Twenty five years after and hospitals for various malignant and nonmalignant
the first cord blood transplant, approximately 3 million cord disorders.
blood units are stored in family cord blood banks all over the
world and 900 cord blood units were released for therapy.1–3 Cord Blood Transplantation in
Over 650,000 umbilical cord blood units have been stored
in public banks for transplants worldwide. Over 30,000 cord
Thalassemia in India
blood transplants have been performed globally.3 Current estimates suggest that approximately 10,000 children
Cord blood stem cells are collected after the birth of are born each year in India with Thalassemia. Transplantation
the baby. After the umbilical cord is clamped and cut, of stem cells offers the only hope of cure for a number of
approximately over 80 mL of cord blood is collected from the diseases of childhood, both malignant and nonmalignant,
umbilical cord in a blood bag with an anticoagulant. This cord including Thalassemia. The preferred donor is a matching
blood is very rich in stem cells, and after removal of red blood sibling, but with a growing number of one child families and
cells, the stem cells are mixed with a cryoprotectant dimethyl only a one in four chance of a sibling being a match, there is an
sulpoxide. These stem cells are stored in liquid nitrogen at increasing need for match unrelated donors. Hematopoietic
-196°C in liquid nitrogen.4 The shelf life of these stem cells is stem cells transplantation offers the only hope of cure for
21 years.5 a number of diseases of childhood both malignant and
These stem cells can be used by the baby, the sibling, nonmalignant including thalassemia and metabolic storage
parents and even grandparents after HLA matching. disorders.3
Placenta and cord blood which was earlier discarded in Lifecell International, India’s first and most accredited
the garbage is now providing lifesaving stem cells "Garbage private Cord Blood Bank offering dual storage facility with
Chapter 57 Cord Blood Banking and Transplantation 205
over hundred collection centers in India and abroad. Thirty cord blood more children will receive cord blood transplants
Cord Blood Transplants have been successfully carried out to cure thalassemia and other inherited disorders.
till date out of which 21 were for thalassemia. The detailed
patient’s outcome was as follows: the youngest patient was Conclusion
2 years and oldest 9 years in age. There were 9 males and
12 females. Standard conditioning regimen was used in The absence of ethical concern and the unlimited supply of
all the patients. One patient had lower GI bleed and febrile cells explain the increasing interest of using cord blood for
neutropenia. Mild hypertension was seen in 5 patients. Major regenerative medicine and cellular therapy.
hypertensive encephalopathy with convulsions due to DMSO After 26 years of the first cord blood stem cell transplant,
toxicity was seen in 1 patient. One patient had hypotension. we anticipate an abundant umbilical cord blood supply,
The other transplant patients had a smooth recovery. On digitalized umbilical cord blood selection, multiple new
follow up 95% of the patients have shown a disease free indications and significantly improved clinical outcomes.
survival. In India, family cord blood banking was started by lifecell
Ideally the cord blood unit should be 6/6 HLA match with International in 2004. In the future, it is expected that cord
the patient, but 5/6 or 4/6 are also accepted.6 The required blood transplantation will be the mainstay in the therapy for
total nucleated and CD 34 cell dose depends on the recipient malignant and nonmalignant disorders. Stem cell science
body weight. The disease free survival rate varies between 80 represents to those afflicted with chronic disease a vehicle
and 95% in beta thalassemia.7 for disease free survival. Moreover regenerative medicine,
cellular therapy and tissue engineering will offer a panacea
for the needy patients.
Cord Blood Transplantation in India
The cost of autologous transplant in India is $ 10,000 to References
$ 12,000 and allogeneic transplant is $ 18,000 to $ 20,000. The
chance of finding a matching family donor is 25%, whereas 1. Ballen KK, Gluckman E, Broxmeyer HE. Umblical cord
it is one in a million in unrelated donors. In India there is blood transplantation: the first 25 years and beyond. Blood.
2013;122:491-8.
no bone marrow donor registry and hence patients in India,
2. Gluckman E, Ruggeri A, Rocha V, et al. Family directed umbilical
face a problem in finding a donor. Approximately 5% of the cord blood banking. Haematologica. 2011;96(11):1701-7.
transplant patients are lost due to fungal infections in India. 3. Broxmeyer HE, Farag SS, Rocha V. Cord blood Haematopoietic
The major transplant centers in India are CMC Vellore, Tata Cell Transplantation. In: Appelbaum FR, Forman SJ, Negrin RS,
Memorial Hospital Mumbai, Army Hospital Delhi Cantt, Antin JH (Eds). Thomas’ Haematopoietic Cell Transplantation.
Tata Medical Centre Kolkata, AIIMS New Delhi and Apollo 5th edn. Oxford, England: Wiley Blackwell; 2013.
Hospitals. Approximately 100 to 125 Umbilical Cord Blood 4. Dhot PS, Nair V, Swarup D, et al. Cord Blood Stem Cell Banking
transplants are carried out in the country annually. and Transplantation. Indian J Pediatric. 2003;70(12):989-92.
5. Broxmeyer HE, Lee MR, Hangoc G, et al. Haematopoietic stem/
progenitor cells, generation of induced pluripotent stem cells
Public Cord Blood Banking and isolation of endothelial progenitors from 21 to 23.5 year
cryopreserved cord blood. Blood. 2011;117(18):4773-7.
Lifecell International has launched the Public Cord Blood 6. Eapen M, Klein JP, Ruggeri AL, et al. Impact of allele level
Programme on 27th July 2014 at Chennai. This will offer HLA HLA matching on outcomes after myeloablative single unit
matched cord blood units for the needy patients as a cure for umbilical cord blood transplantation for haematologic
their ailments.8 malignancy. Blood. 2014;123(1):133-40.
With the availability of increasing umbilical cord blood 7. Dhot PS. Cord blood transplantation for Thalassaemia in India.
supply and numerous new indications being added resulting Parent’s Guide to Cord Blood. 2014;6:2-3.
in markedly improved clinical outcomes in cord blood 8. Appelbaum FR. Pursuing the goal of a donor for everyone in
need. N Engl J Med. 2012;367(16):1555-6.
transplant patients. It is our hope at Lifecell, that by providing
families in India with a means to store their children’s sibling
Erythropoiesis Stimulating
Agents versus Red Cell Transfusion
Sandeep Puri
58
INTRODUCTION Available erythropoiesis stimulating agents
requirements are difficult to predict, but the target rate access thrombosis when ESAs were administered with target
of correction of anemia has been calculated as <1 g/dL to hemoglobin levels of 14 g/dL. Around 2004, it became
2 g/dL per month. When hemoglobin level exceeds the target, apparent that as a result of treatment with ESA’s, patients with
the ESA dose should be reduced without discontinuing the end stage renal disease and cancer chemotherapy induced
treatment so as to avoid the excessive decrease in hemoglobin. anemia were having thromboembolic events. In a review of
There is evidence that risk for mortality is high in patients trials (both independent and industry sponsored) evaluating
with chronic kidney disease when the hemoglobin levels the role of ESAs for treatment of anemia, it was found that
were persistently below 11 g/dL or levels that had significant there is 1.57-fold increased VTE risk and a 1.10-fold increased
fluctuations between the target range and <11 g/dL.4 Thus mortality risk when ESAs were administered to patients with
these agents have generated a ray of hope of an improved anaemia and cancer.14
outcome in an otherwise debilitating disease. In another systematic meta-analysis addressing the
Despite the promising results, the overall effect of benefits and harms of ESAs for anemia, it was concluded that
therapy with ESA on patient outcome is still debated. This is the risk of serious adverse events was significantly higher
probably attributed the documentation of side effects such among recipients of ESAs than among controls.15 There is
as hypertension, seizures, possible cardiovascular events, inconclusive evidence to suggest that the use of ESAs in
vascular access thrombosis, pure red cell aplasia and iron treating anemia in patients with heart failure is beneficial.16
deficiency. Limited evidence suggests that the presence of clinically
evident cardiac disease (congestive heart failure or ischemic
Hypertension heart disease) in ESA-treated patients increased their risk
of nonfatal myocardial infarction. Consequently, extreme
Erythropoietin induced increase in hematocrit in nor caution should be used when using these drugs in patients
motensive subjects is associated with a significant increase with established heart disease.
in resting mean arterial pressure of 6 mm Hg as measured In November 2009, the results from the trial to reduce
by intra-arterial catheter. This has been attributed to cardiovascular events with aranesp therapy (TREAT) were
impaired endothelium dependent vasodilatation, systemic presented.17 Participants were randomly assigned into
vasoconstriction and renal mechanisms (hyporeninemic receiving darbepoetin for a hemoglobin target of 13 g/dL, or
hypoaldosteronism).5 In patients on hemodialysis, the receiving placebo with rescue darbepoetin treatment below a
incidence of hypertension correlated with the dose of hemoglobin of 9 g/dL. There was a significant improvement
erythropoietin. Intravenous erythropoietin doses of 40, 80 in quality of life but a doubling in the risk of stroke in the
and 120 U/kg thrice weekly for 49 weeks were associated with normal hemoglobin arm provided support for an adverse
hypertension in 28%, 32% and 56% of subjects respectively.6 relationship between ESAs and stroke.17
Similar results have been obtained with Darbepoetin.7 In Therefore, the use of ESAs should be carefully reconsidered
rare cases, hypertensive encephalopathy and seizures have in patients with high risk of thromboembolic events such
occurred. Blood pressure response differs markedly in RBC as previous history of thrombosis, surgery, prolonged
transfusion where blood pressure decreases despite similar immobilization and in patients with hypercoaguble states.
increase in hematocrit. Most of the trials have under reported There is no data on preventive use of anticoagulants or
the incidence of hypertension with ESAs due to standard of aspirin.
care escalation of antihypertensive medications during the
trial. Also there has been a question mark over the quality
of trials as in most of the study groups, ultrafiltration during
Pure Red Cell Aplasia
dialysis was increased in line with increase in hematocrit. An extremely rare but serious adverse effect of the long-
term administration of ESAs is pure red cell aplasia reported
Cardiovascular Complications primarily with SC administration. Pure red-cell aplasia was
the most common adverse event associated with the use of
Randomized trials in patients undergoing dialysis have epoetin therapy in the database of the FDA’s Adverse Event
found higher rates of cardiovascular and thromboembolic Reporting System. The production of immunoglobulin (Ig)
complications with higher (13–15 g/dL) vs lower G antibodies against endogenous erythropoietin has been
(10–11.5 g/dL) hemoglobin targets.8-10 Two recent trials: demonstrated in patients with chronic kidney disease.
cardiovascular risk reduction by early anemia treatment Therefore, the resulting anemia is much more severe than
with epoetin beta (CREATE) and correction of hemoglobin prior to the onset of therapy. Bone marrow examination
and outcomes in renal insufficiency (CHOIR), suggest that shows almost complete absence of erythroid precursors,
targeting higher than recommended hemoglobin levels with but normal white cell and platelet precursors. Any patient
ESAs poses a safety risk.11,12 The Normal Hematocrit Study13 who develops a loss of response, severe anemia, and low
of patients with kidney and heart disease also identified a 1- reticulocyte count should be evaluated.18 No pure red cell
to 5-fold increased risk of myocardial infarctions and vascular aplasia has been reported in cancer patients.
208 Section 2 Cellular Therapies
the transfused RBCs. Excess iron is typically present in liver, Impact of RBC Transfusion in Cancer
heart, skin and endocrine organs. Organ toxicity begins when
reticuloendothelial sites of iron storage become saturated. In immunocompromized patients, blood transfusion
Therefore, continuous transfusion therapy in absence of iron may increase the risk for non-Hodgkin lymphoma. Also
chelation can lead to fatal liver/heart dysfunction. there is increased relative risk for development of Chronic
lymphocytic leukemia/small lymphocytic lymphoma.28
Several retrospective studies looked at impact of RBC
Infectious Disease Transmission transfusion on treatment outcome in patients with regional
Current RBC transfusions are relatively safe in regards to cancer. Most of these studies had a curative intent (surgical
transfusion related infections. Viral transmission of HIV 1 treatment) and revealed higher perioperative mortality in
and 2, HTLV, HBV and HCV have been limited by systematic patients receiving RBC transfusions.29
testing of blood products.26 Bacteria may be introduced into There is paucity of data on the rate of progression of
the pack at the time of blood collection from sources such as primary tumor when RBC transfusion is used as a mode of
donor skin, donor bacteremia or equipment used for blood correcting anemia.
processing. This risk is also minimal with RBCs as they are
stored in refrigerator where most of the contaminants are Miscellaneous
nonviable.
Other complications include hypothermia, citrate toxicity,
hyperklemia etc.
Immune Injury
Acute immune injuries have emerged as the leading CONCLUSION
complication of RBC transfusion. Most of these reactions
occur immediately after transfusion. Anemia in chronic diseases is associated with a significant
• Hemolytic transfusion reactions: They are caused by reduction in quality of life. Adequate management of
pre-existing antibodies. Delayed hemolytic transfusion anemia is essential to the core treatment plan. Both
reactions are seen 3–10 days after transfusion of apparently RBC transfusion and ESAs are efficacious means to
cross matched compatible RBCs. In such cases, there has improve hemoglobin levels. However, recent data
been previous allo-immunization to minor RBC antigens, indicates that ESAs, are associated with serious adverse
therefore a history of prior transfusion or pregnancy is effects and have greater morbidity and mortality as
mandatory. compared to RBC transfusion. There is insufficient data
• Febrile nonhemolytic transfusion reactions: It is defined as to suggest improvement in health related quality of life in
1°C rise in temperature during or soon after transfusion. chemotherapy induced anemia. The primary role of ESAs,
It is attributed to reaction between recipient antibodies currently, is to decrease the number of transfusions and
and white cell antigen in the blood product. This too has not replace the same. Robust pharmacovigilance programs
declined with use of leucodepletion techniques. are essential and are expected to shed additional light on
• Allergic blood transfusion reactions: It is seen in 1–3% of the effects of ESAs on survival and disease progression in
RBC transfusion recipients. Anaphylaxis with hypotension, such patients.
bronchospasm, stridor, tachycardia, arrhythmias and GI
symptoms occur with one in 20,000–50,000 transfusions. references
• Transfusion related acute lung injury (TRALI): It develops
1. Jelkmann W. Erythropoietin after a century of research:
within 2–8 hours of transfusion in patients without other younger than ever. Eur J haematol. 2007;78(3):183-205.
risk factors for acute lung injury. It is very rare but an 2. http://www.fda.gov/drugs/DrugSafety/DrugSafetyPodcasts/
important cause of transfusion related mortality. ucm077123.htm.
• Post transfusion purpura: It is a rare but serious 3. Locatelli F, Pisoni RL, Combe C, et al. Anaemia in haemodialysis
complication. There occurs destruction of platelets due to patients of five European countries: Association with morbidity
antibodies against platelet specific antigens. and mortality in the Dialysis Outcomes and Practice Patterns
• Transfusion related graft vs host disease: A rare Study (DOPPS). Nephrol Dial Transplant. 2004;19:121-32.
4. Eckardt KU, Kim J, Kronenberg F, et al. Haemoglobin variability
complication, it occurs 8–10 days post transfusion. It
does not predict mortality in European hemodialysis patients. J
carries a mortality rate of 80–90% resulting from infections Am Soc Nephrol. 2010;21:1765-75.
and bleedind secondary to pancytopenia and liver 5. Lundby C, Thomsen JJ, Boushel R, Koskolou M, Warberg J,
dysfunction. It is related to immune response mediated Calbet JA, et al. Erythropoietin treatment elevates haemoglobin
by donor T lymphocytes against the recipient lymphoid concentration by increasing red cell volume and depressing
tissue.27 plasma volume. J Physiol. 2007;578(Pt 1):309-14.
210 Section 2 Cellular Therapies
6. Pollok M, Bommer J, Gurland HJ, Koch KM, Schoeppe W, 17. Pfeffer MA, Burdmann EA, Chen CY, Cooper ME, de Zeeuw D,
Scigalla P, et al. Effects of recombinant human erythropoietin Eckardt KU, et al. A trial of darbepoetin alfa in type 2 diabetes
treatment in end-stage renal failure patients. Results of a and chronic kidney disease. N Engl J Med. 2009;361:2019e32.
multicenter phase II/III study. Contrib Nephrol. 1989;76:201-11. 18. Bennett CL, Luminari S, Nissenson AR, et al. Pure red cell
7. Kanbay M, Akcay A, Delibasi T, Uz B, Kaya A, Koca C, et al. aplasia and Epoetin therapy. N Engl J Med. 2004;351:1403-8.
Comparison of effects of darbepoetin alfa and epoetin alfa 19. Dicato M, Plawny L. Erythropoietin in cancer patients: pros
on serum endothelin level and blood pressure. Adv Ther. and cons. Curr Opin Oncol. 2010;22:307-11.
2007;24:346-52. 20. Leyland-Jones BEST investigators and study group. Breast
8. Furuland H, Linde T, Ahlmen J, Christensson A, Strombom U, cancer trial with erythropoietin terminated unexpectedly.
Danielson BG. A randomized controlled trial of haemoglobin Lancet Oncol. 2003;4:459-60.
normalization with epoetin alfa in pre-dialysis and dialysis 21. Glaspy J, Smith R, Aapro M, et al. Results from a phase 3,
patients. Nephrol Dial Transplant. 2003;18:353-6. randomized, double-blind, placebo-controlled study of
9. Besarab A, Bolton WK, Browne JK, et al. The effects of normal as darbepoetin alfa for the treatment of anaemia in patients not
compared with low hematocrit values in patients with cardiac receiving chemotherapy or radiotherapy. Paper presented at
disease who are receiving hemodialysis and epoetin. N Engl J the American Association for Cancer Research Annual Meeting
Med. 1998;339:584-90. Proceedings; April 14-18, 2007; Los Angeles, CA.
10. Parfrey PS, Foley RN, Wittreich BH, Sullivan DJ, Zagari MJ, 22. Bohilus J, Schmidlin K, Brillant C, Schwarzer G, Trelle S,
Frei D. Double-blind comparison of full and partial anaemia Seidenfeld J, et al. Recombinant human erythropoiesis
correction in incident hemodialysis patients without stimulating agents and mortality in patients with cancer: a
symptomatic heart disease. J Am Soc Nephrol. 2005;16:2180-9. meta analysis of randomized trials. Lancet. 2009;373:1532-42.
11. Drueke TB, Locatelli F, Clyne N, and CREATE Investigators. 23. Aapro M, Osterwalder B, Scherhag A, et al. Epoetin-beta
Normalization of haemoglobin level in patients with chronic treatment in patients with cancer chemotherapy induced
kidney disease and anaemia. N Engl J Med. 2006;355:2071-84. anaemia: the impact of initial haemoglobin and target
12. Singh AK, Szczech L, Tang KL, and CHOIR Investigators. haemoglobin levels on survival, tumor progression and
Correction of anaemia with epoetin alfa in chronic kidney thromboembolic events. Br J Cancer. 2009;101:1961-71.
disease. N Engl J Med. 2006;355:2085-98. 24. Crémieux PY, Barrett B, Anderson K, et al. Cost of outpatient
13. Besarab A, Bolton WK, Browne JK, et al. The effects of normal as blood transfusion in cancer patients. J Clin Oncol. 2000;18:
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disease who are receiving hemodialysis and epoetin. N Engl J 25. Hess JR. Red cell storage. J Proteomics. 2010;73:368-73.
Med. 1998;339(9):584-90. 26. Bihl F, Castelli D, Marincola F, et al. Transfusion-transmitted
14. Bennett CL, Silver SM, Djulbegovic B, Samaras AT, Blau CA, infections. J Transl Med. 2007;5:25.
Gleason KJ, et al. Venous thromboembolism and mortality 27. Anderson KC, Weinstein HJ. Transfusion-associated graft-
associated with recombinant erythropoietin and darbepoetin versus-host disease. N Engl J Med. 1990;323:315-21.
administration for the treatment of cancer-associated 28. Castillo JJ, Dalia S, Pascual SK. Association between red
anaemia. JAMA. 2008;299:914-24. blood cell transfusions and development of non-Hodgkin
15. Tonelli M, Hemmelgarn B, Reiman T, Manns B, Reaume MN, lymphoma: A meta-analysis of observational studies. Blood.
Lloyd A, et al. Benefits and harms of erythropoiesis-stimulating 2010;116:2897-907.
agents for anaemia related to cancer: a meta-analysis. CMAJ. 29. Vamvakas EC, Blajchman MA. Deleterious clinical effects of
2009;180:E62-71. transfusion associated immunomodulation: Fact or fiction?
16. Lindguist DE, Cruz JL, Brown JN. Use of erythropoiesis Blood. 2001;97:1180-95.
stimulating agents in the treatment of anaemia in patients of
systolic heart failure. J Cardiovasc Pharmacol Ther. 2014; pii
1074248414541841 [Epub ahead of print].
Natural Killer Cells in Tumor
and Transplantation
Role of Natural Killer Cells in disease, which makes these cells uniquely suited for adoptive
immunotherapy.9 Unlike B- and T lymphocytes, NK cells do
Transplant Immunology not express clonally rearranged receptors to detect antigens.
Conventionally ‘transplantation immunology’ has focused Instead, activation is regulated by integration of signaling
on adaptive component of immune system, i.e. T- and B-cell from germ-line encoded activating and inhibitory cell surface
reactions. Only recently natural killer (NK) cells have been receptors.10
found to play an important role after transplantation of Over the past 20 years, NK cells have attracted
solid organs and hematopoietic stem cells. Transplantation substantial interest in the context of hematopoietic stem cell
process, results in tissue injury and inflammation, and as transplantation. While many aspects of NK cell biology in this
a result, upon reperfusion of the graft, the beneficiary’s setting remain poorly understood, donor selection criteria
immune system come across a plethora of soluble and cell in HLA-mismatched transplantation include the potential
surface danger- and stress-signal molecules. of a donor to generate alloreactive NK cells post-transplant.
Natural killer (NK) cells have activating receptors which Retrospective studies showing a survival advantage for
recognize “cell stress” induced by various foreign activities. patients transplanted from an HLA-identical donor carrying
These activating receptors are negatively regulated by the activating KIR receptors have led to large prospective trials
interaction between inhibitory molecules and autologous that aim to preferentially recruit stem cell donors with a
human leukocyte antigens (HLA). With the availability of an beneficial activating KIR gene profile. While these are exciting
array of activating receptors specific for stress-related cellular times for researchers interested in NK cells, much work
events, NK cells are exclusively equipped to detect and react remains to be performed to unravel the many ways in which
to the damage initiated by injury. NK cell activation is a NK cells influence the transplant outcome.
phenomenon which includes coordination of all activating
and inhibitory signals received by the cell.7 The two major Natural Killer Cells and Cancer
components of this system are:
Surveillance
1. Inhibitory signals received by major histocompatibility
complex (MHC) class I molecules. Myriad of reports from mouse model evidence the role of
2. Activating signals received by molecules up regulated in NK cells in the abolition of tumor cells and removal of NK
response to stress and specific to the NK cell lineage. cells functional arm often led to a more aggressive tumor
The activating NK cell receptor, i.e. NKG2D, recognizes growth and metastasis in a tumor induced mouse and higher
stress- or pathogen-derived ligands, whereas inhibitory killer frequencies of spontaneous tumor in chemical carcinogen
immunoglobulin-like receptors (KIR receptors) recognize challenged mice. In humans, correlative studies show that
self, but not allogeneic human leukocyte antigens (HLA)-A, low NK cell activity was correlated with cancer risk which
-B, or -C. NK cell subsets express either KIR receptors or indicates a role for NK cells in tumor surveillance.11 However,
CD94/NKG2A. KIRs are highly polymorphic whereas NKG2A unlike mouse model, low NK cells infiltration in established
are less polymorphic.8 tumors subverts the antitumor immune response. Low
Although NK cell mediated organ damage might be infiltration which was attributed to inefficient homing into
limited, the role of these cells as initiators or perpetuators of malignant tissues could be meliorated by cytokine mediated
adaptive immune responses, well known in other context, immune activation or by occurrence of alloreactive NK cells
has not received much attention in the transplant setting. The provided by allogeneic hematopoietic stem cell transplant.12
stress sensor system of NKG2D-mediated activation has the In the recent past, NKG2D receptor has been identified
potential to override MHC class I inhibition in vitro, suggesting as a seminal NK cell receptor, which in concert with other
the importance of understanding the consequences of ROS receptors like NKp30, NKp44 and NKp46, determine NK cell
injury on NK cells. As we all know, MHC system is inherited, activation toward tumor cells.
similarly the repertoire of KIR genes is also inherited, and Natural killer (NK) cells play an important role in
out of all KIR genes, certain genetic patterns are disease- protection against transplantable tumors. In varying range of
associated. The clinical challenge of infection in the setting experimental models, this has been done by depletion of NK
of immune suppression after transplantation may be as cells before tumor challenge to show more aggressive tumor
important in the setting of cell therapies as it is in organ growth. Other investigators incorporated the strategy of
transplantation, further highlighting the need to understand administering cytokines enhancing NK cell effector functions,
NK cell responses for optimal transplant therapies. e.g. IL-2, IL-12, IFN or IFN inducing agents to exhibit
Interestingly, many studies have shown that the natural enhanced antitumor activity. As a common observation, it
killer (NK) cells are also involved in immunity both against was noted from these experiments that NK cells are more
residual tumor cells and against pathogens in patients efficient at eliminating tumor deficient or low MHC-I on their
undergoing stem cell transplantation. Most importantly, the surface. Despite their well proven antitumor activity and
beneficial effects attributed to NK cells in the post-transplant immune-surveillance, NK cells may not be able to completely
period appear to occur without the risk of graft-versus-host eliminate a tumor alone, but may seek cooperation from other
Chapter 59 Natural Killer Cells in Tumor and Transplantation 213
arms of immune system such as T or B cells. Furthermore, 4. Walzer T, Jaeger S, Chaix J, et al. Natural killer cells: from CD3−
there is increasing evidence that indicate that although NKp46+ to post-genomics meta-analyses. Current opinion in
NK cells alone may not be capable of eliminating tumors, immunology. 2007;19:365-72.
5. Trinchieri G. Biology of natural killer cells. Advances in
they may kill some tumor cells, providing apoptotic tumor
immunology. 1989;47:187-376.
bodies to antigen presenting cells such as Dendritic cells, for 6. Giorda R, Weisberg E, Ip T, et al. Genomic structure and strain-
subsequent activation of T-cell response. Additionally, IFN-γ specific expression of the natural killer cell receptor NKR-P1.
or other cytokines secreted by NK cells in response to tumors The Journal of Immunology. 1992;149:1957-63.
may activate local macrophages and dendritic cells, which in 7. O’Connor GM, Hart OM, Gardiner CM. Putting the natural
return fuel NK cells by cell-cell contact or soluble factor in a killer cell in its place. Immunology. 2006;117:1-10.
positive feed-back manner.13 8. Spahn JH, Li W, Kreisel D. Innate immune cells in
transplantation. Current opinion in organ transplantation.
2014;19:14-9.
Conclusion 9. Terszowski G, Passweg JR, Stern M. Natural killer cell immunity
after transplantation. Swiss Med Wkly. 2012;142:w13700.
Although identified as nonspecific killer cells, the field of
10. Passweg J, Stern M, Koehl U, et al. Use of natural killer cells
NK cells have gone through vast metamorphosis and this in hematopoetic stem cell transplantation. Bone marrow
developing area of investigation indicates that NK cells may transplantation. 2005;35:637-43.
be more critical in facilitating immune response in a manner 11. Wu JD, Higgins LM, Steinle A, Cosman D, Haugk K, Plymate
that previously was not well appreciated. SR. Prevalent expression of the immunostimulatory MHC
class I chain–related molecule is counteracted by shedding
in prostate cancer. The Journal of Clinical Investigation.
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reactivity of mouse lymphoid cells against syngeneic and D, Vollmer E, et al. Malignant and non-malignant lung tissue
allogeneic tumors. II. Characterization of effector cells. areas are differentially populated by natural killer cells and
International Journal of Cancer. 1975;16:230-9. regulatory T cells in non-small cell lung cancer. Lung Cancer.
2. Herberman RB, Nunn ME, Lavrin DH. Natural cytotoxic 2008;59:32-40.
reactivity of mouse lymphoid cells against syngeneic and 13. Piccioli D, Sbrana S, Melandri E, et al. Contact-dependent
allogeneic tumors. I. Distribution of reactivity and specificity. stimulation and inhibition of dendritic cells by natural killer
International Journal of Cancer. 1975;16:216-29. cells. The Journal of Experimental Medicine. 2002;195:335-41.
3. Freud AG, Caligiuri MA. Human natural killer cell development.
Immunological reviews. 2006;214:56-72.
Potential and Hazards
of Gene Therapy
Ajit C Gorakshakar
60
Human gene therapy to cure hereditary disorders is a topic Historical Aspect
of public interest, because genetic diseases account for a
significant amount of human sufferings. More than 3000 If we define gene therapy as treatment of a disease by
genetic diseases are known to cause from defects in single genetic manipulation then treatment of an individual with
gene. Many such diseases are well known like sickle cell thyroid hormones in hypothyroidism or with steroids to treat
anemia, thalassemias, cystic fibrosis, etc. while other diseases inflammation in colitis should be considered as gene therapy
like severe combined immunodeficiency syndromes, the because this involves certain genes being turned up or down
various lipid and carbohydrate storage diseases are less or switched on or off. However, today we consider gene
common. therapy as a procedure to correct defective genes to cure the
Gene therapy is the process where the abnormal or disease disease.
causing gene is corrected at DNA level. The term Gene Therapy It can be done by following ways:
was first introduced at the International Congress of Genetics 1. Replacing mutated gene which causes disease with the
(Yaron et al. 1997). This can be done by actually repairing healthy gene.
the defect of the endogenous gene (targeting a gene) or by 2. Inactivating or knocking out a mutated gene. The gene can
introducing new genetic material in to the genome. be turned off, so it no longer promotes the disease.
The actual process depends on the following variables: 3. Introducing a new gene into body to fight a disease.
• The disease to be treated The first attempt to treat a human disease by introducing
• The gene to be inserted new genetic material in to the cells was carried out in the
• The target cells of insertion early 1970s by Dr Stanfield Rogers to treat argininemia
• The vector used to transmit the gene in three sisters. This was performed before the existence
• The route of administration of the gene. of Ethics committees. In 1980 Dr Cline tried to treat two
Selection of a disease to work on is very important. European patients suffering with beta thalasseima. In each
Generally single gene disorders with recessive inheritance case bone marrow removed from the patient was subjected
were selected for this purpose. Therefore lot of work has to treatment with DNA containing the normal hemoglobin
been done on gene therapy to cure sickle cell anemia or gene. Considering various hazards involved in the procedure,
β-thalassemia where β-globin gene is corrected to produce ethics boards were constituted to critically evaluate the
normal β-chains and in appropriate quantity. protocol before starting the experiments.
This can be done by two ways:
1. Germline gene therapy: In this sperm and ova are Gene Therapy in Transfusion Medicine
genetically altered. This is done by injecting the corrected Transfusion medicine is a branch of science involving
gene directly into a fertilized egg at risk so all the cells in administration of cells or any other biologic material like
the body have altered genetic makeup and the genetic serum, clotting factors, etc. to treat patients. In gene therapy
disease is permanently cured. new biological material is transfused in patient body to treat
2. Somatic gene therapy: This refers to manipulation of body specific disease. Therefore, it comes under the preview of
cells other than germinal tissue. This is performed on fully transfusion medicine. Hematopoietic cells are excellent
developed individuals. This technique does not place the targets for gene therapy. They can be easily accessed and may
whole organism at risk if it fails. So, the experiments can be be included ex vivo with the vectors containing the genetic
repeated on the same organism. repair material. Lastly the disease for which gene therapy
Chapter 60 Potential and Hazards of Gene Therapy 215
strategies are developed, directly affect the transfusion quiescent cells with equally high efficiency. They have broad
medicine department. For example if gene therapies for cell specificity. Majority of human population is exposed
thalassemia or sickle cell anemia are developing and patents to adenoviruses. So antibodies against them are already
are cured, this will reduce the load in blood bank considerably. present in our body. Therefore, these vectors are degraded
within 24 hours even before reaching the target cells. They
Prerequisite for gene therapy quickly activate immune cells which can affect the patients
health. Other disadvantages included short term expression,
The complete human genome sequencing project has transient expression of foreign genes and immunogenicity.
significantly contributed to ‘Gene Therapy’ experiments.
Because this project gave us the information about all the
Retroviruses
genes present in entire human genome. Before gene therapy
experiments are started, one should be aware of complete Small RNA viruses, replicating through DNA intermediates.
knowledge of a disease gene, including it’s molecular The genome consists of very few genes required for virus
structure, function and regulation. This is required to provide replication. While designing the vector, these genes are
the best ‘replacement part’. detected and replaced by the therapeutic gene (up to
10 kb). Moloney marine leukemia virus (MMLV) is the most
Gene delivery systems common retrovirus used for designing vector. It’s limitations
are low vector titer, low transfection efficiency, particle
The main challenge of Gene therapy is the difficulty of instability and inability to transduce nondividing post mitotic
transporting large, fragile and negatively charged DNA cells. To overcome these problems, vectors derived from
molecules into the nucleus of the cell without degradation. So, lentiviruses have been constructed. Lentiviruses are one
one has to create safe and efficient gene delivery vehicles. For type of retroviruses which can infect both dividing as well as
this purpose knowledge about complete understanding of the quiescent cells. This virus ensures long term expression and
interaction mechanism between the target cells and delivery efficient transfer without inflammatory responses.
system is required. Similarly knowledge about intercellular
traffic and targeting mechanism is also very important.
Adeno Associated Viruses
Vector design Single stranded nonenveloped DNA viruses. They have low
risk of insertional mutagenesis ‘Suicide’ gene therapy is
There are many aspects involved in engineering the vector feasible using this virus. It’s genome is small (4 kbp). They
to be used for gene therapy. They are, specific disease to be are able to transduce a wide variety of cells regardless of
treated, the target cells and the therapeutic strategy. The their stage in the cell cycle. They are able to permanently
normal/corrected gene which will be inserted can provide a implant their genes into a particular part on chromosome 19
functional form of a missing or detecting gene or it can act as of humans. However, the main drawback of this vector is its
a suicide gene (in cancer) designed to kill the cell in which size of the genome which significantly limits the amount of
it is expressed. In case of cystic fibrosis in which the disease genetic material that it can carry.
causing gene is nonfunctional, a functional gene is added
while in case of thalassemia the gene for normal product of
Lentiviruses
fully functional β-globin gene is inserted.
They can deliver a significant amount of viral RNA into the
Viral delivery systems DNA of host cells. They have the unique ability to infect non
dividing cells. So, today genetically engineered lentivirus
They consist of viruses which are replication deficient but is widely used as a gene delivery vector. This has some
can deliver DNA for expression. Adenoviruses, retroviruses, advantages over other vectors. They include high efficiency
Adeno Associated viruses and lentiviruses are used as gene infection of dividing and nondividing cells, long-term
delivery vectors (Escors and Breckpot 2010). They have high stable expression of a transgene and low immunogenecity.
gene transfection efficiency but their residual viral elements Lentiviruses have been successfully used for transfection of
can cause insertional mutagenesis and immunological diabetic mice with the gene encoding platelet derived growth
problems. In 1980s gene transfer to mammalian cells factor (PDGF) (Lee et al. 2005) and also correcting hemophilia
was feasible after the development of retroviral vectors in mouse model, by expressing wild type platelet factor VIII
(Yaron et al. 1997). In 2006, melanoma was successfully gene (Shi et al. 2007). They do not require degradation of the
treated using retroviral vectors. nuclear membrane for integration.
3
Blood Safety
Leucoreduction
Kanchan Bhardwaj
61
Leucoreduced blood product/leucoreduction (LR) are the Clinical Indications for
usage of blood products from which the leucocytes have been
removed. Leucoreduced red blood cells and platelets have Leucoreduction
been used to decrease the adverse effects of their transfusion
occurring because of allogenic donor leucocytes. It involves Established Indications
the process which removes leucocytes from the blood • Reduce frequency and severity of recurrent febrile non
components and reduces the incidence of adverse events hemolytic transfusion reactions (FNHTRs) in chronically
associated with the donor leucocytes (Table 1). transfused patients.
• Reduce risk of CMV transmission to susceptible recipients
Approximate leucocytes in various (neonates, transplant recipients, oncology patients, etc.).
• Reduce rate of HLA alloimmunization and platelet
Red blood cell preparations refractoriness among hematology-oncology patients.
Blood components Number of leucocytes
Fresh WB ≈ 109 Potential Benefits of Leucoreduction
PRBCs ≈ 108 – 109 • Reduce risk of transfusion related immuo-modulatory
effect on cancer recurrence and postoperative bacterial
Buffy coat depleted PRBC ≈ 108
infection
Washed RBCs ≈ 107 • Reduce risk of transfusion associated graft-vs-host disease
Leucofiltered ≈ < 5 × 106 (TA-GvHD)
• Reduce organ dysfunction and mortality especially in
Critical antigenic load is the level of leucoreduction patients undergoing cardiac surgery1
to prevent FNHTR is 5 × 108 is one log reduction. It can be • Mortality rates are lower when Leucocyte-depleted blood
achieved by buffy coat removal. is used
Critical immunogenic load is the level of leucoreduction to • Reduction in storage lesions
prevent alloimmunization to HLA antigens or preventing the • Improved chance of finding an organ transplant match
transmission of lymphotrophic viruses the level of leucocyte • Possible reduction of variant Creutzfeldt–Jacob Disease
should be less than 5 × 106, i.e. 3 log reduction. (vCJD) transmission.
products are formed. So, it prevents FNHTR, CMV transmis Freezing and Deglycerolization
sion, HLA alloimmunization and better quality control main
tenance can be done (Table 2). It is a costly and time consuming procedure so not used for
such preparations. It is reserved for storing rare blood groups
cells.
Poststorage Filtration
The filtration of components prior to issue from the laboratory Quality Control of Leucodepletion (Table 3)
or at the bedside of the patient prior to transfusion. During
storage leucocytes breakdown and cytokines accumulate No common standards exist for all nations. The American
which are not removed (Table 2). Association of Blood Bank Standards (AABBS) require that
95% of the units sampled meet the requirement for residual
leucocyte content.7 In Europe, the threshold requirement
Low Leucocyte Apheresis Devices for leucocytes is <1 × 106/unit. Techniques available for
• Based on differences in cell mass of cells to be separated. counting methods specifically designed for the extremely low
• Interface detection system–achieve standard leucodeple concentration of white blood cells found in leucoreduced
tion. blood components are microscopy, flow cytometry, and
• The Amicus apheresis system used for preparing platelet the polymerase chain reaction.8 The relative proportion of
concentrates. The device incorporate three features: residual leukocyte subpopulations in leucoreduced blood
active interface; autoelutrition and fluid flow dynamics. is not addressed by any standards. Different leucocyte
An optical interface detector is positioned within the subpopulations are relevant for different biological effects
separation chamber to monitor changes in the platelet attributed to donor leucocytes. The clinical outcomes
interface. evidence of a particular leucoreduction technique is superior
• Trima accel automated system separates platelets from to any other for the removal of specific population is not
donor whole blood using a centrifugal technology with there.9,10 All the methods of leucoreduction that meet the
saturated fluidized particle—bed dynamics to achieve a threshold standard for total number of residual leucocytes are
natural separation of WBC’s from platelets. considered equivalent. Process problems have been reported
with both leucofiltration and apheresis. Kao11 et al. repor-
ted that 7% of apheresis platelets and 5% pooled platelets
Centrifugation and Buffy Coat Removal
failed and for RBCs 0.3–2.7% of units failed. They also reported
It is done by automated component extractions. This gives a substantial losses of platelets and RBCs during the process of
log 1 reduction. It is enough to prevent FNHTR. It does not leucofiltration. In apheresis the technique can fail. Factors
prevent HLA alloimmunization/viral transmission. As per the like sudden change in rate of flow entering the machine or
definition of LR, this cannot be labeled as LR. In developing pauses in blood flow disturb the centrifugal separation of
countries like India, this is used for reducing FNHTRs. blood resulting in spillover of leucocytes into the platelets.
Table 3: Showing quality control of leucoreduction Pros and Cons of Universal Leucoreduction
Product Standards
• Pros
Leucoreduced packed red Residual leucocytes < 5 × 106 - Patients with selected indications to receive leuko
blood cells (LR PRBCs) Red cell loss < 15% of original depleted units will more likely receive leucoreduced
Leucoreduced apheresis Residual leucocytes < 5 × 106 units.
platelets/single donor Total platelet count ≥ 3 × 1011 - Inventory management is streamlined.
platelets (LR SDP) pH 6.2 • Cons
Leucoreduced random Residual leucocytes < 5 × 105 - Large clinical studies fail to demonstrate clear benefit
donor platelets (LR RDP) Total platelet count ≥ 4.5–5.5 × 1010 of leucoreduction on proposed transfusion–associated
pH 6.2 immunosuppression.
Chapter 61 Leucoreduction 225
- It adds the extra financial burden to the health care 6. Nishimura T, Kuroda T, Mizoguchi Y, et al. Advanced methods
cost. for leukocyte removal by blood filtration. In: Brozovic B (Ed).
- Affect the adequacy of blood supply and interfere with The Role of Leukocyte Depletion in Blood Transfusion Practice:
Proceedings of the International Workshop. Oxford, Blackwell
the supply of blood donors of African ancestry (sickle
Scientific, 1989. pp. 35-40.
cell trait). 7. Silva MA (Ed). Standards for Blood Banks and Transfusion
- It removes physician’s choice from product selection. Services, 23rd edn. Bethesda, Md., American Association of
Blood Banks, 2005.
Leucodepletion—Indian Perspective 8. Van der Meer PF, Gratama JW, van Delden CJ, et al. Comparison
of five platforms for enumeration of residual leucocytes in
In India selective leucodepletion is done. It is done by leucoreduced blood components. Br J Haematol. 2001;115:
adopting Buffy coat method of component preparation which 953-62.
gives 1 log reduction and can minimize FNHTR. In special 9. Triulzi DJ, Meyer EM, Donnenberg AD. WBC subset analysis
cases harvesting component through apheresis technology of WBC-reduced platelet components. Transfusion. 2000;40:
771-80.
and pre storage/post storage leucodepletion in special cases
10. Roback JD, Bray RA, Hillyer CD. Longitudinal monitoring of
like patients with repeated FNHTR, patients on regular trans WBC subsets in packed RBC units after filtration: implications
fusion therapy, immunosuppressed patients–CMV negative for transfusion transmission of infections. Transfusion. 2000;
blood and neonates requiring exchange transfusion. 40:500-6.
11. Kao KJ, Mickel M, Braine HG, et al. White cell reduction in
References platelet concentrates and packed red cells by filtration: a
multicenter clinical trial. The Trap study group. Transfusion.
1. van de Watering LMG, et al. Circulation. 1998;97:562-8. 1995;35:13-9.
2. Vamvaks EC, Blajchman MA. Universal WBC reduction: the 12. Sherrill J Slichter. Leukocyte Reduction and Ultraviolet B
case for and against. Transfusion. 2001;41:691-712. Irradiation of Platelets to Prevent Alloimmunization and
3. Seftel MD. Universal prestorage leucoreduction in Canada Refractoriness to Platelet Transfusions. The Trial to Reduce
decreases platelet alloimmunisation and refractoriness. Blood. Alloimmunization to Platelets Study Group. N Engl J Med.
2004;103(1):333-9. 1997;337:1861-70.
4. Fisk JM, Snyder EL. Universal pre-storage leucoreduction—a 13. Sirchia G, Rebulla P, Parravicini A, Carnelli V, Gianotti GA,
defensible use of hospital resource: the Yale-New Haven Bertolini F. Leukocyte depletion of red cell units at the bedside
Hospital experience. Dev Bio (Basel). 2005;120:39-44. by transfusion through a new filter. Transfusion. 1987;27:402-5.
5. Steneker I. Leukocyte Depletion from Fresh Red Cell 14. Sirchia G, Wenz B, Rebulla P, et al. Removal of white cells
Concentrates by Fiber Filtration: Filtration Mechanism. from red cells of transfusion through a new filter. Transfusion.
Amsterdam, VU University Press, 1992. 1990;30:30-3.
Improving Blood Donor Screening
by Nucleic Acid Technology
Rekha Hans
62
Annually, millions of people worldwide receive blood compo sequences. TMA consists of three main steps: target capture,
nents or blood derived products for different indications, so amplification, and detection.2 It allows for simultaneous
safety of these products remains the major responsibility and testing of multiple viruses in a single test tube, making the
concern of any blood transfusing service. A single donation can assay convenient and faster.
be transfused to three or more different patients which means
screening blood for transfusion transmitted infections (TTIs) Blood donor screening
is a very crucial step before taking the blood units into the
inventory. Although screening of blood for these infections is Blood donor screening by NAT can be divided into four
being practised worldwide, TTIs are still being transmitted and groups3:
reported though their incidence has decreased significantly. 1. Transfusion relevant pathogens for which NAT testing is
done in most countries (HBV, HCV and HIV)—the HIV
virus has doubling time of approximately 17 hours. The
Need for Nucleic acid technology diagnostic window period of 15 days by conventional
The conventionally used screening methods target the method has been reduced to 8–9 days by screening with
human body’s response to a virus (virus induced antibodies), mini pool of up to 96 samples per pool and can further be
so the interval between the donor’s exposure to a virus and reduced to 5–6 days for Individual Donation (ID-NAT).
detectable amount of antibodies/antigens (window period) Similarly, the diagnostic window period for HCV is 80
is missed by these methods of screening, thus creating a days for enzyme immunoassays and has reduced to 6–7
need for a more sensitive and specific method to detect the days with mini pool NAT ( pool size of 96) and further to
presence of virus directly. The German Red Cross Blood donor 4–5 days with ID-NAT.
service, Frankfurt took the lead to develop its first “in-house” Compared to HIV-1 or HCV, the HBV virus has
method of nucleic acid testing of transfusion transmissible doubling time of approximately 2.56 days.4 NAT has
viruses. Now-a-days fully automated FDA certified methods helped in reducing the window period of HBV from
are available for nucleic acid technology (NAT) testing that 38.3 days with serological methods to 24.6 days (ID-NAT).3
has been adopted in many countries for blood screening. The major risk of HBV transmission stems from occult
hepatitis B infections which harbours low viral load in
Nucleic acid technology absence of seroreactivity to HBsAg. With NAT, the risk
of these infections has decreased and this has benefited
The method of NAT is based on amplification of targeted multi-transfused patients like thalassamics and those with
regions of viral ribonucleic acid (RNA) or deoxyribonucleic cancer. NAT also complements serological screening by
acid (DNA). detecting false negative serology results.
The two commonly used NAT-screening technologies are 2. Transfusion relevant pathogens for which testing is done in
polymerase chain reaction (PCR), and transcription-mediated some countries like United States for WNV. Other viruses
amplification (TMA). PCR is a method that amplifies single are HAV, B19V, Chickungunya virus and HIV-2. There are
or small amounts of DNA segments, producing thousands or cost effective issues which needs to be addressed before
millions of copies of specific DNA sequences from the target recommending NAT for these viruses.
sequences.1 TMA is a transcription-amplification process 3. Probably transfusion relevant pathogens but not tested
that uses two enzymes—reverse transcriptase and RNA as part of blood donor screening (HAV, SARS CoV and
polymerase to produce millions of copies of the targeted RNA Influenza virus).
Chapter 62 Improving Blood Donor Screening by Nucleic Acid Technology 227
15. Stolz M, Tinguely C, Graziani M, Fontana S, Gowland P, detection of human immunodeficiency virus-1&hepatitis B&C
Buser A, et al. Efficacy of individual nucleic acid amplification Viruses in Indian blood donors. Indian J Med Res. 2008;127:
testing in reducing the risk of transfusion-transmitted hepatitis 140-7.
B virus infection in Switzerland, a low-endemic region. 19. Jain R, Aggarwal P, Gupta GN. Need for nucleic acid testing
Transfusion. 2010;50:2695-706. in countries with high prevalence of transfusion transmitted
16. Vermeulen M, Lelie N, Sykes W, Crookes R, Swanevelder J, infections. ISRN Hematol. 2012;718671 Epub 2012 Sep 12.
Gaggia L, et al. Impact of individual-donation nucleic acid 20. Chatterjee K, Coshic P, Borgohain M, Premchand, Thapliyal
testing on risk of human immunodeficiency virus, hepatitis B RM, Chakroborty S, et al. Individual donor nucleic acid
virus, and hepatitis C virus transmission by blood transfusion testing for blood safety against HIV-1 and hepatitis B and C
in South Africa. Transfusion. 2009;49:1115-25. viruses in a tertiary care hospital. Natl Med J India. 2012;25:
17. Nantachit N, Thaikruea L, Thongsawat S, leetrakool N, 207-9.
Fongsatikul L, Sompan P, et al. Evaluation of a multiplex human 21. Agarwal N, Chatterjee K, Coshic P, Borgohain M. Nucleic
immunodeficiency virus-1, hepatitis C virus, and hepatitis B acid testing for blood banks: An experience from a tertiary care
virus nucleic acid testing assay to detect viremic blood donors center in New Delhi, India. Transfus Apher Sci. 2013;49:482-4.
in northern Thailand. Transfusion. 2007;47:1803-8. 22. World Health Organization and International federation of Red
18. Makroo RN, Choudhury N, Jagannathan L, Malhotra MP, Cross and Red crescent societies. Safe blood starts with me.
Chaudhary RK, Marwaha N. Multicenter evaluation of World Health Organization. Geneva. 2000. p. 12.
individual donor nucleic acid testing (NAT) for simultaneous
ID-NAT Versus MP-NAT
Rajesh Kumar
63
Blood transfusion provides an essential and life saving support decrease the possibility of transmission via transfusion and
in modern health care, when used appropriately, it saves also detects mutants and occult cases.
lives. However, it can cause acute or delayed complications Although NAT screening cannot completely eliminate the
and also carries the risk of transfusion transmitted infections risk of TTIs, but it has reduced the risk HIV, HCV and HBV
(TTIs) such as human immunodeficiency virus (HIV-1), where it has been implemented. Implementation of NAT has
hepatitis B virus (HBV), hepatitis C virus (HCV) and other introduced not only a new methodology, new logistic but
diseases. Despite the current practice of screening blood with when combine with sensitive serology it provides the most
enzyme linked immuno sorbent assays (ELISA) of different sensitive and specific screening platform for blood screening.
sensitivities and even after implementing the more sensitive, The window period for detection by ID-NAT by ultrio plus is
newest generation of serological test, a considerable residual 4.7 days for HIV-1, 2.2 days for HCV and 14.9 days for HBV
risk of transfusion transmission of these virus remains. and with pools of multiple (MP-6) donors is 11 for HIV-1 and
Although the more sensitive serological tests have shortened 2, 12 days for HCV and 25 days for HBV. The corresponding
the pre-seroconversion window period, they still are not able window periods for serological test are 15–20 days. 2–26 weeks
to identify a number of newly infected blood donors. and 50–150 day respectively. The objective of NAT testing is
In spite of all the precautionary measures used to avoid to provide blood in minimum risk to recipients through the
inoculating infective agents into transfusion recipients; it detection of as few viral copies (Cps) as possible of all known
is possible to transmit disease when blood from a recently genotypes, and sub groups. The NAT tests of high sensitivity
infected donor fails to be identified by routine screening rely on amplification of intended target regions of viral nucleic
tests. This is because of the so called window period, i.e. acids (NA) for detection. The aspects which influence the final
the period in which a donor is infected but before the outcome of analytical sensitivity can be categorized under
condition is detectable by routine methods. This technologic three headings: (a) Sample preparation, (b) Amplification,
limitation puts recipients at a tangible, albeit infrequent (c) Detection. In sample preparation the decisive steps are the
risk for transmissible disease. Since viremia precedes sero volume of infectious sample used for testing. The efficiency
conversion by several days in case of HIV and several weeks of virus disruption to release NA, efficiency of NA extraction,
for hepatitis B and C virus, tests that detect viral nucleic acid efficiency of NA elution if required, and removal of substances
are considered a significant technologic and an addition that interfere in the following steps. The success in the
step in our quest to achieve the goal of zero risk for blood amplification category is ensured by the volume of extracted
transfusion recipients. Nucleic acid amplification test (NAT) NA used for target region amplification, and the ability to
has the potential to detect viremia earlier than current tolerate interfering substances, the detection efficiency is
screening methods which are based on seroconversion. determined by the volume of amplicon used for detection,
NAT is currently being used in selected centers for donor the detection technology, the type and number of probes, and
screening, though it is not mandatory by Drugs and Cosmetics the ability to demarcate signal from noise. The final output
Rules. It is a highly sensitive method of testing blood. It is a of all these factors defines the analytical sensitivity. Nearly all
direct test which detects the viral ribonucleic acid (RNA) and screening assays are qualitative assays scoring for reactives.
deoxyribonucleic acid (DNA) by the amplification method. The analytical sensitivity also known as the limit of
It reduces the window period by detecting low level of viral detection (LoD) is determined through dilution analysis of
genomic materials that are present soon after infection but known standards, followed by probit analysis to predict the
before the body starts producing antibodies in response to a minimum Cps/International Units (IU) that can be detected
virus. This allows for earlier detection of infection and further at a particular frequency. The concentrations detected at
Chapter 63 ID-NAT Versus MP-NAT 231
frequencies of 95% and 50% are typically used for defining affordability of ID-NAT has been the premise for promotion
the LoD. Of great practical relevance is the screening of pool testing. The residual risk of the resulting TTIs places
sensitivity of the NAT assay which is determined by the mode a serious burden on the health-care, finances, society and
of practice. Since serology tests are executed as individual human life. HIV-2 from India was reported from Mumbai
donation testes, this is a situation unique to NAT testing. in 1991; infections have been reported from several states of
Currently NAT assays in blood screening are executed in the India. HIV-2 was detected in high-risk groups and professional
format of (a) Individual donation-(ID), or (b) as multi/mini blood donors. The seroprevalence of HIV-1 (Mono infection)
pool (MP) donations. In ID-NAT testing, similar to serology in a survey conducted in five urban and five rural populations
testing as aliquot of each individual sample is tested directly of Tamil Nadu was 0.1% while dual infection with HIV-1
and provides the screening sensitivity which is equivalent to and HIV-2 was 0.44%. The one of the drawback of Ulrio plus
the analytical sensitivity. In MP-NAT testing predetermined ID-NAT is unable to detect the HIV-2 but detected by Cobas
number of donations from as few as 4 to as large as 96 are TaqScreen assay in the MP6.
equally combined prior to testing. Based on the prevalence However, in India, high prevalence of TTI’s and compo
of infection in the donor population, assuming that the other nent transfusion is not widely accepted, the need for safe
members in the pool do not have virus, the virus concentration blood is of utmost importance to the repeat transfusion
of the original sample is diluted by the factor of the pool size. requiring group of patients with thalassemia, sickle cell
The decreased concentration of the virions in the final testing anemia, malignancy and hemophilia. For blood screening
aliquot impacts the probability of detection. In other words the most sensitive assay in the most sensitive format should
LoD is inversely related to pool size, with larger the pool size be practiced to minimize the risk of TTIs.
greater the loss in sensitivity. Grabarczyk et al.1 observe that
the 95% LoD for HBV being 4 IU/mL for ID-NAT becomes References
24 IU/mL for MP6; and for an ID-NAT assay with a LoD for
5 IU/mL becomes 120 IU/mL for MP24. Lin et al.2 report 1. Grabarczyk P, Letowska M, Kopacz A, Leszewski G, Sulkowska
that 50% LoD of 0.46 IU/mL for HBV ID-NAT translates to E, Brojer E. Effectiveness of various minipool nucleic acid
test systems for hepatitis B virus detection in blood donors.
1.56 IU/mL for MP6.
Transfusion. 2009;49:1494-5.
Several comparative studies evaluating ID-NAT versus 2. Lin KT, Chang CL, Tsai MH, Lin KS, Saldanha J, Hung CM.
pool testing, using the same reagent, thereby excluding the Detection and identification of occult HBV in blood donors
variability of reagent and platforms have been executed. Using in Taiwan using a commercial, multiplex, multi-dye nucleic
the Procleix Ultrio Plus assay Vermeulen et al.3 observed a acid amplification technology screening test. Vox Sanguinis.
decrease in detection of HIV NAT yields as follows: 10% in 2014;106:103-10.
MP4, 20% in MP8, 30% in MP16. Grabarczyk et al.1 compared 3. Vermeulen M, Coleman C, Mitchel J, Reddy R, Van Drimmelen
HBV NAT yields for MP6 with Cobas TaqScreen MPX versus H, Fickett T, et al. Comparison of human immunodeficiency
virus assays in window phase and elite controller samples:
Cobas Ampliscreen in MP24 and observed 12.6 folds increase
Viral load distribution and implication for transmission risk.
for HBV WP and 6.6 fold increases for occult hepatitis B Tranfusion. 2013;53:2384-98.
infection (OBI) NAT yields. In order to understand the 4. Vermeulen M, Coleman C, Mitchel J, Reddy R, Van Drimmelen
performance of the commercial reagents in the commonly H, Fickett T, et al. Sensitivity of individual-donation and
practiced format, the Procleix Ultrio Plus assay in the ID-NAT minipool nucleic acid amplification test options in detecting
format has been compared with the TaqScreen assay in the window period and occult hepatitis B virus infections.
MP6 format. For HIV-1 NAT yields the decrease in detection Transfusion. 2013;53:2459-66.
from ID NAT with Procleix Ultrio Plus assay was 17% in pools 5. Makroo RN, Choudhury N, Jagannathan K, Parihar-Malhotra
of 6 by Cobas Taqscreen assay. For HBV the overall decrease M, Raina V, Chaudhary RK, et al. Multicenter evaluation of
individual donor nucleic acid testing (NAT) for simultaneous
was 30%, with more pronounced decrease during 2nd WP
detection of human immunodeficiency virus–1 and hepatitis B
characterized by low viral load (49% decrease) and OBI (33% & C viruses in Indian blood donors. Indian J Med Res. 2008;
decrease) HBV infection status.4 127:140-7.
The ability to successfully implement ID NAT in India and 6. Agarwal N, Chatterjee K, Coshic P, et al. Nucleic acid testing
its impact on interdicting release of infectious units has been for blood banks: An experience from a tertiary care
demonstrated in a multicenter study with centralized testing centre in New Delhi, India. Tranfus Apher Sci. 2013;49:
and on-site screening studies.5,6 Although, the argument of 482-4.
Occult Hbv Infection:
A Risk for Transfusion
Sheetal Malhotra
64
Introduction the assay and endemicity of HBV infection (individuals from
countries highly endemic for HBV are more likely to develop
Occult hepatitis B infection (OBI) is defined as the long- OBI).7 Various studies have been undertaken to estimate
lasting persistence of hepatitis B virus (HBV) genomes in seroprevalence of OBI in blood donors. A study from Egypt
the liver (with detectable or undetectable HBV DNA in the showed a seroprevalence of OBI as 4.1%.3 A prospective study
serum) of individuals testing negative for the HBV surface undertaken on 31,190 first time voluntary blood donors from
antigen (HBsAg) in the presence (80%) or absence (20%) of Italy showed anti-HBc positivity in 8.3% samples of which
anti-HBc with or without anti-HBs antibodies.1 The amount 10.4% were ‘anti HBc only’. HBV DNA was found in 0.55% of
of HBV DNA in the serum is usually very low (<200 IU/mL) or anti-HBc-positive/HBsAg negative subjects.2 Data from other
intermittently detected making detection of such infections countries shows following figures –8/5973 donors or 0.13%
difficult but donation by such individuals is a potential (with viral load <12u/mL) from Taiwan,8 3/600 or 0.5% donors
source of HBV transmission to recipients.2 It is emerging as a from Saudi Arabia,9 anti-HBc positivity in 42/100 donors of
challenging problem in blood transfusion safety. which 90.5% were positive for HBV DNA from Sudan,10 16
OBI donors/131 anti-HBc positive donors (OBI 12.2%) from
Etiopathogenesis Iran,11 anti-HBc positivity in 167/966 donors (17.28%) with
76 demonstrating anti-HBs positivity and 5 with detectable
Occult hepatitis B infection (OBI) may be observed in the HBV DNA from Pakistan,12 48 OBI donors/72 HBV DNA
window period of acute HBV infection in blood donors and in reactive/HBsAg negative donors (66.7%) from Thailand,13 35
recipients of blood and blood products or in patients with HCV OBI donors/320 HBsAg-negative, anti-HBc and/or anti-HBs-
chronic infection and in immunosuppressed individuals. It positive donors (10.9%) from Lao PDR,14 and 3.7% from US.15
shows the high prevalence in HCV co-infected patients as
HBV activity can be impaired by other infectious agents.3 OBI Seroprevalence in India
is related to infection with variant viruses (S-escape mutants)
undetectable by the available HBsAg tests, but is more Studies on Indian blood donors show an OBI seroprevalence
often caused by the host mechanisms producing a strong of 20–30% in anti-HBc positive donors. A study undertaken
suppression of the HBV replication. It represents the fifth by Bhattacharya et al. on 1027 HBsAg negative donors
phase in the natural history of chronic hepatitis B (HBsAg- revealed that 188 (18.3%) of them were anti-HBc positive, out
negative phase).4 Anti-HBc only/isolated anti-HBc positive of which 21% were positive for HBV DNA.16 Another study
cases without anti-HBs is seen in resolving acute infection, by Choudhary et al. involving 30,853 donors from Cohort 1
low grade chronic infection in the nonreplicative phase, in (24,694 in 2001) and Cohort 2 (6159 in 2000) were screened
case of infection with variant strains of HBV or due to false for anti-HBc and anti-HBs. HBV DNA was detected in 40
positive anti-HBc reactivity.5 out of 147 (27.21%) and 48 out of 230 (20.87%) donors with
anti-HBc only from the two cohorts.17 In a study by Hari
et al. from PGIMER Chandigarh, anti-HBc positivity was seen
Epidemiology in 8.4% (142/1700) of the blood donor samples. HBV DNA was
detected in 1 of 100 anti-HBc reactive donors tested. None of
Global Seroprevalence
the anti-HBc-only samples were positive for HBV DNA.18 In a
The first recorded evidence of OBI and possible transmission study by Doda et al. on 28,134 HBsAg Elisa negative samples,
via blood transfusion is in late 1970s.6 Prevalence of OBI HBV DNA was detected in 18 samples, out of which12 were
varies in different studies due to differences in sensitivity of found to be OBI.19
Chapter 64 Occult Hbv Infection: A Risk for Transfusion 233
Genotype characterization of OBI shows prevalence of Case reports by Lui et al. and Levicnik-Stezinan et al.
genotype C, B and a newly emerging genotype I in Thailand, have shown that blood products from donors with an occult
genotype D in Europe, genotype E in Ghana and genotype B HBV infection can result in TT HBV.25,26 A study in Taiwan
in China.20 In India, HBV genotype D is associated with high showed TT HBV transmission in two of 11 recipients of OBI
occult HBV infections.21 blood became subsequently infected.27 In another study from
the Japanese Red Cross, OBI blood was infectious in 19% of
Clinical Implications of OBI recipients.28 In a prospective sudy involving 247 patients by
Tani et al. from Japan, the residual risk of blood transfusion
The clinical implications of OBI involve different clinical from donors with low anti-HBc titers or high anti-HBc titers
contexts as follows: with high anti-HBs titers was evaluated and none of the
1. Risk of HBV transmission through, blood transfusion, recipients showed reactivity for any of the HBV markers
hemodialysis and organ transplantation. (HBsAg, anti-HBs, anti-HBc and HBV DNA).29A study from
2. Causing cryptogenic liver diseaseas immunosuppression India on 174 multi transfused thalassemic patients, HBV DNA
may induce OBI reactivation and development of acute was detected in 50% of patients with anti-HBc as the only
and sometimes fulminant hepatitis. marker (OBI).30
3. Contributing to the progression of liver disease and HCC.
4. Affecting response to antiviral therapy for patients with Preventive Strategies
chronic HCV infection, contributing to the progression of
the chronic liver disease toward cirrhosis. The above figures indicate that OBI is considerable risk in
Thus, OBI as a cause of chronic liver disease in patients transfusion recipients; this biological entity has implications
with HBsAg-negative status is becoming an important disease for donor and blood screening. Preventive strategies include
entity. OBI has been reported as a potential risk factor for the stringent donor screening including permanent deferral
development of nonhepatic malignancies like pancreatic of donors with a history of jaundice together with a broad
cancer and hematological malignancies.4,22 hepatitis B vaccination strategy.14,23 Symptomatic infection
with HBV may occur in approximately 30–50% of cases,
assuming this symptomatic infection rate also applies to OBI
Risk of TT HBV with OBI cases, a strict deferral policy of donors with a history of hepatitis
HBsAg negative individuals with positive anti-HBc and/or or jaundice would exclude 30–50% of OBI donors.23 Anti-HBc
positive for antibodies against HBsAg may harbor HBV DNA. is a life-long serological marker of previous HBV infection, as
Blood donation by such individuals is a potential source of it is detectable both in acute and chronic infections and after
HBV transmission to the recipients. OBI may be involved viral clearance; this marker, therefore, indicates a current or
in the transmission of HBV by blood transfusion in two past HBV infection. Screening for anti-HBc is recommended
conditions: in several countries in which the prevalence of hepatitis B is
1. “OBI carrier”when the donor is infected with wild-type low.2 Inclusion of anti-HBc antibody screening would exclude
HBV strains suppressed in the irreplication activity. the HBsAg negative/DNA-positive blood units; it would
Chronic occult infection frequently shows fluctuating exclude anti-HBc antibody positive blood with undetectable
levels of viremia (with fluctuating infectivity) with periods HBV DNA leading to unnecessary deferral of blood donors
when HBV DNA is not detectable in the serum. especially in countries with high HBV seroprevalence where
2. The donor is infected with S-escape mutant HBV is able to large number of blood donors are anti-HBc positive.14
replicate but synthesize a mutated HBsAg not detected by The efficacy of the test is also limited by two main factors.
the routinely available diagnostic assays. This is the most First is its lack of specificity. It is postulated that 70–90% of
frequent condition associated with the residual cases of samples containing high levels of anti-HBs antibodies are
hepatitis B related to blood transfusion.4 essentially safe for transfusion unless transfused to immuno
In a study from Australia, OBI residual risk was calculated as compromized recipients. Tani et al. from Japan concluded
approximately 1 in 982,000 per unit transfused. The infectivity in a study that blood components from donors nonreactive
of components derived from donors with OBI depends on a for HBsAg and HBV DNA but reactive for anti-HBc titers of
number of donor and recipient factors, particularly the anti- <1:32 or anti-HBc titers of >1:32 with anti-HBs >200 mIU⁄ mL
HBs level in transfused components.23 The residual risk of are considered suitable for transfusion.29 Anti-HBc screening
OBI estimated by Taira et al. in a study from Japan was 252/ also fails to identify two sources of anti-HBc negative
million donations (1 in 3968 donations). They concluded that potentially infectious blood, in the pre-seroconversion
severe hepatitis can occur by transfusion from OBI donors window period or late chronic infection referred to as “core
despite the comparatively lower viral loads in components window period”.31As anti-HBc positivity is too high to allow
and they identified two fatal post-transfusion fulminant HBV anti-HBc detection to be implemented on a routine basis in
cases associated with OBI donors in look back studies.24 our country, the use of HBsAg testing together with NAT can
234 Section 3 Blood Safety
be adopted as the policy of choice for screening donors in among blood donors in Sudan. J Egypt Public Health Assoc.
order to further reduce and prevent HBV transmission. It is 2013;88(1):14-8.
postulated that samples collected in the post-acute phase of 11. Behzad-Behbahani A, Mafi-Nejad A, Tabei SZ, Lankarani
infection often show very low levels of HBV DNA in the serum KB, Torab A, Moaddeb A. Anti-HBc & HBV-DNA detection in
blood donors negative for hepatitis B virus surface antigen in
(<100 IU/mL) and can, therefore, be missed when testing is
reducing risk of transfusion associated HBV infection. Indian J
carried out in large pools or by less sensitive assays for HBV- Med Res. 2006;123:37-42.
DNA.8 Requirement for NAT testing in such regions is an 12. Bhatti FA, Ullah Z, Salamat N, Ayub M, Ghani E. Anti-hepatits
assay that is very sensitive for the detection and identification B core antigen testing, viral markers, and occult hepatitis B
of HBV DNA and is especially useful in countries with a high virus infection in Pakistani blood donors: implications for
prevalence of HBV where occult HBV infections are quite transfusion practice. Transfusion. 2007;47:74-9.
common and detection can be challenging. In a study by 13. Phikulsod S, Oota S, Tirawatnapong T. Working Group for NAT
Linet al from Taiwan, all 8 of NAT-yield cases were from Studyin Thai Blood Donations: One year experience of nucleic
acid technology testing for human immunodeficiency virus
donors with an occult infection.8 Satoh et al. has introduced
type 1, hepatitis C virus and hepatitis B virus in Thai blood
a new method of concentrating HBV DNA and HBsAg donations. Transfusion. 2009;49:1126-35.
simultaneously to increase the sensitivity of detection tests. 14. Jutavijittum P, Andernach IE, Yousukh A, Samountry B,
By this method, 35 of the 77 anti-HBc-positive and HBsAg- Samountry K, Thammavong T, et al. Occult hepatitis B infections
negative donors were HBV DNA-positive by ID NAT and among blood donors in Lao PDR. Vox Sang. 2014;106:31-7.
a further five donors became HBV DNA-positive by HBV 15. Kleinman SH, Kuhns MC, Todd DS. Retrovirus Epidemiology
concentration. Twenty-seven of 40 occult HBV infections DonorS: Frequency of HBV DNA detection in US blood donors
became HBsAg-positive byHBsAg concentration.32 Still there testing positive for the presence of anti-HBc: implications for
are few documented reports of HBV transmission by donors transfusion transmission and donor screening. Transfusion.
2003;43:696-704.
missed ID NAT, so “zero risk” for HBV is yet to come.33
16. Bhattacharya P, Chandra PK, Datta S, Banerjee A, Chakraborty
S, Rajendran K, et al. Significant increase in HBV, HCV, HIV and
References syphilis infections among blood donors in West Bengal, Eastern
India 2004-2005: exploratory screening reveals high frequency
1. Raimondo G, Caccamo G, Filomia R, Pollicino T. Occult HBV of occult HBV infection.World J Gastroenterol. 2007;13:3730-3.
infection. Semin Immunopathol. 2013;35:39-52. 17. Chaudhuri V, Nanu A, Panda SK, Chand P. Evaluation of
2. Romano L, Velati C, Cambie G, Fomiatti L, Galli C, Zanetti AR, serologic screening of blood donors in India reveals a lack of
et al. Hepatitis B virus infection among first-time blood donors correlation between anti-HBc titer and PCR-amplified HBV
in Italy: prevalence and correlates between serological patterns DNA. Transfusion. 2003;43:1442-8.
and occult infection. Blood Transfus. 2013;11:281-8. 18. Dhawan HK, Marwaha N, Sharma RR, Chawla Y, Thakral B,
3. EL-Ghitany EM, Farghaly AG, Hashsish MH. Occult hepatitis Saluja K, et al. Anti-HBc screening in Indian blood donors: Still
B virus infection among hepatitis C virus seropositive and anun resolved issue. World J Gastroenterol. 2008;14:5327-30.
seronegative blood donors in Alexandria, Egypt. J Egypt Public 19. Doda V, Arora S, Kirtania T. Serological characterization of
Health Assoc. 2013,88:18-31. occult hepatitis B virus infection among blood donors in India.
4. Squadrito G, Spinella R, Raimondo G. The clin; ical significance Transfus Apher Sci. 2014. p. 30.
of occult HBV infection. Annals of Gastroenterology. 2014. pp. 20. Chamni N, Louisirirotchanakul S, Oota S, Sakuldamrongpanish
2715-19. T, Saldanha J, Chongkolwatana V, et al. Genetic characterization
5. Gessoni G, Beggio S, Barin P, Favarato M, Galli C, Valverde S, and genotyping of hepatitis B virus (HBV) isolates from donors
et al. Significance of anti-HBc only in blood donors: a with an occult HBV infection. Vox Sanguinis. 2014. 17. doi:
serological and virological study after hepatitis B vaccination. 10.1111/vox.12178. [Epub ahead of print].
Blood Transfus. 2014;12:s63-8. 21. Shyamala V. Factors in enhancing blood safety by nucleic acid
6. Tabor E, Hoofnagle J, Small wood L, Drucker JA, Pineda- technology testing for human immunodeficiency virus, hepatitis
Tamondong GC, Ni LY, et al. Studies of donors who transmitpost C virus and hepatitis B virus. Asian J Transfus Sci. 2014;8:13-8.
transfusion hepatitis. Transfusion. 1979;19:725-31. 22. Nishikawa H, Osaki Y. Clinical Significance of Occult Hepatitis
7. Emara MH. Occult hepatitis B: the Egyptian situation. Tropical B Infection in Progression of Liver Disease and Carcinogenesis.
Gastroenterology. 2012;33:242-50. Journal of Cancer. 2013;4:473-80.
8. Lin KT, Chang CL, Tsai MH, Lin KS, Saldanha J, Hung CM. 23. Seed CR, Kiely P. A method for estimating the residual risk of
Detection and identification of occult HBV in blood donors transfusion-transmitted HBV infection associated with occult
in Taiwan using a commercial, multiplex, multidye nucleic hepatitis B virus infection in a donor population without
acid amplification technology screening test. Vox Sang. universal anti-HBc screening. Vox Sang. 2013;105:290-8.
2014;106:103-10. 24. Taira R, Satake M, Momose S, et al. Residual risk of transfusion-
9. Bamaqa MS, Azahar EI, AL-Ghamdi AK, Alenzi FQ, Farahat transmitted hepatitis B virus (HBV) infection caused by blood
FM. Nucleic acid amplification technology for hepatitis B virus components derived from donors with occult HBV infection in
and its role in blood donation screening in blood banks. Saudi Japan. Transfusion. 2013;53:1393-1404.
Med J. 2009;30:1416-21. 25. Lui CJ, Lo SC, Kao JH, et al. Transmission of occult hepatitis B
10. Mahmoud OA, Ghazal AA, Metwally Del S, Elnour AM, virus by transfusion to adult and pediatric recipients in Taiwan.
Yousif GE. Detection of occult hepatitis B virus infection J Hepatol. 2006;44:39-46.
Chapter 64 Occult Hbv Infection: A Risk for Transfusion 235
26. Levicnik-Stezinan S, Rahne-Potokar U, Candotti D. Anti-HBs 30. Sabat J, Dwibedi B, Dash L, Kar SK. Occult HBV Infection in
positiveoccult hepatitis B virus carrier blood infectious in two Multi Transfused Thallasemic Patients. Indian J Pediatr. 2014.
transfusion recipients. J Hepatol. 2008;48:1022-5. p. 15.
27. Wang JT, Lee CZ, Chen PJ. Transfusion-transmitted HBV 31. Allain JP. International collaborative studyproposal for
infection in anendemic area: the necessity of more sensitive the characterization of occult hepatitis B virus infection
screening for HBV carriers. Transfusion. 2002;42:1592-7. identified by nucleic acid or anti-HBc screening. Vox Sang.
28. Satake M, Taira R, Yugi H. Infectivity of blood components 2007;92:254-7.
with low hepatitis B virus DNA levels identified in a look back 32. Satoh K, Iwata-Takakura A, Yoshikawa A, Gotanda Y, Tanaka T,
program. Transfusion. 2007;47:1197-1205. Yamaguch T, et al. A new method of concentrating hepatitis B
29. Tani Y, Aso H, Matsukura H, Tadokoro K, Tamori A, Nishiguchi virus (HBV) DNAand HBV surface antigen: an application of
S, et al. Significant background rates of HBV and HCV the method to the detection of occult HBV infection. Vox Sang.
2008;95:174-80.
infections inpatients and risks of blood transfusion from
33. Vermeulen M, Dickens C, Lelie N. Hepatitis B virustrans
donors with lowanti-HB ctitres or high anti-HB ctitres with
mission by blood transfusion during 4 years of individual
high anti-HB stitres in Japan: a prospective, individual NAT
donationnucleic acid testing in South Africa: estimated and
study oftransfusion- transmitted HBV, HCV and HIV infections. observed window period risk. Transfusion. 2012;52:880-92.
Vox Sang. 2012;102:285-93.
Current Status of Bacterial
Detection in Blood Components
Mina Sidhu
65
Abstract unknown transmissible agents. Blood safety and prevention
of transfusion transmitted pathogens are the principal aim
Transfusion of allogenic blood and its components are of transfusion medicine. To improve the blood safety many
a potential source of infection by a variety of known and improvements have been made in Donor selection process,
unknown transmissible agents. Blood safety and prevention Implementation of new technologies like third and fourth
of transfusion transmitted pathogens are the principal aim generation ELISA and nucleic acid amplification techniques
of transfusion medicine. To improve the Blood Safety many (NAT) in detection of transfusion transmitted viral infections,
improvements have been made in Donor selection process, reducing the risk to the minimum, thus increasing the quality
Implementation of new technologies like third and fourth and safety of the Blood Component to the highest level ever.1
generation ELISA and nucleic acid amplification techniques As a result of this success, transfusion transmitted bacterial
(NAT) in detection of transfusion transmitted viral infections. infections (TTBI) have emerged as greatest threat to blood
Bacterial contamination of blood components and prevention safety and is an important transfusion related adverse event.
of transfusion transmitted bacterial infections (TTBIs) still In United States, bacterial contamination is considered the
remains the major challenge in transfusion medicine. Platelet second most common cause of death overall from transfusion
concentrates (PCs) represent one of the highest risks for (after clerical errors) with mortality rate from bacterial
bacterial infection due to storage at room temperature with sepsis ranging from 1:20,000 to 1:85,000. Estimates of severe
continuous agitation. Rate of bacterial contamination of PCs morbidity and mortality range from 100 to 150 transfused
is 1in 1000–3000. In general, bacterial screening of blood individuals each year.2
components is done by conventional or automated culture Platelet concentrates (PCs) represent one of the highest
methods. Many developments have been made in the rapid risks for bacterial infection. This is due to the required
direct and indirect method of bacterial detection mainly for storage conditions for PCs in gas-permeable containers at
PCs including NAT, pan genera detection (PGD). Fluorescent room temperature 22oc to 24oc with constant agitation to
activated cell sorting (FACS), noninvasive O2 detection, etc. for maintain the function and survival, which support bacterial
bacterial detection. The ideal system for detection of bacterial proliferation from low contamination levels to high titers.3
contamination should be highly sensitive and specific, should The risk of bacterial contamination of platelet units is 1 in
be able to generate fast results, detect low levels (as low as 1 1000 to 3000, 50–250 times higher than the combined risk
CFU/blood bag) of bacterial load and affordable. Currently of human immunodeficiency virus, hepatitis B and C virus
no such system is available. Nevertheless various pathogen and human T-cell leukemia virus 1 and 2, clinical sepsis in
reduction techniques are available for PCs and plasma. about 1/20,000 and related fatality in 1/60,000 transfusion.4-6
Therefore, automated culture systems can be complemented The common pathogens isolated in platelet concentrate are
with rapid systems for detection of TTBIs. mainly skin flora, including Staphylococcus epidermidis and
Key words: Transfusion transmitted bacterial contamination, Propionibacterium acnes.
Platelet concentrate, Culture systems, NAT, PGD, FACS. Sepsis associated with the transfusion of red cell blood
component is a rare event because of storage at 2–6oC,
where bacteria generally do not grow. From 1976 through
Introduction September 1998, 26 fatalities were reported to US food and
Transfusion of allogenic blood and its components are drug administration. Most common organism involved was
a potential source of infection by a variety of known and Yersinia enterocolitica.3 Coagulase positive Staphylococcus,
Chapter 65 Current Status of Bacterial Detection in Blood Components 237
ambient O2, as a marker for bacterial growth. The method permeable dye thiazole orange, which stains the nucleic
involves docking an eBDS sample pouch onto the tubing of acids of viable and dead cells. The bacteria are detected
platelet bag, transfer of an aliquot of 2–3 mL of the platelet by flow cytometric analysis. The sensitivity of the assay is
product into the sample pouch, which is then separated from a approximately 105 CFU/mL, but a preincubation step
the platelet unit using a heat sealer. The eBDS pouch is then in a bacterial growth medium increases the sensitivity
incubated at 35 oC for 24 hours with continues agitation. considerably to 10 CFU/mL. Bacterial detection with FACS
Pouches are then removed from the incubator, kept at analysis without the preincubation step takes approximately
room temperature for at least 2 minutes and the probe of 20 minutes. Not all bacteria are easily detected by FACS
the oxygen analyzer inserted into the sample port of the analysis. Sometimes due of the influence of the background
pouch. The oxygen analyzer automatically reads the oxygen signal, the quantification of the bacterium is difficult.32
concentration in the sample pouch. Bacterial contamination The BactiFlow assay detects and enumerates bacteria
is assumed if the O2 reading is less than 12.5%, which is the by fluorescent labeling of viable cells. A nonfluorescent
cut-off value between a positive and negative reading.28,29 fluorochrome passes the cell membrane of cells with intact
Using this technology only aerobes and facultative anerobes membrane integrity and enzymatic activity, and is cleaved
are detected. The sensitivity of the Pall eBDS system is similar by intracellular esterases. To reduce the background noise,
to the BacT/ALERT system in the order of 1–10 CFU/mL.29 the esterase activity of platelets is selectively eliminated by
enzymatic digestion. The analytical sensitivity of this assay is
Microcalorimetry validated to be 300–500 CFU/mL.33
pathogen reduction is that it inactivates a broad range of important milestone in improving bacterial safety of blood
pathogens such as bacteria, viruses, parasites and fungi. components. Due to delay in generation of results and
There are several approaches for pathogen reduction of PCs. problem of false positive cases, newer rapid techniques like
Intercept (Fenwall, formerly Baxter Corporation), Mirasol BactiFlow, PGD and NAT testing can be adopted. The future
(Gambro BCT), and the Theraflex UV pathogen reduction detection of TTBI lies in the tests that are rapid, sensitive and
system (Macopharma). The intercept method uses psoralen are able to detect broad range of bacteria. Undoubtly, NAT
combined with long wave length UV light to inactivate a broad is fast detection system and can be used close to the time
range of contaminating pathogens, including bacteria. The of transfusion. Very low level of bacteria can be detected.
Mirasol technique uses riboflavin (Vitamin B) in combination However, cost effectiveness study should be done to see if
with UV to reduce pathogens in blood products.38 Although implementation of NAT assay is affordable, for the detection
attractive, pathogen reduction of PCs is costly. of Transfusion transmitted bacterial infections.
11. Barker LM, Nanassy OZ, Reed MW, et al. Multiple pH measure- culture system and estimation of false nagatve culture rates.
ment during storage may detect bacterially contaminated plate- Transfusion. 2007;47:1381-9.
let concentrates. Transfusion. 2010;50:2731-37. 27. Boekhorst PA, Beckers EA, Vos MC, et al. Clinical significance of
12. Gkoumassi E, Klein-Bosgoed C, Dijkstra-Tiekstra MJ, et al. bacteriological screening in platelet concentrate. Transfusion.
Noninvasive pH monitoring of platelet concentrates: A large 2005;45:514-9.
field test. Transfusion. 2013;53:2287-92. 28. Pietersz RN, Engelfret CP, Persin KHW. Detection of bacterial
13. Yomohow R, Brecher ME. pH and Glucose test of SDP should contamination in platelet concentrate. Vox Sang. 2007;93:
be discontinued in favour of more sensitive detection method. 260-7.
Transfusion. 2005;45:646-8. 29. Fournier Wirth C, Deschaseaux M, Defer C. Evaluation
14. Mueller MM, Hourfar MK, Huber E, et al. Oxygen measure of enhanced Bacterial Detection System for screening of
ments in platelet fluids—a new noninvasive method to contaminated platelets. Transfusion. 2006;46:220-4.
detect bacterial contaminations in platelets. Transfus Med. 30. Trampuz A, Salzmann S, Antheaume J, et al. Micro
2012;22:211-6. calorimetry: A novel method for detection of microbial
15. Störmer M, Petrescu-Jipa VM, Radojska S, et al. Measuring contamination in platelet products. Transfusion. 2007;47:
platelet aggregation may detect bacterial contamination 1643-50.
in platelet concentrates. 23rd Regional Congress of the 31. Vollmer T, Hinse D, Kleesiek K, et al. The Pan Genera detection
International Society of Blood Transfusion, 2-5 June 2013, immunoassay: A novel point-of-issue method for detection
Amsterdam, The Netherlands. Vox Sang. 2013;105(suppl 1):34. of bacterial contamination in platelet concentrates. J Clin
16. Mitchel KM, Brecher ME. Approaches the detection of bacterial Microbiol. 2010;48:3475-81.
contamination in cellular blood products. Trans Med Rev. 32. Dreier J, Vollmer T, Kleesiek K. Novel flow cytometry-based
1999;13:132-44. screening for bacterial contamination of donor platelet
17. Schmidt M, Weis C, Hect J, et al. Optimized Scansystem platelet preparations compared with other rapid screening methods.
kit with enhanced sensitivity. Detection within 24 hours after
Clin Chem. 2009;55:1492-1502.
spiking. Vox Sang. 2005;89:135-9.
33. Vollmer T, Dreier J, Schottstedt V, et al. Detection of bacterial
18. Ribalt S, Harper K, Grave L, et al. Rapid screening method for
contamination in platelet concentrates by a sensitive flow
detection of bacteria in platelet concentrate. J Clini Microbiol.
cytometric assay (BactiFlow): A multicentre validation study.
2004;42:1903-8.
Transfus Med. 2012;22:262-71.
19. Störmer M, Petrescu-Jipa VM, Radojska S, et al. Measuring
34. Mohammadi T, Pietersz RN, Vandenbroucke-Grarib CM, et al.
platelet aggregation may detect bacterial contamination
Detection of bacteria in platelet concentrate: Comparison of
in platelet concentrates. 23rd Regional Congress of the
broad range real time 16S rDNA polymerase chain reaction
International Society of Blood Transfusion, 2-5 June 2013,
Amsterdam, The Netherlands. Vox Sang. 2013;105(suppl 1):34. and automated culturing. Transfusion. 2005;45:731-6.
20. Eder AF, Kennedy JM, Dy BA, et al. Bacterial screening of 35. Stormer M, Kleesiek K, Dreier J. High volume extraction of
apheresis platelets and the residual risk of septic transfusion nucleic acid by magnetic bead technology for ultrasensitive
reactions: The American Red Cross experience (2004-2006). detection of bacteria in blood components. Clin Chem.
Transfusion. 2007;47:1134-42. 2007;53:104-10.
21. Koopman MM, van’t Ende E, Lieshout-Krikke R, et al. Bacterial 36. Sireis W, Ruster B, Daiss C, et al. Extension of platelet shelf life
screening of platelet concentrates: Results of 2 years active from 4 to 5 days by implementation of a new screening strategy
surveillance of transfused positive cultured units released as in Germany. Vox Sang. 2011;101:191-9.
negative to date. Vox Sang. 2009;97:355-7. 37. Jacobs MR, Bajaksouzian S, Yomtovian R, et al. Detection
22. European Pharmacopeia Update: Microbiological control of of bacteria in Leukocyte-reduced whole blood derived
cellular products. Chapter 2.6.27. platelet units using the Immunetics BacTx Test. Transfusion.
23. Schmidt M, Sireis W, Seifried E. Implementation of bacterial 2010;50:194A.
detection methods into blood donor screening-overview of dif- 38. Pelletier JP, Transue S, Snyder EL. Pathgen inactivation
ferent technologies. Transfus Med Hemother. 2011;38:259-65. techniques. Best Pract Res Clin Hematol. 2006;19:205-42.
24. Murphy WG, Coakley P. Testing platelet components for 39. Wang J, Wang X, Li Y, et al. A novel, universal and sensitive
bacterial contamination. Transfus Apher Sci. 2011;45:69-74. lateral-flow based method for the detection of multiple
25. Nanua S, Weber C, Isgriggs L, et al. Performance evaluation of bacterial contaminations in platelet concentrations. Anal Sci.
the Versa TREK blood culture system for quality control testing 2012;28:237-41.
of platelet units. J Clin Microbiol. 2009;47:817. 40. Rotman B, Cote MA. Application of a real-time biosensor to
26. Benjamin RJ, Wagner SJ. The residual risk of sepsis: Modelling detect bacteria in platelet concentrates. Biochem Biophys Res
the effect of concentration on bacterial detection in two bottle Commun. 2003;300:197-200.
Pathogen Reduction Technology
Kanchan Bhardwaj
66
Pathogen reduction technology is one more positive step acid—specific adduct formation as a primary mechanism
towards blood safety designed to reduce the risks from bacteria, for pathogen inactivation. Psoralen is furocoumarins,
viruses and parasites in donated blood components. This which reversibly intercalate into helical regions of DNA and
system proactively and safely reduces the risk of transfusion— RNA. Under illumination with UVA, psoralens react with
transmitted diseases by inactivating susceptible pathogens pyrimidine bases to form covalent adducts and cross links
and residual leucocytes that may be present in donated with nucleic acids. Bacteria, protozoa, viruses and nucleated
blood components. This offers the potential to inactivate cells modified with psoralens are unable to replicate.2-6
untested for and emerging pathogens before they become Photodynamic method utilizes Riboflavin which produces
a major transfusion risk to patients. While not all pathogens active oxygen species which oxidizes guanine in nucleic
are susceptible to inactivation, this system offers the most acid and prevents replication of the pathogen genome in
comprehensive approach available. The development and addition to nucleic acid reactions as a primary mechanism
adoption of pathogen reduction technology (PRT) continues. for pathogen inactivation.
The ability to reduce transmission of a broad range of infectious
and noninfectious threats remains the primary reason for Platelet concentrates with
PRT.1 The robust inactivation technology that is compatible
with blood compo nent processing procedures, offers the
Amotosalen photochemical
potential for significantly improving transfusion safety. To treatment
be highly effective, a successful technology must inactivate Helinx technology prevents replication of DNA and RNA
pathogens in extracellular, intracellular, and nuclear in pathogens without affecting blood components being
compartments including pathogen nucleic acid sequences treated. Closed system with pooled buffy coat or single
integrated into donor leucocytes. Technology capable of donor platelet concentrate, suspended in approximately 35%
inactivating residual leucocytes may confer additional benefits plasma and 65% platelet additive solution, is connected to a
owing to inhibition of critical leucocyte functions, including container of amostosalen through which it is passed. This is
cytokine synthesis, lymphocyte proliferation and antigen exposed to UVA for 3 minutes after which it is transferred to a
presentation. Nucleic acid targeted pathogen inactivation compound absorption device (CAD) for 4 hours to passively
process offers the potential to inactivate leucocytes as well as reduce the residual amotosalen to low levels (<0.5 µmol/L)
infectious pathogens. after which the platelets are transferred to a final container for
storage for 5–7 days under conventional storage conditions.
Pathogen inactivation in Platelet The Intercept blood system developed by Cerus is the first
Concentrates pathogen inactivating system approved to treat platelets for
transfusion. It has been approved for use in Europe. Safety
The pathogen inactivation in platelet concentrates is profile of treated platelets is similar to that of conventional
divided into two basic groups: nucleic acid targeted and platelets.7-9 Hemovigilance reporting system to examine the
photodynamic methods. These utilize photochemical incidence of acute reactions in the 24 hours post-transfusion
reactions in an ex vivo treatment combining a photo reactive period confirmed a low rate of acute transfusion reaction
compound and ultraviolet light. Nucleic acid targeting using and tolerability of the treated platelets.10 Impact of intercept-
a novel psoralen amotosalen has undergone clinical trials treated platelets based on proteomic analyses have been
and been commercialized. These methods relay on nucleic reported, and the overall interpretation of proteomic findings
Chapter 66 Pathogen Reduction Technology 243
suggests some minor changes although, there is no evidence inhibitor and coagulation factors. It involves pooling of
for a potential clinical concern for transfusion recipients.11 It 100–2500 units of plasma and is not amenable to a single unit
is also being developed for treatment of plasma and red cells. viral inactivation process.
Intercept-treated platelets are in routine use in 21 countries Methylene blue (MB) is used for pathogen reduction
and plasma in routine use in 12 countries.1 for plasma by Macopharma THERAFLEX MB. Based on
hemovigilance data for methylene blue (MB)-treated plasma,
Riboflavin and Ultraviolet light a significantly higher incidence of severe allergic reactions or
other adverse events in comparison with other fresh frozen
treated Platelets plasma types could not be confirmed.17,18 Methylene blue and
This method involves the suspension of platelets in 100% long wavelength visible light has been used in clinical practice
donor plasma. It acts against enveloped viruses, nonenve for preparation of FFP which utilizes active oxygen species.
loped viruses, protozoa and some bacteria. It reduces the It is not effective against intracellular species. Theraflex MB-
infectious pathogen load by 99.9% in > 70% of the pathogens treated plasma is in routine use in 15 countries.1
to the limit of detection. It causes 6 log reduction of A photochemical method using amotosalen and UVA
leucoreduction hence preventing CMV transmission. In this light has been developed to inactivate viruses in single units
the platelets are transferred into an illuminating bag to which of plasma prepared as FFP. Riboflavin treated and subjected
riboflavin is added. This is illuminated in an illuminator for to UVA leads to pathogen inactivation in short time (frozen
4–10 minutes after which it is ready for storage or transfusion. within 8 hours), removing time constraints associated with
Platelet additive solution may be needed to be added after FFP processing.
illumination to platelet concentrates with reduced plasma
content to ensure adequate storage conditions for up to 7 Potential System for Pathogen
days. Functional adequacy of the platelets was maintained.
Terumo BCT Mirasol PRT is based on this principle (CE
Inactivation in Red Cell concentrates
approved). This is also used for plasma before freezing Nucleic acid—targeted processes that do not require light
it. Recent clinical research on Mirasol-treated platelets activation are two groups of compounds which have been
and plasma PRT has focused on studies that quantify the used for pathogen reduction in red cell concentrates viz.
performance of white blood cell inactivation, with strong Inactine (PEN-110) and frangible anchor linker effectors
evidence of advantages over gamma irradiation.12 Mirasol- Frale called S-303. This approach offers the potential to
treated platelets are in routine use in 18 countries and plasma minimize nonspecific damage by active oxygen species to red
in routine use in 11 countries. cells and plasma proteins.19 Published research20,21 on whole
blood PRT currently in development provides evidence of
Methylene blue treated Platelets pathogen reduction, white cell inactivation and reduction in
antigen expression by Mirasol PRT.
Initial success in developing a novel procedure that uses
short-wave ultraviolet light (UVC 200–280 nm) without
Conclusion
addition of a photoactive chemical to treat platelets has
been reported and is currently being evaluated for safety and Both controlled clinical studies and postmarketing surveil
efficacy.13,14 Published information suggests both the UVC lance are necessary to further establish the safety and
platelets treatment intended for apheresis and buffy-coated effectiveness of each PRT method. Recent evidence does not
derived platelets and the more widely available methylene suggest an obvious safety threat for any of the current PRT
blue (MB) treatment for plasma from apheresis and whole methods in routine use. The relative value in terms of patient
blood derived plasma are effective at inactivation of many health and health economics, including cost-utility, of these
parasites, bacteria and viruses.15,16 methods in terms of health gains achieved by PRT remain
insufficiently studied.
Pathogen Inactivation in Fresh Frozen
Plasma Preparation References
Solvent detergent (SD) process for inactivation of enveloped 1. Custer B. Update on pathogen reduction technology. ISBT
viruses in plasma is used. It is not effective against non- Science Series. 2013;8:80-4. Doi10.1111/voxs.12014.
2. Lin L, Dikeman R, Molini B, et al. Photochemical treatment of
enveloped viruses. It is used for factor replacement in
platelet concentrates with amotosalen and UVA inactivates a
congenital and acquired factor deficiencies. As it does not broad spectrum of bacteria. Transfusion. 2004;44:1496-504.
contain von Willebrand factor (vWF), it has been advocated 3. Van Voorhis WC, Barrett LK, Eastman RT, et al. Trypanosoma
in therapeutic plasma exchange (TPE) therapy of thrombotic cruzi inactivation in human platelet concentrates and plasma
thrombocytopenic purpura (TTP). There is mild to marked by psoralen and long-wave length UV. Antimicrob Agents
decrease of antithrombotic proteins: protein S, plasmin Chemother. 2003;47:475-9.
244 Section 3 Blood Safety
4. Eastman RT, Barrett LK, Dupuis K, et al. Leishmania inactiva 13. Picker SM. Pathogen reduction technologies: the best solution
tion in human pheresis platelets by a psoralen (amotlsalen for safer blood? J Blood Disorders Transf. 2012;3(5):5.
HCl) and long-wavelength ultraviolet irradiation. Transfusion. 14. Pohler P, Lehmann J, Veneruso V, et al. Evaluation of the
2005;45:1459-63. tolerability and immunogenicity of ultraviolet C-irradiated
5. Lin L, Hanson CV, Alter HJ, et al. Inactivation of viruses in autologous platelets in a dog model. Transfusion. 2012;52(11):
platelet concentrates by photochemical treatment with 2414-26.
amotosalen and long-wavelength ultraviolet light. Transfusion. 15. Seghatchian J, Tolksdorf F. Characteristics of the THERAFLEX
2005;45:580-90. UV-Platelets pathogen inactivation system—an update.
6. Grass JA, Hei DJ, Metchette K, et al. Inactivation of leucocytes Transfus Apher Sci. 2012;46(2):221-9.
in platelet concentrates by psoralen plus UVA. Blood. 16. Steinmann E, Gravemann U, Friesland M, et al. Two pathogen
1998;91:2180-8. reduction technologies-methylene blue plus light and
7. McCullough J, Vesole DH, Benjamin RJ, et al. Therapeutic shortwave ultraviolet light-effectively inactivate hepatitis C
efficacy and safety of platelets treated with a photochemical virus in blood products. Transfusion. 2012; doi: 10.1111/j.1537-
process for pathogen inactivation: the SPRINT trail. Blood. 2995.2012.03858.x. [Epub ahead of print].
2004;104:1534-41. 17. Bost V, Odent-Malaure H, Chavarin P, et al. A regional
8. Van Rhenen D, Gullikson H, Cazenave JP, et al. Transfusion haemovigilance retrospective study of four types of therapeutic
of pooled buffy coat platelets components prepared with plasma in a ten-year survey period in France. Vox Sang. 2013;
photochemical pathogen inactivation treatment: the euro doi: 10.1111/vox.12007. [Epub ahead of print].
SPRITE trail. Blood. 2003;101:2426-33. 18. Seltsam A, Mueller TH. Updated hemovigilance data do not
9. Snyder E, McCullough J, Slichter SJ, et al. Clinical safety of show an increased risk of allergic reactions to methylene blue-
platelets photochemically treated with amotosalen HCl and treated plasma. J Allergy Clin Immunol. 2013;doi:10.1016/j.
ultraviolet A light for pathogen inactivation. Transfusion. 2005; jaci.2012.11.046. [Epub ahead of print].
45:1846-75. 19. Cancelas JA, Dumont LJ, Rugg N, et al. Stored red blood
10. Osselear JC, Bueno JL, Messe N, Jacquet M, Castro E. Flament cell viability is maintained after treatment with a second
J. Prospective active hemovigilence plan for INTERCEPT generation S-303 pathogen inactivation process. Transfusion.
platelets in Europe: status report. Vox Sang. 2005;89:137. 2011;51(11):2367-76.
11. Prudent M, Crettaz D, Delobel J, et al. Proteomic analysis of 20. Reddy HL, Doane SK, Keil SD, et al. Development of a
Intercept-treated platelets. Journal of proteomics. 2012;76 riboflavin and ultraviolet light-based device to treat whole
(Spec No.):316-28. blood. Transfusion. 2013;53(Suppl 1):131S-6S.
12. Fast LD, DiLeone G, Marschner S. Inactivation of human 21. Pidcoke HF, McFaul SJ, Ramasubramanian AK, et al. Primary
white blood cells in platelet products after pathogen reduction hemostatic capacity of whole blood: a comprehensive analysis
technology treatment in comparison to gamma irradiation. of pathogen reduction and refrigeration effects over time.
Transfusion. 2011;51(7):1397-404. Transfusion. 2013;53(Suppl 1):137S-49S.
Discarding of Blood and
Blood Components
after thawing (4%), donor positive for allo or autoantibody laboratory, selection and transfusion of the recipient. Each of
(0.7%) and icteric bags (0.4%) (Table 6). these processes therefore provide an opportunity to improve
The blood and blood component losses occur all along the efficiency by minimising losses.3
chain of production, right from the recruitment of the donor, The important causes of discarding whole blood/packed
bleeding of the donor, component preparation, storage of cells included reactivity for infectious agents, expiry of blood
the inventory in the blood bank, storage in the hospital or units, under collection and splitting of blood for pediatric
248 Section 3 Blood Safety
patients. Other causes included leakage of blood bags, prevalence areas the use of rapid screening tests for donors
presence of clots or antibodies, hemolysis, lipemia and over can help to reduce the wastage of donated blood.4
collection. Blood collection: Underweight blood bags have an excess
Donor recruitment: Thorough and confidential screening, of anticoagulant that can denature the blood during storage.
counseling and education of blood donors will go a long way Suboptimal weight of blood collected would be unsuitable for
in reducing the number of reactive blood units. Often times component preparation and transfusion. The major factors
this is not possible in an Indian set up because low levels of for under collection at our centre included donor reactions
education and awareness precludes understanding of the like sweating, dizziness and anxiety causing discontinuation
reasons and justification for deferring a donation. In high of the donation process, collapse of the vein and prolonged
Chapter 67 Discarding of Blood and Blood Components 249
Feb 13 2 6 2 6 (2-RBC †) 16
Mar 13 32 8 - 3-lipemic 43
Apr 13 25 10 - 21(9-lipemic 56
12- RBC †)
donation time. Most syncope attacks are vasovagal in nature for component preparation. Thus, selecting a good donor,
and bear no relation to the time since last meal.5 Donor training and monitoring the staffs will help to reduce cases
education and counseling along with a comforting and clean of the underweight/overweight blood units resulting in low
environment is important to allay the anxiety surrounding discard rate.
the donation process. It has been our experience that Splitting: In the case of pediatric and thalassemic patients
healthy individuals hesitate to donate blood because of requiring blood, appropriate volume bags are made by
the superstitions prevalent in our country regarding blood transferring blood from the mother bag to satellite bags
donation, thus promoting the practice of professional donors through a sterile connecting device which is a closed system.6
and middlemen. Calibrated scales and balances should be The remaining mother bag is often discarded due to a lack of
used to accurately measure the volume of blood collected to further demand.
prevent under collection and wastage.
Small manual spring balances or scales for weighing Leakage: The blood bag should be checked for any leakage
are still being used in mobile sessions, while blood mixing or defects prior to the collection of blood. Mishandling of
machines are used in the blood bank. The manual spring blood bags during collection, processing, and storage or
balances require the phlebotomist to monitor the weight manufacturing error may be the major causes of defects
of the blood bags and mix the anticoagulant with the blood and leakages of blood bags. The integrity and sterility of
every 60 seconds during the whole blood donation process. blood bags is mandatory and precautions should be taken
Overlooking this monitoring process during busy hours can to prevent leakages. During centrifugation the bag may be
lead to too much blood going into the collected bags. These exposed to sharp surfaces or forced to a sharp wall junction
problems can be avoided by using the automated blood or corner, resulting in the bag material being stretched too
mixing machines. When sufficient blood has entered the far, causing a tear. The defect and leakage at any part of
bag, an audio visual alarm is activated and the blood flow the plastic blood bags can be detected by visual inspection
is automatically stopped by clamping the tube to prevent during the processing, after pressure in a plasma extractor,
further blood flow into the bag. In overweight blood bags, before freezing, and after thawing.
the amount of anticoagulant is not enough to prevent Lipemia: Gross lipemia visible on inspection was another
the blood from clotting and the clotting process may be important cause of discarding of blood units. Some cases of
initiated. These blood bags with blood clots are unsuitable grossly lipemic components have been traced to donors who
250 Section 3 Blood Safety
had a fatty meal before donation in the study by Mbahi M Blood components like platelets and FFP have their
et al.7 This raises an important concern for donor screening unique issues when it comes to wastage. One important
and education especially because donors are advised to reason is RBC contamination which is detrimental to the
have adequate meals before a donation. Lipemia itself does product quality. At our centre, platelets are prepared both
not affect the safety of a product but might interfere with the by PRP method and apheresis. However, for the purposes
ability to perform viral marker tests.8 The maintenance of a of this study only the platelets produced by the PRP method
donor registry can aid in identifying donors who previously were considered. After centrifugation, the blood bags
donated lipemic blood so that future donations can be should be carefully handled to prevent RBC contamination
deferred until he/she is properly investigated. of plasma and platelets. Subsequently, these centrifuged
blood bags should be carefully reposted onto the plasma
Hemolysis: It is an important parameter for the quality extractor (or blood press) and allow gentle pressure to the
assessment of stored RBCs. It is evaluated by visual centrifuged blood bag from the plasma extractor plate. The
inspection though it can be better identified by photometric separation should be done slowly with close monitoring
methods. Hemolysis of red cell units occurs during processing during the transfer of layers of blood components into the
for component separation and also due to repeated handling satellite bags. The separation is stopped when about 8 mm of
during storage, issue and transport before transfusion to plasma remains above the red cells.12 This technique is labor
the patient.9 RBC hemolysis can be prevented by stringent intensive. Human factor has strong effect on the quality and
temperature regulation between 2 and 6°C, using good quality purity of the blood components preparation.
polyvinyl chloride bags with DEHP as plasticizer which is Identifying the centrifuges causing a high rate of RBC
known to reduce hemolysis, leukocyte reduction and the use contamination, the technician handling the centrifuge and
of additive solutions containing mannitol. During component plasma extractor, the centrifuge speed and time, the date
separation, centrifugation speeds more than 5000G induce and time of component separation can help to resolve the
hemolysis and this should be avoided. Transporting the technical details and improve the quality of the final end
blood bags at appropriate temperature prevents hemolysis of product.
blood after being issued from the blood bank.10 The short shelf life of platelets (5 days) makes it essential
Inventory management and expiry of blood units: Blood to ensure a constant and proportionate production and
bank should arrange the blood units of near expiry in front distribution of platelet units. This is especially tricky in
shelves in freezers and have proper inventory management in situations like dengue months where the demand outstrips
blood bank so that they can be used in judicious way (i.e. first in the supply and other months in the year where more platelets
and first out- FIFO) and wastage can be reduced. Maintaining are separated than the demand and hence get outdated.
a record of donors of rare blood groups like Bombay blood FFP refers to the fluid portion of one unit of human
group who can be contacted for blood donation will help to blood that has been centrifuged, separated, and frozen solid
maintain a reserve for as and when the need arises. at −18 °C (−0 °F) or colder within six hours of collection. The use
To prevent wastage due to out dating a type and screen of FFP has increased over the past few years due to increased
protocol is recommended. This protocol includes screening acceptance of blood component therapy. FFP can be stored
for other antigens apart from routine ABO and Rh typing like in deep freezers for prolonged duration of time (upto 1 year).
ABO (A1 and A2), Rh (etc.), Bombay, Para bombay, MNS For this reason it is important to maintain an inventory and
and Kell, Lewis, Duffy and auto antibodies depending upon carefully manage the limited space in a deep freeze to ensure
protocol and incidence of other groups. If the donor has a that a constant input output cycle is maintained so that the
rare blood group which is not in demand immediately the near expiry units are kept in front and used first, the expired
donation can be deferred and the donor registered with the products are discarded and space is made free for the newly
blood bank so that he/she can be recalled when required. separated components. At our centre, many units of FFP
One study in Hong Kong reduced out dating rate by 11.5% to had to be discarded due to a shortage of storage space in the
1.3% by this protocol.11 deep freezer and other technical issues like power outgages.
Also other factors like decreased clinical use and demand,
Antibody positive: In our centre blood is screened for non availability of plasma fractionation technique and no
antibodies post the donation process. Most of the blood units permission to distribute plasma to other private centres lead
positive for irregular antibodies are discarded because of to discarding of further plasma units.
the lack of demand of corresponding antigen negative blood The FFP should be stored in cardboard or polystyrene
within the expiry period of that particular unit. A system in protective containers that minimize the risk of breakage
which donors are screened for irregular antibodies before of brittle frozen product during storage, handling, and
donation can help improve efficiency by reducing the wastage transportation. Another approach to decrease the leakage
due to presence of antibodies. Also to be emphasized in this and contamination immediately before immersion of the
context is the importance of maintaining donor registries and frozen blood bags in the water bath is that the whole container
promoting regular voluntary donation. should be placed in a sterile plastic bag.
Chapter 67 Discarding of Blood and Blood Components 251
Manoj A Kahar
68
INTRODUCTION Diseases simulating HTR include Paroxysmal Nocturnal
Hemoglobinuria, Autoimmune hemolytic anemia, G-6-P-D
Blood transfusion like administration of any other drug is an deficiency, Malignant Hyperthermia, Hemoglobinopathies
irreversible event that carries potential benefits and risks to and RBC membrane defects.
the recipient. Some typical causes of transfusion associated deaths are:
Definition of adverse transfusion reaction: Any unfavorable • Acute hemolysis (ABO-incompatible blood components)
transfusion – related event occuring in a patient during or • Acute pulmonary edema
after transfusion of blood components. • Bacterial contamination of product
• Delayed hemolytic reactions
• External hemolysis (e.g. temperature exceeded 40°C)
Classification
• Acute hemolysis; damaged blood component (e.g.
Adverse transfusion reaction (ATR) may be immediate nondeglycerolized, improper solution)
(occuring during or within 24 hrs after completion of • Transfusion-associated graft-versus-host disease.
transfusion) or delayed (evident after days, weeks, months The three leading causes of transfusion-related death
or even years after transfusion). Immediate and delayed reported in literature are transfusion related acute lung injury
transfusion reactions are further divided as immune (TRALI), HTRs and bacterial contamination.
(mediated by Antigen-Antibody reaction) or nonimmune.
Pathophysiology of immune hemolysis: In vivo lysis of
Reaction Immediate (Acute) Delayed RBCs can result from the interaction of complements with the
Immune IHTR DHTR RBC membrane (intravascular hemolysis) (Flow chart 1) or
mediated FNHTR PTP the removal of cells sensitized with IgG and/or complement
Allergic TA-GVHD by the reticuloendothelial system (extravascular hemolysis).
Anaphylactic and Anaphylactoid
NCPE-TRALI ACUTE Adverse transfusion reaction
Non immune Bacterial contamination Iron overload WITH IMMUNE CAUSES
mediated Circulatory overload Transfusion
Physical or chemical damage to associated Immediate Hemolytic Transfusion Reaction
RBCs infections
Depletion and dilution of Immediate hemolytic transfusion reaction (IHTR) occurs
coagulation factors and platelets very soon after the transfusion of incompatible RBC. The RBC
is rapidly destroyed, releasing hemoglobin and RBC stromata
It may be impossible to assess the severity of ATR by the in the circulation (Flow chart 2).
symptoms because a life-threatening (acute hemolytic) and • Cause: The majority of hemolytic reactions are caused by
relatively mild (nonhemolytic febrile) transfusion reaction transfusion of ABO incompatible blood, e.g. group A, B or
may initially cause exactly the same symptoms (e.g. fever, AB red cells to a group O patient. Most hemolytic reactions
chills). Hence, any ATR should be considered a potentially are the result of human error such as the transfusion of
life-threatening reaction until clinical observations and/ properly labeled blood to the wrong patient, or improper
or laboratory results establish otherwise. On the other side, identification of pre-transfusion blood samples.
there are many diseases that can cause RBC hemolysis and • Non-immune hemolysis of RBCs in the blood container or
misinterpreted as hemolytic transfusion reaction (HTR). during administration can occur due to physical disruption
Chapter 68 Adverse Transfusion Reactions 253
(temperature changes, mechanical forces, non-isotonic Flow chart 1 Complement activation may be initiated by antibody
fluid). binding to the appropriate antigens on the red cells
• Symptoms: Chills, fever, pain (along IV line, back, chest),
hypotension, dark urine, uncontrolled bleeding due to
DIC.
• Management: Immediately stop transfusion. Notify
hospital blood bank urgently (another patient may also
have been given the wrong blood!). These patients usually
require ICU support and therapy includes vigorous treat-
ment of hypotension and maintenance of renal blood flow.
• To assess the patient’s condition, several laboratory
parameters should be monitored during the next days:
Electrolytes, ABG, Coagulation tests, CBC and Creatinine.
• Prevention: Proper identification of the patient from
sample collection through to blood administration, proper
labeling of samples and products is essential (Figs 1 to 3).
Prevention of non-immune hemolysis requires adherence
to proper handling, storage and administration of blood
products.
– Investigation: Fever can be the initial sign in more Transfusion Related Acute Lung Injury
severe transfusion reactions (hemolytic or bacterial
sepsis) and should be taken seriously. Synonyms: Non-cardiogenic pulmonary edema (NCPE),
– For isolated fever or chills in some patients, the medical Pulmonary hypersensitivity reaction and allergic pulmonary
officer may elect to restart the transfusion. If the fever is edema.
accompanied by significant changes in blood pressure • Transfusion related acute lung injury (TRALI) is a clinical
or other signs and symptoms, the transfusion should be diagnosis of exclusion characterized by acute respiratory
ceased and investigated. distress and bilaterally symmetrical pulmonary edema
– Check for HLA antibodies in patients having repeated with hypoxemia (PaO2/FiO2 < 300 mm Hg), normal central
febrile reactions. venous pressure and normal pulmonary wedge pres
– Prevention: A proportion of patients who have febrile sure developing within 2 to 8 hours after a transfusion.
reactions will have similar reactions to subsequent A CXR shows interstitial or alveolar infiltrates when no
transfusions. Many are prevented by leucocyte filtration cardiogenic or other cause of pulmonary edema exists
(either bedside or pre-storage). (Figs 4A and B).
• Cause: Pulmonary vascular effects are thought to occur
Urticarial (Allergic) Reactions secondary to cytokines in the transfused product or from
interaction between patient white cell antigens and donor
• Cause: Seen in approximately 1% of recipients and caused antibodies (or vice versa).
by foreign plasma proteins. On rare occasions they may • Predisposing factors for TRALI include: Direct lung injury
be associated with laryngeal edema and bronchospasm. (aspiration, pneumonia, toxic inhalation, lung contusion,
Two possible etiologies have been proposed, based on the near drowning) and Indirect lung injury (sepsis, shock,
passive transfer of donor plasma (1) The donor plasma has multiple trauma, burn injury, acute pancretitis, CABG,
an allergen with which IgE, IgG or both antibodies in patient drug overdose)
plasma react. (2) The donor plasma has regains (IgE or IgG • Management: Symptomatic support for respiratory
or both) that combine with allergens in the patient plasma. distress includes oxygen administration and 72% cases
• Management: If urticaria occurs in isolation (without may require intubation and mechanical ventilation.
fever and other signs), slow the rate or temporarily stop Symptoms generally resolve over 24–48 hours. Death
transfusion. If symptoms are bothersome, consider occurs in 5–10% of patients experiencing a TRALI reaction.
administering an antihistamine before restarting the • Suggested prerequisites for performing a laboratory
transfusion. If associated with other symptoms, cease the workup:
transfusion and proceed with investigation. – A laboratory workup of a TRALI case is expensive.
• Investigation: In the case of mild urticarial reactions – Donors may experience anxiety and/or concern that
with no other signs or symptoms, it is not necessary to their blood donation may have resulted in harm to a
submit blood specimens for investigation. It is also usually recipient. Antibodies are most common in multiparous
possible to restart the transfusion. Such a decision should female donors as consequence of prior pregnancies.
be made after assessment by the treating doctor.
• Management: Slowing or temporarily stopping the trans of pretransfusion testing. Appropriate antigen negative
fusion allows citrate to be metabolised. Replacement blood will be supplied.
therapy may be required for symptomatic hypocalcemia • Prevention: Alloimmunization to the D and K (Kell)
or hypomagnesemia. antigens is prevented by the provision of Rh(D) negative
and Kell negative blood for Rh(D) negative, Kell negative
Potassium Effects patients. This is important for females with child-bearing
• Cause: Stored red cells leak potassium proportionately potential as these antibodies can cause severe hemolytic
throughout their storage life, transfusion of such red cells disease of the newborn during pregnancy.
can lead to hyperkalemia. After 28 days storage in citrate, a • At risk groups: Patients with sickle cell disease or major
unit of RBC contains approximately 6 mmol of potassium hemoglobinopathy syndromes who are chronically
per unit. Irradiation of red cells increases the rate of transfused are at greatest risk of alloantibody formation.
potassium leakage. Clinically significant hyperkalemia Prior to commencing transfusion, patients with these
can occur during rapid, large volume transfusion of older condition should have extended red cell phenotyping
red cell units in small infants and children. Transfusion performed (EDTA sample). Blood matched for the
induced hypokalemia is most likely to be caused infusion of patient’s Rhesus and Kell antigens is usually supplied for
intracellular potassium depleted RBC blood components, transfusion.
such as usual RBCs or frozen washed RBCs.
• Prevention: Blood less than 7 days old is generally used Platelet Refractoriness
for rapid large volume transfusion in small infants (e.g. • When thrombocytopenic patients do not achieve the
cardiac surgery, exchange transfusion). expected post-transfusion platelet count increment they
are said to be refractory. This usually occurs in patients
DELAYED Adverse transfusion receiving frequent platelet transfusions.
reaction WITH IMMUNE CAUSES • Cause:
– Clinical causes include; sepsis, DIC, bleeding, fever,
Delayed Hemolytic Transfusion Reaction some drugs, and enlarged spleen.
– Immunological causes include; the development of
• Definition: Delayed hemolytic transfusion reaction (DHTR) antibodies to human leucocyte antigens (HLA) or
is most often the result of an anamnestic response in a human platelet antigens (HPA).
patient who has previously been sensitized by transfusion, • Management: Immunological refractoriness can be mana
pregnancy, or transplant and in whom antibody is not ged by the provision of HLA or HPA matched platelets.
detectable by standard pretransfusion methods. • Prevention: Leukocyte reduction of blood products
• Cause: A DHTR occurs when a patient develops an to levels less than 106/unit reduces the likelihood of
antibody directed against an antigen on transfused alloimmunization. This can be achieved through the use of
red cells. Antibodies implicated in DHTR are Anti JKa, prestorage or bedside leucocyte reduced blood products.
Anti-E, Anti-D, Anti-C, Anti-K, Anti Fya, Anti M (common
antibodies) and Anti-A1, Anti-P1 (uncommon antibodies).
The antibody may cause shortened red cell survival, Post Transfusion Purpura
with clinical features of fever, jaundice and lower than • It is characterized by a rapid onset of thrombocytopenia
expected hemoglobin following transfusion. Most delayed due to anamnestic production of platelet alloantibody.
hemolytic reactions produce few symptoms and may • Post transfusion purpura (PTP) usually occurs in multi
go unrecognized however there are reports of serious parous females. The lag time between transfusion and
consequences in critically ill patients. onset of thrombocytopenia is approximately 7 to 14 days.
• Prevention: An antibody screen is performed as part of • Purpura and thrombocytopenia occur about 1 to 2
pre-transfusion testing. When an antibody is detected, weeks after transfusion. Hematuria, melena, and vaginal
it is identified and appropriate antigen negative blood is bleeding have also been reported.
provided. Sometimes antibodies fall below detectable Pathophysiology:
limits and may not be detected by pretransfusion testing. – Platelet antibody specificity most frequently identified
A medical alert card should be issued to all patients in is HPA-1a (anti-PLA1), others are HLA-A2 and
whom irregular antibodies were found so that they can Lymphocytotoxic antibodies.
present this information at the time of transfusion in a – Platelet alloantibody attaches to the platelet surface
different medical facility. which permits extravascular destruction by the RES in
the liver and spleen. The patient’s autologous platelets
Alloimmunization are destroyed enhancing the thrombocytopenia.
• Patients experiencing alloantibody formation are Management: Three types of therapy have been advocated:
asymptomatic. The alloantibody is discovered at the time Corticosteroids, exchange transfusions, and plasmapheresis.
258 Section 3 Blood Safety
Intra
venous immunoglobulin therapy has also been reticuloendothelial sites of iron storage become saturated.
advocated. Liver and endocrine dysfunction creates significant morbidity
and the most serious complication is cardio toxicity which
Transfusion-Associated Graft Versus causes arrhythmias, and congestive heart failure. Patients
receiving chronic transfusion usually have their iron status
Host Disease monitored and managed by their physician.
• Cause: Transfusion-associated graft versus host disease Management and prevention: Iron chelation therapy is
(TA-GVHD) occurs when donor lymphocytes in cellular usually commenced early in the course of chronic transfusion
blood products engraft in a susceptible transfusion therapy.
recipient. These donor lymphocytes proliferate and
damage target organs especially bone marrow, skin, Infectious Disease Transmission
liver and gastrointestinal tract. The clinical syndrome
comprises fever, skin rash, pancytopenia, abnormal liver • A variety of infectious agents may be transmitted by
function and diarrhea and is fatal in over 80% of cases with transfusion. Definitive evidence of transmission by
a median survival of 21 days after transfusion. The usual transfusion requires demonstration of seroconversion or
onset is 8–10 days post transfusion, with a longer interval new infection in the recipient and isolation of an agent
between transfusion and onset of symptoms in infants. with genomic identity from both the recipient and the
• The most commonly reported setting for Ta-GVHD is implicated donor.
immunocompetent recipients of blood from biologically • Strong presumptive evidence of transfusion transmission
related (directed) or HLA identical donors. The disease includes recipient seroconversion within an appropriate
is also reported in immunologically compromised interval after transfusion, the recognition of appropriate
patients. Death is usually due to infection or hemorrhage infectious markers in an implicated donor on follow-up
secondarily to bone marrow aplasia. investigation, or both.
• Pathophysiology: GVHD is caused by a proliferation • Suspected transfusion-transmitted bacterial or parasitic
of T-cell lymphocytes derived from the donor blood infection (malaria) should be reported urgently in order to
immunologically responding to major and minor histo recall other potentially infectious blood products from the
compatibility antigens in the patients. Patients with cell same donation.
mediated immunodeficiency are at risk of not being able • The risk of acquiring an infectious disease through blood
to reject transfused lymphocytes. Another risk group is transfusion has not been totally eliminated even though
patients haploidentical with that of the donor, these are the level and sensitivity of testing today makes transfusion
usually first degree of reactions. Diagnosis can be made very safe. Physicians/hospital staff should report all
by skin biopsy, liver biopsy or bone marrow examination. instances when an infectious disease is reasonably likely
HLA typing essential to confirm the diagnosis. to have been transmitted by a blood transfusion.
• Prevention: Gamma irradiation of cellular blood products • The requirements for investigating transfusion associated
(whole blood, red blood cells, platelets, granulocytes) for infections (TAI) are found in the Code of Federal
at risk patients. Regulations, Title 21, Section 606.170, and the AABB
Standards.
• The most commonly investigated infectious diseases are:
Immunomodulatory Effects retroviruses, hepatitis viruses, West Nile virus, malaria,
• Some studies suggest a link between blood transfusion babesiosis, and Chagas disease.
and increased risk of infection and cancer recurrence. The transfusion center investigation includes:
However, this is currently considered unproven. • Complete medical history
• Cause: Unknown, possibly mediated by donor white cells • Clinical laboratory data
or plasma. • History of known risk factors for the reported infection.
• Management and prevention: Not known, possibly The objectives of blood center investigations of Tai are:
leucocyte depletion of blood products. • Quarantine of additional infectious components
• Deferral of an “unsafe” donor
DELAYED Adverse transfusion • Notification of the donor and other recipients of the
reaction WITHOUT IMMUNE CAUSES donor’s blood
• Process improvements
Iron Accumulation • Given the risks related to transfusion practice, it is of
paramount importance to have adequate skills and to take
Cause: Iron accumulation is a predictable consequence optimal precautions for the transfusion of blood and blood
of chronic RBC transfusion. Organ toxicity begins when components.
Chapter 68 Adverse Transfusion Reactions 259
Neelam Marwaha
69
Introduction • Bacterial contamination of blood and blood products
constituted a more frequent infectious hazard of blood
Blood and its components prescribed by almost all medical transfusion rather than viral diseases.
and surgical specialists, are considered life-saving but may • The risk of transmitting viral infection by use of screened
give rise to undesirable and unintended adverse effects. blood is almost negligible and is mainly linked to donations
Hence, the safety of the entire transfusion chain, i.e. from occurring in the window period, when sensitive testing
the donor to the recipient needs monitoring. This can only technologies are applied.
be achieved by careful observation and analysis of adverse • Administration of ABO incompatible blood was invariably
events, hence hemovigilance, which is defined as “a set of due to failure to detect incorrect identity of patient or the
surveillance procedures, from the collection of blood and blood unit. Errors occurred both in the blood centre and
its components to the follow-up of recipients to collect and the patient bedside, the latter being more common, i.e.
assess information on unexpected or undesirable effects failure to check patient and blood unit identity at bedside.
resulting from the therapeutic use of labile blood products • Administration of ABO compatible but non-compatibility
and to prevent their occurrence or recurrence”. due to other alloantibodies occurred on two accounts (a)
The objectives of hemovigilance are: failure of clinicians to provide information regarding prior
• To recognize serious adverse events of transfusion transfusions and (b) technical limitations of alloantibody
• To analyze factors leading to such events. detection and typing systems.
• To devise preventive strategies. • Immune non-hemolytic complications of transfusion
• To analyze the outcome of preventive strategies. like transfusion related acute lung injury (TRALI), post
transfusion purpura (PTP) and transfusion-associated
graft versus host disease (TA-GVHD) were unpredictable
Hemovigilance systems and seemed under reported.
Hemovigilance in a country can be organized in different • The causes of transfusion related mortality were TA-GVHD,
ways. However, for logistic reasons, the system has been TRALI, bacterial contamination of blood components
organized based on the existing network for collection of data and incorrect blood/component transfused. Bacterial
related to blood transfusion services in various countries. contamination emerged as a cause for concern.
There are at least three distinct patterns of hemovigilance. • Pre-deposit autologous transfusion also could not
1. Through legislation. guarantee 100% safety as there was occurrence of clerical
2. Voluntary reporting of transfusion events. errors leading to ABO mismatched blood issue and
3. Multidisciplinary regulation. bacterial contamination on storage.
• Transfusion errors: “Getting the right blood/component fresh frozen plasma and cryoprecipitate for one year. In case
to the right patient at the right place at the right time” is platelets have been detected to have bacterial contamination,
a complex organizational process that involves patient’s a rapid alert to the blood center will lead to immediate
bedside sampling through laboratory testing, issuing of removal of other components from that donor unit from the
blood/component and finally bedside transfusion. Errors inventory.
may occur at any stage of the process. Each hospital/ The plasma derivatives like albumin, factor concentrates
institution needs to formulate/adopt guidelines where and immunoglobulin preparations which are prepared
patient identification and blood/component identification through plasma fractionation are covered under pharma
are made in accordance with strict procedures. The covigilance, in most of the countries.
guidelines need to be circulated to all user departments/ Donor reactions have a negative impact on voluntary
clinicians periodically. Use of the computerization in blood donation programme. Such information is relevant for
transfusion services with barcode printers and scanners formulating appropriate donor motivational strategies and
to provide bar-coded identities for patient’s samples post-donation care of donors. Donor vigilance is another
and blood packs and introduction of an IT “block” to the important area and definitions of donor reactions have
administration of blood without confirming identity of already developed by the ISBT working party.
patient and blood unit were suggested. Implementation India had no national hemovigilance system in place till
of computerization has definitely reduced the risks of the year 2012, although the implementation of hemovigilance
transfusion errors. program is included in the National Blood Policy. Few
• Bacterial contamination: Careful donor screening and institutional efforts at hemovigilance have been reported in
proper phlebotomy site preparation are the first essential the scientific literature from the country, but that does not
prerequisites. Storage at recommended temperatures and constitute a system.
specified shelf life must be observed. Visual inspection
of blood/component unit before issue and also before Hemovigilance in India
transfusion is necessary. Bacterial screening of platelet
A centralized and structured hemovigilance programme was
concentration is also being implemented. Automated
launched in India on 10th December 2012. It is an independent
rapid bacterial detection systems have been developed.
programme under the broad ambit of Pharmacovigilance
• Immune complications: Clinician’s awareness regarding
Programme of India (PvPI), which was initiated in July 2010
TRALI and TA-GVHD needs to be increased. Appropriate
with the objective of assuring patient safety and promoting
diagnostic guidelines need to be formulated particularly
public health. The PvPI is being executed by the Indian
for TRALI. Hemolytic transfusion reactions other than
Pharmacopoeia Commission, Ministry of Health and Family
those due to ABO system antigens can best be avoided by a
Welfare (MOHFW), Govt. of India. The data is being collected
type and antibody screen rather than type and cross match
through adverse drug reaction (ADR) monitoring centers set
policy.
up in 90 medical institutions in the country. Trained staff- the
• New blood safety initiatives: Pathogen inactivation
technical associates, have been recruited for these ADRs for
of fresh frozen plasma and platelet concentrates will
data collection and submission. After the successful launch
take care of window phase transmissions and emerging
of PvPI, the hemovigilance programme has been started
infectious threats. Clinical trials are being conducted to
as a collaborative venture by the Indian Pharmacopoeia
evaluate product safety and efficacy. Commission and the National Institute of Biologicals (NIB),
“Near Miss” events are errors which are detected before an autonomous institute under the MOHFW, Govt. of India,
the transfusion of incorrect blood/component, and hence a which ensures quality of biologicals for use in the country. The
major transfusion error can be avoided. Reporting of “Near National Co-ordinating Center for hemovigilance is located
Miss” events helps in identifying “hot spots” for transfusion at NIB, and a Core Committee chaired by the Director, NIB,
errors and is of immense value for hemovigilance purposes. co-ordinates this programme.
The objective of a Rapid Alert System is to take corrective The objectives of the programme are (i) to collect, collate
measures in the shortest possible time. It has been used to and analyse data related to transfusion reactions, (ii) to create
alert the appropriate authorities to take note of appearance awareness amongst healthcare professionals in the country
of clusters of transfusion reactions, defects in disposable for participation in the programme, (iii) to generate evidence
materials in the transfusion chain or defective equipments, based recommendations and forward the same to central
etc. To cite an example in most of the tertiary care hospitals in drugs standards control organisation (CDSCO) for blood
our country, blood components are transfused in preference safety related regulatory decisions, (iv) to communicate
to whole blood. The shelf-life of each component is different; relevant information to all key stakeholders and (v) to create
hence the time of actual transfusion of each component from national and international linkages.
the same donor may be different. Platelets have a short shelf- The Core Committee is assisted by a National Advisory
life (3 to 5 days) while red cells have shelf-life of 35 days and Committee which has a varied representation, reflecting
262 Section 3 Blood Safety
key stakeholders. After detailed discussions amongst all the An Expansion and Consolidation
committee members, the transfusion reaction reporting
Phase (2013-15)
form (TRRF) and a guidance document for filling the
TRRF have been finalized. Reporting has been initiated for • Enroll additional 10 medical colleges
severe transfusion reactions as per ISBT reportable table of • Continue training of hemovigilance human resource
serious adverse events so as to harmonize definitions as per • Identify gaps and address through appropriate training
international norms. Each reporting forms also documents • Link up with international hemovigilance network.
the imputability assessment of the reaction. Software has
been developed by the IT division of NIB and validated for An Expansion and Maintenance
online submission of TRRF from medical institutions. The Phase (2015-16)
responsibility for submission of hemovigilance data has
been given to the medical officer in charge of blood centers • Enroll all remaining medical colleges
/departments of transfusion medicine with assistance from • Continue training of hemovigilance human resource.
the technical associates from the PvPI.
Presently, reporting is voluntary and limited to information Optimization Phase (2017)
related to adverse transfusion reactions. Awareness about the • Create center of excellence for hemovigilance.
programme, its objectives and its non-punitive implications Till date the progress in this programme has been highly
is being generated through a hemovigilance newsletter promising. The transfusion reaction reporting form (TRRF),
accessible at website hemovigilance@nib.gov.in and also a Guidance Document and the software for submitting
through a series of workshops to be held in different regions data online (hemovigilance) are already in place. Fourteen
of the country. sensitization workshops/CMEs have been organized in
The functional units for hemovigilance in a particular different parts of the country. Seventy-nine centers have
center as defined in the guidance document are as follows. already been issued user IDs and password. Thirty-nine
• Medical and nursing staff at the patient bedside blood centers have started submitting their data online. The
• Department of transfusion medicine first issue of the hemovigilance newsletter (January to June
• Hospital transfusion committee 2013) was launched in the first half of 2013 and the third
• Technical associate IPC-PvPI issue (January to June 2014) is on the website and also widely
• Hemovigilance center, NIB disseminated to various medical institutions and blood
• PvPI national co-ordinating center, IPC centers. Updated information is available at the website of
• CDSCO National Institute of Biologicals, NOIDA (www.nib.gov.in).
The entire programme has been structured to collect, The success of any programme depends upon participation,
collate and analyse data for making recommendations to the the more the data we collect; more meaningful will be data
regulators to make policy changes for improving blood safety. analysis and consequent blood safety recommendations for
the country.
Roadmap for Hemovigilance in the Country
suggested reading
The programme is planned to proceed in a phase-wise
manner. An initiation phase (2012-13), an expansion and 1. An Action Plan for Blood Safety. National Aids Control
consolidation phase (2013-15), an expansion and mainte Organisation, Ministry of Health and Family Welfare. Govt. of
nance phase (2015-16) and optimization phase (2017). India. 2003.
2. Bisht A, Singh S, Marwaha N. Hemovigilance program- India.
Asian J Transf Sci. 2013;7:73-4.
An Initiation Phase (2012-13) 3. deVries RRP, Faber JC, Strengers PFW. Hemovigilance: an
effective tool for improving transfusion practice. Vox Sang.
• Develop systems and procedures for data collection 2011;100:60-7.
• Enroll 90 medical colleges 4. Engelfreit CP, Reesink HW. Hemovigilance systems. Vox Sang.
• Initiate software development for national hemovigilance 1999;77:110-20.
5. Faber JC. Heamovigilance in Europe: the European Heamo
database
vigilance network. Transf Clin Biol. 2001;8:285-90.
• Start data collection 6. Hemovigilance Module, Biovigilance component. National
• Development and establishment of training centers Healthcare Safety Network. Centers for Disease Control and
• Training of hemovigilance human resource Prevention USA. http://www.cdc.gov/nhsn/PDFs/hemovig
• Zonal workshop for public awareness of transfusion safety ModuleProtocol_current.pdf (last accessed in Nov 2013).
• Initiate and continue publication of hemovigilance 7. Hemovigilance Programme of India. National Institute
newsletter. of Biologicals and Indian Pharmacopoeia Commission
Chapter 69 Hemovigilance and its Launch in India 263
Collaboration. http://www.nib.gov.in/hemovigilance.html 13. Sharma RR, Kumar S, Agnihotri SK. Sources of preventable
(last accessed in Dec 2013). errors related to transfusion. Vox Sang. 2001;81:37-41.
8. International Hemovigilance Network. http://www.ihn-org. 14. Todd A. Hemovigilance – closing the loop. Vox Sang. 2002;83
com (last accessed on Jan 8, 2014). (Suppl I):13-6.
9. International Society of Blood Transfusion Working Party on 15. Transfusion and Transplantation Reactions in Patients. Dutch
Hemovigilance. Proposed standard definitions for surveillance Hemovigilance system. http://www.tripnet.nl (last accessed
of non infectious adverse transfusion reactions. July 2011. on Jan 8, 2014).
http://www.isbtweb.org/Hemovigilance/ISBT_definitions_ 16. Williamson L, Cohen H, Love E, Jones H, Todd A, Soldan K.
final_2011 (last accessed on Jan 8, 2014). The Serious Hazards of Transfusion (SHOT) Initiative: The UK
10. Pharmacovigilance programme of India for Assuring Drug approach to Hemovigilance. Vox Sang. 2000;78:291-5.
Safety http://www.cdsco.nic.in/pharmacovigilance_intro.htm 17. World Health Organisation Draft Guidelines for Adverse
(last accessed on Jan 8, 2014). Event Reporting and Learning Systems. http://www.who.
11. Rouger Ph, Noizat-Pirena F, Le Pennac PY. Hemovigilance and int/patientsafety/events/05/Reporting_Guidelines.pdf (last
Transfusion Safety in France. Vox Sang. 2000;78:287-9. accessed in Dec 2013).
12. Serious Hazards of Transfusion (SHOT) UK. SHOT
organization. http://www.shotuk.org.
Advances in Serological Assays
for Screening Blood Donors for
Infectious Agents
Paramjit Kaur
70
introduction the infected individual. Serological assays for antigens use
specific antibodies to detect constituents of the infectious
The first infectious disease test performed on blood donations agent (e.g. HBsAg and HIV-1 p24 Ag). Molecular assays
in the United States was a test for syphilis and screening was (nucleic acid amplification tests or NAT) detect the nucleic
introduced in the 1940s. The first assays for hepatitis B surface acid sequences, RNA or DNA of the infectious agent. They
antigens were introduced in the early 1970s. Early 1980s are effective for the detection of recent infections or incident
brought attention and resources to the prevention of more cases while serologic assays for antibodies are effective for
serious consequences of blood transfusion when potential the detection of prevalent cases. Improvements in technology
transmission of acquired immunodeficiency syndrome have brought sensitivity of newer generation serologic assays
(AIDS) by blood transfusion was discovered. With the for antibodies and combined antigen antibody assays closer
evolution of new screening tests, improved donor screening to NAT.3,4
procedures and quality systems ushered in. In addition
multiple layers of safety became part of the regulatory
Evolution of Hepatitis C Virus Assays
framework to ensure safer blood components.1,2
The frequency with which an infection is transmitted to The initial clone discovered was from the NS4 region and the
blood recipients depends upon the length of the asymptomatic derived protein was designated 5-1-1. It was expanded to form
blood borne period, how often blood is donated during this c100-3 antigen that formed the basis of the first-generation
period and the immune status of the recipient population. anti-HCV assay. Second generation assays included the c22
The efficacy of blood donor screening test relies not only on core antigen and the c33c antigen from the NS3 region. Third-
the ability to detect an individual infected by transfusion generation assay further included a protein from NS5 region.
transmissible infection but also on the duration of the Even with improved HCV antibody assays the infectious
window period. The sensitivity of a screening assay depends window period remained around 66 days. HCV core antigen
on its ability to close the window period. EIAs shortened the window period considerably, but high cost
The main types of assay used for blood screening are: was the major limitation. A fourth-generation HCV antigen
Immunoassays (IAs): and antibody assay (combination EIA) further improved
• Enzyme immunoassays (EIAs) testing as two infectious markers of HCV are detected in the
• Chemiluminescent immunoassays (CLIAs) same assay. Serology based assays may detect presence of
• Hemagglutination (HA)/particle agglutination (PA) assays anti-HCV antibodies, HCV antigen or both simultaneously.
• Rapid immunochromatographic assays The testing platforms could be enzyme immunoassays
• Nucleic acid amplification technology (NAT) assays. (EIAs), chemiluminescence (CLIA) or rapid tests. The CLIA
Appropriate evaluation is required in selecting the type of end point detection signal has better sensitivity than enzyme
assay for each TTI, based on critical assay characteristics, such based assays. The improvements in assay performance have
as sensitivity and specificity, positive and negative predictive been termed as “generations” of the assays. In many blood
value as well as cost and ease of use. The epidemiology and centers located within resource-poor countries the laboratory
geographical distribution of the agents are also critical. facilities are limited and rapid tests provide an alternate
screening methodology to EIAs. They are based upon any one
of the following principles; agglutination, immunofiltration
Serological versus Molecular Assays
or immuno-chromatography. The antigens used in the test
Serological assays for antibodies detect antibodies against are usually same as those incorporated in third generation
the infectious agent generated by the immune system of EIAs. Each test strip or cassette has an inbuilt control band
Chapter 70 Advances in Serological Assays for Screening Blood Donors for Infectious Agents 265
for validation. The reported sensitivities with these tests newborn, the need for rapid results, etc. The availability of
ranged from 98 to 100%.5 a fourth-generation test capable of offering an increase in
Signal-to-cut–off ratios for commercially available assays: sensitivity over other antibody tests is yet another evolutionary
CDC has recommended that a person be considered to step. The concept of simultaneous antigen and antibody
have serologic evidence of HCV infection only after an anti- detection is of great importance, as there is a definite need to
HCV screening-test-positive result has been verified by a continue testing for early infection and to apply cost-saving
more specific serologic test (e.g. RIBA) or a nucleic acid test strategies.
(NAT). This more specific, supplemental testing is necessary,
particularly in populations with a lower prevalence of disease, Evolution of Hepatitis B Virus Assays
to identify and exclude false positive screening test results.
However, currently, the majority of laboratories report Serologic testing for hepatitis B virus (HBV) surface antigen
positive anti-HCV results based on a positive screening assay (HBsAg) and antibody to HBV core antigen (anti-HBc) has
alone.6 historically been the foundation of blood screening, while HBV
The recommended anti-HCV testing algorithm has been nucleic acid testing (NAT) was recently developed to detect
expanded to include an option that uses the signal-to- HBsAg-negative, anti-HBc-negative blood units donated
cut–off (s/co) ratios of screening-test-positive results. This during early acute infection. Even single-sample HBV NAT
can serve as an alternative to a supplemental test in some may not substitute for anti-HBc screening, as indicated by
circumstances, minimizing the number of specimens that studies of donors with isolated anti-HBc who have extremely
require supplemental testing and providing a result that has low DNA levels undetectable by standard single-sample NAT
a high probability of reflecting the person's true antibody and who have been associated with transfusion-transmitted
status.7 HBV. Moreover, HBsAg testing may still be needed even in
Signal-to-cut–off ratios are calculated by dividing the the setting of combined anti-HBc and NAT screening. HBsAg-
optical density (OD) value of the sample being tested by the positive units from donors in the chronic stage of infection
OD value of the assay cut-off for that run. Analysis of enzyme may contain very low or intermittently detectable DNA levels
immunoassay and chemiluminescence assay data indicates that single-sample NAT would miss.8,9
that s/co ratios can be used to predict supplemental test- Assays may utilize single or multiple monoclonal antibodies
positive results. A specific s/co ratio can be identified for each and/or polyclonal antibodies to capture and detect antigen
test that would predict a true antibody-positive result (as in a variety of formats including enzyme immunoassays
defined by the results of supplemental testing) ≥95% of the (EIAs), agglutination methods, and microparticle assays with
time, regardless of the anti-HCV prevalence or characteristics fluorescent or chemiluminescent detection systems. Assay
of the population being tested. platforms range from manual to fully automated, random
access, high throughput systems.
Sensitivity of HBsAg assays is most often expressed in
Evolution of Human Immunodeficiency
nano-grams (ng) of surface antigen protein per milliliter.
Virus Assays However, because of the complex nature of HBsAg molecules
The first anti-HIV screening assays, used in the mid1980s, and specificity differences in antibodies used to construct
were based on HIV-1 lysates. The replacement of native HBsAg immunoassays and calibrate reference samples,
viral proteins by recombinant and synthetic peptide-based HBsAg standards vary in their definition of the HBsAg
antigens, and the development of class-specific antibody- ng/mL, making it difficult to compare assay sensitivity
capture and antigen-sandwich assays, resulted in reducing performance unless the same standards are used. WHO has
the infectious window period by 20–30 days. Ag/Ab assays developed an international reference standard for HBsAg
present, however, some limitations. False-negative results with concentration expressed as IU/mL, and although factors
have been observed in primary HIV infection when Ag to convert IU to ng were determined, the WHO reference
declines and IgG Ab are not yet detectable. While HCV standard committee did not formally adopt a conversion
Ag/Ab assays, based on an indirect IA format for Ab detection, factor. The most sensitive assays detect HBsAg levels
are subjected to this second ‘window phase’, this failure is no ≤0.1 ng/mL, but significantly less sensitive methods with
longer observed with the most recent HIV Ag/Ab assays due detection limits >1 ng/mL continue to be used in some
to their format based on sandwich detection of antibodies countries.8
including IgM. HIV Ag/Ab assays exhibit an overall high
sensitivity in the detection of Ab irrespective of the genotype, Syphilis Serology
including HIV-2.4
HIV assays have evolved over the last decade to produce There are two different types of serologic assays for syphilis:
tests which possess novel characteristics and which can 1. Nontreponemal assays, such as the rapid plasma reagin
address those diagnostic issues which remain, e.g. detection (RPR) test, the venereal disease research laboratory
of early infection, indeterminate results, infection in the (VDRL) test, and the automated reagin test (ART), are
266 Section 3 Blood Safety
nonspecific tests that detect “reagin” antibodies directed protein 2 (HRP-2), plasmodial lactate dehydrogenase (pLDH)
against an antigen called cardiolipin. Persons with active or or aldolase—using various rapid immunochromatographic
recently treated syphilis infections generally have reactive formats, and using whole blood as the sample of choice.11
results in nontreponemal tests, while uninfected persons Malaria RDTs are currently used in some clinical settings
or persons successfully treated years earlier usually have and programs. However, before malaria RDTs can be widely
nonreactive nontreponemal test results, but retain specific adopted, several issues remain to be addressed, including
antibody reactivity. improving their accuracy; lowering their cost; and ensuring
2. Treponemal assays include chemiluminescence immuno their adequate performance under adverse field conditions.
assays, enzyme immunoassays (EIA), fluorescent trepo Serology detects antibodies against malaria parasites, using
nemal antibody “absorbed” assays (FTA-ABS), Treponema either indirect immunofluorescence (IFA) or enzyme-
pallidum microhemagglutination assays (MHA-TPA) linked immunosorbent assay (ELISA). Serology does not
and Treponema pallidum particle agglutination assays detect current infection but rather measures past exposure.
(TP-PA). Treponemal assays test for antibodies to However, antibody detection is of limited role in endemic
antigens that are specific to treponemes. Although areas and may lead to high donor deferral.
treponemal assays are useful in identifying recent and
historically remote syphilis infections, they are not useful References
in monitoring the progression of syphilis or response to
1. AABB Technical Manual 17th edition.
antibiotic therapy. Establishments that screen donors 2. Bihl F, Castelli D, Marincola F, et al. Transfusion transmitted
using a nontreponemal assay as the test of record may infections. Journal of translational medicine. 2007.
also use a treponemal diagnostic test for the purposes 3. Bianco C. Current concepts in transfusion transmitted diseases.
of donor counseling or consideration of requalification ISBT Science Series. 2011;6:56-60.
after deferral, and also for demonstrating that the reactive 4. Bianco C. Testing strategies looking forward. ISBT Science
screening test is a biological false positive.10 Series. 2010;5:230-3.
5. Marwaha N, Sachdev S. Current testing strategies for hepatitis
C virus infection in blood donors and the way forward. World J
Malaria Gastroenterol. 2014;20:2948-54.
6. CDC. Guidelines for laboratory testing and result reporting of
Various test kits are available to detect antigens derived
antibody to hepatitis C virus. MMWR. 2003;52(No. RR–3).
from malaria parasites. Such immunologic (“immuno 7. Gupta E, Bajpai M, Choudhary A. Hepatitis C virus: Screening,
chromatographic”) tests most often use a dipstick or diagnosis, and interpretation of laboratory assays. Asian J
cassette format, and provide results in 2–15 minutes. These Transfus Sci. 2014;8:19-25.
“Rapid Diagnostic Tests” (RDTs) offer a useful alternative 8. Kuhns MC, Busch Mp. New strategies for blood donor screening
to microscopy in situations where reliable microscopic for hepatitis B virus. Nucleic acid testing versus immunoassay
diagnosis is not available. The detection of malarial antigen methods. Mol Diag Ther. 2006;10:77-91.
was originally intended as a more rapid and objective 9. Schmidt M, Seifried E. Current concepts in serological testing
alternative to direct microscopy. However, although rapid TTID. ISBT Science Series. 2011;6:61-6.
10. Sena AC, White BL, Sparling PF. Novel Treponema Pallidum
and reasonably objective, their sensitivity is not sufficient for
serologic tests: A paradigm shift in syphilis screening for the
use in a screening context, as their overall sensitivity is still 21st century. Clinical infectious diseases. 2010;51(6):700-8.
less than that of blood films. The assays are generally based 11. Kitchen AD, Chiodini PL. Malaria and blood transfusion. Vox
on the detection of major specific proteins—histidine rich Sanguinis. 2006;90:77-84.
Section
4
Miscellaneous
The Evidence-based
Transfusion Medicine
Kulbir Kaur
71
Introduction medicine and in the recent past efforts have been made to
evaluate transfusion therapy based on:2
Evidence-based medicine (EBM) is the process of systemati- • Randomized controlled trials and their critical appraisal to
cally reviewing, appraising and using clinical research find- optimize transfusion practice
ings to aid the delivery of optimum clinical care to patients.1 • Systematic reviews of assessment of the impact of blood
Evidence-based medicine forms part of the multifaceted transfusion on patient outcomes
process of assuring clinical effectiveness, the main elements • Development of clinical transfusion practice guidelines
of which are (Fig. 1):1 • Implementation of clinical practice guidelines.
• Production of evidence through research and scientific Presently most of the available data shows that current
review transfusion practices in clinical medicine are either sub-
• Production and dissemination of evidence-based clinical optimal, variable and without clear cut guidelines especially
guidelines in developing countries. These variations may be due to lack
• Implementation of evidence-based, cost-effective practice of availability of well-defined guidelines for blood transfusion
through education and management of change in clinical practice, lack of awareness of available guidelines
• Evaluation of compliance with agreed practice guidance or reluctance to follow the available guidelines. Reluctance
through clinical audit and outcomes-focused incentives. to implement them may be at least partly due to perceived
Evidence based transfusion medicine (EBTM) is defined as weaknesses in the evidence base for their recommendations.
judicious and explicit use of presently best available research It has been proved beyond doubt that blood transfusion
evidence in making decisions about the transfusion of blood is a treatment with substantial risks, associated costs and
and blood components to individual patients after estimating limited supplies.3 Despite careful donor selection and the
the risk of benefit and harm on the basis of research on development of highly specific and sensitive techniques
population samples, to inform clinical decision-making of testing, the achievement of zero risk blood still remains
for prescribing blood or blood components to individual an elusive goal. It is important to follow evidence based
patients and to formulate guidelines for their application transfusion practices because transfusion of blood/blood
from individual patients to health care services in general and
to extend to allied health professions.
Considered as a gift of life, blood transfusion is a vital
component of health care delivery system world over, but
the decision to transfuse blood or blood components is
taken under different circumstances. It involves many issues
related to blood donors as well as recipients of blood based
on clinical diagnosis, laboratory investigations, local and
national policies in various health care settings. Sometimes
the decision for blood transfusion has to be taken in
emergency situations while in routine cases, time permits to
make appropriate decisions and follow systematic approach,
but in any given situation the decision-making process should
be guided by the principles of evidence-based transfusion Figure 1 Elements of the evidence-based transfusion medicine
270 Section 4 Miscellaneous
components are sometimes associated with adverse effects the hemoglobin level will decrease, and then perform
like hemolytic transfusion reaction (HTR), transfusion expectant transfusion.7
related acute lung injury (TRALI), anaphylactic reaction, • In the case of neonates and critically ill children, recent
sepsis due to bacterial contamination, febrile nonhemolytic randomized studies, e.g. premature infants in need of
transfusion reaction (FNHTR), transfusion associated transfusion (PINT) study support the use of a restrictive
circulatory overload (TACO), transmission of infections like strategy compared to more liberal criteria in case of
HIV, HBV, HCV, syphilis, malaria, etc. Some studies have also neonates and critically ill children. Table 1 shows the
concluded that transfusion may be an independent risk factor general guidelines for transfusion to infants.8
for increased morbidity, mortality and length of stay in blood/
blood component recipients.4 Evidence-Based Platelet Utilization
As such, most of the research in transfusion medicine has
been carried out to collect evidence and evaluate whether it is
Guidelines
beneficial to follow restrictive or liberal transfusion practices, Platelet transfusions are used to treat bleeding due to
and the analysis of these results has aided in framing certain critically decreased circulating platelet counts or functionally
guidelines with respect to utilization of blood and blood abnormal platelets. Platelets may be used prophylactically
components in order to optimize transfusion outcomes and to prevent bleeding at prespecified low platelet counts.10
resource utilization. Research has also helped in establishing However, platelet transfusions are commonly implicated in
the mechanisms related to many of the adverse transfusion adverse effects like allergic/anaphylactic reactions, FNHTRs,
events and this in turn has facilitated in devising newer transmission of bacterial infection and development of
methodologies to make blood/blood component transfusions platelet refractoriness. Guidelines recommend that:10
safer. Some of the evidence based guidelines with respect to • In general, maintain platelet count above 10,000/mm3 in
transfusion practices have been discussed below: stable, nonbleeding patients.
• Platelet count to be maintained above 20,000/ mm3 in
Evidence-based Red Blood Cells unstable nonbleeding patients.
• Maintain platelet count above 50,000/mm3 in patients
Utilization Guidelines undergoing invasive procedures or actively bleeding.
Red cell transfusion is an extremely common treatment • In the case of critically ill or those undergoing
throughout the world. Blood is administered to improve massive transfusion, a transfusion target of more than
oxygen delivery. An American Society of Anesthesiologists 50,000/mm3 is recommended for acutely bleeding patients
Task Force makes the following recommendations:5 and above 100,000/mm3 for those with multiple trauma or
• Transfusion is rarely indicated when the hemoglobin level CNS injury. The platelet count may fall below 50,000/mm3
is above 10 g/dL and is almost always indicated in patients when more than 1.5–2 blood volumes have been replaced
when the hemoglobin level is below 6 g/dL. with red cells. In the presence of micro vascular bleeding,
• The determination of transfusion in patients whose transfusion may be appropriate when counts are known or
hemoglobin level is 6–10 g/dL should be based on any suspected to be below 100,000/mm3.
ongoing indication of organ ischemia, the rate and • In case of neonates undergoing invasive procedures/minor
magnitude of any potential or actual bleeding, the patient’s surgery or experiencing clinically significant bleeding may
intravascular volume status and risk of complications due be transfused at less than 50,000/mm3. For major surgery
to inadequate oxygenation. or bleeding in the face of additional hemostatic stressors
• The use of alternative measures to reduce allogeneic (e.g. disseminated intravascular coagulation, necrotizing
red cell use should be considered, including pre
operative autologous donation, intraoperative and post-
operative autologous blood recovery, acute normovo Table 1: General guidelines transfusion to infants9
lemic hemodilution, and operative and pharmacologic Maintain HCT between Clinical status
strategies.
Severe cardiopulmonary disease
(e.g.
• The same considerations regarding individualization of 40–45%
mechanical ventilation >0.35 FiO2)
red cell transfusions apply to critical care as perioperative
Moderate cardiopulmonary disease (e.g.
patients. Up to 40%
of the blood volume in a bleeding,
30–35% less intensive assisted ventilation such as
otherwise healthy young adult can be replaced with nasal CPAP or supplemental oxygen)
crystalloid without the need for red cell transfusion.
Transfusion triggers for red cells in critical care must 30–35% Major surgery
be customized on the basis
of individual patient Stable anemia, especially if unexplained
characteristics.6 Clinical judgment is needed to estimate 20–30% breathing disorder or unexplained poor
how much more bleeding may occur and how much lower growth
Chapter 71 The Evidence-based Transfusion Medicine 271
enterocolitis) transfusion is appropriate at counts below appropriate Factor VIII concentrates or Factor VIII
100,000/mm3. concentrates containing FVIII: vWF are not available.
• Platelets should not be used in patients with autoimmune • Do not transfuse cryoprecipitate unless laboratory studies
thrombocytopenia or thrombotic thrombocytopenic confirm deficiency of a specific clotting protein for which
purpura (TTP) except for life-threatening hemorrhage. this component is indicated (e.g. fibrinogen).23
• In case of critically ill patients, with Hypofibrinogenemia/
Evidence-Based Fresh Frozen Plasma dysfibrinogenemia, transfuse for bleeding associated with
fibrinogen levels less than 100 to 120 mg/dL or reduced
Utilization Guidelines functional levels of fibrinogen.24
Fresh frozen plasma (FFP) may be among the most “high risk” • Cryoprecipitate is especially useful when it is not possible
of all blood components11,12 and FFP transfusion may lead to to give enough FFP to provide adequate levels of fibrinogen
adverse events like TRALI, transfusion-transmitted infection, without volume overloading the patient.
fluid overload and allergic complications.13,14 Various studies • Cryoprecipitate has been used for uremic bleeding
but
have shown an association between cases of TRALI and efficacy has not been clearly demonstrated, and
plasma from female donors and therefore, male donors are 1-deamino-8-D-arginine vasopressin (DDAVP) and other
now preferred for production of FFP.15,16 Considering the modalities are preferred.25
risks associated with FFP transfusion, a prophylactic policy
is justified only if the risk of bleeding is greater than the risk Limitations of Evidence-Based
of associated adverse effects. At present, FFP transfusion is
indicated for use in patients with the following conditions:17-22
Transfusion Medicine
• Active bleeding, or before surgery or an invasive Despite the emphasis on following evidence-based practices,
procedure in patients (adults and neonates) with acquired evidence based medicine too has certain limitations.
deficiencies of one or more coagulation factors as • EBM alone cannot provide a clinical decision. Each deci-
demonstrated by an increased international normalized sion will also be based on the individual patient factors,
ratio (INR), prothrombin time (PT), or activated partial available resources, clinical expertise and the costs of the
thromboplastin time (aPTT), when no alternative intervention.
therapies are available or appropriate. • Patients enrolled in clinical trials are not always
• Immediate correction of vitamin K deficiency or reversal of representative of individual patients requiring treatment
warfarin effect in a patient with active bleeding, or before and generalizing to different clinical settings may not be
surgery or an invasive procedure (in conjunction with use appropriate.
of prothrombin complex concentrates). • In EBM there is over emphasis on methodology at the
• DIC or consumptive coagulopathy with active bleeding. expense of clinical relevance.
• Massive transfusion with coagulopathic bleeding. • Evidence base for much of the practice has not developed
• Thrombotic thrombocytopenic purpura (TTP). to the point at which it can be universally applied with
• Active bleeding, or before surgery or an invasive procedure confidence.
in patients with a congenital factor deficiency when no • Even when clear-cut evidence based guidelines are avail-
alternative therapies are available or appropriate. able, there may be a lack of compliance and dissemina-
• Previous common uses of FFP that are now considered tion.26
inappropriate include: volume replacement, correction Some of these limitations can be overcome by increasing
of hypoalbuminemia, nutritional support, and immuno sharing of expertise and resources between local, national
globulin replacement. and international scientific communities and by avoiding
the duplication of research, increasing opportunities to dis-
Evidence-Based Cryoprecipitate seminate the work that
has already been done and planning
Utilization Guidelines future research.2
BK Rana
72
Keywords: Accreditation; Blood bank; Blood centre; expectation of good services from the viewpoint of the service
Improvement; QCI: Quality Council of India; National provider, the patient, and the referrer, as well as the regulatory
Accreditation Board for Hospital and Healthcare Providers; bodies.
Transfusion service.
Types of Blood Banks/Centres
Introduction National Accreditation Board for Hospitals and Healthcare
Blood Safety is now talking of the town. We all are aware that Providers (NABH) has classified blood banks/centres on
blood banks/centres are regulated by Central Drugs Licensing the basis of its management (Government, Indian Red
Authority under Drugs and Cosmetics Acts and Rules. In a Cross, Voluntary/Charitable or any other) and functionality
manner, blood banks/centres are fairly regulated compared (Hospital based or standalone). It means that there are
to medical laboratories; however, it is observed that it’s no restrictions on any licensed blood bank for making
not enough to ensure quality of blood and its products. To application for accreditation on the basis of its management
support the licensing mechanism and ensure that blood and and function.
its products meet the specified requirement and provide
an opportunity for BB to improve on a continuous basis,
the Quality Council of India (QCI) through one of its Board,
Scope of Accreditation
National Accreditation Board for Hospitals and Healthcare This standard covers whole blood and all its products
Providers (NABH) has therefore introduced standards for including molecular typing, HLA typing and therapeutic
Blood Banks/Centres and Transfusion Services in 2007. This procedures. In a way, all those activities for which a license
chapter deals briefly with the standards structured by the can be granted by the licensing authority are covered.
NABH for accreditation of blood banks/centres.
Speeding changing market forces, increasing health
awareness, and the entry of the corporate sector into health
Requirements of Accreditation Standards
delivery have raised the demand for quality in healthcare NABH Accreditation Standards for Blood Banks/Centres and
services. Particularly, when a patient comes to know that Transfusion Services are divided into eleven clauses and each
transfusion can be fatal instead of lifesaving. It became all clause has been further divided into several sub-clauses.
the more critical that a blood or its product must confirm This chapter describe brief outline of these clauses and sub-
to highest quality norms set by regulator who grants license clauses.
to run a blood bank. The phenomenal growth in blood
banking and transfusion services has further enhanced the
Organization and Management
importance of this field. Development of newer technologies
to detect and identify disease markers has made tremendous The blood bank/blood centre should be legal identity and
contribution in the quality of services. NABH accreditation must have a valid license from Central Drugs Standard Control
for hospitals takes into account major aspects of blood Organization (CDSCO) and approved by Drug Controller
banking and transfusion services, although its role and scope General (India), central license approving authority under
are limited. Hence, to assess the quality and safety of blood Drug and Cosmetic Rules-1945 with further amendments. The
and its products and to have a method for the monitoring blood bank is required to define its organization structure in
of quality standards, a basic accreditation program has terms of various positions and their functions (responsibilities
been introduced in the country. These standards reflect the and authorities). Blood bank must at all times comply with all
274 Section 4 Miscellaneous
applicable legal requirements of the country. Staff should be Personnel
bound by the ethical code of their respective profession. The
Management (a person or group of persons responsible for The blood bank/blood centre is required to employ an adequate
managing the functions of the blood bank at which decisions number of individuals qualified by education, training
are made) must define and implement a quality system which and experience. The operation of blood bank/blood centre
at a minimum meets the requirements of NABH accreditation should be conducted under the active direction and personal
standards including its quality policy documented in a quality supervision of competent technical staff. Qualification and
manual. Quality policy contains scope of services, objective of experience for Director/In-charge/ medical officer/in-charge,
quality management system with management commitment supervisors, technicians and nurses must be at the minimum
to comply with the standards and local regulations. Quality as required by licensing authority. Each staff must be given
manual is considered as an apex document which guides a documented job description to ensure that staff Director/
the blood bank for its functions through policies, processes In-charge understands their responsibilities and authority to
and procedures. Staff must understand and follow the discharge their duties appropriately. All personnel employed
documented system as described in the quality manual. should also have training specific to quality assurance and
Management needs to identify a Quality Manager, who will quality management. It is the responsibility of the management
be responsible for the implementation and maintenance of to ensure that all employees are trained to prevent adverse
quality management system, and a Technical Manager, who incidents and/or contain the effects of, and report adverse
will be responsible for technical operation. incidents. Blood Centre should assess the competency of
each staff to perform assigned tasks following training and
periodically thereafter. Retraining and reassessment must
Accommodation and Environment be provided based on competency analysis, when necessary.
The premises, used for operation of a blood bank/blood A pre-employment medical examination and regular
centre and/or preparation of blood components shall be health checkup must be conducted on all the employees.
constructed in such a manner so as to permit the operation It is required not only to protect the employees from deadly
of the blood bank/blood centre and preparation of blood infection but also safeguard the blood centre from certain
components under hygienic conditions and shall avoid entry legal implications. Blood centre must maintain records of the
of insects, rodents and flies. It should be well-lighted and personal information, relevant educational and professional
ventilated. The blood bank/blood centre shall be designed for qualification, training and experience, and competence of
the efficiency of its operation, to optimize the comfort of its all personnel. Staffs has to strictly maintain confidentiality of
occupants and to minimize the risk of injury and occupational information regarding donor/patient/recipient.
illness. Patient/recipients, employees and visitors should
be protected from recognized hazards. A blood bank/blood
Equipment
centre shall have a minimum area as defined by licensing
authority for its operations. It should provide necessary space The blood centre must be in the possession of all the equip
for registration and medical examination, blood collection, ment those are required for blood collection, component
refreshment-cum-rest-room, laboratory for testing of blood preparation, processing, examination and storage, as
transmissible disease markers like hepatitis, syphilis, malaria, appropriate as per the scope of license. It is must that equipment
HIV-antibodies, blood component preparation (if applicable), are properly calibrated, maintained, and monitored for their
sterilization-cum-washing, and store-cum-record room. All functioning and kept in a clean and proper manner and so
these facilities must have effective separation between in placed as to facilitate regular cleaning and maintenance.
order to avoid incompatible activities. Access to areas affecting To protect the equipment from damage and improper use,
the quality of the examinations should be controlled. A good only authorized personnel should operate them. Up-to-date
housekeeping practice must be followed. It should have instructions on the use and maintenance of the equipment
adequate back up facility for maintaining electrical supply (including relevant manuals and direction for use provided by
round the clock for effective operation. The blood bank/ the manufacturer of the equipment) shall be readily available
blood centre should have process to minimize and respond to personnel. It is essential that certain records related to
to environmentally related risks to the health and safety of equipment are maintained. These may be but not limited to
employees (including immunization), donors, volunteers, manufacturer’s name, type, identification and serial number
patient/recipients and visitors. Suitable measures should be or other unique identification, manufacturer’s contact person
taken for monitoring of adherence to biological, chemical and and telephone number, date of receiving and date of putting
radiation safety standards and regulation, where applicable. into a service, current location, where appropriate, condition
Proper communication facilities for communication within when received (new, used or reconditioned), manufacturer’s
and outside the center must be available. instructions, if available, or reference of their retention,
Chapter 72 Blood Bank Accreditation 275
equipment performance records that confirm the equipment used in their processing activities, as well as laboratory sample
suitability for use, maintenance carried out and that planned and donor and patient/recipient records, are identified and
for the future, damage to or malfunction, modification or traceable. In case, donor who is subsequently found to have
repair of the equipment. Blood centre is required to establish been infected with transfusion transmissible infection, the
a programme that regularly monitors and demonstrates blood bank/blood centre shall inform the patient/recipient’s
proper calibration and function of instruments, reagents physician. Appropriate record of such events shall be kept.
and analytical system. It shall also have a documented and The unused components from this unit shall be discarded.
recorded programme of preventive maintenance, which, at All procedures including examination procedures,
a minimum, follows the manufacturer’s recommendation. which meet the needs of the users of blood bank/blood
Calibration of equipment is essential to ensure correct results centre services, should only be used. National guidelines/
and therefore these must be calibrated regularly on scheduled manuals and other regulatory directives shall be followed
basis as described in the standard operating procedure for at all times. In case, in-house procedures are used, these
that particular equipment. Calibration can be done in-house shall be appropriately validated for their intended use and
as well as by outside agency using master calibrators. As fully documented. Record of the results obtained and the
per policy, which NABH is intend to follow soon equipment procedure used for the validation in case in-house developed
must be either calibrated in-house using proper procedures, methods are used, must be kept. All procedures shall be
conditions and master calibrators or by an NABL accredited documented and be available at the workstation for relevant
calibration laboratory in that specific field. This is to ensure staff. It is a good practice if procedures and necessary
unbroken chain of traceability of measurements to national/ instructions can be made available in a language commonly
international standards. understood by the staff.
As a policy, blood bank should try to collect blood from
External Services and Supplies voluntary, nonremunerated, low risk, safe and healthy
donors. Donors are educated prior to collection of blood
There must be documented policies and procedures for the regarding the risk factors of transfusion transmissible
selection and use of purchased external services, equipment infections and maintain a voluntary donor directory.
and consumable supplies that affect the quality of its Pre-donation counselling by the counsellor/staff with
services. Purchased items shall consistently meet the quality appropriate training shall be made available maintaining
requirements. There should be procedures and criteria privacy and confidentiality. A medical officer with minimum
for inspection and acceptance/rejection of consumable MBBS qualification should be reviewing the donor’s health
materials. All supplies and reagents used in the collection, conditions and physical examination of the donor. Prior to
processing, compatibility testing, storage and distribution blood donation, the consent of the donor shall be obtained in
of blood and blood components shall be stored at proper writing with donor’s signature or thumb impression after the
temperature in a safe and hygienic place. A proper inventory procedure is explained and the donor is informed regarding
control system should be in place. This system should include testing of blood for all mandatory tests for safety of recipients.
the recording of lot number of all relevant reagents, control The donor shall be provided an opportunity to ask questions
materials and calibrators, the date of receipt in the center and refuse consent. After donation, if the donor seeks the
and the date the material was placed in service. Evaluation status of transfusion transmitted infection (TTI), the same
of suppliers of critical reagents, supplies and services that may be provided with prior consent.
affect the quality of examinations must be done and a record Blood shall be collected only by a licensed blood bank/
is maintained of these evaluations. blood centre and shall be drawn from the donor by a
qualified physician or under his/her supervision by nurse/
technician trained in the procedure. A physician is to be
Process Control
present on the premises when the blood is being collected.
Policies and validation of processes and procedures: The blood Blood shall be collected by single venipuncture and flow
bank/blood centre is required to have policies, processes and of blood shall be continuous. The blood bags for collection
procedures to ensure the quality of the blood, component, of blood shall be sterile, pyrogen-free and disposable, with
derivatives and services, and ensure that these policies, a closed system of collection as per standards provided by
processes and procedures are carried out under controlled national authority. Necessary drugs and equipment shall
conditions. There should be a mechanism to identify who be available for treatment of donor reaction, if any. Donor
performed each critical step in collection, processing, blood collection staff shall be trained in identification and
compatibility testing and transportation of blood, component management of donor reactions like Syncope (fainting or
and derivatives issued, and when it was performed. It is the vasovagal syndrome), Tetany (twitching or muscular spasm),
responsibility of blood bank/blood centre to ensure that all nausea and vomiting, hematoma, convulsions, and cardiac
blood, components, derivatives issued and critical materials problems.
276 Section 4 Miscellaneous
Blood donation camps shall be organized only by blood analysis or function does not conform to laid down procedure.
bank/blood centres authorized by State Blood Transfusion It allows blood bank to identify deviation, if any, caused in its
Council (SBTC) to augment blood stocks. Adequate publicity operations and take necessary measures.
and information education and communication (IEC)
material shall be made available to the organisations. It is of Performance Improvement
utmost importance that the number of blood units collected
shall commensurate with the actual requirement of blood The blood bank/blood centre must put a policy and procedure
units rather than by social or emotional pressures. in place for addressing complaints, or other feed backs
To ensure blood safety, the blood bank/blood centres received from donors, clinicians, blood camp organizers
shall provide pre- and post-donation counselling services. All or other parties. These can be used as improvement tools.
blood bank/blood centres shall train their donor organisers/ Record of complaints, investigations and corrective actions
medical officers to undertake counseling in the absence of taken by the blood bank/blood centre shall be maintained.
a donor counsellor. Donors who are HIV sero-reactive shall Procedure for corrective action shall include a process of
be referred to a voluntary counseling and testing center investigation to determine root cause of the problem.
(VCTC) for post donation confirmation and counselling or Blood bank/blood centre management shall monitor
may be provided with the facilities for the confirmation and the results of any corrective action taken, in order to ensure
counseling at blood bank/blood centre. For TTI other than that they have been effective in overcoming the identified
HIV, the donor shall be referred for follow-up to concerned problems. Whenever, the investigation casts doubt on
speciality for further management. compliance with policies and procedures, blood bank/blood
In case, components (red blood cells, washed red cells, centre shall ensure that an additional audit is done for that
leucocyte depleted red blood cells, Frozen and deglycerolised area. All corrective actions are documented and recorded
red blood cell concentrate, random donor platelets, with root cause analysis and submitted to management
granulocyte concentrate, single donor plasma, fresh frozen review meeting. Preventive action is a proactive process for
plasma, cryo poor plasma or factor VIII deficient plasma, identifying opportunities for improvement, whenever they
single donor cryoprecipitate) are being prepared, the sterility are identified either technical or concerning the quality
of all components shall be maintained during processing by system. The action plan needs to be developed, implemented
the use of aseptic methods and sterile pyrogen-free disposable and monitored to reduce the likelihood of the occurrence of
bags and solutions. such nonconformities.
A designated area shall be used for storage to limit NABH has recently defined following ten quality indica
deterioration and prevent damage to materials in process and tors for blood banks to monitor. Out if these ten, first five
final products. The access to such areas shall be controlled. are mandatory to monitor and report to NABH every six
Donated blood must be tested for ABO group, unexpected months. It will help NABH and blood banks to keep an eye on
antibodies and transfusion transmitted infection (TTI) which improvement being demonstrated.
include HIV, vital hepatitis, syphilis, and malaria.
Blood can only be issued by the blood bank/blood centre Combined TTI cases
along with the blood cross matching report. In case of (HIV + HBV + HCV + syphilis + MP)
i. TTI% = × 100
emergency, where delay in providing blood may jeopardize Total no. of donors
the patient/recipient’s life, blood or blood components shall ii. Adverse transfusion reaction rate% =
be issued before completion of routine cross matching tests,
No. of adverse transfusion reactions
on receipt of a signed written request of the treating physician × 100
stating that the clinical condition of the patient/recipient is Total number of blood and
sufficiently urgent to require the issuance of blood before component issues
completing ABO and Rh(D) tests and compatibility testing. (All major and minor reactions to be classified as per
Records of such requests shall be retained for 5 years. NHvPI and reported to blood bank)
Transfusion reactions must be monitored and documented iii. Wastage rates =
and a root cause analysis must be carried out to prevent such No. of blood/blood components discarded
incidents in future. × 100
Total no. of blood/blood components issued
To ensure quality, the blood bank/blood centre must
practice internal quality control measures and also participate
iv. Turnaround time (TAT) of blood issues =
in external quality assurance scheme (EQAS)/proficiency
testing programme (PT). Sum of the time taken
× 100
Total number of blood and blood
Identification of Deviations and Adverse Events components cross matched/reserved
The blood bank/blood centre is required to implement a (Time taken to be calculated from the time the request/
defined policy and procedure when any aspect of its test sample is received in the blood bank till the blood is
Chapter 72 Blood Bank Accreditation 277
cross matched/reserved and available for transfusion. Internal Audit and Management Review
Blood Bank shall set upper limits for routine and
emergency issues separately). Management review and internal audits of all elements of the
v. Component QC failures (for each component) = system, both managerial and technical, is to be conducted at
regular intervals but not less than once in twelve months in
No. of component QC failures order to verify that operations continue to comply with the
× 100
Total no. of component tested requirements of the quality management system. Audits are
vi. Adverse donor reaction rate% = planned, organized, and carried out by the quality manager
or designated qualified personnel. Personnel should not
No. of donors experiencing adverse reaction audit their own activities to prevent conflict of interest.
× 100
Total no. of donors The procedures for internal audits need to be defined and
vii. Donor deferral rate% = documented and include the types of audit, frequencies,
methodologies and required documentation. If deficiencies
No. of donor deferrals
× 100 or opportunities for improvement are noted, the blood bank/
Total no. of donation + total no. of deferrals blood centre must undertake appropriate corrective and/or
viii. %of components = preventive actions. All elements of the quality system should
be covered in internal audit at least once every twelve months.
Total component issues
× 100 Blood bank/blood centre management is required to
Total whole blood + component issues review its quality management system and all of its medical
ix. TTI outliers %age = services, including examination and advisory activities, to
ensure their continuing suitability and effectiveness. The
No. of deviations beyond +/– 2SD
× 100 results of such reviews shall be incorporated into a plan that
Total no. of batch assays includes goals, objectives and action plans. A typical period
x. Delays in transfusion beyond 30 minutes after for conducting a management review is once every twelve
issue—sample audit by BB every month. months. The results of internal audits must be a part of the
agenda. Findings and the actions that arise from management
reviews are recorded, and blood bank/blood centre is
Document Control informed of these findings and the decisions made as a result
of the review. Blood bank/blood centre management will
Blood bank/blood centre define document and maintain take necessary actions to address the concerns raised in the
procedures to control all documents and information meeting within an appropriate and agreed-upon-time.
(from internal and external sources) that form its quality
documentation. In this context, ‘document’ is any information
or instructions, including policy statements, text books, Conclusion
procedures, specifications, calibration tables, biological Therefore, every blood bank must aim for ‘Zero Tolerance’
reference intervals and their origins, charts, posters, notices, or ‘Error Free’ operations in its activities to offer better
memoranda, software, drawings, plans, and documents of patient care and ensure patient safety. It is nice to learn
external origin such as regulations, standards or examination that organisations like National AIDS Control Organisation
procedures. (NACO) and State Blood Transfusion Councils are advocating
importance of accreditation. The ISBTI and ISTM are also
playing a vital role in creating awareness amongst blood
Records
bankers and stakeholders.
All records relevant to the quality management system are
uniquely identified and appropriately labeled. Policies, suggested reading
processes and procedures to ensure that records are
1. Accreditation Standards on Blood Banks/Blood Centres
identified, reviewed, retained and that records are created,
and Transfusion Services, 2nd edn, June 2013: National
stored, and archived in accordance with record retention Accreditation Board for Hospitals and Healthcare Providers
policies. A policy, that defines the length of time various (NABH).
records pertaining to quality management system or technical 2. ISO 15189: 2012- Medical Laboratories: Requirements for
records are to be retained, is to be laid down. Quality and Competence.
73
National Plasma Policy for Access
to Plasma Derived Proteins for
Clinical/Therapeutic Use
state blood transfusion services in the country in order 2.5 To ensure proper infrastructure, equipment and
to optimize recovery and utilization of plasma. transportation facilities to have high quality of plasma.
1.2 To evolve processes and methods to standardize 2.6 To direct efforts towards recruitment and retention of
logistics of plasma collection, transport and storage voluntary, non-remunerated blood donors, through
from blood banks to warehouses/Plasma Fractionation education and awareness programs also incorporating
Centres (PFC) and transport of plasma derived products incorporate IEC strategies, NGO involvement and
(PDP) to distribution outlets. special donor registries for hyperimmune products,
1.3 To put in place mechanisms to improve co-ordination etc. as an integral part of voluntary blood donation
and interaction between various BCSUs and plasma programs.
warehouses/PFCs in order to achieve desired end 2.7 To standardize pricing of PDP, with the help of existing
product quality. policies/resources, to ensure not for profit but techno-
1.4 To advocate for effective and judicious clinical use of financial viable and self-sustaining mechanisms.
human plasma and PDP to minimize unwarranted use
of whole blood/ plasma/PDP. Objective 3
1.5 To formulate national guidelines on ‘Clinical use of To take adequate regulatory and legislative steps for monitor-
plasma derived products’ and update as required from ing of activities related to plasma derived products.
time to time.
1.6 To review plasma/PDP utilization by various facilities
periodically at state and national level.
Strategy
1.7 To promote interdepartmental activities with all 3.1 To facilitate the regulatory approval of updated
concerned including other Ministries, stakeholders and methodology with the purpose of increasing plasma
health programs that would help optimize production recovery from donated blood.
and utilization of PDP. 3.2 To review the regulatory framework with respect
1.8 To facilitate access and availability of PDP to cater to to availability/manufacturing and distribution of
special requirement including remote locations will be acceptable quality of PDP for clinical use.
done with closed coordination with DGAFMS. 3.3 To review and update Standards, Drugs and Cosmetics
1.9 To establish latest technology and time to time up Act/Rules and Indian Pharmacopoeia from time to
gradation to bring about self-sufficiency for PDP. time.
1.10 To participate in public private partnership/ 3.4 To periodically review the existing provisions of
collaborations to improve production and improve prevailing regulatory frameworks well as introduce
availability of PDP. stringent penalties for unauthorized/irregular practices
1.11 To evolve mechanisms for periodical review and in plasma processing and delivery of PDP.
evaluate the implementation of the policy across the
country. Objective 4
To encourage Research and Development in the field of
Objective 2 blood components, plasma fractionation and plasma derived
products.
To make available adequate resources to develop and
organize the plasma mobilization throughout the country.
Strategy
Strategy 4.1 To organize capacity building/exposure visits/
hands on training of personnel dealing with plasma
2.1 To support/strengthen the existing network of Blood fractionation, related to all process and quality aspects.
Transfusion Services (BTS) so as to consolidate 4.2 To facilitate research in blood components, plasma
and improve blood and plasma donor base, blood fractionation and PDP in association with recognized
componentization and recovery of good quality of bodies including ICMR, DST and DCGI.
plasma. 4.3 To make available financial support for the conduct
2.2 To allocate resources and funds in existing public health of R and D in processing of plasma and PDPs through
programs as well as advocate for resource allocation by various channels.
corporate sectors, bilateral/international/agencies for 4.4 To collaborate with industry and academia to launch
plasma mobilization. blood products faster and promote Inter-country and
2.3 To additionally strengthen source plasma collection intra-country exchange for training and experience of
through existing BCSU/Apheresis centers within personnel associated with plasma fractionation.
existing network of blood transfusion services. 4.5 To direct efforts towards development of indigenous
2.4 To ensure engagement of trained manpower at all kits/processes and technology, to make them cost
levels to facilitate plasma mobilization. competitive.
280 Section 4 Miscellaneous
4.6 To facilitate evidence based practices in research 5.4 To encourage training programs to ensure proficiency,
involving utilization of human plasma/blood, from accreditation and other changing quality parameters
units unused/discarded from blood banks due to any from time to time.
reason and evolve a regulatory framework thereof. 5.5 To encourage higher standards and uniformity,
External Quality Assessment Scheme (EQAS) shall be
Objective 5 introduced, through the referral laboratories approved
by the National Blood Transfusion Council.
To strengthen Quality Systems in blood transfusion services 5.6 To ensure complete process control with sound
for plasma collection, transportation, processing, production documentation system, to inculcate data sharing and
and distribution of PDP. create opportunities to promote learning and growth.
5.7 To collate and analyze the data and share with all stake-
Strategy holders, regularly as a part of the larger quality manage-
ment initiative in the area of plasma fractionation.
5.1 To set national quality standards covering all aspects
in manpower, equipment, processes, procedures,
Reference
products and quality systems.
5.2 To articulate a continuous all round improvement 1. National Policy for Access to Plasma Derived Medicinal
program in plasma fractionation as part of quality Products from human plasma for clinical therapeutic use:
systems as an endeavor to work towards gold standards. Adden dum to National Blood Policy 2003. Department of AIDS
Control, Ministry of Health and Family Welfare, Government of
5.3 To mandate that Plasma Fractionating Centres allocate
India; Published in June 2014, NACO, 2014.
resources for improving the quality of plasma as a raw
material to linked BCSU in the form of manpower,
equipment, logistics, etc.
Ethical Issues in Transfusion
Medicine
• Donor screening and testing used to be simple: Today’s Indian Scenario of Ethical Issues in
donors are asked intimate questions about their lifestyles
Transfusion Medicine
and put through a battery of laboratory tests. This has had
significant repercussions for the relationships between With the rising awareness of ethical issues in every field of
blood centers, blood donors, physicians and patients. medical care and research in India, awareness is growing in
The blood donor, an ostensibly healthy individual until the field of transfusion medicine as well. But we are nowhere
notified of an abnormal result by the blood center, may near the international code of ethics.
seek a physician’s advice and doubt the creditability of the In the 1990s, in response to public interest litigation a
testing procedure and deferral policies. A more specific Supreme Court order banned professional blood sellers and
test might turn out to be negative and the donor may be directed the government to formulate a national blood policy.
labeled as healthy. This donor might return to the blood The National Blood Transfusion Council, with the National
center asking for compensation for the unnecessary Blood Policy as a tool, and the Drugs Controller, with the help
mental anguish and the expenses incurred and might of the Drugs and Cosmetics Act, now aim to ensure blood
never donate again. safety and ethical transfusion practices in India.
• Blood safety depends partly on the information provided Currently under the Drugs and Cosmetics Act it is manda
by the donor and it is also the donor’s ethical duty to tory to test blood for anti-HIV 1 and 2, anti-HCV, HBsAg, RPR
provide truthful information. It is unethical to wilfully for syphilis and malaria.7 Consent for testing is taken and
conceal information about high-risk behavior or medical the donor is given the option of receiving the results—this is
history.2 mandatory in some countries such as the US and UK.
Until recently donors were not informed because specific
consent for testing was not taken,8 and the screening tests had
ETHICAL ISSUES RELATED TO PATIENTS relatively high false positive rates, which could cause panic.
No confirmatory tests were required. So the donation system
Ethical issues related to patients include access to risk-free was projected as anonymous and unlinked and adequate
safe blood free of charge or need of replacement, informed counseling was not available. The National Blood Policy of
consent for transfusion, the right to refuse the transfusion, 2002 has addressed this gap.9
and the right to be informed if harmed.2 The Code of Medical Ethics that is binding on doctors,
• Consent for transfusion: Consent for transfusion has to be honors confidentiality. However, in a court of law in India,
informed consent. The patient should be informed of the this privilege is not absolute but qualified. Doctors can reveal
known risks and benefits of transfusion, and alternative information in the interest of individual or general welfare of
therapies such as autologous transfusion or erythropoietin. society and when there is no mal-intention.
Only then should the consent be documented. If the Ethical issues are mostly violated in relation to the patient
patient is unable to give prior informed consent, the basis in India. Patients all over the country do not have access to
of treatment by transfusion should be in the best interests safe blood, free of charge, or the option of giving consent and
of the patient. choosing safer alternatives. With the National Blood Policy,
• Right to refusal: The patient’s right to refuse blood a decision was taken to improve transfusion services all over
transfusion should be respected. Some religious sects such the country and create greater awareness about transfusion
as Jehovah’s Witnesses do not accept blood transfusions. issues. The policy must also address all the other issues
Followers of this belief live in India as well and there have in the international code of ethics for blood donation and
been instances of blood refusal here. transfusion to make India achieve international standards.
• Right to be informed if harmed: If the patient has been National AIDS Control Organisation (NACO) is working
transfused blood and components that were not intended hard to improve blood transfusion services (BTS) in India.1
for him/her, whether harmed or not, he/she has the right Now counsellors have been provided for donor counseling
to be informed. Similarly a patient who has inadvertently and notification in almost all blood banks. But various studies
received blood positive for a transfusion transmissible in donor management have shown that there is still more to be
marker has a right to be informed and given due done to attract donors to collect their test reports and do the
compensation. follow-up as suggested by counselor.
Recently hemovigilance programme has been launched
in India by government to monitor blood safety and CMEs
ETHICAL ISSUES RELATED TO BLOOD are being conducted in various medical institutes to apprise
ESTABLISHMENTS clinicians about ethical issues related to patients.
Nidhi Bhatnagar
75
Transfusion services in the India are currently undergoing
dynamic changes in an effort to optimize patient blood
management and ensure rapid availability of the safest, most
compatible blood products possible. Providing precisely
matched blood to transfusion patients to reduce the risk of
adverse events and improve transfusion outcomes remains
the ultimate goal of immunohematology practice. This is
particularly important when managing patients who receive
chronic transfusions, including those with sickle cell disease,
thalassemia, or chronic anemia, who are more likely to
produce alloantibodies.
Historically, red blood cell agglutination testing has been
conducted using conventional tube techniques due in large
part to their simplicity. Although suitable for individual testing, Figure 1: Principle of the gel test column
the tube method presents some limitations, including low agglutination technology
reproducibility, the potential for human error, and inefficient
personnel management. For example, tube shaking methods • Large agglutinates remain on or near the top of the gel
may differ by user and affect the grading and interpretation interface. Smaller agglutinates pass partway through the
of the results. Other challenges include repeated washing gel, depending on size.
steps, resulting in loss of red blood cells (RBCs) and weakly • Unagglutinated cells pass to the base of the microtube.
bound antibody; difficulty detecting mixed-field populations; Column agglutination technique (CAT), methods have
and providing accurate RBC typing in recently transfused emerged in the last two decades, and are now replacing
individuals. Additionally, the limited capacity for automation manual tube methods due in large part to their flexibility,
inherent with tube testing results in increased frequency of efficiency, and reliability. The original technique was based
transcription errors.1 Tube testing also is considered fairly on the principle of gel filtration for separation of red blood
labor-intensive, resulting in high costs and inadequacy for cells from human blood.3 It was found that the principle of
large-scale implementation.2 gel centrifugation could be modified for use as a serological
tool using Sephadex G100 superfine or Sephadex G200
Principle of Gel Technology (Fig. 1) Superfine. Originally described by Yves Lapierre et al.4 in
1985, the aim of this technology is to standardize red blood
• The serum and cell reaction takes place in a microtube.
cell agglutination reactions, and by trapping the agglutinates,
• This microtube consists of a reaction chamber that narrows
to permit simple and reliable reading. The column consists of
to become a column.
special microtubes containing a dextran gel matrix. Red blood
• Each column contains sephadex gel suspended in a buffer
cells and serum or red blood cells alone are dispensed into
solution.
the microtubes, incubated if necessary, and then centrifuged
• Depending on the configuration of the card, the gel is
under strictly controlled parameters. The gel within the
premixed with antisera/AHG/other reagents.
microtubes acts as a sieve, unagglutinated red blood cells
• The sephadex gel matrix acts as a sieve.
form a pellet at the bottom of the microtube, and agglutinated
286 Section 4 Miscellaneous
red blood cells are trapped in the gel. The gel may be neutral or of nonspecific antibodies and false positive reactions. These
contain specific reagents such as AHG or specific antibodies techniques included an automated polybrene technique,
(anti-A, -B, anti-D, anti-Kell, etc.). Reactions are easily visible two-stage papain technique, microtiter plate IAT, and a spin
and may be graded (Fig. 2). tube low ionic strength IAT. Antibody titers showed increased
reaction strength and so titer scores were higher than tube IAT
Practical applications of titers, suggesting an increased sensitivity for the gel system.
Use in a routine hospital blood bank laboratory for red cell
gel technology phenotyping (ABO, Rh, Kell, M and N), direct antiglobulin
When performing the antiglobulin tests, no washing of the testing (DAT), antibody screening and indirect antiglobulin
red blood cells is required because during centrifugation, the test compatibility testing showed that care must be taken to
cells are separated from their suspension medium and serum ensure that no greater than a 1% suspension of red blood cells
as they pass into the microtube. Red cells sensitized by IgG is used and that problems may be encountered in patients
or complement components react with the AHG contained in who are DAT positive.
the gel and the resulting agglutinates are retained within the It has also been shown, however, that serological
matrix. The serum fraction does not, therefore, make contact assessment of drug induced immune hemolytic anemia is
with the AHG impregnated gel, so that neutralization of the considerably improved by using the gel system.7
AHG reagent is avoided. Gel column agglutination, which has gained popularity
This technique also obviates the need to use pre-sensitized in recent years, also has the unique advantage of detecting
control cells to check negative reactions. Neutral cards can mixed-field agglutination patterns, because the high-density
be used as part of antibody screening for a two stage enzyme media allows a clear separation between agglutinated and
treated cell technique. nonagglutinated cells, thus indicating the presence of a dual
Reactions using gel techniques are stable for at least population of red blood cells.8 It was shown that gel column
48 hours and have the added facility of being able to be agglutination is superior to other commonly used serology
photocopied, thereby providing a permanent record for methods, including solid phase red cell adherence assay
future reference. This system is currently marketed as the ID- (SPRCA) in the detection and monitoring of mixed-field
Microtyping System (DiaMed-GB) in 1988. reaction patterns.
Another system is also available based on column Also, in certain situations with antibody and/or
technology-the Ortho Biovue System (Ortho Diagnostic). This complement attached to RBCs, the gel column method
column contains a density gradient comprising a combination appears to be more accurate in estimating the degree of RBC
of a macromolecular density barrier and glass microspheres. sensitization in direct antiglobulin testing.9 On a practical
The system is currently available as microtubes with columns level, the stability of the gel-based agglutination method’s
containing AHG or neutral density gradients. Again, this endpoint is another important advantage of the test. The
system offers the ability to perform a “no wash” antiglobulin results can be read and reviewed at a later time, whereas the
test. endpoint of the manual tube method is stable for less than
It has been shown that the sensitivity of Dia Med ID gel an hour. This stability allows the less-experienced technician
system is superior to that of conventional tube tests without to review ambiguous results with a supervisor and enables
any loss of specificity.4-6 A comparison of different techniques documentation in the patient’s chart with photographs of the
for routine antibody screening and identification showed gel card.
increased antibody detection rate and decrease in the number
Advantages of Gel Technology
• No cell washing is involved
• Each Gel microtube is already being filled up with AHG
reagent with air-tight seal→ so no chance of contamination
of AHG reagent →No erroneous result.
• 25 minute Test, without Hazardous cell washing step-
Labor and Time saving.
• No requirement of sensitized check cells—Time and cost
saving.
• Result remains as it is for at least 48 hours in RT.
• No degradation, easy to supervise. Gel cards can be
scanned or photocopied for permanent documentation.
• Helps in accreditation process.
• Further option for automation is available.
Figure 2: Grading of agglutination • It can help in medicolegal cases.
Chapter 75 Gel Based Technology: Past, Present and Future 287
Past, Present and Future agglutination systems have proven to be highly accurate with
a sensitivity >97.58% and a specificity >99.93%.10,11
In the past when the technology was newly launched in It provides a new approach which may, by its sheer simplicity
market, it was used for performing basically Indirect Coombs of use and standardization of technique, facilitate the working
test. The blood bank technicians found this technique very practices of transfusion laboratories and thus enhance the
convenient, easy to perform, standard, reproducible, and quality of the service provided to both patients and clinicians.
useful from documentation point of view.
At present, this technology is being used globally and well
accepted among all the transfusion medicine fraternity. The References
technology is being currently used in almost 30% of the blood 1. Mistry H. Automation in UK blood transfusion laboratories:
banks across India. Majority of the blood banks are performing Strategies for error reduction. Perspectives in Transfus Med.
Coombs cross match, DCT, ICT, etc. Some of the Blood 2013;3:7-10.
banks are using this technology not only for crossmatch but 2. Rumsey DH, Ciesielski DJ. New protocols in serologic testing:
also for extensive tests like—Antibody screening, Antibody a review of techniques to meet today’s challenges. Immuno
identification, Weak D testing, etc. The technology is being hematology. 2000;16(4):131-7.
3. Kanura T, Kurashina S, Nakao M. A gel filtration technique for
used for blood grouping also as there are different formats of
separation of erythrocytes from Lab Clin Med. 1974;83:840-4.
cards available now like forward grouping only, forward and 4. Lapierre Y, Rigal D, Adam J, et al. The gel test: A new way to detect
reverse grouping, reverse grouping only, newborn grouping, antigen-antibody reactions. Transfusion. 1990;30:109-13.
cards with two clones of Anti-D, A1 and H Lectin cards. 5. Hitzler W, Shomig-Breckner H, Mathias D. Gel centrifugation
At present, the antibody screening test is not a routinely test-a new micro method for blood group typing and antibody
performed in all the blood banks, but, with the help of Gel screening. Arzthide Laboratorium. 1989;35:89-92.
technology this can be performed very easily and blood banks 6. Bromilow IM, Adams KE, Hope J, Eggington JA, Duguid JKlM.
arrange the requisite type of blood to patient easily in case of Evaluation of the ID-gel gest for antibody screening and
identification. Transfusion Med. 1991;1:159-61.
positive antibody screening with the help of own inventory or
7. Salama A, Berghofer H, Mueller-Eckhardt C. Detection of cell-
referring to the other blood banks in near vicinity which are drug (hapten)-antibody complexes by the gel test. Transfusion.
performing the antibody identification and antigen typing of 1992;32:554-6.
the donors. 8. Summers T Jr, Johnson VV, Stephan JP, Johnson GJ, Leonard
Presently, automation is also available for this technology, G.The value of automated gel column agglutination technology
but it is being used for very few parameters. The future of Gel in the identification of true inherited D blood types in massively
technology is the automation of all the immunohematology transfused patients. Transfusion. 2009;49(8):1672-77.
tests. With automation, we can perform the antigen typing 9. Dittmar K, Procter JL, Cipolone K, Njoroge JM, Miller J, Stroncek
of donors and can have a central donor data base, so that DF. Comparison of DATs using traditional tube agglutination
to gel column and affinity column procedures. Transfusion.
in case of emergency, desired donors can be called and safe
2001;41(10):1258-62.
transfusion can save the life of patient. 10. Cid J, Nogués N, Montero R, Hurtado M, Briega A, Parra R.
Thus, column agglutination technology offers several Comparison of three microtube column agglutination systems
technical benefits including pre-dispensed reagents, for antibody screening: DG Gel, DiaMed-ID and Ortho
omission of the washing step in the antiglobulin phase, BioVue. Transfusion Med. 2006;16(2):131-6.
and stability of the reaction in the column. Although there 11. Chang C, Brown M, Davies L, Pointon L, Brown R, Barke D.
are differences in sensitivity and specificity between these Evaluation of Erytra fully automated analyser for routine use in
automated methods, all commercially available gel column transfusion laboratory. Transfus Med. 2014;24(1):33-8.
Automated Blood Grouping
and Antibody Screening
Ashish Jain
76
In 1901, Karl Landsteiner’s elucidation of the ABO blood • Routine turnaround time
group system established the basis for numberless discoveries • Quality control requirements
of new human blood group antigens and antibodies during • Cost for operation
the past hundred years. The discovery and identification of • Ease of operation
human blood groups represented a milestone for a successful • Laboratory information system interface capability and
administration of compatible blood transfusions and cost
therefore founded the introduction of immunohematology • Training requirements and costs
as new scientific subject.1 Immunohematology covers the • Vendor assistance in validation and training
analysis of blood group antigens and antibodies and their • Maintenance and service agreements
interactions in health and disease.2 For many years, the • Mean time between failures.
manually performed tube tests were the only standard Chizhevsky et al.4 demonstrated that using an automated
in immunohematologic laboratories for all routine and system for ABO/Rh(D) typing and unexpected antibody
scientific questions. These tube tests were later followed screening improves productivity compared to using
and supplemented by more sensitive and specific manual the manual methods by decreasing the hands-on time
methods, such as solid phase or gel centrifugation techniques. required. Sarkozi et al.5 reported that the introduction of a
Only since the past few years, partly or fully automated systems robotic system for peri-analytical automation substantially
for blood group typing and cross-matching were introduced improved productivity and decreased operational costs in
in immunohematology to a greater extent.1 Now-a-days, a clinical chemistry laboratory. This system enabled them
an increasing amount of immunohematologic laboratories to significantly increase their workload while reducing
uses automated systems for blood group typing and cross- personnel. A few of the automated platforms available today
matching in addition to the manual work strategies. are described in Table 1.3
Shin et al.6 compared automated and manual results where
Automation in serologic testing they found that the concordance rates were 100%, 99.6%, and
100% with respect to forward ABO typing, reverse typing, and
Automation of serologic testing has many advantages, Rh typing tests, respectively. Lee et al.7 reported concordance
including improved quality management. With fewer manual rates between AutoVue Innova and manual methods of 99.6%
steps, standardization of testing, and the ability to review and 100% for ABO/ Rh(D) typing and unexpected antibody
saved images of test results, errors are reduced and problems screening, respectively. Kim et al.8 analyzed 136 samples
are more easily traced. Purchasing, installing, validating, and using the unexpected antibody screening test and found
using automation present challenges to transfusion service that 2 results were positive only on the AutoVue Innova and
personnel more familiar with manual testing.3 not the LISS/Coombs card. Han et al.9 reported the use of
Factors to be considered in equipment selection should automated equipment dramatically reduced the risk priority
include: 3 number compared to manual processes.
• Platform and test methods
• Physical space and electrical requirements
Solid-phase immunoassays
• Tests to be automated
• Stat processing capability Solid-phase immunoassays are test systems in which one
• Backup testing method and costs of the reactants (either antigen or antibody) is immobilized
Chapter 76 Automated Blood Grouping and Antibody Screening 289
to a solid support before test initiation.10 During testing, the the wells as tightly agglutinated cell buttons. A positive test
immobilized component captures additional reactants in the shows adherence of indicator red cells over part, or all, of the
liquid phase and binds them to the solid phase. Solid-phase reaction surface. A negative test shows a button of indicator
red cell adherence (SPRCA) assays have evolved from several red cells at the bottom of the test well.
techniques as an alternative to conventional hemagglutination
assays. Rosenfield et al.11 were among the first investigators to Erythrocyte-magnetized technology
describe SPRCA techniques for use specifically in the blood
bank laboratory. The use of stable pre-dispensed reagents The erythrocyte-magnetized technology (EMT; Diagast, Loos,
also permits SPRCA assays to evolve into fully automated test France) is an original and innovative technology based on the
procedures.10 Reagent red cell membranes that have been magnetization of red blood cells (RBCs).12 The use of magnetic
immobilized to the surfaces of polystyrene microtitration beads to allow quick transfer of antigens or antibodies with
plates are used to capture erythrocyte-specific IgG antibodies simple magnets has been an innovation force for automated
from patient or donor sera or plasma. After a brief incubation diagnostic devices in fields other than immunohematology.
period, unbound components are washed from the wells This principle allows a highly reliable automation without the
and anti-IgG-sensitized indicator red cells are added. In a need for centrifugation.13 The heart of EMT is the adsorption
positive reaction, centrifugation causes the indicator red cells of paramagnetic (magnetism occurring only in the presence
to adhere immunologically to the captured red cell-specific of an externally applied magnetic field) compounds on the
antibodies (via antibody bridging), resulting in the total or membrane of the RBCs. Thus, when placed on a magnetic
partial adherence of the indicator red cells over the surfaces plate after contact with antibodies, reactive and nonreactive
of the wells. In the absence of detectable antigen-antibody magnetized RBCs are rapidly pulled to the bottom of the well.
interactions (negative test), the indicator red cells are not A final phase of shaking reveals positive or negative reactions
impeded during centrifugation and pellet to the bottom of (Fig. 1).
therefore the identification of antigen negative unit becomes sequence specific primers or by assessing the dosage of RHD
difficult. Molecular methods help to define these antigens. relative to a gene by real-time PCR.7, 8
HPA, HLA and HNA Typing in future. It will not replace serological methods for ABO and
RhD grouping, for antibody detection and identification but
DNA-based methods are being commonly used for typing of should be used for minor antigens typing. In serologically
human platelet antigens (HPA), human leucocyte antigens complex situations, genotyping will support decision making
(HLA) and human neutrophil antigens (HNAs). HPA typed and will contribute to increased quality of red cell reference
products are needed in cases of immunized patients and laboratory work. Genotyping will allow a wider spectrum
neonatal alloimmune-mediated thrombocytopenic purpura. of antigens accessible for routine typing. Donors typed for
Platelet donors can also be typed and called for donation in clinically important different blood group antigens will
case of any of the above conditions. The HPA typed panel of create database for antigen negative inventory which will
donors (registry) is already in place in certain countries. be useful for providing antigen matched blood. This will in
HLA matched donors are required for tissue and organ turn enable electronic selection of units antigen matched
transplants. A marrow registry of typed HLA donors is to recipients at multiple blood group loci, thus significantly
beneficial. Antisera to HLA antigens are difficult to obtain improving transfusion outcomes. Molecular genotyping
and involves tedious typing methods. High throughput assays will complement antigen typing in cases where serology
are now being developed for typing of HPA and HLA antigens. faces difficulties (e.g. positive DAT) or in complex situations
HNA antigens and antibodies are known to play important (e.g. clarification of ABO and Rh variants). Thus molecular
role in pathophysiology of various immune neutropenias genotyping, used as an adjunct to hemagglutination, will be
including transfusion related acute lung injury. Due to a powerful tool that would radically change approaches used
difficulty in handling neutrophils and rarity of the availability to support patients in their transfusion needs.
of antisera, most HNAs are typed using DNA based methods.
References
Limitations of DNA-based Methods 1. Storry JR, Castilho L, Daniels G, et al. International Society of
DNA-based methods have the fundamental weakness of not Blood Transfusion Working Party on red cell immunogenetics
detecting directly the presence of an antigen on the surface of and blood group terminology: Cancun Report (2012). Vox
Sang. 2014;107(1):90-6.
a red cell. Testing by DNA analyses has technical, medical, and
2. Olsson ML, Irshaid NM, Hosseini-Maaf B, et al. Genomic
genetic pitfalls, and is also not approved by the FDA.20,21 From analysis of clinical samples with serologic ABO blood grouping
a practical perspective, not all blood group polymorphisms discrepancies: identification of 15 novel A and B subgroup
can be analyzed; for example, if a large number of alleles alleles. Blood. 2001;98:1585-93.
encode one phenotype, or alleles with a large deletion, or a 3. Kulkarni SS, Vasantha K, Gupte SC, Mohanty D, Ghosh K.
hybrid alleles, or when the molecular basis is not yet known. Potential of commercial anti-D reagents in the identification
Additionally, there is a high probability that not all alleles in of partial D variants in Indian population. Indian J Med Res.
all ethnic populations are known. There are many genetic 2007;125(5):641-4.
4. Kulkarni S, Colah R, Gorakshakar A, et al. Frequency of partial
events that cause apparent discrepant results between
D in Western India. Trans Med. 2008;18(2):91-6.
hemagglutination and DNA test results; the genotype is not 5. Matheson KA, Denomme GA. Novel 3'Rhesus box sequences
the phenotype (e.g. null phenotypes of Rh, Kell and Kidd confound RHD zygosity assignment. Transfusion. 2002;42(5):
systems).22-25 Confirmation by hemagglutination of predicted 645-50.
antigen negativity is recommended using a reagent antibody 6. Kulkarni S, Vasantha K, Ghosh K. RHD zygosity by serological
if available, and/or by cross-matching. Detection of null and molecular methods in the donor population of Mumbai,
phenotypes is more easy and simpler by hemagglutination. India. Vox Sang. 2012;100(suppl 1):217.
The molecular bases associated with a large number of 7. Perco P, Shao CP, Mayr WR, et al. Testing for the D zygosity with
antigens have been reported. However, in many cases the three different methods revealed altered Rhesus boxes and a
new weak D type. Transfusion. 2003;43(3):335-9.
analysis has been restricted to a relatively small number of
8. Chiu WK, Murphy M, Fidler C, et al. Determination of RhD
people. This information is being applied to DNA typing Zygosity: Comparison of a double amplification refractory
with the assumption that such analysis will correlate with mutation system approach and a multiplex real-time quantitative
RBC antigen typing in all populations. Population studies PCR approach. Clinical Chemistry. 2001;47(4):667-72.
from a variety of ethnic backgrounds should be analyzed to 9. Daniels G. Variants of RhD–current testing and clinical conse-
establish the correlation between genotype and the blood quences. BJH. 2013;161:461-70.
group phenotype.26 Till then, caution should be xercised 10. Moise KJJ. Management of rhesus alloimmunization in
when recommending clinical practice based on DNA typing pregnancy. Obstet Gynecol. 2008;112:164-76.
11. Mujezinovic F, Alfirevic Z. Procedure-related complications of
for blood group antigens.
amniocentesis and chorionic villous sampling: a systematic
review. Obstet Gynecol. 2007;110:687-94.
Conclusion 12. Scheffer PG, van der Schoot CE, Page-Christiaens GC, et al.
Noninvasive fetal blood group genotyping of rhesus D, c, E and
Genotyping for RBC polymorphisms will play a more of K in alloimmunised pregnant women: evaluation of a 7-year
important role in the routine work of blood establishments clinical experience. BJOG. 2011;118(11):1340-8.
296 Section 4 Miscellaneous
13. Rozman P, Dovc T, Gassner C. Differentiation of autologous ABO, 20. Reid ME, Rios M. Applications of molecular genotyping to
RHD, RHCE, KEL, JK, and FY blood group genotypes by analysis immunohaematology. Br J Biomed Sci. 1999;56:145-52.
of peripheral blood samples of patients who have recently 21. Reid ME. Applications of DNA-based assays in blood group
received multiple transfusions. Transfusion. 2000;40(8):936-42. antigen and antibody identification. Transfusion. 2003;43:1748-
14. Castilho L, Rodrigues A, Pellegrino Jr J, et al. Blood group geno- 57.
typing for the management of patients with “warm”-induced 22. Avent ND, Reid ME. The Rh blood group system: a review.
hemolytic anemia. Transfusion Clinique and Biologique. Blood. 2000;95:375-87.
2001;8(S):S166. 23. Huang CH, Liu PZ, Cheng JG. Molecular biology and genetics
15. Storry JR, Olsson ML, Reid ME. Application of DNA analysis to of the Rh blood group system. Semin Hematol. 2000;37:150-65.
the quality assurance of reagent red blood cells. Transfusion. 24. Lee S, Russo DCW, Reiner AP, Lee JH, et al. Molecular
2007;47(suppl 1):73-8. defects underlying the Kell null phenotype. J Biol Chem.
16. Kulkarni S, Choudhary B, Gogri H, et al. Quality assurance of 2001;276:27281-9.
reagent red blood cells by DNA analysis. Asian J Transfus Sci. 25. Lucien N, Sidoux-Walter F, Olivès B, Moulds J, Le Pennec PY,
2014;8(suppl 1):6-25. Cartron JP, Bailly P. Characterization of the gene encoding the
17. Reid ME. Applications and experience with PCR bases assays human Kidd blood group/urea transporter protein: evidence
to predict blood group antigens. Transfus Med Hemother. for splice site mutations in Jknull individuals. J Biol Chem.
2009;36(3):168-78. 1998;273:12973-80.
18. Jungbauer C, Hobel CM, Schwartz DW, Mayr WR. High- 26. Reid ME. Applications and experience with PCR bases assays
throughput multiplex PCR genotyping for 35 red blood cell to predict blood group antigens. Transfus Med Hemother.
antigens in blood donors. Vox Sang. 2012;102(3):234-42. 2009;36(3):168-78.
19. Vasantha K, Kulkarni S, Ghosh K. Identification of donors
lacking a combination of clinically significant common blood
group antigens. Vox Sang. 2009;97(suppl 1):96.
Rare Blood Group Registry
Swati Kulkarni
78
Blood group antibodies play an important role in transfusion (frequency varies from 1:200 to 1:1000). Individually some of
medicine during blood transfusion and pregnancy. Though these antigens would not be rare, but combined phenotype
all antibodies are not clinically significant, many have the fits in above mentioned definition. The owner of this rare
potential to be. Importance of the system is based on whether phenotype is able to mount a clinically significant immune
the antibodies can facilitate accelerated destruction of red response against the missing antigens. Thus a rare donor
cells having the corresponding antigen. At present, 339 can be a null phenotype, homozygote for an infrequent
antigens have been discovered on human red cells of which antithetical antigen or combination of antigens. In some
297 are clustered within 33 blood groups systems and the countries all donors with phenotype with a frequency of less
remaining are accommodated in the Collections, 700 Series than 1% of population is considered to be rare. Donor is also
of Low Incidence Antigens or the 901 Series of High Incidence considered rare if his/her red cell antigen profile differs from
Antigens.1 majority of the population.
Blood samples of donors and patients are not routinely According to American Rare Donor Program, a donor
tested for antigens of other blood group system as the phenotype is considered rare if it meets one of the following
antibodies against these antigens are not of regular criteria: (1) Group O and Group A; R1R1, R2R2, R0R0, or
occurrence. As antibody screening and identification is not rr; and K:-1 and Fy(a-) or Fy(b-); and Jk(a-) or Jk(b-); and
performed in all blood banks in India, the problems come S-or s-. (2) Group O and group A; R1R1, R2R2, or rr; and
to light when it is difficult to obtain compatible donors for K:-1; and Fy(a-b-). (3) All ABO groups; negative for high
the patients. Even after the identification of the antibody/ incidence antigens (1:10,000) such as U, Jsb, Kpb, Yta or Ge2.4
antibodies, selection of blood is done by cross matching with According to French regulation, a blood donor is regarded as
large number of donors as rare donor registry is not available rare if its prevalence is equal to or less than 1:250. However,
in our country. A systematic study of screening the regular the whole phenotype of the donor is taken into account in
blood donor population for different blood group system cryopreservation program. For example, Kell homozygous
antigens, identifying rare donors negative for high frequency (KK) donor is considered rare only if he is R1R1 or R2R2 or
antigens (HFA) and those who lack common antigens of rr with homozygous expression of Fya/Fyb, Jka/Jkb and S/s
clinically significant blood group system will help in selecting antigens.5 Thus with the above variation in defining rare blood
donors for patients with multiple antibodies. This registry will groups, it is observed that when blood donor is not available
have immense applicability across the country as the number easily after cross-matching with ABO and RhD, compatible
of multi-transfused patients are rising (Thalassemia, sickle donors can be called rare (mostly negative for single HFA
cell anemia, MDS, aplastic anemia, chronic renal failure, and their combination) and when compatible blood is found
etc.). We can also contribute significantly to World Health after screening large number (more than 1000 donors) can be
Organization International rare donor panel. called very rare donors.
unit obtained from International rare donor programs needs despite its ease of performance, and sensitivity and specificity
to be washed and resuspended, and can in end leads to a blood suitable for what we now consider to be optimal patient care,
unit with less effect compared to a fresh blood unit. Seltsam hemagglutination-based determination of RBC phenotype
et al. reviewed current practice in Germany, Austria and has its limitations. As the serological antisera for identifying
Switzerland and concluded that about one third of patients common antigens of clinically important blood group system
with high frequency antibodies received unsatisfactory like Rh, Duffy, Kell, Kidd, MNS, etc. are costly, small number
transfusion therapy.18 Therefore rare donors should be of donors are typed for few antigens, limiting antigen-negative
identified at national level and only for some very rare or rare donor registries. The most important resource in
phenotypes not available or screened in our population; help screening process is availability of reliable, potent antisera on
should be taken from International rare donor programs. Also large scale and staff devoted to mass antigen screening. Some
the identification of rare donors negative for HFA and their RBC typing reagents are either not available or made from
combination is essential as the frequency of these antigens saved patient’s plasma with the desired antibody specificity.
and antibodies produced by Indian patients will be different. These typing reagents are human derived antibodies, may be
Our preliminary study of screening O group regular donors only weakly reactive, costly, difficult to obtain commercially
have for clinically important blood group antigens have and not sufficient for large scale screening.
shown that approx. 4.74% lacked combination of common Over the past 20 years, the molecular basis of almost all
antigens of Duffy and Kidd blood group systems. Few donors major blood group antigens have been determined, and it is
were very rare, i.e. they lacked Fya, Jka and s antigen with rare now possible to know the phenotypic expression of patients/
Rh phenotype. Some uncommon phenotypes were identified donors blood group by simple PCR methods. Blood group
and used in preparation of reagent panel cells.19 genotyping is already a common practice in many blood
centers and larger Transfusion Services in western countries,
Identification of Rare Donors but only used in cases where serological typing is impossible
due to multiple transfusions or problematic serology.
Identification of rare donors is critical factor in establishing Most blood group antigens result from single-nucleotide
rare donor registry. The rare groups get identified during cross polymorphism (SNP) giving rises to single amino acid
matching, when compatible blood is not available, while substitution which either inactivates the gene product or
performing antibody screening on patient’s or donor’s serum changes its substrate specificity. These SNPs can be identified
and during deliberate screening programs for detection of at molecular level using various molecular techniques like
rare groups. When a patient blood is not compatible with large PCR-SSP, PCR-SSOP, RT-PCR, Sequencing, Pyrosequencing,
number of donors, or has multiple antibodies, the sample Microarrays, etc. Some of these high throughput molecular
should be sent to reference laboratory/regional transfusion techniques make it possible to determine multiple RBC
centers, so that further work up can be performed and rare antigens simultaneously. For example, The Beadchip
blood group identified can be included in registry. (Bioarray Solutions, Warren, NJ), Genome Lab SNP Stream
Mass screening allows many new donors with rare (Beckman Coulter, Fullerton cA) and Blood Chip platform
phenotypes to be identified. Antigen screening for absence (Progenika) have been used to genotype multiple blood
of HFA on mass scale begins with few antigens tested in group antigens.21 In near future, donors and patients will
large number of donors. The donor lacking one or more HFA be genotyped on a mass scale using these technologies.
is then tested for lack of other antigens that may constitute With regard to cost efficiency, many transfusion centers
a rare combination. As the discovery of number of blood opt for conventional multiplex PCR to screen HFA negative
group systems and their antigens is increasing, the question donors. The Vienna Blood Center found that the cost of
arises as to which antigens should the donors be typed supply of antigen-negative blood units for 2% of the blood
for? This depends on the rate of these antigens to induce recipients was approximately 20% of the overall costs of
alloimmunization, their prevalence in discrete population donor immunohematology by serological techniques. Their
and clinical significance of alloantibody. As the allele calculations also revealed that donor antigen typing by DNA
frequency can differ widely in some population, there are screening was the cheapest method to increase antigen
special needs to screen RBC antigens in different regions of negative database.22 Automated methods for mass screening
the world. The data from of rare blood supplied in France and can reduce the technologists load and also use less amount
Austria show the regional aspect in the need of rare blood and of reagent per test. The cost of these equipments, supplies,
indicate that screening for ‘most needed’ rare blood groups maintenance and validation may prohibit the facility from
differ from region to region.18, 20 using this resource, even though it is available.
Sharad Jain
80
“Right blood to the right person at right time in right quantity”. Blood component storage: Whole blood and red cells must
Today is commonly talked about in relation to blood safety. be stored at a temperature of +2°C to +6°C. Whole blood and
Transfusion services are handled by large group of technical red cells never freeze. Never use domestic refrigerator for
persons and series of instruments which plays an important blood storage. Blood storage cabinets are essential for blood
role hence system needs to be evaluated for safety purposes. storage.
Blood transfusion is definitely quite safe due to several Fresh frozen plasma (FFP): The plasma which has been
innovations, advancement in technology, knowledge and separated within 6–8 hours and has been rapidly frozen to–
skills. Blood transfusion saves life, and improves quality of 20°C or colder.
life in large number of critical conditions. It is highly risky like Cryoprecipitate is the cold-insoluble jelly like portion of
other intravenous drugs. plasma remaining after FFP has been thawed. The optimal
Serious hazards of transfusion (SHOT) are a professionally storage temperature is –40°C.
reporting scheme, which collects data on the serious
consequences of the transfusion of blood components in Platelet concentrates: Manual and automated methods are
order: used in preparation of platelet. Concentrates must be kept at
• To educate users for transfusion hazards and their a temperature between +20°C and +24°C in a platelet agitator.
prevention Never refrigerate and keep idle.
• To improve the standards of hospital transfusion practices. Inspection of blood components: Before issue and transpor-
• To form the policy in transfusion services and to aid tation of components from the blood bank, inspection for any
clinical guidelines on the use of blood components. evidence of deterioration, leakage and damage. Possibility of
SHOT assess blood transfusion staff for their competency bacterial contamination could cause severe or fatal reaction
qualification, experience, making records, keeping records, if transfused. Each pack should be checked for hemolysis and
recorded justified reasons for transfusion, procedures contamination during collection for transfusion.
followed during blood transfusion, during transfusion
reaction and several other aspects. Warming blood: Nurses and doctors prefer “warm up” blood
SHOT survey of primary to tertiary care hospital, nursing before transfusion. No warming required if administered
homes and blood banks, reviewed overall transfusion, SHOT at a slow speed. Warming is justified if recipient receiving
reports show several pit falls. Blood safety is a vital issue needs large volumes of blood and component in very short time. It
realization, understanding and immediate attention. Safe prevents cardiac arrhythmia. Blood should be warmed in a
blood transfusion is only possible if procedures are followed specified blood warmer.
rigidly as per SOPs. Fresh frozen plasma should be thawed in the blood bank
Despite several initiatives transfusion errors continues water-bath at a temperature between +30°C and +37°C. The
to occur, as reported during transfusion practices by audit plasma unit should be kept upright and, inside another
worldwide. plastic bag to prevent contamination.
Hospital clinical staff is responsible for: reception of blood Fresh frozen plasma should be infused within 30 minutes
and blood products from the blood bank, correct storage in of thawing. If not immediately required can be stored in a
wards, operating rooms and other clinical areas, monitoring, refrigerator at a +2°C to +6°C and transfused within 24 hours.
operation of blood warmers and safe transfusion of blood and Blood and blood components are to be collected from
blood products. blood bank only for immediate transfusion within 30 minutes.
306 Section 4 Miscellaneous
Normally avoid return of blood to blood bank. If at all is Collect one unit at a time except in a massive transfusion.
essential, return unopened bag within 30 minutes. In case of Document the removal of the blood component by putting
return checklist is followed in blood bank to decide whether the date, time and signature.
it should be put back into stock or discarded. Bag checked
Preadministration procedure: Blood transfusion should be
for temperature by folding the unit around thermometer.
started as soon as possible after blood component collected
Let blood settle in the refrigerator look for hemolysis or any
explaining patient about Blood transfusion. Use I/V cannula
evidence of deterioration in the plasma and red cells.
from 14 G to 24 G depending transfusion rate planned using
SHOT analysis revealed errors during Blood transfusion
170–200 um pore size filter. No drugs to be added in blood
mainly are:
bag. No dextrose, plasma expanders to be given along with
Blood sample collected from wrong patient.
blood transfusion.
Patient details were recorded incorrectly on blood sample
Before each unit record baseline parameters, component
label or the blood request form.
details on tag with expiry date, visual inspection for any signs
Incorrect unit was collected from the blood storage
of discoloration, clumping or leaks.
refrigerator.
Blood components must only be administered by a
Final identity check at the patient’s bedside, prior to registered health care professional who is trained and
transfusion, was omitted or performed incorrectly. competent.
“Only staffs who are trained and competent should The final administration check must be conducted bedside
participate in the blood transfusion process.” by a trained professional administering component.
It is commonly observed that nurses and midwives Repeat identification check details on the patient’s
regularly take responsibility for completing the request form, identification band match details with compatibility label
taking a blood sample for pre-transfusion testing, collecting, attached to the blood component and transfusion form with
administrating blood components and monitoring the each component finally for surety.
patient during the transfusion.
“The decision for blood transfusion must be based on Patient monitoring: Close observations during transfusion
a thorough clinical assessment. The decision to transfuse should be undertaken and documented for every unit
specific components should be documented in the patients’ transfused.
clinical records.” Good record keeping is an integral part of blood safety
practice, following FDA guidelines is essential for safe and
Informed written consent from patient: It is legal, ethical effective care.
and valid document. Consent must be obtained before blood Patient during transfusion must be observed from the
transfusion. The potential risks, benefits must be explained. cabin, look for vague symptoms of transfusion reaction,
Valid consent is recorded in the patient’s clinical records irritable, shivering, and breathless. Check and record
before transfusing blood component. baseline parameters at 15 minute interval. Adjust initial
A patient identification band must be worn by all patients transfusion rate 8–10 drops per minute for first 30 minute,
receiving blood transfusion. Their first name, surname and later increase the rate. Record transfusion notes from starting
date of birth, IPD or OPD as check before collecting blood to end including post transfusion base line.
sample. In any suspected transfusion reaction, first step is to
Collect the required amount of blood into the appropriate stop transfusion and immediately inform medical doctor.
sample tube. Label immediately after collection before Assure and explain patient and their relatives to relieve panic
leaving patient and sign the sample tube. Sample tube and situation. Assess patient’s airway, breathing and circulation
request form details must correspond. Collect only one (ABC). If life-threatening reaction is anticipated, call for
patient sample at a time and never pre-label the sample tube. resuscitation team. Maintain venous line patent with 0.9%
Prior to collecting the blood component ensures patient sodium chloride. Record the patient’s pulse, temperature,
is wearing an identification band, patient consent and respiratory rate, blood pressure, urinary output including
indication for transfusion are recorded in the patient’s color for hemoglobinuria. Urgent evaluation of the blood
clinical record. Suitably trained and competent staff must be required at blood bank, microbiology for assessment of
available for transfusion. Patient’s baseline pulse rate, blood contamination, hematological, biochemical parameters of
pressure, temperature and respiratory rate checked and recipient. Patient needs a close observation and record vitals
recorded in a patient clinical record. at regular interval. If no improvement in clinical condition
Ensure that every blood component collected is checked found, transfer immediately to intensive care unit.
against the patient’s minimum identification along with Check the core identification between the patient and
blood component form, patient compatibility label showing blood component compatibility label. Inspect the component
expiry date on the blood component with patient’s wristband. for unusual clumps, particulate matter or discoloration.
Chapter 80 Importance of Safe Blood Administration Practices 307
Inform the hospital transfusion laboratory; return the unit component, its identification, visual quality check including
and administration set to the transfusion laboratory. Record expiry is essential. Keep close observation of recipient during
the adverse event in the patient’s clinical record. transfusion. Finally at every step documentation and record
The safety of blood transfusion is lying in multiple keeping is a mark of skill and safe transfusion practices.
hands at different levels. The key principles for blood safety
are based on competency, knowledge, experience and Suggested Reading
dedication of staff to work. Automation prevents several
clerical errors. Care is to be taken in identification of recipient 1. Emmanuel JC. The clinical use of blood. Handbook. WHO,
from collection of sample to transfusion. Rationale blood Blood Transfusion Safety, Geneva, 2001.
Strategies to Reduce
Unnecessary Transfusions
committees which devise and implement the blood 3. Thurer RL, Lambert C, Parce P, et al. Variability in transfusion
transfusion policies, prepare educational programmes, practice—beyond cardiac surgery. Transfusion. 2012;52
conduct regular audits and disseminate the guidelines relevant (suppl):121A.
4. Rehm JP, Otto PS, West WW, et al. Hospital wide educational
to their center. A hospital transfusion committee provides a
program decreases red blood cell transfusions. J Surg Res.
mean to implement the above strategies at hospital level and 1998;75:183-6.
have been proven to reduce the unnecessary transfusion as 5. Morrison JC, Sumrall DD, Chevalier SP, et al. The effect of
evident by various studies.12,13 Thus appropriate steps should provider education on blood utilization practices. Am J Obstet
be taken to create hospital transfusion committees.14 Gynecol. 1993;169:1240-5.
In addition a national strategy to check the overall 6. Ayoub MM, Clark JA. Reduction of fresh frozen plasma use
progression of strategies at local level should be done. This with a simple education program. Am Surg. 1989;55:563-5.
7. Handler S. Does continuing medical education affect medical
strategy involves creation of a hemovigilance network
care? A study of improved transfusion practices. Minn Med.
wherein various aspects of blood transfusion are reported 1983;66:167-80.
and guidelines based on newer evidence are prepared. 8. Khan FAH, Kamal RS. Improvement in intraoperative fresh
This network also monitors the blood transfusion practices frozen plasma transfusion practice—impact of medical audits
and based of inappropriate practices formulates the new and provider education. J Pak Med Assoc. 2000;50:253-6.
practices. 9. Toy PT. Effectiveness of transfusion audits and practice
Thus no single strategy could help in reducing the guidelines. Arch Pathol Lab Med. 1994;118:435-7.
10. Kakkar N, Kaur R, Dhanoa J. Improvement in fresh frozen
unnecessary transfusions. A multiapproach strategy with
plasma transfusion practice: results of an outcome audit.
involvement of all right from the clinician to the hospital Transfus Med. 2004;14:231-5.
to the national health agencies is required for reducing the 11. Garrioch M, Sandbach J, Pirie E, et al. Reducing red cell
burden of unnecessary transfusions and saving this precious transfusion by audit, education and a new guideline in a large
resource so that it is available to a needy person in time. teaching hospital. Transfus Med. 2004;14(1):25-31.
12. Torella F, Haynes SL, Bennett J, et al. Can hospital transfusion
committees change transfusion practice? JR Soc Med. 2002;
References 95(9):450-2.
13. Calder L, Woodfield G. The hospital transfusion committee:
1. Theusinger OM, Felix C, Spahn DR. Strategies to reduce the a step towards improved quality assurance. NZ Med J.
use of blood products:a European perspective. Curr Opin 1991;104(921):427-9.
Anesthesiol. 2012;25:59-65. 14. Haynes SL, Torella F. The role of hospital transfusion
2. Taylor SE, Cross MH. Clinical strategies to avoidblood committees in blood product conservation. Transfus Med Rev.
transfusion. Anaesthesia and Intensive Care Medicine. 14:2. 2004;18(2):93-104.
Index
Page numbers followed by f refer to figure and t refer to table.
A Antenatal testing and HDN status in after first centrifugation, layers of 198f
developing countries 62 and blood components, discarding of
Abdominal surgery, previous 90
Antibody 245
ABO
depletion 83 bank accreditation 273
antibodies 81
mediated rejection 78, 82 banks/centres, types of 273
antigens 81
severe 78 cell 190
blood group discrepancies 292
screening, automated 288 growth factors 31
blood-type selection 92
Antifibrinolytics 91 coagulation tests disturbance 18
desensitization 78
Antigen profiling in DAT positive cases 294 collection 248
grouping 92
hemolytic disease of newborn 69 Anti-platelet antibodies 158 component 92, 129
incompatibility Antithymocyte globulins 78 inspection of 305
in BMT 73 Apheresis in donor, adverse effects of 124 irradiated 93
major 73 Aplastic anemia 147, 154, 297 selection of 68
plus minor 73 acquired 3, 152 storage 305
minor 73 congenital 3 therapy 103
transfusions 136 inherited 3 conservation
minor 93 APPT 159 during cardiac surgery 46
ABOI-KT, adverse effects of 85 Aprotinin 30, 164 strategies 40, 57
Accreditation, scope of 273 Arteriovenous malformations 57 disease 182
Acidosis 98 Arthropathy 150 donation
Acquired immunodeficiency syndrome Autoimmune ethics for 281
(AIDS) 264 hemolytic anemia 7, 8-10, 292
gift 282
Adeno associated viruses 215 thrombocytopenia 120
donors
Adenoviruses 215 Autologous
for infectious agents 264
Adult stem cells 184 blood 92
screening 226
Agglutination, grading of 286f donation 40
establishments, ethical issues related to
Agranulocytosis 150 options 40
283
Aldehyde dehydrogenase rich bright cells transfusion 35
film examination 160
(ALDHBR) 195 Auto-recognition 76
for transfusion, selection of 7
Allergic
blood transfusion reactions 209 B group 288, 290f
antigens, typing of 293
reactions 255 Babesia protozoa 138
types 299
type reaction 5 Babesiosis 138
in transfusion practice, role of whole 35
Alloantibody and autoantibody, Bacterial contamination 256, 261
lactate dehydrogenase, elevated 18
differentiation between 294 in blood component 237
loss 91, 102
Allogeneic red blood cell transfusion 46 Bacterial detection
causes of massive 102
in cardiac surgery, associated with 46 direct methods of 238
Allograft rejection 76 massive 102
in blood components 236
Alloimmunization 257 new versus old 301
Bacterial peptidoglycan chromogenic
Alzheimer’s disease 181 RCC seropositivity, reasons for discarding
immunoassay 239
American Society for Apheresis 88 Barrier filtration 222 whole 247t
Amputation free survival 196 B-cell depletion 82 safety 219
Anaphylactic reaction 255 Bernard Soulier syndrome 109 initiatives 261
Anaphylactoid reaction 255 Beta-cell dysfunction 190 sparing surgical techniques 40
Anemia 15 Beta-thalassemia 214 system, intercept 242
correction 40 Bilateral total knee arthroplasty 49 transfusion 20, 21, 30, 144, 206, 281
in cancer patients, management of 15 Bleeding disorders 157 ethics for 281
of chronic renal failure 31 acquired 157 policies, implement 309
secondary to inherited 157 services (BTS) 279
chronic disorders 31 Bleeding time 158, 160 type kidney transplantation 81
zidovudine therapy 31 Blood 36 units, expiry of 250
Angina/ECG changes of ischemia 3 administration practices, importance of units, management of 250
Antenatal screening of thalassemia 65 safe 305 warming methods 97
312 Transfusion Update
Bone marrow 158, 207 Citrate-phosphate-dextrose (CPD) 301 Dermatology 199, 200f
failure 127 adenine (CPDA) 301 Dermatomyositis 134
acquired 3 Clot retraction test 158 Desferrioxamine 149
mononuclear cells (BMMNCs) 185 Clotting factor concentrate 168 Desmopressin 164
transplant (BMT) 141, 179 Clotting time 158 Diabetes 189
Bone morphogenetic protein 198 CMV transmission and leucocytes 224 type 1 189
Brain stem cell 190 Coagulation protein, deficiency of 157 Diabetic
Breath, shortness of 3 Coagulopathy in liver diseases, ketoacidosis 120
management of 162 wound healing 200f
C Cold Discarding FFP, reasons for 249t
Cancer 199 ischemia time 90 Disease, type of 189
surveillance 212 liquid-stored platelets 33 Disseminated intravascular coagulation 13,
Cardiac Column agglutination technique (CAT) 285 30, 56, 127
arrhythmias and failure 145 Combination therapy 152 DNA-based methods, limitations of 295
disease 256 Compatibility testing 140 Donor 67
iron, MRI of 148 Compatible blood components, selection cell cytokines 224
stem cells, resident 184 of 92 confidentiality 282
Cardiopulmonary Complete blood count 17, 18 deferral 282
bypass 44, 45 Congenital amegakaryocytic derived antibodies 93
surgery 127 thrombocytopenia 3, 17 ethical issues related to 282
disease 256 Connective tissue disorder 31 identification of rare 299
Cardiothoracic surgery 200f Cord blood platelet, dose of
Cardiovascular banking 204 random 124
complications 207 transplantation 204 single 125
disease 181 in India 205 platelets, advantages of single 125
in thalassemia in India 204 recruitment 248
surgery 36
Core rewarming, active 97 selection 129
Cataract 180
Coronary specific antibody 79
Cell
artery bypass grafting, primary 44 specific transfusion 77
adhesion 222
heart disease 185 Drug 3
aplasia, pure red 207
intervention 20 induced thrombocytopenia 120
contemplated for cellular regenerative
Cosmetic ophthalmic surgery 200f immune 17
therapy, types of 184
Critical limb ischemia (CLI) 194 Dyserythropoietic anemias, congenital 3
salvage 91
Crosslinked hemoglobin 31 Dyskeratosis congenita 3
stress 212
Cryoprecipitate 104, 164, 174
therapy 155
for neonatal transfusions 69
E
types of 184, 187
utilization guidelines, evidence-based Embryonic stem cell 184
Cellular
271 Endothelial
blood components, irradiation of 67
Culture-derived platelets 33 cell 198
therapies 177
Cyanoacrylates 164 progenitor cells (EPC) 184, 185
Central venous pressure 91
Cytapheresis 135 End-stage kidney disease 81
Centrifugation and buffy coat removal 223
Cytokine transfer/infusion, passive 224 Enhanced bacterial detection system 238
Cerebral
Cytomegalovirus 67 Enzyme
blood flow 26
Cytotoxicity, mechanism of 211 immunoassays 264, 266
ischemia, delayed 26 linked immunosorbent assays (ELISA)
malaria 136
Cerebrovascular surgery 56 D 230, 266
Epidermal growth factor (EGF) 202
Cerus 242 Danazol 30 Epoetin and darbepoetin in adults,
Challenges in gene targeting in human stem D-dimer test 158 guidelines for 16
cells 217 Deferasirox 144, 151 Epsilon aminocaproic acid 30, 91, 164
Chelation therapy 144 Deferiprone 150 Erythrocytapheresis 135
Chemically modified hemoglobin solutions Deferoxamine 144 Erythrocyte-magnetized technology 289
5 Deglycerolization 223 Erythroid colony forming units 31
Chemiluminescence 264 Del phenotypes, identification of 293 Erythropoiesis stimulating agents (ESAs)
Chemotherapeutic agents 18t Dendritic cells 213 38, 206
Chest syndrome, acute 154 Dengue effects of 206
Child-Turcotte-Pugh Score 89 fever 36, 110 versus red cell transfusion 206
Cholelithiasis 145 hemorrhagic fever 110 Erythropoietin 21
Cilostazole 194 infection, grading severity of 111t Escherichia coli 256
Citrate phosphate dextrose adenine (CPDA) shock syndrome 110 Esthetic surgery 199
198 Dentistry 200f External quality assessment scheme (EQAS)
Citrate toxicity 256 Deoxyribonucleic acid (DNA) 226 280
Index 313