Principles & Practice Of ti sa Transfusion Medicine Dr. (Prof.) R. N. Makroo M.B.B.S.; D.1.B.T, M.D. Director & Sr. Consultant, Transfusion Medicine, Molecular Biology & Transplant ImmunologyDr. (Prof.) R. N. Makroo (MBBS, DIBT, MD President Indian Society of Transfusion Medicine, New Delhi PREFACE ‘Transfusion Medicine is an ever evolving and dynamic branch of medical sciences. Itcame into existence with the landmark discovery of ABO blood groups by Karl Landsteiner in 1900. With significant contributions from Levine and Stetson describing the hemolytic disease of the newborn in 1938, Carl Walter developing of the plastic bags for component preparation in 1950, and the understanding of practical application of. apheresis in the 1970's, we have come a long way. Immunohematology and Regenerative Medicine have cropped up as important branches of Transfusion Medicine, wherein the understanding of cell membranes, genes, and molecular biology, combined with fundamental knowledge of the immune system is extrapolated into laboratory bench serology to make safe blood and blood components available for patients. Having being associated with the Indian transfusion arena for over 4 decades, I have seen it blossom from a tiny seedling into a fruit bearing tee. lo all these years I have always felt the need fora standard textbook from authors in this part of the globe that embodies relevant knowledge and experiences. ‘The Ist edition of Principles and Practice Transfusion Medicine was an endeavor towards providing transfusionists and clinicians an insight into the fascinating world of transfusion. Now, the second edition ofthis book gives an update on recent advances and evolving concepts of blood transfusion. As I conceptualized this book, deliberate attempts have been made to keep the text simple and lucid, Most of the information has been provided in a reader friendly format, Questions for Self Assessment have been incorporated atthe end of cach chapter for the benefit ofthe readers. The book lays emphasis on “Blood Sefety” and provides an in depth analysis of methods that can be employed to ensure a safe blood supply. The importance of appropriate and rational use of blood & blood products has been highlighted. The book is suitable for medical graduate and post graduation students, medical laboratory technicians, and even independent and distance learning courses for Blood Bank officers. am also thankful to all my colleagues and friends for their never ending inspiration and support, especially to Dr Soma Agrawal & Dr Mohit Chowdhry for their tireless efforts and invaluable assistance in penning and editing this book all through. I am grateful to my family who has always stood beside me in thick and thin, I dedicate this book to my parents who always have and will continue to shower their blessings on me. Dr. (Prof.) R.N. Makroo aanACKNOWLEDGEMENTS ‘ express my gratitude to all those who have contributed their Valuable inputs in compiling tis publication op Late (Prof) Dr. J. 6. Joly Dr. 2. . Bharucha Dt. K. Ghosh Late Or. RK. Saran Dr. Neelam Mana Oe. Bhavna Arora Dr. Rajesh Gopal Dr. Mohd. Shoukat Dr V.P. Gupta Dr. N, Choudhary Dr. Bharat Singh Or. K. Chaudhary Dr Sabita Biswas Or. Debashish Gupta Or. Shishi Sth Dt. 8. B. Rejachyaksha Dr. M, Gajar Or, Veena Dada Dr. Manisha Srivastava Dr. P, Sunder Late Or. V.L. Ray Dr. Dilip Wan Dr, Anju Verma Dr. Vimarsh Raina Ex. Brig, Dr. ¥. V, Machave Dr Jayashree Sharma Dr. Rema Menon Dr. Kulbir Kaur DLTR Raina Or. G.N. Gupta Dr. Sneh Lata Gupte r, Surekha Devi Dr. Kabita Chatterjee Dr. Rashmi Sood Bhanot Dr. Richa Gupta Or. Rimpreet Walia Dr. Vikas Hegde Dr. Shobini Rajan r. Col. Bhushan Ashthana Dr. Sweta Nayak Dr, Sutesh Kumar Kelman ORR Sharma (Col, Dr. Sarkar Dr. Ageem Tari Ms, Rosamma NL Dr. Brinda Kakkar Dr, Dhaval Fadadu Ms. Asha Katl Bazaz Mr Uday Kumar ThakurCONTENTS aa eae ae CHAPTER 1 Historical Overview of Transfusion Medicine. Over View of Transfusion Medicine in India.......... CHAPTER2 Physiology of Blood.... Introduetion.. Haematopoiesis. I Red Cell Production and Kinetics... Brythropoiesis.......00.0 Erythropoietin Haemogiobin.. Iron Metabolism... The Role of Hepeidin........c00 Platelet Structure, Function and Kinetics Reticulated Platelets (RP). Physiology of Haemostasis... Red Cell and Platelet Metabolism... CHAPTER 3 Basic Principles of Immunohaematology. Immunity... Immune Response..... Antigens....., IgG Antibodies........ IgM Antibodies IgA Antibodies.Naturally Occuring Antibodies....... Immune Antibodies..............000000 Monoclonal Antibodies.. Newer Approaches to Monoclonal Antibody Production... Antigen Antibody Reaction. Complement... Complement in Transfusion Medicine... Factors Affecting Antigen Antibody Reaction. Basic Genetic Concepts with reference to Blood Groups Recent Advances: Dendritic Cell Therapy... Chimeric Antigen Receptor (CAR) f Cell Therapy... Self Assessment and Review Exercise.......--ueom CHAPTER 4 Blood Collection and Processing... ‘Types of Blood Donors. ‘Voluntary Blood Donors Replacement/Related Donors... Professional/Commercial Paid Donors / Donor Selection... Rare Blood Donor. Donor Selection Blood Collectio Advantages of Blood Collection Bags Over Glass Bottles... Methods of Venepuncture (Phlebotomy)... Special Precautions to be Taken While Collecting Blood. Post Donation Care & Advice ‘Adverse Donor Reactions... Processing of Donor Blood........ Donor Notification and Counseling. Self-Assessment and Review Exercise. CHAPTER 5 Preservation and Storage of Blood. Introduction...Anticoagulant Preservative Solutions...... Functions of Various Chemicals Used In Anticoagulant Preservative Solution. ..........0.83 Additive Systems. 84 Storage Temperature for Blood 87 Storage of Blood During Transportation. ...........000 89 Transportation of Blood within the Hospital... Physical and Chemical Changes in Stored Blood. Heparin. Storage of Blood in Frozen State Freezing and Thawing of Red Cells ....... 92) PlateletPreservation.... 93 Platelet Additive Solutions . 91 Self-Assessment and Review Exercise. 9 CHAPTER 6 Blood Component Preparation and Therapy. Introduction. Whole Blood... Fresh Whole Blood.. Preparation of Blood Components... Red Blood Cells.......ssesesssse Leucodepleted Blood Products....... Methods of Leucodepletion. Centrifugation... . Washed Red Blood Celis. Freezing and Deglyceroli Bufty Coat Removal Filtration ‘Types of Filtration... Benefits of Pre-Storage Filtration... Guidelines of Leucocyte Filtration, Fresh Frozen Plasma ......---- =Single Donor Plasma or AHG Poor Plasma. Cryoprecipitate. Cryoprecipitate Pooling... Preparation of PRP and Platelet Concentrate. Buffy Coat Pooling Technology. PlateletCrossmatch.. Reffactoriness to Platelet Transfusion and Management... Guidelines for Platelet Transfusion... Granulocyte Concentrates. Irradiated Blood Components... List of Equipment and Consumables for Blood Component Preparation... Self-Assessment and Review Exercise........ CHAPTER 7 Apheresis (Haemapheresis)........-. 135 Indications of Haemapheresis. 135 Methodology of Haemapheresis....... Intermittent Flow Centrifugation (IFC)......... Continuous Flow Centrifugation General Requirements for Apheresis Procedure. Donor Selection for Apheresis Procedures. Plateletpheresis... Leucopheresi Neocytapheresis.. - 7 Adverse Effects of Apheresis in Donors....... Plasmapheresis/ Plasma Exchange... Therapeutic Leucapheresis.. ‘Therapeutic Thrombopheresis.. Red Cell Pheresis. Advances in Haemapheresis. Double Red Cell Collection..... sess a 162 ALYX Component Collection System for Red Cell Apheresis.Nomogram for Donor Selection..........-.s.++0 164 Photopheresis 167 68 Rheopheresis.......... Self-Assessment and Review Exercise........ CHAPTER 8 ABO Blood Group System. el 69 Introduction... 169 Basic Genetics & Biochemistry of ABO system.. 170 ‘The ABO Antigens in Relation to RBC Membrane. 175 Subgroups of A, AB & B. 2 176 Antibodies of ABO Blood Group System...........::.00 ao 177 Routine ABO Testing Procedure..........ssssssssssssssssnereee on ABO Blood Group Reagents.........ccssssssssetesesseen esses 179) ABO Blood Grouping Procedures...........:s+++ss-steessvsseeeeeeesssee 180 Slide or Tile Method 181 Tube Method....... 181 182 184 +187 189 wed 92 193 193 193 Microplate Method ........... Column Agglutination Method for Grouping... Molecular Blood Grouping and Genotyping... Universal RBCS... Problems in ABO Grouping, Classification of ABO Blood Group Discrepancy... Resolving Discrepancies in ABO Grouping. Problems in ABO Grouping & Their Solution.. Self Assessment and Review Exercise.. 199 CHAPTER 9 The Rh Blood Group System... 10.202 Discovery of Rh System.....-seee+0 202 Clinical Importance of Rh..... 202 Basic Genetics of the Rh System...... 202‘Terminologies for Rh Blood Group System... ‘The Rh Antigen... Rh-Membrane Complex..... Variants of D Antigen. Rh Antibodies. Rh Grouping...... Reagents for Rh (D) grouping. Rh (D) Grouping Procedures... Rh Phenotyping..... Problems in Rh Grouping. Resolving Rh Grouping Problems... Self-Assessment and Review Exercise... CHAPTER 10 Other Blood Group System. Importance of Other Blood Group Systems. Lewis Blood Group System. MNSs Blood Group System. Kell Blood Group System... Duffy Blood Group System. Kidd Blood Group System... Lutheran Blood Group System... TiBlood Group... P Blood Group System... Other Minor Blood Group Systems. Blood Group and Disease Association. Self-Assessment and Review Exercise. CHAPTER 11 Antiglobulin Test. Principle. : Anti-Human Globulin Reagents (AHG). poem i244 20245 Applications of Antiglobulin Test Control Cells for AHG Tests.....--c.ssse Preparation of IgG Coated Positive Control Cells... aoe Direct Antiglobulin Test .. 246 Indirect Antiglobulin Test. 1246 247 247 248 249 Factors Affecting the Sensitivity of LAT... Low Ionic Strength Saline Indirect Antiglobulin Test Sources of Error in Antiglobulin Test. Self-Assessment and Review Exercise. CHAPTER 12 Antibody Screening and Identification....., 250 Methods for Screening of Antibodies... 1ne250 Selection of Screening Cells... 251 f= 25 252 252 254 255 261 263 263 Preparation of Cell Panel for Antibody Screening & Identification. Preservation of Cell Pancl... Techniques of Freezing & Thawing of Reagent Red Cells. Methods of Antibody Identification. Antibody Screening and Identification. Quick Reference for Antibody Identification......... Calculation of Probability. Self-Assessment and Review Exercise..... CHAPTER 13 Compatibility Testing (Pre Transfusion Testing).. Compatibility Testing Procedure. 265 Identification of Recipient's Blood Sample...... 265 Checking The Patient’s Previous Records... 266 Blood Request Form...... 267 ABO and Rh Grouping... 0.269 269 Screening for Irregular Antibodies... Selection of Blood..Cross Matching. . Major Crossmatch Techniques... Compatibility Testing in Emergencies. Electronic Crossmatch (EXM). Radio Frequency Identification in Blood Banking... Labelling and Issue of Blood Self-Assessment and Review Exercise. CHAPTER 14 ‘Transfusion Practice in Clinical Medicine... Introduction... Patient Blood Management... Transfusion Practice in Surgery and Haemorrhage... Autologous Transfusion. Preopertive Autologous Transfusion... Acute isovolumic or Normovolumic Haemodilution. Intraoperative Blood Salvage. Post Operative Blood Salvage... ‘Anaemia and Surgery.. Surgery and Coagulation Disorders... Blood Transfusion in Open Heart Surgery... ‘Vhromboelastogram, Platelet Aggregation Tests. Other Point of Care (POC) Tests. Massive Transfusion... FFP Transfusion... Platelet Transfusion. ecceseeeees Criteria FPR Activation of MTP (Massive Transfusion Protocol)... Blood Bank Procedures... Blood Transfusion in Liver Transplantation. Blood Transfusion in Bone Marrow Transplantation Blood Transfusion in Renal Transplantation.Transfusion Therapy in New Bom Infants. Multiple Transfusions..... Blood Transfusion in Thalassemia... Exchange Transfusion.. Auto-Immune Haemolytic Anaemia... Disseminated Intravascular Coagulation .... Heparin Induced Thrombocytopenia. Glucose-6-Phosphate Dehydrogenase Deficiency...... Haemophi Guidelines for Administration of Blood. Blood Use in War and Disasters... Disaster Preparedness........ ‘Walking Blood Bank. Altematives to Transfusion or Blood Sparing Strategies... PharmacologicalA gents. Recombinant Haematopoietic Growth Factors. Red Cell Substitutes, Platelet Substitutes PlateletDerived Product Photochemically Treated Platelets Outdated Platelet Application. Therapeutic Phlebotomy... Biochemical Tests for Assessing Iron Overload... ‘Step-Wise Algorithm for the Diagnosis of Haemochromatosis. Guidelines for Therapeutic Phlebotomy. Maximum Surgical Blood Ordering Schedule. Self-Assessment and Review Bxercise..... CHAPTER 15 Plasma Fractionation and Plasma Protein Solution (PPS).. 356 356 357 Plasma Fractionation. Cohn’s Ethanol Fractionation.358 sod 61 1362 Chromatographic Separation Albumin. Plasma Substitutes. Colloid Solutions........ care Dextran 40... 363 Dextran 70... 363 363 364 364 366 Hydroxyethyl Starch 450 (HES) . Gelatin Crystalloid Solutions Self-Assessment and Review Exercise. CHAPTER 16 Transfusion Transmitted Diseases... 367 Introduction 367 Viral Hepatitis 367 Hepatitis A Virus (HAV)........ 369 Hepatitis B Virus (HBV). 369 Subtype of HBV .. 370 Clinical Presentation of HBV Infection .. 3B 374 374 376 378 379 10382 383 383 385 386 386 Serological & Biochemical Markers of HBV Infection .. Challenges in HBV Screenin, Hepatitis B Carriers Occult Hepatitis B Virus Infection Screening Tests for Hepatitis-B Virus ..... Non-A, Non-B (NANB) Hepatitis. Hepatitis E (HEV), Hepatitis C (HCV). Screening Test For HCV... Hepatitis D.. seseeee Hepatitis G Virus (HGV).. HepatitisF Virus.. AIDS (Acquired Immuno Deficiency Syndrome). 389 ie sa sutun momanS As| | | | Etiological Agent... Structure of HIV .. Immunopathogenesis of HIV Infection . Clinical Presentation of HIV Infection .. ‘Transmission of HIV Infection Clinical Course of HIV Infection Serological Profile of HIV Infections Tests to Detect HIV Infection ELISA... IndirectELISA.. Competitive ELISA... Sandwich ELISA. seoseeesee Particle Aggtutination Assays/Simple Tests.. Specialized Rapid Assays... Western Blot or Immunoblot......-0sss-esseetessstee Safety Measures for Personnel Working in HIV & Hepatitis Testing Laboratory. Post Exposure Prophylaxis, Occupational Exposure to HIV. Body Fluids to which Universal Precautions Apply. Body Fluids to which Universal Precautions Do Not Apply...... Chemiluminescent Magnetic immunoassay - CMIA.... HIV Testing Strategies. Malaria. ‘Cytomegalovirus (CMV) Infection. Syphilis VDRL Test RPR Test... TPHA.... Human T-Cell Lymphotropic Viruses... Bacterial Contamination of Platelets........ Biomeurex Bact / Alert System... Pall Biomedical BDS System..Verax Platelet PGD Test - Rapid, Point of Care Bacteria Test Emerging Pathogens: Potential Threat to Blood Safety. Newer Hepatitis Virus,.....-.0..+ Epstein Bart Virus... Parvovirus BI9...... West Nile Virus... ChikungunyaVirus...... Dengue Vinws. Impact of Dengue Epidemics on Transfusion Prions.... Protozoa....... Self-Assessment and Review Exercise... CHAPTER 17 Blood Transfusion Reactions....... Haemolysis.. Intravascular Heemolysis. Extravascular Haemolysis.... ‘Types of Transfusion Reactions Haemolytic Transfusion Reaction Causes of Haemolytic Transfusion Reaction .. Prevention & Control of Haemolytic Transfusion Reactions. ‘Measures to be taken in any suspected Transfusion Reaction. Non Haemolytie Transfusion Reactions... Febrile Non Haemolytic Transfusion Reactions (FNHTR) ... Urticarial (Allergic) Transfusion Reactions. Anaphylactic Transfusion Reac : ‘Transfusion Related Acute Lung Injury (TRALI).. ‘Transfusion associated Circulatory Overload (TACO) Graft Versus Host Disease... ‘Transfusion Siderosis (Haemosiderosis) .. SepticemiaHypotensive Transfusion Reaction, 458 Self-Assessment and Review Exercise. 458 CHAPTER 18 Haemolytic Disease of Foetus and New Born (HDFN) 460 Introduction. 460 Etiopathogenesis... Classification of HDN... Pathophysiology.......-.. sec Rh (D) Haemolytic Disease of the New Bom. Factors Influencing the production of Rh Antibodie: Antenatal Assessment of Rh-HDFN. Investigation on New Bom, Antenatal Management of Rh Immunization Post ~ Natal Treatment of Infant. Criteria for exchange Transfusion in Rh-HDEN.. Selection of Blood for Exchange Transfusion in Rh- HDEN. Prevention of HDEN, ABO Haemolytic Disease of New Bom... Investigations in ABO-HDEN. Management of ABO-HDEN, Estimation of IgG Anti-A and Anti -B.. Witebsky partial neutralization test. Inactivation of IgM antibodies by 2 Mercaptoethanol (2ME). Inactivation of IgM Antibodies by Dithiothritol (DTT).. Self-Assessment and Review Exercise...... CHAPTER 19 Quality Assurance In Blood Transfusion Services Introduction. Quality. Quality Control, Quality Assurance & Quality Audit oan copa cE RRR HEQuality Manual... Requirements for Quality Assurance in Blood Transfusion Service... Quality Assurance of Personnel and Premises. Standard Operating Procedures (OPS)... Quality Assurance in Blood Cellection. Component Collection by Apheresis. Quality Assurance of Laboratory Test Quality Control of Reagents... Quality Control of kits used for testing Blood Transmissible Infections. Levey Jenning’s Chart Quality Control of Equipment... Storage and Transport of Blood and Blood Components. Quality Control of Blood Components. Quality Monitoring Tests... Documentation (Record Keeping) & At ISBT 128 Labelling System...... Biomedical Waste Management... Auditing. Quality Audit... Medical Audit. Indicators for Monitoring of BTS... Biosafety and Role of Management in Quality Assurance. Quality Assurance Incident report. Quality Assurance Internal Audit. Self-Assessment and Review Exercise... CHAPTER 20 Cellular Therapies... 527 Human Stem Cells... S27 Adult Stem Cells... 527 Properties of Adult Stem Cells... 12528 ‘Stem Cell Differentiation and Haematopoiesis..‘Haematopoietic Stem Cell (HSC) Transplantation, Peripheral Blood Stem Cell Collection..... Standard Protocol followed During HSC Transplantation. Donor Suitability and Evaiuation, Blood Group and Infectious Disease Testing. Stem Cell Harvesting Procedure (PBSC). Time of Collection. Collection Targets Collection Procedure. Bone Marrow Collection. Umbilical Cord Blood (UCB) Collection... Processing of Haematopoietic Progenitor Cells. Plasma or Red Cell Depletion. Selection of the CD34+ HSCs... Fluorescent - Activated Cell Sorting... Immunoadsorption Systems. Isolex 300i System. CliniMACS.. Avidin-Biotin Selection Physical Parameter Separation. ‘T-Cell Depletion. Cell Processing Protocols. Peripheral Blood Processing... Bone Marrow Processing. ‘Coolmix AS-210. Freezing and Storage..... Liquid Nitrogen Storage... Storage of Untested or Infectious Products. ‘Thawing and Infusion..... .. Transfusion Support in PBSC Transplant... Evaluation and Quality Control Cell Counts...Bacterial and Fungal Cultures... CD34 Analysis/Enumeration.. Cell Viability Assay... Colony-Forming Cell Assay... Haematopoietic stem cell transplant... Role of Stem Cells in Myocardial Infarction and Cardiomyopathy. Mesenchymal Stem Cells... GeneTherapy... Proteomics Applications of Proteomics in Transfusion Medicine. Passenger Lymphocyte Syndrome... Adoptive Immunotherapy... Self-Assessment and Review Exercise.............. CHAPTER 21 SpecialMethods....... Definitions. Examples.......00-++ Titration... corre Methods of Haemoglobin Estimation of Donor. Lectins Polyagglutination.. Saliva Test for A, B & H Substances Bovine Albumin Serum... Enzymes. Low Ionic Strength Salt Solution (LISS). Elution... HeatElution.. Ether Elution. Elution using Chemical Treatment. Adsorption... . Allo-Antibody Adsorption Technique. Gower-L: consisting of zeta and epsilon globin chains ((2e2) > Gower-2: consisting of alpha and epsilon globin chains (a2e2) > Portland: consisting of zeta and gamma globin chains((2y2) Definitive phase- 8 week onwards Between 8-24 weeks of gestation, the predominant Hb is HbE (a2y2) ‘At 24 weeks of gestation, HDF constitutes 90% of total haemoglobin with 10% being adult Hb (HbA; 0262). The predominant site of erythropoeisis is fetal liver and spleen. HbA steadily increases thereafter. At birth the average HDF is 70% of the total haemoglobin and its synthesis decreases rapidly ost-natally, so that by 6 to 12 months of age only a trace is present. At term, the proportion of HbA averages 30%. By 1 year of age, the normal adult haemoglobin pattern appears. HAZ (0262) is the minor adult haemoglobin component. At birth, less than 1% of HbA2 is present, but by 12 months, the normal level of 2% to 4% is attained, Throughout life the normal ratio of HbA to HbA2 is approximately Stages of Erythropoiesis (Figure 2.4) es Figure 2.4: Stages of erythropoiesis; HSC: haematopoletic stem cell, BFU-E: burst forming wit-erythroid, CEU-E: colony forming unit- erythroid, EB: erythroblas, RET: reticulocyte e106 TTTPhysiology of Blood i Requirements for Erythropoiesis Intracellular factors: Expression of 4+ General haematopoietic and erythroid-specific transcription factors ‘¢ Receptors for haematopoietic growth factors ‘+ Proteins: haemoglobin, membrane, membrane cytoskeletal protein, Extracellular factors: 4+ Haematopcietic growth factors in adequate amounts Nutrients (iron, folate, vitamin B12) ‘Stromal cell and matrix support Erythropoietin (EPO) Erythropoietin is the primary erythropoietic factor that acts in concert with various other growth factors (eg., IL-3, IL-6, glucocorticoids, and Stem Cell Factor (SCF); see figure 2.3) involved in the development of cells of erythroid lineage from multi potent progenitors. It is produced in response to tissue hypoxia. Under hypoxic conditions, the kidney will produce and secrete erythropoietin to increase the production of red blood cells by targeting CFU-E, pro-erythroblast and basophilic erythroblast subsets in the differentiation (Figure 2.5). The negative feedback mechanism accounts for the fine control of erythrocyte production as seen in each individual, Another organ that forms @ part of the negative feedback loop and responds to hypoxia is liver, however, it does so only in small amount in the adults. The same cells of the kidney and liver that sense hypoxia are the ones that produce EPO. The mechanism for sensing hypoxia in cells involves a family of specific transcription factors, hypoxia-inducible factors (HIPs). These constitutively synthesized transcription factors for EPO are hydroxylated and proteosomally digested in the presence of oxygen. However, during hypoxia the degradation of HIFs ceases resulting in EPO gene transcription and EPO production, BONE MARROW CHU. ema Enthoid precursors { \ Erythropoietin RBC mass \ © F atmopiece High Cone - Blood Volume Exyropietin * jonglomeraro,Seaorte] CO rsnnary Function 5 foe 0, Afinity KIDNEY iguee 25: Erythropoietin feedback mechanism one ae easorRS NRESE Principles & Practice of transfusion Medicine “The cells ofthe kidney that produce ERO are a subset of cortical interstitial cells adjacent to proximal tubules (juxtatubular cells) and are most likely fibroblasts, In mild anemia, there are only small foci of EPO producing cells present in the inner cortex, however, as anemia progresses more and more number of these cortical cells are recruited to produce EPO so that in moderate anemia, the EPO- producing cells are present in larger areas within the inner half of the cortex and in severe anetia, they are present throughout the renal corte. Erythropoietin has its primary effect on sed blood cel progenitors and precursors “by promoting their survival through protecting these cells from apoptosis. The colony formning unit-erythroid (CPU-E}, expresses maximal erythropoietin receptor density and is completely dependent on erythropoietin for further differentiation. The CFU-E, the pro-erythroblasts and basophilic erythroblasts also express erythropoietin ceceptors (approx. 1000 Epo receptors/cell) and are therefore affected by it. WES imal grace oc Gene station and trangiption of —— Melton SHEE grow ad marration N Bos Om Figure 2.6: Mechenism of action of EPO o120Physiology of Blood i EPO+EPO-R complex Intracellular signaling by JAK-2 certs Ye Endocytosis of EPO/EPO-R complex JIAK-2 chaperons cyoplasmc part af EPO-R _— Phosphorylation ‘Activation of JAK-2 a Phosphoryats tyrosines in EPO-R = transduction Signa Activation of ransctiption- 5 (STATS) RAS-raf- MAP kinase Phosphoinostol-3 kinase ee 7 —_— “Activation of SHP-1 & Activation of pressor of ‘SHP-2 Phosphatases cytokine signaling-3 (SCOS-3) Control phosphorylation & Cease EPO-R signaling Figure 2.7: Flowchart explaining mechanism of action of BPO Mechanism of action of EPO EPO interacts with its receptor EPO-R which are present on CFU-E through Basophil erythroblastic stages of erythroblast differentiation. The binding of EPO to EPO-Rs leads to three major events: 1) hhomodimerization: The process of joining two identical subunits to form a single compound and conformational changes in EPORs, 2) initiation of intracellular signaling by the EPO-Rs, and 3) endocytosis (internalization) of the EPO/EPO-R complex (Figure 2.6 and Figure 2.7) ‘The activation of this cascade causes maturation and differentiation of RBC precursors and an increase production of RBCs HAEMOGLOBIN Haemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood cells. In 1959, ‘Max Perutz determined the molecular structure of haemoglobin by X-ray crystallography. Haemoglobin consists mst of protein the “Bobi” chain), and these protinin tan, are composed of Sequences of amino aci oneE Principles & Practice of Transfusion Medicine A haemoglobin molecule consists of four polypeptide chains Two alpha chains, each with 141 amino acids, gene for which is located on chromosome 16 ‘@ Two beta chains, each with 146 amino acids, gene for which is located on chromosome 11 ‘The protein portion of each of these chains is called “globin’ In this case, a and f refer to the two types of globins. The alpha and beta globin chains are very simi in structure. Each a and 8 globin chain folds into 8 a helical segments (A-H) which, in turn, fold to form globular tertiary structures that look roughly like sub-microscopic kidney beans. Hydrogen bonds stabilize the helical sections inside this ‘protein. The four polypeptide chains are bound to each other by salt bridges, hydrogen bonds, and hydrophobic interactions. ‘The folded hheices form a pocket that holds the working part of each chain, the heme. Ahaere group ating molecule containing 4 pyeaeings (made ofcarbon,nitogen nd hydrogen atoms), with a single Fe’* jon at the centre. Without the iron, the ting is called a porphyrin. In a haeme ‘molecule, the iron is held within the flat plane by four nitrogen ligands from the porphyrin ring, The iron ion makes a fifth bond to a histidine side chain from one of the helices that form the haeme pocket. ‘The iron ion may either be in the Fe" or Fe state, but ferrihaemoglobin (methaemoglobin i. Fe") cannot bind oxygen. In binding, oxygen temporatily oxidizes (Fe) to (Fe), so iron must exist in the +2 oxidation state to bind oxygen. The enzyme methaemoglobin reductase reactivates haemogiobin found. in the inactive (Fe) state by reducing the iron centre. The structure of haemoglobin is shown in figure 18. Polypeptide chain B chain a (tpt Compe) | Figure 28: Structure ofhaemoglobin IRON METABOLISM Iron isan essential micronutrient required by every cell. Its presentin the human body in haemoglobin, myoglobin, and several iron containing mitochondrial respiratory enzyines. It serves as a carrier for oxygen (in haemoglobin) and electrons and acts a8 a catalyst for a variety of oxygenation reactions. ‘The normal body iron concentration is approximately 40 to 50 mg/Kg body weight; women have lower amounts and men have somewhat higher quantities of iron. The iron distribution in the body is as follows: ome i i. |& Physiology of Blood EB Haemoglobin: 30 mg/Kg ‘¢ Haeme compounds (myoglobin and cytochromes); iron-dependent enzymes: 5 to 6 mg/Kg, Storage iron (ferritin and haemosiderin in the liver, marrow, spleen and muscle): 5 mg/Kg in ‘women, 10-12 mg/Kg in men Iron Absorption Iron balance is maintained by controlling iron absorption; iron stores and iron absorption are reciprocally related so that as stores increase, absorption declines, Iron in the presence of pepsin and low pH in stomach is broken into Fe and Fe’ ions. At the mucosal cell surface Fe** is converted to Fe by duodenal cytochrome B and is transported across the cell membrane by divalent metal transporter (DMT). ‘The site of iron absorption is duodenum and upper jejunum. Ferroportin 1 helps in transfer of Fe from mucosal cell into circulation. iren is absorbed as Fe. On the basolateral side hhephaestin converts Fe to Fe" form, which is then released into cisculation to be taken up by iron transporting protein apotransferrin (Figure 2.9). Enteroeyte RCFE) ‘Basolateral (blood) Hepcidin Ferroportin 7 Fev Be & transferrin ” (otilsable Tron) Ceraloplasmin Deyt be duodenal ytochrome b DMTi:dvalent metal tranposter 1 HICPA: hac carter protein 1 Hox: haem oxygenase Ft frsitin Figure 29: [ron absorption across duodenal epithelium Factors promoting Iron absorption © Acidic pH of stomach Ascorbic acid ¢ Haem iron Factors hampering Iron absorption 4 Phytates of cereals + Tannates of tea 4 Phosphates of diet and drugs 56 ean ah¢ Milk Jon is not free im the circulation but exists ss transferrin (bound to apotransferrin). Most of the iron used for red cell haemoglobin production is obtained from haemoglobin breakdown of senescent RBCs (called recycling). When red blood cells reach the end of their lifespan (senescent), they are phagocytized by macrophages (in the spleen, liver, bone marrow). Hydrolytic enzymes in macrophages degrade the ingested RBCs and release haemoglobin, Proteolytic digestion of haemoglobin liberates ‘heme and globins. Globins are broken down to amino acids which can be used for protein production. ‘The iron is released from heme, leaving @ porphyrin ring which is converted to bilirubin, Once iron is released from the heme, it is either utilized by the cell for production of iron containing enzymes and proteins or exported (via fertoportin), of stored as ferritin. In macrophages, ceruloplasmin is a fetroxidase and facilitates the transfer of macrophage iron to transferrin Approximately 3-4 mg of the body iron is bound to serum transferrin which transports iron to erythroblasts in the bone marrow. Under physiological conditions, only about one third ofthe iron- binding capacity of transferrin is saturated with iron. Transferrin has two high-affinity binding sites for Fe thus, transferrin is found as apotransferrin (no iron bound), mono- (with iron bound to one of the two binding sites), or diferric transferrin (holotransferrin, both binding sites occupied), The transport of iron into the cells is regulated by the expression of transferrin receptors on their surface. Virtually all cells can express transferrin receptors which have a high affinity for diferric transferrin. The diferric transferrin/ transferrin receptor complex is internalized by endocytosis, and iron dissociation is induced by the acidic and reductive environment jn the endosome. Iron (in the form of Fe) is then exported from the endosome to the cytosol through divalent metal transporter 1 (DMT1). Finally, the apotransferrin/transferrin receptor complex is brought to the surface where apotransferrin is released. because ofthe significantly lower affinity ofthe transferrin receptor for apotransferrin than for diferric transferrin. Iron so transported is either utilized for haemoglobin production by erythroblasts in the ‘bone marrow or other heme containing compounds by muscle/ liver. The iron which is not utilized is stored as feritin and haernosiderin (Figure 2.10). The body lacks any effective mechanism for the excretion of excess iron, although shedding of intestinal cells is partly effective, Iron exchange is limited so thatthe adult male absorbs and loses only about 0.01 mg/Kg/day (daily turnover). Caruloplasein ‘Transferrin (needs Cu) receptor Macrophage Erythroid progenitors Ms. ots i ie . e So ss om diet Apotransfergin (ace Tantei arresting (SBC soon BONE MARROW Figure 2.10: Ion absorption and transport ose ome cap23% Physiology of Blood bg ‘The Role of Hepcidin Hepcidin regulates the absorption of iron from the gastrointestinal tract as well as its release from its storage sites in macrophages through its effect om levels of ferroportin, the protein responsible for transporting iron out of the enterocyte/macrophages. Hepcidin decreases serum iron by decreasing iron absorption and preventing macrophages from releasing iron (causing iron sequestration), Hepeidin is regulated by iron levels and erythropoiesis. When the body has adequate or increased amounts of iron, hepcidin is up regulated, iron absorption from the intestine is inhibited, and iron is sequestered in its storage sites, the macrophages and hepatocytes and opposite is true in the face of iron deficiency. Active erythropoiesis inhibits hepcidin (allowing iron to be absorbed/released for haemoglobin synthesis). Hepcidin is increased by inflammatory cytokines, particularly IL-6, and reduces available iron during inflammatory processes. Inflammation thus causes a “functional” iron deficiency because iron is not released from macrophages (results in increased iron stores). This contributes to the anaemia of inflammatory disease (Figure 2.11). tna on woe — Inflammatory Cytokine Sa | | Decreased iron availabilty se L = Figure 2.11: The role of Hepciin in iron absorption; Mae: Macrophages PLATELET STRUCTURE, FUNCTION AND KINETICS Structure & Function ‘Piatelets are major components ofthe haemostatic system. Their role in haemostasis was frst established by Bizzozero in 1882. He noted their adhesion and participation in formation of a thrombus in the mesenteric vessels of rabbits and guinea pigs. The nornial platelet count in a healthy adult ranges from 150-400 x, 107/L. The mean platelet diameter is 2-3 jum and the mean cell volume 5.8 fl. The platelets are produced in the bone marrow by fragmentation of the cytoplasm of megakaryocytes. Each megakaryocyte is responsible for the production of around 4000 platelets. The time interval from differentiation of the stem cells to the production of platelets averages about 10 days. Platelet production is under the control of tumoral agents such as thrombopoietin, interleukin-3 and GM- CSR. Platelets are anuclear cytoplasmic fragments. They are disc shaped cells with smooth surfaces. Unlike RBC membrane platelets have several openings which resemble holes in a sponge. These are membrane channels which extend deep into the interior of the cell (igure 2.12). one oe pant tfPrinciples & Practice of Transfusion Medicine va Figare 2.12: Steacture of platelets | The detailed structure of the platelets is explained as follows: Peripheral zone: adhesion and aggregation + Surface coat > Glycocalyx ~ glycoproteins, proteins and mucopolysaccharides + Phospholipid bilayer > Phospholipids asymmetric arrangement and source of arachidonic acid ‘+ Integral proteins > Glycoproteins especially - Ib, 1b, Ila, IX and enzymes Structural zone: structure and support {sol-gel zone) + Microtubules + Cytoskeletal network > Actin » Actin binding protein o186Physiology of Blood ‘+ Cytoplasmic meshwork > Actin > Myosin ‘& Organelle zone: secretion and storage ‘¢ Granules > Dense bodies: non protein functional mediators (for contents see table 2.1) > Alpha granules: proteins (for contents see table 2.1) > Lysosomes: enzymes Mitochondria + Glycogen + Membrane systems: secretion and storage > Surface connected Open Canalicular System (OCS) » Dense tubular system > Fused systems The main function of platelets is to form a primary haemostatic plug. The platelet plug formation involves: platelet adhesion, activation, aggregation and secretion. Table 2.1: Contents of Dense body and Alpha granules ‘ Dense body granules + Adenosine diphosphate (ADP)-agonist for platelets. Recruits and activates new platelets for activation + Adenosine tiphosphate (ATP)- agonist for other ces + Calcium + Catecholarnines (epinephrine, norepinephrine) + Serotonin- vasoconstriction + Pyrophosphate + Magnesium. Note: ADP in dense bodies is non metabolic or storage ADP used in platelet aggregation. ADP in cytoplasm is metabolic ADP providing energy for normal platelet metabolism Alpha granules + Group I haemostatic proteins + Fibrinogen: platelet aggregation and conversion to fibrin + Factor V helps in bin formation + WOF: patelet adhesion + Platinogen activator inhibito(PA-): inhibits frinolysis + Alpha 2 antiplasmin: nibs Sbrinolysis + lasminogen: converted to plasmin for firinolysis + Group I non haemostatic proteins + Platelet specific + B-thromboglobulin: chemotactic for fibroblasts in tissue repair + Platelet factor 4- promotes platelet aggregation + Platelet derived growth factor (PDGF): promotes repair of smooth muscles + Platelet non specific + Albumin and fibronectin (cohesion of ets) + Thrombospondin: platelet aggregation 196Principles & Practice of Transfusion Medicine Platelet Production Platelet production or megakaryocytopoiesis begins with commitment of HSCs towards megakaryocyte lineage, and includes the proliferation, maturation, and terminal differentiation of megakaryocytic progenitors (Figure 2.2). This process is characterized by DNA endoreduplication, cytoplasmic maturation and expansion, and release of cytoplasmic fragiments as citculating platelets. The primary growth factor for the development of megakaryocyte (MK) lineage is thrombopoietin (TPO), which induces concentration-dependent proliferation and maturation of Megakaryocytes (Figure 2.3). Two major transcription factors involved in CMP differentiation are GATA-1, which drives differentiation of Megakaryocyte Erythroid Precursot, and PU.I, which regulates granulocyte-monocyte precursors. It is the down regulation of PU.1 expression in the CMP which is associated with the restriction of difesentiation of CMP to erythroid and Megokaryocyte lineages At present, there are two school of thoughts pertaining to thrombopoiesis, which are not mutually exclusive. One of them suggests that each megakaryocyte produces around 7-9 proplatelets. The proplatelets extend into sinusoidal spaces of the bone marrow, where they detach and fragment Into individual platelets giving rise 10 about 2000 to 5000 new platelets. The other model of platelet production suggests that, within the Megakaryocyte cytoplasm, there are preformed territories with internal membranes demaccating pre-packaged platelets, which are released upon fragmentation of the cytoplasm. RETICULATED PLATELETS (RP) Introduction Reticulated Platelets are the platelets which have been newly released from the megakaryocytes into circulation. These nascent platelets are characterized by their increased RNA content. The percentage of reticulated platelets (RP%) can be thought analogous to the reticulocyte count of RBG and it reflects on the thrombopoietic activity of the bone marrow. The absolute Reticulated platelet count in Circulation with normal platelet counts les in the range of 15,000 to 48,000/4L and normal RP % in adults ranges from 1.1-6.1%. An increase in reticulated platelets is observed whenever there is peripheral destruction of platelets and a decrease in their number is seen in case of hypoproliferative diseases of the marrow such macrow aplasia due to chemotherapy, radiation or aplastic anemia. Reticulated platelets owe their better haemostatic properties to possessing twice as much a-granule content as compared to older circulating platelets along with an increased density of adherent membrane receptors Laboratory Detection ‘Due to the presence of RNA various RNA based assays can be used in the detection of Reticulated platelets. RNA can be stained with dyes such as thiazole orange and detected using flowcytometry. Clinical Uses + Detection of early increases in platelet proditction is useful in detecting marrow recovery as seen after chemotherapy, PBSC/Bone Marrow transplant, and radiation induced thrombocytopenia. 4200 rT Th