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) i 2 Figure 24: Stags of erythropoiesis HSC: haematopoietic stem cel, BFU-E: burt forming wit-eytroi, CEU-E: eins Sait : 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 chain” — amin 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oe Physiology of Blood + There is an increase in the circulating reticulated platelets in ITP. These reticulated platelets which have better haemostatic properties and prevent bleeding in patients with ITP even in the face of very low platelet counts An increase in the percentage of reticulated platelets in patients of allogeneic stem cell transplants is associated with graf-vs host disease (GVHD). aR SERRA ARMac pS) Bo ‘@ An increase in the percentage of reticulated platelets in 2 to 3" trimester of pregnancy is suggestive of risk of preeclampsia + Increase in reticulated platelets with steady state thrombocytosis is suggestive of thrombosis & Reticulated Platelet percentage in excess of 11% is postulated to be diagnostic of thrombocytopenia with increased thrombopoietic activity PHYSIOLOGY OF HAEMOSTASIS Sequence of Events 1. Arteriolat vasoconstriction ‘+ Endothelin secretion ‘+ Reflex neurogenic mechanisms 2. Exposure of subendothelial extracellular matrix (primary haemostasis) ‘¢ Temporary haemostatic plug formation 3, Tissue factor (secondary haemostasis) 4, Permanent plug formation Role of Endothelium Endothelium maintains a default anticoagulant surface by secreting factors that block activity of soluble coagulation factors. Its antiplatelet effects include secretion of endothelial prostacyclins (PGI2), nitric oxide and adenosine diphosphatase (ADPase) along with its net negative charge and its anticoagulant effects include secretion of membrane associated Heparin like molecule, thrombomodulin and tissue plasminogen activator. Its prothrombotic effects include release of vWF which initiates platelet plug formation. Cosstriction of damaged blood vessels is the first step in haemostasis. The damaged vessels will constrict which reduces the amount of blood flow through the area and limits the amount of blood loss. This response is triggered by factors such as a direct injury to vascular smooth muscle, chemicals released by endothelial cells and platelets, and reflexes initiated by local pain receptors. The spasm response becomes more effective as the smount of damage increases. Vascular spasm is much more effective in smaller blood vessels, Role of Platelets 1. Platelet adhesion 2. Activation and shape change anePrinciples & Practice of Transfusion Medicine a 3. Secretion 4. Aggregation Platelet Adhesion It is attachment of platelets to something besides other platelets. When bleeding occurs platelet flow into these subendothelial tissues and attach to collagen. This adhesion takes place with the help of WWF and gplb. ‘The vWF acts as a bridge, attaching to both endothelium and gp1b on platelets. Forming a monolayer of platelets on the exposed collagen, however, this adhesion is short-lived, resulting in. platelet rolling in close proximity to the subendothelium. Activation and Shape Change ‘The binding of gp1b with collagen and vWF results in platelet activation. The Shape change involves the change of shape from disc shaped cells to spheres with appearance tiny projections called pseudopods. It involves proteins in the structural zone, actin and myosin, microtubules. The result of shape change: larger membrane surface area for biochemical reactions and greater contact with other platelets. The activation of platelets results in secretion from platelet granules, appearance (conformational changes) of fibrinogen receptor gp-IIb/IIla, platelet aggregation and clot formation. Changes in the membrane phosphatidyl serine which allows coagulation factors to bind to it, is known as the platelet procoagulant activity. Platelet Secretion Itis the release of platelet contents into the surroundings. It is energy dependent and requires ATP. Platelet Aggregation Itis the attachment of platelets to one another and involves two stages a) Primary aggregation where platelets aihere loosely to one another and it may be reversible, b) Secondary aggregation which occurs as platelet release their own ADP, granule contents and thromboxane A2. Fibrinogen and extracellular calcium are required for this. Fibrinogen acts as a bridge that attaches platelets to one another as well as to the endothelium. It results in formation of a primary haemostatic plug, whereby, a barrier is formed which seals the site of injury. Secondary Hemostasis ‘The platelet plug formed above is fragile. It is anchored firmly to the vessel wall by the process of secondasy haemostasis which begins with fibrin formation around the aggregated platelets. The platelet fibrin mass then contracts to form a firmer more cohesive clot. This is known as clot retraction, ‘The Role of Clotting Factors (Figure 2.13) ‘There are two separate clotting pathways, the intrinsic and extrinsic. These eventually converge to form the common pathway. aneT Physiology of Blood Inn pulway Exile patbway High MW Kaingen—rBrgiin pation yee Tisve fate FV XI rata Ey RRS tae, Mert orgy te, AK xa TP Va complex -vint—Fl_pvitta PFS, Ca¥! Car [ “Tense cope o Lg torn pat a | Protrambinad compe / #1 Prtiroabia) —" > ry Chron) anpicsion = + sate hringgen — "> St pan sa) co Ins rs ike fbn i ee =a ca ! | en —e eee Prarin | | mcrae L Figure 2.13: The clotting cascade- Intrinsic, exteosi andthe final corazon pathway and the Fibrinolysis The Intrinsic Pathway Begins in the blood stream. Its basically activated when blood is exposed to collagen or other damaged surfaces. Factor XII is activated to Xila by exposed collagen. XIla, with the help of HMW kininogen, activates XI to Xla, This can proceed more quickly in the presence of prekallikrein. Xa combines with calcium, and activates IX to IXa. Simultaneously, platelets will release PF3, and also simultaneausly, VIII will be activated to Vita. IXa, (with the help of PF3) will join together with VIIa and form factor X activating factor (‘tenase) aneaa [E_ Principles & practice of Transfusion Medicine ‘The Extrinsic Pathway {i begins in the vessel wall, Damaged endothelial ces will release factor III (tissue factor), and the greater the amount of damage, the more is released. This combines with calcium, and activates factor VI and turns it into factor VIIa. ‘The Common Pathway Factor X is activated, either by VIla or tenase , to form Xa. Factor Xa, with the help of calcium ions, and Va will turn prothrombin into thrombin. Thrombin is factor Tla, Factor V is not activated until it has come into contact with thrombin itself. Thus V is not required for this step, but when present will increase the rate. Thrombin will then convert fibrinogen to fibrin. Fibrin strands will begin to join together, and with the help of XIffa this will cause the cross-linking of fibrin strands, Factor XIII is also activated by thrombin, Once activated, thrombin can act as a ‘catalyst to activate factor 5,8, 9 and platelets, hence the pathway sustains. ‘The Cell Based Model of Coagulation ‘The clotting cascade is activated by tissue injury and low levels of circulating activated clotting factors. It is then propagated by enzyme complexes that efficiently assemble on the phospholipid membrane surface supplied by platelets. Haemostasis according to the cell based model occurs in three (overlapping) phases (Figure 2.14). {Initiation ‘Tissue factor bearing cell 2, Amplification 5. Propagation Figure 2.14: The phases of cell based model of coagulation Initiation Phase + The initiation of coagulation takes place on Tissue factor (TF) bearing cells, such as the fibroblast and TF is expressed on a variety of extravascular cells under normal conditions, and can also be expressed by blood monocytes and endothelial clls in inflammatory states. ryrry eeePhysiology of Blood 4 During the process of haemostasis, a break in the vessel wall allows plasma to come into contact with TR-bearing extravascular cells, Factor VI in plasma bina’ tightly to cellular TF and is rapidly activated by coagulation and non-coagulation proteases to activated factor seven (VTia). The factor Vila/TF complex activates both factor X and factor IX. Factor Xa can activate plasma factor V. The factor X activated by the Jactor Vila/TF complex is rapidly inhibited by Tissue Factor Pathway Inhibitor (TFPI) or antithrombin-III (ATIID) if it leaves the protected environment of the cell surface. However, the factor Xa that remains on the cell surface can combine with factor Va to produce sinsll amounts of thrombin which plays an important role in subsequently activating platelets and factor VIII during the araplification phase. Amplification Phase + Damage to the vasculature allows platelets as well as plasma to come into contact with extravascular tissues, Platelets adhere to extravascular matrix components at the site of injury. The process of binding to matrix proteins partially activates platelets, as well as localizes them near a site of ‘TE exposure, 4 Small amounts of thrombin generated on TP-beasing cells amplify the initial procoagulant signal by enhancing platelet adhesion, fully activating platelets and activating factors V, VIII and XI. “Thus, thrombin acts on the platelet surface to “set the stage” for procoagulant complex assembly. During activation, platelets telease factor V from alpha granules onto their surfaces in a partially activated form. Factor V a is then fully activated by thtarnbin or factor Xa. ‘Platelet activation increases external membrane expression of the negatively charged, procoagulant phosphatidylserine (PS) moiety, the PS on the activated platelet surface enhances both the tenase and prothrombinase reactions. Factor ¥ (fvom platelets) appears to significantly contribute to overall Factor V function, as noted in Factor V-deficient patients after platelet transfusion, Platelet receptor binding simultaneously protects activated factors from circulating inhibitors and orients factors on the plasma membrane such that they interact closely with PS to accelerate thrombin generation. * Von Willebrand factor (vWP)/factor VII binds to platelets and is efficiently cleaved by thrombin to activate factor VIIL and release it from WWE, The factor VIM remains bound to the platelet surface. Now that the platelets have been activated and have activated cofactors V and VIII bound to their surfaces, assembly of the procoagulant complexes and large-scale thrombin generation begins, Propagation Phase ‘® During the propagation phase the “tenasc’ and “prothrombinase” complexes are assembled on the platelet surface, and large-scale thrombin generation takes place, Platelets express high affinity ‘binding sites for factor IX(a), factor X(a), V(a) and factor XI. The “tenase” (factor VIlla/IXa) complexes assemble when factor IXa reaches the platelet surface. The factor IXa can diffuse to platelet surfaces from its site of activation on TF-bearing cells, since itis not rapidly inhibited by Anti Thrombin III or other plasma protease inhibitors, In addition, plasma factos XI can bind to activated platelets, facilitating its activation by thrombin. ‘@ The factor Xla can then provide additional factor (Xa directly on the platelet surface. The factor 1Xa/Villa (tenase complex) activates factor X on the platelet surface where the resulting factor Xa can move directly into a complex with its cofactor, factor Va, as illustrated in Figure 2.14. The ane Port yoE Principles & Prectice of Transfusion Medicine platelet surface factor Xa/Va complexes along with Ca** and platelet membrane PS (prothrombinase complex) can now produce the burst of thrombin necessary fo form a haemostatic fibrin clot ‘This rapid thrombin generation cleaves fibrinogen to fibrin monomers, which combine to form a fibrin matrix integrated with adherent platelets. Factor XIIla, produced by the action of thrombin on either plasma or platelet released Factor XIII, converts the soluble fibrin clot into an insoluble fibrin polymer and effectively incorporates a2-antiplasmin into the clot to inhibit plasmin- ‘mediated dissolution. Finally, the platelet plug undergoes clot retraction, which further protects the platelet fibrin matrices from lysis by plasmin Thrombolysis Plasmin is a proteolytic enzyme that is capable of breaking cross-links between fibrin molecules, which provide the structural integrity of blood clots. Plasmin is produced in an inactive form, plasminogen, in the liver, Although plasminogen cannot cleave fibrin, it still has an affinity for it, and is incorporated into the clot when itis formed. ‘issue plasminogen activator (t-PA) and urokinase are the agents that convert plasminogen to the active plasmin, thus allowing fibrinolysis to occur. t-PA is released into the blood very slowly by the damaged endothelium of the blood vessels, such that, after several days (when the bleeding has stopped), the clot is broken down. This occurs because plasminogen became entrapped within the clot when it formed; as it s slowly activated, it breaks down the fibrin mesh, {PA and urokinase are themselves inhibited by plasminogen activator inhibitor-1 and plasminogen activator inhibitor-2 (PAI-1 and PAI-2). In contrast, plasmin further stimulates plasmin generation by producing more active forms of both tissue plasminogen activator (tPA) and urokinase. Alpha 2-antiplasmin and alpha 2-macroglobulin inactivate plasmin. Plasmin activity is also reduced by thrombin-activatable fibrinolysis inhibitor (TAF1), which modifies fibrin to make it more resistant, to the tPA-mediated plasminogen, Natural Anticoagulants Clotting also is controlled by three general categories of natural anticoagulants, The most important natural anticoagulants are protein C & S, antithrombin and TFPL Antithrombins (eg, antithrombin II) inhibit the activity of thrombin and other serine proteases, namely factors Xa, Xa, Xla, and Xila, Antithrombin III is activated by binding to heparin-like smoleciles ont endothelial cells - hence the clinical utility of heparin adminjstration to limit thrombosis. Protein C and protein S are two vitamin K-dependent proteins that act in a complex to proteolytically inactivate cofactors Va and Villa. Protein C activation by thrombomodilin was described earliess protein § is a cofactor for protein C activity (Fig. 3-6). ‘Tissue factor pathway inhibitor (TFPI) is a protein secreted by endothelium (and other cell types) that inactivates factor Xa and tissue factor—factor Va complexes. e260 TTTPhysiology of lod RED CELL AND PLATELET METABOLISM. ‘The RBCs are highly dependent on glucose as its energy source. Its membrane contains high affinity glucose transporters. ‘The entry of glucose is through facilitated diffusion, Seven such related transporters have been isolated from various tissues. 90% of ATP synthesis in RBC takes place through glycolysis (Figure 2.15), 10% occurs through Pentose Phosphate Shunt/Pathway (PPP; Figure 2.16) There are no mitochondria in RBC and hence there is no ATP production by oxidative metabolism PPP has two major sales: a) formation of NADPH which helps maintain glutathione in reduced state (Figure 2.17) and prevents oxidative stress in RBCs b) synthesis of ribose for nucleotide and nucleic acid formation. Giu008 pep Locks glucose in eet Hexokinase io Ginn phate Popa omen | fs epee Fructose-6phosphate ATP os i Ads phosphate to prepare fr cesvage app Frcs, ipboapate Aldolase Cleavage Dibyzonyacetone phosphate «EE on 4. phosphate Those phate amare aes ge podct iL =] Ghyerldehyde-3-phophate f° rctvates dehydrogenase Pte 1s Rephshghete Fit ATP of tore Byes Kate hs Psphogheomatae [cae rs a Acinates cand phosphate mm | ie ‘ADP Second ATP of lyolysls Pyne kinase aa ATP x a ©0)-<——— Pynvate — Lactate Lactate dehydrogenase aerobic metabolism anaefobic metaboligm Figure 2.15: Re cell metabolism: Giyeolysis bare I pa) i neem |i Principles & Practice of Transfusion Medicine lucose6-phosphate ne gfacore phosphate nappa 4 hvdtogenase Trans3{dolase erythrose 4-phosphate ~ ‘Transketolase elyctraldchyde 3-phosphate <—————— | sedoheptulose Sphosphate “glyceraldehyde 3-phosphate fructose 6 phosphate 6phosphoglconte i 1 6- phosphogluconate wanna» es bus phosphate ew, ribose S-phophate \ ays Sphosphate Trangolae Figure 2.16: Pentose Phosphate Pathway or Hexo ce Mono Phosphate Shunt way 7 XS Clatathione reductase Glutathione peroxidase (GSSG) Ovidized Glutathione ‘igure 2.17: Glutathione metabolism in red cells Platelets {mn platelets the energy production in resting tate is by glycolysis (igure 2.15) and by oxidative Krebs cycle (Figure 2.18). There are about 10-6 0 mitochondna per platelet and glycogen is the principle source of energy for metabolism, $286Ql. Pyruvate | 780A i" Pyruvate deydrogenase oo Acetyl CoA HO ae NADHHY \Osato-acetate cit Crate Synthase map) ase rate dysgesse . Malate sap 4 | marae Ibocitra | rogenase | no | ° vADHHY Oxalors Fumarate x i iydrogenase Succinate dchySeogenase ee FADH, —. Coast « y FAD succina cc-ketogit Coan ff co, \ wietoai SL GTP a succinyl Coa synthetase ee ee cae ppivi Figuse 218: Krebs eye rT citric. A) eyde SELF ASSESSMENT & REVIEW EXERCISE ‘The person should be able to answer these questions after ree nis chapter Which ofthe following is false: 1. Classical pathway of complement is activated by imn omplexes 2. Alternate pathway of complement activation is ac»! by antige at directly on pathogen surface. 3, Factor B and D are involved in the classical pathway, 4. All pathways of complement activation ultimately formation «°. -mbrane attack complex. 6206 TTT. nays NEQo. Principles & Practice of Transfusion Medicine Name the EPO dependent stages of erythropoiesis? Discuss the mechanism of action of EPO? t. Explain Iron metabolism using a well labelled diagram? What is hepcidin and what is its role in iron metabolism? j. Discuss the structure of haemoglobin. Explain the principle metabolic pathways for Red cells and platelets using diagrams? 3. Explain the structure of platelets? What are the various types of granules present in the platelets and their contents? Explain the cei based model of coagulation? QUO. What are reticulated platelets? 4308Introduction ‘The knowledge of basic concepts of immunology especially about antigens, antibodies, antigen- antibody reaction, complement factors affecting the antigen antibody reaction and basic genetics is important in understanding the principles of Immunohaematology. Immune System ‘The immune system is made up of special cells, protein, tissues, and organs that defend individuals against germs and micro-organisms every day. Through a series of interrelated mechanisms called the immune response, the immune system attacks organisms and substances that invade our system. Cells of the Immune System The cells that are part of this defence system can form lines of non specific or specitic defence, The cells of the immune system are derived from pluripotent stem cells that are located within the bone marrow, foetal liver and yolk sac of the foetus. These cells ate found in blood, thymus, spleen, liver, lymph nodes and tissues. The macrophage-monocyte cell system and B and T lymphocytes form the backbone of body's defence mechanisms. Other cells include the NK (natural killer) cells and the newly addressed dendritic cells. Monocyte Macrophage Cell System Blood monocytes ase produced by the bone marrow stem cells. They migrate to the site of inflammation and to various tissues and diferentiate into tissue macrophages. They are often referted to as scavengers or Antigen-Presenting Cells (APC) because they pick up and ingest foreign material and present these antigens to other cells of the immune system such as T cells and B cells. Differentiation of classically activated macrophages requires a priming signal in the form of interferon (IFN) gamma. When the primed macrophage subsequently encounters an appropriate stimulus, such as bacterial lipopolysaccharide, it becomes classically activated. Pathogens and pathogen components are subsequently taken up by phagocytosis and delivered to lysosomes where they are exposed to a variety of degradation enzymes including several cathepsin cysteine proteases. Suitable antigens are processed and loaded onto MHC class If molecules in late endocytic compartments and antigen/MHC complexes along with co-stimulatory B7 family members are presented to T cells, oan CTT ae RTB_ Principles & Practice of Transfusion Medicine Dendritic Cells ‘The dendritic cells, originate in the bone marrow, function as antigen presenting cells (APC). Dendritic cells are profesional antigen presenting cells that are most powerful stimulators of naive T cells. They play a key role in the initiation of immune responses Leucocytes are produced and stored in many locations throughout the body, inchuding the thymus, the spleen, and the bone marrow. For this reason, these organs are called the lymphoid organs. There are also clumps of lymphoid tissue throughout the body, primarily in the form of lymph nodes, which houses the leucocyte, Leucocytes circulate throughout the body between the organs and nodes by means of the lymphatic vessels and also circulate through the blood vessels, In this way, the immune system works in a coordinated manner to monitor the body for germs or substances that might cause problems. ‘nce foreign substances (antigens) invade the body, several types of cells work together to recognize and respond to it. These cells trigger the B lymphocytes to produce antibodies, which are specialized proteins that lock onto specific antigens. Antibodies and antigens fit together in a lock && key fashion Once the B lymphocytes have produced antibodies, these antibodies continue to exist in a person's body, so that if the same antigen is presented (othe imamune system again, the antibodies are already there to do their job. This is also why we use immunizations to prevent geting certain diseases, Although the antibodies can recognize an antigen and lock onto it, they are not capable of destroying it without help. That is the job of T cells. The T ces ae part of the system that destroys antigens that have been tagged by antibodies or cells that have been infected or somehow changed. (This group of T cells is called “killer cells”), T cells ate also involved in helping signal other cells (like phagocytes) to do their job. Antibodies can also neutralize toxins (poisonous or damaging substances) produced by different organisms. Lastly, antibodies can activate a group of proteins called the complements that are also a ‘att of the immune system. Complements assist in killing bacteria, viruses, or infected cells. Leucocytes ‘The two basic types of leucocytes are: 1. Phagocytes, cells that engulf the inwading organisms. The most common type is the neutrophil which primarily fights bacteria 2, Lymphocytes, cells that allow the body to remember and recognize previous invaders and help the body to destroy them, There are two kinds of lymphocytes: the If lymphocytes and the T lymphocytes. Lymphocytes start out in the bone marrow and either stay there and mature into B cells, or leave for the thymus gland, where they mature into T cells. B lymphocytes and T lymphocytes have separate jobs to do: B lymphocytes are like the body's military intelligence system, seeking out their targets and sending defences to lock onto them, T cells are like the soldiers, destroying the invaders that the intelligence system has identified. 320 PTT: apne na eAbd Basic Principles of Immunohaematology Natural Killer Cells (Null Cells) ‘The Natural killer cells, often referred to as NK cells, are members of nonspecific defence ‘mechanism, They function as effector cells that directly kill certain tumor cells such as melanomas, lymphomas and virus-infected cells, most notably herpes and cytomegalovirus-infected cells. NK cells, unlike the CD8+ (killer) T cells, kill their targets without a prior “conference” in the lymphoid organs, Jmmanity Allof these specialized cells and parts of the immune system offer the body protection against diseases. ‘This protection is called - The Immunity. Humans have three types of immunity — innate, adaptive, and passive: Innate Immunity ‘The innate or the natural immunity is present since birth 8 is the first line of defence. It protects the individual from foreign invaders & does not change on repeated exposures to the same antigen, thus considered as non specific. The innate immunity also includes the external barriers of the body, like the skin and mucous membranes (like those that line the nose, the throat, and the gastrointestinal tract), which are our first line of defence in preventing pathogens from entering the body. If this outer defensive wall is broken (like if one gets a cut), the skin aitempts to heal the break quickly and special immune cells on the skin attack the invading germs. Other members of innate immunity are interferons, NK cells & C-reactive proteins, Adaptive Immunity Adaptive irmmmonity has the ability of specificity, recognition, memory and specific reactivity. This type of immunity develops throughout our lives, Adaptive insmunity involves the lymphocytes and develops as children and adults are exposed to diseases or are immunized against them through vaccination, (a) Cellular Immunity Cell mediated immunity or cellular immunity is a localized reaction to an organism, usually an intracellular pathogen, roediated by the lymphocytes and the phagocytes rather than by antibody production, [he T-cells and the macrophages play a major role in cell mediated immunity, either as a result of direct cytotoxicity or through the liberation of lymphokines, Cell mediated cytotoxicity is important in the lysis of virus infected cells and rejection of allo-graft and tumor cells. Other cytotoxic cells involved in this type of immune response are the null cells (NK). (b) Humoral Immunity ‘This form of immunity originates in the B-cells, the specialized cells which come from the bone marrow. B-cells are designed to produce antibodies when stimulated to do so, most commonly by ‘Tcells which recognize antigens and trigger the production of antibodies by the B-cells. The B-cells essentially turn into little antibody factories in the blood, floating around to mop up as many of the invaders as possible. e336 1Principles & Practice of Transfusion Medicine When people develop problems with their humoral immunity, they are more susceptible to developing infection and disease. Conditions like HIV attack the immune system directly to make it less functional, and humoral immunity can also be compromised with the use of certain medications, such as chemotherapy and the drugs used to prepare people for organ transplant. Passive Immunity Passive immunity is “borrowed” from another source and it ass for a short time. For example, antibodies in breast milk provide an infant with temporary immunity to diseases that the mother has been exposed to. This can help to protect the infant against infections during early years of childhood Immune Response ‘The response of the body, when first exposed to a foreign antigen is called primary response. ‘This usually results in production of small amount of circulating antibodies stowly and may take weeks to months before any antibody is demonstrable. Once the person who has been sensitized with a particular antigen, is given a subsequent exposure of the same antigen, it results in secondary response. “This response is much faster and results in production of a large amount of antibody. ‘The primary response often results in the production of IgM antibodies while the secondary response will produce mainly IgG antibodies (Figure 3.1), Secondary eigonse inary response ‘First Second va epithe Ste Figure 3.1; Primary and secondary antibody response Antigens An antigen is @ substance which, when introduced in the body will elicit an immune response ie. results in production of an antibody that will react specifically with that antigen in some observable way. The substance should have a molecular weight (M.W.) more than 40,000-50,000 to act as an antigen, Substances below 40,000 M.W. are called haptens. These generally fil to act as antigens unless coupled to a carrier protein of the size approximately more than 10,000 M.W. Exogenous Antigens A foreign antigen that enters the body is engulfed by the antigen presenting cells (APCs) namely the macrophage, B cell and the dendritic cells. The antigen is processed into’ smaller fragments and expressed on the surface of the APC with the help of MHC class I or II as the case may be. (Macrophage presents it via MHC { and B cell via MHC class II and dendritic cells via both MHC class I and MHC class IL.) Interactions between the MHC complex presenting the antigen and T o3ae ——Basic Principles of immunchaematology helper cells (TH cells) occur thereby activating the T helper cells (CD4+ T cells). Activated helper T cell then differentiates into TH 1 or TH 2 and releases various cytokines. Cytokines are small secreted proteins which mediate and regulate immunity, inflammation, and haematopoiesis. They must be produced de novo in response to an immune stimulus. They generally (although not always) act over short distances and short time spans and at very low concentration. They act by binding to specific membrane receptors, which then signal the cell via second messengers, often tyrosine kinases, to alter its behavior (gene expression). Responses to cytokines include increasing or decreasing expression of membrane proteins (including cytokine receptors), proliferation, and secretion of effector molecules. ‘The THI cells signal the macrophage to destroy the phagocytosed antigen and also cause the activation of cytotoxic T cells. Activated eytatoxic T cells proliferate and differentiate into mature cytotoxic T cells, These bind to infected cells presenting antigen on their surface with MHC class I and cause their destruction. ‘The TH? helper cells interact with B cells causing their proliferation and differentiation. These B cells then produce antibodies responsible for humoral immunity. Endogenous Antigens Antigens that are generated within a cell (cg, viral proteins in any infected cell) are degraded into fragments (e.g. peptides) within the cell and displayed at the surface of the cell nestled within a class I histocompatibility molecule, Here they may be recognized by CD8+ T cells (cytotoxic T cells). Blood Group Antigens ‘he red cell membrane carries blood group antigens which are minute glycoproteins and glycolipids of sufficient molecular weight to act as antigens. The specificity of the blood antigens has been shown to be determined by the sequential addition of sugar residues to a common precursor substance as a result of gene action. The precursor substance, which is located on or within the red cell membrane, is composed of four molecules of three different sugars, viz. D-galactose (2 molecules), Galactosamine (1 molecule) and N-acetyl glucosamine (one molecule). There are two main types of precursor substances known as Type 1 and Type 2 which differ in the terminal linkage (see Chapter 3 on ABO Blood Group Systeri). The action ofthe gene causes the production of an enzyme which in turn causes the addition of another sugar to the basic precursor substance, which determines the specificity of the antigen. For details see the ABO Blood Group System (Chapter 3). ‘The number of antigen sites on the red cells vary according to specificity. Thus there are approximately 1 million ABO antigen sites and 25,000 Rh (D) antigen sites on the red cells, The red cell antigens are present not only of the red cells but some have also been detected on leucocytes, platelets, in body fluid, saliva, milk, seminal fluid, plasma (Lewis antigens) and in most tissues of the body. Antigens of some specificities are confined wholly on the red cell membrane (e.g, Rh antigens). ‘The antigens on the red cells are not always constant throughout life; some antigens are poorly developed at bisth (e.g. 1, Lewis). Some casi be altered in certain disease states e.g. Acquired B antigen in AL individuals usually resulting from the action of gram- negative organisms. A or B antigens may bbe weakened in patients with leukemia. Changes in blood group antigens have also been noted during pregnancy (eg. Lewis) 0356Principles & Practice of Transfusion Medicine Antibodies Antibodies are a group of serum proteins, produced in response to antigeni¢ stimulation and are designated as immunoglobulins (Ig). They are found in the gamma globulin part of plastna proteins. ‘There are five classes of immunoglobulins designated as IgM, IgG, IgA, IgD & IgE of which IgM, IgG & IgA are mainly involved in blood group serology. Table 3.1 gives differences between IgG, IgM & IgA antibodies whereas types of variation is given in table 32. Table 3.1: Dlferences between 16, IgM and IgA immunoglobulin (CHARACTERISTICS ir tg oA Structure ‘Monomer Pentamer Monomer Y shaped (Five leqged srider) (serum Dimer | (secretory) Molecule wt 150000 900000 150000 (Monomer) 300000 (dimer) Sedimentation coeficien | 7S 198 [7s Placental transfer Yes No to Serological behavior incomplete __| complete complete Complement fixation Yes Yes No React optimally st 3PC [20° 37°C J All immunoglobulins have two main features in common: Basic structure of the molecule ‘@ Function of the molecule ie. ability to combine specifically with the corresponding antigen ‘Table 3.2: Types of variations in Immunoglobulins Type of | Refers to ‘Examples Variation Isotypes | isotypes result rom structural changes to the constant region heavy and light | (IQA, IgD, IgE, chains. Genes encoding the various isotypes are ail present inthe human IgG, IgM, x, | genome, and A) Antibodies for isotype determination can be prepared by heterologous, immunization (eg, mouse immusnization with human serum). ‘Allotype | Variations in the amino acid structure of heavy and ight chains unreicted ta [ Gm and Kin ‘antibody specificity. Present in some but not all members of the species, allotypes Antibodies to allotypic variants cam be raised by same-species (homologous, allogeneic) immunization { Idiotype | idiotypic variation isthe result of differences in amino acid sequence of the | Fab regions: variable regions of either heavy or light chains. of antibody Antibodies to idiotypes may recognize sequences either within or outside molecules ‘he antigen-combining site, and can be prepared both by homologous or * heterologous immunization. 4366Basic Principles of immunohaematology Structure of Immunoglobulin Molecule Immunoglobulins are formed of amino acid molecules, linked together by peptide bonds forming amino acid chains. There are two types of chains ie. Light Chains and Heavy Chains. The basic immunoglobulin unit consists of two identical heavy chains (molecular wt. 55, 000) and two identical light chains (molecular wt. 22,500). The four chains are held together by covalent (disulphide) and non covalent bonds to give a “Y” configuration (Figure 3.2). Fab aie 1 Ag ‘wink | ate ‘Ag binding 1 binding Position | Position site site ' Constant {Constant Portion 1 Potton Fe fragment Figure 3.2: Basic Structural Unit of Antibody Molecule ‘The light chains belong to two antigenically different types ie. kappa (K) and Lambda (L) but in any given (Ig) molecule, the two light chains are identical being either Kappa or Lambda. The locus for ‘manufacture of kappa light chains is on chromosome 2; that for lambda chains is on chromosome 22. A single cell clone will synthesize immunoglobulin containing either kappa or lambda chains but never both. The heavy chains of the molecule are different for each class and accordingly the heavy chains of IgA ate alpha (a), of IgG-gamma (y), of IgM-mu (1), of IgD delta (8) and of IgE-epsilon(e). The gene for heavy chain is on chromosome 14 and it contains DNA sequences forall these five peptide sequences, a ‘multiplicity reminiscent of the libraries of V, D, and J sequences available for variable-region structure. ‘A major difference in selection at variable and constant region is that selection of one constant-region segment for expression does not automatically eliminate all the others, whereas rearrangement of variable-region DNA is a one-time event that cannot subsequently be modified, When an unstimulated B cell activates one constant-region segment, this does not prevent its clonal progeny from later synthesizing heavy chains with different constant- region sequences a phenomenon known as isotype switch, As an example we can see that an antibody directed against the surface antigen of Hepatitis B always remains as anti-HBs (result of selection at the variable region), though it is initially IgM in ature it undergoes isotype switch to become IgG type over a course of few months. 6376 — syaa EE Principles & Practice of Transfusion Medline Digestion of an immunoglobulin molecule with enzyme papain spits it into three fragments, 2 Fab fragments. 1 Fe fragment. Fab fragment has antigen binding capacity. Fe Fragment has following functions © Complement fixation Placental transport 4 Reaction with antihuman globulin reagent IgG Antibodies IgG antibodies make up approximately 73% of total immunoglobulins in the body and exist as monomers (Figure 3.3). Light Chain Heavy Chain Figure 33: Structure of IgG Molecule IgG antibodies are smaller molecules with a molecular weight of 150,000 and can readily cross placenta, thus causing the haemolytic disease of new born (HDN). IgG antibodies are also called as ‘incomplete antibodies as they do not cause agglutination of red cells with the corresponding antigens in saline. These antibodies cause only sensitization or coating of the red cells. The life span of Ig antibodies is approximately 60-70 days (T, = 22 days). The optimum temperature for the reaction of. IgG antibodies is 37°C. IgM Antibodies IgM antibodies comprise about 8% of the total immunoglobulins in the body and exist as a pentamer (Figure 3.4). Itis a large molecule with molecular weight of 900,000 and cannot cross the placenta. These antibodies readily agglutinate red cells carrying the corresponding antigens in saline and are also known as, complete antibodies. They offen activate complement during an antigen antibodies reaction, thus causing haemolysis of the red cells rather than agglutination. They have a life span of 10 days. o38e an cop}@ = Basic Principles of Immunohaematology i igure 3.4 Structure of IgM Molecule IgA Antibodies IgA is usually present as a monomer in serum, However, in external secretions IgA occurs as a dimer, the two IgA units being linked to a small glycoprotein known as secretary piece or T component. This makes it resistant to proteolytic enzymes. Naturally Occurring Antibodies Under normal circumstances, the new born has by & large no ABO antibodies. However 10-20 weeks after birth, moderate amount of ABO antibodies are present in the serum. These appear without any specific red cell antigenic stimulus. Because ofthis, these antibodies are commonly called as naturally ‘occurring antibodies. Now it is known that certain bacteria, viruses and foods contain red cell antigen like substances, which are responsible for the production of these antibodies. These antibodies are usually IgM in nature and are present in the individuals lacking the corresponding antigens on the red cells. Immune Antibodies Immune antibodies are usually IgG and are due to immunization either due to pregnancy or transfusion, Monoclonal Antibodies Antibodies selectively react with specific molecules or antigens and thus provide useful reagents for detecting molecules of interest. Antibodies are generated by immunizing a host animal with a foreign molecule or antigen, ‘The cells that respond to and recognize an antigen are called B lymphocytes (B cells), Each B cell produces a unique antibody directed against a single epitope or site on the foreign antigen. Because there are many different B cels in the body, the sera of immunized animals will contain a mixture of antibodies to the antigen. These antibodies are called “polyclonal” because they consist of a variety of antibodies recognizing different sites on the antigenic molecule. It is also possible to create a clonal cell line that can produce large quantities of single “monoclonal” antibody that recognizes a single, specific epitope of an antigen of interest. Hybridoma Technology for Monoclonal Antibody Production ‘The first step in making a hybridoma is to generate antibody producing B cells. This is done by immunizing a mouse against the antigen of interest. Typically, multiple immunizations are performed 43906 tn amee +__ Principles & Practice of Transfusion Medicine over a period of weeks until an appropriate titer is achieved, Intraperitoneal (IP) injections are the ‘most common method for delivering the antigens into the mice. Next, it must be determined if the mouse is producing the antibodies of interest. Test bleeds are performed and examined for the presence of antibodies. Some B cells produce the antibodies that bind to the specific epitope on the antigen of interest; others will prodace “nonspecific” antibodies. Ifthe mouse is producing the desired antibody, its spleen is removed and dissociated in culture medium to release the resident B ces. The culture medium also inchudes cells feom special mouse myeloma cell line. These tumor cells can divide indefinitely but cannot produce antibody. When polyethylene glycol (PEG) is added to the mixture, some of the two types of cells fuse to form a hybridoma, The next step is to separate the fused hybridoma cells from the unfused B cells and myeloma cells. Unfused B cells will die because they lack the ability to survive in culture, leaving only ‘he hybridomas and unfused myeloma cells. ‘The myeloma cells lack an important enzyme, HGPRT (hypoxanthine-guanine-phosphoribosyl- transferase) causing them to die when placed in a special culture medium called Hypoxanthine, Aminopferin, and Thymidine (HAT) medium. In this medium, only fused cells survive because Aminopterin in the medium blocks the de novo purine synthesis pathway. Hence, unfused myeloma calls die, as they cannot produce nucleotides by de novo or salvage pathway. Unfused B cells die as they have a short life span. tn this way, only the B cell-myeloma hybrids survive, These cells produce antibodies (a property of B cells) and are immortal (a property of myeloma cells). The incubeted medium is then diluted inte multi-well plates to such an extent that each well contains only 1 cell. ‘Then the supernatant in each well can be checked for desired antibody. Since the antibodies in a well are produced by the same B cell, they will be directed towards the same epitope, and are known as ‘monoclonal antibodies. (Figure 3.5) Once a hybridoma colony is established, it will contiswally grow in culture medium like RPMI-1640 (with antibiotics and foetal bovine serum) and produce the antibody. ‘The next stage isa rapid primary screening process, which identifies and selects only those hybridomas that produce antibodies of appropriate specificity, The hybridoma culture supernatant, secondary enzyme labelled conjugate and chromogenic substrate, are then incubated, and the formation of « coloured praduct indicates a positive hybridoma, Alternatively, immunocytochemical screening can also be used. 4406gE tsicrmense omuamaniay Mouse immunized Mutant cell ine derived fom a tumor of B lymphocytes Cell making : anti X antibody (calls grow indefinitely in normal B lymphocytes die afer medium, but ge in selective mediam) 2 few days in culture FUON rod pated ample wel Lamers) Wenge] Wee) rat (ale ee allow cells to multiply, then test | supernatant for anti-X antibodies: positive clones provide A continuous source of anti-X antibody ‘iguce 3.5: Production of monoclonal antibodies Newer Approaches to Monoclonal Antibody Production 1. Heterohybridomas: This is a way of generation of human antibodies by taking peripheral blood lymphocytes from individuals sensitized to the RhD antigen and immortalizing them by infecting with Epstein-Barr virus (EBV). Such EBV transformed cells can also be fused with mouse myeloma cells (heterohybridomas) to give better results, 2. “Humanized” Mice: Mice with severe combined immunodeficiency (SCID) can be humanized by infusing human peripheral blood lymphocytes (PBL). Responses have been obtained to tetanus toxoid and group A and Rh-D antigens. This strategy is currently experimental but with modification may produce useful antibodies inthe future, 3. Genetic Engineering: Monoclonal antibodies may be genetically engineered so that the antigen- specific binding site (hyper variable region) is of rat and mouse origin but the remainder of the antibody is human. This procedure has the potential advantage of minimizing the development of anti-mouse/rat antibodies- an important consideration when these are used therapeutically, one nae raya)E Principles & Practice of Transfusion Medicine Antigen Antibody Reaction ‘The most commonly observed antigen antibody reactions which occur in blood group sesclogy (immunohaematology) are as follows: & Agglutination @ Sensitization Haemolysis ‘& Neutralization (Inhibition) ‘The other antigen antibody reactions which can occur in blood group serology, but are rarely used in immunohaematology laboratory are: © Precipitation © Complement fixation immunofiuorescence Radio-immunoassay ELISA (Enzyme linked immunosorbent assay). Agglutination Itis the most common type of antigen antibody reaction observed in blood group serology and is due to the clumping of red cells as an end result of antigen antibody reaction. It occurs in two stages. 4 Sensitization (attachment of antibody) + Agglutination (due to lattice formation) Sensitization is defined as the simple coating or binding of an antibody on the surface of red cell antigen, without bringing about agglutination of red cells in saline (Figure 3.6). As there is no visible agglutination or haemolysis seen in this reaction, an alternative method may be used to demonstrate this type of antigen antibody reaction ie: The use of albumin & The use of proteolytic enzymes + The use of antighobulin reagents Haemolysis Many blood group antibodies on reacting with the antigens on red cells activate the complement resulting in the destruction of red cells with release of haemoglobin from the cells. Antibodies that have this capacity are called haemolysins. In the test system haemolysis of the cells is recognized by the haemoglobin tinged supernatant after centrifugation of the test mixture. IgM antibodies are predominantly complement fixing while IgG antibodies rarely bind complement. | eae[seu Basic Principles of (mmunohaematology Figure 3.6: Red ell sensitization with 1gG Neutralization Soluble form of blood group substances (antigens) have the effect of neutralizing (or inhibiting) the reaction of corresponding blood group antibody. For this type of reaction, soluble antigens are added 1 the serum containing antibody. If the strength of antibody decreases or if it disappears completely, an antigen-antibody reaction can be assumed to have taken place. Mixed Field Agglutination and its significance ‘Haemagglutination involves red blood cells. This is usually due to an antibody or other molecule binding with multiple particles, and joining them. Depending upon strength of reaction, atest reaction is graded and given a score: (Table 3.3) ‘Table 3.3: Grading of agglutination reaction Grading of agglutination reaction tees ‘Complete agglutination of al cel, single large agglutinate, No fee cells tet ‘Numbers of large agglutinates. No free cells ++ ‘Small to medium clumps with few free cells + Fine gramuar appearance visually but definite small clumps per low power fed -ve No agglutination When some cells are agglutinated while others are unagglutinated, it is graded as mixed field agglutination. Mixed field agglutination is best appreciated in the gel card (Figure 3.7) ae [Lae Tl ae] | ae] ey] | 0 fl me Pos Pos Pos Pos Weak Neg Mie field igure 37: Mise Geld reaction in Gl a Significance : 1. Usually mixed-field agglutination means a MIXED-CELL POPULATION. 2. One shoutd try to find cause of mixed field agglutination before selecting blood to transfuse by studying the clinical history and diagnosis, 436 ee rn He NCERSNN A Ya anea fi Principles & Practice of Transfusion Medicine Causes 1, In-cases, when blood samples contain two distinct, separable populations of RBCs, usually because group © RBCs were transfused to a group A (or group B) patient, a mixed field agglutination is seen, 2. Mixed field agglutination occurs when a patient has received a transplant of bone marrow that is ‘of an ABO group different from the patient's own. Such patients may have both type of cells, some of their original type and the type of the bone marrow transplant, 3. DAT may give a mixed filed appearance ina recently transfused individual, where both unsensitized recipient and sensitized donor cells are present in the recipient’ cieculation. 4, Mixed feld agglutination may be seen with RBCs carrying A antigens weakened by diseases such as leukemia or lymphoma. 5. Mixed field agglutination may be seen with Ta polyagglutinable RBCs, ‘these result in persistent ‘mixed field polyagglutination ofthe T-type. Extracts of the seeds of Dolickos bifforus and Bauhinia ‘variegata strongly agglutinate Th-polyagghutinable calls Positively charged polymers such as polybrene aggregate normal erythrocytes but not those deficient in sialic acid such as T- or Tn-polyagglutinable cells. This observation forms the basis of the method of separating polyagglutinable from non-agglutinating cells in a mixed field Ta- polvagglutination, 6. Mixed fied agglutination is characteristically seen when weak subgroups of A and B ie. A3 (and B3) RBCs are tested with anti-A and anti-B. If the agglutinated RBCs are removed and the remaining RBCs again tested with the corresponding antiseras, mixed field agglutination occurs in the residual population as well. 7. Permanent Chimerism due to exchange of blood in twins in utero because of vascular anastomosis, or due to dispermy ie. when two sperms fertilize one egg, gives mixed field agglutination reaction. Complement Complement is a complex group of serum prateins capable of interacting sequentially with the bound antibody and causing biological effects such as immune adherence, phagocytosis and cell Isis. It is often involved in blood group reactions and immunological disorders. The constituent proteins of the system work together to assist, or “complement” the action of antibodies in destroying the bacteria. ‘They form approximately 5 % 10 % of plasma globulins. They are components of the acute phase response and their concentration in blood is increased during infections, injuries, and traumas, The ‘complements ase numbered in the order in which they were discovered. ‘The activity of complement depends upon nine secum protein components (C1-C8) which are present in blood as functionally inactive molecules. The components of the complement are labile and are destroyed, by beating serum at 54°C for 30 minutes, by normal serum inhibitor and as a result of storage bane emma alan ealBasic Principles of immunoheematology History Jules Bordet, in 1896, working at the Pasteur Institute in Paris, demonstrated that the factor in the blood serum capable of killing bacteria could be analyzed into two components: heat stable and heat labile. ‘The heat labile component is what is now known as the “complement” ‘the term complement ‘was introduced by Ehrlich in the late 1890s Complement Nomenclature & “C°~ designation for 9 of the complement proteins ¢ Factor - designation for many alternative pathway components (like factor B) Over bar - indicates an enzymatically active protein or complex & Lower case letters - indicates a proteolytic cleavage fragment (C3a or C5a) ‘& RY designation for receptors in the complement system (CRI or C5aR) Inactive components are designated with an “i” (eg, inactivated C3b is termed iC3b) ‘The complements are numbered in the order in which they were discovered. ‘The activation of complement system occurs by three pathways: @ Classical pathway (C1-C9) & Alternative pathway ~C3, C3b factor B, D, H and I (C5-C3). & Mannose Binding Lectin pathway (MBP) Classical Pathway Attachment, Activation & Amplification Antigen antibody complex initiates the complement cascade which begins with the activation of Cl. lis a large multimeric protein complex composed of 3 subunits: Clq, Clr, and Cls (Figure 3.8). Clr and Cis are serine esterases. Clq binds to Fe portion of the immunoglobulin which is complexed to its corresponding antigen. Bound Clq activates Clr which then activates Cls. Activated Cs cleaves C4 to Cia, Cab complexes with C2, cleaves C2 into C2a & C2b and forms a complex CAbC2a. CAb attaches to the cell membranes. One Clq generates 100 to 200 C4b2a complexes (C3 convertase) which further activate C3 into C3a and Cb to form C4b2a3b (C5 convertase) which facilitates opsonization. One complex of C4b2a activates hundreds of molecules of C3. Sa, C4a and C3a are released into plasma and they act as potent anaphylotoxins. C5a is chemotactic and attracts neutrophils and stimulates inflammation. C4b2a3b (C5 convertase) splits C5 into C5a & C5b (Figure 3.9). ease vePrinciples & Practice of Transfusion Medicine ime clq 1g, Cis, Clr, Figare 3.8: Structure of Clq and CL ANTIGEN + ANTIBODY Actvated’C1 (cls) ee "3 Convertase (CHb28) d \ 7 RE RIRRNNENienRgeEOE —o (Exaile) / Tease ce (€5 Comvertase (CAb2636) ve Phagocytosis cx 6789 ca 356.789 Inactivator ' ¥ ca -Membrance Damage Call Lysis igure 39: Activation of Classical Pathway Attack Complex C5-C9 forms a complex on cell membrane, C5 fixes to cell membrane, followed by sequential addition of C6, C7, C8 and C9 or membrane attack complex which is a tubular structure. This traverses the cell ‘membrane, resulting in increased membrane permeability thereby allowing influx of Na+ and 11,0 and lysis of the cell. Alternate Pathway It provides a mechanism for complement activation when. sufficient antibody is not available ic. primary infection. Components of cell membrane of the micro ~organism such as polysaccharide and lipopolysaccharides initiate the cleavage of C3 to C3b. C3b associates with factor B and activates it to Bb and Ba. C3b Bb complex is C3 convertase. Subsequent steps are as in the classical pathway. $466Basic Principles of Immunohaematology Lectin Pathway ‘This pathway is mediated by circulating proteins called mannose-binding lectin (MBL) - also known as mannose-binding protein or MBP. Mannose-rich glycans are common in microbial glycoproteins and glycolipids but rare in those of humans MBL. The MBL-associated serine proteinases MASP-1 and MASP-2, analogous to Cir and Cls; cleave C2 and Cé, forming C4b/C2a (C3 convertase). The terminal components of complement are then activated. The MBI is equivalent to Clq in the classical complement pathway. 3 is the key component of complement (Figure 3.10) MBP/Ficolin ALTERNATIVE s—$ 5 t Ce CS cs 5.09 ‘TERMINAL Figure 3.10: All the three pathways of complement activation viz, classical, alternative and MBP ulimtely convert 3 into C3a It contains an unusual internal thiol-ester bond which is stable in naive state but becomes highly reactive as a result of conformational changes in the C3 protein structure due to proteolytic cleavage. Regulation of Complement Activation ‘The complement system can cause much cell destruction if it goes unhindered. C3 in the plasma is continuously cleaved at a low rate (C3 tick over). If active C3 (C3b) attaches to the cell surface that lacks complement regulators, it permits rapid amplification of the complement cascade There are various mechanisms that regulate the complement activation. They are as follows: @ Short half-life of + Cb + C3bBb + Cb @ Cl inhibitor + Inhibits the Cis activity Protein S in Serum Binds to C5667 ‘+ Inhibits Formation of the Membrane Attack Complex oars oTPrinciples & Practice of Transfusion Medicine 4 Homologous restriction factor (HRF) or CD89 + Bind to C8 © Inhibits C9 binding. & Factor H + Binds to C3b + Facilitates binding of Factor + cleaves C36 to inactivated iC3b ‘4 leaves C4b to inactive fragments 4 Decay Accelerating Factor + Increased dissociation of C3 convertase (both pathieays) Complement In Transfusion Medicine Complement alone, without immunoglobulin, may be present on washed red cells in certain situations 1. IgM antibodies reacting in vitro occasionally sttach to red cell antigens without agglutinating the cells. IgM coating is difficult 1 demonstrate in Antihuman globulin (AHG) tests, partly because IgM molecules tend to dissociate during the washing process and partly because polyspecific AHG contains little if any anti-IgM activity. IgM antibodies characteristically activate complement, so the reaction of antibody with antigen can best be demonstrated by identifying the several hundred C3 molecules bound to the cell membrane near the site of antibody attachment. 2. About 10-20% of patients with warm AIHA have red cells with a positive Direct Antiglobulin Test (DAT) due to C3 coating alone. No IgG, IgA, or IgM coating is demonstrable with routine procedures 3._ In cold haemagglutinin disease, the cold-reactive autoantibody can react with red cell antigens at temperatures up to 32°C, although it does not cause agglutination. Red cells passing through vessels in the skin at this temperature become coated with autoantibody, which activates complement. If the cells escape haemolysis, they return ta the central circulation, where the temperature is 37° C, and the autoantibody dissociates from the cells, leaving complement components firmly bound to the red cell membrane. The component usually detected by AG reagents is C34. 4, Immune complexes that form in the plasma and bind weakly and non specifically to red cells may cause complement coating. The activated complement remains on the red cell surface after the immune complexes dissociate. C3 remains as the only detectable surface globulin, Polyspecific AHG Polyspecific AHG reagents are used for direct antighabulin testing and, in many laboratories, for routine compatibility tests and antibody detection. These reagents contain antibody to human IgG and to the C3d component of human complement, Other anticomplement antibodies may be present, including anti- C3b, -C4b, ana -Cad. ea rey seu sce:Monospecific AHG Reagent Murine monoclonal anti-C3b,-C3d reagent is a blend of hybridoma-derived antibodies. Monoclonal anti-C3d may be less likely to react with C3b than anti-C3d obtained by animal immunization. Factors Affecting Antigen Antibody Reaction Forces that hold red cells apart in saline suspensions: . Zeta potential (explained below) is now considered only a minor factor. An important physical property that maintains distance between saline-suspended red cells is . the water of hydration. Water molecules tightly bound to hydrophilic macromolecules on the cell surface act as insulating bubbles, preventing close cells from coming together. Red cells also have a low interfacial tension {surface tension) induced by van der Waals forces and, therefore, do not self aggregate Properties of the membrane itself also affect agglutination. Mobility and clustering of antigen: bearing molecules exert an incompletely understood effect. Basic Principles of immunghaematology i= r Pe RUNNERS SSRIS SGM Red Cell Ionic Charge Red blood cells have a negative charge due to neuraminic acid on the red cell membrane which makes them to repel each other. The repulsive force holding the cells apart is called the Zeta-Potential. The distance which keeps the red cells apart is very small but sufficient to prevent the small IgG molecule to bridge the gap and agglutinate the red cells (Figure 3.6). However it causes coating or sensitization of red cells. Large IgM molecules, however, bridge the gap and bring the cells together (Figure 3.11) thus causing agglutination of red cells Figure 3.11: Reaction of red cells with IgM antibody causing agglutination easePrinciples & Practice of Transfusion Medicine Antigen Antibody Ratio ‘The strength of antigen antibody reaction can be altered by either increasing the number of antigen sites or the number of antibody molecules. The most common method is to increase the number of antibody molecules relative to the number of antigen sites available, Usually 2 volumes of serum and one volume of 2-4% cell suspension are used for optimum reaction. Temperature ‘The antibodies broadly are of two types: those reactive at “cold” temperatures (eg, 4-25° C) and those reactive at “warm” temperacares (eg, 30-37°C). Many of these “cold-reactive” antibodies have been associated with IgM antibodies, while their “warm-reactive” counterparts have been associated with IgG antibodies. The temperature of antigen-antibody reactivity is related to the chemical nature of the antigen than with the antibody class as often thought. Carbohydrate antigens are more commonly associated with “cold-reactive” antibodies and protein antigens with *warm-reactive” antibodies. Another factor is the type of reaction, exothermic or entropy driven, that determines the temperature of antigen-antibody interaction. Exothermic reactions are associated primarily with hydrogen bonding, to carbohydrate antigens and generally occur best at low temperatures. Entropy- driven reactions on the other hand are associated with hydrophobic bonding with protein antigens. ‘Temperature variations usually have little or no effect upon then Ko of most warm-reactive antibodies but rather effects its speed of reaction. Warm-reactive antibodies may be detected after incubation below 37 C, but incubation times required would be much longer. Cold-reactive antibodies, on the other hand, are considerably affected by temperature and an increase in temperature results in antigen- antibody dissociation. pH Most ofthe blood group antibodies react best at pH 6.5 to 7.5 and once the pH is outside the range of 5.5 & 85 the antigen antibody reactions are inhibited. The recommended pH for most of haematological reactions is 7.2. Incubation Time ‘The optimum incubation time for all blood group reactions is 60 minutes. However the addition of various enhancing agents (medium) can increase the rate and amount of antibody uptake thus decreasing the incubation time e.g. Low Ionic Strength Saline (L3SS). Freshness of Serum and Red Cells In order to have best antigen antibody reaction it is advisable to use fresh serurt and freshly prepared red cells, ‘The serum that is not being used immediately should be stored at - 20°C or lower, Tonic Strength ‘A decrease in the ionic strength of the medium in which the red cells are suspended increases the rate of the antigen antibody reaction e.g, Low Ionic Strength Saline(LISS) decreases the ionic strength of medium, as0e lees anata capo)