Immunohematology Lecture

Immunohematology Lecture o o Historical Overview The first time a blood transfusion was recorded in history 1492 B antigen = B Without A and B antigens = O 1902 Von Descatello and Sturle discovered red cell antigen which was designated as blood group AB and an associate of Karl Landsteiner 1940 Landsteiner and Weiner discovered another blood antigen, Rh system from the primate, Rhesus monkey Clotting was the principal obstacle to overcome 1869 Braxton Hicks recommended sodium phosphate as anticoagulant 1829 The scientist James Blundell transfused blood to a human patient suffering from postpartum hemorrhage Unger - Designed a syringe-valve apparatus so that transfusion from donor to a patient is possible without the assistance of a physician Hustin reported the use of sodium citrate as an anticoagulant solution for transfusions What is anticoagulant? 1914 1915 Lewisohn determined the minimum amount of citrate needed for anticoagulation and demonstrated its nontoxicity in small amounts 1916 Rous and Turner introduced a citratedextrose solution for the preservation of blood 1930 The function of dextrose was fully understood. 1941 Dr. Charles Drew was appointed director of the first American Red Cross blood bank at Presbytarian Hospital 1943 Loutit and Mollison of England introduced the formula for the preservative Acid-Citrate-Dextrose (ACD) 1957 Gibson introduced an improvised preservative solution called CitratePhosphate-Dextrose (CPD), which was less acidic and eventually replaced ACD as the standard preservative used for blood storage 1901 Karl Landsteiner discovered A and B antigens and classified them into o A antigen = A o Anticoagulant - Chemical agents that prevent invitro blood clotting so it may remain in its liquid state. What is the function of dextrose in blood, specifically, RBC? o Dextrose helps in maintaining the RBC integrity and viability What happens if damaged? o Hemolysis the RBC membrane is Three areas of RBC biology are crucial for normal erythrocyte survival and function: 1. Normal chemical composition and structure of the RBC membrane 2. Hemoglobin structure and function 3. RBC metabolism Note: Defects in any or all of these areas will result in RBCs surviving fewer than the normal 120 days in circulation Notes: o The RBC membrane represents a semipermeable lipid bilayer supported by a mesh-like protein cytoskeleton structure o Phospholipids, the main lipid components of the membrane, are arranged in a bilayer structure comprising the framework in which globular proteins traverse and move. o Proteins that extend from the outer surface and span the entire membrane to the inner cytoplasmic side of the RBC are termed integral membrane proteins. o Beneath the lipid bilayer, the second class of membrane proteins, called peripheral proteins, is located and limited to the cytoplasmic surface of the membrane forming the RBC cytoskeleton. o The biochemical composition of the RBC membrane is approximately o 52% protein o 40% lipid o 8% carbohydrate o o o relationship, known as the hemoglobinoxygen dissociation curve. o In situations such as hypoxia, a compensatory shift to the right of the hemoglobin-oxygen dissociation curve alleviates the tissue oxygen deficit. This rightward shift of the curve, mediated by increased levels of 2,3-DPG, decreases hemoglobin’s affinity for the oxygen molecule and increases oxygen delivery to the tissues. o A shift to the left of the hemoglobinoxygen dissociation curve results, conversely, in an increase in hemoglobinoxygen affinity and a decrease in oxygen delivery to the tissues. With such a dissociation curve, RBCs are much less efficient because only 12% of the oxygen can be released to the tissues. The normal survival rate of RBC in the blood is 120 days RBC Characteristics Deformability Permeability o The permeability properties of the To remain viable, RBC membrane normal RBCs must o Active RBC cation also remain transport prevent Flexible, colloid hemolysis deformable, and o Control the permeable volume of the RBCs Notes: o The dissociation and binding of oxygen by hemoglobin are not directly proportional to the partial pressure of oxygen (pO2) in its environment but instead exhibit a sigmoid-curve Notes: o The RBCs’ metabolic pathways that produce ATP are mainly anaerobic because the function of the RBC is to deliver oxygen, not to consume it. o Glycolysis generates about 90% of the ATP needed by the RBC. Approximately 10% is provided by the pentose phosphate pathway. o RBC metabolism may be divided into the anaerobic glycolytic pathway and three ancillary pathways that serve to maintain the structure and function of haemoglobin: the pentose phosphate pathway, the methemoglobin, reductase pathway, and the LueberingRapoport shunt. Notes: o Luebering-Rapoport shunt pathway permits the accumulation of an important RBC organic phosphate, 2,3diphosphoglycerate. o Hemoglobin’s primary function is gas transport: oxygen delivery to the tissues and carbon dioxide (CO2) excretion. o RBC viability is a measure of in vivo RBC survival following transfusion. o To maintain optimum viability, blood is stored in the liquid state between 1°C and 6°C for a specific number of days, as determined by the preservative solution(s) used. The loss of RBC viability has been correlated with the storage lesion, which is associated with various biochemical changes. Components of Anticoagulants 1. Citrate o Anticoagulant present to promote chelation of ionized calcium in exchange for sodium ion 2. Phosphate o Promotes invitro RBC metabolism o Source of ATP for cell consumption 3. Dextrose o Additional source of ATP 4. Adenine o The major source of ATP Additive Solution • • • Saline - Physiologic suspending medium used in additive solution Dextrose - ATP source Adenine - ATP source • Mannitol (Modified) - Makes the solution an enhanced version of solution; Sugar which retards in vitro storage hemolysis. • Additive solutions (AS) are preserving solutions that are added to the RBCs after removal of the plasma with or without platelets. Benefits of RBC Additive Solutions • Extends the shelf-life of RBCs to 42 days by adding nutrients • Allows for the harvesting of more plasma and platelets from the unit. • Produces a packed RBC of lower viscosity that is easier to infuse o o o o The procedure for freezing a unit of packed RBCs is not complicated It involves the addition of a cryoprotective agent to RBCs that are less than 6 days old Glycerol is used most commonly and is added to the RBCs slowly with vigorous shaking, thereby enabling the glycerol to permeate the RBCs The cells are then rapidly frozen and stored in a freezer. Rejuvenation Solution • Promotes regeneration of 2,3 DPG and ATP levels o 3 days after expiration o 1 to 4 hours at 37°C o Rejuvesol (Cytosol Laboratory) is the only FDA approved rejuvenation solution o It consists of PIPA (Before it was PIGPA) ✓ Phosphate ✓ Inositol ✓ Pyruvate ✓ Adenine ✓ (Glucose) RBC Rejuvenation o To save rare blood components o “AB” negative Red Cell Freezing o Preserve for 10 years o Done for autologous units (autologous transfusion) blood component collected from the patient for his/her future use o Storage of rare blood units o 0% of infection transfusion Notes: Blood Substitutes o Used to replace blood component such as RBC in carrying O2 in different tissue recipient o The substance that are able to carry O2 in the absence of intact red cells Hemoglobin-Based Oxygen Carriers Perfluorochemicals Sex Linkage Homozygous Heterozygous Phenotype Genotype Dominant Trait o Two platelet additive solutions, InterSol and Isoplate, have been approved for use in the United States o The FDA has approved the use of 7-day platelets as long as specific criteria are met Recessive Trait Codominant Review of Genetics • Population Genetics - Concerning genetic traits in large numbers of individuals • Cellular Genetics - This pertains to the cellular organization of genetic material • Molecular Genetics - Based on the biochemistry of genes and the structures that support them. • PCR and FISH o Methods used for determination of DNA markers o Used for the determination of legal outcomes ▪ Forensic Pathology ▪ Paternity Claims Allele Antithetical Terminologies 1 of 2 or more different genes occupying a space locus in chromosome Amorph o Silent Gene - Unable to produce detectable antigen on red cell surface Example: O gene Pair of identical genes Pair of different genes Actual physical manifestation or expression of a gene The genetic make-up of an individual o Expressed on an individual o Caused by dominant genes o Not expressed on an individual in the presence of dominant o It can only be expressed if both are recessive gene o Pair of genes neither are dominant than the other o Both are expressed o 50% Haplotype MOM o 50% Haplotype DAD Early Genetics and Mendel’s Law of Inheritance o The Swedish biologist Carolus Linnaeus started the first classification system of living things in the 17th century and used the unit of “species” as its principal definition o In 1859, Charles Darwin published his epic book On the Origin of Species after many years of intense study of various and diverse life-forms The science of genetics found its modern development in the work of Gregor Mendel, published in 1865. The science of genetics found its modern development in the work of Gregor Mendel, published in 1865. o o Mendel was an Austrian monk and mathematician who used sweet pea plants growing in a monastery garden to study physical traits in organisms and how they are inherited. o He determined that physical traits are due to factors he called elementen within the cell. An antigen that is a product of allelic genes o Characteristics found in the X and Y chromosomes 2 Laws of Inheritance 1st Law Law of Independent Segregation Law of Independent Assortment 2nd Law Law of Independent Assortment Inheritance Patterns o Law of Independent Segregation The interpretation of pedigree analysis requires the understanding of various standard conventions in the representation of data figures. Immunohematology Laboratory o o o o o o Quality Assurance in the Blood Bank First name Last name Identification number Date Birthdate Age Note: The blood samples must be collected in the appropriate tube for the test being performed. Standard Operating Procedure o o o Quality assurance is a broad concept that monitors all aspects of testing. o Quality control is a part of the quality assurance system that includes a set of procedures performed daily or on an established schedule. o o o o o Reagent Quality Control o o o o o o The Quality Assurance Cycle o Patient/Client Preparation o Sample Collection o Personnel Competency Pre-Analytic o Test Evaluations o Sample Receipt and Accessioning o Sample Transport Analytic Post-Analytic o SOP manual should be available in the blood bank laboratory. This manual must be reviewed and revised on a regular basis by administrative personnel. Summarizes procedures Specimen collection Information Quality Control Record keeping Date of testing Source of reagent used Expiration dates Lot number Visual inspection Identification of person testing performing Quality Control Testing • Record Keeping • Reporting Components of Laboratory Quality Assurance o o o o o o o o Personnel Specimen collection and labelling Procedures Reagents Quality of material and instruments Reporting methods Financial components Patient and physician’s satisfaction Specimen Collection o o o o o o General Requirements for Personal and Laboratory Safety Smoking o Hair Eating o Hand washing Drinking o Pipetting by Food mouth Application of o Good cosmetics housekeeping Contact lenses o Glassware o o o Face shield or eye protectors Clothing Shoes o o o Centrifuge Autoclaves Sharps Class F Types of Fire Extinguishers and Their Uses Electrical Water Foam Powder CO2 Wet Chemical Class A Class B Class C Class D Used on paper, wood, coal, cardboard, and other solid fuel fires Used on solid fuel fires as well as flammable liquids Used on any kind of fire except for Class F cooking oils Used on flammable liquids and electrical fires Used on cooking oil fires as well as combustible solid materials o Potassium o Uranium Cooking Oils o Chip pan fires o Deep fat fryers Electrical Goods o Kitchen Appliances o Computers o Phones • Consider all patients’ blood and body fluid to be biohazardous regardless of diagnosis • Can be used on: o Class A Always wash hands before and after contact with patients • Can be used on: o Class A o Class B Wear gloves and waterproof aprons, mask, and goggles if splashing can occur during wound sterilization, endoscopy, dialysis, or postmortem procedures • Wear gloves when handling blood, body fluids, tissues, or contaminated surfaces • Dispose of all needles in puncture-proof boxes, which must be accessible in all rooms • Minimize mouth-to-mouth transmission by keeping mouthpieces readily available on crash carts and in all areas where this need is possible • Clean blood and body fluid spills with a solution bleach (10%) and water or with hospital disinfectant • Immediately report all needle sticks, accidental splashes, and contamination of wounds by body fluids Can be used on: o Class A o Class B o Class C o Class D o Electrical Can be used on: o Class B o Electrical Can be used on: o Class A o Class F Combustible Materials o Paper o Textiles o Wood Flammable Liquids o Petrol o Oil o Paint Flammable Gasses o Butane o Methane Flammable Metals Apparatus and Equipment Review of Immunology o Remembers the infectious agent and can prevent it from causing disease later o Immunity to withstand and resist subsequent exposure to the same foreign substance is acquired Immune System • Includes tissues, organs (thymus, bone marrow), cells, and biological mediators that coordinate to defend a host organism against intrusion by a foreign substance or abnormal cells of self-origin. Immunity • The process by which a host organism protects itself from attacks by external and internal agents Innate or Natural Immunity o Primary lines defense. Acquired or Adaptive Immunity o Supplements protection provided by innate immunity of o Later evolutionary development— seen only in vertebrates Early evolutionary development o Specific o Nonspecific o Specialized o Natural - Present at birth o o Immediately available Acquired by contact with a specific foreign substance o o o o May be physical, biochemical, mechanical, or a combination of defense mechanisms The mechanism does not alter on repeated exposure to any specific antigen Initial contact with a foreign substance triggers the synthesis of specialized antibody proteins resulting in reactivity to that particular foreign substance o Memory o Response improves with each successive encounter with the same pathogen Notes: Innate immunity – inside the body Adaptive immunity - introduced in the body Natural – hindi specified yung sakit na inaadapt. • • • Factors Affecting Antigen-Antibody Reactions Distance Antigen-Antibody Ratio Ph Temperature Immunoglobulin Type o o o o o Antigen Antibody Agglutinogen Agglutinins RBC surface Glycoproteins produced by plasma cells in response to antigen Notes: • Antigen is also called “Agglutinogen”. It is found on the RBC surface. • Antibody is also called “Agglutinins” It is made up of glycoprotein. Two Phases Ag and Ab Interaction Note: • • • • In Blood Banking, the antibodies that are more significant are IgG and IgM. Be familiar with the Molecular Weight of each antibody. IgM – largest immunoglobulin IgG – minimal in size. The only Ig that can transfer to the placenta. Detection of RBC Antigen and Antibody Reaction Hemolysis Hemagglutination RBC Destruction RBC Clumping Sensitization Antibody attach to cell bond antigen Lattice Formation RBC form a visible clump IgG, IgM IgM Notes: • • Complement System Notes: • In hemolysis, the RBC is destructed and produce red coloration in serum/plasma. • In hemagglutination, the RBC is not destructed, not damaged, and not destroyed, but they clumped. (Normal RBC that clumps) --- used in blood typing In sensitization, nagdidikit dikit pa lang ang antibody and RBC (kung saan ang cell bond antigen) In lattice formation, nagdidikit-dikit na sila sa isa’t isa. (formation of visible clumping) Notes: Antibody and antigen are specific to each other. • • Pagnag-attach and antigen at antibody, magkakaroon ng introduction ng C3 convertase. • C3 convertase ay magkakaroon ng hydrolysis, dito pumapasok ang alternative pathway. • C3b and C3a, maghihiwalay • Yung C3b ay didikit sa C5 • Maghihiwalay ulit ang C5, magiging C5a and C5b • Hanggang magdikit dikit silang lahat sa foreign body. (hanggang makapagcause sila ng swelling and bursting) Zeta Potential Theory o RBC surface has sialic acid molecules (Negatively charged) o In saline, the cations are attracted to negative red cell surface forming a repelling cloud around the cell. o Electric repulsion o Potentiator (Agent) - Enhance antigen and antibody reaction. Note: Involved in zeta potential is the electric charge of the molecule. • Lectins o Antibody-like substance derived from plant extract. Specificity Plant Source Anti-A1 Dolichos biflorus Anti-B Banderaea simplicifolia Anti-H Ulex europaeus Anti-M Iberis amara I. umbellate I. sempervirens Madura aurantiaca Anti-N Vicia graminea Bauhinia variegate B. candicans Prolectins o Antibody like substance derived from snail.\ Specificity Snail Source Anti-A Helix pomatia H. aspersa Cepaea nemoralis Anti-A1 Euphrada periomphala Anti-B Salmo irideus Anti-H Eel ABO and H Blood Group Systems • ABO Blood Group System o The most group. clinically significant blood Nagmanifest lang ang O kapag parehas na recessive ang O (OO) Exclusion o Landsteiner Law Blood groups that never appear in the RBC surface of the offspring: 1. Blood bankers mistype your blood 2. Adopted 3. Different father 1. The antigen on the RBC determines the blood groups. If you have antigen A, meaning you have a blood type A. o 2. The corresponding antibody is never found in the individual serum. ABO Blood typing can be used in direct exclusion of paternity claim. o ABO antigen starts to develop on the 37th day o Glycosyltransferase is an enzyme responsible for the transfer of sugar to plasma to precursor substances found on the surface of RBCs o Paragloboside is an oligosaccharide structure composed of 4 interlinked sugar; found on the surface of RBCs; attachment site for immunodominant sugars. - - If you have antigen A, hindi pwedeng meron ka ring Anti-A. 3. The opposite antibody is always present in the individual serum. - If you have antigen A, the antibody present in you is Anti-B. Genetic Concepts in ABO Inheritance o Each parent contributes half of the genetic information to the child in hereditary units called genes. o The genes that code for the A, B, O type is located on the long arm of the certain chromosome o ABO Gene = Chromosome 9 o H Gene = Chromosome 19 Parent Alleles A B O A AA A AB AB AO A B AB AB BB B BO B Gene A gene B gene Glycosyltransferase Nacetylgalactosaminyl transferase Dgalactosyltransferase Immunodominant sugar N-acetyl-Dgalactosamine D-galactose L-fucose o 99.99% of individuals have H gene regardless of blood type O AO A BO B OO O Notes: • Bold texts are the phenotypes, normal texts are the genotypes. • AO phenotype – O is recessive, that is why A ang phenotype na mamamanifest. H gene L-fucosyltransferase o H ag is important for attachment of A and B antigen in paragloboside. o 0.01% - Lacks H antigen: “Bombay phenotype” Oh or hh or H null. Notes: • Blood typing can only exclude but never includes paternal claims. • Person who do not have H gene are called “Bombay phenotype” • For patients with blood type O, the only present gene in their RBC is the H gene. • Blood type B, the genes that can be found are the B and H genes. Blood type A, the genes that can be found are the A and H genes. • • ABH soluble; Antigen – Glycoproteins ABH Insoluble; Antigen - Glycolipids Blood type AB, the genes that can be found on RBC surface are A, B, and H genes. General Characteristics of ABO Antigens o A It can be demonstrated as early as the second month of fetal life (On the 37th day) Weakly expressed, unstable, 1 year after birth for a strong reaction (delayed reaction) B Strongly expressed, stable H Strongly expressed, stable o o They persist throughout life unaltered, however abnormal antigens may be found as acquired characteristics in leukemia, and cancer (Lymphoma, severe infection, hematologic dyscracias, CLL) May be found in secretions of people who are secretors. Glycoprotein in nature and can be found in saliva, plasma, semen, peritoneal fluid, pericardial fluid, and colostrum. • • Notes: • ABH antigens are found in the red cells. • ABH soluble substance is the one being found in the body secretions. • ABH antigens and ABH soluble substance is not the same, because they have different regulating gene. • Present ang ABH antigen at ABH soluble substance sa tao, pero magkaiba ito ng kinalalagyan at pinanggagalingan. ABO Antibodies o Naturally occurring antibody that is detectable 3 to 6 months after birth following exposure to ABO-like antigen in the environment. o Mostly IgM and react best at room temperature or below. o Antisera must be stored in a ref temperature. (commercially prepared antisera) Secretor genes (SeSe or Sese) Non-secretor genes (sese) Interactions of the Sese, Zz, and ABH Genes Note: • Sese System Regulates the formation of H antigen and subsequently of A and B antigen in secretory cells. Zz System Regulates production of H antigen on erythrocytes For plants and animals they are also present with lectin and prolectin. Also, they are absent in some conditions such as: hypogammaglobulinemia, gammaglobulinemia, patients with plasma cells destruction (cases when ABO antibodies may or may not be detected) Group Antigen Antibody O H Anti-A Anti-B Anti-AB A A, H Anti-B B B, H Anti-A AB A, B, H None Notes: • • Kung ano ang present sa antigen, kabaliktaran non ang meron sa antibody mo. Blood type O have an antigen, the H antigen. General Characteristics ABO Antibodies o o o o Not normally present at birth Develop 3 to 6 months after birth Reacts better at room temperature Occur in 2 forms 1. Naturally Occurring Antibodies 2. Immune Antibodies ABO Subgroups o Antigens under the ABO blood group system which reacts less strongly to commercially available typing sera. • Pagginamitan ng Dolichos biflorus (specific for anti-A1) ang magiging reaction ay positive for the subgroup A1, but if you use it sa blood type A, subgroup A2, magnenegative yan kahit magkapareho silang blood type A. Weak Subgroups of A A3 Demonstrate mf reaction with anti-A and most anti-A, B Ax RBCs do not agglutinate with Anti A but do agglutinate in anti-A, B Aend Demonstrate mf reaction but only small amounts of RBCs agglutinate Am RBCs are not agglutinated or weakly agglutinated by anti-A or anti-A, B Ay RBCs are not agglutinated by either anti-A or anti-A, B A el Typically, is unagglutinated by anti-A or anti-A, B Weak Subgroups of B B3 Demonstrate mf reaction with anti B and most anti-A, B Bx Weak agglutination with anti B and anti-A, B Bm Unagglutinated by anti B or anti-A, B B el Extremely rare subgroup, unagglutinated by anti-A or anti-A, B Notes: • Kapag naglagay ka ng Anti-A sa sample na may blood type a, kapag ang reaction ay 4+, most probably the present in their blood type is the sub group A1. • Kapag may maliit na clumps, and ang reaction ay 3+, blood group A, subgroup A2 • Sa Anti-B, negative ang reaction. Confirmatory Test for Weak Subgroups: Adsorption-Elution Test Notes: • Yung may pinakamalakas na reaction ay sa blood type O (dahil isa lang ang kanyang antigen) • The least amount of H-Antigen is the A1B (pinakamahinang reaction) 1. Inadequate identification of blood samples, test tubes, or slides. 2. Cell suspensions are either too heavy or too light 3. Clerical errors 4. Mix-up samples 5. Failure to add reagents/failure to follow manufacturer’s instructions. 6. Contaminated reagents 7. Uncalibrated centrifuge Bombay (OH) Phenotype o The allele h is very rare and does not produce the L- fucose necessary for the formation of H structure. o The genotype hh or H null is also known as the Bombay phenotype. General Characteristics of Bombay Phenotype General Rules to Resolve ABO Discrepancies 1. Absence of H, A, and B antigens 1. Always re-test first 2. Check for clerical/technical errors 3. Weakest reaction is usually the one in doubt. - Recheck the reverse typing 4. Check results of the screening cells 5. Check the patient’s age 6. Check the diagnosis 7. Check the transfusion history 2. Presence of Anti-H, Anti-A, and Anti-B in serum 3. Strong reactivity with Anti-I reagents 4. A recessive mode of inheritance 5. No reaction europaeus) Anti-H lectin (Ulex Group 1 • • Due to weakly reactive or missing antibodies Most commonly encountered • Found in the following: 1. Newborn 2. Elderly patients Notes: 3. Leukemic patients demonstrating hypogammaglobulinemia • Blood group OH, ibig-sabihin mayroon siyang H antigen • Second row yung O null or yung Bombay phenotype. 5. Patients drugs • Sa Bombay phenotype, anti-A, Anti-B, and anti-H, pare-parehong walang reaction. 6. Congenital agammaglobulinemia • Anti-A, Anti-B, Anti-H is a direct blood typing procedure. • The A cell, B cell, and O cell is the indirect or the reverse typing. • Pag Bombay phenotype magrereact sa reverse typing lahat. Technical Errors Resulting to ABO Discrepancies 4. Patients with lymphoma using immunosuppressive 7. Immunodeficient 8. Bone marrow transplant who develop hypogammaglobulinemia from therapy 9. Chimerism - Presence of two cell populations in single individual like in cases of fraternal twins Notes: • In terms of group 1 ABO discrepancies, the forward typing is always correct. • For the reverse typing, usually nakikita ang discrepancies. Notes: • Example: Multiple myeloma, mataas ang IgG Group 2 • Due to missing antigens and is the least frequently encountered. • Paraproteinemia – increased abnormal plasma protein. • Found in: • Pagroblema about protein – group 3 yon 1. Subgroups A or B 2. Leukemias may yield weakened A or B antigens • Group 4 Due to miscellaneous problems are seen in: 1. Polyagglutination 3. Hodgkin’s disease 2. Cold reacting Abs 4. “Acquired B” (in IT obstruction, colon or rectum CA) 3. Unexpected ABO isoantigen • When bacterial enzymes (of Proteus vulgaris) modify Nacetylgalactosamine into Dgalactose) - Most common • Increased permeability of the intestinal wall causes adsorption of B-like bacterial polysaccharides onto red cells of A or B patients 4. Warm autoantibodies 5. RBCs with cis AB phenotype 5. Ab to low incidence Ag 6. Excess blood group-specific soluble substances (BGSS) Note: • • Forward typing in group 2 have the discrepancies. While Reverse typing have the strong reaction Notes: • Patient #1, Anti-A and Anti-B, parehas negative reaction (based in forward typing) – Blood type O • But in reverse typing, A1 cells, B cells, O cells, and autocontrol lahat ay negative. • The patient 1 is a group O newborn or elderly patient (read on the table) • Patient #2 – blood type B Kabaliktaran ng Group 1 Group 3 • Caused by protein or abnormalities resulting to rouleaux formation • Found in: 1. 2. 3. 4. Increased globulin Increased fibrinogen Presence of plasma expanders Wharton’s jelly Print pages 162-163 – blood bank 7th edition Laboratory: Blood Typing Blood Typing o o Laboratory procedure performed to detect or identify unknown antigen or antibodies using commercially prepared typing sera and laboratory prepared known red cell suspension o Used as Quality Control for anti-A and anti-B o Used for subgrouping of weak A and B antigen o Components 1. “O” Serum - Source of monoclonal antibody 2. No dye 2 Procedures: 3. Sodium Azide (0.1%) - Act as preservative with antimicrobial properties 1. Forward Typing 2. Reverse Typing Note: • Reverse/indirect/Serum Grouping Kapag gagawa ng red cell suspension, make sure na papalitan ito after 24 hours. Forward/Direct/Cell Grouping o To identify unknown antigen o Specimen is red cell suspension of patient o Reagent used is commercially prepared typing sera o To identify unknown antibodies o Specimen is serum of the patient o Reagent used is laboratory prepared known red cell suspension Known A RCS o Used to detect anti-A o Components Anti- A Typing Sera o Used to detect A antigen o Components 1. “B” Serum - Source of monoclonal anti-A antibody. 2. Dye - Blue (Patent Blue, Methylene Blue, Thymol, Bromphenol) 1. “A” RBCs (A and H Antigen) 2. NSS - Physiologic suspending or washing solution; Tomato red; Usually 2 to 5% concentration o Known B RCS Used to detect anti-B o Components o 1. “B” RBCs (B and H Antigen) 2. NSS – Physiologic suspending or washing solution; Tomato red; Usually 2 to 5% concentration Known O RCS Used as QC for anti-H o Should give negative reaction o Positive known “O” RCS is seen in the following: 3. Sodium Azide (0.1%) – Act as a preservative with antimicrobial properties o Anti-B Typing Sera Used to detect B antigen o Components 1. “A” serum - Source of monoclonal anti-B antibody 2. Dye - Yellow (Tartrazine, Acriflavin) 3. Sodium Azide (0.1%) - Act as a preservative with antimicrobial properties Anti-AB Typing Sera 1. Bombay (Oh) Phenotype 2. Rouleaux formation 3. Technical error Methods of Blood Typing Tube Method Slide Method Routine Typing o Blood o Rapid/Bedside BT o Forward o Forward/Reverse o Centrifugation required is o The interpretation is within 2 minutes o The interpretation can be up to 1 hour o Neonatal and ICU patients Blood Typing Lahat ng blood type ay nagkakaiba iba sa sinisecrete. may secretor, Lahat sila ya nagrereact sa H substance dahil lahat sila ay may H substance. Mas marami at mataas ang reaction kay H substance. Test for ABH Soluble Substance Saliva Neutralization Test Notes: • First 3 columns after the first column, is a forward blood typing. • The last 3 columns are the reverse blood typing. • Sa reverse typing, kung saan siya nagpositive, hindi iyon ang blood type niya. Kaya tinawag na reverse. Principle o Agglutination-inhibition Positive Rxn o No agglutination Negative Rxn o With agglutination Reagents o o o Anti-sera (Anti-A, Anti-B) Lectin (Anti-H) Known RCS (Known A, Known B, Known O) Specimen o Saliva 1. Saliva must be heated at 56°C for 30 minutes prior to testing to inactivate salivary enzyme ABH Soluble Substance 3 Genes 1. SeSe (Secretor Gene) 2. H gene (Secretor Locus Gene) 3. ABO Gene SOP 2. Saliva must be diluted with NSS before use to decreased non-ABH soluble glycoproteins present in the sample to avoid false reaction. Saliva Neutralization Test • Dito ang tinetest ay yung red cell ng patient, with addition of saliva ay naneuneutralize siya. • Red font, naging negative agglutination nung nadagdagan ng saliva, compared sa without saliva. • The indication is that the patient is positive for agglutination inhibition. Immunohematology: Rh Blood Group Systems • • The Rh blood group is highly complex, and the alloimmunization to Rh blood group antigens can complicate transfusion and pregnancy • The name Rh was retained for the humanproduced antibody • Anti-rhesus formed by the animals was renamed anti LW (Landsteiner and Weiner) • Yung antibodies na ni-test ni Landsteiner at Weiner ay 2 kinds Rh is the second in importance only to the ABO blood group system in terms of transfusion, as the Rh system antigens are very immunogenic • Rh-positive indicates that an individual’s red blood cells possess one particular Rh antigen, the D antigen. • Rh-negative indicates that the red blood cells lack the D antigen. • Caucasian – (Rh negative) mostly occurs. o o o o 1 reacting to human 1 is reacting to animal Nomenclature Genetically Associated Rh positive – presence of antigen D Rh negative – lacks the D antigen o Based on the inheritance of a particular gene. o Fisher Race (DCE) o Weiner (Rh Hr) Non-Genetically Associated o Rosenfield (Alpha Numeric) o ISBT (International Society for Blood Transfusion) Notes: History • 1939 - Levine and Stetson described a hemolytic transfusion reaction in an obstetrical patient. • After Delivering a Stillborn o o o o o o • • Woman required transfusions Husband was the donor The recipient had an acute hemolytic transfusion. Father and fetus possessed a common factor that the mother lacked. Levine and Stetson discovered d antigen through the mother who required transfusion after delivering a still born. (Read sa book) 1940 - Landsteiner and Wiener described an antibody made by guinea pigs and rabbits when they were transfused with Rhesus macaque monkey RBCs and was named anti-Rh after the Rhesus monkey. 1941 – the Rh blood group system was known in the medical field (Landsteiner and Weiner) o Two of the terminologies that are based on the postulated genetic mechanism. • Fisher race • Weiner o The 3rd terminology describe the presence of the given antigen • Rosenfield o The 4th is the result of the ISBT Fisher-Race: DCE Terminology • Based on the theory that antigens of the system are produced by three closely linked sets of alleles and that each gene is responsible for producing a product (or antigen) on the RBC surface. • Each antigen (Ag) and corresponding gene were given the same letter designation. Fisher and Race named the antigens of the system D, d, C, c, E, and e ▪ ▪ ▪ D, C, E sets of Alleles D – presence of the D antigen d – Not true, it only signifies the absence of D antigen. (Amorph) • To date, NO (d) antigen has been found, and it is considered an amorph (silent allele) or the absence of D antigen. • The phenotype (blood type observed during testing) of given red cells is defined by the presence or absence of D, C, c, E, and e. • According to the Fisher-Race proposal, each person inherits a set of Rh genes from each parent (one D, or d, one C or c, and one E or e) • Rh genes are co-dominant, each inherited gene express its corresponding antigen on the red cell. • o Kung ang involve sa Fisher-race ay DCE, kay weiner ay R,C,E. ▪ ▪ ▪ ▪ ▪ ▪ R – indicates the D antigen r – absence of D antigen C – 1 or ’ c – absence of 1, 0, and (‘) E – 2, ” e – absence of 2, and (“) Antibody can recognize single or multiple factors (Ag) The combination of maternal and paternal haplotypes (Possessing half the normal # of chromosome found on the cell) determines one’s genotype (the Rh gene inherited from each parent) and dictates one’s phenotype (the antigens expressed on the red cell that can be detected serologically) • Fisher-Race nomenclature may be converted to Wiener nomenclature and vice versa. It is important to remember that an agglutinogen in the Wiener nomenclature actually represent the presence of a single haplotype composed of three different antigens. Examples: ▪ DCE ▪ DCe ▪ DcE ▪ dCE • Conversion from Wiener to Fisher Race An individual’s Rh phenotype is reported as DCE because Fisher-Race postulated that the C/c locus is lies between D/d and E/e loci. • It is essential to remember that d doesn’t represent an antigen but simply the absence of the D antigen. • C, c, E, e represent actual antigens recognized by specific antibodies. Wiener Rh-Hr Nomenclature • Notes: • • • Each factor is an antigen recognized by an antibody. It is postulated that the gene responsible for defining Rh actually produces an agglutinogen that contains a series of three blood factors in which each factor is an antigen recognized by an antibody. The agglutinogen may be considered the phenotypic expression of the haplotype. Remember: R = Subscript (1, 2, Z, O) R = Superscript (‘,’’, y) • When referring to the Rh Ag (factor) in Wiener nomenclature the single prime ( ' ) refers to either C or c. • The double prime (″) refers to either E or e. • If the r precedes the h (rh′ or rh″) we are referring to C or E Ags respectively. • When the h precedes the r we are referring to either c (hr′) or e (hr″) Ags. ▪ Rho = D • There is no designation for the absence of D Ag. Therefore: D C E c E Rho rh’ rh’’ hr’ hr’’ Rosenfield Alpha/Numeric Terminology Example: Convert the Following • A number is assigned to each antigen of the Rh system in order of its discovery (Rh1 = D, Rh2 = C, Rh3 = E, Rh4 = c, Rh5 = e) • In 1960s Rosenfield proposed a system that assigns a number to each antigen of Rh system in order of its discovery or recognized relationship to the Rh system • This system has no genetic basis but simply demonstrates the presence or absence of the Ags on the red cells • A minus (-) sign preceding a number designated the absence of the Ag • If Ag has not been typed for, its number will not appear in the sequence 1. R1r = DCe/dce = Rhoh’hr” / hr’hr” 2. R0r1 = Dce/dce = Rhohr’hh” / rh’hr” Example: R1 = DCe o o R0 = Dce r′ = dCe E = is indicated by 2 or ( ″ ) e = there is no 2 or ( ″ ) D is assigned = Rh1 C = Rh2 E = Rh3 c = Rh4 e = Rh5 Example: R2 = DcE r″ = dcE o When both C and E are present, the letter Z or Y is used. o It indicates the presence of capital C and Capital E. o RZ = DCE o R1r = DCe/dce • ▪ ▪ rY = dCE r′r =dCe/dce Pag 2 r yung nakalagay, ibig-sabihin ay 2 sets ng DCE. • Pag walang kasunod ang r, automatic small letter c and e. D+, C+, E+ = Rh 1, 2, 3 c- , e- = -4, -5 • If the sample was not tested for e, the designation would be Rh: 1,2,3,-4 • All Rh system antigens have been assigned a number Note: • For cell that typed D+ C+ E+ c- e-, the Rosenfield designation is Rh: 1,2,3,-4,-5 International Society of Blood Transfusion • They adapted a six-digit number for each blood group • • The first three numbers represent the system and the remaining three represent the antigenic specificity The number 004 was assigned to the Rh blood group system, then each antigen assigned to the Rh system was given a unique number to complete the six-digit computer number Old System ISBT o o o Composed of 6 digits First 3 digits = Blood group system Last 3 digits = Specific Antigens o o Rh = 004 ABO = 001 Rh Antigens Rh – means capital hr – small letter Example: o Ror – Dce / dce = Rhohr’hr” / rh’hr” Rh Genetic • RHD and RHCE are two closely linked genes located on chromosome 1 that control the expression of Rh proteins • The gene RHD codes for the presence or absence of the RhD protein, and the second gene RHCE codes for either RhCe, RhcE, Rhce, or RhCE proteins • RHD and RHCE are codominant • Another gene important to Rh antigen expression is RHAG, and it resides on chromosome 6. The product of this is Rh associated glycoprotein (RHAG) Biochemistry of Rh Antigens The number of D Ag sites, measured on a variety of Rh phenotype ISBT New System o Zeros are omitted o Blood group systems are represented by capital letters o Specs blood group antigen represented by numbers, Rh=RH ▪ ▪ ▪ ▪ ▪ Summary D-1 C-2 E-3 C-4 E-5 is • The greatest number of D Ag site are on cells of the rare Rh phenotype D--. D-- cell carry only D Ag and completely lack C c and E e • R2R2 cells possess the largest number of D Ag sites. • IgG, react optimally at 37oC or following the addition of antiglobulin reagent. • Produced after exposure of the individual’s immune system to foreign red cells, either through transfusion or pregnancy. • Rh Abs often persists in the circulation for years. There are instances when an accurate Rh type cannot be determined through routine testing • Rh Abs are considered clinically significant; therefore, Ag negative blood must be provided to any patient with a history of Rh Ab sensitization. 1. If the new born’s cell are coated with maternal IgG anti-D in utero, very few D Ag sites will be available to react with reagent anti D • Rh Abs do not bind complement. For complement to be fixed, two IgG molecules must attach in close proximity to the red cell surface • • Red cell destruction due to Rh Abs is primarily extravascular. This type of hemolysis classically characterizes a delayed hemolytic transfusion reaction • Rh antigens are highly immunogenic, the D antigen is most potent • • Is essential when test donor blood sample. • Blood considered Rh-positive if either the D or Du test is positive • • If any donor blood sample that types Rh o (D) negative by either slide or rapid method must be tested further by indirect anti globulin test (IDAT) Variation of the Rho (D) Antigen • When Rh-positive red cell samples are typed for the D Ag. It is expected that they will react strongly with anti D reagent. However, with certain red cells, the testing must be carried through the AHG phase to demonstrate the presence of the D Ag. Red cells carrying the weaker D Ag have been referred to as having the Du type • Three different mechanisms have been described that can explain the weakened expression of the D Ag Genetic weak D o Inheritance of D genes that code for a weakened expression of the D Ag o The D Ag expressed appear to be complete but few in number. Inheritance of these genes can be tracked from one generation to the next and seen most frequently in black. o IHAT (make sure that the patient has the presence of D antigen If both test results are negative, the donor sample is considered Rh-negative. Notes: • 2. Warm autoimmune hemolytic anemia, Abs are directed against the patient’s own red cell and react as though they are Rh specific Exposure to less than 1 ml of Rh-positive red cells can stimulate Ab production in a Rhnegative person. Determination of Du Status Elution of the sensitizing Ab (removing the Ab) and identifying it as anti D will verify that the infant’s red cell is D positive When it comes to grading, you have the high grade weak D and low grade weak D ▪ High-grade weak D – is not passed by generation. Does not need antihuman globulin test. ▪ Low-grade weak D – inherited. Need anti-human globulin test. C Trans (A position effect or gene interaction) Clinical Considerations The Rh Ag on the red cell is normal, but the steric arrangement of the C Ag in relationship to the D Ag appears to interfere with the expression of the D Ag. o Transfusion Reactions This interference with D expression does not occur when the C gene is inherited in the cis (The location of two or more genes on the same chromosome of a homologous pair). The family study can be distinguished which type of weakened D Ag is being demonstrated. o • Circulating antibody can appear as soon as 10 to 14 days of a primary exposure and within 1 to 7 days after a secondary exposure • Rh mediated hemolytic transfusion reactions, whether caused by primary sensitization or secondary immunization, usually result in extravascular destruction of immunoglobulincoated RBCs • The transfusion recipient may have unexplained fever, a mild bilirubin elevation, and a decrease in hemoglobin and haptoglobin • The direct antiglobulin test (DAT) is usually positive, and the antibody screen may or may not demonstrate circulating antibody D Mosaic o One or more parts of the D Ag are missing o Wiener and Unger postulated that the complete D (Rho ) Ag had four parts designated RhA , RhB , RhC , RhD , a lowercase superscript of a, b, c, and d is used to indicate when the corresponding portion (s) of the mosaic is missing o HTN (Hemolytic Disease of the newborn) and HTR (Hemolytic transfusion reaction) o Example, Rh b means the B portion is absent. Hemolytic Disease of the Fetus and Newborn • Anti D was a frequent cause of fetal death before the implementation of Rh-immune globulin therapy • HDFN caused by Rh antibodies is often severe because the Rh antigens are well developed on fetal cells, and Rh antibodies are primarily IgG, which readily crosses the placenta Notes: • Test used for the determination of D status. o o • test / Immunogenicity of common Rh antigens o • Anti – D (screening test) Indirect anti-human globulin coomb’s test (confirmatory test) Small letter d immunogenic – as the highest Sa blood transfusion, kapag ang donor ay may rbc antigen na wala sa recipient rbc, yung recipient ay magpoproduce ng antibody against sa donor’s antigen Hemolytic Disease of the Fetus and Newborn • There is anemia in the affected fetus or child associated with nucleated red blood cells (NRBCs) in the neonate’s blood • At birth, the child is not severely jaundiced because of the placental clearance system, but jaundice may worsen rapidly and produce kernicterus if not treated promptly • In its severest form, there is a cardiac failure, hepatosplenomegaly, and intrauterine death (hydrops fetalis) • During the pregnancy Rh-positive antibodies can be measured in the maternal serum, and bilirubin can be measured in the amniotic fluid to assess the severity of the hemolysis • At birth, the degree of anemia is the best guide to the severity • The blood film reveals increased nucleated RBCs, reticulocytosis, polychromasia, anisocytosis, spherocytes, and cell fragmentation • Positive maternal indirect Coombs test • Positive neonatal direct Coombs test • Ultrasound - To detect organ enlargement or fluid buildup in the fetus • As a rule, serial maternal antibody titers are monitored until a critical titer of 1:32, which indicates that a high risk of fetal hydrops has been reached • Exchange transfusion may be required soon after birth to prevent kernicterus. Fresh blood should be used • Phototherapy may augment bilirubin breakdown and avoid the necessity of exchange transfusion in mild cases. Laboratory Rh Reagents • Coomb’s Cells (C.C) o Control cellular used when AHG test is negative o • • To confirm that washing has been adequate and the anti-globulin reagent is reactive Control Cell o A group O Rh positive donor’s sample is mixed with 1:10 dilution of reagent anti D and allowed to incubate o After sensitization of RBC, they are washed with saline and suspended to a 50% RBC suspension o These sensitized RBCs then used to confirm the anti-IgG activity of the AHG Coomb’s Cells (C.C) o They can be used to ensure that AHG tests with neg results are not false negative because of the inactivation of the AHG reagent o When AHG test is negative, they should be free AHG reagent in the test tube o When the CC is added, the free AHG in the test should cause agglutination of the sensitized RBCs o This positive reaction is a mixed field in nature because; half of RBCs in the mixture lack IgG on their surface and are free cells 2nd half of RBCs have IgG (CC) and are agglutinated False Reactions with Rh Typing Reagents The most prevalent haplotypes or nomenclature for: 1. Asian = DCE/R1 2. African-American = Dce/ R0 3. Caucasian = dce R1 (di ko alam ano nakacapital, nasa book naman read na lang) Immunohematology: Week 5 Lewis System o Clinically insignificant (not important) o Binds complement o Capable of triggering in vitro hemolysis o LE:007 - code o Enhance by enzyme treatment o Lewis antigens are manufactured by tissue cells and secreted into the body fluids then adsorbed onto the red cell membrane. o Readily neutralized by Lewis blood group substances. Lewis Antigens o Lewis substances are glycoproteins. o Lewis antigens are glycosphingolipids. o They are poorly developed at birth. o Le(a-b-) → Le(a+b-) → (Le(a+b+) → Le(ab+) o Le (a-b+) = true phenotype. o Lea - Exclusively found in secretion. o Leb - Found in secretion and be absorbed onto the cell membrane. Note: Anti-Lea is the most commonly encountered antibody of the Lewis system. Biologic Significance 1. Leb has receptors for Helicobacter pylori. (Leb enhance the attachment of H pylori in the gastric mucosa) 2. Lex antigen is a marker for ReedSternberg cells of Hodgkin’s disease. Inheritance o Le genes are located on the short arm of chromosome 19. o Genes do not code for the production of Lewis antigens but produce a specific Lfucosyltransferase to type I-precursor substance. Note: • Namamana ang Lewis through the inheritance of the short arm chromosome 19. Lewis Phenotype Other Major Blood Group Systems MNSs U o MNS: 002 o Well developed at birth o MNS is inherited as close linkage. o MN is associated with glycophorin A o Ss is associated with glycophorin B o Located on chromosome 4 Le(a+b-) Nonsecretors o MNS antigens are important markers in paternity testing Le(a-b+) Secretors (true phenotype) o Found on the red cells and not in secretions. Le(a-b-) Secretors / Nonsecretors o U - Stands for universal o RBCs with the S or s antigen also have the U antigen. Lewis Antibodies o Naturally occurring IgM; react best at Room temperature or lower temperature. (IgM cannot pass in the placenta) Notes: • Cold blood for testing • IgG – clinically significant • Possibly caused Hemolytic Disease of Newborn. • Mn and S – occurs mostly in individuals • 4 antigens (M,N,S,s) Incidence of Phenotypes P Antibodies Anti-P1 o Naturally occurring IgM antibody in the sera P2. o Can be neutralized with soluble substance in hydatid cyst fluid. Anti-P o MN - 50 % (M=30% and N=20%) o S = 55%, s = 45% o IgM in nature o U- = Common among blacks. o o U+ = Common among whites. Naturally occurring alloantibody in the sera of all Pk individuals. o Autoanti-P (Donath Landsteiner Antibody) ▪ MNSs Antibodies Anti-M and Anti-N Mostly IgM, naturally occurring cold reactive saline agglutinins that do not bind complement or react with enzyme-treated cells. o Anti-M Reaction acidification. o enhanced o Predominantly IgM o Binds complement o Associated with spontaneous abortions in early pregnancy (IgG) o May demonstrate in vitro hemolysis. by Anti-S and Anti-s I o I: 027 o At birth and infant red cells are rich in i antigen (I almost undetectable) May bind complement o Associated with HDN and HTRs (Hemoytic Transfusion Reaction) During the first 18 months of life, i slowly decreases, I increase. o Adult red cells, rich in I and only trace amount of i. o Chronic Dyserythropoietic Anemia Type II or HEMPAS o Mostly IgG o Reactive at 37°C and the AHG phase. o o P P: 003 ▪ P Antigens o IgG biphasichemolysins associated with paroxysmal cold hemoglobinuria. Anti-PP1 Pk (Anti-Tja) Anti-N - Seen in renal patients who are dialyzed with equipment sterilized with formaldehyde. o o P1 Consists of three antigens: P, P1, Pk which are biochemically related to the CHO chain that makes up the ABH and I antigens. o Located on chromosome 22 o P1 antigens are poorly developed at birth. Associated with much greater i activity on red cells than control cord cells. I antibodies o Benign anti-I is a weak naturally occurring saline IgM autoagglutinin detectable only at 4°C. o Pathologic anti-I is a potent cold autoagglutinin that demonstrates high titer reactivity and reacts over a wide thermal range (0 to 30°C) Rare phenotype with decreased Kell system antigen expression and abnormal red cell morphology. o Anti-i o An IgM agglutinin o Reacts optimally at 4°C o Associated with infectious mononucleosis. McLEOD Syndrome Kell o KEL: 006 o Kell antigens in chromosome 7 • Found only on red cells • Well developed at birth. • Not destroyed by enzymes. • The K antigen is rated second only to D antigen in immunogenicity. • • o Chronic but often hemolytic anemia. well-compensated o Reticulocytosis o Acanthocytosis o Muscular dystrophy o Common among males suffering from chronic granulomatous disease. Other Kell Antigens Destroyed or inactivated by sulfhydryl reagents. ▪ DDT ▪ ZZAP ▪ AAT Discovered in mother “Kelleher” and her child has jaundice. o Kell (K), Cellano (k), Penny (Kpa), Rautenberg (Kpb), Williams (Kw), Sutter (Jsa), Matthew (Jsb), Class (KL), Peltz (Ku) o Cellano (k) - occurs in Caucasians and Negroes. o Kell (K) - 9% Caucasians; 3.5% Negroes. over 99% Kell Antibodies Anti-K Duffy o Outside of ABO & Rh o Anti-K is the most common antibody seen in blood bank. o IgG “immune” antibodies reactive in AHG phase. o Fya and Fyb o Can cause both HDN and HTR o Well developed at birth o Long arm of chromosome 1 o Easily destroyed by common proteolytic enzymes o Fy(a-b-) is an important anthropological marker for African blacks. Phenotypes KO or K null Phenotype o Lacks Kell antigens o No membrane abnormality McLEOD Phenotype o Lacks the Kx antigen o Fy: 008 Duffy Antigens • Shown to resist infection caused by the Plasmodium vivax and Plasmodium knowlesi. Lutheran Duffy Antibodies Anti-Fya and Anti-Fyb o • Usually IgG antibodies and react best at the AHG phase. • Both are implicated in delayed HTR and HDN. Kidd o o Well developed at birth, contributing to the potential for HDN. o Found on chromosome 18 o Reactivity treatment. o Jk(a-b-) - resists lysis in 2M urea and occurs in Mongoloids enhanced by Lutheran genes are located on chromosome 19, closely linked with the secretor locus. Lua and Lub o Poorly developed at birth and found at the glycoprotein of RBC, associated with the adhesion function and intracellular signaling activity. o Lu(a-b-) - May result from three different genetic backgrounds. Dominant Lutheran Recessive Lutheran Recessive Sex-Linked Lutheran • • • enzyme Kidd Antibodies o o Kidd Antigens Jka and Jkb LU: 005 Lutheran Antigens Jk: 009 o o Lutheran Antibodies o Anti-Lua • Naturally occurring, reacts best at room temperature. o Anti-Lub • Most are IgG, reactive at AHG at 37°C and AHG Phase. o Anti-Jka and Anti-Jkb o Shows dosage o IgG immune antibodies and antiglobulin reactive o o Bind complement Common cause of delayed HTRs o DI: 010 o Most notorious antibody in blood bank. o 2 sets of anti-thetical antigens (Dia, Dib/Wra, Wrb) and 3 low incidence antigens (Wda, Rba, WARR) o Dia Antigen - Useful tool in anthropological studies of Mongolian Ancestry o Dia and Dib are found in Anion exhange molecule (AE-1) Anti-Dia and Anti-Dib are RBCs stimulated, IgG in nature, do not bind complement and reactive DAT (Direct Anti-globulin Test). Dosage Effect o A phenomenon whereby an antibody reacts more strongly with a red cell carrying a double dose (homozygous inheritance of the appropriate gene) than with a red cell carrying a single dose (heterozygous inheritance) of an antigen. Minor Blood Group Systems Diego o Chido/Rodgers o o H/RG: 011 Consists of 9 antigens. o CH/RG Antigens - Antigenic determinants of human complement C4 (C4a and C4b) o Anti-CH/RG abs - Can be neutralized by fresh human serum. Other Human Blood Group Systems Xg Sytems o XG: 012 o Has only one antigen and 2 resulting phenotypes Xga (+) and Xga (-) o Xga Allele - Located on the short arm of X chromosome; more prevalent in women and associated with multiple transfusion. o Anti-Xga - predominantly IgG, reactive in IAT, binds complement but do not cause HDN & HTR. o Sensitive to enzymes. Bg Systems o Bennett Gooodspeed o Related to human leukocyte antigens on RBCs o Bga - HLA-B7 o Bgb - HLA-B17 o Bgc - HLA-A28 Cromer (Crom: 021) o Associated factor with decay-accelerating Dombrock (DO: 014) o Associated ADP-ribosyltransferase 4 Gerbich (GE: 020) o Produced in glycophorin C and D Indian (IN: 023) o Associated with CD 44 Scianna (SC: 013) o Associated with erythroblast membraneassociated protein. Cartwright o Found on the protein acetylcholinesterase Sd o Sd Antigen o Anti-Sd abs - Demonstrates characteristic shiny, refractile, mixed-field agglutination. o Sd biochemically resembles the protein and can be neutralize with urine. Immunohematology Lecture: Week 6 Donor Selection Governing Agencies Center for Biologics Evaluation and Research Responsible for regulating the collection of blood and blood components for transfusion and for the manufacture of pharmaceuticals derived from blood and blood components. o Telephone (Cell phone/ Mobile number is accepted) o Gender o Age (For Autologous Donation - No Age Restriction) o Date of Birth o Consent to Donate o Additional Information • Name of the patient for whom the blood is intended (Directed donation) • Race of the donor • CMV status (viral infections) Food and Drug Administration Responsible for licensing the collection and processing facilities, the blood products and derivatives, and the reagents used in the processing and testing. Physical Examination Presence of Derivatives in blood products. o General appearance American Association of Blood Banks o Weight: 110 lbs (50 kg) Its mission is to establish and provide the highest standard of care for patients and donors in all aspects of transfusion medicine. • If the donor is less than 110 lbs. • Amount of blood to be drawn. • Allowable Amount = Donor’s weight (lbs)/110 lb x 450Ml • Anticoagulant Needed = AA/100 x 14 • Anticoagulant to Remove = 63mL – AN Global / worldwide Note: • Important to verify first using the donor’s valid ID. o Temperature: Orally should not exceed 99.5°F or 37.5°C. • Autologous donation – donating blood for your own or for your future use. o Pulse: 50-100 bpm • 16 years old below need parental consent. o Blood Pressure: Systolic <180mmHg, diastolic <100mmHg • Rare blood types will be reported to Red Cross. o Hct & Hgb: 38% and 12.5 g/dL • CMV – Cytomegalovirus status • Deferral – rejection / postpone Notes: • Sample computation for blood: Patient = 70 lbs 70 lbs / 110 lbs = 0.64 lbs 0.64 x 450 = 286.4 mL (allowable amount with patient having less than 110 lbs.) • Anticoagulant 286.4 / 100 x 40 = 40.1mL 63 mL (standard) – 40.1 mL = 22.9 mL Registration o Name (First name, Middle name, Surname, Suffix) o Date and Time of Donation o Address Copper Sulfate Method Principle: A drop of whole blood when dropped in a solution of copper sulfate, which has a given SG, will maintain its density for approximately 15 seconds. Specific Gravity is 1.053 = 12.5 g/dL. Hematocrit = if the blood stays at the copper sulfate for 10 seconds. Procedure: 1-2 full drop of blood Observe within 15 seconds. • • o Leukemia, lymphoma o High-risk sexual behavior o High-risk occupation o Chaga’s disease o Babesiosis o o Those receiving growth hormones Symptoms of AIDS, HIV o Those taking tegison for psoriasis o Active Pulmonary Tuberculosis Types of Deferral Temporary Deferral Prospective donor is unable to donate blood for a limited period of time. Indefinite Deferral Prospective donor is unable to donate blood for someone else for an unspecified period of time due to current regulatory requirements. This donor would not be able to donate blood until the current requirement changes. These donors may be eligible to donate autologous blood. • Protocol Permanent Deferral Three Years Deferral o Infected with malaria o Visitors, immigrants, or refugees from an area considered endemic for malaria. o Residents of an area endemic to malaria. 12 Months Deferral o Close contact with a patient with hepatitis o Donors who received blood or blood products, organ or tissue transplant. o Tattoo, ear, and skin piercing o Treated for syphilis or gonorrhea o Positive (+) serologic test for syphilis o Traveled to areas endemic for malaria (do not defer a donor who started antimalarial therapy in preparation for travel to areas endemic for malaria) o Rabies vaccine o Sexual contact with any person who has a high risk of exposure to HIV. Prospective donor will never be eligible to donate blood for someone else. These donors may be eligible to donate autologous blood. Some permanent deferrals may result from the testing performed on a previous donation. • • HIV Patients Patients with cancer 2 Months Deferral o Whole blood donation Indefinitely or Permanently Deferral o History of viral hepatitis o History of jaundice of unknown cause o Past/present abuse of self-injected drugs o Cancer o Abnormal bleeding tendencies (haemophilia and congenital conditions) o Cardiopulmonary diseases 6 Weeks Deferral o Pregnant (Deferred during pregnancy and 6 weeks following a 3rd trimester delivery) o 1st and 2nd trimester abortion or miscarriage need not be a cause for deferral. • No need for deferral 5. Component to be removed out 1 Month Deferral o Those taking Accutane o Vaccination of Rubella (German measles) and Varicella zoster (Chickenpox) o Vaccination of mumps, oral polio, Rubeola (measles), smallpox, yellow fever, influenza 2 Weeks Deferral 3 Days Deferral o Tooth extraction or dental work. 48 Hours Deferral o Participant in pheresis program. o (isang component lang ang “pheresis” kunwari platelets lang) kinuha Until Signs & Symptoms Are Gone o Active cold or flu symptoms. No Deferral o Diphtheria o Cholera o Pertussis o Salk o Tetanus o Hepatitis B vaccine o Typhoid o Influenza 1. Blood is drawn 2. Blood is separated into components by a centrifuge. 3. Needed components are collected into sterile bags. 4. Unused components are returned to the donor. Intermittent and Continuous Flow Centrifugation Intermittent • • 1 arm involved Iniipon muna ipoprocess. yung Hemapheresis Donor Selection Hemapheresis - A type of blood donation where whole blood is withdrawn either from a donor or patient wherein after removal, separation, and retention of the desired cellular elements or plasma, the remaining products are recombined and returned to the donor or patient. 1. 2. 3. 4. Whole Blood Plasma Platelet rich plasma/ Leukocytes Erythrocytes Continuous Flow Centrifugation • • 2 arms involved Processed na blood then Takes place in the OR when 1-3 units of WB are collected and the patient’s volume is replaced with colloid or crystalloid. The blood is reinfused during the surgical procedure. Additional Donor Guidelines Post-Operative Salvage 1. At least 48 hours is the elapsed time after hemapheresis donation. An autologous donation in which a drainage tube is placed in the surgical site and postoperative bleeding is salvaged, cleaned and reinfused. 2. A donor must not exceed more than two times in a week or 24 times in a year unless otherwise allowed by a blood bank physician. 3. A donor must be tested to detect cytopenia. 4. Whole blood donor must elapse at least 8 weeks before donating again. 5. Extracorporeal blood must not exceed 15% of the donor’s total blood volume. Autologous Donor Selection 1. No risk of disease transmission. 2. Phlebotomy process stimulates the bone marrow to increase cell production. 3. Decreases the need for allogeneic blood (magka-ibang tao) and may actually increase the supply. o No age limits o No strict weight requirements o Hgb/Hct should not be less than 11g/dL and 33% Types of Autologous Donations/Transfusions Predeposit Donation Refers to the blood that is drawn sometime before the anticipated transfusion and stored, usually liquid but occasionally frozen. Intraoperative Autologous Transfusion Occurs when blood is collected during surgical procedure and reinfused immediately. Immediate Preoperative Hemodilution Additional info • Vitamins, paracetamol, analgesic, antihistamine – can donate blood (it won’t affect) Immunohematology Transfusion Reaction (TR) o Unwanted and unexpected reactions that occur during or after blood transfusion o Signs and symptoms onset may be Acute Delayed o Occurs within 24 hours Occurs after 24 hours up to 7-14 days Febrile Nonhemolytic Transfusion Reaction (FNHTR) o Increase of temp >1°C after transfusion (PX have 37 °C to 38.5°C - 1°C higher than pre temp of PX, considered FNHTTR) o Mild immunologic reaction because the interaction between recipient's antibody is against the HLA of donor o Signs and Symptoms: Fever and Chills o Management and Prevention: leukocyte filters during transfusion Antibody involvement occurs Immunologic NonImmunologic • • • • lgM (Immediate) lgG (Delayed) lgA (Anaphylactic) lgE (Allergic) May be bacterial or viral o Effects on RBCs may be non-hemolytic or hemolytic [Intravascular (IgM) or Extravascular (IgG)] o After 6 hours and nothing happened, 7th hour had a reaction (Rashes, fever) those are still transfusion reaction Use of Allergic Transfusion Reaction- Urticarial TR o lgE - Mediated. – Allergen + IgE = Histamine o Type 1 Hypersensitivity Reaction o Sign and Symptoms: Urticaria, Hives, Itching, Anaphylaxis o Management and Prevention: Antihistamines • Prevented by administering washed components Anaphylactic (Anaphylactoid) Transfusion Reaction o Acute/Immediate Hemolytic Transfusion Reaction (IHTR) Clinical signs are seen suddenly after exposure of the patient to the IgA protein, often before 10mL of plasma has been infused o o The most severe, life-threatening due to ABO incompatibility Mediator: Plasma Proteins, Antibodies to lgA (Anaphylactic Reaction) o o Associated with intravascular hemolysis Management and Prevention: Transfusion of lgA-deficient components o Signs and Symptoms • Most severe cases may result in DIC and renal failure • Classical Signs - fever, chills, hemoglobinuria, dyspnea, hypotension o Anaphylactic in patients deficient with IgA who have class-specific IgA antibodies o Anaphylactoid in a patient with normal levels of IgA but limited type-specific Anti-IgA that reacts with the light chain of the donor's IgA Acute Transfusion Reactions: Immunologic o Mediators: IgM Antibodies (Usually ABO Antigens), Classical pathway Complement (Intravascular) Non-Cardiogenic Pulmonary Edema aka TRALI o Transfusion Related Acute Lung Injury, TRALI o o Most deadly; Acute onset of hypoxemia and pulmonary edema on Chest-XRAY within 6 hours of transfusion without evidence of cardiac failure Anti-neutrophil antigen antibodies or antiHLA antibodies are primarily responsible (Donor antibodies to recipient WI3Cs) o Causes endothelial damage to pulmonary capillaries o Most consistent finding is Anti-Leukocyte antibodies in donor or patient plasma o Leukocyte antibody that initiates complement-mediated capillary endothelial injury that may also trigger C3a and C5a o o May lead to congestive heart failure and pulmonary edema o Management: Slowing the transfusion rate, aliquots Delayed Adverse Immunologic Caused by endotoxins produced by gramnegative bacteria (Platelet contamination) o Mostly associated (Most often cause) with cold growing Yersinia enterocolitica. Also, with Pseudomonas sp., & Escherichia coli o Bacillus subtilis - Most common contaminant o The incidence of Bacterial Sepsis is highest with Y. enterocolitica o Treatment: IV broad-spectrum antibiotics Transfusion-Associated Circulatory Overload (TACO) Transfusion: o After 24 hours to 5-7 days, post-transfusion S/Sx o Characterized by the accelerated destruction of transfused RBCs o Most commonly associated with Secondary (Anamnestic) Response o The associated hemolysis is generally extravascular. (Liver/ spleen) no activation of complement o Signs and Symptoms: Jaundice decreasing hematocrit level o Management and Prevention: Transfusion of Irradiated Blood Components. T cell from a donor Acute Transfusion Reactions: Non-Immunologic o of Delayed Hemolytic Transfusion Reaction (DHTR) Leukocyte reduced blood is indicated Bacterial Contamination Effects and Transfusion-Associated Graft VS Host Disease (TA-GVHD) o (Irradiate) 25 G rays o These reactions occur when immunologically competent lymphocytes are transfused into an immunocompromised host o Sign and Symptoms: Fever, Liver Problems, Rash, Diarrhea o Management and Prevention: Transfusion of Irradiated Blood Components. T cell from a donor o Related to TRALI – Lungs o TACO is the second most common cause of transfusion o Related deaths were reported to the FDA o Iatrogenic transfusion reaction o Delayed Rare Transfusion Reaction o Due to the fast rate of transfusion o o Patients at risk include children, elderly patients, and patients with normovolemic anemia, cardiac disease, or sickle cells Characterized by Severe Thrombocytopenia 1 week after transfusion due to antibody to platelet specific antigens Post-Transfusion Purpura Usually occurs in multiparous women o o Due to anti-human platelet antigen-1A (HPA1a or anti-PLA1) o Management: IV limiting 2 weeks) immunoglobulin (Self- Delayed Adverse Effects of Transfusion: NonImmunologic Transfusion-Induced Hemosiderosis (Iron overload) o Chronic dependent on transfusion o Iron deposition in vital organs is seen in patients who are thalassemic and with chronic transfusions (Aplastic anemia, congenital hemolytic anemia) Treatment is using neocytes (Young RBCs) to reduce the frequency of transfusion (Ironchelating agents) o • • • • • Warm AIHA o Account for the majority of cases of AIHA (70%) o Antibodies involved are usually IgG and react best at 37°C AHG phase o DAT is always positive (With both IgG and C3D) o Most Common Specificity: Anti-Rh o Site of Hemolysis: Usually extravascular (No cell lysis) o If the antibody screen positive, adsorption and elution studies may be necessary to determine if the antibodies are autoantibodies, alloantibodies, or both Transmission of Disease o Hepatitis B, NANB Hepatitis (HCV), HIV, HTLV1, CMV, EBV o Syphilis - Spirochetes do not survive at ref temperature for 72 hours Cold AIHA o Second most common type of AIHA (20%) o Paroxysmal Cold Hemoglobinuria (PCH) Least common type of autoimmune hemolytic anemia (1-2%) Donath Landsteiner Test o Antibody involved is Autoanti-P o Cold Hemagglutinin Disease (CHD) - Usually with Anti-I specificity, the most common type of hemolytic anemia associated with coldreactive autoantibodies o Site of Hemolysis: Usually intravascular o DAT is positive because of the presence of C3d on the RBCs Hepatitis A- Infection by transfusion requires that the donor has Viremia o Autoimmune Hemolytic Anemias (AIHA) Associated with the presence of autoantibodies that agglutinate, sensitize or lyse one's own RBC Warm AIHA • • • Account for the majority of cases of AIHA Abs involved are usually lgG and react best at 37°C Site of Hemolysis - Usually extravascular Cold AIHA • • • Second most common type of AIHA Abs involved is auto-anti-P lgG Site of Hemolysis - Usually intravascular Drug-Induced AIHA Rarest type of AIHA Drug Adsorption Antibody Immune Complex Type Membrane Modification Methyldopa-induced/Auto-antibody Formation Drug-Induced AIHA o After delivery, the baby develops jaundice due to high unconjugated bilirubin o Risk of neurological increased bilirubin o The extent of fetal RBC destruction depends on the specificity, nature, and concentration of the maternal antibody o HDFN is most commonly caused by Anti-D, Anti-C, and Anti-K Drug Adsorption (Hapten Mechanism) • • Drug binds to any protein which produces a drug-RBC-hapten complex that stimulates antibody IgG formation (Examples: Penicillin, erythromycin, tetracycline) IgG is directed against the drug usually penicillin Immune Complex Type Innocent Bystander) • due to Dependent/ Antibodies to drugs such as Quinine, Quinidine, and Phenacetin form immune complexes with the drugs and activate complement (C3d usually found on the RBCs) Membrane Modification Protein Adsorption) • (Drug damage (Non-Immunologic Drugs such as Cephalosporin modify the RBC membrane and allow the nonspecific adsorption of proteins Drug-Independent (Autoantibody Formation) • DAT is usually positive because of the presence of IgG on RBCs • Production of autoantibodies (Examples: methyldopa [Aldomet], Mefenamic Acid [Ponstel], Levodopa, [L-Dopa] Hemolytic Disease of The Newborn o HDFN occurs when the mother has lgG red cell alloantibodies in her plasma that cross the placenta & bind to fetal red cells expressing the corresponding antigen o 1st Born: Unaffected o 2nd Born: Affected o Immune hemolysis may cause variable degrees of fetal anemia ranging from mild anemia to stillborn (Hydrops Fetalis - Baby dies of heart failure in utero) Most Clinical Feature o AnemiaMost immediate complication of HDFN among fetus o Hyperbilirubinemia (Jaundice) most immediate severe complication of HDFN among newborns. Associated with severe hemolytic anemia o Kernicterus – Severe complication Hyperbilirubinemia among neonates o Hydrops Fetalis - Edema of the fetal. Ultrasound 18th week-20th week of gestation to detect heart failure • Severe liver disease/damage • Severe hypoalbuminemia • CHF severe of Hepatosplenomegaly: Enlargement of liver and spleen, increased filtration of abnormal cell in reticuloendothelial cells. o • • • Less Severe Cases RhD - Mother, RhD + Baby Scenario First Pregnancy • Hydrops Fetalis - Total body swelling Intrauterine death Anemia Postnatal Jaundice - Due to unconjugated hyperbilirubinemia nonbeing cleared by immature liver o In the majority of RhD- mothers, an RhD+ pregnancy does not stimulate maternal production of anti-RhD o Volume of fetal RBCs crossing the placenta is too low to be immunogenic o o Anti-D is not a naturally occurring antibody - Only produced following exposure to RhD antigen Identifying at risk pregnancies via ABO and RhD typing of all new antenatal cases (12-16 weeks) o Monitor mother's blood for production of anti-RhD o Fetal Hb binds to oxygen with higher affinity than adult Hb - Gives the fetus better access to oxygen from the mother's bloodstream Second Pregnancy o 1-12 weeks of gestation, only amounts of IgG cross the placenta o 16 - 20 weeks, enough Anti-D can cross the placenta to cause intrauterine death o Chances of death continue to rise as gestation continues o When a group O RhD- mother is carrying a group A or B RhD+ baby, the chances of the mother making an anti-RhD are reduced o o small Role of The Transfusion Lab HbA HbF Two alpha and two beta subunits Two alpha and two gamma subunits Kleihauer Betke Method o HbF is also more acid-resistant than HbA o Maternal anti-A and anti-B (already present in the maternal circulation and DO NOT have to be made) Treat RBCs with acid • HbA will be 'eluted' • HbF will remain intact o They destroy fetal RBCs that may have crossed the placenta before there is time to mount a response to the RhD antigen Principle of the Kleihauer-Betke (KB) Test Standard method of quantitating fetomaternal hemorrhage o Microscopic examination on low power shows adult cells unstained or "ghost cells" with fetal cells stained bright pink o The degree of feto-maternal hemorrhage can be calculated by observing the ratio of fetal to maternal cells under a microscope Effects on The Baby Most Severe Cases • • • • • • Fetal anemia Extravascular hemolysis Hepatosplenomegaly Subcutaneous edema Pleural and pericardial effusions Kernicterus - Build-up of bilirubin in brain tissue Cell-Free Fetal DNA o Fetal DNA circulates in maternal blood from 6 weeks of gestation o Fluorochrome labeled anti-HbF added to the maternal sample o Fluorescence measured by flow cytometry o Antibody Titers o Measured by serially diluting the plasma and performing the IAT test on each diluted sample o Maternal plasma is diluted by half each time added to test RBCs o Result of titer test is determined by observing which dilution does not result in agglutination Other Indications o o o o Full Blood Count: Decreased RBC & decreased Hb levels due to decreased RBC survival Reticulocytosis: Reticulocyte count increases due to increased production of new RBCs as compensation for the red cell loss Hyperbilirubinemia - Serum bilirubin levels rise: High levels of bilirubin are more of a problem following birth because, in the womb, it is cleared via the placenta Neonatal liver is not mature enough to conjugate the bilirubin Positive Direct Agglutination Test (DAT): Demonstrates fetal red cell sensitization Tests on The Fetus o Antenatal ultrasound scan may detect signs of hydrops fetalis o Doppler ultrasound of the middle cerebral artery allows the clinician to access the speed of blood flow - Increased velocity indicates anemia (This technique has largely replaced fetal blood sampling as an initial test for the detection of fetal anemia - Less invasive) o Amniocentesis may be performed in order to measure bilirubin in the amniotic fluid (Allows estimation of the amount of RBC destruction taking place) Fetal blood sampling allows the evaluation of the degree of fetal anemia by measuring Hb and RBC • Allows for RhD status to be identified • Can be risky - Chance of miscarriage • If the Doppler scan confirms anemia, fetal blood sampling is considered Treatment o If fetal anemia is present, it may be necessary to perform an intrauterine exchange transfusion to replace the fetal blood with fresh blood o Post-natal transfusions o Aims to: • Dilute or remove antibodies and RBCs • Raise Hb level • Lower bilirubin level o Phototherapy o Photo-oxidation of bilirubin o Correction of jaundice circulating Temperature Room 21-24°C Refrigerator 1-6°C Freezer -1 to -6°C Glycerol Specimen 60°C 100°C Immediate Spin Phase 21-24°C Thermophase 37°C Incubation 37°C Note: o o Higher Levels of Glycerol - Mechanical Freezer Lower Levels of Glycerol - Liquid Nitrogen