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Fe A. Bartolome, M.D. Dept. of Pathology & Laboratory Diagnosis IMMUNOHEMATOLOGY •merges aspects of hematology, immunology & genetics •serologic, genetic, biochemical and molecular study of antigens associated with membrane structures on the cellular constituents of the blood •immunologic reactions involving components and constituents all blood IMMUNOLOGIC PRINCIPLES • primary immunological components: antigens & antibodies provides basis for blood bank testing and reactions CARDINAL RULE IN BLOOD BANK: The antigens are found on the surface of red blood cells and the antibodies are found in serum or plasma IMMUNOLOGIC PRINCIPLES ANTIGENS • substances that have the capability to stimulate the production of an antibody • characteristics: 1. Chemical nature – protein, CHO, lipopolysaccharide or nucleic acid 2. Molecular weight > 10,000 daltons 3. Complexity – more complex, > antibody stimulation 4. Stability – if unstable degrade less Ab stimulation 5. Foreign IMMUNOLOGIC PRINCIPLES Chemical composition of antigens: 1. Glycoproteins & lipoproteins – most potent 2. Glycolipids 3. Pure polysaccharides – not immunogenic except in humans and mice 4. Pure lipids & nucleic acids – not immunogenic but can be antigenic serve as haptens IMMUNOLOGIC PRINCIPLES Immunogenicity of Blood Group Antigens A, B and D (Rho) – most immunogenic Kell (K) Duffy: Fya Fyb Kidd: Jka Jkb IMMUNOLOGIC PRINCIPLES ANTIBODIES • also called immunoglobulins • characteristics: 1. Protein 2. Produced in response to stimulation by an antigen 3. Specific for the stimulating antigen • consists of 2 heavy chains & 2 light chains held together by disulfide bonds • produce 3 fragments when cleaved by enzymes 2 Agbinding fragments (Fab) & 1 crystallizable fragment (Fc) IMMUNOLOGIC PRINCIPLES Classification of Blood Group Antibodies: 1. Alloantibodies • Reacts with foreign Ag not present on patient’s own RBC • Most produced as result of immune stimulation via transfusion or pregnancy (usually during delivery) 2. Autoantibodies • Reacts with an Ag on patient’s own cells & with that same Ag on the cells of other individuals ABO BLOOD GROUP SYSTEM • discovered by Karl Landsteiner; locus on chr 9 • single most important blood group for the selection and transfusion of blood • widely expressed tissues & body fluids including red cells, platelets & endothelial cells • three antigens: A, B, H • two major antibodies: anti-A and anti-B • four phenotypes: A, B, AB, O A & B Ag’s autosomal co-dominant (expressed on grp A, B and AB red cells; O phenotype autosomal recessive (most frequent) ABO BLOOD GROUP SYSTEM ABO Antigens • present on the surface of red cells as well as tissue and endothelial cells in the body • found in soluble form in plasma & other body secretions in people known as secretors • inherited in simple Mendelian fashion from an individual’s parents • 3 possible genes that can be inherited: A, B, O • A and B genes produce a detectable product • O gene does not produce a detectable product ABO BLOOD GROUP SYSTEM ABO System Phenotype Antigen Natural antibody Genotype A A only Anti-B AA or AO B B only Anti-A BB or BO AB A and B None AB O None Anti-A, Anti-B OO ABO BLOOD GROUP SYSTEM • A and B genes do not directly produce antigens produce an enzyme called transferase attaches a sugar molecule to the chemical structure of the antigen sugar molecule responsible for specificity • O antigen no transferase no antigen produced • A and B antigens on surface of RBC protrude from outermost layer of cell membrane ABO BLOOD GROUP SYSTEM ABO BLOOD GROUP SYSTEM ABO BLOOD GROUP SYSTEM ABO BLOOD GROUP SYSTEM ABO BLOOD GROUP SYSTEM H Antigen • required to produce either A or B antigens • possible genetic combinations: HH, Hh, or hh • HH or Hh (+) produce H Ag 99.99% of Caucasians • hh does not produce H Ag Bombay phenotype (Oh) • anti-H antibodies rare – found only in individuals with Bombay phenotype ABO BLOOD GROUP SYSTEM Example of determining offspring blood types from known or suspected genotypes: Genotype parent #1 (AO) Genotype parent #2 (AB) A O A AA AO B AB BO Phenotypes of possible offsprings: A, AB, B ABO BLOOD GROUP SYSTEM Frequencies of ABO Blood Groups: Blood Group Frequency O 45% A 41% B 10% AB 4% ABO BLOOD GROUP SYSTEM ABO Subtypes: 1. A variants (A1, A2) • A1 most common (80%) & most antigenic • A1 and A2 differentiated using antisera specific for A1 Ag (anti-A1 lectin) prepared from seed known as Dolichos biflorus (+) reaction with A1 but not A2 • Anti-A reacts with both A1 & A2 but more strongly with A2 ABO BLOOD GROUP SYSTEM ABO Subtypes: 2. Weak A and weak B phenotypes 3. Null phenotypes: (a) Bombay (Oh) • No A, B or H Ag on red cells & secretions • With anti-A, anti-B & anti-H in their sera (b) para-Bombay • Absent or only trace A,B & H Ag’s detected on rbc w/ normal expression in secretions & body fluids ABO BLOOD GROUP SYSTEM ABO Antibodies • Natural antibodies antigenic stimulus is environmental exposure occurs from birth • Newborns without ABO antibodies of their own; begin to produce Ab with detectable titer at 6 months of age • Other characteristics of ABO antibodies: 1. IgM 2. Reacts at room temp. after an immediate spin ABO ROUTINE TESTING (slide or test tube method) DIRECT OR FORWARD TYPING • test for antigens • patient’s cells containing unknown antigens tested with known antisera • antisera manufactured from human sera • antisera used: Antisera Color Source Anti-A Blue Group B donor Anti-B Yellow Group A donor Anti-A,B Clear Group O donor ABO ROUTINE TESTING Anti-A,B • not a mixture of anti-A and anti-B • separate Ab that reacts with both A and B antigens • used in forward grouping for two purposes: 1. confirms the results of the anti-A and anti-B 2. will show a (+) reaction with weak subgroups of A and B that do not react with the anti-A and anti-B ABO ROUTINE TESTING Reaction Patterns for ABO Groups Blood group Agglutination with Anti-A Agglutination with Anti-B A + - B - + AB + + O - - ABO ROUTINE TESTING INDIRECT/REVERSE TYPING • known antigen (cell) vs. unknown antibody (patient’s serum) • serum is combined with cells having known Ag content in a 2:1 ratio • uses commercially prepared reagents containing saline-suspended A1 and B cells ABO ROUTINE TESTING Reaction Patterns for ABO Groups Blood Group Agglutination with A cells Agglutination with B cells A - + B + - AB - - O + + ABO ROUTINE TESTING Stages of Hemagglutination First Stage: • red cell sensitization • Ag and Ab held by non-covalent interactions Second Stage: • formation of stable latticework basis of visible reaction ABO ROUTINE TESTING Grading of Agglutination: Negative (0) No clumps or aggregates Weak (+/-) Tiny clumps or aggregates barely visible macroscopically or to the naked eye 1+ Few small aggregates visible macroscopically 2+ Medium-sized aggregates 3+ Several large aggregates 4+ One solid aggregate ABO ROUTINE TESTING Causes of Discrepancies in ABO Testing: A. Technical 1. Incorrect ID/recording 2. Patient/donor serum not added 3. Reagent contamination 4. Under-/over-centrifugation 5. Hemolysis 6. Warming of test mixture ABO ROUTINE TESTING Causes of Discrepancies in ABO Testing: B. Red Blood Cells 1. Missing or weak A/B antigen 2. Acquired B Ag – colon or gastric CA, intestinal obstruction 3. Polyagglutinable RBC 4. Ab-coated RBC – post-transfusion incompatibility; autoimmune hemolytic anemia 5. Maternal-fetal agglutination – mismatched transfusion ABO ROUTINE TESTING Causes of Discrepancies in ABO Testing: C. Serum 1. Roleaux formation – presence of plasma expanders, monoclonal gamma globulins 2. Anti-A1 3. Unexpected alloantibodies 4. Expected antibody absent – hypogammaglobulinemia, extreme ages, immunosuppression ABO ROUTINE TESTING WHAT TO DO? 1. Wash cells with saline 3-4x and repeat all tests and test for antibodies 2. Test for subgroups of A using anti-A1 and anti-A 3. Use cell panels to detect the specificity of abnormal antibodies Rh BLOOD GROUP SYSTEM • discovered in 1940 by Landsteiner & Wiener • most complex erythrocyte located on chromosome 1 • found exclusively on surface of rbc integral part of red cell membrane • primary antigen if present, consider Rh (+) • lack corresponding naturally-occurring antibodies in serum antigen system; Rh BLOOD GROUP SYSTEM CLASSIFICATION/NOMENCLATURE SYSTEM Wiener • Multiple allele hypothesis • 5 antigens: Rho, rh’, rh”, hr’, hr” • Single locus inheritance system with 8 alternate common alleles coding for agglutinogens 1 individual produces 2 agglutinogens inherited from both parents Rh BLOOD GROUP SYSTEM CLASSIFICATION/NOMENCLATURE SYSTEM Fischer & Race • Three alleles: D/d, C/c and E/e • Five antigens: D, C, E, c, e • d no D locus no antigenic products Rosenfeld • Numerical system • Rh1 to Rh5 Rh BLOOD GROUP SYSTEM Rh Antigens • with three integral membrane proteins 1. RhD 2. RhCcEe 3. Rh-associated glycoprotein (Rh50, RhAG) • D antigen resides in RhD protein most immunogenic followed by c, E, C and e Rh BLOOD GROUP SYSTEM Weak D Antigen (Du) • Rho variant • weak or absent red cell agglutination by anti-D detected only with use of anti-human globulin reagent use bovine anti-D • weakened form caused by 1 of 3 situations: 1. a piece of the D antigen is missing 2. D gene is on a chromosome opposite a C gene (+) steric hindrance 3. Inheritance of a gene coding for less D antigen Rh BLOOD GROUP SYSTEM • Presence of D = presence of Rho factor Rh (+) • Absence of D Rh (-) Rh BLOOD GROUP SYSTEM Testing for Rho (D) Antigen: • use antisera originating from human source • antisera with different constituents use of high protein media necessary to produce agglutination since antigens are an integral part of the red cell membrane less numerous than ABO antigens Rh BLOOD GROUP SYSTEM Testing for Du Variant: • use bovine reagent or albumin-suspended anti-D • incubate at 37oC for 15-60 minutes to facilitate formation of Ag-Ab complex • interpretation: (+) Du consider Rh (+) • women who appear to be Rh (-) should be proven to be Du (-) before they are considered to be eligible to receive transfusion Rh BLOOD GROUP SYSTEM Rh Antibodies • not naturally-occurring immune antibodies produced upon sensitization IgG isotype • reactive at 37oC enhanced with enzyme-treated red cells • can cross the placenta • associated with hemolytic transfusion reaction and hemolytic disease of the newborn (HDN) Rh BLOOD GROUP SYSTEM Rh Typing – slide or test tube method • False (+) results: 1. Drying 2. Roleaux formation 3. Auto-agglutination 4. Patient’s red cells heavily coated with Ab’s 5. Presence of cold agglutinins Rh BLOOD GROUP SYSTEM Rh Typing • False (-) results: 1. Use of old cells 2. Wrong cell concentration 3. Hemolysis 4. Inadequate mixing of cells 5. Inactive typing sera 6. Incorrent temperature 7. Existence of Du variant 8. High concentration of blocking antibodies MINOR BLOOD GROUP SYSTEMS Significance: 1. For medico-legal parenthood studies 2. May cause transfusion reaction or HDN MINOR BLOOD GROUP SYSTEMS Systems with cold-reacting antibodies • Antibodies formed react at temperatures 250C or colder • Not considered clinically significant since any reaction seen in the test tube will not be seen in the warmer temperatures of the body • Not likely to cause a transfusion-related accident MINOR BLOOD GROUP SYSTEMS Systems with cold-reacting antibodies 1. Lewis (Le) System • Antigens: Lea and Leb formed in secretions & absorbed onto surface of rbc later • Antibodies – often encountered in individuals with no antigens; may be present at certain times (e.g. pregnancy) and then disappear 2. MNS System • Antigens are weakly antigenic • Antibodies: naturally-occurring or stimulated by direct exposure MINOR BLOOD GROUP SYSTEMS Systems with cold-reacting antibodies 3. P-p System • P1 antigen most antigenic present on cells of 79% of whites & 94% of African-Americans 4. Ii system • Antigens: I and i both present in all individuals • I antigen – present in large quantities in adults • i antigen – present in large quantities on cells taken from the umbilical cord • Anti-I freq. seen in serum of patient’s with recent infectious mononucleosis MINOR BLOOD GROUP SYSTEMS Systems with warm-reacting antibodies • reactive at 370C in anti-human globulin medium • Clinically significant most likely to cause HDN and HTR 1. Kell (K) – Cellano (k) System • k Ag present in 98% of the white population • antibodies primarily IgG 2. Kidd System • Antigens: Jka & Jkb – not very antigenic • Antibodies stimulated by direct exposure via either pregnancy or transfusion MINOR BLOOD GROUP SYSTEMS Systems with warm-reacting antibodies 3. Duffy System • Antigens: Fya & Fyb • Antibodies stimulated through direct exposure capable of causing HDN and HTR HEMOLYTIC DISEASE OF THE NEWBORN • involves hemolysis of red cells in the fetus and neonate • antibody is present in the mother that corresponds to an antigen on the surface of the red cells of the fetus Ab crosses placenta attaches to fetal Ag hemolyze red cells of fetus • Differential septicemia, syphilis diagnosis: physiologic jaundice, CID, toxoplasmosis, congenital HEMOLYTIC DISEASE OF THE NEWBORN ABO Disease • Most common type • Most cases are mild & do not require exchange transfusion • Most common scenario: mother is group O and infant is group A • Even first baby is affected HEMOLYTIC DISEASE OF THE NEWBORN ABO Disease Features: 1.Spherocytosis 2.Increased reticulocyte count 3.Increased indirect bilirubin in 1st 72 hours of life 4.Jaundice appearing during first 24 hrs of life Good evidence for ABO disease is detection of immune anti-A or anti-B in the cord blood of the newborn. HEMOLYTIC DISEASE OF THE NEWBORN Rh Disease • most severe; Rh (+) fetus & Rh (-) mother • FIRST PREGNANCY Rh (+) baby Ag enters maternal circulation sensitize Rh (-) mother anti-Rh production (IgG) cross placenta enter fetal circulation baby not affected • SUBSEQUENT PREGNANCIES Ab already present in mother enter fetal circulation (+) intravascular hemolysis accumulation of rbc destruction products jaundice or kernicterus (erythroblastosis fetalis) HEMOLYTIC DISEASE OF THE NEWBORN HEMOLYTIC DISEASE OF THE NEWBORN Rh Disease • first baby usually unaffected since it is the first time the mother is exposed to the antigen • occasionally, because of: firstborns are affected either 1. previous maternal exposure (e.g. previous aborted pregnancy) 2. unusually great maternal susceptibility to Rh stimulus during normal pregnancy HEMOLYTIC DISEASE OF THE NEWBORN Rh Disease Characteristics of Erythroblastosis Fetalis: 1. Increased number of circulating nucleated red cells 2. Increased osmotic fragility of cells 3. Increased amount of indirect/unconjugated bilirubin Main Clinical Findings: 1. Anemia - < 15 gm/100 ml or 150 gm/L 2. Rapidly developing jaundice HEMOLYTIC DISEASE OF THE NEWBORN Rh Disease Management: For the mother • RhoGam (Rh Immune Globulin) concentrated anti-D coats Rh (+) fetal cells in maternal circulation recognized by mother’s system as abnormal & removed from circulation prevents maternal immune system from processing the Ag on surface of fetal cells no antibody formed HEMOLYTIC DISEASE OF THE NEWBORN HEMOLYTIC DISEASE OF THE NEWBORN For the mother • RhoGam (Rh Immune Globulin) Dose: routinely administered 2x – at 28 wks AOG & within 72 hrs after birth of an Rh (+) infant Also administered following termination of any pregnancy, after amniocentesis in an Rh (-) mother & following accidental transfusion with Rh (+) red cells HEMOLYTIC DISEASE OF THE NEWBORN For the baby: EXCHANGE TRANSFUSION • Indications: 1. Infant serum indirect bilirubin > 20mg/100 ml (342 mol/L) for fullterm infants OR >15mg/100 ml (257 mol/L) for premature infants 2. Cord blood indirect bilirubin > 3 mg/100 ml (51 mol/L) 3. Cord blood hemoglobin < 13 gm/dL (130 g/L) 4. Maternal Rh antibody titer of 1:64 or more HEMOLYTIC DISEASE OF THE NEWBORN Beneficial Effects of Exchange Transfusion: 1. Removal of bilirubin 2. Removal of sensitized RBCs 3. Removal of incompatible antibody 4. Replacement of compatible RBCs incompatible 5. Suppression of erythropoeisis production of incompatible RBCs RBCs with (reduced HEMOLYTIC DISEASE OF THE NEWBORN Comparison of ABO versus Rh HDN Characteristic First pregnancy Disease predicted by titers Antibody IgG Bilirubin at birth Anemia at birth Phototherapy Exchange transfusion Intrauterine transfusion Spherocytosis ABO HDN Yes No Yes (anti-A,B) Normal range No Yes Rare None Yes Rare Yes Yes (anti-D) Elevated Yes Yes Common Sometimes Rare