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IMMUNOHEMATOLOGY

IMMUNOHEMATOLOGY. Fe A. Bartolome, M.D. Dept. of Pathology & Laboratory Diagnosis. IMMUNOHEMATOLOGY. merges aspects of hematology, immunology & genetics

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IMMUNOHEMATOLOGY

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  1. IMMUNOHEMATOLOGY Fe A. Bartolome, M.D. Dept. of Pathology & Laboratory Diagnosis

  2. 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 all blood components and constituents

  3. 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

  4. 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

  5. IMMUNOLOGIC PRINCIPLES • Chemical composition of antigens: • Glycoproteins & lipoproteins – most potent • Glycolipids • Pure polysaccharides – not immunogenic except in humans and mice • Pure lipids & nucleic acids – not immunogenic but can be antigenic  serve as haptens

  6. IMMUNOLOGIC PRINCIPLES Immunogenicity of Blood Group Antigens A, B and D (Rho) – most immunogenic Kell (K) Duffy: Fya Fyb Kidd: Jka Jkb

  7. 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 Ag-binding fragments (Fab) & 1 crystallizable fragment (Fc)

  8. IMMUNOLOGIC PRINCIPLES • Classification of Blood Group Antibodies: • 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) • Autoantibodies • Reacts with an Ag on patient’s own cells & with that same Ag on the cells of other individuals

  9. 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)

  10. 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

  11. ABO BLOOD GROUP SYSTEM ABO System

  12. 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

  13. ABO BLOOD GROUP SYSTEM

  14. ABO BLOOD GROUP SYSTEM

  15. ABO BLOOD GROUP SYSTEM

  16. ABO BLOOD GROUP SYSTEM

  17. 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

  18. ABO BLOOD GROUP SYSTEM Example of determining offspring blood types from known or suspected genotypes: Genotype parent #1 (AO) A O Genotype parent AAAAO #2 (AB) B ABBO Phenotypes of possible offsprings: A, AB, B

  19. ABO BLOOD GROUP SYSTEM Frequencies of ABO Blood Groups: Blood Group Frequency O 45% A 41% B 10% AB 4%

  20. ABO BLOOD GROUP SYSTEM • ABO Subtypes: • 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

  21. ABO BLOOD GROUP SYSTEM • ABO Subtypes: • Weak A and weak B phenotypes • 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

  22. 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: • IgM • Reacts at room temp. after an immediate spin

  23. 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: • AntiseraColorSource • Anti-A Blue Group B donor • Anti-B Yellow Group A donor • Anti-A,B Clear Group O donor

  24. 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: • confirms the results of the anti-A and anti-B • will show a (+) reaction with weak subgroups of A and B that do not react with the anti-A and anti-B

  25. ABO ROUTINE TESTING Reaction Patterns for ABO Groups

  26. 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

  27. ABO ROUTINE TESTING Reaction Patterns for ABO Groups

  28. 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

  29. 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

  30. ABO ROUTINE TESTING • Causes of Discrepancies in ABO Testing: • 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

  31. 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

  32. 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

  33. ABO ROUTINE TESTING • WHAT TO DO? • Wash cells with saline 3-4x and repeat all tests and test for antibodies • Test for subgroups of A using anti-A1 and anti-A • Use cell panels to detect the specificity of abnormal antibodies

  34. Rh BLOOD GROUP SYSTEM • discovered in 1940 by Landsteiner & Wiener • most complex erythrocyte antigen system; 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

  35. 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

  36. 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

  37. Rh BLOOD GROUP SYSTEM • Rh Antigens • with three integral membrane proteins • RhD • RhCcEe • Rh-associated glycoprotein (Rh50, RhAG) • D antigen resides in RhD protein  most immunogenic followed by c, E, C and e

  38. 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: • a piece of the D antigen is missing • D gene is on a chromosome opposite a C gene  (+) steric hindrance • Inheritance of a gene coding for less D antigen

  39. Rh BLOOD GROUP SYSTEM • Presence of D = presence of Rho factor  Rh (+) • Absence of D  Rh (-)

  40. 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

  41. Rh BLOOD GROUP SYSTEM • Testing for Du Variant: • use bovine or albumin-suspended anti-D reagent • 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

  42. 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)

  43. Rh BLOOD GROUP SYSTEM • Rh Typing – slide or test tube method • False (+) results: • Drying • Roleaux formation • Auto-agglutination • Patient’s red cells heavily coated with Ab’s • Presence of cold agglutinins

  44. Rh BLOOD GROUP SYSTEM • Rh Typing • False (-) results: • Use of old cells • Wrong cell concentration • Hemolysis • Inadequate mixing of cells • Inactive typing sera • Incorrent temperature • Existence of Du variant • High concentration of blocking antibodies

  45. MINOR BLOOD GROUP SYSTEMS • Significance: • For medico-legal parenthood studies • May cause transfusion reaction or HDN

  46. 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

  47. MINOR BLOOD GROUP SYSTEMS • Systems with cold-reacting antibodies • 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 • MNS System • Antigens are weakly antigenic • Antibodies: naturally-occurring or stimulated by direct exposure

  48. MINOR BLOOD GROUP SYSTEMS • Systems with cold-reacting antibodies • P-p System • P1 antigen most antigenic present on cells of 79% of whites & 94% of African-Americans • 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

  49. 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 • Kell (K) – Cellano (k) System • k Ag present in 98% of the white population • antibodies primarily IgG • Kidd System • Antigens: Jka & Jkb – not very antigenic • Antibodies stimulated by direct exposure via either pregnancy or transfusion

  50. MINOR BLOOD GROUP SYSTEMS • Systems with warm-reacting antibodies • Duffy System • Antigens: Fya & Fyb • Antibodies stimulated through direct exposure  capable of causing HDN and HTR

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