Immunohematology            Dr. Julio Hilario Vargas           Department of Physiology               School of Medicine  ...
DEFINICIONEs la parte de la hematología que estudia losprocesos inmunitarios que tienen lugar en elorganismo en relación c...
En primer termino se identificaron sobre los hematíes,pero luego se describieron determinantes antigénicosplaquetarios, le...
ImmunohematologyMerges aspects of hematology, immunology &geneticsSerologic, genetic, biochemical and molecularstudy of an...
CARDINAL RULE IN BLOOD BANK:Antigens are found on the surface of red blood cells and the antibodies   are found in serum o...
IMMUNOLOGIC PRINCIPLES
ANTIGENSSubstances that have the capability to stimulate theproduction of an antibodyCharacteristics:  1. Chemical nature ...
Chemical composition of antigens1. Glycoproteins & lipoproteins – most potentGlycolipids2. Pure polysaccharides – not immu...
Grupos Eritrocitarios y sus Antígenos                      ANTÍGENOS MASGRUPOS SANGUÍNEOS                       IMPORTANTE...
SISTEMAS DE GRUPOS SANGUINEOS    Grispan S. Rev. Medica Hondur. 51:103-14. 1983
Immunogenicity of Blood Group Antigens    A, B and D (Rho) – most immunogenic    Kell (K)    Duffy: Fya         Fyb    Kid...
ANTIBODIES              Also called immunoglobulinsCharacteristics:1. Protein2. Produced in response to stimulation by an ...
Classification of Blood Group AntibodiesAlloantibodies   Reacts with foreign Ag not present on patient’s own      RBC   Mo...
ABO BLOOD GROUP SYSTEMDiscovered by Karl Landsteiner; locus on chr 9Single most important blood group for the selection an...
ABO BLOOD GROUP SYSTEM
ABO BLOOD GROUP SYSTEMABO AntigensPresent on the surface of red cells as well as tissue   and endothelial cells in the bod...
ABO BLOOD GROUP SYSTEMABO SystemPhenotype    Antigen    Natural   Genotype                       antibody   A         A on...
ABO BLOOD GROUP SYSTEM  A and B genes do not directly produceantigens  enzymatic reaction products ofenzymes called glyco...
ABO BLOOD GROUP SYSTEM Red blood cell precursor structure
ABO BLOOD GROUP SYSTEM            Antigen formationH antigen      B antigen        B antigen
ABO Blood Group Systemhttp://www.ncbi.nlm.nih.gov/gv/rbc/xslcgi.fcgi?cmd=bgmut/systems_info&system=abo
ABO BLOOD GROUP SYSTEMH AntigenRequired to produce either A or B antigenspossible genetic combinations: HH, Hh, or hhHH or...
ABO BLOOD GROUP SYSTEMExample of determining offspring blood typesfrom known or suspected genotypes:               Genotyp...
ABO BLOOD GROUP SYSTEMFrequencies of ABO Blood Groups:    Blood Group         Frequency         O                 45%     ...
ABO BLOOD GROUP SYSTEMABO Subtypes:1. A variants (A1, A2)   A1 most common (80%) & most antigenic   A1 and A2 differentiat...
ABO BLOOD GROUP SYSTEMABO Subtypes:2. Weak A and weak B phenotypes3. Null phenotypes:  (a) Bombay (Oh)     No A, B or H Ag...
ABO BLOOD GROUP SYSTEMABO AntibodiesNatural antibodies  antigenic stimulus is   environmental  exposure occurs from birt...
ABO ROUTINE TESTING                (slide or test tube method)DIRECT TYPING- test for antigens- patient’s cells containing...
ABO ROUTINE TESTING    Reaction Patterns for ABO Groups               Agglutination   AgglutinationBlood group            ...
ABO ROUTINE TESTINGINDIRECT/REVERSE TYPING- Known antigen (cell) vs. unknown antibody   (patient’s serum)- Serum is combin...
ABO ROUTINE TESTING    Reaction Patterns for ABO Groups               Agglutination    AgglutinationBlood Group           ...
ABO ROUTINE TESTINGCauses of Discrepancies in ABO TestingTechnical  1. Incorrect ID/recording  2. Patient/donor serum not ...
ABO ROUTINE TESTING     Causes of Discrepancies in ABO TestingB. Red Blood Cells   1. Missing or weak A/B antigen   2. Acq...
ABO ROUTINE TESTING Causes of Discrepancies in ABO TestingC. Serum  1. Roleaux formation – presence of  plasma expanders, ...
Rh BLOOD GROUP SYSTEM- Discovered in 1940 by Landsteiner &   Wiener-   Most complex erythrocyte antigen    system; located...
FRECUENCIAS FENOTIPICAS DEL SISTEMA RH   ANTIGENO Rh        Frecuencia ( %)       D (+)                85        D(-)     ...
Rh BLOOD GROUP SYSTEMCLASSIFICATION/NOMENCLATURE SYSTEMWienerMultiple allele hypothesis5 antigens: Rho, rh’, rh”, hr’, hr”...
Rh BLOOD GROUP SYSTEMCLASSIFICATION/NOMENCLATURE SYSTEMFischer & RaceThree alleles: D/d, C/c and E/eFive antigens: D, C, E...
Comparación de las nomenclaturas para       los Ag del Sistema RhWiener      Fisher –Race    Rosenfield Rho             D ...
Rh BLOOD GROUP SYSTEMPresence 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 di...
Rh BLOOD GROUP SYSTEM             Testing for Du VariantUse bovine     or   albumin-suspended      anti-DreagentIncubate a...
Rh BLOOD GROUP SYSTEMRh Antibodies- Not naturally-occurring  immune antibodies produced upon sensitization  IgG isotype...
Rh BLOOD GROUP SYSTEM  Rh Typing – slide or test tube method              False (+) resultsa. Dryingb. Roleaux formationc....
Rh BLOOD GROUP SYSTEM                 Rh Typing               False (-) resultsa. Use of old cellsb. Wrong cell concentrat...
HEMOLYTIC DISEASE OF THE NEWBORNInvolves hemolysis of red cells in the fetus and   neonateAntibody is present in the mothe...
HEMOLYTIC DISEASE OF THE NEWBORN            Comparison of ABO versus Rh HDNCharacteristic                    ABO          ...
3.inmuno   hematología.inmunologia.2011.dr hilario
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3.inmuno hematología.inmunologia.2011.dr hilario

  1. 1. Immunohematology Dr. Julio Hilario Vargas Department of Physiology School of Medicine National University of Trujillo
  2. 2. DEFINICIONEs la parte de la hematología que estudia losprocesos inmunitarios que tienen lugar en elorganismo en relación con los elementossanguíneos.Uno de los aspectos más importantes de lainmunohematología es el estudio y cuantificaciónde los grupos sanguíneos eritrocitarios que soncomponentes antigénicos presentes en lasuperficie de los hematíes, ya que se relacionadirectamente con la terapéutica transfusional y laprevención de accidentes hemolíticos graves.
  3. 3. En primer termino se identificaron sobre los hematíes,pero luego se describieron determinantes antigénicosplaquetarios, leucocitarios y séricos.Los genes determinantes de los grupos sanguíneostransmiten, generalmente, caracteres codominantes ( seexpresan en homocigotos y heterocigotos).Existen genes “amorfos” que no generan productos quepuedan ser identificados como antígenos ( ej: gen d)Todos los antígenos de los grupos sanguíneos han sidodefinidos serológicamente por la presencia de susanticuerpos correspondientes.
  4. 4. ImmunohematologyMerges aspects of hematology, immunology &geneticsSerologic, genetic, biochemical and molecularstudy of antigens associated with membranestructures on the cellular constituents of the bloodImmunologic reactions involving all bloodcomponents and constituentsPrimary immunological components: antigens &antibodies  provides basis for blood bank testingand reactions
  5. 5. CARDINAL RULE IN BLOOD BANK:Antigens are found on the surface of red blood cells and the antibodies are found in serum or plasma
  6. 6. IMMUNOLOGIC PRINCIPLES
  7. 7. ANTIGENSSubstances that have the capability to stimulate theproduction of an antibodyCharacteristics: 1. Chemical nature – protein, CHO, nucleic acid orlipopolysaccharide 2. Molecular weight > 10,000 daltons 3. Complexity – more complex, > antibodystimulation 4. Stability – if unstable  degrade  less Abstimulation 5. Foreign
  8. 8. Chemical composition of antigens1. Glycoproteins & lipoproteins – most potentGlycolipids2. Pure polysaccharides – not immunogenic except in humans and mice3. Pure lipids & nucleic acids – not immunogenic but can be antigenic  serve as haptens
  9. 9. Grupos Eritrocitarios y sus Antígenos ANTÍGENOS MASGRUPOS SANGUÍNEOS IMPORTANTES ABO A, B, AB, O Rh D, C, c, E, e MNS M, N, S, s, U Lewis Lea, Leb P P1, P2 Lutheran Lua, Lub Kell K, k, Kpa, Kpb Duffy Fya, Fyb Kidd Jka, Jkb
  10. 10. SISTEMAS DE GRUPOS SANGUINEOS Grispan S. Rev. Medica Hondur. 51:103-14. 1983
  11. 11. Immunogenicity of Blood Group Antigens A, B and D (Rho) – most immunogenic Kell (K) Duffy: Fya Fyb Kidd:Jka Jkb
  12. 12. ANTIBODIES Also called immunoglobulinsCharacteristics:1. Protein2. Produced in response to stimulation by an antigen3. 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  2Ag- binding fragments (Fab) & 1 crystallizable fragment(Fc)
  13. 13. Classification of Blood Group AntibodiesAlloantibodies 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
  14. 14. ABO BLOOD GROUP SYSTEMDiscovered by Karl Landsteiner; locus on chr 9Single most important blood group for the selection and transfusion of bloodWidely expressed  tissues & body fluids including red cells, platelets & endothelial cellsThree antigens: A, B, HTwo major antibodies: anti-A and anti-BFour 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)
  15. 15. ABO BLOOD GROUP SYSTEM
  16. 16. ABO BLOOD GROUP SYSTEMABO AntigensPresent on the surface of red cells as well as tissue and endothelial cells in the bodyFound in soluble form in plasma & other body secretions in people known as secretorsInherited in simple Mendelian fashion from an individual’s parents3 possible genes that can be inherited: A, B, OA and B genes produce a detectable productO gene does not produce a detectable product
  17. 17. ABO BLOOD GROUP SYSTEMABO SystemPhenotype Antigen Natural Genotype antibody A A only Anti-B AA or AO B B only Anti-A BB or BO AB A and B None AB Anti-A, O None OO Anti-B
  18. 18. ABO BLOOD GROUP SYSTEM A and B genes do not directly produceantigens  enzymatic reaction products ofenzymes called glycosyltransferases attaches a sugar molecule to the chemicalstructure of the antigen  sugar moleculeresponsible for specificity O antigen  no transferase  no antigenproduced A and B antigens on surface of RBC protrude from outermost layer of cellmembrane
  19. 19. ABO BLOOD GROUP SYSTEM Red blood cell precursor structure
  20. 20. ABO BLOOD GROUP SYSTEM Antigen formationH antigen B antigen B antigen
  21. 21. ABO Blood Group Systemhttp://www.ncbi.nlm.nih.gov/gv/rbc/xslcgi.fcgi?cmd=bgmut/systems_info&system=abo
  22. 22. ABO BLOOD GROUP SYSTEMH AntigenRequired to produce either A or B antigenspossible genetic combinations: HH, Hh, or hhHH or Hh (+)  produce H Ag  99.99% ofCaucasianshh  does not produce H Ag  Bombayphenotype (Oh)Anti-H antibodies rare – found only in individuals with Bombay phenotype
  23. 23. ABO BLOOD GROUP SYSTEMExample of determining offspring blood typesfrom known or suspected genotypes: Genotype parent #1 (AO) A OGenotype parent A AA AO #2 (AB) B AB BOPhenotypes of possible offsprings: A, AB, B
  24. 24. ABO BLOOD GROUP SYSTEMFrequencies of ABO Blood Groups: Blood Group Frequency O 45% A 41% B 10% AB 4%
  25. 25. ABO BLOOD GROUP SYSTEMABO 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
  26. 26. ABO BLOOD GROUP SYSTEMABO Subtypes:2. Weak A and weak B phenotypes3. 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
  27. 27. ABO BLOOD GROUP SYSTEMABO AntibodiesNatural antibodies  antigenic stimulus is environmental  exposure occurs from birthNewborns  without ABO antibodies of their own; begin to produce Ab with detectable titer at 6 months of ageOther characteristics of ABO antibodies: - IgM - Reacts at room temp. after an immediate spin
  28. 28. ABO ROUTINE TESTING (slide or test tube method)DIRECT TYPING- test for antigens- patient’s cells containing unknown antigens tested with known antisera- antisera manufactured from human sera- antisera used:Antisera Color SourceAnti-A Blue Group B donorAnti-B Yellow Group A donorAnti-A,B Clear Group O donor
  29. 29. ABO ROUTINE TESTING Reaction Patterns for ABO Groups Agglutination AgglutinationBlood group with Anti-A with Anti-B A + - B - + AB + + O - -
  30. 30. ABO ROUTINE TESTINGINDIRECT/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
  31. 31. ABO ROUTINE TESTING Reaction Patterns for ABO Groups Agglutination AgglutinationBlood Group with A cells with B cells A - + B + - AB - - O + +
  32. 32. ABO ROUTINE TESTINGCauses of Discrepancies in ABO TestingTechnical 1. Incorrect ID/recording 2. Patient/donor serum not added 3. Reagent contamination 4. Under-/over-centrifugation 5. Hemolysis 6. Warming of test mixture
  33. 33. ABO ROUTINE TESTING Causes of Discrepancies in ABO TestingB. 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
  34. 34. ABO ROUTINE TESTING Causes of Discrepancies in ABO TestingC. Serum 1. Roleaux formation – presence of plasma expanders, monoclonal gamma globulins 2. Anti-A1 3. Unexpected alloantibodies 4. Expected antibody absent – Roleaux hypogammaglobulinemia, extreme ages, immunosuppression
  35. 35. 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
  36. 36. FRECUENCIAS FENOTIPICAS DEL SISTEMA RH ANTIGENO Rh Frecuencia ( %) D (+) 85 D(-) 15 C 70 c 80 E 30 e 98
  37. 37. Rh BLOOD GROUP SYSTEMCLASSIFICATION/NOMENCLATURE SYSTEMWienerMultiple allele hypothesis5 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
  38. 38. Rh BLOOD GROUP SYSTEMCLASSIFICATION/NOMENCLATURE SYSTEMFischer & RaceThree alleles: D/d, C/c and E/eFive antigens: D, C, E, c, ed  no D locus  no antigenic productsRosenfeldNumerical systemRh1 to Rh5
  39. 39. Comparación de las nomenclaturas para los Ag del Sistema RhWiener Fisher –Race Rosenfield Rho D Rh1 rh` C Rh2 rh” E Rh3 h`r c Rh4 hr” e Rh5
  40. 40. Rh BLOOD GROUP SYSTEMPresence of D = presence of Rho factor  Rh (+)Absence of D  Rh (-)
  41. 41. Rh BLOOD GROUP SYSTEM Testing for Rho (D) Antigen- Use antisera originating from human source- Antisera with different constituents  use ofhigh protein media necessary to produceagglutination since antigens are an integral part ofthe red cell membrane  less numerous than ABOantigens
  42. 42. Rh BLOOD GROUP SYSTEM Testing for Du VariantUse bovine or albumin-suspended anti-DreagentIncubate at 37oC for 15-60 minutes to facilitateformation of Ag-Ab complexInterpretation: (+) Du  consider Rh (+)Person who appear to be Rh (-) should beproven to be Du (-) before they are consideredto be eligible to receive transfusion
  43. 43. Rh BLOOD GROUP SYSTEMRh 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 transfusionreaction and hemolytic disease of the newborn(HDN)
  44. 44. Rh BLOOD GROUP SYSTEM Rh Typing – slide or test tube method False (+) resultsa. Dryingb. Roleaux formationc. Auto-agglutinationd. Patient’s red cells heavily coated with Ab’se. Presence of cold agglutinins
  45. 45. Rh BLOOD GROUP SYSTEM Rh Typing False (-) resultsa. Use of old cellsb. Wrong cell concentrationc. Hemolysisd. Inadequate mixing of cellse. Inactive typing seraf. Incorrect temperatureg. Existence of Du varianth. High concentration of blocking antibodies
  46. 46. HEMOLYTIC DISEASE OF THE NEWBORNInvolves hemolysis of red cells in the fetus and neonateAntibody 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 fetusDifferential diagnosis: physiologic jaundice, septicemia, CID, toxoplasmosis, congenital syphilis
  47. 47. HEMOLYTIC DISEASE OF THE NEWBORN Comparison of ABO versus Rh HDNCharacteristic ABO HDNFirst pregnancy Yes RareDisease predicted by titers No YesAntibody IgG Yes (anti-A,B) Yes (anti-D)Bilirubin at birth Normal range ElevatedAnemia at birth No YesPhototherapy Yes YesExchange transfusion Rare CommonIntrauterine transfusion None SometimesSpherocytosis Yes Rare

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