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1Terry Kotrla, MS, MT(ASCP)BB 2010 ABO and H Blood GroupsTerry Kotrla, MS, MT(ASCP)BB2010
2HistoryDiscovered in 1900 by Karl Landsteiner and remains the most important blood group systemMixed blood of colleagues (serum from one, cells from another) togetherDiscovered A, B and OHis student discovered AB in 1902
3Landsteiner’s RuleReciprocal antibodies are consistently and predictably present in the sera of normal people whose rbcs lack the corresponding antigen(s)He was awarded the Nobel Prize in Physiology or Medicine in 1930 for his work
5Red Cell membrane structure Surface of the RBC consists of a bilipid membrane in which large protein molecules are embedded.Composed of phosholipids which are both hydrophilic (heads) and hydrophobic (tails).
8Red Blood Cell Membrane External surface of RBC membrane is coated with a diverse array of glycoproteins, complex carbohydrates, and lipoproteins, imparting antigenic structure to the membrane.
9A and B Antigens Inheritance follows Mendelian genetics Frequency in white population: group O 45%, group A 40%, group B 11% and group AB 4%.Frequencies differ in selected populations and ethnic groupsGroup B higher in Black and Asian populations
10A and B AntigensNot fully developed at birth, fewer copies of the antigen on the cells.Antigens detectable as early as 5 weeks after conceptionHuman typing sera may give weaker, or very rarely negative, reactions
11Biochemistry of A, B, H Antigens ABO antigens are located on RBCs, lymphs, platelets, tissue cells, bone marrow and solid organs.Inheritance results in expression.Antigens ARE NOT direct products of the genes.Genes code for TRANSFERASE which causes transfer of monosaccharide molecule onto a precursor substance on the RBC.
12H GeneCodes for the production of fucosyl transferase that catalyzes the addition of L-fucose,the immunodominant structure of H antigen.Two slightly different structures, known as the type 1 and type 2 precursor chains.The H gene and its allele h are inherited independently of the allelic A, B and O genesH antigen only person is group O.Once L-fucose added A and B gene specified products can be add their sugars.
13A GeneCodes for production of a galactosaminyl transferase that effects the addition of N-acetyl-galactosamine to the preformed H-bearing chains.
14B GeneCodes for production of a galactosyl transferase that effects the addition of D-galactose to the same H-bearing structure.
17A, B and HImmunodominant structure of the H antigen is L-fucose – person is group OImmunodominant structure of A antigen N-acetyl-galactosamine – person is group AImmunodominant structure of the B antigen, D-galactose- person is group B
18Bombay (Oh) If L-fucose is added H, A and B cannot be added. Individual may inherit A or B genes but without H the immunodominant sugars CANNOT be added.Must be homozygous for h, which is an amorph, with no detectable product, similar to d.A Bombay individual has genotype hh but WILL type as Group O.Can only be determined by family studies or testing with lectins
20The H System Two genes: H and h Leads to production of H antigen – precursor molecule for A and B antigens.Ulex europaeus = anti-HNot all H is converted to A or B, varies among the blood groups.Memorize: 0>A2>B>A2B>A1>A1B
21H Antibodies H-like antigens are found in nature. Anti-H occasionally detected in A1, A1B and rarely in B individuals and will cause false positive in the REVERSE reaction.So little H on cells may form anti-HRelatively weakAlmost always reacts at RTConsidered clinically insignificant
22H Antibodies May cause an ABO discrepancy Anti-A Anti-B A1 cells B CellsInterp4+1+
23H AntibodiesIn contrast persons of the rare Oh (hh) phenotype (have no A, B or H antigens on their RBCs) form a potent clinically significant anti H which reacts well over a wide thermal range and with all RBCs except those of other O people.Anti-AAnti-BA1 CellB CellO Cell4+
24Oh Phenotype (Bombay)Occurs when two hh genes are inherited at the Hh locus.Possess normal A or B genes (if they were inherited) but unable to express.Must have H on red cell membraneCan transmit A or B gene to offspringTerm “Bombay” used since first discovered in Bombay, India
25Oh Phenotype (Bombay) Symbol “Oh” denotes this phenotype RBCs not agglutinated by anti-A, -B or –A,BSerum/plasma agglutinates A and B cellsNot recognized until serum tested against group O cells and causes strong agglutination.Have anti-A, -B, -A,B and –HCan only be transfused with Bombay blood <0.01%
26Oh Phenotype (Bombay) Confirmatory testing Anti-H lectin (Ulex europaeus) – negativeAgglutination of A, B, AB and O cellsSerum/plasma will not agglutinate Oh cells.
27Secretor Genes A, B and H antigens may be present in fluids. Controlled by Se and se, secretor genes.Need only one copy of the Se gene.The gene se is an amorph.Not linked to ABO locus, inherited independently
29Substance in Secretions Secretor GenesPersons who have A, B and/or H in secretions are called “secretors”Blood GroupSubstance in SecretionsAA and HBB and HABA, B and HOH
30Secretor GenesSecretor studies helpful in defining weak subgroups or resolving genetic make up of individuals with unusual blood group80% of Caucasians are secretors20% are non-secretors
31Subgroups of A (A1 and A2)Subgroups of A are phenotypes that differ from others of the same ABO group with respect to theamount of A antigen carried on RBCs, and,in secretors, present in the saliva.Variant gene produces a weaker than normal red cell antigen
32Subgroups of A (A1 and A2)Different levels of expression of A (or B) on RBCs are classified into subgroups80% of group A individuals are A1Approximately 20% are A2Transferase produced by A2 gene differs from that produced by A1, less efficient in converting H chains to A
34Difference Between A1 and A2 A1 has more A and less H antigen on the cell.A2 has less A and more H antigenCannot be detected serologicallyA2 can produce anti- A2 – qualitative difference?
35LectinsNaturally occurring materials (usually plant) that react specifically with blood group antigens.Dolichos biflorus – anti-A1Will agglutinate A1 red blood cellsWill not agglutinate A2 red blood cells
36Anti-A1 1-8% of A2 and 22-35% of A2B people will have anti-A1 Causes ABO discrepancy – reverse typeIncompatible crossmatch if donor A1NOT clinically significant unless reactive at 37C or AHG.Clinically significant – ability to cause red cell destruction – donor blood or hemolytic disease of the fetus and newborn.
37Subgroups of A weaker than A2 Occur infrequently, characterized by decreasing numbers of A antigensLess than 1% of total A gene pool
38Classification of Weak A Subgroups Strength of agglutination when tested with:Anti-A1 lectinAnti-A,BAnti-H lectinPresence of anti-A1Presence of A and H in secretions
39Subgroups of ASubgroups of A weaker than A2 (Ael, Aint, A3, Ax, Am, etc) are seen only infrequentlyNOTE: A3 is characterized by mixed field agglutination.
40Subgroups of B Less common than subgroups of A Criteria resembles that used for A subgroupsUsually detected based on forward type, reverse type correctlyDo not make anti-B as commonlyNOTE: B3 characterized by mixed field agglutination
41Antibodies to A and BLandsteiner – individuals do not form antibodies against antigens on their own cells.“Naturally ocurring” is a misnomerAntibody production stimulated by substances in the environment similar to blood group antigensSimply implies antibody production NOT due to actual exposure to red blood cellsAllows both serum/plasma and red cells to determine ABO type – check/balance system
42Development of anti-A and anti-B Antibody production first few months of lifeBabies cannot be reversed typed:Antibodies present in baby from momAre not born with antibodies, detectable at 3 to 6 months of ageOnce produced remain constant until elderlyComplete absence of ABO antibodies exceedingly rare
43Anti-A,B Group O have THREE ABO antibodies: anti-A, -B and –A,B may react more strongly than anti-A and anti-B with some weak A or B subgroupsuse anti-A,B to test group O donorsused to type babies, antigens not being well-developed at birth
44Antibody Characteristics React best at room temperature.Agglutinate saline suspended red cells, no additional reagents are necessaryMay produce hemolysis in vivo and in vitro
45Routine Testing For ABO Group Forward -Test known anti-serum with unknown patient cells (antigens)Reverse – Test unknown serum (antibody) with known A and B antigens (cells0MUST MATCH
46ABO Discrepancies Discrepancy between forward and reverse Helpful observationsStrength of reaction: forward 4+ if antigen present, reverse 2-4+.Unexpected negative in reverseUnexpected positive in forward OR reverseMust delay transfusion until resolved – emergency give out O RBCs and appropriate D type
47ABO Discrepancies Errors divided into two categories TechnicalSampleResults in false positives and negativesSample errors divided into rbc and serum
48ABO Discrepancy – Technical False Negative Failure to add serum or antiserum to a test.Failure to identify hemolysis as a positive reaction.Not using the appropriate serum (or reagent) to cell ratio.Improper centrifugation.Incubation of tests at temperatures above C.Use of inactive reagents.Failure to interpret or record test results correctly.
49ABO Discrepancy – Technical False Positive Over centrifugationUse of contaminated reagent antibodies, RBCs or salineUse of dirty glasswareIncorrect interpretation or recording of results
50Problems Associated With Testing RBC Sample from recently transfused or bone marrow transplant patientABO subgroup or weakened antigens due to disease such as leukemiaAbnormal proteins or Wharton’s jellyIncreased A or B blood group substances inhibit reaction – false negativeAntibodies to dyes in forward reagentsCold autoagglutinins – patient cells may spontaneously agglutinate
51Problems Associated with Serum Weak or negative due to patient’s condition or IV fluidsFibrin clotsRouleauxHigh levels abnormal proteinsUnexpected antibodiesAntibodies to reagent chmicialsNegative or weak reactions in infants < 6 monthsBone marrow transplant ABO non-identical donorTransfused with non-ABO identical plasma products
52Resolving ABO Discrepancies REPEAT THE TESTPatient group A with agglutination of A1 cells test patient cells with anti-A1 and patient serum/plasma with A2 cellsMOST COMMONLY encountered discrepancy.Example testing to prove A2 with anti-A1Anti-AAnti-A1A1 cellsA2 cells4+1-2+
53Resolving ABO Discrepancies Incubate reverse at RT for minutes if false negative suspected.Can incubate at 4C but MUST run autocontrolDecreased antibody most frequent cause of false negative in reverseWash patient and reagent RBCsObtain a new sample
54ABO DiscrepanciesTest against group O adult, group O cord and auto-control to detect cold agglutininsCold agglutinin = anti-I, adult cells have I, cord cells have little or no I at birth.Adult O cellsCord O cellsAuto=control4+0/1+
55Acquired B Phenotype - B (A) Forward = AB, reaction with anti-B weakSerum contains anti-BPhenomena associated withcolon or rectal cancerGram negative sepsisIntestinal obstructionIncreased detection with monclonal anti-B
56Acquired BBacteria (E. coli) have a deacetylating enzyme that effects the A sugar….Acquired B PhenotypeGroup A individualGalactosamine results from the deacetylating reaction, resembling D-galactose (found in Group B individuals). This sugar cross-reacts with the reagent anti-B, giving a weak reaction (but still technically it is “extra”). Patients should receive Group A units. Acquired B usually goes away when the condition resolves.N-acetyl galactosamineGalactosamine now resembles D-galactose (found in Group B)Bacterial enzyme removes acetyl group
57Mixed Field Agglutination Sample has TWO distinct cell populationsA or B patient transfused with group OD pos transfused with D neg OR D neg transfused with D posBMT with different ABO typeA3 or B3 subgroupsChimerism - rarest
59Transfusion Practice Provide serologically compatible blood. Whole blood must be ABO identical - has RBCs AND plasmaFor Red Blood Cells the donor must lack the ABO antigen to which the patient has antibodies.Group O is the universal donor.Group AB is the universal recipient.Donor products such as FFP and Platelet concentrates must lack ABO antibodies to recipient RBCs.AB is the universal donorGroup O is the universal recipient
62Example 1 Anti-A Anti-B A1 Cells B Cells 3+ 1+ Problem: Causes: 1+Problem: Reverse grouping, weakened patient antibodyCauses: Age related or weakened immune systemResolution: Incubate at Room Temperature minutes and respin. Check patient history.Problem:Causes:Resolution:
63Example 2 Anti-A Anti-B A1 Cells B Cells 3+ 1+ 4+ Problem: Causes: 4+Problem: 1+ reaction with anti-B. Appears to have additional antigens.Causes: Acquired B antigenResolution: Patient history – bowel obstruction, carcinoma of colon/rectum. (E. coli)Problem:Causes:Resolution:
64Example 3 Anti-A Anti-B A1 Cells B Cells 2+ 0+ 1+ 4+ Problem: Causes: Problem: Weak forward with anti-A and 1+ reaction with A1 cellsCauses: 1) Subgroup of A (A2 with anti-A1)2) unexpected cold reacting antibody to antigen on reagent A1 cellsResolution: 1) test patient cells with anti-A1 lectin and with patient serum test with A2 cells2) an unexpected cold antibody would be detected in the antibody screenProblem:Causes:Resolution:
65Example 4 Anti-A Anti-B A1 Cells B Cells 3+ Problem: Causes: 3+Problem: missing antigen in forward grouping. Patient appears as group A in reverse groupingCauses: A subgroupResolution: extend incubation time because this may enhance the reaction. Test with a polyclonal or monoclonal blend of anti-A,B (may contain subgroup antigens)…..Problem:Causes:Resolution:
66Example 4 Anti-A,B Patient RBC 1+ Probably a subgroup of A (Ax) if the result was negative (0), adsorption or elution studies with anti-A could be performed (these will help determine what A antigens)
67Example 5 Anti-A Anti-B A1 Cells B Cells 2+mf 3+ Problem: Causes: 2+mf3+Problem: strength of anti-B is weaker than expected; reverse indicates a group B individualCauses: Group B individual transfused with group O cellsResolution: recent transfusion? Bone marrow/stem cell transplant? Find what ABO type the patient was prior to transfusionProblem:Causes:Resolution:
68Example 6 Anti-A Anti-B A1 Cells B Cells 4+ 1+ Problem: Causes: 1+Problem: Forward shows AB individual, Reverse shows weaker “extra” reaction with B cells (looks like a group A)Causes: Possible cold allo- or autoantibody (patient may have an antibody to another blood group system; A1 and B cells may have the antigens to these antibodies) (allo: P, M, N, Lewis) (auto: I or IH)Resolution: screen for antibodies using Screening Cells and an autocontrol (we’ll talk later about Ab screens)Problem:Causes:Resolution:
69Example 7 Anti-A Anti-B A1 Cells B Cells Problem: Causes: Resolution: Problem: Reverse grouping, missing patient antibody (probably group O with no antibodies)Causes: Age related or weakened immune systemResolution: Incubate at Room Temperature minutes and respin. Check patient history.Problem:Causes:Resolution:
70Screening Cells (I and II) Example 6Screening Cells (I and II)Autocontrol (AC)ConclusionPatient Serum 1PosNegCold alloantibodyPatient Serum 2Cold autoantibodyif alloantibody – antibody ID techniquesif autoantibody – special procedures (minicold panel, prewarming techniques