RESOLVING ABO DISCREPANCIES by DR G P Saluja Alchemist Hospitals Ltd Panchkula

Case 1 Blood Sample of 20 years Male for Blood Grouping 20 years through OPD He had donated blood earlier He told that his blood group is “O Rh+ve”

Forward Grouping A Cells : Neg B Cells : Neg Rh : 4+ Blood Group : O Rh +ve

Forward & Reverse Grouping A Cells : Neg ANTI A Neg B Cells : Neg ANTI B 4+ Rh : 4+ Blood Group : O Rh +ve There is Discrepancy between Forward & Reverse Grouping But as the reaction in Anti B is 4+, it shows presence of A antigen

Resolving Discrepancy A1 Lectin : Neg Control : Neg Anti H Lectin : 4+ve O cells : Neg Anti AB : 2+ Mf A2 Cells : Neg Blood group is Neither A1 nor A2.

Absorption & Elution 5th wash gave negative reaction with A cells Elute gave negative reaction with o cells Elute gave Positive reaction with A cells This confirms the presence of A Antigen & hence Weak A blood group Confirmed

CASE 2 Forward Grouping FORWARD GROUPING A CELLS +2 B CELLS +4 Rh NEG BLOOD GROUP AB Rh-ve Control NEG

Forward & Reverse Grouping FORWARD GROUPING REVERSE GROUPING A CELLS +2 ANTI A +2 B CELLS +4 ANTI B NEG Rh NEG Du NEG BLOOD GROUP AB Rh-ve BLOOD GROUP B Rh-ve Control NEG

Antibody Screen Three Cell Panel for Antibody screen is Negative

Resolving Discrepancy Anti-A1 Lectin gave negative reaction. Absorption and Elution technique confirmed the presence of A antigen. The findings therefore are: The blood group therefore is AB Rh-ve with Anti A1 Antibody present Forward Grouping Reverse Grouping A antigen +ve B antigen +ve Anti A1 +ve Anti B -ve

CASE 3 Patient Dharmender 33 yrs Male admitted with Dengue MR 105330 IP No 13/5627 Already operated at Fortis Hospital and blood group given was B Rh-ve Forward Grouping FORWARD GROUPING A CELLS 1+ B CELLS 4+ Rh NEG BLOOD GROUP AB Rh-ve Control NEG

Forward and Reverse Grouping CASE 3 Forward and Reverse Grouping FORWARD GROUPING REVERSE GROUPING A CELLS 1+ ANTI A NEG B CELLS 4+ ANTI B NEG Rh NEG Du NEG BLOOD GROUP AB Rh-ve BLOOD GROUP AB Rh-ve Control NEG

Resolving Discrepancy Anti-A1 Lectin gave negative reaction. Absorption and Elution technique confirmed the presence of A antigen. Blood group was confirmed to be A2B Rh-ve

CASE 4 Pre-operative (TKR) blood grouping was done for R M SINGLA 68 years Male A Cells : 4+ ANTI A Neg B Cells : Neg ANTI B Neg Rh : 4+ Blood Group : There is discrepancy between forward and reverse grouping

Enhancement Method With the Enhancement method the strength of reaction with “B” cells has increased to 2+. Hence it shows presence of Anti B antibodies and the blood group is confirmed as A RH+VE

RBC Precursor Structure Glucose Galactose Precursor Substance (stays the same) N-acetylglucosamine Galactose

Formation of the H antigen RBC Glucose H antigen Galactose H enzyme is fucosyltransferase N-acetylglucosamine Galactose Fucose

H antigen The H antigen is the foundation upon which A and B antigens are built A and B genes code for enzymes that add an immunodominant sugar to the H antigen Immunodominant sugars are present at the terminal ends of the chains and confer the ABO antigen specificity

A and B Antigen The “A” gene codes for an enzyme (transferase) that adds N-acetylgalactosamine to the terminal sugar of the H antigen N-acetylgalactosaminyltransferase The “B” gene codes for an enzyme that adds D-galactose to the terminal sugar of the H antigen D-galactosyltransferase

Formation of the A antigen RBC Glucose Galactose N-acetylglucosamine Galactose N-acetylgalactosamine Fucose

Formation of the B antigen RBC Glucose Galactose N-acetylglucosamine Galactose Galactose Fucose

Genetics The H antigen is found on the RBC when you have the Hh or HH genotype, but NOT from the hh genotype The A antigen is found on the RBC when you have the Hh, HH, and A/A, A/O, or A/B genotypes The B antigen is found on the RBC when you have the Hh, HH, and B/B, B/O, or A/B genotypes

O>A2>B>A2B>A1>A1B H antigen Certain blood types possess more H antigen than others: O>A2>B>A2B>A1>A1B Greatest amount of H Least amount of H

Why do Group O individuals have more H antigen than the other groups? Group O individuals have no A or B genes to convert the H antigen to A or B antigens….that means more H antigen sites

ABO Subgroups ABO subgroups differ in the amount of antigen present on the red blood cell membrane Subgroups have less antigen Subgroups are the result of less effective enzymes. They are not as efficient in converting H antigens to A or B antigens (fewer antigens are present on the RBC) Subgroups of A are more common than subgroups of B

Subgroups of A The 2 principle subgroups of A are: A1 and A2 Both react strongly with reagent anti-A To distinguish A1 from A2 red cells, the lectin Dolichos biflorus is used (anti-A1) 80% of group A or AB individuals are subgroup A1 20% are A2 and A2B

A2 Phenotype Why is the A2 phenotype important? A2 and A2B individuals may produce an anti-A1 This may cause discrepancies when a crossmatch is done (incompatibility) What’s the difference between the A1 and A2 antigen? It’s quantitative The A2 gene doesn’t convert the H to A very well The result is fewer A2 antigen sites compared to the many A1 antigen sites

A1 and A2 Subgroups* A1 A2 4+ Anti-B 900 x103 3+ Anti-B & anti-A1 Anti-A antisera Anti-A1 antisera Anti-H lectin ABO antibodies in serum # of antigen sites per RBC A1 4+ Anti-B 900 x103 A2 3+ Anti-B & anti-A1 250 x103 *Adapted from Flynn, J. (1998). Essentials of Immunohematology

Other A subgroups There are other additional subgroups of A Aint (intermediate), A3, Ax, Am, Aend, Ael, Abantu A3 red cells cause mixed field agglutination when polyclonal anti-A or anti-A,B is used Mixed field agglutination appears as small agglutinates with a background of unagglutinated RBCs They may contain anti-A1

A 1 4+ - +,- A,H 810-1170 A 2 2+ + in 1%-8% 240-290 A3 2+ mf 3+ RBC Pheno- types Reaction of Red Blood Cells with known Antiserum Reaction of Serum with Known Cells Substances present in saliva of secretors Presence of A Transferase in Serum Number of antigen sites RBC X 103 Anti A Anti B Anti A,B Anti H Anti A1 A1 Cells B Cells O Cells A 1 4+ - +,- A,H 810-1170 A 2 2+ + in 1%-8% 240-290 A3 2+ mf 3+ Sometimes 35 Ax -/w Almost always A(trace),H Rarely 5 Aend W mf H No 3.5 Am Yes 1 Ay Trace Ael + 0.7

CONCLUSION Always put up Reverse blood grouping. Blood group should only be confirmed when the findings of the forward grouping match with those in reverse grouping. There can be fatal reaction

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