ABO Subgroups and Discrepancies Nada Jamjoom Faculty of Applied Medical Sciences BLOOD BANK MEDICAL TECHNOLOGY KAU

ABH Antigens H antigen

ABO Antibodies ABO Abs are generally IgM class antibodies. For Group A and Group B individuals the predominant antibody class is IgM. Serum from Group O individuals contains not only anti-A and anti-B but also anti-A,B. Anti-A,B is a mixture of class IgG and IgM. The anti-A,B “immune” antibodies are predominantly IgG.

ABO Antibodies Time of appearance: Generally present within first 4-6 months of life Reach adult level at 5-10 years of age Level off through adult life Begin to decrease in later years: >65 years of age

ABO Subgroups Von Dungern in1911 described 2 different A Ag based on reactions between group A RBCs and anti-A and anti-A1 antisera. Group A red cells that react with both anti-A & anti-A1 are classified as A1. Group A red cells that react with anti-A and not anti-A1 are classified as A2. The majority (80%) of the A and AB population are A1 or A1B; the remaining 20% are A2 or A2B. The production of both types of antigens (A1 and A2) is a result of an inherited gene at the ABO locus & the immunodominant sugar on both A1 and A2 RBCs is N-acetyl-D-galactosamine.

Difference between A1 and A2 The difference between A1 and A2 is both quantitative and qualitative. Quantitative difference Inheritance of an A1 gene elicits production of high concentrations of the enzyme α-3-N-acetylgalactosaminyltranferase, which converts almost all of the H precursor structure to A1 antigens on the red cells. The number of A1 antigen sites is higher than the number of A2 antigens sites on the red cell because A1 gene is a very potent gene that creates large numbers of A1 antigens sites on RBC.

Difference between A1 and A2 Qualitative differences 1 to 8% of A2 individuals produce anti-A1 in their serum 25% of A2B individuals produce anti-A1.

Difference between A1 and A2 Reagent anti-A is a mixture of two Abs ; Anti-A which react with both A1 and A2 cells. Anti-A1 which reacts with A cells but not with A2 cells in simple testing . The seeds of the plant Dolichos biflorus serve as a source of anti-A1 which is known as anti-A1 lectin. This reagent agglutinates A1 or A1B but does not agglutinate A2 or A2B cells.

A1 versus A2 Phenotype Blood Group A1 + A2 Negative Reactions of patient’s red cells with Blood Group Anti-A (from B Sera) Anti-A1 Lectin A1 + A2 Negative A1 has 2 antigens A and A1 A2 has only one, A antigen

Weak A and B Subgroups Other A subgroups: RBC of the A end, A3, Ax, Ay or A el. are only rarely seen in transfusion practice. Subgroup of B: infrequent than the weaker subgroup of A, identified by anti-B and anti-A,B. Subgroups B3 , Bx , Bm and Bel .

ABO Discrepancies A discrepancy occurs when the red cell testing does NOT match the serum testing results In other words, the forward does NOT match the reverse ABO discrepancies are usually technical in nature and can be simply resolved by correctly reporting the testing and carefully checking reagents with meticulous reading and recording of results.

ABO Discrepancies Most of the time, the problem is technical Mislabeled tube Failure to add reagent Either repeat test on same sample, request a new sample, or wash cells Other times, there is a real discrepancy due to problems with the patient’s red cells or serum.

Technical Errors ABO Discrepancies Clerical errors Mislabeled tubes Patient misidentification Inaccurate interpretations recorded Transcription error Computer entry error Reagent or equipment problems Using expired reagents Using an uncalibrated centrifuge Contaminated or hemolyzed reagents Incorrect storage temperatures Procedural errors Reagents not added Manufacturer’s directions not followed RBC suspensions incorrect concentration Cell buttons not resuspended before grading agglutination

ABO Discrepancies Group I Discrepancies Group I discrepancies are between forward and reverse groupings because of weekly reacting or missing antibodies. Group I discrepancies are the most common type. If a reaction in the reverse grouping is weak or missing  Group I discrepancy. It means that the patient has depressed antibody production or cannot produce the ABO antibodies.

Examples of Group I Discrepancies ABO Discrepancies Examples of Group I Discrepancies Newborns Do not form antibodies until they 3-6 months Elderly patients Weakened antibody activity CLL, malignant lymphoma, using immunosuppressive drugs can cause hypogammaglobulinemia (little or no antibody production)

Resolution of Group I Discrepancies ABO Discrepancies Resolution of Group I Discrepancies Eliminate all technical errors Determine patients age, diagnosis Incubate patient’s serum with reagent A1 and B cells for 15 to 30 minutes (RT) to enhance antibody reactions If negative, place serum-cell mixture at 4°C for 15 to 30 minutes

Group II Discrepancies ABO Discrepancies Group II Discrepancies These discrepancies are between forward and reverse groupings because of weakly reacting or missing antigens. Group II discrepancies is the least common type.

Examples of Group II Discrepancies ABO Discrepancies Examples of Group II Discrepancies Subgroups of A and or subgroups of B may be present. Leukemias may yield weakened A or B antigens. Hodgkin’s disease “Acquired B” phenomenon is most often associated with lower GI tract disease Cancer of colon/rectum Intestinal obstruction Gram negative septicemia (i.e. E. coli)

Acquired B ABO Discrepancies Bacteria (E. coli) have a deacetylating enzyme that modify the immunodominant BG A sugar…. Acquired B Phenotype Group A individual N-acetyl galactosamine Galactosamine now resembles D-galactose (found in Group B) Bacterial enzyme removes acetyl group

Resolution of Group II Discrepancies ABO Discrepancies Resolution of Group II Discrepancies Eliminate all technical errors Wash patient’s cells with saline The reaction can be enhanced by incubating the test mixture at RT for up to 30 minutes to increase the association of Ab with Ag. If negative, reduce the temperature to 4°C. Testing the patient’s serum against autologous RBCs gives a negative reaction because the anti-B in the serum does not agglutinate autologous RBCs with the acquired B Ag.

Group III Discrepancies ABO Discrepancies Group III Discrepancies These discrepancies are between forward and reverse grouping due to protein or plasma abnormalities and result in Rouleaux formation. These can be caused by elevated levels of globulin from certain diseases such as multiple myeloma and Hodgekin’s lymphoma. Wharton’s jelly ( a viscous mucopolysaccharide material present on cord bloods).

Resolution of Group III Discrepancies ABO Discrepancies Resolution of Group III Discrepancies Rouleaux or red cells result from a stacking of erythrocytes that adhere in a coin-link fashion giving the appearance of agglutination. To resolve this kind of problem, washing the patient’s red cells with saline or adding a drop or two of saline to the tube in case of rouleaux formation. If the agglutination is true red cell clumping will remain. Cord blood must be washed 6-8 times in forward grouping ONLY.

Group IV discrepancies ABO Discrepancies Group IV discrepancies These kind of discrepancies are between forward and reverse groping due to miscellaneous problems. Polyagglutination can occur due to exposure of hidden erythrocyte Ag (T antigen) in patients with bacterial or viral infection. Bacterial contamination in vitro or vivo produces an enzyme that alters and exposes the hidden Ag on red cell leading to T activation. This problem can be resolved by using monoclonal antibody typing reagent.

Common sources of technical errors resulting in ABO discrepancies Dirty tubes or glassware Common sources of technical errors resulting in ABO discrepancies False Negative False Positive Inadequate identification of samples or test tubes Over centrifugation and over reading Failure to add serum or reagents Use of incorrect reagents or samples Contaminated reagents Cell suspention is too heavy or too light

Example I Forward Grouping Reaction of Patient’s Cells with ABO Discrepancies Example I Forward Grouping Reaction of Patient’s Cells with Reverse Grouping Reaction Patient’s Serum with Anti-A Anti-B A1 cells B cells Patient Negative +++ Patient’s probable group: B Possible discrepancy: Group I discrepancy; because of weakly reacting or missing antibodies.

Example II Forward Grouping Reaction of Patient’s Cells with ABO Discrepancies Example II Forward Grouping Reaction of Patient’s Cells with Reverse Grouping Reaction Patient’s Serum with Anti-A Anti-B A1 cells B cells Patient ++++ ++ Patient’s probable group: A Possible discrepancy: Group III discrepancy; caused by Rouleaux formation

Example III Forward Grouping Reaction of Patient’s Cells with ABO Discrepancies Example III Forward Grouping Reaction of Patient’s Cells with Reverse Grouping Reaction Patient’s Serum with Anti-A Anti-B A1 cells B cells Patient ++++ negative ++ Patient’s probable group: A (subgroup A2) Possible discrepancy: Group II discrepancy; because of weakly reacting or missing antigens

Example IV Forward Grouping Reaction of Patient’s Cells with ABO Discrepancies Example IV Forward Grouping Reaction of Patient’s Cells with Reverse Grouping Reaction Patient’s Serum with Anti-A Anti-B A1 cells B cells Patient ++++ ++ Negative Patient’s probable group: A Possible discrepancy: Group II discrepancy; caused by an acquired B Ag

Example V Forward Grouping Reaction of Patient’s Cells with ABO Discrepancies Example V Forward Grouping Reaction of Patient’s Cells with Reverse Grouping Reaction Patient’s Serum with Anti-A Anti-B A1 cells B cells Patient ++ + ++++ Patient’s probable group: O Possible discrepancy: Group IV discrepancy; caused by T activation.