MLT Exam

This chapter covers ABO biochemistry and genetics, the H and secretor genes, A subgroups and the Bombay phenotype, routine ABO typing, and the four groups of ABO discrepancies with the steps used to resolve each.

ABO antigens are carbohydrate structures built one sugar at a time by glycosyltransferase enzymes. The H gene (FUT1, chromosome 19) encodes a fucosyltransferase that adds L-fucose to the terminal galactose of a type 2 precursor chain on red cells, forming H antigen. H antigen is the required substrate for the ABO genes (chromosome 9). The A gene encodes an enzyme that adds N-acetylgalactosamine to H; the B gene adds D-galactose. The O allele codes for an inactive enzyme, so group O cells carry unconverted H antigen and have the most H.

Memory Aid: A for "Acetyl" (N-acetylgalactosamine); B for "Basic galactose" (D-galactose); H for "Hold the fucose."

The amount of H antigen remaining on red cells follows this order: O > A2 > B > A2B > A1 > A1B. This order explains why anti-H lectin (Ulex europaeus) reacts strongest with group O cells and weakest with A1 and A1B cells, and why an A1 or A1B person may make a weak anti-H or anti-IH.

ABO Groups, Antigens and Antibodies

Group O people make anti-A,B, an antibody that reacts with both A and B cells and has an IgG component that crosses the placenta; that is why ABO hemolytic disease of the fetus and newborn occurs almost entirely in babies of group O mothers. ABO antibodies appear at 3-6 months of age, peak in childhood and may weaken in the elderly.

The secretor gene (Se, FUT2) encodes a fucosyltransferase that acts on type 1 chains in secretions. About 80% of people are secretors (SeSe or Sese) and have soluble A, B and H substances in saliva and other body fluids matching their ABO group. Nonsecretors (sese) still have ABO antigens on their red cells, because red cell H depends on the H gene, not Se. Saliva testing for ABH substances uses hemagglutination inhibition: secretor saliva neutralizes the matching antiserum so that indicator cells no longer agglutinate. A secretor study can help classify a weak A subgroup.

About 80% of group A people are A1 and about 20% are A2. A1 cells have far more A antigen sites and carry the A1 epitope; A2 cells have fewer sites and more H. Dolichos biflorus lectin, diluted appropriately, agglutinates A1 cells but not A2 cells. Anti-A1 occurs in about 1-8% of A2 people and 22-35% of A2B people; it is usually an IgM room-temperature antibody that is clinically insignificant but causes an ABO discrepancy and an incompatible immediate-spin crossmatch with A1 donor cells.

Weaker subgroups (A3, Ax, Aend, Am, Ael) show progressively weaker or absent agglutination with anti-A. A3 classically gives mixed-field agglutination. Ax reacts better with anti-A,B than with anti-A. Am and Ael are detectable only by adsorption-elution studies. Weak B subgroups exist but are rarer.

The Bombay (Oh) phenotype occurs when a person inherits hh: no functional H enzyme, so no H antigen, so no A or B antigen can be made even if A or B genes are present. Bombay cells type as group O in forward typing but do not react with anti-H lectin. The plasma contains anti-A, anti-B and a potent anti-H that is IgM, binds complement and hemolyzes at 37 C. Because group O cells are the richest in H, a Bombay patient's plasma reacts strongly with group O screening cells and with every group O donor. Only Bombay blood, usually from a rare donor registry or siblings, can be transfused.

Bench Scenario: A patient types as group O in forward typing. The reverse typing shows 4+ reactions with A1 and B cells, and the antibody screen shows 4+ reactions at immediate spin, 37 C and AHG with both group O screening cells, with a negative autocontrol. Ulex europaeus lectin does not agglutinate the patient's cells. The pattern points to the Bombay phenotype with anti-H; the technician should not release group O blood, should notify the medical director and pursue rare Oh units.

Forward (cell) typing tests patient red cells with anti-A and anti-B (monoclonal antisera); many labs add anti-A,B on donors. Reverse (serum or plasma) typing tests patient plasma with A1 and B reagent cells. The two must agree before an ABO group is reported. Expected reverse reactions are strong (usually 3+ to 4+). A reverse reaction that is weaker than expected, missing, or extra is a discrepancy.

Bench Scenario: Forward typing shows anti-A 4+, anti-B 0. Reverse typing shows A1 cells 0 and B cells 0. The forward type says group A, but anti-B is missing. If the patient is 2 months old, this is expected; infants do not reliably make ABO antibodies before about 4-6 months, and reverse typing is not performed on neonates. If the patient is an adult, look for hypogammaglobulinemia, chemotherapy, a bone marrow transplant, or old age.

ABO discrepancies are classically divided into four groups. The first step in every case is to repeat the test on the same sample with washed cells and fresh reagents, and to check for clerical or specimen errors; if a new sample is needed, recollect. While the discrepancy is unresolved and transfusion is urgent, give group O red cells (and AB plasma if plasma is needed).

ABO Discrepancy Groups

Rouleaux are stacks of red cells like coins caused by high plasma protein. They look like agglutination in the reverse tube (and in tube-based antibody screens). Saline replacement resolves them: after centrifugation, remove the plasma, replace it with 2 drops of saline, remix and respin; rouleaux disperse while true agglutination remains.

A cold autoantibody such as anti-I can coat the patient's own cells so strongly that they agglutinate spontaneously in forward typing (making the patient look AB-positive) and react with all reagent cells in reverse typing. Warm the specimen to 37 C, wash the cells in warm saline, and repeat. If the plasma still reacts, prewarm the reverse typing or perform cold autoadsorption.

Acquired B occurs when bacterial deacetylase (often from gram-negative organisms in colon cancer, intestinal obstruction or sepsis) converts N-acetylgalactosamine on A cells to galactosamine, which resembles D-galactose. A group A patient then forward-types as AB, with a weak anti-B reaction, but the reverse shows anti-B. The patient's own anti-B does not react with his or her own cells (autocontrol negative). Acidified anti-B (pH 6.0) and many monoclonal anti-B clones do not detect acquired B, and the secretor study shows only A and H substance.

An unexpected cold alloantibody in an A or B patient can react with A1 or B reagent cells. An A2 patient with anti-A1 shows an extra 1+ to 2+ reaction with A1 reverse cells and no reaction with A2 cells; Dolichos biflorus does not agglutinate the patient's cells. If the extra reaction is from anti-M or anti-P1, the antibody screen is usually positive at room temperature as well, and reverse cells lacking the antigen will be nonreactive.

Common Trap: Mixed-field forward reactions in an adult usually mean a recent out-of-group transfusion (group A patient given group O red cells) or a hematopoietic progenitor cell transplant, not a laboratory error. Always check the transfusion and transplant history before calling it a subgroup.

H antigen (fucose added by the H gene) is the precursor for A (N-acetylgalactosamine) and B (D-galactose); group O cells have the most H, A1 and A1B the least.

Dolichos biflorus identifies A1 cells; Ulex europaeus detects H; Bombay cells lack H and the plasma has a dangerous anti-H that hemolyzes all group O cells.

About 80% of people are secretors; secretor status affects saliva, not red cell ABO antigens.

Discrepancies: group I missing reverse, group II weak forward, group III rouleaux and protein problems, group IV cold antibodies, acquired B and other extra reactions.

Repeat with washed cells, check identification and history, and give group O red cells and AB plasma until the type is resolved.