MLT Exam

Blood bank questions on the ASCP BOC exam are rarely pure recall. Expect a reaction grid, a panel described in words, a discrepancy between forward and reverse typing, or a crossmatch that will not come up compatible, followed by a question about the most likely explanation or the best next step. The safest answer pattern throughout this section is the same: confirm patient and specimen identification, correlate every result with every other result, resolve the problem before release, and choose the option that protects the patient (group O red cells and AB plasma while an ABO problem is unresolved, antigen-negative units when an antibody is identified).

This chapter reviews the immune response as it applies to red cell antigens, the properties of IgM and IgG blood group antibodies, complement, the physical factors that control agglutination, and the inheritance patterns used to predict phenotypes and exclude parentage.

A primary immune response follows first exposure to a foreign red cell antigen through transfusion or pregnancy. It has a lag phase of days to weeks, produces mostly IgM at first and then a modest amount of IgG, and the titer is low. A secondary (anamnestic) response follows re-exposure. Memory B cells respond within hours to a few days, the antibody is mainly IgG, the titer climbs high and fast, and the antibody has higher affinity. The anamnestic response explains the delayed hemolytic transfusion reaction: a patient whose antibody had fallen below detectable levels (classically anti-Jka) is transfused with antigen-positive cells, and three to fourteen days later the antibody returns at high titer and destroys the transfused cells.

Not every antibody needs red cell exposure. ABO antibodies, and often anti-M, anti-N, anti-P1, anti-I and Lewis antibodies, are called naturally occurring because they arise from exposure to environmental substances (bacteria, pollen, food) whose carbohydrate structures resemble blood group antigens. Immune antibodies (Rh, Kell, Kidd, Duffy, S, s) appear only after transfusion or pregnancy.

IgM Versus IgG Blood Group Antibodies

Among IgG subclasses, IgG3 is the strongest complement activator, IgG1 is next, IgG2 is weak and IgG4 does not activate complement. IgG1 and IgG3 cause most clinically significant hemolysis.

Exam Tip: When a stem says an antibody reacted only at immediate spin or room temperature and not at AHG, think IgM and usually clinically insignificant (except ABO). When it reacted only at the AHG phase, think IgG and clinically significant until proven otherwise.

The classical complement pathway begins when C1q binds two Fc regions close together on the red cell. One IgM pentamer provides those Fc regions by itself; IgG needs two molecules side by side. If the cascade runs to completion, C5b through C9 form the membrane attack complex and the cell lyses in the circulation (intravascular hemolysis, as in an ABO-incompatible transfusion). If it stops at C3, the cell carries C3b, which is converted to C3d; macrophages in the liver remove C3b-coated cells (extravascular hemolysis), and the remaining C3d coating is what the anti-C3d reagent detects in the DAT. Kidd antibodies are the classic IgG antibodies that bind complement. Because complement needs calcium and magnesium, EDTA specimens prevent in vitro complement activation; that is why EDTA tubes are preferred for the DAT.

Agglutination happens in two stages. In the first stage (sensitization), antibody binds antigen; this is controlled by temperature, pH, incubation time, ionic strength and the antigen-antibody ratio. In the second stage (lattice formation), antibody bridges between cells create visible clumps; this is controlled by the distance between cells, the zeta potential and centrifugation.

Red cells carry a net negative charge, mainly from the carboxyl groups of sialic acid. In saline, positive cations form an ionic cloud around each cell, and the zeta potential is the electrical potential difference that keeps cells about 25 nm apart. IgM spans that distance; IgG does not.

Factors That Enhance or Weaken Agglutination

Common Trap: Enzyme treatment is not a universal enhancer. A panel run with ficin-treated cells will lose anti-Fya, anti-Fyb, anti-M and anti-N reactivity entirely, while Rh and Kidd antibodies react more strongly. The pattern of enhancement or destruction is a clue to specificity.

Memory Aid: Enzymes destroy "MNS Duffy" (M, N, S, Fya, Fyb) and enhance "Rh, Kidd, Lewis, P, I" - the "Really Kind Little People Inside" antibodies.

Most blood group genes are autosomal and codominant: if a gene is inherited, its antigen is expressed. A person who inherits two identical alleles is homozygous (Jka/Jka, phenotype Jk(a+b-)); one who inherits two different alleles is heterozygous (Jka/Jkb, phenotype Jk(a+b+)). The phenotype is what serologic testing shows; the genotype is the actual gene pair, which can often only be predicted. A person typing Jk(a+b-) is presumed Jka/Jka, but a silent allele (Jk) is possible. Xga is the classic X-linked blood group antigen.

Dosage describes stronger reactions with cells from homozygous individuals, which carry a double dose of antigen. Antibodies in the Rh (other than D), Kidd, Duffy and MNS systems commonly show dosage; a weak anti-Jka may react 2+ with Jk(a+b-) cells and not at all with Jk(a+b+) cells. This matters when ruling out antibodies.

Cis and trans describe gene position. Genes on the same chromosome are in cis; genes on opposite chromosomes of a pair are in trans. In the Rh system, a C gene in trans to D (for example Dce/dCe) can weaken D antigen expression, called the position effect.

A Punnett square lists each parent's possible gametes and combines them. An AO father and a BO mother can have children of genotype AB, AO, BO and OO, so each of the four ABO phenotypes is possible with a 25% chance. Two group O parents (OO x OO) can have only group O children.

Parentage testing uses two types of exclusion. A direct (first-order) exclusion occurs when a child has an antigen that neither the mother nor the alleged father has; for example, a K-positive child born to a K-negative mother and a K-negative alleged father. An indirect (second-order) exclusion occurs when a child lacks a gene that the alleged father would have had to pass on; for example, an alleged father who types M+N- (presumed MM) and a child who types M-N+. Indirect exclusions are weaker because silent alleles exist.

IgM antibodies react best cold, agglutinate saline-suspended cells and activate complement efficiently; IgG antibodies react at 37 C, need AHG and cross the placenta.

The anamnestic response causes delayed hemolytic transfusion reactions, classically with Kidd antibodies.

LISS and PEG speed IgG uptake; enzymes enhance Rh, Kidd, Lewis, P1 and I but destroy M, N, S (variable), Fya and Fyb.

Most blood group genes are codominant; Rh (non-D), Kidd, Duffy and MNS antibodies show dosage.

Direct exclusion (child has an antigen neither parent has) is stronger evidence than indirect exclusion.