(Study type: Blood collected in a red-top tube; related body system: Circulatory/hematopoietic and immune systems.)

IAT detects and identifies unexpected circulating antibodies or complement molecules in the patient’s serum. The first use of this test was for the detection and identification of anti-D antibodies using an indirect method. The test is now commonly used to screen a patient’s serum for the presence of any antibodies that may react against transfused RBCs. During testing, the patient’s serum is allowed to incubate with reagent RBCs. The reagent RBCs used are from group O donors and have most of the clinically significant antigens present (D, C, E, c, e, K, M, N, S, s, Fy^a, Fy^b, Jk^a, and Jk^b). Anti-D is the antibody most commonly identified by IAT and is the most potent RBC antigenic initiator of in vivo hemolysis. Antibodies present in the patient’s serum coat antigenic sites on the RBC membrane. The reagent cells are washed with saline to remove any unbound antibody. Antihuman globulin is added in the final step of the test. If the patient’s serum contains antibodies, the antihuman globulin will cause the antibody-coated RBCs to stick together or agglutinate. (See study titled “Blood Typing, Antibody Screen, and Crossmatch” for information regarding blood product transfusion reactions.)

Individuals who are RhD negative do not have the Rh antigen on the surface of their RBCs and will develop alloantibodies to the D antigen if exposed to RhD-positive RBCs either through a transfusion or in the case of a pregnant patient when RhD-positive fetal cells cross the placental barrier. Subsequent exposure to RhD-positive RBCs will likely result in significant hemolysis to the transfused recipient’s own RBCs or to the in utero fetus’s RBCs. Laboratory protocols for “weak D” testing have always been required as part of the compatibility process for transfusions; it was optional for certain other patient populations, most notably for obstetric patients. Obstetric patients with the “weak D” phenotype by serotyping are reported as RhD negative as a means of ensuring they receive Rh(D) immune globulin RhoGAM intramuscular (IM) or Rhophylac IM or IV, thus protecting them from inadvertent alloimmunization by an RhD-positive fetus.

Administration of RhIG (Rh immune globulin) to these candidates is not harmful. The advent of molecular technology has led to blood group genotyping (BGG). RhD genotyping offers the advantages of avoiding unnecessary injections of RhIG and transfusion of Rh-negative blood when Rh-positive products could be safely used instead. Alloimmunization may still occur in Rh-negative typed females of reproductive potential as an unintended consequence of RBC transfusion, unknown miscarriage, or failure to receive the recommended RhIG protocol. In the United States, RhD genotyping is available but not routinely used due to the high cost and longer turnaround time for producing genotyping results vs in-house serological methods. Patient and fetal specimens may be collected and sent to larger reference laboratories for RhD genotyping and/or RhD copy testing. RhD genotyping does not evaluate specimens for the presence or absence of RhD but rather for the presence or absence of the other major Rh antigens associated with coexpression or absence of RhD (C, c, E, e). RhD genotyping also identifies other RBC alloantigens known to cause a hemolytic reaction (e.g., Duffy, Kell, Kidd, MNS). The RhD copy test, also available from larger reference laboratories, uses molecular methods to identify the number of copies of RhD in amniotic fluid or patient whole blood specimens; negative findings presume the fetal/patient phenotype is RhD negative.

The inheritance pattern of the RhD antigen is autosomal dominant; homozygotes (DD) will always pass the RhD antigen gene on to their offspring, and heterozygotes (Dd) will pass the RhD antigen gene to their offspring with a probability of 50%. RhD genotyping is recommended when there is a discrepancy in testing for any patient expected to receive a transfusion (due to variability in reagent system sensitivity or when findings at various points in testing are discordant, e.g., immediate spin stage is negative but IAT is positive) or when the biological father’s Rh type is unknown or cannot be positively confirmed at the time the patient undergoes prenatal blood work.