General

Synonym/Acronym:
Indirect antiglobulin test (IAT), antibody screen.

Rationale
To check recipient serum for antibodies prior to blood transfusion.

Patient Preparation
There are no food, fluid, activity, or medication restrictions unless by medical direction.

Normal Findings
(Method: Hemagglutination) Negative (no agglutination).

Critical Findings and Potential Interventions
N/A

Overview

(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 female 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 women as an unintended consequence of RBC transfusion, unknown miscarriage, or failure to receive the recommended RhIG protocol.

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 a subject’s Rh type is unknown (e.g., paternity cannot be positively confirmed at the time the mother undergoes her prenatal blood work).

Indications

• Detect and identify unexpected antibodies before blood transfusions.
• Detect other antibodies in maternal blood that can be potentially harmful to the fetus.
• Determine antibody titers in Rh-negative women sensitized by an Rh-positive fetus.
• Test for the weak Rh-variant antigen D^u using BGG. To determine if the fetus is at risk, in the case of a positive maternal anti-D antibody titer (i.e., the mother is RhD-negative and has developed RhD alloantibodies), paternal blood is genotyped. If the paternal sample is genotyped as RhD negative, the fetus must be RhD negative and no further action is necessary. If the paternal sample is genotyped as homozygous RhD positive (DD), the fetus (and all future offspring) can be assumed to be RhD positive, there is no need to initiate fetal testing, and the RhIG protocol will be initiated. If the paternal sample is heterozygous or if paternity is not certain, then cell free fetal DNA testing of amniotic fluid is recommended to determine fetal RhD status.

Interfering Factors

Factors that may alter the results of the study

• Drugs and other substances that may cause a positive IAT include anticonvulsants, antidysrhythmics, carbapenums, cephalosporins, chlorpromazine, insulin, methyldopa, penicillins, sulfonamides, TB medications, and tetracyclines.
• Recent administration of blood products or high molecular weight plasma expanders (e.g., dextran) may cause cellular aggregation resembling agglutination in ABO typing.
• A treatment for multiple myeloma using the monoclonal antibody daratumumab (Darzalex®) has been shown to cause false positive indirect Coombs results by binding to the CD38 protein site on reagent RBCs, which causes agglutination and results interpreted as a positive result for unexpected antibodies. Pan-agglutination may persist for up to six months although duration varies by individual. The most important safeguard is good communication. If a transfusion is anticipated, a pre-treatment specimen should be collected for T&S prior to the initiation of treatment. However, there are laboratory protocols for ensuring the availability of blood products for patients receiving this therapy prior to specimen collection for T&S. The blood bank should always be informed when pre- or post-treatment samples are submitted for testing.

Potential Medical Diagnosis: Clinical Significance of Results

Positive Findings In:
Circulating antibodies or medications attach to the patient’s RBCs, and hemolysis occurs. Agglutination is graded from 1+ to 4+ in manual testing systems, with 4+ being the strongest degree of agglutination. Automated testing systems are capable of reporting 1+ to 4+ graded results, providing images of the tested material so laboratory professionals can interpret the results, or providing computer-assisted interpretation of the test results as positive or negative findings.

• Hemolytic anemia (autoimmune or induced by drugs or other substances)
• Hemolytic disease of the newborn (related to ABO or Rh incompatibility)
• Incompatible crossmatch
• Infections (mycoplasma pneumonia, mononucleosis, syphilis)
• Other diseases (e.g., autoimmune) may cause a positive IAT to include chronic lymphocytic leukemia and SLE.

Negative Findings In:

• Samples in which the patient’s antibodies exhibit dosage effects (i.e., stronger reaction with homozygous than with heterozygous expression of an antigen) and reagent erythrocyte antigens contain single-dose expressions of the corresponding antigen (heterozygous)
• Samples in which reagent erythrocyte antigens are unable to detect low-prevalence antibodies
• Samples in which sensitization of erythrocytes has not occurred (true negative, complete absence of antibodies)

Nursing Implications, Nursing Process, Clinical Judgement