Antiglobulin testing, also known as the Coombs test, is an immunology laboratory procedure used to detect the presence of antibodies against circulating red blood cells (RBCs) in the body, which then induce hemolysis. The destruction of these red blood cells (RBCs) by antibodies directed against them is described diagnostically as autoimmune hemolytic anemia (AIHA). Many etiologies fall under this classification.
Antiglobulin testing can be either direct antiglobulin testing (DAT) or indirect antiglobulin testing (IAT). The principle of DAT is to detect for the presence of antibodies attached directly to the RBCs, which takes place by washing a collected blood sample in saline to isolate the patient’s RBCs; this procedure removes unbound antibodies that may otherwise confound the result. IAT, by contrast, is used to detect unbound antibodies to RBCs, which may be present in the patient’s serum. With direct antiglobulin testing, a monospecific or polyspecific reagent is then added to the washed RBCs to detect bound IgG and/or complement C3. In practice, many laboratories will first use the polyspecific reagent that can detect for both IgG and C3; a positive result will then be followed with monospecific testing to characterize the antibody further. For indirect antiglobulin testing, serum from a blood sample gets isolated, and native RBCs removed. The isolated serum sample then gets incubated with foreign RBCs of known antigenicity. Antiglobulin reagent is then added, and the presence of agglutination indicates a positive result.
The collection of a blood sample for antiglobulin testing requires a tube that is anticoagulated with ethylenediaminetetraacetic acid (EDTA); in standard practice, this collection tube traditionally has a lavender, red, or pink top. EDTA is used to chelate serum calcium to prevent in vitro fixation of complement factor C3 that would otherwise lead to a falsely negative result.
Various modifications have been reported for the improvement of the Coombs test, including the use of polyethylene glycol (PEG) and the antiglobulin gel test (AGT). Some advantages in the AGT in comparison to the standard Coombs test include better reproducibility and easy testing. The AGT is the most sensitive test for the detection of anti-RBC antibodies in the serum. It is essential in pre-transfusion testing and the diagnosis of hemolytic disease of the newborn. The AGT was released in Europe in 1988. It became available in the USA in 1995. Lapierre and collaborators developed this technology.
The AGT is routinely used to screen human sera for anti-human RBC antibodies. The procedure is as follows:
Coombs test is necessary when autoimmunity to red blood cells is a consideration in the differential diagnosis, including warm and cold hemolytic anemia.
Reporting of antiglobulin agglutination test results can be on a qualitative or quantitative basis. For qualitative methods, the interpreter examines the test tube and assigns a score based on a graded scale:
In patients with autoimmune hemolytic anemia, the degree of agglutination typically correlates with the severity of hemolysis. If no macroscopic agglutination appears, the sample will be examined microscopically to assure that there are no aggregates. A sample exposed to a reagent that demonstrates aggregates of at least 3 to 5 cells under microscopic examination is considered a positive result. Agglutination typically takes around 5 to 10 minutes to occur after the addition of the reagent. Direct antiglobulin testing may also be measured quantitatively using enzyme-linked immunosorbent assay (ELISA), flow cytometry, or other immunoassay techniques. Quantitative measurement of a sample may be necessary when isolation of a specific antibody is desired, such as in cases of autoimmune hemolysis due to antibodies other than IgG or C3.
When performed correctly and utilized in the appropriate clinical context, direct antiglobulin testing has been shown to demonstrate a positive predictive value of 97 to 99 percent, although a more recent study involving hospitalized patients reported a false positive rate of up to 7 to 8 percent in patients without any evidence of hemolysis clinically or histologically. The majority of these false positives show a low grade of agglutination, though up to 1 percent of these results may demonstrate higher grades of agglutination. In another study consisting of a cohort of healthy, non-hospitalized individuals, the incidence of a positive DAT result without evidence of hemolysis was found to be 0.1 percent; approximately two-thirds of this cohort expressed IgG positivity.
Several other confounding variables can affect the accuracy of DAT and IAT test results:
The patient should be made aware that Coombs testing is a relatively safe procedure. The risks associated with testing are the same as that of standard blood sample collection. Patients do not need to fast before testing.
Antiglobulin testing, particularly qualitative direct antiglobulin testing, is clinically useful in cases where there is clinical suspicion of autoantibody-induced RBC hemolysis. DAT testing typically involves the use of a polyspecific reagent consisting of IgG and complement C3. Indirect antiglobulin testing is clinically useful for the detection of circulating antibodies that have the potential to induce RBC hemolysis; this test is most commonly utilized for RBC phenotyping and in crossmatch screening for blood transfusion. A positive antiglobulin result requires analysis in the clinical context to make an accurate diagnosis. Healthcare costs and the burden of laboratory time can be minimized by first screening with a polyspecific reagent before confirming the antibody with monospecific or quantitative analysis. Rarely, autoimmune hemolysis may be suspected even in the absence of positive DAT testing; in this instance, quantitative DAT testing may help to identify less common antibody subtypes other than IgG or C3. In the absence of other confounding variables (see “Interfering Factors” above), positive antiglobulin testing indicates the presence of hemolysis by antibodies directed against native RBCs. There are several major areas of clinical significance:
Alloimmune-mediated hemolytic transfusion reaction (AHTR): AHTR occurs when a post-transfusion specimen develops a newly found alloantibody. The formation of an alloantibody can occur as quickly as within 2 to 3 days. The development of alloantibodies results in a positive DAT test but may or may not be associated with hemolysis.
ABO blood group typing: in blood transfusions and hematopoietic stem cell transplants, indirect antiglobulin testing can be used to identify the RBC phenotype to minimize the chances of donor incompatibility.
Hemolytic disease of the fetus and the newborn (HDFN): HDFN occurs when maternal IgG forms against fetal antigens, notably the Rh or Kell antigen. The most common type of HDFN is due to ABO incompatibility, which occurs in approximately 15 to 25% of pregnancies and tends to be less severe. The incidence of positive DAT testing in ABO HDFN is very low at around 1 percent, and of that group, only approximately 23% of newborns will develop clinically significant jaundice; hence, DAT is a poor positive predictor of newborns that will require treatment.
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