Direct Antiglobulin Test

The direct antiglobulin test (DAT) is performed to determine if a patient's red cells are coated in vivo with IgG or complement components. DAT is helpful in the work-up of autoimmune hemolytic anemia, drug induced hemolytic anemia, hemolytic disease of the newborn, and hemolytic transfusion reactions.  

The DAT procedure involves obtaining red cells from patient, washing them to remove unbound IgG, and then directly testing them with antiglobulin reagent (anti-IgG and/or anti-complement). If antibody is coating the patient’s red cells, the red cells are agglutinated by antiglobulin. The DAT is extremely sensitive; it can detect as few as 100 IgG and 400 C3d molecules per red cell. A positive result indicates that antibody and/or complement has sensitized the patient's RBCs in vivo.

Approximately 1 in 9,000 healthy persons has a positive direct antiglobulin test with no evidence of hemolysis. Some diseases may be associated with a positive DAT, even though the patient does not appear to be actively hemolyzing their red cells. Examples include chronic lymphocytic leukemia, multiple myeloma, systemic lupus erythematosis, infectious mononucleosis, mycoplasma infection, and AIDS. Different studies have reported that 0.3 to 1.5% of hospitalized patients have a positive DAT. 

The strength of the direct antiglobulin test does not predict the biological activity of antibodies. For instance, some patients with a strongly positive direct antiglobulin test have little hemolysis, while other patients with weakly positive or negative direct antiglobulin test experience severe hemolysis. Also, the strength of the direct antiglobulin test often does not change following treatment, even though the clinical condition greatly improves.

Autoimmune hemolytic anemia is classified as warm or cold autoantibody types based on the temperatures at which the antibodies maximally react with red blood cells in vitro.  Warm autoantibodies are more reactive at 37^oC than at lower temperatures, whereas cold autoantibodies react optimally at 5^oC and less strongly at higher temperatures.  

The diagnosis of AIHA depends on the demonstration of a positive direct antiglobulin test (DAT), indicating the presence of immunoglobulin and/or complement on red blood cells.  In warm autoimmune hemolytic anemia, red cells may be coated with IgG, IgG and complement, or complement alone. In warm AIHA, IgG is found alone in about 60% of cases and in association with complement in about 30% of cases. In contrast, cold autoimmune hemolytic anemia is caused by complement-fixing IgM antibodies that react more strongly in the cold than at higher temperatures.In these cases, the direct antiglobulin test detects only complement. Autoantibodies may appear to have specificity for a particular blood group antigen even though the patients’ red cells express that antigen. 

Many drugs have been associated with a positive DAT. 

Medications Associated with a Positive Direct Antiglobulin Test

Drug induced hemolytic anemia is very rare. The incidence has been estimated to be one case per 1 million individuals. The most common cause of drug induced hemolytic anemia is the 2^nd and 3^rd generation cephalosporins. Of these, cefotetan appears to be the worst offender. The purine analogue, fludarabine, is used to treat chronic lymphocytic leukemia and produces a positive DAT is almost 35% of cases. 

A DAT should be performed whenever there is:

• A physician order
• Hemolytic transfusion reaction investigation
• Hemolytic disease of the newborn investigation
• An antibody panel has a positive autocontrol
• An unexpected positive antiglobulin crossmatch (on donor RBCs)

Results are reported as negative or positive.  Reference value is a negative reaction.  

Specimen requirement is one 5 mL lavender top (EDTA) tube of blood.

References

Parker V, Tormey CA. The Direct Antiglobulin Test: Indications, Interpretation, and Pitfalls, Arch Pathol Lab Med, 2017;141:305-310.

Rottenberg Y, et al. Blood donors with positive direct antiglobulin tests are at increased risk of cancer, Transfusion 2009;49:838-842. 

Go RS et al. How I treat autoimmune hemolytic anemia, Blood 2017;129:2971-2979.

Eule C et al. The Direct Antiglobulin Test for Evaluating Anemia, JAMA published online December 3, 2018.