Direct Antiglobulin Test
The antiglobulin test is either direct (DAT) or indirect (IAT). Medical applications of the DAT and IAT are summarized in the following table.
Medical Applications of the Direct & Indirect Antiglobulin Tests
Direct Antiglobulin Test |
Indirect Antiglobulin Test |
Hemolytic disease of the newborn |
Detection of unexpected antibodies in plasma (Antibody screen) |
Autoimmune hemolytic anemia |
Compatibility testing |
Drug induced red cell sensitization |
Detection of some RBC antigens not demonstrable by other techniques |
Hemolytic transfusion reactions |
The direct antiglobulin test (DAT) is performed to determine if a patient's red cells are coated in vivo with IgG or complement components. In the DAT, red cells are taken from the patient, washed to remove unbound IgG and then directly tested with antiiglobulin reagent (anti-IgG and/or anti-complement). If antibody is coating the patient’s red cells, they are agglutinated by antiglobulin. The DAT is extremely sensitive; it can detect as few as 100 IgG and 400 C3d molecules per red cell.
Approximately 1 in 9000 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.
Autoimmune hemolytic anemia is classified as warm or cold autoantiobody types based on the temperatures at which the antibodies maximally react with red blood cells in vitro. Warm autoantibodies are more reactive at 37oC than at lower temperatures, whereas cold autoantibodies react optimally at 5oC and less strongly at higher temperatures.
Characteristic Serological Findings in Autoimmune Hemolytic Anemias
Type of AIHA |
DAT Result |
Antibody Screen |
Antibody Specificity |
Warm antibody |
IgG, C3 or both |
Positive in 55% |
Nonspecific or Rh |
Cold Agglutinin |
C3 alone |
Positive up to 30oC |
Anti-I or i |
Paroxysmal Cold Hemoglobinuria |
C3 alone |
Biphasic hemolysin |
Anti- P |
In warm autoimmune hemolytic anemia, RBCs may be coated with IgG, IgG and complement, or complement alone. 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. In these cases, the direct antiglobulin test detects only complement.
The following table lists numerous drugs that have been associated with a positive DAT.
Medications Associated with a Positive Direct Antiglobulin Test
Acetaminophen |
Fenoprofen |
6-mercaptopurine |
Sulbactam |
Amoxicillin |
Fludarabine |
Methicillin |
Sulindac |
Amphotericin |
Fluoroquinolones |
Methotrexate |
Sulfonamides |
Ampicillin |
Fluorouracil |
Methyldopa |
Sulfasalazide |
Carbenicillin |
Hydralazine |
Metrizoate contrast |
Tazobactam |
Carbimazole |
Hydrochlorothiazide |
Nafcillin |
Teicoplanin |
Carboplatin |
Ibuprofen |
Norfloxacine |
Temafloxacin |
Cephalosporins |
Insulin |
Oxaliplatin |
Teniposide |
Chlordiazepoxide |
Interferon |
Penicillin G |
Tetracycline |
Chlorpromazine |
Interleukin 2 |
Piperacillin |
Ticarcillin |
Chlorpropamide |
Isoniazid |
Probenacid |
Tolbutamide |
Cisplatin |
Latamoxef |
Quinidine |
Tolmetin |
Clavulanate |
Levodopa |
Quinine |
Triamterene |
Declofenac |
Levofloxacin |
Ranitidine |
Zomepirac |
Diphenylhydantoin |
Mefenamic acid |
Rifampicin |
Zosyn |
Erythromycin |
Mefloquine |
Streptokinase |
|
Etodolac |
Melphalan |
Streptomycin |
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 2nd and 3rd 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)
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 hemolyze extensively. Also, the strength of the direct antiglobulin test often does not change following treatment, even though the clinical condition greatly improves.