Direct Antiglobulin Test

28 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 30^oC	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.

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.

Last Updated on Monday, 18 July 2011