Direct Antiglobulin Test

Direct antiglobulin test is used to demonstrate in vivo coating of red blood cells with IgG antibodies and complement (C3d). The assay uses Coombs reagent incubated with the patient’s washed red blood cells. ^[1]

Normal findings: Negative; no agglutination ^[2]

A direct antiglobulin test is positive in the following situations:

• Warm autoimmune hemolytic anemia (SLE, Evan’s syndrome, idiopathic)

• Hemolytic disease of the newborn

• Alloimmune reactions to recently transfused cells, and

• Drug-induced hemolysis (alpha methyldopa, levodopa, quinidine or high dose of penicillin etc).

Medications may cause the formation of antibodies, either against the medication itself or against intrinsic red blood cell antigens resulting in a positive DAT, immune red cell destruction, or both. Some of the antibodies produced appear to be dependent on the presence of the drug (eg, penicillin, quinidine, ceftriaxone), whereas others are independent of the continued presence of the inciting drug (eg, methyldopa, levodopa, procainamide, cephalosporins, fludarabine). ^[3]

A negative antiglobulin test can result in the following cases:

• Hemolytic anemias caused by intrinsic red cell defect (eg, hemoglobinopathies or G6PD)

See the list below:

• Specimen: Blood anticoagulated with EDTA

• Container: Lavender or red-top vacuum tube

All samples must be sent in a sealed, leak-proof container marked with a biohazard sticker to comply with Occupational Safety and Health Administration (OSHA) safety standards.

Clinical features of hemolysis help to differentiate the hemolytic anemia from other anemias. Clinical characteristics of hemolysis include pallor, icterus, and discoloration of urine, and, in some cases, hepatosplenomegaly. Bony changes due to overactivity of the bone marrow (frontal bossing) may occur, although they are never as severe as they are in thalassemia. ^[4]

The laboratory characteristics of hemolytic anemias are related to hemolysis itself and the erythropoietic response of the bone marrow. Features of hemolysis include increase in unconjugated bilirubin and aspartate transaminase in serum; and urobilinogen both urine and stool. ^[4] In case of intravascular hemolysis, increased hemoglobin in serum resulting in hemoglobinuria, increase in lactate dehydrogenase, and reduced haptoglobin are notable laboratory features. However, serum bilirubin level may be normal or only mildly increased. The main sign of the erythropoietic response from the bone marrow is an elevated reticulocytes count. ^[4]

Direct antiglobulin test is used to demonstrate in vivo coating of red blood cells with IgG antibodies and complement (C3d). The assay uses Coombs reagent incubated with the patient’s washed red blood cells. ^[1]

The DAT detects the presence of antibody or complement (or both) on the surface of red cells. Washed red cells from a patient or donor are tested directly with antihuman globulin (Coombs) reagents. When IgG coats red blood cells showing agglutination immediately, direct antiglobulin test is positive. However, complement or IgA coating may only be demonstrated after incubation at room temperature. ^[3]

See the list below:

• Autoimmune hemolysis

• Hemolytic disease of the newborn

• Drug induced hemolysis

• Transfusion reactions

Red blood cell coating by immunoglobulins or complement in vivo causes positive DAT. Clinically, such red cell coating may or may not be associated with immune hemolytic anemia. Some of these patients of hemolytic disease of the newborn and drug-related hemolytic anemias become sensitized (antibodies against the erythrocytes are formed and bind to the their surface but do not cause hemolysis), but these patients do not exhibit symptoms of disease. ^[5]

Depending on the reagent and technique, the DAT may detect a level of 100–500 molecules of IgG per red blood cell and 400–1100 molecules of C3d per red blood cell. ^[3] Without clinical manifestations of immune-mediated red blood cell destruction, positive DATs have been reported in the range of 1 in 1000 up to 1 in 14,000 blood donors and 1–15% of hospital patients. ^[3]

A false-positive DAT is often seen in patients with hypergammaglobulinemia, eg, in some patients with HIV, multiple myeloma, or lymphoma (lymphoplasmacytic). ^{[3, 1, 6]} Negative DAT is very rare in autoimmune hemolytic anemia. It may be due to a small amount of IgG bound to red cell membrane.

A positive DAT frequently is seen in patients with multiple myeloma; this is due to passive adsorption of the M protein onto the red cells. It is usually observed with immunoglobulin G3-MM, and clinically does not result in hemolysis. ^[7]

Gel microcolumn assay (GMA) is a modified serological technique that has been used for ABO and Rh typing, and DAT to detect alloantibodies. GMA has been found to be more sensitive than conventional tube test for detecting potentially significant antibodies coating erythrocytes in vivo. ^[8]