Antiglobulin (Coombs) test is useful to detect the presence of incomplete antibody (IgG) and/or complement on the RBC membrane.

Principle

RBCs coated with incomplete antibody (IgG) or C3 complement does not cause agglutination of RBCs. Coombs reagent contains antibodies (antiglobulins) against human IgG/IgM/complement. If the RBCs coated by incomplete antibody or complement, are treated with Coombs reagent, the antiglobulins in the reagent will produce/induce agglutination of such RBCs.

 The standard Coombs reagent will not detect IgA or IgE antibodies.

Types of antiglobulin test: There are two types, namely direct and indirect antiglobulin tests.

1. Direct (Coombs) antiglobulin test (DAT)

2. Indirect (Coombs) antiglobulin test (IAT)

DIRECT ANTIGLOBULIN TEST

Direct antiglobulin test (DAT) (direct Coombs test) detects antibodies (IgG) and/or complement coated on the surface of patient’s RBC membrane. Patient’s red cells are used in this test.

Materials Required

•• Patient’s RBCs

•• Coombs reagent

•• Normal saline.

Technique (Fig. 59.1)

•• Patient’s RBCs are obtained from either the clotted fresh blood or blood sample in EDTA/citrate.

•• Take RBCs in a test tube and wash three times in normal saline.

•• Centrifuge (2,500 rpm for 5 minutes) and decant the saline. Prepare a 5% saline suspension of RBCs.

•• Take 2 drops of 5% saline red cell suspension in a test tube and add 2 drops of Coombs (antiglobulin)reagent.

•• Mix well and incubate the test tube at 37°C for 5 minutes.

•• Centrifuge at 1,000 rpm for 1 minute.

•• Gently shake the tube and observe for agglutination.

With each test, run one positive control and one negative control.

Interpretation

Agglutination indicates the presence of antibody on the RBC membrane and interpreted as positive DAT.

Uses of Direct Antiglobulin Test (Causes of Positive Test)

•• Hemolytic disease of newborn (HDN) in which direct Coombs test is performed on the newborn baby’s red cells from the cord blood.

•• Autoimmune hemolytic anemia: To demonstrate in vivo attachment of antibodies to red cells.

•• Drug-induced red cell sensitization.

•• Investigation of hemolytic transfusion reaction.

INDIRECT ANTIGLOBULIN TEST

Indirect antiglobulin test (IAT) (indirect Coombs test) detects the presence of incomplete (IgG) antibodies and/or complement in the patient’s serum. Patient’s serum is used for this test.

Materials Required

•• Patient’s serum

•• O Rh positive RBCs

•• Coombs reagent

•• Normal saline.

Technique (Fig. 59.1)

•• Take 2 drops of patient’s serum in a test tube.

•• Add 3 drops of 5% “O” Rh +ve cell suspension of any normal individual.

•• Incubate the above mixture at 37°C for 10 minutes. During this time the “O” Rh positive RBCs are coated with (lgG) anti-Rh antibodies (if present) in the patient’s serum.

•• Wash with normal saline three times to remove the excess of antibodies. Remove the supernatant.

•• Add 2 drops of Coombs (antiglobulin) reagent to the test tube and incubate at 37°C for 10 minutes.

•• Centrifuge at 2,000 rpm for 1 minute and examine for agglutination. With each test, run one positive control and one negative control.

Interpretation

Agglutination of RBCs indicates the presence of antibodies in the patient’s serum and test is reported as positive for indirect antiglobulin test.

Uses of Indirect Antiglobulin Test

•• Hemolytic disease of newborn: Mother’s serum is tested to detect anti-Rh antibody.

•• Cross-matching for blood transfusion: To detect incompatibility of recipient’s serum with donor’s cells.

•• Phenotyping of red cell antigens using known antisera.

•• Titration of anti-Rh antibodies.

Controls for Antiglobulin Test

•• Positive control: Sensitized (coated by anti-D) O Rh (D) positive cells.

•• Negative control: Normal individual’s RBCs which are nonsensitized. Gel Card Method (Figs 59.2A and B)This is more sensitive for detecting antibodies than conventional Coombs test.

Points to note:

•• Coombs test detects incomplete antibodies (IgG) and/or complements against the RBC membrane.

•• There are two types of Coombs test, namely direct and indirect.

•• In direct Coombs test (DAT), patient’s red cells are used and it is positive in HDN (fetal red cells) and autoimmune hemolytic anemias.

•• In indirect Coombs test (IAT), patient’s serum is tested. It is useful in HDN where the mother’s serum is tested for the presence of antibodies. It is also performed while cross-matching for blood transfusion, to detect incompatibility of recipient’s serum with donor’s cells.