Clinical Relevance of Coomb’s Test (Antiglobulin Test)

Coombs’ test is a simple and ingenious technique. Coombs’ test was first described in 1908 by Moreschi and was later rediscovered by RR Coombs and coworkers in 1945 to demonstrate non-agglutinating antibodies to the red blood cell antigens.

(The antibodies are called ‘non-agglutinating’ antibodies because the antibodies couldn’t cause agglutination, in spite of their binding with the antigens on RBCs. The antibodies are also sometimes referred to as ‘incomplete antibodies’ as the antibodies don’t cause agglutination. But there is no incompleteness in the structure of antibody molecule.)

The non-agglutinating antibodies, in fact, bind to the antigens on the surface of RBCs. But the expected agglutination of RBCs doesn’t occur. When another antibody (called anti-antibody or antiglobulin) against the RBC-bound antibody is added, agglutination occurs. The antiantibodies bind to the Fc regions of RBC-bound antibodies and form a lattice, which leads to the agglutination. The test is called antiglobulin test or Coombs’ test.

There are two types of anti-globulin tests:

1. Direct anti-globulin test (DAT)

2. Indirect anti-globulin test (lAT)

1. Direct anti-globulin test (DAT) is used to detect antibody, which is bound to RBCs in vivo.

Washed RBCs from a patient are mixed with anti-globulin.

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The anti-globulins bind to non-agglutinating antibodies on the surface of RBCs and cause visible agglutination. The test result is DAT positive (Fig. 1.0).

Therefore a positive DAT suggests that the patient’s RBCs are coated with non-agglutinating antibodies and the patient is said to be DAT positive.

i. Positive DAT occurs with RBCs of newborn with hemolytic disease of newborn (the RBCs of HDN are coated with anti-Rh antibodies).

ii. Currently, DAT is used to detect IgG antibodies and C3b bound to the surface of RBCs. In patients presenting with hemolysis, the DAT test is useful in determining whether the hemolysis is of immune etiology or not.

Fig. 1.0: (DAT)

DAT is used to detect the presence of non-agglutinating antibodies bound on the surface of RBCs. A. The individual’s washed RBCs are mixed with the anti-globulin reagent. The anti-globulins bind to the Fc regions of the RBC bound antibodies and cause visible agglutination, and the individual is said to be DAT positive. B. If the RBCs are not coated with antibodies, there will not be agglutination and the individual is said to be DAT negative. DAT is used also to detect the presence of anti-Rh antibody coated RBCs of newborn suspected to suffer from hemolytic disease of the newborn

i. Non-immune causes of hemolysis [such as disseminated intravascular coagulation, red cell enzyme deficiencies [such as glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency), red cell membrane defects (such as hereditary spherocytosis, PNH), and hemoglobinopathies (such as sickle cell anemia, thalassemia)] are DAT negative.

ii. Immune causes of hemolysis (such as autoimmune hemolytic anemia, drug induced hemolysis, hemolytic transfusion reactions) are DAT positive.

iii. A positive DAT due to C3 alone is seen in patients with cold autoantibodies, paroxysmal cold hemoglobinurea, and in some cases of drug induced hemolytic anemias.

2. Indirect agglutination test (IAT) is used to detect the presence of unbound antibodies against RBCs in the serum of a patient.

i. IAT to detect anti-Rh antibodies in mother’s serum:

Mother’s serum is incubated with Rh positive O blood group RBCs. If anti-Rh antibodies are present in the mother’s serum the antibodies will bind to the RBCs. The RBCs are then washed.

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Then the anti-globulin reagent is added.

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If anti-Rh antibodies are present in the mother’s serum, the RBCs would be agglutinated and the test is positive for IAT.

ii. IAT to detect antibodies in the serum of a recipient against the donor RBCs during blood transfusion:

IAT test is widely employed (before blood transfusion) in the cross matching of donor’s RBCs with recipient’s serum to detect the presence of antibodies in recipient, which may react with the donor’s RBCs.

Wash the RBCs of the donor.

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Add the recipient’s serum to the RBCs and incubate at 37°C for 30 minutes.

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Wash the RBCs.

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Add the anti-globulin reagent.

Development of agglutination indicates that the recipient’s serum has antibodies against the donor RBCs and the test is reported as positive for antibodies. A positive IAT result indicates that the proposed donor blood should not be transfused to the recipient in question.

Current anti-globulin test reagent (also called Coombs’ reagent) preparations contain a ‘cocktail’ of monoclonal antibodies directed against human IgG and C3b (Binding of non-agglutinating antibodies to RBCs activate the complement system resulting in the deposition of C3b fragments over the RBCs; and therefore apart from antibodies, the RBCs are coated with C3b fragments also).