Direct Coombs Test (Direct Antiglobulin Test- DAT) - Principle, Procedure, Result, Interpretation

Direct Coombs Test (Direct Antiglobulin Test- DAT)

Direct Coombs Test (Direct Antiglobulin Test- DAT) is used for the detection of antibodies (IgG) or complement system factors (C3) bound to the RBC surface antigens.

In the direct Coombs method, in vivo sensitization of red blood cells (RBCs) with incomplete antibodies takes place. It uses antiserum against human immunoglobulin to agglutinate a patient’s RBC and detects cell-bound antibodies.

This test is done on newborn blood samples, mostly on infants with jaundice for diagnosis of Rh incompatibility and ABO incompatibility. However, is not mandatory for pre-transfusion testing while indirect Coombs test is often done.

Anti-human reagents in Coombs Test

For the Coombs Test, there are three types of reagents that may be used:

• monospecific reagents to detect bound immunoglobulin G (IgG)

• monospecific reagents to detect bound complement (C3)

• polyspecific reagents that can simultaneously detect IgG and/or C3

For the step of screening RBCs, the most commonly used reagents are polyspecific reagents that can simultaneously detect IgG and/or C3. If the result is positive, the RBC sample is then subsequently tested with monospecific reagents, either bound immunoglobulin G or bound complement. This finalizes the identification of a substance that is bound to the RBC surface.

Procedure of Direct Coombs Test (Direct Antiglobulin Test- DAT)

Direct Coombs Test (Direct Antiglobulin Test- DAT) is done by tube method. Meanwhile, automated methods such as solid phase or antiglobulin gel (microcolumn method) are now being commonly used.

The procedure is as follows:

1. Add 5% isotonic saline to the sample blood to obtain a cell suspension.

2. Take a drop of the prepared cell suspension with the help of a clean pipette in a small tube.

3. Wash the blood sample with saline to remove excess, unbound IgG or complement present in the serum.

   * such unbound IgG or complement may preferentially bind and hence inhibit the reactivity of the added reagent.

4. Repeat the third step three times to remove all traces of serum.

5. After the last wash, decant the tube completely.

6. Add two drops of anti-human serum (polyspecific reagent) to the tube.

   * This step should be followed immediately as delay in a test or inadequate RBC washing can cause the dissociation of antibodies from the RBC sample which can result in false-negative reports.

   * If complement reagent is used instead of polyspecific reagent, a second incubation can be done to allow enhancement of weak anti-complement reactions.

7. Mix well and centrifuge at 1500 RPM for 1 minute.

8. Gently agitate the tube to resuspend.

9. Examine the test tubes for visible agglutination – both microscopically and microscopically.

The agglutination is graded from 0 to 4+, with 4+ representing a solid button of agglutination.

Result, Interpretation of Coombs Test

Negative result

Absence of agglutination, both microscopically and macroscopically.

Positive result

The presence of agglutination is interpreted as a positive result.

This means antibodies are bound to RBCs of the test blood sample i.e. immune system is attacking the patient’s RBCs. This condition may be autoimmunity, alloimmunity, or a drug-induced immune-mediated mechanism.