Direct Coombs Test

1) Non-EDTA sample

2) Gross hemolyzed sample

3) Major clot in sample

1) Coomb sera:

• Eryclone (Tulip diagnostic)
• Polyspecific antihuman globulin + monoclonal anti-C3d (AHG titre > 1: 256, anti-C3d titre > 1.8). Store at 2-8^o C. Do not freeze.

2) 0.9% saline (normal saline)

3) Centrifuge machine

4) Test tubes & micropipette

5) O positive sensitised cells

1) Take 0.5 ml of blood to be tested in clean, labelled tube

2) Add 5 ml normal saline to sample. Centrifuge at 1500 rpm for 2 minutes.

Remove all supernatant and allow only cells button to remain. Dislodge the cell button by gently tapping the test tube at bottom. Add 5 ml of normal saline again; repeat this procedure two times.

3) Make a 5% suspension of washed RBC of test sample. (1 drop of washed RBC, 19 drops of normal saline).

4) Add 200 microliters of AHG serum to 100 microliters of 5% sample suspension in a separate test tube.

5) Mix & centrifuge at 1000 rpm for 1 minute.

6) Shake the tube to dislodge the cell button.

7) See for agglutination, grossly as well as microscopically.

8) Keep the test tube at room temperature for 5 minutes. Centrifuge at 1000 rpm for 1 minute.

9) See for agglutination, macroscopic & microscopic

1) Positive control: 100 microliters of pooled O-positive 5% cells + 100 microliters of 1:4 anti-D IgG and 200 microliters of AHG.

2) Negative control: 100 microliters of pooled O-positive 5% cells + 100 microliters of NS and 200 microliters of AHG.

Positive DCT indicate that patient RBCs are coated by incomplete antibody, which may be the cause of hemolysis in vivo. Causes include:

1) Hemolytic disease of newborn

2) Autoimmune hemolytic anemia

3) Drug-induced hemolytic anemia

4) Hemolytic transfusion reaction