Haematology Watch, Vol.5 , Issue 1.
LABORATORY HAEMATOLOGY
Coombs Test: Procedure
Mehmood
Direct Coombs Test
1. Make Coombs check cells (CCC) : IgG coated (by using Anti-D serum and RBCs of RhD immunized) and Complement-coated.
2. Identify patient’s whole blood received in EDTA tube.
3. Give it 3 – 4 washes in 0.9% saline.
4. Make its 2 – 5 % suspension in 0.9% saline.
5. Take 3 tubes: label as Test, Control IgG, and Control C3d.
6. Add 2 drops of polyspecific Coombs serum in test tube and control tubes (containing CCC).
7. Then add 1 drop of respective suspensions in them.
8. Mix gently and centrifuge both at 1000 rpm for 15 seconds.
9. Observe and grade any agglutination/haemolysis macroscopically and then at 20X objective lens.
10. a) If positive in Control tube but negative in test tube, leave the test tube at 25 ˚C for 5 minutes, centrifuge, and observe again. [Agglutination after incubation may be due to complement coating of RBCs.]
b) To confirm a positive result, repeat test using monospecific Coombs serum on test cells to differentiate between IgG & C3d coating.
11. If negative in both Control tubes and Test tube, the Coombs serum is ineffective. Repeat the test by using new Coombs serum.
Indirect Coombs Test
1. Identify clotted sample of the patient.
2. Place 2 -3 drops of patient’s serum in a new test tube.
SALINE PHASE:
3. Make a pool of O blood group RBCs.
4. Make its 2 – 5 % suspension in 0.9% saline.
5. Add 1 drop of this suspension in the test tube containing test serum.
6. Centrifuge at 3500 rpm for 1 minute.
7. Observe and grade any agglutination/haemolysis macroscopically and then at 20X objective.
LISS/ALBUMIN PHASE:
8. Re-suspend the cell mixture gently.
9. Add 1 drop of either Low Ionic Strength Solution (LISS) or Albumin, and incubate at 37 ˚C for 10 or 30 minutes, respectively.
10. Centrifuge at 3500 rpm for 1 minute.
11. Observe and grade any agglutination/haemolysis macroscopically and then at 20X objective.
COOMBS PHASE:
12. Wash the cells 3 – 4 times with 0.9% saline, and completely decant the final wash supernatant.
13. Add 2 drops of Coombs serum to the dry RBC button and re-suspend gently.
14. Centrifuge at 3500 rpm for 1 minute.
15. Observe and grade any agglutination/haemolysis macroscopically and then at 20X objective.
16. a) If no agglutination/haemolysis is found at any phase, record as Negative Indirect Coombs Test.
b) If agglutination/haemolysis is found at any phase, record the result with its strength and phase.