Coombs’ Indirect test

Sample

• This is done on the mother serum.

Indication for Coombs’ Indirect test

1. To diagnose the presence of antibodies in the pregnant mother’s serum.
2. Detection of certain weak antigens in RBCs like D^u.
3. To find weak antibodies or incomplete types of antibodies like those found in Duffy or Kidd blood groups.
4. Detection of incomplete antibodies in serum before pretransfusion screening or may do titration of the antibody (Cross-matching for blood transfusion).
5. Detection of cold agglutinins autoantibodies.
6. To diagnose autoimmune hemolytic anemia.
7. To find the red cell sensitization by drugs.

Precautions of Coombs’ Indirect test

1. This should be done on fresh serum.
2. Some of the drugs that give false-positive results are cephalosporin, antiarrhythmic, insulin, methyldopa, Dilantin, sulphonamides, and tetracycline.
3. The false-positive or negative results are seen due to the following conditions:
   1. Clerical mistakes.
   2. Technical errors like not washing RBCs and failure to add reagents.
   3. Contamination by 5% or 10% glucose in water but not glucose in saline from intravenous tubing.
4. Poor quality of commercial coombs’ reagents.

Pathophysiology of Coombs’ Indirect test

1. The indirect Coombs test detects the circulating antibodies in the serum; these are the free antibodies.

2. The major purpose of this test is to detect if the recipient or the patient has serum antibodies other than the ABO / Rh system to RBC before receiving the blood transfusion.
3. This is the test for screening purposes in routine blood transfusion.
4. This test can diagnose other agglutinins, like cold agglutinins, which are usually associated with Mycoplasmal infection.
5. Circulating antibodies against RBC may be seen in the Rh-negative mother who is carrying an Rh-positive fetus.
   1. There is a mixing of the feto-maternal blood during the first delivery.
   2. When fetal blood goes into mother circulation, fetal blood Rh-Positive will sensitize the mother, and IgG-type antibodies are formed.
   3. These antibodies can cross the placental barrier and enter fetal circulation.
   4. In the second pregnancy, a Type II cytotoxic reaction will destroy the fetal RBCs and lead to hemolytic anemia.

Coombs’ serum:

1. It is prepared by injecting IgG immunoglobulin (antibody) into the rabbit, then from his serum, the anti-IgG antibody is separated, which is called Coombs serum.

The principle of the coombs indirect test:

• It detects free antibodies in the blood (serum).

The procedure of  coombs’ indirect test

1. This test is done in the serum.
2. Add recipient serum (patients) to known blood O cells (donors’ RBCs or screening cells) in a test tube.
3. Mix well and incubate for 15 to 30 minutes at 37 °C.
4. Centrifuge for 15 seconds at 3400 rpm.
   1. Observe for agglutination or hemolysis.
5. Wash the RBC three or four times with saline.
   1. Add a large amount of saline and decant completely.
6. Now add Coombs’ serum and again centrifuge.
7. Mix well.
8. Centrifuge at 3400 rpm for 15 seconds.
9. See for agglutination.
10. If the test is positive, then you will see clumping or agglutination.
11. In case of negative results, examine microscopically.

Normal

• Negative, and there is no agglutination.

A positive coombs’ indirect test indicates:

1. Incompatible blood transfusion.
2. Mother has anti-Rh antibodies.
   1. Erythroblastosis fetalis.
3. Acquired hemolytic anemia.
4. due to drugs and cold antibodies.

 

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