Coombs’ Test:- Part 2 – Coombs’ Indirect test, Anti-globulin indirect test
Coombs’ Indirect test
- This is done on the mother serum.
Indication for Coombs’ Indirect test
- To diagnose the presence of antibodies in the pregnant mother’s serum.
- Detection of certain weak antigens in RBCs like Du.
- To find weak antibodies or incomplete types of antibodies like those found in Duffy or Kidd blood groups.
- Detection of incomplete antibodies in serum before pretransfusion screening or may do titration of the antibody (Cross-matching for blood transfusion).
- Detection of cold agglutinins autoantibodies.
- To diagnose autoimmune hemolytic anemia.
- To find the red cell sensitization by drugs.
Precautions of Coombs’ Indirect test
- This should be done on fresh serum.
- Some of the drugs that give false-positive results are cephalosporin, antiarrhythmic, insulin, methyldopa, Dilantin, sulphonamides, and tetracycline.
- The false-positive or negative results are seen due to the following conditions:
- Clerical mistakes.
- Technical errors like not washing RBCs and failure to add reagents.
- Contamination by 5% or 10% glucose in water but not glucose in saline from intravenous tubing.
- Poor quality of commercial coombs’ reagents.
Pathophysiology of Coombs’ Indirect test
- The indirect Coombs test detects the circulating antibodies in the serum; these are the free antibodies.
- 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.
- This is the test for screening purposes in routine blood transfusion.
- This test can diagnose other agglutinins, like cold agglutinins, which are usually associated with Mycoplasmal infection.
- Circulating antibodies against RBC may be seen in the Rh-negative mother who is carrying an Rh-positive fetus.
- There is a mixing of the feto-maternal blood during the first delivery.
- When fetal blood goes into mother circulation, fetal blood Rh-Positive will sensitize the mother, and IgG-type antibodies are formed.
- These antibodies can cross the placental barrier and enter fetal circulation.
- In the second pregnancy, a Type II cytotoxic reaction will destroy the fetal RBCs and lead to hemolytic anemia.
- 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
- This test is done in the serum.
- Add recipient serum (patients) to known blood O cells (donors’ RBCs or screening cells) in a test tube.
- Mix well and incubate for 15 to 30 minutes at 37 °C.
- Centrifuge for 15 seconds at 3400 rpm.
- Observe for agglutination or hemolysis.
- Wash the RBC three or four times with saline.
- Add a large amount of saline and decant completely.
- Now add Coombs’ serum and again centrifuge.
- Mix well.
- Centrifuge at 3400 rpm for 15 seconds.
- See for agglutination.
- If the test is positive, then you will see clumping or agglutination.
- In case of negative results, examine microscopically.
All the reactions are reported as follows:
|Grade of the reaction||Degree of agglutination|
- Negative, and there is no agglutination.
A positive coombs’ indirect test indicates:
- Incompatible blood transfusion.
- Mother has anti-Rh antibodies.
- Erythroblastosis fetalis.
- Acquired hemolytic anemia.
- due to drugs and cold antibodies.