Indirect Coombs Test

Indirect Antiglobulin Test

Coomb’s test also termed as Antiglobulin Test are of two types:

  1. Direct Coombs test
  2. Indirect Coombs test

Indirect coomb’s test

Indirect coomb’s test is performed to see whether a serum sample has antibodies directed against a particular red blood cells and to detect potential non- agglutinating antibodies in the sample. It is done to detect free-flowing antibodies against certain red blood cells.

It is used in prenatal testing of pregnant women and in testing blood prior to a blood transfusion. Pregnant women get a prenatal antibody screening with an indirect Coombs test. It checks the mother’s blood to see if there are antibodies that could pass to and harm her unborn baby.

This test is performed by mixing serum and red blood cells in vitro to allow attachment of antibody prior to testing of red cells. Approximately 5% of patients have a positive IAT due to IgG antibodies, IgM antibodies, or both.

It detects antibodies against RBCs that are present unbound in patient’s serum. In this case, serum is extracted from blood sample taken from patient which is then incubated with RBCs of known antigenicity; I.e. RBCs with known reference values from other patient blood samples. Finally, anti-human globulin is added. If agglutination occurs, the indirect Coombs test is positive.



Indirect Coomb's Test


  1. Label 3 tubes as Test, Positive Control and Negative Control.
  2. Take 2 drops of test serum in tube labeled as T (Test).
  3. In test tube labeled as PC (Positive control), take 1 drop of anti D serum.
  4. Take 1 drop of normal saline In test tube labeled as NC (Negative control).
  5. Add one drop of 5% cell saline suspension of pooled O Rh +ve cells in each tubes.
  6. Incubate all the tubes at 37°C for 1 hour.
  7. Wash the cells 3 times with normal saline.
  8. Add 2 drops of Anti Human Globulin to each tube.
  9. Keep for 5 minutes and centrifuge at 1500 rpm for 1 minute.
  10. Re-suspend cells and examine macroscopically as well as microscopically for agglutination.

Note Point: If no agglutination is not seen on control tube, test is invalid.



  1. Presence of agglutination means a positive test (means patient’s serum contains Anti D antibodies).


  1. Absence of agglutination means a negative test (patients serum does not contain anti D antibodies).



Indirect Coombs test can be used in following test conditions:

  1. Cross-match testing (patient serum is incubated with donor red cells).
  2. Antigen typing (commercial antiserum is incubated with patient or donor cells) e.g. Typing of erythrocyte antigen.
  3. Antibody identification (patient serum is incubated with group O panel cells).
  4. Antibody detection (patient serum is incubated with group O screen cells).
  5. Detecting presence of Anti D antibodies in a Rh negative woman married to an Rh positive man.


Download Link: Indirect Coomb’s Test.pdf


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