What are Indirect Coombs’ Test and its procedure?

The indirect Coombs test looks for antibodies floating in the blood. These antibodies may act against specific red blood cells. This test is often done to determine if there is a reaction to a blood transfusion.

The indirect Coombs test can be used to determine the presence of antibodies against the Rh factor in the mother’s blood. In this case:

A normal (negative) result means that the mother has not developed antibodies to the fetus’s blood. A negative Coombs test indicates that the fetus is not currently at risk of problems related to Rh incompatibility.

An abnormal (positive) result means that the mother has developed antibodies to the red blood cells of the fetus and has become sensitized. However, even a positive Coombs test only indicates that an Rh-positive fetus may be harmed. Even a positive test result cannot indicate the extent of damage that has occurred or may occur to the fetus.

If tests show elevated antibody levels during pregnancy, the risk of fetal harm may increase. The Rh-negative fetus is not harmed even if the mother is sensitive.  

what is indirect coombs test

The procedure of the Indirect Coombs Test

  1. Label the three test tubes T (test serum), PC (positive control), and NC (negative control).
  2. Add 2 drops of test serum to the tube labeled T (test).
  3. Pour 1 drop of anti-D serum into the tube labeled PC (positive control).
  4. Add 1 drop of saline to the test tube labeled NC (Negative Control).
  5. Add 1 drop of Rho positive cell suspension (D) in 5% saline to each tube.
  6. Incubate all three tubes for 1 hour at 37°C.
  7. Wash cells 3 times with normal saline to remove excess free antibody-free serum (insufficient washing of red blood cells may give negative results).
  8. Add 2 drops of Coombs serum (anti-human serum) to each tube.
  9. Hold for 5 minutes and centrifuge at 1500 rpm for 1 minute.
  10. Resuspend cells and examine macroscopically and microscopically.
What is coombs test procedure

When is Indirect Coombs Test performed?

Blood transfusion preparation

The indirect Coombs test is used to screen for antibodies when preparing blood for transfusion. The blood of the donor and the recipient must be compatible in terms of ABO and Rh D. Donor blood for transfusion is also tested for infections in separate processes.

Antibody screening

The recipient’s blood sample and the blood sample from each unit of donor blood are tested for antibodies using an indirect Coombs test. Each sample is incubated against a broad spectrum of red blood cells that together exhibit the full spectrum of surface antigens (ie, blood types).


The indirect Coombs test is used to test the recipient’s serum sample for antibodies against the donor’s red blood cell sample. This is sometimes called cross-blood matching.

Antenatal antibody screening

The indirect Coombs test is used to screen pregnant women for IgG antibodies that are likely to cross the placenta into the fetal blood and cause hemolytic disease in the newborn.

Why do I need this test?

 The test can be done on your blood before the baby is born. Afterward, it can be done on blood from the umbilical cord and from your baby.

You may need this blood test before a blood transfusion. Health care providers need to know if you have antibodies in your blood that could harm the donor’s red blood cells.

If you do not have antibodies to the donor’s red blood cells, you can safely receive a blood transfusion.

If you have antibodies, the blood will stick together (agglutinate) and cannot be used.

You can also take this test if you are pregnant. This can be a problem for the baby if your blood cells lack a surface protein called the D antigen, but your baby has inherited the D antigen from the father. (The presence of the protein is also called Rh positive, and the absence of the protein is called Rh negative.)

In this case, your body will produce antibodies against the D antigen in the baby’s blood. This can cause your baby to have a severe type of anemia called hemolytic disease. This happens more often with the second child than with the first child.

The analysis can be done on your blood before the baby is born. This can then be done on cord blood and from your baby.

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