What is an Indirect Coombs Test?

What is an Indirect Coombs Test?


When we prepare to do a crossmatch we do a blood type and an antibody screen.  The indirect coombs test, also called the Indirect Antiglobulin test (IAT) is the antibody screen portion of pre-crossmatch testing.

The purpose of the indirect coombs is to determine if a patient has red cell antibodies in their serum (or plasma).   People can develop red cell antibodies to red cell antibodies they do not have on their own red cells.

For example, if a person does not have red cell antigen "E" on his red cells, his body would consider red cells with the "E" present on them as foreign and COULD create Anti-E antibody--although, most likely he won't. These antibodies can form after a patient has been transfused or when a woman is exposed to a foreign red cell antigen on the blood cells of the baby she's carrying.

One can also spontaneously form auto-antibodies in response to certain drugs and medications.

When red cell antibodies attach to red cells they can destroy them, lowering the patient's red cell count and hemoglobin level.

Here is an illustration of the whole concept of the indirect coombs test.

Here's how it's done:

1. We add a couple drops serum (plasma) to a drop of sample red cells with known antigens on them.  For example the red cells in tube 1 may have antigens C and K, the red cells in tube 2 may have antigens E and Fya, the red cells in tube 3 may have antigens Jka and S.

This is just a simple example: each red cell sample actually has several different antigens on them in various combinations.

2. Next we add an enhancement solution like PEG (polyethylene glycol) or LISS (low ionic strength solution)  which facilitates the attachment of any patient antibodies (if present) to its corresponding antigen on the red cells.  The tubes are incubated for about 15 minutes at 37 Celsius to simulate temperature conditions in the human body. The most significant red cell antibodies that cause transfusion reactions or cross the placenta during pregnancy are of the IgG form and react at body temperature. 

3. After incubation, the combination of patient serum (plasma), sample red cells and enhancement is washed with saline.  After washing the only thing left in the tubes are the sample red cells and any patient antibody that may be attached to them.

4.  Anti-IgG is then added to the tubes.  This is antibody TO human IgG antibodies.  I realize this may be kind of confusing but read carefully.   If there are red cell antibodies attached to the sample red cells, the Anti-IgG antibodies will bind to the Patient (IgG) Antibodies which are attached to the SAMPLE red cells.   This causes clumping or agglutination which is visible after spinning the tubes in a centrifuge.

So let's say that there is agglutination in tube #2.    Earlier I said that the red cells in sample tube #2 have antigens E and Fya on them.    This might mean that the patient has Anti-E or Anti-Fya in their serum (plasma) or an antibody to any other red cell antigen on that particular sample cell.

It's possible that two or even all three of the tubes could be positive.  This may mean that the patient has multiple antibodies or that they have an non-specific autoantibody that reacts with everything.

Further extensive testing would then be conducted to determine which antibodies are present.