Yesterday, we looked at the direct antiglobulin test, or DAT. Today, we’ll take it one step further and look at the indirect antiglobulin test, or IAT (which is really just the DAT with an extra step thrown in).

This test is the basis for two important assays in transfusion medicine: (1) the antibody screen (in which serum samples from recipients and donors are screened for the presence of antibodies to red cell antigens), and (2) the cross-match (in which the recipient serum is combined with the potential donor red cells in the laboratory as a final check to make sure there will be no antibody-antigen mismatch in the transfusion).  These tests are done to avoid hemolytic transfusion reactions (which can happen if your patient has a red cell antibody, and you give red cells with the corresponding antigen).

The point of this test is to find out if your patient has antibodies against red cells – either antibodies against any red cell antigen at all (which is what the antibody screen looks for), or antibodies against the particular unit of red cells you have chosen to give the patient (which is what the cross-match is for). The DAT is looking to see if your patient’s red cells are coated with antibody; the IAT is looking to see if your patient has antibodies in his or her serum.

Here’s how it’s done:

1)    Mix a little of your patient’s serum (shown as grey antibodies on the left hand side of the diagram) with a little of the donor red cells (shown as red cells with little blue antigens below the second test tube).
2)    Centrifuge tubes and check for agglutination (depicted in the diagram in the center panel, showing red cells with antibodies attached). Sometimes, the reaction is so strong that you can see agglutination with the naked eye at this point! Usually not, though.
3)     Incubate at 37° C for half an hour, then check again for agglutination again. Sometimes heating the test tube will aid in the agglutination process.
4)    Wash the cells (to remove any plasma proteins that could interfere).
5)    Add anti-human globulin (Coombs’ reagent, discussed under the DAT) (depicted as blue antibodies under the fourth test tube).
6)     Incubate and check again for agglutination (shown as the final panel, way on the right hand side of the diagram).

If there is agglutination in the tube (in any of the steps), you know that your patient must have an antibody to one (or more) of the antigens on the donor red cells in that tube. You can’t give that particular unit of red cells, obviously!

Note: The very nice depiction of the IAT above was created by A. Rad, and can be found on Wikimedia commons at: http://commons.wikimedia.org/wiki/File:Coombs_test_schematic.png.