Indirect antiglobulin test

IAT

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.

Direct antiglobulin test

Coombs_test_schematic

The direct antiglobulin test (also called the Coombs’ test, or the DAT) is an important one for you to know. It’s used mostly in one particular setting: when you have a patient with a hemolytic anemia (one in which the red cells are getting busted open) and you want to know if the hemolysis is immune-related or not. As explained below, the DAT is positive in immune hemolytic anemias and negative in non-immune hemolytic anemias.

The whole point of the DAT is to find out whether there are antibodies and/or complement bound to the surface of the patient’s red cells. In an immune hemolytic anemia, the patient may have antibody, or complement, or both bound to his or her red cells. Since you can’t see the antibody or complement under the microscope, you need a way to determine whether these molecules are present – and that’s what the DAT is for.

Here is how it is done. A small amount of a reagent called Coombs’ reagent, or anti-human globulin (AHG) is added to the patient’s blood in a test tube. This reagent (depicted as blue antibodies in the diagram above) consists of antibodies directed against human antibodies. These antibodies are raised by injecting human antibodies into another animal (a rabbit, or a mouse, or some other non-human), and then collecting the anti-human-antibody antibodies the animal makes (the animal sees the human antibodies as foreign substances, and it makes its own antibodies against them). You also add some antibodies directed against complement to the patient’s blood sample (these are not depicted above).

The cool thing about the Coombs’ reagent is that if the patient’s red cells are coated with IgG, the Coombs’ reagent will bind to this IgG on the red cells, bridging the gap between adjacent red cells, and causing the red cells to clump together (see the right hand side of the diagram above)! You can see this clumping with the naked eye. The same principle works for the anti-complement antibodies; if there is complement bound to the red cells, the anti-complement antibody will bind to it, and the red cells will clump together.

So: if you see clumping in the test tube, the DAT is positive, and that means your patient has an autoimmune hemolytic anemia. The next thing to figure out is which kind of autoimmune hemolytic anemia it is – but that’s the subject of another post.

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