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.
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