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.

How to read a bone marrow biopsy

normal marrow

There are several things you should look for when evaluating a bone marrow biopsy specimen – see if you can see them in the image above.

First, take a look at the cellularity. The white spaces are fat cells that have washed out during processing; the cells in between the fat cells are hematopoietic precursors. The ratio of cells to fat is called the “cellularity.” The marrow above is approximately 30-40% cellular. You need to know the age of the patient to estimate whether the cellularity is normal. Here is a rough guide to cellularity by age:

0-3 months: 100%
3 months – 10 years: 80%
20 years: 65%
30 years: 50%
40 years: 45%
50 years: 40%
60 years: 35%
70 years and over: about 30%

Next, take a look at the composition of the marrow. Myeloid cells make up the largest percentage of the normal marrow cellularity; erythroid cells are second most common. The ratio of myeloid to erythroid cells should be about 2:1 to 4:1. It’s easier to see these cells on an aspirate smear, but you can get a pretty good idea on the marrow section too. Neutrophils and precursors often have eosinophilic, granular cytoplasm; if you look closely, you can see the indented nuclei of metamyelocytes and segmented nuclei of mature neutrophils. Erythroblasts generally have very round, dark nuclei; earlier forms are large, and later forms are small. A few megakaryocytes (large cells with abundant eosinophilic cytoplasm and multiple nuclei) should be sprinkled throughout the marrow too. Lymphocytes normally represent about 10-15% of the marrow cellularity. The above marrow appears to have a myeloid:erythroid ratio of 2:1, and megakaryocytes are normal in number.

Finally, take a look through all the sections to see if you see anything weird, like fibrosis, metastatic carcinoma, lymphoid aggregates, or amyloid deposition. As you scan the sections, you should see evenly-distributed cellularity, with evenly-spread hematopoietic precursors. Anything that deviates from this pattern should be investigated on higher power.