With the direct Coombs test you just add the anti-Ig antibodies (Coombs reagent) to your test solution and watch for agglutination. With the indirect Coombs test you have to add RBCs first, then you add the anti-Ig antibodies, and then watch for agglutination. So there is an extra step with the indirect Coombs test.
Direct Coombs is also called DAT (direct antiglobulin test).
Indirect has an extra step. Direct lets you go directly to the result.
The Coombs test was developed in 1945 by Robin Coombs and colleagues who developed the technique of using antibodies to target other antibodies, thus allowing detection of the targeted antibodies.
I always found this very confusing for some reason, maybe it’s my poor immunology background, but I think I finally understood when I looked into Rh factor testing during my Obs-Gyn rotation. I will use the example of Rh factor testing to hopefully explain the direct and indirect Coombs tests.
If a baby comes out of its mom and is swollen, jaundiced, and having respiratory troubles you may have hemolytic disease of the newborn on your differential. That can happen when mom is Rh factor negative and baby is Rh factor positive. That’s a bad combination because mom might have antibodies in her system which target Rh factor (since she would consider Rh factor to be foreign). These antibodies cross the placenta, stick to baby’s red blood cells (which have Rh factor present) and thus target those blood cells for destruction (since immune systems tend to destroy anything coated by antibodies even if they are important). If enough of baby’s blood cells get destroyed, a whole host of bad things can happen. If you have such a baby on your hands and you want to confirm that this is indeed hemolytic disease of the newborn, you can take some of baby’s blood and do a direct Coombs test on it.
To do that you’ll need Coombs reagent (which is the magic stuff that makes the Coombs test work). Let’s digress a bit to discuss Coombs reagent. Basically, this is a reagent comprised of antibodies which target human antibodies. Any human antibodies. These kind of antibodies are created by injecting animals with human antibodies. Let’s say a sheep for example. The sheep’s immune system may respond to these injected human antibodies by making sheep antibodies that stick to the human antibodies and target them for destruction. These new sheep antibodies which stick to human antibodies can then be harvested from the sheep and put in a bottle labelled “Coombs Reagent” (it’s probably slightly more complicated than that). Note that these antibodies are sheep antibodies so they don’t stick to each other, they just sit there behaving themselves until they are exposed to human antibodies.
Now that we have Coombs reagent we can return to our example. Baby potentially has hemolytic disease of the newborn which means some of mom’s antibodies have crossed the placenta and are putting the beat down on baby’s red blood cells. We can confirm this by taking a sample of baby’s blood. If we are correct, what we should see is baby’s red blood cells (with the Rh factor protein sticking out of them) all smothered in anti-Rh factor antibodies which have come from mom. Now since mom is presumably a human, her antibodies can be targeted by the antibodies in the Coombs reagent.
So in the direct Coombs test you wash baby’s blood cells to remove any antibodies that are just floating around not doing anything. The only antibodies that will remain will be ones that are stuck onto baby’s cells. Next you add the Coombs reagent. If any of mom’s antibodies are stuck to baby’s cells, the Coombs reagent will stick to them. Since antibodies can stick to more than one target you end up with “clumps” forming of Coombs reagent stuck to mom’s anti-Rh factor antibodies stuck to baby’s blood cells stuck to more of mom’s anti-Rh antibodies stuck to more Coombs reagent and so on. This clumping is called “agglutination” and is readily visible, and forms the basis of a positive Coombs test.
So now you know why baby is sick, but how could all of this be prevented? What we really need to know is if mom has any anti-Rh factor antibodies in her bloodstream before baby is even born (or ideally before baby develops Rh factor in utero which is around the start of the 3rd trimester). If she has no antibodies, nothing will cross the placenta and there’s nothing to worry about. Here is where the indirect Coombs test comes in.
This time you take a sample of mom’s blood. Mom, of course, is Rh factor negative (no Rh positive person would make antibodies against their own blood unless they had autoimmune hemolysis which is a whole other story) so her own red blood cells have no Rh factor and no antibodies stuck to them. She may however, have anti-Rh factor antibodies floating around in her blood sample. You then add in some red blood cells known to be Rh factor positive. If mom has any antibodies against Rh factor they will immediately stick to those newly added blood cells. Then you wash the whole concoction to remove any freely floating antibodies that aren’t bound to anything. Then you add the Coombs reagent and watch for agglutination just as in the direct Coombs test. So the indirect Coombs test is exactly the same except you have the extra step of adding in red blood cells which you know are Rh factor positive.
Back to some immunology for a second. The one thing that always got me is the notation “Ig”. That just means “immunoglobulin” which is a fancy word for antibody. So you can use the word “antibody” and the notation “Ig” interchangeably. Also, you can indicate what the antibody targets by saying “anti-X”, where X represents the target. So anti-Ig is an antibody that targets other antibodies. This is why they call Coombs reagent, anti-Ig.
Although we’ve just discussed the use of the Coombs test in Rh incompatibility there are multiple uses for the test clinically. Here is the list from Medscape’s page on the subject:
- Autoimmune hemolytic anemia
- Drug-induced immune hemolytic anemia
- Hemolytic transfusion reactions due to alloantibodies
- Hemolytic disease of the fetus and newborn
- Systemic lupus erythematosus (in the absence of hemolytic anemia)
Lab tests online is a great site with an excellent page on the DAT (Coombs test)
Here is Medscape’s review on the whole subject