The Indirect Antiglobulin Test

Surely everybody in serology knows about the indirect antiglobulin test (IAT) as they probably use some form of it hundreds of times a day. I agree but then everybody also assumes we know what we are talking about and those who don't know get too embarrassed about asking. So let's have a little look at it now.

One of the problems with IgG antibodies is that they can sensitise cells really well but are a bit bit rubbish when it comes to agglutinating them and making that reaction visible to the tester - this wouldn't be so bad if IgG antibodies were not so darned clinically significant with the ability to cause haemolytic transfusion reactions and haemolytic disease of the foetus and newborn.

We need a way of showing that sensitisation reaction - Prof. Robin Coombs (1921-2006) came up with the answer by using anti-human globulin (AHG). AHG are antibodies against antibodies so, rather than sticking to antigens, they stick to antibodies - if those antibodies happen to be stuck to antigens on a cell (sensitisation) then they can cross-link with each other and bring about agglutination. I'd have given him a Nobel Prize for that but what the heck.

Let's have a look at the test then - this should make things a bit clearer.

Take your cell suspension and add the antiserum, incubate it and (if there has been a reaction) you'll get this:

In this picture you can see that there has been a reaction between the antibodies and the red cells (because they have recognised the antigen on the cell surface and stuck to them.

This could be a very strong reaction or a very weak one but they will both look the same - just like a negative result, This is because the antibodies have sensitised the cells (coated them) but are unable to cross-link between the cells and join them up to form agglutination (the clumping together of cells due to antibodies).

We need to add AHG but before we do that there is a very important step - wash the sample.

Why do we need to wash the sample? If the serum is from a patient then there will be LOADS of other immunoglobulins in it protecting the patient from diseases and whatnot - the AHG will stick to thes just as surely as it would stick to those antibodies coating the cell and the AHG, even though it is very concentrated, will be neutralised and you'll get a negative result with or without the cells being sensitised. If the serum sample is actually a specific antiserum you are useing to test the cells then there will be massive amounts of that antibody in there, not all of it will be on the cells and the free antibody will, again, netralise the AHG. Washing the sample (with buffered saline, not water!) removes any unbound material so the only immunoglobulins left in the sample will be those which are bound to the cells (if any).

We can now add te AHG (the cyan antibodies in the picture). these will bind to any antibody in there - as we have washed the sample these antibodies will be bound to the cells.

The AHG can therefore bind to antibodies bound to different cells and cross-link them causing the sensitised cells to agglutinate!

And that is the genius of Coombs!

Of course, as we are all good serologists we need to control the AHG step because who is to say that are result is actually negative or did we forget to add any AHG or not wash the sample so that all of the AHG has been neutralised?

To all of our negative tests we need to add sensitised cells (cells known to be coated with antibody - usually RhD+ cells coated with anti-D because they are cheap and easy to make). When we add these calls, centrifuge the sample and re-read it we should see some agglutination thus proving to ourselves that there was AHG in there in the first place and that there is enough to cause agglutination - it wasn't neutralised! Another way of ensuring that we've put the AHG in is that AHG is often coloured green so it is easy to spot if you've missed a tube.