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A curious case of anti-e


Emelie

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A patient came to the ICU with Hb at 52. It was quickly concluded that he had some sort of hemolytic anemia. He also had an immunodeficiency since birth with low levels of both IgG and IgA, and was wholly uncapable of producing IgM. The doctor requested a monospecific DAT and a screen, and ordered 2 bags of blood. All my lab tests came out as 3+ straight over, including the ctl well, so I proceeded with an antibody identification. Unsurprisingly, the patient's plasma gave 3+ reactions against all panel cells as well as his own rbc's, both with IAT/LISS (gel) and PEG. Autoadsorption x2 gave no other outcome, not even the slightest reduction of reactions. I cross-matched blood (again, 3+ reactions) which the patient received without any reported transfusion reactions.

When I had the results from the cold ab panel on NaCl gel cards it clearly showed an anti-e at all temperatures (4, 20 and 37°C). Since the panel is used to identify IgM antibodies I assume that this is an autoantibody of IgM-type, and not a regular IgG anti-e, possibly caused by the IVIg the patient has received? I have no clue. The hospital where I work isn't very large and we don't do any advanced ab identifications ourselves, neither do we encounter these kinds of results and patients regularly. Is there anybody who has some ideas and would like to enlighten me? Would you have done any further investigation regarding ab detection?

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Hi Emelie,

 

Things like this can be extremely confusing.

 

I am assuming that the anti-e is an autoantibody, or is the patient e negative?

 

If it is an autoantibody, you are correct in thinking that it could have been introduced parenterally in the IVIgG, because they seem to put all sorts of things in that (including high titre ABO antibodies!), although that would not necessarily explain the wide thermal amplitude of the antibody.  One thing I would say though, is that not all cold antibodies are IgM.  I can give you 2 examples straight off that are cold reacting IgG antibodies.  The first is the anti-P (the famous biphasic anti-P) that causes PCH.  The second is an ABO antibody that causes ABO HDN.  If you put a sample of cord blood in the fridge over night, and examine it the next day, you will see agglutination.  The antibody causing this MUST be maternal (otherwise it would be an autoantibody - which the baby could not have made at that stage of life), and, as it has gone through the placenta, MUST be IgG, rather than IgM.

 

If the antibody did not originate in the IVIgG, and it is an autoantibody, I should warn you that the specificities seen in such cases are often "mimicking" antibodies.  In other words, something that looks like a straight forward anti-e, may not be.  Often, if you carry on long enough with repeated adsorptions, you can adsorb out the antibody to exhaustion, using antigen negative red cells (in this case, e negative red cells).

 

Lastly, if the patient has, as you say, a life long immunodeficiency, it may be that the antibody molecules that he does make are "mutations" when compared with other peoples' antibody molecules.  A similar situation can be found in human monoclonal IgM anti-D molecules, which have a V4-34 moiety that is essential to the antibody "working", but is identical to a structure on both anti-I and anti-I.  This is why you should never use a monoclonal anti-D reagent straight from the fridge, as there is a danger that it will react like a true anti-I or anti-I, leading to false positive results.

 

I'm not certain that I have been able to help with this answer (I've probably served only to muddy the waters even further), but I hope I have explained it a little, and may stimulate others to give a more concise, and possibly more accurate answer.

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Hi Malcolm, and thanks for your answer!

As we werent able to phenotype the patient due to his strongly positive DAT (I tried an Rh+K card but ctl turned out a strong 3+ just as everything else), we don't know wether or not he might have a real anti-e rbc ab, but we assume it is an autoantibody or, rather, a mimicking ab due to the strange reaction pattern and that the patient has no record of earlier transfusions.

The haematologist treating him was certain it was cold ab's, but the cold agglutinin test from the laboratory came back negative. Still, we had those findings in our cold ab panel and the patient's rbc's turned into a solid lump as the blood cooled. New samples that were prepared and analyzed in 37°C didn't react differently than the earlier though, and cross-matching e- blood didn't turn out any better than e+. We will try to give the patient e- blood as far as possible, but we don't have that much to choose from in our bloodbank so he will probably receive e+ units further on too since we don't think it is a "true" rbc ab.

It is very interesting to hear what those more experienced and who work with much more sofisticated methods has to say, I learn something new everyday!

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I jus wonder, from your further information, whether or not this patient does have a "cold" autoantibody of wide thermal amplitude, but also has a warm autoantibody too. Mixed warm and cold autoimmune haemolytic anaemia is rare, but does, nevertheless, happen on occasions.

You say that the sample's red cells strongly agglutinate as they cool (the "cold" element of the antibody). Have you got any of the original sample left (pre-transfusion) which you could try washing several times in warmed saline (as near 37oC as possible) and then trying the Rh and K typing again? The reason I ask is that it is quite important to find out if the anti-e is an auto or an alloantibody. The thing is, if it is an autoantibody, AND the patient turns out to be E negative, and you transfuse e negative blood, there is a fair chance, even with his history of immunodeficiency, that he could make an alloanti-E, and then you would have real problems! I know it is expensive, but I honestly think that this case is worthy of an Rh genotype, if you cannot get an Rh type by serology. It may also be worth the money to send it off to a reference laboratory to see if they can resolve the situation. Treatment for cold autoimmune haemolytic anaemia may be different from ttreatment for warm autoimmune haemolytic anaemia, and, if as I suspect, this may be a rare case of mixed cold and warm autoimmune haemolytic anaemia, it is important that the more pathological of the 2 antibodies (whichever that may turn out to be) is treated more agressively.

Whatever you decide to do, I think that it is important that you test the sample (by direct agglutination, but, possibly with the addition of a small amount of albumin) at strict 30oC, just to see if the result of the negative cold agglutinin test was a false negative due to a prozone effect. Certainly, all the results you quote above suggest that there is a cold autoantibody present - and a pretty strong one at that.

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Wow, thanks for the quick reply! I will certainly have another go at the Rh phenotyping if there is any sample left. I did however wash the rbc's in warm PBS doing the monospecific DAT and it didn't reduce the reactions, but I'll keep my fingers crossed (and wash more times)! Unfortunately, the cold agglutinin test is performed at the microbiology lab at our hospital but I'll see if there's anything I can do!

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No, the washing of the DAT in warm PBS should not make any difference, as you are testing with AHG.  When you are doing the Rh and K test, however, I presume that you are using monoclonal agglutinating antibodies that do not require an AHG phase.

 

I just wonder, and I am only wondering, if, as your Microbiology Department performs the cold agglutination tests, whether they would do them in the same way as a Blood Transfusion Department (they may do, I am just wondering out loud, as it were) and whether they are looking for mycoplasma infection (known to cause cold autoantibodies) and that this test was negative, rather than actually looking for red cell agglutination in the test?  I could be totally wrong about this.

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This case is wierd.  I would agree there has to be some sort of cold antibody present if there is visual agglutination in the sample taken from the fridge.  And I also agree it could easily be a cold IgG.  I have seen many cold IgG antibodies!!  But I do wonder if it is really the patient making these antibodies or if these antibodies are coming from the IvIg he is receiving, presumably in large quantities.  If you have the possibility of getting hold of a bottle of the last lot of IvIg he received, it might be worth trying to do an antibody panel (inc A and B) on that.

Also, this could all be a nasty case of drug-induced.  What medication is the patient on.  I guess he's on lifelong antibiotics if he has congenital immunodeficiency.  Many of these can cause severe AIHA.

Also - does he actually have an infection?  Maybe not mycoplasma but something else?

I also wonder whether there are not previous ABO and Rh phenotyping results from before this episode.  I would be especially interested in knowing whether he has ever reacted in the reverse group........

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I washed the cells in order to aquire a neg result in the ctl well when performing the DAT :) I am trying to phenotype Rh+K but now after 2 washes the only thing that's changed is that all the reactions (including ctl) are even stronger and clearer... just out of curiosity, how many times is it reasonable to wash the sample before giving up?

It's alright to wonder! I do not know how Blood transfusion departments perform the cold agglutinin tests, but at our microbiology dept, they titrate the patient's serum with NaCl and 0- blood and cold incubate over night. Titers > 1/16 counts as positive. The test is only performed to identify immunological/haematological disorders, and is not used to diagnose mycoplasma, the use PCR and PAAA for that.

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Galvania - No he did not react in the reverse grouping, we first encountered the patient when we were typing him a couple decades ago but there's not much historical information other than that the lack of anti-B was caused by his inability to create IgM.

Unfortunately the patient passed away today, otherwise it might have been motivated to send samples to another, more sophisticated laboratory for eluating etc earlier on. I suspect his doctors knew he wouldn't last long anyway so they probably weren't very interested in our findings.

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Dear Emelie

That is sad.  I'm really sorry.

But I'm still curious.  I think possibly the cold agglutinin titre was negative because all the antibody was attatched to the patient's red cells before they started the test.

But I would still be VERY interested to know what medication he was on.  The word Cefotetan is rearing its ugly head at me...........

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