Dear colleagues,

Did these cases happen with you, it happened with me 4 times in last three months?

Patients' plasma was pan-reactive grade 3 with all tests done by column agglutination techniques (gel cards Biorad company), includes three screening cells, auto-control, 11 panel cells, also cross match with 4 units same blood group were incompatible grade 3 with all units tested by gel cards.

When we switched to manual tube saline technique, all tests were negative includes antibody screen, reaction with 11 panel cells, and cross match with same unites that were positive with gel, these tests were confirmed macro and microscopic, and also by Coombs' check cells.

What is your interpretation!!!?

Yes Mohammed; almost daily.

We deal with huge numbers of DAT positive samples, and very often we can "get rid" of an auto-antibody reacting by column agglutination technology (CAT), simply by performing the tests by LISS tube IAT. We know that CAT can be very sensitive in detecting such antibodies, and LISS tube IAT less so, so we always run a weak positive control with the tests we perform (both CAT and tube) to ensure that the sensitivity is not reduced to such an extent that we do not detect such antibodies (i.e. we do not miss a genuine weak alloantibody in the palsma).

Yes but I want to continue the story, in those cases, I usually issue units by tube cross match and also observe unit transfusion, no one of these units made any reaction neither acute or delayed and patient usually discharged safely from hospital with good conditions, even with immune-compromised pt. like leukemia or end stage renal disease.

So, shall I consider that reaction with gel is nonspecific reaction, resolved by washing during IAT tube method?

I agree with Malcolm that it is probably a weaker warm auto antibody that gel detects but your tube method does not. I would either call it negative by tube method or call it a weak warm auto.

Malcolm, what exactly is this weak pos control you speak of running with such patients?

It is, if you like, "what it says on the tin".

Our NHSBT Reagents Department produces a weak anti-D, a weak anti-c, a weak anti-K and a weak anti-Fya, each of which (or all of which) can be used as a control to ensure that we are able to detect a very weakly reacting antibody by whatever technique we are using at the time.

These antibodies are genuinely weak, rather than a stronger antibody diluted to make them weak. This is very important, because antibody/antigen reactions are governed by the Law of Mass Action (see Hughes-Jones N. Nature of the reactions between antigen and antibody. Brit med Bull 1963; 19: 171) and the equilibrium constant of a genuine weak antibody is quite different from the equilibrium constant of a diluted stronger antibody.

I think your patient is probably reacting against one of the components in the buffer in the LISS/Coombs cards

I think your patient is probably reacting against one of the components in the buffer in the LISS/Coombs cards

I'm leaning more towards Galvania's explanation. We've all seen antibodies that have reacted to the preservatives used in the screeening cells or the "LISS" dependent antibodies that react only in the presence of LISS or other odd occurences that really had no impact on the transfusion of the patient. The fact that you had 4 in 3 months would suggest you avoid gambling in the near future. It has always amazed me how things like this happen and once again it proves that no technology is perfect and fool proof.

We have seen this where screening, panel same strength...but autocontrol would be negative and crossmatch also compatible....our conclusion was antibody to preservative. We also take different manuf. reagent red cells and dilute it to 0.8% and run it on gel and usually we get negative result.

You can also wash the reagent red cells and re suspend in diluent and you may get negative result if the antibody is not reacting against a LISS but reacting against preservative.

In my experience the crossmatches and auto controls are usually negative with antibodies to gel diluent because they are mostly directed at antibiotics in the preserved cells. Diluent 2 lacks the antibiotics of the pre-diluted cells.

I think your patient is probably reacting against one of the components in the buffer in the LISS/Coombs cards

that you said, same my explanation

I think your patient is probably reacting against one of the components in the buffer in the LISS/Coombs cards

that you said, same my explanation

The gel testing could be picking up a cold autoantibody.

Do a cold antibody screen in tube. Do a DAT.

I've seen this quite often with cold autoantibodies, they seem to like to react stronger in gel from my experience. They react strongly with all cells tested including the auto control in gel, but all tube testing comes out negative with LISS. I have been able to pick up some colds with PEG in tubes though.

The gel testing could be picking up a cold autoantibody.

Do a cold antibody screen in tube. Do a DAT.

Yes I did cold atibody screen but negative.

Yes, like Mabel, I still think it is a weak warm auto.

We see an awful lot of these in our Reference Laboratory.

We also see a few where there is an antibody directed against an antibiotic in CellStab, but we routinely use cells in Diluent2.

that you said, same my explanation

I agree

Yes, like Mabel, I still think it is a weak warm auto.

We see an awful lot of these in our Reference Laboratory.

We also see a few where there is an antibody directed against an antibiotic in CellStab, but we routinely use cells in Diluent2.

Yes Malcolm, but if there is warm auto Ab and removed by tube IAT So I got same benifit that I removed this aouto, let me to know if there is underneath allo warm or not???

From the fact that we have never had a patient have a delayed or immediate haemolytic transfusion reaction using this technique, and the fact that we have been able to detect clinically significant alloantibodies masked by these weak warm auto-antibodies, over many, many years, my answer would be yes.

I am very confused reading this thread, more so especially that we have had a similar problem yesterday.

We were asked to do DAT on a 55y/F patient of Thalassaemia intermedia, who is also known diabetic as well as Hep. C positive. We used Biorad gel card and diluent-2 and it was positive 3+. The consultant asked us to repeat it and our MD asked us to do it by tube technique. By tube we found it ?neg/± but repeated testing with gel was again 3+. The consultant sent us another sample, which gave the same results. Washing the red cells 3 times with NS didn’t change either of the results. How do I report the DAT?

By the way, this patient is multi-transfused & now needed transfusion support again with PRC, we crossmatched (by gel) PRC with no problem. Just out of curiosity we did minor crossmatch again in gel and it was not compatible with 1+ reaction.

My second query is how do we differentiate if the reactivity in gel is due to the patient probably reacting against one of the components in the buffer in the LISS/Coombs cards or genuine weak warm autoab?

Malcolm, are the weak control antibodies commercially available? if not, what other solution do we opt for?

HI aafrin, whether these weak control antisera are available commercially worldwide, I am afriad I do not know, but they are certainly available in the UK through the NHSBT Reagents Department.

Going back to your case, please would you just clarify if the two AHGs you use (the one for the tube technique and the one inn the gel) are the same; both anti-IgG+C3d, both anti-IgG only, or some other combination? Thanks.

Hi Aafrin

Most of the time, your reactions will be real. There aren't so many cases out there that are just reacting non-specifically with something in the Diluent. Also, if you're still getting a 3+ after washing 3 times, that further points to something real. Then, you also have to take the clinical picture and the other results into account. To explain the discrepancy, I would look for something either in a difference between your Coombs reagents, or a problem in one or other of your two techniques that is leading either to false positives or false negatives. When you did the manual test, for example, did you check your negatives with Coombs Control cells? Also, how are your weak positive QC antibodies reacting in both methods? That might give you a clue. On a different tack, have you tried to test the pos DAT in the gel card that allows differentiation between IgG and C3d. It also contains a control well. So that would give you useful information. If you've really got a patient just reacting with the Diluent 2, then your control well will be positive too.

Thanks for the reply Malcolm. Both the AHGs used are IgG+C3d.

Sorry, I didn't see your reply Anna before replying.

Yes, the CCC did give positive reaction in the tube test.

We don't have weak positive QC antibodies, that's why I was asking Malcolm whether they are available commercially.

We have not tested the pos DAT in the gel card that allows differentiation between IgG and C3d. I will have to ask the Biorad, if they can give us one such card or do that test for us.

Thanks a ton for your reply, highly appreciated.

A negative patient control is an excellent idea. If you do not have the cards to differentiate between IgG and C3 with the control do you run your types in gel? The type cards I have experience with have a control for the front type (for those pesky AB pos samples). This would be essential the same as the DAT control. Patient cells and gel with no "reagent".

Trish, we did the blood grouping in gel card and yes the "control" well was negative. I checked. Thanks.