galvania

In my case, temperature, as the screening cells and panel were both from the same manufacturer, therefore the same buffer system

Because I've seen so many of them…………. But actually if your panel is not in the same buffer then pH or ionic strength could be the culprit rather than the temperature. Important to stress that these cold anti-Ms (in that they dont react strictly at 37°C) have no clinical significance. If it happens again, you can try the following: 1. Repeat the panel, incubating for 15mins at RT (on a Coombs card). The results will be stronger in this case. AND 2. Repeat the screen in the following way. Warm the cells to 37°C (best just to use a small aliquot - you dont want to 'cook' the whole bottle). And put the Coombs card (foil still on) in the incubator for 15mins. Put the patient's plasma in the incubator. IN the incubator, pipette 50ul of cells into the appropriate wells followed by the patient's plasma. Incubate for 15mins. At the same time, start the centrifuge empty. This warms the centrifuge up a bit. After 15mins put the incubated card into the centrifuge which will have now stopped and centrifuge immediately. This should negativise the screen results. This is about the nearest you can get to doing a gel test at strictly 37°C

and what is wrong with this patient? what drugs is (s)he on?

It depends a bit on how you work. If you are working manually then it is quite common to pipette a whole series of tests . In this time the cells can cool down enough for the anti-M to latch on, and once it's on it stays on. On the other hand, panels tend to go up individually so the cells stay warmer

I'm no longer working but if this is an antibody then it sounds logical that it could cause non-sp results…..

The DAT MIGHT have a considerable IgA component. Some anti-IgG (and hence also poly) will pick up IgA more than others regardless of method.

might be worth checking to see if she actually has antibodies to IgA

The reverse cells are normally pooled. So the lady's antibody is probably reacting with some of the cells in the pool and not others. Usually the Rh pheno is the same on all the cells in the pools so it probably is not due to the anti-c unless your provider does not ensure that they are all the same, or one of them is a c-variant. Could easily be an anti-M or Lea or Leb though

Also, the polyclonal (human) reagents will give false pos results in samples with pos DATS. But anyway, finding sufficiently good human antibodies to manufacture reagents from is getting harder and harder. So definitely monoclonal

Are you working in gel? If so, repeat in tube. Tube is more sensitive for weak reverse reactions

could you post the article????

This is well known and there are indeed other threads concerning this. Transfused cells may have different densities to the patient's own cells and therefore when you spin the tubes in the centrifuge you may find that the transfused cells and the patient's cells separate. Then, depending on where you sample from in the tube, you will either get only transfused cells, only patients cells or a mixture, giving a mixed field. Typically instruments will select cells from the bottom of the tube, whereas pipetting by hand, one normally samples from the top, giving a discrepancy between the two methods.

what are the antibodies and what is the reason for needing the titration?

If you are seeing a lot of weak results in your anti-D well that subsequently turn out to be negative, I suspect the reason to be one of manipulation rather than anything serological. I would guess that NONE of these babies are group O. I am guessing that you are seeing carryover from your anti-AB. It can happen that if cards are stored somewhere where condensation can take place then drops of antiserum can condense into the reaction chamber and 'jump' into the next well or even next 2 wells when you remove the aluminium. This can cause false positive results. I suggest you check the cards before pipetting in them and see if there are any signs of these drops. Don't use the cards if there are and look for another place to store them

what is the baby's DAT? Is it anaemic? Jaundiced?

Beware of using reagent antisera to do your QC. The buffers in the reagents can interact with the buffers in the cells and, if using anything other than tube, the buffers in the support medium. You may get false positive results

IF it looks like a real reaction and it's only one cell then it may well be an antibody against an LFA. If you send a good sample from the patient together with the cell concerned to your reference centre, they may be able to identify it for you - although that's on an interest-only basis

Oh I quite agree Malcolm - just that those two possibilities need to be taken into account with this type of reaction

or it might have been anti-buffer or cold 'rubbish'......

Cliff - I cant see where to edit my e-mail address. It is now <removed by admin>. And no - I have no problem that anyone in the forum can see this

Sorry, have only just seen this. Now that I am retired >I no longer check my e-mails every day. (yes, that does say day, not hour. I know the youngsters will find that totally unbelievable). Anyway, if you suspect the presence of rouleaux it can be very difficult to differentiate between rouleaux and agglutination in gel although rouleaux does tend to have a pink haze about it. Even more difficult if BOTH are present…. I would not try to elucidate this in gel. I would do this in a tube and look at the result down a microscope (one of the few times where I think use of a microscope is justified) and then you can visually see the difference. As for the other part of the question - normal saline can interfere with the buffers used in the gel itself - regardless of the technique used (IAT, direct agglutination etc). This can lead to unagglutinated cells 'hanging' in the gel and this gives a rosy appearance which can be quite strong. It does not always happen but when it does can be mistaken for a positive result. A negative result can be interpreted as a true negative - saline will not weaken a result in a direct agglutination test. As an aside to Cliff, I didn't get an email notification but that could be because I have not updated my e-mail address!!!!! Will do so as soon as posible. Have changed countries too. Am now in Italy

There is absolutely no problem testing for Ch/Rg in gel. And although it should not be ok to use 50ul plasma instead of 25, actually it does SOMETIMES enhance very weak antibodies. There have been times when I have done this successfully. You will not however find it in the official instructions for antibody screening/identification and this should never be done as a routine technique

Well you carry on doing just that then klsmith, seeing as you had ONE example where it came up with something that would have otherwise been missed, and you clearly think that that justifies it. Actually why not put up enzyme IATs routinely as well. But please do not complain when you have to put up panels on 90% of your samples and get inconclusive results on all of them

This issue has been going on for years. The complement coated cells are produced artificially. The method used is designed to be used to show positive reactivity with anti-C3 in a tube technique. It is not designed to show absence of reactivity in anti-IgG, neither in a tube, or even less in gel. Indeed the method used will ALWAYS show an unwanted positive reaction in anti-IgG if you use gel. CAP are aware of this - enough people have complained about this over the years. Why they don't do something about this is a mystery to me

Except that we are talking about patients here that have anti-B in their own plasma already. We are not talking about patients who have no detectable anti-B in their plasma. So we are talking about a patient who groups as an A in the forward group and has a ++ reaction in B cells, due to anti-B. So if he receives group A plasma, yes, he will receive some anti-B - which will be diluted out by his own plasma which already contains anti-B……….. Realistically, I think it is a question of comparing risks, benefits and the amount of work. In this case, what are the chances that this is an ABwk patient? - Very low What is the risk, if this patient is an ABwk, of transfusing this patient with group A blood? None. On the contrary it is better than transfusing with group AB What is the risk, if this patient is an ABwk, of transfusing this patient with group A plasma? very little as the patient already has a considerable amount of his own anti-B in his plasma What is the risk, if this donor is an ABwk, of transfusing to a group A patient? Very little as the amount of B antigen present is so small How much work do you need to do to be 100% sure that this type of reaction belongs to a patient who is really a group A and not ABwk? As an absolute minimum genotyping, possibly complete sequencing. Long delays and $$$$$$$$$$$$$$$.