Malcolm Needs

Hi Hi-Freq, Yes, that's exactly what we have to do. Say it is an anti-Fya, instead of sending over the required number of Fy(a-) units for them to cross-match, we have to perform the cross-match and then send over the blood. I wouldn't mind, but part of the remit of Red Cell Immunohaematology is education, and part of that education is to allow people to cross-match blood if they are frightened. The point is, if they do the cross-match and then come back to us and say they cannot get compatible blood; fair enough. We'll then gladly do the cross-match for them. It's when they don't make the effort in the first place. Call me cynical if you like, but the vast majority of our 50 odd hospitals will do the cross-match, no problem at all, if we send over antigen negative blood (even if there are multiple antibodies). The majority, if not the vast majority, of our hospitals will perform an "immediate spin" cross-match on a patient with WAIHA if, after alloadsorption, we inform them that there are no detectable underlying atypical alloantibodies and, in an even smaller number of cases, will accept antigen negative blood for an "immediate spin" cross-match, after we've done the serological work-up. A minority, however, will expect us to do the lot, and it is usually the same hospitals (and it is the "usual suspects", if you know what I mean). Anyway, you are quite correct. I should stop moaning. I have a mortgage to pay!!!!!!!!!!!!!!!

Hi There Hi-Freq, I agree with you entirely, and without reservation, that there are blood bankers and non-blood bankers, and that even some very experienced people within the blood bank can, in fact, be non-blood bankers when it comes to samples with antibodies present. Having said that, however, we ask them to ask the doctors/phlebotomists to take sufficient samples so that tests can be performed at the Hospital prior to submission, and to hold some back from this sample, so that they can cross-match when we supply them with antigen negative blood. I realise that the fact that we ask them to ask the doctor/phlebotomist to do this does not mean that they will recieve such samples, but there are times when we receive three samples on the same patient, find that the DAT is now negative, or that the antibody is still an "easy" monospecific, and we then telephone them with the results, only to find that they have sent us all the samples and they have none left with which to cross-match. I wouldn't mind, but some of these occasions have occurred in hospitals (during the day) where the chief in blood bank is ex-Reference staff themselves. I DO TRY TO HAVE MERCY ON THEM, BUT I NEED A RANT SOMETIMES TO RELIEVE THE PRESSURE!!! CONTRARY TO WHAT MY FRIENDS AND COLLEAGUES FREQUENTLY SAY - I'M HUMAN.

Hi Mabel, They are done in gel, and I do agree with you that this is an awfully good method to pick these up - sometimes too good. That having been said, however, it is not that unusual for us to test a sample that shows a pan-agglutinin with papain-treated red cells in gel and, for example, an apparent allo-anti-E by gel IAT with a negative auto by IAT, but then to geta positive DAT and, after performing an eluate, finding that the eluate reacts more strongly with E+ red cells by gel, but still react with E- red cells. Occasionally, very occasionally, we will then go on to alloadsorb the plasma and the apparent anti-E will be adsorbed to extinction by E- red cells. This is a bit of an esoteric test, and we only do it when we are short of work and bored! To be honest though, as these patients are, by definition in this example, E- themselves, we wouldn't bother, and just recommend that E- blood be given (on the grounds that they are often transfusion dependent, or will become so, and we don't want them to form a real allo-anti-E.

I agree with an awful lot of what CKCheng says, but I have certainly seen quite a few cases of auto negative, DAT positive, particularly in cases of either a delayed haemolytic transfusion reaction or a delayed serological transfusion reaction. A DAT is not an onerous test to perform, and is always worth doing in such circumstances I think.

Hey, good for you Lara! Either my thread on Nomenclature is getting through, or I've managed to bore the world into submission (probably the latter!). I can just hear people rushing to their computers to tell me to substitute "certainly" for "probably"!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

I agree with clmergen (and L106's assessment of clmergen's answer). As a reference Service, we do a panel on each sample as a "first line of defence", as the presence of an antibody is why the sample has been sent to us in the first place. Some of these, of course, are DAT+ with free auto-antibody in the plasma. However, what REALLY annoys us is when we receive a second or subsequent sample from the same hospital, when the patient was DAT+ with free auto-antibody in the plasma, and on this occasion the DAT is negative or positive, but there is no longer free auto-antibody (or alloantibody) in the plasma. Very often this is because they have put on to their computer, "Send to the Reference Laboratory" and have not tested the current sample themselves at all before referral. Excuse the pun, but this makes my blood boil.

Within the Reference Laboratories of the NBS in England, we test the panel each day with an weak anti-D, a weak anti-c, a weak anti-K and a weak anti-Fya. This covers a positive and a negative for each cell. I should stress that we use genuine weak antibodies, rather than strong antibodies that have been diluted, because the biological dynamics of genuine weak antibodies, such as the equilibrium constants between antibody/antigen complex and antibody and antigen not complexed (and remember, this state is dynamic) is different for weak antibodies and strong antibodies that have been diluted.

To answer the first point, yes your patient was in danger that night, and so was every other patient with clinically significant atypical alloantibodies. The practitioner should not be allowed to work unsupervised (day or night, and however senior they may be) until a root cause analysis has been performed. This is not a disciplinary measure (although it may sound like it). It is purely to protect patients if the practitioner did something wrong (and I am not accusing him or her of having so done). If the practitioner did follow the procedure and obtained markedly different results from other people, then it is certainly a case for re-training, and he or she should not be allowed to work unsupervised (day or night, and however senior they may be) until they have shown competency in a number of test conditions after the training has been completed. The other thing that must be done after such an incident it to validate the procedure itself. You have to prove that the procedure is correct, and this should be done under stress conditions, if possible (very difficult I know, as "false" stress is almost impossible to do). If the practitioner is found to not have followed the procedure, well...........I think that should become a disciplinary matter (but only if proved). I agree with Rashmi that the pre-warmed (only to 37oC), warm-washed (with saline warmed only to 37oC) LISS tube IAT is the gold standard in the UK. However, I also agree with Mabel that it can be lethal in the wrong hands. It needs to be used on a very regular basis to get experience and maintain competence. I've seen people not mixing the red cells and the saline properly between centrifugation during the washing phase. I've also seen people hammer the tubes on the bench at the reading phase. Not surprisingly, they got false positive results!

Its a pleasure Marion. Don't forget that we are all still learning, however old we (or, at least, I, at 728 years!) might be! I've learned a lot since I've joined BloodBankTalk, and I haven't been here very long.

A bit more detail. The paper I referred to yesterday is: Daniels G, van der Schoot CE, Gassner C, Olsson ML. Report of the Third International Workshop on Molecular Blood Group Genotyping. Vox Sanguinis 2009; 96 (4): 337-343. I still haven't had a chance to see it myself yet, so it may or may not be of use to you Antrita, but it could be worth a try and is almost certainly worth a read in any case.

Well, although I suspect that you are very well aware of this Anna, it all comes down to the fact that Landsteiner and Wiener were working on an antibody made in guinea pigs that had been injected with the blood from the Macaca mulatta (or Rhesus) monkey. The antibody made reacted very much like anti-D, but it soon became apparent that it was not identical to anti-D, and that almost all D Negative individuals are reactive, albeit extremely weakly with this antibody (except newborns, who are D Negative, but react more strongly with the antibody. As a result, in 1963, this antibody was renamed anti-LW (after the work done by Landsteiner and Wiener) and LW became the 16th independent Blood Group System. True anti-D was first described by Levine and Stetson and, because the powers that be did not want confusion between the two (LW and D), it was deemed that the proper name for the Blood Group System should be Rh, rather than Rhesus or rhesus. I agree, it does take more effort to say Rh, rather than Rhesus (as it does to say, for example, Fya, rather than Duffy a, but I think it is worth it. See! I said I was a pedant!

Yes Rashmi, you are correct (otherwise you are controlling precisely nothing). Like Marion, see my comments in the other thread.

Hi Marion, If you write to the RCI Department of your local Blood Centre (which I think is Tooting), we can supply you with what the NBS did to validate these machines (although, having said that, we don't have one at Tooting). I'll forward anything to my colleagues at the various Centres that do, and we'll try to get something to you.

It depends how far you want to send your samples. They certainly perform genotyping at the New York Blood Center and at the International Blood Group reference Laboratory in Bristol, UK, but there must be lots and lots of places all around the world that do so. Vox Sanguinis (I think it was that journal anyway) have just publish the latest ISBT Workshop on Blood Group Genotyping (although I haven't been able to read the paper yet). If you look at the authors names involved in this, and where they are based, that could help you find someone "local" (I use the term in its loosest sense!).

I think the point is that the ISBT Group about red cell antigen nomenclature specifically state that it should be, for example, anti-K, rather than anti-K1, and anti-G, rather than anti-Rh12.....but they also say that it is not anti-Kell or anti-Rhesus12.

We freeze rare cells all the time (about 600 in our collection), but these are from our donors (I am at a Blood Centre). We use something called glycigel. I'll be honest, although I am the Manager, I haven't a clue what this is! I'll find out from my colleague Alan Gray (who does all our rare screening) and then post again. By the way, I meant to say that I agree with every single thing Mabel says.

Anti-Peltz, I mean. Fingers quicker than brain syndrome.

Thanks pluto! Tee hee, but there is such an antibody; it's anti-Ku (anti-K5)!.....or anti-Pelts of course!!!!!!!!!!!!!!!!!!!!!!!!!!!

Only jesting!

Okay, okay, I put my hands up! I am a pedant! Yes, this is an informal setting and so let people use the terminology they want! The only thing I would say is, pluto, do you call anti-Kpb, anti-Kpb or anti-Rautenberg?!

Hi dawntr 50, I tried to give you some sort of answer, but, for some reason, it came out on the thread named "antibody ID", started by Johnny. I don't know why this was, but if you go there, you will be able to read my ramblings.

Over this side of the pond, we tend to use Monkey for M and Nuts for N. We also tend to use Apple for A and Orange for O, but quite often use Bertie for B. Strangely enough, in my lab we do use anti-big K for anti-K and anti-little k for anti-k (but woe betide antone who says anti-Kell or anti-Cellano!).

I forgot to say, incubation of the tests at various temperatures can also help to elucidate antibody specificities in some cases.

I'll give it a go, but I expect others will have more to say. The normal 10 to 12 cell panel should enable you to identify common mixtures of antibodies, such as, for example, anti-E+K, anti-D+C+E, etc, but it is always worthwhile having more than one panel available to you, so that you have extra cells to identify more difficult mixtures. It is also worthwhile using a papain-treated panel, as some antigens are destroyed by such treatment (M, N, Fy(a), Fy( and, usually, S and s). If, therefore, one or more antibodies in the mixture react by IAT with untreated cells, but not with papain-treated cells, then this is a guide that the antibody may be directed against one or more of the above antigens (although that short list is by no means comprehensive; there are many other antigens destroyed by such treatment, but the antibodies against these other antigens tend to be quite rare). The next thing you can try, if you have the reagents available, is to fully phenotype the patient's red cells, as the patient will be unable to produce an atypical alloantibody directed against an antigen they themselves express. This, of course, also assumes that the patient has not been transfused within the previous 3 months (approximately) and does not have a positive direct antiglobulin test. After this, it tends to become something of a lottery, depending upon the reagent sera and red cells that are available to you. If you have sufficient cells, you could try adsorbing the patient's serum/plasma with red cells of known phenotype, and then a) testing the adsorbed plasma with the full panel and/or eluting the antibody(ies) from the cells used to perform the adsorption, and performing an antibody identification on this eluate. Although I mentioned papain treatment of red cells above, there are many other enzymes that can be used, each of which (or, at least, most of which) "cut" amino acid residues at different places and will destroy different antigens. An extremely useful list can be found in the excellent book written by Marion Reid and Christine Lomas-Francis, "The Blood Group Antigen FactsBook", 2nd Edition, 2004, Academic Press, ISBN Number 0-12-586585-6, Library of Congress Catalog Number 2003102995. If you think you have a mixture of IgG and IgM antibodies, you could try treating the patient's serum/plasma with dithiothreitol (DTT), which breaks the J-chains holding together the IgM molecules, leaving you with just the active IgG molecules. You then perform an indirect antiglobulin test using a monospecific anti-IgG reagent. I suspect, however, that the average Hospital Blood Bank will not have all, or even many of these reagents available, and so I really would suggest that, if you get stuck, you send a (large) sample of the patient's blood to a Reference Laboratory; afterall, that is what they are there for! Moving on to dosage, certain antibodies (usually weak examples) will react more strongly, or even only, with red cells apparently expressing homozygosity for a particular antigen (remember, the antigen itself is not homozygous, it is only the gene governing the expression of the antigen that can be homozygous). Commonly, you will find that an anti-M wil react more strongly, or only, with M+N- cells. A similar pattern can often be seen with an anti-Fya and Fy(a+b-) red cells and with anti-Jka and Jk(a+b-) red cells, although, once again, this list is very far from comprehensive. It is well to be aware of the fact that "apparent homozygosity" means exactly what it says. Unless the donor of the red cells in the panel has been genotyped for particular blood group loci, a red cell typing as Fy(a+b-) can, for example, actually be FYA/FY (the FY gene is silent) and so the cell may react with the same strength as an Fy(a+b+) red cell with a particular anti-Fya (they are, after a fashion, heterozygous, or even hemizygous, for the FYA gene, and will express fewer Fy(a) antigen sites on the red cell surface than a genuine FYA/FYA [Fy(a+b-)] red cell). I hope this helps a little, but I am certain that others will be able to put the above in a simpler way, add to it, express different opinions, etc, but it is a start!

There is a very thorough and excellent article/review about this in Nature Biotechnology. Liu QP, Sulzenbacher G, Yuan H, Bennett EP, Pietz G, Saunders K, Spence J, Nudelman E, Levery SB, White T, Neveu JM, Lane WS, Bourne Y, Olsson ML, Henrissat B, Clausen H. Bacterial glycosidases for the production of universal re blood cells. Nature Biotechnology 2007, 25 (4): 454-464. It is not for the faint hearted, however. I had to read it about 27 times before I understood the abstract!!!!!!!!!!!!!!!!!!!!!