Malcolm Needs ☆ Posted July 7, 2010 Share Posted July 7, 2010 Thank You Malcom,I always worried about dilution factor. I mean we could potentially dilute the underlying allo, and therefore get a false negative? I currently recommend adsorptions with PEG, LISS, or WARM but not more than 4x, and still worry about missing something.We have performed multiple control testing over the years with known anitbodies present (weak ones of course), just to see about diluting out antibodies, and we have shown that, in fact, this does not happen.:D:D:D:D Link to comment Share on other sites More sharing options...
AP44924 Posted July 7, 2010 Share Posted July 7, 2010 Thank You. I feel like I was lost, but oe found. Thanks again. Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted July 7, 2010 Share Posted July 7, 2010 Thank You. I feel like I was lost, but oe found. Thanks again.Sounds like a line from a Rod Stewart song!!!!!!!!!!!!!!!!!!!!!!!!!!:D:D:D:D Link to comment Share on other sites More sharing options...
jcdayaz Posted July 8, 2010 Share Posted July 8, 2010 We have performed multiple control testing over the years with known anitbodies present (weak ones of course), just to see about diluting out antibodies, and we have shown that, in fact, this does not happen.:D:D:D:DWhen I worked at my reference lab, we made our own red cell stroma to do adsorptions (absorptions--I always get the grammar wrong). We would do two different ad/absorptions and based on what the stroma was positive/negative for we could rule out/in whatever (pretty much) we needed to. It worked really well. I wish I still had the "recipe" for making the stroma. Is there perhaps anyone out there who has such a "recipe"? How do other people do ab/adsorptions? We have tried the WARM (Ortho, Immucor, don't remember just now) Auto Absorption method and I find it lacking...in several areas. Link to comment Share on other sites More sharing options...
adiescast Posted July 16, 2010 Share Posted July 16, 2010 When I worked at my reference lab, we made our own red cell stroma to do adsorptions (absorptions--I always get the grammar wrong). We would do two different ad/absorptions and based on what the stroma was positive/negative for we could rule out/in whatever (pretty much) we needed to. It worked really well. I wish I still had the "recipe" for making the stroma. Is there perhaps anyone out there who has such a "recipe"? How do other people do ab/adsorptions? We have tried the WARM (Ortho, Immucor, don't remember just now) Auto Absorption method and I find it lacking...in several areas.I have a recipe somewhere for making stroma from a donor unit. Unfortunately, it is not in electronic format, so I can't share it at the moment. Lately we have been saving up reagent screening cells and doing PEG adsorptions with them instead. Preparing stroma from 3 units of red cells was a day and a half chore that I really don't have time for right now. Link to comment Share on other sites More sharing options...
Philip Thornton Posted July 25, 2010 Share Posted July 25, 2010 GREAT POST!! You explained that very sufficiently. Link to comment Share on other sites More sharing options...
adiescast Posted July 26, 2010 Share Posted July 26, 2010 Here is the quick and dirty for the stroma preparation procedure:We used three units of red cells (R1R1, R2R2, rr; one homozygous for Jka, one for Jkb, one for S, one for s; not more than one unit positive for K). Each unit was treated with 10 cc of 1% ficin (freshly prepared) in a waterbath for 15 minutes. Test after incubation with glycine soya to ensure 2+ reaction. Incubate longer if needed. Wash 3 times in a Cobe 2991 with no red cell override. Add 75 cc of digitonin, mix, and incubate in the processor for 5 to 15 minutes. Do three manual washes with superout set to 300cc. Add 75 cc of digitonin, mix, and incubate in the processor for 5 to 15 minutes. Wash until stroma is white (a minimum of 2 liters saline - watch the waste bag, it can't take this much!) Spin times at least 3 minutes gradually increase the superout as the hemolysis is reduced. Additional digitonin may be added if the stroma is not the desired whiteness. Be sure to wash thoroughly afterwards. Test the supernatant with red cells to see if all the digitonin was removed. The stroma may require extra washing before it is used if these cells hemolyze.After wash is completed, add about 200 cc saline. Aliquot into tubes and freeze at -70C Use within 6 months.Credit the staff at Florida's Blood Centers Reference Lab for this procedure. Bb_in_the_rain 1 Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted July 27, 2010 Share Posted July 27, 2010 Just as a matter of interest, if you are treating the red cells with ficin, why does it matter about the S and s antigens, which should be sensitive to such treatment?:confused::confused: Link to comment Share on other sites More sharing options...
adiescast Posted July 27, 2010 Share Posted July 27, 2010 They are not consistently affected by ficin, so you can't count on them being destroyed. Most of the time they are, but the one time you need them to be... Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted July 27, 2010 Share Posted July 27, 2010 They are not consistently affected by ficin, so you can't count on them being destroyed. Most of the time they are, but the one time you need them to be...Well, I know where you are coming from, but, in my experience, and that of better serologists than me (Jill Storrey, for one) the S and s antigens are denatured by ficin (and papain) in all cases, and the antibody actually detected with cells treated in such a fashion are actually examples of auto-anti-U that mimic anti-S or anti-s (usually anti-S, but not exclusively), but you have to look really closely to show this is the case.It's a bit like Rh auto-antibodies against Rh17 and Rh18 that tend to mimic auto-anti-E and auto-anti-e.:redface::redface: Link to comment Share on other sites More sharing options...
adiescast Posted July 27, 2010 Share Posted July 27, 2010 Far be it from me to argue with that kind of authority. I just report what I have observed. We don't have the resources to sort that out here. Do reagent anti-S and anti-s have auto anti-U in them? Also, if the patient sera we are adsorbing has auto-anti-U in it, wouldn't it be adsorbed out completely? U is enhanced by ficin and would be present on all of the adsorbing cells (since they are all tested out to be positive for S or s). Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted July 28, 2010 Share Posted July 28, 2010 Far be it from me to argue with that kind of authority. I just report what I have observed. We don't have the resources to sort that out here. Do reagent anti-S and anti-s have auto anti-U in them? Also, if the patient sera we are adsorbing has auto-anti-U in it, wouldn't it be adsorbed out completely? U is enhanced by ficin and would be present on all of the adsorbing cells (since they are all tested out to be positive for S or s).I am very sorry if I have upset you adiescast; that was the very least of my intentions. I do realise that such activities are the reserve of laboratories such as my own, who have more time and more reagents with which to play.With regard to reagent anti-S and anti-s sera having or not having an element of anti-U in them, the answer is a bit of a lemon. By that I mean that most monoclonal antibodies do actually have specificities in them that are "diluted out" for use, or that do not work at the particular pH at which the reagent is buffered.You may recall the famous case of Garratty, whena patient with acquired-B was detected by an anti-A that, at the pH used, detected the acquired-B antigen, and that after this case, the pH of most anti-A reagents had to be adjusted.Similarly, anti-D reagents have an antibody in them that reacts with an epitope that is common on both D+ red cells and I+ red cells, and that this is why we are cautioned to bring these reagents up to at least room temperature before they are used, in case a D- individual, with a particularly strong I antigen is mis-typed as D+.Finally, yes, the auto-anti-U would be adsorbed out (as would, of course, an alloanti-U), and so, in such cases, we have to "play" with an awful lot of very rare red cells (including S-s-U- and S-s-Uw red cells) to prove that the antibody is actually an auto-anti-U (which is why I agree that only certain laboratories have the time and reagents to muck about like this).Once again, I apologise profusely if I upset you in any way.Malcolm:redface::redface: Link to comment Share on other sites More sharing options...
Liz Posted July 28, 2010 Share Posted July 28, 2010 Thank you Adiescast and Malcolm, I have learnt a lot from your posts on this thread (and many others).Liz Link to comment Share on other sites More sharing options...
KAllen Posted July 28, 2010 Share Posted July 28, 2010 Thank you adiecast. I have been looking for a recipe for making stroma. Link to comment Share on other sites More sharing options...
adiescast Posted July 29, 2010 Share Posted July 29, 2010 I am very sorry if I have upset you adiescast; that was the very least of my intentions. I do realise that such activities are the reserve of laboratories such as my own, who have more time and more reagents with which to play.With regard to reagent anti-S and anti-s sera having or not having an element of anti-U in them, the answer is a bit of a lemon. By that I mean that most monoclonal antibodies do actually have specificities in them that are "diluted out" for use, or that do not work at the particular pH at which the reagent is buffered.You may recall the famous case of Garratty, whena patient with acquired-B was detected by an anti-A that, at the pH used, detected the acquired-B antigen, and that after this case, the pH of most anti-A reagents had to be adjusted.Similarly, anti-D reagents have an antibody in them that reacts with an epitope that is common on both D+ red cells and I+ red cells, and that this is why we are cautioned to bring these reagents up to at least room temperature before they are used, in case a D- individual, with a particularly strong I antigen is mis-typed as D+.Finally, yes, the auto-anti-U would be adsorbed out (as would, of course, an alloanti-U), and so, in such cases, we have to "play" with an awful lot of very rare red cells (including S-s-U- and S-s-Uw red cells) to prove that the antibody is actually an auto-anti-U (which is why I agree that only certain laboratories have the time and reagents to muck about like this).Once again, I apologise profusely if I upset you in any way.Malcolm:redface::redface:That's OK , Malcolm. I didn't intend to sound so snippy. It was actually intended more tongue in cheek (blast the inability to transmit nuances by written word! Should have gone advanced and put in an emoticon...). So when we do an allogeneic adsorption and we see anti-S/s-like differential reactivity, we have taken that to mean that the anti-S/s was not adsorbed out (which leads to the conclusion that the antigen is not completely destroyed by ficin treatment). Is this an incorrect assumption?Thanks for the information about anti-D reagent. I have not heard that before. Is that true for the murine reagent or only human source? Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted July 29, 2010 Share Posted July 29, 2010 That's OK , Malcolm. I didn't intend to sound so snippy. It was actually intended more tongue in cheek (blast the inability to transmit nuances by written word! Should have gone advanced and put in an emoticon...). So when we do an allogeneic adsorption and we see anti-S/s-like differential reactivity, we have taken that to mean that the anti-S/s was not adsorbed out (which leads to the conclusion that the antigen is not completely destroyed by ficin treatment). Is this an incorrect assumption?Thanks for the information about anti-D reagent. I have not heard that before. Is that true for the murine reagent or only human source? I'm so glad I didn't upset you and thanks for the explanation.Well, yes. If there is anti-S or anti-s activity after adsorption with the ficin-treated red cells, this is probably a genuine anti-S or anti-s (as the ficin-treated red cells would have degraded S or s antigen, and would not, therefore, adsorb out a genuine anti-S or anti-s). If, on the other hand, the apparent anti-S or anti-s is adsorbed out by the ficin-treated red cells, then the likelihood is that the anti-S or anti-s is an apparent specificity, but is actually an auto-anti-U, which would be taken out by the ficin-treated red cells. I think that is what I mean, anyway! With regard to the anti-D, I'm going to have to go back to the original paper to check this. I had a very quick look tonight, but I had to abandon the search to go and collect my son Harry from his Kids' Club. Tomorrow, I am in from about 06.30 to, at least, 17.00, because I am on-call (so no early leaving for me!) and I will have a proprer look amongst the detritus that makes up my office and get back to you. Malcolm :):) Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted July 30, 2010 Share Posted July 30, 2010 The original article doesn't seem to differentiate between human-derive and murine derived monoclonal antibodies.The reference is:Thorpe SJ, Ball, C, Fox B, Thompson KM, Thorpe R, Bristow A. Anti-D and anti-i activities are inseparable in V4-34-encoded monoclonal anti-D: the same framework 1 residues are required for both reactivities. Transfusion 2008; 48: 930-940.:):confused::) Link to comment Share on other sites More sharing options...
Liz Posted August 3, 2010 Share Posted August 3, 2010 Thank you Malcolm for the information and reference!Liz Link to comment Share on other sites More sharing options...
jeanne.wall Posted August 4, 2010 Share Posted August 4, 2010 Malcom - Please consider writing a text book - you could impact so many new blood bankers and help those of us that have been around for a while as well. Thanks so much for the excellent description!Jeanne Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted August 4, 2010 Share Posted August 4, 2010 Malcom - Please consider writing a text book - you could impact so many new blood bankers and help those of us that have been around for a while as well. Thanks so much for the excellent description!JeanneThank you for that Jeanne, but my level of knowledge is minute compared with those that have gone before me (Race and Sanger, Mollison, Geoff Daniels, Petter Issitt, Dave Anstee, Lawrie Petz and George Garratty, to name but a few).That having been said, however, the Institute of Biomedical Science in the UK has commissioned a series of primer books on all branches of Pathology with the Oxford University Press. I was asked to write a chapter entitled Human erythrocyte antigens and blood groups in the Transfusion and Transplantation Science book, but I was limited in the number of words I was able to write, and so it does not go into huge detail (to say the least).Unfortunately, the editor tells me that some of the other "authors" (to use the word in its loosest sense in my case) have let us down a bit, in that they have not written their bit, and the whole thing is now way behind schedule, and may even have to be abandoned.:(:( Link to comment Share on other sites More sharing options...
Liz Posted August 4, 2010 Share Posted August 4, 2010 There you go, that means you must write the book, or a series of books. Yes!!!!!!I agree with Jeanne.I disagree with you; your knowledge is far from minute. Waiting for the signed and dedicated first edition (flattering myself).Liz Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted August 4, 2010 Share Posted August 4, 2010 I'll see what I can do - but I'm a bit busy at the moment! Link to comment Share on other sites More sharing options...
Dr. Pepper Posted August 4, 2010 Share Posted August 4, 2010 Current AABB recommendations are to not autoadsorb if the patient has been transfused in the previous three months due to the possibility of adsorbing out alloantibody via the donor cells. I have often wondered if this potential danger has been somewhat overstated.First, although RBCs can last up to 110-120 days, this is not necessarily true of transfused cells. They may already be several weeks old before infusing, they are a mix of all ages when donated, and they can suffer various degrees of damage during storage. If the patient has a rip-roaring AIHA, they may only last as long as the patients' cells (5-10 days). The Tech Manual points does point out that it's difficult to determine how long transfused RBC remain in circulation in patients who need repeated transfusions. AIHA patients, however, should only be transfused if absolutely necessary to tide them over until steroid therapy kicks in. In any event, the three month window is actually shorter; we just don't know how short.Second, if there is alloantibody in the patient capable of harming donor cells, isn't that what's happening in the patient anyway? Whether at the tail end of a primary response or, more likely, in a secondary response, wouldn't the alloantibody be coating the surviving donor cells (that would adsorb out the alloantibody if autoadsorptions were attempted) and perhaps initiating extravascular hemolysis? It's like what we're cautioned about doing in vitro is happening in vivo anyway. In a patient producing increasing amounts of alloantibody, it would seem like the window of danger where there were enough cells left to remove the alloantibody might be relatively short.Third, allogeneic adsorptions are beyond the resources of many smaller blood banks. Adiescast, if you can get enough reagent RBC to do this, that's great, but I can't. Phenotyping sufficient donor units to find what you need is also very expensive and time-consuming. (The reference lab at our blood center has a nice database of phenotyped repeat donors; they will query it to find unit of the appropriate phenotype. If you have them do it, the charges for treating and typing the patient's cells and alloadsorbing mount very quickly.) My reference lab is 15 minutes away but a lot of people don't have that luxury.Fourth, auto or allo, they're already having what amounts to a transfusion reaction.Finally, how many patients actually have concurrant auto- and alloantibody? Issett and Anstee point out that rates of 30-40% might be inflated and that some of that activivity may be autoantibody posing as alloantibody, and that 10-20% rates may be more accurate. I can only recall one case out of perhaps the last 2 dozen we autoadsorbed where the patient had an underlying allo (anti-K). We all have different patient populations with varying degrees of risk.I am not advocating autoadsorbing everyone in disregard of their transfusion history. I endorse the current and prudent practice of autoadsorbing with no previous transfusion history and saving extra cells (sometimes easier said than done) for future autoadsorptions. (I also confess I see little danger in stretching the 3 day post-transfusion window for specimen usability by a day or two if need be, but that's a medical director decision.) And if you need to alloadsorb after that, you or your reference lab, by all means do so.But if you have a recently transfused DAT-positive patient admitted through the ER, in distress with a 3.5 hgb, and it's 8PM on a Friday night, and the reference lab is hours away, I think you can do a PEG autoadsorption and get them blood in an hour or two with a high degree of safety. Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted August 4, 2010 Share Posted August 4, 2010 I agree with a lot of what you say, although quite a few of our patients with auto-antibodies have got alloantibodies, particularly within the Rh Blood Group System. Many of these are quite elderly, and I suspect that they were given Rh mis-matched blood before giving Rh matched blood came into vogue. Link to comment Share on other sites More sharing options...
L106 Posted August 9, 2010 Share Posted August 9, 2010 Malcolm -I agree with Jeanne and Liz's comments, and I would get in line for a copy of a yet-to-be-written book by you!Donna Link to comment Share on other sites More sharing options...
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