a United States Army Medical Department continuing education program. Hist o compatibility testing a panel discussion with dr braun doctor cell dr gary boy and dr species moderated by Roberti. Lord in lieutenant Colonel U. S. Air Force Medical corps assistant chief reno service Wilford Hall U. S. Air Force Medical Center Lackland Air Force Base. We have several good questions that have been sent up here and having quickly glanced at them. I don't have the answers to all of them will begin by. Although this wasn't specifically directed all direct this to dr braun dr brown. Would you use a one haplotype living related donor with a mildly abnormal M. L. C. A mildly abnormal mlc. Well we've used a lot of one haplotype identical uh transplants living related. I still feel that the there are significant advantages in using living related donors and looking at serial renal blood flows in these combinations. It usually turns out that if you use a living related Sibling even mismatched that you've got enough of a renal blood flow buffer. That even if they go through an annual rejection and we've seen two of these you can get them through with a functioning graph. Pulling them out with serum creatinine of less than two and still have about 400 mils of blood flow left to work on. So they can tolerate a serious rejection much better than a cadaver graft will where you might start out with 400 mils of blood flow. And after they get belted with a serious rejection they've got 200 their creatinine is 10 and you can't keep them off dialysis. So I think there's enough of a physiologic buffer in there that you can tolerate a serious rejection in a single haplotype sibling transplant that you can't tolerate in a cadaver graft. Now that's not a an immunological answer or based on typing, which is what the question was aimed at. But I think it's a practical approach to these. And we've looked at some of our living related graphs now who have been functioning for 10 years and they are mismatched, living related in some cases and unmatched cadavers so that they certainly have the potential for long term survival. And the fact of the matter is that we still don't have ways of evaluating the ability of a recipient to tolerate a graft. We still are unsure really of what the significance of blocking factors are beforehand and we still don't know how to evaluate the presence or absence of suppressor cells in these situations. But I certainly would preferentially go ahead with a living related single appetite mismatched sibling transplant from a young age group in preference to a cadaver waiting list. There are two questions that uh are quite interesting. The first is, what is the current status of our liberal transfusion regimen to identify non and or hyper responders or I think what they mean is people who do not form antibodies when sensitized versus people who form a lot of antibodies are sensitized and a very similar question is do you think pre sensitization might be useful because it would identify people who are quote hyper responders and should be and should transfusions uh, and I presume they mean transfusions of whole blood be restarted? Well, I'll ask several people on the panel to comment on that. And I'll begin with myself. We have had several instances in our own transplant program where people did not have identifiable or known or at least willing living related individuals uh for purposes of giving them a kidney. And these people have been put on the cadaver list and over a period of time have received transfusions in the course of a chronic dialysis regimen. And then lo and behold up comes a missing brother or absent father who indeed suddenly has realized that a transplant for the particular patient in hand would be useful and is willing to do it. And we find that we've sensitized this individual to the living related person. So you've eliminated that person and from potentially being transplanted with a living related graft, which is far superior in my mind under any circumstances than any cadaver graft. So, for that reason alone, I don't feel we should liberally transfuse anybody in the fact, even if they are responders, we may at least prevent them from being sensitized against a given individual who they're related to whether or not it's useful to know if someone has the capacity to form antibodies or not to form antibody is as I tried to indicate when I in my talk a matter of opinion and clearly not settled I think that if someone has the capacity to form antibodies and they've never been sensitized the capacity of form and a body is obviously still there when they are first sensitized say by a kidney graft. And it may well indeed although not clearly shown it may well indeed make this individual less likely to have a graft survival compared to someone who doesn't have this capacity. It may indeed be possible that if this individual response is a responder or forms antibody that he would be the best candidate for a for energy and match craft or a good energy match graft versus someone who is not a responder because there's certain individuals who feel that if someone is not one who forms antibody and if these antibodies are H. L. A. Specific which by definition of the H. L. A. Antigens they are. Because the H. L. A. Antigens have been defined by people's reactive reactivity to these antibodies. Therefore that knowing that someone's responder would then make him a candidate to wait for a better graft versus someone who isn't a responder should be transfused that transplanted at the first available organ that comes along and to disregard typing or H. L. A. Matching in these people. I don't think that this has been well established and agreed upon. I think there's arguments for both sides which I may be in a very confused way trying to present to you know I don't feel we should start transfusing people until there's convincing evidence that knowing whether someone forms antibody is worthwhile. Dr. Braun, What might be your opinion on this 1? I agree with you completely. I don't think it's approved at all that you can establish acceptable recipients by attempts to sensitize them in some unknown fashion and it's very uncertain as to whom you should sensitize with what sort of antigens in order to evoke this benevolent situation of acceptability of all kinds of graphs. And until we know that I think the greater likelihood is one of harm and risk to the patient of advantage. I think there's some evidence now that there's a way to compromise to find out what sort of delayed hypersensitivity response an individual has without using uh Lucas cider agent lee antigens. And I'm thinking of the work of mel Williams who did D. N. C. B. A. Coral Benzene delayed hypersensitivity testing on pre transplant patients who are on the waiting list. And if I remember his figures right he found that those individuals who gave a delayed hypersensitivity response to dynamic or Benzene, of them rejected their transplant for as the group that did not show a skin response. Only 27% rejected their transplant interestingly enough. He used only the cutaneous hypersensitivity method. Others have shown that if you look at the in vitro and in stimulation you can detect some individuals who really do have a response to D. N. C. B. That does not show up in the skin test. In other words, perhaps if he had looked at the in vitro response to the 26% of the D. N. C. B. Negatives who rejected the skin grafts, you might have found that some of those actually were DNC responders who were not being identified. So no one knows what the actual molecular analogy is between D. N. C. B. And the H. L. A. Sensitization. But it seems to be an interesting phenomenon and several people have reported. So I guess the answer that I'm giving is that I think we should look into other antigen stimulation responses rather than a sensitization so that we can keep our options open for transplant and not develop hyper acute rejection. Do you have any comments? Yes. The same sort of situation holds in the studies of Coulson and Shumway who have shown that some patients are poor responders in Mlc. Probably based on some sort of circulating inhibitory substance in their serum and in fact this whole discussion really should be aimed at why an individual is not responding. Is it a genetically inherited ability to respond? Is it a acquired inhibitor of some sort? Is it a product of the disease? It could be related to the stage or of disease or the quality of the clinical treatment regimen for that patient. I think those things needed to find and I would agree with dr braun that willy nilly giving blood transfusions as a way to see whether or not someone will respond is an extremely risky way to measure this as yet very poorly understood phenomenon. I must say that in a study that we're doing with first with Dr Belser non with dr salvatore at the university of California in san Francisco were carefully analyzed. 100 patients were actually studying and lymphocyte serum treatment. About 37 of those patients had pre for and antibodies obviously not to the cadaver donor because that would provide a positive cross match and would be excluded but 37% had antibodies against some other H. L. A antigens. And in that series we see no difference between those who had made antibodies and those that did not since you've brought that up. We have a question here for dr brian. And I'll have you comment out to Dr Dr brian, how do you rationalize the disparity and transplant results between institutions regarding preformed antibodies? Mhm. Well there's a whole list of things there, many of them technical um one that depends on a complete analysis of all available positive syrah from the recipient and this is getting to be a horrendous cataloging and library storage type of problem where sometimes in our patients who look presumptively negative on a cross match. We will have to haul out as many as 10 or 12 old syrah in order to actually confirm this many times, we found that patients who are being held on satellite programs elsewhere, who are being screened by another laboratory if they come to us without those serum specimens will have been shown to have spikes of antibody which would give positive reactions with prospective donors with which we were going to transplant them for a kidney. So it one is you must have available to you a tremendous library of syrah on any recipient on any potential recipient, cadaver waiting list. Secondly, you should do all your tests with delusions because undiluted there are some anti complementary factors that interfere. Uh thirdly, you should use some tests of increased sensitivity such as dr Lorton described. And dr deer avoid described with the detection of sensitization either by serological methods or by cellular defined methods. Uh fourth, there's also the possibility that in some individuals and we saw this happen on the table and reported it in transfusion a couple of years ago, patients who may have preformed antibody not directed against the kidney donor may have antibody reacted reacted against lymphocytes in the blood transfusion given at the time of transplant. And we saw a daughter hyper acutely reject her mother's kidney, even though the cross match against the mother was repeatedly negative before and after transplantation. But her white cell antibodies reacted with a unit of two of the three units of blood that she was given at the time of surgery. And because there was a hemorrhagic problem, one unit happened to be given as whole blood with abundant lymphocytes and she hyper acutely rejected on the table. Uh The only antibody that we could find reactive in this situation was the white cell antibody reactive against blood donors, not the kidney donor. This is another uncommon, rare but still uh remote possibility. The fifth reason I would say there's differences between reporting centers and this doesn't hold for the boston group because that's relatively a concise group. But in accumulation of data from uh by dr tara sake. He's relying on the heterogeneity of testing methodologies existing in probably 80 some laboratories, many of whom do or do not adhere to many of the criteria that we discussed this morning. And that makes for a very unstable type of reporting in my estimation, doctor. So you got any comments here's a question which is directed to no one in particular and that is what do biopsies look like And Group three or people who have excessive amounts of antibodies or strong responders as a cause of rejection is its cellular versus funeral. Uh I'm not aware of any published data that delineates this. It's been clearly shown that in individuals who have a chronic vascular rejection that this is humanly mediated, which was shown by the group of Emma at MGH we have seen individuals with high levels of antibody with both acute cellular as well as evidence for human rejection manifested as uh proliferation of their cells and uh also by immunosuppressants. We've seen immunoglobulins on the vessel walls which may or may not be uh significant. Clearly the ones who have hyper acute rejections. Its characteristic of a a policy cellular infiltrate of the interstitial man. Why would anyone else care to comment regarding that question? I'm not aware that these people classically have au Meral pathologic change or a cellular pathologic change. Uh There's a question here that's of interest which I'll throw up into any volunteer on the panel. What is the clinical significance of the two a.m. L. C. And the use of the recipient serum in the test system? Mhm. Any volunteers? The same study that we're undertaking with Belcher's group in SAn Francisco is the location of the first report of the significance of the two a.m. L. See that as we do at two a.m. L. C. In the macro system and you get a stimulation index of greater than eight. That is the response between the two is eight times the control responses that this correlates with rejection. This work was done I think about two years ago was considered highly significant and is highly significant when you look at it statistically data was reviewed carefully by Dr Van Rood who agreed with the significance in looking at the last 100 cases where we've been where our statisticians have been handling all of the data on the case including the M. L. C. S. You know the significance becomes less obvious. I'd like to comment that they had most of their data. That was uh that could be at least the stuff that was published two years ago. Most of the data was that was statistically significant and large enough numbers to calculate in a statistical way we're on the cadaver donors. And indeed it did show that uh they however uh in the living related had only three individuals who had a two way stimulation ratio greater than 1-8. And on the basis of this they felt that this was also significant. I I think that that's very weak. I personally would not refuse to do a living related transplants in individual that had a greater than one day stimulation on two a.m. L. C. Anyone else I'd just like to expand a little further to. And that is that the two a.m. L. C. S. That are being done are being done by a macro mlc system. It's interesting that we moved to a micro mlc system. This correlation apparently doesn't hold at all. Even in Kent Cochran's hands. The fellow who first reported this and also even at the time he first reported this when he tried to do one way ml CS both ways that didn't correlate making an extremely confusing picture. And so I would think that to M. L. C. Sees for decisions about transplantation should be one of those items to be held awaiting further evidence. Question for dr gary boy. We're in the experiments. You sided with the enhanced animals challenged with skin grafts at intervals following the successful kidney graft. And if not what would you predict would happen in the experiments that I showed this morning? These were passively enhanced animals. These animals were not subsequently challenge the skin graft. However, in animals who enhanced by the active process these animals became one and two year survivals survivors. Those animals did get skin grafts and those skin grafts were accepted very well. A question directed to me, the studies of the pelts and Tara sake have found that only about 50% of the transfused patients developed preformed antibodies. In contrast the data from Perkins. Uh I think that This is a variable percentage in various centers and it probably relates to the number of transfusions that people have had and uh the time in catching these individuals following their sensitization. Uh the ones who were greater than 50% were numbers the 85% figure where people would receive greater than 20 transfusions on dialysis. Which is not a common practice in in my experience in talking with others so that I think the amount of exposure is going to determine the percentage of your population where you'll see this. There's another question which is directed to me and that is really Lucas pierce and Williams have incriminated subside. A toxic levels of antibody determined by standard tests as an important factor in graft rejection. Do you believe their results and should their techniques be incorporated in the pre transplant cross match? I myself am not familiar with the sub toxic levels of antibody determination that is being referred to as someone else here on the panel with. I don't know if they're referring to the immunity tests on kidney cells. Uh when Williams was working in hume's laboratory and the group down there was evaluating the sub toxic levels of antibody. They did show that by the immune adherence technique which was number five on dr bronze rating scale that you tested kidney cells from the donor with serum from the recipient. By this technique, they have apparently could pick up more sensitively the presence of antibodies than they could by lymphocytes toxicity. Which is not too surprising because lymphocytes toxicity is not a very sensitive assay. There are very few human serially diluted more than a couple of times. Will still kill human lymphocytes which is distinctly unlike any other side of toxicity system that I know of in biology. So that almost any other technique should be more sensitive in picking up these antibodies than the one that we're using enough. If the question is referring to the the late cross match and the L. G cross match and the anti g mediated cross match in my mind. There's no sufficient data to say that these uh techniques are are clinically significant as far as showing a greater or lesser survival in patients who have been transplanted with these being negative or positive. With the individual who asked this question. Care to comment on Zoltan Lucas has published on subliminal sensitization. Not detectable by standard sido toxicity but the detectable by more sophisticated techniques which employ variants of anti endothelial testing and that's what's really has done. And they've both shown uh people in the retrospective work primarily the reject ear's of their graphs. Uh The syrah from patients who have rejected transplants. Let's start over the pre transplant syrah examined retrospectively uh and patients who have rejected grass were found to be positive by these augmented side a toxic techniques or primarily by using a different target cell. Either kidney cell in the theater or something else. And that's what the question is really intended to get at. The problem with these techniques is preparation of the material or the length of the test required, which we're talking about 68 or 10 hours of cross match technique instead of three or four hours. As with the standard techniques for five hours. With standard techniques of course, Belser suggests, then explains that the fact that his patients do just as well who have antibody as those who don't. Is that his technique for cross matching is better And that's the framework from which the question was intended. The test you're referring to against what ophelia cells and kidney cells. Are these a sido toxic test uh, side of toxicity against kidney cells and then Williams published on immune adherence and Cirelli was against was anti endothelial antibody. I've forgotten what Lucas target cells. Yes, we've been looking at that using lymphocyte blasts with uh Peroni and scripts group and it is true you can pick up a toxic antibodies more sensitively. As I said before, I think you do it by almost any technique because the human lymphocyte is a very resistant cell. The question is, do they correlate with success? And that is a little harder to be sure of. I think all of these techniques should be used in a prospective study. We did a dr philo when he was in our laboratory before he went to University of Indiana, did a very nice study where he pre sensitized dogs and he found that even when he could detect sido toxic antibody at low levels there was no permanent rejection and no permanent loss of kidneys. It took a certain way level of antibody response in order to have permanent damage to kidneys. So that you would question just a little bit if the psycho toxic levels are so low that you can't detect them by our present techniques. Just how much significance is there, really? And when you look at the great variability and clinical results. It's very hard to correlate those kinds of data. I think you're getting at the question that I really wanted you to get at. And that's uh, is there are we talking about too sensitive techniques or false positive cross match is in a sense, in a sense of whether they're clinically significant. Because if you take it to continuing extremes, you will get to the point where no one can be transplanted because they have some kind of positive reaction to one of these ultra sensitive tests. I think that's a very valid point. I mean, what you're saying is ultimately we'll be doing nothing but identical twins. Yeah. Dr gary boy, your test of of self sensitivity Have received a great amount of enthusiasm. How do you feel as far as their significance in renal transplantation is concerned at the present time? Well, at the present time, I think it's still a two. It needs a lot of work. We really haven't done a large enough series of prospective patients to say what the clinical significance is. When we first did this series of tests up in Boston two years. So now we ask about 25 patients prior to transplantation at that time, five patients had a positive LPC reaction prior to transplantation. Three of them lost their kidney in the next two months was a very small number of patients to make any any conclusion of. And since I've been down here in San Antonio, we've also looked at another 20 five Patients and four or five of them again have also been positive and two or three of them have also lost a kidney in a very short period of time. But these survival results are indeed identical to the survival results. Most patients who did not have the positive prior to transplant. So I can't say that this group has a greater risk. I think it's of note that several instances now individuals who are H. L. A identical siblings who are M. L. C. Negative have been found to have sensitivity or pre sensitization to each other. And this has primarily been in patients with a plastic anemia who have received transfusions from H. L. A, identical sibs. But in these instances they've been able to find that they can develop a positive LMC or cell mediated side uh license test as well as an antibody induced cell cell mediated psycho toxicity. And this would imply therefore that the sensitizing uh an agent or agent or inheritance of the sensitizing agent is one which is not uh inherited on the chromosome or a wheel of the H. L. A. System. And since the renal survival and H. L. A identical sibs is pretty uniform throughout the world as being certainly greater than 90%. And excluding technical failures, certainly greater than 95% would imply that at least in certain instances pre sensitization is detected by the L. M. C. Or the A. I. L. M. C. R. R. Sensations that are perhaps not significant with regards to kidney transplant. They may well indeed however have a great significance in bell marrow transplants. And I'll ask dr sell his comments on that. Well. We have only done about 11 or 12 bone marrow transplants and we've looked for these reactions in those patients and in our identical matched but non identical, otherwise not identical. Sibs we've not seen these kinds of reactions but I've been very intrigued by a recent as yet unpublished report from Bruce comida which showed that there are killer lymphocytes present in the marrow of sibs directed against other sibs in the family without pre sensitization and being genetically inherited apparently through chromosomes other than those that handle the mlc locus. And uh this kind of genetically occurring spontaneously occurring CML reaction may well have tremendous significance in the bone marrow transplant situation. We've only recently learned of this and now we and others thomas. I think everyone is now looking for those reactions because they may be extremely important in graft survival. As you know, fully 25% of a plastic anemic patients will reject their graphs despite the fact that they're mlc negative and perfectly identical at the H. L. A. Locust. 25% will reject their graphs. Hist o compatibility testing a panel discussion with dr braun, doctor Cell Dr Gary boy and Dr species was moderated by Roberti Lord in Lieutenant Colonel U. S. Air Force M. C. Assistant Chief reno service. Wilford Hall U. S. Air Force Medical Center, Lackland Air Force Base and produced through the mobile facilities of the television division, Academy of Health Sciences, United States Army, Fort SAm Houston texas.