Mohamed Elrefaei, M.D., Ph.D., director of the Histocompatibility Laboratory at Mayo Clinic and assistant professor of laboratory medicine and pathology at Mayo Clinic College of Medicine and Science, discusses transplant immunology as it relates to heart transplantation, current human leukocyte antigen (HLA) testing and the goal of using non-HLA antibody identification in the future.
from July to July, so we're kind of halfway there. Um so today we have our guest speaker is Mohammed Rafi. Dr. Rafa. It's a graduate from Cairo University Medical School with PhD in immunology in Drexel University. School of Medicine is areas of interest include transplant immunology, history compatibility, infectious diseases out to immunity and allergies. And he will be talking about transplant immunology and HIV testing. Uh, this is a topic of very specific for transplant, but I know that there's some people in the talk. Some more Candies are not specifically work with this area of of our specialty. But doctoral refines gonna make it easy for all of us to understand it and be able to use it for the sake of our patients. So we know for delayed or whatever. Fight you can take over. Thank you for the, uh, thank you for the kind introduction. So, um, again, my name is Melinda. Hi. I'm the director from these two, compared to all the image of the Slavia at the Mayo Clinic in Florida. So what I what I'm going to try to do in this presentation today? Uh, spend the first. You know, few minutes and some few slides. Just having an overview with regard to transplant technology and h and I And, you know, what's the basic science information to help guide through the next part, which I will talk about the services that we provide to the transplant program specifically, you know, with regard to heart transplantation. And, you know, the H L. A lab provides three main services that we will discuss. One obviously is the h l. A typing, and the second is detection identification and following the development, uh, monitoring eventually, uh, anti buys, including donor specific antibodies. And then the third one, which is the cross matching, which we do by full cytology, which is what we, which is a test that we do, you know, at the time of transplant. Um, and the very end I will. I will discuss, um you know what we are? We are in the process of development at this point, which is to discuss a little bit about the role of non each other and Taiwan is and how we're going to hopefully be able to incorporate that in our algorithms of testing in the future. So briefly, um you know What is H L I, which is human? Looks like Auntie gin, which is synonymous with measure. This ticket, that ability complex and that term is used bounce human primates. H l A. I mean, these are collection of black of proteins. They are included by jeans that are present in the short arm from some six. Um, there are many, many nature Lee jeans, that's all. We were going to be significant, but we will focus on the ones that are, and more importantly, there over the years that there have been identified and there's thousands of the wheels. And that's why ancient Lee typing is really critical and trying to determine compatibility when it comes to donor selection. Um, in terms of the structure and the next couple of five that I will show you, but basically and you know, in a cherry molecules made out of two point of chains, Um, for the for the sake of this presentation and transplant, we're focusing too many categories H. L a antigens that are divided into Class one and class two, and we'll discuss differences in the structure. Uh, the importance of HIV is, of course, that they do play a key role in the development and function of both being the and the adaptive immune system. And, of course, in, you know, in rare primary immune deficiency diseases, where there is no h l a antigens, you end up with different versions of scared, um, severe combined immune the vest. So, um, here is a classic pictures. I'm sure all of you have seen, you know, different times. Well, here is the basic structure of the class one. Um, it should be molecule. It may are. Three Alpha hills is they're all they're bound together by themselves. Quite bonds. Uh, what's unique about the class one molecule? Can you guys see my cursor? They're moving it. Or is that just me? Okay, good. So the presence of the beta to my brother and that's a distinguishing feature for the classical molecules. What's important to note is that here is where the peptide binding region is, and that's where the actual EPA tops are present. And from a transparent perspective, that's kind of where the heterogeneity exists. And that's where we're actually, when we do typing, we don't type the whole, you know, the whole gene sequence. We are basically only typing this area here between one and two. And that's where the heterogeneity exists. Important point to remember. And we saw a critical perspective. Is who or what tells express pass for molecules. Uh, transfer molecule class form molecules are expressed from pretty much all nuclear cells that actually includes platelets. So, for, you know, a little perspective. You know, if you have a patient with a history of platelet transfusion that's considered a potential sensitizing event, Um, in contrast, red cells, if you contrast, sometimes fusions a pack themselves tend to not be the red cells do not express. Uh, that's normal. It's considered not nuclear, and they came to be last less satisfied in that sense. Now, compared to class one molecule past two molecules, um, the class two a chilly molecules, they actually they look a little different than they are both alpha and beta chains. They're they're bound by that outside bombs. There is no bigger to micro barbell and expressing the surface of themselves. The important difference here is in contrast to the class one molecules that are expressed and always data cells. The expression of H. L. A Class two is is limited to a group of cells knows as antigen presenting yourselves or professional presenting cells. And these are your B cells, your mom sites, your dendritic cells and your macrophages. And that's important for us when we are assessing, uh, the expression of these cells and using them in our testing. Uh, that's why it's important to understand or remember with, you know, which tells express which acted you. Mm. So this is an important slide to remember. So what is it that we are looking for, uh, again, within each one of these usually regions they are divided into, there are three different low side that express these, um hmm. I antigens. And these are the ones that we are using for typing. So when when you look at the typing of a particular patient transplant recipient or a donor, you want to be able to see what the what the results are within the class one region, there are three, uh, there the three Laws I H L, A, B and C, and then within the class, two reasons. There are three lows. I h l a, D r and D. Q and D. P. And within the within each one of those in the class two is further subdivided into a which is alpha and B, which is beta. So you will see these results on reports and they are also present in the EMR. That's that's an important slide to remember. So in terms of physiological function for H l. A. I mean they as I as I mentioned that they play a really important role in both the immediate and the immune system as an example, The H l A. Classical molecules are really are necessary or are crucial for entity presentation, for example, presenting foreign capitals. Or in that case, uh, is an example of the virus infected so far violent. It is not presented within the context of the H Class one molecule and that on the surface of the virus, in fact, itself and that, you know, allows the C d. T cell to bind through its T cell receptor and results as the city of T cell activation and become science toxic. And then it will attempt to, uh, to device this virus infected cells for the production of chemo games and by and Grand like um, in addition to that that's rule and that the that the immune system within classical molecules are also important for activation of NK cells, natural killer cells. And it has been shown that the binding of specific class for molecules as to to, uh, as we get to the receptors master kill themselves that depending on the combination, that can result in either activation or inhibition. So either tearing out or tearing off of NK cells and obviously in case they are, uh, on the forefront and they, uh, in terms of the dating uses. And in addition, the class two molecules play a very important role in presenting again foreign exogenous peptides. Anything that's like bacterial and that will result in the activation of the CD four T cell. And they felt like the helper of sites. And they will produce cider kinds like I 02 and interfering, Um, and that that kind of turns on the immune system. Overall, obviously, in very evil, all these things are happening something. So it's important to remember that the prince of each way is a good thing and the fact that that we are distinct from each other, or that there's all of Christianity that's good that insurers diversity of the immune response. Um, what's also unique about a Chile is that it has a unique inheritance pattern. So, you know, basic mandalorian genetics generally, and her genes are either considered dominant or recessive terms of their pattern. What happens? H. L. A. Is unique because it's actually needs our normal recessive. It's actually called Cool Donna, which means that each one each one of us has is 50% mom, 50% dad, biologically. And that's why when you look at typing, you will see that for each locus, there's always two numbers and that each number is coming from each parent, and there they are inherited equal. And as a result, in terms of the probability of the outcome, you'll see that there's always four different probabilities in terms of in terms of inheritance parents So and so now we'll talk specifically about you know what the other services that the laboratory provides in terms of, uh, for for transplant program was primary focus in the heart transplant for yeah, as I mentioned, there are three different tests categories, um, typing the antibody identification in the class, right? So as far as typing we have. Everything is done by my wife can dramatically just, you know, based days of serological kind of things are long gone. And, uh, you know, that's not, you know, something that's it's even allowed to be done anymore. Um, an important note here is that, uh, you know, from a clinical perspective, the sample source is important to remember. And I would say in 1990% of the time, we are going to be getting a blood draw top, for example, for, you know, for DNA isolation, however, there are situations where you know, just a simple yellow top would probably would not be would not be acceptable or with yield inaccurate results, especially depending on the main street patient. As an example where I had a case where, uh, potential patient was being worked out for transplant for solo and transplant. But the history of that that patient has received a bone marrow transplant previously. Therefore, we can no longer use a table because I would not you know, we're not, you know, the correct, actually typing um, of of the patients. So we have to go back and get a bucket as well. So that That's just an example of something to the, um there are different types of methods. The one that is used currently, which is the, uh, sequence specific, Uh, over when it is. I probably s S O. P, and and basically we we will get, you know, India isolated again, both including the yellow top. Um, and we use specific primers that comes on with these kids. And, you know, we take the PCR products, which is then the nature and the primers are being stimulated. They are then kind of hybridized in keeping with, uh, these beads that we are present will be expressed unique probes. These probes are basically they bind to the PCR product it compatible, and then you were able to by running it through fluorescent laser to determine depending on which be this software will be able to identify the H type. So it's a pretty robust methods that we can or we use. And this is our, you know, has been our standard method of typing for a long time. And however, we are actually in the process of switching from using sso p two using next generation sequencing and the reason why we want to do that is because it provides higher resolution. The I will explain in a minute why what that means. And that's the question. Significance, um, high resolution, meaning it be able to determine not just what the area is or what is the actual meal of that indigent. In addition, it's high throughput. It could be used in a multiple format that you will need because you know will provide service for all the different transplant programs. Therefore, the turnaround time is just going to hopefully be significant which workers So that we we have We currently have, you know, have validated and get credited for utilizing next generation sequencing. And you know, we're currently using it are Roma or stem cell program, um, service. And hopefully, you know, once once we are ready, we will be able to provide to our self program. Obviously, the heart programming, uh or, um, this is another important slide in terms of different creature when you're looking at reports in the EMR or or even looking at just results from each of the results, um, again back, you know, in 2000 and 10, there has been a standardization of the nuclear feature by which everything needs to appear on in the EMR on any agency report being provided. As you can see here that one first year is the locus Here's regime, which is a or B or C E O d r. And then the first set of numbers here represents your Auntie gin. So that's the type of, uh, you are to, For example, uh, it could be presented as two or in a two, um, and then the next numbers represents the appeals. And that's where the next generation sequence comes in because they will be able to provide you with be a wheel of that particular, uh, particular intelligence, and that becomes really important and determined. You know, whether there are specific antibodies, presidents or not, because for every antigens, there are many, many ideals, and we'll discuss that when we talk about that body is in a few minutes. So, um, so the step in the next set of numbers is that of the wheels, and that's what you you know, This this is so far. Here is what you will usually see all the reports and currently on the all the our bone marrow stem cell reports. We'll show the level typing, and I'm hoping in the very near future that will also be provided for our heart program. The next numbers. All this is not included in any of our reports because they're not significant because the 3rd 3rd numbers to represent synonymous mutations that occur. So changes in the tight sequence that is not translated into a change into an amino acid change. So no real change of protein sequence that has no effects, that why we don't show Africans doesn't have the significance. The last set of numbers here is for the sake of being complete, that these are actual changes that occur outside of the coding regions. So those are the reason we're finding of or potential path ties. Wrap it again, not significant, so we don't show it. The letter end stands for no, and that could be clinically significant so it could be found or could be included. It's not common that there will be a no a real, which means that the patient recipient or donor expresses how has a particular gene. However, because of certain sequence, mutations in the gene is not expressed, so it's not translated into a protein and therefore, you know, we do have to show that that's important to me to know, because if they're, you know, a particular donor, you know, has a gene for an anti for a particular group h l a. But that gene is not expressed that are important to know because the patient making antibodies against it Well, that that would, even though they're technically don't have specific. But then the genes that express so it will. It will then, you know, effect on that decision, because I will. I will suggest that they're not. They're not owners. They will have. These are these antibodies will not have any impact on on there. And the ability in the in the particular, um, the binding of this android is a genius by express. Mhm. So in terms of antibiotic testing, um, it's important to note that these these h l e antibodies are were not born with them, that these are not endogenous there and there are there are antibodies. So a newborn can, Unless there's some have transfer that they will not have any anybody. The sources for Rachel antagonized attempt to be as follows your previous transplant to his mismatch with the donor has mismanaged antigens that's most likely calling sensitization, obviously a multiple females. They with special actions, obviously internalized that can they can also be another call of sensitization. Obviously, you know transfusions, including toilets or or all the steps, uh, at the bottom here responses to, you know, infection viruses, bacteria. That lesson necessarily does not result in the primaries installation, compared to these top three words that would be considered primary sensitization. However, we have noticed that, as we are tracking our potential transfer recipients over time, histories of infection or and organizations has has been shown to be associated with an increase in antibody tigers, which usually tend to be transient. So basically, uh, non specific immune activation can will will result in increases these antibodies so quietly. It's important to note whether, you know, as the patient comes in for power for world draft, for example, to to know whether they have gotten centuries and they have received have received immunizations because that will affect our test results. Um, how this test is done, um, basis. It's a very simple beat essay, its, uh, geologic place and indirectly uniforms essay. It's a multiplex B test. Basically, we have these beads there are coded with different H L A antigens for either a class one or class two, and they are integrated with the patient serum. And then, of course, that a secondary antibody against the you know, whatever the anti antibodies are present in the patient serum. Anti human antibody binds the FC portion, and that secondary antibody tends to be fluorescent labeled. And then again, we will use a laser, you know, and and an instrument, uh, instruments includes, laser admits analyzed. Depending on the internal tags of these speeds, the software work and then determine what's the antibiotic specificity. Uh, for that is present in the patient's serum. And, of course, the higher the fluorescence, the more antibodies are present. And that's how it's being, uh, being reported as many fluorescence intensities is, the higher the fluorescence that that means that there's more antibiotics, uh, in terms of resulting. Uh, it's important to note you will see a term just e p r A. Which is your Cathy. 8% reacted eventually, and the C P r A means, you know, this is an example here where we have a c p. R A of plated too is highly subsidized. Has lots of antibodies that have been detected. C P R a. Is 91%. Now what does that mean? It means that then, in the donor pool and potential donors, all of these antigens will be present in about 91% of potential. So that means that the higher the c p r A. The harder it is, the more pitch the pitch is more sensitized that the most likely that they will be able to, uh, define the compatible bill. Now, why is this important again? This is studies that have been published, and there's many, many studies, but the you know the conclusion is the same. The parents of donor, specific and wise have been associated with negative impact on the heart transplant outcome in terms of survival. Uh, we obviously would like to be able to transplant patients with no donor specific antibodies, and they hope that they would stay that way if they develop the noble DS Staples transplant that will have it affecting their survival over time. Obviously transplanting cases with the presidents of donor specific antibodies to transplant has been shown to have the worst, the worst outcome over time. Um, in addition, another study we have shown in multiple people and this is kind of accepted is that you know, within the presence of these donor specific antibodies, uh, antibiotic is the Q costume in general, the human specific. And that's not just in the heart but also been shown repeated name kidney transplant has been associated with the most. One of the most prevalent, in addition, is the one that's associated with it was the worst outcome. So we when when we're models during patients, both sense when you know when I see that the patients starting to develop, um, donor specific antibodies, um, against the Q specific, especially, are the noble for transplant. That's something that that would be looking seriously, uh, in terms of mechanism. How these donor specific antibodies Why are they important terms of their ability to be injury? Obviously, the you know, the fact that you know the mechanism that will will define or or more along, um common or is the one that they are complement fixing. And I want to show the slide because I want to highlight That's not the only mechanism. So we asked primarily the H l A. You know, the donuts. Can antibodies bind into their age corresponding? Actually intelligence. And you know what? They are confident things that the results of constant activation and that can be, uh, hyperactive reaction and that, you know, and then you get C for this thing. I know it's, you know, I don't, but that's that's something we try to avoid because they can cause, uh, again, severely mentally, any reaction. But however, it's important to note that that is not the only mechanism by which, uh, yes, you can be getting autographs injury. And in addition, you know the binding of these domes incentivized to Chile. They can also result in through the through binding, and the rescue receptor tend to either bottle sites or no bills. You're getting cases. Uh, that can actually result in activation and increased human taxes and recruitment of these employment Ori cells to to the to the power grab. In addition, there is also the binding of these activities can result in increased production of protons or inside guys like I am eight, for example, all of these things our craft injured so antibodies and have more than one mechanism by which they can mediate our draft. I guess that's the message to have time to make. So, um, yeah, there there's a few potential and no points as far as how the associated with these, uh, antibiotic testing is being inherent Hazards. One is the timelines between our ability, you know, when the testing is actually diamond and the transplant actually clears. Obviously, we do try to test people. Uh, well, we do test people on the day of the transplant, obviously, because of limited on the turnaround time, the antibody results from that day will not be available pre transplant because of cold ischemia time. But we are relying primarily on the fact that we're testing patients on a regular basis that our current algorithms we can sum average 60 day. So hopefully you know, when we until, um, this these doors like that fire, uh, and moving forward the stress that we then have example that's less than 60 days old serum samples, smaller sizes, days old, and we can utilized to determine whether there are building our specific antibodies that are done to assess uh, Susan But you know, there is an inherent limitation, Justin, because again it's not done. It's not a real time. Every the next, uh, the next potential carry out their limitation. That we should we are aware of is the fact that there are weekend bodies that, you know, we know that they're there. But they are there. Love versus have recorded, uh, unacceptable. So we don't use them to avoid, uh, donors like him. And that's fine, because if we if we included every potential and that low level antibody will have to be severely limit the the development potential donors. However, one of the important things that we try to assess, um, what's part of that's part of what we do is look to see if there is multiple weekend bodies, because studies have repeatedly shown that the presence of the local we can devise does have another fact. You know, there might there might not be any one particular antibody at high enough levels to cause to cause automatic and this qualification of a particular donor. But then we have to look at this weekend device, determine whether the potential added effect could also have an impact. The third Uh, point. That's important is that even though I'm looking at all the chili antibodies, and even if there aren't any donor specific antibodies, that should not be, um, you know, that should should not be the end of the story, because many studies have shown that there are other antibodies or antibodies against other engines and just listening. A few here and some of these chicken have been shown to be our intelligence, and they do have an important role and immediate rejection and being associated with worse stratified outcome. And these are defined this one, h l e antibodies. And just here is examples. This is again different. Studies have shown that you know, patients that have developed, uh, antibody mediated rejection have had higher models, uh, antibiotics Myers by two most hard, terrifying, similarly patient to have cardiac telegraphic basketball apathy. They have also been shown to be associated with higher levels of either anti mice and and environmental antibodies detected and serum. And that's part of while and want to be able to validate by, uh, find the ability to detect these antibodies. In this year, all these patients and testing of our H. L antibodies. We we do that. We, you know, want to do money over time, mainly because we want to honor their patients post transplant. And that's part of the world care Correlating biopsies was antibody testing to determine whether there is any donor specific antibodies that will protect us. No transplants in addition to other there if there are lower growth hours are increasing. In addition, obviously, if there's going to be, uh, immune intervention rather is part of races or changes. Branson. That's the new monitor. The effect vehicle reduction treatments are developed. Uh, developed the new status overall location transfer? Yeah, um, the last services the cross matching, um, all of our car inspections are done by float psychology. I can't unless you can try to get it open. I can't get it open. Yes. Good. Yeah. I'm sorry. Can you hear me? Yes. Uh, we're okay. So with regard to cross matching, which is the final testing category that we use, Um, so all the prospectors are done by four psychology tree, and these are done perspectively at the day of the transplant. However, because of this, you know how long it takes for the test to be performed. Usually, you know, we cannot provide test results prior to transparent action starting. But you know it has done at least started pre transferred over them. Transform using patients here ourselves. And as you can see here, we are using the T cells and the B cells for protection and post class one and class two. Because, as I mentioned at the beginning, which sells Express, which obviously will be a lot of controls in order to assess the president's force pilot of becoming another application and has received different conditioning regiments that could have an effect on these tests votes and briefly again. As I said in order to determine, you know, we used the lymphocytes, they're isolated from Mexico's. There are stains with either C 19 or 63. The 19 is a Mark European different pen piece of markers. Industries are the soma, and that's what we used to, uh, determining whether it's a cost one or class two. If you see in the in the EMR, you'll see results for cross match. You always see two results was for cost autonomous for past two because because that's how the test is run now, Finally, in terms of future testing. Um, we are. You know, as I mentioned in the previous slides, then on a chilly antibody, testing is really important field that's evolving. And, you know, we currently have a test, and we are using it in the research field. But I am hopeful that if we will be able to be able to provide this just for chemical use, um, this year, that's my objective. Um, there are so many different potential analyze and antigens to be assessed. And the current, you know, test that we're looking at There is up to 30 different animations and quite frankly, some of these I don't actually even Yeah, I know what they're what They're objectives. But some of them have been identified to the SEC with different transplant organs, and that's kind of part of my research. And I am hopeful that as a for those like generation secrets, and we are going to be able to provide, uh, this added testing, um, your algorithm, um, this is my email address. If anybody has any question, please feel free to email me anybody who was a There's a particular tests that you want to see or about 2000 anything. Whether come by to my office and our email. Um, any questions? I did put this slide because I was asked to do that. So that's in the back here for you guys. I'll leave that there. But if there's any questions, uh, please go ahead. Uh, well, thank you very much for that lecture. Definitely. It's a It's a good blood pressure for those who work with transplantations. But, you know, we have some people in the audience who are actually do not work directly with transplantations. Is there any clinical utility of the lab for non transplant purposes? So they guest or guests attendees are here. Is any reason for them to send patients directly to you for any other reason that is not transplant? No, I mean, again, we provide service for typing. Uh, you know, within, um, you know, we're provide typing or, you know, non h l E. For for non transfer purposes, uh, usually get patients who want to be tight because they're, you know, HIV claim, for example, that are gonna be starting on the back in there, and they want to be testing them for, you know, for whether they express p 57 1 we're also looking at, uh, you know, we're typing in for for disease associations. Um, you know, again, we got a lot of these requisitions and don't instead of the heart related, but, you know, disease associations, we also test them for drug hypersensitivity associations. Um, so these are the primary tests that will perform as requested. Okay, thank you for that. The other question that I had and then, uh, I'll let other people kind of came in. Um, so every now and then we'll have patients who are, unfortunately extremely sensitized, and, uh, they have many unacceptable antigens. So, um, and then we started to know When we talk back to our referring physicians, they'll say, Well, what can we do about it? So then we start talking about as a civilization protocols or transplant team relief. Um, we have not been focused on on that topic, but many other institutions, um, in the country are how effective are these desensitization protocols? And, um, you know, is there any particular patient, uh, level of sensitization that we can say? Well, this this patient's gonna do better than this other mhm. That's that's a very good question. And I have worked in this, you know, as form of my previous position when I was in Seattle. So I'm sure you you know about this family Georgian protocols, and especially in the kidney programs, they tend to be more aggressive. We here at Male Florida tend to be pretty conservative until handover selection we don't use. You know, we're We don't try to transplant patients that are highly sensitized with high C p r a. With antibiotics. But, uh, and I have worked and other programs have published that, you know, they can attempt desensitization, and they can also use, you know, they they have no transplants of high risk patients that have doing more specific antibodies as long as they can lower you these antibody levels to make people 5000. Obviously, you have to have a more aggressive posters by monitoring, in addition to higher immune depression for a cool. So yes, that actually has been done because the options to some of these houses by patients tend to become more and more limited, and you have to start taking higher risks if you know, because the alternative is going to be deaf. So to answer your second question, it was regard to limit. That would say, trying to desensitize to go all 5000. I would I personally have experience. And, you know, I'm trying to work with desensitization protocols that the responding to desensitization for class one tends to be much more robust than the class too. And unfortunately, as I mentioned, you know, development Que de esa is is definitely, definitely a bad sign. And they tend to also be very hard to desensitize posters. It requires more more rounds of problems, freeze the higher their higher, deceive the higher big alpine valleys and levels. They have to be more resistant. So uh so, yeah. I mean, I if I have to choose to desensitize the potential patient, I would say somebody was moderate level class one B s a s attempt to have the best response. Alright, great. Thank you very much for that. Uh, any questions from the audience? Yeah. Mhm. All right. Um mo, can you put back the slide on the CMI? So that way people can take a final look and, uh uh huh. Yes. Um all right. Thank you. I think I put the slides in Med Hub. I was asked to do that, so