← Blood, Sweat and Smears - A Machaon Diagnostics Podcast
Loading summary
Transcript49 lines
- [00:06]
A
Hello and welcome to Blood, Sweat and Smears, a podcast produced by Macheon Diagnostics. In this podcast series, we will be discussing thrombosis and hemostasis from the perspective of our host, benign hematologist and medical director of Macheon Diagnostics, Dr. Brad Lewis. Please remember to subscribe and leave a review. With that, I'll turn it over to Dr. Lewis. Brad, take it away.
- [00:31]
B
Hello everyone, this is Brad Lewis from Matrion Diagnostics. Welcome back to our podcast series on classical hematology, Blood, Sweat and Smears. Today, I have to say I'm tremendously looking forward to an interview with Robert Brodsky about a test that he and his lab have recently developed, the modified HAM test. You'll hear more about that in just a moment. You've heard on other podcasts about thrombotic microangiopathies and some of the difficulties in really trying to work towards the diagnosis, particularly of a complement mediated TMA with atypical hus. There is no reliable diagnostic test half the time. Maybe you can make the diagnosis using genetics. Occasionally other tests are used, but there's not been a great test. There's really a gap in our understanding of that. There's been a problem both in finding a good diagnostic test, especially one that might be useful in difficult situations like lupus or pregnancy. For example, we've been looking for a really good test for reliably monitoring the efficacy of complement or C5 blockade in some of these patients who occasionally begin to break through, maybe, and maybe it's some other disease beginning to rear its ugly head. And we would like to be able to predict failure, both to predict it before we stop therapy with eculizumab or altomiris for patients who have atypical hus, and also to be able to monitor them for possible impending failure after we stop. Nothing really great has been in the offing for that kind of work. The modified HEMS test looks as though it may be the answer to many of those questions and some others we'll talk about at the end of the podcast. So Dr. Brodsky could just start us off. Tell us, what is the modified HAMS test?
- [02:11]
C
So I'd like to draw the distinction between the modified HAM test and the newer assay we have, which is a bioluminescent modified HAM test. So the original assay we developed about 10 years or so ago, it was never widely adopted, even though it worked pretty well, mainly because it was very cumbersome and technically difficult to do. It had few cells, there were washes, it only gave one or two time points and the reproducibility of it was not fantastic. And you had to do it multiple times. The big advance here in the bioluminescent assay is we have cells that are bioluminescing constantly until they are starting to be metabolically killed by complement. The advantage of that is that we can take the cells and we can put them in a luminometer after adding patient serum. And there's no washes, so we don't have to worry about losing cell number. That can affect the assay. And the other thing it does is instead of giving one or two time points, this gives a continuous readout every five minutes. So it tends to be a much more robust assay. And we also made several different lines and we often refer to this assay as kind of like a complement biosensor, depending on the complement regulators we knock out. We have cells that are wild type, we have cells that are missing one molecule, like CD46, we have cells that are missing two, like 55 and 59, and then we have cells that are missing all three. So it's really been a big advance over the last year. And this was just recently published in Blood is in first edition.
- [04:01]
B
Could you talk a little bit about sort of how this mham, the bioluminescent modified HAMS test, or the biosensor assay that you have, how it would be used? When would you use an assay like this?
- [04:14]
C
Yeah, there's a lot of potential applications for this. The first and simplest one is to try and turn atypical hus, as you mentioned, or what we often call complement mediated tma, into a diagnosis of inclusion rather than one of exclusion. So as you alluded to, usually when we see a TMA, we get the Adams TS13 and we rule out TTP and we may get a Shiga toxin to rule out that. And then if those are both negative, you think about complement mediated tma, but you're never really sure. And you have to make a decision because these patients can lose their kidney or even their lives pretty quickly if you don't start appropriate therapy. Mutations you can send, but often you get variants that are unknown. You don't know whether these are really contributing to the disease. And it often takes days, weeks to get those results back. And you have the problem that 50% of patients with complement mediated TMA don't have rare variants in one of the complement regulatory genes. So the very simple list use would be a diagnostic assay. And I can imagine the day where, you know, you have a TMA and you, you might want to send off Adam's CS13 and this assay simultaneously. And again that way if your Adam CS13 say comes back, it 86%, your activity and biomham comes back and it shows a lot of cell kill that is consistent with atypical hus, you have your diagnosis. The other very nice thing this assay can do is we can add inhibitors into the serum to show that the complement inhibitor, e.g. iculizumab or rabilizumab, or you pick the complement inhibitor, actually blocks the complement mediated killing. So it gives you an almost an in vitro assay to suggest which complement inhibitor is working. The other thing that this can do is we can, by switching buffers, we can look at the alternative pathway in isolation. One of the things that this paper showed is that much to our surprise and much to the surprise of everyone else, that the stimulus for most cases of complement mediated TMA is coming from the classical pathway and it's polyclonal autoreactive IgM molecules. And this actually kind of makes sense and it explains why 50% of patients don't have mutations or autoantibodies. The mutations or autoantibodies are involved often in the amplification loop of the alternative pathway. It's not that they don't have any role in the disease. They lower the threshold, but they're not the driver, they're an adjunct and they lower your threshold for getting it. So you can have a really strong stimulus and no variance and still develop the disease. Or, or you can have a moderate stimulus with one or two variants and get the disease. So we can resolve whether or not those rare mutations, those rare variants that you get on your panel are really contributing to the pathogenesis of the disease by isolating the alternative pathway. Another potential use of this, and we need to study this more in a prospective manner, is that we've noticed that when you stop people on complement inhibitors with atypical HUs, some of them remain positive and some of them turn negative. So it may give insight into who you can discontinue the drug more safely in. It would make me feel a lot more comfortable stopping the drug in someone who doesn't still have a lot of activity in the pilumin, SNM, ham, as opposed to someone who still has a lot of kill. But that needs to be studied prospectively. There's a lot of ways that this can be used, and I'll mention one last one is monitoring therapy. So, for example, you know, some of these complement inhibitors there's really no good way to monitor the drug levels and this often comes up in TMAs. I often, I just got a referral for a patient who was treated. Their TMA is better, but they're still in renal failure. So what do you do? A, are they being blocked adequately? And B, assuming they are, when should you stop or should you continue with the complement inhibitors? So I think they can be useful in that manner too to show that you're actually blocking the drug. And we can monitor any complement inhibitor, whether it be a classical pathway inhibitor, a terminal pathway inhibitor or an alternative pathway inhibitor, so that, that goes out to diseases other than complement mediated TMAs. So some of the PNH patients who are on some of these alternative pathway inhibitors, there's no, really, there's no assays for that. We can monitor that cold agglutinin disease, if you're on sutinlumab, we can monitor that. So there's going to be a lot of uses for this assay.
- [09:40]
B
Have you looked at it at all in the post transplant TMA setting? There's already been some thought that it may actually be a classical pathway mediated process.
- [09:47]
C
Yeah, we are in the process of doing that and it's. We have studied about 12 or 14 patients. That's a very interesting group of disorders. And most of them are not complement driven. Some of them are, and we're trying to tease these apart, but many of them aren't. Many of these patients are just having severe endothelial injury and it's not really complement driven, but some of them are clearly complement driven. So we're trying to sort those out and I think we'll get greater insight into this.
- [10:28]
B
So this modified hamst, just to go back a little bit, is certainly a tremendous step up really in looking at the complement cascade. We've had assays prior to this where we could measure individual components of the complement cascade, even do functional assays, but only functional assays of a particular in vitro activity of individual components of the Cascade, the soluble C5B through 9. A soluble membrane attack complex assay measures an epiphenomena. It measures a byproduct of the activation of the cascade. And what you've got here is something that's really measuring the activity of the complement cascade itself in an in vitro environment, but it measures the activity of the cascade. So it's measuring something that really we haven't done before. I suppose maybe the Weissland assays does this a little bit, but there's really been nothing like this before.
- [11:18]
C
Well, you're absolutely right there. So none of those assays, including the soluble C5B9 measures complement activation on a cell. So soluble C5B9 is only fluid phase. Soluble C5B9 cannot land on a cell and can't insert in the cell and make a membrane attack complex. The membrane attack complex that lands in the cell usually has about 18 to 20 C9 molecules. Soluble C5B9 has one, maybe up to three C9 molecules and it's complexed with vitronectin or cluster in and it's only a fluid phase marker and it's very hard to even distinguish. We've tried with soluble C5B9 distinguishing between TTP and HUS. They're elevated at the same level in those diseases. You take patients with bad sepsis, graft versus host disease. A lot of these conditions are going to have elevated diable C5B9, but it doesn't tell you that you're getting insertion of the membrane attack complex on a cell leading to cell death.
- [12:35]
B
So in those settings or in other settings, do you think that it's more specific to have a positive modified HAMS test?
- [12:42]
C
Oh, absolutely, absolutely. Especially on the cell lines with a lot of regulators still on. So the cell line that seems to best distinguish it is the cell line where we just knock out CD46 and the cell still has CD55, CD59 and can still bind factor H.
- [13:05]
B
So how confident do you feel using this assay and setting? Somebody comes in and you're and you know, they were septic but now they're getting worse. When you thought they were going to be getting better. If you got back a modified HAM sets, you feel fairly comfortable saying this is a complement mediated tma, whether you call it AHAS or not.
- [13:22]
C
Yeah. So I think certainly we still need more samples. But yes, we feel pretty darn confident in this. If you have a patient that comes in with a TMA and they have a positive bioluminescent MHAM and you can block it with eculizumab or a C5 inhibitor or a C1S inhibitor or whatever. If you can do that in vitro, I'd be shocked if that patient doesn't respond to C5 inhibition.
- [13:55]
B
Fantastic. So that brings me to a couple of other issues. There's a small but significant false positive rate. It appears in the modified HAMS test in our controls. We found something like, I think, as did you, about 10% of patients come up positive who are, as far as we know, perfectly healthy. And the rate would suggest it's not a Real complement mediated process. What I've done is to say, you know, if it clinically looks like it's going to be a tma, that could be a complement mediated TMA and the M ham is positive, that's awfully good evidence that it really is. How do you approach that, that issue?
- [14:33]
C
So it's, it's not so much a false positive and we can really make it even more specific by using our alternative pathway assay with it. So it is true that if you take some younger healthy controls that they will be able to kill the CD46 knockout line almost to the extent that you see an atypical hus and again, I mean you see variation in the strength of this autoreactive IGM amongst healthy controls. However, the statement I made is this is true for most assays. They are as good as the clinical scenario that they're tested in. So if the pretest probability is high, such as a patient that walks in off the street with a TMA and they're positive and it's blocked by a C5 inhibitor, that patient's going to respond. This is a multi hit disease. A lot of these patients can be in remission and still have killing in a bioluminescent M ham, but there's a threshold effect. It's almost like a Starling curve where you flip people over into actually a tma. So those patients probably have a lower threshold for developing a TMA if they have another strong trigger, pancreatitis, an autoimmune disease, a big surgery, but they don't necessarily have it. But if you have a patient that comes in with a TMA and they're positive and they're blocked, that's going to be pretty powerful evidence that A, that's the diagnosis and B that they will respond to C5 inhibitors.
- [16:20]
B
Fantastic. Thank you. So what do you say to people where the patient comes in with one of the confusing presentations? They have a flare of lupus and now their lupus you think is coming under control, but they're developing a worsening TMA in that setting. Now I say the same thing in people who look like they might have HLH or even caps. And then in a moment I'll ask you about pregnancy. So in each of those settings, what, how do you use the M hand test?
- [16:46]
C
You know, again, this is still early, early days in this and for all of these we need prospective studies. I can tell you that most lupus patients, very few lupus patients will have a positive modified HAM test. I don't think any of them have had a positive now lupus patient with a tma. I haven't studied those. Maybe they do caps very positive. But we've reported that before and we understand why they're positive. We're going to have another paper coming out pretty soon showing pathophysiology of CAPS and how that relates to CR1 deficiency. But we still have to study some of these, these other diseases as to whether or not they're positive. Again, if they are and there's a TMA and they respond to a C5 inhibitor, you can be pretty darn sure that they're going to benefit from it. That would be my prediction.
- [17:43]
B
That's fantastic. Yeah, I've been in the position of saying to people, studies haven't been done yet, there's a lot of information we don't have. But really we have the patient and you've got to make a decision one way or another. And with the positive assay, it does give me the confidence to make the decision to start therapy.
- [17:58]
C
I mean, this was published last, last week, I think, or maybe yes, the.
- [18:03]
B
Week before last week.
- [18:04]
C
And the whole point of the assay was really to show that what we thought was the pathophysiology of this disease for 50 years was only partly right. It was actually mostly wrong. So that in and of itself is really important. The other thing that is very clear is that it is probably going to be a fantastic diagnostic assay for complement mediated TMAs and it's certainly going to be very helpful for monitoring drugs. The other conditions that may, you know, be positive, causing complement activation on cell surfaces, we need to study more. And these undifferentiated TMAs we definitely need to study more.
- [18:51]
B
So you actually already alluded to one of my other questions, which was what about using this assay as a way of moving some of these vuss that we see so many of to either positive or negative status.
- [19:02]
C
I think that's going to be very easy because. So, for example, some of these factor H mutations, when we just isolate the alternative pathway, we can see them turn positive. You don't notice them when you have all pathways open because the complement killing from the classical pathway is so strong. But by using a buffer that just has magnesium and no calcium, you isolate the alternative pathway. So you can tell whether that factor H variant, whether that CD46 variant, which you wouldn't expect because it's usually not in the serum, or whether the eye variant is actually contributing to limiting that amplification loop.
- [19:45]
B
You talked a little bit earlier about some of the work with caps. I don't know if you wanted to talk about that anymore. The role of this assay might be in patients coming in with anti phospholipid syndrome at all, or with with CAPS or with tma.
- [19:59]
C
Yeah, I can't talk too much about that yet. It's unpublished, but. But I think we're going to have an even simpler assay for CAPS that's just simply going to involve a simple flow cytometry assay and have the same.
- [20:14]
B
Sort of predictive value of who's going to do well, who's going to do badly.
- [20:19]
C
Exactly, yes.
- [20:20]
B
Very exciting. I think you've talked about most of what I really wanted to talk about here.
- [20:25]
C
You were about to ask me about pregnancy, which is a fascinating subject. One of the things that many people have noticed is that all of these complement driven disorders, pnh, cold agglutinin disease, atypical hus, caps, every one of them gets really bad in pregnancy and it's usually late second to third trimester and then after delivery it settles down. That's called the HELP syndrome. It almost exclusively occurs in the mid to late third trimester. And the treatment for it in most cases is delivery. So what's going on there? We have preliminary data, again, still not published, with this assay that shows that the autoreactive IgM increases in the third trimester of pregnancy and it goes back to normal shortly after delivery. Now why would it do that? Well, the reason is, I think there's a teleologic reason. And again, we're in the process of trying to prove this as well. But one of the things that you want to prepare the mom for and you know, to help the baby is for childbirth. The leading cause of death going back hundreds of years ago was bleeding. Second leading cause of death was infection. And we think this is a normal physiologic function of pregnancy where the autoreactive IGM is going to help control bacterial bacteremia much better than if the IgM levels are low and then after delivery it goes back to normal. And this is probably why you see PNH patients break through their eculizumab or their rabilizumab or whatever complement inhibitor they're on, usually in the late second, third trimester and goes back to normal a week or two after delivery. Same thing with the HELP syndrome. This is why patients get severe preeclampsia. Help in the third trimester and then we have to deliver the baby usually in three or four days. Things start to settle down. We're very interested in studying that. We also have a sense. And again, you're going to need to do very big epidemiologic studies to make any strong statements here. But it appears that younger people have much more autoreactive IgM than people as they age. And what role does that play in endothelial damage in the heart and in the blood vessels? What role does that play in protecting against skin cancer or malignancy? And, you know, so these are also things that we, we really need to study to understand, you know, the role of humoral immunity. We just have not had assays that could really give us answers like this until recently.
- [23:35]
B
So you've answered all my questions, really, about this assay. I think it's an incredibly exciting addition to our armamentarium. We finally do have something that lets us really begin to answer that question in some of these difficult situations like pregnancy or lupus or just the patient who has a number of things going on. And you're trying to ask, could there be a compliment mediated TMA also? And we have a way of asking it and even asking, does one of the complement blocking drugs affect this patient, at least in vitro? Very exciting. Very, very exciting. New assay should be available through mation in, I think, just a few weeks. We're very close to making this test publicly available. Just to toot the mation horn, you mentioned that genetics often isn't available for weeks. Just to say it. We sequence all the genes that are currently known to be relevant to a typical HUS with a two day turnaround time. But like you say, the problem with using that diagnostically is it's only positive 40% of the time or something like that is what it looks like.
- [24:34]
C
No. And sometimes you don't know. I mean, just because there's a rare variant, you don't know that it's affecting function.
- [24:41]
B
Exactly. Yeah. Very often we get our vus, and that's why I was asking about your test as a way of sorting the vuss.
- [24:49]
C
But in conjunction with that, it could be very powerful because you could imagine a default that if a variant is found, you could test whether that variant is functional or not.
- [25:02]
B
Yeah, again, incredibly exciting. Again, thank you very much. I hope you will get out onto the Chesapeake and get that sailing under your.
- [25:10]
C
I will.
- [25:11]
B
All right. Before you get to retirement. Oh, great answers. This was a fantastic, fantastic interview. I. This is for me, an incredible pleasure. All right. Anything else that I didn't ask you that you really wanted to have a chance to talk about? No.
- [25:27]
C
You know, again, I think we have. We still have a lot to learn with this assay, but I think it's going to be useful in so many, you know, I mean, even what's going on in macular degeneration, right?
- [25:38]
B
Yeah.
- [25:39]
C
40% of patients with macular degeneration have variants in factor VIII. Are they functional? We can answer that question now. You know, complement. Yeah. There's just so much. We have so much to do and you can't do everything. You know, that's another disease. And there's all these other kidney diseases. Complement plays a pretty big role. We're working with some of our nephrologists and pathologists to really tease them apart. The other thing that you can use.
- [26:14]
B
So have you looked at the C3GS? Is this essay useful in that setting? Because that also is a difficult diagnostic setting.
- [26:21]
C
The answer is yes, but only three patients. And the answer is yes, it's useful in those patients.
- [26:27]
B
Interesting.
- [26:28]
C
But it's three patients. We need more. There's a lot we have to do. And then complement deficiencies. What if you sequence someone who's getting a lot of infections and you find a factor B mutation? How do you know? Is that why they're getting the infections? Well, let's see if that factor B mutation blocks complement.
- [26:50]
B
Yeah.
- [26:51]
C
Their alternative pathway is not working at all. We can use this assay in that way. So it's going to be useful not only for complement activating disorders, but also for complement deficiency disorders. Now, we have pretty good assays for looking at most of our complement. Assays look at a deficiency in complement. None of them really can look at activation. This can really look at activation on a cell, but. But it can do both.
- [27:17]
B
Very exciting. Okay, thank you very. Thank you very much.
- [27:21]
C
Thank you. Bye bye.
- [27:24]
B
Take care.
- [27:26]
A
That's it for us here at Blood, Sweat and Smears, a podcast produced by Matrion Diagnostics, your reference lab and CRO, specializing in three thrombosis, hemostasis and rare disease. Thank you for listening. And if you have a question or comment or there's a topic you'd like Dr. Lewis to speak to, please send us an email to blood sweatandsmeersacheondiagnostics.com that's M A C H A O N diagnostics.com you can follow Macheon at Twitter at matriondx. Be sure to subscribe to stay in the know. Share this podcast with clinicians you think might appreciate it, and we hope you'll join us next time here at Blood, Sweat and Smears.