🔬 Flow Cytometric Detection of Fibrin(ogen) Amyloid Microclots

see corrisonding Substack episode 

A breakthrough in detection methods might finally give us the diagnostic tool we've been desperately seeking

For years, Long COVID patients have been fighting an invisible war. Not just against their symptoms, but against a medical system that couldn't quite put its finger on what was wrong. "It's all in your head," some were told. "Your bloodwork looks normal," others heard. Meanwhile, millions of people worldwide have been living with a condition that has no clear diagnostic marker, no targeted treatment, and precious little understanding from the very institutions meant to help them.

That might be about to change.

Enter imaging flow cytometry—  this technology can detect and quantify amyloid microclots in blood plasma in about seven minutes per sample. 

Accelerating discovery: A novel flow cytometric method for detecting fibrin(ogen) amyloid microclots using long COVID as a model

A central role for amyloid fibrin microclots in long COVID/PASC: origins and therapeutic implications

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Transcript

0:25

Welcome to the deep dive. Long COVID, it just feels like this enormous sprawling mystery, doesn't it? It really does. Millions affected across the globe, yet, well, so much remains unknown about who actually has it, and maybe more importantly, how we can really help them. It's just a truly frustrating challenge. You have this condition with such widespread impact, but often there's no... clear way to diagnose it or target treatment specifically. And what's truly compelling here, I think, is that while long COVID presents with just this huge array of different symptoms, one particular thread that seems to be emerging as a really key factor is hypercoagulability. Hypercoagulability. Yeah, essentially increased tendency for blood to clot. And this leads to the formation of what are called insoluble amyloid microclots. Okay. So think of them as these tiny, really stubborn blood clots made of a peculiar sticky protein called amyloid. Your body just can't seem to dissolve them easily. And, you know, previous research had already given us some pretty strong hints about their significance. Precisely. And it's this fundamental challenge, this diagnostic gap that makes the latest research so compelling. We're about to delve into a, well, a pretty groundbreaking study that zeroes in on this very problem. It offers a glimpse into a potential future for long COVID diagnosis. It's all about a novel method for detecting these these elusive microclots, which could be a genuine game changer. Absolutely. I mean, imagine moving from this sort of nebulous symptom based diagnosis to having a concrete measurable marker. That's the goal. Right. Definitely. So with that understanding of the the overarching mystery, let's peel back the layers on what's emerging as a key culprit. this hidden clotting problem maybe at the heart of long COVID. We know long COVID is incredibly complex. It's like a tangle of immune dysregulation, maybe autoantibodies, possibly even persistent bits of the virus hanging around. Yeah, all those factors are likely involved. But amidst all this complexity, is there one particular thread that's really emerging as a critical target? Something we can actually aim for in terms of diagnosis and treatment. Yes, absolutely. The presence of these specific clotting pathologies, these fibrinogen amyloid microcloths we mentioned, is indeed becoming a central focus. Okay. When we kind of zoom out to the bigger picture, the origin of these microcloths seems quite direct. The SARS-CoV-2 spike protein itself, it actually has the ability to activate clotting factors in the blood. Just the protein on its own. Yes. And even more directly, it can interact with fibrinogen, which, you know, is the main protein involved in forming blood clots normally. Right. So these interactions seem to be what leads to the formation of these abnormal, highly resistant clots we previously used. fluorescence microscopy to actually see a significant load of these amyloid microclots. Right. You could visualize them. Exactly. And hyperactivated platelets, too, in patients, not just with long COVID, but actually even during the acute phase of the infection itself. That's a crucial point, then. If researchers knew about these microclots, if they could actually see them under a microscope, why haven't we had a widely available diagnostic tool for long COVID until now? What was the bottleneck? Yeah, that brings up a really critical point about the methods themselves. While fluorescence microscopy gave us good insights, you know, into the morphology, the shape and size of these microclots. It wasn't practical. It just wasn't practical for widespread clinical use. Yeah. It lacked, well, statistical robustness, for one, objectivity. And really rapid throughput. Essentially, it's a powerful research tool, absolutely, but not something you could easily or quickly process in a standard pathology lab for diagnosing, you know, potentially thousands of patients. Right. Scalability was the issue. Okay. So if the previous methods kind of hit a wall, what's the scientific equivalent of, I don't know, finding a new key? This brings us to this genuinely novel approach, a new flow cytometric method. This might just be the diagnostic key we've all been searching for. Now, for those listening who aren't familiar, imaging flow cytometry is really innovative because it kind of combines the best of both. worlds. It does. It has the detailed image acquisition, the pictures you get from traditional microscopy, but then it also boasts the incredibly high speed quantitative analysis, the number crunching of traditional flow cytometry. Exactly. So it's like having this super fast, super detailed camera that can analyze thousands of tiny particles in minutes, but then also give you incredibly precise data about them. And what's truly remarkable here are the advantages this method delivers. First, just by combining that high-resolution imaging with rapid analysis, it significantly reduces errors and boosts accuracy. That's huge. Okay, better data. Better, faster data. Second, unlike conventional flow cytometers, it provides spatially resolved information. Meaning? Meaning it can actually give you insights into the shape and structure of these microclots. not just count them. Ah, I see. More detail. Right. But perhaps most significantly, thinking about widespread accessibility. While they used an advanced imaging flow seismometer in this particular study, the results strongly suggest something really important. That the core signals, the things indicating the presence of these microclots, could potentially even be detectable using a conventional flow cytometer. Oh, wow. Okay, that is a big deal. It's a huge deal. Because conventional flow cytometers, well, they've been standard equipment and pathology labs for decades. Right. So it's already out there. Exactly. It makes this a much more widely available technique than something specialized like fluorescence microscopy. Potentially, anyway. Okay, let's unpack the study a bit more then. How did they actually go about exploring this promising new method? What did they do? Sure. The research involved comparing blood samples from two groups, a healthy control group and a group of long COVID patients. They used stored platelet-poor plasma, or PPP samples. So essentially, they took blood plasma, but removed most of the platelets, those tiny cells involved in clotting, so they could specifically look at the actual microclots themselves without interference. That's the standard approach, yeah. Yeah. The core technique involved exposing these plasma samples to a fluorescent dye called theoflavin-T, or THT. Theoflavin-T, okay. Yeah, THT. This guy is special because it specifically binds to those amyloid proteins we talked about earlier. The sticky ones. The sticky ones, yes. These unusual misfolded proteins that basically form the core of these stubborn resistant microclots. This binding makes the microclots glow really brightly under the right light. Making them easy to spot. Making them easily detectable by the imaging flow cytometer. And talk about efficiency. For you, the listener, this is, well, this is a big deal. The total time required to process just a single sample, from putting it in the machine to getting the analysis out, was approximately seven minutes. It's incredibly rapid. Incredibly rapid, especially for a potential diagnostic tool. And within that short time, they were able to detect anywhere from, what, 400 to 3,000 microclots per sample? That's right. Quite a range, but detectable. So here's where it gets really interesting, the striking findings from the data. What did they actually find when they compared the group? Well, the results were quite clear, quite stark, actually. Long COVID patients had a significantly higher concentration of these microclots. We're talking a median of over 52,000 objects per milliliter. 52,000 compared to? Compared to only about 21,000 in the healthy controls, so more than double. Wow. And that difference was statistically significant, so unlikely to be just chance. Okay, so way more clots. What else? And not only were there more of them, but the long COVID patients also had a significantly larger mean area for their microclots. Right. So they were bigger, too. They were bigger on average. So more numerous and, importantly, larger clots circulating. Okay, that sounds problematic. Well, if we look at the bigger picture, the sort of distribution of microclot areas... The long COVID patients showed a much greater number and a much broader range of sizes. Not just bigger on average, but more variety in size. Exactly. Particularly skewed towards the larger categories. Yeah. They saw really dramatic increases in clots in the medium, large, and even very large size ranges. But this leads us to a kind of fascinating insight. while, you know, obviously larger clots are problematic because they could potentially block blood vessels. Sure, that makes sense. The study also found that an increase in the smaller microclots also seems to have significant clinical implications. How so? The smaller ones? Well, these smaller ones might not cause a major blockage on their own, right? They're too small for that. But they can still contribute to platelet activation, kind of waking up the platelets. and also cause damage to the inner lining of blood vessels, the endothelium. Ah, so even the little ones cause trouble downstream. It seems that way, contributing to that sort of background inflammation and vascular issue. And the visual evidence from the imaging flow cytometer itself, the pictures it took, really brought this home, didn't it? It really did. Even at lower magnification, you could apparently see a stark visual difference. Just notable variations in size and fluorescence between the control samples and those from the long COVID patients. That's right. The long COVID clots just look larger and glowed more intensely, which really reflects their amyloid nature. It paints a very clear picture visually as well as statistically. Wow. And what's also quite revealing is that they observed a significant amount of what they called background events that, in the long COVID samples compared to the controls. Background events? Like just noise? Well, not exactly noise. When they looked closely at the bright field images, the regular light images, not the fluorescence, they determined that some of this background could actually be cellular debris. Debris? From what? potentially from damaged endothelial cells, those blood vessel lining cells we mentioned, and maybe even fragments of fibrinogen strands floating around. I see. So signs of damage. Exactly. And this finding aligns perfectly with previous research that has pointed towards endothelial damage being a feature in lung COVID. It sort of reinforces this idea of a systemic vascular component to the condition. Okay. So pulling this all together... What does this all mean for you, the listener, and maybe for the future of long COVID diagnostics? This sounds like it holds real potential, right? It really does. It could become a direct diagnostic tool for long COVID patients and maybe even for individuals with other kinds of clotting pathologies. Indeed. And that accessibility aspect we touched on is truly monumental here. detecting parameters like the microclot concentration, their average size, their size distribution. All those things the machine measures. Precisely. That information could profoundly guide clinical decisions, not just about diagnosis, but also about treatment. How so? Well, clinicians could potentially use this information to tailor therapies to address the specific clotting abnormalities they see in an individual patient. You know, moving away from these broad symptom-based approaches towards something more targeted. Personalized medicine for long COVID clotting issues. Potentially, yes. And it could also allow them to effectively monitor how well treatments are working over time. Are the clot levels going down? Are they getting smaller? Right, tracking progress. And the fact, again, that the study suggests these signals might even be detectable using a conventional flow cytometer. Well, that just points towards a clearer path for much wider implementation. Making it accessible to more patients hopefully sooner rather than later. That's the key. That's incredibly exciting. But, like any good scientific breakthrough, it also comes with some important limitations, For those of us eager for a swift solution, it's crucial to understand what still needs to be done. What are the main caveats here? The next steps. Absolutely. And that's critical. This brings us to the vital next steps. The researchers themselves acknowledge this is a preliminary investigation. A proof of concept. Exactly. A fantastic proof of concept. But really just the beginning. Further research is definitely needed, particularly with larger and much more diverse patient data sets. Bigger studies. Bigger studies, yeah. Yeah. To truly establish standardized reference ranges like what's normal, what's high, and to determine its overall reliability as a diagnostic tool. And crucially... They need to figure out how to differentiate these microclots from maybe similar ones found in other inflammatory conditions or comorbidities that might also present with clotting issues. Right. Make sure it's specific to long COVID or understand when it overlaps. Precisely. And they also need to explore whether there are any significant differences in the results when using fresh blood samples versus the stored samples they used here. That's an important practical question. Okay, so still work to do, but very promising. So just to bring it all together then, this novel rapid cell-free flow cytometric detection method... using imaging flow cytometry, has brilliantly revealed the presence of these amyloid fibrinogen microclots in blood plasma. Yes, it's shown it clearly. This really feels like a potential breakthrough in clinical diagnostics, offering invaluable insights into these specific clotting pathologies that seem so central to long COVID for many. And while it's preliminary, as you said, it truly does lay the foundation for more research into its utility as a proper diagnostic tool. Indeed. It's a crucial step forward, I think, in unraveling what is still a very complex condition. Absolutely. So here's a final thought to leave you with. If this novel method of detecting these specific microclots can unlock better diagnosis and maybe even treatment for long COVID, What broader implications could that have? Could it help us understand and manage other chronic inflammatory conditions where maybe similar clotting pathologies might be at play, just hidden until now? That's a fascinating question. Something to think about until our next deep dive.