Preprint Flow Clotometry: Measuring Amyloid Microclots in ME/CFS, Long COVID, and Healthy Samples with Imaging Flow Cytometry, 2024, Pretorius

https://www.researchsquare.com/article/rs-4507472/v1

Article

Flow Clotometry: Measuring Amyloid Microclots in ME/CFS, Long COVID, and Healthy Samples with Imaging Flow Cytometry

Etheresia Pretorius Stellenbosch University Massimo Nunes Stellenbosch University Jan pretorius Douglas Kell University of Liverpool

https://doi.org/10.21203/rs.3.rs-4507472/v1



Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) has received more attention since the characterization of Long COVID (LC), a condition somewhat similar in symptom presentation and, to some extent, pathophysiological mechanisms.

A prominent feature of LC pathology is amyloid, fibrinolysis-resistant fibrin(ogen) fragments, termed microclots.

Despite prior identification of microclots in ME/CFS, quantitative analysis has remained challenging due to the reliance on representative micrographs and software processing for estimations.

Addressing this gap, the present study uses a cell-free imaging flow cytometry approach, optimized for the quantitative analysis of Thioflavin T-stained microclots, to precisely measure microclot concentration and size distribution across ME/CFS, LC, and healthy cohorts.

We refer to our cell-free flow cytometry technique for detecting microclots as 'flow clotometry'.

We demonstrate significant microclot prevalence in ME/CFS and LC, with LC patients exhibiting the highest concentration (18- and 3-fold greater than the healthy and ME/CFS groups, respectively).

This finding underscores a common pathology across both conditions, emphasizing a dysregulated coagulation system.

Moreover, relating to microclot size distribution, the ME/CFS group exhibited a significantly higher prevalence across all area ranges when compared to the controls, but demonstrated a significant difference for only a single area range when compared to the LC group.

This suggests a partially overlapping microclot profile in ME/CFS relative to LC, despite the overall higher concentration in the latter.

The present study paves the way for prospective clinical application that aims to efficiently detect, measure and treat microclots.

Health sciences/Medical research/Biomarkers/Diagnostic markers

Biological sciences/Biological techniques/Imaging/Fluorescence imaging


Expanded Key Points for Review Only

• We have recently developed a method to detect microclots in plasma samples using cell-free flow cytometry analysis, which we have coined 'flow clotometry'.
• Quantitative data presented in this paper corroborate that microclots are present in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) plasma samples at significant levels when compared to ‘healthy’ participants, and that Long COVID patients exhibit the highest concentrations of microclots, with an 18- and 3-fold change in their median compared to the healthy participants and ME/CFS group, respectively.
• The size distribution of microclots in the ME/CFS group is significantly different from that of healthy participants in all area ranges, but were only significantly different in one area range when compared to the LC group. Hence, the size distribution of microclots in ME/CFS samples is broadly comparable to that of LC samples.
• Importantly, microclots are present in an easily accessible and measurable fraction of blood, and hence the implementation of flow cytometry (clotometry) microclot assessment in the clinical setting is warranted. Such methods can provide a high throughput, and deliver quantitative information regarding both burden and size distribution of fibrinaloid microclots.

Main Key Points

• We have developed a method to detect microclots in plasma samples using cell-free flow cytometry analysis, which has been coined 'flow clotometry'.
• Quantitative data corroborate that microclots are present in myalgic encephalomyelitis/chronic fatigue samples at significant levels when compared to healthy participants

 

Does anyone know if this group have used healthy controls in their previous studies?

ETA: I only ask as if they haven't this could be the first evidence that the blood clots seen in the previous studies were not an artefact of the testing process and were genuinely in the patients blood. That is of course if the healthy control samples were collected/stored/processed/tested exactly the same as the patient samples.

 

Yes. See figures in this post from Hutan in Oct 2021, which is for the original LC microclot paper. That paper had followed their acute Covid microclot paper, which itself followed the pre-Covid evaluation of diabetes. All these amyloid fibrin papers followed their original observation of "dense matted deposits" via electron microscopy, before they tagged them with Thioflavin T to indicate they were amyloid.

The technique used in this pre-print also uses Thioflavin T, but the advance seems to be quantitive assessment by object count (of specific size range), versus the overall area of green signal by image analysis previously. Still not confirming they are circulating in vivo. Perhaps the fibrin PET studies will help answer that question.

They comment in discussion —

 

Strong paper. It provides further evidence for a fascinating observation in the disease; an observation that could suggest interventions. I'm pleased this avenue of enquiry is making progress.

 

I have not read the paper but there still seems to be no acknowledgement that particles of this size, if present in vivo, would have been removed by centrifugation? The sizes they quote are about monocyte size and would be removed by a brief low speed spin - much faster than red cells would.

That appears to mean that these particles develop newly in the plasma when incubated with thioflavine T. They do not appear to be clots in any normal sense, in that they are not composed of an extended fibrin lattice but more like a precipitate of protein of the sort seen when you add trichloroacetate or heat plasma in the Bence-Jones test. It is unclear why this sort of precipitation should occur in vivo and why it should occur at such a chronic low level that no pathology is detectable. Microparticles in venous blood of this sort would be expected chiefly to produce a pneumonitis and ultimately pulmonary hypertension as in pulmonary veno-occlusive disease.

 

This is the interesting question. It might be that a shift in concentration or activation of amyloid-forming proteins occurs in LC and ME/CFS and that this assay is a way to show that, but nothing to do with circulating micro clots in people blocking vessels.

The use of the previous ME/CFS cohort is way less than ideal. If I remember rightly this population had a very atypical mix of other comorbid conditions including several with rheumatoid arthritis. It makes a mockery of the whole thing if they are re-measuring these. These samples will also be very old now and freezing plasma might do all sorts of things to the formation of precipitates.

 

I think they should be more than just aware of this. If I recall the results by Dalton et al, they had shown that this kind of storage completely nullifies any results and makes them useless. Haven't gone through the study yet, but the deciding factor would possibly whether controls had been exposed to Covid which seemed to be a driving force in the results of Dalton et al.

 

If it's a density gradient and the clots are "wispy" then maybe they could stay above cells? But perhaps that's irrelevant at 3000g and most anything particulate should be at the bottom of the tube? Although in counter to that they always make the point that it's platelet-poor plasma not platelet-null plasma, so at least platelets can be in the supernatant, so maybe also things of similar density, esp given that the platelets spread out when activated.

 

But they aren't wispy. We have seen them on immunofluorescence and they are lumps. They are solid protein, so a lot higher density than cells, and they are as big as monocytes. The only thing that would stop these falling to the bottom of the tube even without centrifugation is the red cells - which are removed. These things should sediment about a thousand times faster than a platelet, which is lipid rich.

 

Spot on @Trish

 

A side note. My samples were sent overnight on dry ice and had to be tested within 24hrs.