The Occurrence of Hyperactivated Platelets and Fibrinaloid Microclots in ME/CFS, 2022, Nunes, Pretorius et al

Hi, Chris--thanks for clarifying that! Do you have any more insights into some of the other concerns mentioned in this thread?

 

Then it should be indicated in the paper.
Methodology with fluorescence microscopy is so often inadequate. If one wants work to be taken seriously then it is worth putting the effort into a manuscript to cover these things.

 

They definitely weren't blinded for the platelet spreading or clumping they visually assigned scores of 1-4 to though, as they don't even report values for controls, so it appears they only scored ME platelets.

 

I have asked a haematologist to look at the MS for me. I don't know if Chris has any haematologists to consult. I think we need expert input on this. The input at UCL Div of Med Grand Round on Covid vascular phenomena was that nobody had heard of this work.

 

Covid past infection appears to be excluded, prior to entry, for the healthy controls and the ME/CFS group

One concern is the ME/CFS group, taken from ME/CFS Community within SA, is not a random sample, often patients volunteer for specific reasons related to research being undertaken, eg they like the researchers theories and solutions (eg - a nutraceutical or whatever theory which corresponds with their own theory/hope for an outcome). This then skews the results despite blinding of the person handling the sample (selection bias)

There seems to be a high comorbidity for Leaky gut/gut dysbiosis 40%, Rheumatoid Arthritis 12%. I do not know what the incidence of these illnesses are within the ME/CFS community in SA, so are the a true representation of ME primarily? or is it just the co-morbid illness causing the activated platelets and fibrinaloid microclots (or a combination of the comorbid illness and ME). This could be another area of selection bias skewing results. However these could be a part of the disease pathology for this subsets of patients (but not for all people with ME as we are a heterogenous group of many different causations)

I suppose only a replication in larger patient groups (and international), with a range of diagnosed co-morbidities (with clear criteria) will tease this out. As with all things ME, it is likely to be multifactorial until one day it is not.

 

I asked Douglas Kell about this striking elevated level of % Area Amyloid for type 2 diabetes (relative to controls). He agreed that they had seen this effect for type 2 diabetes and that this means that a metric of “preformed amyloid microclots” cannot - taken in isolation - be diagnostic of ME/CFS or Long Covid. However, he does think that other metrics derived from the microscopy images could be diagnostic - no evidence as yet.

 

Thanks for looking into this, much appreciated.

 

It is a pity we do not have a haematologist here. I have asked for an opinion but it is probably in an in tray.

I will try to explain why, with my limited knowledge of clotting, I cannot make sense of all this. (My limited knowledge does extent to a month working in a clotting lab as a student.)

As I understand it the studies are done on platelet poor plasma. That is plasma that has been prepared (I think) by:
1. Blocking clotting with a calcium trapping agent like citrate.
2. Spinning out red and white blood cels and platelets.

If there are any clots in the 5-100 micron range ('micro clots') they should be spun down with the platelets.

Tiny clots do not normally form in blood or in tubes except maybe as accretions around foreign bodies. Normal clotting in tubes consists of fibrin polymers forming over long distances (millimetres) before interweaving and contracting to form the familiar macroscopic jelly.

It seems as if any clots that were in the patients should have been spun down. So the 'microclots' observed subsequently must form in the tubes over time. In normal circumstances without the calcium trapping agent the whole solution would form a single mass of jelly in a few minutes. It is conceivable that the experiment is done without a calcium trapping agent, using very carefully prepared plastic surfaces but I would be surprised. (I am not sure it changes the puzzle.)

The odd thing then is the formation of microclots when normally in a tube you get a diffuse clot. As soon as a tiny clot forms it extends indefinitely. This suggests to me that the micro clots are forming around some sort of foreign body like dust particles.

The conclusion from all this for me is that these studies do not show the presence of microclots in patients. They show the formation of microclots in plasma from patients under lab conditions. That may indicate something odd about the patient's clotting but if was as simple as that why do these clots not extend? It seems maybe the process is more related to amyloid accretion than fibrin clotting. What implications that might have in living patients is unclear to me.

 

I wonder if the binding of thioflavin T causes amyloid rich micro-aggregates? Patients with a polyclonal anamnestic antibody response after an infection might have Thioflavin T cross-linking antibodies or something like that, although I am doubtful they would.

 

Wouldn't this depend on their density and form? Platelets are presumably much denser than these fibrin polymers, which as I understand it are more like a network in the plasma than discrete objects like platelets. So a centrifuge that would spin out platelets wouldn't necessarily spin out 'micro clots'.

 

It would but these look quite 'heavy' to me. Platelets include quite a lot of lipid, which is lighter than water. Loose uncondensed fibrin clot may have almost the same density as plasma but these particles look like fairly solid protein lumps. Moreover, most of them seem to be hundreds of times larger than platelets.

 

I'd imagine it's pretty hard to get a sense of physical density looking at fluorescent markers, but for what it's worth these images of larger structures from acute Covid patients in their previous paper look fairly loose. What they saw in ME samples, in comparison, was smaller and less impressive.

Are these small (roughly platelet sized) very bright areas actually physically dense or is that a product of the density of binding sites for ThT, the intensity of fluorescence, and the sensor and image processing?

 

Is it possible that there is something in plasma from patients that triggers microclots growth around it, under lab conditions? Something that would trigger the formation of microclots even in plasma with normal clotting activity.

 

To be really honest those pictures look like a random selection of dirty slides. There does not seem to be much consistency in the bright objects. But those that are there have clear enough margins (remember that some may be blurred because of the depth of field) which suggests to me that this is condensed protein. Loose fibrin gels probably would not show up other than as a diffuse haze.

 

I spoke to an academic haematologist with expertise in clotting today. They had not heard of any of the Pretorius work or the methods being used. The more I look at the pictures the less they mean to me.

 

Will he try to check her work or the methods being used? Did he say what he thinks about?

 

Preprint means not reviewed doesn't it? And I've just seen that Amy Proal is one of the authors. I formed an impression of her scientific rigour on this thread:

https://www.s4me.info/threads/hypothesis-piece-by-amy-proal-a-microbiologist-with-me-cfs.134/

Where amongst other things she advocated the quackery that is the "Marshall Protocol" whilst simultaneously misrepresenting it, and confidently asserted that "Electromagnetic radiation from mobile phones and cellphone towers (among other sources) has been shown to lower immunity.", forgetting to add the words "in mice".

So I can't take anything she's involved with seriously, sorry. It's a lazy shortcut I know, but I need lazy shortcuts.

 

A blog, the second part of which covers this preprint.

Peroxisomes, Mitochondria, And Fibrinoid Microclots: Two New Preprints.

https://www.meprecisely.com/2022/07...a-and-fibrinoid-microclots-two-new-preprints/

 

The haematologist did not make any specific comment on the paper, which I had emailed several days before, other than that they had never heard of the investigators. The only other relevant comment made was that 'there is a lot of rubbish about'.

I interpreted the responses as indicating that my haematologist friend would rather not have to give an opinion on the paper. We then discussed the possible relevance of clotting to fatigue and research avenues for about half an hour.

I think that probably speaks for itself.

 

Published.

https://mdpi-res.com/d_attachment/p...armaceuticals-15-00931.pdf?version=1658922364