A nanoelectronics-blood-based diagnostic biomarker for ME/CFS (2019) Esfandyarpour, Davis et al

[Esther12]

It's quite a stark difference between patients and controls, so unless there's been a real screw-up this should lead on to some interesting work. I've got some concerns about the politics of promoting this to the media now, but even if this is something the 'only' provides a useful way of testing for ill-health/fatigue in those suffering from a range of different conditions, that would still be quite a break-through.

Presumably work on this test has been continuing since their paper was submitted, and hopefully they will be getting further data to help work out what is going on.

 

[Octogenarian]

@Ben H My impression is that participation is easy from a subject's point of view...

Nothing is easy for severe, bedbound patients.

 

[Octogenarian]

The crux of it is to prove pwME are not healthy and that any deconditioning is not the primary component of that poor health.

Yes, Ron Davis understands that better than anyone.

 

[NelliePledge]

Am speculating here based on zero science knowledge but would they have been able to get hold of a couple of samples from people with another illness to see what happens so that they might already have seen differences but can’t say anything because it wasn’t formal research/? Or is that not how it happens in reality?

 

[Inara]

I think that maybe more has been added to the "expert" responses on the SMC website. Went to the web archive link, and got a message that the content was being downloaded - I've never had that happen before, though must admit I don't visit the web archive much unless it's links from here, so maybe I'm wrong. Prof Kevin McConway's response seems rather longer than that quoted in early posts in this thread. But here's the bit I wanted to quote:



(my bolding above)

Prof Kevin McConway: “I am a trustee of the Science Media Centre, but I am writing these comments in my capacity as a professional statistician.”

It feels like they dont want ME to be a real disease.

 

[Esther12]

Am speculating here based on zero science knowledge but would they have been able to get hold of a couple of samples from people with another illness to see what happens so that they might already have seen differences but can’t say anything because it wasn’t formal research/? Or is that not how it happens in reality?

Or it could be that they've been doing formal research on this, but are not saying anything about results not submitted as a part of their paper. Taking that cautious approach with public statements probably would be best. I'd hope work like this has been going on, but we've seen how researchers can become prematurely confident in their own findings and fail to do the sort of double checking that others see as important. Fingers crossed that this sort of work has been/is being done.

 

[Hoopoe]

Wessely is probably trying to say, without actually saying it, that the observed effect is due to deconditioning.

 

[NelliePledge]

Or it could be that they've been doing formal research on this, but are not saying anything about results not submitted as a part of their paper. Taking that cautious approach with public statements probably would be best. I'd hope work like this has been going on, but we've seen how researchers can become prematurely confident in their own findings and fail to do the sort of double checking that others see as important. Fingers crossed that this sort of work has been/is being done.

Thanks @Esther12 wasnt sure if I was off the mark.

 

[Sasha]

Ron Davis: [...]

“The second phase is to try and figure out how to diagnose it is ME/CFS and not some other related disease. That’s going to take some time as we have to look through a large number of other diseases, but that is proceeding at the moment.”

I'd have thought that even just having sedentary healthy controls and a single disease control group (e.g. MS) would have been hugely informative. There seems to be something about this that I'm not getting!

 

[NelliePledge]

Wessely is probably trying to say that this is due to deconditioning, without actually saying it.

Very probably indeed

 

[Kalliope]

Article in ScienceAlert with comments from Ron Davis, Simon Wessely, Andrew Lloyd and Robert Naviaux : A Chronic Fatigue Blood Test Looks Increasingly Promising, But It's Still Early Days

ScienceAlert reaches a huge audience. They have 9 million followers on FB.

 

[Sasha]

I am happy that the paper on this test has been published. Would disagree a little on the immediate need to discriminate between PWME and deconditioned otherwise healthy people.

Th Psych lobby is moving away from deconditioning to a sensitivity to exercise or central sensitivity model.

TBH I don't care about the psych lobby at this point - I care about having good science done to get us a valid test and I think that the objection that the test might simply be showing something to do with deconditioning seems entirely valid, regardless of who is bringing it up. For our own sakes, I think we need that comparison to be made.

 

[anciendaze]

I've just skimmed the paper, and find it extremely interesting, and not just for ME/CFS. They are running a provocative test at the cellular level, rather than attempting to wreck the entire patient, as in some tests I've had. Using osmotic stress tallies with widespread experience that patients have serious problems with dehydration or electrolyte imbalances. These are physiological stressors which should not be confused with psychological stress.

Aside: with some training in mechanics of continuous media I was taken aback that medical people generally assumed "stress" was psychological. We were used to distinguishing stress and strain in things like piano wire, which has minimal psychology.

Operating at the cellular level also allows you to deal with localized problems, as in tissues deprived of oxygenated blood by abnormalities in circulation. Nothing in biology requires the effect to cover the entire body.

I doubt that this will clearly distinguish patients according to clinical judgment, because we already know that judgment is subject to real difficulty. It takes a considerable effort to make clinical judgment produce anything like a uniform cohort. This is not a defect in the research, but in clinical evaluation in the absence of biomarkers.

Requiring a biomarker to match clinical assessments, when different schools of thought have trouble agreeing, is a recipe for stagnation. I would also point to severe problems in distinguishing a known serious illness like periodic paralysis in the absence of laboratory tests. Most such patients go decades without a correct diagnosis. That rare illness also depends on problems with electrolytes and ATP production, so it might be detected by this, though I would expect the machine learning algorithms to need different training.

As a separate example of the problem with matching clinical judgment, one not depending on osmotic stress AFAIK, I will also mention the clinical difficulty of diagnosing anti-NMDA receptor encephalitis without detailed laboratory tests. In that case, there is little question "something is wrong", but demanding that testing should support the whole range of clinical descriptions would be ridiculous. The illness can look like all kinds of strange things.

Added: waiting for the biopsychosocial group to understand support-vector machine learning could take a while.

 

[Sasha]

Nothing is easy for severe, bedbound patients.

Agreed, but it's all relative and in terms of getting healthy but sedentary controls and patients from other disease groups, especially if blood has been biobanked, it ought to be fairly easy.

 

[wigglethemouse]

I've only read the paper once, but it seems this test currently requires very custom one-off research equipment, and the test itself takes 3 hours to run, never mind all the before and after prep. So it's a slow, researcher intensive process.

The 40 samples tested in the paper had to be tested within 5 hours of collection. This really limits what you can do from an experimental point of view.

The paper describes some of the the handling challenges / experiments

Furthermore, we explored a number of different preparation and storage methods to minimize the need for freshly drawn blood, and to provide the possibility of using patient samples collected around the globe.

We conducted the assay with samples kept at 4 °C, room temperature, and in a 37 °C storage cabinet, as well as those frozen in -20 °C freezers and in liquid nitrogen (at 200 cells per ìL).

We concluded that plasma used within 5 h of preparation at 200 cells per uL gives the most reliable and reproducible results. The other most successful techniques tested were 24 h storage at room temperature and liquid nitrogen freezing for 1 wk, both of which preserved the pattern of
fresh samples, although the response was slightly attenuated.

So immediate liquid nitrogen freezing and storage is a possibility. But at present it seems they can only test one sample at a time. I believe access to biobank freezers requires special procedures. You can only open the freezers so many times before the samples degrade with the temperature changes. My take is that taking one sample out of a freezer at a time logistically is very difficult. Also those biobank samples will have had to be immediately processed and frozen in a consistent manner.

Anyone on here know more about biobank collection, handling per the supplemental instructions, and liquid nitrogen freezing and retrieval? Would the London biobank samples be usable for example?

 

[wigglethemouse]

Karl Morten touched on blood handling issues and quick degradation in his NZ talk. I believe this was in relation to his work to try and validate the Myhill et al ATP profile test
Transcript : https://www.s4me.info/threads/dr-ka...-his-research-december-2018.7287/#post-130458

if you have a blood sample and you're looking at things in the blood sample it's a living system so in a blood sample they're billions of cells those cells are all alive and they're all doing things and could to have an impact on the results so we looked at this on normal controls so this is um don't worry too much this is just different ways of making a blood to taking blood and then we measure the glucose concentration in the blood so in our blood we should have about five or six milli molar glucose it's your diabetic it'll be higher than that so we start off about five milli molar glucose here and then over 24 hours you can see the blood glucose drops really really low so you're down at 1 millimolar all of the samples that get sent to this testing laboratory or at least 24 hours old so what we're saying is that it's okay to do that but you need to know what's going on and if a patient behaved differently to a control in 24 hours then that could have an effect

 

[jaded]

Of course Henrik had to have a say...

What exact tools is he referring to? Psychobabble ones?

 

[wigglethemouse]

Not really because I cannot work out the geometric relation between probe and a cell? several cells? - how do we know what the impedance is being measured across?

I would also like to see the raw renormalised impedances to see how variable they were.

Good questions @Jonathan Edwards . I would love to learn more about the structure, how many sensors, when to capture a reading from a sensor.... could other structure geometries work better.

I posted this yesterday in case you missed it.

Citation 38 might be relevant....
Nanoelectronic impedance detection of target cells (Dec 2013) Esfandyarpour et al
https://onlinelibrary.wiley.com/doi/abs/10.1002/bit.25171

This previous paper talks more about the nanoneedle measurement

ABSTRACT
Detection of cells is typically performed using optical fluorescence based techniques such as flow cytometry.

Here we present the impedance detection of target cells using a nanoelectronic probe we have developed, which we refer to as the nanoneedle biosensor.

The nanoneedle consists of a thin film conducting electrode layer at the bottom, an insulative oxide layer above, another conductive electrode layer above, and a protective oxide above.

The electrical impedance is measured between the two electrode layers. Cells captured on the surface of the nanoneedle tip results in a decrease in the impedance across the sensing electrodes.

The basic mechanisms behind the electrical response of cells in solution under an applied alternating electrical field stems from modulation of the relative permittivity at the interface. In this paper we discuss, the circuit model, the nanofabrication, and the testing and characterization of the sensor. We demonstrate proof of concept for detection of yeast cells with specificity.

We envision the sensor presented in this paper to be combined with microfluidic pre-concentration technologies to develop low cost point-of-care diagnostic assays for the clinical setting.

ETA : Link to full text of 2013 paper for anyone wanting to understand the technical details of the sensor more.
http://sci-hub.se/10.1002/bit.25171

 

[Amw66]

It wouldn't actually surprise me at all if the nanoneedle test would also respond to other diseases on the same spectrum as ME/CFS. Robert Naviaux for example has looked at suramin use for both ME/CFS and autism and the other drug that made ME/CFS cells behave normal, copaxone, was a drug originally developed for MS. I think it would be incredible news if this test was specific to only ME/CFS patients, but the skeptic in me reckons it won't be. Having said that, this test could still be used to identify that the patient is sick and perhaps whether certain drugs would work or not, regardless if the patient is classified as suffering from ME/CFS or some other condition.

SW's arguments are for some parts sensible to me, for some parts not. The argument about what is a cause and consequence seems like an excuse that could be pulled off every time, even if a valid biomarker was found.

Agree 100%. Indeed, I'd go further and say if this disease was one day to be shown by faulty brain wiring or whatever I wouldn't necessarily have a problem with that: but even if that were to be true it would remain the case that CBT and GET are ineffective, as are denial of medical tests and welfare/benefits (an approach I believe has been attributed to a certain scientist who's been popping up in the media to downplay this paper).

In my view, the biggest harm to ME/CFS patients hasn't been the suggestion this is a mental illness - though that remains entirely unproven. The biggest harm to patients has been that this is an illness that can be cured easily by the patient thinking his or herself better, that the patient can effectively 'snap out of it' and should ignore what their bodies are telling them, that failure to improve is the fault of the patient, and that any complaints around the efficacy of this treatment approach are a symptom of the patient's delusion rather than an indication of the treatment being entirely ineffective.

This

 

[Simon M]

I am sorry to say that I still do not understand what they did or why. I have no idea how many cells they tested or whether they even knew how many cells they were testing. The introduction does not inspire confidence. I really would like to think this study is meaningful but so far I can get no feel for what these lines mean.

Not really because I cannot work out the geometric relation between probe and a cell? several cells? - how do we know what the impedance is being measured across?

I would also like to see the raw renormalised impedances to see how variable they were.

"What" is a lot easier to answer than "why".

They produced "simplified blood" of each patient made up of PBMCs resuspended in plasma, 200 cells per microlitre.

A sample of this was added to the Nanoneedle chip and they then measured in impedance. Impedance is defined as the ratio of applied voltage to the induced current (if that helps).

Each chip has 4000 Nanoneedles, I think, and they used a sample frequency of five times a second for approximately three hours.

Once they had a study baseline measure they increased plasma sodium chloride concentration to 200 mmol per litre, to apply osmotic stress/increase energy demand.

Here are the diagrams from the paper showing the Nanoneedle design, and the captions:

​

(B ) Circuit model of a sensor−solution interface, where Zm-s is media−sensor surface interactions, Zc-c is cell−cell interactions, Zc-s is cell−sensor surface adhesion, Zc is a cell impedance (membrane capacitance Cm, and cytoplasm conductivity of the cells, σcp), and Rs is resistance of the solution. (E and F) SEM images of a nanoelectronic sensor tips, (E) top view

The "why?" Is more complex.

The paper states that "the array directly measures the impedance modulation results from cellular and/or molecular interactions". However, they don't offer any direct evidence of that, though more later on what they think is going on.

Interestingly, they weren't initially trying to develop a diagnostic test. Instead they were trying to set up a cellular model of post exertional malaise, using a high salt environment to ramp up ATP consumption and presumably lead to its depletion. Salt stress had been used to do that in other biological systems.

However the differences between patients and controls were so dramatic that they switched to investigating the Nanoneedle system as a diagnostic test.

They say the exact mechanisms behind the differences remains unclear and is they have a programme of work to try to narrow things down. They suggested several possibilities, including changes involving the endoplasmic reticulum and plasma membrane, and even increased cytokine production in response to the salt stress.

One possibility was change in size/shape in response to the osmotic pressure, but live microscopy at several critical stages in the experiment revealed no visible differences between patient and control cells.


Does that help? I don't understand the details of the different types of impedance measured.