Assessing cellular energy dysfunction in CFS/ME using a commercially available laboratory test, 2019, Morten, Newton et al

[boolybooly]

What jumps out at me from this is that the "MES test does not have the reliability and reproducibility required of a diagnostic test" & "The differences observed by the Myhill group may be down to differences in sample processing time between cohorts".
So the test was unreliable; I assume that this could be due to the poor sampling protocol i.e. not processing samples within the required time (5 hours?).
The fact that they initially used the wrong sample type (neutrophils rather than PBMCs) is also worrying.
I think this emphasises why we need a properly validated diagnostic test delivered by the NIH.

Haven't read the paper by Karl Morten et al in full.

It would be nice and easy if we could assume Tomas et al are the gold standard by which to judge Myhill et al but as I see it, its not the case because the Myhill et al response said Thomas et al may have the protocol wrong and did not take up the offer to learn it from JMH which is a critical issue. So it cannot be considered a reliable replication and the null result does not disprove that there are problems with detection in the replication, more the reverse.

Also, likewise, I believe the Myhill et al response indicates that they consider its not accurate to characterise neutrophils as the "wrong" cell type and suggests they are a perfectly good cell type, simply more difficult to use in the lab because they are liable to morph over short timescales. I know of no argument to contradict this. Would be grateful to know if there is one.

As you mentioned in your previous the Thomas Newton Morten grouping have indicated a competitive interest regarding the provision of testing services/patents. So they do not appear to be completely free of interest which ideally a replicating lab should be due to the unconscious biases which have been mentioned elsewhere in the thread.

So while I absolutely agree the mito test should be given proper scrutiny by the highest authorities, NIH would be ideal, because of the potential it or something like it could have, by that same token it must be subjected to rigorous replication testing by disinterested parties and IMHO this disagreement between two interested parties is the starting point for that, rather than the conclusion. So I will be interested to see how this pans out!

 

[adambeyoncelowe]

It would be nice and easy if we could assume Tomas et al are the gold standard by which to judge Myhill et al but as I see it, its not the case because the Myhill et al response said Thomas et al may have the protocol wrong and did not take up the offer to learn it from JMH which is a critical issue. So it cannot be considered a reliable replication and the null result does not disprove that there are problems with detection in the replication, more the reverse.

Also, likewise, I believe the Myhill et al response indicates that they consider its not accurate to characterise neutrophils as the "wrong" cell type and suggests they are a perfectly good cell type, simply more difficult to use in the lab because they are liable to morph over short timescales. I know of no argument to contradict this. Would be grateful to know if there is one.

As you mentioned in your previous the Thomas Newton Morten grouping have indicated a competitive interest regarding the provision of testing services/patents. So they do not appear to be completely free of interest which ideally a replicating lab should be due to the unconscious biases which have been mentioned elsewhere in the thread.

So while I absolutely agree the mito test should be given proper scrutiny by the highest authorities, NIH would be ideal, because of the potential it or something like it could have, by that same token it must be subjected to rigorous replication testing by disinterested parties and IMHO this disagreement between two interested parties is the starting point for that, rather than the conclusion. So I will be interested to see how this pans out!

Surely Myhill has the same conflict of interest?

I'm just sceptical that there's some mystical process that only her team can do to get the 'right' results. If it wasn't described in the protocol, what could it be? And could it be some process that biases the results?

I think if Myhill wants to continue to use this test, the onus is on her to replicate it and show exactly how it can be replicated by others so they can follow the same process.

 

[Amw66]

Surely Myhill has the same conflict of interest?

I'm just sceptical that there's some mystical process that only her team can do to get the 'right' results. If it wasn't described in the protocol, what could it be? And could it be some process that biases the results?

I think if Myhill wants to continue to use this test, the onus is on her to replicate it and show exactly how it can be replicated for others so they can follow the same process.

Both labs should process samples from the same patients at the same time.
Had this been done ( as suggested) then the picture may be clearer.
Get them in the same place doing the same thing with the same samples. No arguments after that.

 

[JemPD]

Get them in the same place doing the same thing with the same samples. No arguments after that.

 

[borko2100]

I have a feeling that the dysfunction is not in immune cells, but in the brain cells.

 

[FMMM1]

It would be nice and easy if we could assume Tomas et al are the gold standard by which to judge Myhill et al but as I see it, its not the case because the Myhill et al response said Thomas et al may have the protocol wrong and did not take up the offer to learn it from JMH which is a critical issue. So it cannot be considered a reliable replication and the null result does not disprove that there are problems with detection in the replication, more the reverse.

Also, likewise, I believe the Myhill et al response indicates that they consider its not accurate to characterise neutrophils as the "wrong" cell type and suggests they are a perfectly good cell type, simply more difficult to use in the lab because they are liable to morph over short timescales. I know of no argument to contradict this. Would be grateful to know if there is one.

As you mentioned in your previous the Thomas Newton Morten grouping have indicated a competitive interest regarding the provision of testing services/patents. So they do not appear to be completely free of interest which ideally a replicating lab should be due to the unconscious biases which have been mentioned elsewhere in the thread.

So while I absolutely agree the mito test should be given proper scrutiny by the highest authorities, NIH would be ideal, because of the potential it or something like it could have, by that same token it must be subjected to rigorous replication testing by disinterested parties and IMHO this disagreement between two interested parties is the starting point for that, rather than the conclusion. So I will be interested to see how this pans out!

Without wishing to be alarmist here there are reasons why laboratories are regulated e.g.
Industrial Bio-Test Laboratories [https://en.wikipedia.org/wiki/Industrial_Bio-Test_Laboratories]. Things "go wrong" for various reasons --- we need regulators.

If you wish to see a model test then check out the results from Ron Davis's nano-needle test. All of the controls are clearly separated from the people with ME -- ideal situation for a diagnostic test.

In this case you suggest that neutrophils are an appropriate sample type --- where is the data? I.e. the method used to prepare and analyse the samples; plus results which show a clear separation between controls and people with ME? Also, if neutrophils are the basis for a valid method then why change to PBMCs? Where you have a reference method (neutrophils?) and you switch to a new method (PBMCs) then its customary to have the comparison data showing that in fact the new method gives the same result --- is this available?

The data published by Tomas, Newton and others clearly shows that the samples are not sufficiently stable to be sent through the post (72 hours) and then analysed:
"It is possible that differences between patients and controls reflect differences generated during prolonged storage and do not reflect differences at the time of sampling".

I'm wondering if this laboratory is regulated and if so will this be investigated by the regulator - if it is not regulated then why not? We shouldn't have to rely on reviews such as Tomas, Newton and others - however welcome they are.

In my previous post I referred to a method validated by the NIH -- I'm in the UK so I should have written NHS -- although I'd be content with either (NHS/NIH) or (preferably) both.

Might I inquire where our government is i.e. regulators/regulation? Fine speeches in Westminster about ME are one thing but this needs to be investigated and resolved. @EspeMor @Andy

 

[Andy]

This new blog follows the publication recently of a key study from researchers in Oxford and Newcastle who failed in their attempt to validate the Acumen test which is being used commercially to determine mitochondrial function and then treatment in ME/CFS.

It is hoped that the comments below and the report from Dr Morten’s team which explains in more detail what the study was all about and what its’ main findings were, will help address some of the questions still being asked.

However, the report is rather technical and while in our earlier blog we did explain why the authors and the ME Association do not endorse the Acumen test, you might also find that the interview with Dr Karl Morten on Science 4 ME Forums scheduled for 6th September is worth watching.

We would also like to encourage the Myhill Group to respond to the authors of this new study in the usual way via Scientific Reports i.e. the Journal in which the study was published, so that formal discussion might continue.

https://www.meassociation.org.uk/20...n-in-me-cfs-by-dr-karl-morten-21-august-2019/

 

[Robert 1973]

The question would be who would pay for them though.

With any commercial test, I think the onus is on those are are charging for it to prove its reliability. Independent researchers have concluded that the test is unreliable and should not be used in the assessment of people with ME/CFS. If Dr Myhill and her colleagues are still convinced that the test is useful and intend to continue charging patients for it, the onus is on them to prove that it is valid. I don’t know what it would cost to receive and test 20 blinded samples from the ME biobank, but it would surely be a small fraction of the commercial value of the test if they were able to prove that it can accurately distinguish people with ME from healthy controls.

The same applies to any private companies or individuals who are charging for any other tests which have not been independently validated. As Jonathan pointed out in the thread on the German Lyme disease test, “the company has had ample time to do this obvious study and has not. Which to me is a fairly clear indication that they are not interested in any scientific validity for their test, just selling it.” (https://www.s4me.info/threads/lyme-disease-on-bbc-today-programme.10707/#post-191935)

It is surprising to me that we do not have better regulation to prevent the sale of tests and treatments before they have been scientifically validated. As I seem recall @Tom Kindlon has pointed out in the past, if patients were to spend a little bit more on donating to charities funding scientific research instead of paying for dubious private tests and treatments for which there is little or no scientific evidence of efficacy, we might all be better off.

 

[wigglethemouse]

I'd just like to point everyone to @Andy 's post above. The ME Association provided a link to Karl Mortens Feb 2017 report (that he supplied them over two years ago) on his work to replicate the Accumen test.

As it is a report it covers all their experiments and findings and provides more details than are in the paper. They went to a lot of effort in this work.
https://www.meassociation.org.uk/wp...al-Function-in-MECFS-Acumen-Test-21.08.19.pdf

Executive summary
•Using freshly collected blood we were unable to routinely produce a granulocyte layer on the Histopaque gradient used in the Acumen protocol.

•Peripheral blood mononuclear cells (PBMCs) were routinely isolated on a Histopaque gradient from freshly collected blood.

•Other non-gradient approaches showed high levels of neutrophils to be present in fresh blood samples with numbers significantly down when blood was left in the collection tube for 24hrs before processing.

•Granulocytes and PBMCs isolated from the gradient maintain viability following 24hr incubation in cell culture. The inclusion of inhibitors of mitochondrial ATP production had no effect on granulocyte viability but resulted in loss of viability of PBMCs after 7-8 hrs in culture. This suggests the PBMC’s are changing over time and adopting a more mitochondrial energy requiring phenotype. This is consistent with PBMC activation over time something that was also observed in the 24 hr blood sample.

•In freshly isolated blood, PBMC’s show measurable levels of mitochondrial respiration with the mitochondrial contribution to cellular ATP production of between 37-25%. A longitudinal study of PBMC’s isolated from 3 control subjects over 8 weeks showed consistent levels of mitochondrial ATP production and mitochondrial respiration. Mitochondrial respiration levels were low but detectable when large numbers of cells were used.

•In freshly isolated blood samples granulocytes show very little mitochondrial derived ATP production between 14-11% of total ATP levels. Total ATP levels were low and the results varied significantly in the longitudinal control study. Levels of mitochondrial respiration were very low.

•Using ATP as an output of mitochondrial function is problematic when comparing between samples run on separate days. Running consistent standard curves is not always easy even when using a commercial kit. Although mitochondrial respiration levels are low in PBMC’s mitochondrial oxygen consumption may give a more robust assessment of mitochondrial function if used on cells isolated from freshly isolated blood samples

I suspect following this work they engaged with the Newcastle team to see who was right, and that team replicated the Karl Morten team results and not the Accumen results. This would account for the two year delay i.e. it seems they did everything they could to make sure they did not make a mistake before publishing.

One difference I noticed is that this reports states that they had to use sodium azide for 2 hours, where the recent paper used 3 mins per the Accumen protocol. This is what Norman Booth had to say on PR about the use of sodium azide.

John inhibits mitochondrial function with sodium azide, but for only 3 minutes. This short period is adequate and also short enough to prevent apoptosis of any of the neutrophils. In some other studies neutrophils have been inhibited with the toxin Rotenone for as long as 6 hours [4]. If any of the cells are still alive they will have to have found some other pathway to provide them with ATP. As of 1st February 2016 John has made a change in his procedure, and now measures mixed leukocytes containing monocytes as well as neutrophils. There are only small differences from neutrophils only. John is not happy to use lymphocytes alone because of the varying subtypes present and the responses to infection.

Source : https://forums.phoenixrising.me/threads/mitochondrial-and-energy-metabolism-dysfunction-in-me-cfs-—-myhill-booth-and-mclaren-howard-papers.47488/post-785000

 

[wigglethemouse]

One difference I noticed is that this reports states that they had to use sodium azide for 2 hours, where the recent paper used 3 mins per the Accumen protocol. This is what Norman Booth had to say on PR about the use of sodium azide.

I read the recent paper again.

ADP to ATP efficiency.
The efficiency of the conversion of ADP to ATP was monitored in the presence of excess magnesium with and without the complex IV inhibitor sodium azide. ATP concentration was determined as described previously.

Following treatment with the inhibitor the cell suspension solution was centrifuged to remove the inhibitor and the pellet resuspended in SBS (combination of NaCl and KCl phosphate buffered to pH 7.8). This solution was left at room temperature for 3 minutes before being centrifuged again and the supernatant removed.

The pellet was resuspended in SBS and ATP concentration determined as described previously giving the concentration of ATP generated when the inhibitor had been removed. The aim being to compare the amount of ATP generated from cells in the presence of a respiratory chain inhibitor and when it is washed out.

On re-reading it is not clear to me how long the sodium azide was applied for - Norman Booth seemed to think this was important. In addition a NaCl solution is applied. We know from the nanoneedle tests that NaCl can alter PBMC's membrance impedance. Could it be this procedure with the time of sodium azide, or the NaCl addition, where something is different between Accumen and Oxford/Newcastle causing different results? It seems from the Oxford report they applied Sodium Azide for 2 hours!

The Acumen protocol uses 750ìM sodium azide on the neutrophils for 30 minutes in order to knock down mitochondrial ATP production. In our WT cells we need to use almost 3X the concentration of azide for a minimum of 2 hours (without washing) to see a negative effect on ATP concentration.

Hopefully others can take a look and figure this out better than me.

 

[wigglethemouse]

Does anyone know where to obtain the Myhill 2016 protocol? Does it specify the time of Sodium Azide inhibition and the time of NaCl presence? Perhaps it is not included in the Tomas et al paper due to it being proprietary........

A 2016 version of the Myhill protocol was obtained from John McLaren-Howard at Acumen Ltd and followed with two exceptions:

According to Norman Booth on PR (quoted above) the protocol changed on 1 Feb 2016 to measure mixed leukocytes containing monocytes as well as neutrophils. I wonder if they used this version?

 

[Andy]

This paper hasn't stopped the 'test' being sold.

**** Dr Myhill's Mitochondrial Function Profile Test waiting list is open once again. Please email nicky@doctormyhill.co.uk The list will close when it reaches 100 names. #MEcfs #CFS #MyalgicE #MyE #MyalgicEncephalomyelitis #ChronicFatigueSyndrome #PwME #chronicillnesswarrior

 

[Kitty]

#rolleyes...

 

[rvallee]

This paper hasn't stopped the 'test' being sold.

This is not particularly useful for the GMC complaint, though correct on substance may be impacted by bad faith false equivalency and giving doubt to her credibility. Not that "no, u" is a valid defense but when "we prefer the results" for having cheated to get positive results and "those requirements are optional in this case" are accepted then anything can and will be accepted to defend interests and keep away inevitable litigation for as long as possible.

 

[ME/CFS Skeptic]

This is quite frustrating. If 100 people are planning to pay 300 pounds then in total 30.000 pounds are going to be spent on a clinical test that isn't properly validated.

The Tomas et al. study suggested that initial positive results might have been due to the longer processing of ME/CFS samples that were sent through the post. Until someone has clarified the differences between Tomas et al. and the Myhill studies it's inappropriate to use this test in clinical practice.

I'm concerned that many patients won't be aware of the recent Tomas et al. study, published in Scientific Reports. So I think it's worth trying to inform them and highlighting the issue on social media.

 

[ME/CFS Skeptic]

I've posted this on Facebook. Feel free to point out mistakes so I can adjust them:

The team of Sarah Myhill, a ME/CFS doctor in the UK, is currently selling a laboratory test that isn’t properly validated.

It’s called the “Mitochondrial Function Profile” and is actually a collection of tests, the most prominent one being the “ATP profiles” test. Results are usually expressed as a “mitochondrial energy score” or MES. The group of Myhill published three studies about this test where they claim it is a “powerful diagnostic tool.” These results, however, were difficult to interpret and haven’t been replicated by other researchers.

This year a replication study was published in the respected journal Scientific Reports. The authors include ME/CFS expert Julia Newton and Karl Morton, an expert in mitochondrial disease. The results showed that the test “does not have the reliability and reproducibility required of a diagnostic test.” The test showed “no diferences between CFS/ME patients and healthy controls in any of the components of the MES.” The authors also had a plausible explanation for why the Myhill group did find significant differences. They argue that the ME/CFS samples were processed longer as these were sent through the post and that this could have reduced energy efficiency in the cells. When they tested blood from healthy persons with this prolonged processing time, similar deficiencies in energy production were found.

Perhaps that doesn’t fully settle the question. But I think it’s clear that this test shouldn’t currently be offered in clinical practice as we don’t know if it is useful or not. In my view, it’s inappropriate to ask patients to pay 300 pounds for this.

I suspect that many ME/CFS patients will not be aware of the failed replication attempt in Scientific Reports and that they might be inadequately informed when being offered this test. So feel free to share this message with other ME/CFS patients if you think it might be helpful.

The study by Julia Newton and Karl Morton can be read here (for free): https://www.nature.com/articles/s41598-019-47966-z

 

[TheBassist]

Not a scientific observation, but I've just been listening to Myhill being interviewed by Gary Burgess and she talks in a hectoring tone, as if she was addressing a dog. The thrust of her approach seemed to be "it's diet, stop eating carbs and you'll be fine", which seems to me to highly simplistic. What's the consensus here?

 

[Snowdrop]

Not a scientific observation, but I've just been listening to Myhill being interviewed by Gary Burgess and she talks in a hectoring tone, as if she was addressing a dog. The thrust of her approach seemed to be "it's diet, stop eating carbs and you'll be fine", which seems to me to highly simplistic. What's the consensus here?

I don't know about consensus but even if she was absolutely correct about diet it completely misses the point as to the social aspects of this illness. Many cannot afford a change in diet nor can they cook for themselves nor can some even think their way through how to apply a new diet. So what are they to do? Too bad for them.

It seems to me so typical for those people interested in sharing their opinions on our illness that they view things in isolation (because you don't need to spend time understanding) and they apply methods of wellness that (often) require more energy to apply than is available (this includes CBT too)

With this illness the most visible people are the least affected (not to say they are anything like well). And these are the people being considered as if they are the totality of those ill.

While I'm at it there are clinicians who will take credit for the more 'freshly' ill even though it's possible that some do recover without intervention early on.

 

[TheBassist]

I don't know about consensus but even if she was absolutely correct about diet it completely misses the point as to the social aspects of this illness. Many cannot afford a change in diet nor can they cook for themselves nor can some even think their way through how to apply a new diet. So what are they to do? Too bad for them.

It seems to me so typical for those people interested in sharing their opinions on our illness that they view things in isolation (because you don't need to spend time understanding) and they apply methods of wellness that (often) require more energy to apply than is available (this includes CBT too)

With this illness the most visible people are the least affected (not to say they are anything like well). And these are the people being considered as if they are the totality of those ill.

While I'm at it there are clinicians who will take credit for the more 'freshly' ill even though it's possible that some do recover without intervention early on.

I won’t go as far as to say they even mean well in all cases, even those have had, or claim to have had the illness. After all how can someone who is mild claim to have any real understanding of what it means to be severe? I had the recommendation of eating as much freshly prepared food as possible, which would be ideal anyway, but I am getting arthritis in addition to the ME. No way I’ll be preparing a whole load of salads. Most days it’s all I can do to open a can of soup, so unless it’s an actual fix I’m not entertaining that approach. So many of the approaches advocated, or at least the advocates themselves, radiate victim blaming to me

 

[Milo]

Not a scientific observation, but I've just been listening to Myhill being interviewed by Gary Burgess and she talks in a hectoring tone, as if she was addressing a dog. The thrust of her approach seemed to be "it's diet, stop eating carbs and you'll be fine", which seems to me to highly simplistic. What's the consensus here?

If ME was a dietary issue, we’d be all recovered and back to our lives. Give me a break! (Not you, @TheBassist )