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The ATP Profiles test developed by Dr John McLaren-Howard (and the Mitochondrial Energy Score developed from it) has been a big deal for the chronic fatigue syndrome (ME/CFS) community. The test formed the basis for studies dating back to 2009 by Dr. Myhill, Dr. McLaren and Dr. Booth which asserted that mitochondrial dysfunction plays a major role in chronic fatigue syndrome (ME/CFS).

ATP profiles test

The ATP Profiles test results seemed to make perfect sense: the mitochondria in people with ME/CFS simply weren’t producing enough energy (ATP). The Tomas paper, however, suggests the test results were wrong.

Mitochondrial dysfunction subsequently became a central theme in Dr. Myhill’s writings, website and treatment protocols. (See – The Central Cause: Mitochondrial Failure) in ME/CFS and her book (Diagnosis and Treatment of Chronic Fatigue Syndrome and Myalgic Encephalitis, 2nd ed.: It’s Mitochondria, Not Hypochondria). Dr. Myhill’s  website states that the ATP profiles test she’s used extensively (@ 1000 results) provides an objective test which clearly differentiates ME/CFS from healthy people.

“The joy of the ATP profiles test is that we now have an objective test of chronic fatigue syndrome which clearly shows this illness has a physical basis.”

The studies were like a breath of fresh air which provided hope that a cause which made perfect sense (reduced mitochondrial activity!) and an objective test for ME/CFS had been found.  With the emergence of more studies suggesting that problems with energy production were a big deal in ME/CFS, the earlier studies seemed prescient indeed.

Plus, they presented a potentially ground-breaking possibility. If the tests could be validated, the U.K.’s National Health Service could conceivably include them in their list of accepted tests for ME/CFS. Cara Tomas PhD, Dr. Newton MD, PhD and their group reported that it was with that possibility in mind that they attempted to validate the McClaren mitochondrial test.

The Tomas Study

Cara Tomas1, Tiffany A. Lodge2, Michelle Potter2, Joanna L. Elson3,4, Julia L. Newton1,5 Karl J. Morten2. www.nature.com/scientificreports/

Having taken into account a 24 hour delay between blood collection and cell isolation, we have shown decreases in ATP parameters in control cells similar to those seen by the Myhill group in the CFS/ME patients. We suggest that it is potentially the delay between sample collection and cell isolation that is causing the decrease in mitochondrial function previously reported in CFS/ME patients. Tomas et. al. 2019

The test, which employed various factors to assess ATP levels in neutrophils and PBMC’s, failed at all levels. However the samples were tested, ATP concentrations, ATP ratios and ATP to ADP efficiency were the same between healthy controls and ME/CFS patients.

So how did a test which formerly found all 138 ME/CFS patients to have at least some mitochondrial dysfunction apparently get blown up so completely? Tomas believes it came down to a very simple and vexing issue that we’ve come across multiple times before: sample preparation.

ATP concentration drops

Drops in ATP concentration after samples sat in test tubes for 24 hours

Tomas reported that the 24-72 hours that elapsed between when the blood was taken and the test was done changed things –  and suggested that the Myhill group should have expected that it would.

For one, they expected that the neutrophils – the immune cells being assessed in the papers – would become activated and then die. Apparently, once activated, neutrophils survive just a day or two, and they can be activated very easily. Tomas wasn’t worried about a little activation – they were concerned “most of the granulocytes would be lost” in the 24 hour or so delay between sample acquisition and processing.  (Neutrophils are a granulocyte.)

Plus, once neutrophils (like all immune cells) get activated, they burn up a lot of energy – thus potentially depleting ATP. (The MyHill studies used neutrophils but in the Acumen lab McClaren switched from using neutrophils to PBMC’s a couple of years ago. No results from that lab have been published.)

Testing fresh blood vs blood that sat for 24 hours made all the difference. Both ME/CFS patient and healthy control blood which sat for a lengthy period of time had lower ATP concentrations. ME/CFS and healthy control blood which was quickly tested, on the other hand, had normal ATP concentrations.

Tomas did more than test the test, however. They tested whether their concerns – that neutrophils would drop out over time  – were valid. Using flow cytometry to measure the cell types present in fresh blood and in blood which sat over time, they found evidence that neutrophils – the immune cells being tested for ATP concentrations –  were indeed dropping out over time.

Another basic issue – glucose depletion – came to the fore as well. Apparently, unless cells in a test tube are “fed”, their glucose levels are expected to fall over time.  Tomas found that glucose levels in the test tubes which sat did indeed drop significantly – and quickly. It took a mere 2 hours for glucose levels – the main fuel for the mitochondria –  to become significantly depleted. 

glucose level drops

Glucose declines over the 24 hours samples sat in test tubes.

However, these issues didn’t explain why, if MyHill studies were done on both healthy controls (HC) and ME/CFS patients, the HC and ME/CFS samples would be processed differently.  It turned out that the ME/CFS samples were sent “through the post and processed between 24–72 hours” later while the healthy control blood  samples were collected in the laboratory and processed the same day. The Tomas group believes that this difference in sample processing made all the difference.

Tomas et. al. concluded that the ATP profiles test as now run is inaccurate and should not be used or trusted. They asserted that the test should only be run within an hour of sample collection – something that may not be possible, given how the test is conducted now.

The real blow to the test, though, came when Tomas was able to find no differences in ATP concentrations between the ME/CFS patients and the healthy controls, even when the test was run immediately on the samples. It should be noted that the effectiveness of the Myhill protocol does not hinge on the results of the McClaren test. Even if the test does not show mitochondria problems, the mitochondria could still be dysfunctional.

The study was small (10 patients and 13 healthy controls), but the authors believe it was large enough for them to call for doctors and patients to disregard the results of this test.

Dr. MyHil and John McClaren Respond

Dr. Myhill posted several responses to the paper on her website – none of which, unfortunately, addressed the issues raised in the Tomas paper. She reported that the testing was blinded, that the tests are less expensive than they might have been, that approximately 1000 tests have been run and that the tests are routinely run in quadruplicate, etc.

One statement, “The essence of this paper appears to be that tests of mitochondrial function are not relevant in the assessment of patients with CFS/ME,” is clearly incorrect as the Tomas group has found evidence of mitochondrial issues in ME/CFS and would surely welcome a commercial test that would show that. In fact, in the paper Tomas suggested that, “Other tests of
energetic dysfunction (in ME/CFS) could be developed using the Seahorse extracellular flux assay,” (but advised caution until it was clear what role cellular energy production problems play in ME/CFS).

Cellular Energy Production Takes Big Hit in Chronic Fatigue Syndrome (ME/CFS) Study

Dr. Myhill stated that she did offer to take blood from one patient and send samples to both laboratories to be tested the next day.  She reported that the Tomas group’s failure to take her up on her offer left her group with, “little confidence in the ability of the Tomas group to replicate the Acumen test.” Research groups, however, often have strict protocols which don’t allow for deviations, and it’s unclear what one paired test would have shown.

Dr. McClaren did respond to the central issue. He reported that during the initial testing he explored the effects that sample storage might have using samples that were processed within minutes and re-tested 6, 12, 24, 48 and 72 hours later. He stated that, provided that the samples were kept in the original “vacutainer” tubes and not subjected to extremes of temperature, there were only minor changes in the test results up to the 48 hour point. After that, some samples began to degrade, and he uses phase-contrast microscopy to separate out samples which have signs of cell damage.

His statement, “Test situations regarding time since venepuncture and storage/delivery methods continue to be explored” suggests, though, that not all the issues regarding sample processing have been explored.

Conclusion

Myhill ATP test results

Tomas believes differences in sample processing skewed the ME/CFS patients’ results in the three Myhill studies. John McClaren asserted he tested storage effects but noted that he was continuing to study the matter.

It makes sense that McClaren would assess the effects of storage, as he developed the test, and it’s not clear why the two groups have different findings. Still, with the publication of the Tomas paper, the burden of proof now appears to fall on the Myhill group to find a way to rebut the Tomas finding – probably with a peer-reviewed paper which validates their current testing regimen (using PBMC’s).

That may not be easy. Cara Tomas is a recent PhD who’s been racking up research studies and Julia Newton is a well published researcher. Dr. Myhill, a doctor, and John McClaren, the director of the Acumen lab, are more lay researchers whose output appears limited to their three ME/CFS papers.

Is this the case of well-intentioned efforts gone awry? In retrospect, perhaps the Myhill study results always were a bit too impressive for this heterogeneous disease. Only one of 71 ME/CFS patients in the first study had a normal result and all 138 patients in the second study demonstrated mitochondrial abnormalities which correlated with illness severity to boot.

Efforts to understand the role the mitochondria play in ME/CFS continue, but most recent studies from researchers using the Seahorse machine and other approaches have failed to find evidence of reduced ATP production in ME/CFS when the mitochondria are tested in isolation from the blood. Five studies have found normal ATP production, one has found increased ATP production and one (Tomas) has found reduced ATP production.

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