Participant characteristics

Table 1 shows the number of participants, age and gender for the control and CFS/ME groups in this study.

Table 1 Participant characteristics. Full size table

Myhill test

For each of the parameters calculated in this study using the MES protocol results are shown as a combination of fresh and frozen cells for the healthy control and CFS/ME cohorts. There were no significant differences between fresh and frozen neutrophils or PBMCs for any of the parameters in either healthy controls or CFS/MS patients (Supplementary Information). Therefore, the fresh and frozen samples were pooled for the experiments described below and shown in Fig. 1.

Figure 1 ATP parameters calculated using the protocol from the Myhill test. ATP concentration in (A) neutrophils (p = 0.295) and (B) PBMCs (p = 0.059) of CFS/ME patients and healthy controls in the presence of excess magnesium. ATP concentration in the absence of excess magnesium in (C) neutrophils (p = 0.737) and (D) PBMCs (p = 0.947). Ratio of ATP concentration in cells with endogenous magnesium to ATP concentration in cells with excess magnesium in (E) neutrophils (p = 0.337) and (F) PBMCs (p = 0.314). ADP to ATP efficiency in (G) neutrophils (p = 0.054) and (H) PBMCs (p = 0.550). CFS/ME n = 10; control n = 13. Groups were compared using Welch’s t-tests. Full size image

ATP concentration in the presence of excess magnesium

The first experiment investigated the ATP concentration in neutrophils and PBMCs in the presence of excess magnesium in CFS/ME patients and healthy controls (Fig. 1A,B). Magnesium is required for the intracellular production of ATP. The addition of magnesium to the cells eliminates the effect of differing inter-participant magnesium levels. Results showed there to be no difference in the concentration of ATP between CFS/ME patients and controls in either neutrophils (p = 0.295) or PBMCs (p = 0.059).

ATP concentration in the presence of endogenous magnesium

ATP concentration was investigated in the absence of excess magnesium. Magnesium deficiencies have previously been reported in a subset of CFS/ME patients10,11, however, this was not shown in all studies investigating magnesium status in CFS/ME12. Magnesium is required as a co-factor for the production of ATP, therefore, the measurement of ATP production with only endogenous magnesium present in the cells gives a more accurate indication of the ATP actually being produced by the cells as opposed to the potential capacity the cells have for ATP production which is shown when excess magnesium is added13. Results showed there to be no difference in the concentration of ATP between CFS/ME patients and controls in either neutrophils (p = 0.737) or PBMCs (p = 0.947) (Fig. 1C,D).

ATP ratio

A ratio was calculated of ATP with endogenous magnesium to ATP with excess magnesium. This shows the proportion of ATP that is available as an energy supply as it is the proportion of ATP complexed with magnesium to form MgATP. The ratio was calculated for CFS/ME patients and controls in both neutrophils and PBMCs. There was no significant differences in either cell type (p = 0.337 and p = 0.314 respectively), Fig. 1E,F.

ATP to ADP efficiency

The final part of the Acumen protocol studied involved investigating the efficiency of the conversion of ADP to ATP. This gives us an estimate of the efficiency at which ADP can be recycled to form ATP to provide cellular energy. There were no significant differences between the CFS/ME and control cohorts in either the neutrophil (p = 0.054) or PBMCs ADP to ATP efficiency (p = 0.550), Fig. 1G,H. In their protocol Acumen refer to this parameter as ‘ADP to ATP efficiency’ however, in their publications on this work7,8,9 they refer to the same parameter as OXPHOS. The label OXPHOS is misleading as the equation relies on the recovery rate after the inhibitor has been removed and therefore while OXPHOS is a component of the equation, it is not represented by the final numerical value. As long as the % recovery rate is the same between two samples then they will have the same value of ADP to ATP efficiency regardless of the % decrease in ATP caused by the addition of the OXPHOS inhibitor as illustrated in Fig. 2. This parameter cannot be called OXPHOS, as it is in the papers published by the Myhill group.

Figure 2 Graphs of equal ADP to ATP efficiency with different profiles. This shows the importance of looking at the % drop in ATP after the addition of the inhibitor sodium azide to look at the reliance of cells on OXPHOS. Full size image

The % ATP inhibited (the first component of the ADP to ATP efficiency equation) may be used as a more accurate representation of OXPHOS as it shows the % of ATP inhibited when OXPHOS is inhibited and therefore gives an indication of OXPHOS. However, the value of ATP made by mitochondria after the inhibitor is removed (c) will be influenced by substrate flow into the mitochondria. Hence a reduction in (c) may not be a direct effect of mitochondrial dysfunction but could be linked to substrate supply into the mitochondria. % ATP inhibited is used in Booth et al. and Myhill et al.9, although it is still used alongside the “OXPHOS” parameter in these publications8,9. The % ATP inhibited can often tell a vastly different story than ADP to ATP efficiency (called OXPHOS by Myhill et al.) with neutrophils and PBMCs showing similar values for ADP to ATP efficiency (Fig. 1G,H) but very different values for % ATP inhibited by sodium azide (Fig. 3) indicating differing pathways of ATP production in the two cell types. Figure 3 shows that a significantly higher % of ATP is inhibited in the PBMCs than in neutrophils which suggests that PBMCs in freshly isolated blood rely on OXPHOS for ATP production to a much greater extent that neutrophils (p < 0.001). This is in keeping with other previous work14,15,16,17,18 that has shown neutrophils to be predominantly glycolytic cells.

Figure 3 Percentage of ATP inhibited in neutrophils and PBMCs when OXPHOS inhibitor sodium azide was added. Calculated as the amount of ATP after the addition of sodium azide as a percentage of ATP before cells are treated. This shows how reliant the cells are on OXPHOS as an energy source. A lower % ATP inhibition shows a lower reliance on OXPHOS. N = 23. There were no significant differences between CFS/ME and healthy control % ATP inhibition for either cell type (Supplementary Information S3). Full size image

ADP/ATP translocator protein

The part of the protocol investigating ADP/ATP translocator activity was not able to be conducted. Key reagents specified in the Acumen protocol for these experiments were no longer available. Replacements were offered for these products on the manufacturers website, however, as the purpose of this study was to investigate the validity of the Acumen protocol in the diagnosis of CFS/ME, it was decided that it would be inappropriate to use other reagents not specified by the Acumen protocol.

Effect of delayed cell isolation

Acumen analyse blood samples that are 24–72 hr post phlebotomy7. We had concerns that this time frame was too long for neutrophils which would likely become activated19,20 and most of the granulocytes would be lost in that time period which may account for the differences between our results (Fig. 1) and the results shown by Myhill et al.7,8,9. CFS/ME patients and controls could also differ in the degree of neutrophil activation over time and as this is an unknown variable could compromise the interpretation of the results (i.e. assessing changes in the collection tube rather than differences between the CFS/ME patients and controls in vivo). We isolated the cell fractions from fresh blood and blood that had been left for 24 hrs (similar to Myhill test samples) in order to determine the effect of delayed cell isolation. We first analysed the samples by Flow Cytometry (FACS), a useful tool that separates cells based on their size and internal composition (granularity). The results are shown in Fig. 4.

Figure 4 Cells were analysed using FACS. (A) shows whole blood 1 hr after isolation following RBC lysis. Three distinct populations of cells consisting of neutrophils, monocytes and lymphocytes are observed. (B,C) were isolated using HistopaqueTM (B) =PBMC (monocytes plus lymphocytes) and (C) =neutrophil fractions 1 hr after isolation. (D–F) are comparable to (A–C) but 24 hrs after isolation. Full size image

Figure 4B is the 1 hour PBMC fraction from a Histopaque™ separation and the scatter plot shows no neutrophil like cells. Figure 4C is the 1 hour neutrophil layer from a Histopaque™ separation and it shows contamination with lymphocytes and other cellular debris. Figure 4D–F depict cells that were isolated from blood that had spent 24 hrs in heparin at room temperature. The whole white cell fractions in (A) < 1 hr isolation and (D) 24 hr isolation show different proportions of neutrophils and lymphocytes in the respective quadrants suggesting that the longer processing time has altered cell properties. This could change the properties of the cells in the samples isolated at 24 hrs so they no longer reflect cellular function at the time of harvesting. This further complicates the interpretation of the data as the assumption is made that both controls and ME/CFS patients cells behave in the same way after incubation in a blood tube for 24 hrs. In addition, in the Histopaque™ gradients the 24 hr cell populations neutrophil fraction (F) looks quite different to the 1 hr samples (C) with a large drop in neutrophil numbers. Overall, Fig. 4 highlights the importance of isolating the cells from fresh blood. If blood has been left for an extended period of time cells are lost with further processing using Histopaque™, with additional changes in cell morphology possibly due to activation.

Metabolites linked to the TCA and glycolytic cycles are changed significantly with a general increase in mitochondrial respiration as glucose levels are reduced21 and significant changes in metabolism (Morten & Mccullagh Metabolomics unpublished). Once a blood sample is taken as nutrient levels are not replenished and the millions of cells in the sample will deplete nutrients such as glucose over time, which will likely have a significant effect on cellular metabolism. We have studied glucose depletion in control blood samples. In Fig. 5 we demonstrated that in all blood collection systems glucose levels dropped to below 1 mM over 24 hrs. Even after 2 hrs collection blood glucose levels showed a significant drop in glucose levels. 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.

Figure 5 Glucose concentration of whole blood from healthy controls over 24 hours. First reading was within 15 minutes of donation. Control n = 4, groups where compared using Welch’s t-test. Full size image

The results shown in Figs 4 and 5 indicated that these types of experiments should be carried out on cells that had been isolated from fresh blood i.e. <1 hr post sampling, which was not the case for samples used by Myhill et al.7. It should be noted that all of the samples shown in Fig. 1 were isolated immediately after blood collection.

Effect of delayed cell isolation on Myhill test

Given the differences seen between the properties of cells isolated from whole blood at different time points, we repeated the Myhill test using bloods taken from 6 healthy controls with one tube of blood processed immediately and the other tube processed 24 hours after blood collection. The results for each of the parameters for PBMCs and neutrophils are shown in Fig. 6.

Figure 6 ATP parameters in cells isolated immediately and 24 hours after blood collection. ATP concentration in (A) neutrophils and (B) PBMCs of healthy controls in the presence of excess magnesium. ATP concentration in the absence of excess magnesium in (C) neutrophils and (D) PBMCs. Ratio of ATP concentration in cells with endogenous magnesium to ATP concentration in cells with excess magnesium in (E) neutrophils and (F) PBMCs. ADP to ATP efficiency in (G) neutrophils and (H) PBMCs. Control n = 6. Groups were compared using paired t-tests. Full size image

The results show a significant reduction in ATP when the cells were isolated 24 hours after collection in neutrophils (p < 0.001) and PBMCs (p < 0.008) in both the excess and endogenous magnesium experiments. Leaving the blood whole for 24 hours also caused a reduction in the ATP ratio in neutrophils (p = 0.015) but not in PBMCs (p = 0.490). The longer sample processing time significantly lowered ADP to ATP efficiency in both neutrophils (p = 0.050) and PBMCs (p = 0.005). This implies that the lower ATP seen in CFS/ME cells by the Myhill group, may be due to the differences in sample handling between the CFS/ME and control cohorts or a difference in controls and CFS/ME cell function over 24–48 hrs. Our results suggest that the fact that the samples from CFS/ME patients are sent through the post and processed between 24–72 hours after blood is taken, while the control blood are collected in the laboratory on the same day as processing, may be the cause of the differences seen between the two cohorts by the Myhill group. An alternative explanation would be the CFS/ME patient and control samples behave very differently during prolonged storage.

Neutrophils, as used by Myhill et al., are short lived cells that only last for between 5–90 hours in the circulation22. In the context of using neutrophils as an indicator of mitochondrial function it is important to consider that neutrophils in freshly isolated blood sample will be in a non-activated state, which have very different energetic demands to those that have been activated by pathogens23.