Assessing cellular energy dysfunction in CFS/ME using a commercially available laboratory test

Abstract

The mitochondrial energy score (MES) protocol, developed by the Myhill group, is marketed as a diagnostic test for chronic fatigue syndrome/Myalgic Encephalomyelitis (CFS/ME). This study assessed the reliability and reproducibility of the test, currently provided by private clinics, to assess its potential to be developed as an NHS accredited laboratory test. We replicated the MES protocol using neutrophils and peripheral blood mononuclear cells (PBMCs) from CFS/ME patients (10) and healthy controls (13). The protocol was then repeated in PBMCs and neutrophils from healthy controls to investigate the effect of delayed sample processing time used by the Myhill group. Experiments using the established protocol showed no differences between CFS/ME patients and healthy controls in any of the components of the MES (p ≥ 0.059). Delaying blood sample processing by 24 hours (well within the 72 hour time frame quoted by the Myhill group) significantly altered many of the parameters used to calculate the MES in both neutrophils and PBMCs. The MES test does not have the reliability and reproducibility required of a diagnostic test and therefore should not currently be offered as a diagnostic test for CFS/ME. The differences observed by the Myhill group may be down to differences in sample processing time between cohorts.

Conflict of interest statement

The authors declare no competing interests.

Figures

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.

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.

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).

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.

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.

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.