Abstract
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex neuroimmune disorder characterized by numerous symptoms of unknown etiology. The ME/CFS immune markers reported so far have failed to generate a clinical consensus, perhaps partly due to the limitations of biospecimen biobanking. To address this issue, we performed a comparative analysis of the impact of long-term biobanking on previously identified immune markers and also explored additional potential immune markers linked to infection in ME/CFS. A correlation analysis of marker cryostability across immune cell subsets based on flow cytometry immunophenotyping of fresh blood and frozen PBMC samples collected from individuals with ME/CFS (n = 18) and matched healthy controls (n = 18) was performed. The functionality of biobanked samples was assessed on the basis of cytokine production assay after stimulation of frozen PBMCs. T cell markers defining Treg subsets and the expression of surface glycoprotein CD56 in T cells and the frequency of the effector CD8 T cells, together with CD57 expression in NK cells, appeared unaltered by biobanking. By contrast, NK cell markers CD25 and CD69 were notably increased, and NKp46 expression markedly reduced, by long-term cryopreservation and thawing. Further exploration of Treg and NK cell subsets failed to identify significant differences between ME/CFS patients and healthy controls in terms of biobanked PBMCs. Our findings show that some of the previously identified immune markers in T and NK cell subsets become unstable after cell biobanking, thus limiting their use in further immunophenotyping studies for ME/CFS. These data are potentially relevant for future multisite intervention studies and cooperative projects for biomarker discovery using ME/CFS biobanked samples. Further studies are needed to develop novel tools for the assessment of biomarker stability in cryopreserved immune cells from people with ME/CFS.
Highlights
Myalgic Encephalomyelitis (ME), known as Chronic Fatigue Syndrome (CFS), is a multisystem, complex and extremely debilitating chronic illness
The validation of CD25 and CD69 expression in NK cells as potential markers in frozen cells was clearly compromised by the inconsistent and highly variable levels of expression observed in the biobanked cells, which effectively ruled out their use in future Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) studies with frozen samples
Our study suggests for the first time that peripheral blood mononuclear cell (PBMC) sample biobanking impacts some of the previously defined immune markers which may contribute to the diagnosis and prognosis of ME/CFS
Summary
Myalgic Encephalomyelitis (ME), known as Chronic Fatigue Syndrome (CFS), is a multisystem, complex and extremely debilitating chronic illness. The 2015 Institute of Medicine (IOM) committee report estimated that ME/CFS could potentially affect up to 2.5 million people in the US, and as many as 17-24 million worldwide, of whom roughly 90% are undiagnosed [1]. There is no proven specific cause, no accurate and objective diagnostic test, and no universally effective treatment for ME/CFS. Beyond the puzzling post-exertional malaise as the cardinal symptom that cannot be alleviated by rest, ME/CFS patients present a plethora of unspecific symptoms including debilitating fatigue, unrefreshing sleep, gastrointestinal problems, orthostatic intolerance, cognitive impairments, and pain/myalgia that change in frequency and severity over time and differ from patient to patient [2]. The immunological basis of the disease is highlighted by evidence linking it to autoimmunity, and by the potential role of pathogens as triggers [4,5,6], leading to the general belief that some disrupted immunometabolic and neuroinflammatory pathways may be associated with ME/CFS [7,8,9]
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