Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, debilitating disease with inconclusive causes and no laboratory-based diagnostic tests. Oxygen tension (PO2)-regulated red blood cell (RBC) capillary velocity has emerged recently as a new mechanism regulating cerebral capillary perfusion and measures RBC responses to local hypoxia. Such intrinsic RBC responses to PO2 changes may change distinctly under different pathological conditions and thus represent a new RBC-based approach for disease diagnosis. Indeed, our preliminary results showed that RBCs collected from ME/CFS patients exhibited impaired velocity in a microfluidic capillary in response to reduced PO2 and PO2-regulated RBC capillary velocity was improved significantly when patients received craniocervical instability surgery, strongly suggesting that PO2-regualted RBC capillary velocity may represent a new characteristic of ME/CFS that might be used to diagnose ME/CFS and measure ME/CFS progression. Here, we extend our preliminary study and propose an interdisciplinary approach combing microfluidics, machine learning and RBC cytokine assay to examine rigorously the accuracy of PO2-regulated RBC capillary velocity as a new laboratory test for ME/CFS and investigate mechanistically the roles of RBC cytokine signaling in ME/CFS. Aim 1 will measure PO2-regulated RBC capillary velocity using RBCs from 96 participants and compare the results of ME/CFS patients with age, gender and race-matched healthy controls. Furthermore, we will develop machine-learning algorithms to establish a diagnostic classifier to validate PO2-regulated RBC capillary velocity as a new laboratory test for ME/CFS and assess its feasibility to differentiate ME/CFS patients with different disease severity. Mechanistically, we will examine hemoglobin-band 3 interactions in RBCs from ME/CFS patients and examine their correlations with increased oxidative stress in ME/CFS. Aim 2 will quantify the cytokine profile in RBCs from ME/CFS patients and compare them with age, gender and race-matched healthy controls. Furthermore, we will alter the RBC cytokine profile and measure the corresponding changes of PO2-regulated RBC capillary velocity to examine whether RBC cytokine profile plays a role in the modulation of PO2-regulated RBC capillary velocity. These experiments will provide a previously unrecognized RBC cytokine signaling in ME/CFS and add new insights to the immune dysregulation in ME/CFS. Together, the proposed studies exploit new tools and technology to develop a new laboratory test for ME/CFS and reveal mechanistims underlying PO2-regulated RBC capillary velocity in ME/CFS, which we believe will directly improve the diagnosis and treatment of ME/CFS.

Project Narrative Identifying new laboratory tests that can measure the progression of ME/CFS will directly improve the diagnosis and treatment of ME/CFS and contribute to our understanding of mechanisms underlying ME/CFS. This research develops an interdisciplinary approach to assess the recently discovered, oxygen tension-regulated red blood cell capillary velocity as a new laboratory test for ME/CFS and investigate previously unrealized red blood cell cytokine signaling in ME/CFS. Because ME/CFS is a poorly understood, complex, debilitating disease with no specific laboratory diagnostic tests yet, we believe the proposed project is directly relevant to the NIH's mission and should be broadly valuable for research efforts and clinical studies of chronic diseases including recently emerged long COVID.