BACKGROUND: Myalgic encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a poorly understood disease. Amongst others symptoms, the disease is associated with profound fatigue, cognitive dysfunction, sleep abnormalities, and other symptoms that are made worse by physical or mental exertion. While the etiology of the disease is still debated, evidence suggests oxidative damage to immune and hematological systems as one of the pathophysiological mechanisms of the disease. Since red blood cells (RBCs) are well-known scavengers of oxidative stress, and are critical in microvascular perfusion and tissue oxygenation, we hypothesized that RBC deformability is adversely affected in ME/CFS. METHODS: We used a custom microfluidic platform and high-speed microscopy to assess the difference in deformability of RBCs obtained from ME/CFS patients and age-matched healthy controls. RESULTS AND CONCLUSION: We observed from various measures of deformability that the RBCs isolated from ME/CFS patients were significantly stiffer than those from healthy controls. Our observations suggest that RBC transport through microcapillaries may explain, at least in part, the ME/CFS phenotype, and promises to be a novel first-pass diagnostic test.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is arguably the last major disease we know almost nothing about. It is a multi-systemic illness of unknown etiology affecting millions of individuals worldwide, with the capacity to persist for several years. ME/CFS is characterized by disabling fatigue of at least 6 months, accompanied serious fatigue and musculoskeletal pain, in addition to impaired short-term memory or concentration, and unrefreshing sleep or extended post-exertional. While the etiology of the disease is still debated, evidence suggest oxidative damage to immune and hematological systems as one of the pathophysiological mechanisms of the disease. Erythrocytes are potent scavengers of oxidative stress, and their shape changes appreciably in response to oxidative stress and certain inflammatory conditions including obesity and diabetes. The shape of erythrocytes change from biconcave discoid to an ellipsoid due shear flow in microcapillaries that provides a larger specific surface area-to-volume ratio for optimal microvascular perfusion and tissue oxygenation establishing the importance not only of total hematocrit but also of the capacity for large deformations in physiology. Clinically, ME/CFS patients show normal arterial oxygen saturation but nothing much is known about microvascular perfusion. In this work, we tested the hypothesis that the erythrocyte deformability in ME/CFS is adversely affected, using a combination of biophysical and biochemical techniques. We tested the deformability of RBCs using a high-throughput microfluidic device which mimics blood flow through microcapillaries. We perfused RBCs (suspension in plasma) from ME/CFS patients and from age and sex matched healthy controls (n=9 pairs of donors) through a high-throughput microfluidic platform of 5µm width and 3-5 µm height. We recorded the movement of the cells at high speed (4000 fps), followed by image analysis to assess the following parameters: entry time (time required by the cells to completely enter the test channels), average transit velocity (velocity of the cells inside the test channels) and elongation index (ratio of the major diameter before and after deformation in the test channel). We observed that RBCs from ME/CFS patients had higher entry time (~12%, p<0.0001), lower average transit velocity (~17%, p<0.0001) and lower elongation index (~14%, p<0.0001) as compared to RBCs from healthy controls. Taken together, this data shows that RBCs from ME/CFS patients have reduced deformability. To corroborate our findings, we also measured the erythrocyte sedimentation rate (ESR) for these donors which show that the RBCs from ME/CFS patients had lower (~40%, p<0.01) sedimentation rates. To understand the basis for differences in deformability, we investigated the changes in the fluidity of the membrane using a lateral diffusion assay using pyrenedecanoic acid (PDA), and observed that RBCs from ME/CFS patients have lower membrane fluidity (~30%, p<0.01). Apart from the fluidity, Zeta potential measurements showed that ME/CFS patients had lower net negative surface charge on the RBC plasma membrane (~18%, p<0.0001). Higher levels of reactive oxygen species (ROS) in RBCs from ME/CFS patients (~30%, p<0.008) were also observed, as compared to healthy controls. Using scanning electron microscopy (SEM), we also observed changes in RBC morphology between ME/CFS patients and healthy controls (presence of different morphological subclasses like biconcave disc, leptocyte, acanthocyte and burr cells; area and aspect ratio; levels of RBC aggregation). Despite these changes in RBC physiology, the hemoglobin levels remained comparable between healthy donors and ME/CFS patients. Finally, preliminary studies show that RBCs from recovering ME/CFS patients do not show such differences in cellular physiology, suggesting a connection between RBC deformability and disease severity. Taken together, our data demonstrates that the significant decrease in deformability of RBCs from ME/CFS patients may have origins in oxidative stress, and suggests that altered microvascular perfusion can be a possible cause for ME/CFS symptoms. Our data also suggests that RBC deformability may serve as a potential biomarker for ME/CFS, albeit further studies are necessary for non-specific classification of the disease. Disclosures No relevant conflicts of interest to declare.

Red blood cell biomechanics can provide us with a deeper understanding of macroscopic physiology and have the potential of being used for diagnostic purposes. In diseases like sickle cell anemia and malaria, reduced red blood cell deformability can be used as a biomarker, leading to further assays and diagnoses. A microfluidic system is useful for studying these biomechanical properties. We can observe detailed red blood cell mechanical behavior as they flow through microcapillaries using high-speed imaging and microscopy. Microfluidic devices are advantageous over traditional methods because they can serve as high-throughput tests. However, to rapidly analyze thousands of cells, there is a need for powerful image processing tools and software automation. We describe a workflow process using Image-Pro to identify and track red blood cells in a video, take measurements, and export the data for use in statistical analysis tools. The information in this protocol can be applied to large-scale blood studies where entire cell populations need to be analyzed from many cohorts of donors.

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The role played by the nucleus in cell migration remains poorly understood. Recent works have described different ways in which the nucleus controls cell migration in 3D environments, such as, e.g., mechanosensing, force transmission, or restriction of motion in dense extracellular networks. However, the role of the nucleus in 2D cell migration is more controversial. The literature has usually assumed that the nucleus behaves as a passive element without an active role. Hence, most computational models of cell migration do not consider the nucleus. Some papers have shown that intact cells are faster than enucleated cells, despite the biological mechanisms driving cell motion are identical. These results suggest that the presence of the nucleus inside the moving cell cannot be neglected. Here, we study the role played by the nucleus in 2D migration of fish keratocytes by way of a computational model. Our model incorporates the nucleus dynamics into well established computational mechanistic models of keratocyte migration. The model resorts to two phase-field variables to track the cell and the nucleus. Thus, we can locate the actin and myosin dynamics to the cytosol (i.e., inside the cell and outside the nucleus). We employ two force balance equations that account for the forces acting on the nucleus and the actin network. Our simulations show a realistic picture of keratocyte migration on planar substrates. The model results provide insights into the role played by the nucleus in 2D cell migration. 600-PosSpreading Out: Modeling the Physics of Cell-Substrate Interaction in Cell Spreading and Focal Adhesion Evolution