Optics-Based Assessment of Red Blood Cell Adaptations to Ercise: Insights from Ektacytometry and Flow Cytometry

Abstract. Red blood cells (RBC) are highly adaptable, particularly during ercise, when their membranes and cytoskeleton experience various mechanical and biochemical changes. Despite the crucial role of these adaptations, the impact of ercise-induced changes on RBC deformability and their susceptibility to oxidative stress remains poorly understood [1, 2], highlighting the need for further research and implementation of novel methodological approaches. This study investigated the potential of ektacytometry and flow cytometry as label-free optics-based methods to assess the effects of ercise on RBC deformability to dynamically changing flow conditions and erythrocyte response to oxidative stress. Blood samples were collected from five national-level basketball players at three time points: immediately after (IAT), 24 hours (24h), and 48 hours (48h) after training. Five healthy, age-matched sedentary individuals served as controls. Erythrocyte deformability was assessed using ektacytometry, measuring the elongation index (EI) under increasing shear stress based on laser diffraction patterns. To determine the level of oxidative stress, the autofluorescence of RBC was analyzed by using flow cytometry to measure the fluorescence intensities in spectral channels which corresponds to the excitation of 488/10 nm and emission wavelengths: FL1 (530/30 nm) and FL2 (585/40 nm). Both experiments were conducted under the baseline conditions and upon oxidation induced by 0.5 mM tert-butyl hydroperoxide (TBHP). Ektacytometry showed increased RBC deformability in athletes compared to control, evidenced by lower shear stress values at half-maximal EI (IAT= 3.09±7.8, 24h = 2±0.4, 48h = 2.17±0.4, CTRL = 9.36±6.6). Flow cytometry revealed lower autofluorescence in FL1 at IAT (1.92±0.1) and 24h (1.74±0.1) compared to CTRL (2.56±0.2), but highest at 48h (3.04±0.7). In the FL2 channel athletes showed lower autofluorescence than controls (IAT = 2.63±0.1, 24h = 1.81±0.2, 48h = 2.07±0.1 and CTRL = 2.93±0.1). Upon oxidation with TBHP autofluorescence was expectedly higher but followed the same pattern as under the baseline conditions, suggesting the protective effects of ercise against oxidative stress. These findings demonstrated the protective effects of ercise against oxidative stress and enhanced RBC deformability. Overall, the results emphasize the potential of label-free optics-based approaches for fast and useful diagnostics and assessment of ercise adaptations.