Light on abnormal red blood cell subpopulations: Label-free optics-based approach for studying in vitro rigidified...

RBCs deformability plays a crucial role in maintaining proper blood flow and oxygen delivery throughout the body. Conventional ektacytometry fails to differentiate between variations in deformability of RBC subpopulations as the averaging measurement process obscures these differences. In this study, we introduced an approach that integrates label-free optics-based techniques (flow cytometry, phase-contrast, and two-photon excitation fluorescent microscopy) with ektacytometry to evaluate subpopulations that exhibit decreased RBCs deformability upon an in vitro oxidation using 0.5 mM TBHP, as a low-level oxidative agent. We found that flow cytometry can easily detect rigidified and oxidized subpopulations based on FSC/SSC light distribution, as well as RBCs fluorescence intensity and peak area likely originating from hemoglobin photo and/or degradation products. Two-photon excitation fluorescence microscopy proved altered morphology and spatial location of fluorescence intensity signal near the membrane of oxidized RBCs, when compared to control RBCs, indicating a link with the reduced deformability. The proposed label-free optics-based methodology, which combines established techniques with more sophisticated microscopy, emerges as a promising tool for detecting mechano-biological changes in different RBC subpopulations induced by oxidative stress. The findings suggest potential applications in clinical practice for monitoring pathological conditions influenced by physical or environmental stress and as a quality control measure for stored RBCs.