Red blood cell transport velocity in microchannels indicates the growth of abnormal subpopulation under oxidative stress

Abstract

Background Transformation of red blood cells (RBCs, erythrocytes) during oxidative stress is a widespread phenomenon, which can be observed during natural aging of the cells, bank storage, under pathological conditions, and action of xenobiotics. Accumulated disturbances affect the deformability of RBCs, which slow down or even completely prevent their capillary passage. Here we investigated the transport of erythrocytes in microfluidic channels under the graduated oxidative stress. Material and Methods We used the extended towards low oxidant concentrations (0.1-1.5 mM) model of oxidative stress under the action of tert-butylperoxide (tBH), in vitro. In the developed microfluidic device with an array of 1.5x8x200 µm channels, we measured RBCs transit time by optical microscopy with further video analysis. This data was compared with flow cytometry, confocal microscopy, and AFM measurements to monitor abnormalities in erythrocytes under oxidative stress. Results The transit time measurements in the microfluidic device showed that under oxidative stress normal distribution of RBCs velocities became bimodal (Fig. 1). Increasing the concentration of tBh the number of cells with lower transit velocity increased. The flow cytometry and fluorescent microscopy showed that the oxidative stress led to the appearance of the group of cells with a smaller size (Fig. 2) and increased fluorescence of eosin-5-maleimide (EMA) that is caused by the clusterization of the transmembrane complex of band3. Moreover, the AFM force curve measurements indicated that the oxidative stress made the cells 2.9 times more rigid and stickier than the control ones. We assume that such changes directly influence the ability of RBCs to pass through microchannels. Conclusion Our data showed that the oxidative stress influences RBCs non uniformly, which leads to the division of all the cells into two subpopulations that differ in transit velocities in the microchannels, size, and membrane structure. The results show that erythrocytes velocity measurement in microfluidic devices is a promising technique for testing the degree of RBCs microrheological disorders during blood storage and aggressive drug therapy, including chemotherapy.