Figure 15.

Flow cytometry identification of presence and size of RBC vesicles based on the light scatter parameters and quantification of RBC vesicles based on marker for phospholipid PS and glycophorin A (CD235A) using flow cytometry. (A) Light scatter results using side scatter (SSC) as the indicator of shape and forward scatter (FSC) the indicator of size. Top left panel shows light scatter parameters of the mixture of standard calibration beads (7.6, 3, 0.9, and 0.5 μm). Other panels show light scatter parameters of the blood samples before mechanical shear (no-shear), and after being subjected to different frequencies, cycles, and degrees of oscillatory shear. Inspection of the scatter from the calibrated beads and the blood subjected to mechanical shear indicates the increasing presence of vesicles below 1 μm in size. Direct size comparison cannot be made between beads and vesicles, since beads have a higher index of refraction, and therefore lower size threshold, than vesicles (Lacroix et al., 2010; Yuana et al., 2011). (B) Flow cytometry analysis of RBC vesicles extracted from blood before oscillatory shear (no-shear), and after being subjected to different frequencies and number of cycles of oscillatory shear. Only events positive to anti-CD235a and Annexin V were defined as RBC vesicles (Kriebardis et al., 2008). The number of calibration beads was counted to determine the absolute number of RBC vesicles. The number of cells was approximately 10⁷.