Abstract: Objective To investigate the effect and mechanism of extracellular vesicles derived from erythrocytes on erythrocytes storage damage. Methods A sufficient amount of qualified erythrocytes suspension samples were collected to prepare the extracellular vesicle products derived from qualified erythrocytes, determine the particle size distribution of extracellular vesicles, and draw the relationship between storage time and. western blot was used to detect the expression of CD63, TSG101, Cal, Gly-A, and GAPDH in extracellular vesicles. Dilute the erythrocytes suspension using a NaCl concentration gradient, measure the absorbance values of erythrocytes solutions stored for different days, obtain their osmotic fragility related indicators, record the data, and draw a curve between absorbance and erythrocytes storage time. The hemorheology indexes of erythrocytes under different storage times were detected by automatic hemorheometer to determine the deformability of erythrocytes, record the data and draw the relationship curve between the deformability of erythrocytes and different storage times. Finally, using storage time as the independent variable, analyze whether there is a correlation between the diameter of extracellular vesicles and the physical properties of erythrocytes. Results (1) As the storage time of red blood cell suspension increases, the diameter of the main particles of RBC-EVs decreases, but there is an abnormal increase in the main particles of RBC-EVs between 7 and 11 days. (2) RBC-EVs contain proteins from parental cells, and the expressions of CD63, TSG101, Gly-A, and GAPDH are all positive. (3) As the in vitro storage time increases, the osmotic fragility of erythrocytes increases, and their tolerance to low osmotic solutions weakens. (4) As the in vitro storage time increases, the deformation index and rigidity index of erythrocytes increase, and the deformability of erythrocytes decreases. (5) There is a significant correlation between RBC-EVs and the physical properties of erythrocytes, with the storage time of erythrocytes as the independent variable. Conclusion There is an impact between extracellular vesicles derived from erythrocytes and the storage damage of erythrocytes , and the generation of extracellular vesicles is accompanied by the storage damage of erythrocytes simultaneously.

Key words: Erythrocytes, Extracellular vesicles, Storage damage, Oxidative stress