Abstract: Objective To establish biotin labeling for in vitro erythrocyte technology platform to investigate the correlation between the 24-hour post-transfusion recovery （PTR_24h） of leukocyte-reduced suspended red blood cells （RBCs）stored at 4℃ for different times and the in vitro quality indexes such as apoptosis, membrane surface molecules and energy metabolism. Methods We set different NHS-biotin concentrations 0,1,2.5,5,7.5,10,12.5 μg/mL to label mouse red blood cells（about 1×10⁹/mL）,and determined the optimal labeling conditions by flow cytometry. Based on the optimal conditions,the mice RBCs stored at 4 ℃ for different storage times（0,2,4,6,8,10,12,14 d）were biotinylated and then infused into mice. After 24 hours,blood was collected and PTR_24h was calculated by flow cytometry. The changes of CD47 molecule,phosphatidylserine（PS）on the surface of erythrocyte membrane and intracellular ATP content at different storage time were also determined and analyzed. The correlation between PTR_24h in vivo and quality indexes of RBCs in vitro was analyzed by SPSS. Results The optimal concentration of biotin labeling was 7.5 μg/mL in 1×10⁹ RBCs/mL with positive labeling rate as high as 99%. Taking hemolysis rate and erythrocyte apoptosis rate as quality control,biotin labeling did not cause many RBCs rupture（hemolysis rate <1.5%） and obvious apoptosis. Compared to the mean fluorescence intensity（MFI） on fresh RBCs group,CD47 expression significantly declined from day 10（80.4%）（P<0.05）. External exposure of membrane PS increased to （0.7±0.0） % from day 8（P<0.05）. ATP decreased significantly from day 2,and the difference was statistically significant （P<0.05）. The PTR_24h in vivo showed a decreasing trend with extended storage time. PTR_24h values on day 10 （65.8±2.9）% were significantly lower than that of fresh RBCs（94.0± 0.6）%（P<0.05）,and less than 75%. The Pearson product-moment correlation coefficient showed that there was a strong positive correlation between CD47,ATP and PTR_24h,with r=0.954 （P<0.01） and r=0.938 （P<0.01）,respectively. And PS was negatively correlated with PTR_24h （r = -0.777, P<0.05）. Conclusion The expression levels of CD47,PS and ATP content of RBCs stored at 4℃ with different times were strongly correlated with PTR_24h. It can provide a reference for clinical evaluation of blood transfusion effectiveness.

Key words: Biotin labeling, Red blood cells, Post-transfusion recovery