Assessment of survival of aging erythrocyte in circulation and attendant changes in size and CD147 expression by a novel two step biotinylation method.

Three intravenous injections (1mg each) of biotin-X-NHS (BXN) given at 24h intervals labeled all circulating erythrocytes with biotin in C57Bl/6 mice. After 5 days, administration of another i.v. injection of BXN (0.6mg) resulted in the labeling of erythrocytes released in blood circulation after the first biotinylation step, with a lower intensity of biotin. The older erythrocyte population with high intensity of biotin (biotin(high) population) and the later population of newly formed erythrocytes with lower intensity of biotin (biotin(low) population) could be stained with streptavidin-APC (SAv) and identified by flow cytometry. Using the double biotinylation technique, we could examine the survival and age related changes in biotin(low) population of erythrocytes that was released in circulation during a defined time period (5 days). Our results indicate that the percentage of Biotin(low) erythrocytes in circulation remained static for 10 days after the second biotinylation step and than started to decline steadily with time. Mean fluorescence intensity of biotin label on surviving biotin(low) population of erythrocytes however remained stable. These results suggest that after 15 days of release in blood, erythrocytes may undergo random destruction. Furthermore, forward scatter as well as CD147 expression of Biotin(low) population also declined with age. Double biotinylation technique described in this communication offers an easy method for tracking age related changes in populations of erythrocytes released in circulation during a defined period of time.