Identification of Stages of Erythroid Differentiation in Bone Marrow and Erythrocyte Subpopulations in Blood Circulation that Are Preferentially Lost in Autoimmune Hemolytic Anemia in Mouse
Mouse erythrocytes were labeled with biotin in vivo by the two step biotinylation procedure. DIB labeled mice were given intraperitoneal injections of 2x108 rat erythrocytes weekly for 5–6 weeks to induce AIHA. The experimental protocol is given in panel A. Blood samples were collected from control and AIHA-induced mice 3 days after the 5th and 6th injections (6 and 13 days after 2nd biotinylation step). Erythrocytes were stained ex vivo with streptavidin-APC and anti-mouse CD71-PE, and proportions of the different age cohorts were determined. Representative flow histograms showing the proportion of different age groups of erythrocytes after 5 doses of injections are shown in panels B (control) and C (AIHA-induced). Turnover profile of reticulocytes (panel D; p = 0.02, ANOVA), biotinnegative young erythrocytes (panel E; p = 0.046, ANOVA), biotinlow intermediate age group of erythrocytes (panel F; p = 0.010, ANOVA) and biotinhigh old erythrocytes (panels G; p<0.001, ANOVA) in control and AIHA-induced mice have been shown. Each bar on the graph represents mean ± SEM of observations. n = 8 control and 12 AIHA-induced mice. *p<0.05, **p<0.01 and ***p<0.005 for comparison of the groups (Student t-test).