Expanded erythropoietic activity in the BM, extramedullary splenic erythropoiesis, and erythroid oxidative stress are characteristics of SCD mice. (A) Cytofluorimetric analysis of total erythroid precursors from the BM and the spleen of AA and SS mice using a CD44-Ter119 gating strategy in BM and spleen. (B) Erythroblast subpopulations determined by CD44-Ter119 gating strategy. Population I corresponds to proerythroblasts, population II corresponds to basophilic erythroblasts, population III corresponds to polychromatic erythroblasts, and population IV corresponds to orthochromatic erythroblasts. The percentage of subpopulations was analyzed. (C) ROS levels in erythroid precursors from the BM and the spleen were analyzed. (D) Amount of Annexin V⁺ 7AAD⁻ or 7AAD⁺ total erythroblasts (CD45⁻ TER119⁺ CD44⁺ FSC^hi cells) from the BM and the spleen were analyzed. (E) Amount of Annexin V⁺ orthochromatic erythroblasts (E; CD45⁻ TER119⁺ CD71⁺ FSC^low cells) from BM, using the CD71-TER119 cytofluorimetric strategy, as shown in supplemental Figure 4B, was analyzed. All bar graphs are mean ± standard error of the mean (n = 6 for each experiment; 14-16 weeks old; 4 female mice and 2 male mice). *P < .05, Student t test.