Reduced hemolysis and IE in Hbb^th3/+ mice with the Hbb diffuse haplotype. (A) Erythroid indices (y-axis) according to genotype (x-axis) in 8-week-old mice. Hbb^th3/+ single (sing), n = 15; Hbb^th3/+ diffuse (diff), n = 15. (B) Circulating RBC survival determined after biotin labeling. Graph shows mean values ± SD for 7 mice each with the indicated genotypes. Calculated RBC half-life in days are shown in parentheses. Differences between Hbb^th3/+ sing and Hbb^th3/+ diff mice were significant at all time points between days 2 and 14, with a false discovery rate of 0.05 by the Benjamini and Hochberg method. (C) Representative spleens from mice of the indicated genotypes. The scale bar represents 1 cm. Graph on right shows spleen-to-body weight ratios. n = 10 mice for each genotype. (D) Developmental stage distribution of erythroblasts in spleen (left panel) and bone marrow (right panel) determined by flow cytometry for surface antigens and forward scatter (FSC). Ery.A (Ter119^highCD71^highFSC^high), Ery.B (Ter119^highCD71^highFSC^low) and Ery.C (Ter119^highCD71^lowFSC^low) represent increasingly mature erythroblast populations.¹⁰ Representative flow cytometry plots are shown in supplemental Figure 2A. Hbb^th3/+ sing, n = 5 or 6; Hbb^th3/+ diff, n = 5 or 6. (E) Semiquantitative evaluation of splenic and bone marrow erythropoiesis in H&E-stained sections (supplemental Figure 2B and C) assigned by a blinded, board–certified veterinary pathologist (H.S.). Graphs show the levels of erythroid precursors on a 5-point scale (arbitrary units), with 0 being the lowest. n = 6-8 mice for each genotype. All graphs show data as mean value ± SD. Bar graphs were analyzed by 2-sample t test. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001. Hb, hemoglobin; H&E, hematoxylin and eosin; ns, not significant; Retic, reticulocyte; RDW, red cell distribution width; SD, standard deviation.