Erythroid hyperplasia, ineffective erythropoiesis, and hemolysis in humanized γ^HPFHδβ⁰ CA mice. (A) Erythroid hyperplasia in the bone marrow and spleen of CA mice was measured by flow cytometry. Bone marrow and spleen cells from age- and sex-matched CA and wild-type mice were stained with fluorescently labeled antibodies to the erythroid antigen Ter119 and transferrin receptor CD71 and fluorescent-labeled annexin V. Dead cells were excluded by 7-aminoactinomycin D (7AAD) staining. The percentages of proerythroblasts (region I: CD71^HIGH, Ter119^LOW), early erythroblasts (region II: CD71⁺, Ter119⁺), late erythroblasts (region III: CD71⁻, Ter119⁺), and total Ter119⁺ erythroid cells (total Ter119⁺) in the bone marrow and spleen are shown in Table 2. Erythroid populations of humanized HbA mice (α2α1/α2α1 γβ^A/γβ^A) and C57BL/6J wild-type mice did not differ significantly (data not shown). (B) Demonstration of ineffective erythropoiesis in CA mice by apoptosis of erythroid progenitors by annexin V–binding assay.³²  Representative histograms are shown of annexin V staining of early (region II) and late (region III) erythroblasts in CA and control mouse bone marrow and spleen cells from panel A. No antibody control samples were stained with all the antibodies in panel A, except annexin V. Annexin V⁺ cell ratios are quantified in Table 3. Annexin V⁺ erythroid populations of humanized HbA mice (α2α1/α2α1 γβ^A/γβ^A) and C57BL/6J wild-type mice did not differ significantly (data not shown). (C) Bilirubin levels increased significantly in untransfused CA mice compared with age- and sex-matched wild-type control mice, indicating increased hemolysis in CA mice. Bilirubin levels returned to control levels in hypertransfused, but not hypotransfused CA mice. *P < .05; **P < .0001; n = 3 in each group.