ChAc diserythropoiesis, remnant intracellular vesicles, and accumulation of active Lyn during erythroid maturation. (A) (Upper) Cell proliferation of erythroid precursors derived by in vitro liquid culture of CD34⁺ cells isolated from peripheral blood of normal (control cells) subjects and ChAc subjects (n = 6). Data are presented as means ± SD; *P < .05 compared with control cells. Percentage decrease in cell number was 53.2 ± 2.9% (n = 6). (Lower) Morphology of culture day 14 erythroid precursors from healthy controls and ChAc subjects. Cytospins were stained with May-Grunwald-Giemsa. Cells were imaged under oil at 100× original magnification using a Panfluor objective with 1.30 numeric aperture on a Nikon Eclipse DS-5M camera and processed with Digital Slide (DS-L1) Nikon. Images representative of 6 separate experiment at 14 days of culture. Quantification of bi-trinucleated cells is reported in supplemental Figure 5C. (B) Electron microscopy of ChAc erythroid precursors at 14 days of culture. (Upper) Hemoglobinized cells of roundish shape filled exhibit pyknotic nucleus and cytoplasmic organellar remnants, including mitochondria (open arrowhead), profiles of endoplasmic reticulum (arrow), and small (open arrow) and large (solid arrowhead) vesicles. Scale bar, 2 μm. Remnants at higher original magnification are shown in the inset; bar, 1 μm. (Lower left) Small erythroid cell, a portion of cytoplasm is apparently detaching from the cell body: bar, 2 μm. (Lower right) A mature, acanthocytoid erythrocyte shows organelle remnants in the cytoplasm (mitochondrion, open arrowhead, endoplasmic reticulum, arrow); bar, 2 μm. (C) Cytofluorimetric maturation analysis of day 14 erythroid precursors (see also supplemental Materials and methods for gating strategy). This cyto-fluorimetric strategy allows the identification of the following homogenous cell populations: pro-erythroblasts (Pro-E), basophilic erythroblasts corresponding to intermediate erythroblasts (Int-E), and polychromatic and orthochromatic erythroblasts as late erythroblasts (Late-E). Data are expressed as percentages shown as means ± SD (n = 4). (D) Amount of annexin-V–positive cells at 14 days of culture in healthy controls and ChAc subjects. Data are expressed as means ± SD (*P < .05 compared with healthy controls, n = 4). (E) Immunoblot analysis of phospho-Lyn and total Lyn in erythroblasts from healthy (control, Ctrl) subjects and ChAc subjects at 14 days of culture. GADPH was used as loading control. One representative blot from 6 with similar results is presented. Densitometric analysis is presented on the right; data are shown as means ± SD (n = 6; *P < .01 compared with healthy controls).