Increased number of proliferating erythroid cells in human thalassemic specimens. (A) At the end of the 2-phase liquid culture, the absolute expression of Bcl-XL, EpoR, Ki-67, CycA, and Jak2 mRNA relative to 14S ribosomal control RNA was quantified in 11 patients (black) and 6 healthy controls (white). An unpaired t test was used for statistical analysis. Benzidine staining was used to evaluate the level of erythroid differentiation. Both the amounts of β-globin mRNAs and those of cell cycle–related genes were quantified by quantitative polymerase chain reaction assay. We also performed highperformance liquid chromatography (HPLC) to determine variations in the percentages and absolute amounts of adult Hb in treated and nontreated samples (not shown). Error bars represent SD. (B) Time dependence of proliferating erythroid cells from 3 thalassemic patients (light gray, gray, and black) and one control subject (white). Aliquots of cells after various days of culture (second phase), corresponding to different stages of erythroid differentiation, were collected, cytospun, and stained for the proliferative marker Ki-67. Thalassemic patients showed an increased number of proliferating cells. More than 300 cells were counted to obtain the percentage of Ki-67–positive cells in each aliquot. (C) Spleen sections from a healthy subject (traumatic rupture) and a thalassemic patient (transfused thalassemia intermedia) who underwent splenectomy. Top panels: Ki-67 staining (brown; magnification, 100×). Bottom panels: Ki-67 (brown) and a mixture of glycophorin C and alpha-1-spectrin (red; magnification, 400×). A Plan Fluor 10×/0.30 numeric aperture objective and a Plan Fluor 40×/0.75 numeric aperture objective were used, along with the same microscope, camera, and software as in Figure 1.
