Erythroid expansion and phenotype of cultured primary cells from subjects with MPDs. CD34⁺ cells were isolated from peripheral blood from a subject with postthrombocythemic myeloid metaplasia (sample no. 768) and peripheral blood (sample no. 770) and bone marrow (sample no. 769) from a subject with ET. Cells were cultured in parallel with CD34⁺ cells isolated from a healthy bone marrow donor (NBM). Both MPD subjects had the JAK2 V617F⁺ mutation. Cells were cultured in the presence of IL-3, IL-6 and SCF, and EPO was added on day 5 of culture. (A) Cells were cultured in the presence of IL-3, IL-6 and SCF, and EPO was added on day 5 of culture. Cell number is presented as fold-expansion over the starting number of CD34⁺ cells. (B) CD71 and Glycophorin A expression was analyzed on cultured cells. Maturation-specific erythroid gates were drawn in accordance with flow cytometry models of murine and human erythroid development, with high expression of CD71 and moderate expression of glycophorin A in the earliest erythroblasts (group I) with successive increases in glycophorin A expression and loss of CD71 as the cells mature (groups II-IV). Shown are a representative MPD sample and NBM sample, EPO-independent erythroid differentiation in analyzed MPD samples (mean 33.7%, n = 3) was significantly greater than NBM samples (mean 6.6%, n = 2, P = .017). (C) Morphologic characterization of cultured cells. Cytospins of cultured cells were stained with a modified Wright-Giemsa stain. Note that before the addition of EPO on day 5, cells from a subject with ET possessed features characteristic of erythroblasts: round nuclei with clumped chromatin, royal blue cytoplasm and a lack of granulation. These features are more prominent following EPO in both healthy and MPD samples.