Flow cytometric analysis of the ploidy of normoblasts in the peripheral blood of patients with beta-thalassemia.

Abstract

The chronic severe anemia of patients with beta-thalassemia major stimulates extensive erythropoiesis, which results in circulating nucleated normoblasts. We devised a dual staining flow cytometric procedure in order to analyse the cell cycle and ploidy of these normoblasts. Peripheral blood cells of O blood-group type were first stained with Fluorescein Isothiocyanate (FITC)-conjugated anti-H lectin which labels erythroid cells (RBC and normoblasts) by green fluorescence, and then with propidium iodide (PI) which binds to DNA and thereby labels nucleated cells (leukocytes and normoblasts) by red fluorescence. The leukocytes and normoblasts present in the blood sample of thalassemic patients could be distinguished and "gated" based on their green fluorescence. The PI (red) fluorescence, i.e., the DNA histogram of each population, was thus obtained. The results indicated no statistically significant difference in the PI fluorescence of these two populations. Thus, in spite of the abnormal erythropoiesis in beta-thalassemia, the resultant orthochromatic normoblasts are normal with respect to their DNA content.