Flow cytometric analysis of hydroxyurea effects on fetal hemoglobin production in cultures of β-thalassemia erythroid precursors

An increase in fetal hemoglobin (Hb F) ameliorates the clinical symptoms of the underlying disease in the beta hemoglobinopathies-sickle cell anemia and beta-thalassemia (thal). Hydroxyurea (HU) can elevate Hb F production in erythroid cells and is the agent currently in clinical use for patients with sickle cell anemia; it is presently being tested in clinical trials for thalassemia. We have developed a two-phase liquid culture system that mimics the in vivo hematological changes that are observed in patients treated with HU. Adding HU during the second phase of the culture increases the proportion of Hb F, increases the levels of total hemoglobin (Hb) content per cell and increases cell size, but it decreases the numbers of cells and the total amount of Hb produced. In the present study we developed and utilized a double labeling procedure for flow cytometric analysis of the cellular distribution of Hb F. Cells exposed to various concentrations of HU on day 6 of the second phase of the culture were harvested on day 12, and stained simultaneously with fluorochrome-conjugated monoclonal antibodies specific for human glycophorin A, an erythroid specific marker, and human Hb F. Both the percentage of the Hb F-containing cells and their intensity of fluorescence were recorded. The latter value gives a semi-quantitative estimation of the mean cellular Hb F content. The results indicated that cultures derived from different beta-thalassemic patients differ in their response to HU. In most patients, low doses of HU decreased the percentage of Hb F-cells as well as their Hb F content. At high doses, some patients showed an increase in both parameters, while others showed an increase in the percentage of Hb F-cells with minimal increase in their mean Hb F content, while still other patients showed little effect at all. In all patients, high doses of HU caused a decrease in cell numbers. These results suggest that HU has mixed effects on erythroid precursors. Both the two-phase liquid culture and the flow cytometric analysis procedures described herein provide the experimental tools for screening of Hb F-inducing drugs and for evaluating patients' cell response prior to treatment.