Flow cytometry of erythropoiesis in culture: Bivariate profiles of fetal and adult hemoglobin

Abstract

The development of erythroid cells in vivo and in vitro comprises proliferation and maturation, from early committed progenitors to enucleated erythrocytes. An important marker of erythroid maturation is the expression and accumulation of hemoglobin in nucleated red cells. The pattern of hemoglobin accumulation is different in fetal and adult cells: fetal erythroid cells accumulate mostly the fetal form of hemoglobin (HbF, containing γ chains). The possibility of manipulating adult erythroid stem and progenitor cell development toward fetal erythropoiesis is of great clinical interest because β -chain hemoglobin disorders such as sickle-cell anemia and the β -thalassemias are ameliorated by increased HbF production. At about the time of birth, in parallel to and probably caused by the migration of hemopoiesis to the bone marrow, erythroid progenitors switch to β -chain expression to make adult hemoglobin (HbA, containing β chains). This chapter discusses flow cytometry to measure two-parameter hemoglobin profiles (HbF versus HbA or the sickle-cell mutant HbS) of developing nucleated red cells, together with correlated DNA histograms and absolute cell counting by reference beads. The method is suitable for monitoring the erythropoiesis of patients with hemoglobinopathies in response to treatment.