To the Editor:
Fetal nucleated erythrocytes circulating in maternal blood are a potential source of fetal DNA for noninvasive prenatal genetic diagnoses. Nucleated red blood cells (NRBCs) have several advantages over fetal lymphocytes, granulocytes, or trophoblasts. They are nucleated, and thus a source from which DNA can be extracted for direct genetic analysis. Furthermore, they express transferrin receptor (CD71), glycophorin A, and thrombospondin receptor (CD36) on cell surface membrane. Thus, monoclonal antibodies are used for isolation of these cells in conjunction with various sorting techniques. Fetal NRBCs have a limited life span in the maternal circulation . However, the estimated ratio of fetal to maternal cells is extremely small, on the order of 1/10 4 -1/10 9 .
To enrich these fetal NRBCs, we employed a twophase liquid culture system ] that supports the growth and differentiation of earlier human erythroid progenitor cells. In this system erythropoietin-independent and erythropoietin-dependent phases are separated, and it affords advantages in manipulation of culture conditions and components at various stages without termination of the cultures. After informed consent was obtained, mononuclear cells were separated by Ficoll-Hypaque (density 1.077 g/cm 2 ) density gradient centrifugation from 15 mL peripheral blood of a pregnant woman at 10 weeks of gestation and cultured in the first phase in β£-MEM with 10% fetal calf serum, 1.5 mmol/L glutamine, 1% penicillin-streptomycin, 1 g/mL cyclosporin A, 10% conditioned medium at 37Β°C, 5% CO 2 . After 4-5 days, the nonadherent cells were harvested and recultured in β£-MEM containing 30% fetal calf serum, 1% bovine serum albumin, 10 -5 mol β€-mercaptoethanol, 10 -6 mol dexamethasone, 0.3 g/mL transferrin, 10 ng/mL stem cell factor, and 1 U/mL erythropoietin for another 3-4 days. We examined at cellular morphology on cytospinprepared slides, counted benzidine-positive, hemoglobin-containing cells , and estimated the percentages of CD71/glycophorin A positive cells by flow cytometry. We performed Kleihauer-Betke stain, PCR for the presence of SRY , and