Detection of fetal erythroid cells from maternal blood using fluorescence in situ hybridization and liquid culture

Fetal nucleated erythrocytes circulating in maternal blood are a potential source of fetal DNA for noninvasive prenatal genetic diagnosis, However, th e estimated ratio of fetal to maternal cells is extremely small. In order t o enrich these cells, we performed direct culture using a two-phase liquid system. Mononuclear cells were obtained from maternal blood samples at 8-10 (+3) weeks of gestation and cultured in the first phase. After 4-5 days, th e nonadherent cells were harvested and recultured with erythropoietin in th e second phase for another 3-5 days. We examined cellular morphology, and c ounted the number of benzidine-positive cells and the percentage of glycoph orin A/CD71 positive erythroid cells. We also did Kleihauer-Betke stain for Hb F, polymerase chain reaction (PCR) for SRY/DYZ1, chromosome analysis, a nd fluorescence in situ hybridization (FISH). The number of total erythroid cells reached about 0.1 x 10(6)-1.0 x 10(6)/mL with a purity of 84.0-97.3% . Hb F stain showed total erythroid cells of approximately 0.4 x 10(4)-9.8 x 10(4)/mL. Male DNA was detected in one case by PCR. In this case, the XY karyotype was confirmed by FISH and amniocentesis. This approach provides e nriched source of fetal cells for further prenatal genetic analysis without complicated separation or sorting procedures.