Prenatal diagnosis procedures that do not endanger the fetus have been a long-sought goal of medical genetics. Currently used procedures for sampling of fetal cells for prenatal diagnosis, are invasive techniques that carry a small, yet finite, risks to the developing fetus. During pregnancy small number of embryonic RBC penetrate the maternal circulation. Among them are nucleated RBC - normoblasts. Analysis of the DNA of these embryonic normoblasts with specific probes by in situ hybridization and the polymerase chain reaction techniques might provide a non invasive means for diagnosis of genetic abnormalities. Since the percentage of fetal normoblasts, among the maternal cells is extremely low, an enrichment step is essential.We have recently developed procedures for culturing fetal normoblasts derived from erythroid progenitors present in neonatal cord blood and for flow cytometric analysis of fetal cells. The goal of the proposed study is to utilize these procedures to devise techniques for isolating from the maternal circulation pure populations of fetal normoblasts. For this purpose, we will attempt flow cytometric sorting based on simultaneous fluorescent labeling of the nucleus and two fetal specific characteristics - fetal hemoglobin and the surface antigen - "little i". In order to work out the details of the procedure, cultured fetal normoblasts derived form erythroid progenitors present in the neonatal cord blood will be used. The efficiency of the isolation protocol and the quality of the extracted DNA will be assessed by mixing, at various ratios, male cultured fetal normoblasts with normal female adult blood. Following isolation of the Hb F-containing normoblasts their DNA will be extracted and analyzed for specific chromosome Y-specific sequences.Subsequently, we will attempt to isolate fetal normoblasts from blood samples of pregnant women. In Phase II we will adapt these isolation techniques, in conjunction with specific molecular probes, for prenatal diagnosis of sickle cell anemia and thalassemias.Proposed commercial application:Prenatal diagnosis procedures that do not endanger the fetus or the mother have been a long-sought goal of medical genetics. Currently used procedures for sampling of fetal cells for prenatal diagnosis, are invasive techniques that carry a small, yet finite, risks. A safe and inexpensive non invasive method would be applicable for prenatal screening of pregnant women, and should be a cost-effective approach to identify fetuses with genetic abnormalities.National institute of Human Development (NIHD)