FLOW CYTOMETRY AND MOLECULAR GENETICS OF FETAL CELLS

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Fetal cells of many types cross the placental barrier and persist in the maternal blood throughout gestation. Thus, they represent a reservoir of fetal tissue potentially available for genetic and cell surface analysis. One aim of this project is to detect fetal erythrocytes in the maternal circulation using fluorescently labeled antibodies against uniquely fetal antigens (such as Rh-D, i, glycophorin, and membrane-crosslinked HgB F). The labeled erythrocytes may then be physically sorted, based on combinations of fetal antigens, using a fluorescence-activated cell sorter (FACS IV). Accurate quantification of fetomaternal hemorrhage occurring during gestation will provide insight into the sensitization of Rh negative primigravidae and help evaluate the effectiveness of therapy such as Rh immune globulin. Additionally, it will determine whether or not a correlation exists between the extent of fetomaternal transfusion and otherwise unexplained elevations in maternal serum alphafetoprotein. Importantly, since the majority of the FACS-isolated fetal erythrocytes are nucleated, their DNA may be utilized for molecular probing. As a first approach, Y chromosome-specific probes (highly reiterated, hence requiring relatively small amounts of DNA) will be used as a means of identifying male fetal cells in the blood of pregnant women. As other probes become available and the sensitivity of detecting the sequences hybridizing even with non-reiterated probes increases, the inheritance of specific genes in the fetus will be determined. Prenatal genetic diagnosis on material obtained via maternal venipuncture should thereby be facilitated. Chorionic villus biopsy represents another, new method for obtaining fetal cells during the first trimester, permitting both chromosome and DNA analysis. This procedure, which is undergoing active development and evaluation, may ultimately be subject to complications depending on the extent to which it might cause fetomaternal hemorrhage. Women who are blood group incompatible with their fetuses would then be at risk for sensitization if significant fetomaternal transfusion occurs post biopsy. The current project will specifically address this issue. In addition, recombinant DNA technology will be used to differentiate between maternal and fetal DNA, to monitor possible maternal contamination of tissue samples and to provide prenatal genetic diagnosis. In the course of the experiments proposed, it is anticipated that a general fund of knowledge in molecular biology and flow cytometry will be developed.