Fetal nucleated cells circulating in maternal peripheral blood are a noninvasive source of fetal DNA for prenatal genetic diagnoses. The successful isolation of fetal cells from maternal blood depends upon identification of differences between fetal and maternal cell surface antigen expression. To our best knowledge, a monoclonal antibody that binds only fetal blood cells has not yet been identified. We studied antigens recognized by six different monoclonal antibodies for their biologic expression on fetal blood cells as a function of gestational age, and compared their ability to bind fetal but not maternal cells. The results suggest a relationship between gestational age and nucleated cell surface antigen expression. The monoclonal antibodies FB3-2, H3-3, CD71 and 2-6B/6 are suitable reagents for first or early second trimester fetal cell isolation, although FB3-2 and H3-3 are more specific for fetal cells due to significantly lower expression of these antigens on maternal mononuclear cells. The observation that samples from fetuses with chromosome abnormalities or multiple structural anomalies express higher levels of these antigens indicates that these reagents will potentiate the detection of abnormal fetal cells in maternal blood samples.
The purpose of this study was to improve recovery of fetal nucleated erythrocytes (NRBCs) from maternal blood for non-invasive prenatal diagnosis. Peripheral blood samples were obtained from 27 women who had just undergone pregnancy termination at 6 to 23 weeks. Samples were split and mononuclear cells were isolated using Histopaque gradient at densities of 1.090 g/ml and 1.119 g/ml. CD45 depletion using magnetic activated cell-sorting, followed by flow-sorting with antibody to gamma-globin and fluorescence in situ hybridization (FISH) analysis, were used to evaluate the number of fetal NRBCs recovered. In samples separated with the 1.119 g/ml density gradient, the yield of true anti-gamma haemoglobin positive cells (median, 14. 9; range, 0-717.5) was significantly higher than that with the 1.090 g/ml density gradient (median, 4.9; range, 0-532.5). After FISH analysis, in the 14 samples in which the fetal karyotype differed from the mother, the median number of fetal NRBCs separated by the 1. 119 g/ml density gradient was 22.9 (2-717.5), which was significantly higher than that by the 1.090 g/ml gradient (median, 11.5; range, 0-532.5, p=0.022). Increased density of the gradient used for the initial enrichment of fetal cells results in improved fetal cell recovery in fresh post-termination blood samples, which may permit better non-invasive detection of fetal cells in maternal blood.
Objective: To improve the recovery of fetal nucleated erythrocytes (NRBCs) from maternal blood for noninvasive prenatal genetic diagnosis. Methods: Blood samples were obtained from 10 women at 8–22 weeks of gestation. Samples were split and mononuclear cells were isolated using 1.083 and 1.090 g/ml of Percoll solution. Flow sorting with antibody to fetal hemoglobin and fluorescence in situ hybridization (FISH) analysis were used to evaluate the number of fetal cells recovered. Results: In samples separated with the 1.090 density gradient, the yield of true γ-hemoglobin-positive cells (median 21.0, range 2.2–303.8) was 1.9 times higher than that in the 1.083 density (median 11.1, range 1.1–87.5), although it took 2.1-fold longer time to flow sort the γ-hemoglobin-positive cells. In 7 out of 10 cases, the number of γ-hemoglobin-positive cells recovered from the 1.090 density gradient was 3 times or greater than that from 1.083 gradient. After FISH analysis, we detected a median of 13.3 (range 2.2–98.8) fetal NRBCs per 10-ml maternal blood in the 1.090 density gradient, whereas a median of 11.0 fetal NRBCs were detected in the 1.083 gradient (range 1.1–35.0). The number of fetal NRBCs in the 1.090 density was significantly higher than that in the 1.083. Conclusion: Increased Percoll density results in improved fetal cell recovery in fresh posttermination maternal samples. The increased yield of fetal cells using this gradient may permit better noninvasive detection of fetal chromosome as well as DNA abnormalities in maternal blood.
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