Improvement of fetal cell recovery from maternal blood: Suitable density gradient for FACS separation

Abstract

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.