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目的 探讨孕妇外周血中单个胎儿有核红细胞及游离DNA在非创伤性产前诊断的可行性。方法 对 1 1 6例孕妇外周血进行检测。 (1 )对 51例 1 4～ 2 6孕周的妇女外周血经密度梯度离心后用显微操作分离单个有核红细胞。 (2 )提取 65例孕妇 (5～ 40孕周 )外周血血浆DNA。 (3)应用巢式聚合酶链反应 (PCR)扩增单个有核红细胞的男性SRY基因 ,应用引物延伸预扩增 (PEP)法及巢式PCR扩增孕妇血浆游离DNA的SRY基因。结果 (1 )分选单个有核红细胞的成功率为 90 2 0 % (46/ 51 )。(2 )单细胞SRY基因的结果与胎儿实际性别的符合率为 82 61 % (38/ 4 6) ,敏感性为 80 0 0 % (2 4 / 30 ) ,特异性为 87 50 % (1 4 / 1 6)。 (3)游离DNA的SRY基因的结果与胎儿实际性别的符合率为 90 77% (59/65) ,敏感性为 89 1 3 % (41 / 4 6) ,特异性为 94 74% (1 8/ 1 9)。结论 (1 )孕妇外周血中单个有核红细胞及游离的DNA可来自胎儿 ,它可成为产前诊断的胎儿物质来源。 (2 )单细胞分离技术使孕妇外周血中的胎儿有核红细胞的分选纯度几乎达到 1 0 0 % ,解决了母胎细胞混合的难题 ,为非创伤性产前诊断提供了一条新思路。
Objective To investigate the feasibility of single fetal nucleated erythrocytes and free DNA in non-invasive prenatal diagnosis in pregnant women. Methods 116 cases of pregnant women were detected in peripheral blood. (1) Peripheral blood of 51 women with gestational age from 14 to 26 weeks was centrifuged by density gradient to separate single nucleated erythrocytes by micromanipulation. (2) Peripheral blood plasma DNA was extracted from 65 pregnant women (5 to 40 gestational weeks). (3) The male SRY gene of single nucleated erythrocytes was amplified by nested polymerase chain reaction (PCR). The SRY gene of plasma free DNA of pregnant women was amplified by primer-extension pre-amplification (PEP) and nested PCR. Results (1) The success rate of sorting single nucleated erythrocytes was 90 20% (46/51). (2) The coincidence rate of unicellular SRY gene with the actual fetus was 82 61% (38/46), the sensitivity was 80 0% (2 4/30) and the specificity was 87 50% (1 4 / 1 6). (3) The coincidence rate of SRY gene of free DNA with the actual sex of fetus was 90 77% (59/65), the sensitivity was 89 13% (41/46) and the specificity was 94 74% (18 / 1 9). Conclusions (1) The single nucleated erythrocytes and free DNA in the peripheral blood of pregnant women can come from the fetus, which can be the source of fetal material for prenatal diagnosis. (2) The single cell separation technology can make the purity of fetal nucleated red blood cells in pregnant women’s peripheral blood almost 100%, which solves the problem of mixed mother’s cells and provides a new idea for non-invasive prenatal diagnosis.