对孕妇外周血中的单个有核红细胞及游离DNA来源的鉴定

Identification of the origin of single nucleated red blood cells and free DNA in peripheral blood of pregnant women

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 using single fetal nucleated red blood cells (FNRBCs) and free DNA for noninvasive prenatal diagnosis. Methods 116 samples of maternal blood were analyzed. Single FNRBCs were isolated from the peripheral blood samples of 51 pregnant women with the gestational period of 14 to 26 weeks by micromanipulation techniques after density gradient centrifugation. Nested polymerase chain reaction method was used to amplify the SRY gene. Plasma DNA in blood samples of 65 pregnant women with the gestational period of 5～40 weeks were extracted and primer extension preamplification (PEP) and nested polymerase chain reaction were employed to amplify the SRY gene. Chorionic tissue/ amniotic cells were extracted to compare the concordance between the examination result of the meternal blood and that of chorionic tissue/ amniotic cells. Venous blood of healthy men and unpregnant women were used as controls. Results (1) The detection rate of single nucleated red blood cells was 90.20% (46/51). (2) The concordance rate of SRY gene amplification results of single cells with real fetal sex was 82.61 % (38/46), the sensitivity rate was 80 00% (24/30), and the specificity rate was 87.50 % (14/16). (3) The concordance rate of SRY gene amplification results of free DNA with real fetal sex was 90.77% (59/65), the sensitivity rate was 89.13% (41/46), and the specificity rate was 94.74% (18/19). Conclusion (1) The single nucleated red blood cells and free DNA in maternal blood are of fetal origin and can be one of the valuable material sources for prenatal diagnosis. (2) The detection purity of FNRBC by using micromanipulation techniques is nearly 100% and it provides a new way for noninvasive prenatal diagnosis.