孕妇无创产前检测中重复检测的应用价值

The application value of retest on non-invasive prenatal test for pregnant women

目的 探讨重复检测在孕妇无创产前检测(NIPT)的应用价值。方法 2017年10月至2018年12月,在北京市海淀区妇幼保健院产前诊断中心就诊的孕妇中,纳入适宜人群进行NIPT筛查,目标疾病为21-三体、18-三体、13-三体。孕周12~27+6周时进行检测。采血后离心分离血浆、提取游离DNA、文库构建、分析质控、大规模平行测序(MPS)、无创产前胎儿染色体非整倍体分析等步骤。重复检测方法包括重抽血和重建库,需要重复检测的原因主要为检测失败和Z值灰区(目标染色体Z值介于3~4之间)。对NIPT提示胎儿非整倍体高风险的孕妇,行介入性产前诊断。对重复检测病例的原因及结果进行分析。结果 共纳入6110例NIPT检测病例,进行重复检测者315例(5.16%,315/6110),重抽血56例(0.92%,56/6110),重建库259例(4.4%,259/6110)。分析原因,检测失败155例(2.53%,155/6110),Z值灰区160例(2.62%,160/6110)。检测失败的155例中,16例样本不合格(2例凝血,3例时间超期,11例保存温度不当,重抽血未发现高风险病例);67例DNA文库质控不合格(5例文库峰图异常,62例文库浓度低,重建库未发现高风险病例);72例下机数据质控不合格,其中43例GC偏移(重建库后未发现高风险病例),21例胎儿浓度低(重抽血后3例仍然胎儿浓度低,其余18例中1例为高风险),7例多条染色体异常(重抽血后发现1例高风险),1例数据产生不足(重建库后结果为低风险)。Z值灰区160例中,148例重建库后确定高风险者34例;对重建库仍为Z值灰区者的12例孕妇进行了重抽血,确定为高风险者5例;重复检测确定低风险者共121例(75.6%,121/160)。41例染色体高风险孕妇经介入性产前诊断,确诊26例染色体异常。结论 NIPT检测涉及到多个质控环节,必要的重复检测在NIPT实验质控中起到了重要作用,Z值灰区的重复检测可以降低NIPT假阳性率。

Objective To discuss the application value of retest in non-invasive prenatal test(NIPT) for pregnant women. Methods From October 2017 to December 2018, the appropriate population who was treated in the Prenatal Diagnosis Center of The Beijing Haidian Maternal and Child Health Hospital was screened by NIPT. The blood collection,plasma separation by centrifugation, extraction of cf DNA, library construction, analysis of quality control, massively parallel sequencing (MPS), noninvasive prenatal fetal chromosome aneuploidy analysis and other steps were performed at 12~ 27+6 weeks of gestation for target diseases (trisomies 21, 18 and 13). Retest methods were redrawing blood and rebuilding the DNA library to avoid failed detection and Z score grey area (the target chromosome Z score was between 3 and 4). Pregnant women with high risk of fetal aneuploidy by NIPT were performed interventional prenatal diagnosis. The causes and results of retest were analyzed. Results A total of 6 110 cases with NIPT were included, 315 cases (5.16%, 315/6 110) were performed retest, of which 56 cases (0.92%, 56/6110) were redrew the blood, and that of the other 259 cases were rebuilt the DNA library (4.4%,259/6110). The reasons were analyzed that 155 cases were (2.53%, 155/6 110) failed in detection, 160 cases (2.62%, 160/6110) were Z score grey area. The 155 cases of test failure were as follows: 16 cases were unqualified samples (2 cases of blood clotting, 3 cases of expired blood sample and 11 cases of unsuitable storage temperature, and no high-risk case was found in the redrawing test);there were 67 cases of unqualified DNA library quality control (5 cases of abnormal peaks in the DNA library, 62 cases of low library concentration, among which no high-risk case was found in the reconstruction of the library). There were 72 cases of failed sequencing data quality control, including GC content bias in 43 cases (among no high-risk case was found in the reconstruction of the library), low fetal concentrations in 21 cases (fetal concentrations were still low in 3 cases after redrawing, and 1 case was high-risk in the remaining 18 cases), multiple chromosomal abnormalities in 7 cases (1 case was high-risk after redrawing), insufficient sequencing data in 1 case (low risk case was found in the reconstruction of the DNA library). 160 cases of Z score gray area were as follows: 34 cases were identified as high-risk after rebuilding DNA library in 148 cases;12 pregnant women with Z score gray area after rebuilding DNA library were performed redrawing, and 5 cases were identified as high-risk. A total of 121 cases (75.6%, 121/160) were low risk by repeated testing. 41 cases with high risk of target chromosome were performed interventional prenatal diagnosis to confirm 26 cases were abnormal chromosome. Conclusions NIPT detection involves many quality control points, and necessary retest plays an important role in the quality control of NIPT experiment. Repeated detection in the Z-score gray area can reduce the fals e positive rate of NIPT.