改良磁珠筛选法富集母体外周血胎儿游离DNA在无创产前检测中的应用价值

Application value of modified magnetic bead screening for enrichment of cell-free fetal DNA from maternal peripheral blood in non-invasive prenatal testing

目的评估改良磁珠筛选法富集胎儿游离DNA(cell-free fetal DNA,cffDNA)在无创产前检测(non-invasive prenatal testing,NIPT)中的应用价值。方法从2017年10月至2019年12月在北京市海淀区妇幼保健院接受常规NIPT的11 000例孕妇中,回顾性纳入cffDNA浓度偏低(<6.00%),或首次检测Z值灰区(3.00~4.00),或产前介入性产前诊断确定假阳性或生后确定假阴性病例共31例。取首次常规NIPT留存的血浆标本,使用改良磁珠筛选法富集cffDNA进行NIPT(改良富集法NIPT)。采用Wilcoxon秩和检验比较改良富集法NIPT与常规NIPT测得的男胎cffDNA浓度变化。结果 31例孕妇中13例常规NIPT cffDNA浓度偏低,11例常规NIPT 13/18/21-三体假阳性(3例13-三体,4例18-三体,4例21-三体,均经介入性产前诊断证实),6例首次常规NIPT Z值灰区(复测为低风险),1例常规NIPT 21-三体假阴性(生后染色体检查证实)。使用改良富集法后,<150 bp的游离DNA(cell-free DNA,cfDNA)片段得到有效富集,21例男胎的cffDNA浓度由常规NIPT的4.43%(2.45%~17.61%)提升至改良富集法NIPT的13.46%(7.75%~36.64%),差异有统计学意义(Z=-14.22,P<0.01)。13例常规NIPT cffDNA浓度偏低者经改良富集法NIPT,均成功检出为低风险;6例Z值灰区病例及11例假阳性病例中的6例经改良富集法NIPT确定为低风险,与常规NIPT复测结果一致;1例假阴性病例确定为高风险。结论与常规NIPT相比,改良富集法NIPT降低了因cffDNA浓度偏低导致的检测失败率,降低了假阳性率,并成功检测出假阴性病例,显示出更高的检测成功率和更低的假阳性率。

Objective To evaluate the value of modified magnetic bead screening for enrichment of cell-free fetal DNA(cffDNA)in non-invasive prenatal testing(NIPT).Methods This study retrospectively recruited 31 cases with low concentration of cffDNA(<6.00%),Z value in the gray zone(3.00-4.00)at the first detection,or false-positive(confirmed by invasive prenatal diagnosis)or false-negative(confirmed by postnatal chromosome test)results among 11000 pregnant women who underwent routine NIPT in Beijing Haidian District Maternal and Child Health Care Hospital from October 2017 to December 2019.Plasma samples collected for the first-time routine NIPT were used to enrich cffDNA using modified magnetic beads for NIPT(modified NIPT).Wilcoxon rank sum test was used to compare the modified NIPT with the routine NIPT in detecting the cffDNA concentrations of male fetuses.Results Among the 31 pregnant women,there were 13 cases with low cffDNA concentration in routine NIPT,11 having false-positive results in the routine NIPT(three for trisomy 13,four for trisomy 18 and four for trisomy 21,all were confirmed by invasive prenatal diagnosis),six with gray-zone Z values in the first-time NIPT(retesting indicating low risk)and one having false negative result for trisomy 21(confirmed by postnatal chromosome test).Cell-free DNA(cfDNA)fragments less than 150 bp were effectively enriched using the modified magnetic bead screening and the concentration of cffDNA was increased from 4.43%(2.45%-17.61%)in routine NIPT to 13.46%(7.75%-36.64%)in the modified NIPT(Z=-14.22,P<0.01).Results of the modified NIPT indicated that 13 cases with low cffDNA concentration of routine NIPT were successfully detected as low risk,as well as the risks in the six cases with gray-zone Z value and six of the 11 false-positive cases in the routine NIPT were low,which were consistent with the retest results of the routine NIPT,while high risk was found in one false-negative case.Conclusions The modified NIPT could reduce the false positive rate by lowering the failure rate caused by low concentration of cffDNA and is able to identify false-negative cases.Compared with the routine NIPT,it shows a higher success rate and a lower false positive rate.