无创胎儿游离DNA RHE血型基因型诊断技术的准确性和可行性研究  

Accuracy and feasibility of non-invasive cell-free fetal DNA RhE blood group genotyping

作　　者：杨金华 任道菊 李小薇 肖军 游江舟 陈春月 张晓娟 李翠莹 YANG Jinhua;REN Daoju;LI Xiaowei;XIAO Jun;YOU Jiangzhou;CHEN Chunyue;ZHANG Xiaojuan;LI Cuiying(Graduate School of PLA Medical College,Beijing 100853,China;Air Force Clinical College,The Fifth Clinical Medical College,Anhui Medical University,Hefei 230032,China;Department of Blood Transfusion,Air Force Specialty Medical Center,Air Force Medical University,Beijing 100142,China)

机构地区：[1]解放军总医院医学院研究生院,北京100853 [2]安徽医科大学第五临床医学院空军临床学院,安徽合肥230032 [3]空军军医大学空军特色医学中心输血科,北京100142

出　　处：《中国输血杂志》2025年第3期368-374,共7页Chinese Journal of Blood Transfusion

基　　金：2024年度空军军医大学临床研究计划项目(2024LC2406)。

摘　　要：目的 探讨利用孕妇外周血中胎儿游离DNA(Cell-Free Fetal DNA,cff-DNA)进行无创产前诊断胎儿RHE血型基因型的准确性和可行性。方法 选择2023年11月-2024年8月在本院建档的单胎且RhE血型为Rhee的纯合子孕妇134例,应用游离DNA提取试剂盒提取孕妇外周血中游离DNA,通过实时荧光定量PCR(Quantitative Real-time PCR,RT-qPCR)方法检测游离DNA的RHE血型基因型。当标本RT-qPCR扩增信号阴性时,对其进行甲基化RASSF1A基因扩增,扩增结果阳性作为成功提取到胎儿游离DNA的标志。血清学微柱凝胶法检测新生儿外周血的RhE血型表现型作为结果验证。结果 134例孕妇外周血标本中胎儿游离DNA检测的RHE血型基因型与新生儿外周血RhE血型表现型结果一致的有133例,其中RHee基因型90例,RHE基因型43例,诊断符合率为99.3%,灵敏度为97.7%,特异度为100%,约登指数为0.977,ROC曲线下面积0.995,Kappa值0.983,阳性预测值100%,阴性预测值98.9%。1例标本检测失败,经过甲基化RASSFIA基因扩增,验证其失败原因为该例标本未提取到胎儿游离DNA。妊娠早期、中期、晚期诊断符合率分别为93.8%(15/16)、100%(51/51)、100%(67/67),经Fisher精确概率法计算,P>0.05,说明3组妊娠周期间的诊断符合率差异无统计学意义,该方法在不同妊娠时期检测的符合率没有差异。结论 利用孕妇外周血中胎儿游离DNA检测胎儿RHE血型基因型,是1种准确率高,可行性强的无创产前诊断方法,有助于临床诊断抗-E引起的胎儿新生儿溶血病。Objective To explore the accuracy and feasibility of non-invasive prenatal diagnosis of fetal RhE genotype using cell-free fetal DNA(cff-DNA)from maternal peripheral blood.Methods A total of 134 pregnant women with single fetuses and RhE-negative blood group were selected from our hospital from November 2023 to August 2024.Free DNA ex-traction kit was used to extract free DNA from peripheral blood of pregnant women,and the RhE blood group genotype of free DNA was detected by real-time fluorescent quantitative PCR(RT-qPCR).If the qPCR amplification signal of the sam-ple was negative,the methylated RASSF1A gene was amplified,and the positive amplification result was used as a sign of successful extraction of cff-DNA.Serological microcolumn gel method was used to detect the phenotype of RhE blood group in neonatal peripheral blood.Results Among the 134 maternal peripheral blood samples,the cff-DNA detection of RhE blood group phenotypes was consistent with the RhE blood group genotyping of neonatal peripheral blood in 133 cases,inclu-ding 90 cases of Rhee genotype and 43 cases of RhE genotype,with diagnostic concordance rate of 99.3%,sensitivity of 97.7%,specificity of 100%,youden index of 0.977,area under ROC curve of 0.995,the Kappa value of 0.983,positive predictive value of 100%,and negative predictive value of 98.9%.The sample of 1 case failed to be detected.After the amplification of methylated RASSFIA gene,it was confirmed that the reason for the failure was that no cff-DNA was extracted from the sample.The diagnostic concordance rates of the first,second and third trimesters were 93.8%(15/16),100%(51/51)and 100%(67/67),respectively.Fisher′s exact test method was used to calculate the P value,which was P>0.05,indicating that there was no statistical significance in the difference of diagnostic concordance rate among the three pregnancy periods,and there was no difference in the detection concordance rate of this method in different pregnancy peri-ods.Conclusion The use of cff-DNA in maternal peripheral bloo

关 键 词：无创产前诊断 胎儿RhE血型 胎儿游离DNA 胎儿新生儿溶血病 实时荧光定量PCR 

分 类 号：R457.1[医药卫生—治疗学]