Dif-Couvreux D, Houfflin-Debarge V, Delsalle A, Dourieux S, Dubreucq S, Manessier L, Puech F
Service de Diagnostic Anténatal, Hôpital Jeanne-de-Flandre, 2, avenue Oscar-Lambret, 59037 Lille Cedex.
J Gynecol Obstet Biol Reprod (Paris). 2006 Nov;35(7):658-64. doi: 10.1016/s0368-2315(06)76460-2.
The aim of our study was to evaluate the possibility of identifying the fetal RhD status in maternal plasma using conventional hemi nested PCR analysis.
After informed written consent, 20 mL of peripheral blood were collected in 99 D-negative pregnant women either at an amniocentesis for prenatal diagnosis or at a prenatal checkup. Fetal DNA extracted from 400 microL of maternal plasma was analyzed by two different operators with a hemi-nested PCR extending an area of the RhD gene exon 10. The results were compared to the fetal RhD status obtained by PCR amniotic fluid analysis or blood analysis of newborns after delivery. The influence of mother's and baby's phenotype were also studied.
Among the 99 D-negative pregnant women, all Caucasian, 47 were in their second trimester and 52 in their third trimester (mean: 27.20 weeks of gestation +/-8.25). Sixty-nine fetuses were D-positive and thirty D-negative. The sensitivity and specificity of our technique were respectively 100% and 86.7% and 15% of discordant results were observed between the two operators. Four false positives were observed. According to maternal phenotype, a fetal unexpressed RHD gene was suspected in only one case because of a particular fetal phenotype (ddCcEe).
A conventional hemi nested PCR analysis of maternal plasma could be used for accurate fetal RhD status. However this procedure is difficult to apply for routine analysis because of the importance of anti-contamination measures required to obtain good results. Real time quantitative PCR analysis on fetal DNA is more suitable. Whatever the operating procedure used, polymorphism of RhD gene may follow in either false negative from presence of rearranged gene or false positive from occasional presence of a non functional RHD gene.
我们研究的目的是评估使用传统半巢式PCR分析来鉴定母血中胎儿RhD状态的可能性。
在获得书面知情同意后，收集了99名D阴性孕妇的20 mL外周血，这些孕妇要么是在进行羊膜穿刺术以进行产前诊断时，要么是在产前检查时。从400 μL母血中提取的胎儿DNA由两名不同的操作人员使用半巢式PCR进行分析，该PCR扩增RhD基因外显子10的一个区域。将结果与通过PCR羊水分析或分娩后新生儿血液分析获得的胎儿RhD状态进行比较。还研究了母亲和婴儿表型的影响。
在这99名均为白种人的D阴性孕妇中，47名处于孕中期，52名处于孕晚期（平均孕周：27.20周±8.25）。69名胎儿为D阳性，30名胎儿为D阴性。我们技术的敏感性和特异性分别为100%和86.7%，两名操作人员之间观察到15%的不一致结果。观察到4例假阳性。根据母亲的表型，仅在1例中因特殊的胎儿表型（ddCcEe）怀疑胎儿RHD基因未表达。
对母血进行传统半巢式PCR分析可用于准确鉴定胎儿RhD状态。然而，由于要获得良好结果需要采取严格的防污染措施，该方法难以应用于常规分析。对胎儿DNA进行实时定量PCR分析更合适。无论采用何种操作程序，RhD基因的多态性可能导致因重排基因的存在而出现假阴性，或因偶尔存在无功能的RHD基因而出现假阳性。
