Thurik F F, Ait Soussan A, Bossers B, Woortmeijer H, Veldhuisen B, Page-Christiaens G C M L, de Haas M, van der Schoot C E
Department of Experimental Immunohematology, Sanquin Research, Amsterdam and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
Department of Immunohematology Diagnostics, Sanquin Diagnostic Services, Amsterdam, The Netherlands.
Prenat Diagn. 2015 Aug;35(8):754-60. doi: 10.1002/pd.4600. Epub 2015 May 19.
We aim to elucidate causes of false-positive fetal RHD screening results obtained with cell-free DNA.
Fetal RHD screening was performed in 32,222 samples from RhD-negative women by multiplex real-time PCR in triplicate for RHD exons 5 and 7 using cell-free DNA isolated from maternal plasma obtained in the 27th gestational week. PCR results were compared with cord blood serology in 25,789 pregnancies (80.04%). False-positive cases were analyzed. Known biological causes (RHD variant genes), technical causes of discordance, and errors around blood sampling were investigated with leukocyte DNA from maternal and cord blood, and cell-free DNA from stored maternal plasma.
Not only RHD but also Y-chromosome (DYS14) sequences were present in four plasma samples from RHD-negative women bearing an RHD-negative girl. Sample mix-up and other sampling errors could be excluded in three samples.
These results indicate that false-positive fetal RHD screening results can be caused by cell-free DNA fragments in maternal plasma derived from a third cell line that is not representative for either the maternal genome or the genome of the vital fetus. We propose that remaining (cyto)trophoblasts of a vanishing twin are the underlying mechanism, and we estimate a frequency of this phenomenon of 0.6%.
我们旨在阐明通过游离DNA获得的胎儿RHD筛查假阳性结果的原因。
对32222例RhD阴性女性的样本进行胎儿RHD筛查，在孕27周时从母体血浆中分离游离DNA，采用多重实时PCR对RHD外显子5和7进行一式三份检测。将25789例妊娠（80.04%）的PCR结果与脐血血清学结果进行比较。对假阳性病例进行分析。利用母体和脐血的白细胞DNA以及储存的母体血浆中的游离DNA，研究已知的生物学原因（RHD变异基因）、不一致的技术原因以及采血周围的误差。
在怀有RHD阴性女孩的RHD阴性女性的四份血浆样本中，不仅存在RHD序列，还存在Y染色体（DYS14）序列。在三个样本中可以排除样本混淆和其他采样误差。
这些结果表明，胎儿RHD筛查假阳性结果可能是由母体血浆中来自第三种细胞系的游离DNA片段引起的，该细胞系既不代表母体基因组，也不代表存活胎儿的基因组。我们认为消失双胎残留的（细胞）滋养层是潜在机制，我们估计这种现象的发生率为0.6%。
