Londero D, Stampalija T, Bolzicco D, Castro Silva E, Candolini M, Cortivo C, Dreossi C, Fantasia I, Pecile V, De Angelis V
Department of Transfusion Medicine, ASUI-Udine, Udine, Italy.
Unit of Fetal Medicine and Prenatal Diagnosis, Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy.
Transfus Med. 2019 Dec;29(6):408-414. doi: 10.1111/tme.12605. Epub 2019 Jun 17.
This study aimed to validate non-invasive RHD genotyping of cell-free fetal DNA (cff-DNA) using different DNA extraction methods and of fresh and frozen extracted cff-DNA.
Non-invasive RHD genotyping of cff-DNA predicts fetal RhD phenotype, allowing for the rational implementation of antenatal immunoprophylaxis and representing a big step forward in the management of RhD-immunised women. Validation of a diagnostic method is mandatory before its clinical application.
RhD-negative pregnant women were recruited at different gestational ages. The cff-DNA extraction was carried out using manual and automatic methods in order to improve cff-DNA yield and optimise the extraction. Fetal RHD genotyping was performed using a commercial real-time polymerase chain reaction (PCR) kit, and the results were compared with postnatal serological RhD determination on cord blood.
Overall, 133 plasma samples were examined for the validation process, and a total of 423 tests were performed. No differences have been observed between the two extraction methods or between fresh or frozen cff-DNA regarding cff-DNA stability and quality parameters. There was 100% concordance between fetal RHD genotyping of cff-DNA and RhD phenotype on cord blood for both extraction methods on both fresh and frozen cff-DNA.
Our study shows the reliability of automatic and manual cff-DNA extraction methods and the possibility of freezing extracted cff-DNA when performing RHD genotyping. This result might be relevant for improving laboratory work and organisation through the development of a standardised procedure for fetal RHD genotyping on cff-DNA, laying the foundations for evidence-based use of anti-D Ig prophylaxis in RhD pregnant women.
本研究旨在验证使用不同DNA提取方法对游离胎儿DNA（cff-DNA）进行无创RHD基因分型，以及对新鲜和冷冻提取的cff-DNA进行无创RHD基因分型的情况。
cff-DNA的无创RHD基因分型可预测胎儿RhD表型，有助于合理实施产前免疫预防，是RhD免疫女性管理方面的一大进步。在临床应用诊断方法之前，必须对其进行验证。
招募不同孕周的RhD阴性孕妇。采用手动和自动方法进行cff-DNA提取，以提高cff-DNA产量并优化提取过程。使用商业实时聚合酶链反应（PCR）试剂盒进行胎儿RHD基因分型，并将结果与脐血产后血清学RhD测定结果进行比较。
总体而言，共检测了133份血浆样本用于验证过程，总共进行了423次检测。在cff-DNA稳定性和质量参数方面，两种提取方法之间以及新鲜或冷冻cff-DNA之间均未观察到差异。对于新鲜和冷冻cff-DNA，两种提取方法的cff-DNA胎儿RHD基因分型与脐血RhD表型之间的一致性均为100%。
我们的研究表明了自动和手动cff-DNA提取方法的可靠性，以及在进行RHD基因分型时冷冻提取的cff-DNA的可能性。这一结果可能与通过开发cff-DNA胎儿RHD基因分型的标准化程序来改善实验室工作和组织相关，为RhD孕妇基于证据使用抗D免疫球蛋白预防奠定基础。
