Clinical Services and Research, Australian Red Cross Blood Service, Kelvin Grove, Brisbane, QLD, Australia.
Clinical Services and Research, Australian Red Cross Blood Service, Kelvin Grove, Brisbane, QLD, Australia.
Pathology. 2017 Dec;49(7):757-764. doi: 10.1016/j.pathol.2017.08.010. Epub 2017 Oct 31.
Non-invasive fetal RHD genotyping in Australia to reduce anti-D usage will need to accommodate both prolonged sample transport times and a diverse population demographic harbouring a range of RHD blood group gene variants. We compared RHD genotyping accuracy using two blood sample collection tube types for RhD negative women stratified into deleted RHD gene haplotype and RHD gene variant cohorts. Maternal blood samples were collected into EDTA and cell-free (cf)DNA stabilising (BCT) tubes from two sites, one interstate. Automated DNA extraction and polymerase chain reaction (PCR) were used to amplify RHD exons 5 and 10 and CCR5. Automated analysis flagged maternal RHD variants, which were classified by genotyping. Time between sample collection and processing ranged from 2.9 to 187.5 hours. cfDNA levels increased with time for EDTA (range 0.03-138 ng/μL) but not BCT samples (0.01-3.24 ng/μL). For the 'deleted' cohort (n=647) all fetal RHD genotyping outcomes were concordant, excepting for one unexplained false negative EDTA sample. Matched against cord RhD serology, negative predictive values using BCT and EDTA tubes were 100% and 99.6%, respectively. Positive predictive values were 99.7% for both types. Overall 37.2% of subjects carried an RhD negative baby. The 'variant' cohort (n=15) included one novel RHD and eight hybrid or African pseudogene variants. Review for fetal RHD specific signals, based on one exon, showed three EDTA samples discordant to BCT, attributed to high maternal cfDNA levels arising from prolonged transport times. For the deleted haplotype cohort, fetal RHD genotyping accuracy was comparable for samples collected in EDTA and BCT tubes despite higher cfDNA levels in the EDTA tubes. Capacity to predict fetal RHD genotype for maternal carriers of hybrid or pseudogene RHD variants requires stringent control of cfDNA levels. We conclude that fetal RHD genotyping is feasible in the Australian environment to avoid unnecessary anti-D immunoglobulin prophylaxis.
在澳大利亚，为了减少抗-D 的使用，需要对非侵入性胎儿 RHD 基因分型进行研究，以适应样本运输时间延长和不同人群的特点，这些人群携带着不同的 RHD 血型基因变异体。我们比较了两种血液样本采集管类型对 RhD 阴性女性的 RHD 基因分型准确性，这些女性分为缺失 RHD 基因单倍型和 RHD 基因变异体队列。分别从两个地点（一个在州际）采集 EDTA 和无细胞（cf）DNA 稳定（BCT）管中的母体血液样本。采用自动化 DNA 提取和聚合酶链反应（PCR）扩增 RHD 外显子 5 和 10 以及 CCR5。自动化分析标记母体 RHD 变体，然后通过基因分型进行分类。样本采集和处理之间的时间从 2.9 到 187.5 小时不等。EDTA 样本的 cfDNA 水平随时间增加（范围 0.03-138ng/μL），而 BCT 样本则没有（0.01-3.24ng/μL）。对于“缺失”队列（n=647），除了一个未解释的假阴性 EDTA 样本外，所有胎儿 RHD 基因分型结果均一致。与脐带 RhD 血清学相匹配，BCT 和 EDTA 管的阴性预测值分别为 100%和 99.6%，阳性预测值均为 99.7%。总体而言，37.2%的受试者携带 RhD 阴性婴儿。“变异”队列（n=15）包括一个新的 RHD 和八个混合或非洲假基因变体。基于一个外显子，对胎儿 RHD 特异性信号进行回顾性分析，发现三个 EDTA 样本与 BCT 不一致，这归因于由于运输时间延长而导致的母体 cfDNA 水平升高。对于缺失单倍型队列，尽管 EDTA 管中的 cfDNA 水平较高，但 EDTA 和 BCT 管中采集的样本的胎儿 RHD 基因分型准确性相当。预测携带混合或假基因 RHD 变体的母体胎儿 RHD 基因型的能力需要严格控制 cfDNA 水平。我们得出结论，在澳大利亚环境中进行胎儿 RHD 基因分型是可行的，可以避免不必要的抗-D 免疫球蛋白预防。
