Immunohaematology and Transfusion Medicine Service Metropolitan Area of Bologna, "S. Orsola-Malpighi" Polyclinic, Bologna, Italy.
Immunohaematology and Transfusion Medicine Service, "Ospedale degli Infermi", Rimini, Italy.
Blood Transfus. 2018 Nov;16(6):514-524. doi: 10.2450/2018.0270-17. Epub 2018 Apr 9.
Foetal RHD genotyping can be predicted by real-time polymerase chain reaction (qPCR) using cell-free foetal DNA extracted from maternal plasma. The object of this study was to determine the diagnostic accuracy and feasibility of non-invasive RHD foetal genotyping, using a commercial multiple-exon assay, as a guide to appropriate administration of targeted antenatal immunoprophylaxis.
Cell-free foetal DNA was extracted from plasma of RhD-negative women between 11-30 weeks of pregnancy. The foetal RHD genotype was determined non-invasively by qPCR amplification of exons 5, 7 and 10 of the RHD gene using the Free DNA Fetal Kit RhD. Results were compared with serological RhD cord blood typing at birth. The analysis of diagnostic accuracy was restricted to the period (24-28 weeks) during which foetal genotyping is usually performed for targeted antenatal immunoprophylaxis.
RHD foetal genotyping was performed on 367 plasma samples (24-28 weeks). Neonatal RhD phenotype results were available for 284 pregnancies. Foetal RHD status was inconclusive in 9/284 (3.2%) samples, including four cases with RhD maternal variants. Two false-positive results were registered. The sensitivity was 100% and the specificity was 97.5% (95% CI: 94.0-100). The diagnostic accuracy was 99.3% (95% CI: 98.3-100), decreasing to 96.1% (95% CI: 93.9-98.4) when the inconclusive results were included. The negative and positive predictive values were 100% (95% CI: 100-100) and 99.0% (95% CI: 97.6-100), respectively. There was one false-negative result in a sample collected at 18 weeks. After inclusion of samples at early gestational age (<23 week), sensitivity and accuracy were 99.6% (95% CI: 98.7-100) and 95.5% (95% CI: 93.3-97.8), respectively.
This study demonstrates that foetal RHD detection on maternal plasma using a commercial multiple-exon assay is a reliable and accurate tool to predict foetal RhD phenotype. It can be a safe guide for the appropriate administration of targeted prenatal immunoprophylaxis.
通过从母体血浆中提取游离胎儿 DNA，实时聚合酶链反应 (qPCR) 可预测胎儿 RHD 基因分型。本研究的目的是确定使用商业性多外显子检测方法进行非侵入性胎儿 RHD 基因分型的诊断准确性和可行性，作为指导适当进行靶向产前免疫预防的依据。
在妊娠 11-30 周的 RhD 阴性妇女的血浆中提取游离胎儿 DNA。使用 Free DNA Fetal Kit RhD 通过 qPCR 扩增 RHD 基因的外显子 5、7 和 10 对胎儿 RHD 基因型进行非侵入性检测。结果与出生时脐带血血清学 RhD 定型进行比较。诊断准确性分析仅限于通常进行胎儿基因分型以进行靶向产前免疫预防的时期（24-28 周）。
对 367 份血浆样本（24-28 周）进行了 RHD 胎儿基因分型。有 284 例妊娠获得了新生儿 RhD 表型结果。9/284（3.2%）例样本的胎儿 RHD 状态不确定，包括 4 例 RhD 母体变体。登记了 2 例假阳性结果。敏感性为 100%，特异性为 97.5%（95%CI：94.0-100）。诊断准确性为 99.3%（95%CI：98.3-100），当包含不确定结果时，降低至 96.1%（95%CI：93.9-98.4）。阴性和阳性预测值分别为 100%（95%CI：100-100）和 99.0%（95%CI：97.6-100）。在 18 周采集的样本中存在 1 例假阴性结果。纳入早孕期（<23 周）的样本后，敏感性和准确性分别为 99.6%（95%CI：98.7-100）和 95.5%（95%CI：93.3-97.8）。
本研究表明，使用商业性多外显子检测方法在母体血浆中检测胎儿 RHD 是一种可靠且准确的预测胎儿 RhD 表型的工具。它可以作为适当进行靶向产前免疫预防的安全指导。
