| Summary: | Noninvasive fetal <i>RHD</i> genotyping is an important tool for predicting RhD incompatibility between a pregnant woman and a fetus. This study aimed to assess a methodological approach other than the commonly used one for noninvasive fetal <i>RHD</i> genotyping on a representative set of RhD-negative pregnant women. The methodology must be accurate, reliable, and broadly available for implementation into routine clinical practice. A total of 337 RhD-negative pregnant women from the Czech Republic region were tested in this study. The fetal <i>RHD</i> genotype was assessed using two methods: real-time PCR and endpoint quantitative fluorescent (QF) PCR. We used exon-7-specific primers from the <i>RHD</i> gene, along with internal controls. Plasma samples were analyzed and measured in four/two parallel reactions to determine the accuracy of the <i>RHD</i> genotyping. The <i>RHD</i> genotype was verified using DNA analysis from a newborn buccal swab. Both methods showed an excellent ability to predict the <i>RHD</i> genotype. Real-time PCR achieved its greatest accuracy of 98.6% (97.1% sensitivity and 100% specificity (95% CI)) if all four PCRs were positive/negative. The QF PCR method also achieved its greatest accuracy of 99.4% (100% sensitivity and 98.6% specificity (95% CI)) if all the measurements were positive/negative. Both real-time PCR and QF PCR were reliable methods for precisely assessing the fetal <i>RHD</i> allele from the plasma of RhD-negative pregnant women.
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