Fetal Sex and RHD Genotyping with Digital PCR Demonstrates Greater Sensitivity than Real-time PCR
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2015-11-01Author
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<jats:title>Abstract</jats:title> <jats:sec> <jats:title>BACKGROUND</jats:title> <jats:p>Noninvasive genotyping of fetal RHD (Rh blood group, D antigen) can prevent the unnecessary administration of prophylactic anti-D to women carrying RHD-negative fetuses. We evaluated laboratory methods for such genotyping.</jats:p> </jats:sec> <jats:sec> <jats:title>METHODS</jats:title> <jats:p>Blood samples were collected in EDTA tubes and Streck® Cell-Free DNA™ blood collection tubes (Streck BCTs) from RHD-negative women (n = 46). Using Y-specific and RHD-specific targets, we investigated variation in the cell-free fetal DNA (cffDNA) fraction and determined the sensitivity achieved for optimal and suboptimal samples with a novel Droplet Digital™ PCR (ddPCR) platform compared with real-time quantitative PCR (qPCR).</jats:p> </jats:sec> <jats:sec> <jats:title>RESULTS</jats:title> <jats:p>The cffDNA fraction was significantly larger for samples collected in Streck BCTs compared with samples collected in EDTA tubes (P &lt; 0.001). In samples expressing optimal cffDNA fractions (≥4%), both qPCR and digital PCR (dPCR) showed 100% sensitivity for the TSPY1 (testis-specific protein, Y-linked 1) and RHD7 (RHD exon 7) assays. Although dPCR also had 100% sensitivity for RHD5 (RHD exon 5), qPCR had reduced sensitivity (83%) for this target. For samples expressing suboptimal cffDNA fractions (&lt;2%), dPCR achieved 100% sensitivity for all assays, whereas qPCR achieved 100% sensitivity only for the TSPY1 (multicopy target) assay.</jats:p> </jats:sec> <jats:sec> <jats:title>CONCLUSIONS</jats:title> <jats:p>qPCR was not found to be an effective tool for RHD genotyping in suboptimal samples (&lt;2% cffDNA). However, when testing the same suboptimal samples on the same day by dPCR, 100% sensitivity was achieved for both fetal sex determination and RHD genotyping. Use of dPCR for identification of fetal specific markers can reduce the occurrence of false-negative and inconclusive results, particularly when samples express high levels of background maternal cell-free DNA.</jats:p> </jats:sec>
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