Development and validation of multiplex real-time PCR assay for noninvasive prenatal assessment of fetal RhD status and fetal sex in maternal plasma

Elena Ordoñez, Laura Rueda, M. Paz Cañadas, Carme Fuster, Vincenzo Cirigliano

Research output: Contribution to journalArticleResearchpeer-review

2 Citations (Scopus)

Abstract

© 2013 S. Karger AG, Basel. Objective: Noninvasive prenatal detection of RhD status and fetal sex is becoming part of daily practice in clinical laboratories. We evaluated a high throughput procedure for automated DNA extraction and developed a multiplex real-time PCR (rt-PCR) for the simultaneous detection of three fetal loci in a single reaction to assess fetal sex and RhD status in maternal plasma. Methods: An automated DNA extraction method was evaluated together with a new multiplex rt-PCR assay for the simultaneous detection of exons 5 and 7 of the RHD gene together with the Y chromosome marker DYS14 in maternal plasma. The test was evaluated on 60 samples of known fetal genotype obtained from RhD-negative pregnant women before being applied prospectively on 158 consecutive clinical cases. Results were compared with newborn phenotypes. Results: Automated DNA extraction allowed successful analysis of all samples. DYS14 was detected in 118 cases (male fetuses) and both RHD exon 5 and 7 were detected in 148 samples. In 70 samples neither RHD exon 5 nor RHD exon 7 were detected (RhD-negative fetuses). Absence of all three sequences (female RhD-negative fetuses) was assessed in 33 samples. All prenatal results were in concordance with postnatal RhD status and fetal sex without false- positive or -negative results. Conclusion: The automated DNA extraction procedure coupled with a novel multiplex rt-PCR assay proved accurate, efficient and reliable allowing rapid and high throughput noninvasive determination of fetal sex and RhD status in clinical samples.
Original languageEnglish
Pages (from-to)13-18
JournalFetal Diagnosis and Therapy
Volume34
Issue number1
DOIs
Publication statusPublished - 1 Jan 2013

Keywords

  • Maternal circulation
  • Noninvasive prenatal diagnosis
  • Polymerase chain reaction
  • RHD genotyping

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