title: Development and validation of multiplex real-time PCR assay for noninvasive prenatal assessment of fetal RhD status and fetal sex in maternal plasma.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Departament de Genètica Molecular, Labco Diagnostics, Barcelona, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Ordoñez
authors.forename: Laura
authors.initials: L
authors.lastname: Rueda
authors.forename: M Paz
authors.initials: MP
authors.lastname: Cañadas
authors.forename: Carme
authors.initials: C
authors.lastname: Fuster
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Cirigliano
mesh_terms: D005260:Female; D006801:Humans; D008297:Male; D062145:Maternal Serum Screening Tests; D011247:Pregnancy; D060888:Real-Time Polymerase Chain Reaction; D012204:Rh-Hr Blood-Group System / Q000097:blood*; D012732:Sex Determination Analysis
chemical_list: D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen