title: Non-Invasive Prenatal RHD Genotyping Using Cell-Free Fetal DNA from Maternal Plasma: An Italian Experience.
abstract: BACKGROUND
  This study assessed the diagnostic accuracy of a non-invasive approach to fetal RHD genotyping using cell-free fetal DNA in maternal plasma and a combination of methodological strategies.
  
  
  METHODS
  Real-time PCR (qPCR) was performed on 216 RhD-negative women between weeks 10+0 and 14+6 of gestation (1st qPCR). qPCR was repeated (2nd qPCR) to increase the amount of each sample for analysis, on 95 plasma aliquots that were available from first trimester blood collection (group 1) and on 13 samples that were collected between weeks 18+0 and 25+6 of gestation (group 2). qPCR was specific for exons 5 and 7 of the RHD gene (RHD5 and RHD7). The results were interpreted according to the number of positive replicates of both exons.
  
  
  RESULTS
  1st qPCR: diagnostic accuracy was of 93.3%. Diagnostic accuracy increased from 90.5% (1st qPCR) to 93.7% (2nd qPCR) in group 1 and from 84.6% (1st qPCR) to 92.3% (2nd qPCR) in group 2. These increments were not statistically significant.
  
  
  CONCLUSIONS
  Our approach to RHD genotyping in early pregnancy yielded high diagnostic accuracy. Increasing the amount of DNA analyzed in each sample did not improve significantly the diagnostic accuracy of the test.
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital of Perugia, Perugia, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Picchiassi
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital of Perugia, Perugia, Italy.
authors.forename: Gian Carlo
authors.initials: GC
authors.lastname: Di Renzo
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital of Perugia, Perugia, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Tarquini
authors.affiliation: Department of Internal Medicine, University Hospital of Perugia, Perugia, Italy.
authors.forename: Vittorio
authors.initials: V
authors.lastname: Bini
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital of Perugia, Perugia, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Centra
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital of Perugia, Perugia, Italy.
authors.forename: Luana
authors.initials: L
authors.lastname: Pennacchi
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital of Perugia, Perugia, Italy.
authors.forename: Fabiana
authors.initials: F
authors.lastname: Galeone
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital of Perugia, Perugia, Italy.
authors.forename: Mara
authors.initials: M
authors.lastname: Micanti
authors.affiliation: Department of Obstetrics and Gynecology, University Hospital of Perugia, Perugia, Italy.
authors.forename: Giuliana
authors.initials: G
authors.lastname: Coata
keywords: Fetal DNA; Hemolytic disease of the fetus and newborn; Prenatal RHD genotyping