title: Evaluation of prenatal RHD typing strategies on cell-free fetal DNA from maternal plasma.
abstract: BACKGROUND
  The discovery of cell-free fetal DNA in maternal plasma led to the development of assays to predict the fetal D status with RHD-specific sequences. Few assays are designed in such a way that the fetus can be typed in RHDpsi mothers and that RHDpsi fetuses are correctly typed. Owing to the limited knowledge about the mechanism responsible for the presence of fetal DNA in maternal plasma, precautions in developing prenatal genotyping strategies must be made.
  
  
  METHODS
  Real-time quantitative (RQ)-polymerase chain reaction (PCR) assays were developed for prenatal diagnostic use with cell-free fetal DNA from maternal plasma. An RQ-PCR assay on RHD exon 5 (amplicon 361 bp), negative on RHDpsi, was developed with genomic DNA and evaluated with cell-free fetal DNA. A previously published RHD exon 5 RQ-PCR (amplicon 82 bp) was duplexed with an in-house developed RHD exon 7 RQ-PCR and evaluated with cell-free fetal DNA from pregnant D-RHDpsi+ women.
  
  
  RESULTS
  The RHD exon 5 361 bp assay showed on cell-free plasma DNA from D- women carrying a D+ fetus, low amplification levels, resulting in high Ct values and false-negative results. Owing to fragmentation of cell-free plasma DNA, too few DNA stretches of sufficient length (> 360 bp) are present. The RHD exon 5 82 bp and exon 7 RQ-PCR duplex was evaluated with RHDpsi+ cell-free plasma DNA and showed complete specificity and maximal sensitivity.
  
  
  CONCLUSIONS
  Assays designed for prenatal genotyping should be developed and evaluated on cell-free plasma DNA. Prenatal RHD typing is accurate with the RHD exon 5 82 bp and exon 7 duplex strategy.
authors.affiliation: Sanquin Research, Amsterdam, the Netherlands.
authors.forename: Martine G H M
authors.initials: MG
authors.lastname: Grootkerk-Tax
authors.forename: Aicha Ait
authors.initials: AA
authors.lastname: Soussan
authors.forename: Masja
authors.initials: M
authors.lastname: de Haas
authors.forename: Petra A
authors.initials: PA
authors.lastname: Maaskant-van Wijk
authors.forename: C Ellen
authors.initials: CE
authors.lastname: van der Schoot
mesh_terms: D001788:Blood Grouping and Crossmatching / Q000379:methods*; D004247:DNA / Q000097:blood*; D005091:Exons; D005260:Female; D005312:Fetal Blood / Q000276:immunology*; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction; D011247:Pregnancy / Q000097:blood*; D012204:Rh-Hr Blood-Group System / Q000235:genetics*
chemical_list: D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen; D004247:DNA