title: A multiplex PCR for non-invasive fetal RHD genotyping using cell-free fetal DNA.
abstract: OBJECTIVE
  To design a protocol for non-invasive prenatal diagnosis of fetal Rhesus D (RhD) status.
  
  
  METHODS
  A total of 112 single lymphocytes were used to test the efficiency of the assay. The protocol was validated using blood samples from 84 RhD-negative pregnant women at 7-24 weeks of gestation. Cell-free DNA (cfDNA) was enzymatically digested using AciI and analyzed by a polymerase chain reaction (PCR) that allowed simultaneous amplification of RHD exons 7 and 10, SRY, RASFF1A and ACTB.
  
  
  RESULTS
  On the one genome-equivalent level, the efficiency of the protocol was ≥ 94.6% for each locus amplified. Conclusive results from the first set of PCRs were obtained for 79 cases with one false-positive. In five cases the analysis was repeated and, subsequently, all cases were accurately diagnosed.
  
  
  CONCLUSIONS
  The proposed protocol is rapid, applicable in most molecular diagnostic laboratories and provides the basis for non-invasive examination of fetal RhD with 96.7% specificity and 100% sensitivity.
authors.affiliation: Department of Medical Genetics, Athens University School of Medicine, Athens, Greece.
authors.forename: Georgia
authors.initials: G
authors.lastname: Tounta
authors.forename: Christina
authors.initials: C
authors.lastname: Vrettou
authors.forename: Aggeliki
authors.initials: A
authors.lastname: Kolialexi
authors.forename: Nikolas
authors.initials: N
authors.lastname: Papantoniou
authors.forename: Aspasia
authors.initials: A
authors.lastname: Destouni
authors.forename: George T
authors.initials: GT
authors.lastname: Tsangaris
authors.forename: Aris
authors.initials: A
authors.lastname: Antsaklis
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Kanavakis
authors.forename: Ariadni
authors.initials: A
authors.lastname: Mavrou
mesh_terms: D001788:Blood Grouping and Crossmatching / Q000379:methods*; D005260:Female; D005333:Fetus; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D008297:Male; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D015203:Reproducibility of Results; D012204:Rh-Hr Blood-Group System / Q000235:genetics*; D055748:SOXB1 Transcription Factors / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics
chemical_list: C411762:RASSF1 protein, human; D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen; D055748:SOXB1 Transcription Factors; D025521:Tumor Suppressor Proteins