title: Pre-analytical conditions in non-invasive prenatal testing of cell-free fetal RHD.
abstract: BACKGROUND
  Non-invasive prenatal testing of cell-free fetal DNA (cffDNA) in maternal plasma can predict the fetal RhD type in D negative pregnant women. In Denmark, routine antenatal screening for the fetal RhD gene (RHD) directs the administration of antenatal anti-D prophylaxis only to women who carry an RhD positive fetus. Prophylaxis reduces the risk of immunization that may lead to hemolytic disease of the fetus and the newborn. The reliability of predicting the fetal RhD type depends on pre-analytical factors and assay sensitivity. We evaluated the testing setup in the Capital Region of Denmark, based on data from routine antenatal RHD screening.
  
  
  METHODS
  Blood samples were drawn at gestational age 25 weeks. DNA extracted from 1 mL of plasma was analyzed for fetal RHD using a duplex method for exon 7/10. We investigated the effect of blood sample transportation time (n = 110) and ambient outdoor temperatures (n = 1539) on the levels of cffDNA and total DNA. We compared two different quantification methods, the delta Ct method and a universal standard curve. PCR pipetting was compared on two systems (n = 104).
  
  
  RESULTS
  The cffDNA level was unaffected by blood sample transportation for up to 9 days and by ambient outdoor temperatures ranging from -10 °C to 28 °C during transport. The universal standard curve was applicable for cffDNA quantification. Identical levels of cffDNA were observed using the two automated PCR pipetting systems. We detected a mean of 100 fetal DNA copies/mL at a median gestational age of 25 weeks (range 10-39, n = 1317).
  
  
  CONCLUSIONS
  The setup for real-time PCR-based, non-invasive prenatal testing of cffDNA in the Capital Region of Denmark is very robust. Our findings regarding the transportation of blood samples demonstrate the high stability of cffDNA. The applicability of a universal standard curve facilitates easy cffDNA quantification.
authors.affiliation: Department of Clinical Immunology, Section 2034, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.
authors.forename: Frederik Banch
authors.initials: FB
authors.lastname: Clausen
authors.forename: Tanja Roien
authors.initials: TR
authors.lastname: Jakobsen
authors.forename: Klaus
authors.initials: K
authors.lastname: Rieneck
authors.forename: Grethe Risum
authors.initials: GR
authors.lastname: Krog
authors.forename: Leif Kofoed
authors.initials: LK
authors.lastname: Nielsen
authors.forename: Ann
authors.initials: A
authors.lastname: Tabor
authors.forename: Morten Hanefeld
authors.initials: MH
authors.lastname: Dziegiel
mesh_terms: D001800:Blood Specimen Collection / Q000379:methods; D004247:DNA / Q000097:blood / Q000235:genetics / Q000276:immunology*; D005260:Female; D005333:Fetus / Q000276:immunology* / Q000378:metabolism; D005865:Gestational Age; D006801:Humans; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D015203:Reproducibility of Results; D012204:Rh-Hr Blood-Group System / Q000235:genetics / Q000276:immunology*; D013696:Temperature; D013997:Time Factors; D014186:Transportation
chemical_list: D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen; D004247:DNA