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).
CONCLUSION: 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.