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).

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.

Figure 1. Effects of blood sample transportation time on DNA levels in maternal plasma.

The detection of DNA extracted from maternal plasma taken from blood samples (collected in 6-mL EDTA-tubes) subjected to 2–9 days of transport. DNA quantities are shown as Ct values. The total DNA concentration was assessed by detecting GAPDH (n = 110). Fetal DNA concentration was assessed by detecting RHD (n = 52). The boxes represent 25–75%, the line represents the median Ct values, and the whiskers are minimum and maximum values. The number of samples used to detect fetal and total DNA concentrations (n = fetal/total) was as follows: Day 2 (n = 0/3), Day 3 (n = 4/10), Day 4 (n = 8/17), Day 5 (n = 10/20), Day 6 (n = 12/29), Day 7 (n = 12/20), Day 8 (n = 4/8), and Day 9 (n = 2/3).

Figure 2. Effect of ambient outdoor temperatures on fetal DNA in maternal plasma.

Fetal DNA is shown as RHD Ct values against groups of 5-day averages of average daily temperatures, in degrees Celsius. A total of 1539 samples with RHD positive result were divided into groups based on 5-day averages of average daily temperatures, −5–0°C (n = 264), 0–5°C (n = 83), 5–10°C (n = 440), 10–15°C (n = 370), 15–20°C (n = 295), and 20–25°C (n = 87). See the Materials and Methods section for a detailed description of temperature calculations. Line inside box, median; limits of box, 75^th and 25^th percentile; whiskers, 2.5^th and 97.5^th percentiles.

Figure 3. Effect of ambient outdoor temperatures on the total DNA level in maternal plasma.

The effect of ambient outdoor temperatures is shown as the mean average degrees Celsius calculated for samples grouped in GAPDH Ct value intervals. See the Materials and Methods section for a detailed description of temperature calculations. Neither the mean average temperatures in intervals 20–24 nor those in intervals 24–33 were significantly different from each other. However, the mean average temperatures in intervals 21–24 were significantly different from the mean average temperatures in intervals 24–30.

Figure 4. Levels of cffDNA during pregnancy.

The cffDNA levels were estimated by the universal standard curve; the cffDNA levels increased with increasing GA at blood sampling. The data shows a marked increase after GA 30 weeks.

Figure 5. Distribution of blood samples according to GA at sampling.

The proper GA at sampling is scheduled at 25 weeks. GA ranged from 10 to 39 weeks.