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Detection of cell-free fetal DNA in plasma of pregnant women using multiplex quantitative fluorescent-PCR

ObjectiveTo test the sensitivity and specificity of retrieved cell-free fetal DNA (cff-DNA) from maternal plasma in the first and second trimesters for future use in clinical practice as a safe noninvasive prenatal diagnostic tool. MethodsMaternal blood was drawn from 25 women from the outpatient antenatal clinic of the National Research Centre between 10+0 and 12+6 weeks of gestation for quantitative fluorescent-PCR (QF-PCR). DNA was extracted from maternal buffy coat and from 0.8 ml cell-free maternal plasma samples using a QIAamp DNA Blood Mini Kit. All women were followed up until delivery according to the antenatal unit protocol, when neonatal blood was collected for a confirmatory QF-PCR. PCR amplifications were performed using the Amp FlSTR Identifier PCR Amplification Kit, which coamplifies the repeat regions of 15 short tandem repeat loci. Segments of the X and Y homologous genes were also amplified for sex identification. ResultsParticipants included women aged between 25 and 37 years, with a mean age of 29.3±0.6 years. Overall, 28% of the women were primigravidae, 40% were in their second pregnancy, 20% in their third, and only three women were pregnant for the fourth or fifth time. Determination of fetal sex was successfully achieved in all cases by amplification of the X/Y homologous region amelogenin. Male fetuses (n=10) were identified by the detection of the Y-derived sequence together with the X-specific product. In all cases, the ratio between these two fluorescent peaks also allowed the assessment of the sex chromosome copy number and maternal contamination. For chromosomes 2, 3, 5, 7, 8, 11, 12, 13, 16, 18, 19, 21, and X and Y, 24 out of 25 cff-DNA samples yielded a normal QF-PCR result, whereas in neonatal blood all 25 samples yielded a normal result with a heterozygosity of 81.86% for all loci. The ratio between the two corresponding peaks in all cases was very close to 1 : 1. Ninety-five percent of plasma sampled for each marker had a range of (x¯±1.96 SE). The markers were positively skewed with a median above 1.1. Four markers had a median above 1.0, which made fetal DNA easily detectable. The QF-PCR technique had a sensitivity of 100% and a specificity of 96.15% with an accuracy of 0.98; the likelihood ratio of a positive result was 20.97 with a 96%, positive predictive value. The false-negative value was 1, with a false-negative rate of 0.038. Fetal DNA samples were amplified successfully using the QF-PCR technique, and full results were obtained 48 h after DNA extraction. All neonatal samples were identical to that of the circulating cff-DNA, except for one sample of fetal DNA that showed some degree of maternal contamination. ConclusionCirculatory cff-DNA can be easily enriched from the plasma of pregnant women as early as 10 weeks’ gestation and onward. This technique relies on informative markers and the availability of parental DNA. Therefore, it can be used as a routine method for prenatal screening for common chromosomal or sex-linked aneuploidies.