Reliable noninvasive prenatal testing by massively parallel sequencing of circulating cell-free DNA from maternal plasma processed up to 24 h after venipuncture

• The effects of plasma processing times on downstream NIPT procedures are studied.
• Fetal trisomy 21 could be reliably detected, even when processing after 48 h.
• By enrichment the effect of processing time on the fetal fraction is minimized.
• The percentage of ccffDNA should be determined in the correct sample fraction.

ObjectivesCirculating cell-free fetal DNA (ccffDNA) in maternal plasma is an attractive source for noninvasive prenatal testing (NIPT). The amount of total cell-free DNA significantly increases 24 h after venipuncture, leading to a relative decrease of the ccffDNA fraction in the blood sample. In this study, we evaluated the downstream effects of extended processing times on the reliability of aneuploidy detection by massively parallel sequencing (MPS).Design and methodsWhole blood from pregnant women carrying normal and trisomy 21 (T21) fetuses was collected in regular EDTA anti-coagulated tubes and processed within 6 h, 24 and 48 h after venipuncture. Samples of all three different time points were further analyzed by MPS using Z-score calculation and the percentage of ccffDNA based on X-chromosome reads.ResultsBoth T21 samples were correctly identified as such at all time-points. However, after 48 h, a higher deviation in Z-scores was noticed. Even though the percentage of ccffDNA in a plasma sample has been shown previously to significantly decrease 24 h after venipuncture, the percentages based on MPS results did not show a significant decrease after 6, 24 or 48 h.ConclusionsThe quality and quantity of ccffDNA extracted from plasma samples processed up to 24 h after venipuncture are sufficiently high for reliable downstream NIPT analysis by MPS. Furthermore, we show that it is important to determine the percentage of ccffDNA in the fraction of the sample that is actually used for NIPT, as downstream procedures might influence the fetal or maternal fraction.