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


METHODS
Whole blood from pregnant women carrying normal and trisomy 21 (T21) fetuses was collected in regular EDTA anti-coagulated tubes and processed within 6h, 24 and 48h 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.


RESULTS
Both T21 samples were correctly identified as such at all time-points. However, after 48h, 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 24h after venipuncture, the percentages based on MPS results did not show a significant decrease after 6, 24 or 48h.


CONCLUSIONS
The quality and quantity of ccffDNA extracted from plasma samples processed up to 24h 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.
Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands.
Karen
K
Buysse
Lean
L
Beulen
Ingrid
I
Gomes
Christian
C
Gilissen
Chantal
C
Keesmaat
Irene M
IM
Janssen
Judith J H T
JJ
Derks-Willemen
Joep
J
de Ligt
Ilse
I
Feenstra
Mireille N
MN
Bekker
John M G
JM
van Vugt
Ad
A
Geurts van Kessel
Lisenka E L M
LE
Vissers
Brigitte H W
BH
Faas
D000782:Aneuploidy; D004247:DNA / Q000097:blood*; D004314:Down Syndrome / Q000175:diagnosis / Q000235:genetics; D005260:Female; D005820:Genetic Testing / Q000379:methods*; D005865:Gestational Age; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D018962:Phlebotomy; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D015203:Reproducibility of Results; D013997:Time Factors
D004247:DNA
Circulating cell-free fetal DNA; MPS; Massively parallel sequencing; Maternal plasma; NIPT; Noninvasive prenatal testing; Processing time; ccffDNA; circulating cell-free fetal DNA; massively parallel sequencing; noninvasive prenatal testing