Cell-free DNA in maternal plasma and serum: A comparison of quantity, quality and tissue origin using genomic and epigenomic approaches

Abstract

Objectives: The objectives of this study were to compare the concentrations, size profiles and major tissue contributors of cell-free DNA (cfDNA) in plasma and in serum.

Design and methods: Thirteen pregnant women in the third trimester were recruited for this study. We collected EDTA-plasma and serum samples using various collection tubes. We determined their cfDNA concentrations and fetal cfDNA fractions using a zinc-finger X (ZFX)/zinc-finger Y (ZFY) droplet digital polymerase chain reaction (ZFX/ZFY ddPCR) assay. We used paired-end massively parallel sequencing (MPS) to measure plasma and serum cfDNA sizes at single-base resolution. We deconvoluted the genome-wide bisulfite sequencing data with reference to the methylation profiles of different tissues.

Results: The concentrations of cfDNA collected in Sarstedt Serum Z tubes were found to be significantly higher than those in Greiner Bio-One Vacuette® Z Serum Separator Clot Activator tubes or Vacuette® Z Serum Clot Activator tubes. The concentrations of fetal cfDNA were significantly reduced in samples collected in the Vacuette® serum collection tubes. Fetal cfDNA fractions were significantly reduced in all sera compared to plasma. MPS of serum cfDNA revealed a right shift of the size distributions compared to plasma. Methylation-based tissue mapping of serum cfDNA revealed an increase of cfDNA from neutrophils and B cells but not T cells.

Conclusions: The use of different serum collection tubes has a significant impact on serum cfDNA concentrations. This effect is likely mediated through the combined effect of genomic DNA release from white blood cells and DNA degradation or removal.

Keywords: Digital PCR; Epigenetics; Massively parallel sequencing; Noninvasive prenatal testing; Plasma cell-free DNA; Pregnancy; Serum cell-free DNA.