Table 1.

Pre-analytical factors that can influence outcomes of ccfDNA analysis

Pre-analytical step affecting ccfDNA	Challenge	Recommendation	Quote
Blood collection tube	Ex vivo release of genomic DNA from leukocytes; PCR inhibition	Use of a dedicated ccfDNA stabilization tube	[48••]
		Use of EDTA tube, in case no dedicated ccfDNA stabilization tube is available	[49]
Time between blood collection and plasma processing	Ex vivo release of genomic DNA from leukocytes	Must be verified and validated in combination with downstream application In case of unstabilized EDTA blood, processing should be performed within 2 to 6 h	[44•, 48••, 50, 51•]
Plasma or serum	Ex vivo release of genomic DNA from leukocytes	Use of plasma	[48••, 52, 53, 54]
Plasma processing protocol	Incomplete separation of cellular fraction; mechanical lysis of blood cells	For EDTA blood, use double centrifugation protocol with low and high speed centrifugation Follow ccfDNA tube manufacturer’s instructions	[44•, 48••, 55, 56]
Plasma storage	Reduction of yield; increased fragmentation	Do not store plasma at 2–8 °C for longer than 24 h Freeze at − 20 °C or − 80 °C Avoid repeated freezing/thawing cycles	[44•, 48••, 51•]
DNA purification method	Suboptimal compatibility with blood collection tube; low yield due to DNA loss; isolated ccfDNA lengths bias	Validation and verification of DNA purification method Use of an integrated system of kit and tube Follow ccfDNA tube manufacturer’s instructions	[43, 48••, 58–60]
DNA quantification	Over quantification due to impurities and detection limit in spectrophotometry	Use of qPC- based methods rather than spectrophotometry	[43, 48••, 59]
DNA storage	Reduction of yield Increased fragmentation	Store ccfDNA at − 20 °C or below Avoid repeated freezing and thawing	[48••, 51•]