Table 8.

Evidence-based methodological recommendations for processing and analysis of cfDNA. *insufficient data available to comment on other methods (e.g. digital PCR).

Methodological factor	Recommendation	Justification
Blood specimen type	Plasma for analytical techniques that are sensitive to a wild-type DNA background; plasma or serum for all other techniques.	cfDNA levels in serum appear significantly greater than in plasma due to contamination from genomic DNA.
Storage conditions of blood and time to processing	Store EDTA blood at either room temperature or 4 °C and process within 6 hours. Specialist tubes should adhere to manufacturers' guidelines.	Storage of blood at room temperature does not influence cfDNA yield relative to storage at room temperature. cfDNA levels increase in EDTA blood processed after 6 hours.
Centrifugation speed and time of blood	Spin blood and then re-spin plasma prior to storage for cfDNA isolation (double spin). Centrifugation speed and temperature (room temperature or 4 °C) is not critical.	Plasma can be contaminated with cells from the buffy layer. The second spin helps to minimize the potential for contamination of plasma with cells from the buffy layer.
Method of cfDNA isolation and quantification	Follow manufacturers recommendation for cfDNA isolation. Quantify by qPCR or fluorometry.	Quantification of cfDNA shows good agreement by qPCR, dPCR and fluorometry.*