Circulating cell-free DNA undergoes significant decline in yield after prolonged storage time in both plasma and purified form

Circulating DNA (cirDNA) is generally purified from plasma that has been biobanked for variable lengths of time. In long-term experiments or clinical trials, the plasma can be stored frozen for up to several years. Therefore, it is crucial to determine the stability of cirDNA to ensure confidence in sample quality upon analysis. Our main objective was to determine the effect of storage for up to 2 years on cirDNA yield and fragmentation.We stored frozen EDTA plasma and purified cirDNA from 10 healthy female donors, then quantified cirDNA yield at baseline, and at regular intervals for up to 2 years, by qPCR and Qubit. We also compared cirDNA levels in non-haemolysed and haemolysed blood samples after 16 months of storage and tested the effect of varying DNA extraction protocol parameters.Storage up to two years caused an annual cirDNA yield decline of 25.5% when stored as plasma and 23% when stored as purified DNA, with short fragments lost more rapidly than long fragments. Additionally, cirDNA yield was impacted by plasma input and cirDNA elution volumes, but not by haemolysis.The design of long-term cirDNA-based studies and clinical trials should factor in the deterioration of cirDNA during storage.