The influence of blood sample processing on blood-based DNA methylation signatures

Stability is crucial for the clinical applicable biomarker such as DNA methylation profiles. However, the influence of various blood processing on the DNA methylation signatures have been barely studied. Here, we systematically evaluated the impact of temporary storage and frozen and thaw on the levels of DNA methylation. The methylation intensities of 13 CpG loci from 53 individuals (42 healthy participants and 11 lung cancer patients) whose blood samples were processed by up to 14 various protocols were quantitatively determined by the mass spectrometry, obtaining more than 8,000 quantitative data. We disclosed a trend of accumulatively increased DNA methylation along with time when the blood from healthy people were stored for up to 96 h at room temperature (RT), whereas the storage at 4°C had much weaker effects or no impact. On the contrary, the methylation patterns in the blood of lung cancer patients were more stable at both RT and 4°C even for 96 h. Multiple cycles of frozen and thaw could result in demethylation, but was more significant to the blood than to the extracted DNA. Our study indicated that the blood processing in vitro could influence the DNA methylation signatures and introduce unexpected biases.