Guidelines for pre-analytical conditions for assessing the methylation of circulating cell-free DNA

Methylation analysis of circulating cell-free DNA (cirDNA), as a liquid biopsy, has a significant potential to advance the detection, prognosis, and treatment of cancer, as well as many genetic disorders. The role of epigenetics in disease development has been reported in several hereditary disorders, and epigenetic modifications are regarded as one of the earliest and most significant genomic aberrations that arise during carcinogenesis. Liquid biopsy can be employed for the detection of these epigenetic biomarkers. It consists of isolation (pre-analytical) and detection (analytical) phases. The choice of pre-analytical variables comprising cirDNA extraction and bisulfite conversion methods can affect the identification of cirDNA methylation. Indeed, different techniques give a different return of cirDNA, which confirms the importance of pre-analytical procedures in clinical diagnostics. Although novel techniques have been developed for the simplification of methylation analysis, the process remains complex, as the steps of DNA extraction, bisulfite treatment, and methylation detection are each carried out separately. Recent studies have noted the absence of any standard method for the pre-analytical processing of methylated cirDNA. We have therefore conducted a comprehensive and systematic review of the important pre-analytical and analytical variables and the patient-related factors which form the basis of our guidelines for analyzing methylated cirDNA in liquid biopsy.