Optimization of bile preparation for liquid biopsy in cholangiocarcinoma focusing on circulating tumor DNA and protein stability

Liquid biopsy using bile offers a promising non-invasive approach for molecular analysis in cholangiocarcinoma (CCA). However, the stability of key biomarkers, such as proteins and circulating DNA (ctDNA), at room temperature has not been fully elucidated. This study investigates the temporal stability of proteins and ctDNA in bile samples under room temperature conditions to optimize pre-analytical handling for molecular diagnostics. Bile samples were collected from six patients diagnosed with CCA. Protein concentrations, enzyme activity (E-Cadherin and N-Cadherin), and mutant KRAS ctDNA levels were assessed at 1-, 3-, 5-, and 7-hour intervals using quantitative assays and droplet digital PCR (ddPCR). Proteins and enzyme activity demonstrated no significant degradation over the 7-hour room temperature storage period (P > 0.05). Similarly, mutant KRAS ctDNA levels remained stable without significant changes (P > 0.05), confirming the preservation of molecular integrity in bile samples. This study demonstrates that bile samples can maintain the stability of proteins and ctDNA for up to 7 h at room temperature. These findings provide critical insights into bile sample handling, supporting its application in liquid biopsy and molecular diagnostics for CCA.