Pre-analytical standardization and multi-biofluid harmonization for liquid biopsy biomarkers in lung cancer: From discovery to clinical laboratory implementation.

Lung cancer remains the leading cause of cancer-related mortality worldwide, with early detection being critical for improving survival outcomes. Liquid biopsy using circulating biomarkers from blood, urine, saliva, sputum, exhaled breath condensate (EBC), and sweat has emerged as a promising non-invasive approach for lung cancer diagnosis, prognosis, and treatment monitoring. However, despite an abundance of biomarker discovery studies, clinical translation has been critically hampered by the absence of standardized pre-analytical protocols, inter-platform analytical discordance, and a lack of systematic frameworks for integrating biomarkers across multiple biofluids. This review identifies and addresses a major unmet need in the field: the harmonization of pre-analytical, analytical, and post-analytical workflows across diverse non-invasive biofluids for lung cancer liquid biopsy. We systematically evaluate pre-analytical variables specific to each biofluid, including collection methods, processing timelines, storage conditions, and matrix-specific confounders such as hemolysis, salivary contamination, and volatile compound degradation. We compare analytical platforms, including qRT-PCR, next-generation sequencing, mass spectrometry, ELISA, and electrochemical biosensors, and assess their cross-platform reproducibility for key analyte classes: circulating tumor DNA, microRNAs, proteins, metabolites, volatile organic compounds, and extracellular vesicles. Furthermore, we propose a structured clinical implementation roadmap encompassing external quality assessment schemes, commutable reference materials, multi-center validation requirements, and regulatory considerations. We conclude that the integration of multi-biofluid biomarker panels with artificial intelligence-assisted interpretation, combined with rigorous pre-analytical standardization, holds the greatest promise for translating liquid biopsy from bench to bedside in lung cancer care.