Analytical approaches for the isolation and characterisation of circulating tumour cells in clinical practice

Circulating tumour cells (CTC) are rare cells shed from primary and metastatic tumours into the bloodstream, representing a minimally invasive source of information on tumour biology, dissemination, and therapeutic resistance. Their detection, isolation, and characterisation have emerged as critical tools for precision oncology, serving as robust prognostic and predictive biomarkers that reflect metastatic potential, treatment response, and overall survival. CTC isolation and analysis are challenging due to their extremely low frequency—often one cell per million blood cells—and their phenotypic heterogeneity. Current strategies include enrichment-based methods, exploiting physical or molecular properties for selective capture, and non-enrichment approaches, which preserve cellular integrity for downstream molecular, functional, and imaging analyses. Key performance metrics for isolation platforms include capture efficiency, enrichment, purity, throughput, recovery, and cell viability. Validation often relies on spiked cell lines, though these models may overestimate performance compared with heterogeneous patient-derived CTCs. Integrative approaches combining multiple methodologies are essential to fully characterize CTC populations and overcome individual limitations. Technological advances, including microfluidics, single-cell sequencing, and artificial intelligence-driven analysis, have enhanced the sensitivity and specificity of CTC detection, enabling comprehensive molecular and functional profiling. The FDA-approved CellSearch® system remains the clinical gold standard, primarily in metastatic disease, but emerging platforms aim to enable early detection, real-time disease monitoring, and the identification of subpopulations with high metastatic potential. Future directions in CTC research focus on early cancer detection, longitudinal monitoring of therapeutic response, and personalized treatment adaptation. Integration with other circulating biomarkers, such as circulating tumour DNA and exosomes, promise enhanced prognostic and diagnostic accuracy. Standardization of protocols and validation in clinical samples will be critical for translating these technologies into routine oncology practice. • CTCs reflect metastasis, resistance, and survival. • Detection methods are becoming more sensitive and specific. • Combining morphology, molecular, and functional analysis is more comprehensive. • Standardized protocols are needed for clinical use. • Future: early detection, real-time monitoring, and personalized medicine.