Robust Isolation and Multiplexed Identification of Circulating Rare Cells via Continuous Centrifugal Microfluidics and Hydrogel Immunocytochemistry.

Circulating rare cells (CRCs), including circulating tumor cells (CTCs) and circulating cancer-associated fibroblasts (cCAFs), are promising biomarkers for cancer diagnosis and prognosis. However, their extremely low abundance and phenotypic heterogeneity make efficient isolation and multiparametric analysis technically challenging. Here, we present a continuous centrifugal microfluidic workflow for the sequential isolation and identification of CRCs from whole blood. The cartridge comprises five interconnected chambers with controllable valves that enable precise handling of density gradient medium and blood layers during centrifugation. Based on a previously validated enrichment platform, the present workflow integrates hydrogel-based cell immobilization with multiplexed immunocytochemistry to support the downstream identification and characterization of multiple CRC subtypes, including CTCs and cCAFs. Following isolation, cells are immobilized within a hydrogel matrix to support cell retention during repeated immunostaining and imaging-based analysis. Using this workflow, both CTCs and cCAFs were identified from the same sample, demonstrating the feasibility of integrated analysis of multiple circulating rare-cell populations within a single liquid-biopsy workflow. This platform provides a practical and reproducible approach for downstream rare-cell characterization in liquid biopsy applications.