Stable Wet-Fixed S-MPF Preparation for Enhanced Circulating Tumor-associated Cell (C-TAC) Analysis

BACKGROUND/AIM: High-quality cytopreparation is essential for the morphological analysis of circulating tumor-associated cells (C-TACs). While the Soft Micro Pore Filter (S-MPF) method is effective for cell recovery, technical challenges such as air bubble entrapment and uneven cell distribution remain. We aimed to establish a stable and reproducible cytopreparation protocol by precisely controlling airflow and fluid dynamics using a three-way stopcock. MATERIALS AND METHODS: A modified preprocessing workflow was developed integrating a three-way stopcock to eliminate air bubbles through priming. We evaluated: (i) processing time and leukocyte residual rate, (ii) recovery and transfer rates using spiked HeLa cells, and (iii) the preservation of large 3D C-TAC clusters from lung cancer patient samples compared to a provisional method. RESULTS: The modified method substantially improved filtration time (from 2.91 to 0.39 min) and reduced the leukocyte residual rate (from 7.3% to <0.1%). The cell transfer rate to slides was highly stable at 98.7% [coefficient of variation (CV)=11.8%]. Furthermore, large 3D clusters consisting of ≥15 cells were successfully transferred from clinical samples while maintaining their diagnostic morphological features. CONCLUSION: The integration of a three-way stopcock provides a robust and standardized workflow for S-MPF-based cytopreparation. This protocol substantially enhances the reliability of C-TAC morphological diagnostics in routine clinical practice.