# A fixation-compatible protocol for intracellular and surface marker-based detection of circulating tumor cells in hepatocellular carcinoma

## TL;DR

This study introduces a reliable method to detect liver cancer cells in blood using both surface and internal markers, even after preserving the cells.

## Contribution

A fixation-compatible protocol is developed for detecting hepatocellular carcinoma CTCs using both intracellular and surface markers.

## Key findings

- Fixed samples showed comparable staining performance to fresh samples with minimal cell recovery loss.
- CTC-derived DNA revealed tumor-specific CTNNB1 mutations not found in plasma cfDNA.
- Fixation enabled intracellular staining without compromising surface marker detection.

## Abstract

Circulating tumor cell (CTC) detection in hepatocellular carcinoma (HCC) is limited not only by the rarity of CTCs but also by a heavy reliance on cell surface markers such as EpCAM, which are variably expressed or lost during tumor progression. Detecting intracellular markers, such as cytokeratin offers an important complementary and comprehensive strategy but remains technically limited in flow cytometry due to the need for fixation and permeabilization, which often lead to cell loss and surface epitope damage. In this study, we systematically evaluated the feasibility of using fixed samples for flow cytometry, using HepG2 cells, PBMCs, and CTCs from patients with HCC. Our results demonstrate that fixation enabled intracellular staining without compromising cell surface marker detection, even after short-term storage at 4 °C and long-term storage at -80 °C. Fixed samples, particularly fixed unfrozen, exhibited comparable staining performance to fresh samples with only a 7–10% reduction in cell recovery. Clinical validation in HCC patients confirmed successful CTC detection, and tumor-specific CTNNB1 mutations were identified in CTC-derived DNA but not in matched plasma cfDNA. These findings support fixed CTC sample workflows as a reliable and practical approach for CTC analysis in HCC.

The online version contains supplementary material available at 10.1038/s41598-025-23698-1.