Complementary detection strategies for circulating tumor cells in breast cancer: clinical implications of combining immunofluorescence and cytopathological staining

Tanja Jesenko ^1,2, Cvetka Grasic Kuhar ^1,2, Ziva Pisljar ^1,2

⁰Institute of Oncology Ljubljana, Ljubljana, Slovenia.

Frontiers in Oncology +

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August 7, 2025

View abstract on PubMed

Summary

Combining Giemsa and Immunofluorescence (IF) staining offers a more accurate assessment of circulating tumor cells (CTCs) in metastatic breast cancer. This integrated approach improves the detection and characterization of CTCs, enhancing diagnostic precision.

Area Of Science

Oncology
Cell Biology
Biomarker Discovery

Background

Circulating tumor cells (CTCs) are crucial biomarkers for monitoring metastatic breast cancer and treatment response.
Detecting CTCs is challenging due to their low numbers, diverse phenotypes, and non-standardized detection methods.
Cytopathological Giemsa and Immunofluorescence (IF) staining provide complementary information for CTC characterization.

Purpose Of The Study

To develop and compare an IF staining protocol with Giemsa staining for CTC detection and characterization in metastatic breast cancer.
To evaluate the utility of IF staining for distinguishing epithelial, mesenchymal, hybrid, and hematopoietic cells.
To assess the combined diagnostic potential of Giemsa and IF staining for CTC analysis.

Main Methods

Developed an IF staining protocol using antibodies against Cytokeratin (CK), vimentin (VIM), and CD45.
Compared IF staining with Giemsa staining for CTC detection rates and morphological detail in 29 metastatic breast cancer patients.
Utilized a three-color antibody panel for phenotypic discrimination and structural analysis of CTCs.

Main Results

Giemsa staining detected a higher number of CTCs than the IF protocol.
Potential loss of fragile cells during IF staining and faint nuclear signals in IF-stained samples may have contributed to lower detection rates.
IF staining confirmed CTC identity, enabled detailed phenotypic discrimination, and revealed CK polarization patterns suggestive of intravasation.

Conclusions

Giemsa and IF staining are complementary, not mutually exclusive, for CTC analysis.
Relying on a single detection method may underestimate the true CTC burden.
An integrated strategy combining Giemsa and IF staining enhances the comprehensive evaluation of CTC populations and improves diagnostic precision in metastatic breast cancer.

Keywords:

biomarker breast cancer circulating tumor cells cytopathological detection immunofluorescence-based detection