Circulating tumor cells (CTCs) hold recognized prognostic value in various cancers, including breast cancer, where their presence correlates with survival outcomes. However, the typical 24 h window for blood processing and CTC isolation poses a logistical challenge, particularly for multicenter studies. This study aimed to evaluate cryopreservation at different stages of CTC isolation and immunocytological detection to extend the blood sample processing period. Using spiked peripheral blood samples with MDA-MB-231, SKBR3, and MCF7 breast cancer cell lines, four distinct cryopreservation points were assessed: following Ficoll gradient separation, immunomagnetic separation, cytocentrifugation, and cytokeratin labeling. Our findings demonstrated that cryopreservation of the mononuclear and granulocytic cell fraction after double-density Ficoll gradient separation was the only viable method for subsequent CTC detection. This approach allowed for consistent recovery of CK+ CTCs, with an average recovery rate of over 81% after one year of cryopreservation. In contrast, cryopreservation at later stages resulted in undetectable CTCs or only cellular debris. In conclusion, cryopreservation following density gradient centrifugation is a feasible strategy for delaying CTC isolation and immunocytological analysis in breast cancer research, facilitating its application in multicenter clinical trials. [ABSTRACT FROM AUTHOR]

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