Investigation of a storage method for peritoneal and pleural effusion cytology specimens for long-term preservation of antigenicity in immunocytochemical staining

Introduction Additional immunocytochemical staining is frequently required in routine cytodiagnosis to refine presumptive diagnoses. However, optimal conditions for preserving antigenicity in cytological specimens over time remain unclear. This study evaluated the effects of preparation method, storage temperature, and storage duration on antigen preservation in clinical cytology specimens and cultured malignant tumor cells. Methods Residual sediments from 12 pleural or ascitic fluid cytology specimens were fixed in BD CytoRich Red for 1 h and processed using the SurePath liquid-based cytology method. Immunocytochemical staining performed immediately after preparation served as the baseline control. Specimens were stored as glass smears or in preservative fluid at 4 °C or −20 °C for 1 week, 1 month, or 3 months. Ki-67 and calretinin were used as primary antibodies. To reflect routine cytology practice, Papanicolaou-stained preparations were also included. Cultured malignant tumor cell lines (TCC-Meso-1, MCF-7, and KATO III) were prepared as Papanicolaou-stained, coverslipped slides and stored at room temperature. Results In clinical glass smears, neither Ki-67 nor calretinin showed a significant decline compared with baseline across storage conditions. In preservative fluid, Ki-67 positivity differed from glass smears and was associated with storage temperature; 4 °C storage resulted in significant reductions from 1 week onward. At −20 °C, reductions were limited, although selected short-term comparisons with baseline reached statistical significance. In clinical Papanicolaou-stained preparations, Ki-67 showed a significant time-dependent decrease evident at 3 months, with a similar pattern observed for calretinin. In FDR-adjusted comparisons with the non-stored baseline, Pap-stained samples exhibited the greatest reductions in rate ratios, whereas most cooled non-Pap preparations retained relatively preserved immunopositivity. In cultured Pap-stained cell preparations, Ki-67 reductions were modest and tumor type–related, with no decrease observed in TCC-Meso-1 cells. Conclusion Fixation followed by glass smear preparation and low-temperature storage (4 °C or −20 °C) allows relatively stable preservation of Ki-67 and calretinin immunoreactivity. Storage at −20 °C may be preferable when longer preservation periods are required. In contrast, delayed immunocytochemical staining of Papanicolaou-stained specimens may result in tumor type–related variability in Ki-67 immunoreactivity and therefore should be interpreted with caution.