Usefulness and Limitations of Cryopreservation for Immunocytochemical Staining of Canine Cytological Specimens for Detection of Cytokeratin and Vimentin

Simple Summary Immunocytochemistry is a useful diagnostic tool in cytology; nonetheless, compared with pathological tissue specimens, cytological specimens have a crucial issue with long-term storage of antigenicity. A convenient and effective method is required for the storage of air-dried smears, which are commonly used for cytology, especially in small animal medicine. In the present study, we evaluated the usefulness of cryopreservation of air-dried smears for immunocytochemical staining for cytokeratin and vimentin, which can help distinguish between epithelial and non-epithelial tumors. The study results revealed that immunoreactivity for cytokeratin and vimentin could be detected for at least 33 months in unfixed smear samples stored in a freezer, and demonstrated the usefulness and effectiveness of cryopreserved air-dried smears in veterinary immunocytochemistry. Immunocytochemistry is an advanced diagnostic tool for identifying the origin of tumor cells. This study aimed to highlight the usefulness of cryopreserved, air-dried cytological samples in detecting cytokeratin and vimentin. Air-dried cytological smear samples were prepared from a total of 39 resected canine tumors and stored in a medical freezer without fixation. The duration of cryopreservation ranged from 2 to 56 months. The same tumors were processed for routine histopathological examination. Based on the morphological diagnosis, cryopreserved FNA smears from epithelial tumors were stained by enzymatic immunocytochemistry (ICC) for cytokeratin; those from mesenchymal and melanocytic tumors were stained by ICC for vimentin. To ascertain the positivity of tumor cells to the selected markers, tissue paraffin-embedded sections were also stained by immunohistochemistry (IHC) for the same markers. Immunoreactivity for cytokeratin was detected in cryopreserved cytological smears for a maximum of 46 months. Immunoreactivity for vimentin was clearly detected for 33 months. Smears stored at room temperature for 1 week did not show any signals under immunocytochemical examination. Thus, immunocytochemistry for cytokeratin and vimentin can be safely applied to air-dried smears cryopreserved in a freezer for at least 33 months.