The argyrophilic nucleolar organizer regions (AgNORs) are cellular proliferation markers, crucial for predicting the clinical course and aggressiveness of tumors. The purpose of this study was to establish an easy and practical AgNOR staining method in the cytology of dogs and cats. Air-dried cytological slides were prepared from dogs (n = 14) and cats (n = 12). Ethanol, acetone, and formalin were tested as fixatives for AgNOR staining. Subsequently, various methods of Romanowsky-based counterstains were tested before and after AgNOR staining. Clear and strong AgNOR spots were observed with all fixatives, and post-May-Grünwald staining was the best counterstaining method. The established method showed clear AgNOR spots even in the long-term storage samples and Romanowsky-stained ones.

Epithelial-mesenchymal transition (EMT) plays a crucial role in metastasis of epithelial tumors; however, it is challenging to detect EMT by cytology. In the present study, EMT was visualized by fluorescenceimmunocytochemistry (FICC). Air-dried smears from epithelial tumors of dogs (n = 22) and cats (n = 9) were stained using mouse monoclonal anti-E-cadherin and rabbit monoclonal anti-vimentin antibodies.Enzymatic immunohistochemistry (IHC) revealed that 51.6 % (8/22 in dogs, 8/9 in cats) of the cases showed EMT. In dogs, FICC could detect EMT in 62.5 % (5/8) of those cases. In cats, FICC could detect EMT in 100 % (8/8) of the cases. In conclusion, the present FICC method could successfully detect EMT using conventional air-dried cytology smear slides.

CD20 and CD3 are considered reliable markers for B and T cells, respectively. This study aimed to develop a rapid multiple immunofluorescence (RMIF) method for the detection of CD20 and CD3 on a single cytology slide. Air-dried smears were prepared using samples collected from dogs (n=26) and cats (n=6). Immunosignal detection using the newly developed method required 60 min. Clear immunosignals for CD20 and CD3 were detected in 24 of 26 samples in dogs and in all 6 cats. As the RMIF (CD20/CD3) method can detect markers of both B and T cells simultaneously on a single cytology smear, it would be an efficient tool for the immunophenotyping of canine and feline lymphoma samples.

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.

Immunocytochemistry is an advanced diagnostic tool used to identify the origin of tumor cells. The present study aimed to clarify the usefulness of cryopreserved, air-dried cytology samples for the detection of cytokeratin and vimentin. Air-dried smear samples were prepared from canine tumors and stored at –30°C or room temperature without fixation. The duration of cryopreservation varied from 2 months to 4 years and 8 months. Formalin-fixed paraffin sections were also prepared from these tumors. These samples underwent enzymatic immunocytochemistry and immunohistochemistry for the detection of cytokeratin and vimentin. Immunoreactivity for cytokeratin was detected in samples cryopreserved for a maximum of 3 years and 10 months. Immunosignals for vimentin were clearly detected for 2 years. Expression of cytokeratin and vimentin was not detected in samples stored at room temperature for 1 week. Immunoreactivity was observed in all specimens using immunohistochemistry. These findings suggest that immunocytochemistry for cytokeratin and vimentin can be safely applied to air-dried smears that have been cryopreserved at –30°C for at least 2 years. This simple method may be useful for retrospective cytological studies and/or re-evaluation of cytology results.