Rapid Multiple Immunofluorescent Staining for Detection of Cytokeratin and Vimentin for Use in Diagnostic Cytology in Canine Neoplastic Diseases
Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
Immunocytochemistry (ICC) is an advanced tool used in the field of veterinary diagnostic cytology. Detection of diagnostic markers, such as cytokeratin and vimentin, helps to determine if the tumor cells are of epithelial or non-epithelial origin. However, standard enzyme-based ICC has limitations in clinical usage, because it is time-consuming and often results in non-specific staining. A more convenient and reliable method is therefore needed.
The purpose of the present study was to develop a rapid multiple immunofluorescent (RMIF) method for detection of cytokeratin and vimentin from a single cytological smear. The practical utility of the method was then evaluated in clinical samples.
Air-dried smear samples were prepared from dogs (n=13). Mouse monoclonal anti-human cytokeratin (AE1/AE3) and rabbit monoclonal anti-human vimentin (SP20) were used as primary antibodies, followed by fluorochrome-conjugated secondary antibodies. RMIF protocols were developed by modifying staining procedures by including a type of fixation. Immuno-signals detected by RMIF-ICC were compared with those obtained in enzyme-based ICC.
RMIF-ICC detected immuno-signals within 45 minutes. The specificity of signals was clearly higher than that of enzyme-based ICC. Epithelial, non-epithelial, and mesothelial cells were clearly distinguishable in a single smear of pleural effusion. In a smear of a lymph node with metastasis of epithelial tumor, the RMIF method was also able to differentiate the metastatic epithelial cells from the lymphocytes.
The RMIF method can be a useful tool for diagnostic cytology in veterinary medicine.