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To evaluate its usefulness as a differential diagnostic marker of canine hemangiosarcomas, the expression of claudin-5 molecule was studied in different canine tumours of vascular origin. Methods and results: Ninety two canine neoplastic tissue samples obtained from necropsies and biopsy specimens were routinely processed and stained immunhistochemically for claudin-5. The neoplastic endothelial cells of canine hemangiosarcomas, hemangiomas, and lymphangiomas showed a strong membrane immunoreactivity for claudin-5, but the other investigated canine malignant and benign tumours, including fibrosarcomas, myxo-, leiomyo-, cardiac rhabdomyo-, neurofibro-, synovial-, osteo-, and chondrosarcomas, spindle cell melanomas, hemangiopericytomas, benign fibroblast proliferations, and leiomyomas were negative for this endothelial marker. In these non-vascular canine tumours intense immunostaining was detected in the endothelial cells of the incorporated intratumoural vessels and neovasculature. The canine splenic hematomas induced by hemangiosarcomas were distinguished from splenic hematomas induced by non-neoplastic lesions by the means of claudin-5 protein. In hemangiosarcomas the percentage of positive neoplastic endothelial cells was higher, and stronger when using the claudin-5 molecule compared to CD31 and vWf. Conclusion: The results show that claudin-5 molecule can be used as a new differential marker, and could also be of a diagnostic value in the differential diagnosis of canine hemangiosarcomas from sarcomas of other origin with hemorrhages or increased vascularization. Claudin-5 could help to reveal neoplastic proliferation of endothelial cells causing splenic hematomas and differentiate these tumours from non-vascular neoplastic splenic lesion. 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