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Summary In a cytological study, cytomorphological characteristics of osteosarcoma and reactive mesenchymal bone proliferations (callus) were determined in 25 dogs with osteosarcoma (OSA) (group I) aged 1- 13 years (median 7.2 years) and 20 dogs (group II) aged 4 months- 7 years (median 2 years) that were admitted for removal of surgical bone implants after uncomplicated healing of bone fractures. Cytologic tissue imprint biopsies were performed in all patients and diagnosis of bone tumours was confirmed by histological examination of open surgical biopsies. The slides were examined for general cytologic criteria, incidence of cell populations and the appearance of osteoid. In addition, specific cytologic criteria of cytoplasm (cell size, nucleus:cytoplasm-ratio, distinctiveness of cell borders, colour, eosinophilic granulation), nucleus (nuclei per cell, nuclear size, chromatin pattern, frequency of mitoses, colour of nucleus and nucleoli, nucleoli per nucleus) in osteoblasts, fibroblasts and osteoclasts were evaluated. The appearance of criteria of malignancy was also examined. Histological examination of bone tumours revealed three subtypes of osteosarcoma, osteoblastic (n=17), chondroblastic (n=5) and fibroblastic (n=3). While osteoblasts and other cell populations in group II were mostly intact, mild to moderate cellular necrosis occurred frequently in patients with osteosarcoma. Cellularity was higher in group I than in group II. In both groups mild to moderate amounts of an eosinophilic osteoid- like extracellular substance were detected. Haematopoetic precursor cells occurred only in one dog with osteosarcoma, but were found in 15 dogs in group II. Osteoblasts (median number group I: 86%, group II: 90.5%) and fibroblasts (median number group I: 9%, group II: 4.5%) were the predominant cell forms in both groups. Cell borders of osteoblasts were less distinct in group I than in group II. The cytoplasm of osteoblasts was pale blue to blue with a more pronounced basophilia in group II. In 48% of the patients in group I, but in none of the animals in group II, osteoblasts showed a slight to moderate eosinophilic cytoplasmic granulation. In both groups osteoblasts contained one red to pale blue nucleus. There were one or two grey-red to blue nucleoli in group II whereas in group I, 44% of the patients had osteoblasts with more than 2 nucleoli per nucleus. Comparing the two groups, cell diameter was higher in group II while the nucleus diameter was significantly higher in group I. Therefore, the median nucleus: cytoplasm ratio was higher in group 1 (1:2.0) than in group 11 (1:3.5). While 23/25 patients with OSA showed mitotic osteoblasts, these could only be found in one of the group II dogs. Osteoblasts in group I had frequently a clumped chromatin pattern and had significantly more criteria of malignancy (median: 6 criteria per patient) than those in group II (median: 2 criteria per patient), including anisonucleosis (76%), angular nucleoli (68%), macronucleolization (52%), aberrant mitoses (44%), nuclear moulding (48 %), thickening of the nuclear membrane (40%) and cytoplasmic vacuolisation (40%). In summary, the cytologic criteria presented in this study may be helpful in the objective diagnosis of canine osteosarcoma. Further studies are needed to determine the sensitivity and specificity of these criteria in the diagnosis of osteosarcoma.