Cytology of Neoplasia
World Small Animal Veterinary Association World Congress Proceedings, 2006
Alan H. Rebar1; Rose E. Raskin2, DVM, PhD, DACVP
1Senior Associate Vice President for Research, Executive Director, Discovery Park, Purdue University; 2Professor of Veterinary Clinical Pathology, Dept of Veterinary Pathobiology, Purdue University, School of Veterinary Medicine, West Lafayette, IN, USA

Neoplastic processes may be either benign or malignant. It is virtually impossible to differentiate between benign neoplasias and hyperplasia cytologically.

Malignant neoplastic processes are identified through the recognition of specific malignant criteria exhibited by the cell population under consideration. Criteria of malignancy may be loosely classified into four categories: 1) General criteria of malignancy; 2) Nuclear criteria of malignancy; 3) Cytoplasmic criteria of malignancy; and 4) Structural criteria of malignancy. These are summarized in Table 1.

General criteria of malignancy refer to the appearance of the cell population as a whole. Malignant neoplastic processes are generally characterized as a uniform population of pleomorphic cells; that is, cells usually appear to be of a single cell type (for example, all mast cells or all spindle-shaped connective tissue cells) which exhibit variable cell size and variable nuclear size. These features can generally be recognized at low magnification and represent the first suggestion that the cytologic diagnosis is one of malignant neoplasia. While such pleomorphism in a uniform population of cells is easy to recognize in uncomplicated cases, the presence of large numbers of inflammatory cells may mask neoplasia. Therefore, whenever an inflammatory response is associated with a population of non-inflammatory cells, the non-inflammatory cells should be carefully scrutinized for evidence of malignancy. Inflammatory cells are commonly encountered in large numbers in cytologic preparations from oral or skin neoplasms with ulcerated surfaces.

Nuclear criteria of malignancy are the most important criteria employed in identifying a malignant neoplasm cytologically. Evaluation of nuclei of malignancy is best performed under oil immersion. Features suggesting malignancy (in Romanowsky stained preparations) include multiple nucleoli, large irregularly shaped nucleoli, coarse chromatin patterns (areas within the nucleus which stain intensely as well as other areas which are virtually unstained or only poorly stained), irregularities and indentations in the nuclear membrane and variable nuclear/cytoplasmic ratios among the cells seen. Mitotic figures per se are not a criterion of malignancy as they may be seen in hyperplastic cell populations (e.g., lymph node hyperplasia); however, abnormal mitoses (e.g., three or more planes of division) are a feature of malignancy. Multi-nucleated tumor cells are occasionally seen, but giant cells may also occur in inflammatory reactions (granulomatous inflammation). Since the nuclear criteria of malignancy are the most important in establishing the diagnosis of malignant neoplasia, the author suggests that 3-4 such nuclear alterations be identified before the diagnosis of malignancy is suggested.

Cytoplasmic criteria of malignancy are considerably less important in establishing a diagnosis of malignant neoplasia but do provide supportive evidence. Cytoplasmic criteria include cytoplasmic basophilia, cytoplasmic vacuolation and variation in amounts of cytoplasm. Cytoplasmic features of malignancy suggest the primitive nature of the neoplastic population; for example, cytoplasmic basophilia results from a high cytoplasmic content of RNA, a constant feature of young proliferating cells.

After the nuclear and cytoplasmic criteria of malignancy have been used to establish the cytologic diagnosis of malignant neoplasia, the structural features of the neoplastic cells may be evaluated in an attempt to further classify the tumor as a carcinoma, sarcoma, or discrete cell neoplasm. Carcinomas are neoplasms of epithelial cell origin. Normal epithelial cells are generally adherent to one another and this property is generally reflected in cytologic preparations from epithelial cell neoplasms. Carcinoma cells are generally round to oval, and are arranged in sheets and clusters. Cells from neoplasms of glandular epithelium (adenocarcinomas) are often arranged in acinar patterns around a central lumen. Adenocarcinoma cells also often contain large vacuoles containing secretory product.

Sarcomas are neoplasms of cells of connective tissue origin. Connective tissue cells generally are embedded in a matrix which they themselves secrete. Consequently, aspirates or imprints from sarcomatous masses are generally less cellular than cytologic preparations made from epithelial or discrete cell neoplasms. Structurally, connective tissue cells are usually spindle-shaped or flame-shaped (cells with tails) and this morphology is also typical of sarcomatous cells.

The sarcomas of veterinary significance include osteosarcoma, fibrosarcoma, liposarcoma, hemangiosarcoma, melanosarcoma and chondrosarcoma. Osteosarcoma and chondrosarcoma cells are more commonly flame-shaped than spindle-shaped. In addition, aspirates from these two neoplasms may contain considerable matrix material--eosinophilic osteoid in the case of osteosarcoma and metachromatic (purple) chondroid in the case of chondrosarcoma. The cytoplasmic margin of osteosarcoma cells is often irregular and vacuolated. Melanosarcoma has several distinguishing cytologic features. Of principal importance is the presence of brown to black cytoplasmic granules (melanin) of variable size and shape. In addition, melanosarcomas are often comprised of cells of two shapes--spindle-shaped and round to oval (epithelioid). Fibrosarcoma, hemangiosarcoma, and liposarcoma may be indistinguishable cytologically. All are comprised of basically spindle-shaped cells. Aspirates from hemangiosarcoma usually contain considerably more blood than the other sarcomatous masses. Liposarcoma cells may contain large lipid-filled vacuoles which may be demonstrated with lipid stains such as oil red O or the sudan stains. Illustrations of the various sarcoma cells can be found elsewhere.

The discrete cell neoplasms constitute a rather large group of tumors of veterinary importance. Included among the discrete cell neoplasms are malignant lymphomas, mast cell tumors histiocytomas and transmissible venereal tumors. Structurally, discrete cell neoplasms are seen cytologically as neoplasms comprised of individual round or oval cells with no adherence between cells and no ordered arrangement of cells (such as cluster formation).

Table 1. Cytologic criteria of malignancy.

Malignant Criteria

Cytologic Features


Uniform population of pleomorphic cells--can be assessed at low magnification


Abnormal mitoses
Variable nuclear size
Variable nuclear/cytoplasmic ratios
Multiple nucleoli
Large irregularly shaped nucleoli
Coarse chromatin patterns
Irregular prominence of nuclear margin




Carcinoma--round to oval cells arranged in sheets of acinar patterns
Sarcoma--spindle shaped cells
Discrete cell tumor--individual round or oval cells

Speaker Information
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Rose E. Raskin, DVM, PhD, DACVP
Purdue University
School of Veterinary Medicine
West Lafayette, Indiana, USA

Alan H. Rebar
Purdue University
West Lafayette, Indiana, USA