DVM, MS, DACVP (Clinical Pathology)
Dr. Fraser received her DVM degree from Purdue University. Following veterinary college, she completed a clinical pathology residency and master’s degree at the University of Georgia. Her professional interests include diagnostic cytology, particularly hematopathology and hematopoietic neoplasia, comparative pathology, coagulation, and teaching. Dr. Fraser is passionate about collaborative pathology, education, inclusivity, and lifelong learning. She strives to improve the lives of animals through outstanding service, scientific collaboration, and compassion. She is currently a clinical pathologist working with Zoetis Global Diagnostics.Read Articles Written by Carisa Fraser
DVM, DECVIM-CA (Oncology), DACVP (Clinical Pathology)
Dr. Meichner received her DVM from the Ludwig-Maximilian University Munich, Germany. Following a small animal rotating internship, she completed a residency in medical oncology (ECVIM), followed by a residency in clinical pathology (ACVP) at North Carolina State University. Dr. Meichner is currently an assistant professor in the Department of Pathology, College of Veterinary Medicine, University of Georgia, as well as director of the Clinical Pathology and Clinical Flow Cytometry Laboratory. Her research interests focus on immunology and canine cancer, especially the characterization of canine hematopoietic tumors and the investigation of alternative treatment strategies.Read Articles Written by Kristina Meichner
In small animal cytology, “lumps and bumps” refer to lesions that manifest within the cutaneous and subcutaneous layers of the skin, resulting in raised, conspicuous masses. These masses may be neoplastic or nonneoplastic and generally fall into 1 or more recognized categories1-3:
- hyperplastic tissue
- inflammation/cellular infiltrates
- cystic or fluid-filled masses
These lesions are ideal for cytologic sampling using fine needle aspiration (FNA), as they are accessed without advanced imaging (e.g., ultrasonography), and sedation or anesthesia is typically not necessary; they require only minimal equipment for sample acquisition; and they are often of high diagnostic yield.1,3
Utility of Small Animal Cytology
Although the overall incidence of skin tumors in dogs and cats is challenging to determine, skin is a common site for neoplasia in small animal species. In the presence of a mass, cytology can often accurately distinguish neoplastic from nonneoplastic lesions, determine general categories of neoplasia, and lead to a more specific diagnosis.4 Multiple studies comparing cytologic with histologic diagnoses for neoplastic and nonneoplastic lesions have demonstrated the utility and reliability of cytology in veterinary medicine.2,5
High concordance rates have been reported between cytology and histology in the evaluation of canine and feline cutaneous and subcutaneous lesions.2,5 One retrospective study of 243 cytologic samples (198 from dogs and 45 from cats) agreed with histologic diagnoses on the presence or absence of neoplasia in 90.9% of cases when evaluated by clinical pathologists.2 The same study reported a sensitivity of 89.3%, specificity of 97.9%, positive predictive value of 99.4%, and negative predictive value of 68.7% for the cytologic diagnosis of neoplasia.2 An obvious advantage of achieving a cytologic diagnosis is avoiding additional diagnostics, thereby saving money for the client and enabling the prompt initiation of appropriate therapies.3 However, clients should be aware that biopsy and histologic evaluation may still be required for a more specific diagnosis. It is important to consider the experience of the evaluator and the influence of inflammation, which may mask or mimic neoplasia.4
Cytology, like all diagnostic tests, is not without limitations. The lack of tissue architecture restricts the amount of information cytology can provide, particularly when overlapping cytologic features (e.g., hyperplastic versus benign neoplastic lesions) exist. Acquiring a satisfactory sample also influences the usefulness of diagnostic cytology; inadequately cellular samples composed up to 16.8% of all cases in 1 study.2 Therefore, appropriate sample acquisition, preparation technique, and screening for cellularity, along with resampling if needed, are recommended to avoid nonrepresentative or nondiagnostic samples.3 Detailed descriptions of how to obtain cytologic samples via FNA are provided elsewhere.6,7
It is also important to remember that lesion type also affects the likelihood of a representative result.8 Knowledge of accurate and detailed patient history and mass characteristics significantly influences the cytologic interpretation and is critical (BOX 1).8 In many cases, histopathologic tissue evaluation should be pursued for a more definitive classification, with or without cytochemical, immunocytochemical, or immunohistochemical stains. Cytologic interpretation does not negate the need to submit a biopsy sample, as each yields essential information.
Approach to Sample Evaluation
The first step in evaluating a cytologic sample from a skin mass is assessing the entire slide at low-power magnification (4× to 10× objective) for adequate cellularity, distribution, cellular preservation, and staining quality (FIGURE 1).3,9,11 Inadequately cellular samples may reflect poor sampling technique. Other considerations include cystic or poorly exfoliative lesions, particularly if resampling produces similar results. In such cases, a tissue biopsy may be required. If only cellular debris or necrotic tissue is identified, sampling a different site or histopathology of the mass is recommended.
Once sample quality has been confirmed, the next step is to evaluate all slides to determine the predominant cell population. Slides should be first assessed at low magnification to identify specific cell patterns or structures, which may provide information about the process or tissue type present.11 High-power magnification (40×, 50×, or 100× objective) is then useful to scrutinize cellular morphology further, categorize the predominant cell type present (i.e., inflammatory or tissue cells), and identify criteria of malignancy or infectious agents.
Principal cell types determine classification, and evaluation for underlying causes (e.g., infectious or noninfectious etiologies) is warranted (TABLE 1). If only inflammatory cells are noted, inflammation would be suspected. An exclusively monomorphic tissue cell population with no significant inflammation suggests a hyperplastic or neoplastic population. Neoplasms are grouped into 4 general categories to assist in interpreting or generating appropriate differentials based on cellular arrangements and cytomorphology (TABLE 2).
Although the 4 basic categories of skin masses (i.e., hyperplasia, cystic, inflammation, and neoplasia) are based on the overall underlying process, it is essential to remember that they are not mutually exclusive. The same lesion may fall into more than 1 category (e.g., cystic adenoma, inflammatory carcinoma), and sampling multiple areas may reveal additional diagnostically significant information.
While cytology is most effective in differentiating neoplastic from nonneoplastic lesions, concurrent inflammation can induce dysplastic epithelial changes and/or reactive changes to fibrous tissue, mimicking neoplasia.2,5,8 As such, extreme caution should be exercised in the presence of inflammation before diagnosing neoplasia, and consultation with a veterinary pathologist is advised. Overtly malignant neoplasms demonstrate specific structural changes that can be observed by light microscopy at high-power magnification (40× to 100× objective) as morphologic atypia, representing the basis of cytologic criteria of malignancy (TABLE 3).9,10,15
Features of Common Cutaneous and Subcutaneous Lesions
Normal/Hyperplastic/Benign Neoplastic Tissue
Benign proliferative lesions (FIGURE 2A) are often cytologically and grossly indistinguishable, demonstrating absent to minimal cytologic criteria of malignancy. The same is true for certain malignant neoplasms, which can morphologically resemble a benign population (e.g., apocrine gland anal sac adenocarcinoma) despite an aggressive biologic behavior (FIGURE 2B). In these cases, histopathologic evaluation is required for differentiation when neoplasia is suspected clinically and further characterization is desired.
Cystic or Fluid-Filled Lesions
Cystic or fluid-filled skin lesions are often tumor-like lesions and may be associated with tissue injury from trauma (e.g., seroma, hygroma, hematoma, necrosis, abscess). Cytologic samples typically contain few nucleated cells (except for abscess) due to an absence of lining tissue cell exfoliation, and location can be beneficial for further differentiation. Seromas, for example, are often found at sites of previous tissue disturbance (e.g., prior surgery sites), hygromas are associated with bony prominences/pressure points, and follicular or glandular cysts are located within the skin. Follicular cysts demonstrate characteristic cytologic features such as cholesterol crystals, anucleate squamous epithelial cells, and keratin. Similar features are found in cystic follicular or keratin-producing tumors (e.g., pilomatricoma, trichoblastoma); therefore, distinction using cytology alone is not always possible. Necrosis and fibroplasia are also seen in various lesions as a response to tissue injury, inflammation, or neoplasia.
Not surprisingly, hematomas (FIGURE 2C) contain an abundance of erythrocytes, as well as blood-derived leukocytes and vacuolated macrophages, some demonstrating erythrophagia. Heme-breakdown products, hemosiderin, and hematoidin appear within 12 to 24 hours, suggesting chronicity. Notably, some neoplasms have large blood-filled cavities, and neoplastic cells may not exfoliate (e.g., hemangioma, hemangiosarcoma, telangiectatic osteosarcoma); thus, the absence of overtly neoplastic cells does not exclude the possibility of neoplasia.
As mentioned, inflammation may be due to infectious or noninfectious processes, and determining the predominant cell type aids in generating diagnostic differentials. Although infectious etiologic agents are not always identified, cellular composition (e.g., predominately neutrophilic versus neutrophilic and macrophagic) and characteristics such as epithelioid macrophages (FIGURE 3A) and multinucleated giant cells (FIGURE 3B) can provide clues to the type of agents that might be present, helping to select the next appropriate step (e.g., bacterial versus fungal culture). In blastomycosis (FIGURE 3C), for example, fungal yeast elicits a mixed population of neutrophils, some demonstrating degenerate features (karyolytic, karyorrhectic, or pyknotic nuclei), and macrophages (including epithelioid macrophages and multinucleated giant cells), representing pyogranulomatous inflammation. Thus, seeing this type of inflammation should alert slide evaluators to consider the possibility of a fungal or bacterial agent (Figure 3D), diligently look for infectious organisms, and, if not found, pursue additional testing (e.g., repeated aspiration, biopsy with histology, fungal culture, serology).
Neoplastic populations are categorized based on cytomorphology (TABLE 2) and include (1) round/discrete cells, (2) epithelial cells, (3) mesenchymal cells, and (4) bare/naked nuclei (FIGURE 4). Melanocytic neoplasms, which can demonstrate variable morphology and be challenging to classify cytologically, are a notable exception.16
Cells should also be evaluated for cellular atypia (i.e., general and nuclear criteria of malignancy; TABLE 3, FIGURE 5). However, malignant cells cannot be distinguished from benign cells based on a single feature. Only when the majority of cells demonstrate numerous criteria (typically at least 3 to 5 nuclear criteria) in an adequately cellular and preserved sample should malignancy be suspected. Nuclear criteria are more significant than general criteria and unlikely to be influenced by external factors such as inflammatory mediators. Biopsy and histopathologic evaluation should be considered if ambiguity or concurrent inflammation exists.
Criteria of malignancy can aid in predicting the biological behavior of a neoplasm (benign or malignant). It is, however, critical to recognize that morphology is not always indicative of biological behavior, and dysplastic features induced by inflammation/ulceration/necrosis can appear similar. Alternatively, as previously mentioned, a benign cytologic appearance does not always indicate a concurrent benign biologic behavior. In such cases, histopathology or repeat aspiration may be necessary if the mass persists following the resolution of inflammation or if there are other indicators that this neoplasm has a more aggressive clinical course (e.g., nodal or distant organ involvement, known malignant behavior despite morphology).
Cytology is a tool that can be used to determine the underlying pathology of skin masses, resulting in a definitive diagnosis or an appropriate list of differentials. It is not a replacement for biopsy and histopathology; rather, these approaches should be used together, with or without cytochemical, immunocytochemical, or immunohistochemical stains. By understanding the limitations of cytology, the importance of sample quality, and the role of cytology in the workup of patients, veterinarians can use the knowledge gained through cytologic assessment to inform clinical decision-making regarding further diagnostics or treatment considerations for these lesions.
- Sharkey LC, Seelig DM, Overmann J. All lesions great and small, part 1: diagnostic cytology in veterinary medicine. Diagn Cytopathol. 2014;42(6):535-543. doi:10.1002/dc.23097
- Ghisleni G, Roccabianca P, Ceruti R, et al. Correlation between fine-needle aspiration cytology and histopathology in the evaluation of cutaneous and subcutaneous masses from dogs and cats. Vet Clin Pathol. 2006;35(1):24-30. doi:10.1111/j.1939-165x.2006.tb00084.x
- MacNeill AL. Cytology of canine and feline cutaneous and subcutaneous lesions and lymph nodes. Top Companion Anim Med. 2011;26(2):62-76. doi:10.1053/j.tcam.2011.02.004
- Johnson MC, Meyer AN. Cytology of skin neoplasms. Vet Clin North Am Small Anim Pract. 2017;47(1):85-110. doi:10.1016/j.cvsm.2016.07.006
- Cohen M, Bohling MW, Wright JC, Welles EA, Spano JS. Evaluation of sensitivity and specificity of cytologic examination: 269 cases (1999–2000). JAVMA. 2003;222(7):964-967. doi:10.2460/javma.2003.222.964
- Moore AR. Preparation of cytology samples: tricks of the trade. Vet Clin North Am Small Anim Pract. 2017;47(1):1-16. doi:10.1016/j.cvsm.2016.07.001
- Schlemmer SN. Obtaining a sample for cytology using fine needle biopsy. Today’s Vet Pract. 2023;13(1):54-60.
- Sharkey LC, Dial SM, Matz ME. Maximizing the diagnostic value of cytology in small animal practice. Vet Clin North Am Small Anim Pract. 2007;37(2):351-372. doi:10.1016/j.cvsm.2006.11.004
- Meinkoth JH, Cowell RL, Tyler RD. Cell types and criteria of malignancy. In: Valenciano AC, Cowell RL. Cowell and Tyler’s Diagnostic Cytology and Hematology of the Dog and Cat. 5th ed. Elsevier; 2020:18-43.
- Raskin RE. General categories of cytologic interpretation. In: Raskin RE, Meyer D, Boes KM. Canine and Feline Cytopathology: A Color Atlas and Interpretation Guide. 4th ed. Elsevier; 2023:14-34.
- Masserdotti C. Architectural patterns in cytology: correlation with histology. Vet Clin Pathol. 2006;35(4):388-396. doi:10.1111/j.1939-165x.2006.tb00154.x
- Holmes E, Raskin RE, McGill P, Szladovits B. Morphologic, cytochemical, and ultrastructural features of gray eosinophils in nine cats. Vet Clin Pathol. 2021; 50(1): 52-56. doi:10.1111/vcp.12950
- Myers A, Meason-Smith C, Mansell J, et al. Atypical cutaneous cryptococcosis in four cats in the USA. Vet Dermatol. 2017;28(4):405-e97. doi:10.1111/vde.12423
- Oldenhoff W, Grooters A, Pinkerton ME, Knorr J, Trepanier L. Cutaneous pythiosis in two dogs from Wisconsin, USA. Vet Dermatol. 2014;25(1):52-e21. doi:10.1111/vde.12101
- Fischer AH, Zhao C, Li QK, et al. The cytologic criteria of malignancy.
J Cell Biochem. 2010;110(4):795-811. doi:10.1002/jcb.22585
- Smedley RC, Sebastian K, Kiupel M. Diagnosis and prognosis of canine melanocytic neoplasms. Vet Sci. 2022;9(4):175. doi:10.3390/vetsci9040175