Practical diagnostic cytology for practitioners

Introduction

Cytology (i.e., the study of lesions at the cellular level) is highly effective and widely used by veterinary practitioners in the diagnosis and assessment of various pathologies, including inflammation, injury, infection, hyperplasia and neoplasia. It is relatively easy, inexpensive, reliable, minimally invasive and rapid (Box 1), and the technique is commonly applied to fine-needle aspirates of unexplained lumps, swellings or other lesions anywhere on the external body (Figure 1). In addition, it can be used to assess cells retrieved by scrapings from skin lesions, swabs of mucosal surfaces, and direct imprints onto glass slides of lesions, tissue biopsies or tissue surfaces. Furthermore, it is commonly applied to fluids from washes of nasal, tracheal, bronchial and alveolar spaces, from catheterization of the bladder and prostate, and to aspirates of body cavity and joint fluids. Increasingly, ultrasound-guided, fine-needle aspiration of masses, or organs such as the liver, spleen and kidney, is used to assess internal lesions. 

Box 1. Advantages of diagnostic cytology.

Pros and cons of cytology

Cytological findings are highly correlated with those from tissue biopsy for histopathology. However, the limitations of the technique when compared to histology are the far greater yield of cells and the greater architectural and cross-cell interaction information given by the latter. Some significant architectural features may be seen with cytology, such as palisading of cells in basal cell tumors, association with capillaries in lipomas and hemangiopericytomas, acinar and tubular formations and extracellular matrix in secretory tissue, and papillary formations in epithelial tissues. However, the cell-level and subcellular information obtained with cytology may be required for the diagnosis of some conditions, such as with hemangiopericytoma, and well-prepared cytological specimens are free from the shrinkage and other artefacts of formaldehyde fixation found with histology. This may be relevant in diagnoses based largely on cell-size evaluation, such as lymphoma.

For most cytology findings, the skills and knowledge required for acquiring and processing a sample, and then evaluating it microscopically, are relatively basic for veterinary practitioners, although a careless approach can invalidate a sample (Box 2). Inflammatory lesions are probably most easily identified (Box 3) as they are based largely on recognition of the types of incoming blood cells and formation of macrophages, and assessment of the severity. Cells from certain tissues are readily recognizable by a few different morphological features – for example, around 80% of skin tumors are of only 10 types – and practitioners can rapidly learn to diagnose the most common cytology findings, and refer on to the specialist those that require more experience. In addition, a vast array of helpful textbooks, manuals, atlases and relevant, practitioner-oriented literature has become widely available in the last couple of decades, both in paper copy and digitally on the internet. 

Box 2. 5 reasons for non-diagnostic cytological preparations. 

Box 3. An algorithmic approach to cytological examination of a cyst, lump or bump.

Technology and knowledge have markedly developed as well in this time, especially for acquisition, analysis and storage of high-quality microscopic images. A microscope, mobile phone, computer, color atlas and clinical centrifuge are the only equipment needed once a sample is acquired, smeared and stained. It is also noteworthy from a business and financial perspective that cytology has become an important component of clinical pathology in the operation of veterinary clinics. Interestingly, supervisors and examiners in the specialty of clinical pathology often note that residents spend the most of their time, gravitate to, and perform and score best in the subspeciality of cytology, when compared to the other clinical pathology fields of biochemistry, general pathology, hematology, and laboratory management.

Effective sample acquisition and imaging

Typically, for a single lesion, clinicians will send 1-4 smears. These are evaluated for cellularity, cell preservation, and the presence of inflammation (Box 4) and malignant features (Box 5). Diagnostic quality samples will have sufficient cellularity and be correctly fixed, with the cells well-distributed and with minimal blood contamination. Samples are acquired usually by aspiration, but occasionally by simple needle insertion when cells are separated, such as in lymphomas. Aspiration using a 22-23G needle and a 20 mL syringe is done using a few back-and-forth movements to retrieve cells from a greater and more representative area of the lesion. The aspirated contents are expressed onto slides with the needle held at a 45-degree angle. Smearing is ideally done using a second slide positioned on top of the first one and retracted slowly horizontally, with minimal vertical pressure, and followed by rapid drying by waving the slides in the air for ~30 seconds, or placing in front of a brisk fan or hair dryer. This prevents cell shrinkage and dissolving.

Box 4. Cytological assessment of inflammation.

Box 5. Criteria of malignancy (> 3 needed).

Light microscopy examination is best performed with a trinocular microscope equipped with a computer-linked digital camera for multiple image capture. Alternatively, a mobile phone camera can be used with the zoom function to get close-up images of the cells (Figure 3). Photomicrographs should be digitally optimized by adjusting for brightness, contrast, and the white balance of the background, which will remove the typical distorting yellow background color. Images should be cropped to focus on relevant, diagnostic features. Various free image-processing software packages allow collages of 4-to-16 optimized images of different diagnostic features to be made automatically to give a more comprehensive and accurate representation of the smear, and thereby facilitate diagnosis (Figure 4). Diagnostic image collages can be digitally transferred into reports as jpeg files, shared live in real-time conferencing programs, or stored for future comparisons or study.

Cytology starts by gross examination of a slide to preliminarily assess the sufficiency or excess of cells, any contaminating blood/lipid, any macroscopic structures (e.g., cell clumps, larvae), and any obvious smear/drying artefacts. Then direct microscopy is done at 3 different levels of magnifications in a stepwise, iterative process; 

I) ~10-20x to comprehensively scan for large structures, gain architectural information, and identify the most diagnostic areas, 

II) ~40-60x to further refine and resolve the detail, 

III) 100x oil for in-depth cellular/sub-cellular detail.

Cytology and neoplasia

A review of samples submitted to the authors’ university laboratory provides some interesting statistics and demonstrates the usefulness of cytology when investigating a possible tumor. Around 95% of 7,560 cases (of which 62% were internal submissions, 38% from external clinics) were from dogs. In total, 14% of samples were non-diagnostic due to insufficient recovery of intact cells (Box 2), and 19% of cases with readable slides were neoplastic (64% were malignant), the remainder being mostly inflammatory.

Of the three cytological neoplasia categories, mesenchymal tumors had the highest overall prevalence (42%), of which 98% were canine and 2% feline, followed by round cell tumors (32%), of which 88% canine/12% feline, and epithelial tumors (26%), of which 93% canine/7% feline. Canine cancers were 30% lymphomas, 27% carcinomas, 26% sarcomas, 13% mast cell tumors, and 4% neuroendocrine tumors. Canine benign tumors were 65% lipoma, 16% adenomas, 7% histiocytomas, and 5% hemangiopericytomas. There were too few benign feline tumors to evaluate, but there were 75 malignant feline tumors: 52% lymphomas, 30% carcinomas, 9% sarcomas, 4% mast cell tumors, and 3% plasma cell tumors.

The top five tumors represented 84% of the total tumors diagnosed in the dog population and 98% in cats. Lipoma was the most common cytological diagnosis of canine neoplasms, twice more frequently diagnosed in external practices, but was uncommon in cats (n=1). Mast cell tumors were twice as common in dogs (8%) than in cats (4%). Lymphoma, the most common feline neoplasm, was twice more commonly diagnosed in cats than in dogs. More than one third of cancers were sarcomas and carcinomas, and were three times more frequently diagnosed in samples from our specialty practice. A description of the most common tumors can be found by clicking on this link.

Criteria of malignancy

Diagnosis of malignant tumors (cancer) is critical. Cytologically, most malignancies can be effectively diagnosed by finding at least 3 specified criteria in a substantial proportion of cells (Box 5). Benign tumors are characterized by the similarity of appearance of cells to each other. In contrast, cancerous cells are characterized by a wide variation in shape and size of the cells, their nuclei and nucleoli. These tend to be characterized by terms using Greek prefixes (e.g., pleo, aniso, macro, xeno, hyper) and it can be helpful to remember these criteria.

Classifications of tumors

Malignant tumors in cytology are divided, according to their origin and morphology, in three groups: 

• Mesenchymal tumor (sarcomas if malignant) samples are usually of low-to-moderate cellularity, with cells found singly or in loosely cohesive clusters with poorly defined cell borders, occasionally associated with a pink, extracellular matrix. Individual cells have a spindle appearance with mildly elongated, oval nuclei and moderate amounts of pale-basophilic cytoplasm frequently appearing as uni-/bipolar extensions. Pin-point, fine, red cytoplasmic granulation is occasionally noted.
• Epithelial tumors (carcinomas if malignant) tend to have round to polygonal cells with a round nucleus and abundant basophilic cytoplasm; the cells exfoliate in clusters. The cell border is more obvious than in a sarcoma, but less obvious than in round cell tumors.
• Discrete round cell tumors are highly exfoliative; thus, sampling frequently retrieves high numbers of discrete, solitary cells. Occasionally, they aggregate in large, multi-layered clusters with little-to-no cellular detail. Most types of round cells are prone to rupture, thus extra care is needed during sample processing.

Conclusion

Acquiring and processing a sample, and evaluating it microscopically, are relatively basic skills that can be easily applied in the clinic, and cytology is playing an increasingly important part in small animal diagnostics. Inflammatory lesions are the most easily identified as they are based largely on recognition of blood cell types, but tumors commonly have typical characteristics that will also allow identification in many cases. Clinicians can rapidly learn to diagnose the most common cytology findings, and refer on to the specialist those that require more experience.

Further reading

• O’Brien PJ, Lumsden JH. The cytological examination of body cavity fluids. Sem. Vet. Med. Surg. Small Anim. 1988;3:140-156.
• Metcalfe LVA, O’Brien PJ, Papakonstantinou S, et al. Malignant melanoma in a grey horse: case presentation and review of equine melanoma treatment options. Ir. Vet. J. 2013;66:22-26.
• Papakonstantinou S, O’Brien PJ. High content imaging for the morphometric diagnosis and immunophenotypic prognosis of canine lymphomas. Cytometry: Part B – Clinical Cytometry Doi: 10.1002/cytob.21170; 2014.
• Domingos M, Davies AM, O’Brien PJ. Application of high content analysis in clinical cytology for translational safety biomarkers of drug-induced toxicity for lymphoma chemotherapy. Basic Clin. Pharmacol. Toxicol. 2014;115:145-153.
• Balan M, O’Brien PJ, McCullough M. Marked paraneoplastic basophilia accompanying eosinophilia in a cat with alimentary T-cell lymphoma. J. Feline Med. Surg. Open Reports 2017;3(2):1-6.
• Balda IO, O’Brien PJ, Mullins RA, et al. Intraoperative impression smear cytology to guide successful treatment of a large renal cyst in a dog: a case report. J. Vet. Sci. 2022;23(2):e34.
• Martínez-Caro J, O’Brien PJ. Novel, diagnostic, cytomorphometric profile of canine, classical haemangiopericytoma: including nuclear criteria of malignancy. Comp. Clin. Pathol. 2023;32(2):299-310.