Intraoral cytology

Ensure you know the fundamentals

When a dog or cat presents with an intraoral or extraoral mass, the veterinarian has many options, including treating medically, removing a small sample for histologic examination or performing a wide excision to effect a cure. Determining if the lesion is secondary to inflammation or malignant neoplasia can be most helpful when choosing further diagnostic and treatment plans. Becoming familiar with the fundamentals of oral cytology and using it in your dental practice can be invaluable.

Photo 1: A cotton swab used to obtain cellular sample from a mandibular swelling in a 5-month-old Labrador Retriever.

Obtaining a sample

Diagnostic cytology samples from the oral cavity can be collected using a swab, scrape, touch impression or fine needle. A swab of the exfoliated cells from an oral lesion (Photo 1) can be rolled onto a clean slide for microscopic examination.

Unfortunately, not all masses exfoliate diagnostic samples. Rubbing a swab over an encapsulated mass usually will not yield a diagnostic/cellular sample.

Impression smears of lesions can be frustrating to interpret, because they often reflect superficial infection and inflammation but not the underlying cause of the mass. If in doubt, both impression smears and aspirates should be attempted, with careful labeling of slides as to which represents what type of preparation.

For a fine-needle biopsy, two methods are commonly used to obtain diagnostic samples: non-aspiration and aspiration techniques. With the non-aspiration variety, pull the plunger back several milliliters to allow air to enter the syringe. Then place a 20-ga needle multiple times into the tissue to harvest core samples. Push the sample out on a slide, and compress it for preparation. For the aspiration method, insert a needle into the mass, and retract the syringe plunger to create negative pressure. Redirect the needle after releasing pressure several times without exiting the mass. Remove the needle from the syringe, and then fill it with air. Reattach the needle, and expel the sample from the needle onto a clean slide. Stain the slide with a modified Wright's stain (Diff-Quik—Dade Behring) (Photos 2A-2D).

Photo 2A: A mature cat with a left facial enlargement.

Photo 2B: Fine-needle aspiration of the enlarged facial area.

Photo 2C: A mixed inflammatory cell population consisting mainly of neutrophils with two macrophages.

Photo 2D: Resolution of the facial enlargement after removal of multiple root fragments.

Examining the sample

Evaluating stained cells from oral masses is not difficult. A high-quality microscope is essential. The 40X objective can be replaced with 50X oil immersion objective for improved cytologic evaluation. First scan the slide under low magnification to identify areas of increased cellularity. Once an interesting area has been identified, use 50X (oil) and 100X (oil) for closer examination.

Examination of the cells should yield a diagnosis of an inflammation, neoplasia or a mixed-cell population (both inflammatory and neoplastic). Good biopsy technique, smear fixation and staining technique are essential, as is proper use of a high-quality microscope. A negative (e.g., tumor-negative) report is less reliable than a positive report. This is an important concept to embrace. The result is only as good as where the sample was taken from.

If the reaction is inflammatory, try to further classify it as to type (neutrophilic, eosinophilic, lymphocytic or macrophagic) and to identify etiologic agents (Photo 3). Neutrophils may appear with degenerative changes affecting the nucleus, occurring once the neutrophil has left the circulation to fight an infection; the resulting appearance is nuclear swelling. Toxic changes to the neutrophil affect the cytoplasm and manifest as Döhle bodies, cytoplasmic basophilia and foaminess. These changes occur during formation of the neutrophil in the marrow and suggest altered development that can be associated with sepsis. If you see toxic change in neutrophils in inflammatory lesions, expect to also see it on neutrophils in a complete blood count since it developed in the marrow.

Photo 3: Cytologic examination of a sample of the lesion in Photo 1 reveals a mixed inflammatory reaction.

Unlike inflammation, neoplastic samples generally contain homogeneous populations of a single cell type. Although mixed-cell populations are sometimes seen, these usually involve a neoplastic area with concurrent inflammation (Photo 4).

Photo 4: A mixed-cell population in a case of feline oral squamous cell carcinoma.

Neoplasia must be differentiated as either benign or malignant. Benign neoplasia is represented by proliferating cells that often resemble the tissue from which they arose and lack criteria of malignancy (Photos 5A and 5B). The cells are of the same type and are relatively uniform in appearance. Cells that demonstrate at least three criteria of malignancy help to determine a cytologic diagnosis of malignancy (Photos 6A and 6B).

Photo 5A: Inflamed enlargement on the attached gingiva in a 4-year-old dog.

Photo 5B: Cytologic examination of a sample reveals reactive fibroplasia.

Photo 6A: A mobile maxillary first molar in an 11-year-old German Shepherd.

Photo 6B: Cytologic examination of a sample reveals sheets of cohesive cells with malignant criteria (anisokaryosis, pleomorphism, high nuclear/cytoplasm ratio, coarse nuclear chromatin) indicative of squamous cell carcinoma.

Criteria of malignancy can include any of the following:

• Anisokaryosis—unusual variation in overall nuclear size (Photo 7).

• Pleomorphism—variability in the size and shape of the same cell type (Photo 8).

• High or variable nuclear/cytoplasm ratio—specifically an increased ratio of nucleus to cytoplasm (Photo 9).

• Increased mitotic activity—mitosis is rare in normal tissue, and cells usually divide evenly in two; any increase in the presence of mitotic figures or cells that are not dividing equally would be considered malignant criteria.

• Coarse chromatin pattern—coarser than normal and may appear ropelike or cordlike.

• Nuclear molding—a deformation of nuclei by other nuclei within the same cell or adjacent cells.

• Multinucleation—multiple nuclei within a cell.

• Nucleoli that vary in size, shape and numbers—anisonucleoliosis, angular nucleoli or multiple nucleoli.

Photo 7: Anisokaryosis.

Photo 8: Pleomorphism.

Photo 9: Increased nuclear/cytoplasm ratio.

Samples that have been classified as malignant should be further evaluated to determine the cell type involved. The basic tumor categories seen in mammals include epithelial, mesenchymal, round cell and neuroendocrine.

Epithelial cell tumors are carcinomas. The samples tend to be highly cellular and often exfoliate in clumps or sheets. Mesenchymal cell tumors are sarcomas and are usually less cellular. The cells tend to exfoliate singly or in wispy spindles. Round cell tumors exfoliate but usually are not in clumps or clusters. Oral round cell tumors include lymphoma, mast cell, plasma cell and melanoma. Cytology samples should be sent to a board-certified pathologist for confirmation.

Summary of microscopic findings

Inflammatory cells (neutrophils, macrophages)

> 85% neutrophils = suppurative

< 50% macrophages = active pyogranulomatous

> 50% macrophages = chronic pyogranulomatous

> 10% eosinophils = eosinophilic

Tissue cells

• Malignant if more than three of the malignant criteria listed above are present

• Epithelial (clusters, clumps) = carcinoma or adenocarcinoma (Photos 10A and 10B)

• Mesenchymal (single, spindly) = sarcoma (Photos 11A and 11B)

• Round cell, mast cell, melanoma, plasma cell (Photos 12A and 12B)

Photo 10A: A swollen and inflamed area surrounding a previously extracted canine in a 13-year-old Poodle.

Photo 10B: Cytologic examination of a sample is consistent with carcinoma (squamous cell).

Photo 11A: A mandibular mass in 10-year-old Rottweiler.

Photo 11B: The aspirate reveals osteosarcoma. Two mitotic figures as well as eosinophilic material consistent with osteoid are shown. The large, multinucleated cell is an osteoclast. (Photomicrograph courtesy of Dr. Rebekah Gunn-Christie.)

Photo 12A: A palatal mass in a 13-year-old mixed-breed dog.

Photo 12B: Cytologic examination of a sample reveals round cells with multiple criteria of malignancy as well as pigment consistent with oral melanoma.

Mixed population = inflammatory and tissue cells (Photo 12C)

Photo 12C: The same oral melanoma case in Photos 12A and 12B sampled in an area of inflammation. Note the inflammatory and neoplastic cells.

The author thanks Rebekah Gunn-Christie, DVM, Dipl. ACVP, for years of cytologic help as well as review of this manuscript.

Suggested reading

• Sugerman, PB, Savage NW. Exfoliative cytology in clinical oral pathology. Aust Dent J 1996;41(2):71-74.

• Sirois M. Cytology and urinalysis. Principles and practice of veterinary technology. St. Louis, Mo: Mosby, 2004.

• Cowell R, Tyler R, Meinkoth J. Diagnostic cytology and hematology of the dog and cat. 3rd ed. St. Louis, Mo: Elsevier, 2008.

• Baker R, Lumsden J. Color atlas of cytology of the dog and cat. St. Louis, Mo: Mosby, 2000.

• Raskin R, Meyer D. Atlas of canine and feline cytology. 2nd ed. Philadelphia, Pa: WB Saunders, 2010.