Cytologic diagnoses that every practicing veterinarian should be able to make (Proceedings)

One of the most commonly used diagnostic modalities in veterinary medicine is cytology. Samples can be acquired quickly and easily, using instruments (e.g. needles, syringes, scalpel blades, etc.) that are readily available in any veterinary practice from the single veterinarian hospital to the largest referral centers. The strengths of cytology lie in the rapidity with which the samples can be obtained, processed and evaluated. In addition, the overall cost for cytologic evaluation of tissue is generally more affordable compared to histopathology. Although reference laboratories are becoming more and more available, there are numerous lesions that, with practice, should be easily diagnosed in-house, thus maximizing the turn-around and profit of that cytologic specimen.

Before one starts to evaluate cytologic specimens in-house, a few important suggestions should be heeded. First and foremost is the equipment to use. New, unused, sterile tools (i.e. needle, syringe, blade, etc.) must be used for sample acquisition. This is followed by high quality, dedicated stains. The stain(s) used for staining cytologic (and hematologic) samples must only be used for this purpose. Skin scrapings, ear swabs and other "dirty" samples should have their own stain, otherwise cross contamination can occur. These dedicated stains must also be kept fresh and replaced when exhaustion is suspected or observed. Of equal important is the microscope to use. Binocular eye pieces are important, as is a conformation that is easy to use and comfortable for the user. If serious diagnostic cytology and hematology are going to be performed in a practice, a dedicated microscope should be used. If properly cared for, it will outlive your career. Resolution and clarity are greatly affected by the objectives that a microscope uses. Types include (from cheapest to most expensive) achromatic, planachromat, fluorite/semi-apochromat and apochromat. A solid investment in the equipment used will be rewarded in the long term.

Learning to confidently evaluate cytologic specimens is similar to any other infrequently used skill. Practicing and volume are key. When starting to build a skill set, it can be extremely useful to stain one slide of a sample such as an aspirate or an impression smear of a biopsy. Evaluate it in-house and document the findings, then submit the remainder for evaluation by a pathologist. When the final report is released, compare findings, and if applicable, review the slide for discrepancies. If there are enough slides, it can be extremely useful to coverslip, catalog and keep slides for future reference. By repeating these steps, one can improve their cytologic skills.

Diagnostic Materials and Artifacts

The first thing every sample must be evaluated for is its quality. Poorly cellular samples prevent adequate assessment and thus conclusive interpretation. One important rule to adhere to when evaluating cytology is to only utilize intact cells with intact nuclei when making judgments. Nuclear debris and nuclear streaming can signal lysed cells, as can nuclei that have begun to break apart, forming what some refer to as "basket cells" because of the wicker pattern that can form. Blood contamination / hemodilution is another major hurdle that must be cleared. Factors that must be assessed to determine if blood contamination is present or whether or not there is in vivo inflammation include: the presence of platelets and fibrin, the morphology of the neutrophils and the ratio of leukocytes to erythrocytes can all be useful tools to help differentiate.

Artifacts are another distraction that must be overcome to fully evaluate smears. Almost without exception, nearly every type of stain can form particulate precipitate, which can then get into a smear. This material should be variably sized, often a dull color and definitely extracellular. It is often mistaken for bacteria; however, bacteria are relatively uniform in size, have crisp, distinct margins and often form organized, regular patterns. Another consideration for bacteria, is the presence of absence of a cellular response.

Another confounding artifact is gel. Ultrasound and lubricating gel often takes on an amorphous to particulate shape and a bright magenta color. In addition to obscuring the smear, gel can also cause discoloration due to it taking up an abundance of stain.

Infectious Etiologies

1. Bacterial sepsis- Regardless of whether the lesion is ulcerated skin, a hypoechoic lesion in the liver or synovial fluid, the identification of bacterial sepsis is important, and allows rapid treatment. It is infrequently the sole lesion present and thus other components of the smear and case must always be taken into consideration. Besides the resident population of cells (e.g. hepatocytes, squamous epithelial cells, etc.), the bulk of these lesions are made up of neutrophils, often in a background of necrotic debris. The neutrophils are generally markedly degenerate, as exhibited by karyolysis, karyorrhexis and pyknosis. Intracellular bacteria must be visualized to considered cytologically septic. Prior antibiotic use can make finding bacteria impossible. A Gram stain can be used to classify the bacteria seen; however, a control slide is critical.

2. Blastomyces dermatitidis- Aspirates, impressions of swabs from these dermal lesions are generally highly cellular. They often display classic pyogranulomatous inflammation, which includes neutrophils (often degenerate), foamy mononuclear macrophages, smooth mononuclear epithelioid macrophages and multinucleated foamy giant cells. Most times, the fungal spherules are plentiful and not difficult to identify. If more sparse, the thicker portions of the smear often have a higher yield. They are round and range in size from 7-40 micrometers in diameter. They have a dark blue, thick, refractile cell wall and occasionally exhibit the characteristic broad based budding.

3. Histoplasma capsulatum- These lesions are generally very cellular. Lung aspirates, BAL/TTW specimens, liver aspirates and rectal scrapes are all excellent samples to evaluate, looking for this fungus. Many times, suppurative to macrophagic inflammation is present, with numerous intracellular organisms seen. The yeast measures 2-4 micrometers in diameter and has a colorless wall, with a purple nucleus. They should be round in shape and not confused for Sporothrix schenckii, which tends to be slightly smaller and more elongate in shape. It is common for there to be several within macrophages or a few within neutrophils. If organisms can't be found in tissue samples, yet histoplasmosis is still suspected, a peripheral blood smear and/or buffy coat preparation should be evaluated before more aggressive tactics are employed.

Figure 1. Histoplasma capsulatum in a lymph node, 100x. Diff-Quik. Yeast are free in the background and within macrophages (arrows)

Figure 1

4. Cryptococcus neoformans- The presentation of these samples is variable in that there can be a marked inflammatory response or almost none. Inflammation can be suppurative, granulomatous or eosinophilic. The organism itself can range in size from 2-20 micrometers; however the thick, non-staining (with traditional polychromatic stains) capsule can reach nearly 200 micrometers in diameter.

5. Coccidioides immitis- Pyogranulomatous inflammation is common with this organism. The yeast ranges in size from 20 - 200 micrometers in diameter. They have a double refractile wall and can contain myriad endospores. These endospores are similar morphologically to Histoplasma sp. they are 2 - 5 micrometers in diameter

Figure 2. Cryptococcus neoformans with suppurative inflammation, 40x, Modified Wright's

Figure 2

Neoplasia

1. Benign cutaneous histiocytoma- Not to be confused with malignant tumors bearing a similar sounding name, these lesions is dogs are readily diagnosable via cytology. Grossly, these lesions are commonly seen around the head, neck and limbs. These samples are heavily cellular and consist of large numbers of discrete, round cells that have a round nucleus set in a moderate amount of pale blue cytoplasm set in a background of finely granular protein. Occasionally, the cytoplasm is light enough that it appears as a negatively staining halo around the intact nucleus. The cells themselves are monotonous and rather non-descript. If a large population of small lymphocytes is also present, spontaneous regression is taking place.

2. Mast cell tumors- As they can present clinically as just about anything, it is always worth aspirating every skin mass that is seen. Early detection of mast cell tumors (MCT) can improve prognosis and increase the number of treatment options available. The main feature of this sample is large numbers of mast cells. These discrete round cells have a round nucleus set in a moderate to large amount of light blue cytoplasm that is variably filled with dark purple granules. Eosinophils, reactive fibroblasts and occasional flecks of collagen are also often present. Described based on their level of differentiation, not grade. On occasion, some stains may not reveal the granules as readily as others. If a round cell tumor is diagnosed, yet the cytomorphology does not fit any of the others, consider the use of another stain or special stains such as Giemsa or toluidine blue.

Figure 3. Mast Cell Tumor, 100x, Modified Wright's

Figure 3

Miscellaneous

1. Sialocele- These samples are variable in appearance. Initially, they are low cellularity samples that are made up of foamy salivary epithelial cells that are nearly always seen as singlets in a background of erythrocytes and small globs of mucus. Windrowing is often very prominent. Occasional signs of hemorrhage are also present, with erythrophagia, hematoidin or less frequently hemosiderin. If the lesion is compromised, secondary suppurative inflammation can be present and the windrowing can decrease.

2. "Benign skin lesion". This is a blanket cytologic interpretation for a host of skin lesions, all of which are benign. These lesions produce smears that are often very busy. They generally contain a large amount of keratin, which presents in many forms (flakes, bars and particles) in a heavy background of assorted debris. Occasional small, tight clusters of basal-type epithelial cells are seen. In the more cyst-like lesions, cholesterol crystals are frequently noted. Inflammation is generally absent unless the lesion ruptures, exposing keratin to the subcutis, which leads to sterile granulomatous inflammation. Differential diagnoses include: epidermal inclusion cyst, epidermoid cysts, follicular cysts, cornifying epitheliomas, keratinizing acanthomas, dermoid cyst, intracutaneous cornifying epitheliomas and any one of a number of follicular tumors. Differentiation requires biopsy with histologic evaluation; however, biologically, these entities behave similarly.

3. Lipoma- Extremely common cytologic diagnosis that is made easily and rapidly. Grossly, the smears often appear as lipid or water droplets that do not dry after an appropriate length of time. Microscopically, the smears are usually sparsely cellular and consist of several aggregates of adipocytes in a clear to light background of blood and cell-free lipid. Adipocytes have abundant, clear cytoplasm with thin cell margins. Occasionally, clumps of cells aggregate with capillaries associated with them.

Figure 4. Lipoma, 20x, Modified Wright's. Not the capillaries running through the clumps of adipocytes

Figure 4

4. Sebaceous adenoma- Aspiration of these wart-like lesions typically results in a highly cellular sample. Epithelial cells are generally seen in cohesive clusters, which are rarely arranged in acinar structures, as well as singlets, often in a light background of blood. The bulk of the epithelial cells are large cells that have abundant foamy cytoplasm. The vacuoles are fine and similar in size. These cells are monomorphic and sometimes accompanied by small cells that have dark blue cytoplasm and have high N:C ratios. These cells represent reserve cells. Occasionally, necrotic material can be found in the center of the lesions. Cytologic differentiation between a sebaceous adenoma and sebaceous hyperplasia is impossible.

References

Baker R, Lumsden JH. Color Atlas of Cytology of the Dog and Cat. St Louis, Mosby. 2000.

Cowell RL Ed. Cytology I. Veterinary Clinics of North America: Small Animal Practice. 32(6). November 2002.

Cowell RL Ed. Cytology II. Veterinary Clinics of North America: Small Animal Practice. 33(1). January 2003.

Cowell RL, Tyler RD. Diagnostic Cytology and Hematology of the Horse. 2nd Edition. St. Louis. Mosby. 2002.

Raskin RE, Meyer DJ. Atlas of Canine and Feline Cytology. W.B. Saunders. Philadelphia. 2001.

Thrall MA. Cytologic Examination of Cutaneous and Subcutaneous Lumps and Lesions. \Veterinary Medicine. 95(3): 224-241. 2000.