Stains

Romanowsky stains are standard and, in general, are excellent for observing the morphologic characteristics of cells, organisms, and other structures. The major artifact is stain precipitate, which can mimic clusters of bacterial cocci. Quick stains (such as Diff-Quik) are often adequate but do not stain cytoplasmic features as well as do parent stains, especially methanolic stains. In some instances, for example, mast cell granules do not stain with quick stains, and the presence of these cells may go undetected if other stains are not used. Also, quick stains must be maintained well, or the stains themselves may contain organisms such as Malassezia from previously stained specimens or from contamination. Other stains that may be used as adjuncts include stains for fungal organisms, such as periodic acid-Schiff (PAS) and Gomori methenamine silver stain (GMS). Gram stain may be used to determine if bacteria are Gram-positive or Gram-negative. Gram-stained slides can be tricky to read and require experience to avoid misinterpretation. Indirect fluorescent antibody (IFA) staining requires special reagents and a fluorescence microscope.

Microscopic Evaluation

Ocular specimens are often small in volume, and therefore examination of the entire sample is easy. The observer should be familiar with the normal cytologic and histologic characteristics of the tissue sampled and recognize cellular patterns, other structures, and background material.1,2 Identification of specific types of inflammatory cells permits classification of inflammation as neutrophilic (synonyms include suppurative and purulent); eosinophilic (often accompanied by mast cells); lymphocytic–plasmacytic; mixed, including pyogranulomatous; and granulomatous. If neoplasia is suspected on the basis of the presence of a mass and a homogenous population of noninflammatory cells, the observer should be able to identify the cell type (epithelial, mesenchymal or connective tissue, and discrete round cells) and the cytologic features of benign and malignant tumors. It is important to recognize that neoplasms can induce an inflammatory response. Finally, the observer should be familiar with the cytologic characteristics of cysts, acute and chronic hemorrhage, and degenerative diseases.

When identifying cell types, it is essential to examine cells in an area where they can be evaluated individually. However, thick collections of material—often consisting of clustered epithelial cells, aggregates of mesenchymal cells, or necrotic material—tend to be understained, and cells with granules that stain more readily than other components (mast cell and eosinophil granules), naturally pigmented elements (melanin), bacteria, and fungal hyphae may be visualized within or on top of the thick tissue (Figure 9-1). Inclusions found in epithelial or inflammatory cells may be normal elements, artifacts of treatment, or evidence of the pathologic process or etiology (Table 9-1). Normal tissue also may be present.

Once a category is identified, a more specific diagnosis may be possible. For example, search for an etiologic agent is indicated if inflammation is present. At the very least, the category can guide additional testing or therapy. Special cytologic features of neutrophils, epithelial cells, and extracellular material (Table 9-2) often provide additional information about the pathologic process; misinterpretation of these features (e.g., mistaking free mast cell granules for bacterial cocci) could lead to erroneous conclusions.

Blepharitis

Blepharitis may be focal or diffuse and acute or chronic; bacterial, mycotic, parasitic, allergic, or immune-mediated blepharitis may occur. The objectives in cytologic examination of lesions of blepharitis are to characterize the type of exudate (neutrophilic, lymphocytic–plasmacytic, eosinophilic, or granulomatous) and search for the causative agent. Agents that may be encountered in scrapings are Sarcoptes spp., Demodex spp., dermatophytic yeast, and bacteria. Demodex folliculorum causes minimal exudation. Bacterial blepharitis, particularly staphylococcal blepharitis, has a neutrophilic exudate. Certain fungi, such as Blastomyces dermatitidis, cause either a primarily neutrophilic or a pyogranulomatous exudate, whereas others cause a granulomatous exudate composed of macrophages, including epithelioid forms, and giant cells. Foreign bodies may elicit a pyogranulomatous or granulomatous response (Figure 9-2).

Immune-mediated disease usually is characterized by a neutrophilic exudate, but eosinophilic types also occur. The presence of either bacteria or a primarily neutrophilic exudate does not exclude allergic and immune-mediated causes, especially if the lesion is ulcerated. In cats, eosinophilic plaques may manifest as periocular blepharitis. A fine-needle aspirate contains primarily eosinophils, some mast cells, and a mixture of other white blood cell (WBC) types.

Discrete Masses

Discrete masses on the eyelids may be neoplastic (benign or malignant) or nonneoplastic. Among neoplasms, benign sebaceous gland tumors (sebaceous adenoma, sebaceous epithelioma) are the most common type on canine eyelids. The glands of Zeis and Moll at the eyelid margin and the meibomian glands, which lie beneath the palpebral conjunctiva and open at the lid margin, are all of the sebaceous type; tumors arising from them are similar to cutaneous sebaceous gland tumors. The cells are readily recognized by their voluminous vacuolated cytoplasm that nearly obscures small rounded nuclei (Figure 9-3). The malignant counterpart of these tumors is rare on the eyelids.

Other tumors frequently encountered on the eyelids and readily diagnosed by cytologic examination include melanoma (benign and malignant), histiocytoma (Figure 9-4), lymphoma, mast cell tumor (low and high grade) (Figure 9-5), papilloma, and squamous cell carcinoma. Squamous cell carcinomas are frequently ulcerated. In mast cell tumors, mast cell granules sometimes are not visible if quick stains are used (see Figure 9-5, B). Other carcinomas and connective tissue tumors (fibrosarcoma, hemangiosarcoma, histiocytic sarcoma) occur less frequently and are discussed in Chapter 2.

Nonneoplastic discrete masses unique to the eyelid include the hordeolum, a localized purulent lesion of sebaceous glands, and the chalazion, a lipogranuloma of the meibomian gland. FNA of these lesions yields numerous foamy macrophages and a few giant cells and lymphocytes. The macrophages are apparently phagocytosing glandular secretory product; cytophagia is not prominent. Variable numbers of sebaceous epithelial cells also are found. Differentiating a hordeolum or chalazion from sebaceous gland adenoma by cytologic examination may be difficult if the latter has ruptured internally and caused secondary inflammation. A hordeolum or a chalazion may contain inspissated secretory product or mineralized debris that appears as amorphous granular material on cytologic preparations.

Ocular idiopathic adnexal granulomas may simulate neoplasms, be bilateral, and be a component of systemic granulomatous disease.³ Systemic histiocytosis of Bernese Mountain Dogs causes periocular granulomatous masses.^4,5 True cysts can occur on the eyelids and typically contain foamy macrophages and cholesterol crystals from epithelial degeneration (Figure 9-6).

Neutrophilic Conjunctivitis

Canine and feline conjunctivitis frequently is neutrophilic and results from bacterial or viral infections, allergic disease, or other causes. Pseudomembranous (ligneous) conjunctivitis is neutrophilic.⁸ Cytologic evaluation may not reveal the cause. Neutrophils may be nondegenerate or degenerate (Figure 9-11); in cats, the latter are rarely encountered. In both dogs and cats, intact neutrophils may be found within squamous cells, and the significance of this finding is unknown. Mucus is a common component of neutrophilic exudates and may cause cells to be aligned in rows on the smear.

The exudate of canine neutrophilic conjunctivitis often contains bacteria, regardless of the primary cause. Bacteria are often large or small cocci and less frequently rods (Figure 9-12). The dilemma is determining whether the bacteria are of primary importance or are merely opportunistic. Normal bacterial flora of the canine conjunctival sac have been described.⁹ Keratoconjunctivitis sicca is a common canine disorder causing neutrophilic exudate in which bacteria frequently are encountered. The disease is diagnosed readily by the Schirmer tear test. In contrast to that of dogs, the exudate of feline neutrophilic conjunctivitis rarely contains bacteria. When observed, bacteria should be considered clinically significant in feline conjunctivitis.

Distemper is the most important viral cause of canine neutrophilic conjunctivitis. Canine distemper is diagnosed on the basis of its classic clinical signs and fluorescent antibody staining of conjunctival smears. Canine distemper inclusion bodies in epithelial cells are found rarely (Figure 9-13), and a search for them has limited diagnostic value.

A common cause of feline neutrophilic conjunctivitis is herpesvirus infection. Diagnosis is confirmed by PCR analysis, fluorescent antibody staining of conjunctival smears, or viral isolation. Multinucleate epithelial cells may be found, but intranuclear inclusion bodies are seen rarely, if ever, cytologically.

Neutrophils also predominate in the conjunctival exudate of feline chlamydial infection. In experimental Chlamydophila felis infections, organisms were found on day 6 postinoculation, after clinical signs first appeared.¹⁰ Solitary, large (3 to 5 micrometers [µm]), basophilic particulate forms initially are found in the cytoplasm of squamous epithelial cells (Figure 9-14). The particulate nature of the initial body is an important observation to distinguish C. felis from incidental foci of homogeneous cytoplasmic basophilia found in squamous epithelial cells (Figure 9-10); organisms also may appear as aggregates of coccoid basophilic bodies (elementary bodies), measuring 0.5 to 1 µm in diameter (Figure 9-15).¹¹ In experimental infections, organisms rarely were found by day 14 after inoculation, and in chronic conjunctivitis, intracytoplasmic organisms are present only infrequently.10,12 Chlamydial conjunctivitis may be confirmed by PCR analysis or fluorescent antibody staining. Chlamydiae other than C. felis also may play a role in ocular disease in cats.¹³

Feline mycoplasmosis, another cause of neutrophilic conjunctivitis, may be diagnosed by finding the organisms on epithelial cells on routinely stained smears. In one study, mycoplasmosis was diagnosed in nine naturally infected cats by isolation and identification of Mycoplasma spp. Of samples from 16 eyes, the organisms were found on Romanowsky-stained smears from 15 eyes, suggesting a high degree of diagnostic sensitivity for routine cytologic evaluation in Mycoplasma infection.¹⁴ Other studies have found cytologic examination to be less reliable in the diagnosis of mycoplasmosis.¹¹ The basophilic organisms, 0.2 to 0.8 µm long, may be found in clusters adherent to the outer limits of the plasma membrane or over the flattened surface of squamous epithelial cells (Figure 9-16). They also may be seen in clusters between cells. Mycoplasma organisms should not be confused with melanin granules (Figure 9-9).

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