Stains

Romanowsky stains are standard and, in general, are excellent for observing morphologic characteristics of cells, organisms, and other structures. The major artifact is stain precipitate, which can mimic clusters of bacterial cocci. The quick stains (such as Diff-Quik) are often adequate, but do not stain cytoplasmic features as well as the parent 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, including periodic acid–Schiff (PAS) and Gomori’s methenamine silver stain, and Gram’s stain to determine if bacteria are gram positive or 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 characteristics of the tissue sampled and recognize cellular patterns, other structures, and background material. 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 based on the presence of a mass lesion 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 important to look in an area where individual cells can be evaluated. However, thick collections of material, often consisting of clustered epithelial cells or necrotic material, tend to be understained, and cells with granules that stain more readily than other components (mast cells and eosinophils), 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.

Figure 9-1 Thick understained tissue from two corneal scrapes, in which significant structures/cells can be visualized. A, Eosinophils and eosinophil granules (arrows). B, Fungal hyphae (arrows). (Wright’s stain, original magnification 200×; insets 600×.)

Table 9-1 Inclusions In or On Cells from Ocular Tissue

Once a category is identified, a more specific diagnosis may be possible. For example, a 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 are listed in Table 9-2 and often provide additional information about the pathologic process; misinterpretation of these features (e.g., mistaking free mast cell granules for bacterial cocci) can lead to erroneous conclusions.

Table 9-2 Special Cytologic Features and Their Significance

Blepharitis

Blepharitis may be focal or diffuse and acute or chronic. Causes may be bacterial, mycotic, parasitic, allergic, or immune mediated. The objectives in cytologic examination 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, 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 (macrophages, including epithelioid forms, and giant cells). Foreign bodies can elicit a pyogranulomatous or granulomatous response (Figure 9-2).

Figure 9-2 Pyogranulomatous inflammation in the eyelid of a dog. Note neutrophils and epithelioid macrophages, including binucleate forms. Lymphocytes and small numbers of red blood cells also are present. (Wright’s stain, original magnification 600×.)

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 be manifested as periocular blepharitis.¹ A fine-needle aspirate reveals 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.² 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.

Figure 9-3 Sebaceous gland epithelial cells from a well-differentiated sebaceous gland tumor on a canine eyelid. (Romanowsky-type stain, original magnification 1000×.)

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 (Figure 9-5, B). Other carcinomas and connective tissue tumors (fibrosarcoma, hemangiosarcoma, histiocytic sarcoma) occur less frequently and are discussed in Chapter 2.^2,3

Figure 9-4 Discrete round cell tumor with cytologic characteristics of a histiocytoma on the eyelid of a dog. (Wright’s stain, original magnification 600×.)

Figure 9-5 A, Well-granulated mast cell tumor on the eyelid of a dog. (Wright’s stain, original magnification 600×.) B, The same specimen stained with a quick stain. Note that mast cell granules did not stain. (Diff-Quik stain, original magnification 600×.)

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. Fine-needle aspiration 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 (Figure 9-6). Differentiating a hordeolum or chalazion from sebaceous gland adenoma by cytologic examination may be difficult if the latter has internally ruptured and caused secondary inflammation. Hordeolum or chalazion may contain inspissated secretory product or mineralized debris that appears as amorphous granular material on cytologic preparations.

Figure 9-6 A giant cell, macrophages, lymphocytes, and a sebaceous gland epithelial cell (lower left) from a chalazion on a canine eyelid. (Romanowsky-type stain, original magnification 1000×.)

Idiopathic ocular adnexal granulomas may simulate neoplasms, be bilateral, and be a component of systemic granulomatous disease.⁴ Systemic histiocytosis of Bernese mountain dogs causes periocular granulomatous masses.^5,6 True cysts can occur on the eyelids and typically contain foamy macrophages and cholesterol crystals from epithelial degeneration (Figure 9-7).

Figure 9-7 Cytocentrifuged material from a cyst on the eyelid of a dog. Note the foamy macrophages and cholesterol crystals. (Wrights stain, original magnification 200×, inset 600×.)

Neutrophilic Conjunctivitis

Canine and feline conjunctivitis frequently is neutrophilic and occurs with bacterial, viral, allergic, or other causes. Pseudomembranous (ligneous) conjunctivitis is neutrophilic.¹⁰ Cytologic evaluation may not reveal the cause. Neutrophils may be nondegenerate or degenerate (Figure 9-12). In cats, the latter are rarely encountered. In both species, intact neutrophils may be found within squamous cells (Figure 9-13), and the significance of this finding is unknown. Mucus is a common component of neutrophilic exudates and may cause cells to appear in rows on the smear.

Figure 9-12 Conjunctival scrape from a dog with neutrophilic bacterial conjunctivitis. Both well-segmented nondegenerate neutrophils and degenerate neutrophils with swollen nuclei are present. (Wright’s stain, original magnification 600×.) Inset, A degenerate neutrophil with two thin bacterial rods. (Wright’s stain, original magnification 1000×.)

Figure 9-13 Conjunctival smear from a dog with neutrophilic conjunctivitis. Intact neutrophils are found in the cytoplasm of a squamous cell (center), and numerous neutrophils, lymphocytes, and a goblet epithelial cell (lower left center) are present. (Romanowsky-type stain, original magnification 1000×.)

The exudate of canine neutrophilic conjunctivitis often contains bacteria, regardless of primary cause. The bacteria are often large and/or small cocci and less frequently rods (Figure 9-14). The dilemma is whether the bacteria are of primary importance or are merely opportunistic. Normal bacterial flora of the canine conjunctival sac have been described.¹¹ 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.

Figure 9-14 Conjunctival scrape from a dog. Note many neutrophils and bacterial rods (long arrow) and cocci (short arrows). (Wright’s stain, original magnification 1000×.)

Distemper is the most important viral cause of canine neutrophilic conjunctivitis. Canine distemper is diagnosed by its classic clinical signs and fluorescent antibody staining of conjunctival smears. Canine distemper inclusion bodies in epithelial cells are found rarely (Figure 9-15), and a search for them has limited diagnostic value.

Figure 9-15 Conjunctival scrape from a dog. Variably sized distemper viral inclusions (arrows) are found within epithelial cells. Note neutrophils and small bacterial rods and cocci. (Wright’s stain, original magnification 1000×.)

(Photomicrograph by Judith Taylor; from Young KM, Taylor J: Laboratory Medicine: Yesterday Today Tomorrow: Eye on the cytoplasm. Vet Clin Pathol 35:141, 2006. Reprinted with permission of the American Society for Veterinary Clinical Pathology.)

A common cause of feline neutrophilic conjunctivitis is herpesvirus infection (Figure 9-16). Diagnosis is confirmed by a PCR assay,^12,13 fluorescent antibody staining of conjunctival smears, or viral isolation. Multinucleated epithelial cells may be found, but intranuclear inclusion bodies rarely are seen.^7,14

Figure 9-16 Nondegenerate neutrophils, squamous cells, plasma cells (top left center), and a lymphocyte (lower right) from a cat with neutrophilic conjunctivitis. Other smears were fluorescent antibody-positive for feline herpesvirus. (Romanowsky-type stain, original magnification 1000×.)

Neutrophils predominate also in the conjunctival exudate of feline chlamydial infection. In experimental Chlamydophila felis infections, organisms were found by postinoculation on day 6 when clinical signs first appeared.¹⁵ Solitary, large (3 to 5 μm), basophilic particulate forms initially are found in the cytoplasm of squamous epithelial cells (Figure 9-17). 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 (see Figure 9-11).⁸ Organisms also may appear as aggregates of coccoid basophilic bodies (elementary bodies), 0.5 to 1 μm in diameter (Figure 9-18). In the experimental infections, organisms rarely were found by day 14 postinoculation,¹⁵ and intracytoplasmic organisms are present only infrequently in chronic conjunctivitis.⁷ Chlamydial conjunctivitis may be confirmed by PCR or fluorescent antibody staining. Chlamydiae other than C. felis also may play a role in ocular disease in cats.¹⁶

Figure 9-17 Initial body of Chlamydophila felis in the cytoplasm of a squamous cell in a conjunctival scrape from a cat. (Romanowsky-type stain, original magnification 1000×.)

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