Normal Histology and Cytology

The dorsal and ventral eyelids are thin extensions of facial skin that meet at the lateral and medial margins, called canthi. Two to four rows of lashes are found on the upper eyelid at the free margin of the dog, while the cat has a row of cilia (Samuelson, 1999). The outermost layer resembles typical skin, with keratinized squamous epithelium and numerous hair follicles that lie in close association with sebaceous and modified sweat glands. Striated muscle fibers course through the deeper layers. The innermost layer, the palpebral conjunctiva, is lined by pseudostratified columnar epithelium containing numerous goblet cells. Near the margins of both eyelids at the posterior end are the meibomian or tarsal glands. These large sebaceous glands lie adjacent to the palpebral conjunctiva and contribute to the lipid component of the tear film.

Inflammation

Blepharitis refers to inflammatory conditions of the eyelid. Cytologically it is characterized by the predominant cell type. Neutrophilic or purulent blepharitis most often involves bacteria such as Staphylococcus and Streptococcus sp.; however, immune-mediated disease or foreign body reactions may result in purulent inflammation. Eosinophilic inflammation should be considered for allergic reactions, some autoimmune conditions, parasitic migration (Cuterebra sp.), or conditions associated with collagen degeneration. Dermatophytosis or other fungal infections may be associated with granulomatous inflammation containing macrophages alone or mixed with other cell types.

Neoplasia

Diffuse neoplasms of the eyelids involve squamous cell carcinoma (especially in cats), sarcoma, mast cell tumor, and lymphoma. Focal presentations involve sebaceous gland adenoma/adenocarcinoma (canine), papilloma (canine), mast cell tumor (feline), melanoma (canine), squamous cell carcinoma (feline), cutaneous basilar epithelial neoplasm (feline), and histiocytoma (canine). Benign tumors predominate but occasionally malignant tumors such as apocrine sweat gland tumors can invade the globe and extensively damage the eye (Hirai et al., 1997).

Cyst

Chalazion, or meibomian cyst, refers to the granuloma formed from the irritating effects of ruptured sebaceous secretions or blockages caused by tumors. The leakage of meibomian cyst material incites an inflammatory response similar to a foreign body reaction. In this situation, numerous foamy macrophages, few giant cells, neutrophils, lymphocytes, amorphous debris, and sebaceous epithelium are present.

Normal Histology and Cytology

The palpebral conjunctiva is lined by a pseudostratified columnar epithelium containing goblet cells. Goblet cells appear as distended cells with an eccentric nucleus. The cytoplasm may contain clear vacuoles or red-blue granules. Mucus is common on cytologic specimens, appearing as lightly basophilic amorphous strands. The palpebral conjunctiva is continuous with the bulbar conjunctiva, which reflects onto the globe and joins the corneal epithelium. The junction of the two conjunctivae creates a sac termed the fornix. This blind sac is lined by stratified cuboidal epithelium containing many goblet cells. The bulbar conjunctiva consists of sheets of stratified nonkeratinized squamous epithelium, mostly intermediate and superficial with less than 1% goblet cells as detected by impression cytology (Bolzan et al., 2005). There are no significant numbers of inflammatory cells; however, mucosa-associated lymphoid tissue is located below the squamous layer of the palpebral conjunctiva near the fornix, where goblet cells are absent.

Hyperplasia

Conditions such as keratoconjunctivitis sicca, vitamin A deficiency, chronic disease, and trauma from mechanical irritants result in increased cell numbers, sometimes with evidence of metaplasia. These specimens contain many keratinized epithelial cells and goblet cells (Fig. 15-1A) (Murphy, 1988). Increased pigmentation of the epithelium may also occur such that the cytoplasm contains numerous fine, black-green melanin granules (Fig. 15-1B).

FIGURE 15-1 Conjunctival scraping. Cat. Same case A-B. A, Goblet cell hyperplasia. Chronic conjunctival disease in this animal resulted in increased numbers of goblet cells. Two are shown (arrow) characterized by columnar shape, eccentric nucleus, and pale foamy cytoplasm. (Wright-Giemsa; HP oil.) B, Pigmentation and hyperplastic epithelium. Two cells are present with abundant fine black-green cytoplasmic granules. Also note the hyperplastic epithelium with increased nuclear-to-cytoplasmic ratio. (Wright-Giemsa; HP oil.)

Inflammation

The predominant cell type present characterizes the conjunctivitis. Neutrophilic conjunctivitis may be associated with infectious agents such as bacteria and viruses as well as noninfectious causes. One should suspect a bacterial origin if degenerative changes are present. With viruses such as feline herpesvirus and chronic canine distemper virus, nondegenerate neutrophils predominate (Fig. 15-2A). Multinucleated epithelial cells (Fig. 15-2B) were found in approximately half of the cases with feline herpesvirus (FHV-1) infection diagnosed by culture or immunofluorescent assay (Naisse et al., 1993). Eosinophils have been associated with FHV-1 infection (Fig. 15-3A&B) (Volopich et al., 2005). Intranuclear inclusions in FHV-1 infection are occasionally encountered in conjunctival smears (Volopich et al., 2005) but are more readily seen in histologic sections. A polymerase chain reaction (PCR) test has greater sensitivity for detection of the virus than other tests (Stiles et al., 1997). Canine distemper inclusions appear pink with alcohol-based Romanowsky stains but purple with aqueous-based Wright stains.

FIGURE 15-2 A, Suppurative conjunctivitis. Conjunctival scraping. Cat. The bulbar conjunctiva is hyperplastic with many nondegenerate neutrophils and few lymphocytes present in this young cat with chronic conjunctivitis. Infection with feline herpesvirus (FHV-1) was suspected. (Aqueous-based Wright; HP oil.) B, Herpesvirus infection with suppurative conjunctivitis. Conjunctival scraping. Cat. Many nondegenerate neutrophils are present along with reactive epithelium, including multinucleation (center). This case was confirmed previously for herpesvirus infection by polymerase chain reaction. One pigmented epithelial cell is also noted. (Wright-Giemsa; HP oil.)

FIGURE 15-3 Conjunctival brushing. Cat. Same case A-B. A, Eosinophilic conjunctivitis. Eosinophils predominate in the inflammatory response of this suspected herpesvirus infection with hypersensitivity in an adult cat. The small, deeply basophilic cells are the basal epithelium. Clinical signs exhibited were sneezing, tearing, blepharospasms, chemosis, and hyperemia. (Wright; HP oil.) B, Mixed cell conjunctivitis. In this field nondegenerate neutrophils predominate in addition to the notable increase in eosinophils. A goblet cell (upper right) is present with columnar shape and pale foamy cytoplasm. (Wright; HP oil.)

Other infectious causes include feline chlamydiosis, which presents as variably sized discrete basophilic to bright magenta bodies in the cytoplasm of epithelium within first 2 weeks of infection. Small elementary bodies (0.3 μm) are shed that infect new cells and grow into larger (0.5 to 1.5 μm) initial bodies (Greene, 1998). The initial bodies (Fig. 15-4A&B) proliferate to become a large, membrane-bound reticulate body containing many elementary bodies (Fig. 15-5). In the Naisse et al. study (1993), visible epithelial inclusions were present in only a third of the cases positively identified by immunofluorescent assay as Chlamydia psittaci, currently called Chlamydophila felis. Besides fluorescent antibody detection, cell inoculation, enzyme-linked immunosorbent assay (ELISA), PCR procedures, or immunochemistry may be used (Volopich et al., 2005; von Bomhard et al., 2003). In one case reported, chlamydial infection in a cat was possibly transmitted from a macaw (Lipman et al., 1994). In another study, of 226 conjunctival samples, 39% were positive for non–C. felis chlamydial DNA, identified as Neochlamydia hartmannellae, in cytologic samples with eosinophilic inflammation (von Bomhard et al., 2003). Infection with feline Mycoplasma sp. presents as small basophilic granules similar to the elementary bodies of chlamydial infections with the exception that they are adherent to the surface membrane (Fig. 15-6A&B). One study from Belgium indicated mycoplasmal infection had an incidence of 25% in cats with conjunctivitis (Haesebrouck et al., 1991).

FIGURE 15-4 Chlamydiosis. Conjunctival scraping. Cat. Same case A-B. A, Three small basophilic initial bodies are present in the cytoplasm of one epithelial cell of this 10-month-old kitten. (Wright-Giemsa; HP oil.) B, Degenerating epithelial cells contain several small basophilic granular initial bodies. Another cat in the house and the owner have conjunctivitis. Nondegenerate neutrophils and small lymphocytes are also present in this sample. (Wright-Giemsa; HP oil.)

FIGURE 15-5 Chlamydiosis. Conjunctival scraping. Cat. One week earlier this cat developed pyrexia, conjunctivitis, rhinitis, and oral ulcers. The indicated cell (arrow) contains a perinuclear reticulate body filled with numerous elementary bodies. Diagnosis was confirmed by fluorescent antibody testing. (Wright)

(Photo courtesy of John Kramer, Washington State University; presented at the 1989 ASVCP case review session.)

FIGURE 15-6 Mycoplasmosis. Conjunctival scraping. Goat. Same case A-B. A, This animal had keratoconjunctivitis clinically. Numerous small gray granules are associated with the cell and few organisms are found extracellularly in the background. Nondegenerate neutrophils are frequent. Stain precipitate or cellular debris is present below the cell. (Wright-Giemsa; HP oil.) B, Note the numerous organisms over the cytoplasm and nucleus. These granules are adherent to the surface membrane and extend into the background. (Wright-Giemsa; HP oil.)

Noninfectious causes include keratoconjunctivitis sicca and allergic conditions. Eosinophilic conjunctivitis has been associated with a hypersensitivity reaction in the cat. In this situation eosinophils are seen commonly (Fig. 15-7A). Mast cell infiltrates may also be so numerous that there is concern for a conjunctival mast cell tumor (Fig. 15-7B). Lymphocytes and plasma cells have been associated with allergic conditions as well as with early canine distemper infection or with chronic inflammation of the conjunctiva.

FIGURE 15-7 Eosinophilic conjunctivitis. Conjunctival scraping. Cat. Same case A-B. A, Bilateral conjunctivitis is present in this animal. An eosinophil is shown at the upper right and a mast cell is at left center. Note the mast cell granules in the background. This response occurred with a suspected hypersensitivity reaction of noninfectious causes. (Wright-Giemsa; HP oil.) B, Many mast cell granules are free in the background. Two mast cells (arrows) and two pigmented epithelial cells are present in the center. The numerous mast cells present cause concern for a mast cell tumor, but they appear fully mature and benign. (Wright-Giemsa; HP oil.)

Neoplasia

Epithelium is frequently atypical and hyperplastic as a result of severe inflammation; therefore, neoplasia may be difficult to diagnose confidently. Neoplasms found associated with the conjunctival area include squamous cell carcinoma, papilloma, melanoma, lymphoma, hemangiosarcoma, and mast cell tumor. An uncommon location of transmissible venereal tumor has been the conjunctivae of the upper and lower eyelids (Boscos et al., 1998).

Miscellaneous Finding

Large amorphous basophilic inclusions within the cytoplasm of squamous epithelium have been attributed to ophthalmic ointment use, particularly those medications containing neomycin (Prasse and Winston, 1999; Young and Taylor, 2006).

Normal Histology and Cytology

The third eyelid, or nictitating membrane, is a large fold of conjunctiva protruding from the medial canthus. It contains a T-shaped cartilaginous plate surrounded by glandular epithelium that is serous in the cat and seromucoid in the dog (Samuelson, 1999). The palpebral and bulbar surfaces are covered by nonkeratinized stratified squamous epithelium. The free margin of the membrane is pigmented and, cytologically, fine green-black melanin granules can be found within the epithelium. Numerous variably sized lymphoid aggregates with overlying epithelium having microvilli or microfolds (M cells) on the apical surface are associated with the bulbar surface of the nictitating membrane (Giuliano et al., 2002). The stroma also contains fibrous connective tissue.

Inflammation

With follicular conjunctivitis there is a mixed population of lymphoid cells that resembles a hyperplastic lymph node (Fig. 15-8A&C). Plasma cell infiltrates (plasmacytic conjunctivitis or plasmoma) have been seen in German Shepherd dogs. These thickened depigmentating lesions are composed of numerous well-differentiated plasma cells.

FIGURE 15-8 Follicular hyperplasia. Nictitating membrane. Cat. Same case A-C. A, Numerous small lymphocytes predominant. (Aqueous-based Wright; HP oil.) B, Higher magnification to demonstrate the presence of a plasma cell (arrow) and several intermediate lymphocytes in addition to the small lymphocytes. (Aqueous-based Wright; HP oil.) C, Reactive epithelium accompanies the reactive lymphoid population. (Aqueous-based Wright; HP oil.)

Neoplasia

These are similar to those seen in the conjunctivae. Adenocarcinomas, papillomas, and malignant melanomas are most common in dogs. A report of an adenocarcinoma affecting the gland of the nictitating membrane in a cat demonstrated the presence of malignant epithelial cells from impression smears of excisional biopsy material (Komaromy et al., 1997). Squamous cell carcinoma, when present on the nictitating membrane, is considered an extension from the eyelid. It appears similar to those found in the skin often with nonseptic purulent inflammation (Fig. 15-9A-E). Dogs and cats most commonly present with hemangiosarcoma and hemangioma within the nonpigmented epithelium of the nictitating membrane compared with the conjunctivae. Exposure to ultraviolet light is suspected to be a risk factor for these endothelial neoplasms (Pirie et al., 2006; Pirie and Dubielzig, 2006).

FIGURE 15-9 Squamous cell carcinoma. Nictitating membrane. Cat. Same case A-E. A, Several weeks duration of erythema and edema along with proliferative lesions on the conjunctiva and third eyelid were present in this 14-year-old cat. Sheets of epithelium appear with some features of malignancy, including anisokaryosis, multinucleation, variable nucleocytoplasmic ratio, and coarse chromatin. Many nondegenerate neutrophils are noted without evidence of sepsis. (Aqueous-based Wright; HP oil.) B, In addition to the previously mentioned malignant features there is perinuclear vacuolation, a feature often associated with malignant squamous epithelium. The presence of severe suppurative inflammation likely accounts for some of the dysplastic changes in the epithelium. (Aqueous-based Wright; HP oil.) C, Two giant epithelial cells with multiple nuclei. The presence of very bizarre morphologic changes and the absence of sepsis further supports the malignant, not dysplastic nature of the epithelium. (Aqueous-based Wright; HP oil.) D, This section is taken from the junction between the palpebral conjunctiva on the left and the nictitating membrane on the right. Note the marked disorganization of the mucous membrane and dermis. This tumor is thought to originate from the eyelid with extension to the nictitating membrane. (H&E; IP.) E, Two keratin pearls that are often associated with malignant squamous epithelium appear in the center and help to identify the neoplasm. (H&E; HP oil.)