13.2.2.1 Periocular and Retrobulbar Lesions

Cytological evaluation of lesions associated with the dermal and subcutaneous tissues surrounding the eye can provide valuable diagnostic information. Impression smear cytology (pressing a glass slide to a lesion) should be accompanied by fine needle aspiration or scraping, as impression smears only represent the superficial surface of the lesion. Neoplastic cells may not be present superficially. Additionally, when ulceration is present, secondary infections are common which can lead to insufficient treatment. Lastly, in ulcerated lesions, atypical squamous epithelial cells are common, representing dysplastic and hyperplastic populations. However, these cells can mimic neoplastic populations, resulting in misdiagnoses, especially with novice microscopists.

Fine needle aspiration of retrobulbar lesions has the benefit of being minimally invasive and providing rapid information. However, not all neoplasms and pathological processes can be diagnosed with cytological evaluation. Some lesions are poorly cellular or poorly exfoliative, providing only small number of cells for cytological evaluation (e.g., hemangioma, certain sarcomas). Therefore, additional diagnostic tests such as advanced imaging and biopsy with histologic examination may be warranted.

13.2.2.2 Cornea and Conjunctiva

There are several methods for collecting cytological specimens from the conjunctiva and cornea which can be broadly divided into debridement and impression techniques. Debridement involves scraping, swabbing, or brushing and recommended techniques vary according to anatomical location (cornea versus conjunctiva) and disease. Debridement instruments include the following: sterile cotton‐tip applicator swab or calcium alginate swab, Kimura spatula, handle edge of a disposable scalpel blade, and cytobrush (Figure 13.2a,b). Impression cytology can be performed using a glass slide or, preferably, a cellulose filter. In general, collecting samples with a cotton‐tip applicator swab or directly with a glass microscope slide is not recommended if other collection methods are available. Collection with cotton‐tip applicators results in poor cytological specimens due to poor cellularity and occasional contaminating fibers. Scraping (Kimura spatula or handle edge of a disposable scalpel blade) and brushing (cytobrush) collection methods are most common in the horse, though methods involving impression cytology have been reported [10, 21–23].

Scraping of corneal ulcers and other corneal lesions should be taken at the margin of the lesion to prevent further damage to the cornea and to limit necrotic cellular debris. When scraping, gentle pressure should be applied whilst scraping several times in the same direction (not back and forth) until material is collected on the end of the collection device [24]. The material can then be gently applied to the center of a clean glass slide, spreading the material thin. Cytobrush samples are collected by turning the bristled tip 3–4 times over the margin of the lesion. The cytobrush is then gently rolled onto a microscope slide. It is important to note that fungal organisms are often found in the deeper corneal layers [25] so when keratomycosis is suspected, aggressive scraping may be warranted. Care should be taken in circumstances in which stromal loss is present, as inadvertent corneal perforation can occur. Additionally, if the ulcer has started to epithelialize, it may be necessary to gently remove the superficial epithelium before collection to allow for diagnostic samples [24].

A recent study compared three common debridement methods for collecting corneal samples in horses with ulcerative keratitis: cytobrush, Kimura platinum spatula, and the handle edge of a scalpel blade [23]. All three techniques provided clinically useful samples and results; however, using the handle edge of a scalpel blade provided the most intact cells and diagnostic samples [23]. This method is also practical as scalpel blades are easily available, sterile, and affordable [23].

Impression cytology most often refers to the use of a cellulose filter, typically applied to the cornea (or conjunctiva) to collect cytological specimens [26]. This method removes the most superficial layers of cells, which can then be stained and examined microscopically or used for molecular analysis (PCR) [26, 27]. The method is increasing in popularity in human ophthalmic centers, and in veterinary research and academic settings [10, 21, 22, 26]. In brief, cellulose filters are applied directly to the area of interest either alone or attached to a plastic tube to ease application [10, 22, 26]. During collection, the eyelids must be held open to avoid introducing tear fluid, which will inhibit cell collection [26]. The filter is left in place for 5–10 seconds and then removed. The cellulose filters can then be stained with various methods and mounted onto slides and coverslipped. Details of this collection method are described elsewhere [10, 21, 22, 26].

There are several benefits of the impression cytology technique. Several studies show that impression cytology of healthy and diseased cornea (and conjunctiva) provides sufficient numbers of well‐preserved cells for cytological evaluation, similarly to cytobrush samples [10, 22]. The method appears to be more comfortable for the patient and causes less irritation and epithelial cell damage when compared to the cytobrush method [22]. Therefore, impression cytology technique may be useful for fragile or deep corneal lesions or for sensitive horses. Impression cytology also maintains cellular/tissue architecture and can better demonstrate the proportion of goblet cells, and therefore may be preferred in research settings or for goblet cell quantification [10, 21].

There are also several limitations to impression cytology, especially in a clinical setting. To begin, the collection materials may not be readily available to practitioners because they are not kept in stock for other diagnostic and treatment protocols (versus scalpel blades). The collection process may also be difficult because the eyelids must be kept open [10]. Staining and processing of the membrane can be technically demanding and may not allow for long‐term storage or easy transport to a diagnostic laboratory. Additionally, impression cytology samples limit evaluation of fine cellular detail and small infectious agents due to the thicker preparations [21, 22]. Therefore, when neoplasia or bacterial etiologies are suspected, impression cytology may not be the ideal collection method [21].

Samples of the conjunctiva can be collected similarly to corneal lesions (Figure 13.2a,b). When diffuse conjunctival disease is present, the lower palpebral conjunctiva is the preferred collection site [24]. When conjunctival lesions are scraped with a scalpel handle edge or Kimura spatula, it is important to avoid the lid margin [24]. Conjunctival cytobrush samples are also collected from the lower palpebral conjunctiva and fornix in a manner similar to corneal sample collection (described above) and this method yields samples of high cellularity [28]. Larger conjunctival biopsy samples can be taken by grasping tissue adjacent to the lesion of interest and using scissors or a scalpel to excise the lesion (Figure 13.2c) [24]. Before placing the tissue sample into formalin, impression smears can be made for cytological evaluation. The tissue should be gently pressed onto a clean glass slide in several separate places make several imprints of the specimen. This sample can then be evaluated for rapid information as well as to complement histological examination of the formalin fixed tissue. Additionally, these tissue samples can be used to detect Onchocerca microfilaria (described below) [24].

13.2.2.3 Aqueous and Vitreous Humor

Ocular paracentesis refers to the sampling of aqueous and vitreous fluid and usually requires general anesthesia [24]. Ocular paracentesis is rarely performed in the field, as there are many associated risks, including but not limited to hemorrhage, lens perforation, endothelial damage, corneal edema, retinal detachment, and introduction of microorganisms. Where required, the procedure may be performed in standing horses with the use of deep sedation, periocular nerve blocks (including retrobulbar block), and topical corneal anesthesia [29–31].

The most common indication for ocular paracentesis is to determine an underlying etiology for uveitis. Sampling of the aqueous and/or vitreous is performed only after other less invasive diagnostic methods (e.g., complete blood count [CBC], blood culture, serology) have failed to yield a diagnosis and when inflammation is not resolving with supportive care. Vitreous paracentesis, specifically, may be considered in horses with marked vitreous opacification, exudative retinal separations, or suspected infectious endophthalmitis [24].

Aqueous humor paracentesis is performed by approaching the anterior chamber through the dorsotemporal limbus (Figure 13.2d). The conjunctiva and cornea should be cleaned with 5% aqueous povidone‐iodine solution and rinsed with sterile 0.9% saline [24]. The eyelids should be retracted using a speculum. Thumb forceps are used to grasp the bulbar conjunctiva near the point of entry. A 27 or 30 gauge needle attached to a 1 mL syringe (with the plunger seal already broken) is inserted into the bulbar conjunctiva approximately 3–4 mm away from the limbus [24]. The needle is gently threaded under the conjunctiva towards the limbus until it enters the anterior chamber parallel to the surface of the iris. Then 0.2–0.5 mL of aqueous humor is gently and slowly aspirated [24]. The needle is then slowly withdrawn and gentle pressure is applied over the exit wound [24, 29]. After collection, a freshly prepared smear should be made on a glass slide in a blood smear fashion. The remaining fluid should be stored in a nonadditive sterile tube for potential culture.

Vitreous paracentesis is performed through a pars plana approach [24]. The ocular surface should be prepared as described for aqueous humor collection. A 23–25 gauge needle attached to a 1 mL syringe (with the plunger seal already broken) is inserted in the dorsolateral quadrant of the eye approximately 10–12 mm behind the limbus [32, 33]. A small amount (typically no more than 0.2 mL) of vitreous humor is gently and slowly aspirated [24]. The needle is then slowly withdrawn and gentle pressure is applied over the exit wound [24]. Slide preparation and submission of remaining fluid are the same as for sampling of aqueous humor.

In addition to cytological evaluation, fluid collected from the aqueous or vitreous can be analyzed for bacterial and fungal culture and susceptibility, protein measurement, antibody titers (e.g., Borrelia and Leptospira spp.), and PCR (e.g., Borrelia and Leptospira spp., equine herpesvirus [EHV], antigen receptor rearrangement for lymphoid neoplasms) [30, 34–40]. Diagnostic laboratories should be contacted ahead of collection to ensure proper sample collection, handling, and shipping, especially for specialized diagnostic tests such as PCR and immunofluorescence assay. For cytological evaluation, submission of both freshly prepared slides and collected fluid in nonadditive tubes is preferred. This allows pathologists to evaluate the gross appearance of the fluid, perform cell counts or access cellularity, determine total protein, and prepare concentrated slides.

13.3.1.1 Normal Anatomy and Cytological Findings

The outermost portion of the eyelids and normal cytology are similar to haired skin in other anatomical locations (see Chapter 12). The innermost aspect of the eyelid is lined with palpebral conjunctiva (see normal conjunctival cytology features below). At the eyelid margin, Meibomian gland orifices can be seen with the aid of magnification. These glands are numerous and extend 5–7 mm into the eyelid and are surrounded by fibrous connective tissue, giving the eyelid structural support. Meibomian glands are sebaceous, producing the outermost lipid component of the tear film [28].

13.3.1.2 Inflammatory Lesions

Blepharitis can be caused by infectious and noninfectious etiologies. When an etiological agent is not identified cytologically but there are large numbers of inflammatory cells, bacterial and fungal cultures may be warranted. If the lesion does not resolve with supportive treatment for the inflammation, biopsy with histological examination may also be indicated. Differential diagnoses for inflammation in the eyelids and periocular regions may be similar to those described in Chapter 12, with a few conditions unique to the eyelids. Inflammatory lesions are most commonly categorized by the predominant inflammatory cell. Mixed cell inflammation with no apparent predominant cell type can be more difficult to characterize cytologically due to the variety of differential diagnoses.

13.3.1.2.1 Neutrophilic Inflammation

Neutrophil‐predominant inflammation in the eyelids and periocular lesions is most commonly caused by bacterial infections, whether primary or secondary to another etiology (e.g., contaminated foreign body or ulcerated neoplasm). Additional considerations for significant neutrophilic inflammation include, but are not limited to, fungal infections, trauma, sterile foreign body reactions, Meibomian cysts, and pemphigus foliaceus.

Bacterial infections in the eyelid usually occur secondary to trauma or foreign body reactions, ulcerated neoplasms, and occasionally following placement of subpalpebral lavage (SPL) systems (Figure 13.3a). Cytologically, bacterial infections typically exfoliate large numbers of nondegenerate to degenerate neutrophils (Figure 13.3b). Degenerate neutrophils are intact cells with swollen, pale nuclei. It is important not to mistake lyzed cells (which will have swollen, pale nuclei) with degenerate neutrophils. Degenerate neutrophils occur with certain bacterial infections but the lack of degenerate neutrophils does not rule out bacterial infections. Finding intracellular bacteria confirms a bacterial infection. However, cytology cannot always determine if a bacterial infection is a primary or secondary process. When there are large numbers of neutrophils present but no bacteria are found, bacterial culture is indicated as a more sensitive detection method.

Fungal infections affecting the eyelids can include opportunistic and systemic fungi [24]. Dermatophytosis in horses can be caused by Trichophyton or Microsporum spp., and lesions can be observed on the eyelids. Grossly, the lesions are often alopecic, dry to exudative, with marginal crusting. In some cases, small masses may develop. Fungal organisms can be identified with cytological examination of samples with impression smears, skin scrapings, or fine needle aspirate of masses. Cytological examination of stained specimens from dermatophytosis lesions reveals mixed cell inflammation, typically predominated by neutrophils with fewer macrophages, multinucleated giant cells, and possibly eosinophils (Figure 13.4). Arthrospores (arthroconidia) and fungal hyphae are occasionally seen (Figure 13.4a–c). Arthrospores are smaller than red blood cells (approximately 2 × 4 μm), round to slightly elongate, and deeply basophilic with a thin nonstaining border. Unstained wet mounts of hair shafts in mineral oil or saline can also be examined for arthrospores or fungal hyphae [24].

Meibomian cyst, or chalazion, develops when there is blockage of the Meibomian glands. These are typically noninfectious but secondary infections may occur. Grossly, the lesions are on the edge of the eyelid, small, yellow to white, and often painful. On cytological examination, there are numerous nondegenerate neutrophils and macrophages (Figure 13.5). Some Meibomian gland epithelial cells may be seen but can be difficult to differentiate from macrophages.

Demodex spp. are presumed to inhabit the hair follicles and Meibomian glands of the eyelids and rarely may cause mild blepharitis [41]. Microscopic evaluation of Meibomian gland secretions (by gentle expression) or alopecic areas of the eyelid (via skin scraping) can reveal Demodex mites, which are species specific. Inflammation may be mild but predominated by neutrophils. Additionally, these mites may be incidental findings in samples collected from the eyelid or cornea (Figure 13.6).

Pemphigus foliaceus is another consideration for pustules on the eyelid with marked neutrophilic inflammation and no apparent bacteria. Cytological examination may reveal large numbers of nondegenerate neutrophils and acantholytic cells. Acantholytic cells are squamous epithelial cells that have lost intercellular connections to other cells and appear cytologically as densely basophilic, individual, round to oval cells. Small numbers of acantholytic cells can be seen with a variety of etiologies (e.g., bacterial dermatitis, dermatophytosis). Increased numbers of these cells can be suggestive of an immune‐mediated process such as pemphigus foliaceus; however, the diagnosis of pemphigus foliaceus requires multiple biopsies of intact pustules and histological examination.

13.3.1.3 Neoplastic Lesions

Common eyelid tumors in the horse include squamous cell carcinoma (SCC) and sarcoid [5, 29, 44, 45]. Less common eyelid tumors include papilloma, melanoma, lymphosarcoma, hemangioma, hemangiosarcoma, perivascular wall tumors (e.g., hemangiopericytoma), fibroma, fibrosarcoma, mast cell tumor, adenoma, myxosarcoma, and basal cell carcinoma [5, 44, 46, 47]. When neoplastic lesions are suspected in the ocular and periocular tissues, thorough palpation of regional lymph nodes and cytological evaluation may be indicated.

13.3.1.3.1 Squamous Cell Carcinoma

Horses at higher risk for SCC are Paints, Appaloosas, Haflingers, quarter horses, Thoroughbreds, Belgian draft horses, and any horses with poorly pigmented eyelid margins [2, 5, 48–50]. SCCs can have varying gross appearances depending on the duration and progression of the neoplasm (Figure 13.8a). They can be small, white, elevated plaques or exophytic masses with distinct margins, or they can be larger, pink, ulcerated masses with irregular margins. Ocular SCCs are locally invasive and can have a high recurrence rate after treatment (~42%) [5, 51]. Metastasis is rare with reports ranging from 6% to 15% but can occur to the lung or regional lymph nodes [51].

Squamous cell carcinomas tend to be highly exfoliative with fine needle aspiration, providing modest to large numbers of cells for cytological evaluation (Figure 13.8b). Cells are most commonly arranged as single cells but variably sized, cohesive, sometimes disorganized clusters are also seen. Cell borders are variably distinct. The presence of elongated squamous cells, or tadpole cells, may increase suspicion for SCC but nuclear atypia is still required to diagnose malignancy [52].

The amount of cytoplasm present may vary depending on the degree of differentiation of the neoplasm [52]. More well‐differentiated tumours will have many angular cells with lower N:C ratios; however, the N:C ratio is still increased compared to normal squamous cells and rounded squamous epithelial cells are still present. Poorly differentiated tumors will have increased round cells with higher N:C ratios [52]. Marked anisocytosis (variation in cell size) is typically common. Colorless refractile perinuclear cytoplasmic granules are commonly observed and may be increased in SCCs compared to reactive (nonneoplastic) populations. Dyskeratosis (dark staining cytoplasmic rings encircling the nucleus) may be observed [52]. Emperipolesis, the presence of intact cells (typically neutrophils) within the cytoplasm, may be seen. Anisokaryosis (variation in nuclear size) is common and multinucleation may be observed. Rounded squamous epithelial cell nuclei will typically have coarse chromatin and multiple prominent nucleoli. Macronuclei (>5 μm) may also be observed. Mitotic figures may or may not be present (Figure 13.8c). Secondary neutrophilic inflammation is commonly observed with SCCs, especially with concurrent ulceration and secondary bacterial infections. Although the general guidelines are to have at least five criteria of malignancy before making a diagnosis of malignant neoplasia, the overall cytological and clinical findings need to be considered.

Most of the cytological findings described above (emperipolesis, tadpole cells, dyskeratosis) are not pathognomonic for SCC and can be seen with reactive populations as well. Squamous epithelial cells can display significant cytological atypia due to benign hyperplasia and dysplasia secondary to inflammation. Therefore, a cytological diagnosis of SCC should only be made by an experienced pathologist when there is significant cellular atypia and a compatible clinical presentation. Other lesions that may exfoliate squamous epithelial cells include benign hyperplastic lesions, keratinizing cysts and neoplasms, papillomas, and carcinomas with squamous differentiation. With the exception of carcinomas with squamous differentiation, the remainder of these lesions should contain squamous epithelial cells with minimal nuclear atypia [52]. Distinguishing SCC from carcinomas with squamous differentiation can sometimes be difficult even with biopsy and histological examination, depending on the sample type, quality of the specimen, and morphology of the neoplastic cells.

When a diagnosis of SCC is made, evaluation of regional lymph nodes may be warranted. Studies in other species have shown that bilateral lymph node removal and histopathology are required to rule out metastasis [53]. However, this aggressive work‐up is not always clinically and financially feasible. Cytological examination of regional lymph nodes may provide some valuable diagnostic information with minimally invasive methods, especially if regional lymph nodes are enlarged or firm.

13.3.1.3.3 Other Eyelid Neoplasms

Lymphoma of the eyelid most often presents clinically as a nonpainful, diffuse thickening of the eyelid and palpebral conjunctiva (Figure 13.10a) [54]. This form of lymphoma is often associated with high rates of mortality relative to horses with other forms of extraocular lymphoma (e.g., lymphoma of the cornea, third eyelid, or nodular forms of conjunctival lymphoma), which all seem to be more amenable to excision [54–58].

Cytologically, a normal lymphoid population should be heterogeneous with a predominance of small, well‐differentiated lymphocytes. Lymphocyte size can be determined by comparing lymphocytes to neighboring red blood cells (RBCs) and neutrophils. Small lymphocytes will typically have a nucleus about the size of 1–1.5 equine RBCs. The equine neutrophil is approximately 12 μm in diameter. Immature lymphocytes are typically 12–15 μm in diameter. Unlike other neoplasms, the cytological diagnosis of lymphoma is often based on the size of lymphocytes and not on standard criteria of malignancy. A predominance of immature lymphocytes (>50%) in multiple areas of (preferably) multiple slides and sources is consistent with lymphoma. In horses, some lymphomas may be more heterogeneous, limiting the cytological diagnosis. However, when intermediate to large cells predominate, a confident diagnosis can be made on cytological specimens (Figure 13.10b). Additional information on lymphoma in horses can be found in Chapter 15.

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