The eyelids are lined by the skin externally and the conjunctiva on the inner surface. Eyelid skin, its cytological sampling and microscopic examination are similar to those of other sites. Scraping is the most commonly used technique for cytological diagnosis and may be performed by skilful cytopathologists, whereas lesions of the canthus or the rim should be performed by ophthalmologists. Scrapings may be performed without anaesthesia using a small platinum spatula (Swedish Dissector) or the blunt side of a scalpel blade. When suspected lesions are not ulcerated, it may be necessary to lift or remove the external epidermal layer covering the lesion. This can be achieved by using the sharp edge of a scalpel blade to gently cut the superficial layer of the epidermis and allow the scraping of the deeper one. FNA has also been used to diagnose eyelid masses.¹⁰ Smears are prepared as previously described and may be fixed in alcohol and stained by the Papanicolaou method or air-dried and stained with May-Grünwald Giemsa (MGG) or Diff-Quik. As in other regions, MGG or Diff-Quik staining are generally preferable for the demonstration of bacteria and to diagnose lymphoid and mesenchymal tumours, while the Papanicolaou (PAP) method is preferable for epithelial or melanocytic tumours.

Squamous papillomas are the most frequent benign tumours of the eyelid. They may be sessile or pedunculated and are composed of finger-like projections of acanthotic and parakeratotic epithelium arising from a fibro-vascular stalk. Cells may show atypical nuclei with koilocytotic modifications, as observed in HPV infections.

The cytological features of adnexal tumours are described in Chapter 28. Eyelid hidroadenomas¹¹ and eccrine poromas¹² diagnosed by FNA have been reported. Strangely enough, the cytological features of sebaceous adenoma, which represents the most frequent adnexal tumour of this area, have not been described.

Basal cell carcinoma (BCC) is the most frequent malignant tumour of the eyelid. It arises as a single lesion after prolonged exposure to sunlight or as multiple lesions in patients with xeroderma pigmentosum. The clinical presentation of BCC may be nodular and/or ulcerative or sclerosing (morphea type). BCCs rarely metastasise, but grow slowly, eroding surrounding tissues, proximal structures and even the globe. Timely diagnosis and treatment are therefore mandatory because a large BCC may present surgical problems as far as excision and plastic reconstruction are concerned; conversely, small lesions may be conveniently treated by cryotherapy, photodynamic therapy or pharmacologically (imiquimod, fluorouracil). Adequate scraping has to reach the dermis; prompt or disproportionate bleeding may suggest the presence of a tumour. Smears (Fig. 32.2) generally show the following:

Fig. 32.2 Basal cell carcinoma of the eyelid. Scraping cytology sample shows dense groups of small basal cells; note the nuclear cohesion, the hyperchromasia and the partial peripheral palisading arrangement of the nuclei (Diff-Quik).

Squamous cell carcinoma is less frequent than BCC. Scraping samples are better observed with Papanicolaou-stained smears.

Malignant melanomas are less frequent than squamous cell carcinomas and their cytological diagnosis is requested less frequently. Cytological features and diagnostic criteria of melanoma are reported in Chapter 28. Pigmentation of the eyelid margins, occurring in association with conjunctival melanoma, is an ominous clinical sign.

Sebaceous carcinomas are found almost exclusively in eyelid skin and arise from the meibomian or other local sebaceous glands, more frequently on the upper eyelid. They appear clinically as firm subcutaneous nodules resembling a chalazion or other lesions, thus delaying the diagnosis and resulting in relatively high morbidity and mortality. Sometimes the tumour growth results in a pagetoid or carcinoma in situ-like involvement of the conjunctiva causing persisting unilateral conjunctivitis before the correct diagnosis is made. The tumour has a lobular pattern and is formed by cells with variable sebaceous differentiation depending on their grade of differentiation. Sebaceous carcinomas may involve adjacent structures or metastasise to cervical or preauricular lymph nodes. FNA shows hyperchromatic nuclei, prominent nucleoli and abundant cytoplasm which contains small fat vacuoles.¹³ FNA of lymph node metastases from sebaceous carcinomas has also been described.¹⁴

Conjunctival epithelium is a stratified non-keratinising epithelium formed by cuboid-columnar cells and containing mucus-producing goblet cells (Fig. 32.3). These cells, together with lachrymal and meibomian glands, maintain hydration and lubrication of the cornea and conjunctiva by producing the tear film. Conjunctival epithelium starts from the eyelid rim, lines the inner eyelid layer, turns at the bottom and continues covering the sclera up to the limbus where the cornea merges with the sclera. The limbus is an important clinical area as it is the point of insertion of the needle for anterior chamber paracentesis. Cytological samples may be obtained by scraping, brushing or imprint cytology.

Fig. 32.3 Normal conjunctival cells in conjunctivitis. Scrape cytology sample showing loosely attached conjunctival cells with wide and well defined cytoplasm. Crashed granulocytes are interspersed among the cells (Diff-Quik).

Conjunctival scraping requires local anaesthesia and should be performed by trained ophthalmologists. Conjunctival brush may also be used under local anaesthesia using a small cytobrush (Cytobrush- S, Medscand, Malmo, Sweden).¹⁵ Imprint cytology of the conjunctiva can be performed by placing cellulose acetate strips on the surface of the conjunctiva or the cornea and pressing gently.¹⁶ The strip is then removed, fixed in absolute ethanol or glacial acetic acid and stained using PAS/Papanicolaou stain.¹⁶ More recently, Biopore membranes have been used^17–19 and specific training in interpreting the corresponding samples has been suggested.²⁰

Allergic and vernal conjunctivitis are quite common and characterised by a prevalence of eosinophils in the inflammatory infiltrate. Bacterial and viral infections are generally diagnosed on the basis of the clinical presentation or sometimes by microbiology. Nonetheless, cytological features may be helpful for a presumptive diagnosis, mainly in clinically unexpected cases. Infections by Verruca vulgaris, measles and herpes may be suggested by specific cytological features as described in Chapter 21. Exhaustive descriptions of the cytological features of the most frequent viral infections are available in the literature.^7–921 Trachoma is a ‘historical’ bacterial conjunctival infection caused by Chlamydia trachomatis; it has almost disappeared but may still be a cause of blindness in underdeveloped countries. The cytological features are almost the same as those described in the female genital tract and are characterised by cytoplasmic basophilic inclusions with halos (Fig. 32.4). Cytological features of Candida, Aspergillus, mucormycosis and phycomycetes conjunctivitis have also been described;^7–9 diagnosis and treatment must be timely and effective because of the risk of corneal damage.

Fig. 32.4 Conjunctival scraping showing Chlamydia trachomatis inclusion bodies forming a dark granular cluster in the cytoplasm, partly surrounding the nucleus (arrow) (MGG).

Benign epithelial tumours are principally represented by papillomas, which are composed of a fibrovascular stalk covered by acanthotic squamous epithelium. Cytological diagnosis is rarely requested; papillomas often recur after surgical excision.

Malignant conjunctival tumours are principally represented by squamous cell carcinoma and its precursors; the term ocular surface squamous neoplasia (OSSN) is generally used to describe a spectrum of lesions, ranging from mild dysplasia to carcinoma in situ, which may arise on the conjunctiva and cornea. Carcinogenetic factors are mainly represented by human papillomavirus types 16 and 18, which have been detected in many of these lesions by PCR. Exposure to ultraviolet light or mustard gas, as observed in exposed soldiers,²² may induce squamous cell carcinoma or precancerous lesions; conjunctival and corneal carcinomas are also more frequent among patients with acquired immune deficiency syndrome (AIDS).^7,8 Squamous cell carcinomas are often observed on the bulbar conjunctiva, mostly at the limbus, and the clinical presentation is not always clear. Pre-invasive lesions show increased vascularity and thickening of the epithelium, which may be initially misdiagnosed as inflammatory or degenerative processes. Sometimes they may be present without any clinical evidence²⁰ or appear as gelatinous and ill-defined lesions. Smears show immature cells with increased nucleus/cytoplasm ratio; the nuclei are enlarged and hyperchromatic with irregular contours, coarse chromatin and prominent nucleoli. Many high-grade lesions and some invasive carcinomas may show superficial keratinisation, which makes the cytological diagnosis more difficult (Fig. 32.5), mainly on Biopore membrane samples.^17,19 Therefore, the presence of hyperkeratotic cells and a few dysplastic cells may suggest intramucosal high-grade lesion or squamous cell carcinoma. The differential diagnosis must include conjunctival squamous metaplasia (abundant cytoplasm, reduced nuclear/cytoplasmic ratio, pyknotic nuclei) and parakeratosis (dyskeratotic cells and keratohyaline granules). A relatively high correlation between cytology and the corresponding histological diagnoses has been found, while the greatest probability of false negatives is in keratinising squamous cell carcinoma.^16,20

Fig. 32.5 Ocular surface squamous neoplasia. Conjunctival scraping cytology showing isolated malignant nuclei interspersed among superficial keratinising cells (PAP).

Lymphomas may arise from conjunctiva-associated lymphoid tissue (CALT); cytopathological features are similar to those of other mucosa-associated lymphomas (MALT) (see Chs 5, 14). Chlamydia psittaci has recently been associated with ocular adnexal lymphomas.

Melanocytic naevi of the conjunctiva are classified in almost the same way as those of the skin, i.e. as junctional, sub-epithelial and compound. Microscopically they are formed by pigmented polygonal cells and are more cellular than their epidermal counterparts. Conjunctival naevi may increase in size, as may occur in puberty, or change shape, as in the case of ‘irritated’ naevi or in malignant transformation. Primary acquired melanosis (PAM) may be considered as the conjunctival counterpart of cutaneous Hutchinson lentigo. The histological features of PAM are quite variable even within the same lesion, and range from small monomorphous melanocytes, which line the basal layer, to atypical nucleolated epithelioid cells. Pagetoid infiltration of the epithelium is indicative of malignant transformation in most cases. Malignant melanomas may arise de novo or by transformation of pre-existing naevi (Fig. 32.6) or acquired melanosis.

Fig. 32.6 Superficial melanoma of the scleral conjunctiva showing dyschromia and irregular borders.

(Courtesy of Dr L Zeppa, Benevento, IT.)

Cytological findings: conjunctival melanoma²³

• Dispersed tumour cells with eccentric nuclei

• Coarsely granular chromatin and prominent nucleoli

• Occasional or prominent cytoplasmic melanic granules

• Occasional spindle-shaped cells.

Imprint cytology and Biopore membrane have given excellent diagnostic results, predicting the histological diagnosis by detection of superficial atypical melanocytes and their proportion relative to the conjunctival cells (Fig. 32.7).²⁴

Fig. 32.7 Conjunctival melanoma. Biopore membrane sample showing numerous hyperpigmented melanoma cells and few conjunctival cells in the upper left corner. Atypical nuclei are recognisable only in the lower right part of the group because of the abundance of melanin (MGG).

(Courtesy of Dr S Keijser, Leiden, NL.)