Histology of the Normal Eyelid

At the level of the tarsus, from anterior to posterior, the eyelid is composed of skin, orbicularis oculi muscle, tarsal plate, and palpebral conjunctiva (Fig. 6.3). Surgical dissection through an incision along the gray line of the eyelid margin can be made between the orbicularis and the tarsus, functionally dividing the eyelid into anterior and posterior lamellae. The skin of the eyelid is thinner compared with other body sites. It consists of an epidermis of keratinizing stratified squamous epithelium, which also contains melanocytes and Langerhans cells (antigen-presenting dendritic cells). The epidermis has four layers with the stratum spinosum and basale collectively called the Malpighian layer. The epithelial cells or keratinocytes are normally connected by intercellular bridges. Underlying the epidermis is the dermis made of loose collagenous connective tissue, which contains dermal appendages such as hair follicles, pilosebaceous units (cilia/eyelashes with associated glands of Zeis), eccrine sweat glands, and apocrine glands of Moll (Fig. 6.4). The orbicularis oculi muscle is striated skeletal muscle that functions in eyelid closure. Muscles that elevate the upper eyelid are the levator palpebrae superioris, of which only the aponeurotic portion is present in the eyelid, and the Müller smooth muscle. The tarsal plate is composed of dense fibrous connective tissue and contains the sebaceous Meibomian glands. Present near the superior border of the tarsal plate are the accessory lacrimal glands of Wolfring. The accessory lacrimal glands of Krause are found in the conjunctival fornices. The palpebral conjunctiva is firmly adherent to the posterior surface of the tarsus. The conjunctiva is composed of nonkeratinized stratified squamous epithelium with goblet cells that produce the mucin layer of the tear film, overlying a loose collagenous stroma (substantia propria) (Fig. 6.5). The eyelids and conjunctiva are supplied with lymphatics, which drain into the preauricular, parotid, and infra-auricular lymph node basins. Conjunctival sites that contain an abundance of goblet cells are the fornices and the caruncle. The caruncle differs from the rest of the conjunctiva because it contains hair follicles, sebaceous glands, and accessory lacrimal glands (Popoff glands).

There is a rich supply of glandular elements within the eyelid that secrete their products in various ways (Table 6.2). Holocrine glands such as sebaceous glands that secrete oily waxy sebum have no lumens and shed the entirety of their cells along with their secretions. Apocrine glands secrete sweat by decapitation of the apical portion of their cells. Eccrine sweat glands and lacrimal glands secrete without losing any part of their cells. Glandular elements and skin are the precursors of carcinoma of the eyelid.

Histology of the Normal Lacrimal Gland and Lacrimal Drainage System

The lacrimal gland is located within the lacrimal gland fossa in the superotemporal quadrant of the orbit. The gland is anatomically divided into orbital and palpebral lobes by the aponeurosis of the levator muscle. Histologically, lacrimal glands are composed of acini and ducts. The secretory acini consist of cuboidal epithelium surrounding a central lumen. The epithelial cells bear eosinophilic periodic acid Schiff (PAS)–positive vesicles called zymogen granules that contain lysozyme, lactoferrin, and immunoglobulin A (IgA) secreted in tears (Fig. 6.6). The ducts, which lie within the fibrovascular stroma, are lined by cuboidal epithelium and a second layer of flat myoepithelial cells. Small aggregates of lymphocytes and plasma cells are normally scattered in the interstitium. The lacrimal gland is considered a minor salivary gland, and the classification of epithelial lacrimal gland tumors is based on the scheme used in other salivary glands (e.g., the parotid).

The lacrimal drainage system is composed of the upper and lower punctum, lacrimal canaliculi, lacrimal sac, and nasolacrimal duct. Histologically, the tubular canaliculus is lined by nonkeratinizing stratified squamous epithelium with 10 to 12 cell layers. There is a transition to pseudostratified columnar epithelium with goblet cells and cilia in the lacrimal sac. The nasolacrimal duct has histologic features similar to those of the sac, with seromucinous glands present in the subepithelial connective tissue.⁴

Histology of the Normal Orbit

The orbit is a pyramid-like or pear-shaped structure limited by a bony wall that protects the eyeball and its surrounding soft tissue. Tissues within the orbit include the globe, optic nerve and meningeal sheaths, extraocular striated muscles, tendons, adipose, fascia, blood vessels, peripheral nerves, and cartilaginous trochlea (Fig. 6.7). Connective tissue septa divide the orbital fat into lobules. Normal mature bone, called lamellar bone, has organized sheets of collagen visible as a pink matrix. Bone fragments may also appear dark purple on hematoxylin and eosin (H&E) staining as a result of the presence of calcium phosphate. Benign and malignant neoplasms of bone and soft tissue may arise from these various structures in the orbit, and their general features are described in Table 6.3.

Histology of the Normal Globe

The histology of the normal globe will not be detailed here. The reader may refer to other sources for more in-depth discussion.^2,5–7

In incisional biopsy, only a portion of the tumor is sampled; in excisional biopsy, the entire lesion is removed. Care­ful handling of tissue by minimizing electrocautery or thermal cautery and avoidance of crushing the specimen are critical to maintain good tissue architecture and cytologic detail. Appropriate orientation of the specimen, proper documentation (e.g., descriptions and drawings of the excision site), and labeling of margins with suture tags or markers are crucial during tissue submission. For routine histologic analysis, excised tissue should immediately be placed in a fixative to avoid autolysis. The most commonly used fixative is 10% neutral buffered formalin. Formalin is a solution of 40% formaldehyde in water buffered with phosphates that crosslinks protein, lipid, and carbohydrates preventing enzymatic degradation of tissue. Nucleic acids, however, can be denatured by formalin. It is important that the tissue be completely immersed in an adequate amount of fixative (approximately 10–20 times the volume of fixative). Once the tissue is fixed, gross examination is performed by a pathologist or a pathology assistant. Larger pieces may be dissected to select representative portions of tissue, or smaller pieces of whole tissue may be submitted. For orientation and identification of surgical margins, it is sometimes necessary to ink the surgical specimen margins with different colors during the gross examination so that the margins can be discerned even after the specimen is cut. For tumors containing bone or specimens with significant calcification such as many phthisical eyes, decalcification before processing is accomplished by using a solution of acid combined with ethylene-diamine-tetraacetic acid.

Specimens are then processed through increasing concentrations of alcohol, followed by xylene, to remove most of the water from the tissue and replace it with paraffin. Organic solvents used in the process dissolve lipid and some synthetic materials such as polymethylmethacrylate (e.g., in PMMA intraocular lenses), polypropylene, and silicone. Silk, nylon, and other sutures do not dissolve during processing. Once dehydrated, the tissue is embedded in paraffin for mechanical stabilization, which allows cutting of sections. The sections are cut using a microtome, usually at a thickness of 4 to 6 microns. The cut section is then placed on a glass slide, sometimes with the aid of a tissue adhesive or heated in an oven to secure the thin paraffin section to the slide. The cut section is colorless except for areas of indigenous pigmentation. Various tissue dyes are used to stain the tissue for identification, the most widely used being H&E. Other tissue stains used are listed in Table 6.4. These special stains have a basic chemical affinity for certain components in the tissue itself, organisms or acellular material that may highlight special features that will aid in the diagnosis, such as Congo red for amyloid, Masson’s trichrome for collagen, or Gomori methenamine silver for fungi (Fig. 6.8). After staining, the tissue section is then ready for microscopic examination.

The entire processing of routine specimens typically takes 10 to 12 hours and is usually performed overnight. Therefore, it is unreasonable for a surgeon to expect an interpretation of a specimen sent for permanent sections to be available on the same day as the biopsy. Techniques used for rapid processing of special specimens are reserved for cases that require emergent handling; however, the quality of histologic preparation after rapid processing is usually inferior to that of standard processed tissue. The highest quality tissue sections are those produced by histotechnologists who are experienced and knowledgeable in handling eye specimens.

Gross Examination of Evisceration, Enucleation, and Exenteration Specimens

Evisceration procedures are usually used as treatment for blind painful eyes or endophthalmitis. The specimens submitted would be the cornea, which should be bisected, and the remaining intraocular contents, including the uvea, retina, and the vitreous. Enucleation surgeries are performed as treatment for blind and painful eyes, panophthalmitis, or intraocular tumors. Guidelines for examination of eyes with retinoblastoma⁸ and uveal melanoma⁹ have been recommended. Whole eye specimens from enucleations require careful examination under a stereomicroscope, recording of gross findings, and macrophotography. An excellent system for gross examination of the eye has been detailed by Roth and Foos.¹⁰ Globes should be fixed promptly in 10% neutral buffered formalin using a volume 20 times that of the eye (approximately 300 mL). The adult eye measures approximately 24 mm in diameter, and formalin diffuses at a rate of about 1 mm/hr; therefore, an eye should be suspended in formalin for at least 24 hours before processing to ensure adequate fixation and tissue stabilization. The dimensions of the globe (anteroposterior × horizontal × vertical) and the length of the optic nerve should be recorded. Ruptures of the sclera or cornea or extrabulbar extensions of intraocular tumors should be noted. The right eye can be distinguished from the left eye by identifying posterior landmarks: the optic nerve is situated nasally, and the inferior oblique muscle inserts as a fleshy attachment temporally over the area of the macula. The superior oblique muscle marks the superior pole; it is tendinous and inserts slightly temporal to the 12 o’clock meridian (Fig. 6.9). Once the laterality of the eye is determined, it may be further oriented according to the anterior insertions of the extraocular muscles. The medial, inferior, lateral, and superior rectus muscles insert progressively further away from the limbus, forming the Spiral of Tillaux. The globe should be transilluminated with bright light before sectioning. This helps identify intraocular lesions such as a tumor that blocks the transilluminated light and casts a shadow (Fig. 6.10). The shadow can be outlined with a marking pencil on the sclera and can be used to guide the gross dissection of the globe. Globes are cut with a blade from back to front with the plane of section beginning adjacent to the optic nerve (Fig. 6.11). The globe is by standard cut into three sections: a central section containing most of the cornea, iris and pupil anteriorly and optic nerve posteriorly, called the pupil-optic nerve section (P-O section), and two other dome-shaped caps of tissue, called the calottes (Fig. 6.12). In routine cases, eyes are cut open in the horizontal plane to include the macula in the same section as the pupil and the optic nerve. Globes may also be opened vertically or obliquely, depending on the location of areas of interest. If the eye contains a wound, the wound should be perpendicular to and included in the P-O section. The globe can also be cut open coronally, separating the anterior and posterior compartments. Clinical funduscopic features and ciliary body tumors can be visualized directly with this technique.