Laboratory Techniques and Special Stains
SPECIMEN HANDLING IN THE OPHTHALMIC PATHOLOGY LABORATORY
This chapter discusses the basic principles of practical ocular histopathology and summarizes techniques used in the handling, gross dissection, and submission of enucleated eyes and other ocular specimens for routine histopathologic examination. Special histochemical and immunohistochemical stains also are discussed.
Fixation
Enucleated eyes are routinely fixed for at least 24 hours by total immersion in a relatively large volume (50 to 100 mL) of 10% neutral buffered formalin. The large volume of fixative is necessary because the solution must penetrate through the sclera. Bouin solution (bright yellow in color) should not be used because it hardens the sclera, making eyes extremely difficult to section. Alcohol-based fixatives are also unsatisfactory because they precipitate protein in the vitreous and interfere with gross examination. Enucleated eyeballs should be briefly rinsed in running tap water to remove excess fixative prior to handling.
Specimen Orientation
Determination of laterality (right or left eye) is a prerequisite for proper specimen orientation. The laterality of an eye is determined by identifying key anatomic landmarks (Fig. 16-1). These include the long posterior ciliary vessels, which appear as blue lines on the posterior sclera on either side of the optic nerve in the horizontal plane; the cornea, whose horizontal diameter is usually a millimeter longer than its vertical diameter; and the insertions of the inferior and superior oblique muscles. The inferior oblique muscle lacks a tendon; its muscular fibers insert directly into the sclera in the inferotemporal quadrant. The nasal end of the muscular inferior oblique insertion lies close to the fovea. The shiny tendinous insertion of the superior oblique muscle is located superiorly and temporally and is marked by parallel white bands. It is an excellent landmark for the superior pole of the eye. After the horizontal meridian and the superior pole have been located, the nasal and temporal sides of the eye are readily identified. It is important to remember that both oblique muscles insert on the temporal side of the eye, and their fibers run nasally. The temporal arc of the posterior sclera is longer than the nasal arc because the optic nerve enters the eye 15 degrees nasal to the posterior pole. The angulation of the optic nerve stump cannot be relied upon to determine laterality, however, because it often bends temporally after fixation.
 FIG. 16-1. Orientation of globe, posterior landmarks. The temporal insertions of the oblique muscles are helpful landmarks. The inferior oblique lacks a tendon, and its muscular fibers insert inferonasally near the fovea. The tendinous insertion of the superior oblique muscle is located superotemporally. The long posterior ciliary vessels are often evident as blue lines on either side of the optic nerve in the horizontal plane. |
Measurement
After orientation, the eye is measured using caliper or a millimeter ruler. Standard measurements include the anteroposterior, horizontal, and vertical diameters of the eye; the length of the segment of optic nerve attached; the horizontal and vertical diameters of the cornea; and the diameter of the pupil (Fig. 16-2A). Measurements should be
made and recorded in a standardized sequence, that is, AP, horizontal, and vertical. Important pathologic features such as wounds, lacerations, and tumors should also be measured and their location noted. Most normal human eyes are slightly <1 inch in diameter (24 to 25 mm), and the normal cornea is ˜1 cm in diameter (11×10 mm).
made and recorded in a standardized sequence, that is, AP, horizontal, and vertical. Important pathologic features such as wounds, lacerations, and tumors should also be measured and their location noted. Most normal human eyes are slightly <1 inch in diameter (24 to 25 mm), and the normal cornea is ˜1 cm in diameter (11×10 mm).
 FIG. 16-2. A. Ocular measurements. Standard measurements include the A-P, horizontal and vertical diameters of the eye, the length of the optic nerve segment, the horizontal and vertical diameters of the cornea, and the size of the pupil. B. Transillumination of globe. Transillumination is used to localize ciliochoroidal melanoma in enucleated eye. Arrow points to round shadow cast by choroidal part of pigmented tumor. The pupil glows brightly. |
It is easier to section an eye after the attached segment of optic nerve is removed. The nerve should sectioned a millimeter or two posterior to the sclera to avoid “buttonholing” a deeply cupped nerve head in a severely glaucomatous eye. A transverse section of the optic nerve should always be submitted if the nerve has not been cut flush with the globe. The surgical margin should be marked with an indelible colored pencil or India ink before the nerve is removed. The optic nerve segment should always be removed before the globe is opened to preclude potential contamination of the surgical margin with intraocular tumor. This is especially important if the eye contains retinoblastoma, which tends to invade the optic nerve. Surgeons occasionally submit the optic nerve in a separate container. Blood staining and crushing serve to identify the true surgical margin of such specimens.
INSPECTION AND DESCRIPTION
The general character and consistency of the eye are noted. Descriptive terms that commonly are used include normal, soft, collapsed, ruptured, lacerated, hard, rubbery, phthisical, etc. Before it is opened, the external surface of the eye should be carefully and systematically examined with a dissecting microscope, starting anteriorly. The cornea is usually slightly hazy in fixed or postmortem specimens. The presence of corneal opacities, scars, vascularization, ulcers, band keratopathy, wounds, incisions, and sutures should be described and measured when appropriate. The shape and size of the pupil, the color of the iris, and the presence and location of iridectomies, surgical iris colobomas, and ectropion of the iris pigment epithelium should be recorded. If the cornea is hazy, iris defects are readily disclosed by transillumination.
The rest of the globe should be carefully inspected with the dissecting microscope looking for signs of surgical or nonsurgical trauma or extraocular tumor extension. The presence, type, location, and size of wounds, scars, vitrectomy ports, sutures, and prosthetic devices such as retinal implants, explants, and encircling bands and tube shunts should be precisely noted. The use of “clock hours,” for example, “between 1 and 3 o’clock” is helpful in the description of wounds and other important lesions. Drawing a diagram of the findings on the pathology protocol facilitates description and permits reconstruction of the gross description if the dictation is lost.
TRANSILLUMINATION
Eyes are transilluminated before they are opened to disclose occult pathology that may not be evident on external examination. Transillumination is performed by holding the eye directly on the surface of a bright light in a darkened chamber. Transillumination is especially helpful during the orientation of eyes that contain intraocular tumors, particularly uveal malignant melanomas (Fig. 16-2B). Pigmented melanomas usually cast a dark shadow on the sclera, which should be localized and measured. When clinical data are absent, transillumination may disclose signs of previous surgery, for example, an iridectomy, that may be an indication for vertical sectioning. Eyes that are filled with blood or contain dense gelatinous exudate often transmit light poorly, as do specimens from heavily pigmented individuals. Staphylomas and cyclodestructive procedures cause increased light transmission.
DISSECTION
Microscopic sections are cut from a paraffin block that contains a short cylindrical segment of the eyeball called the pupil-optic nerve or P-O segment. The P-O segment includes most of the cornea, iris, and pupil anteriorly and the optic nerve posteriorly (Fig. 16-3B).
When tissue is submitted for histopathological examination, care must be taken to ensure that the microscopic sections will include important lesions such as tumors, lacerations, or incisions that must be described and diagnosed. The presence and location of the ocular pathology determines how the eye is opened. As routine practice, eyes are opened horizontally if they do not contain wounds or lesions located in other quadrants. In addition to the pupil and optic nerve, horizontal P-O sections include the macula, as well as the pupil and optic nerve. In the past, many eyes that had prior surgery were opened vertically to include wounds that typically were located at the superior limbus. This category still includes eyes that have had filtering procedures for glaucoma. Surgical techniques have changed, however, and many cataract wounds now are located horizontally. These will be included in routine horizontal P-O sections. If an eye with a small superior incision is opened horizontally, the microscopic sections will not include the wound and the pathologist will be unable to document its presence. Eyes that have had tubes shunts for glaucoma should be opened perpendicular to the insertion of the tube into the anterior chamber. The epibulbar reservoir of the shunt must be carefully dissected and removed from the eye prior to sectioning the eye.
Traumatic lacerations and intraocular tumors can occur anywhere. Such eyes are opened along the meridian that includes the main part of the lesion. The localization of intraocular tumors by transillumination greatly aids sectioning. If the eye does not permit transillumination, the location of tumor sometimes can be determined by careful palpation
SECTIONING TECHNIQUE
I currently section eyes using one half of a standard doubleedged razor blade that has been snapped in half within its protective wrapper (Fig. 16-3A). The two ends of the blade are held between the apposed surfaces of the thumb and forefinger, and a gentle sawing and pushing motion is used. During the initial cut, the eye is positioned cornea side down on the cutting block and steadied with the nondominant hand. Guidelines drawn on the eye with colored pencil facilitate sectioning and assure that the eye maintains proper orientation. The initial cut is begun just external to the dural sheath of the optic nerve and should enter the periphery of the anterior chamber anteriorly. The two dome-shaped caps of tissue that are removed are called calottes (French for visorless cap). Ideally, the first calotte should include about one fourth to one fifth of the peripheral anterior chamber. The central P-O segment should be about 8 to 10 mm thick. During removal of the second calotte, the globe is placed cutsurface-down on the cutting board. Eye protection (glasses, goggles, or face shield) should be worn during the initial cut to protect against occasional squirts or splashes of fixative and intraocular contents that can occur if the intraocular pressure is high or the eye is grasped too firmly.
Any material that flows from the eye during sectioning (blood, crystals, pigment-tinged fluid, or silicone oil) should be described. Gritty, hard, or even impenetrable intraocular material may be encountered. When this happens, possibilities include intraocular bone, a calcified cataractous lens, or some older types of prosthetic intraocular lenses. Intraocular bone is an extremely common finding in phthisical eyes. Eyes that contain bone must be decalcified before they are submitted. In some cases, so much bone is present that calcification is a prerequisite for sectioning.
 FIG. 16-3. A. Sectioning technique for globe. Globe is sectioned with a razor blade. It is positioned corneal side down during initial cut. B. P-O segment and calottes. The central P-O segment, which is submitted for sectioning includes the pupil and optic nerve. |
Every specimen should be examined carefully and systematically with the dissecting microscope and macrophotography performed if indicated. Disturbing light reflexes can be minimized by immersing the specimen in 60% “grossing” alcohol during gross examination and
macrophotography. Placing the flat-bottomed cylindrical dish on a plate of dark blue Plexiglas provides an aesthetic background for the gross photographs, which is enhanced if bottom of the dish is coupled to the plastic with a thin layer of alcohol. The calottes usually are retained in alcohol as “wet tissue” and may be retrieved if additional sections or studies are necessary. Both calottes are “bread-loafed” and submitted entirely, if the eye contains retinoblastoma to disclose possible choroidal invasion by tumor.
macrophotography. Placing the flat-bottomed cylindrical dish on a plate of dark blue Plexiglas provides an aesthetic background for the gross photographs, which is enhanced if bottom of the dish is coupled to the plastic with a thin layer of alcohol. The calottes usually are retained in alcohol as “wet tissue” and may be retrieved if additional sections or studies are necessary. Both calottes are “bread-loafed” and submitted entirely, if the eye contains retinoblastoma to disclose possible choroidal invasion by tumor.
ROUTINE HISTOPATHO LOGY: DEHYDRATION, EMBEDDING, AND MICROTOMY
The P-O segment is embedded in a block of paraffin wax to facilitate the cutting of microscopic sections. Because water and wax are not miscible, the aqueous fixative and the water in the tissue must be removed before the specimen can be embedded. This process is called dehydration. Tissue is dehydrated by passing it through increasing concentrations of ethanol (“graded alcohols”) until absolute ethanol is reached. The dehydrated tissue is then transferred to xylene, which is miscible with both absolute alcohol and paraffin and then infiltrated with molten paraffin under vacuum. After infiltration, the specimen is placed in a plastic mold that is filled with molten paraffin. The paraffin is cooled and hardens into a block, which fits into the chuck of a rotary microtome. The support provided by the surrounding paraffin matrix allows the tissue to be sectioned thinly (5 to 8 µm). The thin slices of paraffin and tissue are then floated onto the surface of a heated water bath, which causes the paraffin to expand. The tissue sections are then mounted on glass slides, which are heated in an oven to promote tissue adherence.
STAINING
Most human tissues are relatively transparent unless they contain endogenous pigment (Fig. 16-4A). To facilitate examination,
microscopic slides are stained with dyes such as hematoxylin and eosin (H&E) that color certain tissue components. Because aqueous solutions of these stains usually are used, the tissue sections must be deparaffinized and rehydrated before they can be stained. This is done by immersing the slides in successive baths of xylene, absolute alcohol, and then decreasing concentrations of alcohol and water. After staining, the tissue subsequently is dehydrated and coverslipped.
microscopic slides are stained with dyes such as hematoxylin and eosin (H&E) that color certain tissue components. Because aqueous solutions of these stains usually are used, the tissue sections must be deparaffinized and rehydrated before they can be stained. This is done by immersing the slides in successive baths of xylene, absolute alcohol, and then decreasing concentrations of alcohol and water. After staining, the tissue subsequently is dehydrated and coverslipped.
 FIG. 16-4. Staining, H&E stain. A. Unstained section. Presence of endogenous pigment allows identification of RPE and choroidal melanocytes. B. Hematoxylin-stained section (same area as A). Basic dye stains DNA in cellular nuclei, highlighting retinal nuclear layers and inflammatory cells in choroid. C. Eosin-stained section (same area as A). Acidic dye stains cytoplasm, erythrocytes in choroidal vessels and proteinaceous subretinal fluid. D. H&E-stained section (same area as A). A shallow retinal detachment and chronic nongranulomatous choroiditis are present. Photoreceptor atrophy denotes chronic process. (A. Unstained section ×100, B. Hematoxylin ×100, C. Eosin ×100, D. H&E ×100.) |
In most laboratories, histopathologic sections routinely are stained with H&E (Fig. 16-4D). Hematoxylin is a basic dye that binds to acidic materials including the DNA in the nuclei of cells (Fig. 16-4B). Eosin (tetrabromofluorescein) is acidic and stains basic substances such as proteins pink (Fig. 16-4C).
H&E staining provides helpful diagnostic color cues. Lesions composed of cells with scanty cytoplasm tend to look blue or basophilic in microscopic sections. Examples of basophilic lesions include infiltrates of lymphoid and inflammatory cells, retinoblastoma, and basal cell carcinoma. Necrosis, calcification, and DNA deposition in eyes with retinoblastoma also appear blue. In contrast, cells that have abundant cytoplasm (e.g., squamous cells or epithelioid histiocytes) appear pink. Squamous cell carcinomas usually are eosinophilic. Normal ocular structures composed largely of connective tissue such as the cornea and sclera appear eosinophilic in H&E sections. Protein-rich subretinal fluid, lens material, amyloid, and exfoliation material also are eosinophilic.
The periodic acid-Schiff (PAS) stain is used routinely in most ophthalmic pathology laboratories because it vividly highlights the thick basement membranes of the eye (lens capsule and Descemet membrane). PAS stains materials that contain unsubstituted vicinal glycol groups (CHOH-CHOH). The vicinal glycol groups are oxidized to dialdehydes (CHO-CHO) by periodic acid, and the Schiff reagent (leucofuchsin) reacts with the dialdehydes forming complexes that range in color from red to magenta. In addition to basement membranes and aggregates of basement membrane material such as guttae and cuticular drusen, PAS stains glycogen, some mucins (e.g., conjunctival goblet cells), and many but not all yeast and fungal hyphae (Fig. 16-5D).
SPECIAL HISTOCHEMICAL STAINS
A variety of special histochemical stains occasionally are ordered in the ophthalmic pathology laboratory to demonstrate the presence of microorganisms and highlight special structures and materials in histologic sections. Standard histochemical stains rely on chemical reactions to demonstrate the presence of substances. All special stains must be used in conjunction with positive control slides that contain the sought-for organisms or materials. These control slides confirm that the special stains are working.
Special stains for microorganisms are ordered most often. These include modifications of the Gram stain for bacteria (Brown and Hopps or Brown and Brenn stains) (Fig. 16-5A), the Gomori methenamine-silver (GMS) stain for fungi (Fig. 16-5C), and several acid-fast stains for mycobacteria. The Dieterle and Warthin-Starry silver impregnation techniques for spirochetes, Bartonella, and Legionella occasionally are ordered (Fig. 5-11). Fungi and yeast are impregnated with silver and appear black against a green counterstain in the GMS stain. The Ziehl-Neelsen and Fite-Faraco acid-fast stains are used to demonstrate the acid-fast organisms that cause tuberculosis, leprosy, and nocardiosis. The organisms appear red (“red snappers”) against a blue background (Fig. 16-5B).
Several histochemical stains are used to demonstrate the presence of metals in tissue. These include the Perls Prussian blue reaction for iron and the von Kossa and Alizarin red stains for calcium. The iron stain is used to demonstrate the deposition of iron in ocular epithelial structures in hemosiderosis and siderosis (Fig. 4-11B) and in corneal iron lines (Fig. 6-20F,G). The iron deposits are blue. The von Kossa stain for calcium actually stains the anionic material that binds with calcium (Fig. 12-6D). This silver stain stains calcium deposits black. The Alizarin red stain forms complexes with calcium, which appear red against a green counterstain. The ruthenium red stain and rhodanine stains for copper are relatively nonspecific and are used infrequently.
The Masson trichrome stain stains collagen blue and cellular tissue red. It is used to demonstrate the presence of fibrosis (Figs. 9-10B and 15-4C). Masson trichrome also is the histochemical stain of choice for granular corneal dystrophy (Fig. 6-21D). Stains for acid mucopolysaccharides (AMP) include alcian blue, Hales colloid iron for AMP, and mucicarmine. The first two stain mucin blue because the histochemical reaction is based partially on the iron stain. AMP stains are used to demonstrate mucin production in tumors and in the assessment of corneal dystrophies. They are the stains of choice for macular corneal dystrophy (Fig. 6-28C,D). The Verhoeff-van Gieson stain for elastic tissue demonstrates the internal elastic lamina in arteries (e.g., temporal artery biopsies) and the excessive production of elastic components (elastotic degeneration) in pterygium and pinguecula (Fig. 5-16C). In the latter, the elastotic degeneration stains black, but the reaction is not quenched by pretreatment with elastase. The reticulin stain occasionally is used in the assessment of certain neoplasms such as hemangiopericytoma and lymphomas.
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