Figure 3-3 A, The goal of sectioning is to obtain a pupil–optic nerve (PO) section that contains the maximum area of interest. B, Two caps, or calottes, are removed to obtain a PO section. C, The first cut is generally performed from posterior to anterior. D, The second cut will yield the PO section. (Illustration by Christine Gralapp.)

The globe can also be opened coronally with separation of the anterior and posterior compartments. The tumor can be visualized directly with this technique, and a section including the maximum extent of the tumor may then be obtained.

Processing and Staining

Fixatives

The most commonly used fixative is 10% neutral buffered formalin. Formalin is a 40% solution of formaldehyde in water that stabilizes protein, lipid, and carbohydrates and prevents postmortem enzymatic destruction of the tissue (autolysis). In specific instances, other fixatives may be preferred, such as glutaraldehyde for electron microscopy, ethyl alcohol for cytologic preparations, and Michel medium for immunofluorescence studies. Table 3-1 lists examples of some commonly used fixatives.

Formalin diffuses rather quickly through tissue. Because most of the functional tissue of the eye is within 2–3 mm of the surface, it is not necessary or desirable to open the eye. Opening the eye before fixation may damage or distort sites of pathology, making histologic interpretation difficult or impossible. The adult eye measures approximately 24 mm in diameter, and formalin diffuses at a rate of approximately 1 mm/hr; therefore, globes should be fixed at least 12 hours prior to processing. It is generally desirable to suspend an eye in formalin in a volume of approximately 10:1 for at least 24 hours prior to processing to ensure adequate fixation. Different institutions may use different protocols, and preoperative consultation is critical.

Tissue Processing

The infiltration and embedding process removes most of the water from the tissue and replaces the water with paraffin. Organic solvents used in this process will dissolve lipid and may dissolve some synthetic materials. Routine processing usually dissolves intraocular lenses made of polymethylmethacrylate (PMMA), polypropylene, and silicone, although the PMMA may fall out during sectioning. Silk, nylon, and other synthetic sutures do not dissolve during routine processing. Specimens are routinely processed through increasing concentrations of alcohol followed by xylene or another clearing agent prior to infiltration with paraffin. Alcohol dehydrates water, and xylene replaces alcohol prior to paraffin infiltration. The paraffin mechanically stabilizes the tissue, making possible the cutting of sections.

The processing of even a “routine” specimen usually takes a day. Thus, 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 for the rapid processing of special surgical pathology material are generally reserved for biopsy specimens that require emergent handling. Because the quality of histologic preparation after rapid processing is usually inferior to that of standard processed tissue, it should not be requested routinely. Surgeons should communicate directly with their pathologists about the availability and shortcomings of these techniques.

Table 3-1

Tissue Staining

Tissue sections are usually cut at 4–6 µm. A tissue adhesive is sometimes used to secure the thin paraffin section to a glass slide. The cut section is colorless except for areas of indigenous pigmentation, and various tissue dyes—principally hematoxylin and eosin (H&E) and periodic acid– Schiff (PAS)—are used to color the tissue for identification (Fig 3-4). Other histochemical stains used in ophthalmic pathology are alcian blue or colloidal iron for acid mucopolysaccharides, Congo red for amyloid, Gram stain for bacteria, Masson trichrome for collagen, Gomori methenamine silver stain for fungi, and oil red O for lipid. A small amount of resin is placed over the stained section and covered with a thin glass coverslip to protect and preserve it. Table 3-2 lists some common stains and gives examples of their use in ophthalmic pathology.

Figure 3-4 The section of a melanoma at the far left is colorless except for mild indigenous pigmentation in the tissue. Moving to the right, note the slides stained with hematoxylin only, eosin only, and both hematoxylin and eosin. (Courtesy of Hans

E. Grossniklaus, MD.)

Table 3-2