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Fixation and processing of ocular tissues

Fixed ocular tissues are submitted as (i) whole globes with attached orbital tissues from exenteration or transpalpebral enucleation (Figures 1.0 and 1.1); (ii) globes without orbital tissues (transconjunctival enucleation) (Figure 1.2); (iii) intraocular contents from evisceration specimens (Figures 1.3 and 1.4); (iv) prolapsed tissues following penetrating trauma; or (v) excisional or incisional biopsies of proliferative lesions (Figure 1.5). The clinical pathologist receives exfoliative (scrapings or impression smears) or aspirated specimens. Each of these samples has unique preparation requirements in order to optimize the pathologist's examination (Torcyznski, 1981).

Figure 1.0 A bisected canine globe that was exenterated due to a large retrobulbar tumor that is indenting the sclera (black arrows), the retina is detached (gray arrows) and the vitreous is compressed and contains asteroid hyaloid bodies (white arrow) (formalin fixation).

Figure 1.1 This bisected canine globe has endophthalmitis and was removed by transpalpebral enucleation. This globe contains white purulent exudate, and the iris is extending into a corneal defect (white arrows). Unless they are likely to contain lesions, the eyelids and other periocular soft tissues would ordinarily be removed during transconjunctival enucleations prior to fixation or if they are left on (transpalpebral enucleation and exenterations), they may be trimmed when the globe is sectioned (formalin fixation).

Figure 1.2 A bisected canine globe removed by transconjunctival enucleation, the technique used for the vast majority of globes submitted for histologic assessment. The retina is detached (white arrows) and noticeably thin (formalin fixation).

Figure 1.3 A canine evisceration sample bisected in preparation for histologic processing. Many are not as cohesive as this and arrive as multiple disconnected fragments. Evisceration samples like this should be sectioned in multiple planes to ensure that all intraocular tissues are present for histologic examination (retina, uvea, and lens) (formalin fixation).

Figure 1.4 Low-magnification histologic image of a typical evisceration specimen. When contrasted to sections from enucleated globes, such samples provide limited (albeit sometimes still useful) diagnostic information. The predominance of pigmented uveal tissue of which the ciliary body is most abundant is routine (hematoxylin and eosin stain, formalin fixation, and subgross magnification).

Figure 1.5 Low-magnification histologic section of a formalin-fixed third eyelid tumor in a dog, confirmed histologically as a minimally invasive adenocarcinoma arising from the gland of the third eyelid (hematoxylin and eosin stain, formalin fixation, subgross magnification).

Tissues other than globes are generally fixed as is routine for nonocular tissues with 10% neutral buffered formalin, while cytology specimens should be submitted as air-dried smears on glass slides. Globes require greater attention; it is important that they be fixed promptly to ensure rapid fixation of the internal structures and minimize autolytic changes. The ideal fixative would (i) penetrate cornea and sclera rapidly; (ii) preserve normal tissue relationships; (iii) allow for detailed gross examination; (iv) minimize embedding, trimming, and cutting artifacts; (v) enable any number of histochemical or immunohistochemical stains; and (vi) permit ultrastructural evaluation in those admittedly few cases in which it might be necessary. As no such perfect fixative exists, selection becomes a compromise influenced by availability, ease of use, and the quality of fixation required for the specific application (Yanoff et al., 1965; Yanoff and Fine, 1967; Yanoff, 1973; Menocal et al., 1980; Margo and Lee, 1995).

Fixatives

The most common method of fixation for light microscopic evaluation of globes is immersion in 10% neutral buffered formalin. This fixative has the advantages of being widely available even in private practice (supplied free by most diagnostic laboratories), and permits good macroscopic evaluation of submitted globes. It allows prolonged storage of globes without danger of excessive hardening, and the fixative is most often used with immunohistochemical protocols. Although it is claimed to penetrate tissue quite rapidly, in actuality fixation via cross-linking of proteins is quite slow (24-48?h) with resulting predisposition to retinal autolysis as well as other artifacts; although often referred to as "formalin artifacts," they are in fact a reflection of slow fixation and are not specifically related to formalin. These changes can be minimized by injecting a small volume of formalin into the vitreous prior to immersion, and they are not encountered in retinas from evisceration samples that are in immediate contact with the formalin.

Several other fixatives have specific advantages and disadvantages (Table 1.1). A mixture of 1% buffered formaldehyde/1.25% glutaraldehyde perhaps best approaches the requirements for an ideal fixative, but its limited stability, special storage requirements, and slow penetration of intact globes make it unsuitable as a fixative for use in private practice. Both Davidson's fixative and Bouin's fluid provide much more rapid penetration of the globe and thus much better retinal fixation than 10% formalin, and they harden the globe to greatly facilitate the trimming required for histologic processing. For eyes with boney ossicles, either of these acid fixatives provides the added convenience of simultaneous decalcification. Unfortunately, however, they both opacify the tissues and make macroscopic inspection and photography of the fixed globe less rewarding than with formalin (see Table 1.2). Because Bouin's fluid causes yellow discoloration of the tissue (and everything else it touches!), and because globes must be removed from the fixative and transferred to formalin within 24?h to prevent excessive hardening and poor subsequent histologic staining, we regard Davidson's fixative as the best ocular fixative for use in clinical ophthalmology practice and general diagnostic laboratories.

Table 1.1 Ocular fixatives and their advantages and disadvantages.

Formaldehyde 1% formaldehyde/1.25% glutaraldehyde Bouin's fluid Glutaraldehyde Triple fixatives (paraformaldehyde, glutaraldehyde formaldehyde, and osmium) Zenker's fluid Davidson's fixative Maintenance of tissue relationships (neurosensory retina-RPE) Marginal Good Good Poor by immersion of the globe as penetration is marginal, so globes must be sectioned and then small pieces immersed Excellent; however, globes are typically opened Good Excellent Preservation of membranes for ultrastructure Poor Very good Poor Excellent Excellent Good Very good Immuno compatibility Good Good Good; however, different epitope retrieval is required Good Good; however, different epitope retrieval is required Good; however, different epitope retrieval is required Good Advantages and disadvantages Cost and, availability Uncommonly used because of availability and stability Better penetration of globe Excellent fixation of small tissue sections or small globes with thin sclera (mice, rats) destined for electron microscopy Uncommonly used except in research; Uncommonly Very good immersion fixative Use limited by toxicity of mercury Poor penetration of the globe and marginal tissue fixation Obscuration of focal lesions at grossing Osmium is toxic and expensive Obscuration of focal lesions at grossing Stains everything yellow

Table 1.2 Histochemical stains frequently used in ophthalmic pathology.

Target Stain Result General purpose Hematoxylin and eosin Proteins - pink Nucleic acid - purplish blue Calcium - blue Basement membranes (mucopolysaccharides and glycoproteins) Periodic acid-Schiff (PAS) Dark pink/purple Collagen Masson's trichrome Blue Acid mucopolysaccharides Alcian blue Blue Iron Perl's Prussian blue Blue Calcium Von...