Techniques for fixing, processing and cutting sections from eyes
In order to gain the most information from an eye removed surgically or at autopsy, some special procedures
and techniques are required. Globes received as surgical specimens should be accompanied by a requisition
which details the reason for enucleation and clinical findings as clinicopathologic correlation is
important.
Fixing the eye
Routinely, eyes can be fixed in formalin for 48 hours, rinsed in water for 8-24 hours and then placed in 60%
ethanol. They should remain in the latter for 24 hours prior to processing but can be stored indefinitely
in alcohol. There is no need to inject fixative into the eye. The only exception to this rule is if the
eye is from an autopsy and a sample of vitreous was required for medicolegal purposes. Once some vitreous
has been removed, the eye will collapse. Therefore, for it to fix in a proper shape it can be worthwhile to
carefully inject just enough formalin into the globe to return it to its original shape. It is desirable to
use the same needle hole to remove vitreous and reinflate the globe. The best location to insert a needle
is the pars plana of ciliary body which is 3.5 mm from the limbus. This site is the least likely to produce
damage to internal ocular structures.
Occasional eyes may need to be received and opened in the fresh state. For example, samples for genetic
studies need to be obtained from retinoblastomas, endophthalmitis may require cultures, and research
protocols may necessitate fresh tissue. The globe should be transilluminated to help identify the
tumor/lesion in question. The procedure used is outlined below. A cut into the globe parallel to the
cornea-optic nerve axis can be made over the lesion using a razor blade, keeping in mind that once the eye
is fixed the cut will need to be extended to produce a pupil optic nerve section as outlined below. Fresh
eyes are usually very difficult to cut as they are soft so the least amount of sectioning to obtain the
required tissue is recommended.
Grossing the specimen
The globe should first be examined externally. It is necessary to orient the specimen and this can be
accomplished using inferior oblique and superior oblique muscle insertions plus the long posterior ciliary
vessels as guides (see diagram 1). The inferior oblique has a muscular insertion on the posterior temporal
(lateral) part of the globe just below the horizontal (indicated by the long posterior ciliary vessels which
appear as two bluish streaks on either side of the optic nerve). The superior oblique has a tendinous
insertion superiorly and slightly temporally.
Once the specimen is oriented, several measurements need to be taken in millimeters. These are horizontal,
vertical and anteroposterior measurements of the globe; horizontal and vertical measurements of the cornea,
optic nerve length and diameter; and diameter of the pupil. Other features to check include abnormalities
on the surface of the cornea - transparency, ulcers, nodules, vascularization, etc.; presence and type of
material in anterior chamber; abnormalities of iris and/or pupil; surface abnormalities on the remainder of
the globe such as lacerations, hemorrhage, pigment, tumor; abnormal dimensions or color of the optic nerve.
If melanoma is suspected clinically, location of vortex veins should be noted as they will need to be
sampled. They can be seen on the superior and inferior aspects of the globe, usually as 4 structures
located at 11, 1, 5 and 7 o'clock when one views the globe from a posterior aspect. Vortex veins can be
sampled in two ways. The first method involves marking their location with ink so that the technologist can
easily find them. Then, the eye is sectioned as outlined below. If the vortex veins happen to be in the
pupil optic nerve portion of eye, it is necessary to ensure that when the technologist cuts sections from
this block, some include the vortex veins. If the vortex veins are in the calottes, a strip of sclera
including the vein can be cut and submitted separately. The second method involves using a razor blade to
shave off the vortex veins together with a partial layer of sclera regardless of their location.
Before the eye is opened, it should be transilluminated to look for masses, defects in the pupil and iris
and abnormalities in pigmentation involving retinal pigment epithelium and choroid which will influence
where cuts are made. Transillumination requires an intense localized light from behind the globe in a
darkened environment. If blood or other dense material fills the globe or if it is phthisical, it will not
transilluminate.
Based on clinical information, external and transillumination findings, one can make cuts into the globe
using a razor blade. The standard technique involves 2 parallel cuts made approximately 2 mm from either
side of the optic nerve. One cut will extend to the limbus and the other approximately 1 mm into the cornea
so as to see into the anterior chamber (see diagram 2). This technique creates a pupil-optic nerve section
and 2 caps (calottes). By not cutting farther into the cornea/anterior chamber, one minimizes the chance of
dislocating the lens or having it pop out of the specimen. The plane of section chosen should represent the
pathology seen grossly. The eye is cut by holding it against a flat surface either with the cornea and
optic nerve facing laterally or with the optic nerve facing up and the cornea facing down. The cut is made
from back to front using a razor blade and light pressure. Once the eye is opened, any abnormalities can be
described. Eyes which are phthisical may contain calcium and/or metaplastic bone making them difficult to
cut. Decalcification may be required.
The optic nerve should have a cross section cut from it and blocked on the surgeon's cut surface. If tumor
is suspected, the tissue cut from roughing into the block should be saved and stained until a full section
of optic nerve has been reached. If the optic nerve has been severed flush with the globe, it can be
helpful to mark the location of the optic nerve with ink to aid the technologist cutting the sections.
For processing, eyes have to be placed into large tissue cassettes; they do not fit into the ones routinely
used for surgical pathology. It is useful to discuss each eye with the technologist who will be embedding
and cutting the specimen to ensure that all abnormal areas of the globe are well represented in sections for
microscopy.
Processing the specimen
Eyes can be processed using an automatic tissue processor. The suitable processing schedule is as follows:
70% ethanol 3 hours; 95% ethanol 1 hour; 95% ethanol 1 hour; 100% ethanol 1 hour; 100% ethanol 1 hour; 100%
ethanol 1 hour; 100% ethanol 1 hour; xylol 30 minutes; xylol 30 minutes; xylol 30 minutes; wax 1 hour 30
minutes; wax 1 hour 30 minutes.
Blocking and cutting the eye
When blocking the eye specimen, a large enough mold should be used to insure extra wax on all sides of the
eye. There is no specific number of sections that has to be cut although various levels should sample any
pathology present. A few sections should include the centre of the optic nerve. The sections should be cut
between 6 and 8 microns. The greater the thickness of the tissue, the greater the tendency for it to lift
from the slide, particularly if it contains much blood. The tissue needs to be well soaked on a slightly
melted ice tray before cutting. Also, the block should be soaked for 30 minutes between ribbons to ensure
that the lens cuts without fracturing.
Two water baths are required, one at room temperature, the other at around 44-46°C to which gelatin has been
added. (Sprinkle approximately ½ tsp. of powdered gelatin in bottom of waterbath before adding water.)
However, the temperature of the waterbath depends on the type of the embedding medium and its melting point.
We use Paraplast x-tra, product no. 8889-503002 with a melting point of 50-54°C. The ribbon should be
placed on a room temperature water bath, where creases can be removed. Separate the sections and transfer
to a hot water bath where they can relax before picking them up with AAS (3-aminopropyl-triethoxysilane)
treated slides. At 44-46°C, the wrinkles usually present in the sclera are eliminated and the cornea and
any blood present stays attached to the slide. At higher temperatures the cornea and any blood in the eye
have a tendency to float from the slide in the staining procedure, despite using AAS slides. Each eyeball
may react differently and the temperature may have to be adjusted accordingly.
Keep the slides in a 37°C oven overnight, then transfer to a 60°C oven for 30 to 60 minutes before staining.
Routine sections are stained with hematoxylin and eosin and 1 section with periodic acid-Schiff reagent.
