Tuesday, December 27, 2005

Common Specimens in Eye Pathology-Gross

Examination of Common Specimens in Eye Pathology: Cornea, Conjunctiva, Epiretinal Membrane, Eyelids, Temporal arteries

Cornea --
Place the specimen in water and examine it under the dissecting microscope. Scoop the cornea out with blunt curved forceps other than pinching it between the teeth of the forceps. Note scars, vessels, or lesions on the surface. Note depth of lesions. Measure the diameter. Then place cornea on a flat surface with epithelium facing down (in order to protect the endothelium). Do not place it on paper towel as this will easily peel the surface layers off! Bisect the cornea with a blade (in drawer under the sharp box) and record the central and peripheral thickness. Submit half for processing and save the other half in formalin. Order PAS stain on all corneas.

The microscopic description should encompass the findings of the epithelium, basement membrane, Bowman's layer, stroma, Descemet's membrane and endothelium. Example:
Gross Description: Specimen, in formalin, labeled "OS cornea," consists of a translucent disc measuring 7 mm in diameter and 1.2 mm in central thickness and 1.4 mm in peripheral thickness. The epithelial surface is irregular and shows tiny blebs. Centrally the epithelium is missing. The specimen was bisected and cut sections reveal thickening centrally to the stroma. The specimen was submitted in cassette A.
Microscopic: Section shows epithelium with hydropic change, subepithelial bullae, and an irregular Bowman's layer. The epithelium is detached in many areas from Bowman's layer. There is stromal edema, mild inflammatory infiltrate consisting of lymphocytes and neutrophils, and stromal scarring. Descemet's membrane is artifactually absent in one large area due to tangential sectioning. The endothelium is absent.

FINAL DIAGNOSIS: cornea, "OS" (penetrating keratoplasty)
- bullous keratopathy with acute and chronic stromal keratitis
-severe endothelial attenuation

Descemet's membrane (DESEK specimens):
Samples that are surgically removed from stripping of Descemet's membrane appear as a translucent sheet that will probably be invisible in solution. The specimen remains invisible under a dissecting microscope, so prior to even examining the specimen simple steps can be taken to visualize the sample. The steps in processing (see reference) are to add an orange tissue binding dye (eosin or mecurochrome) to the solution and make it visible. DESEK may be received from the operating room in saline or formalin. If the specimen arrives in saline then formalin is simply added to the specimen container with a drop of mecurochrome. After 4-8 hours the orange membrane is removed carefully without tearing and placed in the middle of one side of a tea bag. Gently place the tea bag side with the membraneon water and the specimen will miraculously unfold. If not then submerge the tea bag and the membrane will float and unfold. The tea bag can be used to pick up the unfolded membrane for processing. Be sure to look carefully under the dissecting microscope for guttata (tangential lighting helpful).
Fold the edges of the tea bag; the edges away from the specimen. Place the folded tea bag in a nylon bag (just to be sure it is not lost in process). Instruct the technician to bisect the membrane after processing and to place on the embedded edge.

Surgical preparation of conjunctival biopsies-- Biopsies of the mucous membranes tend to curl when placed unsupported in fixative which makes it difficult to embed the specimen. To prevent curling, it should be gently spread on a small piece of paper towel, moistened with saline, with the stromal side against the paper, before placing it in fixative. The specimen, now adherent to the paper, is then gently floated on the surface of the fixative, making certain that the epithelial side is down and submerged in fixative. In a few hours, the specimen will be fixed flat and can either be removed for processing or can be processed while still adherent to the paper (excess paper can be trimmed before further processing). This procedure has the further advantage that any orientation information for the pathologist can be written by the surgeon on the paper (using ball-point pen or pencil). A suture can be used to help orient the specimen.

Conjunctiva is grossed in with the attention to margins. If a neoplastic process is even remotely possible, then ink the margins. If the specimen is less than 1 mm in diameter, it can be submitted without bisection. An accurate diagram should be made of the lesion before showing it to the pathology faculty.

If the conjunctiva comes with an orienting suture and is greater than 2 mm in length, then the attending pathologist will instruct the resident in preservation of proper orientation for microscopy. The principles are exactly the same as for skin biopsies with provided orientation (see below).

For pterygia, the specimens can be flattened and sectioned in a plane to capture the cornea in section. Requesting the histotechnologist to slice the specimen every mm after paraffin infiltration but prior to embedding will reduce the need to take interval sections (deeper sections) with the microtome and will greatly reduce the workload of the technician. Slicing after paraffin infiltration also results in properly oriented sections if the conjunctiva has been unfolded on the hot plate (melting the paraffin) before embedding. The histotechnologist should be trained in unfolding the conjunctiva at the embedding station prior to making these 1 mm slides.

For a diagnosis of suspected ocular cicatricial pemphigoid the conjunctival biopsy should preserved in Zeus media, rather than fixed, and processed by rapid freezing, followed by frozen section, and direct immunofluorescence for C3, IgG, IgA, IgM and fibrinogen.

GROSS DESCRIPTION: The specimen on the patient above was received in formalin, labeled "L. caruncle lesion biopsy," measuring 4 mm x 3 mm x 2 mm in maximal dimensions. The external surface shows hair follicles, an irregular cystic and brown pigmented surface. The undersurface is irregular yellow and waxy white in appearance. On cross section pigmented tissue is present beneath the epithelium. The specimen is inked (green), bisected, and submitted in cassette A.

MICROSCOPIC: Sections show conjunctiva with nonkeratinized stratified squamous epithelium, with goblet cells, hair shafts and sebaceous glands. There are nests of pigmented cells confined to the substantia propria with oval to round nuclei. Cells deeper in the lesion are smaller than those in the superficial portion.

FINAL DIAGNOSIS: Conjunctiva, "left caruncle" (biopsy)- subepithelial nevus

Epiretinal Membrane
The specimen usually arrives in saline. At least four times the volume of formalin must be added to the bottle. If the specimen is free-floating in fixative and difficult to see, place one small drop of mecurochrome dye in the fixative and wait for 3-24 hours to color it. These steps will aid both you and the technicians processing the specimen. After overnight fixation, remove the specimen, place it in an embedding bag, and submit it for sectioning.

Orbital Biopsies
Contact the attending for all orbital tumors. If the surgeons have not discussed the case with the pathology staff, call and get information on the clinical diagnosis and operation planned. If a biopsy of the orbit is suspected to be a lymphoid lesion, a complete lymphoma work up is indicated. See "Procedures for Handling Lymphoproliferative Lesions". Other orbital biopsies should be measured and described in detail with attention given to the surface and encapsulated architecture. The specimen is inked (if the surgeon attempted a complete removal) and bisected. A portion of the bisected specimen is saved in case electron microscopy is desired. Another portion is submitted for paraffin embedding.

Eyelid Lesions (the wedge resection)
Measure specimen carefully and examine for any lesions. Full thickness specimens require precise orientation and marking inks to preserve orientation after processing. If tumor is clinically suspected, discuss with the pathology faculty before processing.
In general, pentagonal wedge excisions have 3 margins that need to be inked: lateral, medial and posterior, i.e. where the surgeon has made cuts in the eyelid. The tarsal conjunctival surface and eyelid skin surface are not margins and do not need to be inked. The most common mistake made by those unfamiliar with anatomy of the eyelid is to ink the conjunctival surface thinking this is a posterior margin. Covered by epithelium, obviously it is not a margin at all. The closest margins to the tumor should be determined grossly. Several options exist to proceed thereafter; here are 4 examples.
1. Each margin, medial, lateral and posterior can be inked, removed in strips and processed separately. If there is tumor in the strips then step sectioning may give an idea of how close the tumor is from the margin but it is not as accurate as method 3 below. This is technically difficult, requires excellent fixation and should be done only after consulting the attending pathologist. It is the method of choice for very large tumors where there is good clearance from all edges and the strips are likely to be negative.
2. Each margin can be inked a different color, the specimen bisected in an anterior to posterior direction and the bisected section edge inked with orange dye for the histotechnologist to embed down in the cassette. Step sections by the histotechnologist will go through the tumor to approach the margins. This preserves of anatomic features of the eyelid. To be sure that the ink holds fast, it is best to bisect the specimen after processing. If the ink does not hold the orientation can still be preserved but this will require the input of the experienced pathologist and histotechnician. This technique is most commonly used because it is relatively simple to remove a thin slide from the center for possible electron microscopy. It also has the disadvantage of giving only relative distances that the tumor resides from the margin. This is an excellent method for small tumors in a small specimen where the margins will be clear in just a few sections.
3. Each margin can be inked a different color and the specimen bisected medial to lateral and embedded on the bisected edge. This has the advantage of providing the distance of the tumor from the medial to lateral margins but has the disadvantage of receiving sections without standard anatomic features. In this method step sections are taken to examine these margins that are usually of the most interest. It is the method of choice for tumors that obviously approach the medial and lateral margins and one needs precise distances to report to the surgeon.
4. After inking each margin a different color the specimen is processed for paraffin and embedding and may be breadloafed in a plane parallel to the nearest margins and embedded on the cut edge. In this case it is critical to embed the specimen in the same orientation and to retain in the block the order that it has been cut at the gross table. For the best fidelity this is best cut and embedded after processing. A crack histotechnolgist is quite capable of handling this with a bit of training. This is the most cost effective way to examine the entire eyelid (all breadloafed sections) in one slide, provided the specimen has been carefully oriented and the tumor does not approach the margin. But if the specimen is embedded inaccurately, then all margin analysis may be lost. This is not a good method when the histology lab is off site or the technician du jour is doing the embedding rather than one dedicated to eye specimens.

5. Hybrid techniques. Although a bit advanced for this forum, there are very efficient ways to preserve the anatomy, give excellent distances measurements to the tumor with high fidelity, and allow central sections to be processed for ultrastructure. Involved in these methods are special gross sectioning in a number of planes, re-embedding the specimen, and a very knowledgeable histotechnologist and a lot of free time.

Skin Biopsies with Provided Orientation

Occasionally skin ellipses or biopsies may come with orientation for margin analysis. These will generally be oriented with a suture at 12:00 o'clock and the requisition will ask for margins. In order to provide orientation, consult the attending pathologist. Our standard practice is to ink the entire undersurface green and the edge containing the suture (superior) with yellow ink. An adjacent side is inked black. The suture is left in place until after paraffin infiltration. At that stage the specimen is checked to see if the marking ink remains after processing. If the ink is present all of the margins can be identified by the absence or presence of yellow or black ink. The specimen is then cut along the dotted line or in multiple planes parallel to the dotted line and aligned in order in a cassette (see embedding instructions). Inking opposite sides different colors, the typical neophyte's approach, will not permit adequate orientation. In the example to the left the skin ellipse is marked with a suture at 12:00 o'clock. The lesion is denoted as tan. Green has been used to ink the entire undersurface but in this view is only visible on the left edge because black and yellow ink have been used to cover adjacent edges and are overlayed on the green. When the specimen is sectioned in the plane of the dotted line, the left edge will be always identified as having only the green ink. The right edge is always identified as having the black ink. The superior edge will contain the yellow ink. In this way the orientation is preserved through microscopy. However, if by mistake the left edge is inked yellow and right edge black, one will be unable to tell the superior margin from the inferior margin in microscopic sections. This is a concept that may be difficult for the beginning Ophthalmology resident and Eye Pathology fellow until they learn to think in three dimensions.

Temporal Arteries
Temporal arteries are submitted to confirm the clinical diagnosis of temporal arteritis. A negative biopsy does not exclude the disease. The benefit of confirming the diagnosis by biopsy may make the procedure very worthwhile in the long term if the clinical diagnosis should be questioned later. Because the pathologic lesions of temporal arteritis are often focal with skip areas it is important that the artery be completely sampled. Our current standard procedure at the gross bench is to measure the length and diameter of the artery and make one clean bisection. The cross section is examined under the dissecting microscope to identify a narrow lumen or nodular thickening (signs of granulomatous arterities). The artery, now bisected is then sent to the histology lab for processing. Instructions for the histology technician include: Artery, make cross sections every 1 mm (no greater than every 2 mm), and embed on the cut edges. Make 1 section and stain for hematoxylin and eosin. Once the artery has been processed and infiltrated with paraffin the histology lab technician will make thin cross sections, no greater than every 2 mm (and preferably 1 mm). All cross sections are placed on the cut edge in a metal embedding cassette and a single paraffin block is made. A single slide is cut from this block, which will sample the entire artery at 1-2 mm intervals. For expediency this section will be stained and examined. Most positive cases will be evident on this first slide and can be immediately reported. If no arteritis is seen, then more sections are taken according to the following instructions to the histology technician. Make step sections deeper every 50th section (five-micron thick section) through the block. This allows sampling every 250 microns (or .25 mm), which should pick up skip lesions without difficulty and leave a large margin of error. In our recent experience the number of sections received is exactly the number that would be calculated from the length of the vessel. (See the classic work of Campbell's group at the Mayo Clinic). We monitor this in every case as will be evident below.

Indications for RUSH processing for the biopsy. If necessary the histologic processing can be ordered RUSH!, which will result in the sections returning by the next day or even sooner. Several factors generally preclude any need to send the cases for rapid or "rush" processing. First, there is some controversy as to the value of temporal artery biopsies in affecting clinical treatment. Several studies have shown that clinical treatment is unaffected. The patients have usually been started on steroids by the time the biopsy is performed. Therapy the will not be withdrawn immediately because a clinical response must be measured. Additionally, the sensitivity of temporal artery biopsy in not 100%; sensitivity in this disorder is hard to estimate because there is no gold standard for the diagnosis. However, Bayesian analysis of bilateral temporal artery biopsies suggests sensitivity could approach about 87% at best , (IOVS 2007;48:675-80). Therefore a negative temporal artery biopsy does not exclude the disorder. Also, the step sections will take additional processing time, so rushed initial sections will not always expedite the case. There are instances when rush processing may be desired. For example, if for some reason an irregularity in judgement has lead the clinician to perform the biospy and at the same time withhold treatment until a result is available (generally not the standard of care), and the clinician informs you of this, then by all means proceed with rush processing. If a long holiday is coming up (the laboratory will be closed) or a patient is going on a trip and needs to have instruction for dosing immediately then rush processing may help. Each instance must be considered on individual basis. It is important to have clinical input to make this decision. The need for a rush case should be discussed with the attending pathologist and orchestrated by talking to the lead histology technician.
Sample report:
CLINICAL DESCRIPTION: 75 year old male with history of headache for 2 months. Exam shows tenderness of the temporal artery. ESR= 75 mm
GROSS DESCRIPTION: Specimen, in formalin, labeled "biopsy from left temporal artery" consists of a tubular structure, measuring 10 mm (length) x 2.5 mm (diameter) and is bisected. Under the dissecting microscope the cross section shows narrowing of the lumen and nodular thickening of the vessel wall. The specimen is submitted after processing in cassette A.

MICROSCOPIC EXAM: Step sections at 250 micron intervals stained with EVG and Trichrome show a moderate sized artery with a narrowed lumen and severe mural granulomatous inflammation. The inflammation involves the thickened intima, media and adventitia. The inflammatory infiltrate is composed of collections of histiocytes with accompanying lymphocytes, plasma cells and eosinophils. The EVG stain shows fragmentation of the internal elastic lamina, with obliteration of short segments. .
FINAL DIAGNOSIS: Artery, "left temporal" (biopsy)- granulomatous arteritis

How does one monitor intervals at which step sections are made by the laboratory? At the end of the step sectioning we simply add up the number of total sections that were made of the artery on all slides. We compare that number to the expected number, which we calculate. For example in a 10 mm artery that was cut at 250 micron intervals then 10,000 microns/250 microns/section= 40 sections are expected. The interval actually cut is given in our final report for cases in which step sections were performed.



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