GROSS DESCRIPTIONS, FIXATION

FUNDAMENTAL CONSIDERATIONS IN EYE PATHOLOGY

Clinical History- The importance of adequate clinical information before processing tissue cannot be overemphasized. Collection of clinical history begins as soon as the specimen is scheduled for the operating room. The operative schedule is reviewed 1 day prior to the date of the operation by the resident and history obtained. Specimens, historical information and the approach to fixation are discussed with the attending. In this way, the fixation selection errors are minimized by the operating room staff and surgeon. Common mistakes are formalin for lymphoma when flow cytometry is needed, formalin for conjunctiva suspected of ocular cicatricial pemphigoid, etc.
Surgical biopsies must have the relevant clinical information in the appropriate place on the Pathology Consultation Request before the gross work-up begins. Proper biochemical or ultrastructural evaluation often depends upon appropriate treatment of the specimen at the outset - this is especially important in Lymphoproliferative lesions (see Reference 33), in body fluids or "washings," and in lesions that may require electron microscopic analysis (formalin contains methyl alcohol and other ingredients that wreak havoc with the ultrastructure of cells).
Fixation of Tissue- Proper fixation is of paramount importance in processing tissue for histopathological evaluation. The OR and pathology laboratory in the main hospital (RRMC) leaves tissues unfixed until the pathologist sees them. Keep this in mind for cases that may be delayed when sent to us. They all should be either fixed at the time of surgery or sent to us immediately. For the JSEI OR our current protocol is to provide formalin for most cases with important exceptions. The exceptions are conjunctiva for ocular cicatricial pemphigoid, selected enucleations, orbital tumors and intraocular tumors and corneas from Dr. Casey. In addition any conjunctival specimen from a surgeon that is not familiar with placing the specimen flat on filter paper. Specimens can be assessed for the proper fixative. The resident on service must seek the associated clinical histories on the day the specimen is received. This is generally done by first looking at the OR schedule the day prior to surgery and identifying possible cases that will need special processing. The specimen is then transferred to an appropriate solution or fixative. Here a few common examples for suspected diagnoses:
1. Lymphoma- fresh for flow cytometry, touch prep; modified carnoy's for DNA extraction. Orbital and conjunctival cases are generally sent to hematopathology fellows fro processing in main pathology. They are not accessioned at JSEI!
2. Ocular Cicatricial Pemphigoid- remain in refrigerated maleimide buffer (Zeus media) and sent immediately to immunohistochemistry for immunofluorescence. Zeus media can be obtained from the immunohistochemistry lab. The composition includes N ethyl maleimide, magnesium sulfate, potassium citrate and ammonium sulfate.
3. Sebaceous carcinoma- 4% paraformaldehyde for possible osmication (lipid fixation).
4. Pseudoexfoliation- glutaraldehyde for electron microscopy.
5. Retinoblastoma- sample fresh frozen or in modified Carnoys for gene sequencing, eye for formalin fixation after material removed for DNA analysis.

The following factors are critical: the thickness of the tissue that the fixative must penetrate (tissue blocks should be no greater than 2 mm in thickness and thinner blocks are preferred; the quantity of fixative (tissue should be immersed in a minimum of 10x its volume fixative), and the temperature (refrigerator cooling, will markedly retard autolysis! Freezing disrupts cells). There are special considerations for whole eyes after fixation (see processing of enucleated specimens).
The most common fixatives in use are:
FORMALIN - 10% formalin is a solution containing 40% formaldehyde that is buffered with phosphates (approx. neutral pH) and is an economical fixative with rapid penetration. The rate of fixation is about 1 mm /hour.
Chemical reactions occur between formaldehyde and amino acids as well as with nucleic acids. The DNA is denatured by breaking interchain hydrogen bonds, particularly at the AT-rich regions. Formaldehyde cross links both proteins and DNA by forming methylene bridges between amino groups. Hydrolysis of N-glycosylic bonds can result in release of free pyrimidine and purine residues and a reaction to 2-deoxy-D ribose. Phosphodiester bonds may be hydrolized, (purinic and apyrimidinic). DNA isolated in fomalin fixed tissue exhibits nonreproducible sequence alterations. Formalin is time honored and give excellent standard morphology for paraffin sectioning.

GLUTARALDEHYDE and PARAFORMALDEHYDE fixatives are generally used for electron microscopic studies. The size of the blocks (one mm or less) and rapid cooling of tissue are even more critical here.

PARAFORMALDEHYDE (4%) is very versatile because it is somewhat reversible. It can be used for paraffin embedded tissues, electron microscopy, and immunoelectronmicroscopy. It is not suitable for lymphoid work ups. Paraformadehyde is simply a polymer of formaldehyde.

ALCOHOL - 95% or absolute must be used for substances that are water soluble (e.g. glycogen). Other substances in use include MERCURIC CHLORIDE (Zenker's fixative), PICRIC ACID (Bouin's Solution), etc.

Alcohol based fixatives such as modifications Carnoy's solution (8 parts methanol and 1 part of glacial acetic acid) or (3 parts ethanol 1 part glacial acetic acid) are excellent for DNA preservation. The former, also called modified methacarn, is quite good for RNA preservation and preserves morphology reasonably well (Cox et al. 2006). Alcohol fixation works by precipitating proteins and stopping enzymatic degradation. Although nuclear fixation is quite good, nucleoli are more prominent with alcohol fixation and one must adjust criteria for atypia.

Decalcification- is accomplished with acid solutions in combination with EDTA. For dense bone a rapid decalcification solution is used. Decalcification is required for tumors containing bone and phthisis bulbi specimens and occasional other specimens. The decalcification solution is generally kept beneath the sink. Decalcification is always done prior to processing but after fixation.

Freezing Tissue- is indicated for lymphoid work ups when PCR of infectious material is contemplated at a later date and for evaluation of research type specimens. Each procedure requires a slight variation of the freezing procedure and the pathology attending should always be consulted.
Handling of Tissue- After fixation an important aspect of processing tissue is to handle the tissue with care, being careful not to crush with forceps, not allowing the tissue to dry during blocking, and permitting adequate space in cassettes to allow free access of dehydrating and infiltrating fluid during processing. Specimens that have been properly fixed in formalin are examined in water in a deep petri dish under the dissecting stereomicroscope. The water dilutes the formalin to a level where the concentration of formalin in the air above the water is undetectable (hence providing safety) as well as rinses out the formalin for the next step which will be 50% ethanol. Forceps are chosen for examination which are appropriate for each sample in handling the tissue. In general eye pathologists use forceps that much smaller (as the 3 on the left in the photo) than those forceps used by general pathologist, on the right. Forceps without teeth are generally preferable for delicate conjunctiva; curved blunt end forceps are best for scooping up corneas without damaging endothelium or epithelium; very fine forceps are necessary for epiretinal membranes. The specimens will be examined under a stereomicroscope with external illumination from a movable focusable light source that permits a range of lighting techniques.
Inking tissue samples- It is important to understand the orientation of each sample if that is discernible. In order to process the specimen one needs to know if it is possible that it contains a neoplastic lesion. Adequate history, thorough knowledge of diseases, location, and the gross appearance are all used in making the decision that a specimen in inked. If uncertain then ink the specimen (ink don't think!). The usual reasons for inking the specimens include identifying surgical margins in the case of neoplasms, providing orientation for the technician and orienting a lesion on microscopic examination. See also embedding instructions to understand how ink place on the cut face will be translated to a slide and the example of a skin ellipse with orientation to understand the principles of inking to preserve margins.
Suspected Neoplasms: Surgeons in general would like to know what margins are involved if they were not cleared. To do this the pathologist needs to ink the margins at the gross bench appropriately. In general, if no orientation is discernible (e.g. a eyelid biopsy without lashes, grey line, margin or conjunctiva) and was not provided by the surgeon with a marking suture or diagram then the undersurface is inked green and the specimen is submitted for appropriate cutting after processing and then embedding and microtomy. If orientation for the specimen is provided then it is critical to identify and ink the margins with different colors so that the proper orientation can be discerned once the specimen is cut. In other words to which margin did the tumor extend? In general the under surface is inked green and 2 adjacent sides of the specimen are inked with black and yellow marking inks. In this way each margin is able to analyzed under the microscope.

PLEASE CONSULT THE ATTENDING PATHOLOGIST BEFORE PROCESSING SPECIMENS WITH WHICH YOU ARE UNFAMILIAR. IF YOU FAIL TO INK THE SPECIMEN APPROPRIATELY AND IT IS PROCESSED THAN INFORMATION MAY BE PERMANENTLY LOST.

BIOPSY EMBEDDING BAGS
USE BIOPSY BAGS FOR SMALL SPECIMENS LESS THAN 3 MM IN ANY DIMENSION AND MAKE SURE BIOPSY BAGS ARE FOLDED ON ALL EDGES SO THE SPECIMEN DOES NOT FLOAT FROM THE CASSETTE DURING PROCESSING. THERE ARE TWO TYPES OF EMBEDDING BAGS. THE NYLON BAGS DO NOT FOLD EASILY AND DO NOT STAY FOLDED. THE PAPER (TEA) BAGS FOLD AND RETAIN THEIR FOLD WHEN WET. SO WET THEM SLIGHTLY BEFORE YOU PLACE THE TISSUE IN AND THE AGAIN AFTER YOU HAVE FOLDED ALL SIDES. DOUBLE BAG ANY CRITICAL SPECIMENS THAT ARE SMALL. IF TEA BAGS ARE USED, THEY SHOULD BE QUARTERED WITH SCISSORS FIRST FOR SMALL SPECIMENS BECAUSE IF THE VOLUME IS TOO LARGE, FINDING THE SPECIMEN WILL BE COMPLICATED FOR THE TECHNICIAN. CAREFUL FOLDING AND WETTING OF THE QUARTERED BAG IS IMPERATIVE.

To saveguard cassettes from losing specimens that slip through the cassette slats, we employ a system of transfer containers. Each day cassettes with tissue are placed in a yellow topped container, with the current date. 50% ethanol is filled just to cover the casettes. The specimens are taken to the main lab in these containers but the solution is not thrown out. The containers are returned to us the next day with the 50% ethanol for our inspection in case a specimen has slipped out. The resident is obliged to make sure these containers are clean before use, and to write the correct date with a marking pen.

Labeling Cassettes:
At the current time we use a color coded system in surgical pathology for cassettes. Eye pathology cassettes are red in color and are labeled in a very specific way. The name of the patient is written in pencil on the side of the cassette. The surgical number beginning with a J for Jules Stein precedes the S number of the surgical case. Each cassette must bear the suffix of a single letter starting with A and proceeding in sucession in the alphabet (A,B,C...) This cassette letter does not necessarily correspond to the specimen number or letter assignment. LABEL THE CASSETTES IN PENCIL OR WITH A SPECIAL PERMANENT MARKING INK MADE SPECIFICALLY FOR CASSETTE MARKING (e.g. STATMARK PEN) AS MOST INK WASHES OFF IN THE ORGANIC SOLVENTS USED IN PROCESSING. PUT THE NAME OF THE PATIENT ON THE SIDE OF THE CASSETTE IN CASE THE PENCIL IS ACCIDENTALLY SMEARED AND WRITE CLEARLY.


GROSS ONLY (IDENTIFICATION) CASES - general instructions

Certain categories of surgical specimens only require a careful gross description. In general, you should be confident that you can identify the tissue in the gross and that it is normal. If you cannot, microscopy should be performed. Specimens in which only a gross exam is usually performed include:
- eyelid tissues from blepharoplasty that are clearly completely normal
- most extraocular muscles from strabismus surgery (provided striations are seen)
- crystalline lenses
- intraocular lenses
- hardware from retinal detachment repair
- Ahmed valve

Use the templates in the computer available for each of the above specimens. After a careful stereo microscope examination and description, the case is signed out as indicated in the following examples:

FINAL DIAGNOSIS: Tissue type, "specimen label" (surgical procedure),
-1st diagnosis
-2nd diagnosis

FINAL DIAGNOSIS: Muscle and fibrous tissue , "right lateral rectus muscle" (resection) –muscle and tendon

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