Step 2. Dissecting the Specimen

Step 2. Dissecting the Specimen

The Cutting Station

The cutting station should be clean and orderly. Most routine dissections require a ruler, a scale, a scalpel with disposable blades, scissors, forceps, a probe, and a long sectioning knife. At the begin-ning of each day, the prosector should make cer-tain that these tools are well maintained, clean, and within easy reach. Between dissections, these instruments and the cutting table itself should be rinsed clean of fluids and tissue fragments. This practice will help eliminate contamination of a specimen with tissue fragments from a prior dissection. Similarly, sectioning blades should be rinsed regularly during a dissection so that frag-ments of a friable tumor are not inadvertently transferred throughout the specimen or to other cases. Nothing is worse than not being sure if a minute fragment of cancer on a slide was a “pickup” from another case.

No more than a single specimen should be on the cutting table at any one time. Although it may seem time efficient to work on multiple speci-mens simultaneously, this dangerous practice openly invites the loss and mislabeling of speci-mens. For example, a small biopsy specimen is easily overlooked and discarded when overshad-owed by a large and messy specimen on the same cutting table, while specimens of similar size and shape may easily be confused and mislabeled.

Handling of Tissues

While all tissues are to be handled cautiously and gently, small specimens in particular are suscepti-ble to ill-treatment. Small and delicate tissue frag-ments may be crushed during transfer to a tissue cassette, they may desiccate if not placed in fixa-tive in a timely manner, and they even may be lost during processing if they are not easily seen.

These problems can be minimized by adhering to a few simple guidelines:

1. Small specimens should never be forcibly squeezed between the ends of a forceps or the tips of the fingers. Instead, small specimens should be gently lifted from the specimen con-tainer using the end of a wooden applicator stick or pickups. Alternatively, small speci-mens can be filtered directly into a tissue bag, avoiding instrumentation altogether.

2. Small specimens should be quickly placed in fixative. Ideally, most small specimens (i.e., less than 1 cm) should reach the surgical pa-thology laboratory already in fixative. This re-quires that physician offices, biopsy suites, and operating rooms be supplied with appropriate fixatives, and that all personnel involved be instructed as to their proper use. Sometimes delays in fixation are necessary, as when a frozen section is required or when special tissue processing is indicated. In these in-stances, the tissue should be kept damp in saline-soaked gauze. Never leave small tissue fragments exposed to the air on the cutting table, and never place these small fragments directly on a dry paper towel. These prac-tices are sure to hasten tissue desiccation.

3. For extremely small specimens, the journey from specimen container to histologic slide is a treacherous one, and they may be lost at any point along the way. For this reason, it is a wise practice to identify these small tissue frag-ments first and then mark the fragments so that they can be found more easily by the his-totechnologist. Before the specimen container is even opened, check its contents for the size and number of tissue fragments, and record these in the gross description. If no tissue is seen or if inconsistencies with the requisition form are noted, carefully open the specimen container and thoroughly examine its surfaces (including the undersurface of the lid) for ad-herent tissue fragments. If no tissue is found or if discrepancies persist, the submitting physi-cian should be notified immediately, and the outcome of this investigation should be docu-mented in the surgical pathology report.

Once all of the tissue is identified in the speci-men container, efforts should be taken to ensure that it safely reaches the histology laboratory and that it is easily identified for embedding and sectioning. Minute tissue fragments should be wrapped in porous paper or layered between porous foam pads before they are placed in the tissue cassette. Before these fragments are sub-mitted to the histology laboratory, they can be marked with eosin or mercurochrome so that they are easier for the histotechnologist to see.

Inking the Specimen

Various inks and colored powders can be used to mark critical points on the specimen. These dyes and powders may help orient both the gross specimen and the histologic section. For example, colored tattoo powder sprinkled on the outer sur-face of a cystic mass can be used to distinguish between the outer and inner aspects of the cavity. Similarly, India ink can be painted on the surgical margins so that they can be easily recognized at the time of histologic examination. Indeed, many times the critical distinction of whether a neo-plasm extends to the surgical margin depends entirely on the absence or presence of ink.

Given the important implications of an inked surface, these inks should be carefully and judi-ciously applied to the gross specimen. Keep in mind that just as the effective use of inks can facil-itate the histologic interpretation, the careless and improper use of these inks can befuddle the mi-croscopic findings. Consider, for example, the im-plications of sloppily applied ink that runs across a surface where it does not belong. The following guidelines outline the proper application of inks:

·   If possible, apply ink before sectioning the specimen.

·   Do not use excessive ink.

·   Dry the surface of the specimen with paper towels before applying ink. When applied to a dry surface, ink is more likely to stick to the desired surface and less likely to run onto other areas of the specimen.

·   Allow the ink to dry before further processing the specimen. Do not cut across wet ink, as the knife is likely to carry the ink onto the cut surface.

Opening and Sectioning the Specimen

The manner of opening and dissecting specimens is variable, depending on the type of specimen and the nature of the lesion. Bone marrow bi-opsies may simply be placed directly into a tis-sue cassette without any further manipulation, while the dissection of complex bone resections may require a multistep process involving special chemical reagents, imaging machines, and bone saws. Although this manual provides specific dis-section guidelines for most of the specimens you will encounter, a few general guidelines underlie many of the instructions.

First, localize the lesion before sectioning the specimen. One effective way to localize the lesion is simply to palpate the specimen. For example, a small peripheral lung tumor may be readily appreciated simply by palpating the lung paren-chyma, and a colorectal tumor can usually be detected by probing the lumen of the specimen with a gloved finger. Sometimes further measures are needed to localize the lesion. Review of speci-men radiographs, for example, may be necessary to uncover the size and location of a lesion when it involves a bone. Once the lesion is localized, the specimen can be sectioned in the plane that best demonstrates the pathology.

Second, open the specimen in such a way as to expose the lesion while maintaining its relation-ships to surrounding structures. In general, the walls of hollow structures (e.g., large bronchi, stomach, intestines) should be opened along the side opposite the lesion to maintain the structural integrity of the lesion and to preserve important anatomic relationships. For tumors involving solid organs, the specimen should be cut along the longest axis of the tumor to demonstrate the tumor’s greatest surface area.

Third, remember to dissect and examine the entire specimen. Often, the dissection is so fo-cused on a localized lesion that the rest of the specimen is not examined. Incomplete dissections represent lost opportunities to fully disclose the extent of a lesion and to uncover unsuspected pathologic processes.

Fixing the Specimen

Before tissue is processed in the histology labora-tory, it should be well fixed. Some institutions prefer to fix the specimen before it is dissected and sampled, while other institutions would rather you dissect and sample the specimen in the fresh state. Each method has its advantages and disadvantages. Specimen fixation greatly fa-cilitates tissue sectioning. For example, tissue fixation permits thin sectioning of fatty tissues, and it helps to preserve the structural detail of thin-walled cysts, mucosa-lined organs, and fria-ble tumors. One major disadvantage of specimen fixation is simply that it takes time. Fixation of larger specimens may require submersion in for-malin for a full day or longer. Delays caused by fixation can be eliminated by dissecting and sampling the specimen while it is fresh. Although this practice may compromise the quality of the histologic sections, it can significantly reduce case turnaround time. Another major disadvan-tage of specimen fixation is that certain diagnos-tic studies require fresh, unfixed tissues. This demand for unfixed tissues is rapidly expanding owing to recent advances in genomic assays that require undegraded DNA and/or RNA. Most of the dissection descriptions in this manual include a step for fixation. Simply skip this step if your institution does not fix speci-mens before dissection or if fresh tissue needs to be collected for appropriate studies.

Even when one chooses to fix a specimen, a limited dissection of the fresh specimen is usually necessary. Fixative will not diffuse into the center of an unopened specimen, especially if the speci-men is large and fatty. To overcome this, hollow organs and large cysts should be opened and solid tissues should be sectioned. Furthermore, a limited dissection is usually needed to expose a lesion if fresh tissue is required for frozen section evaluation, ancillary diagnostic studies (e.g., cyto-genetic studies, flow cytometry), research pur-poses, or storage in a tissue bank. These important decisions regarding the distribution of tissue must be made while the specimen is fresh, not after the specimen has been submerged in fixative.

Storing the Specimen

The tissue remaining after a specimen has been thoroughly dissected and sampled should not be discarded. Instead, it should be stored in such a way as to ensure easy retrieval and reconstruction of the specimen. Tissues for storage should be placed in a well-sealed container that holds enough fixative to cover the specimen. For a given case, separate parts should be stored in separate containers. Each container should be clearly la-beled with the surgical pathology number, the part number, the patient’s name, the patient’s medical record number, and a biohazard warning when indicated. Specimens that may be of special interest, either from a teaching, diagnostic, or medicolegal perspective, should be so designated and stored in a permanent storage area. Medical devices likewise should be placed in a properly labeled container and segregated into an area where they can be stored for long periods of time and easily retrieved. Unlike routine tissue specimens, storage of these prosthetic devices does not require fixation.

When preparing a specimen for storage, antici-pate the need to return to the specimen at a later time, either to review the gross findings or to submit more sections for histology. Although the specimen may be quite fragmented and distorted following its dissection, efforts should be made to reconstruct the specimen before placing it on a storage shelf. There are many examples of how this can be done. Lymph nodes and their associ-ated soft tissues can be separately wrapped and labeled according to their respective regional levels. Residual slices of a serially sectioned organ can be fastened together in their original posi-tions. Important landmarks can be designated with tags or safety pins. These simple methods of reconstructing the specimen can become in-valuable later, when, for example, you have to return to a colectomy to find more lymph nodes, to a prostatectomy to submit additional slices of prostate tissue, or to a mastectomy to sample a specific quadrant of the breast.