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Chapter: Surgical Pathology Dissection : Laboratory Techniques

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Laboratory Techniques: Intraoperative Consultation

Important decisions in the operating room are frequently based on intraoperative consultation with a pathologist.

Intraoperative Consultation

Important decisions in the operating room are frequently based on intraoperative consultation with a pathologist. These intraoperative consulta-tions often require the rapid microscopic exami-nation of fresh tissue. This examination can be accomplished either by the preparation of cyto-logic slides or by the preparation of histologic slides using the frozen section technique.

Cytologic slides can be prepared from fresh specimens by one of three methods. Impression smears are produced by touching a microscopic slide to the cut face of the tissue. 

This procedure is also known as a touch preparation. The slide can be air dried or immediately fixed in alcohol for subsequent staining. The crush preparation is performed by taking a small (1-mm cube) piece of tissue and crushing it between two glass slides. The crush preparation can be fixed or air dried, then stained. Extremely hard tissues can be scraped with a sharp blade and the scrapingsdrawn across another slide in much the same way that a blood smear is prepared. Each of these techniques can be used on different tissues with varying degrees of success in terms of prepara-tion artifacts and quantity of cells obtained. For example, crush preparations work best on very soft tissues, while scrapings are needed on very firm tissues.

The frozen section is another procedure fre-quently used in intraoperative consultations. The details of preparing a frozen section vary greatly from one laboratory to another; however, one should become familiar with a few general concepts.

The first step is to select and prepare a piece of the specimen to freeze. Select a section that demonstrates the interface of the lesion with normal tissue, and try to avoid fatty or calcified tissues. The section should not be greater than 2 × 2 cm, and wet tissue should be gently blotted dry to avoid the formation of ice crystals. Very small pieces are easily lost in opaque embedding medium, so they should be stained with a drop of eosin or India ink before sectioning.

The second step is to freeze the tissue. Tissues are usually frozen on a tissue chuck in freezing medium by immersion in liquid nitrogen. This technique will lead to the formation of ice crys-tals, which can distort the histology. Some labo-ratories therefore prefer to use either refrigerated units with isopentane or cryostats equipped with specialized heat extractors.

Once the specimen is frozen, it is ready for sectioning. A variety of sectioning artifacts can be reduced with a few simple tricks. For example, sections that have been overly frozen will crum-ble when sectioned. In these cases, simply warm up the block by pressing a gloved thumb firmly onto its surface. (Be careful not to cut your finger on the cryostat blade.) Inadequate freezing, on the other hand, will cause the sec-tions to stick and bind. In these cases, the speci-men can be frozen to the appropriate temperature using a cooling spray, such as Histofreeze. Lines and knife marks can be avoided by using a clean and extremely sharp blade. Finally, loosely set screws can contribute to vibration artifacts, so if the sections look like corduroy pants, tighten all of the screws holding the block.

Once the section has been cut, it should be placed on a glass slide, fixed, and stained. A vari-ety of stains are employed in different labora-tories (see earlier for the H&E technique); but whichever stains are used, remember to take your time and follow the staining protocol. Too fre-quently, staining procedures are rushed, and the slides to be stained barely touch the staining solution. The resultant slides can be impossible to interpret or, ironically, may take longer to interpret than a slide stained correctly. Also, when one rushes, one often transfers solutions from one Coplin jar to the next. As a result, solu-tions are contaminated, and the quality of subse-quent frozen sections is diminished. This problem can be reduced simply by touching the edge of the slide to the edge of the jar before transferring the slide to a new solution. Another problem en-countered during staining is that tissue can fall off of the slide. If this happens, try using sialinated or other specially treated slides. The ultimate goal in preparation of a frozen section is to render a timely and accurate intraoperative diagnosis. Re-member to save the piece of tissue that was frozen so that it can serve as a frozen section control for diagnostic and quality control purposes.

 

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