A Comparison of Four Mohs Tissue Preparation ... - Wiley Online Library

A Comparison of Four Mohs Tissue Preparation Methods Using Porcine Skin WILLIAM LEAR, MD, FRCPC, NORMA ANDERSON, HT(ASCP),y JENNIFER AKEROYD, BA, MIR, BScN,z y AND DANIEL BERG, MD, FRCPC

OBJECTIVE Mohs surgery relies on high-quality, rapid tissue preparation and processing. This study evaluated four currently performed tissue preparation and processing methods for speed of processing and depth of cut into the tissue block to achieve a complete high-quality section. METHODS The following four methods were tested: cryoEMBEDDER, float, heat sink, and slide. Standardized specimens of porcine skin were used to ensure uniformity. We measured the time required for a technician to flatten, embed, and cut to the first complete section of each specimen. Additionally, we measured the depth in microns required to cut into an embedded specimen to achieve a complete section. RESULTS There were advantages and disadvantages of each method, and our findings suggest that the heat sink and float methods are more time efficient but that the slide and cryoEMBEDDER methods require less cutting into the specimen to obtain a complete section. The cryoEMBEDDER device used in this study was loaned by cryoEMBEDDER (Salt Lake City, Utah).

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ohs surgery relies on a number of technical steps to achieve its high cure rate and tissuesparing properties. The tissue must be prepared in a manner that allows the full epidermis, dermis, and fat to be observed in a single en face section, ensuring that 100% of the surgical margin can be evaluated microscopically. The tissue preparation should minimize the depth that is required to be cut into the specimen or tissue block to get that full en face section to minimize false-positive readings. To obtain several sections for examination and because many specimens are not perfectly flat, any given specimen must be cut into to a certain depth to obtain a complete section.

Few studies assessing tissue-preparation methods have been reported in the literature. Tissue-processing methods reported in the literature include the conventional or heat sink approach,1 the Cryocup,1,2 the CryoHist,1,2 and the Miami Special.2 Bakhtar and Hanke,1,2 assessing tissue-processing time and

slide quality, found the CryoHist to be a superior vehicle for frozen section preparation, but based on a survey of the practices of Mohs surgeons,3 we chose to evaluate only the more common Mohs preparation methods of dermatologic surgeons, namely, the slide, float, and heat sink methods, as well as the cryoEMBEDDER (cryoEMBEDDER, Salt Lake City, UT) method. Aside from the cryoEMBEDDER, any of these methods can be performed without the purchase of additional pieces of equipment in any appropriate Mohs laboratory. We are not aware of a study that has assessed the time required to obtain a full en face specimen and the depth required to cut into the specimen to obtain that section. This study assesses these criteria.

Methods and Materials To obtain a large surface of skin with uniform physical properties for each method being analyzed,

Silver Falls Dermatology, Salem, Oregon; yDepartment of Dermatologic Surgery, University of Washington, Seattle,

Washington; zFaculty of Nursing, Oregon Health and Sciences University, Portland, Oregon & 2010 by the American Society for Dermatologic Surgery, Inc. Published by Wiley Periodicals, Inc. ISSN: 1076-0512 Dermatol Surg 2010;36:1419–1425 DOI: 10.1111/j.1524-4725.2010.01651.x 1419

C O M PA R I S O N O F F O U R M O H S T I S S U E P R E PA R AT I O N M E T H O D S

fresh pig bellies were used as the source of tissue. A large area of pig belly skin was excised from animals being used in an unrelated anesthesia study. The fresh specimen was excised as one large specimen and immediately chilled until use. The same author (WL) excised circular samples 1.0 cm in diameter and of a depth to the fat from the larger sample using the standard beveled Mohs technique. Each specimen was randomly allocated to one of the four methods. For each method, three specimens were processed as a single piece of tissue (one-piece), and three specimens were also bisected and processed as two pieces (two-piece, to better assess commonly used laboratory variations). In total, therefore, six samples were processed for each of the four methods (three one-piece and three two-piece), for a total of 24 specimens. The same surgeon (WL) mapped and marked each piece of tissue in the same manner.

Description of the Four Techniques cryoEMBEDDER Method: The specimen was placed with the surgical margin down on a cryoEMBEDDER disc that is on the top portion of the cold embedding device. The center of the tissue was pushed down to ensure that it laid flat on the disc, and then the margins were ‘‘teased down’’ until

Four methods of tissue processing (cryoEMBEDDER, float, heat sink, and slide) were evaluated; each method is described below. We borrowed the cryoEMBEDDER from a representative for the company. None of the authors have any financial interest in the cryoEMBEDDER. The embedding medium used in the preparation of all samples was Optimal Cutting Temperature Compound (OCT) (Skura Finetek USA, Inc., Torrence, CA). We chose to work with a single histotechnician (NA, our most experienced technician, with more than 5 years of full-time Mohs experience and more than 20 years experience with histology and frozen sections) to eliminate technician variability in the results. In addition, this technician had used all of the methods (except the cryoEMBEDDER) before the study. She became proficient with the cyroEMBEDDER system over a 1-month period before the study using videos, direct communication with the company representatives, and practice using pig belly tissue.

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Figure 1. (A) Teasing the epidermal edges of the specimen onto the prechilled cryoEMBEDDER disc. (B) Connecting the upper portion of the prechilled chuck and cryoEMBEDDER apparatus with the lower portion. (C) The tissue became solidified in the embedding medium of the chuck and was removed from the apparatus.

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Figure 2. (A) Mounting tissue specimen deep-side up on a chuck prepared with prechilled embedding medium. (B) Lowering prechilled heat extractor onto tissue in an effort to ‘‘flatten’’ the surgical margin. (C) Heat extractor removed from tissue.

the epidermis laid flat; the tissue freezes onto the cold disc. OCT was applied to a chuck and allowed to freeze inside the cryostat and then placed in the bottom portion of the embedding device. OCT was applied to the tissue-containing disc and the chuck, and the two embedding device parts were then immediately aligned. The embedding device was next placed back in the cryostat until the OCT was solidly frozen. The two parts were separated and the disc removed. The embedded tissue remaining on the chuck was then ready for sectioning (Figure 1). Float Method: OCT was applied to a cutting chuck and allowed to freeze in the cryostat. The chuck was then removed from the cryostat for another application of OCT. The tissue was placed surgical margin–side up on the OCT-containing chuck before the second application of OCT froze. The edges were

then gently teased up, and the chuck was placed in the cryostat. A chilled heat sink was placed on top of the tissue. The heat sink was then removed from the frozen specimen, leaving the embedded tissue on the cutting chuck ready to section (Figure 2). Heat Sink Method: OCT was applied to a cutting chuck and allowed to freeze in the cryostat. The specimen was placed surgical margin–side down on a chilled heat extractor. The margins were teased down onto the cold heat extractor until the tissue was stuck to the cold heat sink. OCT was then applied to the tissue on the heat extractor, covering it entirely. The heat extractor with the tissue was then immediately inverted onto the chuck containing OCT, and they were allowed to freeze together. The heat extractor was then pulled away from the chuck, leaving the tissue-containing chuck prepared for sectioning (Figure 3).

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Figure 3. (A) Teasing epidermal edges onto prechilled heat extractor. (B) Inverting tissue and heat extractor onto prechilled chuck with embedding medium. (C) Heat extractor removed from tissue.

Slide Method: The specimen was placed on a glass slide with its deep surgical margin against the slide. Visualization through the clear slide ensured that the tissue was flat on the slide and enabled us to remove air pockets from the bottom of the specimen that would have resulted in holes in the processed and stained tissue. The slide was placed on top of a large cotton swab that had been dipped in liquid nitrogen. The epidermis was then gently pressed down with

tweezers or the back of a scalpel blade while the tissue froze onto the slide (Figure 4). Once the deep margins were frozen flat, the slide was placed in the cryostat for 1 minute before being covered with OCT, which helps keep the tissue frozen and prevents it from lifting away from the slide while warm OCT is applied to the tissue. Additional OCT was then applied to the

Figure 4. (A) Using liquid nitrogen–dipped cotton swab to chill glass slide, tease epidermal edges of specimen onto slide. (B) Inverting chilled tissue and slide onto prechilled chuck with embedding medium. (C) Warmed back of slide with hand to release it from the tissue.

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TABLE 1. Depth Required to Cut into the Block to Obtain a Complete Section mm, Mean 7 Standard Deviation Method

Slide

cryoEMBEDDER

Heat Sink

Float

One piece Two pieces

853 7 227 370 7 104

833 7 261 600 7 230

960 7 433 930 7 375

1,160 7 485 870 7 160

OCT-prepared cutting chuck; the two were then put together by inverting the slide onto the chuck. A bulls-eye leveler was placed on top of the slide to ensure that the tissue was parallel to the knife. A heat extractor was then placed on top of the slide while allowing the tissue to continue to cool in the cryostat. Once the OCT was solidified, the slide was removed from the chuck, assisted by the warmth of the technician’s palm on the slide. For each method, we chose to assess tissue preparation and processing time, in seconds, and the depth, in microns, required to cut into the block to obtain a complete specimen. Time was measured from the moment the surgeon gave the inked specimen(s) to the technician until the first complete section of tissue was obtained from the block. The same experienced technician handled all specimens. Thus, our measure of time captured the time it took the technician to relax and flatten, embed, and cut the first complete section of tissue for each specimen. This was the chosen end point for time, because all specimens were treated the same, regardless of the depth required to obtain a full section. Sections from each specimen were obtained every 60 mm into the block up to a maximum of 1,040 mm. These sections were processed and stained using hematoxylin and eosin in an autostainer. The sections were examined microscopically to deter-

mine the lowest depth into the block at which a complete section was obtained. A complete section was defined as having at least 95% of the epidermis and a complete representation of dermis and fat without any artifacts.

Results Results were tabulated and analyzed and are presented in detail in Tables 1 and 2. The same data are graphically presented in Figures 5 and 6. The one-piece method generally required that the block be cut deeper into to obtain a full section. This difference was especially pronounced for the slide method, for which two-piece preparation required 50% less depth to be cut into the block to obtain a complete section. The heat sink and float methods were the most time efficient, but both preparation methods required cutting deeper into the block than with the slide and cryoEMBEDDER methods.

Discussion Past research has evaluated the number of micrometer slices required to examine the deep margin and

TABLE 2. Time Required to Prepare Mohs Specimen Using Each Method Seconds, Mean 7 Standard Deviation

One piece

Slide

cryoEMBEDDER

Heat Sink

Float

698 7 64

528 7 16

364 7 31

390 7 87

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1800

One piece 800

1400

700

1200

600

Time (seconds)

Depth (microns)

1600

900 One piece Two piece

1000 800 600

500 400 300

400

200

200

100

0 Slide

CE

HS

Float

0 Slide

CE HS Preparation Method

Preparation Method

Float

Figure 5. Graph of depth in microns required to be cut into block to obtain a full Mohs specimen for each method and for one- versus two-piece preparation.

Figure 6. Time in seconds required to obtain a full one-piece Mohs specimen from each method.

95% of the epidermal border, as well as tissue preparation time and quality of slides.

Because of the use of normal pig skin, we could not evaluate the fidelity (ability to detect tumor) of the methods in our study. The study showed that, when the tissue was bisected and processed as two pieces, less depth was required to obtain a complete section. On the other hand, and although not measured directly in this study, the time to process two pieces of tissue is clearly significantly more than the time to process one. Also, in comparing the use of one-piece and two-piece methods, theoretically, the two-piece method has a higher chance of giving false-positive deep margins, owing to the ability of nonmarginal

Each method and approach in our study has its own set of unique advantages and disadvantages, as outlined in Table 3. Each Mohs surgeon and laboratory needs to choose a method that works well for them, taking into account these advantages and disadvantages. In some laboratories, facility with the use of multiple methods would provide maximal flexibility in tissue handling.

TABLE 3. Advantages and Disadvantages of Preparation Methods Studied Method

Advantages

Disadvantages

cryoEMBEDDER

Requires purchase of cryoEMBEDDER Requires more processing time

Heat sink

Less depth into tissue to obtain a full section May be faster than slide method for one-piece specimens Less cutting into block to obtain full section High degree of control in laying down tissue Requires no extra equipment Relatively quick

Float

Relatively quick

Slide

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Requires most processing time

Requires cutting more into the block to obtain complete section Requires cutting more into the block to obtain complete section Tissue sections had more artifacts than with other methods

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epidermal and dermal tissue at the cut edge to inadvertently fold down and into the section. The main limitation in this study was the use of normal skin and thus the inability to assess fidelity of sections. In addition, this study was small and would be improved with a larger number of specimens. Although advantageous in providing consistency, having one histotechnician perform all techniques, in theory, may introduce bias because of a difference in her technical skill with each method. We felt that our histotechnician was experienced or adequately trained in all techniques, limiting this bias. Finally, other commonly used methods should be subject to further study to better categorize comparative advantages and disadvantages.

References 1. Bakhtar O, Close A, Davidson TM, Baird S. Tissue preparation for MOHS’ frozen sections: a comparison of three techniques. Virchows Arch 2007;450:513–8. 2. Hanke CW, Leonard AL, Reed A. Rapid preparation of highquality frozen sections using a membrane and vacuum system embedding machine. Dermatol Surg 2008;34:20–5. 3. Silapunt S, Peterson R, Alcalay J, Golderg L. Mohs tissue mapping and processing: a survey study. Dermatol Surg 2003;29:1109–12.

Address correspondence and reprint requests to: William Lear, MD, FRCPC, Private Practice, Silver Falls Dermatology, 1430 Commercial Street SE, Salem, OR 97302, or e-mail: [email protected]

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