Processing Adipose-Rich Mohs Samples: A Comparative Study of Effectiveness of Pretreatment With Liquid Nitrogen Versus Flash Freezing Spray

Abstract

Processing of adipose-rich Mohs micrographic surgery (MMS) specimens poses challenges that may preclude complete margin evaluation. In this setting, the value of additional freezing methods using various cooling agents has not been previously investigated. The aim of this study is to compare the frozen section quality of high-adipose Mohs specimens processed without additional cooling treatments versus those pretreated with 1,1,1,2-tetrafluoroethane (TFE) or liquid nitrogen (LN2). A set of 3 sections were each taken from 24 adipose-rich Mohs micrographic surgery specimens. A section from each set was subjected to either no additional cooling treatment (control), two 10-second pulse sprays of 1,1,1,2-tetrafluoroethane, or three 2-second pulse sprays of LN2. After staining, 2 blinded raters evaluated slide quality based on the presence or absence of the following features: margin completeness, nuclear clearing, epidermal or adipose folding, holes, or venetian blind-like artifacts. Pretreatment of the sample with LN2 produced a significantly (P < 0.001) greater number of high-quality slides (19/24) compared to pretreatment with 1,1,1,2-tetrafluoroethane (1/24) and no additional treatment (0/24). The adjunctive use of LN2 spray before tissue embedding circumvents the challenges of processing "thick" (high-adipose) specimens and facilitates the production of high-quality frozen section slides during Mohs micrographic surgery.

Reported Outcome Measures in Mohs Micrographic Surgery in Studies With Defined Techniques for Embedding and Processing of Tissue: A Systematic Review

“…In 20 studies, investigators made efforts to decrease histologic artifacts (33%), with those from 7 studies defining it as decreased freezing artifact for embedding medium, 5,[31][32][33]40,41,59 from 4 studies defining decreased artifact as decreased crush artifact during mounting, 14,31,49,67 from 1 study defining it as decreased venetian blind-type artifacts, 68 from 7 studies defining decreased artifact as decreased folds and air bubbles during tissue flattening, 12,54,55,58,[68][69][70] from 3 studies defining it as decreased holes in subcutaneous tissue, 17,20,68 and from others defining it as decreased floaters when staining or marking borders of tumor margin and avoidance of contamination during sectioning. 14,22,29,30,38 One study group assessed the overall appearance of slides using 3 criteria: the ability to visualize normal melanocytes, the ability to visualize atypical melanocytes, and the overall ability to see epidermal and dermal structures.…”

Optimal Preparation of Adipose Tissue in Frozen Sections for Mohs Micrographic Surgery

“…This requires the histotechnologist to first cut into the block so the spray may freeze an adequate portion of the tissue. 1,3 The spray is then discharged, which may agitate shavings from previously cut specimens introducing a safety hazard and risk of contamination. In our experience, coolant sprays cause tissue contraction away from the plane of sectioning, creating an uneven surface.…”

“…Such sections from tissue rich in adipose are best obtained at −28°C to −32°C, significantly lower than the −15°C to −20°C ideal for most other tissues. 3 Because of low thermal conductance, adipose freezes at a slower rate than epidermis, increasing the risk of forming ice crystals during freezing which appear as holes in the adipose on completed sections. 1,4 To mitigate this risk, we set our cryostats to −35°C and use Peltier plates (Figure 2), which are surfaces in the cryostat chamber thermoelectrically cooled to −55°C.…”