Abstract
Mohs surgery uses en face frozen section analysis (FSA) with complete margin examination for the excision of select basal cell carcinomas (BCC), obtaining excellent cosmetic outcomes and extremely low recurrence rates. However, Mohs with FSA is time-consuming because of the need to iteratively perform cryosectioning on sequential excisions. Fluorescent microscopies can image tissue specimens without requiring physical sectioning, potentially reducing the time to perform Mohs surgery. We demonstrate a protocol for nonlinear microscopy (NLM) imaging of surgical specimens that combines dual agent staining, virtual H&E rendering, and video rate imaging. We also introduce a novel protocol that enables micron-level co-registration of NLM images with FSA histology, and demonstrate that NLM can reproduce similar features similar to FSA in BCC specimens with both negative and positive surgical margins. We show that the fluorescent labels can be extracted with conventional vacuum infiltration processing, enabling subsequent immunohistochemistry on fluorescently labeled tissue. This protocol can also be applied to evaluate the performance of NLM compared with FSA in a wide range of pathologies for intraoperative consultation.
Highlights
Keratinocytic carcinoma is the most common form of cancer in the US, with estimates as high as 5.4 million cases in 2012 [1], more than all other forms of cancer combined [2]
We demonstrate nonlinear microscopy (NLM) images of Mohs surgical margins that are precisely co-registered with frozen section analysis (FSA) histological sections, enabling the same tissue features to be visualized on both modalities
We demonstrated a staining protocol and NLM imaging on Mohs specimens of basal cell carcinomas (BCC) and squamous cell carcinoma (SCC) which generates images comparable to FSA histology and does not interfere with IHC
Summary
Keratinocytic carcinoma is the most common form of cancer in the US, with estimates as high as 5.4 million cases in 2012 [1], more than all other forms of cancer combined [2]. The incidence of keratinocytic carcinoma has increased 350% over the last 2 decades due to aging populations and increased sun exposure [1]. If specimens could be rapidly prepared and imaged, the surgeon could operate on one patient at a time, dramatically reducing each patient’s surgical time. This would reduce patient anxiety and the amount of analgesic required, while simplifying clinic workflows. New technologies that enable rapid evaluation of specimens would reduce labor and processing delay as well as patient discomfort
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