In vivo optical imaging-guided targeted sampling for precise diagnosis and molecular pathology.

Conventional tissue sampling can lead to misdiagnoses and repeated biopsies. Additionally, tissue processed for histopathology suffers from poor nucleic acid quality and/or quantity for downstream molecular profiling. Targeted micro-sampling of tissue can ensure accurate diagnosis and molecular profiling in the presence of spatial heterogeneity, especially in tumors, and facilitate acquisition of fresh tissue for molecular analysis. In this study, we explored the feasibility of performing 1-2 mm precision biopsies guided by high-resolution reflectance confocal microscopy (RCM) and optical coherence tomography (OCT), and reflective metallic grids for accurate spatial targeting. Accurate sampling was confirmed with either histopathology or molecular profiling through next generation sequencing (NGS) in 9 skin cancers in 7 patients. Imaging-guided 1-2 mm biopsies enabled spatial targeting for in vivo diagnosis, feature correlation and depth assessment, which were confirmed with histopathology. In vivo 1-mm targeted biopsies achieved adequate quantity and high quality of DNA for next-generation sequencing. Subsequent mutational profiling was confirmed on 1 melanoma in situ and 2 invasive melanomas, using a 505-gene mutational panel called Memorial Sloan Kettering-Integrated mutational profiling of actionable cancer targets (MSK-IMPACT). Differential mutational landscapes, in terms of number and types of mutations, were found between invasive and in situ melanomas in a single patient. Our findings demonstrate feasibility of accurate sampling of regions of interest for downstream histopathological diagnoses and molecular pathology in both in vivo and ex vivo settings with broad diagnostic, therapeutic and research potential in cutaneous diseases accessible by RCM-OCT imaging.

Topical Aluminum Chloride and Monsel's Solution Block Toluidine Blue Staining in Mohs Frozen Sections: Mechanism and Solution.

BACKGROUND: A diminished-staining artifact is observed in some Mohs frozen sections that are stained in toluidine blue (T-blue). Such an artifact, not yet described in the literature, may interfere with a Mohs surgeon's accurate reading. The authors hypothesize that topical hemostatic agents, aluminum chloride, and Monsel's solution are the causative factors. OBJECTIVE: To evaluate the aforementioned topical hemostatic agents as a potential cause of the nonstaining artifact, to propose the mechanism associated with this phenomenon, and to develop a method to prevent or rectify the problem. MATERIALS AND METHODS: Leftover Mohs frozen sections and specimens were treated with aluminum chloride or Monsel's solution and processed with routine Mohs histology. RESULTS: Nonstaining artifact is reproduced in aluminum chloride or Monsel's solution-treated ex vivo skin specimens. The authors found that ethylenediaminetetraacetic acid (EDTA), a chelating agent, can reverse the staining blockage. Such a finding suggests that aluminum or ferric cations bind to tissue and subsequently inhibit T-blue from interacting with the tissue. Direct binding of ferric cations to the tissue section is demonstrated with Prussian blue iron staining. CONCLUSION: By rinsing Mohs frozen sections in an EDTA solution before T-blue staining, the authors could prevent hemostatic agent-induced nonstaining. Applying an EDTA wash and restaining the slides can correct the same artifact.