In vivo reflectance confocal microscopy of shave biopsy wounds: feasibility of intraoperative mapping of cancer margins.

BACKGROUND: Reflectance confocal microscopy (RCM) images skin at cellular resolution and has shown utility for the diagnosis of nonmelanoma skin cancer in vivo. Topical application of aluminium chloride (AlCl(3)) enhances contrast in RCM images by brightening nuclei. OBJECTIVES: To investigate feasibility of RCM imaging of shave biopsy wounds using AlCl(3) as a contrast agent. METHODS: AlCl(3) staining was optimized, in terms of concentration vs. immersion time, on excised tissue ex vivo. RCM imaging protocol was tested in patients undergoing shave biopsies. The RCM images were retrospectively analysed and compared with the corresponding histopathology. RESULTS: For 35% AlCl(3) , routinely used for haemostasis in clinic, minimum immersion time was determined to be 1 min. We identified three consistent patterns of margins on RCM mosaic images by varying depth: epidermal margins, peripheral dermal margins, and deep dermal margins. Tumour islands of basal cell carcinoma were identified at peripheral or deep dermal margins, correlating on histopathology with aggregates of neoplastic basaloid cells. Atypical cobblestone or honeycomb patterns were identified at the epidermal margins in squamous cell carcinomas, correlating with a proliferation of atypical keratinocytes extending to biopsy margins. CONCLUSIONS: RCM imaging of shave biopsy wounds is feasible and demonstrates the future possibility of intraoperative mapping in surgical wounds.

Detection of basal cell carcinomas in Mohs excisions with fluorescence confocal mosaicing microscopy.

BACKGROUND: High-resolution real-time imaging of human skin is possible with a confocal microscope either in vivo or in freshly excised tissue ex vivo. Nuclear and cellular morphology is observed in thin optical sections, similar to that in conventional histology. Contrast agents such as acridine orange in fluorescence and acetic acid in reflectance have been used in ex vivo imaging to enhance nuclear contrast. OBJECTIVES: To evaluate the sensitivity and specificity of ex vivo real-time imaging with fluorescence confocal mosaicing microscopy, using acridine orange, for the detection of residual basal cell carcinoma (BCC) in Mohs fresh tissue excisions. METHODS: Forty-eight discarded skin excisions were collected following completion of Mohs surgery, consisting of excisions with and without residual BCC of all major subtypes. The tissue was stained with acridine orange and imaged with a fluorescent confocal mosaicing microscope. Confocal mosaics were matched to the corresponding haematoxylin and eosin-stained Mohs frozen sections. Each mosaic was divided into subsections, resulting in 149 submosaics for study. Two Mohs surgeons, who were blinded to the cases, independently assessed confocal submosaics and recorded the presence or absence of BCC, location, and histological subtype(s). Assessment of confocal mosaics was by comparison with corresponding Mohs surgery maps. RESULTS: The overall sensitivity and specificity of detecting residual BCC was 96.6% and 89.2%, respectively. The positive predictive value was 92.3% and the negative predictive value 94.7%. Very good correlation was observed between confocal mosaics and matched Mohs frozen sections for benign and malignant skin structures, overall tumour burden and location, and identification of all major histological subtypes of BCC. CONCLUSIONS: Fluorescent confocal mosaicing microscopy using acridine orange enables detection of residual BCC of all subtypes in Mohs fresh tissue excisions with high accuracy. This observation is an important step towards the long-term clinical goal of using a noninvasive imaging modality for potential real-time surgical pathology-at-the-bedside for skin and other tissues.

Confocal mosaicing microscopy in skin excisions: a demonstration of rapid surgical pathology.

Precise micro-surgical removal of tumour with minimal damage to the surrounding normal tissue requires a series of excisions, each guided by an examination of frozen histology of the previous. An example is Mohs surgery for the removal of basal cell carcinomas (BCCs) in skin. The preparation of frozen histology is labour-intensive and slow. Confocal microscopy may enable rapid detection of tumours directly in surgical excisions with minimal need for frozen histology. Mosaicing of images enables observation of nuclear and cellular morphology in large areas of surgically excised tissue. In skin, the use of 10-1% acetic acid as a reflectance contrast agent brightens nuclei in 0.5-5 min and enhances nuclear-to-dermis contrast and detectability of BCCs. A tissue fixture was engineered for precisely mounting surgical excisions to enable mosaicing of 36 x 36 images to create a field of view of 12 x 12 mm. This large field of view displays the excision at 2x magnification, similar to that routinely used by Mohs surgeons when examining frozen histology. Comparison of mosaics to histology demonstrates detectability of BCCs. Confocal mosaicing presently requires 9 min, instead of 20-45 min per excision for preparing frozen histology, and thus may provide a means for rapid pathology-at-the-bedside to expedite and guide surgery.