Immunofluorescence and confocal microscopy for ex-vivo diagnosis of melanocytic and non-melanocytic skin tumors: A pilot study.

BACKGROUND: Ex-vivo confocal laser scanning microscopy (ex-vivo CLSM) offers rapid examination of freshly excised tissue. During the conventional examination immunohistochemistry enables to distinguish various cell types. The possibility of immunofluorescent techniques could enhance the accuracy of the diagnosis performed by ex-vivo CLSM. METHODS: The tissue probes from various skin tumors were stained with FITC-labeled S-100A10, Melan-A and anti-Ber-EP4 antibodies before examination with ex-vivo CLSM in the fluorescence and reflectance modes. Results were compared to negative controls and conventional histopathology. The staining protocols were evaluated by establishing a scoring system according to the signal intensity found in ex-vivo CLSM. RESULTS: S100 immunostaining was successful in 55.6%. Dilution of 1:200 resulted in the best possible evaluation of the tumor. The best suitable protocol was protocol B (phosphate buffered saline [PBS], without blocking agent). Melan A immunostaining was positive in 66.7%, the best dilution was 1:500 and protocol B (PBS, without blocking agent) was the most suitable. Ber-EP4 immunostaining presented a signal in 85.7%, the best dilutions were 1:200 and 1:500 and protocol A (PBS, with blocking agent) showed most optimal results. CONCLUSION: The use of fluorescent-labeled antibodies in ex-vivo CLSM is possible and could improve intraoperative diagnostics of skin tumors.

Identification of ex-vivo confocal laser scanning microscopic features of melanocytic lesions and their histological correlates.

Ex-vivo confocal laser scanning microscopy (CLSM) offers rapid tissue examination. Current literature shows promising results in the evaluation of non-melanoma skin cancer but little is known about presentation of melanocytic lesions (ML). This study evaluates ML with ex-vivo CLSM in comparison to histology and offers an overview of ex-vivo CLSM characteristics. 31 ML were stained with acridine orange or fluorescein and examined using ex-vivo CLSM (Vivascope2500® ; Lucid Inc; Rochester NY) in reflectance and fluorescence mode. Confocal images were correlated to histopathology. Benign and malignant features of the ML were listed and results were presented. Sensitivity and specificity were calculated using contingency tables. The ML included junctional, compound, dermal, Spitz and dysplastic nevi, as well as various melanoma subtypes. The correlation of the confocal findings with histopathology allowed the identification of different types of ML and differentiation of benign and malignant features. The study offers an overview of confocal characteristics of ML in comparison to histology. Ex-vivo CLSM does not reproduce the typical in-vivo horizontal mosaics but rather reflects the vertical histological presentation. Not all typical in-vivo patterns are detectable here. These findings may help to evaluate the ex-vivo CLSM as an adjunctive tool in the immediate intraoperative diagnosis of ML. Superficial spreading malignant melanoma. Histopathology (H&E stain; 200×) correlated to the reflectance (RM; 830 nm) and fluorescence mode (FM; 488 nm) in the ex-vivo CLSM (Vivablock® by VivaScan® , acridine orange).