Evaluation of digital staining for ex vivo confocal laser scanning microscopy

Abstract

BackgroundEx vivo confocal laser scanning microscopy (CLSM) is a novel diagnostic tool for the fast examination of native tissue. However, CLSM produces black/white/green images, depending on the refraction indices of the tissue structures, complemented by nuclear fluorescence staining, which the vast majority of Mohs surgeons and dermatopathologists are not trained to interpret. Digital staining is applicable to ex vivo CLSM investigations to simulate the images of conventional slides stained with haematoxylin and eosin (H&E).ObjectivesThe aim of our study was to evaluate in detail the appearance of human skin structures using digitally stained ex vivo CLSM images and compare the results to that of conventional H&E slides of the same specimen.MethodsAfter providing informed consent, 26 patients donated their Burow's triangles (healthy skin) that resulted from plastic reconstruction after the R0 excision of skin tumours. After being investigated by ex vivo CLSM, including automated digital staining (VivaScope 2500M‐4G, MAVIG GmbH), the specimens were fixed in formalin, embedded in paraffin and stained with H&E.ResultsAlmost all skin structures in the digitally stained ex vivo CLSM images morphologically resembled the structures in the histopathological images acquired from H&E slides. Due to the high refraction index of melanin, the hair shafts appeared bright pink, and the melanocytes and melanophages were poorly imaged, resulting in a strong pink appearance that vastly differed from the appearance of conventional H&E‐stained histopathology.ConclusionsDigital staining of ex vivo CLSM images is an easy and highly useful tool to facilitate the interpretation of black‐field images generated by confocal laser scanning microscopy for dermatopathologists and Mohs surgeons who are familiar with H&E staining. Unlike the pigmented structures, the cutaneous and subcutaneous structures had excellent visualization with only minimal differences from their appearance on H&E slides.

Ex‐vivo fluorescence confocal microscopy with digital staining for characterizing basal cell carcinoma on frozen sections: A comparison with histology

“…In accordance with the first study on DS of healthy skin [24], we could visualize the main anatomical structures, as in H&E slides, with high concordance rates. Fat tissue was visible in every mode as an aggregation of empty, lobulated spaces with bright (blue‐lilac in DS) nuclear elements pushed to the periphery; muscle fibers were displayed as elongated elements with bright (blue‐lilac in DS) nucleus, while connective tissue in scars had a pink appearance in DS.…”

“…As a consequence, the user can experience a direct and intuitive comparison to the standard H&E features. A pilot study on healthy skin reported a very high correlation between digitally stained FCM mosaics and H&E sections, although no statistical analysis was provided [24]. The authors could correlate the morphology of main skin structures, including appendages, and even noticed a better visualization of adipocytes and hair follicles.…”

Routine application of ex vivo confocal laser scanning microscopy with digital staining for examination of surgical margins in basal cell carcinomas

“…This is achieved via the similarity of tissue reflection with eosin staining and replacement of the nuclear hematoxylin staining by acridine orange fluorescence [27]. Digital HE staining has already been comprehensively evaluated in healthy skin samples [28].…”

Exploring the utility of Deep Red Anthraquinone 5 for digital staining of ex vivo confocal micrographs of optically sectioned skin

“…While dermal fibers can be visualized because of their autofluorescence at 488 nm (vs 638 nm) in control unstained tissues, accentuation of dermal fibers (collagen, elastin) in AO images was prominently visible after staining, suggesting some degree of nonspecific staining. Histology correlation studies, as well as prospective clinical investigations to evaluate diagnostic accuracy (sensitivity, specificity, positive and negative predictive value), would be needed to definitively test and prove DRAQ5's enhanced diagnostic value relative to that of AO [24, 25].…”