[Possibilities of multiphoton microscopy for the diagnosis of the vulvar lichen sclerosus]. / Vozmozhnosti mul'tifotonnoi mikroskopii dlya diagnostiki skleroticheskogo likhena vul'vy.

BACKGROUND: Vulvar lichen sclerosus (VLS) is a chronic and recurrent dermatosis of an inflammatory nature with severe focal atrophy of the skin. Connective tissue changes are polymorphic and are still not taken into account in histological diagnostics due to the difficulty of interpreting routine histological methods. In this work, we use multiphoton microscopy (MPM) as a new imaging technique that provides detailed information about the organization of collagen fibers in the dermis based on a non-linear second harmonic generation (SHG) process. OBJECTIVE: To determine the degree of connective tissue damage in lichen sclerosus using standard histological techniques and to reveal the diagnostic capabilities of multiphoton microscopy. MATERIAL AND METHODS: We studied 42 biopsies with a histopathological diagnosis of VLS and 10 biopsies of normal vulvar skin. Histological, histochemical and immunohistochemical evaluation was used in comparison with MPM data. Quantitative analysis included the determination of the thickness, length of collagen fibers and the average intensity of the SHG signal. RESULTS: A comprehensive study of the skin showed 4 groups of changes that can be regarded as the degree of the dermis damage: initial, mild, moderate, severe. The affected area at the initial and mild degree has subtle changes, however, it is reliably identified by quantitative analysis of the SHG signal. So, the initial degree is characterized by thin (1.3-1.8 µm) long (56-69 µm) collagen fibers, with a moderate degree, the fibers are thickened (3.4-4.3 µm) and fragmented (22-37 µm). The affected area in moderate and severe cases undergoes homogenization, which is associated with the deposition of extremely thin (0.6-0.9 µm) short (16-28 µm) collagen fibers and the expression of type V collagen. CONCLUSION: Multiphoton microscopy in the second harmonic generation mode is a reliable method for identifying collagen fibers in tissues. The study made it possible to identify 4 degrees of the dermis damage in vulvar lichen sclerosus.

Cross-Polarization Optical Coherence Tomography for Clinical Evaluation of Dermal Lesion Degrees in Vulvar Lichen Sclerosus.

The aim of the study was to identify different degrees of dermal lesions in vulvar lichen sclerosus (VLS) using cross-polarization optical coherence tomography (CP OCT) based on attenuation coefficient to detect disease early manifestations and to monitor the effectiveness of treatment. Materials and Methods: The study included 10 patients without pathology and 39 patients with VLS diagnosed histologically. CP OCT was performed in vivo on the inner surface of the labia minora, in the main lesion area. From each scanning point, a 3.4×3.4×1.25-mm3 3D data array was obtained in 26 s. CP OCT examination results were compared with histological examination of specimens stained with Van Gieson's picrofuchsin.Quantitative analysis of OCT images was performed by measuring the attenuation coefficient in co-polarization and cross-polarization. For visual analysis, color-coded charts were developed based on OCT attenuation coefficients. Results: According to histological examination, all patients with VLS were divided into 4 groups as per dermal lesion degree: initial (8 patients); mild (7 patients); moderate (9 patients); severe (15 patients). Typical features of different degrees were interfibrillary edema up to 250 µm deep for initial degree, thickened collagen bundles without edema up to 350 µm deep for mild degree, dermis homogenization up to 700 µm deep for moderate degree, dermis homogenization and total edema up to 1200 µm deep for severe degree.Pathological processes in dermis during VLS like interfibrillary edema and collagen bundles homogenization were visualized using CP OCT method based on values of attenuation coefficient in co- and cross-polarization channels. However, CP OCT method appeared to be less sensitive to changes of collagen bundles thickness not allowing to distinguish thickened collagen bundles from normal ones with enough statistical significance. The CP OCT method was able to differentiate all degrees of dermal lesions among themselves. OCT attenuation coefficients differed from normal condition with statistical significance for all degrees of lesions, except for mild. Conclusion: For the first time, quantitative parameters for each degrees of dermis lesion in VLS, including initial degree, were determined by CP OCT method allowing to detect the disease at an early stage and to monitor the applied clinical treatment effectiveness.