LT6274B - Imaging of melanocytes by confocal reflection microscopy method in histological skin sections - Google Patents

Abstract

The invention relates to a system for skin melanocytes reflection testing in histological skin sections using confocal reflection microscopy method, and for testing its reflective properties of healthy human skin and melanocytic skin tumors, moles and melanoma, as well as the use of system.

Description

FIELD OF THE INVENTION The invention relates to the imaging of melanocytes in histological sections of the skin using confocal reflection microscopy, and to the properties of melanocyte reflection in healthy human skin and melanocytic skin tumors - birthmarks and melanomas. The present invention also relates to such imaging as a non-invasive diagnostic method.

Technical level

Confocal Reflection Microscopy (CAM) is a new non-invasive method for in vivo skin testing. Since the laser beam passes through the skin cells and other skin structures with different light-breaking properties (reflection index), the reflected light is captured and the information is decoded by computer hardware to obtain a two-dimensional image. The microscope allows you to present fairly detailed images from different skin levels / depths used in histological studies. The first studies were conducted to identify populations of different skin cells at different levels of the skin.

In vivo confocal reflection microscopy is a promising new technology used in non-invasive diagnostics of skin melanoma (1-2). In medical practice, the study was launched in 1995 when M Rajadhyaksha and co-authors in the Journal of Investigative Dermatology reported that melanin gives a strong contrast to the skin by examining it in vivo with a confocal reflection microscope (3). However, to date, there are no accurate data on melanocyte reflectance properties in skin in vivo confocal reflection microscopy. According to various authors, melanocyte may appear as a bright branched cell (4). In a joint agreement, the in vivo diagnosis of melanoma by confocal reflection microscopy is based on the discovery of melanocytes with contrasting cytoplasm and hypocontrast nuclei (5). An ex vivo or histological cut-off confocal microscopy method, in conjunction with immunohistochemical methods, could provide additional information on skin melanocyte reflection.

US2012170828 describes the use of confocal reflection microscopy for the evaluation of melanoma using 3D confocal images and the use of computer hardware.

US2008319322 describes a non-invasive in vivo study for evaluating or monitoring nerve diseases associated with diabetes, HIV and other diseases. A known method is described in VV02013001113, wherein confocal microscopy provides histological images of X-gal strain, also using fluorescence, thereby obtaining localization and propagation directions of X-gal at the cellular level. However, a completely different material is being studied. US2011206254, which describes a skin pigmentation assay using a diffuse reflection image. This method is used to determine the distribution of hemoglobin and melanin, as well as to determine the reflection of skin surface components. The drawback is that using this method there is no possibility to detect melanocyte reflection properties. It is known that EP2068888 is a non-invasive mechanism for determining mechanoreceptor levels, using confocal microscopy in the cell, but this method does not investigate the properties of reflection of melanocytes. OBJECT OF THE INVENTION The object of the invention is to perform and evaluate ex vivo visualization of skin melanocytes using the confocal reflection microscopy method and to investigate their reflection in healthy human skin and melanocytic skin tumors - birthmarks and melanomas.

Confocal reflection microscopy is one of the non-invasive skin test methods that help to distinguish different microstructures by the differences in light reflectance. In medicine, it is used for ex vivo research on human tissues, i. to examine histological tissue sections, or in vivo, i.e. Tissue research at the cellular level in the current time, avoiding invasion into the body. In vivo confocal microscopy studies are currently performed in the skin, eyes, mucous membranes of the internal organs (eg stomach).

Melanin-containing cells are contrasted by confocal reflection microscopy. Melanocyte is a melanin synthesizing skin cell. Melanin synthesis takes place in melanosomes during these transition stages from I to IV. Immunohistochemical methods can be used to detect different stages of melanosomes in melanocytes. Melan-A reacts with all (i.e., I-IV stages) melanosomes, HMB45 specifically reacts with non-mature stage II melanosomes (5). Melanosomes of stage III-IV have melanin. According to the melanosomal stage, the maturity of melanocytes is evaluated immunohistochemically. Melanocytic atypical melanocytes are characterized by impaired puberty. Reflection properties of atypical melanocytes are particularly important for differential diagnosis of benign and malignant melanocytic skin tumors.

A non-invasive skin melanocyte imaging system incorporating (a) a confocal image microscope for obtaining the data of the melanocytes of the desired skin region has been used to detect melanocyte reflection; (b) immunohistochemical staining to obtain an immunohistochemical image and (c) a computer for imaging skin melanocytes. Melanocyte cytoplasm and kernel reflection were determined by the intensity of the immunohistochemical assay. Melanocytes were identified by computer according to the light intensity characteristics of the melanosomes contained therein.

It was also found that the computerized melanocyte cytoplasm reflection properties depend on the maturity of melanosomes. In the epidermal layer, melanocytes, which are located between keratinocytes, were more likely to be hypocontrast than brightly contrasting, the contrasting nucleus was visible both in healthy skin melanocytes and in non-cytocytes, and melanoma cells (atypical melanocytes) were both hypo-, echo-, and hyper-contrast keratinocytes, concluding that melanocyte cytoplasm reflection properties differ depending on the maturity of melanosomes.

Melanocyte cytoplasm, which has non-primitive melanosomes of stage I-II, is permeable to light and the nucleus is contrasting, while melanocytic cytoplasm with mature III-IV melanosomes, which have strong light-breaking properties, is bright and the nucleus dark.

The skin melanocyte imaging system is designed to determine the properties of melanoma reflection in order to use the information obtained to evaluate or monitor the condition of a patient, the use comprising: (a) a confocal image microscope for imaging skin melanocytes; (b) immunohistochemical staining for determining the maturity of the reaction; and (c) a computer for analyzing digital images.

Melanocytes and non-cytocytes, which form cell nests, were identified by computer according to the light intensity characteristics of the melanosomes forming them. The present invention is used to evaluate melanocyte reflection in both healthy skin and melanocytic skin tumors. The present invention can be applied to the study and detection of keratoma, melanoma, mole, lentigo.

Drawings

FIG. 1. Histological incision of normal skin (53-year-old patient; localization - internal wrist surface; skin phototype I-II): (A) photo taken ex vivo by confocal reflection microscopy; (B) Image of the results of an immunohistochemical study of HMB45. The arrows mark the melanocytes of the basal epidermal layer, which are not cytoplasmic (i.e., dark) and have a slight contrast in the nuclei.

FIG. 2. Spitz Uncle: Clinically 3 mm diameter amelanotic papule with clear edges, 56 years old on the inner wrist surface; skin phototype II: (A) photo taken ex vivo by confocal reflection microscopy; (B) Image of the results of an immunohistochemical study of HMB45.

FIG. 3. Melanoma iri situ (pigmental tumor, 53-year-old patient; thigh; skin phototype II): (A) Ex vivo photo taken by confocal reflection microscopy; (B) Image of Melan-A immunohistochemical results (melanocytes are Melan-A positive, their cytoplasm brown). Most melanocytes (melanoma cells) are hypo- or isocontrastal to keratinocytes.

FIG. 4. Left - in vivo KAM image (0.5 x 0.5 mm; depth - 30 pm below Art. Cumumum) at the surface of the epidermal - dermal joint; right - correlating histological view of the same micropreparation of the same tumor (HE dyeing, 20x). Iri vivo KAM Signs: Dense cell clusters deform the dermal papilla (dark circular structures). An asthma of skin architectonics is observed, along with the proliferation of collagen fibers (white dermis papillas) and melanophages (single reflective cells visible in dark dermal papillas) in papillary dermis.

Diagnosis of Histopathology: Dysplastic Bumblebee with Moderate Cytological Atypical.

The most appropriate implementation options

Study Procedure 1. Patient Selection (VUOI suspected birthmark or skin melanoma); 2. Skin Tumor Dermoscopy and In vivo Confocal Skin Microscopy (VUOI) 3. Skin Tumor Excision (VUOI); 4. Investigations of morphology of skin tumors, diagnosis of definitive pathology and scanning of histological preparations (VPC); 5. In vivo analysis of skin confocal skin microscopy and histology images; 6. Analysis of accumulated data by statistical research methods; 8. Experimental investigation of histological skin incision confocal reflection microscopy.

Following surgical / excision biopsy and histological examination of the skin tumor, the subjects were divided into three groups based on the final diagnosis of pathology established by the State Pathology Center (VPC): Group I - patients diagnosed with benign melanocytic skin tumors (melanocytic moles) with no cytological atypical symptoms ( n = 25), Group II patients diagnosed with dysplastic moles (n = 69) and Group III patients with skin melanoma (n = 44). Based on TNM classification, 9 of the 44 skin melanomas were stage 0 (melanoma in situ), 35 - invasive i. Stages I-IV; average skin melanoma thickness according to Breslovv -1.35 mm.

The in vivo skin confocal reflection microscopy study of selected atypical skin tumors was performed using a VivaScope 1500 apparatus (Lucid, USA), following the manufacturer's recommendations for a standard test procedure (MAVIG Training Course Vilnius, 2010, Appendix 2). In the course of the study procedure, disposable objective glasses were applied to the skin for attachment to an in vivo confocal microscopy machine, glued to the skin on one side, and to a metal ring attached to the confocal system (photo). For skin immersion, Crodamol oil (US) is applied to the skin surface, and an ultrasonic gel is used between the lens slide and the laser. During the study, confocal reflection microscopy images were scanned across the entire, but not larger than, 8 mm tumor area at 30, 60, and 90 pm from the skin surface. If the tumor was larger than 8 mm, the confocal microscopy study was performed mainly on the tumor site with dermoscopic signs.

Changes in skin structure characteristic of dysplastic dysplasia are seen in the in vivo confocal skin imaging system for analyzing and evaluating Vivablock images 30, 60 and 90 μ 30η from the skin surface. The typical honeycomb image of the normal epidermis is estimated at 30 pm from the skin surface (Figure 1). Its absence, i.e. The disordered cell placement when there are no clear cell boundaries was seen as "a characteristic loss of epidermal structure" (Figure 2). At the same level, light atypical cells, branched or large (greater than 20 pm) are seen as "pediatric infiltration" (Figure 3). Cell sockets, their homogeneity and boundaries were evaluated at depths of 60 and 90 µm from the surface of the skin. Those who do not have clear boundaries or integrity cells are considered to be "disco-lean" or atypical single-cell sockets (Figure 4). Meanwhile, nests with lightly separable boundaries of normal monomorphic cells are referred to as "dense cell nests" (Figure 5).

Morphology studies of skin tumors, diagnosis of the final pathology and scanning of histological preparations

The final diagnosis of the pathology and the signs of melanocyte atypical disease were determined by the State Pathology Center after the assessment of morphology studies by a pathologist. Images of haematoxylin and eosin stained histology preparations were scanned at a 20x magnification of Aperio ScanScope GL lens slide scanner (Aperio Technologies, Vista, CA, USA).

In vivo analysis of skin confocal skin microscopy and histology images

An analysis of the in vivo skin confocal microscopy images of the tumors investigated and the images of the micropreparations of the same tumors stained by H / E were performed by a dermatologist. Diagnostic criteria / traits of melanocytic skin tumors in vivo skin confocal reflection microscopy described by other investigators for characterizing cytological atypical were analyzed.

Five criteria for in vivo confocal reflection microscopy were used for statistical analysis: epidermal structure, pediatric infiltration, dense, non-homogeneous, or dyslexic cell sockets. They were analyzed and compared with the hematoxylin and eosin-stained tumor micropreparations scanned in histological images. The correlation between in vivo confocal reflection microscopy and H / E stained micropreparation images is shown in Fig.

Signs of cytological atypical atypical atopic skin confocal reflection microscopy typical of melanocytic atypic were assessed in benign, dysplastic and malignant melanocytic skin tumors.

Preparation of histological skin sections

Paraffinic formalin fixed tissue column is cut into 3 μητι thickness microtome. The incisions are then immersed in a hot water bath, transferred to a microscope slide and dried in a hot oven.

Ex vivo Confocal Reflection Microscopy

Histological skin incisions are used for ex vivo confocal microscopy. With this method, they are tested on a Nikon Eclipse ΤΕ2000Γ microscope with C1si confocal scanning system (lens: x60 Plan Apo VC oil, 1.4 numerical aperture). For illumination use a 515 nm wavelength argon ion laser, a standard 12-bit RGB detector for detection and a 500-530 nm pass-through filter. Image processing software Nikon EZ-C1 Bronze version 3.80 and ImageJ 1.45.

Immunohistochemical examination of these sections is performed after receiving histological images of the cutaneous confocal sections.

Immunohistochemical studies

Immunohistochemical studies with monoclonal mouse antibodies melanosomes (HMB45) and Melan-A are performed using multimeric technology based on detection system ultraView Universal DAB (Ventana, Tucson, AZ). HMB45 (clone HMB45; DAKO, Glostrup, DK) and Melan-A antibodies (clone A103; DAKO, Glostrup, DK) are coated using a 1: 200 dilution solution (exposure time 32 minutes) followed by autoimmune Ventana BenchMark Ultra (Ventana , Tucson, AZ) and Cell Conditioning (Standard Conditioner CC1) 95 ° C for 64 minutes.

Ex vivo analysis of skin confocal skin microscopy and histology images

Because in vivo skin confocal microscopy confocal reflection can not tell which cell is keratinocyte, and which melanocyte, we performed ex vivo confocal microscopy and immunohistochemical examinations of skin histological increments to evaluate melanocyte reflection.

Ex vivo confocal microscopy of healthy skin (Fig. 1), Spitz birthmark (Fig. 2) and skin melanoma (Fig. 3) was performed. The obtained images (Fig. 1-3A) are compared with immunohistochemical images (Fig. 1-3B). The arrows are marked with melanocytes. Their cytoplasm and nucleus reflection was evaluated in comparison to the reflection of keratinocytes.

Cytoplasmic cells (nevomelanocytes) have contrasting nuclei, and the contrast of cytoplasm by confocal reflection microscopy is blurred, directly correlated with a stronger HMB45 reaction (in the right picture a positive nevomelanocyte response stains the cytoplasm brown). The arrowheaded melanocytes are hypocontrastic by confocal reflection microscopy compared to the around keratinocytes.

Ex vivo confocal reflection microscopy is used to visualize both skin keratinocytes and melanocytes, and in combination with immunohistochemical methods to evaluate skin melanocyte reflection in both healthy skin and melanocytic skin tumors.

According to the results of the study, melanocytes, which are located between keratinocytes in the epidermis layer, were more likely to be hypocontrast than brightly contrasting, the contrasting nucleus was visible both in healthy skin melanocytes and in the non-cytotoxic, and melanoma cells (atypical melanocytes) were both hypo-, tertiary, and hypertrophic. for keratinocytes, it is concluded that the properties of melanocyte cytoplasm reflection differ according to the maturity of melanosomes. Melanocytes with non-invasive (stage I-II) melanocytes may have both a hypocontrast and a light-transmissive cytoplasm and a contrasting nucleus.

LITERATURE 1. Bush, KJ, Charles C, Lohmann CM, Marghoob A, Goldgeier M, Halpern AC. Detection of intraepidermal malignant melanoma in vivo by confocal scanning laser microscopy. Melanoma Res. August 2002; 12 (4): 349-55. 2. Gerger A, Viltil M, Langsenlehner U, Richtig E, Hom M, Weger W, et al. Diagnostic image analysis of malignant melanoma in vivo confocal laser scanning microscopy: A preliminary study. Skin Res Technol. August 2008; 14 (3): 359-63. 3. Rajadhyaksha M, Grossman M, Esterovvitz D, Webb RH, Anderson RR. In vivo confocal scanning laser microscopy of human skin: Melanin provides strong contrast. J Invest Dermatol. 1995 Jun; 104 (6): 946-52. 4. Bush Bush, Charles C, Lee G, Halpern AC. Morphologic features of melanocytes, pigmented keratinocytes, and melanophages by in vivo confocal scanning laser microscopy. Mod Pathol 2001; 14 (9): 862-8. 5. Scope A, Benvenuto-Andrade C, Agero AL, Malvehy J, Puig S, Rajadhyaksha M, et al. In vivo reflectance confocal microscopy imaging of melanocytic skin lesions: Consensus terminology glossary and illustrative images. J Am Acad Dermatol. 2007 Oct; 57 (4): 644-58. 6. Naoki Oiso and Akira Kavvada (2011). The Stage of Melanogenesis in Amelanotic Melanoma, Melanoma in the Clinic - Diagnosis, Management and Complications of Malignancy, Prof. Mandi Murph (Ed.), ISBN: 978-953-307-571-6, InTech, DOI: 10.5772 / 18628.

Claims (13)

DEFINITION DEFINITION A skin melanocyte imaging system comprising: (a) a confocal image microscope for imaging skin melanocytes; (b) immunohistochemical reactions to determine the maturity of melanocytes; (c) a computer for digital image analysis, characterized in that melanocytes, as cells of various maturity, are identified in digital images according to the light intensity characteristics of melanosomes. 2. The system of claim 1, wherein the melanocyte cytoplasm reflection properties of the computer digital images depend on the maturity of the melanosomes. 3. The system according to claim 1 or 2, characterized in that the system is adapted to differentiate melanosome according to the melanocyte cytoplasm containing non-mature I-II melanosome, which is permeable to light and the nucleus is contrasting. 4. The system according to any one of claims 1 to 3, characterized in that the system is adapted to differentiate the melanosome according to the melanocyte cytoplasm having mature melanosomes of stage III-IV, which have strong light-breaking properties which are bright and the nucleus is dark. The use of a skin melanocyte imaging system according to claim 1 for determining melanocyte reflectance properties for use in evaluating a patient's condition or in vivo monitoring for use in a CAM, the use of which comprises: (a) using a confocal image microscope for imaging skin melanocytes; (b) using immunohistochemical reactions to determine the maturity of melanocytes; (c) using a computer for digital image analysis; characterized in that melanocytes, as cells of various maturity, are identified in digital images according to the light intensity characteristics of melanosomes. Use according to claim 5, characterized in that the melanocyte cytoplasm reflection properties of the computer digital images depend on the maturity of the melanosomes. Use according to claim 5, characterized in that the system is adapted to differentiate melanosome according to the melanocyte cytoplasm containing non-mature I-II melanosome, which is permeable to light and the nucleus is contrasting. 8. Use according to claim 5, characterized in that the system is adapted to differentiate melanosome by melanocyte cytoplasm having mature melanosomes of stage III-IV, which have strong light-breaking properties which are bright and the nucleus is dark. 9. The use according to claim 5, wherein the condition is healthy skin. 10. The use according to claim 5, wherein the condition is a melanocytic skin tumor. 11. Use according to claim 10, wherein the melanocytic skin tumor is keratoma, melanoma, moles, lentigo. 12. Use according to claim 11, wherein the melanocytic skin tumor is a birthmark. 13. Use according to claim 11, wherein the melanocytic skin tumor is melanoma.