Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
  • A61B5/0062—Arrangements for scanning
  • A61B5/0068—Confocal scanning
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/44—Detecting, measuring or recording for evaluating the integumentary system, e.g. skin, hair or nails
  • A61B5/441—Skin evaluation, e.g. for skin disorder diagnosis
  • A61B5/444—Evaluating skin marks, e.g. mole, nevi, tumour, scar
• • G—PHYSICS
  • G06—COMPUTING; CALCULATING OR COUNTING
  • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
  • G06T7/00—Image analysis
  • G06T7/0002—Inspection of images, e.g. flaw detection
  • G06T7/0012—Biomedical image inspection
• • G—PHYSICS
  • G06—COMPUTING; CALCULATING OR COUNTING
  • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
  • G06T2207/00—Indexing scheme for image analysis or image enhancement
  • G06T2207/30—Subject of image; Context of image processing
  • G06T2207/30004—Biomedical image processing
  • G06T2207/30024—Cell structures in vitro; Tissue sections in vitro
• • G—PHYSICS
  • G06—COMPUTING; CALCULATING OR COUNTING
  • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
  • G06T2207/00—Indexing scheme for image analysis or image enhancement
  • G06T2207/30—Subject of image; Context of image processing
  • G06T2207/30004—Biomedical image processing
  • G06T2207/30088—Skin; Dermal

Definitions

• the invention deals with melanocyte imaging in histological skin sections by using reflectance confocal microscopy and with reflective melanocyte properties in healthy human skin as well as in melanocytic skin tumours such as nevi or melanomas.
• This invention is also related to the imaging that is used in non-invasive diagnostics.
• Reflectance confocal microscopy is a new non-invasive method for analysis of skin in vivo . Since the laser beam passes through skin cell and other skin structures possessing different light refraction properties (refractive index), the reflected light is caught, the information is processed by the computer and a two-dimensional image is obtained. A microscope provides sufficiently detailed images from different skin levels/depths which are used in histological studies. The first studies were carried out for determining healthy-skin cell populations existing at different cell levels.
• Patent application US2012170828 describes the use of reflectance confocal microscopy for diagnosing melanoma by using 3D confocal images and a corresponding computer software.
• Patent US2008319322 describes non-invasive in vivo study used for diagnosing and monitoring neurological diseases related to diabetes, HIV and other disorders.
• WO2013001113 is described a method in which histological images of X-gal strains are obtained by using confocal microscopy and fluorescence, thus determining X-gal sediment localization and dissemination direction at the cell level.
• the material under study is different.
• Patent US2011206254 describes a study of skin pigmentation by using diffuse reflection image. This method is used for determining the distribution of hemoglobin and melanin, as well as for determining the reflection of skin surface components. The drawback of this approach is that it cannot be used for determining the properties of melanin reflection.
• EP2068888 non-invasive system for determining mechanoreceptors also applies confocal microscopy, however, it does not analyze the properties of melanocyte reflection.
• the goal of our invention is to perform and evaluate the skin melanocyte imaging ex vivo by using the method of reflectance confocal microscopy and to study their reflection in healthy human skin as well as in melanocytic skin tumours - nevi and melanomas.
• Reflectance confocal microscopy is a method of non-invasive skin examination which helps to distinguish different microstructures according to the differences in light reflection. In medicine it is used for examining human tissues ex vivo , i.e., examining histological tissue sections, or in vivo , i.e., examining tissues at the cell level at the current moment without invasion into the organism. At present, in vivo reflectance confocal microscopy studies are carried out in such areas as skin, eyes, or mucous membranes of internal organs (e.g., stomach).
• Melanocytes containing melanin are contrasting when studied by reflectance confocal microscopy.
• Melanocyte is a skin cell which synthetizes melanin. Melanin synthesis takes place in melanosomes during their I-IV stages of maturity. Immunohistochemical methods can be applied for determining different melanosome stages in melanocytes.
• Melan-A reacts with all the melanosomes (i.e., in I-IV stages)
• HMB45 reacts specifically with immature melanosomes in stage II (5).
• III-IV stage melanosomes contain melanin.
• the maturity of melanocytes is immunohistochemically evaluated according to the stage of melanosomes.
• the atypical melanocytes of melanoma are characterized by dysfunctional maturation.
• the reflective properties of atypical melanocytes are extremely important in differential diagnosing of benign and malignant skin tumours of melanocytic origin.
• a system of non-invasive skin melanocyte imaging in skin was used. It consists of (a) a reflectance confocal microscope, used for obtaining the melanocyte reflection data of a particular skin area; (b) immunohistochemical staining for confirmation of melanocytic origin of cells and getting an immunohistochemical image, and (c) a computer for imaging the skin melanocytes.
• the system testing showed that the melanocyte cytoplasm and the nucleus reflection depend on the intensity of the possitivity on the immunohistochemical examination. Melanocytes were identified by the computer according to the characteristics of the existing melanosomes' light intensity.
• melanocyte cytoplasm reflection properties shown by the computer also depended on the stage of melanosome maturity.
• the melanocytes distributed among keratinocytes in the epidermis were hypocontrasting rather than sharply contrasting, a contrasting nucleus was observed both in healthy skin melanocytes and in nevus nevocytes, while the melanoma cells (atypical melanocytes) were hypo-, iso- and hyper-contrasting with respect to keratinocytes, therefore, it may be concluded that the different reflection properties of melanocyte cytoplasm depend on the melanosome maturity stage.
• the melanocyte cytoplasm with immature I-II stage melanosomes is light-conducting, and the nucleus is contrasting, while the melanocyte cytoplasm with mature III-IV stage melanosomes, which possess high light refraction properties, is light, and the nucleus is dark.
• the system of skin melanocyte imaging in skin is used for determining the properties of melanoma reflection, so that the obtained information could be used for diagnosing the patient's state or observation, where the application consists of:
• the melanocytes under study and the nevocytes (nests forming cell) were identified by the computer according to the light intensity characteristics of the contained melanosomes.
• the present invention is used for evaluating melanocyte reflection both in healthy skin and in melanocytic skin tumours.
• This invention can be used for examining and diagnostics of keratoma, melanoma, nevus or lentigo.
• FIG. 1 Histological section of normal skin (53 year old patient; localization - inner surface of the wrist; skin phototype I-II):
• Spitz nevus clinically a 3 mm diameter amelanotic papula with clear edges, on the inner surface of the wrist of a 56 year old female patient; skin phototype II:
• Fig. 3 Melanoma in situ (pigmented tumour, 53 year old female patient, thigh skin, skin phototype II):
• Fig. 4 Left - in vivo RCM obtained image (0.5 x 0.5 mm; depth - 30 ⁇ m below str. corneum) at the surface level of the epidermis-dermis junction; right - a correlating histological image of the same tumour micro preparation (HE staining, 20x).
• In vivo RCM characteristics clusters of dense cells deform dermal papillas (dark circular structures). Atypical skin architectonics has been noted, together with proliferation of collagen fibres (whitish derma papillas) and melanophages (single light-reflecting cells seen in dark derma papillas) in papillary dermis.
• the histopathological diagnosis a dysplastic nevus with a medium degree of cytological atypia.
• In vivo reflectance confocal microscopy examination of the selected samples of skin tumours with atypical features was carried out by using VivaScope 1500 apparatus (Lucid, JAV), following the manufacturer's recommendations for standard examination procedures (MAVIG training courses, Vilnius, 2010, Appendix 2).
• VivaScope 1500 apparatus Lucid, JAV
• MAVIG training courses Vilnius, 2010, Appendix 2
• disposable objective lenses were used for fixing skin to in vivo reflectance confocal microscopy apparatus: on the one side they were glued to the skin with fabric glue, on the other side - to the metal ring which was fixed to the confocal system with magnets (see the photo).
• Crodamol oil USA was trickled on the skin for immersion, and ultrasound gel was applied between the objective lense and the laser.
• the images of reflectance confocal microscopy were scanned through the whole area of the tumour, but not larger than 8 mm, at 30, 60 and 90 um from the skin surface. If the tumour was larger than 8 mm, confocal microscopy was carried out in the tumour place with the most obvious features of dermoscopic atypia.
• the skin structure changes characteristic of dysplastic nevi observed when examining skin in vivo by reflectance confocal microscopy imaging system, were analysed and evaluated in digital mosaic images (Vivablock) at 30, 60 and 90 um from the skin surface.
• the typical 'honeycomb' structure image of normal epidermis was evaluated at 30 um from the skin surface (Fig. 1). Its absence, i.e., chaotic positioning of cells without clear cell borders, was evaluated as 'loss of typical epidermic structure' (Fig. 2).
• Fig. 3 At the same level light-colour atypical cells, dendritic or large (over 20 um), can be seen. They are evaluated as 'pagetoid infiltration' (Fig. 3).
• the final diagnosis of pathology is made and the features of melanocyte atypia are determined at the National Centre of Pathology after the doctor-pathologist evaluates the results of the morphological study.
• the images of histological preparations stained with hematoxilin and eosin (HE) are scanned with the Aperio ScanScope GL objective lense scanner (Aperio Technologies, Vista, CA, JAV) at 20x magnification.
• cytological atypia of in vivo reflectance confocal microscopy and characteristic of melanocytic atypia were evaluated in benign, dysplastic and malignant melanocytic skin tumours.
• Parafin embedded formalin fixed tissue sample was cut into 3 ⁇ m-thick sections on a microtome. Then the sections were immersed in a hot water bath, after that transferred onto a microscope glass and dried in a hot oven.
• HMB45 monoclonal mouse antibodies against melanosoma
• Melan-A is carried out by using multimeric technology based on the detection system ultraView Universal DAB (Ventana, Arlington, AZ).
• HMB45 clone HMB45; DAKO, Glostrup, DK
• Melan-A antibodies clone A103; DAKO, Glostrup, DK
• 1:200 diluted solution due to the exposure - 32 minutes
• automated immunostainer Ventana BenchMark Ultra Ventana, Arlington, AZ
• CC1 standard conditioner
• Fig. 1 Ex vivo reflectance confocal microscopy of healthy skin (Fig. 1), Spitz nevus (Fig. 2) and cutaneous melanoma (Fig. 3) was performed. The obtained results (Fig. 1-3 A) were compared with immunohistochemical images (Fig. 1-3 B). Arrows mark melanocytes. The reflection of their cytoplasm and nucleus was evaluated by comparing it with keratinocyte reflection.
• Nevus cells have contrasting nuclei, while the cytoplasm contrast, when studied through a reflectance confocal microscope, is not bright, and directly correlates with a stronger HMB45 reaction (Fig. 2 picture on the right: a positive nevomelanocyte reaction stains the cytoplasm in brown colour).
• the melanocytes marked with arrows are hypo-contrasting compared to the surrounding keratinocytes when studied through a reflectance confocal microscope.
• melanocytes located among keratinocytes in epidermis were hypo-contrasting rather than sharply contrasting
• the contrasting nucleus was seen both in healthy skin melanocytes and in nevocytes
• melanoma cells typically melanocytes
• melanoma cells typically melanocytes
• the properties of melanocyto cytoplasm reflection differ depending on the melanosome maturity stage.
• Melanocytes with immature (I-II stage) melanosomas may possess both hypocontrasting and light-conducting cytoplasm and a contrasting nucleus.

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  • 2014-09-09 LT LT2014100A patent/LT6274B/lt not_active IP Right Cessation
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