Confocal Microscopy

From dermoscopedia
Main PageConfocal Microscopy
5.00
(2 votes)
 Author(s): Giovanni Pellacani
Annotations
Description This page has not yet been summarized.
Author(s) Giovanni Pellacani
Responsible author Giovanni Pellacani→ send e-mail
Status unknown
Status update August 30, 2017
Status by Ralph P. Braun


Introduction:

In vivo reflectance confocal microscopy (RCM) is a non-invasive imaging technique able to generate quasi-histologic resolution images of the skin. Since RCM provides, similarly to dermoscopy, horizontal sections of the skin up to the superficial dermis and works, similarly to histopathology, at cellular level resolution, it provides the bridge between RCM and histopathology. For this reason the application of RCM has been used in conjunction with dermoscopy in order to obtain information concerning the cyto-architectural substrate of the different dermoscopic pattern and to help to gain a more precise diagnosis in difficult lesions. RCM can be used in different fields of dermatology. In fact, it can be applied in inflammatory skin diseases, or used for the study of the skin in different pathophysiologic conditions, or employed for the study of efficacy of cosmetic products or topical drugs. However, since the contrast is mainly provided by presence of organelles and of light reflecting structures, with the highest index given by melanin, RCM is commonly used for diagnosis of skin tumours. In different settings, the use of RCM for the diagnosis of dermoscopically difficult lesions resulted in a significant improvement of specificity, thus showing to be able to save a consistent number of unnecessary excisions.


Technology:

RCM is basically a confocal microscope able to detect the light reflected by subcellular structures at a desired focal point that can be used to in vivo and non-invasively study the skin. It uses a low-power laser source (diode laser at 830 nm wavelength) that penetrates the skin. The light is reflected from subcellular structures at a desired focal point and filtered through a pinhole before entering the detector in order to reject all the light coming back from out-of-focus planes (confocal principle). The optical system consists of 30 x 0.9 n.a. lens. This process allows to obtain grey-scale horizontal images of an area of 0.5 / 0.8 square mm of the skin up to a depth of approximately 250 μm, giving a lateral resolution of approximately 1 μm. At a cellular level, melanocytes, pigmented keratinocytes, melanin-containing cells (such as melanophages) and metabolically activated inflammatory cells strongly reflect 830 nm laser light and appear bright. Nowadays there is only one producer of RCM device, Caliber ID (Rochester, NY, USA), that produces 2 different models of RCM for in vivo / skin applications: Vivascope 1500(r) and Vivascope 3000 (r). The 2 devices do not differ in the confocal technology, thus they provide similar quality images of the skin, but in their mechanical asset. In fact, confocal optical system of Vivascope 1500 is mounted with an automatic and controlled moving system that enables to acquires consecutive images on the same plan, and mount them together in order to obtain a mosaic of up to 8 x 8 mm. To avoid the misalignment of the single images during the acquisition process, the head of the device should be maintained connected to the skin through a bridging metallic ring, that on a side is stitched on the skin via an adhesive window, and on the opposite side is connected with the RCM probe through magnets. On the other hand, Vivascope 3000 (r) is an hand-held tool containing the confocal optical system which can be directly applied and manoeuvred on the skin, immediately providing the images of the area where it is applied on. Although with Vivascope 1500(r) the preparation needed for the acquisition is time consuming (average 10 min per a full set of images for one lesion), the possibility to study mosaics including the entire lesion is needed to provide a confident diagnosis, especially in case of melanocytic tumours.

The aspect of Normal Skin

Keratinocytes appear as polygonal cells with decreasing size from top (surface/stratum corneum) to bottom (basal layer). Moreover the nucleus is not present inside the stratum corneum cells. The brighter intercellular contours give rise to the so called "honeycombed pattern" corresponding to unalterated keratinocytes. At the dermoepidermal junction, rings of basal keratinocytes surround dark circular structures (dermal papillae) containing bundles of grossly reticulated fibers (collagen) and sometimes showing a canalicular structure (blood capillary). In normal human skin, melanocytes and pigmented keratinocytes cannot be distinguished, as well as other cell population, such as Langerhans cells, cannot be detected when in their baseline/quiescent status. Appendageal structures, such as pilosebaceous units, eccrine glands and sebaceous glands, can be alsoseen by RCM

Application in skin cancer diagnosis

RCM represents an adjunctive diagnostic aid for the Clinician asked to diagnose lesions presenting equivocal clinical and/or dermoscopic features. Even though histopathology remains the gold standard for the diagnosis of skin cancers, dermoscopy and RCM are able to improve the early recognition of a skin malignancy and reduce unnecessary excisions of benign lesions in order to rule out a melanoma. To improve diagnostic accuracy, however, minimizing the risk to miss a melanoma, RCM is indicated as a second-level examination; in fact its systematic application into the diagnostic process greatly improved specificity, with consequent reduction of unnecessary excisions of benign lesions, in different prospective interventional studies.

Melanoma

RCM criteria for melanoma diagnosis are mainly three:

1. identification of atypical cells (corresponding to atypical melanocytes) within the epidermis

RCM "Atypical cells" correspond to large cells with variable shapes (round, oval, stellate, dendritic, polygonal or bizarre) with a bright grainy cytoplasm and dark nucleus located within the epidermal layers, and/or infiltrating the upper dermis, sparse and isolated, or forming aggregates (nests). When located in the epidermal layers, upper than basal layer, these cells are also called "pagetoid cells" for their strong correspondence with pagetoid melanocytes upon histopathology. Presence of more than 5 roundish/polymorphic pagetoid cells per square mm, or numerous densritic cells (more than 10 cells in a contest of numerous filaments corresponding to dendrites), are strong clues for melanoma diagnosis. However, numerous dendric cells could still represent a pitfall since presence of numerous activated Langerhans cells could be a confounder. When detected at basal layer number and cell polymorphism should be taken into account, since a few (less than 5 per square mm) roundish not pleomorphic cells could be frequently observed in mild dysplastic nevi.

2. architectural disorder at the dermal-epidermal junction.

Architectural disorder correspond to internal evident dishomogeneity of the lesion architecture (with a chaotic assembly of different structures), and/or presence of the so called "non-edged papillae" extended over the 10% of the lesion area. "Non-edged papillae" defines the presence of areas within a mosaic acquired at the dermal epidermal junction where the border between the epidermis and dermis is not clearly outlined, and they are correlated with increasing strength to variably sized junctional nests, discohesive junctional nests and flattened rete ridge: 'non-edged papillae are actually the end result of any process that obscures the basal keratinocytes, which create the “edge.” Thus, both a disorderly melanocytic proliferation occupying the dermal-epidermal junction, discohesive junctional nests and non-melanocytic causes, such as a lichenoid or interface inflammatory infiltrate, could result in non-edged papillae'

3. atypical/irregular aggregates of melanocytes

Solid proliferation of the tumour within the dermis forming large aggregates (nests or cells distributed in sheets) are clues of melanoma when they present large atypical and pleomorphic cells or they have a "cerebriform" clusters (correspond to cellular clusters consisting of confluent amorphous aggregates of low reflecting cells exhibiting granular cytoplasm without evident nuclei and ill defined borders, being the aggregates brain-like in appearance, showing a fine hyporeflective «fissure» like aspect). The only presence of large round, but monomrphic, cells in a contest of a nest should not be considered a clue for melanoma, since it represents a common observation in compound/intradermal nevi, especially in congenital nevi. Benign lesions (especially melanocytic nevi) can be therefore recognized first for the lack of melanoma suggestive features. Junctional nevi usually are characterized by a predominance of a ringed pattern (presence of bright rings at the dermal-epidermal junction) due to the increased number of melanocytes predominantly proliferating in single cells at the junction. Compound nevi shows ringed pattern and/or meshwork pattern (presence of fusiform or ovoidal enlargements in the interpapilary spaces corresponding to junctional nests) and nests (compact with no atypical/pleomorphic cells) within dermal papillae. Intradermal nevi

Basal cell carcinoma

RCM is an excellent tool that provides an accurate non-invasive diagnosis of basal cell carcinoma (BCC). Tumour cells are visible in the contest of aggregates round to oval in shapes or elongated cords, surrounded by polygonal cells disposed along the periphery (palizading), usually separated from the sourrounding stroma by a dark cleft. Detection of tumour-specific aspects corresponds to an almost 100% specific diagnosis. Presence of pigment inside tumour cells enanches the aspect facilitating the visualization of tumour specific structures. Whereas, lack of pigment leads to presence of dark silhouettes. Other prameters identified for BCC diagnosis are the presence of elongated monomorphic nuclei, polarization of the nuclei along the same axis, prominent inflammatory infiltrate, increased dermal vasculature and pleomorphism of the overlying epidermis. RCM helps also in the differentiation between superficial, nodular and infiltrative BCC subtypes. Superficial BCCs are characterized cords connected to the epidermis with peripheral palisading, thickened collagen surrounding the cords and streaming of the epidermis. Nodular BCCs often present tumor islands, clefting and peripheral palisading. Infiltrative BCCs are characterized by dark silhouettes and abundant collagen sorrouding the islands.

other non melanoma skin cancer

RCM helps the diagnosis of other non-melanoma skin neoplasm. Benign non-melanoma skin neoplasms: solar lentigo is characterized by polycyclic papillary contours, corresponding to elongation and fusion of epidermal cristae. Seborrheic keratosis presents roundish onion-like reflecting structure (corneal cysts) in a contest of thickened epidermis. In addition, abundant inflammatory inflitrate, mainly melanophages, is seen in lichen planus-like keratoses. Epithelial carcinomas (namely actinic keratosis and squamous cell carcinomas): these tumours are almost constantly characterized by alteration of the epidermal honeycombed pattern, with irregular cells variable in size and shape, frequently presenting large nuclei, corresponding to dyskeratotic keratinocytes. Moreover, parakeratosis (showing up as round targetoid structures or very reflecting polygonal cells in the corneum layer) is frequently observed. Presence of hyporeflecting tumor nests infiltrating the dermis is the most specific feature for invasive squamous cell carcinoma, although in presence of marked cytological atypia and thickened epidermis a precise differential diagnosis is often difficult.

Evidence for RCM in skin cancer diagnosis

Numerous studies tested the diagnostic value of RCM mainly showing accurate melanoma and basal cell carcinoma diagnosis. There are evidences that RCM improves diagnostic specificity when integrated with dermoscopy. Implementation of RCM in the clinical setting in prospective interventional studies recently showed the possibility to reduce the excision of benign lesions up to the 70%, with a significant cost-benefit in a micro-cost analysis. The use of RCM allows in-vivo tissue imaging, refining the management of the patients in an integrated diagnostic workflow. RCM is a largely demonstrated non-invasive diagnostic tool that improves the diagnosis of melanoma and basal cell carcinoma, saving a consistent number of unnecessary excisions when used routinely during the clinical visit and following dermoscopic examination.

Applications in inflammatory diseases

RCM has been used for the evaluation of several inflammatory skin diseases, identifying specific confocal features correlated with histopathology. The distribution of the inflammatory infiltrate, the involvement of the adnexal structures and the presence of parakeratosis, acanthosis, papillomatosis and dilated blood vessels are the main features used for the interpretation of RCM images of inflammatory diseases. Large comparative studies testing the diagnostic accuracy of RCM features in setting of complex differential diagnosis are still missing. However, the data provided by RCM are useful for confirming a clinical diagnosis, patient management and therapeutic follow-up, as well as clinical studies on drug efficacy.

Cookies help us deliver our services. By using our services, you agree to our use of cookies.