Ex vivo confocal microscopy performs real-time assessment of renal biopsy in non-neoplastic diseases.

BACKGROUND: Ex vivo confocal microscopy is a technique for tissue examination, which generates images of fresh samples with an optical resolution comparable to those obtained by conventional pathology. The objective of this study was to evaluate the feasibility of using ex vivo confocal microscopy in fusion mode (reflectance and fluorescence) and the H&E-like digital staining that is obtained for the analysis of non-neoplastic kidney biopsies. METHODS: Twenty-four renal samples acquired from autopsies were scanned in a 4th generation ex vivo confocal microscopy device. The imaging process was completed in an average of three minutes. RESULTS: Confocal images correlated very well to the corresponding conventional histological sections, both in normal tissue and in chronic lesions (glomerulosclerosis, fibrosis and tubular atrophy). The ex vivo confocal microscopy protocol did not add artifacts to the sample for the ulterior study with light microscopy, nor to the histochemical or immunohistochemical studies. CONCLUSION: The ease and speed of grayscale and fluorescence image acquisition, together with the quality of the H&E-like digitally stained images obtained with this approach, suggest that this technique shows promise for use in clinical nephrology and renal transplantation.

The smart approach: feasibility of lentigo maligna superficial margin assessment with hand-held reflectance confocal microscopy technology.

BACKGROUND: Lentigo maligna may be challenging to clear surgically. OBJECTIVE: To evaluate feasibility of using superficial skin cuts as RCM imaging anchors for attaining negative surgical margins in lentigo maligna. METHODS: Included patients presented with lentigo maligna near cosmetically sensitive facial structures. We evaluated, with hand-held-RCM, microscopic clearance of melanoma beyond its dermoscopically detected edges. Evaluated margins were annotated using shallow skin cuts. If a margin was positive at 'first-step' RCM evaluation, we sequentially advanced the margin radially outward at that segment by 2-mm intervals until an RCM-negative margin was identified. Prior to final surgical excision, we placed sutures at the outmost skin cuts to allow comparison of RCM and histopathological margin assessments. Primary outcome measure was histopathological verification that RCM-negative margins were clear of melanoma. RESULTS: The study included 126 first-step margin evaluations in 23 patients, median age 70 years (range: 43-91). Seventeen patients (74%) had primary in-situ melanoma and six (26%) invasive melanoma, mean thickness 0.3 mm (range 0.2-0.4 mm). Six cases (26%) showed complete negative RCM margins on 'first-step', 11 (48%) were negative at 'second-step', and four (17%) at 'third-step'. In two additional cases (9%), margins clearance could not be determined via RCM due to widespread dendritic cells proliferation. The RCM-negative margins in all 21 cases proved clear of melanoma on histopathology. Of the 15 cases that returned at 1-year follow-up, none showed any residual melanoma on dermoscopic and RCM examinations. Interobserver reproducibility showed fair agreement between bedside RCM reader and blinded remote-site reader, with Spearman's rho of 0.48 and Cohen's kappa of 0.43; using bedside reader as reference, the remote reader's sensitivity was 92% and specificity 57% in positive margin detection. CONCLUSIONS: Margin mapping of lentigo maligna with hand-held-RCM, using superficial skin cuts, appears feasible. This approach needs validation by larger studies.

Diagnostic accuracy of optical coherence tomography in actinic keratosis and basal cell carcinoma.

BACKGROUND: Early diagnosis of non-melanoma skin cancer (NMSC) is potentially possible using optical coherence tomography (OCT) which provides non-invasive, real-time images of skin with micrometre resolution and an imaging depth of up to 2mm. OCT technology for skin imaging has undergone significant developments, improving image quality substantially. The diagnostic accuracy of any method is influenced by continuous technological development making it necessary to regularly re-evaluate methods. OBJECTIVE: The objective of this study is to estimate the diagnostic accuracy of OCT in basal cell carcinomas (BCC) and actinic keratosis (AK) as well as differentiating these lesions from normal skin. METHODS: A study set consisting of 142 OCT images meeting selection criterea for image quality and diagnosis of AK, BCC and normal skin was presented uniformly to two groups of blinded observers: 5 dermatologists experienced in OCT-image interpretation and 5 dermatologists with no experience in OCT. During the presentation of the study set the observers filled out a standardized questionnaire regarding the OCT diagnosis. Images were captured using a commercially available OCT machine (Vivosight ®, Michelson Diagnostics, UK). RESULTS: Skilled OCT observers were able to diagnose BCC lesions with a sensitivity of 86% to 95% and a specificity of 81% to 98%. Skilled observers with at least one year of OCT-experience showed an overall higher diagnostic accuracy compared to inexperienced observers. CONCLUSIONS: The study shows an improved diagnostic accuracy of OCT in differentiating AK and BCC from healthy skin using state-of-the-art technology compared to earlier OCT technology, especially concerning BCC diagnosis.

Cost-benefit of reflectance confocal microscopy in the diagnostic performance of melanoma.

BACKGROUND: The sub-optimal diagnostic accuracy for melanoma leads to excise a high number of benign lesions, with consequent costs. Reflectance confocal microscopy (RCM) improves diagnostic specificity, thus possibly inducing a reduction in unnecessary excisions and related costs. OBJECTIVE: To estimate the influence of RCM on number of benign lesions needed to excise (NNE) a melanoma, in term of clinical outcomes and costs per patient. PATIENTS AND METHODS: Skin neoplasms excised by the dermatology public service in the Province of Modena were retrieved form centralized pathology database. Differences in NNE between the territorial service (using dermoscopy only) and the University Hospital (adding also RCM to the patients' workflow) were calculated and cost analysis was performed through a micro-costing approach. RESULTS: A large reduction in benign lesions excised at University Hospital was evident, leading to NNE of 6.25 for University Hospital, compared to 19.41 for Territorial Dermatology. Since 4320 unnecessary excisions can be saved every million inhabitants, an overall yearly saving of over 280,000 Eur can be expected from the use of RCM. CONCLUSIONS: The systematic use of RCM was dramatically affecting the number of benign lesions excised, and this can be translated in a significant cost-benefit advantage.

A new approach for presurgical margin assessment by reflectance confocal microscopy of basal cell carcinoma.

BACKGROUND: Surgical excision represents the most common elective treatment for basal cell carcinoma (BCC). Several noninvasive approaches have been proposed for in vivo determination of tumour margin, in order to achieve radical removal. OBJECTIVES: To propose a new approach through the combination of dermoscopy and reflectance confocal microscopy (RCM) for lateral margin detection in BCC. METHODS: Ten patients with lesions clinically suggestive of nonpigmented BCCs with ill-defined margins were enrolled. All BCCs were dermoscopically evaluated first and the ill-defined margins were marked with a superficial cut and then inspected using RCM. RESULTS: RCM evaluation showed BCC foci beyond the presurgical marker in three out of 10 lesions. Histology confirmed the RCM results: the presence of BCC features across the cut, corresponding to two superficial BCCs and a morpheaform BCC. CONCLUSIONS: This new procedure helped to improve the identification of proper margins for surgical excision in nonpigmented BCC with clinically and dermoscopically ill-defined margins.

Amplicon-based next-generation sequencing: an effective approach for the molecular diagnosis of epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa (EB) is caused by mutations in genes that encode proteins belonging to the epidermal-dermal junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, the current methods available for diagnosing EB involve immunohistochemistry of biopsy samples and transmission electron microscopy followed by single-candidate gene Sanger sequencing (SS), which are labour-intensive and expensive clinical pathways. OBJECTIVES: According to the recently published recommendations for the diagnosis and treatment of EB, the assessment of the mutational landscape is now a fundamental step for developing a comprehensive diagnostic path. We aimed to develop a customized, cost-effective amplicon panel for the complete and accurate sequencing of all the pathogenic genes already identified in EB, and to minimize the processing time required for the execution of the test and to refine the analysis pipeline to achieve cost-effective results from the perspective of a routine laboratory set-up. Next-generation sequencing (NGS) via the parallel ultra-deep sequencing of many genes represents a proper method for reducing the processing time and costs of EB diagnostics. MATERIALS AND METHODS: We developed an EB disease-comprehensive AmpliSeq panel to accomplish the NGS on an Ion Torrent Personal Genome Machine platform. The panel was performed on 10 patients with known genetic diagnoses and was then employed in eight family trios with unknown molecular footprints. RESULTS: The panel was successful in finding the causative mutations in all 10 patients with known mutations, fully confirming the SS data and providing proof of concept of the sensitivity, specificity and accuracy of this procedure. In addition to being consistent with the clinical diagnosis, it was also effective in the trios, identifying all of the variants, including ones that the SS missed or de novo mutations. CONCLUSIONS: The NGS and AmpliSeq were shown to be an effective approach for the diagnosis of EB, resulting in a cost- and time-effective 72-h procedure.

Physicians' concerns towards prescription adherence and treatment effectiveness in the clinical management of actinic keratosis.

AIM: We report concerns toward prescription adherence and treatment effectiveness in the clinical management of actinic keratosis (AK) in Italy. METHODS: We carried out a cross-sectional web-based survey among Italian dermatologists across Italy. Physicians were asked to answer a self-administered questionnaire about their concerns around AK therapy and barriers to patients' adherence. Each physician also profiled his last patient and answered items concerning his experience with topical treatments and the suitability of current and future treatment options for the profiled patient. RESULTS: Fifty practitioners answered the survey. Most dermatologists agreed that field-therapy is a key element for the management of AK in most patients, and 76% (N.=38) agreed that topical treatments were the best option in such cases given their ability to target subclinical lesions. However most interviewee underlined the importance of fostering patients' adherence and minimizing side effects in order to maximize benefits from therapy. CONCLUSION: We showed that features of current therapeutic options for field-directed therapy (namely long duration of treatment, intensity and duration of local skin reaction) raise practitioners' concerns toward patients' prescription adherence and real-world effectiveness.

Hereditary trichilemmal cysts: a proposal for the assessment of diagnostic clinical criteria.

Trichilemmal cysts (TCs) can occur as sporadic lesions or in hereditary-familial settings with autosomal dominant transmission. These entities have not been widely analyzed in their peculiar aspects yet. The aim of this study was to describe a cohort of patients with diagnosis of TCs through a clinical and biomolecular characterization, intended to highlight some effective diagnostic criteria for their identification. Among 149 cases of this study, 24 cases of TCs (16.1%) arose in patients with at least one first-degree relative with diagnosis of TCs. Peculiar findings concerning hereditary lesions included the multiple presentation with an early onset age. On the basis of clinical evaluation, we propose a panel of clinical and histologic criteria for the diagnosis of hereditary TCs, which includes: (i) the diagnosis of TCs in at least two first-degree relatives or in three first- or second-degree relatives in two consecutive generations; (ii) at least one of the patients with TCs diagnosed <45 years; and (iii) the diagnosis of multiple or giant (>5-cm lesions) or rare histopathologic features (proliferating and ossifying) TCs.

In vivo assessment of chronological ageing and photoageing in forearm skin using reflectance confocal microscopy.

BACKGROUND: Skin ageing is a complex process due to intrinsic chronological factors (chronoageing) and extrinsic environmental factors. The primary extrinsic factor is cumulative ultraviolet (UV) exposure, and is therefore termed photoageing. The current standards for measuring cumulative sun damage are biopsy histology and skin microtopography. However, skin biopsies are too invasive for population studies and skin replicas render only superficial skin architecture data. Reflectance confocal microscopy (RCM) is a noninvasive imaging tool that allows for in vivo imaging of the skin at quasihistological resolution. OBJECTIVES: To define and identify RCM features associated with chronological ageing and photoageing on the forearm in two age groups with different skin phototypes and to assess whether these results agree with previous findings. METHODS: We obtained RCM images of dorsal and volar nonlesional skin of the lower forearm of 75 individuals with skin Fitzpatrick phototypes I-III in two age groups (20-30 years and 50-60 years). From each participant and body site, 21 RCM features were assessed and statistically significant differences between the two age groups and different forearm sites determined. RESULTS: RCM enabled identification of changes in architecture, cell morphology and extracellular matrix (collagen) at the level of the epidermis, dermoepidermal junction and papillary dermis. Changes that were correlated with chronological ageing and which were aggravated on the UV-exposed dorsal forearm were: loss of small skin furrows resulting in wider and less intersecting furrows; irregularity of the epidermal honeycomb pattern; irregularly distributed (mottled) pigmented keratinocytes/melanocytes; irregularity of the papillary rings and/or effacement of the rete ridges; and loss of thin collagen fibres and presence of collagen clods. CONCLUSION: We have tested previously reported and new parameters for skin ageing evaluation by RCM, and identified 15 statistically significant RCM features that can be used to quantify ageing and photoageing in forearm skin noninvasively.

Computer description of colours in dermoscopic melanocytic lesion images reproducing clinical assessment.

BACKGROUND: The assessment of colours is essential for the diagnosis of malignant melanoma (MM), both for pattern analysis on dermoscopic images, and when employing semiquantitative methods. OBJECTIVES: To develop a computer program for colour assessment in MM images mimicking the human perception of lesion colours, and to compare the automatic colour evaluation with one performed by human observers. METHODS: A colour palette comprising six colour groups (black, dark brown, light brown, blue-grey, red and white) was created by selecting single colour components inside melanocytic lesion images acquired by means of a digital videomicroscope, and was implemented in the image analysis program. Subsequently, colours were assessed by the computer program on 331 melanocytic lesion images composing our image database, and the results were compared with the evaluation of lesion colours performed by the clinician. RESULTS: The black, white and blue-grey colours were more frequently found in MMs than in naevi, both by the clinicians and by the computer. In MM images we observed 4.27 +/- 1.14 colours (mean + or - SD) per lesion, as opposed to 3.22 +/- 0.68 in naevi. The correlation between clinical and computer evaluation of the colours was very good, with a value of 0.781 for overall assessment. CONCLUSIONS: This innovative method for automatic colour evaluation, reproducing clinical assessment of melanocytic lesion colours, may provide numerical parameters to be employed for computer-aided diagnosis of MM.

Water sorption-desorption test and moisture accumulation test for functional assessment of atopic skin in children.

Sorption-desorption and moisture accumulation tests are simple and quick methods for the in vivo functional analysis of stratum corneum hydration kinetics. The aim of this study was to evaluate the hydration dynamics of the uninvolved and affected skin of children with atopic dermatitis and to compare them with the skin of healthy children. The study investigated 45 children. The dynamic tests were performed using the corneometer CM820. Numerical parameters were calculated. With the sorption-desorption test, eczematous skin showed lower water accumulation during the sorption phase, whereas water was released more slowly during the desorption phase. With the moisture accumulation test, increases in water accumulation velocity and in water accumulation were observed in atopic children. Dynamic tests showed that the stratum corneum of unaffected atopic skin was less hydrated but more easily hydratable than normal skin. Conversely, despite a lower absorption capability, eczematous skin showed a greater avidity to retain water. New functional parameters (water-sorption capacity and accumulated water decay) are proposed to describe more precisely the hydration kinetics of eczematous skin.