Biopsy outperforms reflectance confocal microscopy in diagnosing and subtyping basal cell carcinoma: results and experiences from a randomized controlled multicentre trial.

BACKGROUND: Reflectance confocal microscopy (RCM) is a noninvasive method for skin assessment, allowing entire lesion evaluation up to the papillary dermis. RCM is a potentially attractive alternative to punch biopsy (PB) in basal cell carcinoma (BCC). OBJECTIVES: To determine the diagnostic accuracy of RCM vs. PB in diagnosing and subtyping BCC, and to study patient satisfaction and preferences. METHODS: Patients with a clinically suspected primary BCC were randomized between RCM and biopsy. Conventional surgical excision or follow-up were used as reference. Sensitivity and specificity for BCC diagnosis and subtyping were calculated for both methods. BCC subtype was stratified based on clinical relevance: aggressive (infiltrative/micronodular) vs. nonaggressive (superficial/nodular) histopathological subtype and superficial vs. nonsuperficial BCC. Data on patient satisfaction and preferences were collected using a questionnaire and a contingent valuation method. RESULTS: Sensitivity for BCC diagnosis was high and similar for both methods (RCM 99·0% vs. biopsy 99·0%; P = 1·0). Specificity for BCC diagnosis was lower for RCM (59·1% vs. 100·0%; P < 0·001). Sensitivity for aggressive BCC subtypes was lower for RCM (33·3% vs. 77·3%; P = 0·003). Sensitivity for nonsuperficial BCC was not significantly different (RCM 88·9% vs. biopsy 91·0%; P = 0·724). Patient satisfaction and preferences were good and highly comparable for both methods. CONCLUSIONS: Biopsy outperforms RCM in diagnosing and subtyping clinically suspected primary BCC. This outcome does not support routine clinical implementation of RCM, as a replacement for PBs in this patient group.

Noninvasive objective skin measurement methods for rosacea assessment: a systematic review.

BACKGROUND: Rosacea assessment and therapy monitoring can be challenging to standardize, as most clinical evaluation systems are prone to interobserver variability and not always validated. Therefore, objective, reliable and preferably noninvasive measurement tools are needed. OBJECTIVES: To give insight into available noninvasive imaging techniques and biophysical methods in rosacea by performing a systematic review. METHODS: PubMed, Embase, Cochrane and Web of Science databases were searched until 1 September 2018 in accordance with PRISMA guidelines, to identify studies providing original data about objective noninvasive imaging and/or biophysical skin measurement techniques for diagnosis, assessing severity or therapy monitoring of adult patients with cutaneous facial rosacea. Risk of bias of included articles was assessed with the Cochrane Risk of Bias tool, Quality in Prognosis Studies tool, and the Newcastle-Ottawa Scale. RESULTS: A total of 78 studies were included, describing 14 imaging and biophysical methods. Widespread information about (sub)surface cutaneous morphology and functionality was obtained. Methodological study quality was relatively low and interstudy outcome variability was large. Several tools show promising value in research settings: for treatment follow-up Demodex mites are countable with reflectance confocal microscopy, spectrometry can quantify erythema, and rosacea severity could be objectified with skin hydration- and transepidermal water loss measurements. CONCLUSIONS: This systematic review describes the spectrum of noninvasive imaging and biophysical methods in rosacea assessment, giving multifaceted information about structure and properties of rosacea skin, especially useful for research purposes. Larger studies with good methodological quality are needed to create validated protocols for further implementation into research. What's already known about this topic? Rosacea is a chronic inflammatory skin disease with a variety of clinical manifestations. Most clinical evaluation systems are subjective, not always validated, and subsurface skin processes remain unnoticed. Currently, different types of noninvasive measurement tools are available for rosacea assessment and therapy monitoring, but a comprehensive overview is lacking. What does this study add? Seventy-eight publications were included, describing 14 imaging and biophysical tools, providing a wide range of information about rosacea skin morphology and functionality. Reflectance confocal microscopy and spectrometry are especially promising in therapy monitoring and skin barrier measurements for rosacea severity assessment. Larger studies with better methodological quality are needed to create validated protocols for implementation into research.

Interrater and intrarater agreement of confocal microscopy imaging in diagnosing and subtyping basal cell carcinoma.

BACKGROUND: Reflectance confocal microscopy (RCM) imaging can be used to diagnose and subtype basal cell carcinoma (BCC) but relies on individual morphologic pattern recognition that might vary among users. OBJECTIVES: We assessed the inter-rater and intrarater agreement of RCM in correctly diagnosing and subtyping BCC. METHODS: In this prospective study, we evaluated the inter-rater and intrarater agreement of RCM on BCC presence and subtype among three raters with varying experience who independently assessed static images of 48 RCM cases twice with four-week interval (T1 and T2). Histopathologic confirmation of presence and subtype of BCC from surgical excision specimen was defined as the reference standard. RESULTS: The inter-rater agreement of RCM for BCC presence showed an agreement of 82% at T1 and 84% at T2. The agreements for subtyping BCC were lower (52% for T1 and 47% for T2). The intrarater agreement of RCM for BCC presence showed an observed agreement that varied from 79% to 92%. The observed agreements for subtyping varied from 56% to 71%. CONCLUSIONS: In conclusion, our results show that RCM is reliable in correctly diagnosing BCC based on the assessment of static RCM images. RCM could potentially play an important role in BCC management if accurate subtyping will be achieved. Therefore, future clinical studies on reliability and specific RCM features for BCC subtypes are required.

Measurement of skin surface biomakers by Transdermal Analyses Patch following different in vivo models of irritation: a pilot study.

BACKGROUND/PURPOSE: FibroTx Transdermal Analyses Patch (TAP) is a novel technology for non-invasive measurements of protein biomarkers on the skin surface, in vivo. The aim of this study was to explore the potential of TAP in detecting skin surface biomarkers following mild perturbations, in vivo, using two experimental models: tape stripping, mimicking acute barrier disruption, and histamine iontophoresis, mimicking acute and local inflammation at minimal skin barrier insult. METHODS: Tape stripping and histamine iontophoresis were performed in two separate experiments on the volar forearm of healthy volunteers (n = 27 and n = 10, respectively). Biomarker levels were assessed with TAP at baseline and up to 72 h after stimulation. Functional (transepidermal water loss -TEWL- and a* value) and morphological (confocal reflectance microscopy -RCM) assessments were added in the tape stripping and histamine iontophoresis experiments, respectively. RESULTS: Cytokines IL-1α and IL-1RA and the antimicrobial peptide hBD-1 showed distinct dynamics, despite substantial inter-individual variation in levels, with an increase following tape stripping and a decrease following histamine iontophoresis. These dynamics could be related to the assessments made by TEWL and RCM. In the tape stripping experiment, additional biomarkers could be detected. CONCLUSION: TAP measurements, especially IL-1α, IL-1RA, and hBD-1, from the skin surface were sensitive enough for monitoring dynamic changes in the skin in the two models of skin perturbation. We conclude that TAP holds promise for non-invasively unraveling the dynamics of processes related to skin perturbation and repair.

The influence of different shavers on the skin quantified by non-invasive reflectance confocal microscopy.

BACKGROUND: The impact of personal care devices on skin is mainly assessed using subjective tools. However, new objective, accurate non-invasive in vivo imaging techniques have been developed. The aim of this study was to evaluate the ability of reflectance confocal microscopy (RCM) in quantifying morphological impact of shavers on skin. Furthermore, tape stripping (TS) as method to study morphological impact of shavers was evaluated. METHODS: In 12 healthy male subjects, for two consecutive days, a split-face test was performed in the neck; on one side a shaver was applied, while the other side was exposed to TS. The stratum corneum (SC) thickness was quantified using RCM and sensory observations were evaluated using questionnaires. RESULTS: Shavers with a different impact on skin, can be discriminated by RCM; shaver B removed more SC after application than the skin friendlier shaver A. Furthermore, the changes in SC thickness induced by TS corresponded well to that of the shavers. CONCLUSION: RCM is able to quantify the impact of different shavers on skin. Besides, TS appeared to be a suitable model mimicking the mechanical impact of shavers on skin. RCM in combination with the TS model appeared to be a suitable minimally invasive model to obtain morphological and cell biological data on skin-material interactions caused by different personal care devices.

A four-phase strategy for the implementation of reflectance confocal microscopy in dermatology.

BACKGROUND: Reflectance confocal microscopy (RCM) is gradually implemented in dermatology. Strategies for further implementation and practical 'hands on' guidelines are lacking. OBJECTIVE: The primary outcome was to conduct a general strategy for further implementation of RCM. The secondary outcome was the diagnosis of psoriasis and differentiation of stable from unstable psoriatic plaques by means of the 'hands on' protocol, derived from the strategy. METHODS: We used a four-phased model; an exploring phase, a systematic literature search, a clinical approach and, finally, an integration phase to develop a clinical guideline for RCM in psoriasis. Receiver operating characteristic curve statistics was applied to define the accuracy for the diagnosis of unstable psoriasis. RESULTS: A general strategy for further implementation of RCM and practical approach was developed to examine psoriasis by RCM and to distinguish stable from unstable psoriasis. Unstable psoriasis was diagnosed by epidermal inflammatory cell counts with a sensitivity and specificity of 91.7% and 98.3%, respectively, and with an accuracy of 0.92 (area under the curve). In addition, a monitoring model was proposed. CONCLUSION: This is the first study that shows a method for implementation of RCM in dermatology. The strategy and hands on protocol for psoriasis may serve as a model for other dermatological entities and additionally may lead to specialized ready-to-use RCM protocols for clinical dermatological practice.

The value of in vivo reflectance confocal microscopy in the diagnosis and monitoring of inflammatory and infectious skin diseases: a systematic review.

In vivo examination of the skin by reflectance confocal microscopy (RCM) has been performed for about 20 years, leading to a broad spectrum of imaged infectious and inflammatory skin diseases (ISD) with many described RCM features. We systematically reviewed all available literature concerning ISD evaluated by RCM. Furthermore, we assessed the accuracy of the features and defined recommendations for future studies after indicating the limitations in the current published literature. PubMed, Embase, Cochrane Library and Web of Science databases were searched for literature. All studies on RCM and ISD were reviewed and quality assessment was determined by using the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) checklist. The literature search revealed 77 eligible studies for inclusion. Different RCM features in a broad spectrum of ISD have been described. Further, RCM has been used for monitoring treatment and evolution of ISD, as well as for diagnostic purposes. This systematic review provides an overview of the broad spectrum of ISD imaged by RCM. Although RCM seems to be a promising monitoring and diagnostic tool for ISD, studies with appropriate methodological quality are necessary to create adequate guidelines and protocols for further implementation of RCM in clinical practice.

Application of leukotriene B4 and reflectance confocal microscopy as a noninvasive in vivo model to study the dynamics of skin inflammation.

BACKGROUND: Application of leukotriene B4 (LTB4) is an established in vivo model that locally induces skin inflammation. Currently in this model, a biopsy is inevitable. In vivo reflectance confocal microscopy (RCM), a noninvasive imaging technique, could overcome this limitation. To find out to what extent RCM may be an in vivo investigative and diagnostic tool in neutrophilic conditions, we studied the dynamics of polymorphonuclear leukocytes (PMN) migration from dermis to stratum corneum using an established LTB4 model. METHODS: Leukotriene B4 was topically applied on the skin of the lower back of seven volunteers. The skin sites were evaluated by RCM for three consecutive days with a 24 h time interval. For histological correlation, 3-mm punch biopsies were obtained. The tissue sections were hematoxylin-eosin and immunohistochemical stained. Minimal and average epidermal thickness was measured. RESULTS: Reflectance confocal microscopy imaging showed highly reflective ill-defined particles with a granular content throughout the epidermis 24 h after application of LTB4. Over time, the appearance of these cells changed throughout the epidermis. Epidermal thickness increased over time, and the measurements based on the RCM images corresponded very well with the histological images. CONCLUSIONS: Reflectance confocal microscopy was able to visualize PMN migration, accumulation, and degeneration over time in the used LTB4 model. The noninvasive character and the possibility to obtain multiple in vivo images from the same location over time make that RCM in combination with this model a useful tool to study the dynamics and function of PMN in inflammatory processes in the skin.

Combining tape stripping and non-invasive reflectance confocal microscopy : an in vivo model to study skin damage.

BACKGROUND: Evaluation of (immuno)histological and cell biological changes in damaged skin requires often an invasive skin biopsy, making in vivo models inappropriate to study skin damage. Reflectance confocal microscopy (RCM) might overcome this limitation. Therefore, we evaluated the use of a tape-stripping model in combination with RCM to provide morphological data on skin damage and recovery. METHODS: In 25 volunteers, a tape-stripping stimulus was applied. The skin was imaged with RCM during 1 week and 3 mm punch biopsies were obtained. RESULTS: Strong correlations between epidermal thickness determined by RCM and conventional histological measurements were found. RCM thickness measurements correlated well with epidermal proliferation. The 10× or 15× repeated tape-stripping resulted in skin damage similar to acute stripping. Mild repeated tape-stripping showed no skin damage. CONCLUSION: Overall, we demonstrated that non-invasive RCM in combination with tape-stripping could be used as model to obtain morphological and cell biological data on skin-material interactions.

Reflectance confocal microscopy: non-invasive distinction between actinic keratosis and squamous cell carcinoma.

BACKGROUND: Early recognition of squamous cell carcinoma (SCC) is difficult. Non-invasive reflectance confocal microscopic (RCM) imaging of the skin is a promising diagnostic technique. Although several RCM features for SCC and AK have been described, it is not determined whether RCM has the ability to distinguish between SCC and actinic keratosis (AK). OBJECTIVE: To determine in vivo reflectance confocal microscopic features that are specific for making a distinction between AK and SCC. METHODS: In 24 patients, 30 lesions clinically suspicious for AK or SCC were selected for RCM imaging. Following the imaging procedure, a 3 mm skin biopsy was obtained for confirmation of the histopathological diagnosis. Two observers evaluated the RCM images according to a literature based list of RCM features. The obtained data were evaluated by an univariate and forward multivariate logistic regression analysis, kappa analysis and independent T-test. RESULTS: The univariate logistic regression showed statistically significant odds ratios for several RCM features, including architectural disarray in the stratum granulosum, architectural disarray in the spinous layer and nest-like structures in the dermis. The forward multivariate logistic regression analysis showed that the combination of these features increased the ability to make the correct diagnosis AK and SCC non-invasively. The interobserver agreement between a starting and an experienced RCM observer ranged from poor to no agreement. CONCLUSION: This study revealed specific RCM features that can distinguish between AK and SCC, stimulating further prospective, large cohort research in this field. This will result in correct, efficient and adequate diagnosis and treatment of clinically difficult to distinguish AK and SCC lesions.

Prospective differentiation of clinically difficult to distinguish nodular basal cell carcinomas and intradermal nevi by non-invasive Reflectance Confocal Microscopy: a case series study.

BACKGROUND: Clinical differentiation between a nodular basal cell carcinoma (nBCC) and a benign intradermal nevus can be difficult. Even with additional dermoscopic evaluation, a correct diagnosis may be difficult. Currently, histopathological examination of a biopsy is the gold standard to differentiate between these lesions. However, this is an invasive technique and sampling errors can occur. In vivo Reflectance Confocal Microscopy (RCM) is a non-invasive technique to evaluate a skin lesion at a microscopic level. RCM features of nBCCs and intradermal nevi have been described in research setting. However, the use of RCM for prospective differentiation between difficult to diagnose nodules into nBCCs and intradermal nevi in clinical practice has not been demonstrated yet. OBJECTIVE: In this study, we aim to address a common clinical scenario; to differentiate clinically and dermoscopically difficult to distinguish nodules, into nBCCs and intradermal nevi by RCM. MATERIAL AND METHODS: Six patients with clinically and dermoscopically difficult to distinguish nodular skin lesions were evaluated by RCM to differentiate prospectively between nBCCs and intradermal nevi. In five out of six cases, a 3 mm punch biopsy was obtained to confirm the RCM diagnosis. RESULTS: Observed RCM features that allowed differentiation between nBCCs and intradermal nevi were the dermal-epidermal junction patterns, the appearance of the nests and the degree of vascularization. CONCLUSIONS: This case series study demonstrates the value of non-invasive in vivo RCM imaging in routine patient care, with respect to the prospective diagnosis of clinically difficult to distinguish nBCCs and intradermal nevi. Subsequently, biopsies of benign lesions in cosmetic areas could be avoided.

The potential of the skin as a readout system to test artificial turf systems: clinical and immunohistological effects of a sliding on natural grass and artificial turf.

The purpose of this study was to investigate the interaction of skin with natural grass and artificial turf at clinical, histological and immunohistochemical level. Therefore, 14 male volunteers performed slidings on dry natural grass, wet natural grass and artificial turf. Directly and 24 h after the slidings, a clinical picture and a 3-mm punch biopsy of the lesion were taken. Paraffin sections (6 µm) were hematoxylin-eosin stained. Immunohistochemistry was performed for CD3, hBD-2, K16, K10, Ki67 and HSP70. Clinically, a sliding performed on artificial turf caused less erythema but more abrasion compared to natural grass. At histological level, artificial turf or dry natural grass damaged the stratum corneum the most. Directly after the sliding, CD3, hBD-2, K16, K10, Ki67 and HSP70 expression was normal. 24 h after a sliding on artificial turf or dry natural grass, an increase of K16, hBD-2 and HSP70 expression was observed. In this pilot study it was not possible to clearly distinguish between skin damage induced by a sliding on artificial turf and natural grass. However, small differences at clinical and histological level seem to exist. This demonstrates the potential of the skin as readout system to evaluate artificial turf systems and mechanical skin damage.

Successful Regulatory T Cell-Based Therapy Relies on Inhibition of T Cell Effector Function and Enrichment of FOXP3+ Cells in a Humanized Mouse Model of Skin Inflammation.

BACKGROUND: Recent clinical trials using regulatory T cells (Treg) support the therapeutic potential of Treg-based therapy in transplantation and autoinflammatory diseases. Despite these clinical successes, the effect of Treg on inflamed tissues, as well as their impact on immune effector function in vivo, is poorly understood. Therefore, we here evaluated the effect of human Treg injection on cutaneous inflammatory processes in vivo using a humanized mouse model of human skin inflammation (huPBL-SCID-huSkin). METHODS: SCID beige mice were transplanted with human skin followed by intraperitoneal (IP) injection of 20-40 × 106 allogeneic human PBMCs. This typically results in human skin inflammation as indicated by epidermal thickening (hyperkeratosis) and changes in dermal inflammatory markers such as the antimicrobial peptide hBD2 and epidermal barrier cytokeratins K10 and K16, as well as T cell infiltration in the dermis. Ex vivo-expanded human Treg were infused intraperitoneally. Human cutaneous inflammation and systemic immune responses were analysed by immunohistochemistry and flow cytometry. RESULTS: We confirmed that human Treg injection inhibits skin inflammation and the influx of effector T cells. As a novel finding, we demonstrate that human Treg injection led to a reduction of IL-17-secreting cells while promoting a relative increase in immunosuppressive FOXP3+ Treg in the human skin, indicating active immune regulation in controlling the local proinflammatory response. Consistent with the local control (skin), systemically (splenocytes), we observed that Treg injection led to lower frequencies of IFNγ and IL-17A-expressing human T cells, while a trend towards enrichment of FOXP3+ Treg was observed. CONCLUSION: Taken together, we demonstrate that inhibition of skin inflammation by Treg infusion, next to a reduction of infiltrating effector T cells, is mediated by restoring both the local and systemic balance between cytokine-producing effector T cells and immunoregulatory T cells. This work furthers our understanding of Treg-based immunotherapy.