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.

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.

The effect of adalimumab on key drivers in the pathogenesis of psoriasis.

BACKGROUND: The use of recently introduced biologics targeting specific immune mechanisms has identified crucial steps in the pathogenesis of psoriasis. Studying the dynamics of changes of these target mechanisms in sequential skin biopsies during treatment with biologics may reveal potential biomarkers. Correlation between clinical parameters and the expression of specific genes during treatments may identify markers indicative of treatment response. OBJECTIVES: This observational open-label study aimed to provide an overview of important cell biological changes in lesional skin during treatment with adalimumab, and their relationship to clinical improvement. METHODS: Ten patients with moderate-to-severe plaque psoriasis were included and treated with adalimumab for 16 weeks. At baseline, and after 10 days and 16 weeks of treatment clinical scores were assessed and biopsies were taken to examine gene expression at the mRNA and protein level. RESULTS: The expression of marker genes for innate immunity, and epidermal differentiation and proliferation was rapidly restored to normal levels, whereas genes of the adaptive immune system showed a delayed decrease. The static and dynamic course of CD1a+ Langerhans cells and Ki67+ nuclei showed a significant strong correlation to the Psoriasis Area and Severity Index score. No correlation between interleukin-17 expression and clinical scores was found. CONCLUSIONS: The innate immune system is affected during adalimumab treatment well before the changes in the adaptive immune system become apparent. We may speculate that the addition of a treatment with an early effect on adaptive immunity to adalimumab may result in superior effectiveness compared with monotherapies.

High discordance between punch biopsy and excision in establishing basal cell carcinoma subtype: analysis of 500 cases.

BACKGROUND: Basal cell carcinoma (BCC) is the most frequently occurring cancer in humans. Worldwide incidences rise about 10% each year, increasing the burden on dermatologists, general practitioners and pathologists as well as increasing costs for the health care system. Increasingly non-surgical treatment options are used in the treatment of BCC, without histological confirmation of BCC subtype, potentially resulting in under-treatment. OBJECTIVE: We evaluated the diagnostic accuracy of a punch biopsy for the BCC histological subytpe in a primary BCC and the prevalence of biopsy-based under-diagnosis of aggressive subtypes. Accuracy of a punch biopsy was defined as concordance of the diagnosis of subtype of BCC at punch biopsy and excision. METHODS: A retrospective chart-review was performed of primary BCC, which were proven by punch biopsy and subsequently treated by excision. The first 100 consecutive BCCs per year during the years 2004-2009 were included, yielding a total of 500 evaluated BCCs. RESULTS: The overall accuracy of punch biopsy for BCC subtype at excision was 69%, in single-type BCC 83% (n = 343) and in mixed-type BCC 37% (n = 157). Accuracy varied substantially according to BCC subtype, being highest in the superficial subtype (84%) and subsequently in infiltrative (69%), nodular (63%) and micronodular subtype (38%). In 11% of all cases, an unsuspected more aggressive subtype was present. CONCLUSION: Punch biopsy has a high accuracy in single-type BCCs and a considerably lower accuracy in mixed-type BCCs for establishing BCC subtype compared to excision. The presence of an unsuspected aggressive subtype could explain therapy failure of non-surgical treatments like imiquimod or photodynamic therapy.

Reflectance confocal microscopy: an effective tool for monitoring ultraviolet B phototherapy in psoriasis.

BACKGROUND: In vivo reflectance confocal microscopy (RCM) is a novel, noninvasive imaging technique which enables imaging of skin at a cellular resolution comparable to conventional microscopy. OBJECTIVES: We performed a pilot study to evaluate RCM as a noninvasive tool for monitoring ultraviolet (UV) B phototherapy in psoriasis. METHODS: In six patients with psoriasis, lesional and nonlesional skin was selected for RCM imaging using a standardized protocol. Well-known histological features of psoriasis were visualized: parakeratosis, acanthosis, agranulosis, papillomatosis, presence of epidermal inflammatory cells, increased number of papillary capillaries and increased capillary blood flow. RCM imaging was performed before the first irradiation with UVB phototherapy, after nine irradiations, at clearance and 12 weeks after clearance. In four patients, 4-mm punch biopsies were obtained and stained with haematoxylin-eosin. Additionally, immunohistochemical staining was performed with monoclonal antibodies specific for CD31, CD3, filaggrin, K16, Ki67 and CD1a for correlation to RCM images. RESULTS: There was a high correlation between clinical, RCM and histological features. Normalization of RCM and histological features corresponded highly to clinical improvement of psoriasis. CONCLUSIONS: This study is the first to establish the use of RCM as an effective tool for noninvasive monitoring of UVB phototherapy in patients with psoriasis. Potentially, RCM could be used in many other skin diseases for monitoring therapeutic response on a cellular level in a clinical or research setting.

Cellular features of psoriatic skin: imaging and quantification using in vivo reflectance confocal microscopy.

BACKGROUND: In vivo reflectance confocal microscopy (RCM) is a novel, exciting imaging technique. It provides images of cell-and tissue structures and dynamics in situ, in real time, without the need for ex vivo tissue samples. RCM visualizes the superficial part of human skin up to a depth of 250 µm. In psoriasis, an erythematosquamous skin disease, we evaluated well known histological features of stable psoriasis vulgaris (PP) with RCM. RCM images were correlated to morphological and cell biological findings in routine HE and immunohistochemical stained histology with CD3 and antifilaggrin antibodies. METHODS: Lesional and nonlesional skin of eight patients with PP were evaluated with RCM, after which 4-mm punch biopsies were taken and cut vertically in two equal parts. One part was processed in the conventional vertical way, the other horizontally (en face) for optimal correlation to RCM images. We evaluated and quantificated nine histopathological features of psoriasis: parakeratosis, epidermal and dermal inflammatory infiltrate, diminished or absent stratum granulosum, epidermal thickening, thinning of the suprapapillary epidermal plate, increased height of the papillary dermis, increase in number of dermal papillae and increase in number and volume of papillary capillaries. RESULTS: Quantification and evaluation of cell biological and histological features of PP with RCM correlated highly to evaluation in HE, CD3 and filaggrin-stained histology. CONCLUSIONS: RCM is a novel technique which can be used for real time, cytometric evaluation and quantification of PP features. RCM might be suited equally for cytometric evaluation of other superficial tissues.