Correlation of image analysis features and visual morphology in melanocytic skin tumours using in vivo confocal laser scanning microscopy

Background/purpose: In vivo confocal laser scanning microscopy (CLSM) represents a novel imaging tool that allows the non-invasive examination of skin cancer morphology at a quasi histological resolution without biopsy. Previous studies dealt with the search for diagnostic, but subjective visual criteria. In this study we examined the correlation between objectively reproducible image-analysis features und visual morphology in melanocytic skin tumours using CLSM.
Methods: Eight hundred and fifty-seven CLSM tumour images including 408 benign nevi and 449 melanoma images were evaluated. Image analysis was based on features of the wavelet transform and classification tree analysis (CART) was used for classification purposes. In a second step, morphologic details of CLSM images, which have turned out to be of diagnostic significance by the classification algorithm were evaluated.
Results: CART analysis of the whole set of CLSM images correctly classified 97.55% of all melanoma images and 96.32% of all nevi images. Seven classification tree nodes seemed to indicate benign nevi, whereas six nodes were suggestive for melanoma morphology. The visual examination of selected nodes demonstrated that monomorphic melanocytic cells and melanocytic cell nests are characteristic for benign nevi whereas polymorphic melanocytic cells, disarray of melanocytic architecture and poorly defined or absent keratinocyte cell borders are characteristic for melanoma.
Conclusion: Well-known, but subjective CLSM criteria could be objectively reproduced by image analysis features and classification tree analysis. Moreover, features not accessible to the human eye seem to contribute to classification success.     

In vivo reflectance confocal microscopy: automated diagnostic image analysis of melanocytic skin tumours

Background In vivo reflectance confocal microscopy (RCM) has been shown to be a valuable imaging tool in the diagnosis of melanocytic skin tumours. However, diagnostic image analysis performed by automated systems is to date quite rare. Objectives In this study, we investigated the applicability of an automated image analysis system using a machine learning algorithm on diagnostic discrimination of benign and malignant melanocytic skin tumours in RCM. Methods Overall, 16 269 RCM tumour images were evaluated. Image analysis was based on features of the wavelet transform. A learning set of 6147 images was used to establish a classification tree algorithm and an independent test set of 10 122 images was applied to validate the tree model (grouping method 1). Additionally, randomly generated ‘new’ learning and test sets, tumour images only and different skin layers were evaluated (grouping method 2, 3 and 4). Results The classification tree analysis correctly classified 93.60% of the melanoma and 90.40% of the nevi images of the learning set. When the classification tree was applied to the independent test set 46.71 ± 19.97% (range 7.81–83.87%) of the tumour images in benign melanocytic skin lesions were classified as ‘malignant’, in contrast to 55.68 ± 14.58% (range 30.65–83.59%; t‐test: P < 0.036) in malignant melanocytic skin lesions (grouping method 1). Further investigations could not improve the results significantly (grouping method 2, 3 and 4). Conclusions The automated RCM image analysis procedure holds promise for further investigations. However, to date our system cannot be applied to routine skin tumour screening.     

In vivo confocal laser scanning microscopy in the diagnosis of melanocytic skin tumours

Summary Melanoma of the skin represents one of the greatest challenges in early or preventive detection. Whereas surgical excision in early stages of melanoma development is almost always curative, delayed recognition puts the patient at risk for destructive growth and death from disease once the tumour has progressed to competence for metastasis. The worldwide introduction of dermoscopy has led to improved diagnostic accuracy for melanocytic skin tumours. Whereas dermoscopy has probably reached the method's inherent potential diagnostic accuracy because of the lack of cellular level evaluation, further improvements could be expected by in vivo confocal laser scanning microscopy. In vivo confocal microscopy represents a novel imaging tool that allows the noninvasive examination of skin cancer morphology in real time at a 'quasihistopathological' resolution viewing microanatomical structures and individual cells. Numerous morphological confocal features of melanocytic skin tumours have been described and histopathological correlates of confocal structures have been previously elucidated. Recently, several studies have evaluated the diagnostic accuracy of in vivo confocal microscopy for melanocytic skin tumours, investigating approximately 50 000 tumour images. Remarkably, sensitivity superior to the diagnostic accuracy achieved with dermoscopy could be reached by this imaging modality. These studies represent a significant contribution to the body of research necessary for the evaluation and implementation of in vivo confocal microscopy in clinical practice to avoid many currently unnecessary biopsies. In vivo confocal microscopy probably augurs a sea change in the way we evaluate melanocytic skin tumours in the future and will ultimately move the art of histological diagnosis closer to the bedside.     

Reflectance confocal microscopy of facial lentigo maligna and lentigo maligna melanoma: a preliminary study

Background Facial lentigo maligna (LM) and lentigo maligna melanoma (LMM) may be difficult to diagnose clinically and dermoscopically. Reflectance confocal microscopy (RCM) enables the in vivo assessment of equivocal skin lesions at a cellular level. Objectives To assess cytomorphological and architectural RCM features of facial LM/LMM. Methods Four women and eight men aged 58–88 years presenting with facial skin lesions suspicious of LM/LMM were included. In total, 17 lesion areas were imaged by RCM before biopsy. The histopathological diagnosis of LM was made in 15 areas; the other two were diagnosed as early LMM. Results A focal increase of atypical melanocytes and nests surrounding adnexal openings, sheets of mainly dendritic melanocytes, cord‐like rete ridges at the dermoepidermal junction (DEJ) and an infiltration of adnexal structures by atypical melanocytes were found to be characteristic RCM features of facial LM/LMM. Areas with a focal increase of atypical melanocytes and nests surrounding adnexal openings were observed at the basal layer in three cases. The remaining cases displayed these changes at suprabasal layers above sheets of mainly dendritic melanocytes. Cord‐like rete ridges at the DEJ and an infiltration of adnexal structures by atypical melanocytes were observed in all cases. Previously described criteria for RCM diagnosis of melanoma, such as epidermal disarray, pleomorphism of melanocytes and pagetoid spreading of atypical melanocytes, were additionally observed. Conclusions We observed a reproducible set of RCM criteria in this case series of facial LM/LMM.     

In vivo reflectance confocal microscopy image interpretation for the dermatopathologist

Reflectance confocal microscopy (RCM) is a technology utilized for bedside diagnosis of cutaneous pathology by non‐invasive, in vivo, cellular‐level imaging. With the recent establishment of reimbursement codes by the US Centers for Medicaid and Medicare Services, RCM is now likely to be employed by clinical dermatologists and impact decision making on skin cancer management. Dermatopathologists, therefore, would benefit from learning how to interpret RCM images and how RCM findings correlate with histopathological criteria of diagnosis. This review briefly explains the principles behind RCM image acquisition, describes the key RCM features of normal skin, and delineates the RCM characteristics of frequently observed benign and malignant neoplasms.     

In vivo reflectance confocal microscopy findings in a case of atypical fibroxanthoma

Clinical and dermoscopic features of atypical fibroxanthoma (AFX) are mostly non-diagnostic, so other in vivo diagnostic tools may give additional clues for accurate clinical diagnosis, such as in vivo reflectance confocal microscopy (RCM). However, there has been scarce data on RCM features of AFX in the literature, in only clear cell type. Herein we present a case of epithelioid cell predominant type AFX with RCM findings.     

Characterization and evaluation of pigment distribution and response to therapy in melasma using in vivo reflectance confocal microscopy: a preliminary study

Background Melasma is a frequent skin disorder characterized by the appearance of abnormal pigment (melanin) deposits in different layers of the skin. Melasma has been classified into epidermal, dermal and mixed types using Wood’s lamp, and the type and extent of the pigment deposits determine the type and invasiveness of the treatment. Aims The aims of this study were to carry out a preliminary evaluation of the effective usefulness of reflectance confocal microscopy (RCM) in pigment distribution definition and subsequent re‐classification of melasma types. Moreover, RCM therapeutical follow‐up efficiency to combination therapy with pyruvic acid and hydroquinone was also tested. Materials and methods A small group (n = 15) of patients previously diagnosed with facial melasma were selected and their pigment distribution was evaluated by RCM. In seven of these patients therapeutical follow‐up was performed. Results The results of the study suggest that RCM is more accurate than techniques previously used in the diagnosis of melasma, thus providing precise information on the location and extent of pigment deposits. Discussion and conclusion The non‐invasive nature of this technique suggests that RCM may be a suitable tool for treatment monitoring, providing additional information not only on the evolution of the disorder but also on the possible occurrence of therapeutical side or adverse effects.     

The use of confocal laser-scanning microscopy in microsurgery for invasive squamous cell carcinoma

BACKGROUND: Ex-vivo confocal laser-scanning microscopy offers rapid imaging of excised tissue specimens without conventional histotechnical procedures. As vertical sections are prepared, morphological features can be assessed according to standard criteria used in conventional histopathology. OBJECTIVES: To validate the diagnostic confocal examination of squamous cell carcinoma (SCC) in microscopy-guided surgery. METHODS: Four independent observers received standardized instructions about diagnostic confocal microscopy features of SCC. Subsequently, 120 confocal images of fresh excisions from SCC or normal skin, imaged using a commercially available, near-infrared, reflectance confocal laser-scanning microscope, were evaluated by each observer. RESULTS: General morphology, such as location, size and shape of the cancer area could be visualized by the imaging system. Furthermore, densely packed and irregularly organized nuclei and nuclear atypia could be delineated. Overall, a sensitivity of 95% and a specificity of 96.25% were achieved by the four observers (positive predictive value 96.25%, negative predictive value 95.23%). CONCLUSIONS: This study provides a set of well-described morphological criteria with obvious diagnostic impact which should be used in further investigations. In the future, confocal laser-scanning microscopy may guide microsurgery of any skin cancer.     

Combined in vivo reflectance confocal microscopy and digital dermoscopy for follow up of patients at high risk of malignant melanoma: A prospective case series study

Digital dermoscopy (DD) follow up is a useful strategy for monitoring patients at high risk of melanoma. Reflectance confocal microscopy (RCM) is a valuable second‐level examination after dermoscopy for the evaluation of difficult to diagnose lesions. The aim of this study was to assess the value of RCM in routine DD monitoring of patients at high risk of melanoma. In this prospective study, performed over 22 months, changing melanocytic lesions were detected by DD and excised. RCM imaging was performed before surgical excision of all the lesions, and histopathology used as the gold standard diagnostic test. Eighty‐seven lesions, including 11 thin melanomas, were studied. Dermoscopic evaluation at follow up revealed a significant association between melanoma and asymmetry in two axes (P = 0.035). Enlargement and other changes in structure or color did not significantly differ between nevi and melanomas. Widespread pagetoid cells, non‐edged papillae, irregular and dishomogeneous junctional clusters, and sheet‐like structures were significantly associated with malignancy (P < 0.001). RCM allowed accurate diagnosis of melanoma in 10 of 11 cases (90.9%). The remaining case was classified as a dysplastic nevus. Forty‐six lesions (52.8%) in which RCM revealed no atypia were deemed unnecessarily removed. This study was limited by sample size. In conclusion, combined DD and RCM facilitates the recognition of thin malignant melanomas and reduces unnecessary excisions.     

Histological classification of melasma with reflectance confocal microscopy: a pilot study in Chinese patients

Objective: To investigate the histological classification of melasma with reflectance confocal microscopy (RCM) in vivo. Methods: Two hundred and ten cases with facial melasma lesions were enrolled. After informed consent, the target melasma lesion of 10 patients were imaged with RCM and then biopsied as well. Under the RCM scanning, the distribution of the melanin determined the histological types, and then, the results of RCM images were compared with those of the histopathology. The other 200 cases were tested only with RCM. Results: For the 10 cases imaged and biopsied, compared with that of the perilesional normal skin, the amount of melanin was significantly increased in the epidermis in all lesions under RCM, while three cases also found melanin in the dermis. Thus, seven of the10 patients were categorized as the epidermal type while the other three as mixed ones, and the results were well correlated with those of the histopathology. Of the other 200 patients, 143 cases 71.5%) were categorized as the epidermal type while the other 57 (28.5%) cases as mixed ones. Limitations: If more melasma cases are biopsied, the data will be more convincing. Conclusion: RCM in vivo analysis shows complete coherence with histopathology results, which could be an alternative for the classification of melasma, and based on the results of RCM imaging, melasma is classified into two major types: the epidermal type and mixed type.     

Digital image enhancement for in vivo laser scanning microscopy

BACKGROUND/PURPOSE: In vivo confocal laser scanning microscopy (CLSM) is a new method that provides skin images in horizontal plane at a level of resolution that allows to view microanatomic structures. This study examines whether certain digital image-processing steps can increase the visibility of various structures in CLSM. STUDY DESIGN: Fifty images were taken from normal skin of 25 probands, and 39 image enhancement procedures were created. Eight procedures that seemed to provide some quality enhancement were deliberately selected for further evaluation. Subsequently, a collection of random pairs of the original image and an image submitted to any of the eight selected procedures was rated by five independent observers. RESULTS: In three of the eight procedures tested, the modified image was significantly preferred to the original image (chi2-test,: P< or =0.001). In particular, smoothing, shading correction, delineate and grey-level normalization in various combinations were helpful in showing the characteristic honeycomb pattern, pigmented basal cell layer, cell borders and the nuclei more clearly. CONCLUSION: Digital image processing may help to increase visibility of in vivo CLSM images.     

In vivo confocal laser scanning microscopy for non-invasive diagnosis of pemphigus foliaceus

Background/purpose: In vivo confocal laser scanning microscopy (CLSM) is a modern non-invasive method for investigation of the skin that allows real-time visualization of individual cells and sub-cellular structures at resolution similar to the one provided by routine histopathology. Our aim was to investigate the potential of CLSM for non-invasive diagnosis of pemphigus foliaceus (PF).
Methods: Pre-existing and mechanically induced lesions in two cases of PF were examined by means of CLSM, parallel to routine histology, direct immunofluorescence microscopy and enzyme-linked immunosorbent assay performed in the same patients.
Results: The morphological features characteristic for PF, namely an intraepidermal blister with acantholytic cells in the blister cavity, were readily detectable by means of CLSM. The findings were consistent in both patients and across the investigated lesions. The confocal images were consistent with the routine histology of the pre-existing lesions. No differences in the confocal images of pre-existing lesions compared with mechanically induced ones were observed.
Conclusions: Our findings suggest the potential of CLSM as a non-invasive tool for the diagnosis of pemphigus and differentiation of its subtype. Although at present the method cannot replace the current diagnostic standards for pemphigus, it may be successfully used as in vivo non-invasive screening tool to facilitate the diagnosis and point to the need for further investigation of the patient.     

反射式共聚焦显微镜在炎症性皮肤病诊疗中的应用

反射式共聚焦激光扫描显微镜(RCM)作为一种在体无创性皮肤检查设备,能够实时动态反应细胞形态及皮肤结构的变化,在某些皮肤病中其图像特征与组织病理学检查结果具有高度的一致性,本文就RCM在炎症性皮肤病的诊断、疗效评价和疾病进展中的应用作一综述。     

Confocal laser-scanning capillaroscopy: a novel approach to the analysis of skin capillaries in vivo

Background: New techniques for diagnostics and therapy in dermatology are becoming increasingly non-invasive, among which confocal laser-scanning microscopy (CLSM) is the most prevalent. It allows visualization of cellular structures of the skin up to a depth of 300 μm in vivo . Until now, most studies have been conducted on pathologically altered skin, mostly oncologic lesions. We now present a detailed analysis of capillaries located in the upper dermal papillae.
Methods: Multiple measurements were performed on the dorsal and ventral surface of the right forearm of 30 healthy volunteers (22–88 years) under standard conditions (room temperature, body position, time of day). Images were obtained with the Vivascope 1500 (Lucid) under standard settings and analyzed using the freeware ImageJ with a customwritten macro plugin. The following parameters of the capillaries in vivo were measured: area, perimeter, circularity and maximum diameter.
Results: Statistical analysis showed that all four parameters were constant within a narrow range, regardless of the body site, sex and age. In this physiological study, we can clearly demonstrate that by confocal laser-scanning capillaroscopy (CLSC), it is possible to visualize and measure skin capillaries at the extremities in a reproducible manner.
Conclusion: This new approach offers a considerable advantage compared with nailfold capillaroscopy, which can only be performed at the proximal nail segment, and over histological analysis, which can be hampered by fixation artifacts resulting in altered size and shape of the vessels to be analyzed. CLSC could allow for precise analysis of in vivo skin vasculature in systemic and proliferative diseases of the skin.     

Wavelength effects on contrast observed with reflectance in vivo confocal laser scanning microscopy

Background/purpose: The ability to optically section live biological tissue in vivo with laser light is made possible by confocal laser scanning microscopy (CLSM). In this work, the effects of changing the wavelength of incident light used for CLSM imaging of human skin are reported and analyzed.
Methods: Optical phantoms and the skin of eight human volunteers were imaged using CLSM systems having three different incident light wavelengths (405, 785, and 830 nm).
Results: Qualitative and quantitative differences were observed between images obtained at each wavelength, despite the proximity of the two near infrared 785 and 830 nm wavelengths. Furthermore, the penetration depth achieved with the 405 nm CLSM permitted imaging into the papillary dermis.
Conclusion: The laser wavelength used in CLSM reflectance imaging is important to properly understand and resolve different biological structures within human skin.