Dermatology: Practical and Conceptual Original Article | Dermatol Pract Concept. 2025;15(3):5253 1 Diagnostic Accuracy of Magnified Dermoscopy and Reflectance Confocal Microscopy in Assessing Melanocytic Lesions Stefania Guida1,2, Silvana Ciardo3, Shaniko Kaleci3, Francesca Farnetani3, Marco Spadafora4, Giulia Radi5, Renato Rossi6, Elisa Molinelli7, Sabrina Longhitano3, Claudio Conforti8, Carmen Cantisani9, Camilla Chello8, Oriana Simonetti7, Anna Maria Offidani7, Pietro Rubegni10, Franco Rongioletti1,2, Caterina Longo3,4, Elisa Cinotti10§, Giovanni Pellacani9§ 1 School of Medicine, Vita-Salute San Raffaele University, Milan, Italy 2 Dermatology Clinic, IRCCS San Raffaele Scientific Institute, Milan, Italy 3 Dermatology Unit, Surgical, Medical and Dental Department of Morphological Sciences Related to Transplant, Oncology and Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy 4 Azienda Unità Sanitaria Locale-IRCCS di Reggio Emilia, Skin Cancer Center, Reggio Emilia, Italy 5 Azienda Sanitaria Territoriale Pesaro-Urbino, Pesaro, Italy 6 Department of Dermatology, Skin Center Senigallia, Ancona, Italy 7 Dermatological Clinic Department of Clinical and Molecular Sciences, Polytechnic University of the Marche Region, Ancona, Italy 8 IDI-IRCCS, Dermatological Research Hospital, Rome, Italy 9 Dermatology Clinic, Department of Clinical Internal, Anesthesiological and Cardiovascular Sciences, Sapienza University of Rome, Rome, Italy 10 Dermatology Section, Department of Medical, Surgical, and Neurological Sciences, Santa Maria Alle Scotte Hospital, Siena, Italy §equally contributed Key words: Magnified dermoscopy, Reflectance confocal microscopy, Diagnostic accuracy, Melanoma, Melanocytic lesions Citation: Guida S, Ciardo S, Kaleci S, et al. Diagnostic accuracy of magnified dermoscopy and reflectance confocal microscopy in assessing melanocytic lesions. Dermatol Pract Concept. 2025;15(3):5253. DOI: https://doi.org/10.5826/dpc.1503a5253 Accepted: February 9, 2025; Published: July 2025 Copyright: ©2025 Guida et al. This is an open-access article distributed under the terms of the Creative Commons Attribution- NonCommercial License (BY-NC-4.0), https://creativecommons.org/licenses/by-nc/4.0/, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original authors and source are credited. Funding: None. Competing Interests: None. Authorship: All authors have contributed significantly to this publication. Stefania Guida: data curation, conceptualization, writing- original draft preparation; Silvana Ciardo: data curation, image collection, draft revision; Shaniko Kaleci: statistical analysis; Francesca Farnetani, Marco Spadafora, Giulia Radi, Renato Rossi, Elisa Molinelli, Sabrina Longhitano: data curation; Claudio Conforti: original draft revision; Carmen Cantisani, Camilla Chello: data curation; Oriana Simonetti, Anna Maria Offidani, Pietro Rubegni, Franco Rongioletti, Caterina Longo: supervision; Elisa Cinotti, Giovanni Pellacani: conceptualization, writing-revision. Corresponding Author: Stefania Guida, Vita-Salute San Raffaele University, Via Olgettina 58, 20132 Milan, Italy. Phone: +39 02 2643 7354. E-mail: guida.stefania@hsr.it 2 Original Article | Dermatol Pract Concept. 2025;15(3):5253 Introduction Magnified dermoscopy (MD), or optical super-high magnifi- cation dermoscopy, is an imaging technique that offers up to 400x magnification, allowing for the visualization of cellular and structural details not visible with conventional 10-20x dermoscopy [1,2] MD has been applied to various skin le- sions, particularly for differentiating between nevi and mela- nomas, with criteria such as irregular cell shape and size being identified as significantly associated with malignant lesions [1,2]. Despite growing interest in its potential applications in routine skin cancer diagnosis, data on MD remain limited. Noteworthy, reflectance confocal microscopy (RCM) is a non-invasive diagnostic technique enabling the visualiza- tion of cells and architecture on different skin layers; as an adjunctive tool, it has been proven to reduce unnecessary excisions and significantly improve melanoma recognition, as compared to dermoscopy, in a recent randomized control trial [3]. However, the impact of MD in diagnostic accuracy of melanocytic lesions has not yet been provided, and a di- rect comparison of the adjunctive role of MD and RCM as compared to conventional dermoscopy is currently lacking. This retrospective study aimed to share our experience with dermoscopically equivocal pigmented lesions, analyz- ing their characteristics and evaluating the diagnostic accu- racy of melanoma detection using conventional dermoscopy, 400x MD, and RCM. Materials and Methods Study Population Conventional dermoscopy and MD (400x) images of pa- tients presenting to the Dermatology Unit at Policlinico of Modena, Italy, with flat, dermoscopically equivocal pig- mented lesions—diagnosed as either nevi or melanoma— were retrospectively analyzed. Diagnoses were confirmed through histopathological examination or a two-year clin- ical follow-up. Data on patient age, sex, anatomical lesion location, and histopathological results were also collected. Dermoscopy and Magnified Dermoscopy Dermoscopy images were captured by a technician with over 10 years of experience in both dermoscopy and reflectance confocal microscopy (RCM) using a DermLite Photo device (3Gen, San Juan Capistrano, CA, USA) and evaluated based on the revised 7-point checklist criteria [4]. MD images were obtained using a commercially available Fotofinder Medicam 1000 system (Fotofinder System, Bad Birnbach, Germany) and analyzed following the criteria established by Cinotti et al [2]. MD was performed after a quick exploration of the lesion at lower magnification (usually 70x magnification) to highlight the areas of interest. Then, as previously described [2], at least four images at 400x were taken from the most diagnostically suggestive parts, since MD enables the visual- ization of small areas at a time of the entire lesion (field of view of 1 x 0.5625 mm). Reflectance Confocal Microscopy RCM images were collected with Vivascope 1500® (MAVIG GmbH). RCM criteria included previously described features at the epidermis: atypical cells (presence, shape [dendritic or roundish] and distribution [focal or widespread]); at the dermal-epidermal junction (DEJ): atypical junctional nests, me- dusa head-like structures, sheet-like structures; at the dermis: heterogeneous dermal nests, and inflammation [5-7]. Introduction: Magnified dermoscopy (MD), or optical super-high magnification dermoscopy, is an emerging technique in dermatology. Objectives: The study aimed to evaluate the distribution of conventional dermoscopy, MD, and re- flectance confocal microscopy (RCM) features in dermoscopically equivocal pigmented lesions and to estimate their diagnostic accuracy. Methods: A retrospective analysis of conventional dermoscopic (20x), MD (400x), and RCM im- ages of dermoscopically equivocal pigmented lesions, diagnosed as either nevi or melanoma, was performed. Distribution of features, sensitivity, and specificity for dermoscopy, MD, RCM, and a com- bination of these last two with conventional dermoscopy was estimated. Results: A total of 74 nevi and 20 melanomas were included in the analysis. A positive correlation was observed between seven-point checklist in conventional dermoscopy and the diagnosis of melanoma. With MD, a significant correlation between dots, non-edged papillae, and melanoma was observed, but the technique did not have a significant impact on diagnostic accuracy as compared to traditional dermoscopy. On the other hand, RCM, alone or in combination with traditional dermoscopy, proved to increase diagnostic accuracy, in particular, specificity for melanoma diagnosis. Conclusions: RCM has a defined role in increasing diagnostic accuracy of doubtful dermoscopic le- sions, while the role of MD in clinical practice has yet to be defined, and methodologic standardization as well as a revision of terminology is encouraged to improve the recognition of features. ABSTRACT Original Article | Dermatol Pract Concept. 2025;15(3):5253 3 Statistical Analysis Statistical analysis was carried out with the SPSS software version 24 (SPSS, Armonk, NYC, US). Demographic, clinical, dermoscopic, MD, and RCM variables were included in the analysis. Continuous variables (patients [N], mean, standard deviation [SD]) were compared using an unpaired student’s t test (two groups). Categorical variables (frequency [N, %]) were compared using Pearson’s chi-squared test. Sensitivity, specificity, and 95% confidence intervals (CI) were calculated for melanoma diagnosis with either conven- tional dermoscopy, MD, or a combination of dermoscopy and MD as well as with RCM alone or in combination with dermoscopy. In addition, the receiver operating characteristic (ROC) curve was constructed with pulled evaluations of the three evaluators and the area under the curve (AUC) obtained. According to the literature, AUC of 0.5 was considered non- discriminant, 0.7 to 0.8 was considered acceptable, 0.8 to 0.9 was considered excellent, and more than 0.9 was consid- ered outstanding [8]. Alpha level was set at less than 0.05. Results Study Population and Dermoscopy An overall number of 104 lesions, one for each patient, were retrieved. Eight lesions located on the face and two acral lo- cated on the feet were excluded, leading to the inclusion of 74 nevi and 20 melanomas, with a mean Breslow thickness of 0.17 ± 0.2 (range 0-0.6). The distribution of demographic data, dermoscopic evaluation, MD, and RCM features are reported in Table 1. Intuitively, we observed a significant correlation between increased age, size, parameters of the seven-point checklist, and melanoma diagnosis (Table 1). Magnified Dermoscopy Features We found some significant correlations between MD fea- tures and the final diagnosis of melanocytic lesions. Accord- ingly, about 24% of melanomas versus 2% nevi showed dots (P=0.005), while 38% melanomas versus 16% nevi showed a network without edged papillae (P=0.03) (Figure 1). Reflectance Confocal Microscopy A significant correlation between melanoma and RCM fea- tures, including atypical cells with either dendritic or round- ish shape showing widespread distribution and atypical junctional nests, was observed (Table 1, Figure 2). Diagnostic Accuracy Results related to diagnostic accuracy are reported in Table S1 and Figure 2. Sensitivity for melanoma diagnosis with dermoscopy reached 80% (95% CI: 59.5–93.3), while specificity was 65% (95% CI: 54.5–75.3), very similar to results observed with MD combined with dermoscopy, with an acceptable AUC (Table S1, Figure 3). Notably, RCM alone or in combination with dermoscopy reached the highest sensitivity (85%, 95% CI: 65.6–96.0) and specificity (81%, 95% CI: 71.2–88.9 and 79%, 95% CI: 69.6–87.8, respectively), with excellent AUC (Table S1, Figure 3). Discussion In recent decades, dermoscopy has emerged as an import- ant tool in dermatology, particularly in the diagnosis of skin cancers, earning the reputation as being the dermatologist’s stethoscope [9]. More recently, interest has grown in magni- fied dermoscopy (MD), a technique utilizing 400x magnifica- tion to visualize structures not detectable with conventional 20x dermoscopy [10]. While MD has been applied to skin cancer diagnostics, and criteria distinguishing nevi from mel- anoma have been identified [1,2], this paper presents the first report of diagnostic accuracy of MD as compared to and integrated with dermoscopy and compared to RCM with or without dermoscopy. The current study highlighted that the diagnostic ac- curacy of dermoscopy and its combination with MD was similar, while with RCM, we observed a slight increase in sensitivity and a substantial increase in specificity, resulting in improved diagnostic accuracy, as indicated by an excellent AUC and a reduction in false negative cases. Our results are in line with data from recent meta- analyses highlighting a pooled accuracy for melanoma diag- nosis of 88% sensitivity for dermoscopy and 90% for RCM and 38–49% specificity for dermoscopy and 42–77% for RCM, revealing a sensitivity and specificity for dermoscopy of 80% and 65% and RCM of 85% and 81%, respectively [11-13]. As a matter of fact, in our study, RCM showed the highest diagnostic accuracy for melanoma, in particular in terms of specificity, with an increase of about 10% in speci- ficity, as compared to dermoscopic approach. This result can be related to the unique possibility of exploring the skin in different layers separately and of identifying qualitative and quantitative signs of atypia due to the good cellular resolu- tion of the instrument [5,7]. Furthermore, in this study, we observed a correlation be- tween melanoma and the presence of dots and non-edged papillae observed with MD. While the association of non- edged papillae, referred to ill-defined network lines, with melanoma appears intuitive, dots have not previously been associated with a specific diagnosis; this is thus a novel finding. Indeed, as dots have not been correlated to specific anatomic or dermoscopic structures, further investigations should clarify this aspect. According to previous studies, melanoma diagnosis was associated with the presence of 4 Original Article | Dermatol Pract Concept. 2025;15(3):5253 Table 1. Demographic Characteristics and Dermoscopic and MD Features, according to Melanoma or Nevi Diagnosis. Melanoma N=20 Nevus N=74 Total N=94 p-value Demographic characteristics Sex, n (%) female 13 (65) 37 (50) 50 (53.2) 0.233 male 7 (35) 37 (50) 44 (46.8) Age, mean ± SD (range) 59.1 ± 15 (36-93) 44.9 ± 15.4 (8-80) 47.8 ± 16.4 (8-93) 0.001 Skin site, n (%) neck 1 (5) 4 (5.4) 5 (5.3) 0.307 trunk 12 (60) 56 (75.7) 68 (72.3) limbs 7 (35) 14 (18.9) 21 (22.3) Size, mean ± SD (range) mm 9.3±4.8 (3-20) 5.6±2.6 (1-12) 6.2±3.4 (1-20) 0.001 Dermoscopy features Prevalent pattern, n (%) reticular 6 (30) 24 (32.4) 30 (31.9) 0.309 globular 1 (5) 10 (13.5) 11 (11.7) homogeneous 1 (5) 3 (4.1) 4 (4.3) Peripheral globules/starburst 0 4 (5.4) 4 (4.3) Two-component 7 (35) 27 (36.5) 34 (36.2) Multi-component 5 (25) 6 (8.1) 11 (11.7) Atypical pigmentation, n (%) 18 (90) 41 (55.4) 59 (62.8) 0.004 Blue-white veil, n (%) 12 (60) 11 (14.9) 23 (24.5) 0.001 Atypical vessels, n (%) 1 (5) 6 (8.1) 7 (7.4) 1 Irregular streaks, n (%) 5 (25) 4 (5.4) 9 (9.6) 0.019 Irregular dots/globules, n (%) 11 (55) 38 (51.4) 49 (52.1) 0.772 Irregular blotches, n (%) 13 (65) 24 (32.4) 37 (39.4) 0.008 Regression, n (%) 14 (70) 39 (52.7) 53 (56.4) 0.166 MD features Keratinocytes, n (%) 18 (90) 67 (90.5) 85 (90.4) 1.000 Roundish melanocytes, n (%) 11 (55) 39 (52.7) 50 (53.2) 0.855 Dendritic melanocytes, n (%) 2 (10) 11 (14.9) 13 (13.8) 0.728 Melanophages, n (%) 6 (30) 16 (21.6) 22 (23.4) 0.432 Cell irregularity, n (%) 8 (40) 19 (25.7) 27 (28.7) 0.209 Cell distribution, n (%) 12 (60) 57 (77) 69 (73.4) 0.126 Dots, n (%) 5 (25) 2 (2.7) 7 (7.4) 0.004 Roundish nests, n (%) 1 (5) 12 (16.2) 13 (13.8) 0.287 Structureless, n (%) 15 (75) 47 (63.5) 62 (66) 0.430 Vessels, n (%) 7 (35) 29 (39.2) 36 (38.3) 0.732 Hyperkeratotic concentric areas, n (%) 3 (15) 7 (9.5) 10 (10.6) 0.439 Network with edged papillae, n (%) 9 (45) 46 (62.2) 55 (58.5) 0.167 Network without edged papillae, n (%) 8 (40) 12 (16.2) 19 (21.3) 0.021 RCM features Atypical cells, n (%) 16 (80) 6 (8.1) 22 (23.4) 0.001 Widespread atypical cells, n (%) 2 (10) 1 (1.4) 3 (3.2) 0.001 Original Article | Dermatol Pract Concept. 2025;15(3):5253 5 Melanoma N=20 Nevus N=74 Total N=94 p-value Dendritic cells, n (%) 13 (65) 6 (8.1) 19 (20.2) 0.001 Roundish cells, n (%) 1 (5) 0 1 (1.1) 0.001 Atypical junctional nests, n (%) 11 (55) 9 (12.2) 20 (21.3) 0.001 Medusa head like structures, n (%) 1 (%) 0 1 (1.1) 0.213 Sheet-like structures, n (%) 0 1 (1.4) 1 (1.1) 1.000 Heterogeneous dermal nests 2 (10) 1 (1.2) 3 (3.2) 0.113 Inflammation 0 5 (6.8) 5 (5.3) 0.581 Table 1. Demographic Characteristics and Dermoscopic and MD Features, according to Melanoma or Nevi Diagnosis. (continued) Figure 1. Pictures of conventional dermoscopy (CD) and magnified dermoscopy (MD) in a nevus and a melanoma. A) CD showing a pigmented lesion with different shades of brown color, irregular network and reticular depigmentation, with a histopathologi- cal diagnosis of nevus, and B) corresponding MD showing edged papillae/well-defined network lines (stars). C) CD showing a pigmented brown lesion with irregular network and focal reticular depigmentation, with a histopathological diagnosis of melanoma 0.3 mm Breslow, and D) corresponding MD showing non-edged papillae/ill-defined network lines (asterisks) and dots (arrows). scattered, large, irregular (in shape and size), dendritic or roundish violet/blue pigmented cells in melanoma, while edged papillae (or well-defined network lines) were more commonly associated with nevi in MD [1,2]. This study represents the first attempt to clarify the im- pact of the emerging MD in melanoma diagnosis, showing that currently, MD diagnostic accuracy is comparable to conventional dermoscopy. One potential explanation for this finding is the recently reported lack of consensus among evaluators regarding the identification of MD features [14]. Accordingly, a notable challenge lies in the terminology used for MD, which shares similarities with features observed in RCM and conventional dermoscopy, potentially causing confusion for evaluators experienced with different imaging modalities. To date, no comprehensive alignment between the features of conventional dermoscopy, histology, RCM, and MD has been established, highlighting the need for stan- dardized terminology to define MD criteria. However, the retrospective design of the study may pres- ent certain limitations. Indeed, only images of small areas from each skin lesion can be captured with MD (while RCM Vivascope 1500 enables the visualization of the full lesion), impairing a comprehensive evaluation of the entire lesion. Additionally, the images in the current study were acquired 6 Original Article | Dermatol Pract Concept. 2025;15(3):5253 Figure 2. A) Conventional dermoscopy of a melanoma showing an irregular pig- mented network; B) corresponding magnified dermoscopy showing non-edged papillae/ ill- defined network lines (asterisks) as well as edged papillae/well-defined network lines (stars); C) corresponding reflectance confocal microscopy (RCM) image at epidermal level showing atypical cells (blue circles) and D) RCM of the same lesion at dermo- epidermal level showing atypical cells (blue circle) and non-edged papillae (blue stars). Figure 3. Receiver operating characteristic (ROC) curve for dermoscopy, magnified dermoscopy (MD), reflectance confocal microscopy, and the area under the curve (AUC) obtained. by an investigator without prior experience in MD, suggest- ing that diagnostic accuracy could be enhanced through pro- spective studies conducted by experienced investigators with better area selection for imaging. Another significant challenge with MD is related to the quality of images necessary for reliable feature identification. Current guidelines for image acquisition are lacking, further complicating the proper application of this technique. Original Article | Dermatol Pract Concept. 2025;15(3):5253 7 Dermatol. 1993; 28(6): 923-7. DOI: 10.1016/0190-9622(93) 70131-c. PMID: 8496455. 11. Pezzini C, Kaleci S, Chester J, Farnetani F, Longo C, Pellacani G. Reflectance confocal microscopy diagnostic accuracy for malig- nant melanoma in different clinical settings: systematic review and meta-analysis. 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