Visualization of keratin with diffuse reflectance and autofluorescence imaging and nonlinear optical microscopy in a rare keratinopathic ichthyosis

Funding Information: Funding: This work was supported by grants from the National Research, Development and Innovation Office of Hungary—NKFIH (FK_131916, 2019 (Semmelweis University, M.M.)), EFOP-3.6.3-VEKOP-16-2017-00009 (P.A., S.B.); the New National Excellence Program of the Ministry for Innovation and Technology (ÚNKP-20-4-II-SE-7 (N.K.), ÚNKP-19-3-I-SE-78 (L.F.)); and the European Regional Development Fund project “Time-resolved autofluorescence methodology for noninvasive skin cancer diagnostics” (no. 1.1.1.2/16/I/001, agreement no. 1.1.1.2/VIAA/1/16/014 (I.L., A.L., M.L.)). Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Keratins are one of the main fluorophores of the skin. Keratinization disorders can lead to alterations in the optical properties of the skin. We set out to investigate a rare form of keratinopathic ichthyosis caused by KRT1 mutation with two different optical imaging methods. We used a newly developed light emitting diode (LED) based device to analyze autofluorescence signal at 405 nm excitation and diffuse reflectance at 526 nm in vivo. Mean autofluorescence intensity of the hyperkeratotic palmar skin was markedly higher in comparison to the healthy control (162.35 vs. 51.14). To further assess the skin status, we examined samples from affected skin areas ex vivo by nonlinear optical microscopy. Two-photon excited fluorescence and second-harmonic generation can visualize epidermal keratin and dermal collagen, respectively. We were able to visualize the structure of the epidermis and other skin changes caused by abnormal keratin formation. Taken together, we were able to show that such imaging modalities are useful for the diagnosis and follow-up of keratinopathic diseases.publishersversionPeer reviewe

Autofluorescence imaging of the skin is an objective non-invasive technique for diagnosing pseudoxanthoma elasticum

Funding Information: Funding: This work was supported by grants from the National Research, Development and Innovation Office of Hungary—NKFIH [FK_131916, 2019 (Semmelweis University, M.M.); K_132695, 2019 (Semmelweis University, T.A.)], EFOP-3.6.3-VEKOP-16-2017-00009 (P.A., S.B.) the ÚNKP-20-4-II-SE-7 (N.K.) and ÚNKP-20-3-I-SE-24 (S.Z.) New National Excellence Program of the Ministry For Innovation and Technology from the source of the National Research, Development and Innovation Fund of Hungary and the European Regional Development Fund projects “Time-resolved autofluorescence methodology for non-invasive skin cancer diagnostics” [No. 1.1.1.2/16/I/001, agreement No. 1.1.1.2/VIAA/1/16/014 (A.L.)] and “Development and clinical validation of a novel cost effective multi-modal methodology for early diagnostics of skin cancers” [No. 1.1.1.2/16/I/001 agreement No. 1.1.1.2/VIAA/1/16/052 (I.L.)] and COST action CA16115 EuroSoftCalcNet. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Pseudoxanthoma elasticum (PXE) is a rare multisystemic autosomal recessive connective tissue disease. In most cases, skin manifestations of PXE are the first to develop, followed later by severe ocular and cardiovascular complications. In our present study, in addition to dermoscopy, we introduced novel techniques, autofluorescence (AF) and diffuse reflectance (DR) imaging for the assessment of affected skin sites of five PXE patients. PXE-affected skin areas in most skin sites showed a previously observed pattern upon dermoscopic examination. With the novel imaging, PXE-affected skin lesions displayed high AF intensity. During our measurements, significantly higher mean, minimum and maximum AF intensity values were found in areas of PXE-affected skin when compared to uninvolved skin. Conversely, images acquired with the use of 660 and 940 nm illumination showed no mentionable difference. Our results demonstrate that AF imaging may be used in the in vivo diagnostics and quantification of the severity of the skin lesions of PXE patients. In addition, it is a safe, fast and cost-effective diagnostic method. AF imaging may be also used to objectively monitor the efficacy of the possible novel therapeutic approaches of PXE in the future.publishersversionPeer reviewe

Quantitative multispectral imaging differentiates melanoma from seborrheic keratosis

Funding Information: This work was supported by grants from the EFOP-3.6.3-VEKOP-16-2017-00009 (S.B., P.A.) EFOP-3.6.3-VEKOP-16 (S.B.) the ÚNKP-20-4-II-SE-7 (N.K.) and ÚNKP-20-3-I-SE-24 (S.Z.) New National Excellence Program of the Ministry For Innovation and Technology from the source of the National Research, Development and Innovation Fund of Hungary and the European Regional Development Fund projects “Time-resolved autofluorescence methodology for non-invasive skin cancer diagnostics” [No. 1.1.1.2/16/I/001, agreement No. 1.1.1.2/VIAA/1/16/014 (A.L.)] and “Development and clinical validation of a novel cost effective multi-modal methodology for early diagnostics of skin cancers” [No. 1.1.1.2/16/I/001 agreement No. 1.1.1.2/VIAA/1/16/052 (I.L.)] and the National Research, Development and Innovation Office of Hungary—NKFIH (FK_131916, 2019 (Semmelweis University, M.M.)). Publisher Copyright: © 2021 by the authors.Melanoma is a melanocytic tumor that is responsible for the most skin cancer-related deaths. By contrast, seborrheic keratosis (SK) is a very common benign lesion with a clinical picture that may resemble melanoma. We used a multispectral imaging device to distinguish these two entities, with the use of autofluorescence imaging with 405 nm and diffuse reflectance imaging with 525 and 660 narrow-band LED illumination. We analyzed intensity descriptors of the acquired images. These included ratios of intensity values of different channels, standard deviation and minimum/maximum values of intensity of the lesions. The pattern of the lesions was also assessed with the use of particle analysis. We found significantly higher intensity values in SKs compared with melanoma, especially with the use of the autofluorescence channel. Moreover, we found a significantly higher number of particles with high fluorescence in SKs. We created a parameter, the SK index, using these values to differentiate melanoma from SK with a sensitivity of 91.9% and specificity of 57.0%. In conclusion, this imaging technique is potentially applicable to distinguish melanoma from SK based on the analysis of various quantitative parameters. For this application, multispectral imaging could be used as a screening tool by general physicians and non-experts in the everyday practice.publishersversionPeer reviewe

Nonlinear optical microscopy is a novel tool for the analysis of cutaneous alterations in pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE, OMIM 264800) is a rare autosomal recessive disorder with ectopic mineralization and fragmentation of elastin fibers. It is caused by mutations of the ABCC6 gene that leads to decreased serum levels of inorganic pyrophosphate (PPi) anti-mineralization factor. The occurrence of severe complications among PXE patients highlights the importance of early diagnosis so that prompt multidisciplinary care can be provided to patients. We aimed to examine dermal connective tissue with nonlinear optical (NLO) techniques, as collagen emits second-harmonic generation (SHG) signal, while elastin can be excited by two-photon excitation fluorescence (TPF). We performed molecular genetic analysis, ophthalmological and cardiovascular assessment, plasma PPi measurement, conventional histopathological examination, and ex vivo SHG and TPF imaging in five patients with PXE and five age- and gender-matched healthy controls. Pathological mutations including one new variant were found in the ABCC6 gene in all PXE patients and their plasma PPi level was significantly lower compared with controls. Degradation and mineralization of elastin fibers and extensive calcium deposition in the mid-dermis was visualized and quantified together with the alterations of the collagen structure in PXE. Our data suggests that NLO provides high-resolution imaging of the specific histopathological features of PXE-affected skin. In vivo NLO may be a promising tool in the assessment of PXE, promoting early diagnosis and follow-up

Quantitative Multispectral Imaging Differentiates Melanoma from Seborrheic Keratosis

Melanoma is a melanocytic tumor that is responsible for the most skin cancer-related deaths. By contrast, seborrheic keratosis (SK) is a very common benign lesion with a clinical picture that may resemble melanoma. We used a multispectral imaging device to distinguish these two entities, with the use of autofluorescence imaging with 405 nm and diffuse reflectance imaging with 525 and 660 narrow-band LED illumination. We analyzed intensity descriptors of the acquired images. These included ratios of intensity values of different channels, standard deviation and minimum/maximum values of intensity of the lesions. The pattern of the lesions was also assessed with the use of particle analysis. We found significantly higher intensity values in SKs compared with melanoma, especially with the use of the autofluorescence channel. Moreover, we found a significantly higher number of particles with high fluorescence in SKs. We created a parameter, the SK index, using these values to differentiate melanoma from SK with a sensitivity of 91.9% and specificity of 57.0%. In conclusion, this imaging technique is potentially applicable to distinguish melanoma from SK based on the analysis of various quantitative parameters. For this application, multispectral imaging could be used as a screening tool by general physicians and non-experts in the everyday practice

Altered Glycosylation of Human Alpha-1-Acid Glycoprotein as a Biomarker for Malignant Melanoma

A high-resolution HILIC-MS/MS method was developed to analyze anthranilic acid derivatives of N-glycans released from human serum alpha-1-acid glycoprotein (AGP). The method was applied to samples obtained from 18 patients suffering from high-risk malignant melanoma as well as 19 healthy individuals. It enabled the identification of 102 glycan isomers separating isomers that differ only in sialic acid linkage (α-2,3, α-2,6) or in fucose positions (core, antenna). Comparative assessment of the samples revealed that upregulation of certain fucosylated glycans and downregulation of their nonfucosylated counterparts occurred in cancer patients. An increased ratio of isomers with more α-2,6-linked sialic acids was also observed. Linear discriminant analysis (LDA) combining 10 variables with the highest discriminatory power was employed to categorize the samples based on their glycosylation pattern. The performance of the method was tested by cross-validation, resulting in an overall classification success rate of 96.7%. The approach presented here is significantly superior to serological marker S100B protein in terms of sensitivity and negative predictive power in the population studied. Therefore, it may effectively support the diagnosis of malignant melanoma as a biomarker