BACKGROUND:Loss-of-function FLG mutation (FLGmut) carriers are at an increased risk of developing atopic dermatitis (AD), characterized by earlier onset and more severe disease. AD is driven by a complex interplay between skin barrier function, TH2 and TH2-dominant immune dysregulation, and dysbiosis. Results from the Short-Term Topical Application for Prevention of Atopic Dermatitis study suggest 2 early initiating AD pathogenetic pathways: an FLGmut-related skin barrier deficiency pathway and an immune function-related inflammatory pathway. The Short-Term Topical Application for Prevention of Atopic Dermatitis study suggested that early preventative intervention with specialized emollients for barrier function augmentation may benefit newborns with FLGmut. This requires early identification of FLGmut carriers, for which noninvasive Raman spectroscopic determination of natural moisturizing factor (NMF) levels in the stratum corneum of the thenar eminence provides a surrogate marker. OBJECTIVE:To identify strategies for early identification of infants with FLGmut. METHODS:FLG sequencing was performed on 253 infants, and NMF concentrations were measured in the stratum corneum of the palmar eminence (pSC-NMF) using noninvasive Raman spectroscopy at 6 time points after birth. Furthermore, the pSC-NMF concentrations were obtained from both parents of 150 infants. RESULTS:Babies are born with little to no NMF. In the first days after birth, NMF levels rapidly increase and 65% of newborns with FLG wild type already reach pSC-NMF concentrations, which excludes them as FLGmut carriers with high specificity. At 2 weeks of age, FLGmut carriers could be distinguished from newborns with FLG wild type with high sensitivity (97%) and specificity (97%). In addition, parent pSC-NMF concentrations offer the possibility to exclude their newborn as FLGmut carriers with high specificity. CONCLUSION:Noninvasive Raman spectroscopy enables the accurate early identification of infants with FLGmut.

We have developed a method to determine the limit of detection (LoD) for quantitative measurement of exogenous analytes in the outer layer of the human skin by in vivo confocal Raman spectroscopy. The method is in accordance with the guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use that have been adopted by regulatory authorities such as the American Food and Drug Administration and the European Medicines Agency. The method can be applied in silico so that the limit of detection can be assessed before starting a skin penetration study, for example, in areas of pharmaceutical formulation, pharmacokinetics, or toxicokinetics. This can significantly reduce the need for expensive and time-consuming feasibility studies. This paper describes the method to calculate this LoD as well as the experimental and methodological factors that can influence the calculation of the LoD.

Background: A preference for type 2 immunity plays a central role in the pathogenesis of atopic dermatitis (AD). Dupilumab, an mAb targeting the IL-4 receptor a (IL-4Ra) subunit, inhibits IL-4 and IL-13 signaling. These cytokines contribute significantly to IgE class switch recombination in B cells, critical in atopic diseases. Recent studies indicate IgG+CD23hiIL-4Ra+ type 2 memory B cells (MBC2s) as IgE-producing B-cell precursors, linked to total IgE serum levels in atopic patients. Total IgE serum levels decreased during dupilumab treatment in previous studies. Objective: We sought to assess the effects of dupilumab treatment in comparison with alternative therapies on the frequency of MBC2s and the correlation to total IgE levels in pediatric patients with AD. Methods: Pediatric patients with AD, participating in an ongoing trial, underwent randomization into 3 treatment groups: dupilumab (n = 12), cyclosporine (n = 12), and topical treatment (n = 12). Plasma samples and PBMCs were collected at baseline (T0) and at 6 months after starting therapy (T6). Flow cytometry was used for PBMC phenotyping, and ELISA was used to assess total IgE levels in plasma. Results: Our findings revealed a significant reduction in MBC2 frequency and total IgE levels among patients treated with dupilumab. In addition, a significant correlation was observed between MBC2s and total IgE levels. Conclusions: Systemic blocking of the IL-4Ra subunit leads to a decrease in circulating MBC2 cells and total IgE levels in pediatric patients with AD. Our findings unveiled a novel mechanism through which dupilumab exerts its influence on the atopic signature. (J Allergy Clin Immunol 2024;154:1333-38.)

Loss-of-function (LoF) filaggrin (FLG)-mutations are the strongest genetic risk factor for atopic dermatitis (AD). The STOP-AD study showed early initiation of daily specialized emollient, used only until 2 months, reduces the incidence of AD in the first year of life in high-risk infants. 321 newborns were recruited, randomized 1:1 between intervention group (IG), receiving emollient treatment and control group (CG). FLG status was determined by Microfluidics PCR for full coverage of FLG repeat alleles. 257 infants with genotyping completed the study, 119 in IG and 138 in CG. 12 month cumulative AD-incidence was 46% in the CG vs. 33% in the IG (p=0.03). 44 (17.4%, evenly split between groups) were LOF-FLG mutation carriers, who had significantly higher AD-prevalence of 56% and 59% at 6 and 12 months respectively in the CG, compared to 32% at 6m (p =0.02) and 34 at 12m % (p=0.01) in WT-FLG. In IG group the LOF-FLG babies had AD rates nearly the same as WT-FLG infants at 6 and 12m: 19% and 14% respectively compared to 15% (p=NS)and 14% (p=NS) in WT-FLG babies. Daily specialized emollient use until 2 months may be especially beneficial for LoF FLG-mutation carriers, reducing their AD-prevalence and incidence to the level of the treated FLG-wildtype group. Identification of FLG status soon after birth may be beneficial in deciding which child may benefit most from targeted preventive strategies.

Summary of cutaneous adverse events at the different time point for each patient included in this study. n/a: not available

Loss-of-function filaggrin (LoF-FLG)-mutation carriers are at higher risk of early, more severe Atopic Dermatitis (AD) than wild type (WT-FLG) infants . We present a strategy for early discrimination between WT-FLG and LoF-FLG-mutation carriers, based on Raman spectroscopic analysis of Natural Moisturizer Factor (NMF) content in the stratum corneum of the thenar eminence. The single center STOP-AD study recruited 321 newborns, 260 completed the study. NMF-content was measured, once <4 days after birth, at 2, 4, 8, 26 and 52 weeks. FLG-mutation status was determined by microfluidics PCR on genomic DNA for full FLG-coverage. NMF-analysis was performed for both parents of 134 newborns. Parental NMF-content > 0.35 [gNMF/gprotein], predicted WT-FLG newborns with 100% specificity and 72% sensitivity. Newborn NMF-content > 0.38 [gNMF/gprotein] at 0-4 days predicted WT-FLG with 100% specificity and 43%-sensitivity. Combined analysis of parental and newborn NMF-content predicted WT-FLG with 100% specificity and 87% sensitivity. At 2 weeks NMF-content >0.38 [gNMF/g protein] predicted LoF-FLG-mutation carriers with 100% sensitivity and 92% specificity. Parental NMF and newborn NMF-measurement at 0-4 days could inform families that their newborn child was in a relatively lower risk group for development of AD and could be used to exclude 87% of WT-FLG newborns from any preventive treatment being proposed. Families of the remaining group of newborns, containing 100% of LOF-FLG mutation carriers, could be informed of potentially increased risk of AD and could be offered potential preventive treatment. Repeat NMF-analysis at 2 weeks could exclude almost all remaining FLG-wildtypes from further proposed preventive treatment

Near-infrared (NIR) fluorescence imaging using exogenous fluorescent agents provides whole-field images in real-time to assist the surgeon in the excision of a tumor. Although the method has high sensitivity, the specificity can sometimes be lower than expected. Raman spectroscopy can detect tumors with high specificity. Therefore, a combination of both techniques can be advantageous. A complication that must be addressed is that the NIR spectral region is favored by both techniques for (in vivo) tissue analysis. When fluorescence and Raman emissions spectrally overlap, it becomes challenging or impossible to detect the Raman signal. In this paper, by avoiding this overlap, we describe a Raman spectroscopy setup capable of recording high-quality Raman spectra from tissue containing NIR exogenous fluorescent agents. We identify an optimal wavelength interval (900-915 nm) for Raman excitation, which avoids both excitation of fluorescent dyes and Raman signal self-absorption by the tissue. In this way, Raman spectroscopy can be combined with the currently most-used NIR fluorescent dyes. This combined novel setup could pave the way for clinical trials benefiting from both fluorescence imaging and Raman spectroscopy to avoid positive margins in cancer surgery.

Background In head and neck oncological surgery the goal is to achieve a complete tumor resection with acceptable remaining function and appearance. For oral cavity squamous cell carcinoma (OCSCC) only 15% of the resections are reported as adequate. Since 2013, we have performed intraoperative assessment of resection margins (IOARM) in our institute, based on palpation and visual inspection of the resected specimens by pathologist and surgeon. This has resulted in an improvement of adequate resection margins from 15% to 50%, underlining the importance of IOARM. However, this method is subjective, labor intensive, and logistically challenging. Objective Our aim is to develop an objective method for fast and reliable IOARM based on Raman spectroscopy (RS). Methods RS is a non-destructive objective optical technique that provides information about the molecular composition of tissues. It can discriminate between healthy tissue and tumors. We developed a prototype Raman instrument employing a fiber-optic needle probe. The fiber-optic needle is driven into the OCSCC specimen, from the resection surface towards the tumor. Based on the Raman spectra collected along the insertion path, the location of the tumor border can be determined. From this the resection margin can be determined. Results First tests of the method show that the instrument accurately predicts the achieved resection margins. Per location the measurement and assessment takes 5 seconds. Conclusions This development signifies an important step towards a fast and objective IOARM. The fast measurement time enables an objective inspection of the margins achieved at a large number of locations of the resection surface. In head and neck oncological surgery the goal is to achieve a complete tumor resection with acceptable remaining function and appearance. For oral cavity squamous cell carcinoma (OCSCC) only 15% of the resections are reported as adequate. Since 2013, we have performed intraoperative assessment of resection margins (IOARM) in our institute, based on palpation and visual inspection of the resected specimens by pathologist and surgeon. This has resulted in an improvement of adequate resection margins from 15% to 50%, underlining the importance of IOARM. However, this method is subjective, labor intensive, and logistically challenging. Our aim is to develop an objective method for fast and reliable IOARM based on Raman spectroscopy (RS). RS is a non-destructive objective optical technique that provides information about the molecular composition of tissues. It can discriminate between healthy tissue and tumors. We developed a prototype Raman instrument employing a fiber-optic needle probe. The fiber-optic needle is driven into the OCSCC specimen, from the resection surface towards the tumor. Based on the Raman spectra collected along the insertion path, the location of the tumor border can be determined. From this the resection margin can be determined. First tests of the method show that the instrument accurately predicts the achieved resection margins. Per location the measurement and assessment takes 5 seconds. This development signifies an important step towards a fast and objective IOARM. The fast measurement time enables an objective inspection of the margins achieved at a large number of locations of the resection surface.

Confocal Raman spectroscopy (CRS) enables the real-time profiling of substances penetrating to the skin without sample pre-treatment or labelling. Until now, CRS had been used as a semi-quantitative method, which posed challenges for evaluating topical formulations and assessing bioequivalence. We present a novel approach of CRS for quantitative analysis of skin delivery. The quantitative CRS in vivo has been correlated with the well-established in vitro Franz-diffusion experiments, indicating the potential of CRS for determining skin delivery. We anticipate CRS providing a rapid and non-invasive method that will be an attractive alternative to the clinical studies currently used in bioequivalence testing.

We present the first clinical integration of a prototype device based on integrated auto-fluorescence imaging and Raman spectroscopy (Fast Raman device) for intra-operative assessment of surgical margins during Mohs micrographic surgery of basal cell carcinoma (BCC). Fresh skin specimens from 112 patients were used to optimise the tissue pre-processing and the Fast Raman algorithms to enable an analysis of complete Mohs layers within 30 minutes. The optimisation allowed >95% of the resection surface area to be investigated (including the deep and epidermal margins). The Fast Raman device was then used to analyse skin layers excised from the most relevant anatomical sites (nose, temple, eyelid, cheek, forehead, eyebrow and lip) and to detect the three main types of BCC (nodular, superficial and infiltrative). These results suggest that the Fast Raman technique is a promising tool to provide an objective diagnosis “tumour clear yes/no” during Mohs surgery of BCC. This clinical integration study is a key step towards a larger scale diagnosis test accuracy study to reliably determine the sensitivity and specificity in a clinical setting.

This study reports on the effects of insertion velocity, needle tip geometry and needle diameter on tissue deformation and maximum insertion force. Moreover, the effect of multiple insertions with the same needle on the maximum insertion force is reported. The tissue deformation and maximum insertion force strongly depend on the insertion velocity and the tip geometry. No correlation was found between the outer diameter and the maximum insertion force for small needles (30G - 32G). The endurance experiments showed no remarkable difference in the maximum insertion force during 100 insertions.

The composition of topical and transdermal formulations is known to determine the rate and the extent of drug delivery to and through the skin. However, to date, the role of excipients in these formulations on skin delivery of actives has received little attention from scientists in the field. Monitoring skin absorption of both drug and vehicle may provide insights into the mechanism by which excipients promote permeation and may facilitate the design of effective and safer products. Previously, we have investigated the use of quantitative Confocal Raman Spectroscopy (CRS) to investigate the delivery of an active to the skin, and we also reported the first fully quantitative study that compared this method with the well-established in vitro permeation test (IVPT) model. To further explore the potential of quantitative CRS in assessing topical delivery, the present work investigated the effects of commonly used excipients on the percutaneous absorption of a model drug, ibuprofen (IBU). Permeation of IBU and selected solvents following finite dose applications to human skin was determined in vitro and in vivo by Franz diffusion studies and quantitative CRS, respectively. The solvents used were propylene glycol (PG), dipropylene glycol (DPG), tripropylene glycol (TPG), and polyethylene glycol 300 (PEG 300). Overall, the cumulative amounts of IBU that permeated at 24 h in vitro were similar for PG, DPG, and TPG (p > 0.05). These three vehicles outperformed PEG 300 (p < 0.05) in terms of drug delivery. Concerning the vehicles, the rank order for in vitro skin permeation was DPG ≥ PG > TPG, while PEG 300 did not permeate the skin. A linear relationship between maximum vehicle and IBU flux in vitro was found, with a correlation coefficient (R2) of 0.95. When comparing in vitro with in vivo data, a positive in vitro–in vivo (IVIV) correlation between the cumulative permeation of IBU in vitro and the total amount of IBU that penetrated the stratum corneum (SC) in vivo was observed, with a Pearson correlation coefficient (R2) of 0.90. A strong IVIV correlation, R2 = 0.82, was found following the linear regression of the cumulative number of solvents permeated in vitro and the corresponding skin uptake in vivo measured with CRS. This is the first study to correlate in vivo permeation of solvents measured by CRS with data obtained by in vitro diffusion studies. The IVIV correlations suggest that CRS is a powerful tool for profiling drug and vehicle delivery from dermal formulations. Future studies will examine additional excipients with varying physicochemical properties. Ultimately, these findings are expected to lead to new approaches for the design, evaluation, and optimization of formulations that target actives to and through the skin.

The goal of head and neck oncological surgery is complete tumor resection with adequate resection margins while preserving acceptable function and appearance. For oral cavity squamous cell carcinoma (OCSCC), different studies showed that only 15%-26% of all resections are adequate. A major reason for the low number of adequate resections is the lack of information during surgery; the margin status is only available after the final histopathologic assessment, days after surgery. The surgeons and pathologists at the Erasmus MC University Medical Center in Rotterdam started the implementation of specimen-driven intraoperative assessment of resection margins (IOARM) in 2013, which became the standard of care in 2015. This method enables the surgeon to turn an inadequate resection into an adequate resection by performing an additional resection during the initial surgery. Intraoperative assessment is supported by a relocation method procedure that allows accurate identification of inadequate margins (found on the specimen) in the wound bed. The implementation of this protocol resulted in an improvement of adequate resections from 15%-40%. However, the specimen-driven IOARM is not widely adopted because grossing fresh tissue is counter-intuitive for pathologists. The fear exists that grossing fresh tissue will deteriorate the anatomical orientation, shape, and size of the specimen and therefore will affect the final histopathologic assessment. These possible negative effects are countered by the described protocol. Here, the protocol for specimen-driven IOARM is presented in detail, as performed at the institute.

Objective The depth of invasion (DOI) is considered an independent risk factor for occult lymph node metastasis in oral cavity squamous cell carcinoma (OCSCC). It is used to decide whether an elective neck dissection (END) is indicated in the case of a clinically negative neck for early stage carcinoma (pT1/pT2). However, there is no consensus on the cut-off value of the DOI for performing an END. The aim of this study was to determine a cut-off value for clinical decision making on END, by assessing the association of the DOI and the risk of occult lymph node metastasis in early OCSCC. Methods A retrospective cohort study was conducted at the Erasmus MC, University Medical Centre Rotterdam, The Netherlands. Patients surgically treated for pT1/pT2 OCSCC between 2006 and 2012 were included. For all cases, the DOI was measured according to the 8 th edition of the American Joint Committee on Cancer guideline. Patient characteristics, tumor characteristics (pTN, differentiation grade, perineural invasion, and lymphovascular invasion), treatment modality (END or watchful waiting), and 5-year follow-up (local recurrence, regional recurrence, and distant metastasis) were obtained from patient files. Results A total of 222 patients were included, 117 pT1 and 105 pT2. Occult lymph node metastasis was found in 39 of the 166 patients who received END. Univariate logistic regression analysis showed DOI to be a significant predictor for occult lymph node metastasis (odds ratio (OR) = 1.3 per mm DOI; 95% CI: 1.1–1.5, p = 0.001). At a DOI of 4.3 mm the risk of occult lymph node metastasis was >20% (all subsites combined). Conclusion The DOI is a significant predictor for occult lymph node metastasis in early stage oral carcinoma. A NPV of 81% was found at a DOI cut-off value of 4 mm. Therefore, an END should be performed if the DOI is >4 mm.

To the Editor, Atopic dermatitis (AD) is a common inflammatory skin disease among children with increasing prevalence in the past decades. The strongest and most widely replicated genetic risk factor for AD is a null mutation in the filaggrin gene (FLG) located on chromosome 1q21. FLG encodes the protein filaggrin, which is involved in the formation and homeostasis of the skin barrier.1 Previous research showed that AD patients with a mutation in FLG have a different phenotype, characterized by early onset disease with persistence into adulthood, increased severity and increased risk of asthma and allergic sensitization.1 Additionally, it has been suggested that patients with a mutation in FLG respond differently to immunosuppressive treatment compared to wild-type patients.1 This suggests that FLG mutation profiling could be used to stratify patients in terms of clinical course and to develop personalized treatment strategies. However, genotyping is time consuming and expensive, and DNA collection poses ethical considerations, which hampers its use in daily practice. Recent literature suggests that decreased concentrations of filaggrin-derived components of the natural moisturizing factor (NMF) are a proxy for the presence of FLG-null mutations.2, 3 During the terminal differentiation of keratinocytes, profilaggrin is dephosphorylated and enzymatically degraded into a highly hygroscopic mixture of amino acids and amino acid derivatives, including pyrrolidone carboxylic acid (PCA), histidine and its metabolite urocanic acid (UCA).2 The free amino acids and their derivatives constitute the majority of NMF in the stratum corneum (SC).2 A previous study in a selected Irish paediatric population of AD patients showed that NMF could discriminate between FLG mutation carriers and wild-type with a sensitivity of 98.73% and a specificity of 86.89% using 1.07 arbitrary units (a.u.) as the cut-off value.4 It has not been investigated whether this cut-off value could be applied to assess FLG mutation status in different clinical cohorts. The aim of our study was to screen the entire encoding region of FLG for potential mutations and to validate whether NMF could be used as a biomarker for FLG genotype in an unselected multi-ethnic clinical cohort of children with mild-to-severe disease. We conducted a cross-sectional study at the tertiary referral centre for Pediatric Dermatology in the Erasmus University Medical Center (Erasmus MC)-Rotterdam, the Netherlands. Study procedures were approved by the Medical Ethical Committee of the Erasmus MC (MEC-2017-370). All patients and/or their parents (guardians) signed informed consent. Children (0–18 years) diagnosed with AD according to the UK Working Party criteria consulting the dermatologist between June 2018 and September 2019 were eligible to participate in this study. FLG mutations were determined on DNA isolated from buccal swabs using Isohelix SK-1S swabs (Cell Projects Ltd). Single-molecule molecular inversion probes (smMIPS) and barcoded next-generation sequencing (NGS) were performed to screen the entire encoding region of FLG for all mutations resulting in premature protein termination as previously described.5 All patients with a mutation in FLG were referred to as patients with ≥1 mutation(s) (FLG−). Wild-type patients were referred to as FLG+. The NMF content was measured in the SC of the thenar eminence (nonlesional skin) using confocal Raman spectroscopy (gen2- SCA Skin Composition Analyzer; RiverD International B.V.) as described previously.4 All investigators were blinded for FLG genotype until the end of the study. Acute AD severity was assessed using the Eczema Area and Severity Index (EASI) score on the same day as the NMF measurements. A receiver operating characteristic (ROC) curve was constructed to measure the diagnostic ability of NMF to predict FLG mutation status (wild-type versus ≥1 mutation) by mapping the sensitivity versus 1-specificity for all possible values of the cut-off point. The optimal cut-off point was determined by maximizing the Youden function. A multivariate linear regression model was used to examine the association between EASI (as independent variable) and NMF content, corrected for FLG mutation status, age and sex. A total of 101 patients were included in this study (Figure S1). We identified 12 different FLG mutations, corresponding to 30 (30%) patients with ≥1 mutation in FLG (Table S1). This included 25 heterozygous mutation carriers and 5 patients with more than one mutation in FLG. There were no differences in age, sex, ethnicity and disease severity between both groups (Table S2). FLG+ patients had a median NMF of 1.26 a.u. (IQR 1.18–1.37), compared with a median NMF level of 0.82 a.u. (IQR 0.56–0.94) in FLG− patients (Figure 1, p < .01). Furthermore, the NMF content in patients with a single mutation in FLG was significantly different compared to both wild-type patients and patients with 2 mutations in FLG (p < .01, Figure S2). ROC curves were constructed to test the diagnostic ability of NMF measured using Raman spectroscopy. The optimal cut-off value for NMF to distinguish between FLG− and FLG+ patients was 1.03 a.u. with an area under the curve (AUC) of 0.93 (95% CI 0.87–0.99) (Figure 2). This resulted in a sensitivity of 97%, specificity of 87%, positive predictive value of 76% and negative predictive value of 98% (Table S3). We did not find a significant association between the EASI score and NMF measured at the thenar eminence (corrected for FLG mutation status, age and sex) (beta −0.555 (95% C.I. −0.005–0.003), p = .58, Table S4). The use of the cut-off value of 1.03 a.u. resulted in high sensitivity and specificity and was very close to the previously determined cut-off value of 1.07 a.u.4 Interestingly, 13% of the wild-type patients had low NMF content in the SC, consistent with previous literature.4 Since we used the smMIP-NGS technique, this could not be attributed to a rare or new mutation in FLG. Our results underline the importance of other factors affecting the NMF content in the SC, which are not a direct result of a FLG mutations. Previous research showed a positive correlation between intragenetic copy number variation (CNV) and the amount of filaggrin breakdown products.1 In addition, there is an important role for proteases in this multistage breakdown process (e.g. bleomycin hydrolase (BH) and caspase-14), which can be less effective due to variants influencing enzyme activity or the effect of external humidity.1 Furthermore, the presence of other mutations in the epidermal differentiation complex (EDC) could account for low NMF values and should be investigated further. Since Raman spectroscopy has the potential to measure the functional consequences of all mechanisms and mutations leading reduced NMF, this could be more useful in daily practice than genetic analysis alone. Our results showed no correlation between EASI score and NMF value, making the nonlesional skin of the thenar eminence a suitable location to predict FLG mutation status without direct interference of acute disease severity. Previous studies have shown that filaggrin degradation products in the SC of the nonlesional skin of the forearm are affected by both a mutation in FLG and disease severity.6 The effect of disease severity can be attributed to the activated immune system in both lesional and nonlesional skin. Previous research, using a tape stripping technique, showed an up-regulation of markers for AD severity including T helper 2 (Th2)-skewed markers (interleukin [IL]-13, CCL17, CCL22, IL-5) in the nonlesional skin of the forearm, which was associated with a reduced NMF content.6 The current results did not show a correlation between acute disease severity and NMF measurement on the thenar eminence. This might be explained by the thicker SC with a slower turnover time as compared to the SC on the forearm, making the thenar eminence less susceptible to an acute up-regulation of the immune response. Determining both cytokines and NMF values in the SC of thenar eminence and correlating this to disease severity could support our hypothesis. Major strengths of this study are the application of the non-invasive clinically compatible NMF measurement in an unselected multi-ethnic patient cohort and the use of a novel technique to detect all mutations leading to a premature protein termination in FLG. Especially in this multi-ethnic population, screening of only the most common mutations is not sufficient and will lead to an underrepresentation of FLG mutations. A limitation to mention is that patients were instructed not to apply any topical therapies on the thenar eminence 24 h before the measurement, but information on the use of these ointments during the rest of the week prior to the visit was missing. Previous research has suggested that topical steroid treatment could decrease the level of NMF in the SC of both mice and humans.7 Future research should focus on other factors, next to FLG-null variants, leading to a reduction in the NMF. Furthermore, it is of interest to validate the cut-off value of 1.03 a.u. in other clinical cohorts and to evaluate its use as a predictor for treatment outcomes to enable personalized treatment. In conclusion, we validated that the NMF content in the SC of the thenar eminence can be used as biomarker for FLG status in an unselected clinical cohort of children with AD from different ethnical backgrounds. This will be useful to identify patients with high risk for a more severe phenotype and to stratify patients in future clinical studies to evaluate treatment response. This project is part of the European Reference Network (ERN)-SKIN. We thank D. Karadavut, L.D.N. Bos and C.E. Meccanici for the inclusion of the patients and retrieving information on treatment history from the electronic patient files. We want to acknowledge Sten P. Willemsen (statistician) for constructing Receiver Operating Characteristic (ROC) curves. Last, we want to thank Sanja Kezic for critically reading the manuscript. M.M.F.v.M., M.S.J., A.E.M.N., M.B.B., L.M.P., M.V.G. S.G.M.A.P. have no conflict of interest to declare. P.J.C. and G.J. P.: The gen2-SCA device is manufactured by RiverD International B.V. MMFvM, PJC, LMP and SGMAP designed the study. MMFvM, MSJ and AEMN were responsible for the collection of data. MvG supervised the filaggrin mutation analysis. MMFvM performed the data analysis and wrote the manuscript. SGMAP supervised the study. All authors critically commented on the manuscript. Study procedures were reviewed and approved by the Medical Ethics Committee of the Erasmus MC University Medical Center Rotterdam, the Netherlands (MEC-2017-370). Signed informed consent was obtained from all participants. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The presence of penetrated oils in the stratum corneum (SC), oil-induced occlusion of the SC and formation of occluding homogeneous film on the skin surface are discussed in relation to their influence on results of water profile calculations using conventional and newly proposed extended methods. It is shown that the conventional method does not determine the water profiles in treated skin correctly due to the superposition of Raman bands of SC's proteins and penetrated and remnant oils.

BACKGROUND:Carriers of loss-of-function mutations in the filaggrin gene (LoF FLG) have less natural moisturizing factor (NMF) in their stratum corneum (SC) and an increased risk of atopic dermatitis (AD). Natural moisturizing factor can be measured noninvasively by Raman spectroscopy. The use of Raman-derived NMF at birth to screen for FLG genotype could inform targeted AD prevention, but values in neonatal populations are largely unexplored.OBJECTIVE:To examine the associations between Raman-derived neonatal NMF measurements and FLG genotype.METHODS:Natural moisturizing factor was measured by Raman spectroscopy in the SC of the thenar eminence within 4 days of birth in 139 term neonates. Filaggrin genotyping was performed for 117 neonates (84%).RESULTS:The mean (SD) NMF was 0.37 (0.11) g/g protein, with values increasing across the first 3 days (day 1 vs 3: 0.29 [0.09] vs 0.43 [0.08, P < .001]). Twelve infants (10.3%) were carriers of LoF FLG, all heterozygous. Natural moisturizing factor was lower in LoF FLG carriers compared with wild-type (0.27 [0.08] vs 0.38 [0.11] g/g protein, P ≤ .001). Natural moisturizing factor had good discriminatory power for FLG genotype (area under the receiver operating curve [AUROC]: 0.79; 95% CI: 0.66, 0.91; P ≤ .001). This improved after correcting day 1 and 2 measurements to day 3 (AUROC: 0.83; 95% CI: 0.75, 0.92; P < .001).CONCLUSION:This study suggests that Raman-derived NMF measured in the early postnatal period may have the potential to classify by FLG genotype. The full translational value of this needs to be determined.

The skin is a barrier between the environment and the body. When a product is applied to the skin, which constituents are penetrating, how much, how fast, how deep? Until now no technology was available to answer that question. Based on in vivo Raman spectroscopy, we have developed a method which enables quantitative determination of the skin penetration of molecular substances, expressed in g/cm2 of skin surface. The method is applicable to water-soluble, alcohol-soluble and lipid-soluble compounds. Quantitative in vivo analysis of dermal product penetration is important in product development, in toxicology and in dermatological research.