Dermatology: Practical and Conceptual Original Article | Dermatol Pract Concept. 2024;14(4):e2024231 1 Differential Diagnosis of Irritant Versus Allergic Contact Dermatitis Based on Noninvasive Methods Panagiota Gkagkari1, Anna Tagka2, Alexandros Stratigos2, Vangelis Karalis1, Aikaterini Kyritsi1, Andreas Vitsos1, Michail Christou Rallis1 1 Section of Pharmaceutical Technology, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens, Athens, Greece 2 First Department of Dermatology-Venereology, National and Kapodistrian University of Athens, Andreas Syggros Hospital, Athens, Greece Key words: Irritant Contact Dermatitis, Allergic Contact Dermatitis, Antera® 3D Camera, Transepidermal Water Loss, Oxidative Stress Citation: Gkagkari P, Tagka A, Stratigos A, et al. Differential Diagnosis of Irritant Versus Allergic Contact Dermatitis Based on Noninvasive Methods. Dermatol Pract Concept. 2024;14(4):e2024231. DOI: https://doi.org/10.5826/dpc.1404a231 Accepted: June 19, 2024; Published: October 2024 Copyright: ©2024 Gkagkari 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. Corresponding Author: Anna Tagka, MD, First Department of Dermatology-Venereology, National and Kapodistrian University of Athens, Andreas Syggros Hospital, 5 Ionos Dragoumi Str., 11621 Athens, Greece; Tel. +30-210-726-5100. E-mail: annatagka3@gmail.com Introduction: Irritant contact dermatitis (ICD) is characterized by direct injury to the epidermal cells, activating the innate immune response. Allergic contact dermatitis (ACD), in contrast, is delineated by a delayed hypersensitivity reaction of type IV. Despite the distinct etiopathogenic mechanisms under- pinning each condition, the differentiation between them presents a significant diagnostic challenge. Objective: This study aimed to determine whether a combination of clinical evaluation and noninvasive measurements—encompassing oxidative stress, erythema, hydration, melanin content, transepidermal water loss (TEWL), hemoglobin concentration, and skin texture and volume—could distinguish ICD from ACD. Methods: Two cohorts, each comprising 21 patients, were evaluated: one diagnosed with ICD and the other with ACD. All participants underwent biophysical and clinical assessments, along with Antera® 3D evaluations. Tape strips were utilized for skin sampling, and oxidative stress levels were measured via fluorescence assessments. Results: ICD prompted an almost immediate inflammatory reaction (peaking at 24 hours), whereas ACD incited a delayed response (72 hours). Noninvasive evaluated parameters such as hemoglobin concentration, skin texture and volume, melanin content, erythema, and TEWL showed significant differences between the ICD and ACD cohorts (P < 0.05). The allergens amcinonide, nickel sulphate, cobalt chloride, budesonide, PPD, and thiuram mix were found to induce elevated levels of oxidative stress. Conclusions: The evaluation of patients with noninvasive parameters, including transepidermal water loss (TEWL), hemoglobin concentration, and skin texture and volume, could markedly aid in distin- guishing irritant contact dermatitis from allergic contact dermatitis (ACD). Nevertheless, the study was constrained by a limited sample size. ABSTRACT 2 Original Article | Dermatol Pract Concept. 2024;14(4):e2024231 Introduction Contact dermatitis manifests in two primary forms: irritant contact dermatitis (ICD), accounting for 80% of cases, and allergic contact dermatitis (ACD), comprising the remaining 20% [1,2]. ICD is distinguished by direct damage to kerati- nocytes, initiating the activation of the innate immune system and leading to the release of a cascade of pro- inflammatory cytokines to mediate the damage [3-7]. These cytokines stim- ulate the activation of epidermal Langerhans cells, dermal dendritic cells, and endothelial cells. Experimentally, sodium lauryl sulfate (SLS) induces damage analogous to that ob- served in ICD [3,8]. Conversely, ACD is typified by a delayed hypersensitiv- ity reaction of type IV (Gell-Coombs), induced by dermal contact with haptens or non-protein allergens. Acute man- ifestations include erythema, vesicles, and blisters, while chronic exposure may result in lichenification accompanied by cracks and fissures [9]. The mediation of ACD involves T cells that recognize small chemical molecules or metal ions within the major histocompatibility complex (MHC) [10]. These electrophilic chemicals penetrate the skin and interact with extracellular and intracellular proteins, a property un- derpinning their capability to activate both innate immune and T cell responses [11-13]. Following contact with a specific allergen, allergen- specific T cells proliferate and differentiate into effector T cells, which enter the circulation [14]. The repeated application of a specific allergen on the skin leads to the re- cruitment of these T cells back to the dermal layer, inducing apoptosis in keratinocytes and culminating in ACD [15,16]. Notably, skin lesions in ACD manifest upon re-exposure to the allergens, with metals, preservatives, antibiotics, and fra- grances being the most common allergens linked to sensiti- zation within the general population [17]. Objectives This study endeavored to enhance the clinical differential evaluation of ACD and ICD by integrating the analysis of noninvasive parameters. The investigation focused on sev- eral key indicators of skin health as response to irritants or allergens, including oxidative stress levels, erythema, skin hy- dration, melanin content, transepidermal water loss (TEWL), hemoglobin concentration, and the texture and volume of the skin fraction affected. Oxidative stress, for instance, can provide insights into the cellular damage and inflammatory response triggered by contact with irritants or allergens. Sim- ilarly, changes in TEWL, erythema, and skin hydration offer quantitative measures of the skin's barrier function and in- flammatory status. By comparing these parameters between individuals diagnosed with ACD and those with ICD, the study aimed to identify distinctive patterns that could aid in their differentiation. Incorporating such noninvasive assessments into the di- agnostic process seems promising for improving the accuracy of ACD and ICD diagnosis. By providing a more nuanced understanding of the skin's response to irritants and aller- gens, healthcare professionals could tailor treatment strate- gies more effectively. Moreover, this approach aligns with the growing emphasis on minimally invasive techniques in der- matological diagnosis. As the study progresses, the findings could contribute valuable insights into the pathophysiologi- cal distinctions between ACD and ICD, ultimately enhancing patient care and therapeutic outcomes. Methods SLS Gauze A gauze impregnated with a 6% w/v solution of SLS (Farmalabor, Italy) was applied to the forearm skin for 24 hours. The clinical manifestations were evaluated follow- ing the removal of the SLS-impregnated gauze. Patch Test Patch testing remains the definitive standard for diagnosing ACD. Following the European Society of Contact Dermati- tis guidelines for patch testing, substances were applied to the upper back and removed after 48 hours. The evaluations were conducted 72 hours post-application, utilizing a Eu- ropean baseline and supplemental series of allergens (aller- gEAZE® Patch Test Chambers, SmartPractice Canada). Study Design All conducted procedures adhered to the Good Clinical Practice (GCP) guidelines as outlined in Directive 2001/20/ EC, the US Federal Code of Users (21 CFR Part 312), and the International Conference on Harmonization (ICH). The research was performed in alignment with the Declaration of Helsinki principles (Directive 2001/83/EC; ICH Issue E9 1996; Directive 2001/20/EC; Directive 2002/98/EC; Directive 2003/63/EC; ICH E(6) R1; 21 CFR Part 312; WHO 2008). Approval for the protocol was granted by the Institutional Scientific Review Board of the Andreas Syg- gros Hospital, NKUA,University Medical School (Protocol Nr. 3612/2021). Participants Forty-two participants (21 ACD and 21 ICD diagnosed pa- tients) were recruited from the 1st Department of Derma- tology and Venereal Diseases, Andreas Syggros University Hospital, between June 2021 and December 2021. All pa- tients provided informed consent. Exclusion criteria included Original Article | Dermatol Pract Concept. 2024;14(4):e2024231 3 pregnancy, the use of anti-inflammatory and antihistaminic medications, sweating, and physical activity. Inclusion crite- ria encompassed a history of acute, subacute, and chronic dermatitis and the manifestation of rashes following expo- sure to allergens and irritants. Clinical Assessment All participants successfully completed the study. Medical history and demographic data such as sex, age, body mass index (BMI), phototype (Fitzpatrick skin type), medical con- ditions, family history, and smoking habits were collected and found to be homogeneous (Table 1). Contact Dermatitis Evaluation The International Contact Dermatitis Research Group (ICDRG) criteria were employed to assess the intensity of positive reactions in ACD patients, ranging from weak pos- itive reactions, characterized by erythema, infiltration, and possibly papules (1+), to extreme positive reactions, marked by intense erythema, infiltration, and coalescing vesicles (3+). For ICD patients, the clinical evaluation was based on the reaction within 24 hours and the clinical assessment of irritation. Oxidative Stress Evaluation Prior to and at the time of patch evaluation, two tape strips (Standard D100 Squame Discs, USA) were applied to the patients’ skin and stored at -20°C. The samples were then processed with 500 μl of H2O HPLC (Fischer Chemical U.K. Limited), 200 μl of 90% MeOH (Fischer Chemical U.K. Limited, Bishop Meadow) Na2EDTAx2H2O (Lach-Ner, s.r.o., Turkey), 75 μl of 10 mg/ml BHT (Sigma Chemical Co., USA) in Ethanol HPLC (Acros Organics, Germany), and 1.5 μl of 10 mM Desferal (Ciba-Geigy Switzerland) in H2O HPLC. Following vortex mixing (MSI Minishaker IKA, USA) at 2,500 rpm for one minute and centrifugation at 9,000 rpm for eight minutes at 4°C, 50 μl from each vial solution and 100 μl of saline were transferred to a 96-well plate (Costar – Corning, USA), with three wells per solution. Three wells received 150 μl of saline as a control. Subsequently, 10 μl of 30 μM 5-(and-6)-chloromethyl-2',7'-dichlorodihy- drofluorescein diacetate, acetyl ester (CM-H2DCFDA) etha- nolic solution (Molecular Probes -Thermo Fisher Scientific USA) was added to each well. The plate was then vortexed at 300 rpm for 10 minutes and placed in a Fluostar BMG (Germany) reader with the excitation filter set at 485 nm and emission at 520 nm, with a gain of 85. The plate underwent incubation at 37°C for 50 minutes prior to measurements. Skin Analysis Skin assessments were conducted using an Antera® 3D cam- era (Miravex, Dublin, Ireland) at 0 hours and 72 hours in ACD patients and at 0 hours and 24 hours in ICD patients. Hemoglobin concentration and skin texture were evaluated using Antera® 3D software (Miravex, Dublin, Ireland). Measurements of Skin Biophysical Parameters In both ACD and ICD patients, skin parameters, includ- ing hydration, TEWL, erythema, and melanin, were mea- sured using noninvasive biophysical methods at 0 hours and 72  hours and at 0 hours and 24 hours, respectively. Hydration levels were gauged using a Corneometer CM 820 (Courage + Khazaka electronic GmbH, Cologne, Germany), based on changes in the dielectric constant, with data Table 1. Characteristics of Patients at Baseline. Characteristics of Patients Allergic Contact Dermatitis Irritant Contact Dermatitis Demographics Median Age 46 50 Sex (%) Male 9.52 9.52 Female 90.48 90.48 BMI classification (%) Normal weight 76.19 66.67 Overweight 23.81 23.81 Underweight - 4.76 Morbid obesity - 4.76 Fitzpatrick skin type (%) I 4.76 23.81 II 57.14 61.9 III 33.33 14.29 IV 4.76 - Current smoker (%) 42.86 38.1 Characteristics of Disease Patient atopic dermatitis history (%) 47.62 28.57 Family atopic dermatitis history (%) 28.57 28.57 Disease (%) Primary (%) 9.52 - Recurrence (%) 90.48 - Exposure to contact allergens (%) 80.95 - Abbreviation: BMI: body mass index. 4 Original Article | Dermatol Pract Concept. 2024;14(4):e2024231 responsible for 19.05% of reactions and textile dye mix 6.6% for 14.29%; thiuram mix 1%, fragrance mix I 8%, and PPD 1% each accounted for 9.52%. Cobalt chloride 1%, 4-aminoazobenzene 1%, thiomersal 0.1%, amcin- onide 0.1%, fragrance mix II 14%, colophonium 20%, budesonide 0.01%, and formaldehyde 2% each contributed to 4.76% of reactions (Figure 1B). Oxidative Stress Oxidative stress, indicated by increased fluorescence inten- sity, was significantly elevated in both ICD (P < 0.001) and ACD (P < 0.001) patients (Figure 2). The mean fluorescence increase in ICD patients was 6.586%, while in ACD patients, it was 5.515%. There were no significant differences between the two groups at the time of evaluation. All allergens led to a significant rise in oxidative stress, with the most substantial increases observed for amcinonide, nickel sulfate, cobalt chlo- ride, budesonide, PPD, and thiuram mix allergens (Table 2). Antera® Measurements Analysis using Antera® 3D software (Figure 3) revealed pronounced inflammatory activity in both ICD and ACD patients. All Antera® 3D parameters, including hemoglobin concentration, skin texture, and volume values, were sig- nificantly higher in ICD patients within 24 hours (Figure 4, P < 0.001). Likewise, these parameters were significantly ele- vated at 72 hours in ACD patients (Figure 4, P < 0.001). The median hemoglobin concentration, skin texture, and volume increased in both groups as the intensity of inflammation escalated. The percentage difference in median hemoglobin concentration was 94.95% in ACD and 47.14% in ICD patients. Notably, significant differences were observed be- tween ACD and ICD patients at 72 hours and 24 hours, re- spectively (Figure 4A, P < 0.001). The percentage difference in the median texture value was 66.39% in ACD compared to 37.73% in ICD patients. The distinction between ACD and ICD patients at the time of evaluation was statistically significant (Figure 4B, P < 0.05). Similarly, significant differ- ences were noted between ACD and ICD patients in terms of volume (Figure 4C, P < 0.05). Biophysical Measurements Transepidermal water loss (TEWL) is directly associated with skin barrier functionality and indirectly with the inten- sity of inflammation. Significantly higher TEWL levels were noted in ICD patients at 24 hours and in ACD patients at 72 hours (Figure 5, P < 0.001). The median TEWL showed a 20% increase in ACD and a 406.42% increase in ICD patients. Noteworthy differences were identified between ACD and ICD patients at the time of analysis (Figure 5, P < 0.05). recorded in arbitrary units. The skin barrier function (TEWL) was evaluated using a Tewameter TM 210 (Courage + Khazaka electronic GmbH, Cologne, Germany) by mea- suring the density gradient of water evaporation from the skin, with estimations based on the mean value of the flux density of water (in g/m2/h). Erythema and melanin levels were quantified using a Mexameter MX 18 (Courage + Khazaka electronic GmbH, Cologne, Germany), with data also recorded in arbitrary units. Before each measurement, the treated area was cleansed with 0.9% sodium chloride solution and dried with sterile gauze. Statistical Analysis The statistical analysis commenced with the determination of descriptive criteria and estimation of scatter metrics. Nor- mality testing was performed prior to statistical comparisons among two or more groups. The Shapiro-Wilk test was uti- lized to assess the normality of data distribution. Upon re- jection of the nominal normality hypothesis, the data were deemed to deviate from a normal distribution. When data appeared normally distributed, parametric procedures were employed. However, in all instances within this study, as the data were found not to be normally distributed, non- parametric methods were applied. The Mann-Whitney U test was employed for comparisons between two indepen- dent groups, for instance, to evaluate the effect of sex. For pairwise comparisons between groups, such as the compari- son of skin biophysical parameters across two different time points, the Wilcoxon signed-rank test was used. The type I error (significance level) was set at 5% for all analyses within this study. A result was deemed significant if the estimated p-value (P) was less than the significance level, with p-values of < 0.05 denoted as statistically significant and indicated by the symbols (*) for P < 0.05, (**) for P < 0.01, (***) for P < 0.001, and (****) for P < 0.0001. All statistical analyses were conducted using SPSS® (v.25, IBM, Chicago, IL, USA). Results Clinical Assessment ICD Patients All patients exhibited erythema at the time of evaluation (24 hours). ACD Patients The clinical assessment of ACD patients at 72 hours, based on the ICDRG criteria, is illustrated in Figure 1. Strong re- action (+ + +) according to the ICDRG scale was presented by PPD 1%, textile dye mix 6.6%, and 4 - aminoazoben- zene 1% (Figure 1A). Nickel sulfate 5% w/w solution was Original Article | Dermatol Pract Concept. 2024;14(4):e2024231 5 ICDRG scale [13]. The findings highlight nickel sulfate (figure 1B) as the most prevalent sensitizer, aligning with observations made by Dekoven et al. [17,19]. Fluorescence-based oxidative stress assessment revealed no significant differences between ACD and ICD patients (Figure 2). However, oxidative stress levels increased in ICD patients after 24 hours and in ACD patients after 72 hours (P < 0.05), with significant elevations observed in response to moderate sensitizers such as amcinonide and budesonide and to strong sensitizers like cobalt chloride, nickel sulfate, PPD, and thiuram mix allergens (Table 2) [20-23]. PPD, commonly found in hair dyes and black henna tattoos, enhances the formation of reactive oxygen species (Table 2), thereby elevating oxidative stress in human ke- ratinocytes, a finding consistent with our results [24,25]. Discussion All participants in this study exhibited signs of skin inflam- mation. In patients with ICD, the inflammatory response significantly subsided 48 hours following the application of SLS, indicative of ICD’s characteristic direct harm to ke- ratinocytes, which promptly triggers the innate immune re- sponse [18]. Conversely, the reaction to allergens in ACD is typically not instantaneous. Despite ACD’s more severe clin- ical presentation compared to ICD, the response is delayed, often taking up to 72 hours to manifest [18]. According to the ICDRG criteria, allergens are cat- egorized based on the degree of reaction within 72 hours (Figure 1A). PPD, textile dye mixtures, and 4-aminoazobenzene elicited the most potent reactions, corroborating the Figure 1. (A) Clinical assessment of ACD patients after 72 hours. Incidents presented (+) 4.76%, (++) 71.43 % and (+++) 23.81%. (B) The most common allergens were, in order, nickel sulfate solution 5 % w/w and textile dye mix 6.6% w/w. 6 Original Article | Dermatol Pract Concept. 2024;14(4):e2024231 Our observation regarding the increase in oxidative stress caused by nickel sulfate (Table 2) also supports the notion of enhanced lipid peroxidation, as evidenced in previous studies [27-29]. The Antera® 3D camera facilitated the evaluation of in- flammation intensity in ICD and ACD, demonstrating that mean hemoglobin concentration and skin texture values Figure 2. Mean value of fluorescence for ICD and ACD patients. Fluorescence values were significantly increased for both groups at the time of evaluation (***P < 0.001). Figure 3. Antera® 3D images of ACD and ICD patients. (A) Textile dye mixture: a 21-year-old female patient with allergic contact dermatitis. (B) PPD: a 21-year-old female patient with aller- gic contact dermatitis. (C) A 45-year-old female patient irritated with SLS 6% in the forearm. (D) A 55-year-old female patient irritated with SLS 6% in the forearm. Table 2. Increase in Allergen Oxidative Stress at 72 Hours Expressed in Fluorescence Units. Allergens Mean Increase in Fluorescence Units Formaldehyde 2% 420 Thiuram mix 1% 1537 Nickel sulfate 5% 0 Budesonide 0.01% 2936 Amcinonide 0.1% 2677 Colophonium 20% 0 Thiomersal 0.1% 2155 PPD 1% 2046 Textile Dye Mix 6.6% 1323 Textile Dye Mix 6.6% 1528 Fragrance Mix II 14% 1195 Fragrance Mix I 8% 576 Nickel Sulfate 5% 3049 Cobalt chloride 1% 3703 PPD 1% 1576 Textile Dye Mix 6.6% 463 4-aminoazobenzene 1% 598 Nickel sulfate 5% 2965 Thiuram mix 1% 4990 Nickel sulfate 5% 3936 Fragrance mix I 8% 0 Original Article | Dermatol Pract Concept. 2024;14(4):e2024231 7 Limitations Given the limited sample size of 21 patients each for ACD and ICD, further research is necessary to definitively ascer- tain whether biophysical measurements, Antera® 3D camera imaging, and oxidative stress analysis can distinguish be- tween ICD and ACD inflammation effectively. Conclusions Numerous distinctions were identified between ACD and ICD responses. ICD prompts an almost immediate inflam- matory reaction (peaking at 24 hours), whereas ACD incites a delayed response (72 hours). The reduction in skin bar- rier function was substantially greater in ICD than in ACD. All Antera® 3D parameters (hemoglobin concentration, skin texture, and volume) were significantly elevated in ACD compared to ICD patients. No statistically significant differ- ences in oxidative stress were noted between ACD and ICD patients. Nickel sulfate emerged as the most common sen- sitizer, while PPD, textile dye mix, and 4-aminoazobenzene triggered the most severe inflammatory responses. Aller- gens such as amcinonide, nickel sulfate, cobalt chloride, budesonide, PPD, and thiuram mix were found to induce significant oxidative stress. Acknowledgments The authors would like to acknowledge Mrs Lagiokapa Polyxeni, Pesli Maria and Kostaki Maria for their valu- able help. References 1. Thyssen JP, Johansen JD, Linneberg A, Menné T. 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