Improving skin cancer detection by Raman spectroscopy using convolutional neural networks and data augmentation.

Background: Our previous studies have demonstrated that Raman spectroscopy could be used for skin cancer detection with good sensitivity and specificity. The objective of this study is to determine if skin cancer detection can be further improved by combining deep neural networks and Raman spectroscopy. Patients and methods: Raman spectra of 731 skin lesions were included in this study, containing 340 cancerous and precancerous lesions (melanoma, basal cell carcinoma, squamous cell carcinoma and actinic keratosis) and 391 benign lesions (melanocytic nevus and seborrheic keratosis). One-dimensional convolutional neural networks (1D-CNN) were developed for Raman spectral classification. The stratified samples were divided randomly into training (70%), validation (10%) and test set (20%), and were repeated 56 times using parallel computing. Different data augmentation strategies were implemented for the training dataset, including added random noise, spectral shift, spectral combination and artificially synthesized Raman spectra using one-dimensional generative adversarial networks (1D-GAN). The area under the receiver operating characteristic curve (ROC AUC) was used as a measure of the diagnostic performance. Conventional machine learning approaches, including partial least squares for discriminant analysis (PLS-DA), principal component and linear discriminant analysis (PC-LDA), support vector machine (SVM), and logistic regression (LR) were evaluated for comparison with the same data splitting scheme as the 1D-CNN. Results: The ROC AUC of the test dataset based on the original training spectra were 0.886±0.022 (1D-CNN), 0.870±0.028 (PLS-DA), 0.875±0.033 (PC-LDA), 0.864±0.027 (SVM), and 0.525±0.045 (LR), which were improved to 0.909±0.021 (1D-CNN), 0.899±0.022 (PLS-DA), 0.895±0.022 (PC-LDA), 0.901±0.020 (SVM), and 0.897±0.021 (LR) respectively after augmentation of the training dataset (p<0.0001, Wilcoxon test). Paired analyses of 1D-CNN with conventional machine learning approaches showed that 1D-CNN had a 1-3% improvement (p<0.001, Wilcoxon test). Conclusions: Data augmentation not only improved the performance of both deep neural networks and conventional machine learning techniques by 2-4%, but also improved the performance of the models on spectra with higher noise or spectral shifting. Convolutional neural networks slightly outperformed conventional machine learning approaches for skin cancer detection by Raman spectroscopy.

Incidence and profile of skin cancers in patients following ultraviolet phototherapy without psoralens: A retrospective cohort study.

BACKGROUND: Psoralen + ultraviolet-A (PUVA) is associated with photocarcinogenesis. However, carcinogenic risk with other ultraviolet phototherapies remains unclear. OBJECTIVE: Evaluate whether phototherapy without psoralens increases skin cancer risk. METHODS: Retrospective cohort study of patients treated at a teaching-hospital phototherapy center (1977-2018). Skin cancer records were validated against pathology reports. Age-standardized incidence rates (ASIRs) of skin cancer were evaluated for gender, skin phototype, diagnosis, ultraviolet modality, anatomical site; and compared to provincial population incidence rates (2003). RESULTS: In total, 3506 patients treated with broadband-ultraviolet-B, narrowband-UVB and/or combined UVAB were assessed with a mean follow-up of 7.3 years. Majority of patients had psoriasis (60.9%) or eczema (26.4%). Median number of treatments was 43 (1-3598). Overall, 170 skin cancers (17 melanoma, 33 squamous cell carcinoma and 120 basal cell carcinoma) occurred in 79 patients. Patient-based and tumor-based ASIR of skin cancer was 149 (95% CI: 112-187)/100,000 and 264 (219-309)/100,000 person-years, respectively. There was no significant difference between tumor-based ASIRs for melanoma, squamous cell carcinoma, and basal cell carcinoma compared to the general population; or in phototherapy patients with-psoriasis or eczema; or immunosuppressants. No cumulative dose-response correlation between UVB and skin cancer was seen. LIMITATIONS: Treatment and follow-up duration. CONCLUSION: No increased risk of melanoma and keratinocyte cancer was found with phototherapy.

Baseline Characteristics of Canadian Patients in the Psoriasis Longitudinal Assessment and Registry (PSOLAR).

BACKGROUND: The Psoriasis Longitudinal Assessment and Registry (PSOLAR) is a global, prospective, longitudinal, disease-based registry. It serves as a post-marketing safety commitment with a focus on patients with moderate to severe plaque psoriasis who are candidates for systemic therapy. OBJECTIVES: To describe the baseline disease demographics and clinical characteristics of a Canadian subgroup of participants enrolled in PSOLAR. METHODS: Baseline demographic/disease characteristics, medical histories, and previous psoriasis treatments for Canadian patients in PSOLAR were summarized using descriptive statistics. RESULTS: There were 1896 patients analyzed in the Canadian subgroup at 37 clinical sites, accounting for 15.7% of the global PSOLAR population. Baseline disease and clinical characteristics were as expected for a moderate to severe psoriasis population and were generally similar to the global PSOLAR population. Two distinctions were noted in the Canadian subgroup versus those enrolled globally: a higher proportion of patients were overweight/obese (84.7% vs. 80.4%) and male (61.4% vs. 54.7%). In addition, the Canadian subgroup had numerically higher historical peak disease activity (PGA score 3.35 vs. 3.1) and longer disease duration (22.3 years vs. 17.5 years). Canadian PSOLAR patients reported a variety of comorbidities, including psoriatic arthritis (31.5%), hypertension (34.6%), hyperlipidemia (24.3%), mental illness (24.1%), and inflammatory bowel disease (1.6%). CONCLUSION: The Canadian subgroup of PSOLAR patients was generally similar to those enrolled globally with respect to baseline disease demographics and clinical characteristics. Multiple comorbidities are noted in the Canadian subgroup, underscoring the need for a holistic approach to the treatment of psoriatic patients.

Barriers and Facilitators in the Creation of a Surveillance System for Solar Radiation-Induced Skin Cancers.

Outdoor workers are exposed to many hazards, including solar ultraviolet radiation (UVR). Identifying, reporting, analyzing and tracking the exposures or health outcomes of outdoor workers have not generally been formally considered. This article aims to summarize the best practices/strategies for creating an occupational sun exposure or skin cancer surveillance system for outdoor workers and to understand the key barriers and facilitators to the development of such a system. For the design of a successful occupational safety and health (OSH) surveillance system five occupational surveillance strategies are summarized: exposure registry, disease registry, disease screening/medical surveillance, sentinel event surveillance, and disease surveillance via data linkage. Ten key considerations are identified, including identifying a clear goal, a defined target population and stakeholder involvement, five critical barriers are highlighted including underreporting and funding, and five vital facilitators are recognized including communication/collaboration and a simple reporting process.

Tailored Sun Safety Messages for Outdoor Workers.

Background: Messaging surrounding skin cancer prevention has previously focused on the general public and emphasized how or when activities should be undertaken to reduce solar ultraviolet radiation (UVR) exposure. Generic messages may not be applicable to all settings, and should be tailored to protect unique and/or highly susceptible subpopulations, such as outdoor workers. The primary objective of this study was to develop a set of tailored, practical, harm-reducing sun safety messages that will better support outdoor workers and their employers in reducing the risk of solar UVR exposure and UVR-related occupational illnesses. Methods: We adapted a core set of sun safety messages previously developed for the general population to be more applicable and actionable by outdoor workers and their employers. This study used an integrated knowledge translation approach and a modified Delphi method (which uses a survey-based consensus process) to tailor the established set of sun safety messages for use for outdoor worker populations. Results: The tailored messages were created with a consideration for what is feasible for outdoor workers, and provide users with key facts, recommendations, and tips related to preventing skin cancer, eye damage, and heat stress, specifically when working outdoors. Conclusion: The resulting tailored messages are a set of evidence-based, expert- approved, and stakeholder-workshopped messages that can be used in a variety of work settings as part of an exposure control plan for employers with outdoor workers.

Follow-up of Patients With Keratinocyte Carcinoma: A Systematic Review of Clinical Practice Guidelines.

Importance: Patients treated for cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC), collectively called keratinocyte carcinoma (KC), are at risk for recurrence, metastasis, and additional primary cutaneous malignant neoplasms. It is unclear how often patients should be seen for follow-up skin examination after initial treatment of KC. Objective: To summarize the recommendations and evaluate the methodological quality of clinical practice guidelines for dermatologic follow-up of patients with BCC and invasive SCC. Evidence Review: PubMed, MEDLINE, and Embase were searched for relevant articles published from January 2010 to March 2022. Search terms included guideline, squamous cell carcinoma, and basal cell carcinoma. National or international guidelines containing recommendations for follow-up frequency after a diagnosis of localized cutaneous KC were included. Quality was assessed using the 6 domains of the Appraisal of Guidelines Research and Evaluation II (AGREE II) tool: (1) scope and purpose; (2) stakeholder development; (3) rigor of development; (4) clarity of presentation; (5) applicability; and (6) editorial independence. The Preferred Reporting Items for Systematic Review and Meta-analysis Protocols (PRISMA-P) was used to guide study reporting. Findings: Among the 14 guidelines meeting eligibility criteria, there was little consensus on the appropriate follow-up frequency after initial KC treatment. Overall duration of follow-up ranged from a single posttreatment visit to lifelong surveillance. Most guidelines stratified their recommendations by recurrence risk. For low-risk BCC and guidelines that did not stratify by risk, follow-up recommendations ranged from every 6 to 12 months. For high-risk BCC, 1 guideline suggested follow-up every 3 months, while 4 recommended every 6 months. For low-risk SCC, 5 guidelines recommended annual follow-up; 3 guidelines, every 6 months; and 1 guideline, every 3 months. For high-risk SCC, recommendations included a range of follow-up frequencies, spanning every 3 months (n = 5 guidelines), 4 months (n = 1), 6 months (n = 6), or annually (n = 4). One guideline did not use risk stratification and recommended annual screening. The highest scoring AGREE II domain was "scope and purpose," which assessed the guideline's overall objectives, and the lowest scoring was "applicability," which assessed barriers and facilitators to implementation. Conclusions and Relevance: The findings of this systemic review highlight variations in follow-up recommendations for patients after initial treatment for KC. Randomized clinical trials are needed to define an optimal follow-up regimen.

Polarization memory rate as a metric to differentiate benign and malignant tissues.

Non-invasive optical methods for cancer diagnostics, such as microscopy, spectroscopy, and polarimetry, are rapidly advancing. In this respect, finding new and powerful optical metrics is an indispensable task. Here we introduce polarization memory rate (PMR) as a sensitive metric for optical cancer diagnostics. PMR characterizes the preservation of circularly polarized light relative to linearly polarized light as light propagates in a medium. We hypothesize that because of well-known indicators associated with the morphological changes of cancer cells, like an enlarged nucleus size and higher chromatin density, PMR should be greater for cancerous than for the non-cancerous tissues. A thorough literature review reveals how this difference arises from the anomalous depolarization behaviour of many biological tissues. In physical terms, though most biological tissue primarily exhibits Mie scattering, it typically exhibits Rayleigh depolarization. However, in cancerous tissue the Mie depolarization regime becomes more prominent than Rayleigh. Experimental evidence of this metric is found in a preliminary clinical study using a novel Stokes polarimetry probe. We conducted in vivo measurements of 20 benign, 28 malignant and 59 normal skin sites with a 660 nm laser diode. The median PMR values for cancer vs non-cancer are significantly higher for cancer which supports our hypothesis. The reported fundamental differences in depolarization may persist for other types of cancer and create a conceptual basis for further developments in polarimetry applications for cancer detection.

Assessment of sun-safety education behavior via spectrophotometric evaluation: A preliminary study.

BACKGROUND: Biases inherent in self-assessment of sun exposure and sun-safe behavior may lead to inaccurate conclusions about the effectiveness of sun-safety educational programs. OBJECTIVES: We aimed to compare self-reports to objective measures of sun exposure, when examining the effectiveness of passive versus active educational interventions. METHODS: From May to June 2018, 73 participants recruited at a dermatology clinic were sequentially assigned to receive sun-safety education through one of 3 modes: interactive online module, video, or no education. A baseline Sun Exposure and Behavior Inventory (SEBI) questionnaire was administered, and spectrophotometric measurements of sun-exposed and sun-protected areas were taken and reported in the CIE L*a*b* color space. Participants were followed 4-8 and 16 weeks after the initial visit where the SEBI was re-administered, and serial measurements of skin color were taken. The change in SEBI scores and L*a*b values, as calculated by the individual typology angle (ITA°), was analyzed. RESULTS: There was a significant increase in skin darkening in all the groups at 4-8 and 16 weeks follow-up. There was no statistically significant difference between the groups in the magnitude of color change. However, subjectively at 4-8 weeks post-intervention, participants in the interactive module and video groups had significantly improved self-reported SEBI scores compared to control (p < .05, Kruskal-Wallis). By 16 weeks, only the interactive module group showed significant improvement in SEBI scores compared to control (p < .05, ANOVA). CONCLUSION: In determining the effectiveness of sun-safety programs, spectrophotometric evaluation of sun-induced skin pigmentation can allow for a more complete evaluation of self-reported sun exposure and sun-protective behavior.

Constructing a portable optical polarimetry probe for in-vivo skin cancer detection.

SIGNIFICANCE: Management of skin cancer worldwide is often a challenge of scale, in that the number of potential cases presented outweighs the resources available to detect and treat skin cancer. AIM: This project aims to develop a polarimetry probe to create an accessible skin cancer detection tool. APPROACH: An optical probe was developed to perform bulk tissue Stokes polarimetry, a technique in which a laser of known polarization illuminates a target, and the altered polarization state of the backscattered light is measured. Typically, measuring a polarization state requires four sequential measurements with different orientations of polarization filters; however, this probe contains four spatially separated detectors to take four measurements in one shot. The probe was designed to perform at a lower cost and higher speed than conventional polarimetry methods. The probe uses photodiodes and linear and circular film polarizing filters as detectors, and a low-coherence laser diode as its illumination source. The probe design takes advantage of the statistical uniformity of the polarization speckle field formed at the detection area. RESULTS: Tests of each probe component, and the complete system put together, were performed to evaluate error and confirm the probe's performance despite its low-cost components. This probe's potential is demonstrated in a pilot clinical study on 71 skin lesions. The degree of polarization was found to be a factor by which malignant melanoma could be separated from other types of skin lesions.

Sun Protection Use at Work and Leisure by Outdoor Workers in Alberta, Canada.

OBJECTIVE: To assess the prevalence and determinants of sun protection behaviours among outdoors workers at work and leisure in Alberta, Canada. METHODS: We collected outdoor workers' demographics, skin cancer risk factors, job information, and sun habits at work and leisure using self-completed questionnaires. For both settings, we compared use of specific behaviours and modelled determinants of sun protection behaviour scores. RESULTS: At work, wearing a sleeved shirt (81% often/always) and hat (73%) were most prevalent, while seeking shade (12%) and applying sunscreen (36%) were least prevalent. Workers had higher sun protection scores at work than leisure. Hours spent outdoors was a strong determinant for both models. Additional leisure model predictors were eye colour, sex, skin type, and job group. CONCLUSIONS: Differences in behaviours across settings were observed and should be considered when developing solar UVR exposure reduction initiatives.

Solar ultraviolet radiation exposure among outdoor workers in Alberta, Canada.

BACKGROUND: Outdoor workers are at risk of prolonged and high solar ultraviolet radiation (UVR) exposure, which is known to cause skin cancer. The objectives of this study were to characterize the UVR exposure levels of outdoor workers in Alberta, Canada, and to investigate what factors may contribute to their exposure. METHODS: This study collected objective solar UVR measurements from outdoor workers primarily in Alberta during the summer of 2019. Workers were recruited via the management or health and safety teams from building trade unions and employers. Calibrated, electronic UVR dosimeters were worn by workers on their hardhats, wrists, or lapels for five working days. Data on workers' demographics, jobs, sun protection behaviors, and personal risk factors were collected using questionnaires, and meteorological data for each sampling day were noted. Mean daily exposure measured as the standard erythemal dose (SED) was calculated and compared to the international occupational exposure limit guideline (1.3 SED). Marginal models were developed to evaluate potential determinants of occupational solar UVR exposure. RESULTS: In total, 883 measurements were collected from 179 workers. On average, workerswere exposed to 1.93 SED (range: 0.03-16.63 SED) per day. Just under half of workers (45%) were exposed to levels exceeding the international exposure limit guideline. In the bivariate analyses, landscape and maintenance workers, as well as trade and recreation workers, had the highest levels of exposure (average: 2.64 and 1.84 SED, respectively). Regional variations were observed, with the "other" cities/regions (outside of Edmonton and Calgary) experiencing the highest average levels (2.60 SED). Workers who placed the dosimeters on their hardhats experienced higher levels compared to the other groups. Exposure was highest on sunny and mixed days. Education, trade, city, dosimeter placement, forecast, hair colour, and number of hours outside were included in the final exposure model, of which trade, dosimeter placement, forecast, and number of hours outside at work were statistically significant. CONCLUSIONS: Exposure to elevated solar UVR levels is common among outdoor workers in Alberta. The study findings can help inform future monitoring studies and exposure reduction initiatives aimed at protecting workers.

Strategic Task and Break Timing to Reduce Ultraviolet Radiation Exposure in Outdoor Workers.

Objectives: Public health messaging about sun avoidance strategies is often not practical for outdoor workers. The objective of this study was to use personal monitoring data to determine when peak UVR exposure occurs for outdoor workers, estimate how much UVR could be reduced by altering the timing of shady tasks or breaks during peak exposure times, and descriptively compare these to peak periods of ambient UVR. Ultimately, we aim to provide evidence-based sun avoidance recommendations for outdoor workers in British Columbia, Canada. Methods: UVR exposure data [standard erythemal dose (SED)] were collected during the 2013 summer months in Vancouver, using personal electronic dosimeters that sampled once per minute for an average of 4.4 working days (range: 1-7 days). Mixed-effect models were used to estimate the 60-, 30-, and 15-min time intervals at which maximum exposure occurred for the months of July and August. Using these time intervals, UVR exposure during peak periods was summarized as SED and as a percentage of the total daily exposure. Ambient UVR was also collected using data from the nearest Brewer spectrophotometer station and parallel analyses were conducted. Results: There were 73 workers and 321 participant-days available for analysis. Models indicated that periods of maximum exposure for 15-, 30-, and 60-min intervals began at 12:28, 12:17 pm, and 11:52 am, respectively, for sunny days in July. These periods were similar in August. The median exposure during these time periods and the potential for reducing UVR was 0.03 SED (2.8% potential daily exposure reduction), 0.09 SED (7.1%), and 0.18 SED (15.9%), respectively. However, there was a large range in exposure estimates as some workers experienced up to 84.8% of their exposure in the peak 60-min interval. Conclusion: Skin cancer prevention messaging does not include practical messages for outdoor workers and providing times of peak UVR help to identify times when the greatest reductions in exposure can occur. Prevention measures including shady breaks, increased sun protection, and task reorganization during these peak times are recommended during these peak times to reduce UVR exposure among those at highest risk.

Non-Melanoma Skin Cancer Risk Among Patients in the Psoriasis Longitudinal Assessment and Registry (PSOLAR)

To the Editor: Patients with psoriasis are at increased risk of developing non melanoma skin cancer (NMSC), including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC).1,2 The risk is especially elevated among those who previously received systemic treatment or phototherapy.2 Systemic treatments, including biologic therapies and methotrexate (MTX), are effective in managing immune-mediated diseases; however, they may increase susceptibility to NMSC due to immunosuppression or other factors.3

Relationship between sun safety behaviours and modifiable lifestyle cancer risk factors and vitamin D levels.

BACKGROUND: Sun exposure is the most important environmental risk factor for causing skin cancer. PURPOSE: This study examines the relationship between sun protection behaviours and modifiable lifestyle risk factors for other cancers as well as vitamin D levels. METHODS: Cross-sectional data were analysed from two large national health surveys (n = 31, 445 and n = 5604). Sun exposure and protection were characterized by the presence of sunburn, duration of sun exposure, frequency of seeking shade, frequency of wearing a hat and frequency of wearing sunscreen. Using Statistical Analysis System (SAS) software 9.3.1, multivariate logistic regression models were compiled. RESULTS: Unhealthy behaviour practices were associated with sunburns or infrequent sun protection behaviour, such as cigarette consumption (either current or former smokers), second-hand smoke exposure, not having a regular doctor, higher level of alcohol consumption, street drug usage and low levels of fruit/vegetable consumption. Approximately one-quarter of individuals had less than the recommended value of serum vitamin D levels (<50 nmol/L), despite 39.2% of these individuals reporting ≥1 hour of sun exposure. CONCLUSION: Modifiable lifestyle risk factors for other cancers are correlated with infrequently practicing sun protection behaviours for skin cancer prevention. Therefore, cancer prevention campaigns can aim to target all these risk factors associated with different cancers. Sun exposure is not a reliable source to obtain recommended vitamin D levels and that other sources (eg. fish, egg yolk, fortified drinks and supplements) are a safer and more reliable option.

A Qualitative Analysis of Canadian Indoor Tanning Policies.

BACKGROUND: The International Agency for Research on Cancer classifies artificial tanning devices as Group 1 human carcinogens. Studies have shown that use of indoor tanning before age 35 can increase the risk of melanoma development by 75%. It has therefore been recommended that indoor tanning use be restricted in individuals younger than age 18. OBJECTIVES: This study aims to review the state of provincial indoor tanning policies, especially in regards to use by youth across Canada, and what strategies are being implemented to enforce them. METHODS: Focused interviews were conducted with representatives from the provincial Ministries of Health across Canada in May and June 2014. Follow-up interviews were performed between February and May 2017. RESULTS: As of January 2018, regulations are in effect in all Canadian provinces restricting indoor tanning by minors and requiring display of signage warning of the risks of indoor tanning by salons. However, there are discrepancies among the provinces on how and if tanning salons are monitored and how and if these regulations are enforced. CONCLUSIONS: While implementing youth bans on indoor tanning is a promising start, all Canadian provinces need to ensure that efforts are being undertaken to ensure compliance with these policies to effectively combat the rising incidence of skin cancer among the Canadian population.

Prescribing Home Narrowband UVB Phototherapy: A Review of Current Approaches.

Conventional, full-body phototherapy equipment is costly and therefore patients are usually treated in dermatology centres. Such office-based therapy is often not feasible for those patients who live far away from a phototherapy centre due to lost time and wages, inability to travel because of extensive skin disease, or prohibitive travel costs. Home phototherapy has emerged as a modality that meets the needs of those patients. Our aim was to review available studies on UV sources, treatment protocols, efficacy, and safety of home phototherapy. A literature review was conducted on PubMed using the terms "home" AND "phototherapy" AND ("guide" OR "approach" OR "review" OR "protocol"). From the data extracted, narrowband UVB (311 nm) offers the best balance between safety and efficacy and is recommended for home phototherapy by most authors. Treatment is safe and possible adverse effects are related to overexposure (erythema, blistering). The usual treatment protocol was administering treatments on alternating days, including weekends, with dosing based on the patient's Fitzpatrick skin type. We also provide information on the available home phototherapy systems in Canada and their reimbursement. Home phototherapy is underused in Canada. Narrowband UVB phototherapy sources equipped with a 3-dimensional panel provides a practical and safe option.

Degree of optical polarization as a tool for detecting melanoma: proof of principle.

Determining the optical polarization properties of a skin lesion is a proposed method to differentiate melanoma from other skin lesions. We developed an in vivo Stokes polarimetry probe that fires a laser of known polarization at the skin and measures the Stokes parameters of the backscattered light in one shot. From these measured Stokes parameters, we can calculate the degree of polarization (DOP). Through testing on rough skin phantoms, a correlation between backscattered DOP and skin roughness was identified for both linear and circular input polarization, the latter of which was found to be more useful. In a pilot clinical trial of 69 skin lesions in vivo, it was found that the mean DOP for melanoma (linear input on melanoma: 0.46 ± 0.09) was greater than that of other lesions (linear input on all other lesions: 0.28 ± 0.01). This separation is greater for circular polarized input light, and it is likely that circular polarized light's greater sensitivity to surface roughness contributes to this result. In addition, all skin lesions demonstrated a stronger depolarizing effect on circular polarized light than linear polarized light. We have identified DOP as a potentially useful measurement to identify melanoma among other types of skin lesions.

Melanin quantification by in vitro and in vivo analysis of near-infrared fluorescence.

Objective measurements of melanin can provide important information for differentiating melanoma from benign pigmented lesions and in assessing pigmentary diseases. Herein, we evaluate near-infrared (NIR) fluorescence as a possible tool to quantify melanin. Various concentrations of in vitro Sepia melanin in tissue phantoms were measured with NIR fluorescence and diffuse reflectance spectroscopy. Similar optic measurements were conducted in vivo on 161 normal human skin sites. Diffuse reflectance spectroscopy was used to quantify the melanin content via Stamatas-Kollias algorithm. At physiologic concentrations, increasing in vitro melanin concentrations demonstrated higher fluorescence that was linearly correlated (R2  = 0.99, p < .001). At higher concentrations, the fluorescence signal plateaued. A linear relationship was also observed with melanin content in human skin (R2  = 0.59, p < .001). Comparing the fluorescence and reflectance signals with in vitro and in vivo samples, the estimated melanin concentration in human skin ranged between 0 and 1.25 mg/ml, consistent with previous quantitative studies involving invasive methods.