In vivo spectroscopy in dermatology: methods and new fields of application

BACKGROUND: The human skin is an easily accessible organ on which non-invasive examination methods can be applied. Remittance spectroscopy uses registration of the proportion of reflected light from the skin surface. Optical characteristics of skin are determined by scattering, reflection and absorption. The remittance is influenced by various test conditions and topography. Spectroscopic methods are applied for determination of microcirculation, measuring of erythema and pigmentation and in pharmacology and toxicology of applied drugs. CONCLUSION: The standardization of spectroscopic measurement of human skin is essential to reduce error sources. In future a better investigation of skin structures and molecules, which are responsible for optical characteristics of skin and a more precise evaluation of spectroscopic signals are necessary to get more information about the state of skin and to develop new fields of applications, such as early diagnosis of skin diseases or monitoring of metabolic processes.     

The minimal melanogenesis dose/minimal erythema dose ratio declines with increasing skin pigmentation using solar simulator and narrowband ultraviolet B exposure

Purpose: To investigate the relation between pre‐exposure skin pigmentation and the minimal melanogenesis dose (MMD)/minimal erythema dose (MED) ratio after a single narrowband ultraviolet B (nUVB) and solar simulator (Solar) exposure. Background: In fair‐skinned individuals, it is well known that the UV dose to give pigmentation (MMD) after a single exposure to UVB is larger than the UV dose to elicit erythema (MED) (MED<MMD), but it remains to be established if this is true also in dark‐skinned individuals. Methods: Eighty‐four volunteers with a wide variation in skin pigmentation (Fitzpatrick skin types I–V) were included. Results: After a single Solar or nUVB exposure we found that the ratio MMD/MED depends on skin pigmentation. In light‐pigmented individuals, up to 1.9 MED is required to induce pigmentation (MMD). The MMD/MED ratio is about 1.5 in medium‐pigmented and dark‐pigmented individuals. In very brown‐pigmented individuals the MMD/MED ratio is 1 (MED=MMD). This connection was most pronounced for facultative skin at wintertime. The ratio was almost stable for constitutive pigmentation with MMD/MED=1.3. The ratios were almost independent of skin type. Conclusion: The ratio MMD/MED is highly dependent on skin pigmentation after a single exposure to Solar or nUVB and is independent of skin type.     

Epidermal thickness,skin pigmentation and constitutive photosensitivity

The important factors for UV sensitivity in humans are considered to be the skin pigmentation and the epidermal thickness. In this study on 73 Caucasians (age 20–85 years), we investigated in UV unexposed buttock skin the relationship between the UV sensitivity and constitutive skin pigmentation and thickness of the stratum corneum and the cellular part of the epidermis, in 34 normal people and in 39 skin cancer patients (20 patients with cutaneous malignant melanoma and 19 patients with basal cell carcinoma of the skin). Skin pigmentation was measured by skin reflectance spectroscopy, and UV sensitivity by phototest with a solar simulator. Thicknesses of the stratum corneum and the cellular part of the epidermis were determined by light microscopic evaluation of skin biopsies from the phototest areas. We found that epidermal thickness was independent of skin type and was not correlated to constitutive skin pigmentation. Thickness of the stratum corneum was statistically not different in normal persons and in skin cancer patients (P=0.4l) and was independent of gender (P=0.61) and age (P=0.56), while thickness of the cellular epidermis decreased with age (P<0.01). Stratum corneum thickness was found to be of minor importance for the constitutive UV sensitivity (accounting for on average 11% of the total photoprotection), which was mainly determined by the constitutive skin pigmentation (goodness-of-fit for correlation r=0.83). A theoretical model for the relationship of UV dose to induction of clinical erythema grade and skin pigmentation and thickness of the stratum corneum was developed. Objective measurements of skin pigmentation in UV unexposed skin by skin reflectance spectroscopy in Caucasians, normal people and people with cutaneous malignant melanoma and basal cell carcinoma of the skin predicts the constitutive UV sensitivity with a high degree of precision.     

The effects of heating and cooling on ultraviolet radiation-induced erythema and pigmentation in human skin

Background/Purpose: As most biochemical systems are affected by temperature, thermal changes before or after ultraviolet (UV) irradiation could influence skin vascular blood flow changes and inflammatory responses. In this study, our aim was to investigate the influence of thermal changes on UV-induced acute skin reactions, namely, erythema and pigmentation.
Methods: Our volunteers consisted of 10 males, with ages ranging from 22 to 24 years and with Fitzpatrick's skin type III or IV. Skin temperatures were changed with a 45°C heating pad or by ice pack application before or after UV irradiation (control, 1 minimal erythema dose (MED), 2 MED) and then changes in erythema and pigmentation were measured by a Minolta Spectrophotometer CM-2002.
Results: The present study demonstrates that both heating and cooling increase skin erythema and reduce pigmentation, and that the timing of heating and cooling influences the UV-induced skin reaction. Pre-heating and post-cooling groups showed more UV-induced erythema than the post-heating and pre-cooling groups, respectively.
Conclusion: Our results indicate that alteration of skin surface temperature could modulate UV-induced erythema and pigmentation responses.     

In vivo measurement of the photostability of sunscreen products using diffuse reflectance spectroscopy

BACKGROUND/AIM: The issue of the photostability of sunscreens has been frequently raised because of the possible loss of photoprotection, mainly in the UVA range, during sun exposure. Up to now, in vitro techniques have been mainly proposed to evaluate photostability. These techniques have generated controversial debates concerning the predictive value of these in vitro observations in relation to the in vivo reality during sun exposure. METHODS: Diffuse reflectance spectroscopy (DRS) is a recently developed technique that allows measurement of the UVA efficacy of sunscreen products in vivo on human volunteers. The absorption spectrum of the product is obtained by measuring the change in reflection of the skin with and without product. From this absorption spectrum, the UVA protective efficacy of the test product can be calculated for an appropriate source and a given biological action spectrum. We have used the DRS technique in vivo to determine the photostability of sunscreen products by measuring reflection spectra in the UVA range (320-400 nm) before and after product application and before and after UV exposure of the test products. Comparison between these spectra or between the corresponding calculated UVA protection factors has made it possible to determine the remaining level of protection in the UVA range after exposure. This study was designed to compare in vivo the protective efficiency and the photostability of three marketed sunscreen products (SPF 23-30) after solar-simulated exposure for SPF testing or after actual sun exposure. These in vivo data were then compared to in vitro photoinstability results. RESULTS: According to the in vitro measurements, one sunscreen product was found photostable and two products photo-unstable. After UVe exposure for in vivo SPF determination, a decrease in UVA absorption and UVA-PF was observed for the two photo-unstable products, while the photostable product did not present any decrease in UVA absorption. These results were confirmed through exposure to actual sun. CONCLUSION: Our study confirms the prediction of the in vitro methods previously used to assess the photostability of sunscreen products. In addition, DRS provides a powerful new tool for the in vivo simultaneous evaluation of photostability and estimation of the UVA protection factor of sunscreen products performed during the test for SPF determination.     

Digital image analysis of facial erythema over time in persons with varied skin pigmentation

Background/purpose: Selected chemotherapeutic agents used for the treatment of cancer are known to cause skin toxicities. One group of agents, epidermal growth factor receptor (EGFR) inhibitors, characteristically precipitates an acneform rash. Currently, no standard of care exists for the management of the rash resulting from EGFR inhibitors. In order to objectively evaluate any management strategy, a method to quantify the rash is required. The purpose of this paper is to describe a method to quantify the erythema of a facial rash through the use of digital photography and image analysis. Methods: A Canfield OMNIA System using a Canon PowerShot Pro1 camera was used to obtain high‐resolution digital images of facial rashes. Digital images were recorded in Joint Photographic Experts Group format, corrected for brightness and white balance and color. A method was developed to analyze digital images of erythema independent of the range of skin pigmentation. Results: Two examples are given to illustrate the method developed and its utility. Conclusion: An inexpensive and portable method is described for objectively monitoring the development of facial erythema in subjects of the full range of skin pigmentation. This method can be used clinically to examine the development and resolution of facial rash erythema in response to treatment.     

Minimal erythema dose in UV-shielded and UV-exposed skin predicted by skin reflectance measured pigmentation

Aims: To investigate the relationships between epidermal thickness, age, skin pigmentation and UV sensitivity in sun-exposed skin and skin not exposed to the sun in healthy people without skin cancer or skin diseases. Methods: Phototesting with a xenon arc solar simulator was performed in 137 healthy Caucasians in buttock skin un-exposed to UV (27 children, 34 young adults and 32 older adults) and in skin of the back exposed to UV (44 young adults). The pigmentation of the phototest sites was measured objectively by a skin reflectance spectrometer before phototesting. Thickness of the stratum corneum and the cellular epidermis were measured in skin biopsies from the phototest sites. All measurements were performed in the winter and spring months. Results: Stratum corneum and cellular epidermis were thinner at the back than at the buttocks (P<0.01). Thickness of the stratum corneum at the back or the buttocks was not related to the degree of skin pigmentation (P=0.62 and P=0.20, respectively). Thickness of the stratum corneum at the buttocks was unaffected by gender (P=0.42) and age (P=0.83) whereas cellular epidermis decreased with age (P<0.01) and was thinner in females than in males (P<0.01). In spite of the higher pigmentation at the back than at the buttocks, the minimal erythema dose (MED) was lower at the back than at the buttocks (x=2.7 and x=2.2 SED's, respectively; P=0.04). Given the same degree of skin pigmentation, there was no difference in the MED in buttock skin in children, young adults and older adults un-exposed to UV (P= 0.61). Prediction of the MED in un-exposed buttock skin and in exposed skin of the back by a theoretical model based on an exponential function of the measured skin pigmentation was found to provide good estimates of the MED determined by phototest. Conclusion: Skin pigmentation at un-exposed buttock skin can reliably predict the constitutive UV sensitivity in healthy Caucasian children and adults and is recommended in surveys where phototesting cannot be performed.     

Measurement of cutaneous erythema by means of photodensitometry: application to cutaneous photosensitivity

We have developed a photodensitometry method to evaluate the intensity of cutaneous erythema objectively. The method measures the optical density of photographic slides of cutaneous erythema. It combines techniques used commonly but separately by investigators: diffuse transmittance spectroscopy (which is a variant of diffuse reflectance spectroscopy) and photography. We have used this method to study photosensitivity in 22 volunteers who received increasing doses of ultraviolet radiation to the back. Our work confirms the usefulness of an important parameter in photobiology: the regression slope of the curve representing the erythema index, a function of the logarithm of the dose applied.     

In vivo estimation of pigmentation in ultraviolet-exposed hairless mice

A new in vivo method of visual scoring of pigmentation in hairless hr/hr mice with a C3H/Tif background is described. The mice were placed under a bank of 6 Philips TL08 fluorescent ultraviolet A (UVA) tubes in a dark room, and the pigmentation of the skin was compared with a Kodak Gray Scale with 20 different shades from white to black. The radiation from the tubes changed both the color of the back of the mouse and the gray scale into purple hues. The purple color of the back of each mouse could then be classified as one of 20 shades on the gray scale. An experiment was conducted exposing 3 groups of 20 mice to different doses of UV radiation from Philips TL01 tubes. One group of 20 mice was not irradiated and served as control. The pigmentation of each mouse was scored by one investigator every 2–3 weeks. After a few weeks of exposure a clear distinction between the groups was seen. To evaluate the inter- and intrapersonal variation of the method, 30 mice with various degrees of pigmentation were scored independently and blindly by two investigators. This was done twice during the study with a few days' interval. No interpersonal difference was found, but one investigator scored differently the first and second time by only 0.5 points. The described method provides a reproducible in vivo method, with very good discrimination, for estimation of pigmentation in hairless mice.     

In vivo quantification of epidermis pigmentation and dermis papilla density with reflectance confocal microscopy: variations with age and skin phototype

Reflectance confocal microscopy (RCM) may help to quantify variations of skin pigmentation induced by different stimuli such as UV radiation or therapeutic intervention. The objective of our work was to identify RCM parameters able to quantify in vivo dermis papilla density and epidermis pigmentation potentially applicable in clinical studies. The study included 111 healthy female volunteers with phototypes I-VI. Photo-exposed and photo-protected anatomical sites were imaged. The effect of age was also assessed. Four epidermis components were specifically investigated: stratum corneum, stratum spinosum, basal epidermal layer and dermo-epidermal junction. Laser power, diameter of corneocytes and upper spinous keratinocytes, brightness of upper spinous and interpapillary spinous keratinocytes, number of dermal papillae and papillary contrast were systematically assessed. Papillary contrast measured at the dermo-epidermal junction appeared to be a reliable marker of epidermis pigmentation and showed a strong correlation with skin pigmentation assessed clinically using the Fitzpatrick's classification. Brightness of upper spinous and interpapillary spinous keratinocytes was not influenced by the skin phototype. The number of dermal papillae was significantly lower in subjects with phototypes I-II as compared with darker skin subjects. A dramatic reduction in the number of dermal papillae was noticed with age, particularly in subjects with fair skin. The method presented here provides a new in vivo investigation tool for quantification of dermis papilla density and epidermal pigmentation. Papillary contrast measured at the dermo-epidermal junction may be selected as a marker of skin pigmentation for evaluation in clinical studies.     

人工紫外线诱导正常皮肤色素斑的初步研究

目的:建立人工紫外线诱导正常皮肤色素斑的模型。方法:采用日光模拟器人工光源,以连续紫外线(波段290～400nm)照射健康受试者上臂内侧皮肤,观察不同照射剂量及照射后不同测量时间局部皮肤黑素测量值的变化。结果:照射后1周,2.0倍最小红斑量(MED)照射剂量组产生黑素的强度高于1.5MED组(P<0.05),与2.5MED组的差异无统计学意义(P>0.05);2.0MED组红斑消退及表面光滑度较2.5MED组轻(P<0.05),与1.5MED组差异无统计学意义(P>0.05)。紫外线照射后1周,各剂量组黑素测量值(L值)均达到高峰,其后随时间延长迅速下降(P<0.05),在第4、5周下降趋势变缓(P>0.05);而红斑测量值(a值)在紫外线照射1周时达到高峰后呈持续递减趋势(P<0.05)。结论:2.0MED照射剂量是紫外线在正常皮肤诱导色素斑的最佳剂量,照射后1～4周是观察色素变化的最佳时机。     

Melanin differentially protects from the initiation and progression of threshold UV-induced erythema depending on UV waveband

BACKGROUND/PURPOSE: This study aimed to determine the relationship between various measures of constitutive skin pigmentation and erythema caused by solar-simulated UV (ssUV), 290 and 310 nm UV. METHODS: Skin pigmentation was assessed clinically by skin typing as well as objectively by measurement of the melanin index (MI) by reflectance spectroscopy. Subjects having Fitzpatrick skin types I-IV were exposed to graded doses of ssUV and either narrowband 310 nm (n=70) or 290 nm (n=69) UV, and assessed 24 h after exposure. Minimal erythema dose (MED) was assessed visually as the lowest dose that caused minimally perceptible erythema. Susceptibility to further development of erythema with higher exposure doses was measured by the gradient of erythema dose-response curves. This was determined by linear regression using reflectance spectrometry data beyond the MED. RESULTS: Although there was considerable variation within each skin type, MI and ssUV MED increased with increasing Fitzpatrick skin type. MI correlated with ssUV MED and 310 nm UV MED, but not 290 nm UV MED. There was also a significant negative correlation between MI and erythema dose-response gradients caused by ssUV, 310 and 290 nm UV. CONCLUSION: Melanin situated near the basal epidermis may not protect from the initial development of threshold erythema caused by 290 nm UV because it penetrates poorly past the stratum corneum and is not well absorbed by melanin in vivo compared with 310 nm UV. Higher erythemal 290 nm UV doses may reach basal epidermal melanin, which may then afford protection against further 290 nm UV erythema.     

Depth profile of diffuse reflectance near-infrared spectroscopy for measurement of water content in skin

BACKGROUND/PURPOSE: The penetration depth of light in diffuse reflectance near-infrared spectroscopy for measuring water content in skin is assessed both from theoretical and experimental points of view. METHODS: The Monte Carlo simulation was implemented to investigate the dependencies of the light penetration depth on a source-detector distance. To compare with the simulation results, an in vivo experiment for water contents of skin was performed introducing two different optical fiber probes. RESULTS: It is found that the minimum separation between a source and detector fibers influences largely the measurement depth. The larger separation leads to a deeper measurement depth at a particular wavelength. The measurement depth is also influenced fairly by the absorption coefficient of the tissue. The larger absorption coefficient results in a shallower measurement depth. CONCLUSION: The correlations between the water contents measured by the optical and capacitance techniques were discussed. The dependencies of the light penetration depth on the source-detector geometry and wavelength are presented.     

Time course of ultraviolet-induced skin reactions evaluated by two different reflectance spectrophotometers: DermaSpectrophotometer and Minolta spectrophotometer CM-2002

BACKGROUNDS: Many attempts have been made to quantify ultraviolet (UV)-induced erythema and pigmentation, but most studies have been focused on the initial changes of reaction for a few hours or days and neglected the later events. METHODS: : A time course of skin colour changes induced by fluorescent sunlamp with a broad band of UVA and UVB radiation was evaluated in 15 Korean male volunteers using two different reflectance spectrophotometers for 28 days. The results were presented by E (erythema)- and M (melanin)-index as well as values converted to the L*a*b* system recommended by the CIE (Commission Internationale de l'Eclairage). RESULTS: The mean individual typology angle of the subjects was 46.6 degrees, which indicated "light" group in constitutional skin colour category. A day after UV exposure, the L* and b* values decreased significantly, following the colour direction of persistent pigment darkening. The values went in the opposite direction persistently until after the 1st week, when maximum tanning was obtained. They then shifted toward their original positions, parallel to the constitutive melanization axis. The a* index showed a significant increase toward the mean colour of haemoglobin on day 1. It returned to its original value along the constitutive melanization axis. The E-index showed a maximum value at day 1, then returned to baseline. The value of M-index reached a peak at day 7. There was no significant difference between the two instruments, but each has its own characteristic features. CONCLUSION: These promising quantitative methods should enable us to achieve objective measurement of the dermatophysiologic changes and to evaluate the efficacy of therapeutic modalities on skin disorders without the inherent errors associated with subjective judgement. Our results provide standard data on a time course of UV-induced skin erythema and pigmentation.     

Minimal erythema dose and minimal melanogenesis dose relate better to objectively measured skin type than to Fitzpatricks skin type

Background: Fitzpatrick skin type (FST I–IV) is a subjective expression of ultraviolet (UV) sensitivity based on erythema and tanning reactivity after a single exposure. Pigment protection factor (PPF) is an objective measurement of skin sensitivity in all skin types after a single exposure. Methods: The aim was to compare FST and PPF with clinically determined minimal erythema dose (MED) and minimal melanogenesis dose (MMD) in 84 persons with skin types I–V both after single and multiple exposures (one, four, five, six, or 12) to buttock and back skin. Results: FST was better correlated to MED than to MMD, and FST correlated better to constitutive than to facultative pigmented areas after multiple exposures rather than to a single exposure. PPF was generally much better correlated to MED and MMD than FST especially after a single exposure and multiple exposures with steady‐state pigmentation. Multiple regression analyses showed that MED was the only significant, or most important determinator, of both FST and PPF. The correlation coefficient was highly significant for PPF (r²=82). Conclusions: PPF is a better predictor of the individual UV sensitivity (linear relation) than FST (only 4 grades) and PPF can substitute FST.     

Pigmentation above the constitutive level: An indicator of excimer laser radiation-induced erythema in Koreans

Ultraviolet (UV) irradiation induces skin erythema, but it is not clear which factors have the greatest effects on UV sensitivity. Six healthy Korean adult men were enrolled and their melanin index (MI) and increment of erythema index (ΔEI) were measured. In each individual, 12 different sites were selected and 36 spots were irradiated with a single shot of monochromatic excimer laser with a dose of 350 mJ/cm(2) . The sites were categorized into three groups based on the cumulative sun exposure: UZ, unexposed zones; FEZ, frequently exposed zones; and IEZ, intermittently exposed zones. The sun exposure indexes (SEI) were also calculated based on previously described methods. ΔEI, MI and SEI were measured and calculated. The ΔEI of UZ was significantly higher than that of FEZ, but lower than that of IEZ. In general, there was a significant relationship between ΔEI and MI (R(2) = 0.135). However, IEZ did not show significant results. In contrast, there was a stronger relationship between ΔEI and SEI (R(2) = 0.344). Overall, the values were significantly higher for the SEI (0.541 [UZ], 0.281 [IEZ] and 0.228 [FEZ]) than for MI (0.311 [UZ], 0.011 [IEZ] and 0.073 [FEZ]). There were significant site variations in UV sensitivity along with skin pigmentation. In addition, significant differences were observed according to the exposure frequency. The SEI was found to be strongly correlated with UV sensitivity. These results suggest that the induced level of pigmentation above the constitutive level will be a better indicator for UV sensitivity than baseline MI.     

Quantitative skin color measurements in acanthosis nigricans patients: colorimetry and diffuse reflectance spectroscopy

Tristimulus colorimetry and diffuse reflectance spectroscopy (DRS) are white‐light skin reflectance techniques used to measure the intensity of skin pigmentation. The tristimulus colorimeter is an instrument that measures a perceived color and the DRS instrument measures biological chromophores of the skin, including oxy‐ and deoxyhemoglobin, melanin and scattering. Data gathered from these tools can be used to understand morphological changes induced in skin chromophores due to conditions of the skin or their treatments. The purpose of this study was to evaluate the use of these two instruments in color measurements of acanthosis nigricans (AN) lesions. Eight patients with hyperinsulinemia and clinically diagnosable AN were seen monthly. Skin pigmentation was measured at three sites: the inner forearm, the medial aspect of the posterior neck, and anterior neck unaffected by AN. Of the three, measured tristimulus L*a*b* color parameters, the luminosity parameter L* was found to most reliably distinguish lesion from normally pigmented skin. The DRS instrument was able to characterize a lesion on the basis of the calculated melanin concentration, though melanin is a weak indicator of skin change and not a reliable measure to be used independently. Calculated oxyhemoglobin and deoxyhemoglobin concentrations were not found to be reliable indicators of AN. Tristimulus colorimetry may provide reliable methods for respectively quantifying and characterizing the objective color change in AN, while DRS may be useful in characterizing changes in skin melanin content associated with this skin condition.     

Topographic measurement of skin color by narrow-band reflectance spectrophotometer and minimal erythema dose (MED) in Koreans

Background/aims: Narrow-band reflectance spectrophotometer is one of the objective and quantitative devices for measuring the skin colors. There has been some controversy concerning the relationship between the objectively measured skin color and cutaneous responsiveness to ultraviolet radiation, including minimal erythema dose (MED). The aims of this study were to compare the color of Korean brown skin with that of Caucasians by objective measurement with the narrow-band reflectance spectrophotometer, and to determine whether the skin color has any correlation with MED in Koreans, and demonstrate differences in this correlation according to the anatomical sites. Methods: With narrow-band reflectance spectrometer, skin colors were measured at 17 body sites of 20 healthy Korean male volunteers after measuring MED for the same subjects. Results: The constitutional skin color, as measured by the melanin index of the nonexposed sites, showed a statistically significant correlation with MED values, whereas the facultative skin color did not. The Korean brown skin showed higher melanin index and lower erythema index compared with that of Caucasians. Conclusions: We confirmed significant differences in the correlation between MED and melanin indices, depending on the anatomic sites. This suggests that selection from various sites, even among the nonexposed sites, is quite important and deserves further evaluation.