Diagnosis of malignant melanoma and basal cell carcinoma by in vivo NIR-FT Raman spectroscopy is independent of skin pigmentation.

There is a general need for methods to obtain fast in vivo diagnosis of skin tumours. Raman spectroscopy measures molecular structure and may thus have potential as a tool for skin tumour diagnosis. The purpose of this study was to investigate how skin pigmentation influenced the Raman spectra and skin tumour diagnostics in vivo. We obtained Raman spectra in vivo from the normal skin of 55 healthy persons with different skin pigmentation (Fitzpatrick skin type I-VI) and in vivo from 25 basal cell carcinomas, 41 pigmented nevi and 15 malignant melanomas. Increased skin pigmentation resulted in a higher spectral background caused by fluorescence, which could be removed by background correction. After background correction, we found only a negligible effect of pigmentation on the major spectral bands, and the comparison of the intensity of these bands allowed us to differentiate between normal skin and the different skin lesions independent of skin type. The diagnosis of skin lesions is possible due to significant (p < 0.05) differences found in the water band around 3250 cm(-1), the protein specific band around 1250 cm(-1) (amide-III) and the amide-III ratio that describes the protein/lipid ratio by comparing bands around 1250 cm(-1) with bands around 1300 cm(-1). We have shown that NIR-FT Raman spectroscopy is useable for malignant melanoma and basal cell carcinoma diagnostics in vivo and that pigmentation of the skin or lesion does not influence the diagnosis, but larger data sets are required to establish accurate diagnostic power.

Daylight-mediated photodynamic therapy of moderate to thick actinic keratoses of the face and scalp: a randomized multicentre study.

BACKGROUND: Photodynamic therapy (PDT) is an attractive therapy for nonmelanoma skin cancers and actinic keratoses (AKs). Daylight-mediated PDT is a simple and tolerable treatment procedure for PDT. Methyl aminolaevulinate (MAL)-PDT is approved for the treatment of thin or nonhyperkeratotic AKs on the face and scalp. However, thick AK lesions are often treated as well when present in the field-cancerized treatment area. OBJECTIVES: In a randomized multicentre study to evaluate efficacy of daylight-mediated PDT for different severity grades of AKs. METHODS: One hundred and forty-five patients with a total of 2768 AKs (severity grades I-III) of the face and scalp were randomized to either 1½ or 2½ h exposure groups. After application of a sunscreen (sun protection factor 20) and gentle lesion preparation, MAL was applied to the entire treatment area. Patients left the clinic immediately after application and exposed themselves to daylight according to randomization. Daylight exposure was monitored with a wrist-borne dosimeter. RESULTS: No difference in lesion response was found between the 1½ and 2½ h exposure group. The mean lesion response rate was significantly higher in grade I lesions (75·9%) than in grade II (61·2%) and grade III (49·1%) lesions (P < 0·0001). Most grade II (86%) and III AKs (94%) were in complete response or reduced to a lower lesion grade at follow-up. Large variations in response rate of grade II and III AKs were found between centres. No association was found between response rate and light dose in patients who received an effective light dose of > 3·5 J cm(-2). CONCLUSIONS: Daylight-mediated PDT of moderate to thick AKs was less effective than daylight-mediated PDT of thin AKs especially in some centres. However, nearly all thicker lesions (grades II and III) were reduced to a lower lesion grade at 3 months after a single treatment of daylight-mediated PDT.

Effects of ageing on dermal echogenicity.

BACKGROUND/AIMS: Changes in the dermis associated with ageing can be detected by high-frequency skin ultrasonography. In photoaged skin, this technique shows a subepidermal low echo-genic band (SLEB) that is probably an ultrasound manifestation of elastosis and oedema in the papillary dermis. Since some authors found an association between age and SLEB thickness or its echogenicity on exposed sites, it has been proposed to use these parameters to quantify skin photoageing. METHODS: To determine whether SLEB can be used as a quantitative marker of ageing, its prevalence was determined on forearm skin in a group of 55 individuals (age 18-57 years). The size of SLEB has been measured by quantifying the number of low echogenic pixels in the subepidermal area, which is an accurate method for assessing SLEB severity. RESULTS: The prevalence of SLEB increased with age, but SLEB was also present in young subjects. The echogenicity of the subepidermal area did not show any age dependence. However, when a ratio of echogenicity between upper and lower dermis was calculated, a linear dependence on age was found. CONCLUSIONS: This study indicates that skin echogenicity measured as a ratio between the upper and lower dermis may be used to objectively estimate photoageing.

Ultrasound in dermatology. Part I. High frequency ultrasound.

This paper addresses the basic terminology of modern ultrasound equipment for the examination of the skin, the technical limitations of currently available equipment and the normal skin structure. Different applications for dermatosonography are discussed, and the paper summarises current knowledge about common sources of variation in the ultrasound image of the skin, such as skin tumours and selected skin diseases.

Hydration of human nails investigated by NIR-FT-Raman spectroscopy.

The human nail, although it is usually stable against outer influences, becomes soft and flexible after soaking in water. Frequent washing increases brittleness of nails. Hydration of nails is thought to be the most important factor influencing the physical properties of nails and possibly acts through changes in keratin structure. Here NIR-FT-Raman has been used to examine molecular structural changes of intact moisten nails. Raman spectra were obtained both in vitro from nail samples and in vivo before and after soaking in water. The water uptake of normal nail samples during the first 15 min was reflected in the increasing intensity ratio of the nu(OH)/nu(CH(2)) bands. A saturating effect appeared soon after 10 min which is explained by a defined water holding capacity. R(nu) representation of the low frequency range of the Raman spectra showed that mainly bound water is found both in dry and in wet nails. This implies water-protein interaction. Protein backbone vibration at 932 cm(-1) indicating alpha-helical proteins increased in intensity in the wet nails. The nu(S-S) which is sensitive to changes in conformation of proteins showed a 4% higher intensity. Additional protein-water interactions could lead to a slight change of the dihedral angle of the C-S-S-C bonds and to geometric changes in coiling behavior of the alpha-helical protein. Suggesting a separation between matrix proteins and fiber proteins giving them a greater freedom of flexibility. The in vivo spectra detected from the distal part of the nail resembled spectra in vitro. Raman spectra of the proximal part of the nail showed that it was fully saturated with water. The proximal part of the nail did not show changes in water content and protein structure during nail moisturizing in the Raman spectra. Our results suggest that the softening of the nail following hydration may be due to changed matrix protein molecular structure induced by water.

Water and protein structure in photoaged and chronically aged skin.

Changes in the structural proteins and hydration during aging is responsible for altered skin morphologic and mechanical properties manifested as wrinkling, sagging, loss of elasticity, or apparent dryness. To gain insight into the age-related alterations in protein conformation and water structure, we obtained Raman spectra from the sun-protected buttock skin representing chronologic aging and the sun-exposed forearm skin representing combined effects of photoaging and chronologic aging. Ten aged individuals (five men, five women; age range 74-87) and 10 control young individuals (five men, five women; age range 22-29) entered the study. In the photoaged forearm skin the positions of protein-specific amide I, amide III, and CH stretching bands were shifted, suggesting increased protein folding. In contrast, major changes were seen only in the amide I peak in chronologically aged skin. The intensity of the 3250 cm(-1) OH stretching band was increased in photoaged skin (but not in chronologically aged skin) indicating an increased water content. R(v) representation of the low-frequency region of Raman spectra was applied to determine water structure. In the young skin and chronologically aged skin water was mostly present in the bound form. In the photoaged skin, however, an increase in intensity at 180 cm(-1) was noted, which reflects an increase in the not-protein bound water (tetrahedron water clusters). In conclusion, it seems that proteins in the photoaged skin are more compact and interact with water to limited degree. Impairment in protein hydration may add to the understanding of ultrastructural, mechanical, and biochemical changes in structural proteins in the aged skin.

Side effects from the pulsed dye laser: the importance of skin pigmentation and skin redness.

The pulsed dye laser is the treatment of choice for port-wine stains. In this study we evaluate the importance of preoperative skin pigmentation and skin redness for the development of side effects from one treatment with the pulsed dye laser. A risk assessment is performed and skin reflectance measurement objectifies postoperative pigmentary changes. Fourteen human volunteers (skin types I to V) were laser-treated on the inside of the proximal brachium. Photographic documentation was used for blinded, clinical evaluation of side effects 3 and 6 months postoperatively. Skin was artificially reddened using topical application of 10% nicotinic acid cream. The development of pigmentary alterations and texture changes depended on the preoperative pigmentation and redness degrees. The risk of inducing clinically visible pigmentary alterations and texture changes increased with higher preoperative skin pigmentation and redness degrees, and with the application of increasing laser doses. Pigmentary alterations were induced at a lower fluence level than texture changes. The risk of side effects was higher 3 months postoperatively than 6 months postoperatively, substantiating a gradual disappearance of side effects. Skin reflectance measurements documented postoperative hyperpigmentation that faded partially from 3 to 6 months postoperatively. This is the first human experimental model for port-wine stains which provides quantitative data on the relationship between preoperative skin colours and postoperative clinically disturbing side effects.

Removal of dermal edema with class I and II compression stockings in patients with lipodermatosclerosis.

BACKGROUND: Lipodermatosclerosis is a sequela of deep venous insufficiency and a risk factor for the occurrence of venous leg ulceration. Medical compression stockings facilitate leg ulcer healing and prevent occurrence of ulcers resulting from removal of edema. Although the exact level of compression necessary for removal of dermal edema in patients with deep venous insufficiency has not been established, garments providing high compressive values of 30 to 40 mm Hg have been recommended. Dermal edema can be visualized by high-frequency ultrasonography. OBJECTIVE: We used ultrasound imaging to study whether a lower level of compression (class I 18 to 26 mm Hg vs class II 26 to 36 mm Hg) is effective in removal of dermal edema. This question is important because the use of hosiery with a lower compression class would enhance compliance and enable treatment of patients with mixed arteriovenous disease. METHODS: In 11 patients skin images were obtained with 20 MHz ultrasound from the malleolar region in lipodermatosclerotic skin and corresponding normal skin of the contralateral leg. The ratio of low echogenic pixel number to total pixel number (LEP/TP), which correlates with dermal water, was measured before and after 5 days of applied compression in two treatment courses where classes of compression were switched randomly. Ankle circumference was also measured. RESULTS: We found that LEP/TP was 33% higher in lipodermatosclerotic skin than in the matched normal skin indicating presence of skin edema. Application of class I and II compressive hosiery resulted in LEP/TP decrease by 17% (95% confidence interval, 0.07 to 0.26) and 14% (95% confidence interval, 0.04 to 0.21), respectively, suggestive of dermal edema reduction. No statistically significant difference in efficacy of dermal edema removal between class I and II was found. No changes in ankle circumference after application of both classes of compression was observed. CONCLUSION: Application of light and moderate compression results in a partial edema removal from the dermis in lipodermatosclerosis in the absence of measurable reduction in leg circumference. Class I compression is as effective as class II for elimination of dermal edema. These data indicate that light compression may be a useful modality for patients with deep venous insufficiency and lipodermatosclerosis who are not eligible for treatment with garments having higher compressive forces.

Structure of water, proteins, and lipids in intact human skin, hair, and nail.

Raman spectroscopy is a nondestructive analytical method for determining the structure and conformation of molecular compounds. It does not require sample preparation or pretreatment. Recently, near-infrared Fourier transform Raman spectroscopy has emerged as being specially suited for investigations of biologic material. In this study, we obtained near-infrared Fourier transform Raman spectra of intact human skin, hair, nail, and stratum corneum. We disclosed major spectral differences in conformational behavior of lipids and proteins between normal skin, hair, and nail. The amide I and III band location indicated that the majority of proteins in all samples have the same secondary alpha-helix structure. Positions of (S-S) stretching bands of proteins revealed a higher stability of the disulfide bonds in the hair and the nail. Analysis of vibrations of protein -CH groups showed that in the hair and the nail the proteins are apparently highly folded, interacting with the surroundings only to a small degree. The position of lipid specific peaks in spectra of hair, nail, and stratum corneum suggested a highly ordered, lamellar crystalline lipid structure. A greater lipid fluidity was found in whole skin. Assessment of the structure of water clusters revealed that mainly bound water is present in the human skin, stratum corneum, and nail. In conclusion, structural changes of water, proteins, and lipids in intact skin and skin appendages may be analyzed by Raman spectroscopy. This technique may be used in the future in a noninvasive analysis of structural changes in molecular compounds in the skin, hair, and nail associated with different dermatologic diseases.

Changes in skin redness, pigmentation, echostructure, thickness, and surface contour after 1 pulsed dye laser treatment of port-wine stains in children.

BACKGROUND: The pulsed dye laser is the treatment of choice for children with port-wine stains (PWSs). Evaluation of treatment outcome and adverse effects is traditionally based on subjective clinical scoring systems. We intend to objectify treatment results and adverse reactions after 1 treatment with the pulsed dye laser. DESIGN: A before-and-after trial using skin reflectance to detect changes in skin redness and pigmentation, ultrasonography to evaluate changes in echostructure and skin thickness, and 3-dimensional surface contour analysis to detect changes in surface texture. PATIENTS: Twelve children with PWSs. SETTING: A university dermatological department. RESULTS: The skin reflectance-determined change in skin redness correlated with the clinical response (r = 0.46, P < .002). The percentage of reflectance-determined lightening was equal for pink, red, and dark red PWSs (median, 42.9%). Skin pigmentation increased after laser treatment (P < .007). Ultrasonography revealed lower dermal echogenicity of preoperative PWSs than of postoperative PWSs (P < .007) and healthy skin (P < .001). An increase in echogenicity reflected a decrease in the dermal water (blood) content. Variations were found in the dermal localization of the PWS. Skin thickness was significantly higher in the PWS before treatment than after (P < .001). The preoperative lesional thickness correlated inversely with the ultrasound-assessed treatment response (r = 0.35, P < .04). The surface contour parameters decreased significantly after laser treatment, indicating a flattening of the skin surface. The contour changes correlated positively with treatment response. By clinical evaluation, no hypopigmentation or texture changes were detected. CONCLUSION: The evaluation of treatment outcome and adverse effects is refined by the use of skin reflectance, ultrasonographic, and surface contour analysis.

Quantitative evaluation of chronological ageing and photoageing in vivo: studies on skin echogenicity and thickness.

Skin ageing is divided into chronological ageing and photoageing due to the cumulative effects of solar ultraviolet radiation. It is, however, difficult to measure the degree of photoageing and chronological ageing in humans in vivo. Here, we have evaluated the usefulness of ultrasonography for measurement of chronological ageing and photoageing in vivo. Twenty megahertz ultrasonography was performed in 90 individuals (29 men, 61 women, age 18-94) to describe age-related changes in sun-exposed regions with different levels of sun exposure (dorsal and ventral forearm, forehead, ankle) and non-exposed buttock skin. Skin thickness and skin echogenicity in different layers of the dermis were measured in ultrasound images. Additionally, cutaneous photodamage was scored clinically. Age-related changes were dependent on body site as well as layer of the dermis. A progressive, age-related decrease in echogenicity of the upper dermis was found in sun-exposed regions (dorsal forearm, forehead), but not in moderately exposed regions (ventral forearm, ankle). In the buttock an increase in echogenicity was observed. The echogenicity of the lower dermis increased in all examined sites. Skin thickness increased with age in the forehead and buttock, but decreased in the extremity skin. Our findings show that photoageing causes a decrease in echogenicity in the upper dermis. In contrast, chronological ageing is associated with an increase in echogenicity in the lower dermis. Although both increases and decreases in skin thickness were observed in different anatomical regions, there was no general relationship between skin thickness and age. Dermal echogenicity was deemed valuable for in vivo study of chronological ageing and photoageing.

Skin temperature of UV-induced erythema correlated to laser Doppler flowmetry and skin reflectance measured redness.

BACKGROUND/AIMS: The sensitivity of human skin to UV radiation is investigated by visual grading of the resulting erythema reactions 24 h after exposure to a series of increasing UV doses. Visual erythema assessment is, however, subjective and depends on pigmentation and redness of the adjacent un-irradiated skin and can be aided by skin reflectance spectroscopy and laser Doppler blood flow measurements. Erythema is accompanied by a raised skin temperature, and this reaction might be utilised as a simple objective measurement of UV sensitivity. METHODS: Sixteen patients with cutaneous malignant melanoma, 16 patients with basal cell carcinoma, and 36 healthy people were phototested with simulated sunlight on previously UV un-exposed buttock skin. The resulting erythema reactions were graded visually 20-24 h post-exposure and measured by skin reflectance spectroscopy and laser Doppler flowmetry, and the surface skin temperature was determined in the erythema reactions and in adjacent un-irradiated skin by a contact thermometer. RESULTS: Skin surface temperature in UV-induced erythema reactions was dose dependent, was statistically identical in skin cancer patients and in healthy people, and was age independent. The average temperature increase in barely perceptible erythema was 0.7°C (SD=1.1°C), and in bright red erythema it was 3.5°C (SD=2.0°C). Skin surface temperature increases were correlated to measurements by skin reflectance spectroscopy and by laser Doppler flowmetry. CONCLUSIONS: Skin surface temperature changes can be used as a simple objective measurement of UV sensitivity in healthy people and in skin cancer patients and may be particularly useful in heavily pigmented people where visual assessment of erythema is difficult or impossible.

Distinctive molecular abnormalities in benign and malignant skin lesions: studies by Raman spectroscopy.

Near-infrared Fourier transform Raman spectroscopy is an analytical, nondestructive technique that provides information about the molecular structure of the investigated sample. The molecular structure of proteins and lipids differ between neoplastic and normal tissues and therefore Raman spectroscopy has been considered promising for the diagnosis of cancer. We aimed to compare the molecular structure of normal skin, benign and malignant skin lesions by the near-infrared Fourier transform Raman spectroscopy. Biopsies were obtained from the following skin lesions: skin tag, dermatofibroma, seborrhoeic keratosis, actinic keratosis, keratoacanthoma, basal cell carcinoma, squamous cell carcinoma, nevus intradermalis, nevus compositus, dysplastic nevus and lentigo maligna. Control skin was harvested from the vicinity of these lesions. In the Raman spectra, the secondary structure of the proteins was reflected by the amide vibrations of peptide bonds. The principal lipid vibrations were twisting and wagging (CH2) and CH stretching vibrations. Histologically distinguishable lesions showed specific combinations of band changes indicating alterations in the protein conformation and in the molecular structure of the lipids. Histogenetically related lesions (actinic keratosis and sqamous cell carcinoma) produced similar but not identical patterns of spectral changes. Because the examined skin lesions produced reproducible and unique spectra, we suggest that Raman spectroscopy will be useful for diagnosis of skin lesions.

Ultrasound examination of hair follicles in hidradenitis suppurativa.

OBJECTIVE: To describe the in vivo skin echostructure, hair follicle shape, and dermal thickness in hidradenitis suppurativa. DESIGN: Qualitative and quantitative assessment of high-frequency (20-MHz) B-mode ultrasound images of lesional and paralesional skin. SETTING: University hospital. PATIENTS: Age- and sex-matched outpatients with hidradenitis suppurativa (n = 15) and healthy control subjects (n = 13). Median age was 34 years (range, 31-38). RESULTS: Clinically normal paralesional hair follicles in hidradenitis have an abnormal shape. The follicles appear to be wider in the deep dermis, the difference being statistically significant in the genitofemoral region (P = .007). Patients with hidradenitis have larger follicles in the axilla than controls (P = .002). Mature acne and hidradenitis lesions are indistinguishable, but both are different from epidermal cysts. Mean axillary and genitofemoral skin was significantly thicker in patients than in controls. CONCLUSIONS: In vivo ultrasonography shows characteristic differences in the shape of hair follicles in hidradenitis. The general underlying abnormalities appear to occur in the deep part of the follicle. The mature lesions are indistinguishable from acne, but are clearly different from epidermal cysts. A thickened skin may play a pathogenic role in the development of hidradenitis.

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 = 0.41) 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.

Swelling of the temporal region: a case of benign masticatory muscle hypertrophy.

A 35-year-old Caucasian woman had bilateral indolent swelling of the temporal regions. Imaging studies with nuclear magnetic resonance, ultrasonography and histopathological investigation revealed enlargement of the temporal and masseter muscles, with no pathology in the skin or subcutaneous tissue. A small prolactinoma of the pituitary gland was incidentally found. The condition, designated benign masticatory muscle hypertrophy, should be distinguished from similar diseases affecting the skin and the subcutaneous tissue of the scalp. The cause remains unknown.

Sebum excretion in hidradenitis suppurativa.

BACKGROUND: Clinical and histological similarities between hidradenitis suppurativa and acne have been pointed out. The possible role of sebaceous glands in hidradenitis has not previously been investigated. Acne treatment, in particular is however not effective in hidradenitis. No previous information was found on regional sebum excretion in hidradenitis. OBJECTIVE: Investigate the sebum excretion (SE) and markers of cutaneous virilization in hidradenitis patients. METHODS: Sebutapes and scores of acne, hirsutes and alopecia in 16 women with hidradenitis suppurativa were compared with 16 healthy controls. RESULTS: The SE and the number of active glands followed a similar pattern in both groups (face > axillae/genitofemoral fold, p < 0.0001) but no significant differences were seen between the two groups. No differences were seen in the median Body Mass Index, number of obese persons in either group or any of the clinical markers of virilization studied. CONCLUSION: No significant axillary, genitofemoral or facial seborrhea was found in hidradenitis. It is suggested that SE is not an important factor in the development of hidradenitis, and this may help explain the generally unsatisfactory therapeutic effect of retinoids in this disease. The results further suggest that general cutaneous virilization is not a feature of hidradenitis.