Neuropeptide and neuronal marker studies in vitiligo

Neuropeptide and neuronal marker immunoreactivity was studied in skin biopsies from lesional and marginal areas in 12 patients with vitiligo, and in seven normal controls. The vitiligo was active in seven, static in two, and of unknown activity in three. Antibodies against general neuronal marker PGP 9.5 (PGP 9.5), substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY), were used. The epidermis, dermo-epidermal junction, papillary and reticular dermis, and appendages, were assessed semiquantitatively for reactivity with each antibody. Staining with PGP 9.5 in the upper dermis was assessed quantitatively by image analysis. An increase in reactivity against NPY antibody was seen in five of 10 cases (three with active vitiligo) in the marginal areas, and in three of 12 subjects (all with active vitiligo) in the lesional vitiligo areas. VIP antibody reactivity showed a minimal increase in the marginal and lesional vitiligo areas (in two cases each, both of whom had active vitiligo), SP and CGRP reactivities did not differ from normal. PGP 9.5 staining was minimally increased at the dermo-epidermal junction and lower Malpighian layer in biopsies from marginal areas in three of 10 subjects (all with active vitiligo). Quantitative analysis of PGP 9.5 reactivity in the upper dermis showed no difference between vitiligo and normal biopsies. These findings support the concept of neuronal or neuropeptide involvement in vitiligo, and in particular suggest that NPY may have a role in the pathogenesis of the disease.     

PGP 9.5 distribution patterns in biopsies from early lesions of atopic dermatitis

Peripheral nerve fibres are often increased in lesional skin of atopic dermatitis (AD) patients. We attempted to study nerve fibre profiles, using PGP 9.5 as neuronal marker, in early AD lesions in 10 patients, as compared to non-lesional skin in the same patients and skin from healthy controls. The number of PGP 9.5-positive nerve fibre profiles was not different in the biopsies taken from normal-looking AD skin and healthy controls. The total number of PGP 9.5-positive nerve fibre profiles in the whole skin sections was higher in both the epidermis and the dermis in the group of skin biopsies taken from early lesions of AD patients. Further, the number of epidermal PGP 9.5-positive dendritic cells was increased in AD skin. It seems reasonable that PGP 9.5-positive nerve fibres and PGP 9.5-positive dendritic cells have pathological roles in AD. The findings might serve as a basis for further studies in evaluating novel diagnostic and therapeutic approaches.     

Cutaneous PGP 9.5 distribution patterns in hidradenitis suppurativa

The aim of this study was to investigate the presence and density of the nerve fibre-marker protein gene product 9.5 (PGP 9.5), with immunohistochemistry, in skin from patients with hidradenitis suppurativa (HS). Punch biopsies were obtained from 16 patients; 10 with involvement of the groin and six with axillary disease. Specimens were taken from HS lesions in the groin or axilla, clinically non-involved skin and from 12 healthy control subjects. Coded slides were observed in the microscope and PGP 9.5 positive nerve fibre profiles (profiles) as well as PGP 9.5 positive cells (cells) were counted. The overall impression was that the median number of profiles was decreased in lesional epidermis, yet statistically significant only in the groin (p = 0.0014). The median number of profiles in dermis was significantly decreased in lesional skin of the axilla, whereas in the groin there were contradictory findings with significantly increased number of profiles in upper dermis and non-significant in mid and lower dermis. The number of cells with strong immunofluorescence was few or absent in epidermis, but increased in dermis in the lesional skin. This difference was statistically significant throughout the dermis in specimens from the groin (p < 0.01) and showed the same trend, although not significant, in the axilla. The PGP 9.5 immunofluorescent cells were not yet further investigated, so it is not exactly known what cell type they represent. In conclusion, despite several study limitations, the findings indicate that PGP 9.5 positive nerve fibres could be involved in the pathogenesis of HS. Both regarding the profiles and the cells, further studies remain to show if these differences are primary events, or secondary to e g chronic inflammation, which is considered a major issue of HS.     

Bird's eye view observations of the subepidermal nerve network of normal guinea pig skin

This study was undertaken to visualize the subepidermal nerve networks immunohistochemically. Specimens were obtained from the normal skin of the back, abdomen and nose of seven adult male Hartley guinea pigs, and immersed in 1 M NaCl solution. Dermal sheets were obtained by separating the dermis and epidermis, followed by fixation in Zamboni's fixative. The dermal sheets were sectioned parallel to the separated surface. Using the immunoperoxidase technique and immunofluorescence, the sections were immunostained with primary antibodies to S 100 protein (S100), protein gene product 9.5 (PGP 9.5), neuron-specific enolase (NSE), substance P (SP) and calcitonin gene-related peptide (CGRP). In double-labeled immunofluorescence of PGP 9.5 and SP or CGRP, the sections were viewed under a confocal laser scanning microscope. In skin of the back and abdomen, networks of S100-, PGP 9.5- and NSE-positive fibers were observed, some of which showed a multicentric arrangement. The outermost structures were formed by the thickest fibers which were 5-10 micro m thick, the outer networks consisted of fibers 3-6 micro m thick, and the inner networks consisted of fibers 1-3 micro m thick. From these networks, single fibers approximately 0.5 micro m thick branched out and terminated in free endings. The SP- and the CGRP-positive substances appeared as granules on the PGP 9.5-positive fibers. These results confirm that the dermis has a three-layered sensory nerve plexus, i.e. deep, superficial and subepidermal. In the skin of the nose, however, nerve networks were made up of only thick fibers which were probably situated in the subpapillary dermis.     

Time course effect and sequential morphological changes of subepidermal nerve fibers in guinea pig skin after application of histamine ointment: a laser scanning confocal fluorescence microscopic and electron microscopic study

This study was undertaken to elucidate the morphological effects of histamine on subepidermal nerve fibers. A 10% histamine ointment was topically applied to the back skin of 17 adult male Hartley guinea pigs. Biopsy specimens were obtained at times from 5 min to 24 h, and were examined by conventional immunofluorescence (IF), laser scanning confocal fluorescence microscopy (LSCM) and transmission electron microscopy. On IF and LSCM, marked diminutions in the immunoreactivity of protein gene product 9.5-immunoreactive (PGP 9.5-IR) fibers as well as of substance P-immunoreactive (SP-IR) and calcitonin gene-related peptide-immunoreactive (CGRP-IR) substances were observed 5 min after histamine application. By 30 min, immunoreactivity of PGP 9.5, SP and CGRP was completely lost. By 2 h, however, immunoreactivity of PGP 9.5-IR fibers and CGRP-IR substances started to show recovery. By 4 h, immunoreactivity of PGP 9.5, SP and CGRP had almost recovered, but the recovery time for each substance was slightly different (PGP 9.5 first, CGRP next, and SP last). By 6 h after histamine application, immunoreactivity of all these substances had fully recovered. Ultrastructurally, 5 min after histamine application, axonal and mitochondrial swelling and glycogen deposition were seen in the axons of subepidermal nerve fibers. By 30 min, severe axonal degeneration had occurred in some of the axons. It was only by 4 to 6 h that almost normal ultrastructural features were observed. Schwann cells and perineurial cells did not show any pathological changes. These findings demonstrate that 10% histamine ointment produced organic changes in the axons in the subepidermal nerve fibers of guinea pig skin, but these morphological changes were short-lived, reversible and transitory.     

Phototherapy reduces the number of epidermal and CGRP-positive dermal nerve fibres

The purpose of this study was to gain an understanding of why phototherapy relieves itching. Skin samples (3 mm punch biopsies) from non-inflamed gluteal skin of 10 patients undergoing phototherapy were compared before and after 20 treatments. All the cutaneous nerve fibres here visualized by antibodies against PGP 9.5, sensory nerve fibres by antibodies against calcitonin gene-related peptide (CGRP) and capsaicin-sensitive primary nociceptive afferents by antibodies against VR1-receptor. Following treatment, the number of PGP 9.5-positive nerve fibres in the epidermis was reduced from 193 +/- 52 to 102 +/- 34 (p < 0.0001) and the number of CGRP-immunoreactive nerve fibres, which occurred only in dermis, was reduced from 28 +/- 15 to 22 +/- 7 (p = 0.04). The VR1-immunoreactive nerve fibres, some of them containing immunoreactivity to CGRP, were not affected. The success of phototherapy in combating itch may at least partly be linked with the reduction in the number of epidermal nerve fibres. The reduction in the number of CGRP-immunoreactive nerve fibres in the dermis may contribute to the beneficial effects of UV irradiation on the inflammatory process.     

Unexpected diminished innervation of epidermis and dermoepidermal junction in lichen amyloidosus

Background  Lichen amyloidosus is a localized, chronic, pruritic skin disease characterized by deposition of amyloid in the papillary dermis. The pathogenesis of the pruritus of lichen amyloidosus is largely unknown.
Objectives  To determine any change in the nerve fibre density in lichen amyloidosus lesions as an explanation for itch.
Methods  Using an antibody to protein gene product (PGP) 9.5, the immunohistochemical analysis of the skin biopsies of 30 Hispanic patients with clinicopathologically proven lichen amyloidosus and of 11 healthy Hispanic controls matched for age, sex and site was performed.
Results  Unexpectedly, the mean amount of PGP9.5 stain, a measure for nerve fibre amount, for the healthy controls was higher than the lichen amyloidosus group both in the epidermis ( P  <   0·0019) and dermoepidermal junction ( P  <   0·0064). No change was observed in the papillary dermis. Furthermore, the proportion of area covered by PGP9.5 showed a significant decrease in the epidermis ( P  <   0·0024) and dermoepidermal junction ( P  <   0·0075) in lichen amyloidosus compared with healthy controls. Age, gender and body site were found not to be influencing factors in nerve fibre amounts in lichen amyloidosus samples.
Conclusions  We speculate that the severe pruritus observed in lichen amyloidosus might be the result of the hypersensitivity of the remaining nerve fibres as a response to an unexplained neurodegeneration of the absent nerve fibres.     

Symptoms of notalgia paresthetica may be explained by increased dermal innervation.

Notalgia paresthetica is a sensory neuropathy characterized by infrascapular pruritus, burning pain, hyperalgesia, or tenderness. To assess whether the symptoms may be caused by alterations in the cutaneous innervation, skin from the affected area of patients (n = 5) was compared with controls (n = 10) comprising the contralateral unaffected area from the same patients and site-matched biopsies of normals, using immunohistochemistry. Frozen sections were immunostained with antisera to the neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and neuropeptide with tyrosine, and to the general neural marker PGP 9.5 and the glial marker S-100 to show the overall innervation and glial cells, respectively. No discernible change in the distribution of neuropeptide-immunoreactive axons was found, but all of the specimens from the affected areas had a significant increase in the number of intradermal PGP 9.5-immunoreactive nerve fibers compared with unaffected areas from the same patients and normal controls. Epidermal dendritic cells immunoreactive for S-100, possibly Langerhans cells, were substantially increased. It is concluded that there is an increase in the sensory epidermal innervation in the affected skin areas in notalgia paresthetica, which could contribute to the symptoms, and that neural immunohistochemistry of skin biopsies could be helpful in the diagnosis of the disease.     

Expression of PGP 9.5 in granular cell nerve sheath tumors: an immunohistochemical study of six cases

BACKGROUND: Protein gene product 9.5 (PGP 9.5) is expressed in brain at 20 to 50 times the levels detected in other organs. Immunohistochemical studies reveal this protein is localized to both central and peripheral neurons. Recently, PGP 9.5 is reported to be a useful marker for cellular neurothekeomas. Herein we test whether PGP 9.5 is a new marker for granular cell nerve sheath tumors. MATERIAL AND METHODS: An immunohistochemical analysis for PGP 9.5 expression was carried out on all cases with the diagnosis of granular cell nerve sheath tumor seen over a 2-year period. In addition, we compared expression of PGP 9.5 with other accepted markers for neuroectodermal tumors including anti-S-100 protein and NKI/C3 monoclonal antibodies. RESULTS: Six granular cell nerve sheath tumors were diagnosed in over 80,000 dermatopathology specimens in the two-year period. These cases were all positive for PGP 9.5 as well as for S-100 protein and NK1/C3. CONCLUSION: These findings identify PGP 9.5 as a new immunohistochemical marker for use in the diagnosis of granular cell tumors. They also strengthen the histogenetic relationship between granular cell nerve sheath tumors and tumors of Schwann cell or perineurial origin.     

Neuronal changes in psoriasis exacerbation

Background The nervous system contributes to inflammatory skin diseases.
Objective The aim of this investigation was to study the neuronal contribution to psoriasis at the remission and exacerbation phases.
Methods We examined the expression of the neuronal markers protein gene product 9.5 (PGP 9.5), growth-associated protein-43 (GAP-43) and substance P, in addition to its receptor (R), neurokinin-1R (NK-1R) in psoriatic skin from seven female patients at remission and exacerbation, using immunohistochemistry.
Results The number of epidermal PGP 9.5 immunoreactive nerve fibres in the involved skin during exacerbation was decreased ( P  < 0.01) compared to involved skin at remission and non-involved skin at the exacerbation phase. GAP-43-positive nerve fibres were decreased ( P  < 0.05) in the involved skin in contrast to non-involved skin, during exacerbation. Substance P expression was seen on both immunoreactive nerve fibres and cells with a down-regulation ( P  < 0.01) in the number of positive nerve fibres in the involved skin compared to non-involved skin, at the exacerbation phase. The number of substance P-positive cells was slightly lower in the involved skin at exacerbation than at remission. The number of NK-1R immunoreactive cells was increased ( P  < 0.01) in the involved skin in contrast to non-involved skin, at the exacerbation phase.
Conclusion Our findings suggest a crosstalk between the nervous system and inflammation during psoriasis exacerbation in the form of an altered expression of nerve fibres, substance P and its NK-1R.     

Neuropeptides and general neuronal marker in psoriasis an immunohistochemical study

Nerve fibres immunoreactive to antibodies to vasoactive intestinal polypeptide (VIP) and substance P (SP) were increased in lesional psoriatic skin when assessed semi-quantitatively. Biopsies from psoriatic plaques on the arm were studied in 13 patients and compared with biopsies from non-lesional areas (in three of the same psoriatic subjects) and from normal skin in seven non-psomtic controls. Immunohistochemical methods were used on cryocut skin sections to demonstrate the neuropeptides SP, VIP, calcitonin gene-related peptide and neuropeptide Y, and the general neuronal marker protein gene product (PGP) 9.5. The immunofluorescence was examined by semiquantitative and, for PGP 9.5, by quantitative methods. VIP reactive nerve fibres were increased at areas of eccrine sweat glands throughout the dermis, at the dermo-epidermal junction, and in the epidermis, in psoriasis lesional skin. SP reactive nerve fibres were increased at the dermo-epidermal junction, where the nerves ran parallel with and perpendicularly through the junction. PGP 9.5 reactive nerve fibres showed an increase at the dermo-epidermal junction, in the papillary dermis, and at the eccrine sweat glands in lesional psoriatic skin but not in non-lesional, or in control skin. These findings support the hypothesis that neuropeptides may be involved in the pathogenesis of psoriasis.     

Expression of PGP9.5 on Langerhans' cells and their precursors

Langerhans' cells are epidermal dendritic cells, derived from blood precursors. Their main function is antigen presentation to T-cells. They are able to express neuronal proteins, such as neuron-specific enolase or substance P-receptor. They are closely associated with nerve fibres. PGP9.5 is the most specific neuronal protein in the epidermis. Epidermal Langerhans' cells can express PGP9.5 if denervated. Using flow cytometry, we found that cultured CD34+ precursors did not express PGP9.5, whereas suspensions of fresh or cultured Langerhans' cells could express this neuronal protein. Precursors of Langerhans' cells are not able to express PGP9.5, suggesting that they are not mature enough or that the capacity to express PGP9.5 may be acquired only in the epidermis. The function of PGP9.5 on Langerhans' cells and mature dendritic cells remains unknown. PGP9.5 might be related to dendritic cell maturation or to the lack of contacts with nerve endings.     

PGP 9.5 expression in cutaneous keratoacanthomas and squamous cell carcinomas

The aim of the study was to investigate the expression of PGP 9.5 in cutaneous keratoacanthomas (KAs) and squamous cell carcinomas (SCCs). Thirty-one cases of KA (10 in the growth stage, 9 in the mature phase and 12 in the involution stage) and 36 SCCs including 13 well differentiated cases, 12 moderately differentiated tumors, 7 poorly differentiated lesions and 4 pseudoadenoid entities were investigated. PGP 9.5 expression was positively correlated with tumor stage (P < 0.001) and potential perineural invasion (P < 0.001). There was no significant difference in the distribution of patients presenting variable levels of PGP 9.5 staining with regard to maximal tumor size and the extent and degree of stromal invasion. PGP 9.5 expression proved closely associated with tumor aggressiveness and is classified as a marker for predicting the outcome of resection-treated skin cancer patients.     

Neuron-specific PGP9.5 expression in rat hair follicle development and cycle

Protein gene product PGP9.5 is a neuron-specific ubiquitin C-terminal hydrolase. We found that it also has immunoreactivity in the hair follicle of the Wistar rat dorsal skin and its expression patterns change with the development and cycle. During the morphogenesis, the PGP9.5 was expressed in the hair germ and hair peg elongated from the epidermis, and became restricted in the outer root sheath as the development progressed. In catagen, however, the PGP9.5 was detected in the tailing epithelial strand of the regressing proximal follicle epithelium, and in the keratinocytes directly contacted with the club hair, but rarely in the outer root sheath. With the beginning of the anagen of the second hair follicle, the PGP9.5 was again expressed in the second hair germ, and in the keratinocytes surrounding the remaining club hair and of distal follicle of the first hair. These findings showed that PGP9.5 is not specific to the neuron but is also involved in the hair follicle, and should provide new insight into the development and regression of the hair follicle.     

PGP 9.5 neuronal marker may differentiate immunohistochemically HIV-related from Mediterranean and immunosuppression-associated Kaposi’s sarcoma

Mediterranean Kaposi’s sarcoma (MKS), HIV-related KS (HIV-KS) and immunosuppression-associated KS (IS-KS), caused by human herpes virus 8 (HHV-8), share similar histological features. The aim of this study was to investigate differences in epidermal nerve fibers (ENFs) between the three KS types and controls. Skin biopsies from 23 HIV-KS, 16 MKS, 28 IS-KS patients and 18 controls, age-gender matched, were immunostained with PGP 9.5; ENFs in upper epidermal layer (EL) and penetrating the basement membrane were measured. The mean number of nerve fibers penetrating ENFs was significantly lower in HIV-KS (p < 0.001) compared to all other groups. MKS and IS-KS had comparable ENFs but lower than controls (p < 0.00 1). In the upper EL all groups had comparable ENFs and lower than controls. In conclusion, HIV-KS can be distinguished histologically from other types, by counting ENFs. Moreover, KS is associated with decreased ENFs, which may be a histological reflection of nerve damage. This is even more pronounced in HIV-KS patients and could be explained by a neurotoxic action of HHV-8, HIV, and their co-existence.     

Eosinophil cationic protein- and eosinophil-derived neurotoxin/eosinophil protein X-immunoreactive eosinophils in prurigo nodularis

Abstract It is known that eosinophils are actively involved in allergy and inflammation. The granular components of eosinophils, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin/eosinophil protein X (EDN/EPX), play an important role in such allergic and inflammatory processes. Prurigo nodularis is a chronic inflammatory skin disease with obvious cutaneous nervous involvement. To detect ECP and EDN/ EPX expression in the eosinophils and their relation to nerve fibres in prurigo nodularis, ECP and EDN/EPX single-labelling immunofluorescence, and ECP and PGP 9.5 double-labelling immunofluorescence, were performed. In prurigo nodularis lesional skin, the ECP- and EDN/EPX-containing cells, which were mainly distributed in the upper dermis, were significantly increased in number compared to their numbers in uninvolved and normal skin. The immunoreactivity of ECP and EDN/EPX in prurigo lesional skin was stronger than in uninvolved skin or control skin. The PGP 9.5-immunoreactive nerves were also increased in number in the areas where there were increased eosinophils. The nerves were in close proximity to eosinophils, and occasionally even seemed to be in contact. The present results indicate that the cutaneous nerves and the ECP- and EDN/EPX-containing eosinophils are possibly involved in the pathogenesis of the disease. The close relationship of nerves and eosinophils indicates that the cutaneous nerves may influence eosinophil function in the chronic inflammatory states of prurigo nodularis. ECP and EDN/EPX could thus be released to the local tissue and modulate the inflammation of the prurigo nodularis lesion. Received: 28 August 1999 / Received: 2 January 2000 / Accepted: 20 March 2000     

Altered cutaneous innervation in psoriatic skin as revealed by PGP 9.5 immunohistochemistry

Summary There is conflicting evidence in the literature as to whether cutaneous nerves are altered in psoriasis or not. In this study, antibodies to protein gene product (PGP) 9.5 were used to visualize cutaneous nerves in biopsies from involved and uninvolved skin of nine patients with psoriasis and from normal skin of eight healthy controls. A profound reduction in the epidermal nerve fibre density was observed in the involved psoriatic skin. These intraepidermal nerve fibres were also mostly short and found in the basal layer. Only a few nerve fibres were found in the suprabasal layer and they were non-varicose, long fibres going straight up without branching. In the uninvolved skin of psoriatic patients, the distribution and number of the intraepidermal nerve fibres was similar to that observed in normal skin. In the dermis, the distribution and the number of the nerve fibres showed no differences between involved psoriatic skin, uninvolved psoriatic skin, and normal skin. The results support previous studies in which alterations of cutaneous nerves in psoriasis have been described.     

Middermal elastolysis. Report of a case and immunohistochemical studies on the dermal distribution of fibrillin, vitronectin and amyloid P component.

A 39-year-old woman with demarcated wrinkled areas, histologically characterized by absence of elastic fibers in the middle and upper reticular dermis, is described. Immunoreactivity of vitronectin and amyloid P component, present at the periphery of elastic fibers in normal skin in adults, was absent from the middermis of lesional skin as were orcein stained fibers. C9 neoantigen immunoreactivity, associated with elastic fibers in sun-exposed skin of middle-aged and elderly individuals, was present in conjunction with elastic fibers in papillary and lower reticular dermis in lesional skin but was absent in the middermis. In contrast, a fibrillin immunoreactive network was present throughout the dermis, indicating that the elastin-associated microfibrils are retained in the absence of amorphous elastin in lesional skin of middermal elastolysis.     

Probable Mechanisms of Loss of Merkel Cells in Completely Depigmented Skin of Stable Vitiligo

Vitiligo is frequently associated with segmental involvement. It spares paralyzed limbs in transverse myelitis. There is spontaneous repigmentation of vitiligo lesional skin (VL) in diabetic neuropathy. Increased neuropeptide in VL and adjacent normal skin of vitiligo (ANS) along with thickened nerve fibers showing ultrastructural abnormalities all indicate a neural pathogenesis (1). Loss of Merkel cells has been observed in early vitiligo lesional skin (2). Recently, catecholamines have been found to play a major role in initiating the cascade of events leading to loss of melanocytes (1, 3, 4). To investigate the presence of Merkel cells in the completely depigmented skin of stable vitiligo (SV), a study was undertaken using TROMA 1, a monoclonal antibody specific for adult Merkel cells. No TROMA 1-positive cells were observed in SV, whereas normal numbers of these cells were seen in ANS. This new finding further supports the hypothesis of neural involvement in vitiligo.     

Role of In Vivo Reflectance Confocal Microscopy in Determining Stability in Vitiligo: A Preliminary Study

Background:

Vitiligo is an acquired pigmentary disorder. In vivo reflectance confocal microscopy (RCM) reproducible imaging technique has already been reported to be useful in the diagnosis of other skin diseases.

Objective:

To define RCM features of vitiligo on different clinical stages.

Materials and Methods:

A total of 125 patients with a clinical diagnosis of vitiligo were included in this study. After informed consent, lesional skins of those vitiligo patients were characterized by using RCM. Five patients with inflammatory cell infiltration observed at the edge of skin lesions and another 5 patients without inflammatory cell infiltration were selected. Biopsies were performed at same sites of the RCM examination areas for histological and immune-histological analysis.

Results:

In the active stage of vitiligo, the RCM examination revealed that the bright dermal papillary rings presented at the dermoepidermal junction level in normal skin lost their integrity or totally disappeared, border between vitiligo lesion and normal skin became unclear, and highly refractile cells that referred to infiltrated inflammatory cells could be seen within the papillary dermis at the edge of the lesions. In the stable stage of vitiligo, the RCM showed a complete loss of melanin in lesional skin and a clear border between lesional and normal skin.

Conclusion:

A simple clinical examination with RCM may reliably and efficiently allow evaluation of the stability status of vitiligo lesions.