DOI: 10.1111/j.1468-3083.2010.03633.

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ORIGINAL ARTICLE

Characterization and evaluation of pigment distribution and response to therapy in melasma using in vivo reectance confocal microscopy: a preliminary study
M Ardigo,,* N Cameli, E Berardesca, S Gonzalez

San Gallicano Dermatological Institute, Rome, Italy Memorial Sloan-Kettering Cancer Center, New York, NY, USA *Correspondence: M Ardigo. E-mail: ardigo@ifo.it

Abstract
Background Melasma is a frequent skin disorder characterized by the appearance of abnormal pigment (melanin) deposits in different layers of the skin. Melasma has been classied into epidermal, dermal and mixed types using Woods lamp, and the type and extent of the pigment deposits determine the type and invasiveness of the treatment. Aims The aims of this study were to carry out a preliminary evaluation of the effective usefulness of reectance confocal microscopy (RCM) in pigment distribution denition and subsequent re-classication of melasma types. Moreover, RCM therapeutical follow-up efciency to combination therapy with pyruvic acid and hydroquinone was also tested. Materials and methods A small group (n = 15) of patients previously diagnosed with facial melasma were selected and their pigment distribution was evaluated by RCM. In seven of these patients therapeutical follow-up was performed. Results The results of the study suggest that RCM is more accurate than techniques previously used in the diagnosis of melasma, thus providing precise information on the location and extent of pigment deposits. Discussion and conclusion The non-invasive nature of this technique suggests that RCM may be a suitable tool for treatment monitoring, providing additional information not only on the evolution of the disorder but also on the possible occurrence of therapeutical side or adverse effects. Received: 26 August 2009; Accepted: 29 January 2010

Keywords
melanin, melasma, pigment, reectance confocal microscopy, therapy

Introduction
Melasma is a common multi-factorial skin disorder characterized by hyperpigmentation of the skin. Whereas it affects both genders, it exhibits stronger prevalence in women of all ethnicities, particularly Oriental, Hispanic, Arab and North African. Melasma is more frequent during pregnancy and in women between 20 and 50 years of age.1,2 Melasma can be caused or aggravated by prolonged sun exposure, as well as high levels of sexual hormones during pregnancy or extensive use of birth control pills.36 Other risk factors include use of certain drugs, such as tetracycline or antimalarial drugs.7,8 Abnormal melanocyte activation caused by a combination of the stimuli referred above [e.g. combination of hormones and ultraviolet (UV) light] induces an abnormal aggregation and distribution of melanosomes in the keratinocytes of the junctional and spinous layers, accumulation of pigment in the dermis caused by incontinentia pigmenti, and phagocytosis by dermal macrophages, constituting the major clinical traits in the diagnosis of melasma.1,9 Treatment of melasma requires an accurate clinical evaluation, taking into consideration the anatomical location of the affected

area, skin phototype and age. This information must be crosscorrelated with complete medical records including duration of disease, pattern of onset, as well as the identication of possible aggravating factors. Only when the previous data have been adequately analysed, the severity of the disease can be assessed, guiding the selection of the most effective therapy, which ranges from active principles targeting melanogenesis to the physical removal of melanin deposits within the epidermis and dermis.10,11 The leading factor in the determination of the prognosis and therapeutic approach is the identication of the location and extent of the abnormal deposits of pigment. Use of the Woods lamp has allowed classifying melasma into three main sub-types: epidermal (augmented pigmentation under Woods lamp); dermal (lack of obvious increase in pigmentation under UV light and presence of melanin and melanophages widespread in the dermis) and mixed type (which can be considered as a mosaic of the two aforementioned types).2,12 In vivo reectance confocal microscopy (RCM) is a relatively novel technique that allows real time, non-invasive imaging of the supercial layers of the skin, interpreting the light reectance

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indexes of the different skin structures.1316 This technique has been used for the evaluation of several inammatory, neoplastic and melanocytic skin conditions and may constitute an excellent alternative to invasive biopsy collection in the diagnosis of several skin disorders.1721 In RCM, efcient detection depends on the presence of intrinsic microstructure-specic contrast caused by differential composition of the tissues. Contrast is mainly generated by keratin, melanin, haemoglobin and cellular organelles. Melanin is the best endogenous contrast agent in pigmented skin and melanocytes because of its high refractivity, which causes strong backscattering, allowing a precise identication of melanocytes, pigmented keratinocytes and melanophages.1316 In this regard, RCM can be used as non-invasive histological tool to localize precisely abnormal deposits of pigment, providing additional information to characterize the different sub-types of melasma. In addition, RCM constitutes a valid tool for management and monitoring of the evolution of the disorder and response to therapy. In this study, we report the pilot use of RCM in the diagnosis of melasma in a group of 15 patients. RCM was used to assess the sub-type of melasma in the patient group and also to perform a preliminary evaluation of the response to therapy in seven of 15 cases.

patients; skin phototype IV: six patients; skin phototype V: two patients; skin phototype VI: one patient. Seven of the 15 subjects recruited for the study also volunteered for treatment follow-up using RCM. The relevant features of these patients are shown in Table 1. Ten healthy volunteers with an even distribution of skin phototypes showing no trait of melasma were recruited as control group to obtain information about pigment distribution in normal skin in the same anatomical site. The research protocol was submitted to and approved by the Ethics committee of the San Gallicano Dermatological Institute of Rome.
Protocol design, study and clinical evaluation

Patients and methods

Patients

Clinical examination, photographic documentation and incorporation into medical records were performed in all the cases. In six patients, Woods lamp classication was also performed (Table 2). In melasma patients, we selected a target hyperpigmented skin area located on the cheek for RCM examination. Control patients were also subjected to RCM in comparable areas of the cheek to evaluate the distribution of pigment in relation with each skin phototype. Selection of control group composed by subject of different phototype was performed to rule out anatomical bias affecting the evaluation of real pigment level deposition. Based on pigment location and assessment of the severity of the disorder, 7 of 15 patients were selected for treatment and followup evaluation by RCM (Table 3).
Reectance confocal microscopy imaging

Fifteen patients, ages ranging 2862 years (average = 43 years), exhibiting clinical evidence of melasma for at least 5 months prior to commencement of the study, were recruited from the Outpatient Services at San Gallicano Dermatological Institute of Rome. Patients signed informed consent prior to their inclusion in the study. The distribution of skin phototypes, according to the Fitzpatrick denition,22 was as follows: skin phototypes IIII: six

The RCM microscope employed was a Vivascope 1500 (Lucid Technologies, Rochester, New York, NY, USA), which is commercially available for clinical in vivo imaging. The system operates with a diode Class 3A Laser (European version) with a peak of emission at 830 nm and under 35 mW of power at tissue level. A 30 0.9 NA water immersion objective lens was used. The RCM

Table 1 Relevant clinical parameters of the patients included in the study

Patient 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12 13. 14. 15. Gender F F F F F F F F F F F F F F F Age 32 33 34 56 45 54 52 28 44 34 62 39 42 51 44 Skin phototype III IV III IV IV V III IV II III VI III IV IV V Diagnosis ethiology Melasma idiopathic Melasma pregnancy Melasma pregnancy Melasma pregnancy Melasma pregnancy Melasma idiopathic Melasma idiopathic Melasma hormonal Melasma idiopathic Melasma pregnancy Melasma idiopathic Melasma pregnancy Melasma hormonal Melasma idiopathic Melasma hormonal Location Face Face Face Cheeks Face Cheeks Face Face Face Cheeks Face Face Cheeks Face Face Time (months) 8 5 12 24 7 13 6 9 25 21 9 6 12 12 8

Face: typical skin site (forehead, upper lip, cheeks).

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Table 2 Semi-quantitative assessment of the degree of pigment in the different skin layers in melasma patients and Woods lamp classication
Patient Woods lamp classication N.D. Dermal type N.D. N.D. Epidermal type N.D. N.D. N.D. Epidermal type Mixed type Epidermal type Dermal type N.D. N.D N.D. Pigment determination (brightness) Epidermis DEJ +++ +++ +++ ++ +++ ++ +++ +++ +++ ++ +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ ++ +++ ++ +++ +++ +++ +++ +++ ++

Confocal scoring

Dermis + ++ ++ + +++ + ++ ++ +++ + +++ ++ ++ + ++

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Three different skin compartments [epidermis, dermoepidermal junction (DEJ) and dermis] were scored using a qualitative scale of pigmentation, including the following categories: absent ()) low (+), medium (++) or high (+++). Evaluation of pigment intensity in melasma patients was referred to the control group, in which RCM images were collected from equivalent areas of normal skin in the cheek.
Melasma treatment follow-up

DEJ, dermoepidermal junction; N.D., not determined. Degree of pigment: ), absent; +, low; ++, medium; +++, high.

Using RCM to assess the precise location of the abnormal deposits of pigment and the severity of the disorder, seven patients were selected to undergo to treatment. Therapy consisted of six cycles of skin peeling with 50% piruvic acid every day for 2 weeks, followed by topical application of Kligmans formula containing 2% hydroquinone, applied daily for a total of 5 months of treatment.2326 The efcacy of the treatment was evaluated using semiquantitative assessment by RCM performed at the beginning of the treatment, and as indicated for the duration of the treatment (Table 3). The anatomical site investigated with RCM during the follow-up was identied by photographic reference.
Statistical analysis

was attached to the skin patient using an adhesive ring to reduce motion artefacts during examination. For the interfaces, Crodamol STS (IVN Nettetal, D-41334 Nettetal) was used between the adhesive window and the skin and ultrasound gel between the adhesive window and the objective lens. This system has been described elsewhere.14 Mosaic images of the spinous layer were rst collected and used to select the most refractile area of the epidermis. In the selected area, single images (0.5 by 0.5 mm) from the different layers of the skin (from the cornied, outer layer to the papillary dermis) were collected and used for descriptive analysis. Skin areas presented RCM changes compatible with lentigines as the presence of increased density and diameter of dermal papillae (DP) were excluded from the evaluation.

Statistical analysis of the semi-quantitative estimation of pigmentation at the different skin layers was performed using the Wilcoxon matched pair test (signicance P < 0.06) (Table 3).

Results
RCM features of control group

Table 3 Comparison between the semi-quantication of pigment amount before and after treatment in the ve cases treated for 5 months. Two patients were lost to follow-up
Before treatment Epidermis DEJ Dermis Case 1 Case 3 Case 6 Case 13 Case 15 +++ +++ ++ +++ +++ +++ +++ +++ +++ ++ + ++ + ++ ++ After treatment Epidermis DEJ ++ ) + ) + + + + ++ + Dermis + + + ) +

Reectance confocal microscopy evaluation of the control group cheeks skin disclosed the presence of different distribution and semi-quantication of pigment according to the different phototypes. In particular, presence of rare, bright keratinocytes (+, ++) was visualized in stratum spinosum only in higher phototypes (IV and V). Brightness of papillary rims, seen as highly refractile rims around adnexal structures at the level of the DEJ, was semi-quantied from + to +++ according to increasingly phototypes. Dermal pigmentation was rarely seen and is generally present only in phototypes V and VI.
RCM features of melasma

DEJ, dermoepidermal junction. Wilcoxon matched pair test (signicant: P < 0.06): Epidermis P < 0.007; DEJ P < 0.0079; dermis P < 0.095.

The presence of pigment located at all the different levels of the skin (epidermis, DEJ, upper dermis) of melasma patients was rst considered. Then, a semi-quantitative analysis using RCM was also performed (Table 2). At the level of the epidermis, we observed elevated degree of pigment seen as highly refractile keratinocytes with very prominent nuclei distributed within the spinous layer (Fig. 1a). At the DEJ, presence of strongly visible papillary rings around the DP composed by sequence of brighter cellular structures was seen (Fig. 1b), corresponding to activated melanocytes and junctional keratinocytes receiving packed

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(a)

(d)

(b)

(e)

(c)

(f)

Figure 1 Initial employment of reectance confocal microscopy (RCM) imaging to visualize the epidermis, dermoepidermal junction (DEJ) and dermis of control vs. melasma patients. Representative RCM images of the pigment distribution in different layers of the skin (a, d: epidermis; b, e: DEJ; c, f: upper dermis) in a skin photo-type III patient diagnosed with melasma (ac), or a skin photo-type III control patient (df ). Arrows in (c) indicate the presence of melanophages.

melanosomes. Moreover, abnormal presence in the supercial dermis of fuzzy round or polygonal refractile structures within dermal collagen bundles was also seen. These structures were consistent with melanophages lled with melanin originated in the DEJ (Fig. 1c). We also observed an increase in pigmented keratinocytes in epidermis (Figs 2,3). Such increases were catalogued as medium

(++) or high (+++) in 4 15 (26.6%) and 11 15 (73.3%) of the cases respectively. In 5 15 cases (33.3%), we found evidence of dendritic, highly refractile cellular structures in the stratum spinosum (Fig. 2); these cells could be interpreted as activated Lengerhans cells more than melanocytes because of their monomorphic, multipolar dendritic shape and because of their location above the DEJ, at the level of the spinous layer. Supporting this

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(a)

(b)

(c)

(d)

Figure 2 Identication of melasma traits in a patient using reectance confocal microscopy (RCM). Clinical picture (a) and corresponding RCM images of the epidermis, (b) dermoepidermal junction (DEJ) (c) and dermis (d) of a representative melasma patient. (b) Exhibits numerous dendritic cells in the spinous layer.

interpretation, activated, dendritic melanocytes in sun-exposed or in UV-treated skin areas, as well as in non-malignant tumoral lesions have been previously described on RCM to be located exclusively at the level of the DEJ in sun-exposed site.1320 At the DEJ, highly (+++) refractile papillary rings were seen in most patients (Figs 2,3); only 3 15 cases (20%) exhibited medium (++) levels of DEJ pigmentation. Presence of melanophages in the upper dermis was extremely variable (Figs 2,3). In 5 15 (33.3%) cases, RCM revealed sparse dermal pigmentation (+); 7 15 (46%) patients showed medium degree (++) of presence of dermal melanophages, whereas the remaining three cases (20%) exhibited high levels (+++) of dermal pigmentation. Surprisingly, we found no correlation between the classication of melasma using UV light (Woods lamp) and the prevalent distribution of pigments as assessed by RCM (Table 2).
Monitoring the response to melasma treatment using RCM

After the rst RCM evaluation, melasma therapy was followed in seven patients using RCM. Patients were examined twice, once

after 2 months, and once at the end of the treatment (5 months). Two patients discontinued therapy because of adverse reactions to the treatment: one patient developed excessive irritation after the rst peeling using piruvic acid; the other discontinued treatment after the second peeling procedure. The remaining ve patients nished the treatment protocol (Table 2). Reectance confocal microscopy assessment of the response to treatment revealed a statistically signicant decrease in brightness, mainly at the epidermis and DEJ. The Wilcoxon matched pair test returned P values of P < 0.007 (epidermis) and P < 0.0079 (DEJ). In particular, in 2 5 cases, RCM revealed a major reduction in pigmented keratinocytes in the epidermis, as well as a signicant reduction in brightness around DP. Such reduction was noticeable in the rst clinical follow-up visit, at the end of the six topical applications of piruvic acid (Fig. 3). We also observed complete disappearance of bright large keratinocytes in the upper epidermal layer and normal brightness of the papillary rings in 2 5 patients; the other three patients still showed small traces of pigment, but a signicant improvement nonetheless compared with the initial assessment by RCM. The

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(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

(k)

(l)

Figure 3 Reectance confocal microscopy (RCM)-based assessment of the response of melasma patients to piruvic acid hydroquinone treatment. Clinical pictures (a, e, i) and corresponding RCM images of the epidermis (b, f, l), dermoepidermal junction (DEJ) (c, g, m) and dermis (d, h, n) of a melasma patient before treatment (ad), or treated with piruvic acid (peeling) followed by topical hydroquinone 2% after 2 months (eh) and 5 months (in). A progressive reduction in brightness (i.e. pigmentation) in all the skin layers was observed; some features of inammation were also detected, including overall redness (i), dilated blood vessels (n, red arrows) and perivascular inammatory cells (n, yellow arrows) after 5 months of treatment.

effect of the treatment on dermal pigmentation was less appreciable; we founded only a partial, non-signicant (P < 0.095) reduction in the number of bright polygonal structures in the upper dermis corresponding to melanophages after conclusion of the treatment. Finally, three of the ve remaining patients receiving treatment exhibited marked signs of inammation, including vasodilatation in the upper dermis associated with dermal inltration of bright, round cells corresponding to inammatory cells (Fig. 3). These microscopic features were consistent with our ndings through clinical examination, which revealed mildly erythematous areas on the areas of the skin subjected to treatment.

Discussion
Reectance confocal microscopy is a non-invasive tool for in vivo microscopic analysis of the skin, mostly well-suited for the evalua-

tion of pigment-related disorders, such as hyperpigmentation. Recent advances in imaging acquisition and collection have boosted the quality of RCM-based diagnosis; its resolution is now close to conventional histology when assessing normal tissue and superior when structural identication is based on observation of endogenous pigments.16,20 Previous characterization of melasma using UV illumination (Woods lamp) has classied melasma into epidermal, dermal and mixed sub-types.2,9,12 This technique is not microscopic, but based on the quantication of the different levels of uorescence according to the depth of the pigment, thus being limited by its singlevalued nature, which does not take into account the contributions of different layers. It also neglects the effect of the different skin phototypes on the refraction of UV light. Reectance confocal microscopy permits a more exible categorization, as it allows the identication and semi-quantifying

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of the amount of pigment in every layer of the skin (stratum spinosum, DEJ and upper dermis), classifying every melasma into a single microscopic mixed phenotype, with a greater epidermal, DEJ or dermal component. These ndings are also supported by the physiopathology of the disease itself, which has revealed that excess pigment is produced by activated melanocytes at the DEJ and distributed to junctional and epidermal keratinocytes; later, pigment drops down into the dermis. Concomitant inammation results in elevated numbers of macrophages in the upper dermis, which phagocyte the pigment produced in the DEJ. The differences between the Woods lamp classication and RCM are best illustrated in one of the cases of this study; case 5 (see Table 2) was classied as an epidermal melasma using the Woods lamp. However, RCM revealed high levels of pigment at every layer of the skin analysed. The opposite was also evident in our study, e.g. case 12 classied as dermal melasma according to the Woods lamp test, but showing prominent amounts of pigment mainly in the epidermis and at the DEJ and less in the dermis when observed with RCM (Table 2). In addition to melasma, RCM revealed microscopic features consistent with sun-induced photodamage in 10 15 patients (66.6%). These features included irregularly shaped DP corresponding to elongated rete ridge (marked undulations of the DEJ), thicker collagen and elastic bres in the upper dermis, observed through RCM as thick, bright linear, dermal structures, and increased number of bright dendritic, Langerhans cells above the DEJ in the spinous layer. The presence of Langerhans cells in photo-damaged skin has been previously reported using optical microscopy.20 The appearance of evidence of photodamage in this study seemed not to correlate with the age of the patients (data not shown). Reectance confocal microscopy-based follow-up examination of patients undergoing treatment revealed a more signicant effect in the outer layers of the skin (epidermis and DEJ), being non-signicant in the dermis. The rst part of the treatment, piruvic acid peelings, exerted a marked reduction in pigment in the epidermis; a similar effect at the DEJ was only noticeable after several applications of hydroquinone-containing ointment, which inhibits melanin synthesis. It is reasonable to speculate that the lack of effect of the treatment in the dermis is because the pigment found in the dermis is originated at the level of DEJ and captured by dermal macrophages; the turnover of pigment elimination in dermal macrophages is slow and insensitive to inhibition of synthesis as dermal macrophages do not produce pigment. Finally, RCM also revealed that treatment with hydroquinone caused the appearance of features consistent with local inammation, including vasodilatation and presence of perivascular inammatory cells in the upper dermis. A previous study has shown the feasibility of visualizing blood ow and inltration of immune cells by RCM.27 The capability of revealing signs of adverse effects

such as irritation is a very valuable asset to prevent and manage the risk of post-inammatory pigmentation reactions caused by the use of hydroquinone, which has a well-characterized pro-inammatory effect.23 In summary, this preliminary study reveals important advantages in the use of RCM for the non-invasive diagnosis, classication, prognosis and management of melasma. Employment of RCM renders the previous categorization of melasma into epidermal and dermal rather obsolete, revealing that every case of melasma is truly a mixed type with different degrees of epidermal or dermal presence of the pigment. Furthermore, RCM bears promise for the non-invasive follow-up of the disorder and to monitor the response to therapy. Further studies will establish the actual capability of RCM for the diagnosis, characterization and monitoring of multiple pigmented skin disorders.

Acknowledgements
The authors thank Dr Miguel Vicente-Manzanares for editorial assistance in the preparation of the manuscript. The authors did not receive any funding for writing the manuscript and publication of this study.

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