In‐depth study of Wood's lamp examination combined with reflective confocal laser scanning microscopy for the guidance of vitiligo staging and treatment

Both Wood's lamp and reflective confidential laser scanning microcopy are helpful for the diagnosis and treatment of vitiligo. However, there is few research that contains large samples and consistent observations.


| INTRODUC TI ON
Vitiligo is a common skin disease in which the mucous membranes lose pigmentation, which seriously affects the physical and mental health of affected patients, particularly when the vitiligo lesions are located in exposed areas.The skin lesions appear as limited or generalized white patches of varying forms with clear or unclear borders, often without obvious conscious symptoms.Vitiligo is a multicausal disease associated with autoimmune, neuropsychiatric, and chemical (certain phenols and catechol) stimulation. 1The incidence of vitiligo is often not related to age of onset or sex.Although vitiligo is hereditary, the genetic mechanism is not yet fully understood. 2The onset of concomitant leukodystrophy and vitiligo is associated with various systemic diseases. 3Current vitiligo treatment 4 is based on medications (hormones, calcium phosphatase inhibitors, topical corticosteroids), laser therapy, and transplantation, 5 and the treatment varies at different stages of development.Currently, the diagnostic tools for vitiligo are reflective confocal laser scanning microscopy (RCM), Wood's lamp examination, and dermatoscopy, 6 which are used together to reduce misdiagnosis.RCM has become the main diagnostic tool for vitiligo due to its noninvasive and real-time features and proven consistency with histopathological findings. 7The aim of the present study was to investigate the correlation between RCM and Wood's lamp findings in the diagnosis of vitiligo and guidance of individualized treatment.

| MATERIAL S AND ME THODS
The study cohort comprised 215 patients with vitiligo who attended the outpatient clinic of Wuhan First Hospital from October 2021 to June 2022.The study cohort included patients from all over China, comprising 110 males and 105 females aged from 3 months to 65 years old.The average vitiligo disease activity (VIDA) score was 2.85; there were 60 patients with a VIDA score of −1 to 0, and 155 patients with a VIDA score of 1-4.The numbers of lesions identified as progressive or stable based on Wood's lamp examination and RCM are described below.(3) age ≤65 years; (4) voluntary study participation and provision of written informed consent.

| General data of 215 patients (215 lesions) who met the eligibility criteria were analyzed
The patient's name, sex, and age, lesion site, area, stage, and type, VIDA 9 score, medical history, family history, concomitant symptoms (e.g., pruritus, halo nevus, white hair), and isomorphic reaction were recorded.Photographs of the lesions were taken under natural light to assess the color and border clarity of the lesions under the naked eye.

| Wood's lamp imaging
The Wood's lamp was preheated for 1 min before use.The lesions were examined in a dark room with the Wood's lamp parallel to the skin lesion.A clear bright blue-white picture of the skin lesion appeared in the display and was photographed, and the boundary of the skin lesion was outlined.A random point on the border of the lesion was selected, along with points at 0.75 cm inside and outside the lesion, giving a total of three detection points for each lesion: one within the lesion, one at the lesion border, and one on the neighboring normal skin.

| RCM imaging
The patient was lying down with the tissue ring fixed with a paste window at the selected site.Distilled water was used as the medium between the adhesive ring and the skin, and medical ultrasound coupling agent was used as the medium between the lens and the adhesive ring to ensure that the probe was close to the skin and perpendicular to the skin surface; scanning was started after fixing the position.The clinician first determined the zero point, then scanned the cross-section downward in a layer-by-layer manner to the basal layer (true epidermal junction) to select and save the best images.Sixteen consecutive images were acquired.The scanning observation area was 500 × 500 μm, and the scanning range was 3 × 3 mm (in the XY horizontal direction).The longitudinal scan was from the stratum corneum to the true epidermal junction with a thickness of 5 μm per layer; the horizontal scan comprised images of the pathological changes at the basal layer and true epidermal junction.The same three detection points examined under the Wood's lamp were examined with RCM.

| Statistical methods
Measurement data are expressed as mean ± standard deviation.The t-test for two independent samples was used for measurement data, while the chi-squared test was used to compare measurement data between two groups, with a significance level of α = 0.05.Kendall's tau-b correlation analysis was used for correlation studies, with a significance level of α = 0.01.All data were analyzed using SPSS 26.0 software.

| Lesion staging
The staging was based on the VIDA score 9,10 that combines the clinical features, Wood's lamp features, and isomorphic reac- Lesions that were porcelain white with clear borders under the Wood's lamp and were categorized as grade 1 lesions were considered to be at a stable stage.The lesion was considered to be in the slowly progressive stage if it was an off-white color with clear borders under the Wood's lamp or was categorized as grade 2. Rapidly progressive lesions were defined as trichromatic, confetti-like (Figure 4), hypopigmented lesions with blurred margins, and inflammatory signs that were categorized as grade 3.
Reflectance confocal microscopy was used to observe the pigment distribution, pigment ring integrity, presence of inflammatory cells (lymphocytes and melanophages), and melanocyte regeneration.The following indexes were recorded. 14

| Analysis of lesions under the naked eye and Wood's lamp
The characteristics of 215 vitiligo lesions under the naked eye and Wood's lamp are shown in Table 1.The lesion color and boundary definition were significantly different under the naked eye versus under the Wood's lamp (p < 0.05).

| Comparison of RCM findings, Wood's lamp findings, and staging determined by expert consensus
Group I was staged based on a comprehensive assessment of the clinical features, VIDA score, Wood's lamp findings, and isomorphic response.Group II was staged based on the RCM findings.Group III was staged based on the Wood's lamp findings.As shown in Table 2, the staging did not significantly differ between group II (RCM findings) and group I (expert consensus) (χ 2 = 3.63, p > 0.05) or between group I and group III (χ 2 = 3.60, p = 0.05).No significant differences were observed between the three staging methods in recognizing the stable and progressive stages.RCM seemed to be more sensitive than the Wood's lamp in performing disease staging, although this difference between methods was not statistically analyzed.

| Confetti-like and trichrome lesions
The Wood's lamp examination revealed 15 confetti-like lesions.The difference between the three staging methods was not significant (χ 2 = 2.25, p = 2.25, Fisher's exact test).Seven lesions appeared confetti-like under the naked eye, of which three were in the slowly progressive stage and four were in the rapidly progressive stage.
When the confetti-like lesions were visible to the naked eye, the disease was in the progressive stage; when they were not visible to the naked eye but were visible with the Wood's lamp, the disease was in the rapidly progressive stage.
The lesions that were trichromatic under the naked eye were also trichromatic under the Wood's lamp and were all in the rapidly progressive stage.Certain lesions that are not easily observed with the naked eye can be visualized with the help of the Wood's lamp, which can help in selecting the appropriate treatment.Relying solely on visual observation to determine the stage of vitiligo development was inaccurate, and aided diagnosis with RCM and Wood's lamp examination may substantially improve the diagnostic accuracy.

| RCM image analysis (Figure 5)
In rapidly progressive lesions, there were large numbers of melanocytes and lymphocytes at the lesion border and at the three detection points, with more inflammatory cells at the border and fewer inflammatory cells at 0.75 cm inside the lesion.In slowly progressive lesions, there was lymphocytic infiltration at the border or a small number of phagocytic melanocytes at 0.75 cm outside the border.
Lesions in the stable and recovery stages generally had less inflammatory cell infiltration than those in the progressive stages.When the lesion boundary was blurred under the Wood's lamp, it was also difficult to find a clear border under RCM owing to extensive inflammatory infiltration.

| Lesion grade and disease stage (Table 3)
Among grade 3 lesions under the Wood's lamp and the naked eye, RCM revealed that the inflammatory cells were in a highly active state, and treatment was required to suppress further expansion of lesions as soon as possible.The grade 2 lesions had the possibility of further expansion, while the grade 1 lesions were in a relatively stable state.Kendall's tau-b correlation analysis showed that the three lesion grades were associated with the disease development, and their staging had a linear correlation (p < 0.01).

TA B L E 1
Comparison of vitiligo lesion characteristics under the naked eye and Wood's lamp.

| DISCUSS ION
In clinical practice, relying only on visual observation of vitiligo lesions to judge the stage of the disease and guide treatment easily results in misjudgment, which also affects the efficacy of treatment.Previous studies have mostly used visual observation and patient recall for the diagnosis of vitiligo, 16,17 which frequently causes misjudgment of lesions with atypical and inconspicuous clinical features.The treatment of vitiligo is based on the presence of Koebner's phenomenon, 18 hypopigmentation, trichromatosis, 19 and confetti-like depigmentation, as these features have long been thought to be associated with disease activity in vitiligo. 20Therefore, the presence of certain skin manifestations may indicate disease activity. 21The VIDA score is a recall-based patient-reported outcome measure that is considered unreliable but remains the most cited measurement tool by researchers.
However, the VIDA score is dependent on the patient's subjective perception, and the uncertainty of the disease course has Wood's lamp images to observe the features of vitiligo lesions to provide a diagnostic basis. 22Numerous studies have confirmed that the diagnosis of vitiligo using RCM is reliable and consistent with the histopathological findings. 21,23sions that appeared gray-white or trichromatic under the Wood's lamp were in the rapidly progressive stage; lesions with clear borders under the Wood's lamp needed further analysis by RCM for the stage to be determined; lesions with blurred borders under the Wood's lamp were in the rapidly progressive stage; lesions that were visible under the naked eye and under the Wood's lamp were in the rapidly progressive stage.
Vitiligo lesions are a bright bluish-white color under the Wood's lamp. 24Under RCM, epidermal protrusions encircle dermal papillae, forming bright rings ("pigment rings") located in the basal layer; progressive vitiligo lesions have incomplete loss of pigment rings.RCM also reveals highly refractive cells, including lymphocytes and melanocytes.The inflammatory cell infiltration is usually seen at the edge of a progressive lesion. 25RCM observation of progressive vitiligo lesions shows reduced pigmentation of the basal layer compared with the surrounding normal skin, the general presence of a basal cell ring, pigment deficiency in some of the basal rings, and infiltration of phagocytic melanocytes 26,27 and inflammatory cells in the dermal papillae and superficial layers.Inflammatory cell infiltration is usually seen at the edges of progressive lesions. 12,28RCM observation may predict the progression of the disease, and the presence of inflammatory cell infiltration at 0.75 cm beyond the lesion border may indicate that the lesions will progress and spread.Our determination of the extent of inflammatory cell infiltration at the site of the lesion may help guide clinical individualized and targeted treatment.
Studies have shown that when the boundary of the skin lesion is unclear under the Wood's lamp, the lesion is progressive. 26,29 the present study, we categorized the lesions into three grades based on observations under the Wood's lamp and the naked eye.
This grading method had a linear correlation with the developmental stage of the disease and is theoretically feasible.Studies have shown that covering most of the white area in the middle of the lesion and treating only the edges of the lesion and the surrounding skin has a treatment efficacy of 89.5%, 30 which is 67.6% higher than the efficacy of treating all areas of the lesion.Therefore, when performing topical drug treatment and laser treatment on vitiligo lesions, the treatment area of rapidly progressive and slowly progressive lesions should be extended outward by 0.75 cm according to RCM images. 31e area without inflammatory infiltration in the middle of the lesion is not treated with light and other treatments.The Wood's lamp and RCM have become important tools for vitiligo diagnosis.
The main limitation of the present study is the small sample size.
The findings require confirmation in a larger sample size.

( 1 )
vitiligo diagnosed in accordance with the "Diagnostic and treatment criteria for melasma and vitiligo (2010 version)" formulated by the Pigmentology Group of the Chinese Society of Integrative Medicine and Dermatology in 2010 8 ; (2) lesion diameter >1.5 cm;

( 1 )
poor compliance, loss to follow-up, or self-termination of treatment; (2) administration of systematic treatment, glucocorticoids, immunosuppressants, laser treatment, and other vitiligo treatments within the last 2 months; (3) serious heart, liver, kidney, or neurological disease; (4) photosensitive diseases or history of photosensitivity; (5) lesions in areas where the RCM probe cannot be placed, such as the eyelids and the perinasal area; (6) inability to receive drug treatment owing to special circumstances; (7) incomplete data collection that affected the judgment of treatment efficacy.

tions. 11 ,
12 VIDA score criteria: (1) new lesions or expansion of original lesions within the last 6 weeks (+4 points); (2) new lesions or expansion of original lesions in the last 3 months (+3 points);(3)   new lesions or expansion of original lesions in the last 6 months (+2 points); (4) new lesions or expansion of original lesions in the last 1 year (+1point); (5) stable for at least 1 year (0 points);(6)   stable for at least 1 year with spontaneous pigment regeneration (−1 point).Lesions that met any one of the following three criteria were defined as being in the progressive stage: (1) VIDA score >1; (2) inflammatory, trichromatic, confetti-like lesions, or hypopigmented patches with indistinct white margins; (3) appearance of an isomorphic reaction in the last 1 year.Lesions that met all the following criteria were defined as being in the stable stage: (1) VIDA score of 0; (2) appearance of porcelain white spots with clear borders or pigmentation and other clinical features; (3) no isomorphic reaction in the past 1 year; (4) white spot lesion with clear borders under Wood's lamp examination, and a smaller lesion area under the Wood's lamp than under the naked eye.In the present study, we observed the lesion color under the Wood's lamp, assessed the clarity of the border, 13 and compared the size of the lesion under the naked eye versus under the Wood's lamp.The following indexes were recorded.(1) Color under the Wood's lamp (Figure 1): tricolor, off-white, and porcelain white; (2) boundary status of the lesion (Figure 2): no border, blurred border, and clear border; (3) lesion size under the Wood's lamp and under the naked eye (Figure 3) assessed as grade 1 (lesion boundary the same under F I G U R E 1 Photographs of vitiligo lesions under the Wood's lamp.(A) Trichrome lesion; (B) off-white lesion; (C) porcelain white lesion.F I G U R E 2 Images showing vitiligo lesion borders under the Wood's lamp.(A) Lesion with clear borders; (B) lesion with indistinct borders.the Wood's lamp and the naked eye); grade 2 (maximum distance between the boundary under the Wood's lamp and the nearest two border points under the naked eye ≤0.75 cm); grade 3 (minimum distance between the boundary under the Wood's lamp and the nearest two border points under the naked eye >0.75 cm or the boundary point could not be found).
(1) Pigmentation status of the lesioned skin: a score of +1 indicated the presence of residual pigment, while −1 indicated the complete loss of pigment; (2) pigment ring integrity: a score of +1 indicated uneven distribution and reduction, while −1 indicated the complete absence of a pigment ring; (3) inflammatory cell infiltration: a score of +2 indicated that melanophagocyte infiltration was detected at the lesion edge; +1 indicated that lymphocytes were detected; −1 indicated that there were no inflammatory cells.A total score of <1 was considered to represent the stable phase, ≥1 represented the progressive phase, and ≥2 represented the rapidly progressive phase. 15 | RE SULTSThe characteristics of the RCM images were consistent with the characteristics of the Wood's lamp images.

F I G U R E 3
The upper row shows photographs of vitiligo lesions under natural light, while the lower row shows photographs of vitiligo lesions under the Wood's lamp.(A) Grade 3 lesion; (B) grade 2 lesion; (C) grade 1 lesion.F I G U R E 4 Confetti-like skin lesion.

F I G U R E 5 3
Reflective confocal laser scanning microscopy images.(A/B) Reduced pigmentation of the basal layer compared with the surrounding normal skin, the basal cell ring is largely present, and a large number of melanophagocytic and inflammatory cell infiltrates are visible around the dermal papillae and superficial blood vessels; (C) reduced pigmentation of the basal layer compared with the surrounding normal skin, the basal cell ring is largely present, and sparse melanophagocytic and inflammatory cell infiltrates are visible around the dermal papillae and superficial blood vessels; (D) reduced basal layer pigmentation compared with the surrounding normal skin, the basal cell ring is roughly present with uneven pigmentation distribution, and there is sparse inflammatory cell infiltration around the dermal papillae and superficial blood vessels; (E) reduced basal layer pigmentation compared with the surrounding normal skin, the basal cell ring is roughly present, and there is sparse inflammatory cell infiltration around the dermal papillae and superficial blood vessels; the basal rings shown in A and B are mostly seen at the border of rapidly progressive lesions, the basal rings shown in C and D are mostly seen at the border of slowly progressive lesions or normal skin around progressive lesions, and the basal ring in 5E is mostly seen within stable vitiligo lesions.Grading and staging of the lesions under the Wood's lamp and under the naked eye.a large impact on the accuracy of the VIDA score.Furthermore, the clinical findings are dependent on the physician's experience, and macroscopic features are often deceptive.Studies have not yet confirmed that the clinical features and Wood's lamp findings are consistent with the histopathological findings.In the present study, RCM images were used in combination with macroscopic

Group Total Three colors Off-white Porcelain white No boundary found Blurred boundaries Clear boundaries
Comparison of reflectance confocal microscopy, Wood's lamp, and vitiligo disease activity staging results.