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 Table of Contents  
Year : 2020  |  Volume : 7  |  Issue : 1  |  Page : 2-6

Evaluation of active and stable stages of vitiligo using S-100 and human melanoma black-45 immunostains

1 Department of Pathology, Government Medical College and Hospital, Chandigarh, India
2 Department of Cytology and Gynecological Pathology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Dermatology and Venereology, Government Medical College and Hospital, Chandigarh, India

Date of Submission11-Feb-2019
Date of Decision27-Oct-2019
Date of Acceptance10-Mar-2020
Date of Web Publication02-Jun-2020

Correspondence Address:
Reetu Kundu
Department of Cytology and Gynecological Pathology, Postgraduate Institute of Medical Education and Research, Sector 12-A, Chandigarh - 160 012
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijdpdd.ijdpdd_44_19

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Background: Vitiligo is the most prevalent pigmentary disorder occurring worldwide. In most cases, the diagnosis is made by clinical examination alone. The disease status (active/stable) needs to be assessed to make appropriate therapeutic decisions. Objective: The aim of the present study was to evaluate the histopathological and immunohistochemical features in active and stable lesions. Materials and Methods: Biopsies from vitiliginous areas from 50 patients (25 each of clinically active and stable vitiligo); with hematoxylin and eosin, Masson Fontana (MF), S-100, and human melanoma black-45 (HMB-45) stained sections were studied. Results: Age of the patients ranged from 7 to 69 years (mean age: 33.6 ± 15.73 years). Of 50 patients, 27 (54%) were male and 23 (46%) were female. All the cases showed variable degree of basal hypopigmentation. Histopathological findings, epidermal spongiosis, basal vacuolar degeneration, dermal melanophages, and dermal lymphomononuclear cells were commonly observed in active lesions as compared to the stable ones. On MF staining, 23/25 cases (92%) of active vitiligo showed complete loss of basal melanin. Quantitative analysis of HMB-45 immunostaining in stable and active vitiligo revealed mean number of positive melanocytes as 2.52 ± 1.0/high power field (hpf) and 0.08 ± 0.28/hpf, respectively, while on S-100 immunostaining the mean values of positive Langerhans cells were 1.70 ± 0.38/hpf and 7.78 ± 4.11/hpf, respectively. Conclusion: The demonstration of overall reduction in the number of HMB-45-positive melanocytes and increase in S-100-positive Langerhans dendritic cells in the active vitiligo lesions is facilitated by immunohistochemistry. The technique is of immense help in differentiating active and stable stages of vitiligo, thus guiding therapy.

Keywords: Active lesions, human melanoma black-45, immunohistochemistry, S-100, stable lesions, vitiligo

How to cite this article:
Kaur G, Punia RS, Kundu R, Thami GP. Evaluation of active and stable stages of vitiligo using S-100 and human melanoma black-45 immunostains. Indian J Dermatopathol Diagn Dermatol 2020;7:2-6

How to cite this URL:
Kaur G, Punia RS, Kundu R, Thami GP. Evaluation of active and stable stages of vitiligo using S-100 and human melanoma black-45 immunostains. Indian J Dermatopathol Diagn Dermatol [serial online] 2020 [cited 2021 Oct 17];7:2-6. Available from: https://www.ijdpdd.com/text.asp?2020/7/1/2/285800

  Introduction Top

Vitiligo is a common acquired chronic hypomelanotic skin disease characterized by the development of progressive depigmented macules resulting from the destruction or nonfunctioning of melanocytes in the epidermis.[1] A recent meta-analysis showed the pooled prevalence of vitiligo to be 0.2% and 1.8% among population/community-based studies and hospital-based studies, respectively.[2]

The histopathological findings in vitiligo vary according to the stage of the disease.[3] In the early stage, a few mononuclear cells infiltrate the dermoepidermal junction. The number of melanocytes, decreases and they finally disappear from vitiliginous skin as the disease progresses.[4] In established lesions, the affected skin appears normal apart from the absence of melanin pigment from the basal layer.[5] Melanin can be highlighted using Masson Fontana (MF) special stain or immunohistochemical markers.[4],[5] Vitiligo lesions at this stage have shown on immunohistochemistry that melanocytes are usually absent from the basal layer, although they may be present in reduced numbers and can show degenerative changes.[6]

Although no universal consensus exists on the optimum duration of nonprogression for the disease to be labeled as stable clinically, staging vitiligo lesions as active and stable remains crucial for making therapeutic decisions.[7] This distinction is facilitated by the use of immunohistochemical markers and there exists a paucity of studies on this from the Indian subcontinent. Therefore, the present study was planned to analyze the immunohistochemical findings in active and stable stages of vitiligo using S-100 and human melanoma black-45 (HMB-45) immunostains with calculation of relative ratios of melanocytes and dendritic cells in addition to the routine histomorphological assessment. The presence of dormant and activated melanocytes in active and stable lesions was also evaluated.

  Materials and Methods Top

The present cross-sectional study was conducted in the department of pathology, in a tertiary care hospital in collaboration with the department of dermatology. The study protocol was approved by the Institutional Ethics Committee and informed consent was obtained from all the patients. The study included 50 consecutive patients of vitiligo (25 each of active and stable disease) diagnosed clinically and confirmed on histopathology.

Clinical criteria used for the categorization of stages of vitiligo are as under the following:

  1. Active vitiligo – Recent development of new lesions, extension of the old lesions in size and shape, ill-defined borders, and presence of the Koebner phenomenon was taken as active stage
  2. Stable vitiligo – No development of new lesions during the past 6 months, lesions stationary in size during the past 6 months, hyperpigmented and well-defined borders and absence of the Koebner phenomenon was taken as stable vitiligo.

Skin biopsies

The biopsies optimally fixed in 10% neutral buffered formalin were processed routinely and embedded in paraffin blocks. Sections of 2–3 μm thickness were cut for hematoxylin and eosin (H and E) staining, MF staining, HMB-45, and S-100 immunostaining. Adequate positive and negative controls for special staining and immunohistochemistry were put. Section from hyperpigmented skin of nipple-areola of the breast was used as a positive control for MF and HMB-45. The positive control for S-100 was section from schwannoma. The negative control sections were where the primary antibody was omitted and substituted with buffer.

Histomorphological and Masson Fontana assessment

The assessment of H and E stained sections in all 50 cases was done on histopathology. Skin biopsies were evaluated for epidermal spongiosis, basal cell layer vacuolization, dermal melanophages, and dermal lymphocytic infiltrate. On MF staining, the sections were examined for the presence or absence of black-colored melanin pigment in the basal layer of the epidermis.

Immunohistochemical analysis

The epidermal cells showing cytoplasmic positivity for HMB-45 were taken positive. In the epidermis, the total number of HMB-45+ cells was counted using a large field (×40) objective and the average HMB-45+ cell count per high-power field was calculated and the results were interpreted as mean ± standard deviation The epidermal cells showing both nuclear and cytoplasmic positivity for anti-S-100 were taken as S-100 positive and quantified in a similar fashion.

Statistical analysis

The patient characteristics were described in two groups – active and stable. The distribution of the measurable data (HMB-45-positive melanocytes in epidermis, S-100-positive dendritic cells in epidermis, and the relative ratio of melanocytes to dendritic cells) was tested for its normality using Kolmogorov–Smirnov Test. The association of the categorical/classified data within the two groups was calculated using the Chi-square test.

  Results Top

Clinical profile

The age of the patients ranged from 7 to 69 years (mean age: 33.6 ± 15.73 years). Of 50 patients, 27 (54%) were male and 23 (46%) were female. Male-to-female ratio was 1.2:1. Of 25 patients with active vitiligo, 14 (56%) were male and 11 (44%) were female. Among 25 cases of stable, 52% were male and 48% were female. The lesions were located on lower limbs 16 (32%), upper limbs 16 (32%), abdomen 6 (12%), back 6 (12%), chest 4 (8%), forehead 1 (2%), and axilla 1 (2%).

Histopathological and Masson Fontana findings

All the cases of vitiligo showed variable degree of basal hypopigmentation, ranging from mild to moderate to marked, depending on the clinical stage of the disease. The various other histopathological findings included epidermal spongiosis, vacuolar degeneration of basal cell layer, dermal melanophages, and dermal lymphomononuclear cell infiltrate. All these features were commonly observed in active lesions as compared to stable ones. The differences between the two clinical stages were statistically significant (P = 0.000–0.003).

On MF staining, 23/25 (92%) cases of clinically active vitiligo showed complete loss of melanin pigment in the basal layer of the epidermis [Figure 1] with only 2 (8%) cases demonstrating the rare presence of melanin. On the other hand, all the 25 cases of stable vitiligo showed rare to normal presence of melanin pigment in the basal layer [Figure 2].
Figure 1: Active vitiligo. (a) Complete absence of melanin in basal cell layer with mild spongiosis (H and E, ×400). (b) The absence of melanin pigmentation on Masson Fontana (MF, ×400). Inset shows normal melanin pigment in the positive control section (MF, ×400). (c) human melanoma black-45 immunostaining showing no melanocytes in the basal layer (IHC, ×400). (d) Epidermis showing increased number of Langerhans cells on S-100 immunostaining (IHC, ×400)

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Figure 2: Stable vitiligo. (a) Decreased melanin pigment in basal cell layer (H and E, ×400). (b) Mild lymphocytic cell infiltrate in the papillary dermis (H and E, ×400) Inset shows absence of melanin in the lesional area flanked by perilesional areas showing melanin preservation (MF, ×200). (c) Occasional presence of human melanoma black-45 positive melanocytes (arrow) in the basal layer (IHC, ×400). Inset shows normal melanin pigmentation in the positive control section on human melanoma black-45 immunostaining (IHC, ×400). (d) Epidermis showing occasional S-100 positive Langerhans cells (arrows) on immunostaining (IHC, ×400)

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Immunohistochemical findings

Quantification of human melanoma black-45 positive melanocytes

The use of HMB-45 marker revealed the presence or absence of black melanin pigment in the basal layer of epidermis. Quantitative analysis of HMB-45 immunostaining showed a larger mean number of positive melanocytes, i.e., 2.52 ± 1.0/hpf in patients with stable vitiligo [Figure 3]. The mean value for HMB-45-positive melanocytes was significantly lower in active vitiligo, i.e., 0.08 ± 0.28/hpf. The difference between active and stable stages of vitiligo was statistically significant (P = 0.000).
Figure 3: Quantitative analysis of human melanoma black-45 and S-100 immunostaining in active and stable stages of vitiligo

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Quantification of S-100 positive Langerhans cells

Immunohistochemistry for S-100 marker demonstrated the presence of Langerhans cells in the epidermis. These cells had an irregular appearance with long and thin cytoplasmic prolongations, often localized in the suprabasal layers between the keratinocytes. On quantitative analysis, the mean value of S-100-positive Langerhans cells was 7.78 ± 4.11/hpf in clinically active cases, whereas in patients with stable vitiligo, the mean value was 1.70 ± 0.38/hpf. The difference was statistically significant for S-100 marker between the two clinical stages of vitiligo (P = 0.000).

The relative ratio of melanocytes and dendritic cells was also calculated in all the 50 vitiligo cases [Table 1]. In the actively spreading lesions, the relative ratio was significantly lower and ranged from 0.00 to 0.15 (mean: 0.010 ± 0.036). On the other hand, the ratio in stable cases had a range of 0.56–3.75 (mean: 1.558 ± 0.807) (P = 0.000).
Table 1: Comparison of histopathological and immunohistochemistry findings between active and stable vitiligo

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  Discussion Top

“Vitiligo” is derived from the Latin word “vitium” which means a defect. The word has documentation in the book, De-Medicina, by the Roman physician Celsus.[8] It is an acquired common pigmentary disorder of the skin characterized by well-circumscribed milky white macules that vary in diameter from a few millimeters to several centimeters. The exact etiopathogenesis of vitiligo remains elusive; however, it is multifactorial encompassing various factors such as genetic predisposition, neuropeptides, autoimmunity, cytokines, macrophage migration inhibitory factor, oxidative stress, humoral and cell-mediated immunity, and some newer theories of melanocytorrhagy.[9],[10],[11],[12],[13]

At present, there is no uniform clinical classification of vitiligo; however, according to the Vitiligo European Task Force, the disease is best classified initially into either segmental vitiligo or nonsegmental vitiligo, which is also referred to as generalized.[14] Vitiligo activity is an important step during the development or the progression of the disease as vitiligo is characterized by episodes of stability and activity.[15] The activity status of vitiligo lesions is to be assessed to guide therapy. Activity is the main contraindication to surgical management of vitiligo as the use of grafting techniques during activity may lead to the development of new lesions.[16] Some assessment of stability and activity based on a combination of history and clinical scoring is possible.[17]

Definitive diagnosis and assessment of disease progression or regression is ensured by histopathological and immunohistochemical evaluation. The present work was a prospective study carried out on 50 clinically and histopathologically diagnosed cases of vitiligo with expression of MF, HMB-45, and S-100 antibodies in active and stable stages of vitiligo. The histopathological changes were studied and correlated with disease activity by history and clinical examination. Histopathological findings were in congruence with the existing literature.[18],[19],[20] It was seen that epidermal spongiosis was significantly associated with disease activity. The presence of dermal lymphocytic infiltrate and basal cell layer vacuolization were also significantly higher with clinically active vitiligo in the lesional biopsy specimens. In the dermis, melanophages were observed phagocytizing melanin that was released during the degenerative process of melanocytes during the active phase of vitiligo.

The absence of functioning melanocytes in the vitiliginous skin is the key feature differentiating it from the skin with normal coloration.[19] This can be verified by MF staining, specific for melanin or by the use of dihydroxyphenylalanine technique for tyrosinase demonstration. In the current study on MF, 23/25 (92%) cases of clinically active vitiligo showed complete loss of melanin pigment in the basal layer of epidermis with only 2 (8%) cases demonstrating rare presence of melanin. Active and inactive melanocytes can be detected using immunohistochemical markers for HMB-45, Mel-5, NKI/beteb, and Melan-A.[21],[22],[23] Immunohistochemistry for HMB-45 was done in the present study in clinically active and stable stages of vitiligo. The quantification of HMB-45 + showed a relatively larger number of positive cells in the stable phase as compared to the active phase of the disease. This difference between the two phases was statistically significant. The findings in active vitiligo correlated well with those observed in a previous study by Anbar et al.[24]

The use of CD1a immunohistochemistry has been assessed earlier by Hann et al.[25] to see the expression of Langerhans cells in the clinical stages of vitiligo. In the current study, S-100 immunohistochemical marker was used to demonstrate increase in epidermal Langerhans cells in cases with active vitiligo lesions in comparison to the stable vitiligo. S-100 was used as it was cost-effective and easily procured. The P = 0.000 makes this difference between the active and stable stage of vitiligo statistically significant. The scarcity of studies regarding the use of S-100 in demonstration of Langerhans cells in active lesions makes the present study all together relevant.

Some recent studies have evaluated the role of keratinocytes in vitiligo using immune markers, aquaporin-3, and E-cadherin.[26] Immunohistochemical expression of several adhesion molecules has also been studied to detect impairment in adhesion, which exists in vitiliginous skin.[27] Topical corticosteroids, immunomodulators, and narrow-band ultraviolet B are current modalities in the treatment of the disease.[28] Pigment cell transplantation is a new substitute for segmental/stable nonsegmental vitiligo. S100B, a marker of melanocyte cytotoxicity, has been recently proposed as a novel potential marker which could serve as a new target for treatment.[29] Research is underway to discover more efficient targeted therapies in vitiligo.

  Conclusion Top

The study shows that HMB-45 and S-100 are valuable antibodies in demonstration of melanocytes and Langerhans cells, respectively, in active and stable stages of vitiligo thereby helping in making therapeutic decisions. The detection of some melanocytes in depigmented skin in stable vitiligo indicates that residual melanocytes may be preserved in long-standing vitiligo lesions.

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Conflicts of interest

There are no conflicts of interest.

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