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Activity status in vitiligo lesions: diagnostic clues

Awad, Sherif S.; Moftah, Noha H.

Journal of the Egyptian Women's Dermatologic Society: January 2014 - Volume 11 - Issue 1 - p 55–61
doi: 10.1097/01.EWX.0000438130.76693.71
Original articles
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Background Assessment of the activity status of vitiligo lesions is essential to make appropriate therapeutic decisions. Up till now, the activity of vitiligo is determined by the appearance of new lesions, which is mostly provided by patients’ words.

Objective The objective of this study was to provide clues for dermatologists to determine the activity status in vitiligo lesions.

Patients and methods This prospective analytical study included 157 nonsegmental vitiligo cases in addition to regressing vitiligo in 10 patients under treatment. Dermatological examination was carried out. Skin biopsy specimens were taken from the margin of active and stable vitiligo lesions and stained with hematoxylin and eosin and Masson-Fontana. Immunohistochemical examination was performed for 20 active vitiligo lesions for CD3, CD20, and CD68.

Results Active vitiligo patches showed depigmented convex festooning edges, pigmented spikes, poorly defined borders, a reticulated or trichrome pattern, hypopigmentation, an inflammatory raised edge or a quadrichrome pattern in the following descending frequencies: 84.7, 82.4, 76.5, 55.3, 36.5, 17.6, 1.2, and 1.2%. Meanwhile, all stable vitiligo patches showed sharply demarcated borders. All regressing vitiligo patches had pigmented convex festooning edges and depigmented spikes. Histopathologically, active vitiligo lesions showed mononuclear cells (sparse, moderate or marked levels in 24.7, 65.9, or 9.4% of the lesions), epidermotropism in 100%, vacuolated keratinocytes in 78.8% and melanophages in 72.9% of the cases. All active vitiligo lesions were positive for CD3 and CD68 and negative for CD20.

Conclusion The study suggested that the morphology of the edge and the color of the vitiliginous patch can reflect the actual activity of the disease. Histopathologically, attack of epidermal keratinocytes by inflammatory infiltrates may be considered as a diagnostic clue for disease activity.

Department of Dermatology, STDs and Andrology, Al-Minya University, Al-Minya, Egypt

Correspondence to Sherif S. Awad, MD, Department of Dermatology, STDs and Andrology, Al-Minya University, Al-Minya, Egypt Tel: +20 122 734 0233; e-mail: sherifu@rocketmail.com

Received October 17, 2013

Accepted November 2, 2013

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Introduction

Vitiligo, a common acquired pigmentary disorder, is an enigmatic disease with different clinical presentations and unpredictable prognosis 1. Vitiligo activity is a prominent step during the development or the extension of the disease as vitiligo is characterized by episodes of stability and activity 2.

One of the striking features of vitiligo is the absence of clinical symptoms and signs of inflammation. Meanwhile, an inflammatory response could be detected at the progressing edge of the depigmented lesions inducing destruction of melanocytes and indicating activity 3. However, there is no agreement on the reliability of skin pathology for assessing lesional activity 4.

Disease activity is sometimes determined by vitiligo disease activity, which is the scoring system for classifying disease activity in relation to time 5. However, it is obtained from the patient himself/herself, whose assessment is not accurate as they might miss remote patches in nonaccessible body parts or miss minute changes of activity.

Ines et al.2 denied the availability of clinical signs or routine blood tests to determine vitiligo activity. On reviewing the literature, there has been a lack of previous studies evaluating and correlating the clinical signs of disease activity. The main aim of this work was to provide clues for dermatologists to determine the activity status in vitiligo lesions.

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Patients and methods

Patients

This prospective analytical study included 157 nonsegmental vitiligo cases with an active or stable course in addition to regressing vitiligo in 10 patients under treatment. The course of the disease was determined according to patients’ history. Active vitiligo showed either enlarged old lesion/lesions or development of new lesion/lesions within the last month. Stable vitiligo showed no change in the size of old lesions or no new lesions 1 year before examination. Regressing vitiligo showed a decrease in the size of vitiligo lesions within the last month. All patients were recruited from the Dermatology Outpatient Clinic, Al-Minya University Hospital, during the period from December 2009 till the end of December 2012. The study was approved by the Research Scientific Committee, and patients’ informed consents were obtained. Patients’ clinical data were collected with clinical assessment of lesions by two blinded independent dermatologists for the following criteria: the edge (configuration, sharpness and color) and the vitiliginous patch (pattern and color).

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Skin biopsy

Skin biopsy specimens, using 4-mm punch probes, were obtained from the margin of active (either enlarged or recent ones) and stable vitiliginous lesions. Moreover, skin biopsy specimens were obtained from 10 healthy volunteers. Each biopsy was routinely processed to be stained with hematoxylin and eosin and Masson-Fontana 6. A light microscope [Accu-Scope #3025 five headed (A3025-5); Olympus, Tokyo, Japan] with a built-in camera (digital camera E-330 SLR; Olympus) was used to examine and photograph the sections.

Assessment of inflammatory infiltrates was performed using a computer-assisted image analyzer (AnalySIS; Five Olympus Soft Imaging Solutions GmbH, Münster, Germany). Mononuclear cells in the upper dermis were evaluated as sparse, moderate or marked after examining 10 high-power fields (HPF ×400) and obtaining the mean number of cells/HPF in each section. A section with mononuclear cells of less than or equal to 30/HPF was classified as the sparse group, more than 30 and less than or equal to 150 as the moderate group, and more than 150/HPF as the marked group.

Biopsy specimens from 20 active vitiligo lesions were stained by three rabbit monoclonal antihuman antibodies [CD3 (at a dilution of 1 : 25, code no.: M7254; DAKO Corporation, Carpinteria, California, USA), CD20 (at a dilution of 1 : 200, code no.: M0755; DAKO Corporation), and CD68 (at a dilution of 1 : 100, code no.: M 0814; DAKO Corporation)]. The ready-to-use detection system (code no.: K0673, LSAB2 system, DAB; DAKO Corporation) was used according to the manufacturer's instructions. All tissue sections were stained under similar conditions to ensure equal staining quality.

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Statistical analysis

Data were statistically analyzed using SPSS for Windows, Version 16.0.1 (SPSS Inc., Illinois, Chicago, USA). Statistical analysis included descriptive analysis as mean±SD for quantitative variables and frequency and % for qualitative variables. The unpaired t-test was used for comparing quantitative parametric variables and the χ2-test was used for comparing qualitative variables. Significance was expressed in terms of the P-value, which was considered significant when it was 0.05 or less.

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Results

Clinical examination

According to patients’ history, 85 patients showed active vitiligo lesions and 72 patients showed stable vitiligo lesions in addition to regressing vitiligo in 10 patients. Intense itching was described by five active vitiligo patients (5.9%) as a symptom preceding the appearance of initial depigmentation. One patient (1.2%) complained of erythema, and clinical examination revealed an erythematous raised margin of the patch.

There was complete concordance (100%) of the observations of the two blinded dermatologists regarding clinical signs. The active vitiliginous edges showed characteristic depigmented convexity and festooning towards the pigmented skin in 72 of 85 patients (84.7%). Meanwhile, stabbing strands or spikes of pigmented skin pointing towards the center of the depigmented patches were demonstrated in 70 patients (82.4%). Vitiliginous patches with poorly defined borders and erythematous raised margins were observed in 65 patients (76.5%) and one patient (1.2%), respectively (Figs 1 and 2; Table 1).

Figure 1

Figure 1

Figure 2

Figure 2

Table 1

Table 1

We noted several signs apart from the usual milky vitiligo depigmentation, including hypopigmentation, which appeared in 15 of 85 patients (17.6%). The reticulate pattern of pigmentation or Chinese letter appearance appeared in 47 patients (55.3%). The trichrome pattern, with three different shades of color, was present in 31 patients (36.5%). In addition, the quadrichrome pattern was identified in one patient (1.2%) (Figs 1 and 2; Table 1).

In all 10 regressing vitiligo lesions, pigmented convex and festooned edges were also seen but in the opposite direction towards the center of depigmented patches. Meanwhile, spikes were also demonstrated but as strands of hypopigmentation or depigmentation pointing to the pigmented side (Fig. 1). In all stable vitiligo lesions, sharp margins between vitiliginous and nonvitiliginous skin were apparent with the absence of all previous signs (Table 1).

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Histopathological examination

Mononuclear cells were demonstrated in the upper dermis of all active lesions (100%) varying from sparse to moderate and marked degrees in 21 of 85 (24.7%), 56 of 85 (65.9%), and eight of 85 active lesions (9.4%), respectively. Epidermotropism with encroachment of mononuclear cells into basal keratinocytes and vacuolated keratinocytes was observed in 85 of 85 (100%) and 67 of 85 active lesions (78.8%), respectively. With the use of Masson-Fontana stain, melanophages were identified in 62 of 85 active vitiligo lesions (72.9%) (Fig. 3; Table 2).

Figure 3

Figure 3

Table 2

Table 2

With regard to stable vitiligo lesions, sparse and moderate degrees of infiltrates were demonstrated in the upper dermis in 66 of 72 (91.7%) and six of 72 stable lesions (8.3%), respectively, with the absence of marked inflammatory infiltrates, epidermotropism and vacuolated keratinocytes in all stable biopsy specimens. Meanwhile, melanophages were identified in six of 72 stable vitiligo lesions (8.3%) (Table 2). All control biopsy specimens from healthy volunteers showed sparse infiltration only.

There was a statistically significant increase in moderate inflammatory infiltrates and melanophages in active than in stable vitiliginous lesions (P=0.0001). With regard to sparse infiltration, there was a statistically significant increase in either stable vitiligo (P=0.0001) or control normal biopsies (P=0.0001) compared with active vitiligo. Meanwhile, there was no statistically significant difference in sparse inflammatory infiltration between stable vitiliginous patches and control normal biopsy specimens (P=0.08).

All immune-stained biopsy specimens were positive for CD3 and CD68, but negative for CD20 (Fig. 4).

Figure 4

Figure 4

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Discussion

Assessment of the activity status of vitiligo lesions is mandatory to make therapeutic decisions. During activity, one of the main targets is to stop propagation through halting the immunological process using appropriate therapy 7,8. Accordingly, missing the time of activity may lead to a loss of opportunity of using the appropriate drug in its due time to stop disease progression. In contrast, activity is a main contraindication to surgical management of vitiligo as the use of grafting techniques during activity may lead to the development of new lesions at both the donor and the recipient sites 9,10.

Possible assessment of stability and activity may rely on a combination of history, serial digitized photographs and clinical scoring 4, which are time consuming and complicated processes. Accordingly, we are in need for simple clinical methods to assess the activity of vitiligo lesions.

The clinical changes recorded in this study are possible clues for a dermatologist to decide whether vitiligo is in activity or not. Among the most frequent findings during activity were the depigmented convex festooning edges (84.7%) and pigmented spikes (82.4%). Normally, the melanocyte is known to be in contact with 36 keratinocytes in basal and suprabasal layers through its dendrites forming ‘the epidermal melanin unit’ 11. Accordingly, the central existence of melanocytes within its functioning pigmenting areas may explain the appearance of the convexity and the festooning in active vitiligo patches as centrifugal depigmentation may occur when the melanocyte vanishes during macule progression. The remaining melanin between the two convex depigmenting areas will provide the pigmented spikes.

In contrast, repigmenting vitiligo patches under treatment showed pigmented convexity and festooning directed towards the depigmented areas (100%). This is because the newly formed melanocytes, evolving from hair bulge areas, provide melanin, which spreads centrifugally to neighboring keratinocytes, leading to the appearance of perifollicular pigmentation 12. With subsequent obliteration of depigmentation, a convex pigmented edge, which is demonstrated at the periphery due to melanization from nondestroyed marginal melanocytes, appeared with the pigmented perifollicular islands spreading centrifugally with another convex edge. Finally, both convex edges meet each other obliterating the white areas. Hence, the spikes are hypopigmented or depigmented, projecting towards the pigmented areas.

We also observed vitiligo patches with poorly defined borders in 76.5% of active vitiligo lesions, which were not found in any stable lesions. Instead, sharp delineated borders were found during stability in all lesions (100%).

Generally, melanosomes, containing melanin, are actively transported along melanocytic dendrites to neighboring keratinocytes, and then they move outward within the keratinocytes with gradual degradation till they are exfoliated through the stratum corneum 13. Accordingly, the concentration of melanin decreases in superficial layers with its higher concentrations within basal keratinocytes, which have very low mitotic activity. Meanwhile, the constitutive level of melanin production and delivery is in equilibrium with melanin loss through upper keratinocytes shedding 14. This equilibrium is lost in vitiligo when melanocytes are destroyed; hence, melanin concentration in the epidermis decreases, and subsequently hypopigmentation can be seen during activity (17.6%). The clinical finding of hypopigmented vitiliginous patches was described before by Sharqui 15.

Reticulated pattern of pigmentation or Chinese letter appearance was detected during activity (55.3%) and this is explained by an overlap of the remaining melanin in areas supplied by several groups of melanocytes.

Trichrome and quadrichrome vitiligo lesions were found in 36.5 and 1.2% of active vitiligo patients, respectively, demonstrating the decrease in epidermal melanin levels during activity, with sharp lines between the color differences. This was clinically described previously 16,17. Parsad et al.18 reported another possible explanation for the color changes, in which a decrease in the tyrosinase activity leads to death/inactivation of melanocytes, resulting in a switch in melanin formation from dark eumelanin towards pheomelanin with its lighter hue.

Vitiliginous lesions are asymptomatic, although itching may precede or accompany the onset of lesions in a few patients 19. In the current study, itching was elicited in five cases, who complained of it before the initial appearance of depigmentation.

Histopathologically, this study showed a statistically significant increase in moderate inflammatory infiltrates in active (65.9%) than in stable (8.3%) vitiliginous patches, which was mostly associated with absent marked infiltrates in stable lesions. These results are similar to those of previous studies 20–22, which reported a greater increase in inflammatory infiltrates in active than in stable vitiligo. In the current study, all immune-stained biopsy specimens of active vitiligo were positive for CD3 and CD68, but negative for CD20, indicating the presence of T lymphocytes and histiocytes, respectively, with the absence of B lymphocytes.

In this study, there was no statistically significant difference in sparse infiltrates between stable vitiliginous patches and control normal biopsies, indicating that there was no histopathological difference regarding inflammatory infiltrates between stable and control biopsies. This finding is in agreement with some authors, who failed to detect frank inflammatory changes in stable vitiligo patches 23.

Among histopathological signs of activity were epidermotropism (100%) and vacuolated keratinocytes (78.8%), which were absent in biopsy specimens of stable vitiligo lesions. This is similar to the findings of Le Poole and Norris 24. Meanwhile, these two findings were evident with all active vitiligo lesions showing sparse inflammatory infiltrates.

With regard to melanophages, there was a statistically significant increase in active (72.9%) than in stable (8.3%) vitiliginous lesions. The melanophages that are still present in stable lesions may be due to remaining melanin in vitiligo lesions. To the best of our knowledge, this study is the first one to mention that melanophages were present during activity. Vacuolar degeneration of basal cells leads to drop of melanin before their upward epidermal movement. After that, dermal histiocytes engulf the dropped pigment leading to the appearance of these melanophages. A previous literature described vitiligo with blue hue, which was not found in this study; yet it may be explained by the Tyndall phenomenon after dermal melanin drop 25.

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Conclusion

We suggested that the morphology of the edge and the color of a vitiliginous patch can reflect the actual activity of the disease and can be used as clues to start immune-suppressor drugs and to avoid introduction of unneeded surgical procedures. Vitiligo patches with depigmented convex and festooned edges, pigmented spikes, a poorly defined border, a reticulated pattern, a trichrome pattern, hypopigmentation, an inflammatory raised edge and a quadrichrome pattern, in this sequence of frequency, could be considered as markers of clinical activation. Meanwhile, amelanotic vitiligo lesions with sharply demarcated borders could indicate the stability status of a lesion. Histopathologically, epidermotropism, vacuolated keratinocytes, and melanophages in a descending frequency may be considered as diagnostic clues for the disease activity.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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Keywords:

active; regressing; stable; vitiligo

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