Melanocyte Density Research Articles

An Immunohistochemical Analysis of Pseudomelanocytic Nests Mimicking Melanoma In Situ: Report of 2 Cases

Several case reports have discussed the difficulty in differentiating junctional pseudomelanocytic nests as a result of lichenoid inflammation from a true melanocytic neoplasm. Even immunohistochemistry can be misleading in these cases with both Melan-A and Mart-1 frequently resulting in false positivity as a result of nonspecific labeling of nonmelanocytic cells containing melanosomes. We present a series of 2 similar cases which were initially misdiagnosed as melanoma in situ likely as a result of Mart-1 positivity of the pseudomelanocytic nests. However, in our review, a significant lichenoid reaction was apparent at the dermal-epidermal junction. Staining with microphthalmia-associated transcription factor (MITF) showed a normal density of melanocytes along the dermal-epidermal junction and failed to uniformly label the Mart-1-positive pseudomelanocytic nests. In both patients, medications frequently resulting in fixed drug eruptions were identified, and a final diagnosis of fixed drug eruption was rendered in both cases. In light of these findings we suggest MITF is a more useful marker for evaluating lentiginous proliferations along the dermal-epidermal junction particularly when dealing with the differential diagnosis of lichenoid reaction with pseudomelanocytic nests versus melanoma in situ.

Rapid Frozen Section Immunostaining of Melanocytes by Microphthalmia-Associated Transcription Factor

Mohs micrographic surgery (MMS) has increasingly become an accepted therapy for melanoma in situ on chronically sun damaged skin (CSDS). However, melanocytes are difficult to locate in frozen material on hematoxylin and eosin. In addition, determining the cut-off between the melanoma and the "atypical melanocytic hyperplasia" in CSDS can be challenging in frozen or formalin-fixed paraffin-embedded sections, with or without immunohistochemistry (IHC). In this article, we report the use of a rapid, 35-minute protocol using microphthalmia-associated transcription factor (MITF) IHC for identifying melanocytes in frozen tissue for its potential use in MMS. In contrast to melanoma antigen recognized by T cells (MART-1), MITF is a nuclear stain, which simplifies identification of melanocytes and quantification of melanocytic parameters. In this study, MITF IHC in frozen sections yielded equivalent melanocyte nuclear diameter and density measurements compared with formalin-fixed paraffin-embedded sections. Nuclear diameter measurements obtained with MITF were similar to that previously reported with MART-1, but the melanocyte density figures were lower. Reliable labeling of melanocytes in frozen sections required the use of diaminobenzidine (DAB) chromogen with Giemsa counterstaining and a buffer devoid of surfactant. Our experience with MITF IHC indicates that it is a dependable immunostain in frozen sections, and may prove to be useful in MMS as an adjunct to hematoxylin and eosin and MART-1 IHC for interpretation of margins for melanoma in situ on CSDS.

A split‐face study of intense pulsed light on photoaging skin in Chinese population

Intense pulsed light (IPL) is regarded as the gold standard of nonablative photo-rejuvenation. Yet there is still a need to observe its efficacy and safety on dark skin using a split-face module. Twenty-four Chinese women with photoaging were enrolled in this study. Patients were randomized to receive four IPL treatments at 3- to 4-week intervals on one side of face, with the other side spared as control. Changes of photoaging were evaluated using a global evaluation, an overall self-assessment, a Mexameter and a Corneometer. Skin biopsies were taken after four sessions of treatment on one side of face. The melanocyte density and the contents of melanin, collagen fibers, and elastic fibers were stained and used to evaluate the improvement on dyschromia and other signs of photoaging. The global scores of photoaging on treated side decreased significantly from 3.02 to 1.22, while it remained unchanged on the untreated side. Twenty-one of 24 patients (87.5%) rated their improvement as excellent or good. The difference on the values of melanin index and erythema index on treated side were significantly larger than those on untreated side after the 1st session, the 4th session and at 3-month follow-up (P<0.05). The melanin contents were significantly decreased and the collagen fibers were obviously increased only on treated side (P<0.05). Adverse effects of treated side were limited to mild pain and transient erythema. Using this split-face module, IPL treatment is proved both clinically and histologically to be effective in treating photoaging skin in Chinese population. Adverse effects were minimal and acceptable.

Independent regulation of hair and skin color by two G protein‐coupled pathways

Hair color and skin color are frequently coordinated in mammalian species. To explore this, we have studied mutations in two different G protein coupled pathways, each of which affects the darkness of both hair and skin color. In each mouse mutant (Gnaq(Dsk1), Gna11(Dsk7), and Mc1r(e)), we analyzed the melanocyte density and the concentrations of eumelanin (black pigment) and pheomelanin (yellow pigment) in the hair or skin to determine the mechanisms regulating pigmentation. Surprisingly, we discovered that each mutation affects hair and skin color differently. Furthermore, we have found that in the epidermis, the melanocortin signaling pathway does not couple the synthesis of eumelanin with pheomelanin, as it does in hair follicles. Even by shared signaling pathways, hair and skin melanocytes are regulated quite independently.

Time-kinetic study of repigmentation in vitiligo patients by tacrolimus or pimecrolimus

New topical immunomodulators have been reported to cause repigmentation of vitiligo lesions. However, time-kinetics of such repigmentation in different anatomic locations is not well known. We performed a randomized double-blind placebo control study with tacrolimus versus the vehicle and a nonrandomized control study with pimecrolimus to evaluate the time to reach significant pigmentation, its duration and extent in treated areas. Antioxidant status of serum was also assessed. Twenty patients, in the tacrolimus study, had one pair of lesions on different localizations, and 20 on face and/or upper limbs for pimecrolimus. The extent of repigmentation was evaluated by slides and mapmakings at baseline and every 4 weeks during 7 months. Adverse events were recorded. The derivatives of oxygen metabolites, the ferric reducing ability of serum and vitamin E were assessed. Three groups of patients were identified with the tacrolimus study. Eight had no significant change in response characterized by a parallel increase of repigmentation or none in treated and control areas. Nine had a better repigmentation to tacrolimus at fifth month of treatment. Three had a marked repigmentation in control areas at the end of treatment. Repigmentation was significant on the face compared to upper-limbs with pimecrolimus from fourth to seventh month. A significant reduction of oxidative stress and an increase in antioxidant capacity in serum of patients treated with topical tacrolimus was observed, while those treated with pimecrolimus did not show any significant changes but an increase in vitamin E. Our work defines three periods in repigmentation, triggering during the first 4 months, increase in pigmentation with tacrolimus and a plateau or a sustained repigmentation. The continuity of the treatment seems necessary to ensure a prolonged repigmenting effect and even an enhanced one, such as the one we observed on the face with pimecrolimus. The extent of repigmentation was more significant on the face compared to other locations probably due to differences in melanocyte density. Furthermore, we did not find any relationship between repigmentation and the duration of vitiligo. Tacrolimus was able to reduce the systemic oxidative stress independently from its repigmenting capacity. Both drugs were well tolerated.

Pathology of conjunctival melanocytic neoplasms

Abstract Purpose To describe the classification, grading and staging of conjunctival melanocytic proliferation. Methods We have audited our experience with conjunctival melanomas, using a novel mapping system and have found shortcomings in the current Tumour Node Metastasis (TNM) staging system. We have also reviewed our cases of intra‐epithelial melanocytic neoplasia and confirmed other authors’ impressions that conjunctival ‘primary acquired melanosis with atypia’ is histologically similar to cutaneous in situ melanoma. To improve objectivity in the reporting of conjunctival intra‐epithelial melanocytic neoplasia, we propose a scoring system based on pattern of melanocytic infiltration, density of melanocytes &amp; degree of cellular atypia. Results The term ‘conjunctival melanosis’ should be used only to describe the slit‐lamp appearance of hyperpigmentation. Histologically, this abnormality should be categorized as ‘hypermelanosis’ or ‘melanocytosis’. Hypermelanosis can either be primary or secondary to ocular or systemic disease. Benign melanocytosis comprises conjunctival melanocytic hyperplasia and naevi. Malignant melanocytosis is essentially melanoma, which is primary (in situ or invasive) or secondary (i.e., spreading to conjunctiva from adjacent tissues) or rarely metastatic. We suggest that the TNM staging system for conjunctival melanoma should be revised to: (1) include a Tis stage; (2) take account of superficial extent, invasion of adjacent tissues and caruncular involvement, in stages TI to TIII; and (3) to sub‐categorize TIV disease so that there is better correlation with likely mortality. Conclusion We have revised the classification of conjunctival melanocytic proliferations &amp; improved the grading and staging of melanoma. These developments should be useful in treatment &amp; research.

Histologic Distinction Between Subungual Lentigo and Melanoma

The distinction between a benign subungual pigmented macule (lentigo) and an early lesion of melanoma in situ can be difficult. To identify histologic parameters of potential diagnostic value, we retrospectively reviewed biopsies and excisions of 35 pigmented nail lesions. We studied 20 melanomas (10 invasive and 10 noninvasive) and 15 benign subungual melanotic lentigines. Ten specimens of normal nail apparatus obtained for reasons other than melanonychia were also examined as controls. The parameters, which were analyzed, included the density of melanocytes, the presence of multinucleated cells, pagetoid spread, cytologic atypia, inflammation, and the distribution of melanin pigment. The density of melanocytes was measured as the number of cells per 1 mm stretch of subungual dermo-epithelial junction [=melanocyte count (MC)]. The MC for invasive melanomas was as follows: mean=102, median=92.5, and range 52 to 212. For noninvasive (only in situ) melanoma, the mean MC was 58.9, median 51, and range 39 to 136. For benign subungual melanotic macules, the mean MC was 15.3, median 14, and range 5 to 31. In normal controls, the mean MC was 7.7, median 7.5, and range 4 to 9. Qualitative features associated with in situ melanoma and useful for its distinction from benign subungual melanotic macules included the presence of confluent stretches of solitary units of melanocytes, multinucleated melanocytes, lichenoid inflammatory reaction, and florid pagetoid spread of melanocytes.

Melanocyte localization and distribution in human cholesteatoma.

Melanocytes in skin are derived from the neural crest and colonize the epidermis in the first trimester of gestation. Melanocytes have been observed in the nasopharyngeal, inner ear and oral mucosa and should therefore be present in the middle ear mucosa. To identify and determine the distribution of melanocytes in human cholesteatoma and normal meatal skin in Caucasian adults. Human cholesteatoma (n=18) and normal meatal skin samples (n=10) were investigated immunohistochemically with anti-HMB-45 and MART-1 antibodies. Localization and distribution of melanocytes were assessed in the epidermis and cholesteatoma using an automatic analyzing system. Regular skin exhibited melanocytes within the epidermis and accounted for 10% of the total cell number. They occurred partly as membrane-bound clusters. Cholesteatoma matrix melanocytes were observed in the basal layer and exhibited an oval or roundmorphology. Decreased numbers of melanocytes in the basal layer correlated with keratinization within cholesteatoma samples. Melanocytes revealed monomorphous nuclei, abundant cytoplasm containing particles of melanin. Found adjacent to glands and blood vessels, melanocytes were also scattered among the mesenchymal cells. Accounting for 2-6% of the total cell number within the squamous epithelium, melanocyte density was significantly lower in cholesteatoma tissue than in skin. The melanocyte distribution pattern was different when comparing the epithelia of skin and cholesteatoma. The presence of melanocytes in cholesteatoma may be due to an ingrowth, consequently controlled by keratinocyte-derived signals. In terms of the pathogenesis of cholesteatoma, neither squamous metaplasia nor melanocyte metaplasia can be excluded by our data.

The Effects of Dickkopf 1 on Gene Expression and Wnt Signaling by Melanocytes: Mechanisms Underlying Its Suppression of Melanocyte Function and Proliferation

Dickkopf 1 (DKK1), which is expressed at high mRNA levels by fibroblasts in the dermis of human skin on the palms and soles, inhibits the function and proliferation of melanocytes in the epidermis of those areas via the suppression of beta-catenin and microphthalmia-associated transcription factor (MITF). In this study, we investigated the protein expression levels of DKK1 between palmoplantar and non-palmoplantar areas and the effects of DKK1 on melanocyte gene expression profiles and on Wnt signaling pathways using DNA microarray technology, reverse transcriptase-PCR, Western blot, 3-dimensional reconstructed skin, immunocytochemistry, and immunohistochemistry. DKK1-responsive genes included those encoding proteins involved in the regulation of melanocyte development, growth, differentiation, and apoptosis (including Kremen 1, G-coupled receptor 51, lipoprotein receptor-related protein 6, low-density lipoprotein receptor, tumor necrosis factor receptor super-family 10, growth arrest and DNA-damage-inducible gene 45beta, and MITF). Of special interest was the rapid decrease in expression of MITF in melanocytes treated with DKK1, which is concurrent with the decreased activities of beta-catenin and of glucose-synthase kinase 3beta via phosphorylation at Ser9 and with the upregulated expression of protein kinase C alpha. These results further clarify the mechanism by which DKK1 suppresses melanocyte density and differentiation, and help explain why DKK1-rich palmoplantar epidermis is paler than non-palmoplantar epidermis via mesenchymal-epithelial interactions.

The Density and Distribution of Melanocytes Adjacent to Melanoma and Nonmelanoma Skin Cancers

The determination of clear margins during Mohs surgery for melanoma in situ arising on sun-damaged skin is often made difficult by the presence of background atypical melanocytic hyperplasia. To determine the density and distribution patterns of melanocytes adjacent to melanoma and nonmelanoma skin cancers. 180 skin specimens obtained during the routine repair of defects resulting from the removal of melanoma and nonmelanoma skin cancers were analyzed using H and E-stained permanent sections to determine the quantity and distribution of epidermal melanocytes. The mean melanocyte density was 7.97 melanocytes per 1 mm of epidermis (SD,+/-6.7). Contiguous melanocytes were found in 30 (16.7%), atypical melanocytes were observed in 8 (4.4%), and follicular extension of melanocytes was observed in 11 (6.1%) of the specimens. These features were significantly associated with higher melanocyte densities (p< .001) and were more commonly observed in specimens from patients with melanoma. There is a high degree of variability in melanocyte densities seen adjacent to melanoma and nonmelanoma skin cancers. Contiguous melanocytes, atypical melanocytes, and follicular melanocytes can be seen in the sun-damaged skin surrounding both melanoma and nonmelanoma skin cancers, but especially with melanoma. Because some of the features of melanoma in situ can be seen in chronically sun-damaged skin, the Mohs surgeon should be cautious when assessing the margins for melanoma in this setting.

The Density and Distribution of Melanocytes Adjacent to Melanoma and Nonmelanoma Skin Cancers

The Density and Distribution of Melanocytes Adjacent to Melanoma and Nonmelanoma Skin Cancers

Melanocytes in Long-standing Sun-Exposed Skin

To help distinguish early melanoma from normal sun-damaged skin by quantifying the density, confluence, and depth of follicular penetration of melanocytes in long-standing sun-exposed skin of the face and neck. Case series. Referral center. Random selection of 149 patients undergoing Mohs surgery for basal cell and squamous cell carcinomas of the face and neck. Frozen-section slides were made from long-standing sun-exposed normal skin and stained with MART-1 (melanoma antigen recognized by T cells 1 staining) immunostain. The number, confluence, and depth of penetration of melanocytes along the follicular epithelium were quantified per high-power field (original magnification x 400, equivalent to 0.5 mm of skin). Confluence was categorized by the number of adjacent melanocytes (none, 0-1; mild, 2; moderate, 3-6; and severe, >6). The mean number of melanocytes per high-power field was 15.6 (range, 6-29). Confluence was severe in 1.0% of the specimens, moderate in 34.0%, mild in 54.0%, and absent in 11.0%. Focal areas of increased melanocyte density occurred in 24.2% of the specimens; in these areas, the mean number of melanocytes per high-power field was 20.3 (range, 7-36) and the confluence of melanocytes was severe in 13.0%, moderate in 50.0%, and mild in 37.0%. The mean depth of follicular epithelium penetration by melanocytes was 0.38 mm. Pagetoid spread and nesting of melanocytes were not seen. Nonspecific scattered MART-1-staining dermal cells were in half of the slides. Melanocytes in long-standing sun-exposed skin have an increased density and a confluence that is often moderate (3-6 adjacent melanocytes), but they do not exhibit pagetoid spread or nesting. Nonspecific MART-1-staining dermal cells should not be interpreted as invasive melanoma.

Café-au-lait spots in neurofibromatosis type 1 and in healthy control individuals: hyperpigmentation of a different kind?

Solitary café-au-lait spots are quite common in the general population but multiple café-au-lait macules (CALM) are often indicative of an underlying genetic disorder. The frequency of having more than five CALM is rare in normal individuals and is therefore considered as a cut-off for the diagnosis of neurofibromatosis type 1 (NF1). The etiopathogenesis of these macules is still very obscure. In this study we compared epidermal melanocyte and dermal mast cell numbers between four groups: control normal and control CALM skin, and NF1 normal and NF1 CALM skin and elaborated a possible role for stem cell factor (SCF) in CALM formation. The groups were analyzed by immunohistochemistry for numerical analysis of the melanocyte and mast cell population and by ELISA, western blot analysis and real-time quantitative PCR for further determination of the role of SCF. We found a significant increase in melanocyte density in NF1 CALM skin compared with the isolated CALM in control individuals. However, both groups displayed a similar increase in mast cell density. In addition, we found increased levels of soluble SCF in NF1 CALM and in NF1 normal fibroblast supernatant. We conclude that SCF is an important cytokine in NF1 skin, but that additional (growth) factors and/or genetic mechanisms are needed to induce NF1-specific CALM hyperpigmentation.

Factors determining the optimal body site and method for obtaining punch biopsies of human skin as a tissue in which to assess pharmacodynamic and pharmacokinetic endpoints in drug development studies

There are potential advantages to detecting pharmacodynamic (PD) and pharmacokinetic (PK) endpoints in a tissue-based compartment such as the skin during the development of molecularly targeted drugs. We explored regional differences between inner arm, inner thigh, lower back and buttocks in 12 healthy male Caucasian volunteers in the tolerability of skin biopsy procedures; the Ki67 proliferation index; the frequency of detecting hair follicles and sweat glands; and the percentage of melanocytes. We also explored the amounts of tissue and protein obtained, and two separate methods of splitting biopsies for processing in mutually exclusive media. Biopsies from all body sites were well tolerated. The subjective ranking order was inner arm > buttocks = back > thigh. There were no statistically significant differences in the Ki67 labelling index (P > 0.05). The frequency of detecting sweat glands was the same in all body sites, but the frequency of detecting hair follicles was higher in back and buttock, compared to arm and thigh. The percentage of melanocytes was significantly lower in the buttocks compared to the back and thigh (P < 0.05), but not compared to the arm (P = 0.07). A 4-mm punch biopsy yielded a mean of 16.8 mg of tissue (range: 9-28 mg) and 160 microg of protein (range: 80-270 microg). In vivo sample splitting, by following a 2-mm punch with a 4-mm overpunch, had a shorter time from devascularisation to immersion into processing medium than ex vivo dissection of a 4-mm sample, which may be of importance to the assessment of labile endpoints. We conclude that multiple punch biopsies of the skin are feasible, with the buttocks representing the studied body site with the optimal balance between tolerability, hair follicle density and melanocyte density for obtaining tissue in which to assess PD and PK endpoints during drug development studies.

Mechanisms of Skin Tanning in Different Racial/Ethnic Groups in Response to Ultraviolet Radiation

Ultraviolet radiation stimulates pigmentation in human skin, but the mechanism(s) whereby this increase in melanin production (commonly known as tanning) occurs is not well understood. Few studies have examined the molecular consequences of UV on human skin of various racial backgrounds in situ. We investigated the effects of UV on human skin of various races before and at different times after a single 1 minimal erythemal dose UV exposure. We measured the distribution of DNA damage that results, as well as the melanin content/distribution and the expression of various melanocyte-specific genes. The density of melanocytes at the epidermal:dermal junction in different types of human skin are remarkably similar and do not change significantly within 1 wk after UV exposure. The expression of melanocyte-specific proteins (including TYR (tyrosinase), TYRP1 (tyrosinase-related protein 1), DCT (tyrosinase-related protein 2), MART1 (melanoma antigens recognized by T-cells) gp100 (Pmel17/silver), and MITF (micropthalmia transcription factor)) increased from 0 to 7 d after UV exposure, but the melanin content of the skin increased only slightly. The most significant change, however, was a change in the distribution of melanin from the lower layer upwards to the middle layer of the skin, which was more dramatic in the darker skin. These results provide a basis for understanding the origin of different skin colors and responses to UV within different races.

Histological variation of Japanese skin with aging

Histological variation of Japanese skin with aging

Repigmentation in vitiligo universalis: Role of melanocyte density, disease duration, and melanocytic reservoir

Recent reports suggest that vitiligo lesions are not totally devoid of melanocytes. We report an interesting observation made in the vitiliginous skin of an Indian patient being treated for pemphigus vulgaris with dexamethasone cyclophosphamide pulse therapy. Our observations indicate that melanocytes are never completely absent in the depigmented epidermis and that these melanocytes can recover their functionality in vivo and in vitro under an appropriate stimulus.

Ultrastructural characterization of the distribution of melanin and epidermal macrophages in photodamaged skin.

We examined the characteristics of melanin distribution, the possible mechanisms underlying the histological incontinence of pigment, and the significance of epidermal macrophages in photodamaged skin. We used electron microscopy to compare and quantitate melanin distribution in various types of cells and structures, to qualitatively observe associations of melanin granules with melanophages, and to examine morphological differences of epidermal macrophages in sun-exposed versus sun-protected facial skin. Melanin-containing cells (such as Langerhans' cells) and melanin-containing structures (such as colloid bodies) in photodamaged skin were more numerous than in sun-protected skin, in proportion to differences in melanocyte density and in epidermal melanin content. Although the precise mechanism(s) of histological incontinence of pigment in photoaging skin appear to be very complicated, it is certainly one of the morphological hallmarks of photodamaged facial skin, and the degeneration of keratinocytes (noted by their electron-lucent properties), a feature characteristic of photoaging, contributes to that process. Furthermore, the increased numbers of epidermal macrophages in sun-exposed skin may be associated with photoaging processes (probably through their phagocytic function) as well as alterations of the cutaneous immune system.

MORE EFFORTS FOR TISSUE-ENGINEERING SKIN

A251 Aims: Submit a review of Tissue-engineering skin, and provide new sight for future efforts. Reviews: Requirements for functions of tissue-engieering skin are resemblance to native human skin including three aspects: protection, perception and regulation. Protective function consists of barrier, UV light absorption, immune surveillance and mechanical stability. There are some permeability differences between artificial skin and native skin which may be ascribed to the different composition and ultrastructure of stratum corneum(SC) lipids, and absence of hair. The UV light absorption ability has a direct ratio with the pigmentation of healed cultured skin substitutes, which is frequently irregular and unpredictable. Paracrine factors from skin cells and the extracellular matrix act as local environmental signals for the the pigmentation which is proportional to growth density of melanocytes. Immune surveillance requires the complete engraftment of native skin cells and a functional vascular plexus which the available artificial skin lacks. Many factors contribute to inevitable contraction of cultured skin substitutes, such as the effect of keratinocytes and fibroblasts, thickness of dermis, immaturity of the dermal-epidermal junction and materials. The perception and regulation functions have almost been studied only in experiments because of difficult transplantation of necessary elements like touch domes and skin appendages. Immune rejection is the main reason for limitting the popularity of allogeinic grafts. In the process immune cells, cytokines and blood supply work. CD80 on keratinocytes might bring the ultimate rejection of allogeneic keratinocytes. The host inflammatory response to some extents acts as a more significant role than immune reaction. Prospects: Tissue-engineering skin needs further developments. This may be achieved through improvement of culture conditions. crosslink agents, certain cytokines, phorbol ester and vitamine C can be added to culture medium to enhance mechanical stability of artificial skin and accelerate its formation. Perfection of cultured skin substitutes requires complete engraftment of the celluar part and a functional vascular plexus which may conflict with requirement of diminishing antigenicity. Here improvement of culture condition appears to be significant through which cells can move from the peripheral healthy region without transplantion. Stem cell is another attractive topic. Due to the difficult acquirement and culture, a stem cell bank can be built. Skin is perhaps the most accessible of all somatic tissues for therapeutic gene transfer. Gene therapy for cultured skin grafts develops quickly. The goal includes accelerating wound healing and curing diseases and the key is how to prolong expression of the transfected gene. As artificial skin makes a transition from research to clinical practice, the uniform criterions for qualitative and quantitative analysis of cultured skin substitutes must be established for evaluation of materials, composition, performance and safety.Figure

Efficacy of autologous transplantation of noncultured epidermal suspension in two different dilutions in the treatment of vitiligo

Previous reports have shown the effectiveness of a basal layer cell suspension in the treatment of stable vitiligo; however, there has been no mention in the literature until now of the optimal number of melanocytes required in an autologous noncultured epidermal suspension for transplantation to obtain early and acceptable results. In this randomized prospective study, we compared the efficacy of two different dilutions to determine the optimum density of melanocytes required for transplantation to achieve early and cosmetically acceptable results. We enrolled 20 patients with stable vitiligo with a body surface area involvement of< 10%. Fifty per cent of patients in Group A (where the recipient area transplanted was approximately three times the size of the donor area, and the density of melanocytes transplanted per square millimeter was approximately 231.60+/- 27.03) showed marked (> 75%), 20% moderate, 10% mild, and 20% minimal repigmentation (or no response). In Group B (where the recipient area was increased to five-fold the donor area), none of the patients showed marked, 10% showed moderate, 20% showed mild, and 70% showed either minimal or no repigmentation. The density of melanocytes transplanted in this group was 154.90+/- 27.65/mm(2). The extent of repigmentation was significantly greater (P< 0.05) in Group A than in Group B, and the density of melanocytes in the suspension was also significantly greater (P< 0.01) in Group A than in Group B. We contend that the minimum number of melanocytes required to produce satisfactory repigmentation is probably in the range of 210-250/mm(2).