The Effect of Melanin Bleaching on Immunohistochemical Staining in Heavily Pigmented Melanocytic Neoplasms

Department of Dermatopathology, St. John's Dermatology Centre, St. Thomas' Hospital, London, U.K.
American Journal of Dermatopathology (Impact Factor: 1.39). 09/1998; 20(4):357-61. DOI: 10.1097/00000372-199808000-00006
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The accumulation of excessive amounts of melanin in melanocytic lesions can obscure cellular morphology and can further hinder immunocytochemical procedures. We have used a modification of the potassium permanganate/oxalic acid melanin-bleaching technique, involving much reduced bleaching times, in order to remove melanin granules prior to incubation with primary antibody. We have assessed a panel of antibodies applicable to the evaluation of melanocytic lesions and in addition have also assessed antibodies that may be more useful in research. The study attempts to determine which antigens may be affected by bleaching and which are not. Antigens S100, HMB 45, NKIC3, CD34, and L26 are relatively unaffected by this procedure. Factor-VIII-related antigen and vimentin and CD68 antigens produced enhanced staining. In contrast, antigens CD3, CD31, and CD45RO were abolished. In addition, smooth muscle actin and desmin antigens demonstrated considerable nonspecific background staining and were not reliable in this study. This technique demonstrates that a fairly wide range of antigens are preserved after bleaching and that distinction between melanocytes and melanophages can reliably be performed using the conventional immunocytochemical chromogen 3,3-diaminobenzidine and without the need for elaborate counterstaining.

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    • "It was also documented that even at lower concentrations of KMnO4 (0.25% and 0.1%), tissue deterioration was evident [8]. On another note, there are also myriads of reports on the drawbacks of KMnO4 bleaching on immunostaining that hinder any further investigations [15], [29]. The latter bleaching agent has been shown to alter the specificity and sensitivity of antigenic epitopes of the tissues used for a variety of antibodies, apart from mediocre tissue conservation [30], [31]. "
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    ABSTRACT: Purpose The removal of excessive melanin pigments that obscure ocular tissue morphology is important to address scientific questions and for differential diagnosis of ocular tumours based on histology. Thus, the goal of the present study was to establish an effective and fast melanin bleaching method for paraffin and frozen mouse and human ocular tissues. Methods Paraffin-embedded and frozen ocular specimens from mice and human donors were subjected to bleaching employing two methods. The first employed potassium permanganate (KMnO4) with oxalic acid, and the second 10% hydrogen peroxide (H2O2). To determine optimal bleaching conditions, depigmentation was carried out at various incubation times. The effect of diluents used for 10% H2O2 was assessed using phosphate-buffered saline (PBS), and deionized water. Three different slide types and two fixatives, which were ice-cold acetone with 80% methanol, and 4% paraformaldehyde (PFA) were used to determine the optimal conditions for better tissue adherence during bleaching. All tissues were stained in hematoxylin and eosin for histological evaluation. Results Optimal bleaching was achieved using warm 10% H2O2 diluted in PBS at 65°C for 120 minutes. Chromium-gelatin-coated slides prevented tissue detachment. Adherence of cryosections was also improved with post-fixation using 4% PFA and overnight air-drying at RT after cryosectioning. Tissue morphology was preserved under these conditions. Conversely, tissues bleached in KMnO4/oxalic acid demonstrated poor depigmentation with extensive tissue damage. Conclusions Warm dilute H2O2 at 65°C for 120 minutes rapidly and effectively bleached both cryo- and paraffin sections of murine and human ocular tissues.
    PLoS ONE 07/2014; 9(7):e102512. DOI:10.1371/journal.pone.0102512 · 3.23 Impact Factor
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    ABSTRACT: We describe a form of junctional melanocytic neoplasm with a massive production of melanin accumulated in the dermis. The pigment is stored in macrophages, which are by far the most numerous cellular component of the lesion. Another peculiar aspect is the occasional presence of a few melanocytes scattered in a pagetoid pattern above the dermo-epidermal junction in the spinous layer. The histological picture of this lesion is similar to a form of "tumoral melanosis" induced by a regressed malignant melanoma. The lesion had a worrisome clinical picture, its dark colour constituting a clinico-pathological diagnostic problem. The main clinical clues to the benign nature of this entity are the small lateral diameter, the uniform distribution of the pigment and the stability of the lesion over time; moreover, the patients are alive and well after a considerable length of time. Although a regressed dysplastic or malignant lesion cannot be totally excluded from a scientific point of view, we conclude that there is no sound morphological or clinical evidence that the lesion is other than biologically benign. The lesion is most likely another peculiar variant of epithelioid and spindle cell naevus.
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    ABSTRACT: Though acral lentiginous melanoma (ALM) is a major type of malignant melanoma, no immunohistochemical study on this type of melanoma has been reported. The purpose of this study is to analysis the immunohistochemical findings of ALM using routinely used immune markers. An immunohistochemical study was performed on paraffin sections of 20 ALMs using S-100 protein, HMB-45, MART-1, vimentin, epithelial membrane antigen (EMA) and CAM 5.2. S-100 protein (95%) was found to be a more sensitive marker than either HMB-45 (80%) or MART-1 (70%) for recognizing ALM. Melanin bleaching was useful for recognizing heavily pigmented ALM using both S-100 protein and HMB-45. The intensity of HMB-45 correlated well with the melanin content. However, there was no significant correlation between the intensity of S-100 protein and the melanin content. One and two out of 20 cases stained focally with EMA and CAM5.2, respectively, but these cases stained also with HMB-45 and/or S-100 protein. S-100 protein and HMB-45 were relatively sensitive markers for recognizing ALM. Despite the occasional positivity for the epithelial markers in ALM, all epithelial marker-positive cases stained also with HMB-45 and/or S-100 protein. Therefore, we recommend that the panel of antibodies used for recognizing ALM should contain at least S-100 protein and HMB-45.
    International Journal of Dermatology 03/2003; 42(2):123-9. DOI:10.1046/j.1365-4362.2003.01583.x · 1.31 Impact Factor
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