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
Source: PubMed


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: The effect of melanin bleaching using permanganate/oxalate and dilute hydrogen peroxide on subsequent immunohistochemical staining of heavily pigmented melanocytic neoplasms is investigated. Permanganate/oxalate precluded the use of some antibodies but allowed much faster bleaching times, whereas dilute hydrogen peroxide enabled a full range of antibodies to be used, yet bleaching times were far longer. Each technique has advantages; however, the choice of method should be determined by the nature of the information needed to make a diagnosis and the speed at which a report is required.
    British journal of biomedical science 02/1999; 56(3):188-93. · 1.30 Impact Factor
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    ABSTRACT: Distinguishing heavily pigmented melanocytes from melanophages on routine hematoxylin and eosin slides can be difficult. Melanin bleaching with potassium permanganate solution is a traditional means of removing melanin from tissues and can be used before immunohistochemical staining to remove any pigment that might be confused with the brown chromogen diaminobenzidine. Azure B stains melanin granules green-blue, easily contrasts with diaminobenzidine, and may be used as a counterstain on unbleached sections after immunohistochemical staining. To our knowledge, studies comparing melanin bleaching with azure B counterstaining in the immunohistochemical evaluation of malignant melanomas have not been performed. Paraffin sections from 33 heavily pigmented malignant melanomas were bleached with a 3.0-g/L potassium permanganate solution, immunohistochemically stained for S-100 and HMB-45, and counterstained with hematoxylin. Unbleached sections were similarly stained for S-100 and HMB-45 and counterstained with azure B. To establish optimal permanganate concentrations, a variable number of sections were bleached with lower permanganate concentrations ranging from 0.125 to 2.5 g/L. S-100 antigenicity was preserved at all permanganate concentrations, whereas HMB-45 antigenicity was abolished at concentrations of 0.5 g/L and greater. At permanganate concentrations from 0.125 to 0.5 g/L, both antigenicities were preserved; however, melanin was incompletely removed. Complications of bleaching included tissue damage and loss of cytologic detail. Positive immunohistochemical staining was observed in azure B counterstained sections. Azure B stained melanin greenblue and was easily distinguished from the brown diaminobenzidine chromogen, regardless of the antibody tested. Neither tissue damage nor loss of cytologic detail was observed. We conclude that the use of azure B counterstaining is superior to permanganate bleaching in the histologic evaluation of heavily pigmented cutaneous malignant melanomas.
    Modern Pathology 12/1999; 12(12):1143-7. · 6.19 Impact Factor
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