The effect of melanin bleaching on immunohistochemical staining in heavily pigmented melanocytic neoplasms.
ABSTRACT 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.
- SourceAvailable from: Christoph Brochhausen[Show abstract] [Hide abstract]
ABSTRACT: 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.PLoS ONE 01/2014; 9(7):e102512. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The prognostic value of mitotic rate in melanoma is increasingly recognized, particularly in thin melanoma in which the presence or absence of a single mitosis/mm can change staging from T1a to T1b. Still, accurate mitotic rate calculation (mitoses/mm) on hematoxylin and eosin (H&E)-stained sections can be challenging. Antimonoclonal mitotic protein-2 (MPM-2) and antiphosphohistone-H3 (PHH3) are 2 antibodies reported to be more mitosis-specific than other markers of proliferation such as Ki-67. We used light microscopy and computer-assisted image analysis software to quantify MPM-2 and PHH3 staining in melanoma. We then compared mitotic rates by each method with conventional H&E-based mitotic rate for correlation with clinical outcomes. Our study included primary tissues from 190 nonconsecutive cutaneous melanoma patients who were prospectively enrolled at New York University Langone Medical Center with information on age, gender, and primary tumor characteristics. The mitotic rate was quantified manually by light microscopy of corresponding H&E-stained, MPM-2-stained, and PHH3-stained sections. Computer-assisted image analysis was then used to quantify immunolabeled mitoses on the previously examined PHH3 and MPM-2 slides. We then analyzed the association between mitotic rate and both progression-free and melanoma-specific survival. Univariate analysis of PHH3 found significant correlation between increased PHH3 mitotic rate and decreased progression-free survival (P=0.04). Computer-assisted image analysis enhanced the correlation of PHH3 mitotic rate with progression-free survival (P=0.02). Regardless of the detection method, neither MPM-2 nor PHH3 offered significant advantage over conventional H&E determination of mitotic rate.The American journal of surgical pathology 04/2013; · 4.06 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Thin melanomas are frequently associated with brisk lymphocytic infiltrate. Pigmented melanocytes are difficult to distinguish from melanophages, which are usually seen interspersed among lymphocytes on routine hematoxylin and eosin (HE) stained slides. As the presence of melanocytes in the papillary dermis characterizes the lesion as Clark II requiring the Breslow index, it is important to identify these cells properly and overcome such technical limitations. Even using immunohistochemistry staining for Melan-A and DAB as chromogens, this distinction is still difficult because the brown pigment formed by the chromogen DBA can not be easily differentiated from the brown melanin granules. We have introduced a simple modification on the technique, by replacing hematoxylin with Giemsa as counterstain. In this regard, the melanin pigment was decorated in green-blue while the Melan-A positive melanocytes were colored brown. Negatively stained melanophages contain only course green-blue granules of melanin in their cytoplasm. Thus, we were able to identify Melan-A positive cells in the papillary dermis accurately, determining microinvasion (Clark II) in 31 (77,5%) out of 40 in situ (Clark I) melanomas associated with brisk infiltrate. This technique is useful to distinguish melanophages and melanocytes interspersed among the lymphocytic infiltrate associated to thin melanomas, allowing detection of early invasion and avoiding Clark levels and Breslow index misinterpretation.Jornal Brasileiro de Patologia e Medicina Laboratorial 04/2006; 42(2):143-148.