potassium-permanganate and Melanoma

Pathologists diagnose diseases by observing the histologic and cellular morphology microscopically. However, the high pigmentation in melanin-containing tumors can hide the tumor cell structures, making diagnosing challenging. Previously, hydrogen peroxide and potassium permanganate were utilized for melanin bleaching with several limitations. For instance, hydrogen peroxide has a weak bleaching ability, and the process is time-consuming (12 h). Meanwhile, potassium permanganate affects the antigenicity of antigens and is unsuitable for immunohistochemical (IHC) staining. In this study, the hypochlorous acid (HClO) solution was applied to hematoxylin-eosin and IHC staining of melanin tissue sections. The study discovered that 1% HClO could completely bleach melanin particles in tumor tissues in a short period (19.95 ± 2.53 min) without compromising the hematoxylin-eosin staining. In addition, 2% HClO was utilized for bleaching at room temperature for 61.17 ± 4.32 minutes after the tissue was incubated with 3,3'-diaminobenzidine in IHC staining. This treatment effectively removed melanin without negatively impacting 3,3'-diaminobenzidine signal expression, thus ensuring that the sections met the necessary diagnostic requirements. Therefore, this method could facilitate pathologists in disease diagnosis of melanin-containing tissues.

Topics: 3,3'-Diaminobenzidine; Eosine Yellowish-(YS); Hematoxylin; Humans; Hydrogen Peroxide; Hypochlorous Acid; Melanins; Melanoma; Potassium Permanganate; Staining and Labeling

This study investigated the effects of trichloroisocyanuric acid (TCCA) on the bleaching and morphology of melanin-containing pathological sections. The pathological sections of 27 patients with high melanin content were bleached with 0.5% potassium permanganate, 10% hydrogen peroxide, and different concentrations of TCCA. Significant differences were found among the blank control group, 1% TCCA group (P < 0.0001). The hematoxylin-eosin (HE) score of the "recovery pH" HE staining group after treatment with 1% TCCA was similar to that of the "Conventional HE" scheme group (P > 0.05). The morphological diagnostic scores of 50 cases of pathological sections with different melanin content before and after TCCA bleaching were compared. The results showed a significant difference in the diagnostic score between the middle- and high-melanin content groups before and after 1% TCCA bleaching (P < 0.05). Immunohistochemical staining was performed on meningeal melanoma tissue. For this, 8% TCCA solution was used to remove melanin after Ki67, S100, and β-catenin immunohistochemical staining. After bleaching with TCCA, the staining and positioning of each marker with different localization were accurate and the background was clear. The same results were also shown with EBER-ISH. This study concluded that 1% TCCA could be used for HE staining of pathological sections containing melanin, and "restore pH" HE scheme as the staining method after TCCA melanin removal. Further, 8% TCCA was used for bleaching after immunohistochemical DAB staining. Melanin can be completely removed, and sections can meet diagnostic needs.

Topics: beta Catenin; Eosine Yellowish-(YS); Hematoxylin; Humans; Hydrogen Peroxide; Immunohistochemistry; Ki-67 Antigen; Melanins; Melanoma; Potassium Permanganate; Triazines

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.

Topics: Antigens, Neoplasm; Azure Stains; Humans; Immunoenzyme Techniques; Melanins; Melanocytes; Melanoma; Melanoma-Specific Antigens; Melanophores; Neoplasm Proteins; Pigmentation; Potassium Permanganate; S100 Proteins; Skin Neoplasms; Staining and Labeling

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.

Topics: Actins; Antigens, CD; Antigens, CD20; Antigens, CD34; Antigens, Differentiation, Myelomonocytic; Antigens, Neoplasm; CD3 Complex; Desmin; Humans; Immunohistochemistry; Leukocyte Common Antigens; Melanins; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Oxalates; Oxidation-Reduction; Pigmentation; Platelet Endothelial Cell Adhesion Molecule-1; Potassium Permanganate; Reproducibility of Results; S100 Proteins; Sensitivity and Specificity; Skin; Skin Neoplasms; Vimentin; von Willebrand Factor

To investigate how nuclear morphometric variables, tumor thickness (measured according to Breslow), invasion depth (classified according to Clark), nuclear DNA content and type of DNA histogram are associated with each other in primary malignant melanomas of the skin.. Image analysis DNA cytometry and nuclear morphometry were performed on 85 primary skin melanomas. The relationships of size, sphericity and DNA content of melanoma cell nuclei; melanoma thickness; and Clark level were analyzed in detail. The effect of melanin bleaching on DNA cytometry results was studied.. Melanoma thickness correlated with nuclear size in aneuploid, but not diploid, melanomas. The prevalence of aneuploidy did not increase with tumor thickness. In aneuploid melanomas the proportion of cells with higher-than-diploid and higher-than-tetraploid DNA content increased with tumor size.. Aneuploidy is as common in thin as in thick melanomas. Genetic instability in aneuploid melanomas correlates with melanoma thickness. This correlation in aneuploid melanomas partially explains the correlation between nuclear size and melanoma thickness. In diploid melanomas no correlation was observed between nuclear size and melanoma thickness. DNA cytometry is a valuable tool for studies on the background of phenotypic changes in skin melanomas.

Topics: Adult; Aged; Aged, 80 and over; Aneuploidy; Cell Nucleus; Diploidy; DNA, Neoplasm; Female; Humans; Image Cytometry; Image Processing, Computer-Assisted; Male; Melanins; Melanoma; Middle Aged; Potassium Permanganate; Skin Neoplasms

This study addresses two questions: i) which antigens can withstand bleaching by 2.5 g/L of potassium permanganate followed by 10 g/L of oxalate, before immunohistochemical staining; and ii) are any other steps in the immunohistochemical staining technique resistant to bleaching? A panel of 10 antigens was stained immunohistochemically and the results compared with staining performed with a bleaching step interpolated at different steps in the procedures. Four antigens (HMB-45, S-100, factor VIII-related antigen and collagen type IV) were unaffected by bleaching; two antigens (CD-20 and CD-45) had their staining enhanced by bleaching; one had the staining reduced (hsp27); and in three it was abolished (CD-45Ro, CD-31 and Ulex/anti-ulex antibody) by bleaching. Two antibodies (UCHL-1 and L-26) showed evidence for altered specificity following bleaching. None of the steps after application of the primary antibody was resistant to bleaching. Three chromagens used for peroxidase demonstration-amino ethyl-carbazole, diaminobenzidine and chloro-naphthol-were also found to be sensitive to bleaching. While some antigens were resistant to the effects of bleaching, some were not, and no other step in the immunohistochemical procedure could withstand bleaching.

Topics: Antigens, Neoplasm; Coloring Agents; Female; Humans; Immunoenzyme Techniques; In Vitro Techniques; Male; Melanins; Melanoma; Oxalates; Oxalic Acid; Potassium Permanganate; Uveal Neoplasms

Information on the composition of melanins is obtained by analysis both of 4-amino-3-hydroxyphenylalanine (AHP) after hydriodic acid degradation and of pyrrole-2,3,5-tricarboxylic acid (PTCA) after potassium permanganate oxidation. Analysis of thiazole-4,5-dicarboxylic acid (TDCA) and pyrrole-2,3-dicarboxylic acid (PDCA) after permanganate oxidation, provides additional information on the composition, TDCA on pheomelanin residues, and PDCA on indolic residues without carboxy groups. Using model melanins formed from dopa and cysteinyldopa in different proportions, we found the TDCA/(PTCA+PDCA) ratio to yield a reliable estimate of the relative proportions of pheomelanin and eumelanin. The PDCA/PTCA ratio reflects the relationship between indole residues with and without carboxy groups. We have analyzed degradation products from cultures of IGR 1, an extensively studied melanoma cell line. Cell cultures were harvested after 2, 4, and 7 days. Culture media were changed after 2 days in all series, and also after 4 days in one series harvested at 7 days. Cells without medium change had seven times the amount of melanin found in cultures with medium change. The PDCA/PTCA ratio decreased with increasing amounts of melanin. With increased melanization, eumelanin is increased relatively more than pheomelanin. The cell content of 5-S-cysteinyldopa (5-S-CD) was similar in all cultures, while 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MICA), a eumelanin precursor metabolite, was found in increased amounts of media of heavily pigmented cultures.

Topics: Chromatography, Liquid; Cysteinyldopa; Dicarboxylic Acids; Humans; Hydrolysis; Mass Spectrometry; Melanins; Melanoma; Monophenol Monooxygenase; Neoplasm Proteins; Oxidation-Reduction; Potassium Permanganate; Pyrroles; Sulfur; Thiazoles; Tumor Cells, Cultured; Tyrosine

With the increasing use of immunoperoxidase technics, it may be difficult to differentiate between the dark staining of 3,3'-diaminobenzidine (DAB) compound reaction product and melanin pigment. The latter may be particularly observed in skin. Samples of both normal skin and melanotic malignant melanoma were treated for the removal of melanin by standard technics both before and after a peroxidase-antiperoxidase (PAP) immunohistologic sequence. Many methods for the removal of melanin pigment resulted in diminished DAB staining intensity. Some also caused cellular disruption. However, the method of choice was found to be treatment with 0.25 g/dL potassium permanganate and 1 g/dL oxalic acid before the immunoperoxidase sequence.

Topics: 3,3'-Diaminobenzidine; Humans; Immunoenzyme Techniques; Melanins; Melanoma; Oxalates; Oxalic Acid; Potassium Permanganate; Skin Neoplasms

A method for the quantitative analysis of eumelanin and pheomelanin in tissues, e.g., hair and melanoma, is described. The method is simple and rapid because it does not require the isolation of melanins from the tissues. The rationale is that permanganate oxidation of eumelanin yields pyrrole-2,3,5-tricarboxylic acid (PTCA) which may serve as a quantitatively significant indicator of eumelanin, while hydriodic acid hydrolysis of pheomelanin yields aminohydroxyphenylalanine (AHP) as a specific indicator of pheomelanin. The degradation products, PTCA and AHP, can be readily analyzed by high-performance liquid chromatography. Chemical degradations of synthetic melanins, prepared from dopa, 5-S-cysteinyldopa, and their mixtures in various ratios, gave PTCA and AHP in yields that correlated with the dopa/5-S-cysteinyldopa ratio. The PTCA/AHP ratio as well as the contents of PTCA and AHP reflected well the type of melanogenesis in hair and melanomas. The amounts needed for each degradation were 0.5 mg of melanin, 2 mg of hair, and 5 mg of tissue samples. As many as 20 samples can be analyzed within 3 working days.

Topics: Animals; Chemical Phenomena; Chemistry; Chromatography, Liquid; Guinea Pigs; Hair; Humans; Hydrolysis; Melanins; Melanocytes; Melanoma; Mice; Mice, Inbred C57BL; Oxidation-Reduction; Potassium Permanganate

In this study, a method is provided for analyzing quantitatively the content and the class of melanin pigments in the tissues, e.g., hair and melanoma. The method is simple and rapid because it does not require the isolation of melanins from the tissues. The rationale was that permanganate oxidation of eumelanin yields pyrrole-2,3,5-tricarboxylic acid (PTCA) as its major pyrrolic product, which may serve as a quantitatively significant indicator of eumelanin, while hydriodic acid hydrolysis of pheomelanin yields amino-hydroxyphenylalanine (AHP) as a specific indicator of pheomelanin. The degradation products, PTCA and AHP, were determined by high-performance liquid chromatography. Sepia melanosome-melanin and synthetic 5-S-cysteinyldopa-melanin served as reference standards of eumelanin and pheomelanin, respectively. Our method provided data that corresponded well to the content and class of melanins in normal hair. Based on this control study, it was found that the melanins in the melanosomes of both B16 and Harding-Passey (HP) melanomas were eumelanic and that the melanin content in B16 melanosomes was more than 10 times higher than that in HP melanosomes, though these two melanosomes revealed distinct colors and ultrastructures, i.e., brown-black, eumelanosome-like granules in B16 and reddish- or light-brown, pheomelanosome-like granules in HP.

Topics: Animals; Fishes; Hair; Humans; Hydrolysis; Melanins; Melanoma; Mice; Neoplasms, Experimental; Oxidation-Reduction; Potassium Permanganate; Proline