Melanins are naturally occurring pigments in both normal and pathologic tissues. Two common bleaching processes are potassium permanganate followed by oxalic acid treatment and dilute hydrogen peroxide (H2O2) process. The potassium permanganate/oxalic acid method is faster and more easily incorporated in conventional daily immunostaining protocols, whereas the dilute H2O2 method requires 24 hours. This study aimed to reduce melanin bleaching time by using a 10% H2O2 dilution. First, reaction time was reduced to 30 minutes by raising the temperature to 65°C. Second, containers with high thermal conductivity were used to improve bleaching effectiveness. Experimental comparisons of melanin treatments with H2O2 contained in an iron jar, a glass coplin jar, and a plastic steel jar obtained bleaching time of 20, 30, and 40 minutes, respectively. These modifications of the conventional bleaching method significantly improve the speed and efficiency of the procedure and are recommended when performing immunohistochemical studies.

The method is particularly effective for removing melanin pigment to facilitate histopathological examination of cytomorphology and for obtaining an unmasked tissue section for immunohistochemical analysis.

Diagnosing melanocytic lesions is among the most challenging problems in the practice of pathology. The difficulty of physically masking melanin pigment and the similarity of its color to commonly used chromogens often complicate examination of the cytomorphology and immunohistochemical staining results for tumor cells. Melanin bleach can be very helpful for histopathological diagnosis of heavily pigmented melanocytic lesions. Although various depigmentation methods have been reported, no standardized methods have been developed. This study developed a fully automated platform that incorporates hydrogen peroxide-based melanin depigmentation in an automated immunohistochemical analysis. The utility of the method was tested in one cell block of malignant melanoma cells in pleural effusion, ten ocular melanoma tissue samples, and ten cutaneous melanoma tissue samples. Our results demonstrated that the proposed method, which can be performed in only 3 hours, effectively preserves cell cytomorphology and immunoreactivity. The method is particularly effective for removing melanin pigment to facilitate histopathological examination of cytomorphology and for obtaining an unmasked tissue section for immunohistochemical analysis.

Introduction. The difficulty in diagnosis of severe melanocytic lesions is a problem to be overcome in pathological practice. Melanin bleaching is an effective approach to ameliorate melanin disturbances in severely pigmented lesions. Although various methods for improving melanin pigmentation in immunohistochemical staining have been reported, these depigmentation methods still need to be optimized and standardized. In this study, the coloring efficiency of 3,3′-diaminobenzidine (DAB) and alkaline phosphatase (AP) after melanin depigmentation was compared under the automatic immunohistochemical staining platform. Methods. The applicability of the optimized depigmentation method was validated in 10 formalin-fixed paraffin-embedded (FFPE) blocks of ocular melanoma tissues. Specimens were demelaninized with 10% hydrogen peroxide at 60°C for immunohistochemical staining (Melan-A and SOX10), and tissue chromogenic staining was performed with DAB and AP detection systems, respectively. Results. The optimized depigmentation method including immunohistochemistry (IHC) could be completed in 3 h, effectively preserving cell morphology and immunoreactivity. Among these, the color-rendering effect and contrast of AP are better than DAB. Conclusion. This optimized method can effectively remove melanin and improve the accuracy of IHC staining interpretation. AP staining has better visibility and readability without the interference of residual melanin. The comparison results showed that after melanin depigmentation, the immunohistochemical staining agent was replaced with red AP, which avoided the misjudgment caused by brown DAB when melanin depigmentation was incomplete. This improved method can be applied to future histopathological and immunohistochemical staining of melanin-deposited tissues.