Wan Weisong, Wang Le, Zeng Yuye, Hu Yuchang, Liu Yufei
The First Clinical Medical College of China, Three Gorges University, Yichang, 443000, China.
Institute of Pathology, China Three Gorges University, Yichang, 443000, China.
Sci Rep. 2025 Feb 10;15(1):5001. doi: 10.1038/s41598-025-89186-8.
This study seeks to address the challenge of melanoma identification in immunohistochemical (IHC) diagnosis, which is complicated by the similar coloration of melanin and DAB (Diaminobenzidine) staining, by introducing methylene blue counterstaining as an innovative solution. We compared the effectiveness of methylene blue counterstaining with that of the traditional hydrogen peroxide bleaching method in the diagnosis of melanoma. The study included 46 paraffin-embedded melanoma samples, and the staining efficacy for markers such as Melan A, HMB-45, PRAME, and Ki-67 was assessed via both methods. The results demonstrated that methylene blue counterstaining effectively converted the brownish-yellow melanin granules to a deep green color, significantly enhancing contrast and clarity with DAB staining. The average contrast and clarity scores for the methylene blue counterstaining method were 1.96 ± 0.21 and 1.91 ± 0.28, respectively, which were significantly greater than those of the conventional IHC group and the hydrogen peroxide bleaching group (P < 0.01). Furthermore, methylene blue counterstaining did not cause noticeable tissue damage or cellular morphology distortion, with tissue integrity scores comparable to those of the conventional IHC group (P > 0.05). Although the contrast and clarity also improved in the hydrogen peroxide bleaching group, it resulted in a significant decrease in tissue integrity (P < 0.01). This study is the first to apply methylene blue counterstaining in melanoma IHC analysis, demonstrating its advantages in enhancing staining quality, simplifying procedural workflows, and preserving antigenicity. This method provides a novel and effective tool for the pathological diagnosis of melanoma, potentially improving diagnostic accuracy and reliability.
本研究旨在应对免疫组织化学（IHC）诊断中黑色素瘤识别的挑战，黑色素和二氨基联苯胺（DAB）染色的相似颜色使这一挑战变得复杂，为此引入亚甲蓝复染作为一种创新解决方案。我们比较了亚甲蓝复染与传统过氧化氢漂白方法在黑色素瘤诊断中的有效性。该研究纳入了46个石蜡包埋的黑色素瘤样本，并通过这两种方法评估了Melan A、HMB - 45、PRAME和Ki - 67等标志物的染色效果。结果表明，亚甲蓝复染有效地将棕黄色的黑色素颗粒转化为深绿色，显著增强了与DAB染色的对比度和清晰度。亚甲蓝复染法的平均对比度和清晰度评分分别为1.96±0.21和1.91±0.28，显著高于传统免疫组化组和过氧化氢漂白组（P<0.01）。此外，亚甲蓝复染未造成明显的组织损伤或细胞形态畸变，组织完整性评分与传统免疫组化组相当（P>0.05）。虽然过氧化氢漂白组的对比度和清晰度也有所改善，但它导致组织完整性显著下降（P<0.01）。本研究首次将亚甲蓝复染应用于黑色素瘤免疫组化分析，证明了其在提高染色质量、简化操作流程和保留抗原性方面的优势。该方法为黑色素瘤的病理诊断提供了一种新颖有效的工具，有望提高诊断的准确性和可靠性。
