Objective: To seek the optimal melanin-removal method for hematoxylin and eosin (HE) staining, immunohistochemistry and molecular detection. Methods: Thirty-eight paraffin tissue samples of malignant melanoma diagnosed at the Fujian Cancer Hospital, Fuzhou, China between January 2018 and March 2022 were collected and used to make a tissue microarray. Melanin in these cases was removed using warm hydrogen peroxide, double oxidation depigmentation, modified potassium permanganate-oxalic acid or trichloroisocyanuric acid, followed by HE staining. The cases were divided into two cohorts: one was subject to the one of the above four methods to remove melanin first, followed by immunohistochemistry (SOX-10, Ki-67, HMB45 and Melan A), while the other was subject to immunohistochemical staining first and then a melanin removal. Following that, seventeen melanin-rich paraffin tissue samples were collected and depigmented using the methods described above. DNA extraction was then done, followed by assessments of DNA content and quality. Moreover, the completeness of melanin removal, the effect on HE and immunohistochemical staining, and the quality of DNA were compared between the depigmented methods. Results: Regarding the effectiveness of melanin removal, the modified potassium permanganate-oxalic acid and the warm hydrogen peroxide methods were the most effective, and both showed residual melanin in only 5.26% (2/38) of the cases. The trichloroisocyanuric acid method showed residual melanin in 10.53% (4/38) of the cases. The worst was the double oxidation depigmentation method, which showed pigment residue in 15.79% (6/38) of the cases. For HE staining, the percentage of good staining with the warm hydrogen peroxide method was 92.11%, higher than the other three methods. For immunohistochemical staining, the mean staining scores of immunohistochemistry first followed by melanin removal with modified potassium permanganate-oxalic acid, double oxidation and trichloroisocyanuric acid were 20.84, 26.63 and 35.02, respectively. These immunohistochemical staining scores were higher than those of melanin removal first followed by immunohistochemistry (8.70, 15.41 and 21.22, respectively). The mean staining score of melanin removal by warm hydrogen peroxide method followed by immunohistochemistry was 33.57, superior to that of immunohistochemistry followed by the melanin removal (19.96). Moreover, the staining scores of HMB45, MelanA and Ki-67 with immunohistochemical staining followed by trichloroisocyanuric acid method were 36.45, 33.79, and 36.24, respectively, while the staining score of SOX10 with melanin removal by warm hydrogen peroxide followed by immunohistochemistry was 34.39. The DNA was significantly degraded by modified potassium permanganate-oxalic acid, double oxidation depigmentation and trichloroisocyanuric acid, whereas the mean concentration of DNA extracted after melanin removal by hydrogen peroxide method was 59.59 μg/L, substantially higher than that of DNA extracted without melanin removal (30.3 μg/L, P=0.001). The A260/A280 of DNA extracted after melanin removal by hydrogen peroxide was between 1.8 and 2.0 in all cases, and the A260/A230 was above 2.0 in sixteen cases, suggesting high purity of DNA. However, the DNA extracted without removing the melanin showed poor purity, with A260/A280 below 1.8 in eight cases and A260/A230 below 2.0 in sixteen cases. Conclusions: Warm hydrogen peroxide showed the least melanin residue, superior HE staining and a minimal effect on DNA purity/quality compared to the other three methods. It thus appears most suitable for PCR, NGS and other molecular detection. Melanin removal with trichloroisocyanuric acid after immunohistochemical staining has the least melanin residual, and thus could be the most convenient and efficient. However, it is noted that the efficacy of the same depigmentation method varies with different antibodies. Therefore, the optimal depigmentation method should be selected based on the specific markers of interest.
目的： 探索适用于HE染色、免疫组织化学染色和分子检测的最佳脱色素方法。 方法： 收集2018年1月至2022年3月福建省肿瘤医院病理科38例富含黑色素的黑色素瘤石蜡标本，制成组织芯片，分别采用水浴过氧化氢法、双重氧化脱色素法、改良高锰酸钾法、三氯异氰尿酸法脱黑色素后行HE染色。之后将切片分成2个队列，第1个队列分别采用上述4种方法脱色素后再行免疫组织化学染色（SOX10、Ki-67、HMB45和Melan A）；第2个队列先行同样指标的免疫组织化学染色后再分别采用上述4种方法脱色素。另收集其中色素含量较高的17例石蜡标本，采用上述4种方法脱黑色素后提取DNA，进行DNA浓度及质量的测定。比较分析不同脱色素方法的脱色素能力，以及对HE和免疫组织化学染色的效果、DNA浓度和质量的影响。 结果： 在脱黑色素能力方面，改良高锰酸钾法和水浴过氧化氢法脱色素效果最好，均仅5.26%（2/38）有少量色素残留；其次是三氯异氰尿酸法，10.53%（4/38）有大量色素残留；最差的是双重氧化脱色素法，15.79%（6/38）有大量色素残留。在HE染色方面，水浴过氧化氢法脱色素后染色好的比例达92.11%，优于其他3种方法。在免疫组织化学染色方面，改良高锰酸钾法、双重氧化脱色素法和三氯异氰尿酸法，先免疫组织化学染色后脱色素的染色评分均值分别为20.84、26.63和35.02，均优于先脱色素后免疫组织化学的染色评分均值（8.70、15.41和21.22）；水浴过氧化氢法，先脱色素后免疫组织化学的染色评分均值为33.57，优于先免疫组织化学染后脱色素的染色评分均值（19.96）。HMB45、Melan A和Ki-67这3个指标采用先免疫组织化学染色后三氯异氰尿酸脱色素的染色评分分别为36.45、33.79和36.24，效果最佳；SOX10采用先水浴过氧化氢法脱色素后免疫组织化学染色的评分为34.39，效果最佳。在DNA浓度及质量方面，采用双重氧化脱色素法、改良高锰酸钾法及三氯异氰尿酸法脱色素后，DNA降解严重；而水浴过氧化氢法脱色素后提取的DNA平均浓度为59.59 μg/L，显著高于未经脱色素处理的平均DNA浓度（30.3 μg/L，P=0.001）；DNA的吸光度值A260/A280均在1.8~2.0之间，A260/A230 16例在2.0以上，而未经脱色素处理提取的DNA中8例A260/A280<1.8，16例A260/A230<2.0。 结论： 对富含黑色素的黑色素瘤组织进行HE染色和分子检测时，宜选用水浴过氧化氢法脱色素；免疫组织化学染色后采用三氯异氰尿酸法脱色素，色素残留少，染色效果佳，且方便快捷。同一种脱色素方法对不同的抗体染色效果不同，可根据要检测的抗体选择最佳的脱色素方法。.