Background: Metastatic melanoma in sentinel lymph nodes is often elusive to detect with morphology alone. Per American Joint Committee on Cancer staging guidelines, a single atypical melanocyte in lymph node qualifies as metastasis, whether identified by morphology or immunohistochemistry, but single cell staining must be convincing. We propose that the use of a second immunohistochemical run performed on a single slide will allow for more confident diagnosis of micrometastases. Materials and Methods: We designed a technical study to determine whether a second antibody application on previously stained slides can successfully detect the same population of cells. Melanocytic neoplasms were stained with SOX-10 using Ventana Benchmark Ultra stainers, coverslipped, and examined, followed by coverslip removal and application of MART-1 (Ventana A103). The order of antibody application and chromagen detection kit (AP-RED vs. DAB) was reversed to establish reliability and robustness of the protocol. Results: All melanocytes marked with SOX-10 and MART-1, and produced a range of staining quality that varied based on order of stain application and chromagen kit were used. The optimal combination was red MART-1 applied first followed by brown SOX-10 applied second. Conclusions: Consecutive staining of melanocytes with SOX-10 and MART-1 may improve diagnostic confidence of melanocyte identification, particularly in detection of single cell, micrometastases in sentinel lymph nodes or in situations where dual immunohistochemical stains may be unavailable.

中文翻译：

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单张玻片上的连续免疫组织化学染色可有效确认黑素细胞的来源

背景：前哨淋巴结中的转移性黑色素瘤通常难以仅用形态学检测。根据美国癌症联合委员会分期指南，淋巴结中的单个非典型黑素细胞符合转移，无论是通过形态学还是免疫组织化学鉴定，但单细胞染色必须令人信服。我们建议在单张载玻片上进行第二次免疫组化检测，可以更可靠地诊断微转移。材料和方法：我们设计了一项技术研究，以确定在先前染色的载玻片上应用二抗是否可以成功检测到相同的细胞群。黑色素细胞肿瘤使用 Ventana Benchmark Ultra 染色机用 SOX-10 染色，盖上盖玻片并检查，然后去除盖玻片并应用 MART-1 (Ventana A103)。抗体应用和色原检测试剂盒（AP-RED 与 DAB）的顺序颠倒，以建立协议的可靠性和稳健性。结果：所有黑色素细胞都用 SOX-10 和 MART-1 进行标记，并产生一系列染色质量，根据染色应用的顺序和染色原试剂盒的使用而有所不同。最佳组合是先涂红色 MART-1，然后再涂棕色 SOX-10。结论：用 SOX-10 和 MART-1 连续染色黑色素细胞可以提高黑色素细胞鉴定的诊断信心，特别是在检测单细胞、前哨淋巴结微转移或双重免疫组化染色可能不可用的情况下。结果：所有黑色素细胞都用 SOX-10 和 MART-1 进行标记，并产生一系列染色质量，根据染色应用的顺序和染色原试剂盒的使用而有所不同。最佳组合是先涂红色 MART-1，然后再涂棕色 SOX-10。结论：用 SOX-10 和 MART-1 连续染色黑色素细胞可以提高黑色素细胞鉴定的诊断信心，特别是在检测单细胞、前哨淋巴结微转移或双重免疫组织化学染色可能无法使用的情况下。结果：所有黑色素细胞都用 SOX-10 和 MART-1 进行标记，并产生一系列染色质量，根据染色应用的顺序和染色原试剂盒的使用而有所不同。最佳组合是先涂红色 MART-1，然后再涂棕色 SOX-10。结论：用 SOX-10 和 MART-1 连续染色黑色素细胞可以提高黑色素细胞鉴定的诊断信心，特别是在检测单细胞、前哨淋巴结微转移或双重免疫组织化学染色可能无法使用的情况下。