Supplemental Digital Content is available in the text. Diagnostic assays for molecular alterations highly correlated with prognosis, predictive efficacy or safety of therapeutics are valuable clinical tools and in some cases approved as companion diagnostics (CDx) by the Federal Food and Drug Administration. For example, assays that determine echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) translocation status have been approved as CDx assay for therapies that target this molecular alteration. Characterizing the parameters that may compromise diagnostic accuracy for molecular biomarkers is critical for optimal patient care. To investigate the impact of pre-analytical handling and processing of tumor tissue on commonly used diagnostic immunohistochemistry-based assays for ALK and mesenchymal epithelial transition protein [c-mesenchymal epithelial transition (c-MET)], we investigated the effects of cold ischemia, fixative type, fixation time, and cut-slide age on staining consistency and intensity using human lung xenograft tumor tissue. Cold ischemia times for up to 5 to 6 hours for c-MET or ALK, respectively had minimal impact on staining. The optimal fixation conditions for both assays were found to be at least 6 hours and up to 48 hours for c-MET or 72 hours for ALK, in 10% neutral buffered formalin and Zinc formalin. The ALK antigen demonstrated marked staining intensity differences across non-neutral buffered formalin fixative types and times. Finally, cut-slide age influenced assay performance for both ALK and c-MET, with maximum stability observed when cut slides were stored at ambient temperatures (30°C) for no longer than 3, and 5 months, respectively. This study highlights the potential for pre-analytical factors to confound diagnostic test result interpretation.

中文翻译：

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分析前条件对肺标志物抗原性的影响

文本中提供了补充数字内容。与预后、预测疗效或治疗安全性高度相关的分子改变的诊断分析是有价值的临床工具，在某些情况下被联邦食品和药物管理局批准为伴随诊断 (CDx)。例如，确定棘皮动物微管相关蛋白样 4 (EML4)-间变性淋巴瘤激酶 (ALK) 易位状态的检测方法已被批准作为针对这种分子改变的疗法的 CDx 检测方法。表征可能影响分子生物标志物诊断准确性的参数对于优化患者护理至关重要。为了研究肿瘤组织的分析前处理和处理对 ALK 和间充质上皮转化蛋白 [c-间充质上皮转化 (c-MET)] 的常用诊断性免疫组织化学测定的影响，我们研究了冷缺血的影响，使用人肺异种移植肿瘤组织染色一致性和强度的固定剂类型、固定时间和切片年龄。c-MET 或 ALK 的冷缺血时间分别长达 5 至 6 小时，对染色的影响最小。在 10% 中性缓冲福尔马林和福尔马林锌中，发现两种测定的最佳固定条件是 c-MET 至少 6 小时至 48 小时或 ALK 72 小时。ALK 抗原在非中性缓冲福尔马林固定剂类型和时间之间表现出显着的染色强度差异。最后，切片年龄影响 ALK 和 c-MET 的测定性能，当切片切片在环境温度 (30°C) 下分别储存不超过 3 个月和 5 个月时，观察到最大稳定性。这项研究强调了分析前因素混淆诊断测试结果解释的可能性。