BACKGROUND Cancer metastasis is the primary cause of cancer-related deaths and remains incurable. Current clinical methods for predicting metastatic recurrence are not sensitive enough to detect individual cancer cells in the body; therefore, current efforts are directed toward liquid biopsy-based assays to capture circulating and disseminated tumor cells (CTCs and DTCs) in the blood and bone marrow, respectively. The most promising strategy is fluorescence-based immunostaining using cancer cell-specific markers. However, despite recent efforts to develop robust processing and staining platforms, results from these platforms have been discordant among groups, particularly for DTC detection. While the choice of cancer cell-specific markers is a large factor in this discordance, we have found that marker-independent factors causing false signal are just as critical to consider. Bone marrow is particularly challenging to analyze by immunostaining because endogenous immune cell properties and bone marrow matrix components typically generate false staining. For immunostaining of whole tumor tissue containing ample cancer cells, this background staining can be overcome. Application of fluorescent-based staining for rare cells, however, is easily jeopardized by immune cells and autofluorescence that lead to false signal. RESULTS We have specifically found two types of background staining in bone marrow samples: autofluorescence of the tissue and non-specific binding of secondary antibodies. We systematically optimized a basic immunofluorescence protocol to eliminate this background using cancer cells spiked into human bone marrow. This enhanced the specificity of automated scanning detection software. Our optimized protocol also outperformed a commercial rare cell detection protocol in detecting candidate DTCs from metastatic patient bone marrow. CONCLUSIONS Robust optimization to increase the signal-to-noise ratio of immunofluorescent staining of bone marrow is required in order to achieve the necessary sensitivity and specificity for rare cell detection. Background immunofluorescent staining in bone marrow causes uncertainty and inconsistency among investigators, which can be overcome by systematically addressing each contributing source. Our optimized assay eliminates sources of background signal, and is adaptable to automated staining platforms for high throughput analysis.

中文翻译：

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骨髓播散性肿瘤细胞免疫荧光检测的优化。

背景癌症转移是癌症相关死亡的主要原因并且仍然无法治愈。目前预测转移性复发的临床方法不够灵敏，无法检测体内的单个癌细胞；因此，目前的努力是针对基于液体活检的分析，以分别捕获血液和骨髓中的循环和播散性肿瘤细胞（CTC 和 DTC）。最有希望的策略是使用癌细胞特异性标志物的基于荧光的免疫染色。然而，尽管最近努力开发强大的处理和染色平台，但这些平台的结果在各组之间不一致，特别是对于 DTC 检测。虽然癌细胞特异性标志物的选择是造成这种不一致的一个重要因素，我们发现导致错误信号的与标记无关的因素同样重要。通过免疫染色分析骨髓特别具有挑战性，因为内源性免疫细胞特性和骨髓基质成分通常会产生假染色。对于含有大量癌细胞的整个肿瘤组织的免疫染色，可以克服这种背景染色。然而，对稀有细胞应用基于荧光的染色很容易受到免疫细胞和导致错误信号的自体荧光的危害。结果 我们在骨髓样本中特别发现了两种类型的背景染色：组织的自发荧光和二抗的非特异性结合。我们系统地优化了一个基本的免疫荧光方案，以使用掺入人类骨髓的癌细胞来消除这种背景。这增强了自动扫描检测软件的特异性。我们的优化方案在检测来自转移性患者骨髓的候选 DTC 方面也优于商业稀有细胞检测方案。结论 需要进行稳健优化以提高骨髓免疫荧光染色的信噪比，以实现稀有细胞检测所需的灵敏度和特异性。骨髓中的背景免疫荧光染色会导致研究人员之间的不确定性和不一致，这可以通过系统地解决每个贡献源来克服。我们优化的分析消除了背景信号源，