Background The Fluorescence In Situ Hybridization (FISH) technique is a very useful tool for diagnostic and prognostic purposes in molecular pathology. However, clinical testing on patient tissue is challenging due to variables of tissue processing that can influence the quality of the results. This emphasizes the necessity of a standardized FISH protocol with a high hybridization efficiency. We present a pretreatment protocol that is easy, reproducible, cost-effective, and facilitates FISH on all types of patient material simultaneously with good quality results.During validation, FISH analysis was performed simultaneously on formalin-fixed paraffin-embedded, fresh frozen and cytological patient material in combination with commercial probes using our optimized one-fits-all pretreatment protocol. An optimally processed sample is characterized by strong specific signals, intact nuclear membranes, non-disturbing autofluorescence and a homogeneous DAPI staining. Results In our retrospective cohort of 3881 patient samples, overall 93% of the FISH samples displayed good quality results leading to a patient diagnosis. All FISH were assessed on quality aspects such as adequacy and consistency of signal strength (brightness), lack of background and / or cross-hybridization signals, and additionally the presence of appropriate control signals were evaluated to assure probe accuracy. In our analysis 38 different FISH probes from 3 commercial manufacturers were used (Cytocell, Vysis and ZytoLight). The majority of the patients in this cohort displayed good signal quality and barely non-specific background fluorescence on all tissue types independent of which commercial probe was used. Conclusion The optimized one-fits-all FISH method is robust, reliable and reproducible to deliver an accurate result for patient diagnostics in a lean workflow and cost-effective manner. This protocol can be used for widespread application in cancer and non-cancer diagnostics and research.

中文翻译：

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在福尔马林固定石蜡包埋、新鲜冷冻和细胞学载玻片上促进荧光原位杂交的万能预处理方案。

背景荧光原位杂交（FISH）技术是一种非常有用的工具，用于分子病理学中的诊断和预后目的。然而，由于组织处理的变量会影响结果的质量，因此对患者组织进行临床测试具有挑战性。这强调了具有高杂交效率的标准化 FISH 协议的必要性。我们提出了一种预处理方案，该方案简单、可重复、具有成本效益，并有助于同时对所有类型的患者材料进行 FISH，并获得高质量的结果。在验证过程中，同时对福尔马林固定石蜡包埋、新鲜冷冻和细胞学进行 FISH 分析患者材料与商业探针结合使用我们优化的一刀切预处理方案。经过优化处理的样品的特点是强特异性信号、完整的核膜、无干扰的自发荧光和均匀的 DAPI 染色。结果 在我们的 3881 名患者样本的回顾性队列中，总体 93% 的 FISH 样本显示出高质量的结果，从而导致患者诊断。所有 FISH 都在质量方面进行了评估，例如信号强度（亮度）的充分性和一致性、缺乏背景和/或交叉杂交信号，另外还评估了适当控制信号的存在以确保探针的准确性。在我们的分析中，使用了来自 3 个商业制造商（Cytocell、Vysis 和 ZytoLight）的 38 种不同的 FISH 探针。该队列中的大多数患者在所有组织类型上都显示出良好的信号质量和几乎没有非特异性背景荧光，而与使用哪种商业探针无关。结论 优化的一刀切 FISH 方法稳健、可靠且可重复，以精简的工作流程和具有成本效益的方式为患者诊断提供准确的结果。该协议可广泛应用于癌症和非癌症诊断和研究。