Teng Xiaojing, Zhang Shuhong, Liu Wei, Bi Kuo, Zhang Lei
Department of Pathology, Beijing Friendship Hospital, Capital Medical University (Lymphoma Diagnostic Center, Beijing Institute of Clinical Medicine), Beijing, 100050, People's Republic of China.
Virchows Arch. 2017 May;470(5):567-573. doi: 10.1007/s00428-017-2097-z. Epub 2017 Feb 25.
Fluorescence in situ hybridisation (FISH) is a molecular cytogenetic technique, which is regularly applied to formalin-fixed paraffin-embedded (FFPE) tissue sections of a variety of cancers to assess chromosomal aberrations. However, high-quality FISH requires optimal enzymatic digestion, and insufficient digestion is not noted until the hybridisation signals are evaluated in the fluorescence microscope. As a consequence, FISH results may be unreliable, and the experiment might have to be repeated. To solve this problem, we developed a new method for real-time evaluation of enzymatic tissue digestion. Termination of enzyme activity at the proper time facilitates successful hybridisation, and experiments do not have to be repeated. We first performed FISH on 20 FFPE samples, which had been pepsin digested for different times, and this revealed distinct morphological changes within the nucleus and perinuclear space that were detectable by light microscopy. These observations suggested that the presence of intact and clear bare nuclei, surrounded by a translucent perinuclear space, might serve as an indicator of adequate digestion. We developed a protocol for assessment of this indicator, based on morphological features, and applied this to a collection of 400 tissue samples, partly of breast cancer and partly of different types of lymphoma, prior to FISH. The FISH success rate was 99.5% (398/400), which was significantly higher than that of the conventional method. In all successful cases, morphological signs of adequate digestion were paralleled by easily interpretable FISH signals. This new method for the real-time assessment of digestion quality improved the success rate of FISH and in addition was simple and rapid.
荧光原位杂交（FISH）是一种分子细胞遗传学技术，常用于多种癌症的福尔马林固定石蜡包埋（FFPE）组织切片，以评估染色体畸变。然而，高质量的FISH需要最佳的酶消化，而在荧光显微镜下评估杂交信号之前，消化不足是不易察觉的。因此，FISH结果可能不可靠，实验可能需要重复进行。为了解决这个问题，我们开发了一种实时评估组织酶消化的新方法。在适当时间终止酶活性有助于成功杂交，实验无需重复。我们首先对20个经胃蛋白酶消化不同时间的FFPE样本进行FISH，结果显示细胞核和核周空间内有明显的形态学变化，这些变化在光学显微镜下即可检测到。这些观察结果表明，完整且清晰的裸核周围环绕着半透明的核周空间，可能是消化充分的一个指标。我们基于形态学特征制定了一种评估该指标的方案，并在FISH之前将其应用于400个组织样本，其中部分为乳腺癌样本，部分为不同类型的淋巴瘤样本。FISH成功率为99.5%（398/400），显著高于传统方法。在所有成功的案例中，消化充分的形态学迹象与易于解读的FISH信号是并行的。这种实时评估消化质量的新方法提高了FISH的成功率，而且简单快速。
