Second Department of Pathology, School of Medicine, University of Brescia, Italy.
Eur J Histochem. 2012 May 4;56(2):e12. doi: 10.4081/ejh.2012.12.
Fixation is a critical step in the preparation of tissues for histopathology. The objective of this study was to investigate the effects of different fixatives vs formalin on proteins and DNA, and to evaluate alternative fixation for morphological diagnosis and nucleic acid preservation for molecular methods. Forty tissues were fixed for 24 h with six different fixatives: the gold standard fixative formalin, the historical fixatives Bouin and Hollande, and the alternative fixatives Greenfix, UPM and CyMol. Tissues were stained (Haematoxylin-Eosin, Periodic Acid Schiff, Trichromic, Alcian-blue, High Iron Diamine), and their antigenicity was determined by immunohistochemistry (performed with PAN-CK, CD31, Ki-67, S100, CD68, AML antibodies). DNA extraction, KRAS sequencing, FISH for CEP-17, and flow cytometry analysis of nuclear DNA content were applied. For cell morphology the alternative fixatives (Greenfix, UPM, CyMol) were equivalent to formalin. As expected, Hollande proved the best fixative for morphology. The morphology obtained with Bouin was comparable to that with formalin. Hollande was the best fixative for histochemistry. Bouin proved equivalent to formalin. The alternative fixatives were equivalent to formalin, although with greater variability in haematoxylin-eosin staining. It proved possible to obtain immunohistochemical staining largely equivalent to that following formalin-fixation with the following fixatives: Greenfix, Hollande, UPM and CyMol. The tissues fixed in Bouin did not provide results comparable to those obtained with formalin. The DNA extracted from samples fixed with alternative fixatives was found to be suitable for molecular analysis.
固定是组织病理学制备的关键步骤。本研究旨在探讨不同固定剂与福尔马林对蛋白质和 DNA 的影响，并评估替代固定剂在形态诊断和核酸保存方面的应用。将 40 个组织用六种不同的固定剂固定 24 小时：金标准固定剂福尔马林、历史固定剂 Bouin 和 Hollande 以及替代固定剂 Greenfix、UPM 和 CyMol。组织进行染色（苏木精-伊红、过碘酸雪夫、三色、阿尔辛蓝、高铁二胺），并用免疫组织化学（用 PAN-CK、CD31、Ki-67、S100、CD68、AML 抗体）检测其抗原性。进行 DNA 提取、KRAS 测序、CEP-17 的 FISH 检测以及核 DNA 含量的流式细胞术分析。对于细胞形态，替代固定剂（Greenfix、UPM、CyMol）与福尔马林相当。正如预期的那样，Hollande 被证明是最适合形态学的固定剂。Bouin 得到的形态与福尔马林相当。Hollande 是最适合组织化学的固定剂。Bouin 与福尔马林相当。替代固定剂与福尔马林相当，尽管在苏木精-伊红染色方面存在更大的变异性。Greenfix、Hollande、UPM 和 CyMol 等固定剂可以获得与福尔马林固定后基本相当的免疫组织化学染色结果。Bouin 固定的组织未提供与福尔马林固定相当的结果。从用替代固定剂固定的样本中提取的 DNA 适用于分子分析。
