Mostegl Meike M, Richter Barbara, Dinhopl Nora, Weissenböck Herbert
Institute of Pathology and Forensic Veterinary Medicine, Department of Pathobiology, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria.
J Vet Diagn Invest. 2011 Nov;23(6):1212-6. doi: 10.1177/1040638711425584. Epub 2011 Oct 20.
Chromogenic in situ hybridization (ISH) is a commonly used tool in diagnostic pathology to detect pathogens in formalin-fixed, paraffin-embedded (FFPE) tissue sections. Prolonged formalin fixation time was identified to be a limiting factor for the successful detection of nucleic acid from different pathogens, most probably due to the cross-linking activity of formalin between RNA, DNA, and proteins. Therefore, in the current study, the influence of formalin fixation time on ISH signal intensity of 2 viral (Porcine circovirus-2 [PCV-2] and Porcine respiratory and reproductive virus [PRRSV]) and 2 protozoal agents (Cryptosporidium serpentis and Tritrichomonas sp.) was evaluated. Tissue samples were fixed in 7% neutral buffered formaldehyde solution, and at defined intervals, pieces were embedded in paraffin wax and subjected to pathogen-specific ISH. For all 4 pathogens, the signal intensity remained comparable with the starting ISH signal for different periods of fixation (PCV-2: 6 weeks, PRRSV: 23 weeks, C. serpentis: 55 weeks, Tritrichomonas sp.: 53 weeks). Thereafter, the signal started to decline until loss of nucleic acid detection. The influence of increased proteinase K concentrations for inverting the formalin-induced cross-linking activity was examined compared with the standard protocol. With all 4 infectious agents, a 4-fold proteinase K concentration restored the ISH signals to a level comparable with 1 day of fixation. In conclusion, the influence of prolonged formalin fixation on the intensity of detected ISH signal highly depends on the analyzed infectious agent and the pretreatment protocol.
显色原位杂交（ISH）是诊断病理学中常用的工具，用于检测福尔马林固定、石蜡包埋（FFPE）组织切片中的病原体。已确定延长福尔马林固定时间是成功检测不同病原体核酸的限制因素，这很可能是由于福尔马林在RNA、DNA和蛋白质之间的交联活性。因此，在本研究中，评估了福尔马林固定时间对2种病毒（猪圆环病毒2型[PCV-2]和猪繁殖与呼吸综合征病毒[PRRSV]）和2种原生动物病原体（蛇隐孢子虫和三毛滴虫）ISH信号强度的影响。组织样本固定于7%中性缓冲甲醛溶液中，在规定的时间间隔，将组织块包埋于石蜡中，并进行病原体特异性ISH检测。对于所有4种病原体，在不同固定时间内信号强度与起始ISH信号相当（PCV-2：6周，PRRSV：23周，蛇隐孢子虫：55周，三毛滴虫：53周）。此后，信号开始下降直至核酸检测失败。与标准方案相比，研究了增加蛋白酶K浓度对逆转福尔马林诱导的交联活性的影响。对于所有4种感染因子，蛋白酶K浓度增加4倍可将ISH信号恢复到与固定1天相当的水平。总之，延长福尔马林固定对检测到的ISH信号强度的影响高度依赖于所分析的感染因子和预处理方案。
