Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.
J Clin Pathol. 2011 Nov;64(11):960-7. doi: 10.1136/jclinpath-2011-200152. Epub 2011 Jun 29.
In surgical pathology, formalin-fixed paraffin-embedded tissues are increasingly being used as a source of DNA and RNA for molecular assays in addition to histopathological evaluation. However, the commonly used formalin fixative is carcinogenic, and its crosslinking impairs DNA and RNA quality.
The suitability of three new presumably less toxic, crosslinking (F-Solv) and non-crosslinking (FineFIX, RCL2) alcohol-based fixatives was tested for routine molecular pathology in comparison with neutral buffered formalin (NBF) as gold standard. Size ladder PCR, epidermal growth factor receptor sequence analysis, microsatellite instability (MSI), chromogenic (CISH), fluorescence in situ hybridisation (FISH) and qPCR were performed.
The alcohol-based non-crosslinking fixatives (FineFIX and RCL2) resulted in a higher DNA yield and quality compared with crosslinking fixatives (NBF and F-Solv). Size ladder PCR resulted in a shorter amplicon size (300 bp) for both crosslinking fixatives compared with the non-crosslinking fixatives (400 bp). All four fixatives were directly applicable for MSI and epidermal growth factor receptor sequence analysis. All fixatives except F-Solv showed clear signals in CISH and FISH. RNA yield and quality were superior after non-crosslinking fixation. qPCR resulted in lower Ct values for RCL2 and FineFIX.
The alcohol-based non-crosslinking fixatives performed better than crosslinking fixatives with regard to DNA and RNA yield, quality and applicability in molecular diagnostics. Given the higher yield, less starting material may be necessary, thereby increasing the applicability of biopsies for molecular studies.
在外科病理学中，福尔马林固定石蜡包埋组织除了用于组织病理学评估外，还越来越多地被用作 DNA 和 RNA 分子检测的来源。然而，常用的福尔马林固定剂具有致癌性，其交联作用会损害 DNA 和 RNA 的质量。
本研究将三种新型推测毒性较低、非交联（FineFIX、RCL2）的醇基固定剂与中性缓冲福尔马林（NBF）作为金标准进行比较，以评估其在常规分子病理学中的适用性。进行了大小梯 PCR、表皮生长因子受体序列分析、微卫星不稳定性（MSI）、显色原位杂交（CISH）、荧光原位杂交（FISH）和 qPCR。
与交联固定剂（NBF 和 F-Solv）相比，醇基非交联固定剂（FineFIX 和 RCL2）可获得更高的 DNA 产量和质量。交联固定剂（NBF 和 F-Solv）的大小梯 PCR 产生的扩增子大小较短（300bp），而非交联固定剂（400bp）。所有四种固定剂均可直接用于 MSI 和表皮生长因子受体序列分析。除 F-Solv 外，所有固定剂在 CISH 和 FISH 中均显示出清晰的信号。非交联固定后，RNA 产量和质量更高。qPCR 结果显示，RCL2 和 FineFIX 的 Ct 值较低。
在 DNA 和 RNA 产量、质量以及分子诊断中的适用性方面，醇基非交联固定剂的性能优于交联固定剂。鉴于更高的产量，可能需要更少的起始材料，从而增加了活检在分子研究中的适用性。
