Petras Melissa L, Lefferts Joel A, Ward Brian P, Suriawinata Arief A, Tsongalis Gregory J
Department of Pathology, Dartmouth-Hitchcock Medical Center, Dartmouth Medical School, Hanover, NH, USA.
Diagn Mol Pathol. 2011 Dec;20(4):189-93. doi: 10.1097/PDM.0b013e318211d554.
DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue is usually more degraded and contains more polymerase chain reaction (PCR) inhibitors than DNA isolated from nonfixed tissue. In addition, the tumor size and cellular heterogeneity found in tissue sections can often impact testing for molecular biomarkers. As a potential remedy to this situation, we evaluated the use of Whatman FTA paper cards for collection of colorectal tumor samples before tissue fixation and for isolation of DNA for use in a real-time PCR-based KRAS mutation assay. Eleven colon tumor samples were collected by making a cut into the fresh tumor and applying the Whatman FTA paper to the cut surface. Matched FFPE tissue blocks from these tumors were also collected for comparison. KRAS mutation analysis was carried out using the Applied Biosystems 7500 Fast Real-time PCR System using 7 independent custom TaqMan PCR assays. Of the 11 colon tumors sampled, 6 were positive for KRAS mutations in both the Whatman FTA paper preparations and corresponding FFPE samples. Whatman FTA paper cards for collection of colorectal tumor samples before tissue fixation and for isolation of DNA have many advantages including ease of use, intrinsic antimicrobial properties, long storage potential (stability of DNA over time), and a faster turnaround time for results. Extracted DNA should be suitable for most molecular diagnostic assays that use PCR techniques. This novel means of DNA preservation from surgical specimens would benefit from additional study and validation as a dependable and practical technique to preserve specimens for molecular testing.
从福尔马林固定石蜡包埋（FFPE）组织中分离出的DNA通常比从非固定组织中分离出的DNA降解程度更高，且含有更多的聚合酶链反应（PCR）抑制剂。此外，组织切片中发现的肿瘤大小和细胞异质性常常会影响分子生物标志物的检测。作为应对这种情况的一种潜在补救措施，我们评估了使用Whatman FTA纸卡来收集结直肠肿瘤样本，即在组织固定前收集样本并用于分离DNA，以用于基于实时PCR的KRAS突变检测。通过切开新鲜肿瘤并将Whatman FTA纸应用于切口表面，收集了11个结肠肿瘤样本。还收集了这些肿瘤匹配的FFPE组织块用于比较。使用Applied Biosystems 7500 Fast实时PCR系统，通过7种独立的定制TaqMan PCR检测方法进行KRAS突变分析。在采集的11个结肠肿瘤样本中，Whatman FTA纸制备物和相应的FFPE样本中有6个KRAS突变呈阳性。用于在组织固定前收集结直肠肿瘤样本并分离DNA的Whatman FTA纸卡具有许多优点，包括使用方便、具有内在抗菌特性、储存潜力大（DNA随时间的稳定性）以及结果周转时间更快。提取的DNA应该适用于大多数使用PCR技术的分子诊断检测。作为一种可靠且实用的技术来保存用于分子检测的标本，这种从手术标本中保存DNA的新方法将受益于更多的研究和验证。
