Reid Kate Megan, Maistry Sairita, Ramesar Raj, Heathfield Laura Jane
Division of Forensic Medicine and Toxicology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory, 7925, South Africa; MRC/UCT Human Genetics Research Unit, Division of Human Genetics, Department of Pathology, Institute of Infectious Diseases and Molecular Medicine, Faculty of Health Science, University of Cape Town, Anzio Road, Observatory, Cape Town, South Africa.
Division of Forensic Medicine and Toxicology, Department of Pathology, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory, 7925, South Africa.
Forensic Sci Int. 2017 Nov;280:181-187. doi: 10.1016/j.forsciint.2017.09.020. Epub 2017 Oct 13.
Molecular analyses in a post-mortem setting are becoming increasingly common, particularly in cases of sudden unexplained death, with the aim of identifying genetic mutations which may be responsible for causing death. In retrospective investigations, the access to suitable autopsy biological samples may be limited, and often formalin fixed paraffin embedded (FFPE) tissue is the only sample available. The preservation of tissue in formalin is known to damage DNA through crosslinking activity. This results in the extraction of severely fragmented DNA of variable yields, which subsequently reduces the ability to perform downstream molecular analyses. Numerous studies have investigated possible improvements to various aspects of the DNA extraction and amplification procedures from FFPE tissue and this review aims to collate these optimization steps in a cohesive manner. A systematic review was performed of three major databases, which identified 111 articles meeting the inclusion criteria. Five main areas for optimization and improvements were identified in the workflow: (1) tissue type, (2) fixation process, (3) post-fixation, (4) DNA extraction procedure and (5) amplification. It was found that some factors identified, for example tissue type and fixation process, could not be controlled by the researcher when conducting retrospective analyses. For this reason, optimization should be performed in other areas, within the financial means of the laboratories, and in accordance with the purposes of the investigation. Implementation of one or more of the optimization measures described here is anticipated to assist in the extraction of higher quality DNA. Despite the challenges posed by FFPE tissue, it remains a valuable source of DNA in retrospective molecular forensic investigations.
在尸检情况下进行分子分析正变得越来越普遍，尤其是在不明原因猝死的案例中，目的是识别可能导致死亡的基因突变。在回顾性调查中，获取合适的尸检生物样本可能会受到限制，福尔马林固定石蜡包埋（FFPE）组织往往是唯一可用的样本。已知福尔马林对组织的保存会通过交联活性破坏DNA。这导致提取出产量各异的严重片段化DNA，进而降低了进行下游分子分析的能力。众多研究探讨了对FFPE组织DNA提取和扩增程序各方面可能的改进，本综述旨在以连贯的方式整理这些优化步骤。对三个主要数据库进行了系统综述，确定了111篇符合纳入标准的文章。在工作流程中确定了五个主要的优化和改进领域：（1）组织类型，（2）固定过程，（3）固定后处理，（4）DNA提取程序和（（5）扩增。结果发现，一些确定的因素，如组织类型和固定过程，在进行回顾性分析时研究人员无法控制。因此，应在实验室的经济能力范围内，根据调查目的，在其他领域进行优化。预计实施本文所述的一项或多项优化措施将有助于提取更高质量的DNA。尽管FFPE组织带来了挑战，但在回顾性分子法医调查中，它仍然是DNA的宝贵来源。
