Thway Khin, Wren Dorte, Lee Jasmin, Thompson Lisa, Fisher Cyril, Gonzalez David
Sarcoma Unit, Royal Marsden Hospital, London, UK.
Molecular Diagnostics, Royal Marsden Hospital, Sutton, Surrey, UK.
J Clin Pathol. 2017 Jan;70(1):20-24. doi: 10.1136/jclinpath-2016-203691. Epub 2016 May 20.
Molecular genetic analysis is now a routine ancillary diagnostic modality to the histopathological diagnosis of soft-tissue neoplasms, many of which harbour characteristic gene fusions detectable by reverse transcription-PCR (RT-PCR). As the final diagnosis often depends on the molecular result, it is important to obtain the optimal yield of patient RNA.
We assessed the most reliable method of providing formalin-fixed, paraffin-embedded material for optimal RNA yield by comparing three consecutive periods in which different preparations (5×10 μm scrolls, 5×5 μm sections and 1×10 μm sections) were used for RNA extraction for RT-PCR, with its technical success rate.
For '2011', '2012' and '2013', RT-PCR technical failure rates were 13.4%, 4.4% and 7.9%, respectively. The percentage of failed referral cases was 71.4%, 85.7% and 31.3%, and the proportion of core biopsy to excision specimens was 3:15, 2:5 and 13:3.
This study shows that the effectiveness of RNA extraction and purification is dependent on both specimen type and the tissue sectioning strategy. The failure rate has improved over recent years, particularly for large specimens as large numbers of thick 10 μm scrolls can saturate RNA extraction columns. In contrast, recent technical fails are more frequent in core biopsies, where 1×10 μm sections are insufficient for adequate RNA extraction. While previous technical fails occurred mostly in referred cases, this appears no longer the case due to the better fixation and processing of specimens in external surgical pathology departments because of the widespread recognition of the importance of molecular diagnostics as an important part of the patient pathway.
分子遗传学分析如今是软组织肿瘤组织病理学诊断的常规辅助诊断手段，其中许多肿瘤含有可通过逆转录聚合酶链反应（RT-PCR）检测到的特征性基因融合。由于最终诊断往往取决于分子检测结果，因此获得最佳的患者RNA产量很重要。
我们通过比较三个连续时期，评估了提供福尔马林固定、石蜡包埋材料以获得最佳RNA产量的最可靠方法，在这三个时期中，使用不同的制备方法（5×10 μm切片条、5×5 μm切片和1×10 μm切片）进行RNA提取用于RT-PCR，并评估其技术成功率。
2011年、2012年和2013年的RT-PCR技术失败率分别为13.4%、4.4%和7.9%。转诊病例的失败率分别为71.4%、85.7%和31.3%，粗针活检标本与切除标本的比例分别为3:15、2:5和13:3。
本研究表明，RNA提取和纯化的有效性取决于标本类型和组织切片策略。近年来失败率有所改善，特别是对于大标本，因为大量10 μm厚的切片条会使RNA提取柱饱和。相比之下，最近粗针活检中的技术失败更为频繁，其中1×10 μm切片不足以进行充分的RNA提取。虽然以前的技术失败大多发生在转诊病例中，但由于分子诊断作为患者诊疗路径重要组成部分的重要性得到广泛认可，外部外科病理科对标本进行了更好的固定和处理，情况似乎已不再如此。
