细胞学中全转录组和靶向 RNA 测序分析的前分析效应：在保存前延长胸腔积液标本储存时间对其的影响。
Pre-analytical effects on whole transcriptome and targeted RNA sequencing analysis in cytology: The effects of prolonged time in storage of effusion specimens prior to preservation.
机构信息
Department of Pathology and Genomic Medicine, Houston Methodist, Houston, Texas, USA.
Department of Pathology and Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
出版信息
Cytopathology. 2023 Nov;34(6):551-561. doi: 10.1111/cyt.13304. Epub 2023 Sep 15.
OBJECTIVES
To investigate the pre-analytics of the molecular testing of cytology specimens, we studied the effects of time in refrigerator storage (4°C) of malignant effusions on RNA sequencing (RNAseq) results.
METHODS
Ten effusion specimens were stored in a refrigerator (4°C) for different durations (day 0, 1, 4, and 7). All specimens were prepared as cytospins fixed in either Carnoy's solution or 95% ethanol (EtOH) and in an RNA preservative for a fresh frozen (FF) high-quality reference. Whole transcriptome (wt) and targeted (t)RNAseq of two multigene expression signatures were performed. We then compared transcript expression levels (including mutant allele fraction) according to pre-analytical variables using a concordance correlation coefficient (CCC) and a mixed effect model.
RESULTS
Sequencing results were mostly stable over increasing time in storage. Cytospins fixed in Carnoy's solution were more concordant with FF samples than cytospins fixed in 95% EtOH at all timepoints. This finding was consistent for both wtRNAseq (averages: day 0 CCC = 0.98 vs 0.91; day 7 CCC = 0.88 vs 0.78) and tRNAseq methods (averages: day 0 CCC = 0.98 vs 0.81; day 7 CCC = 0.98 vs 0.90). Cytospins fixed in Carnoy's solution did not show significant changes in expression over timepoints or between expression signatures, whereas 95% EtOH did.
CONCLUSION
RNAseq can be accurately performed on effusion specimens after prolonged refrigerator storage. RNA extracted from scraped cytospin slides fixed in Carnoy's solution was marginally superior to 95% EtOH fixation, but either method had comparable analytic performance to high-quality FF RNA samples.
目的
为了研究细胞学标本分子检测的分析前因素，我们研究了恶性积液在冰箱（4°C）中冷藏保存不同时间（第 0、1、4 和 7 天）对 RNA 测序（RNAseq）结果的影响。
方法
将 10 份积液标本分别在冰箱（4°C）中储存不同的时间（第 0、1、4 和 7 天）。所有标本均制备成细胞涂片，分别用卡诺氏液或 95%乙醇（EtOH）固定，并置于 RNA 保存液中，作为新鲜冷冻（FF）高质量参考。对两个多基因表达标志物进行全转录组（wt）和靶向（t）RNAseq。然后，我们使用一致性相关系数（CCC）和混合效应模型根据分析前变量比较转录物表达水平（包括突变等位基因分数）。
结果
随着储存时间的增加，测序结果大多保持稳定。在所有时间点，用卡诺氏液固定的细胞涂片与 FF 样本的一致性均优于用 95%EtOH 固定的细胞涂片。该发现既适用于 wtRNAseq（平均值：第 0 天 CCC=0.98 比 0.91；第 7 天 CCC=0.88 比 0.78），也适用于 tRNAseq 方法（平均值：第 0 天 CCC=0.98 比 0.81；第 7 天 CCC=0.98 比 0.90）。用卡诺氏液固定的细胞涂片在时间点之间或在表达标志物之间的表达没有显著变化，而 95%EtOH 固定的细胞涂片则有显著变化。
结论
经过长时间冰箱储存后，可准确对积液标本进行 RNAseq。用卡诺氏液固定的刮取细胞涂片提取的 RNA 略优于 95%EtOH 固定，但两种方法的分析性能均与高质量 FF RNA 样本相当。
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