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

Abstract 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.