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.
Original language | English (US) |
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Pages (from-to) | 551-561 |
Number of pages | 11 |
Journal | Cytopathology |
Volume | 34 |
Issue number | 6 |
DOIs | |
State | Published - Nov 2023 |
Keywords
- cytology processing
- cytopathology
- effusion
- fixative
- liquid
- molecular
- RNA sequencing
ASJC Scopus subject areas
- Pathology and Forensic Medicine
- Histology