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.
Cytopathology
; 34(6): 551-561, 2023 Nov.
Article
em En
| MEDLINE
| ID: mdl-37712171
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.
Texto completo:
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Base de dados:
MEDLINE
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Cytopathology
Assunto da revista:
PATOLOGIA
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Estados Unidos