Reverse transcription–polymerase chain reaction molecular testing of cytology specimens: Pre‐analytic and analytic factors

Abstract

The introduction of molecular testing into cytopathology laboratory practice has expanded the types of samples considered feasible for identifying genetic alterations that play an essential role in cancer diagnosis and treatment. Reverse transcription–polymerase chain reaction (RT‐PCR), a sensitive and specific technical approach for amplifying a defined segment of RNA after it has been reverse‐transcribed into its DNA complement, is commonly used in clinical practice for the identification of recurrent or tumor‐specific fusion gene events. Real‐time RT‐PCR (quantitative RT‐PCR), a technical variation, also permits the quantitation of products generated during each cycle of the polymerase chain reaction process. This review addresses qualitative and quantitative pre‐analytic and analytic considerations of RT‐PCR as they relate to various cytologic specimens. An understanding of these aspects of genetic testing is central to attaining optimal results in the face of the challenges that cytology specimens may present. Cancer Cytopathol 2017;125:11‐19. © 2016 American Cancer Society.

How to Prepare Cytological Samples for Molecular Testing

“…Even with optimised digestion and heating steps, however, it is not possible to completely remove all chemical modifications such as residual methyl groups from FFPE-extracted RNA. 14 Cellularity is evaluated by examining an H&E-stained section prepared from the CB; thus, the percentage of tumour cells in deeper sections of the CB used for molecular testing is assessed in an extrapolative fashion inferred but not actually known. When the tumour cellularity is high and more than sufficient for testing, paraffin scrolls can simply be placed directly into a tube for extraction without microdissection, and cellularity assessment of an H&E section taken after the scrolls (postcurl section) may be unnecessary.…”

“…2 The advantage of a short acquisition time for molecular processing is mostly required to ensure high-integrity RNA for some molecular applications such as complementary DNA labelling for microarray analysis and transcriptome analysis. 14 In contrast, RT-PCR or quantitative RT-PCR analysis for fusion gene detection is more tolerant of partially degraded RNA because the design can be based on an analysis of smaller regions of RNA. 14 For long-term storage, aliquots can be frozen at 80°C RNA later or similar RNA stabilising solutions and stored in freezers 34 ; fresh cells can also be stored at room temperature for months in Whatman filter paper cards (GE Healthcare Life Sciences, Buckinghamshire, England).…”

How to prepare cytological samples for molecular testing

“…Non-cross-linking alcoholic reagents may yield superior results as RNA fixatives in comparison with aldehydes because they cause little chemical change and usually provide higher quality nucleic acids for molecular testing than do FFPE sections 14. Several studies using previously stained cytology smears have shown that molecular testing can be performed successfully using both Diff-Quik as well as Papanicolaou-stained slides.…”

“…In addition, residual material from CytoLyt samples has been shown to feature optimal RNA integrity being suitable for nucleic acid isolation and subsequent analysis by reverse transcription-PCR (RT-PCR) 31. However, RNA degradation was reported in specimens stored for 12 months at room temperature, and long-term storage requires –80°C 14. Interestingly, also, exfoliative oral cytology specimen has shown an adequate RNA preservation 31…”

“…The advantage of a short acquisition time for molecular processing is mostly required to ensure high-integrity RNA for some molecular applications such as complementary DNA labelling for microarray analysis and transcriptome analysis 14. In contrast, RT-PCR or quantitative RT-PCR analysis for fusion gene detection is more tolerant of partially degraded RNA because the design can be based on an analysis of smaller regions of RNA 14.…”