Evaluation of RNA and DNA extraction from liquid‐based cytology specimens

Fujii, Tomomi; Asano, Aya; Shimada, Kaoru; Tatsumi, Yoshihiro; Obayashi, Chiho; Konishi, Noboru

doi:10.1002/dc.23524

Cited by 25 publications

(28 citation statements)

References 23 publications

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“…Cytologic samples provide high‐quality DNA for molecular testing and are comparable, or even superior to, formalin‐fixed, paraffin‐embedded surgical pathology specimens for the evaluation of clinically relevant mutations . Liquid‐based cytology (LBC) makes it possible to perform additional techniques, such as immunocytochemistry and in situ hybridization, and also allows for molecular testing using DNA and RNA extracted from the remaining fixed cells . Some studies have demonstrated that LBC systems provide excellent cell preservation for routine cytopathology and can be used for DNA‐ and RNA‐based molecular testing …”

Section: Introductionmentioning

confidence: 99%

“…4,5 Liquid-based cytology (LBC) makes it possible to perform additional techniques, such as immunocytochemistry and in situ hybridization, and also allows for molecular testing using DNA and RNA extracted from the remaining fixed cells. [4][5][6] Some studies have demonstrated that LBC systems provide excellent cell preservation for routine cytopathology and can be used for DNA-and RNAbased molecular testing. 4,5 The 2015 World Health Organization classification 7 classifications.…”

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confidence: 99%

“…[4][5][6] Some studies have demonstrated that LBC systems provide excellent cell preservation for routine cytopathology and can be used for DNA-and RNAbased molecular testing. 4,5 The 2015 World Health Organization classification 7 classifications. 8 These guidelines classify the major ADC subtypes according to the predominant histological pattern (primary pattern) and the estimation of the percentage of other ADC patterns that may be present in a resected specimen (in increments of 5%; secondary pattern).…”

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confidence: 99%

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Genotyping and cytomorphological subtyping of lung adenocarcinoma based on liquid‐based cytology

Tanaka

Sakamoto

Suzuki

et al. 2019

Diagnostic Cytopathology

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Background Liquid‐based cytology (LBC) samples allow immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and molecular testing of nucleic acids to be performed in the remaining fixed cells. The current study aimed to examine the relationship between gene mutational status and cytomorphological features in primary lung adenocarcinoma (ADC) using LBC materials. Methods Forty consecutive patients with primary lung ADC underwent surgical resection in our hospital. Cytological material was obtained by scraping the cut‐surface of the lesion, and samples were fixed and stored as LBC materials using CytoRich Red. Epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations, anaplastic lymphoma kinase (ALK), and c‐ros oncogene 1 (ROS1) gene rearrangements were detected, and cytomorphological studies were performed. Results Twenty cases (50%) were positive for EGFR mutation and four (10%) were positive for KRAS mutation. ALK gene rearrangement was identified in one case (2.5%) by IHC and FISH, and ROS1 gene rearrangement was identified in one case (2.5%) by IHC and real‐time polymerase chain reaction. The KRAS‐positive group included higher proportions of cases with an inflammatory background (100%), predominantly papillary architecture (75%), and papillary‐type ADC pattern (75%) compared with the EGFR‐positive group and the other group, which included ALK and ROS1 gene rearrangements. Conclusions LBC material is suitable for use in molecular testing. Differences in major gene aberrations detected by this method might predict specific cytomorphological features.

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Section: Introductionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Genotyping and cytomorphological subtyping of lung adenocarcinoma based on liquid‐based cytology

Tanaka

Sakamoto

Suzuki

et al. 2019

Diagnostic Cytopathology

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“…However, shipping and storage of frozen RNA is expensive, requires special handling and Ultra-Low-Temperature freezers, and is time sensitive. The extraction of DNA and RNA from liquid-based cytology specimens has previously been studied [6,7].…”

Section: Main Textmentioning

confidence: 99%

Improvement in RNA quantity and quality in cervico-vaginal cytology

Chong

Han

Lee

et al. 2020

Virol J

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The separation of exfoliated cells from the brushes used during cervico-vaginal smears is difficult, a problem which may affect the quality of ribonucleic acid (RNA) extracted. We compared the results of RNA extraction from cervicovaginal cytology samples according to the type of tubes, preservative solutions, and storage temperature. The samples included exfoliated cervico-vaginal cytological specimens from patients with human papilloma virus 16, positive for cervical intraepithelial neoplasia or cervical cancer. Exfoliated cells were obtained by shaking a brush in a conventional rigid vial tube or squeezing the brush in a soft vial tube. RNA quantity and quality were compared between the two tubes. The concentration and purity of RNA (A260/A280 and A260/A230 ratios) was compared amongst five groups: Group 1, standard frozen storage; Group 2-4, RNA stabilization reagents with room temperature [RNAlater RNA Stabilization Reagent, RNAprotect cell Reagent and AllProtect Tissue Reagent]; and Group 5, Surepath Preservative fluid. To demonstrate the utility of the extracted RNA for PCR-based cDNA synthesis, GAPDH and E6 were targeted and gel band densities of GAPDH and E6 were measured. The median RNA concentration was significantly higher in the soft tubes compared with the rigid tubes (100.2 vs. 7.1 ng/μL, p = 0.0209). The purity of the RNA was higher in soft vial tubes than in rigid vials, as measured by A260/280 and A260/ 230 ratios. The RNA concentration, purity, and GAPDH density of groups 1, 2 and 3 were significantly higher than those of groups 4 and 5. Moreover, E6 density of group 1 and 2 was significantly higher than that of group 3, 4 and 5. The use of soft tubes enhanced the mRNA quantity and quality in cervico-vaginal cytology. The products of mRNA extraction using RNAlater RNA Stabilization Reagent and RNAprotect Cell Reagent at room temperature were comparable to those obtained by conventional frozen storage. Our protocol improved the yield and quality of RNA and might produce better results for molecular analysis in cervico-vaginal cytology.

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“…Some commercial preservative solutions for LBC include formaldehyde to improve the imaging of specimens; however, the percentage of formaldehyde in LBC preservation solutions usually is lower than that in common fixative solutions such as buffered formalin for FFPE specimens. Recently, Fujii et al reported that genomic DNA extracted from human urothelial carcinoma and cervical cancer cell lines that were fixed in several commercial LBC solutions could be used in genetic analyses, although DNA expression decreased with longer fixation times.…”

Section: Introductionmentioning

confidence: 99%

Reducing DNA damage by formaldehyde in liquid‐based cytology preservation solutions to enable the molecular testing of lung cancer specimens

Matsuo

Yoshida

Yamashita

et al. 2018

Cancer Cytopathology

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Background Liquid‐based cytology (LBC) is a useful cytopathological method, and LBC lung adenocarcinoma specimens may be used for genetic analysis in the near future. In the current study, the authors determined whether LBC specimens can be used for epidermal growth factor receptor (EGFR) mutation analysis in human lung adenocarcinoma cell lines. Methods Genomic DNA was extracted from 3 lung adenocarcinoma cell lines that were fixed in LBC preservation solution using 2 protocols (one for cultured cells and one for tissues) of a DNA extraction kit. Different fixation times were tested for each protocol: 30 minutes, 1 hour, and 1 to 9 days. As controls, cells also were fixed in 10% formalin or 95% ethanol. The authors investigated the effect of fixation time on DNA fragmentation, polymerase chain reaction (PCR) amplification, and EGFR mutation detection. Results The DNA yield of LBC specimens tended to decrease depending on fixation time. When using the DNA extraction protocol for tissues, PCR amplification was successful after 9 days of fixation, although extracted genomic DNA that was fixed for >1 hour demonstrated fragmentation. Mutation analyses using the Cycleave PCR method were successful after 7 days of fixation. The DNA extraction protocol for tissues was appropriate for lung adenocarcinoma cell lines that were stored for >1 day in a preservative solution. The results of the current study demonstrated that EGFR mutations can be detected on day 7 using lung adenocarcinoma cell lines fixed in CytoRich Red preservative. Conclusions When LBC specimens are used for targeted molecular genetic testing, the appropriate preservative solution and extraction protocol first should be determined.

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Evaluation of RNA and DNA extraction from liquid‐based cytology specimens

Cited by 25 publications

References 23 publications

Genotyping and cytomorphological subtyping of lung adenocarcinoma based on liquid‐based cytology

Genotyping and cytomorphological subtyping of lung adenocarcinoma based on liquid‐based cytology

Improvement in RNA quantity and quality in cervico-vaginal cytology

Reducing DNA damage by formaldehyde in liquid‐based cytology preservation solutions to enable the molecular testing of lung cancer specimens

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