Gloria H. Sura MD , Kevin Tran MS , Alexander J. Trevarton PhD , Michal Marczyk PhD , Chunxiao Fu PhD , Lili Du PhD , Jiaxin Qu PhD , Rosanna Lau MS , Amy Tasto PhD , Rebekah E. Gould MS , Agata Tinnirello PhD , Bruno V. Sinn MD , Lajos Pusztai MD, PhD , Christos Hatzis PhD , W. Fraser Symmans MD
{"title":"Ficoll-Hypaque 和 CytoLyt 乳腺癌恶性渗出物血液清除技术的比较分析:对 RNA 质量和测序结果的影响。","authors":"Gloria H. Sura MD , Kevin Tran MS , Alexander J. Trevarton PhD , Michal Marczyk PhD , Chunxiao Fu PhD , Lili Du PhD , Jiaxin Qu PhD , Rosanna Lau MS , Amy Tasto PhD , Rebekah E. Gould MS , Agata Tinnirello PhD , Bruno V. Sinn MD , Lajos Pusztai MD, PhD , Christos Hatzis PhD , W. Fraser Symmans MD","doi":"10.1016/j.jasc.2024.11.001","DOIUrl":null,"url":null,"abstract":"<div><h3>Introduction</h3><div>To optimize RNA sequencing (RNA-seq) outcomes, we investigated preanalytical variables in malignant effusions containing metastatic breast cancer. We compared 2 processing methods—Ficoll-Hypaque density gradient enrichment and CytoLyt hemolysis—focusing on their effects on RNA quality, transcript abundance, and variant detection from cytospin slides, relative to fresh-frozen samples. Additionally, we compared read-based and Unique Molecular Identifier (UMI)-based library preparation methods.</div></div><div><h3>Materials and methods</h3><div>Thirteen malignant effusion specimens from metastatic breast cancer were processed using both the Ficoll-Hypaque and Cytolyt methods. RNA was extracted from fresh-frozen samples stored in RNA preservative and from cytospin slides fixed in Carnoy's solution. RNA quality was evaluated using RNA integrity number (RIN) and the percentage of fragments >200 bases (DV200). Sequencing was conducted with both read- and UMI-based methods.</div></div><div><h3>Results</h3><div>Purified RNA was more fragmented by the Cytolyt method (mean RIN: 3.56, DV200: 78.97%), compared to the Ficoll-Hypaque method (mean RIN: 6.29, DV200: 88.08%). Sequencing data had high concordance correlation coefficient (CCC) for measurements of gene expression, whether from Cytolyt or Ficoll-Hypaque treated samples, and whether using the UMI- or read-based sequencing methods (read-based mean CCC: 0.967 from Cytolyt versus 0.974 from Ficoll-Hypaque, UMI-based mean CCC: 0.972 from Cytolyt versus 0.977 from Ficoll-Hypaque).</div></div><div><h3>Conclusions</h3><div>Despite the increased RNA fragmentation with the Cytolyt, RNA-seq data quality was comparable across Cytolyt and Ficoll-Hypaque methods. Both clearing methods are viable for short-read RNA-seq analysis, with read and UMI-based approaches performing similarly.</div></div>","PeriodicalId":38262,"journal":{"name":"Journal of the American Society of Cytopathology","volume":"14 2","pages":"Pages 91-101"},"PeriodicalIF":0.0000,"publicationDate":"2025-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Comparative analysis of Ficoll-Hypaque and CytoLyt techniques for blood removal in breast cancer malignant effusions: effects on RNA quality and sequencing outcomes\",\"authors\":\"Gloria H. Sura MD , Kevin Tran MS , Alexander J. Trevarton PhD , Michal Marczyk PhD , Chunxiao Fu PhD , Lili Du PhD , Jiaxin Qu PhD , Rosanna Lau MS , Amy Tasto PhD , Rebekah E. Gould MS , Agata Tinnirello PhD , Bruno V. Sinn MD , Lajos Pusztai MD, PhD , Christos Hatzis PhD , W. Fraser Symmans MD\",\"doi\":\"10.1016/j.jasc.2024.11.001\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><h3>Introduction</h3><div>To optimize RNA sequencing (RNA-seq) outcomes, we investigated preanalytical variables in malignant effusions containing metastatic breast cancer. We compared 2 processing methods—Ficoll-Hypaque density gradient enrichment and CytoLyt hemolysis—focusing on their effects on RNA quality, transcript abundance, and variant detection from cytospin slides, relative to fresh-frozen samples. Additionally, we compared read-based and Unique Molecular Identifier (UMI)-based library preparation methods.</div></div><div><h3>Materials and methods</h3><div>Thirteen malignant effusion specimens from metastatic breast cancer were processed using both the Ficoll-Hypaque and Cytolyt methods. RNA was extracted from fresh-frozen samples stored in RNA preservative and from cytospin slides fixed in Carnoy's solution. RNA quality was evaluated using RNA integrity number (RIN) and the percentage of fragments >200 bases (DV200). Sequencing was conducted with both read- and UMI-based methods.</div></div><div><h3>Results</h3><div>Purified RNA was more fragmented by the Cytolyt method (mean RIN: 3.56, DV200: 78.97%), compared to the Ficoll-Hypaque method (mean RIN: 6.29, DV200: 88.08%). Sequencing data had high concordance correlation coefficient (CCC) for measurements of gene expression, whether from Cytolyt or Ficoll-Hypaque treated samples, and whether using the UMI- or read-based sequencing methods (read-based mean CCC: 0.967 from Cytolyt versus 0.974 from Ficoll-Hypaque, UMI-based mean CCC: 0.972 from Cytolyt versus 0.977 from Ficoll-Hypaque).</div></div><div><h3>Conclusions</h3><div>Despite the increased RNA fragmentation with the Cytolyt, RNA-seq data quality was comparable across Cytolyt and Ficoll-Hypaque methods. Both clearing methods are viable for short-read RNA-seq analysis, with read and UMI-based approaches performing similarly.</div></div>\",\"PeriodicalId\":38262,\"journal\":{\"name\":\"Journal of the American Society of Cytopathology\",\"volume\":\"14 2\",\"pages\":\"Pages 91-101\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2025-03-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of the American Society of Cytopathology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S2213294524002266\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"Medicine\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of the American Society of Cytopathology","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2213294524002266","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"Medicine","Score":null,"Total":0}
Comparative analysis of Ficoll-Hypaque and CytoLyt techniques for blood removal in breast cancer malignant effusions: effects on RNA quality and sequencing outcomes
Introduction
To optimize RNA sequencing (RNA-seq) outcomes, we investigated preanalytical variables in malignant effusions containing metastatic breast cancer. We compared 2 processing methods—Ficoll-Hypaque density gradient enrichment and CytoLyt hemolysis—focusing on their effects on RNA quality, transcript abundance, and variant detection from cytospin slides, relative to fresh-frozen samples. Additionally, we compared read-based and Unique Molecular Identifier (UMI)-based library preparation methods.
Materials and methods
Thirteen malignant effusion specimens from metastatic breast cancer were processed using both the Ficoll-Hypaque and Cytolyt methods. RNA was extracted from fresh-frozen samples stored in RNA preservative and from cytospin slides fixed in Carnoy's solution. RNA quality was evaluated using RNA integrity number (RIN) and the percentage of fragments >200 bases (DV200). Sequencing was conducted with both read- and UMI-based methods.
Results
Purified RNA was more fragmented by the Cytolyt method (mean RIN: 3.56, DV200: 78.97%), compared to the Ficoll-Hypaque method (mean RIN: 6.29, DV200: 88.08%). Sequencing data had high concordance correlation coefficient (CCC) for measurements of gene expression, whether from Cytolyt or Ficoll-Hypaque treated samples, and whether using the UMI- or read-based sequencing methods (read-based mean CCC: 0.967 from Cytolyt versus 0.974 from Ficoll-Hypaque, UMI-based mean CCC: 0.972 from Cytolyt versus 0.977 from Ficoll-Hypaque).
Conclusions
Despite the increased RNA fragmentation with the Cytolyt, RNA-seq data quality was comparable across Cytolyt and Ficoll-Hypaque methods. Both clearing methods are viable for short-read RNA-seq analysis, with read and UMI-based approaches performing similarly.