TY - JOUR
T1 - Molecular testing opportunities on cytology effusion specimens
T2 - the pre-analytic effects of various body fluid cytology preparation methods on RNA extraction quality and targeted sequencing
AU - Sura, Gloria H.
AU - Tran, Kevin
AU - Fu, Chunxiao
AU - Du, Lili
AU - Marczyk, Michał
AU - Martinez, Yadira
AU - Tinnirello, Agata A.
AU - Gould, Rebekah E.
AU - Lau, Rosanna
AU - Symmans, W. Fraser
N1 - Publisher Copyright:
© 2022
PY - 2023/1/1
Y1 - 2023/1/1
N2 - Introduction: RNA sequencing (RNAseq) analysis is emerging as a clinical research or diagnostic approach for cytologic samples, but there is need for formal comparison of different sample preparation methods in the cytology laboratory to identify which pre-analytic methods could provide alternatives to formalin-fixed paraffin-embedded (FFPE) sections. Materials and methods: We prepared 13 malignant effusions (metastatic estrogen receptor-positive breast cancer) in the cytology laboratory using 6 routine cytologic methods: FFPE cell block, Carnoy's solution, 95% ethanol (EtOH), air-dried and Diff-Quik, ThinPrep, and SurePath preparations. Measurements of RNA quality, expression of 2 multigene expression signatures, molecular subtype, and 4 common activating mutation sites in each preparation were compared with fresh frozen (FF) cell pellet in RNA preservative using distribution of fragment length and concordance correlation coefficient (CCC). Results: The fraction of RNA fragments measuring 200 bases or more (DV200) were 24% higher from cytospins fixed in Carnoy's solution or 95% EtOH than DV200 from FFPE cell blocks. SurePath samples failed RNAseq quality control. There was high concordance of gene expression measurements with FF samples using cytospins fixed in Carnoy's solution, 95% EtOH, Diff-Quik (CCC = 0.829, 0.812, 0.760, respectively), or ThinPrep (CCC = 0.736), but lower using FFPE cell block (CCC = 0.564). The proportion of mutant transcripts was concordant between FF and any cytologic preparation methods. Conclusions: Cytospin preparations fixed with Carnoy's or 95% ETOH then Papanicolaou stained produced RNAseq results that were equivalent to FF samples and superior to FFPE cell block sections.
AB - Introduction: RNA sequencing (RNAseq) analysis is emerging as a clinical research or diagnostic approach for cytologic samples, but there is need for formal comparison of different sample preparation methods in the cytology laboratory to identify which pre-analytic methods could provide alternatives to formalin-fixed paraffin-embedded (FFPE) sections. Materials and methods: We prepared 13 malignant effusions (metastatic estrogen receptor-positive breast cancer) in the cytology laboratory using 6 routine cytologic methods: FFPE cell block, Carnoy's solution, 95% ethanol (EtOH), air-dried and Diff-Quik, ThinPrep, and SurePath preparations. Measurements of RNA quality, expression of 2 multigene expression signatures, molecular subtype, and 4 common activating mutation sites in each preparation were compared with fresh frozen (FF) cell pellet in RNA preservative using distribution of fragment length and concordance correlation coefficient (CCC). Results: The fraction of RNA fragments measuring 200 bases or more (DV200) were 24% higher from cytospins fixed in Carnoy's solution or 95% EtOH than DV200 from FFPE cell blocks. SurePath samples failed RNAseq quality control. There was high concordance of gene expression measurements with FF samples using cytospins fixed in Carnoy's solution, 95% EtOH, Diff-Quik (CCC = 0.829, 0.812, 0.760, respectively), or ThinPrep (CCC = 0.736), but lower using FFPE cell block (CCC = 0.564). The proportion of mutant transcripts was concordant between FF and any cytologic preparation methods. Conclusions: Cytospin preparations fixed with Carnoy's or 95% ETOH then Papanicolaou stained produced RNAseq results that were equivalent to FF samples and superior to FFPE cell block sections.
KW - Cytology processing
KW - Cytopathology
KW - Effusion
KW - Fixative
KW - Liquid
KW - Molecular
KW - RNA sequencing
UR - http://www.scopus.com/inward/record.url?scp=85140783515&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85140783515&partnerID=8YFLogxK
U2 - 10.1016/j.jasc.2022.09.003
DO - 10.1016/j.jasc.2022.09.003
M3 - Article
C2 - 36270909
AN - SCOPUS:85140783515
SN - 2213-2945
VL - 12
SP - 10
EP - 19
JO - Journal of the American Society of Cytopathology
JF - Journal of the American Society of Cytopathology
IS - 1
ER -