Abstract
Raman spectroscopy can provide a rapid, label-free, nondestructive measurement of the chemical fingerprint of a sample and has shown potential for cancer screening and diagnosis. Here we report a protocol for Raman microspectroscopic analysis of different exfoliative cytology samples (cervical, oral and lung), covering sample preparation, spectral acquisition, preprocessing and data analysis. The protocol takes 2 h 20 min for sample preparation, measurement and data preprocessing and up to 8 h for a complete analysis. A key feature of the protocol is that it uses the same sample preparation procedure as commonly used in diagnostic cytology laboratories (i.e., liquid-based cytology on glass slides), ensuring compatibility with clinical workflows. Our protocol also covers methods to correct for the spectral contribution of glass and sample pretreatment methods to remove contaminants (such as blood and mucus) that can obscure spectral features in the exfoliated cells and lead to variability. The protocol establishes a standardized clinical routine allowing the collection of highly reproducible data for Raman spectral cytopathology for cancer diagnostic applications for cervical and lung cancer and for monitoring suspicious lesions for oral cancer.
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Acknowledgements
This work was financially supported by Enterprise Ireland co-funded by the European Regional Development Fund (ERDF) and Ireland’s EU Structural Funds Programme 2007–2013, Health Research Board Collaborative Applied Research Grant CARG2012/29 to CERVIVA (www.cerviva.ie) and Science Foundation Ireland (12/IP/1494). I.B. and D.O’D. were supported by Dublin Institute of Technology Fiosraigh Postgraduate Scholarships.
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F.M.L. conceived the idea for the manuscript and co-wrote, compiled and finalized the manuscript. D.T., I.B. and D.O’D. co-wrote the manuscript and compiled the figures. H.J.B., F.B., A.M., F.O’C., S.N., A.M., S.F., S.G., C.M.H., C.M.M. and J.J.O’L. revised and edited the manuscript.
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Key papers using this protocol
Bonnier, F. et al. Anal. Methods 6, 7831–7841 (2014): https://doi.org/10.1039/C4AY01497A
Behl, I. et al. Anal. Methods 9, 937–948 (2017): https://doi.org/10.1039/C6AY03360A
Duraipandian, S. et al. Sci. Rep. 8, 15048 (2018): https://doi.org/10.1038/s41598-018-33417-8
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Traynor, D., Behl, I., O’Dea, D. et al. Raman spectral cytopathology for cancer diagnostic applications. Nat Protoc 16, 3716–3735 (2021). https://doi.org/10.1038/s41596-021-00559-5
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DOI: https://doi.org/10.1038/s41596-021-00559-5
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