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Raman spectral cytopathology for cancer diagnostic applications

Nature Protocols volume 16, pages 3716–3735 (2021)Cite this article

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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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Fig. 1: Schematic overview of the procedure.
Fig. 2: Bright-field images of stained and unstained cervical and lung exfoliated cells.
Fig. 3: Raman spectra from cervical exfoliated cells before and after pretreatment and preprocessing.
Fig. 4: Typical results from cervical exfoliated cells.
Fig. 5: Raman spectra from oral exfoliated cells before and after preprocessing.
Fig. 6: Typical results from oral exfoliated cells.
Fig. 7: Raman spectra from lung exfoliated cells before and after pretreatment and preprocessing.
Fig. 8: Typical results from lung exfoliated cells.

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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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Author notes

  1. These authors contributed equally: Damien Traynor, Isha Behl, Declan O’Dea.

Authors and Affiliations

  1. Centre for Radiation and Environmental Science, FOCAS Research Institute, Technological University Dublin, Dublin, Ireland

    Damien Traynor, Isha Behl, Declan O’Dea & Fiona M. Lyng

  2. School of Physics & Clinical & Optometric Sciences, Technological University Dublin, Dublin, Ireland

    Damien Traynor, Isha Behl & Fiona M. Lyng

  3. School of Biological and Health Sciences, Technological University Dublin, Dublin, Ireland

    Declan O’Dea & Alison Malkin

  4. EA 6295 Nanomédicaments et Nanosondes, Université de Tours, Tours, France

    Franck Bonnier

  5. St James’s Hospital, Dublin, Ireland

    Siobhan Nicholson, Finbar O’Connell & Aoife Maguire

  6. Oral Medicine Unit, Dublin Dental University Hospital, Trinity College, Dublin, Ireland

    Stephen Flint, Sheila Galvin & Claire M. Healy

  7. Discipline of Histopathology, University of Dublin Trinity College, Dublin, Ireland

    Cara M. Martin & John J. O’Leary

  8. Emer Casey Molecular Pathology Research Laboratory, The Coombe Women and Infants University Hospital, Dublin, Ireland

    Cara M. Martin & John J. O’Leary

  9. CERVIVA Research Consortium, Dublin, Ireland

    Cara M. Martin, John J. O’Leary & Fiona M. Lyng

  10. FOCAS Research Institute, Technological University Dublin, Dublin, Ireland

    Hugh J. Byrne

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Contributions

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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Correspondence to Fiona M. Lyng.

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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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