Oral squamous cell carcinoma ranks as the 15th most common cancer worldwide. The present study was undertaken to standardise a protocol for the analysis of oral exfoliated cells using Raman microspectroscopy. For this purpose, samples were obtained from two different sites, based on prevalence of disease (ventral side of the tongue and buccal mucosa).Different oral rinsing agents were employed and it was concluded that non-alcoholic mouthwash adequately removes food debris. Samples were collected using various collection tools and compared. It was observed that endo-cervical brushes yielded cells from deeper layers of the epithelium. Furthermore, monolayer formation of cells was carried out adopting cytospin and ThinPrep techniques and only the ThinPrep method provided flat and separated cells on the glass slide. Raman spectra were acquired from the nuclear and cytoplasmic regions of the cell using an XploRA confocal Raman instrument (HORIBA Jobin Yvon) with a 532nm laser as source. Glass spectral contamination was removed using non negatively constrained least squares (NNLS) algorithms. Corrected spectra were subjected to principal components analysis (PCA) which was able to differentiate the nucleus and cytoplasm regions of the cell; based on nucleic acid and protein features respectively. However, no classification of the two anatomically different sites was observed according to PCA or PCA-LDA (Linear discriminant analysis) using either the nuclear or cytoplasmic spectra.Nevertheless, the study has developed a standardised protocol for sample collection, sample preparation, spectral acquisition and data processing for future studies of oral exfoliated cells based on Raman microspectroscopy.
Oral squamous cell carcinoma (OSCC) is one of the most common malignancies worldwide, and new protocols for routine and early detection are required.Raman spectroscopy is an optical based method that can provide sensitive and non-invasive real time detailed information on the biochemical content of a sample like saliva, through the unique vibrations of its constituent molecules and this is sensitive to changes associated with disease. A comprehensive systematic review of the available scientific literature related to Raman spectroscopy of human saliva for diagnosis of OSCC was performed. The 785 nm laser line was most applied wavelength along with principal components analysis associated with linear discriminant analysis. The main salivary components possibly associated with the presence of OSCC were proteins and lipids. Measurement in the liquid physical state, and with no addition of nanoparticles for signal enhancement, seemed to best conserve the salivary integrity. However, in terms of sampling protocols, no differentiation was generally made between stimulated and non-stimulated saliva. Raman spectroscopy of saliva holds a promising future for clinical applications such as early detection of OSCC. However, more systematic analyses are still required for a better elucidation regarding sampling procedure, storage and degradation.
K E Y W O R D Soral cancer, oral dysplasia, Raman spectroscopy, saliva, vibrational spectroscopy
| INTRODUCTIONOral squamous cell carcinoma (OSCC) is one of the most frequently encountered malignant tumours worldwide, and its incidence is expected to reach around 350 000 new cases Abbreviations: CCD, charge coupled device; ELISA, enzyme-linked immunosorbent assay; MALDI-Q-TOF, ionisation-quadrupole-time-offlight; OSCC, oral squamous cell carcinoma; PCA-LDA, principal components analysis associated with linear discriminant analysis; PLSDA, partial least squares discriminant analysis; SERS, surface-enhanced Raman spectroscopy; SVM, support vector machine.
This study demonstrates the efficacy of Raman micro‐spectroscopy of oral cytological samples for differentiating dysplastic, potentially malignant lesions from those of normal, healthy donors. Cells were collected using brush biopsy from healthy donors (n = 20) and patients attending a Dysplasia Clinic (n = 20). Donors were sampled at four different sites (buccal mucosa, tongue, alveolus, gingiva), to ensure matched normal sites for all lesions, while patient samples were taken from clinically evident, histologically verified dysplastic lesions. Spectra were acquired from the nucleus and cytoplasm of individual cells of all samples and subjected to partial least squares‐discriminant analysis. Discriminative sensitivities of 94% and 86% and specificity of 85% were achieved for the cytoplasm and nucleus, respectively, largely based on lipidic contributions of dysplastic cells. Alveolar/gingival samples were differentiated from tongue/buccal samples, indicating that anatomical site is potentially a confounding factor, while age, gender, smoking and alcohol consumption were confirmed not to be.
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