Salivary Raman Spectroscopy: Standardization of Sampling Protocols and Stratification of Healthy and Oral Cancer Subjects

Abstract

Minimally invasive cancer detection using bio-fluids has been actively pursued due to practical limitations, though there are better suited noninvasive and online in vivo methods. Saliva is one such clinically informative bio-fluid that offers the advantages of easy and multiple sample collection. Despite its potential in cancer diagnostics, saliva analysis is challenging due to its heterogeneous composition. Recently, there has been an upsurge in saliva exploration using optical techniques. Forms of saliva such as precipitate and supernatant have been monitored, but this sampling method needs to be standardized due to the obvious loss of analytes in processing. In that context, present work details the comparison of four different saliva sampling methodologies, i.e., air-dried, lyophilized, pellet, and supernatant using Raman spectroscopy collected from 10 healthy samples. Composition-driven spectral features of all forms were compared and classified using principal component analysis and linear discriminant analysis. Analysis was carried out on all four groups in the first step. In the second step, groups of pellet and supernatant , and air-dried and lyophilized were analyzed. Findings suggest that pellet and supernatant exhibit discrete spectroscopic features and demonstrate high classification efficiency, which is indicative of their distinctive biochemical composition. On the other hand, air-dried and lyophilized forms showed overlapping spectral features and low classification, suggesting these forms retain majority spectroscopic features of whole saliva and are less prone to sampling losses. Thus, this study indicates air-dried and lyophilized forms may be more appropriate for saliva sampling using Raman spectroscopy providing the comprehensive information required for cancer diagnosis. Furthermore, the method was also tested for the classification of oral cancer and healthy subjects ( n = 27) which yielded 90% stratification. The findings of the study indicate the utility of minimally invasive salivary Raman-based diagnostics in oral cancers.