Laser-induced autofluorescence (LIAF) and diffuse reflection spectroscopy (DRS) are two emerging noninvasive optical tools that have shown immense potential to detect oral cavity pre-cancer. In a recent study, we have used spectral ratio reference standards (SRRS) of LIAF intensity ratios F500/F635, F500/F685, and F500/F705 for grading of tissues belonging to sites other than dorsal side of tongue (DST), lateral side of tongue (LST), and vermillion border of lip (VBL) that exhibited similar spectral shape for normal and abnormal tissues. This led to dismal diagnostic accuracies, and for the three LIAF-SRRS, normal tissue values were often misclassified as squamous cell carcinoma (SCC), which means that the true negatives were being wrongly identified as true positives. This study examines the applicability of the site-specific diffuse reflection spectral intensity ratio (R545/R575) of the oxygenated hemoglobin bands to classify different DST lesions and compares the results obtained with those obtained using LIAF-SRRS. DRS-SRRS of R545/R575 differentiated benign hyperplastic DST tissues from normal tissue with a sensitivity of 86% and specificity of 80%, which were indistinguishable using LIAF-SRRS. Further, in distinguishing hyperplastic tissues from premalignant dysplastic lesions, DRS-SRRS gave a sensitivity of 90% and a specificity of 86%, as compared to sensitivity of 89% and specificity of 72% shown by the three LIAF-SRRS together. The diagnostic accuracy and statistical adequacy of the two techniques were assessed by receiver operating characteristic curve (ROC-Curve) analysis. Three LIAF ratios gave a low overall ROC area under curve (ROC-AUCs) of 0.521, whereas the DR ratio (R545/R575) has shown an improved accuracy of 0.970 in differentiating different tissue types. While distinguishing hyperplastic from dysplastic tissues, the DR ratio gave a higher discrimination accuracy of 0.9. Based on these findings, it can be concluded that the DRS-SRRS technique by virtue of its low cost and higher diagnostic accuracies could be a viable alternate to LIAF-SRRS for <i>in vivo</i> screening of tongue pre-cancers and grading of different tissue types.

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