Comparative Evaluation of Toluidine Blue and Acridine Orange for Mast Cell Detection in Different Histopathological Grades of Oral Submucous Fibrosis: A Pilot Study

Background: Oral submucous fibrosis (OSMF) is an areca nut-induced potentially malignant disorder characterized by progressive fibrosis and limited mouth opening. Mast cells are known to play a crucial role in fibrosis through the release of mediators such as transforming growth factor-beta, fibroblast growth factor, vascular endothelial growth factor, heparin, and chymase. Accurate identification of mast cells across different histopathological grades may provide insight into disease progression and prognostic significance. Aim: This study aimed to evaluate the efficacy of toluidine blue and acridine orange stains in detecting mast cells in various histopathological grades of OSMF and to compare their reliability for mast cell quantification. Materials and Methods: Histopathologically diagnosed cases of OSMF were graded according to Passi et al . (2017), with normal oral mucosa serving as control. Three sections from each grade (I–IV) were analyzed. Toluidine blue staining was performed for mast cell identification under light microscopy, whereas acridine orange staining was assessed under fluorescent microscopy. Ten random high-power fields per section were evaluated for mast cell count. Picrosirius red staining was used to assess collagen fiber deposition and degree of fibrosis. Results: A progressive increase in mast cell count was observed with advancing histopathological grades of OSMF using toluidine blue staining. Acridine orange demonstrated limited specificity, with inadequate detection in early grades and weaker staining intensity in advanced grades. Collagen deposition correspondingly increased from Grade I to Grade IV. Conclusion: Mast cell density correlates positively with the severity of fibrosis in OSMF, underscoring its role in disease progression. Toluidine blue remains a reliable and cost-effective method for mast cell detection, whereas acridine orange shows limited diagnostic utility in routine histopathology. Larger studies are warranted to validate these findings.