COMPARATIVE EVALUATION OF THE EFFICACY OF FIXATIVE AGENTS AND DIFFERENT STAINS FOR EVALUATING MAST CELLS MANUALLY AND DIGITALLY

Introduction- Mediators of inflammation are generated by mast cells. According to research articles, mast cell identification is based on the differential staining of secretory granules but it is always not possible to distinguish reliably between mast cells and basophils in tissues.1 Very few study are done till date to assess the Oral lesions using photometric analysis. So in the present study, RGB was used to compare the efficacy of Stains with the aid of computerised photometric analysis using RGB scoring. Aim - To evaluate and compare the efficacy of fixative agents (10% Neutral Buffered Formalin and Absolute Methanol) and stains (Hematoxylin and Eosin, Toluidine blue and May Grunwald Giemsa stain) for intensity of stain to identify mast cells manually under Light microscope by three independent observers and digitally by Photo Processing software using RGB values in Adobe Photoshop 7.0. Materials and methods - 100 randomly selected Oral Lichen Planus cases were diagnosed using WHO criteria for histopathological examination. Samples were collected by doing punch biopsy and divided into 2 study groups. Group A – 50 tissue samples were fixed in 10% Neutral buffered Formalin for minimum period of 24 hours. Group B – 50 tissue sections fixed in Absolute Methanol for 15 min. Tissues from both the groups were further divided into three parts and stained with H&E, Toluidine blue and MGG stains respectively. The mounted sections of both Group A and Group B were evaluated on the basis of intensity of stain for identification of mast cells by three independent observers under light microscope and digitally using Adobe Photoshop version 7.0 using RGB values for each stained slide respectively and data was statistically analysed. Results - Present study shows non-significant difference in intensity of stain in Group A and Group B between Toluidine Blue and MGG stain whereas difference between Toluidine Blue and H&E stain, MGG and H&E stain was statistically significant for manual observations obtained by three independent observers. Evaluation of intensity of color based on RGB values shows statistically significant difference for Red and Blue color on H&E stained sections. For Toluidine Blue and MGG stained sections, there is statistically significant difference in the color intensity of Red, Green and Blue color with p value less than 0.005.In Group A there was non-significant difference in intensity of Green color between H&E and MGG stain while statistically Significant difference for red, green and blue color between Toluidine Blue, MGG and H&E stain with p value less than 0.005.In Group B, statistically significant difference in the color intensity of Red, Green and Blue color for H&E, Toluidine blue and MGG stain was observed. Conclusion –It was found that 1. Absolute Methanol is more effective in fixation as compared to 10% Neutral buffered formalin which is otherwise the gold standard on account of its effectiveness, low cost and consistent results to identify the mast cells. 2. The efficacy of H&E stain is the lowest among H&E, Toluidine blue and MGG stain to identify the mast cells. 3. The intensity of Red, Green and Blue color shows good contrast among H&E, Toluidine blue and MGG stain when fixed with Absolute Methanol as compared when fixation is done with 10% Neutral Buffered Formalin to identify the mast cells.