Comparison and Evaluation of Mitotic Figures in Oral Epithelial Dysplasia using Crystal Violet and Feulgen Stain

Abstract

Background: Routine staining procedures often pose a problem in differentiating a mitotic cell from an apoptotic cell, deteriorating the reliability of histology grading. Although various new methods have been recommended for identifying mitotic figures (MFs) in tissues, the time factor and cost makes them less feasible. Thus, an attempt was made to evaluate the efficacy of crystal violet and Feulgen reaction in identifying MFs and also to see for any variation in the number of MFs in various grades of Epithelial dysplasia. Objectives:1. Using crystal violet and Feulgen stain in the identification and counting of MFs on diagnosed cases of epithelial dysplasia and thereby to evaluate their efficacy. 2. To evaluate the variation in the number of MFs in various grades of epithelial dysplasia. Materials and methods:The study sample includes retrieval of 30 formalin fixed paraffin embedded tissue sections diagnosed for different grades of epithelial dysplasia (WHO grading system, 2005) from the archives, Department of Oral Pathology, MSRDC, Bengaluru. Ten tissue sections each of mild, moderate and severe epithelial dysplasia were stained with H&E, Feulgen and 1% crystal violet stains and the number of MFs were counted. Five cases of cervical carcinoma were taken as control. Stained sections were compared, and data obtained was statistically analyzed using the Kruskal-Wallis test.Results: A significant increase in the number of MFs (p = 0.02) was observed in Feulgen stained sections as compared to H&E stain. Conclusion:Feulgen stain can be considered as a simple, reliable, cost-effective and reproducible method of staining MFs.

Evaluation of Mitotic Figures and Cellular and Nuclear Morphometry of Various Histopathological Grades of Oral Squamous Cell Carcinoma: Comparative study using crystal violet and Feulgen stains

“…Similarly, Rao et al also noted a significant increase in the number of MFs in Feulgenstained sections as compared to 1% crystal violetstained samples. 10 A similar study by Palaskar et al evaluating the effectiveness of H&E, toluidine blue, Giemsa, crystal violet and Feulgen stains in oral epithelial dysplasia samples concluded that all of the aforementioned stains were useful in the identification of MFs, although Feulgen stains yielded superior results. 19 In a study evaluating oral epithelial dysplasia and OSCC, Nikita et al reported that Feulgen stains provided a greater contrast when staining MFs as compared to H&E, toluidine blue, Giemsa and crystal violet stains; in addition, the MFs were easier to see at lower magnifications in comparison to the other stains.…”

“…The Feulgen stain is a modified histochemical stain that is simple, cost-effective, reliable and allows for differentiation between apoptotic bodies, karyorrhexis and MFs, thereby excluding false-positive results. 2,5,10,11 Furthermore, the purple-coloured chromatin material against a green cytoplasm facilitates the identification of MFs, even at lower magnifications. Multiple studies have reported using Feulgen staining to study nuclear and cellular morphometry and chromatin frameworks in oral cytosmears or paraffin-embedded tissue sections of various potentially malignant lesions, such as oral hyperplasia, dysplasia and OSCC.…”

“…9 Special stains such as Feulgen and 1% crystal violet stains which depend on the acid hydrolysis of DNA can be used to identify chromosomal material in cells and tissues. 10 Previous studies have utilised haematoxylin and eosin (H&E), Giemsa, crystal violet, toluidine blue and Feulgen stains to investigate chromatin patterns and identify MFs in brain, uterus and breast tissue. 2,5,11 However, few studies have employed Feulgen stains to stain paraffin-embedded sections of oral dysplasia and OSCC cells.…”

“…2,5,11 However, few studies have employed Feulgen stains to stain paraffin-embedded sections of oral dysplasia and OSCC cells. 8,10 This study aimed to compare the number of MFs assessed quantitatively in normal mucosa and OSCC samples using Feulgen and 1% crystal violet stains. A secondary objective was to evaluate the mitotic count among various grades of OSCC.…”

Image analysis assisted study of mitotic figures in oral epithelial dysplasia and squamous cell carcinoma using differential stains

“…Special stains like gallocyanin, Nissl stain, Giemsa and toluidine blue, Feulgen have also been employed for studying mitotic figures. 9,13,35 Switching over to a modified histochemical stain that is cost effective and statistically reliable in its potential would be a wise option and Crystal Violet proved to be promising in this regard.…”