title: Can Feulgen Stain be a Reliable Biomarker over PAP Stain for Estimation of Micronuclei Score?
abstract: BACKGROUND
  Malignant transformation of the Potentially Malignant Lesions (PML) in the oral cavity is associated with elevated mortality rate because of its aggressive and exceedingly invasive nature. Meticulous diagnosis and prompt therapy of PML may help prevent malignant conversion in oral lesions. Carcinogenic insult to oral cells results in chromosomal damage and formation of Micronuclei (Mn), before the development of clinical symptoms.
  
  
  OBJECTIVE
  To determine the genotoxic effect of smoking and chewing tobacco on target tissue using Mn assay and to evaluate the prevalence of other nuclear anomalies associated with it and to determine the reliability of feulgen stain for Mn assay over Papaincolau (PAP) stain.
  
  
  METHODS
  PAP and feulgen staining was done to study Mn in individuals who were having tobacco habits (smoking and chewing) without lesion (n=30), individuals who were having tobacco habit (smoking and chewing) with PML (n=30) and apparently healthy subjects (n=30). Data was analysed for statistical significance using SPSS 17.0 by Kruskal - Wallis Test and Bonferronii test.
  
  
  RESULTS
  Tobacco habits in the form of smoking and chewing have mutagenic effects on human chromosomes which is indicated by increased frequency of Mn in oral exfoliative cells. The mean Mn frequency using feulgen stain was found to be 12.27 with lesion, 10.23 with without lesion and 3.87 in controls. Whereas, metanucleated analysis revealed no significant correlation with the formation of Mn. Non-specific DNA stain (PAP) showed high numbers of Mn cells in all the groups compared to feulgen. Statistically significant difference (p<0.0001) was observed when both the stains were compared for Mn numbers.
  
  
  CONCLUSIONS
  These findings indicate that the individuals having tobacco habits (smoking and chewing) with lesion have high number of Mn cells, thus supporting the assay to be used as a reliable biomarker to assess the genotoxic effect of tobacco in the oral mucosa. The reason for almost twice as high Mn in PAP stained smears is suggestive of cell injury which is collimated by formation of keratin bodies, resulting in its misinterpretation as Mn, leading to false positive results. Hence, it was concluded that PAP stain can be used to identify abnormal cytological changes resulting from mutagenic agent but not to interpret Mn.
authors.affiliation: Senior Lecturer, Department of Oral Pathology, Tatyasaheb Kore Dental College and Research Centre , Kolhapur, Maharashtra, India .
authors.forename: Manish
authors.initials: M
authors.lastname: Kumar
authors.affiliation: Professor and Head of Department, Department of Oral Pathology, Kanti Devi Dental College , Mathura, Uttar Pradesh, India .
authors.forename: Umesh Chandra
authors.initials: UC
authors.lastname: Prasad
authors.affiliation: Senior Lecturer, Department of Oral Pathology, NIMS , Jaipur, Rajasthan, India .
authors.forename: Betina
authors.initials: B
authors.lastname: Chandolia
authors.affiliation: Professor, Department of Oral Pathology, Surendra Dental College , Sriganganagar, Rajasthan, India .
authors.forename: S M
authors.initials: SM
authors.lastname: Manjunath
authors.affiliation: Professor, Department of Oral Pathology, Gurunanak Dental College and Research Institute , Sunam, Punjab, India .
authors.forename: Shiva
authors.initials: S
authors.lastname: Basu
authors.affiliation: Demonstrator, Department of Oral Pathology, Tatyasaheb Kore Dental College and Research Centre , Kolhapur, Maharashtra, India .
authors.forename: Silvie
authors.initials: S
authors.lastname: Verma
keywords: Cytological stains; Nuclear anomalies; Potentially malignant lesion; Tobacco habit