micronucleus ( mn ) is the name given to the small nucleus that form whenever a chromosome or a fragment of a chromosome is not incorporated into one of the daughter nuclei during cell division . the damaged chromosomes , in the form of acentric chromatids or chromosome fragments , lag behind in anaphase when centric elements move towards the spindle poles . after telophase , the undamaged chromosomes , as well as the centric fragments , give rise to regular daughter nuclei . the lagging elements are included in the daughter cells , too , but a considerable proportion is transformed into one or several secondary nuclei , which are , as a rule , much smaller than the principal nucleus and are therefore called micronuclei . bigger micronuclei ( mni ) result from exclusion of whole chromosome following damage to the spindle apparatus of the cell ( aneugenic effect ) , whereas smaller mni result from structural aberrations , thereby causing chromosomal fragments ( clastogenic effect ) . an evaluation of the literature shows that a variety of different stains are used in mni studies . among the dna - specific stains , the ones most widely used are feulgen and acridine orange ; in some experiments , 4,6-diamidino-2-phenylindole ( dapi ) and propidium iodide were also used . about 30% of the studies in epithelial cells were conducted with nonspecific stains ( giemsa , may - grnwald - giemsa , papanicolaou and less frequently orcein and hematoxylin and eosin ) . however , only little attention has been , until now , given to the effect of different staining procedures on the results of mn assays . it has been established that in the head and neck region mni are found at increased frequencies from normal mucosa to potentially malignant disorders ( pmds ) to carcinoma , suggesting ever increasing chromosomal instability . with this view in mind , the present study was carried out to investigate the effect of different stains on mni frequencies in exfoliated oral epithelial cells . oral exfoliated cells from patients with pmds ( i.e. , cases ) were taken and oral exfoliated cells were also taken from normal healthy subjects ( i.e. , controls ) . mni frequencies were compared using three different stains , i.e. , feulgen stain , papanicolaou ( pap ) stain and hemotoxylin and eosin ( h and e ) stain . study sample consisted of 60 subjects and was divided into two groups as follows : group i ( controls ) : control group comprised of 15 healthy subjects with clinically normal oral mucosa . group ii ( cases ) : comprised of 45 patients [ 15 oral submucous fibrosis ( osmf ) , 15 lichen planus and 15 leukoplakia ] clinically diagnosed as having one of the pmds of the oral cavity . age - and sex - matched healthy subjects having no obvious oral lesions or habits of consumption of tobacco , other tobacco - related substances , or potentially toxic substances were selected as control group . patients with provisional or confirmed diagnosis of any cancer were not included in the study . written informed consents from these patients were taken for the procedures to be carried out on them . oral mucosal cells were scraped from lesional tissue in cases and from the buccal mucosa of control group using a slightly moistened cytobrush ( medscand medical ab , malmo , sweden ) . the smears were fixed with commercially available spray fixative ( available with the rapidpap kit ) for 15 minutes . all the slides were observed under a light microscope using low magnification ( 400 ) for screening and high magnification ( 1000 ) for counting of mni . the most commonly used method , i.e. , the zigzag method , was followed for screening of slides . one thousand cells with intact nuclei and cell boundaries were counted on each slide . for the purpose of designating an extra nuclear body as a mn , the following criteria given by tolbert et al . were considered : 
 ( a)rounded smooth perimeter suggestive of a membrane;(b)less than a third the diameter of the associated nucleus , but large enough to discern shape and color;(c)staining intensity similar to that of the nucleus;(d)texture similar to that of nucleus;(e)same focal plane as nucleus ; and(f)absence of overlap with , or bridge to , the nucleus . 
  rounded smooth perimeter suggestive of a membrane ; less than a third the diameter of the associated nucleus , but large enough to discern shape and color ; staining intensity similar to that of the nucleus ; texture similar to that of nucleus ; same focal plane as nucleus ; and absence of overlap with , or bridge to , the nucleus . only those structures fulfilling the above - mentioned criteria were recorded as mni [ figure 1 ] . the total number of mni observed in 1000 intact epithelial cells was calculated as the mni frequency . kruskal - walli 's ( kw ) anova test was used for multiple group comparisons and pairwise comparison was done by wilcoxon 's signed rank test . the mann - whitney test was applied for two group comparisons ( cases v / s . photomicrographs showing micronuclei ( arrow ) in oral exfoliated epithelial cells ( a : feulgen stain , b : pap , c : h and e , 1000 ) oral mucosal cells were scraped from lesional tissue in cases and from the buccal mucosa of control group using a slightly moistened cytobrush ( medscand medical ab , malmo , sweden ) . the smears were fixed with commercially available spray fixative ( available with the rapidpap kit ) for 15 minutes . all the slides were observed under a light microscope using low magnification ( 400 ) for screening and high magnification ( 1000 ) for counting of mni . the most commonly used method , i.e. , the zigzag method , was followed for screening of slides . one thousand cells with intact nuclei and cell boundaries were counted on each slide . for the purpose of designating an extra nuclear body as a mn , the following criteria given by tolbert et al . were considered : 
 ( a)rounded smooth perimeter suggestive of a membrane;(b)less than a third the diameter of the associated nucleus , but large enough to discern shape and color;(c)staining intensity similar to that of the nucleus;(d)texture similar to that of nucleus;(e)same focal plane as nucleus ; and(f)absence of overlap with , or bridge to , the nucleus . 
  rounded smooth perimeter suggestive of a membrane ; less than a third the diameter of the associated nucleus , but large enough to discern shape and color ; staining intensity similar to that of the nucleus ; texture similar to that of nucleus ; same focal plane as nucleus ; and absence of overlap with , or bridge to , the nucleus . only those structures fulfilling the above - mentioned criteria the total number of mni observed in 1000 intact epithelial cells was calculated as the mni frequency . kruskal - walli 's ( kw ) anova test was used for multiple group comparisons and pairwise comparison was done by wilcoxon 's signed rank test . the mann - whitney test was applied for two group comparisons ( cases v / s . photomicrographs showing micronuclei ( arrow ) in oral exfoliated epithelial cells ( a : feulgen stain , b : pap , c : h and e , 1000 ) the mean age of the controls was found to be 35.1  10.5 years , whereas for pmds it was 39.1  15.1 years . any statistical significance of age between the cases and controls was ruled out by unpaired student t - test ( p = 0.26 ) . the mean age of the subjects with osmf was found to be 29.9  10.0 years , for lichen planus it was 40.5  15.6 years and for leukoplakia it was 46.8  14.9 years . it was observed that subjects with leukoplakia had the highest mean age and the ones with osmf had the lowest mean age as compared to the other two pmds . among group i ( control ) , out of 15 subjects , 9 ( 60% ) were males and 6 ( 40% ) were females . in group ii ( cases ) , out of 45 subjects , 35 ( 77.8% ) were males and 10 ( 22.2% ) were females . any statistical significance of gender between the cases and controls was ruled out by the way of the chi square test ( p = 0.18 ) . among the different pmds , except for lichen planus ( f  60% , m  40% ) , the same pattern of males predominance was seen in osmf ( m  100% , f of mni/1000 cells ) between the three staining procedures together , kw anova test was used independently for cases [ table 1 ] and controls [ table 2 ] . the kw anova was followed by wilcoxon 's signed rank test for inter staining procedure comparison in cases and controls . statistically significant ( p < 0.01 ) results were obtained for each comparison being made independently [ figure 2a ] . comparison of micronuclei frequency between the three staining procedures together in cases ( kruskal walli 's anova test followed by wilcoxon 's signed rank test ) comparison of micronuclei frequency between the three staining procedures together in control ( kruskal - wallis anova test followed by wilcoxon 's signed rank test ) ( a ) comparison of mni frequency ( no . of mni/1000 cells ) between the three staining procedures together in cases and controls , ( b ) comparison of the mni frequency ( no . of mni/1000 cells ) between the cases and controls for each of the three staining procedures the mann - whitney test was used for comparison of the mni frequency ( no . of mni/1000 cells ) between the cases and controls for each of the three staining procedures [ table 3 ] . statistically significant results ( p < 0.01 ) were obtained in each of the staining procedures [ figure 2b ] . comparison of micronuclei frequency between cases and control for each of the three staining procedures ( mann - whitney test ) micronucleus ( mn ) represent small , additional nuclei formed by the exclusion of chromosome fragments or whole chromosomes lagging at mitosis . mn rates , therefore , indirectly reflect chromosome breakage or impairment of the mitotic apparatus . about 30 years ago , stich et al . developed a protocol for mn assays with exfoliated buccal mucosa cells , which was widely used in occupational and lifestyle studies . it was repeatedly emphasized that this noninvasive method might be a suitable biomonitoring approach for the detection of increased cancer risk in humans because more than 90% of all human cancers are of epithelial origin . the level of baseline chromosome damage in untreated cancer patients and also in various pmds is much higher than in cancer - free controls . therefore , mni scoring can be used as a biomarker to identify different preneoplastic conditions much earlier than the manifestations of clinical features and might specifically be utilized in the screening of high - risk population for a specific cancer . approximately more than 200 studies with epithelial cells have been published in the last 30 years and apart from cells of the oral mucosa , cells from other organs , such as nasal mucosa , cervix , bladder , esophagus and bronchi , were also used . however , until now only little attention has been given to the effect of different staining procedures on the results of mn assays . according to the results of the detailed survey of the current methodological and data acquisition statuses of laboratories , that have published papers on the buccal mn assay ( discussed at eighth workshop of the human micronucleus ( humn ) project held at antalya , turkey , 2007 ) , more than 50% of the laboratories which participated in the survey used feulgen stain and only one of the laboratory had used h and e stain . according to the same survey the second most common stain used was may - grnwald - giemsa ( mgg ) stain , however ayyad et al . found better results for counting of mni using pap stain as compared to mgg stain.thus in the present study an attempt was made to compare feulgen stain ( the most commonly stain used for mn assay ) , pap stain ( the most commonly used stain in routine exfoliative cytology ) and h and e stain ( the most easily available stain ) . in the present study , among the cases taken together as a whole , feulgen stain showed the least mni frequency and h and e showed the maximum count , while the value obtained with pap was intermediate between the former two . the possible explanation for the lowest count with the feulgen stain could be its high dna specificity and a clear transparent appearance of the cytoplasm which enables easy identification of mni . one shortcoming of this staining method apart from being highly technique sensitive ( as observed during this study ) is that it is relatively lengthy and may lead to the underscoring of mni . the increased mni frequency observed with dna nonspecific stains ( pap and h and e in this study ) can be due to any of the following reasons : 
 misinterpretation of nuclear anomalies like karyorrhexis , karyolysis , condensed chromatin , and binucleates as mni , as significant correlation was observed between these anomalies and mni count using dna nonspecific stains in a study carried out by nersesyan et al.formation of keratin granules that are found in degenerated cells with nuclear anomalies . these round cytoplasmic bodies , which are formed as a consequence of cell injury , do not contain dna and may be classified as mni with nonspecific stains.contamination by the bacteria that are commonly found in the mouth can interfere with mni scoring . bacteria can be differentiated from mni by their characteristic shape , smaller size , color , staining intensity , and their presence upon and between buccal cells on the slide.another common confounding issue is the small dye granules that may sometimes resemble mni but usually have a slightly different refractility and color intensity . misinterpretation of nuclear anomalies like karyorrhexis , karyolysis , condensed chromatin , and binucleates as mni , as significant correlation was observed between these anomalies and mni count using dna nonspecific stains in a study carried out by nersesyan et al . these round cytoplasmic bodies , which are formed as a consequence of cell injury , do not contain dna and may be classified as mni with nonspecific stains . contamination by the bacteria that are commonly found in the mouth can interfere with mni scoring . bacteria can be differentiated from mni by their characteristic shape , smaller size , color , staining intensity , and their presence upon and between buccal cells on the slide . another common confounding issue is the small dye granules that may sometimes resemble mni but usually have a slightly different refractility and color intensity . in the present study , these factors were conscientiously taken care of , to minimize the possibility of scoring these mimickers . the explanation for a relatively higher count seen with h and e as compared to pap stain warrants further evaluation . since the scientific literature lacks sufficient relevant data , in relation to the use of h and e stain for mni count in oral exfoliated cells , further detailed studies are required to ensure the validity of this stain . the mean mni frequency noted in cases was significantly higher ( p < 0.01 ) than the one noted in controls , using each of the three stains [ figure 2b ] . these results are in accordance with the ones observed in many studies carried out so far in pmds.[7101721 ] casartelli et al . concluded that the gradual increase in mni frequency from normal mucosal to precancerous lesion to carcinoma suggested a link of this biomarker with neoplastic progression . according to samanta and dey , the various possible explanations for mni formation in preneoplastic conditions include chromosomal aberrations , chromosome loss / breakage , mitotic apparatus dysfunctions , aneuploidy and genetic instability . according to belien et al . multiples of 1000 epithelial cells should be observed for more standard results for evaluation of micronuclei , which necessitates automation.since automation will hamper the cost effectiveness of this screening test , when applied to masses , so the authors of the present study restricted to 1000 cells and found statistically significant results . the results of our study are in contrast to the ones observed in a similar study carried out by nerseseyan et al . , according to which , a statistically significant difference in mni counts between smokers and nonsmokers was observed only with dna nonspecific stains and not with the dna - specific stains . the researchers ( nerseseyan et al . ) validated their results by concluding that smoking does not induce a significant increase in mni counts . except for the feulgen stain , none of the stains used in our study were common to the stains used in the above mentioned study , so any justification for the differences being observed can not be optimally elaborated . a note on comparison between the two is made here only to emphasize the dna specificity of feulgen stain that was being observed in both the studies . being a dna - specific stain , values with feulgen stain were found to be lowest in measure . because of the possibility of mis - interpretation of nuclear anomalies ( e.g.,karyorrhexis , karyolysis , condensed chromatin , and binucleates ) and keratin granules as mni with nonspecific stains , a higher count was observed with pap and h and e stain . taking into consideration the drawbacks of feulgen stain ( i.e. , technique sensitivity and time consuming procedure as compared to the other two staining procedures ) on the one hand and at the same time statistically significant results being observed between the cases and controls in this study , using all the three stains , whether it be a dna - specific stain ( i.e. , feulgen stain ) or dna nonspecific stain ( pap and h and e stain ) , on the other hand , this study intends to lay a foundation for further detailed comparative studies to ensure the validation of dna nonspecific stains in mn assays .
background : micronucleus ( mn ) represents small , additional nuclei formed by the exclusion of chromosome fragments or whole chromosomes lagging at mitosis . 
 mn rates , therefore , indirectly reflect chromosome breakage or impairment of the mitotic apparatus . during the last few decades , micronuclei (  mni  ) in oral exfoliated epithelial cells 
 are widely used as biomarkers of chromosomal damage , genome instability and cancer risk in humans . however , until now only little attention has been given to the effect of different staining procedures on the results of these mn assays.aim:to compare the mni frequencies in oral exfoliated epithelial cells using three different stains , i.e.,feulgen stain , papanicolaou stain ( pap ) and hemotoxylin and eosin stain ( h and e).materials and methods : oral exfoliated cells from 45 cases of potentially malignant disorders ( 15 oral submucous fibrosis , 15 lichen planus and 15 leukoplakia ) and 15 controls with healthy mucosa , were taken and mni frequencies ( no . of mni/1000 cells ) were compared using three different stains.results:mean mni frequency in cases was found to be 3.8 with feulgen stain , 16.8 with pap and 25.9 with h and e. in controls , mean mni frequency was 1.6 with feulgen stain , 7.7 with pap and 9.6 with h and e stain . statistically significant value ( p < 0.01 ) were observed when the three stains were compared together using kruskal walli 's anova test.conclusion:feulgen being a dna - specific stain gave the least counts , although statistically significant results from the comparison of mni frequency between cases and controls were obtained with all the three stains .