A comparative study for selectivity of micronuclei in oral exfoliated epithelial cells

Abstract

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.

Can Feulgen Stain be a Reliable Biomarker over PAP Stain for Estimation of Micronuclei Score?

“…al., revealed raised Mn frequency in Non-specific DNA stains in contrast to specific DNA stains which were in agreement with our results [12]. Results of Grover et al, were also in accordance with our results, they showed increase Mn frequency in PAP and H&E stains compared to feulgen [38]. [Table/ Fig-9] summarize different studies that have been done up till now to compare the DNA specific and non-specific stains.…”

“…This apparently increased frequency of Mn observed in PAP stained smears in our study as well as other studies, which have used other non-specific stains like MGG and Giemsa may indicate false positive appearances [12,38,39]. Our investigation specifies that PAP stained nuclei are not dependable and should be used with caution, only for metanucleated analysis.…”

Micronucleus Evaluation of Exfoliated Buccal Epithelial Cells Using Liquid-Based Cytology Preparation

“…In both groups studied, the Papanicolaou and Giemsa stains showed greater frequencies of MN compared to Feulgen/fast green, as illustrated in table 2 and figure 1. Grover et al [33] reported similar results when comparing Feulgen/fast green and Papanicolaou stains, with significant differences between the case and control groups. Nersesyan et al [34] obtained similar results when comparing Giemsa to Feulgen/fast green; the frequency of MN for Giemsa was significantly higher, measuring 5-fold greater for the case group compared to the control.…”

“…The preference for this method is associated with its DNA specificity, thereby leaving an unstained, clear cytoplasm that facilitates the detection of MN [32,33,34]. Approximately 30% of the epithelial cell studies used DNA-non-specific stains including Giemsa and Papanicolaou [33,35]. In the present study, the LBC method was used to assess the frequency of MN in buccal epithelial cell smears stained with Giemsa, Papanicolaou and Feulgen/fast green.…”

“…A survey conducted by Bonassi et al [32] found that over 50% of the participating laboratories used Feulgen/fast green stain. The preference for this method is associated with its DNA specificity, thereby leaving an unstained, clear cytoplasm that facilitates the detection of MN [32,33,34]. Approximately 30% of the epithelial cell studies used DNA-non-specific stains including Giemsa and Papanicolaou [33,35].…”