for al 2004 found MN frequency in precancerous (0.63%)

for the DNA damage and to work out the intensity of DNA
damage chromosomal aberrations and micronuclei are excellent markers.

Parvathi devi et al 2011 found step wise increase in
frequency of MN cells, Normal (0.06%), precancerous (0.12%) and cancerous
(0.45%), which indicates cytogenetic damage to epithelium. Haldar et al 2004
found MN frequency in precancerous (0.63%) and cancerous (1.36%). Palve DH et
al 2008 (288) also concluded that gradual increase of MN from normal (0.21% to
cancerous lesions (1.84%). Casartelli et al 2000 observed MN frequencies in
exfoliated buccal cells in normal mucosa, precancerous lesions and squamous
cell carcinoma. They concluded that the gradual increase in MN counts from
normal mucosal to precancerous lesions to carcinoma suggested correlation of
micronuclei as biomarker with neoplastic progression.

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Himadri Kalita et al 2013 observed increased micronuclei
count (4.2 ±0.96 in females and 6.6±1.95 in males) in buccal cells of betel
quid chewers. Smita Jyoti et al 2013 conducted a study on 25 OSMF patients
(gutka chewers along with smoking) using acridine orange stain found a
significant increase in MN count when compared to gutka chewers and control

In our study the frequency of micronuclei per cell in
participants with oral lesions was 2.37 with a standard deviation of 1.100

Nuclear specific stains and micronuclei

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-Grünwald-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

We used feulgan fast, acridine orange, H&E stain and Pap
but found that micronuclei identification and counting was much easier when we
used feulgan fast green stain since it is a nuclear specific stain. Using
acridine orange was a cumbersome procedure since the slides can be viewed only
under a fluorescent microscope. The stain disappears and slides cannot be
preserved for a long time. Pap is a routine stain in exfoliative cytology and
we did not observe any difference in the scoring pattern of the micronuclei. It
was difficult to score micronuclei with H&E stain as an excess of eosin stain
in any part of the slide was a confounding factor.


OSF is a potentially malignant disease of oral cavity and is
most commonly found in Asian countries. Reichart et al 2006 suggested that as a
result of transmigration of populations, an increasing number of OSF cases are
being found in other countries. It constitutes one of the major oral health
problems in countries like India (Kumar KK et al 2007). In this study, 33 OSF
patients were studied. In our study the ratio of male participants was more
than the female participants. Hazarey et al 2005from Nagpur also reported that
most of their patients were with a similar male to female ratio of 5:1. Zhang
et al 2006 from China suggested that the prevalence of betel quid chewing is
highest in the Hunan and Hainan provinces (64.5% to 82.7%) with signs of OSF in
0.9% to 4.7% of the population and the 30 to 49 years age group being the most
commonly affected. Our study showed similar results where the prevalence of OSMF
was high and the participants were males belonging to the age group of 20-49

               Areca nut, often the cause of OSMF
is often wrapped in the leaf of a tropical creeper, Piper beetel commonly known
as the betel leaf or paan. The usage of paan is widespread in the Indian
subcontinent, mostly in the Hindi speaking heartland of North and Central
India. In the Allahabad region, consumption of a unique preparation called
dohra is widespread (Mehrotra et al 2003). It is popular in the district as
well as neighbouring regions of Jaunpur and Pratapgarh. It is a mixture of
tobacco, slaked lime, areca nut and other ingredients like catechu (katha),
peppermint and cardomom (illayachi) etc. It is a wet preparation and marketed
without any brand name. About 200 mg product is kept in plastic bag and a
rubber band is applied. One packet is sold for as less as one rupee. Users
consume tobacco (Surti/Zarda) with dohra according to their level of addiction.

               In this study, 80% of the
patients, chewed paan masala with tobacco (gutkha) 20% pan masala without
tobacco, 14% of the patients used betel quid. Kumar et al reported from Chennai
that 81% of their patient’s had the habit of chewing raw areca nut/commercial