Efficacy of Oral Exfoliative Cytology in Diabetes Mellitus

Efficacy of Oral Exfoliative Cytology in Diabetes MellitusEFFICACY OF ORAL EXFOLIATIVE CYTOLOGY IN DIABETES MELLITUS PATIENTS-A frail MICROSCOPIC CONFOCAL MICROSCOPIC STUDYAbstractIntroduction Diabetes Mellitus has develop a global problem. By observe the wellness status of these individuals, diabetic complications tether to morbidity can be prevented.Aim We aimed to analyze alte rations in the syllable structure and cytomorphometry of buccal epithelial carrells of casing 2 Diabetes Mellitus patients use oral exfoliative cytology proficiency and determine its importance in public health screening, diagnosis and monitoring of diabetes mellitus.Material Methods The say was carried out in 100 sheath 2 Diabetes Mellitus patients and 30 healthy individuals. Smears were interpreted from the set buccal mucosa and stained by the Papanicolaou technique. Staining with Acridine orange was carried out to view qualitative changes with confocal optical maser see microscope (LSM -510 Meta).The cytomorphometry was evaluated using photograph PRO PLUS 5.5 software with growing L.C camera. All findings were statistic whollyy analyzed.Results The results grounded that with adjoin in fasting plasma glucose levels, there is operative incr peace of mind in nuclear area, decrease in cytoplasmatic area, and increase in nuclear cytoplasmic ratio (PConclusion In the present workplace, we free-base probatory alterations in the cytomorphometry and cytomorphology of buccal epithelial cells of Type 2 Diabetes Mellitus patients. This discipline supports and extends the view that these cellular changes can alert the clinician to the scuttle of diabetes and aid in monitoring of diabetes throughout the sprightliness of the patient.Keywords Confocal laser scan microscope Cytomorphometry Diabetes mellitus Oral exfoliative cytology.IntroductionDiabetes mellitus (DM) is non a single disease entity precisely rather a group of metabolic disorders sharing the common a t a lower placelying feature of hyperglycaemia. Hyperglycemia results from defects in insulin discrimination, insulin action or both. The chronic hyperglycemia and resultant metabolic deregulation of carbohydrate, fat and protein may be associated with secondary maltreat in multiple organ systems, especially the kidneys, eyes, nerves and broth vessels.1Recent estimates indicate that the number of pack with DM in 2010 is 285 zillion and is pass judgment to be 438 million in 2030. The global prevalence rate is 6.6% in 2010 and is expected to rise to 7.8% in 2030.2India leads the world with largest number of diabetic subjects earning the indefinite distinction of being termed the diabetes capital of the world. According to the Diabetes Atlas 2009 produce by the International Diabetes Federation, the total number of people with diabetes in India is 50.8 million and is expected to rise to 87 million in 2030.3 It can no longer be considered a disease of affluent nation al champ ion, it has become a global problem, a major epidemic of the twentieth century, and one which shows no sign of abating. 3DM is the jumper lead campaign of end-stage nephritic disease, adult-onset blindness and traumatic lower extremity amputations. The prevalence of DM is increase sharply in the developing world as more people adopt a sedentary lifestyle, with India and China being the largest contributors to the worlds diabetic load.2Oral Exfoliative cytology is a relatively simple and non-invasive clinical technique which has the potential to be developed as a routine investigation for screening of DM. It can be used chair-side during routine dental examination.4The unhomogeneous alterations in the cytomorphology of the oral mucosa in diabetes and characterization of these changes give clinicians a more accurate image of what really happens during diabetes. So in our study, the cytomorphometric and cytomorphologic changes were compared jibe to the different glucose levels of diabetes. In addition to light microscope, we used confocal laser scanning microscope, to ascertain the mixed cytomorphologic changes seen under light microscope. It is possible that the degree of cellular change depends on the progression of diabetes. Hence the present study was undertaken as this research area has received little attention to date.Materials MethodsSelection of subjects The study was carried out in Type 2 DM patients attending the diabetic outpatient department of General Medicine of Sri Ramachandra University, Chennai, India. These patients were under regular monitoring of melodic phrase sugar levels and subsequent treatment. The experimental group included 100 Type 2 DM patients and the control group consisted of 30 healthy individuals unaffectionate of whatever systemic diseases with clinically normal oral mucosa. Study groups The intact study sample was grouped for statistical analysis, based on the young Fasting Plasma Glucose (FPG) levels as follows se parate I FPG 110-150mg/dl Group II FPG 150-200mg/dl Group cardinal FPG 200mg/dl Control FPGExclusion criteria Individuals with tog of tobacco use in whatsoever form, habitual alcohol intake, any other systemic illness, clinically evident nutritional deficiencies like anemia, straw man of oral sepsis were excluded from the study. After selection of the patient, informed consent was obtained and the procedure was carried out. In addition, the biochemical hematological measurements were carried out to exclude anemia other systemic disease.Smear collection and preparation Patients were asked to rinse their mouth to remove any debris. Following this, with a gentle scraping motion, cells were scraped from clinically normal appear right buccal mucosa. The scrapings were then evenly smeared onto the glass slide and immersed in 95% isopropyl alcohol in a coplin jar, for half an hour.Staining technique Smears from all the samples were stained by the Papanicolaou technique ( soft diet). For few samples two smears were taken from the same site, one stained using PAP and other using Acridine orange(AO), a fluorescent colour, to view with confocal laser scanning microscope. In cases with precise high plasma glucose levels smear was stained with Periodic Acid Schiff (PAS) technique to assess presence of candida.Cytomorphometric assessment The cytomorphometric analysis was done using IMAGE PRO PLUS 5.5 software with Evolution L.C camera. In each of the PAP stained slide, ten fields were chosen by systematic sampling in a step wise manner, moving from left to right and then down and across in order to avoid step the same cells again. Cells with clearly defined cellular outlines were only chosen those that were clumped, overlapped or folded were excluded for analysis. The cells were projected on to the monitor via the camera at 40x magnification and images were captured. In the software main menu, the function Measurement mode was selected and the icon specifying po lygon was enabled to analyze the area of interest. The nuclear area (NA) and cytoplasmic area (CA) were obtained by drawing around the nuclear and cell boundaries using the cursor. The areas were recorded in square microns. The nuclear cytoplasmic ratio (N/C) was calculated for all the cells. The various parameters calculated were by the Image analysis software thereby trim down the subjective error.Cytomorphologic assessment The PAP PAS stained slides were viewed under light microscope whereas the AO stained slides were viewed under confocal laser scanning microscope (LSM-510 meta).Morphologic assessment included inflammatory component, cell retroversion, micronuclei, binucleation, intracytoplasmic inclusion, candida, keratinization, intracytoplasmic microorganisms any other changes.ResultsOn comparison of the cytomorphometry between the various study groups, the results showed a meaning(a) increase in the NA N/C ratio (pDiscussionDM has become a global problem. It is not a d isease but a metabolic disorder. Early signal detection and subsequent monitoring of this disorder will definitely improve the health of individuals suffering from diabetes. By monitoring the health status of the individuals, diabetic complications leading to morbidity can be prevented, thus producing a healthy society.In the present study we aimed to analyze alterations in the morphology and cytomorphometry of buccal mucosal cells of Type 2 diabetics using exfoliative cytology technique and determine its importance in public health screening and monitoring of DM.The study showed a evidentiary steady increase in NA with increase in relationship sugar level (Figure1) from control group (Mean NA-51.03m2) to the diabetic Group III (Mean NA-81.21 m2). This finding concurs with other previously reported studies who reported a pregnant increase in NA in diabetic patients.5-7 However all these studies did not categorize the patients based on the blood glucose levels. Similar studies us ing cytomorphometry adjudge been done to analyze the effect of alcohol, tobacco, radiotherapy on buccal mucosa. 8-12 Nucleus contains the genomic deoxyribonucleic acid, histones and several proteins. The nuclear size can therefore be altered by change in the content of desoxyribonucleic acid or proteins. There is usually twice as much protein as DNA in a nucleus.13 Hyperglycemia induces a compensatory increase in insulin secretion which in turn causes increase in protein formation. Insulin has several mitogenic functions, including initiation of DNA synthesis in certain cells. These may account for the increase in NA seen in diabetes patients.14Our study revealed a decrease in CA with increase in blood sugar level i.e.GroupIII mean CA was 2270.63 m 2 and control mean CA was 2771.6 m2 (Figure1). This finding is distant to studies by Alberti et al 5 and Shareef et al6 who found that CA did not show any significant difference in diabetics and Hassan et al7 who reported a significant increase in CA. However this finding concurs with Prasad et al15 who reported increase in cell diam and cytoplasmic diameter with increase in glycemic status. Ogden et al10 let reported a similar reduction in cell diameter in patients with habit of alcoholism. The reduction in CA in our study could be due to the dehydrated condition of the diabetics. Increased blood glucose causes dehydration, polyuria polydipsia, intracellular extracellular dehydration. Glucose does not diffuse easily through the pores of the cell membrane causing an increase in osmotic pressure. This increase in osmotic pressure in extracellular fluid causes osmotic transfer of urine out of the cells explaining the reduction in CA.16The comparison of N/C ratio between control and experiment groups showed a steady increase in N/C ratio (from Control-0.0188 to Group III-0.0369) with increasing blood glucose levels(Figure1) .This may be due to a real increase in NA and decrease in CA in diabetic patients, rathe r than bonnie a deviation from normal. This finding concurs with Rivera et al17 and Prasad et al15 who withal reported increase in N/C ratio.In the qualitative changes we found that micronuclei (MN) was present in 44 cases which was statistically significant (p=0.018). MN are known biomarkers of genome alter and has been studied in buccal cell systems. They provide a well-to-do and reliable index of both chromosome breakage and chromosome loss.MN is found in cells that have completed nuclear division.MN was judged according to criteria by Tolbert et al. 18 Another biomarker of genome damage is nuclear bud formation which was also visualized but not statistically analyzed.16 The nuclear bud has the same morphology and staining properties as the nucleus, however, its diameter may range from a half to a fourth of that of the main nucleus. Hyperglycemia results in formation of Advanced Glycation End products (AGE), endothelial nitric oxide synthase uncoupling, activation of prote in kinase C activation of polyol pathway. This in turn causes activation of reactive oxygen species which results in induction of aerophilic attempt. Oxidative focusing is an imbalance between the production of reactive oxygen species and the biological systems efficacy to readily detoxify the reactive intermediates or to repair the resulting damage. The effects of oxidative stress depend upon the size of these changes, wherein a cell can pommel small perturbations and regain its original state. However, more severe oxidative stress can cause cell death and even moderate oxidization can trigger apoptosis, while more intense stresses may cause necrosis. Oxidative stress has been found to cause genomic damage in DM. 19 This supports the observation of MN in diabetic patients. Few other studies also showed increase in MN in lymphocyte cultures of type 2 diabetics.20, 21This is the first reported study in which an attempt has been make to study qualitative changes of buccal smear using confocal microscopy. The use of fluorescence dye (Acridine Orange) and visualization under confocal laser scanning microscope enhanced the demonstration of nuclei and micronuclei. They also helped to avoid false dictatorial results. Statistical analysis revealed that cell degeneration was present in 44 cases out of 100 cases. It was statistically significant (p=0.05) implying that increase in blood glucose level leads to oxidative stress and showed increase in degeneration and necrosis. The various qualitative changes included karyorrhexis, pyknosis, apoptosis chromatin abnormalities. Previous studies by Alberti et al5 Shareef et al6 also demonstrated qualitative changes of karyorrhexis. Another significant qualitative change found was the presence of keratinization. This finding is in unison with an earlier study by Zimmermann et al22 who stated that endocrine disorders like DM had increased keratinized cell count in the buccal mucosa. The increase in this keratinizatio n may be a compensation for decreased salivary flow.5 Binucleation was present in 45 cases out of 100 but showed no statistically significant result. The significance of these binucleate cells is unknown, but they are probably indicative of failed cytokinesis following the last nuclear division in the master(a) cell layer. Studies by Alberti et al5 and Shareef et al6 also showed similar findings of binucleation. Increased number of polymorphonuclear leukocytes was detect in the experiment group than chronic inflammatory cells. Increase in inflammation could be due to decreased salivary flow found in diabetics owing to hypofunction of the salivary glands. Hassan et al7 in their study found that relative incidence of inflammation was higher in diabetic smears. Figure2 Figure3 shows the various qualitative changes viewed under light microscope and confocal microscope.Conclusion From our study, we could conclude that diabetes produces significant alterations in the cytomorphometry and cytomorphology of buccal epithelial cells. The use of confocal laser scanning microscopy demonstrated qualitative changes with higher steadiness and clarity and helped in reducing the possibility of recording false positive and/or false negative observations. Clinical significance Exfoliative cytology is helpful in diabetic patients who have aversion and fear to needle pricks as it is easy and can be carried out regularly .The minimal time and ease of the procedure is beneficial in mass screening and public health awareness programme. They also aid in monitoring of diabetes mellitus throughout the lifetime of the patient, thereby decreasing the morbidity and preventing long term complications.AcknowledgementsAuthors would like to give thanks the Central Research Facility Department of General Medicine of Sri Ramachandra University, Chennai. We would also like to thank the lab assistant Department of Oral pathology and statistician for their valuable help in the study. No co nflict of interest. No line of descent of funding.