|Year : 2018 | Volume
| Issue : 2 | Page : 398-402
Correlation between fasting blood sugar and cytomorphometric values of diabetic patient's buccal mucosa exfoliative cytology
Rahul Agrawal1, Naresh Kumar2, Kanupriya Gupta1, Tej Bali Singh3
1 Department of Oral and Maxillofacial Pathology, Faculty of Dental Sciences, Varanasi, Uttar Pradesh, India
2 Department of Oral and Maxillofacial Surgery, Faculty of Dental Sciences, IMS, BHU, Varanasi, Uttar Pradesh, India
3 Department of Community Medicine, IMS, BHU, Varanasi, Uttar Pradesh, India
|Date of Web Publication||8-Mar-2018|
Prof. Naresh Kumar
Dean and Head, Faculty of Dental Sciences, IMS, BHU, Varanasi - 221 005, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Objectives: Type-2 diabetes mellitus (DM) has considerable prevalence in India. A noninvasive diagnostic tool will be more appropriate in conditions like DM. In this study, we intend to find a difference in cytomophometric values, and glycogen accumulation (if any) in buccal mucosa exfoliated cells of type-2 DM patients when compared to nondiabetic healthy individuals, and establish its diagnostic role.
Methodology: In the present study, 36 known DM patients with at least 1-year history (case group) and 36 healthy, age- (5 year interval) -matched patients (control group) were included in the study. Patients with any other systemic disease were excluded from the study. Buccal mucosa exfoliative cytology smears were prepared from all 72 patients and stained with Papanicolaou (PAP) stain. The cytomorphometric analysis was performed to evaluate nuclear area (NA), cytoplasmic area (CA), and cytoplasm to nuclear area ratio (CNR) of cells in the smear. The data were tabulated and statistically analyzed using Chi-square test, Student's t-test, Karl Pearson's correlation coefficient and significance (two tailed) test.
Results: Statistically significant difference was found in NA and CNR in exfoliative cytology of DM patient group when compared to control group. Fasting blood sugar of DM patients was significantly correlated with NA and CNR of buccal mucosa exfoliated cells.
Conclusion: The cytomorphometric changes in exfoliated buccal mucosa cells of type-2 DM patients can have a diagnostic value which needs to be explored further.
Keywords: Cytomorphometry, diabetes mellitus, exfoliative cytology
|How to cite this article:|
Agrawal R, Kumar N, Gupta K, Singh TB. Correlation between fasting blood sugar and cytomorphometric values of diabetic patient's buccal mucosa exfoliative cytology. J Can Res Ther 2018;14:398-402
|How to cite this URL:|
Agrawal R, Kumar N, Gupta K, Singh TB. Correlation between fasting blood sugar and cytomorphometric values of diabetic patient's buccal mucosa exfoliative cytology. J Can Res Ther [serial online] 2018 [cited 2019 Nov 20];14:398-402. Available from: http://www.cancerjournal.net/text.asp?2018/14/2/398/209958
| > Introduction|| |
Diabetes mellitus (DM) is an ancient metabolic disorder dating from 1500 BC, as a disease accompanied by polyurea, demanding due treatment. It is basically characterized by chronic hyperglycemia associated with disturbances in the metabolism of carbohydrates, proteins, and lipids, as a result of an absolute or relative deficiency of insulin.
The broad categories of DM are designated as type-1 (insulin dependent DM) and type-2 (noninsulin dependent DM). Type-1 is further classified as type-1A DM resulting from autoimmune beta-cell destruction which usually leads to insulin deficiency, whereas type-1B DM which is also characterized by insulin deficiency, where the etiology is unknown. Type-2 DM is a heterogeneous group of the disorder usually characterized by variable degrees of insulin resistance, impaired insulin secretion and increases glucose production. Distinct genetic and metabolic defect in insulin action and secretion give increase to the common phenotype of hyperglycemia in type-2 DM. The prevalence of type-2 DM is higher in certain pacific islands, intermediate in country such as India and the United States and relatively low in Russia and China.
Several studies have examined the deleterious effects of diabetes on oral mucosa. It was reported that diabetes adversely affects the morphology of cheek mucosa, which may compromise tissue function to favor the occurrence of oral infections and neoplasia., In DM, some important changes of beneficial nature take place which is associated with hyperplasia and increase glycogen deposits in cells of the oral epithelium.
The morphologic and functional changes in oral mucosa can be studied at the cellular level by using Exfoliative cytology which can help in diagnosis with better patient acceptability. Using cytomorphometric analysis, various parameters such as cytoplasmic area (CA), nuclear area (NA), and cytoplasmic to nuclear area ratio (CNR) can be evaluated. CA, NA, and CNR have shown to be significant in the diagnosis of oral and systemic diseases.
Cytomorphometric analysis of exfoliated cells could be a noninvasive diagnostic marker for DM. Thus, the purpose of this study was to demonstrate cytological changes using morphometric analysis in the exfoliated oral mucosal cells of type-2 diabetic patients, to establish its role in diagnostic criteria.
| > Methodology|| |
Patients for the study were obtained from the outpatient department of Faculty of Dental Sciences, IMS, BHU. Case group, including 36 patients with a history of DM for a minimum period of 1 year and control group comprising 36 age- (5 yr interval)-matched healthy patients without any history of diabetes were included in the study. Patients suffering from any other systemic disease, with any tobacco/alcohol associated habits, or with any obvious oral mucosal lesions were excluded from the study.
After obtaining a written informed consent, the detailed information about the history of DM including duration, type, medication etc., was recorded. Fasting blood sugar (FBS) levels was estimated and recorded for all the patients from case group and control group. Hemoglobin percentage was estimated to rule out anemia.
Patients were asked to rinse the oral cavity with water. Scrapings were obtained using a wooden spatula moistened with normal saline. Using a gentle scraping motion exerting little pressure, cells were scraped from the apparently normal buccal mucosa of both case and control groups and smears were prepared. From each patient, two smears were prepared and stained as per PAP staining technique.
The PAP stained smears were subjected to cytomorphometric analysis using Vimage 2014 (Image analysis software) with research microscope (Nikon CiL Eclipse). Fifty unfolded cells with a clear outline, and predominant staining in each prepared smear were selected in a step-wise manner moving the microscope stage from left to right and then down and across to avoid measuring the same cell again.
Cell area was measured in square microns (μm2). For measurement, the cell perimeter was traced with digitalized cursor using interactive measurement tool and the software automatically calculated the cell area.
NA was measured in square micron (μm2) automatically on tracing the nuclear boundary. Cytoplasmic area (CA) was calculated using the formula: CA = cell area – NA. CNR was another parameter calculated by using the formula: CNR = CA/NA.
In each slide, cytology by a number of fifty cells in five microscopic fields with the magnification of 40-fold was examined in terms of the characteristics of the nucleus (the presence of bi- or multi-lobed nucleus, karyorrhexis, vacuolization of cytoplasm, the presence of inflammation in persons with diabetes type-2 and the healthy persons. However, these were not subjected to any statistical analysis.
The data were tabulated and statistical analysis was performed. Chi-square test was used to find out the significant association between study variables and study groups. Student's t-test was used to determine the significance difference in the mean values between cases and control groups. Karl Pearson's correlation coefficient was also calculated between the study variables separately for the cases and control groups. 5% level of significance at two tailed tests was used at significance difference level.
| > Results|| |
In cases group, of 36 patients, the mean age was 52.56 ± 8.06 years with maximum patients, i.e., 14 (38.9%) were more than 55 year of age. Whereas in control group, of 36 patients (mean age = 49.14 ± 7.78 years) the maximum number, i.e., 13 (36.1%) were from 35 to 45 years of age range. In cases group, of 36 patients the majority, i.e., 23 (63.9%) were males. Whereas in control group, 26 (72.2%) were males.
Of 36 case patients, the maximum number, i.e. 20 (55.6%) had a history of DM <5 years, and the majority, i.e. 35 (97.2%) of patients were on oral hypoglycemic drugs, whereas only 1 (2.8%) patient was on insulin. Seventeen cases (47.2%) had FBS levels in the range of 126–135 mg/dl.
Twenty-three patients, (63.9%) had NA ranged from 85.01 to100 μm2. Whereas in control group, 26 (72.2%) had NA ranged from 55.01 to 70.00 μm2. Chi-square value for NA was found to be 50.389 and the value of P < 0.001, showing significant association.
In cases group, maximum number of patients, i.e., 21 (58.3%) had CA ranged from 2300.01 to 2800.00 μ. Whereas in control group, maximum number of patients, i.e., 26 (72.2%) had CA ranged from 2300.01 to 2800.00 μ. Chi-square value for CA was found to be 6.332 and P = 0.097 showing nonsignificant association.
Most of the cases group patients, i.e. 18 (50%) had CNR ranged from 20.01 to 30.00. Whereas in control group, 35 patients (97.2%) had CNR ranged from >30. Pearson's Chi-square value for CNR was found to be 22.562 and P < 0.001 showing strong significant association.
Mean values of NA, CA, and CNR for cases group and control group were calculated and statistically significant difference was found in the mean values of NA and CNR between the two groups. There was no statistically significant difference between CA of cases and control groups [Table 1].
|Table 1: Mean±standard deviation of variables in cases and control group|
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The correlation between FBS and NA (r = −0.493, P = 0.002) and FBS and CNR (r = 0.484, P = 0.003) was found to be statistically significant. CA did not show any statistically significant association with FBS levels of case group patients [Table 2].
|Table 2: Karl Pearson's correlation coefficient between fasting blood sugar levels and nuclear area, cytoplasmic area, and cytoplasm to nuclear ratio in cases group|
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In an attempt to note the qualitative changes in the exfoliated epithelial cells in case and control group, it was seen that the inflammatory component was higher in the diabetic group compared to healthy group, with 35/36 showing inflammatory component compared to healthy group, where only 5/36 showed inflammatory cells.
| > Discussion|| |
Dentists come across various oral manifestations of this disease. If they have a proper understanding of these manifestations, disease can be diagnosed. Early diagnosis is helpful in control of blood sugar level at an early stage to prevent various complications. Several studies have examined the deleterious effects of DM on oral mucosa. It was reported that DM adversely affects the morphology of oral mucosa, which may compromise tissue functions to favor the occurrence of oral infections and oral neoplasia., These effects can be studied at the cellular level using oral exfoliative cytology.
Exfoliative cytology is a simple and noninvasive technique. With the advancement in the field of quantitative exfoliative cytology, there is a reemergence of oral exfoliative cytology as a powerful diagnostic tool.
In recent years, important advances have occurred in the determination of diagnosis for the disease DM and also new strategies for its treatment. The study is an attempt to study morphometric and cytologic changes in the exfoliated cells of apparently normal buccal mucosa in type-2 DM patients.
In the present study, 36 known type-2 DM patients were taken as case group, whereas control group comprised 36 healthy age- (5 year interval)-matched individuals. In a similar study done by Alberti et al. 10 type-2 diabetic patients were taken as case group, whereas control group comprised ten healthy individuals.
The age of individuals in this study ranged from 38 to 70 years. In case group, the mean age of the cases was 52.56 ± 8.06 years of which 38.9% of cases were more than 55 years of age. In control group, mean age of the individuals was 49.14 ± 7.78 years of which 36.1% of cases were in 35–45 years of age [Table 1] and [Table 3]. On statistical analysis, no significant difference between the mean ages of the two groups was found which showed that the two groups were suitable for comparison and the age variable was matched.
|Table 3: Distribution of patients according to demographics, history of disease and investigation variables in case and control groups|
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The case group comprised 63.9% of males and 36.1% of females, showing male predilection. In a study on Indian population by Hong et al. 49.1% were males and 50.9% were females showing slight female predilection. In a similar study in the United States population, it was reported that the prevalence of diabetes in adults was slightly higher in women and increased significantly with age. The male predilection in the present study may be a chance finding.
The case group comprised patient with a history of diabetes ranging from 1 year to 25 years with a mean duration of 6.02 ± 4.48 years. Nearly 55.6% of patients had the disease for <5 years. In another study by Alberti et al. the duration of disease was >1 year in 80% of diabetic patient.
In this study, only one patient in the study group was on insulin. Rest all patients were on oral hypoglycemic drugs. In the study of Alberti et al. the study group comprised patients with either oral hypoglycemic or insulin.
In the present study, patients were considered diabetic when the FBS level was ≥126 mg/dl (as per the American Diabetic Association 2000 criteria for the diagnosis of DM). Whereas patients with FBS levels <110 mg/dl were considered normal. Patients with FBS level >110 mg/dl, but <126 mg/dl were considered impaired fasting glucose and not included in the study. The FBS levels in case group ranged from 126 to 207 mg/dl with an average of 143.53 ± 18.72 mg/dl. 47.2% of diabetics had FBS levels in the range from 126 to 135 mg/dl. In a similar study by Alberti et al. the mean blood sugar levels in the study group was 137 ± 49.8 mg/dl.
In the present study, fifty unfolded cells were analyzed in a step-wise manner in PAP stained smears. Cowpe et al. published their quantitative assessment on oral smears and suggested that 50 cells are sufficient to provide a consistent assessment of a variety of oral mucosal site. PAP stained smears were subjected to cytomorphometric analysis to assess NA, CA and CNR.
The results of the present study showed that NA of the exfoliated cells from the buccal mucosa of case group ranged from 70.59 to 105.89 μm2 with a mean value of 87.76 ± 8.68 μm2. Whereas in control group, the NA ranged from 53.79 to 88.89 μm2 with a mean value of 66.88 ± 6.50 μm2. On statistical analysis, significant difference was found in mean values between the two groups. The NA values are consistent with the study done by Alberti et al. (diabetic group was 86.5 ± 12.30 μm2 and that of control was 61.3 ± 16.60 μm2) and Jajarm et al. where the mean NA was significantly higher in diabetic group. The test of correlation was found to be statistically significant with moderate association (r = 0.493, P = 0.002) when the NA of the case group was correlated with FBS levels [Table 2].
Increase in NA in the buccal mucosa of type-2 diabetic patients could be due to delay in keratinization process caused by decreased cellular turnover. In diabetics, the glycation of proteins, lipids, and nucleic acids increases with sustained hyperglycemia causing much greater accumulation of advanced glycation end products in the walls of large vessels as well as basement membrane of the microvasculature. The effect of this is a progressive narrowing of the vessel lumen, decreased perfusion of affected tissues and decrease turnover which may cause a delay in keratinization process of epithelium. This delay in the differentiation process of epithelium leads to increase in the cells which present a large nucleus as a primary characteristic.,,
In the present study, the CA of the exfoliated cells from the buccal mucosa of the case group ranged from 1873.23 to 3520.09 μm2 with a mean of 2693.60 ± 406.65 μm2. Where as in control group the CA ranged from 2029.86 to 3204.81 μm2 with a mean of 2594.77 ± 264.36 μm2. On statistical analysis, the difference in mean CA between the two groups was not significant. The CA values are in accordance with that of Alberti et al., according to them, CA in the diabetic group did not exhibit statistically significant difference (P > 0.05) when compared to control group. The CA in diabetic group was 3562.2 ± 438.00 μm2 and that of control group was 3940.7 ± 900.64 μm2. In our case group, the CA was correlated with the FBS levels. The test of correlation was found to be statistically nonsignificant (r = 0.258, P = 0.128), [Table 2].
The preservation of CA in diabetic group may be a compensatory mechanism to maintain the cell function in stress conditions like DM. It can be explained on the fact that in a cell the enzymes that are normally inactive often can be activated when needed. As previously mentioned, there is decreased perfusion of affected tissues and decrease turnover and the cell may remain in a stressful situation. When most of the adenosine triphosphate (ATP) has been depleted in the cell, a considerable amount of cyclic adenosine monophosphate (c-AMP) is found as a breakdown product of ATP. The presence of this c-AMP in turn immediately activates the glycogen splitting enzyme, phosphorylase liberating glucose molecule that are rapidly metabolized to provide energy which is used for replenishment of ATP stores. The c-AMP acts as an enzyme activator for phosphorylase and control intracellular ATP concentration, thus maintaining the functions even in stressful condition. In this way, constant cross feed between the synthetic systems results in the almost equal amount of substance in the cell at all times. The enzyme system can either be activated or inhibited according to the need of the cell. This regulatory mechanism most often function as feedback control system that continuously monitor the cell biochemical composition and make correction as needed even in adverse condition.
In the present study, the CNR of the exfoliated cells from the buccal mucosa of the case group ranged from 21.35 to 47.71 with a mean value of 31.02 ± 6.12, whereas in control group, the CNR ranged from 27.76 to 49.27 with a mean of 39.05 ± 4.84 [Table 1]. On statistical analysis, significant difference was found in the mean values of CNR between the two groups. The CNR values are in consistent with the findings of Alberti et al. and Jajarm et al. noted the mean CNR in diabetic group was 39.04 ± 0.553, whereas in control group, it was 44.07 ± 0.492, according to which the mean CNR was significantly lower in diabetic group than controls. In our study group, the CNR was correlated with the FBS levels [Table 2]. The test of correlation was found to be statistically significant association (r = 0.484, P = 0.003).
Ziskin et al., in a study on the oral epithelium of diabetic patients reported that in diabetics some important changes of beneficial nature take place which are associated with hyperplasia and glycogen deposits.
| > Conclusion|| |
The present study showed an increase in NA but CA did not present statistically significant difference whereas the CNR was diminished significantly in diabetics. Other nuclear changes, inflammation can collectively alert clinician for possibility of diabetes. The cytomorphometric alterations demonstrated in this study suggests that even though the oral mucosa of diabetic patient appears clinically normal, cytological changes are present. The general understanding of the alterations in the cellular pattern of oral mucosal cells in diabetic patients may help us to develop noninvasive diagnostic tool for DM in future. Further studies with larger sample size and corelating treatment modality with the cytomorphometric changes should be taken up so that patient can receive better therapeutic approach.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]