|Year : 2012 | Volume
| Issue : 1 | Page : 33-37
Cytomorphometric analysis of exfoliative buccal cells in type II diabetic patients
M Suvarna1, C Anuradha2, K Kiran Kumar3, P Chandra Sekhar3, K Lalith Prakash Chandra3, BV Ramana Reddy3
1 Department of Oral Pathology, St. Joseph's Dental College and Hospital, Eluru, India
2 Department of Oral Pathology, Sree Sai Dental College and Research Institute, Srikakulam, India
3 Department of Oral Pathology, SIBAR Institute of Dental Sciences, Takkellapadu, Guntur, Andhra Pradesh, India
|Date of Web Publication||21-Mar-2012|
B V Ramana Reddy
Department of Oral Pathology, SIBAR Institute of Dental Sciences, Takkellapadu, Guntur - 522 509, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Aim: To evaluate the quantitative and qualitative changes in cytological buccal smears of type II diabetic patients by using the parameters like nuclear area (NA), cytoplasmic area (CA), and cytoplasmic/nuclear ratio (C/N).
Materials and Methods: The study was done in 40 known type II diabetic patients and 40 healthy individuals. Buccal smears were taken and subjected to Pap-stain. NA and CA of 20 integral cells in the smear were measured using Image Pro-Express Version 6.0 image analysis system by Media Cybernetics Inc., Bethesda, MD, USA. The C/N ratio was then calculated. For comparing cytomorphometric parameters (NA, CA, C/N ratio), t-test was used.
Results: There was a statistically significant increase in average NA and significant decrease in the C/N ratio in diabetics when compared to non-diabetic healthy individuals. The average CA did not show any statistical difference between the two groups. The morphologic alteration seen in diabetic group was the presence of acute inflammation.
Conclusion: Exfoliative cytology is an additional tool to aid in diagnosis of diabetes mellitus, apart from the regular standard tests.
Keywords: Cytoplasmic area, diabetes mellitus, nuclear area
|How to cite this article:|
Suvarna M, Anuradha C, Kumar K K, Sekhar P C, Lalith Prakash Chandra K, Ramana Reddy B V. Cytomorphometric analysis of exfoliative buccal cells in type II diabetic patients. J NTR Univ Health Sci 2012;1:33-7
|How to cite this URL:|
Suvarna M, Anuradha C, Kumar K K, Sekhar P C, Lalith Prakash Chandra K, Ramana Reddy B V. Cytomorphometric analysis of exfoliative buccal cells in type II diabetic patients. J NTR Univ Health Sci [serial online] 2012 [cited 2019 Jun 26];1:33-7. Available from: http://www.jdrntruhs.org/text.asp?2012/1/1/33/94173
| Introduction|| |
Diabetes mellitus is a clinical syndrome characterized by hyperglycemia due to absolute or relative deficiency of insulin, associated with long-term damage, dysfunction, and failure of various organs.  Diabetes has emerged as a major healthcare problem worldwide. The World Health Organization (WHO) in the year 2009 has put the number of persons with diabetes at approximately 170 million, a figure expected to rise to 366 million by 2030.  The new classification system identifies four types of diabetes mellitus: Type I, Type II, other specific types, and gestational diabetes.  Type II diabetes mellitus accounts for approximately 95% of diabetic cases and occurs usually in patients over 40 years of age.
The oral complications of uncontrolled diabetes mellitus are devastating, which may include, but are not necessarily limited to, gingivitis and periodontal disease; xerostomia and salivary gland dysfunction; increased susceptibility to bacterial, viral and fungal (oral candidiasis) infections; caries; periapical abscesses; loss of teeth; impaired ability to wear dental prostheses (related in part to salivary dysfunction); taste impairment; lichen planus; and burning mouth syndrome. 
Several clinical and paraclinical techniques are available for oral mucosal changes. Incisional or excisional biopsy is the most reliable technique for definitive diagnosis. However, in diabetes, changes in blood glucose and the disease itself reduce the viability of such techniques. 
Oral exfoliative cytology is a quick, simple, less technically demanding, painless, non-invasive laboratory procedure for microscopic investigation of different kinds of oral diseases. As a diagnostic tool, it has got an immense value in mass screening programs of high-risk adult population.  It can be done chair-side during routine dental examination. The use of oral exfoliative cytology in the past was limited due to the subjective nature of its interpretations and high false-negative results. These limitations were overcome by the introduction of quantitative methods such as image analysis systems, especially in the assessment of cytomorphometric cellular alterations. 
However, use of this method to evaluate quantitative and qualitative changes in oral epithelial cells in diabetes is debatable. A few studies have used exfoliative cytology to evaluate changes in the oral mucosa in diabetes mellitus and have shown that this disease can produce alterations in oral epithelial cells that are detectable by cytomorphometric analysis. 
The aim of the study is to evaluate the quantitative and qualitative changes in cytological smears of type II diabetic patients by using parameters like nuclear area (NA), cytoplasmic area (CA), and cytoplasmic/nuclear ratio (C/N).
| Materials and Methods|| |
The present study was carried out in 40 type II diabetic patients with a mean history of 5-6 years and 40 normal healthy individuals as controls, all in the age range between 35-75 yrs. Diagnostic criteria used were the presence of diabetic symptoms and fasting serum glucose levels more than 126 mg/dL or 7 mmol. Control group consisted of healthy individuals free of any systemic diseases, with clinically normal oral mucosa. Individuals with smoking and tobacco chewing habits, habitual alcohol intake, presence of oral sepsis, presence of other systemic diseases, and presence of clinically evident nutritional deficiencies were excluded from the study. Written informed consent was obtained and the pro-forma inventory was completed detailing name, age, gender, and relevant medical history.
All smears were made using a sterile metal spatula. With a gentle scraping motion, scrapped from the clinically normal appearing buccal mucosa behind the commissural area and were stained using Pap technique. Each smear was assessed with the image analysis system (IMAGE PRO-EXPRESS VERSION 6.0) under research microscope (BX51M) and digital CCD camera at ×40 magnification.
Pap-stained smears were observed in a stepwise manner, moving from left to right and then down and across, in order to avoid measuring the same cells again. An average of 20 clearly defined cells selected, in each case, were projected on to the monitor via the camera at ×40 magnification and images were captured. Cytomorphometric analysis was done by selecting the function "Measurement mode0" with the icon specifying "Polygon", and thereafter the auto-trace function was also enabled. Using the mouse, the cursor is placed at a point on the perimeter of the image of the cell on the screen and traced for a short distance.
On double-clicking the mouse, the tracing automatically follows the perimeter and completes the outline. This outline of the region of interest calculates the number of pixels in the cell image and displays the area in square microns. Similarly, the perimeter of the nucleus was traced and the area determined. The measurements of NA and CA that were automatically displayed were recorded and the C/N ratio was calculated for all 20 cells, and the averages were derived for each individual cell.
All the above parameters were calculated by the image analysis software, thereby considerably reducing the subjective error. Qualitative analysis/morphological assessment of the 15 stained smears from each group was performed to note any findings such as associated inflammatory component. All the results were indicated as mean±SD for quantitative variables and as percentage for categorical variables. Variables were compared between two groups using " Pearson correlation co-efficient test ", and the comparison between two means was done using the t-test. SPSS software version 13.0 was used. P values <0.05 were considered significant.
| Results|| |
Results showed that NA was significantly (P = 0.000** [P = <0.001 - Significant]) increased in diabetic patients compared to healthy individuals, and there was a significant reduction in the C/N ratio in diabetic individuals compared to control group with a P-value of 0.000** (P = <0.001 - Significant]). The average CA between the two groups did not show any statistical significance (P=0.826) [Figure 1],[Figure 2],[Figure 3] and [Figure 4] and [Table 1].
|Figure 1: Pap stained cytological smear representative of diabetic group (×40)|
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|Figure 2: Pap stained cytological smear representative of normal healthy group (×40)|
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|Figure 3: Pap stained cytological smear of diabetic group showing squamous cell with enlarged nucleus (×40)|
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|Figure 4: Pap stained cytological smear of normal healthy group showing squamous cell (×40)|
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|Table 1: Comparison of diabetic and healthy individuals with respect to all the parameters|
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An attempt was made to note the qualitative changes in the exfoliated epithelial cells and was studied in 15 cases of each group. It was seen that the inflammatory component was higher in the diabetic group compared to healthy group, with 14/15 showing inflammatory component compared to healthy group, where only 5/15 showed inflammatory cells [Figure 5],[Figure 6] and [Table 2].
|Figure 5: Pap stained cytological smear of diabetic group showing squamous cell associated with inflammatory cell infiltrate (×40)|
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|Figure 6: Pap stained cytological smear of normal healthy group showing squamous cells devoid of associated inflammatory cell infiltrate (×40)|
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|Table 2: Comparison of diabetic and healthy individuals with respect to inflammatory component|
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| Discussion|| |
The dentist plays a key role in diagnosis and therapeutics of diabetes mellitus. Responsibility in the detection of diabetes and in referral of uncontrolled diabetics to proper medical care is important. Considerable work has been done in the field of exfoliative cytology for the detection of precancerous lesions, and the effect of tobacco and alcohol on oral mucosa.
The use of oral exfoliative cytology was limited due to the subjective nature of its interpretations and high false-negative results. These limitations in the assessment of cellular alterations are conquered by the introduction of quantitative methods such as image analysis that is easier to use and more reliable. In this study, cytomorphometric assessment of the exfoliated buccal mucosal cells was done to determine the effect of diabetes on clinically normal mucosa.
In the present study, it was found that there was a significant increase in the mean NA and reduction in the C/N ratio among the diabetic groups when compared to the healthy controls. We did not find any significant difference in the CA between two groups. These results were inconsistent with the studies done by Jajarm HH et al.,  Alberti S et al., and Ban Tawfeek Shareef et al. On the contrary, Jajarm HH et al.  found CA enlargement in the diabetic group and he explained it could be related to the disparity in the number of analyzed cases.
Prasad H et al.,  noted a clear and definite increase in nuclear diameter, and decrease in cell diameter and cytoplasmic diameter in uncontrolled diabetes. A qualitative change, such as increased inflammatory cell infiltrate, was also observed. These findings were inconsistent with the studies mentioned above. Other qualitative changes they observed were randomly distributed chromatin with a porous aspect, as well as figures of binucleation and occasional karyorrhexis. In addition to these morphometric changes, intracytoplasmic inclusions, micronuclei, perinuclear halo, and keratinized squames were also noted in their study.
In the present study, we have avoided all the other possible causes that can give rise to increase in NA (as mentioned in the exclusion criteria for patient sample selection). The possible hypothesis for explaining the increase in mean NA is as follows: An increased glucose level directly favors cell growth because of its pivotal role in metabolic processes. An actively growing cell is characterized by a prominent and large nucleus. Another factor that causes an increase in the nuclear area in diabetic patients is the increased susceptibility to trauma of oral mucosa, for which xerostomia also may play a role.
So, the activity of the basal cells has to be enhanced to replenish the loss of cells occurring at the other end by increasing the proportion of actively dividing cells that are also characterized by a prominent and large nucleus. With an increase in the NA, there should be a concomitant increase in the CA. According to Frost K,  the amount of cytoplasm of the cell makes decreases relative to the amount of nucleoplasm in the actively proliferating cells. This results in an actual increase in nuclear size that may be related to an increase in the nuclear contents required for replication.
Although the qualitative and quantitative changes found in the oral smears of type II diabetic patients are features that point to malignancy, it can be differentiated from the latter by the diminished C/N ratio and uniformity in the nuclear configuration. A high percentage of diabetic patients present with xerostomia and the complaint of dry mouth. This results in a dry, atrophic mucosa with accompanying mucositis as well as opportunistic infection with an increase in polymorphonuclear leucocytes, in response to the microbial colonization. Studies of Chavez EM et al., have shown that subjects with poorly controlled Diabetes had significantly lower stimulated parotid salivary flow rates. 
In diabetic patients, it is a well-established fact that there is a neutrophil chemotactic defect. In an attempt to overcome this deficiency, a positive feedback mechanism acts, resulting in an increase in the inflammatory component. Increased inflammation may also be secondary to adverse hormonal, microvascular, and neuronal changes.
| Conclusion|| |
Based on the results obtained, we are encouraged to venture with a suggestion that estimation of CA, NA, and C/N ratio by image analysis can be used as an effective aid in the diagnosis of diabetes mellitus. Also, confounding factors like smoking and alcohol habits in diabetic patients may influence the final result of C/N ratio. In other words, exclusion criteria have to be strictly adhered to. Further prospective studies have to be conducted with a larger sample size, and comparison with other conditions causing similar cytomorphometric changes is also necessary to determine the predictive value of this method.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]
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