Abstract
Background
The degree of metabolic control in diabetes mellitus influences the susceptibility of patients to oral diseases. It is mandatory to regularly monitor glycaemic status, however invasive methods may be contraindicated or intolerable to diabetic individuals. Thus, cytology, being a simple, non-invasive and rapid procedure, is a promising protocol for assessing diabetic status and assisting in management.
Aim
To assess the number of PAS positive glycogen containing cells and associated cellular changes in buccal smears of type II diabetics and correlate the findings with their serum glucose levels.
Settings and design
The study was conducted at the out patient Department of Oral and Maxillofacial Pathology, KLES Institute of Dental Sciences, Belgaum.
Materials and methods
Fifty known cases of type II diabetes mellitus and 50 healthy individuals were included in the study. Fasting blood glucose levels were estimated and buccal smears stained with Periodic Acid Schiff stain. The observed cellular changes were correlated with the glycaemic status of each patient.
Statistical analysis
Statistical evaluations such as Student’s t test (P < 0.01—very significant; P < 0.001—highly significant), correlation-coefficient and probability values were computed.
Results
Smears of diabetic patients depicted an increase in the number of PAS positive cells in significant correlation to their glycaemic status. Cellular and nuclear morphological alterations were also found in squames of diabetic individuals.
Conclusion
Cytological findings and clinical observations, suggest a correlation between the extent of these changes and clinical parameters like glycaemic control. Further studies in this aspect can help in improving the reliability of oral cytology as a diagnostic tool in diabetes.
Keywords: Diabetes mellitus, Glycaemic status, Exfoliative cytology
Introduction
India has earned the dubious distinction of ‘Diabetes capital of the world’. The ‘Asian Indian Phenotype’ is an important contributing factor which makes Indians more susceptible to diabetes. Thus early screening protocols for high risk groups and periodic monitoring of glycaemic status of known diabetics will greatly reduce the burden of this multi system disorder [1, 2].
In diagnostic technology, the race for the next generation of bloodless, painless and accurate techniques in evaluation of glycaemic status has begun. Exfoliative Cytology, a simple, rapid and inexpensive procedure used to study physiologically desquamated cells has proved an efficient diagnostic adjunct in some oral lesions [3, 4].
Glycogen a polysaccharide of glucose which serves as an energy reserve in cells is regulated by two hormones—epinephrine and glucagon. In diabetes these hormones fail to break down glycogen, resulting in its erratic accumulation [5, 6].
Various cyto-chemical studies have been done to evaluate the expression of glycogen in normal embryonic tissue, wound healing, keratinization and inflammation, but with varying results [7].
This study was undertaken to assess the presence (quantity and quality) of glycogen and other cellular changes in the exfoliated buccal cells of Type II diabetics and correlate them with the serum glucose levels, to establish exfoliative cytology as a diagnostic adjunct in Diabetes Mellitus.
Materials and Method
Fifty diagnosed cases of type II Diabetes Mellitus (study group) and fifty healthy, age and sex matched volunteers (control group) were included in the study. The patients were selected randomly from those visiting the out patient department of Oral and Maxillofacial Pathology, KLES Institute of Dental Sciences, Belgaum, for a routine blood examination. Patients with frank oral lesions and other systemic disorders were excluded from the study. The patients were briefed on the study and a detailed case history with specific details on duration of diabetes and mode of glycaemic control were recorded. A signed consent was obtained and the patients were recalled for a fasting blood sample collection.
Two ml of the patient’s venous blood was drawn under aseptic measures and glucose levels estimated via enzymatic photo-colorimetry (Biozyme Liquid Glucose Kit, Biomedix, Bangalore). The values were undisclosed to the observers to eliminate bias.
A thorough examination of the hard and soft tissues of the oral cavity was done and the buccal mucosa was wiped using moistened gauze to remove excess debris and saliva. Scrapings were taken using a disposable wooden spatula and transferred on to a clean glass slide. Smears were prepared in a circular pattern and stained with Periodic Acid Schiff technique.
Smears were analyzed for—
PAS positive cells Uniformity in area assessed was maintained in each slide. 20 separate fields and an approximate 100 cells were viewed at a magnification of 100×. Magenta colored glycogen positive cells (Fig. 1) were counted with the common consensus of two observers and the results tabulated.
Cellular alterations Cells were screened under the oil immersion objective and features such as cytoplasmic changes, nuclear changes, inflammatory cells and microbial colonies were analyzed and the observations tabulated.
Fig. 1.
Periodic Acid Schiff stain of a buccal smear of a diabetic patient (×40)
The obtained results were then compared with serum glucose levels for each patient.
Results
The obtained results of the study group were tabulated under seven sub headings as per the observations made. The entries were recorded in increasing order of the glycaemic status of the patients (Tables 1, 2).
Table 1.
Master chart of diabetic patients, case numbers one to twenty-five
| Case no. | PAS +ve cells/staining pattern | Increased nuclear size | Karyolysis/karyorrhexis | Binucleation | Inflammatory cells | No. of microbes | Serum glucose levels (mg/dl) |
|---|---|---|---|---|---|---|---|
| 1 | 28/S | 0 | 0 | 0 | 0 | 10 | 138 |
| 2 | 30/S | 3 | 0 | 1 | 7 | 11 | 139 |
| 3 | 30/S | 3 | 1 | 0 | 6 | 15 | 140 |
| 4 | 32/S | 4 | 0 | 1 | 7 | 20 | 140 |
| 5 | 30/S | 3 | 0 | 1 | 6 | 28 | 140 |
| 6 | 35/S | 5 | 1 | 1 | 6 | 25 | 140 |
| 7 | 35/S | 4 | 0 | 0 | 8 | 20 | 140 |
| 8 | 32/S | 5 | 0 | 0 | 7 | 25 | 143 |
| 9 | 32/S | 4 | 0 | 0 | 7 | 35 | 143 |
| 10 | 35/S | 4 | 0 | 1 | 7 | 30 | 145 |
| 11 | 34/S | 6 | 1 | 1 | 8 | 25 | 145 |
| 12 | 32/S | 5 | 1 | 0 | 7 | 22 | 145 |
| 13 | 32/S | 3 | 0 | 0 | 7 | 25 | 145 |
| 14 | 45/S | 5 | 1 | 0 | 7 | 32 | 145 |
| 15 | 30/S | 5 | 1 | 0 | 8 | 30 | 145 |
| 16 | 35/S | 4 | 1 | 0 | 7 | 35 | 145 |
| 17 | 45/S | 5 | 0 | 1 | 8 | 28 | 145 |
| 18 | 32/S | 5 | 0 | 0 | 9 | 30 | 145 |
| 19 | 30/S | 6 | 0 | 0 | 8 | 32 | 146 |
| 20 | 34/S | 4 | 1 | 1 | 8 | 30 | 146 |
| 21 | 30/S | 6 | 1 | 0 | 8 | 30 | 147 |
| 22 | 45/D | 6 | 0 | 0 | 9 | 35 | 149 |
| 23 | 47/S | 5 | 0 | 1 | 9 | 32 | 149 |
| 24 | 45/S | 5 | 0 | 0 | 9 | 35 | 149 |
| 25 | 44/S | 6 | 1 | 1 | 10 | 35 | 150 |
Table 2.
Master chart of diabetic patients, case numbers twenty-six to fifty
| Case no. | PAS +ve cells/staining pattern | Increased nuclear size | Karyolysis/karyorrhexis | Binucleation | Inflammatory cells | No. of microbes | Serum glucose levels (mg/dl) |
|---|---|---|---|---|---|---|---|
| 26 | 47/S | 6 | 1 | 0 | 9 | 37 | 150 |
| 27 | 47/S | 6 | 1 | 1 | 8 | 38 | 150 |
| 28 | 44/S | 5 | 1 | 0 | 8 | 30 | 150 |
| 29 | 46/S | 6 | 1 | 0 | 8 | 30 | 150 |
| 30 | 48/D | 5 | 1 | 1 | 8 | 37 | 150 |
| 31 | 44/D | 6 | 4 | 0 | 8 | 39 | 150 |
| 32 | 45/D | 6 | 1 | 0 | 9 | 35 | 150 |
| 33 | 50/D | 5 | 1 | 1 | 8 | 30 | 150 |
| 34 | 48/D | 6 | 1 | 0 | 8 | 30 | 150 |
| 35 | 50/D | 5 | 1 | 1 | 8 | 38 | 151 |
| 36 | 45/D | 6 | 2 | 1 | 9 | 38 | 155 |
| 37 | 50/D | 6 | 1 | 0 | 10 | 36 | 155 |
| 38 | 56/D | 8 | 1 | 1 | 9 | 35 | 157 |
| 39 | 51/D | 5 | 3 | 1 | 9 | 35 | 158 |
| 40 | 52/D | 6 | 1 | 0 | 10 | 36 | 159 |
| 41 | 56/D | 8 | 0 | 1 | 12 | 39 | 160 |
| 42 | 55/D | 8 | 1 | 1 | 11 | 38 | 160 |
| 43 | 51/D | 5 | 0 | 1 | 11 | 35 | 160 |
| 44 | 55/D | 8 | 1 | 1 | 12 | 38 | 160 |
| 45 | 57/D | 8 | 2 | 0 | 12 | 40 | 160 |
| 46 | 55/D | 6 | 1 | 0 | 10 | 35 | 160 |
| 47 | 59/D | 7 | 1 | 1 | 13 | 38 | 161 |
| 48 | 58/D | 8 | 0 | 0 | 15 | 39 | 162 |
| 49 | 62/D | 9 | 0 | 1 | 17 | 43 | 165 |
| 50 | 54/D | 7 | 0 | 0 | 12 | 33 | 295 |
S speckled, D diffuse
The fasting serum glucose levels of the diabetic patients ranged from 135 to 165 mg/dl with an exceptional case of uncontrolled glycaemic status (295 mg/dl). The study group was divided into three class intervals based on the estimated glucose levels. As per the observations recorded in the buccal smears, each feature was then compared to and correlated with the serum glucose levels (Table 3).
Table 3.
Comparison of number of PAS positive cells with serum glucose levels
| PAS positive cells | Serum glucose levels (mg/dl) |
|---|---|
| 28–45 | 135–145 |
| 30–50 | 146–155 |
| 51–62 | 156–165 |
The PAS positive cells stain a dark magenta pink color. Only those cells that stained intensely were taken into account. In some cells, the entire cytoplasm took up the stain while others depicted a speckled distribution. The glycaemic status was also evaluated based on these two staining patterns (Table 4).
Table 4.
Comparison of staining pattern of PAS positive cells with serum glucose
| PAS positive cells | Staining pattern | Serum glucose levels |
|---|---|---|
| 28–47 | Speckled | 135–150 |
| 45–62 | Diffuse | >150 |
Few inflammatory cells, predominantly lymphocytes were found in close proximity to and infiltrating the buccal squames (Table 5).
Table 5.
Comparison of number of inflammatory cells with serum glucose levels
| Inflammatory cells | Serum glucose levels (mg/dl) |
|---|---|
| 0–9 | 135–145 |
| 8–10 | 146–155 |
| 9–17 | 156–165 |
Cells with nuclear size double or more comparatively, than a standard nucleus were taken into account (Table 6).
Table 6.
Comparison of cells with increased nuclear size and serum glucose levels
| Cells with increased nuclear size | Serum glucose levels (mg/dl) |
|---|---|
| 0–6 | 135–145 |
| 4–6 | 146–155 |
| 5–9 | 156–165 |
Micro-organisms mainly cocci and bacilli were found in most buccal smears. They were accounted for, depending on the number of squames colonized by microbes (Table 7).
Table 7.
Comparison of number of microbial colonies with serum glucose levels
| No. of microbial colonies | Serum glucose levels (mg/dl) |
|---|---|
| 10–35 | 135–145 |
| 30–39 | 146–155 |
| 32–43 | 156–165 |
Discussion
In 1997, the expert committee on ‘Diagnosis and Classification of Diabetes Mellitus’ had enlisted certain diagnostic criteria. Positive findings include symptoms such as polyuria, polydypsia and unexplained weight loss and a RBS ≥200 mg/dl or a FBS ≥126 mg/dl or a PBS ≥200 mg/dl. The normal values fall within the range 80–110 mg/dl for FBS and less than 140 mg/dl for PBS [8].
The ADA recommendations for diagnosis of DM focus on the FBS, while WHO focuses on the Oral Glucose Tolerance Test. The glycated hemoglobin (HbA1c) and fructosamine is also still useful for determining blood sugar control over time. However, practicing physicians frequently employ other measures in addition to those recommended. In July 2009, the International Expert Committee (IEC) recommended the additional diagnostic criteria of an HbA1c result for DM. This committee identified the range of HbA1c levels 6.0 % and <6.5 % to categorize those at high risk of developing DM [9].
In this study, 50 known cases of type II diabetes (diagnosed using ADA criteria), 25 males and 25 females in the age group 35–50 years were included. The duration of diabetes ranged from 2 to 10 years and the FBS levels ranged from 135 to 165 mg/dl. All 50 patients were on oral hypoglycemic drugs. Fifty age and sex matched, healthy individuals were included in the control group. Serum glucose levels were evaluated and buccal smears prepared and stained for each patient.
In the study group, the smears revealed a minimum of 28 and a maximum of 62 PAS positive cells in patients with corresponding glucose levels ranging between 135 and 165 mg/dl. The varying numbers of PAS positive cells were consistent with the serum glucose levels (r = 0.4420 and P = 0.0013). Although the significance is unclear, the implication is that these cellular changes lower the mucosal barrier, promoting infections and affecting regeneration and repair. Advanced glycosylation end products (AGEs) observed in diabetic patients accelerate cell ageing and aged cells are known to exhibit such changes [10].
Two staining patterns of PAS positivity were observed; speckled and diffuse (Figs. 2, 3). In patients with blood glucose levels ranging between 135 and 150 mg/dl, the cells predominantly took up a speckled appearance, while in patients with blood glucose levels higher than 150 mg/dl, the cells took up a more intense and diffuse stain. Literature search revealed no relevant data.
Fig. 2.
Speckled Periodic Acid Schiff staining pattern in an individual cell (×100)
Fig. 3.
Diffuse Periodic Acid Schiff staining pattern in an individual cell (×100)
A significant increase in inflammatory cells (Fig. 4) were found in buccal smears of patients with higher glycaemic status (r = 0.4457 and P = 0.0012). This may be due to diminished salivary flow in diabetes related to hypo function of salivary glands secondary to hormonal, vascular and neuronal changes [10, 11].
Fig. 4.
Lymphocytic infiltration of buccal squames (PAS stain, ×100)
It is known that in the epithelium, stratum intermedium has the highest glycogen reserve. An increase in cellular glycogen is a result of reactionary acanthosis, often associated with chronic inflammation. In diabetic patients there is a neutrophil chemotactic defect. To overcome this, a positive feed back mechanism acts, resulting in an increased inflammatory component [10–13].
Poor glycaemic control in patients makes the oral cavity more vulnerable to microbial infections [14, 15]. In this study too, an increase in the number of bacterial micro organisms (Figs. 5, 6) infesting individual cells was observed in patients with higher glycaemic status; r = 0.284 and P = 0.046. Only one case of candidiasis was reported.
Fig. 5.
Microbial colony of cocci and bacilli (PAS stain, ×100)
Fig. 6.
Fungal hyphae (PAS stain, ×100)
Of the cellular features noted, the number of cells showing increase in nuclear size (Fig. 7) was found to be higher in patients with high serum glucose levels; r = 0.3915 and P = 0.0049. Other findings like karyolysis, karyorrhexis and binucleation (Fig. 8) were also noted. Studies in cytomorphometry have yielded similar results. These changes have been attributed to the increased cellular age of diabetics. Atherosclerosis in diabetes leads to ischemia which in turn causes a decline in cell turnover and thus a greater concentration of aged cells [10].
Fig. 7.
Increase in nuclear size (PAS stain, ×100)
Fig. 8.
Binucleation (PAS stain, ×100)
The overall process of aging in an individual is greatly affected by genetic factors, diet, social conditions and age related diseases, such as diabetes. In addition, there is good evidence that cellular alterations induced by aging are an important component of aging in an organism. Cellular aging is a progressive decline in the proliferative capacity and life span of cells; the effects of continuous exposure to exogenous factors cause the progressive accumulation of cellular and molecular damage. Morphological alterations in aging cells include irregular and abnormally lobed nuclei and pleomorphism [16–18].
The smears of the control group revealed a minimum of 5 and a maximum of 18 PAS positive cells in patients with corresponding blood glucose levels of 60–115 mg/dl. Comparatively the PAS staining intensity of the buccal squames in the control group was lighter than those of the study group. No significant findings other than a few micro organisms were seen.
Conclusion
The frequent use of invasive techniques in diabetic patients is losing viability due to variations in blood glucose levels and in the disease itself. Recent advances in quantitative techniques have refined the potential role of cytology with an appraisal of its value in oral diagnosis. Noteworthy changes in the mucosa of diabetics, will give the clinician a more accurate image of what happens in diabetes. Identifying these changes can also help in early diagnosis and screening of diabetic patients. The future scope of this study lies in assessing a larger sample size, evaluating and comparing cellular glycogen expression in early diagnosed cases and treated cases of type II diabetes. Further research must be done to investigate various cytomorphological changes taking place as the disease progresses. Thus, oral cytological changes observed in patients with diabetes mellitus could be associated to cellular metabolic disturbances. A cytological profile assessment in diabetic individuals would be a simple method to evaluate glycaemic status and treatment efficacy.
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