|Year : 2022 | Volume
| Issue : 2 | Page : 161-165
Estimation of serum immunoglobulins as a diagnostic marker in Oral Submucous Fibrosis (OSMF) patients
Ragini Gupta1, Manu Dhillon1, Soumendu B Maiti1, Lalit Narayan Singh1, Joy Lakhina1, Arpan Manna2
1 Department of Oral Medicine and Radiology, Divya Jyoti College of Dental Sciences and Research, Modinagar, Uttar Pradesh, India
2 Department of Oral Medicine and Radiology, Teerthanker Mahaveer Dental College and Research Centre, Moradabad, Uttar Pradesh, India
|Date of Submission||19-Jan-2021|
|Date of Decision||04-Apr-2022|
|Date of Acceptance||18-May-2022|
|Date of Web Publication||22-Jun-2022|
Department of Oral Medicine and Radiology, Divya Jyoti College of Dental Sciences and Research, Modinagar, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Oral submucous fibrosis (OSMF) is a potentially malignant disorder affecting the oral mucosa, now accepted globally as a disease. The Indian subcontinent has high malignant transformation potential with multifactorial etiologies and unclear pathogenesis. Immunological pathogenesis has been hypothesized as a causative factor for oral submucous fibrosis. Aim: To quantitatively evaluate IgG and IgM in serum of oral submucous fibrosis, thereby observing any possible association of these immunoglobulins in the pathogenesis of this disease. Material and Methods: A case-control study was done among 60 subjects, including 30 patients with clinically confirmed OSMF and 30 in the control group. Two milliliters of blood was collected from both groups. Quantitative analysis of serum IgG and IgM was done by turbidometric immunoassay. The results were analyzed by unpaired student's t-test and one-way analysis of variance (ANOVA). Results: All patients of OSMF showed increased serum IgG compared to the control group, and the differences were found to be statistically significant (P >.05). On comparing IgM levels between the control and study group, differences were statistically nonsignificant (P < 0.457). Conclusion: The elevation of immunoglobulin levels supports the concept of autoimmunity.
Keywords: Immunoglobulin G, Immunoglobulin M, oral submucous fibrosis
|How to cite this article:|
Gupta R, Dhillon M, Maiti SB, Singh LN, Lakhina J, Manna A. Estimation of serum immunoglobulins as a diagnostic marker in Oral Submucous Fibrosis (OSMF) patients. J Indian Acad Oral Med Radiol 2022;34:161-5
|How to cite this URL:|
Gupta R, Dhillon M, Maiti SB, Singh LN, Lakhina J, Manna A. Estimation of serum immunoglobulins as a diagnostic marker in Oral Submucous Fibrosis (OSMF) patients. J Indian Acad Oral Med Radiol [serial online] 2022 [cited 2022 Jun 29];34:161-5. Available from: https://www.jiaomr.in/text.asp?2022/34/2/161/347912
| Introduction|| |
Oral submucous fibrosis is considered the most potentially malignant disorder of the oral cavity, with a high rate of malignant transformation It is an insidious, chronic disease affecting any part of the oral cavity and sometimes the pharynx, occasionally preceded by and associated with vesicle formation and is always associated with a juxta-epithelial inflammatory reaction followed by progressive hyalinization of the lamina propria. The sub-epithelial and sub-mucosal myofibrosis lead to stiffness of the oral mucosa and deeper tissues with progressive limitation in the opening of the mouth and protrusion of the tongue, thus causing difficulty in eating, swallowing, and phonation. Arecoline alkaloid present in areca nut (betel nut) is identified as a significant etiopathogenic for OSMF. Its increased release of cytokines and growth factor transforming growth factor-beta (TGF-β) ultimately leads to further atrophy, ulceration, and fibrosis. Immunological pathogenesis has been hypothesized to be involved in the pathogenesis of oral submucous fibrosis. During the last three to four decades, only a few studies have been carried out on the immune responses in OSMF.
This study was designed to evaluate IgG and IgM levels in the serum of oral submucous fibrosis patients, thereby observing any possible association of these immunoglobulins in the pathogenesis of this disease.
| Material and Methods|| |
The present research was undertaken to evaluate serum immunoglobulins as a diagnostic marker for oral submucous fibrosis among 60 subjects, which included 30 patients with clinically confirmed OSMF and 30 in the control group who reported to the Department of Oral Medicine and Radiology. Sample estimation was conducted using G power software. The study was followed according to the ethical principles of the Helsinki Declaration. Before starting the study, permission from the Institutional Ethical Committee (DJCDSR/IEC/14/10) was obtained. Informed consent was obtained from patients to participate in this study. Age and sex-matched subjects with no adverse habits and no oral lesions and systemic diseases constituted the controls. Patients with systemic infection or a history of allergy, asthma, liver diseases, and autoimmune diseases were excluded from the study group. Patients with physiological conditions such as pregnancy and menopause were also excluded.
After recording the case history on pilot-tested Proforma and conducting the clinical examinations, all the OSMF patients were staged according to Mathur RM and Jha T criteria (1993) [Graph 1]. Immunoturbidimetric Assay (AU5811 from Beckman colter) was used to analyze serum immunoglobulins.
Methodology for taking blood samples
All the standardization protocols were followed during blood sample collection and serum separation and storage. The patient's forearm was rested on the laboratory table comfortably. The ante-cubital fossa was exposed, and the tourniquet was applied about half an inch to two inches above the ante-cubital fossa. Blood samples were collected by median cubital vein puncture. The area was rendered aseptic with 70% ethyl alcohol. Using a 5-mL sterile disposable syringe and 23-gauze needle, a needle puncture was made and maneuvered to enter the median cubital vein, and 2 mL of blood was withdrawn. Then, the tourniquet was relieved, and the needle was removed simultaneously. Spirit-soaked cotton was placed on the needle puncture site on the forearm, and instructions were given to apply finger pressure for about 10 min and dispose of the cotton.
Methodology for serum separation and storage
Blood from the syringe was transferred to a vacutainer. The vacutainer was centrifuged at 4000 rpm for 15 min, and serum was separated from the clotted blood. Separated serum was transferred to a new container and sent for estimation of immunoglobulins to the laboratory (AU5811 from Beckman colter). Each sample was analyzed by immuno-turbidimetry technique to find the serum's immunoglobulins (IgG, IgM) levels afterwards the results from the laboratory were analyzed statistically to obtain the mean, standard deviation (SD), comparison, and comparison probability chance value.
Statistical analysis methods
The descriptive statistics were obtained for the variables above and included mean and standard deviation. The data thus obtained were analyzed using Statistical Package for Social Science (SPSS) software version 17 for windows. A comparison of mean values among the study group, control group, and serum immunoglobulins in different clinical stages was obtained using ANOVA unpaired student's t-test. (P < 0.05). All the values where P values were less than. 05 were considered statistically significant.
| Results|| |
The 30 study group patients were aged 15–74 years with a mean age of 37.47 ± 16.24 years. [Table 1] shows the demographic details of the subjects. The study and control groups had an equal number of male and female patients. It was noted that most subjects in our study chewed only gutka (70%). In our study, all 30 (100%) study subjects had a burning sensation, 27 (90%) had difficulty in mouth opening, and 15 (50%) had difficulty swallowing. The cases were staged as per the staging given by Mathur RM and Jha T (1993). Out of 30 OSMF patients, 3 (10%) were in stage I, 13 (43.33%) were in stage II, and 14 (46.67%) were in stage III. A maximum number of cases, i.e., 14 (46.67%), were in stage III of OSMF.
[Table 2] and [Graph 1] show a comparison of serum immunoglobulin levels in the study and control groups. Serum IgG levels in the control group were in the range of 712.0–1593.9 mg/dL with a mean of 1076.96 ± 239.74 mg/dL. Serum IgG levels in the study group were in the range of 1007.0–1794.0 with a mean value of 1304.83 ± 206.14 mg/dL. When the comparison of IgG levels between the control and the study group was made using the ANOVA test, the difference was found to be statistically significant with P < 0.000.
The serum IgM levels in the control group were in the range of 43.0–192.9 mg/dL with a mean of 101.78 ± 37.82 mg/dL. The serum IgM levels in the study group were in the range of 40.0–230.0 mg/dL with a mean of 100.63 ± 43.38 mg/dL. When the comparison of IgM levels between the control and the study group was made using the ANOVA test, no statistically significant difference (P > 0.457) was found.
[Table 3] and [Graph 2] compare serum immunoglobulins in controls and various stages of OSMF. When IgG levels were compared between the control group and the different stages of OSMF, it was found to be 1076.96 ± 239.74 mg/dL in controls and 1350.67 ± 117.75 mg/dL, 1238.92 ± 252.03 mg/dL, and 1356.21 ± 162.35 mg/dL in stage I, stage II and stage III, respectively, which was statistically significant (P = 0.02).
When IgM levels were compared between the control group and different stages of OSMF, it was found to be 101.78 ± 37.82 mg/dl in the control group and 72.33 ± 32.72 mg/dl, 111.69 ± 51.73 mg/dl, and 96.43 ± 35.18 mg/dl in stage I, stage II and stage III, respectively, which was not statistically significant. (P = 0.46).
| Discussion|| |
Immunoglobulins are glycoproteins synthesized by plasma cells and, to some extent, by lymphocytes. IgG, IgA, and IgM constitute the predominant immunoglobulins in decreasing order of concentration in serum. During the last three decades, only a few studies have been carried out on the immune response of OSMF. The results of the previous studies on serum immunoglobulins in OSMF were varied. Hyperimmunoglobulinemia is invariably associated with OSMF. Therefore, this study aimed to estimate the levels of serum IgG and IgM to assess the role of humoral immune response in patients with OSMF. In our study, 30 subjects were in the age range of 15–74 years with a mean age of 30.80 ± 12.08, which is comparable to the mean age of 29.98 years in a study conducted by Pinakapani R (2009) and 30.66 years in a study by Taneja L (2015). However, most of the patients in our study were in the second and third decades of life, which can be attributed to social encounters, economic liberty, the popularity of refined areca nut products, and easy availability of the product. These findings are similar to previous studies conducted by Trivedi CR (2000) and Patidar K (2011). In our study, among 30 OSMF patients, 29 were males, and 1 was female, thus showing male predominance, which was also reported by Patidar K (2011) and Taneja L (2015).
This male predominance in our study could be due to the gutkha chewing habit, which is mostly practiced by younger men in this part of the country. In general, it was found that of the 30 OSMF subjects, 21 patients chewed gutkha alone or in combinations. Gutkha is a mixture of areca nut, tobacco, lime, catechu, and flavoring compounds marketed in small sachets or pouches. Our observation of 21 patients who chewed gutkha is comparable to the observation of Sinor's. P.N et al. (1990). We also noted OSMF in the patients who chewed raw areca nut only, which is comparable to the study of Meher R (1994) and Ranganathan (2004).
In the present study, a burning sensation was present in all the 30 (100%) subjects, which is comparab to the study of Sedat H.A (1988). This could be due to epithelial atrophy, which could be due to poor perfusion of adjacent connective tissue with blood.
In our study, 90% had difficulty in opening their mouth, which is comparable to the studies by Caniff J.P et al. (1986) and Rajendran R (1994). Also, 56.60% had difficulty swallowing. This could be due to the extension of fibrosis to the pharynx and esophagus, according to Sinor et al. (1990).
In our study, 3 (10%) subjects were in stage I, 13 (43.33%) in stage II, and 14 (46.67%) were in stage III, which is comparable to the studies by Pinakpani R (2009) and Taneja L (2015).
In the present study, the mean serum IgG levels in the control group were 1076.96 ± 239.74 mg/das compared to the study group (1304.83 ± 206.14 mg/dL) with P < 0.000, which was statistically significant and is consistent with Pinakapani R (2009), Patidar K (2011), and Taneja L (2015).
The serum IgM levels in the control and study groups were 101.78 ± 37.82 mg/dL and 100.63 ± 43.38 mg/dL, respectively. The IgM levels did not show any significant alterations in the present study. Similar observations were reported by Chaturvedi (1991) and Pinakapani R (2009). On the contrary, Shah et al. (1994) and Taneja L (2015) have reported a significant increase in serum IgM levels in OSMF.
The serum IgG levels were increased in all stages of OSMF as compared to the control group, which was statistically significant (P = 0.02). The above observations are similar to those reported by Chaturvedi et al (1991) and Pinakapani R (2009). The malignant potential of OSMF has been evaluated by various authors and discussed in the present literature. The immunologic alterations observed in OSMF are almost similar or nearer to the alterations observed in oral cancer. So, it is reasonable to assume that OSMF can be an intermediate stage in transforming a normal cell into oral malignancy. Hence, the immunologic follow-up of the OSMF patients will be beneficial for the early detection of the transformation process of OSMF to oral cancer.
There were no significant differences in IgM levels when compared between the OSMF patients and control group study participants. Similar findings were also observed by Chaturvedi and Marathe in1988 and by Taneja L in 2015. Chiang et al.(2002) showed that there is a significant increase in the number of T-lymphocytes and macrophages and a predominance of CD4 lymphocytes over CD8 lymphocytes in the subepithelial connective tissue of OSMF specimens. Macrophages and B-lymphocytes are the minor immunocompetent cells in the subepithelial tissue and are only occasionally found in the epithelium of OSMF specimens. It was implied that cellular immune response might play an important role in the pathogenesis of OSMF. This could be the reason for insignificant differences in values of serum IgM and controls in our study. The limitations of this study were the small sample size and unequal gender distribution. Therefore, studies involving different geographical areas with larger sample sizes are needed to be carried out.
| Conclusion|| |
The increased IgG level is a marker of inflammation in an individual. It may reflect the existing disease condition in the body. The increase in serum IgG level in the present study indicates the chronic inflammatory nature of the condition and the possible role of active immunity in the pathogenesis of this disease, indicating an underlying autoimmune phenomenon involved. Future recommendations include a large sample size, equal distribution of genders, and subjects from different demographic and geographical areas.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Ray JG, Chatterjee R, Chaudhuri K. Oral submucous fibrosis: A global challenge. Rising incidence, risk factors, management, and research priorities. Periodontol 2000 2019;80:200-12.
Gupta S, Jawanda MK. Oral submucous fibrosis: An overview of a challenging entity. Indian J Dermatol Venereol Leprol 2021;87:768-77.
Tekchandani V, Thakur M, Palve D, Mohale D, Gupta R. Co-relation of clinical and histologic grade with soft palate morphology in oral submucous fibrosis patients: A histologic and cephalometric study. J Dent Specialities 2015;3:68-75.
Cui L, Cai X, Huang J, LI H, Yao Z. Molecular mechanism of oral submucous fibrosis induced by arecoline: A literature review. J Clin Diag Res 2020;14:1-5.
Prajapati KJ, Chawda JG. Estimation of major immunoglobulins in smokers and gutkha chewers. J Oral Maxillofac Pathol 2016;20:219-23.
] [Full text]
Mathur RM, Jha T. Normal oral flexibility – A guideline for OSMF. JIDA 1993;64:139-43.
Pinakapani R, Shambulingappa P, Shashikanth MC. Salivary coagulopathy and immunoglobulins in oral submucous fibrosis. J Indian Acad Oral Med Radiol 2009;21:62-6. [Full text]
Taneja L, Bagewadi A, Keluskar V, Arya V. Estimation of major immunoglobulin levels in patients with oral submucous fibrosis. J Cranio Max Dis 2015;4:16-20. [Full text]
Trivedy CR, Warnakulasuriya KA, Peters TJ, Senkus R, Hazarey VK, Johnson KW. Raised tissue copper levels in oral submucous fibrosis. J Oral Path Med 2000;29:241-8.
Patidar KA, Parwani RN, Wanjari SP. Correlation of salivary and serum IgG, IgA levels with total protein in oral submucous fibrosis. J Oral Sci 2011;53:97-102.
Sinor PN, Gupta PC, Murti PR, Bhonsle RB, Daftary DK, Mehta FS, et al
. A case-control study of oral submucous fibrosis with special reference to the etiologic role of areca nut. J Oral Pathol Med 1990;19:94-8.
Maher R, Lee AJ, Warnakulasuriya KA, Lewis JA. Role of areca nut in the causation of oral submucous fibrosis: A case-control study in Pakistan. J Oral Pathol Med 1994;23:65-9.
Ranganathan K, Uma Devi M, Elizabeth JK, Kumar K, Saraswathi TR. Oral submucous fibrosis: A case-control study in Chennai, South Indian. J Oral Pathol Med 2004;33:274-7.
Seedat HA, Van Wyk CW. Betel nut chewing and submucous fibrosis in Durban. S Afr Med J 1988;74:568-71.
Canniff JP, Harvey W, Harris M. Oral submucous fibrosis: Its pathogenesis and management. Br Dent J 1986;160:429-34.
Rajendran R. Oral submucous fibrosis: Etiology, pathogenesis and future research. Bull WHO 1994;72:985-96.
Chaturvedi VN, Sharma AK, Chakrabarti S. Salivary coagulopathy and humoral response in oral submucous fibrosis (OSMF). J Indian Dent Assoc 1991;62:51-3.
Shah N, Kumar R, Shah MK. Immunological studies in oral submucous fibrosis. Ind J Dent Res 1994;5:81-7.
Chaturvedi VN, Marathe NG. Serum globulins and immunoglobulins in oral submucous fibrosis. Indian Practitioner 1988;41:399-403.
Chiang CP, Hsieh RP, Chen TH, Chang YF, Liu BY, Wang JT, et al
. High incidence of autoantibodies in Taiwanese patients with oral submucous fibrosis. J Oral Pathol Med 2002;31:402-9.
[Table 1], [Table 2], [Table 3]