Evaluation and Comparison of Genomic DNA Extraction Methods and PCR Optimization on Archival Formalin-Fixed and Paraffin-Embedded Tissues of Oral Squamous Cell Carcinoma.

Recovery and amplification of nucleic acids from archived formalin-fixed tissue samples is the most developing field in retrospective genetic studies. We compared different deparaffinization methods and DNA isolation techniques, and intergroup comparisons were performed to evaluate the effectiveness of different storing methods for archival OSCC samples based on obtained mean DNA quantity, quality, and PCR amplification of the P53 gene. The study comprised 75 archival histologically diagnosed OSCC samples which were divided into Group I: Formalin-fixed paraffin-embedded tissue blocks and Group II: Long-term formalin-fixed tissue. A comparison of different deparaffinization methods showed that xylene deparaffinization is an efficient method to obtain suitable DNA. Comparing different DNA isolation techniques illustrated that the conventional phenol-chloroform method gives better integrity to DNA in contrast with the kit method. Comparison between FFPET and long-term FFT samples demonstrated that samples fixed in formalin overnight and embedded in wax yield better quality and quantity DNA in comparison with long-term samples fixed in formalin. To obtain suitable integrity of DNA, tissue samples should be stored by fixing in formalin overnight followed by preparation of paraffin tissue blocks, deparaffinization by xylene, and subjecting them to the conventional phenol-chloroform DNA isolation protocol.

Peripheral adenomatoid odontogenic tumor in a cloak of an epulis: report of a rare case.

BACKGROUND: Adenomatoid odontogenic tumor constitutes an uncommon benign odontogenic tumor which is well-known as "two-thirds tumor" (two-thirds of adenomatoid tumors occur in the maxilla, two-thirds occur in young females, two-thirds of the cases are associated with un-erupted teeth and two-thirds of the affected teeth are canines). Larger part of these present as intra - osseous tumors while peripheral counterparts are extremely rare. Peripheral adenomatoid odontogenic tumor is a unique entity which generally presents as a slow growing gingival swelling with minimal bone involvement. This often leads to its erroneous diagnosis as a simple gingival lesion and the real diagnosis is only revealed after its microscopic evaluation. It exhibits a marked predilection for maxillary gingiva of incisor region and most commonly affects the younger females. CASE PRESENTATION: We report a case of 25 years old female patient of African ethnicity who presented with a 2 × 2 cm mass attached to the left mandibular gingiva in cuspid- bicuspid region which is an unusual site for AOT. It was accompanied by slight bone loss. With the differential diagnosis of gingival epulis and peripheral ossifying fibroma; surgical excision was performed. The light microscopic examination of the specimen aided the final diagnosis of Adenomatoid odontogenic tumor with the histopathological features identical to its intra osseous counterpart. CONCLUSION: In this case, the tumor is present on the mandibular gingiva in a 25 years old patient which is an atypical location and age for this tumor; also, the only individual case reported in an African patient. Only, a meager number of peripheral adenomatoid odontogenic tumor cases have been logged so far with majority of them occurring in maxillary gingiva. Furthermore, an ambiguity still prevails regarding its true origin and possible biological course. Hence, reporting of similar cases should be encouraged to facilitate the better understanding of its varied epidemiological details and clinical presentation.

Candidal Prevalence in Diabetics and its Species Identification.

CONTEXT: Candida species is a part of the normal mouth flora. Diabetes mellitus is a predisposing factor for the onset of oral candidiasis. AIM: The objective of this study was to estimate salivary glucose in patient with diabetes and healthy individuals, to determine total candidal counts as well as different candidal species in the saliva of patients with diabetes and nondiabetics. SETTINGS AND DESIGN: A sample size of 80 patients was taken. Out of 80 patients, 30 patients were uncontrolled diabetes patients (Group I), 30 patients were controlled diabetes patients (Group II), and 20 patients were healthy individuals (Group III). SUBJECTS AND METHODS: From all the salivary samples, salivary glucose estimation was done using the glucose oxidase-peroxidase method. Candidal colony-forming units (CFUs) were determined from all the salivary samples. CHROMagar Candida medium was used for the identification of various Candida species. STATISTICAL ANALYSIS USED: One-way ANOVA was used to test for differences between the means of the three groups. Pearson's correlation coefficient test was used to evaluate the relationships between the variables. RESULTS: The mean salivary glucose levels were higher in Group I followed by Group II and Group III. The mean candidal CFU was significantly higher in Group I than in Group II and Group III. CONCLUSION: Candida glabrata was the predominant species found and more so in patients with diabetes and needs further study. Other common species isolated was C. albicans. Species identification can help in better treatment strategies and gain good control over the disease.

Comparison of efficacy of local anesthetic solution, distilled water and normal saline as emergency fixatives.

CONTEXT: Adequate tissue fixation is fundamental to good quality histological sections. Owing to undesirable effects of 10% buffered formalin, its availability in clinics is questionable. Thus, the present study was conducted with a novel approach to fixation, together with the scope of finding fixative properties of more commonly used reagents available at the clinics. AIMS: The present study was aimed to compare the efficacy of local anesthetic solution, normal saline (NS) and distilled water (DW) with that of 10% neutral-buffered formalin. SETTINGS AND DESIGN: It is a single-blinded study where histological assessment of fixation was done to assert if the tissues procured were sufficient or insufficient for the clinical diagnosis with/without any problems. SUBJECTS AND METHODS: Forty soft-tissue specimens obtained from 2 goat tongue were used. Tissues each were directly immersed in local anesthesia, DW, NS solution and formalin for 12 and 24 h each and labeled as Group I and Group II, respectively. The sections were evaluated for staining quality and were subjected to statistical analysis. STATISTICAL ANALYSIS USED: Kruskal-Wallis test was employed to assess the differences in histological quality scores. Comparison between the tissues of the two groups was estimated with Mann-Whitney U-test. Kappa Statistic was used to measure the interobserver variation. RESULTS: There was a significant difference (P ≤ 0.05) in the efficacy of all the three emergency fixatives. CONCLUSIONS: On the basis of the results obtained, local anesthetic solution can be used as an emergency fixative.

Effect of Conventional and Microwave Tissue Processing Technique on DNA Integrity: A Comparative Molecular Analysis.

BACKGROUND: Methods of diagnostic molecular biology are routinely applied on formalin-fixed, paraffin-embedded tissues processed via conventional method. Recently, there has been a growing interest to use microwave technology in histopathology laboratories to overcome the deficiencies of the conventional processing method. Thefore, this study was aimed to compare and analyze the quality and quantity of DNA obtained from tissues processed by conventional and microwave tissue processing techniques and to further ascertain the applicability of the latter for PCR (polymerase chain reaction based research). METHODS: Thirty fresh tissues of oral squamous cell carcinoma (OSCC) were included, and each sample was cut into two equivalent halves. One tissue half was processed by conventional manual method whereas the other half was processed using a domestic microwave oven. DNA was obtained from all the tissues which were then subjected to Polymerase chain reaction (PCR) to evaluate GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) gene expression. RESULTS: The results revealed better DNA yield from microwave processed tissue while the quality of the DNA was alike from both the techniques. CONCLUSION: On the basis of the results obtained, it can be concluded that DNA produced by microwave processed tissues was similar to that obtained by conventional processing technique in terms of quantity and quality. Thus, microwave processed tissue samples can be successfully used for further molecular studies and researches.

Vinegar as a disinfectant of extracted human teeth for dental educational use.

CONTEXT: Extracted human teeth are routinely used in dentistry to learn technical and preclinical skills. Centers for Disease Control and Prevention (CDC) has adopted guidelines for infection control of extracted teeth used for research and teaching, requiring that teeth be sterilized before use. Many of the proposed disinfection methods starting from use of formalin, sodium hypochlorite and to autoclaving have their own drawbacks and may not be practical. AIM: To assess the effectiveness of vinegar for disinfection/sterilization of extracted human teeth. MATERIALS AND METHODS: A total of 80 extracted non-carious human teeth were kept in seven disinfectant media-10% formalin, 3% hydrogen peroxide, 5.25% sodium hypochlorite, 70% alcohol, vinegar, neem extract and normal saline. Ten samples were placed in each disinfectant individually for a period of 7 days, at room temperature. In all, 10 teeth were treated with microwave irradiation at 650 W for 3 min. Later, teeth from each group were placed individually in separate test tubes containing 10 ml of tryptic soy broth at 37°C for 48 h to observe the evidence of growth of microorganisms. Semiquantitative analysis of all the samples was done in Clade agar at 37°C for 48 h. STATISTICAL ANALYSIS USED: The number of teeth disinfected in each group was compared using Chi square test. RESULTS: 10% formalin, 3% hydrogen peroxide and vinegar were totally effective. The result was statistically significant with a Chi square value of 61.414 and P < 0.001. CONCLUSIONS: Vinegar can be used as an effective disinfectant medium for extracted human teeth.

Non-hereditary cherubism.

Cherubism is a self-limiting non-neoplastic autosomal dominant fibro-osseous disorder of the jaw usually found in children between 2-5 years of age. It occurs predominantly in boys and is characterized clinically by bilateral swelling of cheeks due to bony enlargement of the jaw that gives the patient a typical cherubic look. Cherubism may occur as solitary cases or in many members of family, often in multiple lesions. Radiographically the lesions appear as multilocular bilateral radiolucent areas. The present case report describes a 13-year-old female cherubic child with progressive swelling of cheeks.

Quick response code in acrylic denture: will it respond when needed?

Use of acids and other caustic substances in destroying the human body to avoid personal identification is drawing great deal of attention in recent times. With rapid industrial growth, incidence of industrial disasters has increased. In an event of chemical industry disaster, human identification can become a challenge. Embedding bar codes and other electronic means for identification in denture have proved to be a novel innovation in identification of edentulous patients. However, the validity of this method in events of chemical extremes has not been assessed. Hence, the study was undertaken to know the effect of different acids on dentures embedded with quick response codes. Results of this study indicate that simple bar code can be readable up to 33 h after acid treatment. With minimal armamentarium, bar code can be generated incorporating large data into it, providing it to be a cheap and reliable means of denture identification.

Assessment of tissue eosinophilia as a prognosticator in oral epithelial dysplasia and oral squamous cell carcinoma-an image analysis study.

Association of tissue eosinophilia with oral squamous cell carcinoma has shown variable results ranging from favourable to unfavourable or even having no influence on prognosis. Also, very few studies have been done to know the role of eosinophils in premalignancy. So the present study investigated role of eosinophilic infiltration in oral precancer and cancer and its possible use as a prognosticator. 60 histopathologically proven cases (20 cases each of metastatic and nonmetastatic oral squamous cell carcinoma and oral leukoplakia with dysplasia of various grades) were included. Congo red is used as a special stain for eosinophils. Each specimen slide was viewed under high power in 10 consecutive microscopic fields for counting of eosinophils. As a result, a significant increase in eosinophil count was found in oral carcinomas compared to dysplasia. Nonmetastatic cases showed higher counts than metastatic carcinomas. So, it is concluded that eosinophilia is a favourable histopathological prognostic factor in oral cancer. Moreover, higher eosinophil counts in carcinoma group compared to dysplasia group proved that they might have a role in stromal invasion thus suggesting that quantitative assessment of tissue eosinophilia should become a part of the routine histopathological diagnosis for oral precancer and OSCC.

Stromal myofibroblasts in nonmetastatic and metastatic oral squamous cell carcinoma: An immunohistochemical study.

BACKGROUND AND AIMS: Myofibroblasts are one of the important components of the tumor microenvironment which could possibly play an important role in tumor progression. The purpose of this study was to compare the presence of α-smooth muscle actin (α-SMA) and CD34 positive fibroblasts in nonmetastatic and metastatic oral squamous cell carcinoma and to evaluate their role in tumor metastasis. MATERIALS AND METHODS: Ten cases each of histologically proven metastatic and nonmetastatic oral squamous cell carcinoma formed the study group. The tissue sections were stained immunohistochemically for α-SMA and CD34. The stromal spindle cells positive for these markers in the study groups were counted and compared. RESULTS: α-SMA positive cases were more in the metastatic group and CD34 positive cases were found to be more in the nonmetastatic tumors. CONCLUSIONS: Though difference in the staining pattern was statistically nonsignificant, the inverse relationship between α-SMA and CD34 positive cells is indicative of dynamic nature and the influence of tumor stroma in tumor progression and metastasis.

Dens invaginatus (Type III B).

Dens invaginatus or 'dens in dente' is a developmental malformation of the tooth resulting from infolding of the dental papilla before calcification. This article presents a case of dens invaginatus occurring in maxillary right lateral incisor of a 45-year-old male patient. The patient presented with pain and clinically missing maxillary right canine. The tooth was found to be non-vital. Radiographic examination revealed the tooth-in-tooth appearance of lateral incisor with a dilated pulp chamber. The crown of impacted canine was found within the pulp chamber of lateral incisor. Owing to this unique clinical presentation, both the lateral incisor and the impacted canine were extracted. Histopathologic examination confirmed the diagnosis of Dens invaginatus Type III B. A brief review on etiopathogenesis, radiographic features and treatment of dens invaginatus has also been included.

Pemphigus vulgaris--a report of three cases.

Pemphigus vulgaris (PV) is a potentially life-threatening illness that manifests in the mouth and on skin. In a majority of patients it affects the oral mucosa and is sometimes difficult to diagnose when only mucosal involvement is present. In an attempt to highlight the proper treatment plan of this potentially fatal disorder, the authors document a report of three cases. These patients were prescribed conventional steroids which brought about partial relief but early recurrence with discontinuation of the drug. Subsequent management of these patients with azathioprine along with corticosteroids improved the outcome of the disease with longer remission periods. In this case series, the steroid sparing effect of azathioprine was achieved successfully and hence needs to be considered as a primary drug in management of PV.

Cancer Therapy: A Continuance of Health Burden.

BACKGROUND: Cancer diagnosis coupled with emotional impact converge to create one of the most difficult physical and emotional periods of life. Cancer treatment causes plethora of short and long term complications which can be so debilitating that patient may interrupt treatment. Pretreatment oral assessment and supportive oral care during and after cancer therapy can increase quality of life and supportive care costs. METHODS: Study was conducted on 189 patients (86: head and neck cancer cases, group I and 103: other than head and neck cancer cases, group II) receiving cancer therapy. Patients were subjected to clinical assessment and findings were recorded in specially designed proforma and complete oral (objective and subjective) and constitutional findings were recorded. RESULTS: Among the patients undergoing chemotherapy in both groups, prevalence of oral findings was found to be highest with methotrexate whereas constitutional symptoms was found to be highest with doxyrubicin. Whereas in radiotherapy patients subjective and objective oral symptoms increased from 10th - 30th fractionated dose of radiations and then subsequently decreased and constitutional symptoms were found to be consistent in all fractionated dosages with lowest at 50th fraction. Under combined chemo and radiotherapy patients, constitutional symptoms were highest than the oral findings. CONCLUSIONS: Cancer therapy can greatly damage the normal tissues and diminish patients quality of life and often leads to serious clinical sequelae. Therefore, therapy induced damage should be anticipated and prevented whenever possible and managed early.

An anthropometric analysis of facial height, arch length, and palatal rugae in the Indian and Nepalese population.

INTRODUCTION: A country such as India abounds with diverse population groups with distinct anthropometric characteristics. Among these, numerous Nepalese population groups are present in different states of India comprising one of the most common immigrant races. The aim of the study is to compare two distinct races, Indians and Nepalese on the basis of facial height proportions, arch length and palatal rugae patterns and assess their significance in racial identification. MATERIALS AND METHODS: A total of 120 subjects comprising of 60 Indians and 60 Nepalese were selected, with each group including 30 males and 30 females. Facial heights were measured using sliding digital calipers, arch lengths with the help of a brass wire and rugae patterns were traced on dental casts obtained with alginate impressions. RESULTS: Facial height measurements did not give significant results for racial or gender identification of given races. Differences between arch length parameters were found to be significant between the two population groups. Secondary and fragmentary palatal rugae forms were found to be more common in Nepalese than Indians. CONCLUSION: The Indian and Nepalese have similar anthropometric characteristics with regard to facial height. However, arch length and palatal rugae characteristics vary between the two races.

Dental cementum in age estimation: a polarized light and stereomicroscopic study.

Dental hard tissues are good candidates for age estimation as they are less destructive and procedures to determine age can be easily performed. Although cementum annulations and cementum thickness are important parameters in this regard, they are seldom used. This study was undertaken to review the methods, difficulties in execution of techniques, and accuracy of cementum thickness and annulations in estimating the age. Unstained and stained ground sections of tooth were used to measure cemental thickness and count cemental annulations based on which age was estimated and was compared with known age. Although there was positive relation between cemental thickness and annulations with age, only in 1-1.5% of cases, age could be predicted with accuracy.