Computational protocol: Characterization of muscle alteration in oral submucous fibrosis-seeking new evidence

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Protocol publication

[…] The present study was undertaken at the Department of Oral Pathology, Maulana Azad Institute of Dental Sciences, New Delhi, India with prior approval of institutional ethical review committee. Informed consent was obtained from patients and controls participating in the study. A careful clinical examination of patients reporting to the department with signs and symptoms of OSF was performed with special concern to inter-incisal mouth opening. The distance between the incisal edges of maxillary right central incisor and mandibular right central incisor with patient’s mouth fully opened, was measured using vernier caliper. Three measurements were carried out in each case and the average value was calculated to give the final inter-incisal distance. The clinical staging scheme proposed by Lai et al. is as follows ():Group A: More than 35mmGroup B: Between 30 to 35 mmGroup C: Between 20 to 30 mmGroup D: Less than 20 mmTo make each interval exclusive for statistical analysis, the following clinical staging model was used:Group A: ? 35 mmGroup B: Between 30 to 34 mmGroup C: Between 20 to 29 mmGroup D: ? 19mmAge and sex matched healthy individuals were taken as controls. Incisional biopsy was then performed after taking patient’s (and controls’) consent. In both the study and the control groups, the biopsy site was chosen to be 1 cm behind the commissure of the mouth in order to remove any bias in the study due to difference in the depth of muscle at different locations in the buccal mucosa. All the tissues were fixed in 10% neutral buffered formalin for 12 hours prior to processing.The sections were stained with hematoxylin and eosin and observed under microscope. All the tissues which were fragmented or did not show muscle fibres due to inadequate depth of the biopsy were excluded from the study. Exclusion criteria also included any associated dysplastic changes since hyperplasia may occur in such cases which may provide erroneous results while measuring distance. The samples were collected over a time period of six months. Stained sections in the study group were histopathologically graded as very early, early, moderately advanced and advanced OSF according to the criteria given by Pindborg et al. (). The Motic image analysis software (Motic Images Plus 2.0) was used to calculate the distance of muscle from the rete ridge of the overlying epithelium. The shortest distance was measured from the crest of most superficial muscle fibre seen in a randomly chosen field to the bottom of deepest rete ridge. Three different histopathologists reviewed the slides simultaneously on image analysis software to remove any observer bias. The mean of distance in five such fields was referred as the muscle-epithelial distance for that particular case.Wherever in doubt, Van Gieson stained sections were used to clearly delineate muscle fibres. Muscle-epithelial distance was also calculated in the control tissues for comparison.While observing the histopathological sections, several degenerative changes in the muscle fibres were noted. Out of these, the four most commonly observed changes were:a. Highly eosinophilic muscle fibres with loss of striationsb. Fragmentationc. Nucleus internalizationd. Multiple nucleiEach of the above mentioned observation was recorded as present or absent in every tissue section.Statistical analysis was performed using Post-hoc Bonferroni Test to compare muscle-epithelial distance amongst various clinical groups as well as amongst different histopathological groups. One-way ANOVA test was used to compare clinical mouth opening and histopathological grade. […]

Pipeline specifications

Software tools MUSCLE, ReTe
Application Nucleotide sequence alignment
Diseases Oral Submucous Fibrosis