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OI0293 Salivary exosomes: comparison between methods of isolation
OOOO Volume 117, Number 5 Methods: All trainees within the British Isles were sent an online questionnaire and allowed access to an existing ePortfolio. Two trainee focus groups were conducted to help design the oral medicine ePortfolio template. Qualitative and quantitative data would be collected from both questionnaires and focus groups. Quantitative data would be presented using descriptive statistics, and qualitative results would be processed using the “content analysis” method. Results: Of all trainees, 61% (11 of 18) responded to the questionnaire; 72.7% (8 of 11) thought the specialty requires standardized portfolios. Dominant themes from qualitative data were identified. Beneficial themes included practical benefits (e.g., remote access, less paperwork) and workforce benefits (e.g., national standardization of training). Negative themes included concerns about ePortfolio limiting trainer input and dissatisfaction with requirement for trainees to self-fund the ePortfolio. Suggestions from the trainees about the design of the ePortfolio included an emphasis on practicality and simplicity and a requirement for a voluntary pilot study to further refine the ePortfolio before full implementation. Evidence of deeper learning and reflective practice were demonstrated by the trainees attending the focus groups. Conclusions: The results of this action research project suggest that there is a strong desire among oral medicine trainees within the British Isles to implement an ePortfolio. The focus group setting appeared to be successful in producing a pragmatic design for the oral medicine ePortfolio and in promoting deeper learning and reflective practice within the participating trainees.
OI0293 SALIVARY EXOSOMES: COMPARISON BETWEEN METHODS OF ISOLATION Marilena Vered, Ayelet Zlotogorski-Hurvitz, Gavriel Chaushu, Tuula Salo, Dan Dayan, Department of Oral Pathology and Oral Medicine, Tel Aviv University, Tel Aviv, Israel Objectives: Exosomes are nanoparticles (30-100 nm) secreted by most cell types. It was the aim of our study to isolate for the first time salivary exosomes using a chemically based agent (Exoquick (EQ)-TC; Systems Biosciences, CA, USA) and to compare their characteristics to those isolated by ultracentrifugation (UC). Methods: Whole saliva (5-10 mL) from healthy individuals was collected under standardized conditions, kept at 4 C, and centrifuged (3000 g for 15 minutes), and the supernatant was frozen ( 70 C) until further use. Exosomal precipitates (ppt) obtained by EQ were termed EQppt, and those resulting from UC (120 000 g for 3 hours) were termed UCppt. EQ was added to saliva in a ratio of 1:2, incubated overnight at 4 C followed by centrifugation (5000 g for 30 minutes). Exosomes isolated with EQ were characterized by ELISA and Western blotting molecular tests for assessment of the exosomal markers CD63, CD9, and CD8 and compared with those isolated by UC. Morphologically, the comparison was done by atomic force microscopy (AFM) and electron microscopy (EM). Results: All the tests were done using a saliva volume of 0.5 mL. EQppt was considerably larger than UCppt. ELISA tests found no significant differences in the concentrations of the exosomal markers in EQppt when compared with UCppt. Using the same markers, Western blotting showed thinner bands in EQppt than UCppt. Examination with AFM found that EQppt contained only a few single particles of exosomal size alongside with aggregates of varying size, unlike in UCppt. EM of EQppt showed only a few particles with classical exosomal size and
ABSTRACTS Abstracts e351 bilayered morphology together with aggregates and debris of other biologic precipitates. UCppt showed purer exosomes almost without debris or aggregates. Conclusions: Although isolation of exosomes by EQ is quicker and easier, it provides an impure precipitation when compared with exosomes isolated by UC.
OI0306 DOES NARROW BAND IMAGING ADD VALUE IN ORAL MEDICINE? Giulia Otaviani, Margherita Gobbo, Marta D’Ambros, Serena Zacchigna, Roberto Di Lenarda, Matteo Biassotto, Dental Science Department, Division of Oral Medicine and Pathology, Trieste, Italy Objectives: The study of angiogenesis, an early and key event in carcinogenesis, could improve early detection of malignant/premalignant oral lesions. Narrow band imaging (NBI) is an optical technique that is used both to perform objective examination of the oral cavity, with the aid of different grades of magnification, and to visualize intrapapillary capillary loops (IPCLs) through a 415-nm light source. The aim of this study was (1) to define the sensitivity and specificity of NBI in detecting dysplastic and neoplastic lesions and (2) to evaluate its learning curve. Methods: The NBI-guided technique was used to photograph and biopsy 91 white, red, or white-red oral lesions after detecting IPCL abnormalities. Lesions were classified by an expert blind rater (main rater) and by 17 students (nonexpert raters) after adequate instructions, according to Takano’s IPCL classification. Lesions were classified into mild/moderate/severe dysplasia or neoplasia. Histopathologic retrievals were used as a reference standard. Dysplastic oral lesions were induced in 40 mice. Lesions were analyzed according to IPCL classification and biopsied. Comparison between histopathologic retrievals and clinically determined grade of dysplasia (mild/moderate/severe dysplasia or neoplasia) was performed. Kendall W was used to compare clinical/histologic findings and expert/nonexpert evaluations. Results: In the clinical study, the concordance between the main rater and reference standard was 72.5%; the k coefficient of the main rater was 0.74 (95% CI). Nonexpert raters had 21.7% concordance with the reference standard and 26.8% with the main rater. NBI examination found 57% to 96% sensibility and 85% to 99% specificity, depending on grades of dysplasia. The highest values were found in neoplasia detection. In the animal study, sensitivity and specificity of NBI-guided grading were 95.7% and 98.5%, respectively. Conclusions: Our findings support (1) that high sensitivity/ specificity can be obtained both in patients and in mice and (2) that NBI has a steep learning curve. We can hypothesize that NBI could be of great help in the early detection of oral lesions, performing guided biopsies and follow-up of suspicious oral lesions, although guidelines and education courses could help in rendering it more reproducible.
OI0317 EPIDEMIOLOGIC, CLINICAL, AND RADIOGRAPHIC EVALUATION OF JAW LESIONS Juliane Piragine Araujo, Celso Augusto Lemos Junior, Decio Santos Pinto Junior, Gustavo Henrique Campos Rodrigues, Fabio Abreu Alves, Stomatology, Dentistry School, São Paulo University, São Paulo, Brazil Objectives: This study evaluated the prevalence and the clinical and radiographic features of jaw lesions in a single institution. Methods: A prospective study of descriptive character evaluated clinical and radiographic features of all lesions arising
OI0293 SALIVARY EXOSOMES: COMPARISON BETWEEN METHODS OF ISOLATION Marilena Vered, Ayelet Zlotogorski-Hurvitz, Gavriel Chaushu, Tuula Salo, Dan Dayan, Department of Oral Pathology and Oral Medicine, Tel Aviv University, Tel Aviv, Israel Objectives: Exosomes are nanoparticles (30-100 nm) secreted by most cell types. It was the aim of our study to isolate for the first time salivary exosomes using a chemically based agent (Exoquick (EQ)-TC; Systems Biosciences, CA, USA) and to compare their characteristics to those isolated by ultracentrifugation (UC). Methods: Whole saliva (5-10 mL) from healthy individuals was collected under standardized conditions, kept at 4 C, and centrifuged (3000 g for 15 minutes), and the supernatant was frozen ( 70 C) until further use. Exosomal precipitates (ppt) obtained by EQ were termed EQppt, and those resulting from UC (120 000 g for 3 hours) were termed UCppt. EQ was added to saliva in a ratio of 1:2, incubated overnight at 4 C followed by centrifugation (5000 g for 30 minutes). Exosomes isolated with EQ were characterized by ELISA and Western blotting molecular tests for assessment of the exosomal markers CD63, CD9, and CD8 and compared with those isolated by UC. Morphologically, the comparison was done by atomic force microscopy (AFM) and electron microscopy (EM). Results: All the tests were done using a saliva volume of 0.5 mL. EQppt was considerably larger than UCppt. ELISA tests found no significant differences in the concentrations of the exosomal markers in EQppt when compared with UCppt. Using the same markers, Western blotting showed thinner bands in EQppt than UCppt. Examination with AFM found that EQppt contained only a few single particles of exosomal size alongside with aggregates of varying size, unlike in UCppt. EM of EQppt showed only a few particles with classical exosomal size and
ABSTRACTS Abstracts e351 bilayered morphology together with aggregates and debris of other biologic precipitates. UCppt showed purer exosomes almost without debris or aggregates. Conclusions: Although isolation of exosomes by EQ is quicker and easier, it provides an impure precipitation when compared with exosomes isolated by UC.
OI0306 DOES NARROW BAND IMAGING ADD VALUE IN ORAL MEDICINE? Giulia Otaviani, Margherita Gobbo, Marta D’Ambros, Serena Zacchigna, Roberto Di Lenarda, Matteo Biassotto, Dental Science Department, Division of Oral Medicine and Pathology, Trieste, Italy Objectives: The study of angiogenesis, an early and key event in carcinogenesis, could improve early detection of malignant/premalignant oral lesions. Narrow band imaging (NBI) is an optical technique that is used both to perform objective examination of the oral cavity, with the aid of different grades of magnification, and to visualize intrapapillary capillary loops (IPCLs) through a 415-nm light source. The aim of this study was (1) to define the sensitivity and specificity of NBI in detecting dysplastic and neoplastic lesions and (2) to evaluate its learning curve. Methods: The NBI-guided technique was used to photograph and biopsy 91 white, red, or white-red oral lesions after detecting IPCL abnormalities. Lesions were classified by an expert blind rater (main rater) and by 17 students (nonexpert raters) after adequate instructions, according to Takano’s IPCL classification. Lesions were classified into mild/moderate/severe dysplasia or neoplasia. Histopathologic retrievals were used as a reference standard. Dysplastic oral lesions were induced in 40 mice. Lesions were analyzed according to IPCL classification and biopsied. Comparison between histopathologic retrievals and clinically determined grade of dysplasia (mild/moderate/severe dysplasia or neoplasia) was performed. Kendall W was used to compare clinical/histologic findings and expert/nonexpert evaluations. Results: In the clinical study, the concordance between the main rater and reference standard was 72.5%; the k coefficient of the main rater was 0.74 (95% CI). Nonexpert raters had 21.7% concordance with the reference standard and 26.8% with the main rater. NBI examination found 57% to 96% sensibility and 85% to 99% specificity, depending on grades of dysplasia. The highest values were found in neoplasia detection. In the animal study, sensitivity and specificity of NBI-guided grading were 95.7% and 98.5%, respectively. Conclusions: Our findings support (1) that high sensitivity/ specificity can be obtained both in patients and in mice and (2) that NBI has a steep learning curve. We can hypothesize that NBI could be of great help in the early detection of oral lesions, performing guided biopsies and follow-up of suspicious oral lesions, although guidelines and education courses could help in rendering it more reproducible.
OI0317 EPIDEMIOLOGIC, CLINICAL, AND RADIOGRAPHIC EVALUATION OF JAW LESIONS Juliane Piragine Araujo, Celso Augusto Lemos Junior, Decio Santos Pinto Junior, Gustavo Henrique Campos Rodrigues, Fabio Abreu Alves, Stomatology, Dentistry School, São Paulo University, São Paulo, Brazil Objectives: This study evaluated the prevalence and the clinical and radiographic features of jaw lesions in a single institution. Methods: A prospective study of descriptive character evaluated clinical and radiographic features of all lesions arising