| Home Country | India
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| Host Country | United Kingdom
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| Fellowship End date | 01 October 2009
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| Contact Email | bbrashier@crfindia.com
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Summary of Fellowship ExperienceThe research fellowship for sputum induction technique was completed in 3 months and comprised of 4 phases.
Phase 1:
This phase was be for 7 days, wherein I had initial overview and accompanied reading of all the sputum induction and sputum sampling techniques, and various techniques required for studying and identifying the sputum cells, sputum inflammatory mediators, cytokines and chemokines and their clinical and research implications.
Phase 2:
This was the critical phase of the fellowship training which was on 2 months duration and comprised of training in the sputum induction, sputum processing, immunocytochemistry and in situ hybridization, analyzing fluid phase mediators of sputum, cellular protein estimation by ELISA technique, DNA extraction from sputum, reverse transcription and polymerase chain reactions to study cellular mRNA transcriptional changes in sputum.
During this training, I learnt the following:
• Facilities and equipments required for sputum induction.
• Nebulisers required for adequate sputum induction.
• Various techniques to prepare hypertonic saline, particularly 3% and 4.5% in sterile conditions and their possible outcomes and effects on the sputum cells, along with their safety profile.Safety of sputum induction in different severities of COPD and asthma.
• Possible adverse events that could occur during the course of the test and the predictors and mechanism which determine the adverse events during sputum induction and the procedures to reduce the adverse event.
• Techniques and durations of inhalation, and stratification of cells and fluid phase inflammatory markers at early and late phases of sputum induction.
• Sputum sample homogenization and reagents required for homogenization of sputum, and duration and the right temperature which affectively homogenizes the sputum
• Sputum filtration methods to separate it from mucus and debris.
• Counting of total and differential sputum cells and staining techniques to check the viability of these cells. Preparing slides and assuring quality of slides and cell counting.
• Techniques to separate the fluid phase of sputum and its storage.
• Cytospins preparations.
• Immuno-cyto-staining and in-situ hybridization techniques in the cells of induced sputum.
• Analysis of fluid phase components of induced sputum with methods like bioassays, enzyme assays and immunoassays.
• Protein estimation in the sputum by ELISA techniques and DNA extraction from sputum and nasal cell pellets, reverse transcription and polymerase chain reactions to study cellular mRNA transcriptional changes in sputum.
Phase 3
This phase of fellowship was important as my training was verified by my guide and supervisors. This exercise went on for for 15 days, wherein I did the following
• Conduct sputum induction on 10 COPD and 10 asthmatic subjects.
• Process the induced sputums, sampled them, separate the cells and fluid phase mediators, prepare cytospins, stain the cells, count the cells and assay the fluid phase mediators.
• Studied the inflammatory mediators such as IL8, IL6. MIG and gro alfa using ELISA technique
All these procedures were under the observation of my trainer, Peter Fenwick, who finally verified my techniques in accordance international standards and his, satisfaction.
I had very nice time in England. I had gone with my family, made many friends in the lab and enjoyed British culture and food