Graduation Year


Document Type




Degree Granting Department

Public Health

Major Professor

Stuart Brooks, M.D.

Committee Member

Robert Haight, M.D., M.S.P.H.

Committee Member

Thomas Truncale, M.D., M.P.H.


Sputum, Induction, IOS, Spirometry, Methylene blue


Sputum induction by inhalation of hypertonic saline has been used for more than15 years. It has become one of the most intriguing methods to study airway inflammation. It is the only direct, non-invasive method for measuring airway inflammation indices.Sputum induction has been used in the diagnosis of many respiratory illnesses including asthma, chronic pulmonary obstructive disease, tuberculosis, chronic cough, lung cancer and Pneumocystis Carinii on patients who are unable to produce sputum spontaneously. There are currently many different methods used worldwide to induce sputum, but there is a lack of one generally accepted gold standard method.

The proposed protocols for sputum induction proved to be safe, simple and produced satisfactory amount of expectorate. However, it did not contain enough cells from the lower respiratory tract and was contaminated by squamous cells when compared to another method based on the work of F. E. Hargreave.

Investigation demonstrated that the use of impulse oscillometry, which requires no effort from patients, needs further research with larger study samples before it could be used instead of spirometry to evaluate airway obstruction.

Initial methylene blue stain of the fresh expectorate smear was shown to be useful tool for identifying grossly contaminated sputum samples by squamous epithelial cells.Our first study group included 20 volunteers in good health. Sputum was induced by inhalation of 3% saline mist created by ultrasonic nebulizer at maximum output (4ml/min). Sputum induction intervals lasted 4-5 minutes with cumulative duration of induction about 4-15 minutes which was tolerated well. Lung function was evaluated for obstruction at baseline and every 5 minutes with spirometry and impulse oscillometry.The whole expectorated sample was processed and slides were stained with HEMA 3stain. With this method we were able to collect a mean of 6.1 ml expectorate. The mean total cell count was 804 000 with high proportion of squamous cells.

The second study group included 5 volunteers in good health. This method utilized 3%, 4% and 5% saline mist for inhalation, 7 minutes each. Ultrasonic nebulizer was set at low output of 0.9 ml/min. This procedure was also tolerated well without major adverse effects. Lung function was evaluated at baseline and every 7 minutes for obstruction. Only dense portions of expectorate were selected and processed. Slides were stained with Wright stain. This method produced much more total cells with a mean of 3 385 000 per gram of sputum which came from the lower airways and were not contaminated by squamous cells.

The second method was far superior producing adequate sputum sample with cells from the lower airways and minimal squamous cell contamination and will be used in our Breath Lab.