M C Ronchi1, G Galli, R Zonefrati, A Tanini, G Scano, R Duranti. 1. Department of Internal Medicine, Section of Immunoallergology and Respiratory Diseases, University of Florence, Italy. m.ronchi@dmi.unifi.it
Abstract
BACKGROUND: Sputum examination is being increasingly used as a non-invasive method for studying airway inflammation. However, the application of sputum still presents some methodological problems and the results of sputum analysis may be substantially flawed by salivary contamination, cell and mucus debris. In addition, much work is needed to deepen the possibility of extensive application of cell and molecular biology techniques to sputum analysis. OBJECTIVE: In an attempt to improve the technique of sputum processing, we investigated the effect of: (i) 20 and 11 microm filtration in addition to 40 microm on salivary contamination; (ii) Percoll density gradient centrifugation on sputum slides quality; (iii) a culture medium (Minimum Essential Medium containing HEPES 22 mm, pH 7.4: MEM) as washing and suspension solution compared to PBS on cell viability. METHODS: Induced sputum samples were obtained in 37 asthmatics. 21 samples were processed as selected sputum and 16 samples as entire expectorates. After dithiotreitol (DTT) homogenization, each specimen was aliquoted in two parts of equal volume. One portion was processed with the usual method, the other using a modified method: cell pellet was suspended in sterile MEM, filtered through 40, 20 and 11 microm net filters and separated from the residual debris by Percoll gradient centrifugation. RESULTS: As compared to the current sputum processing this method resulted in: (i) no selective bronchial cellular loss; (ii) a significant decrease of salivary contamination, particularly in entire expectorates in which squamous cells were reduced from 47 (36) to 15.5% (20) as median values and interquartile range; (iii) a higher proportion of good quality cytospins; (iv) maintenance of cell viability over the time (88% vs. 81% in MEM and PBS, respectively) 1 h after sample collection. CONCLUSION: In the present study we demonstrated that the proposed method is feasible and makes it possible to overcome most of the technical limits met with the commonly used method, pointing to a potential extension of induced sputum application for more sophisticated techniques.
BACKGROUND: Sputum examination is being increasingly used as a non-invasive method for studying airway inflammation. However, the application of sputum still presents some methodological problems and the results of sputum analysis may be substantially flawed by salivary contamination, cell and mucus debris. In addition, much work is needed to deepen the possibility of extensive application of cell and molecular biology techniques to sputum analysis. OBJECTIVE: In an attempt to improve the technique of sputum processing, we investigated the effect of: (i) 20 and 11 microm filtration in addition to 40 microm on salivary contamination; (ii) Percoll density gradient centrifugation on sputum slides quality; (iii) a culture medium (Minimum Essential Medium containing HEPES 22 mm, pH 7.4: MEM) as washing and suspension solution compared to PBS on cell viability. METHODS: Induced sputum samples were obtained in 37 asthmatics. 21 samples were processed as selected sputum and 16 samples as entire expectorates. After dithiotreitol (DTT) homogenization, each specimen was aliquoted in two parts of equal volume. One portion was processed with the usual method, the other using a modified method: cell pellet was suspended in sterile MEM, filtered through 40, 20 and 11 microm net filters and separated from the residual debris by Percoll gradient centrifugation. RESULTS: As compared to the current sputum processing this method resulted in: (i) no selective bronchial cellular loss; (ii) a significant decrease of salivary contamination, particularly in entire expectorates in which squamous cells were reduced from 47 (36) to 15.5% (20) as median values and interquartile range; (iii) a higher proportion of good quality cytospins; (iv) maintenance of cell viability over the time (88% vs. 81% in MEM and PBS, respectively) 1 h after sample collection. CONCLUSION: In the present study we demonstrated that the proposed method is feasible and makes it possible to overcome most of the technical limits met with the commonly used method, pointing to a potential extension of induced sputum application for more sophisticated techniques.
Authors: Frauke Pedersen; Frederik Trinkmann; Mustafa Abdo; Anne-Marie Kirsten; Klaus F Rabe; Henrik Watz; Simonetta Baraldo; Marina Saetta; Jens M Hohlfeld; Olaf Holz Journal: Int J Chron Obstruct Pulmon Dis Date: 2021-03-01