BACKGROUND: Induced sputum is the most commonly used method to analyze airway inflammation in cystic fibrosis (CF) patients ex vivo. Due to the complex matrix of the sample material, precise and reliable analysis of sputum constituents depends critically on preanalytical issues. OBJECTIVES: Here we compared the commonly used method for sputum processing by dithiothreitol (DTT) with a novel mechanical method in regard to basal cellular parameters, neutrophil markers and glutathione (GSH) levels. METHODS: Sputum samples from CF patients were processed in parallel with or without the use of DTT. The key improvement of the mechanical method was the processing in many very small aliquots. Cellular and humoral markers were assessed and compared according to Bland-Altman. RESULTS: Total cell count, cell viability, differential cell count, neutrophil elastase levels and flow cytometrically analyzed neutrophil markers (CD63, CD11b, DHR) did not differ between the two methods. Intracellular and extracellular GSH levels were significantly higher in DTT-treated samples (p = 0.002). CONCLUSION: The mechanical sputum-processing method presented had a similar yield of cells and fluids as the conventional DTT method and the advantage of omitting the introduction of reducing agents. This method allows a more reliable analysis of redox-dependent airway inflammation in sputum cells and fluid from CF patients than methods utilizing DTT.
BACKGROUND: Induced sputum is the most commonly used method to analyze airway inflammation in cystic fibrosis (CF) patients ex vivo. Due to the complex matrix of the sample material, precise and reliable analysis of sputum constituents depends critically on preanalytical issues. OBJECTIVES: Here we compared the commonly used method for sputum processing by dithiothreitol (DTT) with a novel mechanical method in regard to basal cellular parameters, neutrophil markers and glutathione (GSH) levels. METHODS: Sputum samples from CFpatients were processed in parallel with or without the use of DTT. The key improvement of the mechanical method was the processing in many very small aliquots. Cellular and humoral markers were assessed and compared according to Bland-Altman. RESULTS: Total cell count, cell viability, differential cell count, neutrophil elastase levels and flow cytometrically analyzed neutrophil markers (CD63, CD11b, DHR) did not differ between the two methods. Intracellular and extracellular GSH levels were significantly higher in DTT-treated samples (p = 0.002). CONCLUSION: The mechanical sputum-processing method presented had a similar yield of cells and fluids as the conventional DTT method and the advantage of omitting the introduction of reducing agents. This method allows a more reliable analysis of redox-dependent airway inflammation in sputum cells and fluid from CFpatients than methods utilizing DTT.
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Authors: M Kolpen; T Bjarnsholt; C Moser; C R Hansen; L F Rickelt; M Kühl; C Hempel; T Pressler; N Høiby; P Ø Jensen Journal: Clin Exp Immunol Date: 2014-07 Impact factor: 4.330
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Authors: Andreas Hector; Michael S D Kormann; Ines Mack; Philipp Latzin; Carmen Casaulta; Elisabeth Kieninger; Zhe Zhou; Ali Ö Yildirim; Alexander Bohla; Nikolaus Rieber; Matthias Kappler; Barbara Koller; Ernst Eber; Olaf Eickmeier; Stefan Zielen; Oliver Eickelberg; Matthias Griese; Marcus A Mall; Dominik Hartl Journal: PLoS One Date: 2011-09-20 Impact factor: 3.240
Authors: Theodore G Liou; Frederick R Adler; Natalia Argel; Fadi Asfour; Perry S Brown; Barbara A Chatfield; Cori L Daines; Dixie Durham; Jessica A Francis; Barbara Glover; Theresa Heynekamp; John R Hoidal; Judy L Jensen; Ruth Keogh; Carol M Kopecky; Noah Lechtzin; Yanping Li; Jerimiah Lysinger; Osmara Molina; Craig Nakamura; Kristyn A Packer; Katie R Poch; Alexandra L Quittner; Peggy Radford; Abby J Redway; Scott D Sagel; Shawna Sprandel; Jennifer L Taylor-Cousar; Jane B Vroom; Ryan Yoshikawa; John P Clancy; J Stuart Elborn; Kenneth N Olivier; David R Cox Journal: BMC Med Res Methodol Date: 2019-04-26 Impact factor: 4.615