Novel method to process cystic fibrosis sputum for determination of oxidative state.
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.


OBJECTIVE
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).


CONCLUSIONS
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.
Hector
A
A
Paediatric Pneumology, Children's Hospital, University of Munich, Lindwurmstrasse 4, Munich, Germany.
Jonas
F
F
Kappler
M
M
Feilcke
M
M
Hartl
D
D
Griese
M
M
D000328:Adult; D015703:Antigens, CD / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D039481:CD11b Antigen / Q000378:metabolism; D002452:Cell Count; D002470:Cell Survival; D002851:Chromatography, High Pressure Liquid; D003550:Cystic Fibrosis / Q000276:immunology*; D004229:Dithiothreitol; D005260:Female; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D019272:Leukocyte Elastase / Q000378:metabolism; D008297:Male; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D023062:Receptors, Interleukin-8A / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism; D013183:Sputum / Q000378:metabolism*; D060149:Tetraspanin 30
D015703:Antigens, CD; D015415:Biomarkers; D039481:CD11b Antigen; C554586:CD63 protein, human; C509295:ITGAM protein, human; D007202:Indicators and Reagents; D010980:Platelet Membrane Glycoproteins; D023062:Receptors, Interleukin-8A; D012235:Rhodamines; D060149:Tetraspanin 30; C058319:dihydrorhodamine 123; D019272:Leukocyte Elastase; D005978:Glutathione; D004229:Dithiothreitol