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. 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. Copyright (C) 2009 S. Karger AG, Basel