Novel Method to Process Cystic Fibrosis Sputum for Determination of Oxidative State

Abstract

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.

Culture-free diagnostics of Pseudomonas aeruginosa infection by silver nanorod array based SERS from clinical sputum samples

“…These frozen aliquots of processed sputum samples were obtained by a non-invasive method from subjects with CF after signing informed consents with Institutional Review Board approval. Sputum processing was performed by the staff 27 and as discussed in detail in the Supplementary Note 1.…”

Innate immunity in cystic fibrosis lung disease

“…The phenotypical characterization of neutrophils in CF airways is hampered by several facts: (i) heterogeneity among CF patients (extent of neutrophilic inflammation, infections), ability to sample sputum (impeding sputum studies in CF infants younger than about 5/6 years of age), (iii) pre-analytical processing of complex airway fluid material (sputum, see [197] for details) and cleavage of surface markers essential for neutrophil subtype characterization. A few studies used advanced optimized flow cytometric approaches to dissect airway neutrophil subpopulations [8,185,[198][199][200].…”

The Effect of Dithiothreitol on the Transcriptome of Induced Sputum Cells

“…In order to obtain an overview of the general effects of DTT, pooled samples (600 ng each) of four RNA preparations from these patients - processed either with or without DTT - were analyzed. Processing of the sputum samples without DTT was performed by adding 8 volumes of PBS with subsequent mechanical homogenization of the sputum sample according to the method of Hector et al [5]. Processing of the sputum samples with DTT followed a standard protocol adding 4 volumes of DTT to the sputum cell pellet.…”