AAethodo ogoica improvements for measuring eicosanoids and cytokines in exhaled breath condensate

Lund University, Lund, Skåne, Sweden
Respiratory Medicine (Impact Factor: 3.09). 02/2006; 100(1):34-8. DOI: 10.1016/j.rmed.2005.04.007
Source: PubMed


Exhaled breath condensate (EBC) is simple to collect and as such a non-invasive method that has attracted substantial interest in the last few years. However, several methodological concerns have been raised and it has been difficult to reproduce results between different centres. Because of low concentrations of inflammatory markers, potential loss in the sampling system may have great influence. The aim of the present study was to se if evaporation and plastic coating could facilitate detection.
Through methodological improvements, we have now made it possible to measure EBC concentrations of eicosanoids and cytokines in our system. Due to absorbance of both fatty acid derivates and proteins to several plastics, the first step is coating of all surfaces with bovine serum albumin and Tween 20. Since several assays are sensitive to these factors, the methodology has to be standardised to avoid false results. Secondly, larger amounts of EBC have to be vacuum-dried, and thereafter resolved in the respective assay buffers. The EBCs have to be concentrated 5-10 times, depending on samples and assay sensitivity.
Due to these improvements we can measure, for example, cysteinyl-leukotrienes, leukotriene B4, prostaglandin E and 8-isoprostane. High sensitivity assays have also made it possible to measure cytokines, for example, interleukin (IL)-1beta, IL-8 and IL-13.
We are aware of different results from other labs. However, it seems essential to coat and to concentrate the samples in order to achieve reliable and measurable results.

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    • "In a sitting position, wearing a noseclip, they breathed tidally for 15 min through a two-way non-rebreathing valve, which also served as a saliva trap. In order to avoid loss of molecules from inflammatory markers due to adhesion to the walls, the tubes were coated with 1% bovine serum albumin and 0.01% Tween 20 for 30 min according to previous optimization procedures (Tufvesson and Bjermer 2006). The EBC samples were immediately frozen at –70 °C. "
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    • "EBC is an attractive non-invasive method for assessing airway diseases but its clinical use is still hampered by methodical limitations [5]. There is accumulating evidence that the physical surface properties of the collecting devices influence the marker measurement [9,12-14,22]. Moreover, for some markers the applied temperature was identified as a major confounder [10,11]. "
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    ABSTRACT: The collection of exhaled breath condensate (EBC) is a suitable and non-invasive method for evaluation of airway inflammation. Several studies indicate that the composition of the condensate and the recovery of biomarkers are affected by physical characteristics of the condensing device and collecting circumstances. Additionally, there is an apparent influence of the condensing temperature, and often the level of detection of the assay is a limiting factor. The ECoScreen2 device is a new, partly single-use disposable system designed for studying different lung compartments. EBC samples were collected from 16 healthy non-smokers by using the two commercially available devices ECoScreen2 and ECoScreen at a controlled temperature of -20 degrees C. EBC volume, pH, NOx, LTB4, PGE2, 8-isoprostane and cys-LTs were determined. EBC collected with ECoScreen2 was less acidic compared to ECoScreen. ECoScreen2 was superior concerning condensate volume and detection of biomarkers, as more samples were above the detection limit (LTB4 and PGE2) or showed higher concentrations (8-isoprostane). However, NOx was detected only in EBC sampled by ECoScreen. ECoScreen2 in combination with mediator specific enzyme immunoassays may be suitable for measurement of different biomarkers. Using this equipment, patterns of markers can be assessed that are likely to reflect the complex pathophysiological processes in inflammatory respiratory disease.
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