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Effect of sample processing on the analysis of IL4, IL5, and IL13 in sputum supernatants

 
: Holz, Olaf; Müller, Meike; Ignatenko, Stanislav; Kappeler, Dominik; Buhl, Roland; Beeh, Kai-Michael; Kornmann, Oliver; Kirsten, Anne; Homburg, Ursula; Garn, Holger; Krug, Norbert; Hohlfeld, Jens Michael

American Journal of Respiratory and Critical Care Medicine 189 (2014), Art. A4255
ISSN: 1073-449X
ISSN: 0003-0805
ISSN: 1535-4970
American Thoracic Society (ATS International Conference) <2014, San Diego/Calif.>
English
Abstract
Fraunhofer ITEM ()

Abstract
Introduction: The analysis of cytokines and mediators in sputum supernatants may be affected by endogenous proteases and the processing of sputum with dithiothreitol (DTT) during homogenization. Sputum extraction with PBS (Respiration 2012), dialysis (ARJCCM 2008) or the addition of protease inhibitors (ERJ 2001) has been suggested to improve the recovery of mediators from sputum supernatants. Depending on the antibodies used the effect of these interfering factors may also vary with the analytical systems. Here we report our experience with the MesoScale (MSD)-platform for the analysis of IL4, IL5, and IL13.
Methods: In contrast to published data we observed higher levels of IL4, IL5 and IL13 in DTT treated compared to PBS extracted samples in pre-tests with the MSD system. Therefore we used pooled DTT-treated sputum samples for spiking experiments (healthy subjects, with and without protease-inhibitor (PI), spiked with recombinant IL4, IL5, and IL13 in two concentrations and from 26 asthmatic subjects undergoing an allergen challenge (ClinicalTrials.gov Identifier: NCT0174376). The MSD 10-plex ECL assay (MesoScale Discovery, USA) was used for cytokine analysis.
Results: The median recovery of the 1000 and 20 pg/mL spike from diluent was 78/100% for IL4, 17/18% for IL5 and 60/112% for IL13, respectively. The recovery from spiked DTT treated sputum samples was 25/24% for IL4, 17/9% for IL5, and 38/2% for IL13. PI did not significantly affect these values. Using DTT-treated sputum samples we were able to show a significant increase for IL5 and IL13, but not for IL4, in sputum obtained after allergen challenge compared to the patients' baseline sputum.
Conclusion: Despite the low recovery in the spiking experiments, which do not fullfill standard acceptance criteria, it was possible to detect the expected allergen-induced increase in IL5 and IL13 in DTT-treated sputum supernatants. Thus, the time consuming extraction of sputum samples can be avoided for this type of analyses.

: http://publica.fraunhofer.de/documents/N-332366.html