Julia Hentschel1, Ulrike Müller2, Franziska Doht2, Nele Fischer2, Klas Böer3, Jürgen Sonnemann4, Christina Hipler5, Kerstin Hünniger6, Oliver Kurzai6, Udo R Markert7, Jochen G Mainz2. 1. CF-Center, Pediatrics, Jena University Hospital, Germany. Electronic address: Julia.Hentschel@med.uni-jena.de. 2. CF-Center, Pediatrics, Jena University Hospital, Germany. 3. Institute for Clinical Chemistry and Laboratory Diagnostics, Jena University Hospital, Germany. 4. Pediatric Haematology and Oncology, Pediatrics, Jena University Hospital, Germany. 5. Department of Dermatology, Jena University Hospital, Germany. 6. Septomics Research Center, Friedrich Schiller University, Germany; Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany. 7. Department of Obstetrics, Placenta Laboratory, Jena University Hospital, Germany.
Abstract
BACKGROUND: Non-invasive sampling of airway epithelial-lining-fluid by nasal lavage (NL) is an emerging method to monitor allergy, infection and inflammation in patients with respiratory diseases. However, the influences of collection-, processing- and storage-methods have not been sufficiently evaluated and standardized. METHODS: Influences of repeated NL, centrifugation setups, repeated freezing and thawing, and protease inhibitors on mediator concentration were evaluated in healthy controls and CF patients, which serve as a model for chronic bacterial infection and inflammation. Polymorphonuclear leukocyte elastase (NE)/myeloperoxidase (MPO)/interleukin (IL)-1/IL-6/IL-8 and tumour necrosis factor alpha (TNF) concentrations were measured using ELISA and Multiplex Bead-Arrays. RESULTS: NL-repetition within 0.5-4h markedly decreased NE, IL-8 and MPO-concentrations for up to 70%. NL centrifugation up to 250×g for cellular differentiation did not significantly influence mediator concentration in native and processed NL fluid. NL freezing and thawing markedly decreased IL-8 and MPO concentrations by up to 50% while NE remained stable. In contrast to preceding reports, storing at -70°C for ≥5 years led to significantly reduced mediator concentrations in NL compared to contemporary analyses, being most pronounced for IL-1β, IL-6 and TNFa. Storing of samples in the presence of protease inhibitors led to an increase in marker concentration for IL-8 (+27%) and MPO (+15%) even after one year of storage. CONCLUSIONS: NL is an easy and robust technique for inflammation monitoring of the upper airways. For the first time we have shown that diagnostic NL should be performed only once daily to get comparable results. Whereas NL-fluid can be stored unprocessed at -70°C for cytokine analysis over 1-2 years with protease inhibitors supporting stability, ≥5 years storage as well as repeated freezing and thawing should be avoided.
BACKGROUND: Non-invasive sampling of airway epithelial-lining-fluid by nasal lavage (NL) is an emerging method to monitor allergy, infection and inflammation in patients with respiratory diseases. However, the influences of collection-, processing- and storage-methods have not been sufficiently evaluated and standardized. METHODS: Influences of repeated NL, centrifugation setups, repeated freezing and thawing, and protease inhibitors on mediator concentration were evaluated in healthy controls and CFpatients, which serve as a model for chronic bacterial infection and inflammation. Polymorphonuclear leukocyte elastase (NE)/myeloperoxidase (MPO)/interleukin (IL)-1/IL-6/IL-8 and tumour necrosis factor alpha (TNF) concentrations were measured using ELISA and Multiplex Bead-Arrays. RESULTS: NL-repetition within 0.5-4h markedly decreased NE, IL-8 and MPO-concentrations for up to 70%. NL centrifugation up to 250×g for cellular differentiation did not significantly influence mediator concentration in native and processed NL fluid. NL freezing and thawing markedly decreased IL-8 and MPO concentrations by up to 50% while NE remained stable. In contrast to preceding reports, storing at -70°C for ≥5 years led to significantly reduced mediator concentrations in NL compared to contemporary analyses, being most pronounced for IL-1β, IL-6 and TNFa. Storing of samples in the presence of protease inhibitors led to an increase in marker concentration for IL-8 (+27%) and MPO (+15%) even after one year of storage. CONCLUSIONS: NL is an easy and robust technique for inflammation monitoring of the upper airways. For the first time we have shown that diagnostic NL should be performed only once daily to get comparable results. Whereas NL-fluid can be stored unprocessed at -70°C for cytokine analysis over 1-2 years with protease inhibitors supporting stability, ≥5 years storage as well as repeated freezing and thawing should be avoided.
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