title: Influences of nasal lavage collection-, processing- and storage methods on inflammatory markers--evaluation of a method for non-invasive sampling of epithelial lining fluid in cystic fibrosis and other respiratory diseases.
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.
authors.affiliation: CF-Center, Pediatrics, Jena University Hospital, Germany. Electronic address: Julia.Hentschel@med.uni-jena.de.
authors.forename: Julia
authors.initials: J
authors.lastname: Hentschel
authors.affiliation: CF-Center, Pediatrics, Jena University Hospital, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Müller
authors.affiliation: CF-Center, Pediatrics, Jena University Hospital, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Doht
authors.affiliation: CF-Center, Pediatrics, Jena University Hospital, Germany.
authors.forename: Nele
authors.initials: N
authors.lastname: Fischer
authors.affiliation: Institute for Clinical Chemistry and Laboratory Diagnostics, Jena University Hospital, Germany.
authors.forename: Klas
authors.initials: K
authors.lastname: Böer
authors.affiliation: Pediatric Haematology and Oncology, Pediatrics, Jena University Hospital, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Sonnemann
authors.affiliation: Department of Dermatology, Jena University Hospital, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Hipler
authors.affiliation: Septomics Research Center, Friedrich Schiller University, Germany; Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Hünniger
authors.affiliation: Septomics Research Center, Friedrich Schiller University, Germany; Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Jena, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Kurzai
authors.affiliation: Department of Obstetrics, Placenta Laboratory, Jena University Hospital, Germany.
authors.forename: Udo R
authors.initials: UR
authors.lastname: Markert
authors.affiliation: CF-Center, Pediatrics, Jena University Hospital, Germany.
authors.forename: Jochen G
authors.initials: JG
authors.lastname: Mainz
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D015415:Biomarkers / Q000032:analysis; D016022:Case-Control Studies; D002498:Centrifugation / Q000592:standards; D002648:Child; D002675:Child, Preschool; D003550:Cystic Fibrosis / Q000175:diagnosis / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005615:Freezing; D006801:Humans; D007249:Inflammation; D007375:Interleukin-1 / Q000032:analysis; D015850:Interleukin-6 / Q000032:analysis; D016209:Interleukin-8 / Q000032:analysis; D019272:Leukocyte Elastase / Q000032:analysis; D008297:Male; D008875:Middle Aged; D017817:Nasal Lavage Fluid / Q000737:chemistry*; D009195:Peroxidase / Q000032:analysis; D011480:Protease Inhibitors / Q000737:chemistry; D013048:Specimen Handling / Q000592:standards*; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis
chemical_list: D015415:Biomarkers; C508600:IL6 protein, human; D007375:Interleukin-1; D015850:Interleukin-6; D016209:Interleukin-8; D011480:Protease Inhibitors; D014409:Tumor Necrosis Factor-alpha; D009195:Peroxidase; D019272:Leukocyte Elastase
keywords: Centrifugation; Cystic fibrosis; Cytokines; Cytology; Nasal lavage; Sampling technique