title: Method validation for preparing urine samples for downstream proteomic and metabolomic applications.
abstract: BACKGROUND
  Formal validation of methods for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A protocol for processing of a biospecimen (urine) was validated for fitness-for-purpose in terms of key downstream endpoints.
  
  
  METHODS
  Urine processing was optimized for centrifugation conditions on the basis of microparticle counts at room temperature (RT) and at 4°C. The optimal protocol was validated for performance (microparticle counts), and for reproducibility and robustness for centrifugation temperature (4°C vs. RT) and brake speed (soft, medium, hard). Acceptance criteria were based on microparticle counts, cystatin C and creatinine concentrations, and the metabolomic profile.
  
  
  RESULTS
  The optimal protocol was a 20-min, 12,000 g centrifugation at 4°C, and was validated for urine collection in terms of microparticle counts. All reproducibility acceptance criteria were met. The protocol was robust for centrifugation at 4°C versus RT for all parameters. The protocol was considered robust overall in terms of brake speeds, although a hard brake gave significantly fewer microparticles than a soft brake.
  
  
  CONCLUSIONS
  We validated a urine processing method suitable for downstream proteomic and metabolomic applications. Temperature and brake speed can influence analytic results, with 4°C and high brake speed considered optimal. Laboratories and biobanks should ensure these conditions are systematically recorded in the scope of accreditation.
authors.affiliation: 1 Integrated BioBank of Luxemburg (IBBL) , Luxembourg .
authors.forename: Wim
authors.initials: W
authors.lastname: Ammerlaan
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Trezzi
authors.forename: Conny
authors.initials: C
authors.lastname: Mathay
authors.forename: Karsten
authors.initials: K
authors.lastname: Hiller
authors.forename: Fay
authors.initials: F
authors.lastname: Betsou
mesh_terms: D000328:Adult; D055252:Cell-Derived Microparticles / Q000378:metabolism*; D002474:Cell-Free System; D003404:Creatinine / Q000652:urine*; D015891:Cystatins / Q000652:urine*; D005260:Female; D006801:Humans; D008297:Male; D055442:Metabolome; D008875:Middle Aged; D020543:Proteome; D015203:Reproducibility of Results; D013696:Temperature; D059349:Urine Specimen Collection / Q000379:methods*
chemical_list: D015891:Cystatins; D020543:Proteome; D003404:Creatinine