title: Method validation for preparing serum and plasma samples from human blood for downstream proteomic, metabolomic, and circulating nucleic acid-based applications.
abstract: BACKGROUND
  Formal method validation for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. Serum and plasma processing protocols were validated for fitness-for-purpose in terms of key downstream endpoints, and this article demonstrates methodology for biospecimen processing method validation.
  
  
  METHODS
  Serum and plasma preparation from human blood was optimized for centrifugation conditions with respect to microparticle counts. Optimal protocols were validated for methodology and reproducibility in terms of acceptance criteria based on microparticle counts, DNA and hemoglobin concentration, and metabolomic and proteomic profiles. These parameters were also used to evaluate robustness for centrifugation temperature (4°C versus room temperature [RT]), deceleration (low, medium, high) and blood stability (after a 2-hour delay).
  
  
  RESULTS
  Optimal protocols were 10-min centrifugation for serum and 20-min for plasma at 2000 g, medium brake, RT. Methodology and reproducibility acceptance criteria were met for both protocols except for reproducibility of plasma metabolomics. Overall, neither protocol was robust for centrifugation at 4°C versus RT. RT gave higher microparticles and free DNA yields in serum, and fewer microparticles with less hemolysis in plasma. Overall, both protocols were robust for fast, medium, and low deceleration, with a medium brake considered optimal. Pre-centrifugation stability after a 2-hour delay was seen at both temperatures for hemoglobin concentration and proteomics, but not for microparticle counts.
  
  
  CONCLUSIONS
  We validated serum and plasma collection methods suitable for downstream protein, metabolite, or free nucleic acid-based applications. Temperature and pre-centrifugation delay can influence analytic results, and laboratories and biobanks should systematically record these conditions in the scope of accreditation.
authors: 1 Integrated BioBank of Luxembourg (IBBL) , Luxembourg .
authors: Wim
authors: W
authors: Ammerlaan
authors: Jean-Pierre
authors: JP
authors: Trezzi
authors: Pierre
authors: P
authors: Lescuyer
authors: Conny
authors: C
authors: Mathay
authors: Karsten
authors: K
authors: Hiller
authors: Fay
authors: F
authors: Betsou
mesh_terms: D001800:Blood Specimen Collection; D002498:Centrifugation; D006801:Humans; D055432:Metabolomics / Q000379:methods*; D009696:Nucleic Acids / Q000097:blood*; D010949:Plasma / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D044967:Serum / Q000378:metabolism*; D013696:Temperature
chemical_list: D009696:Nucleic Acids