title: Relevance of pre-analytical blood management on the emerging cardiovascular protein biomarkers TWEAK and HMGB1 and on miRNA serum and plasma profiling.
abstract: BACKGROUND
  Disease-independent sources of biomarker variability include pre-analytical, analytical and biological variance. The aim of the present study was to evaluate whether the pre-analytical phase has any impact on the emerging heart disease TWEAK and HMGB1 protein markers and miRNA biomarkers, and whether peptidome profiling allows the identification of pre-analytical quality markers.
  
  
  METHODS
  An assessment was made of sample type (serum, EDTA-Plasma, Citrate-Plasma, ACD-plasma, Heparin-plasma), temperature of sample storage (room temperature or refrigerated), time of sample storage (0.5, 3, 6 and 9h) and centrifugation (one or two-step). Aliquots of all processed samples were immediately frozen (-80°C) before analysis. Proteins were assayed by ELISAs, miRNA expression profile by microarray and peptidome profiling by MALDI-TOF/MS.
  
  
  RESULTS
  Temperature, time and centrifugation had no impact on TWEAK and HMGB1 results, which were significantly influenced by matrix type, TWEAK levels being significantly higher (F=194.7, p<0.0001), and HMGB1 levels significantly lower (F=36.32, p<0.0001) in serum than in any other plasma type. Unsuitable miRNA results were obtained using Heparin-plasma. Serum miRNA expression profiles depended mainly on temperature, while EDTA-plasma miRNA expression profiles were strongly affected by the centrifugation method used. MALDI-TOF/MS allowed the identification of seven features as indices of pre-analytical serum (m/z at 1206, 1350, 1865 and 2021) or EDTA-plasma (m/z 1897, 2740 and 2917) degradation.
  
  
  CONCLUSIONS
  Serum and EDTA-plasma allow the analysis of both proteins and miRNA emerging biomarkers of heart diseases. Refrigerated storage prevents an altered miRNA expression profile also in cases of a prolonged time-interval between blood drawing and processing.
authors.affiliation: Department of Medicine - DIMED, University of Padova, Italy. Electronic address: daniela.basso@sanita.padova.it.
authors.forename: Daniela
authors.initials: D
authors.lastname: Basso
authors.affiliation: Department of Medicine - DIMED, University of Padova, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Padoan
authors.affiliation: Comprehensive Biomarker Center GmbH, Heidelberg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Laufer
authors.affiliation: UOC Immunotrasfusionale, University-Hospital of Padova, Italy.
authors.forename: Vittorio
authors.initials: V
authors.lastname: Aneloni
authors.affiliation: Department of Medicine - DIMED, University of Padova, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Moz
authors.affiliation: Comprehensive Biomarker Center GmbH, Heidelberg, Germany.
authors.forename: Hannah
authors.initials: H
authors.lastname: Schroers
authors.affiliation: Department of Medicine - DIMED, University of Padova, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Pelloso
authors.affiliation: Comprehensive Biomarker Center GmbH, Heidelberg, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Saiz
authors.affiliation: Comprehensive Biomarker Center GmbH, Heidelberg, Germany.
authors.forename: Medea
authors.initials: M
authors.lastname: Krapp
authors.affiliation: Department of Medicine - DIMED, University of Padova, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Fogar
authors.affiliation: Department of Medicine - DIMED, University of Padova, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Cornoldi
authors.affiliation: Department of Medicine - DIMED, University of Padova, Italy.
authors.forename: Carlo-Federico
authors.initials: CF
authors.lastname: Zambon
authors.affiliation: Department of Medicine - DIMED, University of Padova, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Rossi
authors.affiliation: Department of Medicine - DIMED, University of Padova, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: La Malfa
authors.affiliation: UOC Immunotrasfusionale, University-Hospital of Padova, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Marotti
authors.affiliation: Comprehensive Biomarker Center GmbH, Heidelberg, Germany; Eurofins Medigenomix GmbH, Ebersberg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Brefort
authors.affiliation: Department of Cardiology, Angiology, and Pulmonology, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Tanja M
authors.initials: TM
authors.lastname: Weis
authors.affiliation: Department of Cardiology, Angiology, and Pulmonology, University Hospital Heidelberg, Heidelberg, Germany.
authors.forename: Hugo A
authors.initials: HA
authors.lastname: Katus
authors.affiliation: Department of Medicine - DIMED, University of Padova, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Plebani
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000097:blood; D002318:Cardiovascular Diseases / Q000097:blood* / Q000175:diagnosis; D000074049:Cytokine TWEAK; D005260:Female; D024243:HMGB1 Protein / Q000097:blood*; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000097:blood*; D008875:Middle Aged; D048069:Tumor Necrosis Factors / Q000097:blood*
chemical_list: D015415:Biomarkers; D000074049:Cytokine TWEAK; D024243:HMGB1 Protein; C585491:HMGB1 protein, human; D035683:MicroRNAs; C109743:TNFSF12 protein, human; D048069:Tumor Necrosis Factors
keywords: HMGB1; Heart diseases; MALDI-TOF/MS; MicroRNA; Pre-analytics; Proteomics; TWEAK