title: The pre-analytical processing of blood samples for detecting biomarkers on protein microarrays.
abstract: Specimen collection method and quality insurance are pivotal in biomarker discovery. Pre-analytical variables concerning blood collection and sample handling might affect analytical results and should be standardised prior application. In this study, we examine pre-analytical characteristics of blood samples using protein microarray. The influences of 1) standby times until centrifugation (1 h, 4 h, 24 h and 48 h), 2) four blood collection methods, and 3) IgG purified from those samples on differentially reactive antigens between samples ("DIRAGs") were investigated. Spearman correlation analyses of intra-individual arrays demonstrated remarkable differences (0.75-0.98 vs. 0.5-0.75) of antibody reactivities within and between serum and plasma samples. Class comparison showed that reactive antigen profiles were best preserved using IgG purified samples of serum tubes without separation gel as after 24h 83% of the 1h baseline DIRAGs were re-found. Testing dilution series with protein microarrays and Luminex xMap® Technology, we found linear correlations (R(2) = 0.94-0.99) between IgG concentration and read-out when using purified IgG instead of serum. Therefore, we highly recommend standardising pre-analytics and proposing the use of purified IgG for autoantibody immune-profiling with protein microarrays to reduce potential unspecific binding of matrix proteins abundant in serum and plasma samples.
  
  
  CONCLUSIONS
  Although purified IgG cannot completely compensate the influence of pre-analytics, in highly parallel immune-profiling IgG enables reduction of unspecific effects, which occur when using serum or plasma for analysis on protein microarrays. Reproducibility problems due to pre-analytical processing of blood samples might explain discrepant results reported in various biomarker studies.
authors: Austrian Institute of Technology AIT, Health & Environment - Molecular Diagnostics, Vienna, Austria. Electronic address: sandra.rosskopf.fl@ait.ac.at.
authors: Sandra
authors: S
authors: Rosskopf
authors: Austrian Institute of Technology AIT, Health & Environment - Molecular Diagnostics, Vienna, Austria. Electronic address: istvan.gyurjan.fl@ait.ac.at.
authors: István
authors: I
authors: Gyurján
authors: Austrian Institute of Technology AIT, Health & Environment - Molecular Diagnostics, Vienna, Austria.
authors: Regina
authors: R
authors: Soldo
authors: Austrian Institute of Technology AIT, Health & Environment - Molecular Diagnostics, Vienna, Austria. Electronic address: johana.luna.fl@ait.ac.at.
authors: Johana A
authors: JA
authors: Luna-Coronell
authors: Austrian Institute of Technology AIT, Health & Environment - Molecular Diagnostics, Vienna, Austria. Electronic address: klemens.vierlinger.fl@ait.ac.at.
authors: Klemens
authors: K
authors: Vierlinger
authors: Department of Obstetrics and Gynaecology, Medical University of Vienna, Vienna, Austria. Electronic address: christian.singer@meduniwien.ac.at.
authors: Christian F
authors: CF
authors: Singer
authors: Department of Obstetrics and Gynaecology, Medical University of Vienna, Vienna, Austria. Electronic address: christine.rappaport@akhwien.at.
authors: Christine
authors: C
authors: Rappaport
authors: Department of Obstetrics and Gynaecology, Medical University of Vienna, Vienna, Austria. Electronic address: nina.pecha@meduniwien.ac.at.
authors: Nina
authors: N
authors: Pecha
authors: Austrian Institute of Technology AIT, Health & Environment - Molecular Diagnostics, Vienna, Austria. Electronic address: andreas.weinhaeusel@ait.ac.at.
authors: Andreas
authors: A
authors: Weinhaeusel
mesh_terms: D053000:Analytic Sample Preparation Methods; D015415:Biomarkers / Q000097:blood*; D001800:Blood Specimen Collection; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology / Q000302:isolation & purification*; D040081:Protein Array Analysis / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D015415:Biomarkers; D007074:Immunoglobulin G
keywords: Biomarker; DIRAGs; Immune-proteomics; Immunoglobulin G; Pre-analytics; Protein microarray