title: Methodical and pre-analytical characteristics of a multiplex cancer biomarker immunoassay.
abstract: OBJECTIVE
  To test the methodical and pre-analytical performance of a new multiplex cancer biomarker panel using magnetic beads.
  
  
  METHODS
  The MILLIPLEX(®) MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 comprises the tumor markers carcinoembryonic antigen, alpha-fetoprotein, total prostate-specific antigen, cancer antigen 15-3, cancer antigen 19-9, cancer antigen 125, cytokeratine 19-fragment, β-human chorionic gonadotropin, human epididymis protein 4, osteopontin, prolactin, the cell death and angiogenesis markers soluble Fas, soluble Fas-ligand, tumor necrosis factor related apoptosis-inducing ligand, vascular endothelial growth factor and the immunological markers interleukin-6 (IL-6), IL-8, tumor necrosis factor-α, transforming growth factor α, fibroblast growth factor-2, macrophage migration inhibitory factor, leptin, hepatocyte growth factor, and stem cell factor. We determined intra- and inter-assay imprecision as well as dilution linearity using quality controls and serum pools. Furthermore, the stability of the 24 biomarkers examined in this panel was ascertained by testing the influence of different storage temperatures and time span before centrifugation.
  
  
  RESULTS
  For all markers measured in the synthetic internal quality controls, the intra-assay imprecision ranged between 2.26% and 9.41%, while for 20 of 24 measured markers in the physiological serum pools, it ranged between 1.68% and 12.87%. The inter-assay imprecision ranged between 1.48%-17.12% for 23 biomarkers in synthetic, and between 4.59%-23.88% for 18 biomarkers in physiological quality controls. Here, single markers with very low concentration levels had increased imprecision rates. Dilution linearity was acceptable (70%-130% recovery) for 20 biomarkers. Regarding pre-analytical influencing factors, most markers were stable if blood centrifugation was delayed or if serum was stored for up to 24 h at 4 °C and 25 °C after centrifugation. Comparable results were obtained in serum and plasma for most markers. However, great changes were observed for single markers.
  
  
  CONCLUSIONS
  MILLIPLEX(®) MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 assay is a stable and precise method for detection of most biomarkers included in the kit. However, single markers have to be interpreted with care.
authors.affiliation: Natalie Hermann, Katja Dreßen, Christopher Jakobs, Stefan Holdenrieder, Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, 53127 Bonn, Germany.
authors.forename: Natalie
authors.initials: N
authors.lastname: Hermann
authors.affiliation: Natalie Hermann, Katja Dreßen, Christopher Jakobs, Stefan Holdenrieder, Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, 53127 Bonn, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Dreßen
authors.affiliation: Natalie Hermann, Katja Dreßen, Christopher Jakobs, Stefan Holdenrieder, Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, 53127 Bonn, Germany.
authors.forename: Frank A
authors.initials: FA
authors.lastname: Schildberg
authors.affiliation: Natalie Hermann, Katja Dreßen, Christopher Jakobs, Stefan Holdenrieder, Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, 53127 Bonn, Germany.
authors.forename: Christopher
authors.initials: C
authors.lastname: Jakobs
authors.affiliation: Natalie Hermann, Katja Dreßen, Christopher Jakobs, Stefan Holdenrieder, Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, 53127 Bonn, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Holdenrieder
keywords: Cell death markers; Cytokines; Methodical evaluation; Multiplex immunoassay; Tumor marker