Original Article
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World J Methodol. Dec 26, 2014; 4(4): 219-231
Published online Dec 26, 2014. doi: 10.5662/wjm.v4.i4.219
Methodical and pre-analytical characteristics of a multiplex cancer biomarker immunoassay
Natalie Hermann, Katja Dreßen, Frank A Schildberg, Christopher Jakobs, Stefan Holdenrieder
Natalie Hermann, Katja Dreßen, Christopher Jakobs, Stefan Holdenrieder, Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, 53127 Bonn, Germany
Frank A Schildberg, Institutes of Molecular Medicine and Experimental Immunology, University Hospital Bonn, 53127 Bonn, Germany
Author contributions: Hermann N and Dreßen K contributed equally; Hermann N, Dreßen K and Holdenrieder S designed the present study and coordinated the logistic process; Hermann N, Dreßen K and Holdenrieder S were responsible for defined blood sampling and storing; Hermann N, Dreßen K, Schildberg FA and Jakobs C were responsible for immunoassay measurements; Statistical analysis was performed by Hermann N and Dreßen K; Hermann N, Dreßen K and Holdenrieder S were involved in the interpretation of the data, the conception of the manuscript as well as the revision; all authors read and approved the final manuscript.
Correspondence to: Stefan Holdenrieder, MD, Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Sigmund-Freud Str. 15, 53127 Bonn, Germany. stefan.holdenrieder@uni-bonn.de
Telephone: +49-228-28712126 Fax: +49-228-28712159
Received: December 2, 2013
Revised: September 11, 2014
Accepted: October 14, 2014
Published online: December 26, 2014
Processing time: 386 Days and 10.8 Hours
Abstract

AIM: 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.

CONCLUSION: 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.

Keywords: Multiplex immunoassay; Tumor marker; Cytokines; Cell death markers; Methodical evaluation

Core tip: In this study, the methodological quality of a new research-use-only multiplex magnetic bead assay, particularly designed for cancer diagnosis, was evaluated. This attractive panel includes 24 biomarkers: established as well as auspicious tumor markers and markers deriving from the fields of apoptosis, immunology and angiogenesis. Herewith, the complexity and multifactorial background of a cancer disease is depicted. Measurements were performed with physiological serum pools and intra- and inter-assay imprecision as well as dilution linearity were assessed. Furthermore, the influence of preanalytical factors was investigated.