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 apoptosisinducing 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 ℃ and 25 ℃ 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.© 2014 Baishideng Publishing Group Inc. All rights reserved.Key words: 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,
Differential diagnosis of CRC can be improved by new biomarker classes and their combination assessed by novel multiplex immunoassay.
Multiple factors contribute to the development and progression of breast cancer. Markers of tumor growth and invasion, cell death, immune activation, and angiogenesis can be assessed in parallel by a novel multiplex immunoassay panel. The diagnostic performance of a multiplex cancer biomarker magnetic bead panel comprising 24 tumor associated parameters was evaluated in sera of 154 women including 77 patients with breast cancer, 10 with precancerous lesions, 31 with benign breast diseases, and 36 healthy controls. Marker levels were log-transformed for variance stabilization. Significance testing was done using t-test or Wilcoxon rank-sum test with correction of p values for multiple testing. Furthermore, receiver operating characteristic analyses were performed. Serum levels of several biomarkers were significantly (p ≤ 0.001) higher in cancer patients than in healthy controls, particularly alpha-fetoprotein, cancer antigen 15-3, cancer antigen 19-9, migration inhibitory factor, carcinoembryonic antigen, cancer antigen 125, hepatocyte growth factor, soluble Fas, tumor necrosis factor-α, stem cell factor, and osteopontin. As most markers were also elevated in benign breast diseases, only cancer antigen 15-3 showed significant differences to cancer patients (p ≤ 0.001). The resulting areas under the curve in receiver operating characteristic curves for discrimination between benign and malignant breast diseases achieved 0.71 with a sensitivity of 33.8% at 95% specificity. Multiplexing enables parallel analysis of different biomarker classes for cancer detection. Established cancer antigen 15-3 proved to be most relevant for differential diagnosis.
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