Clinical Chemistry
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Clinical Chemistry 52: 1284-1293, 2006. First published May 11, 2006; 10.1373/clinchem.2006.067595
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(Clinical Chemistry. 2006;52:1284-1293.)
© 2006 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Development and Validation of a Multiplex Add-On Assay for Sepsis Biomarkers Using xMAP Technology

Kristian Kofoed1,2,a, Uffe Vest Schneider1, Troels Scheel1, Ove Andersen1,2 and Jesper Eugen-Olsen1

1 Clinical Research Unit and2 Department of Infectious Diseases, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark.

aAddress correspondence to this author at: Clinical Research Unit 136, H:S Hvidovre Hospital, Kettegaard Allé 30, DK-2650 Hvidovre, Denmark. Fax 45-3632-3797; e-mail kristian.kofoed{at}hh.hosp.dk.

Background: Sepsis is a common and often fatal disease. Because sepsis can be caused by many different organisms, biomarkers that can aid in diagnosing sepsis and monitoring treatment efficacy are highly warranted. New sepsis markers may provide additional information to complement the currently used markers.

Methods: We used a combination of in-house and commercially available multiplex immunoassays based on Luminex® xMAP technology to assay biomarkers of potential interest in EDTA-plasma samples.

Results: A 3-plex assay for soluble urokinase plasminogen activator receptor (suPAR), soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), and macrophage migration inhibiting factor (MIF) was developed and validated in-house. This 3-plex assay was added to a commercially available interleukin-1ß (IL-1ß), IL-6, IL-8, granulocyte/macrophage colony-stimulating factor, and tumor necrosis factor-{alpha} human cytokine panel. No cross-reactivity was observed when the assays were combined. Correlation between values obtained with the 8-plex, the 5-cytokine panel, the 3 in-house 1-plex assays, and a suPAR ELISA ranged from 0.86 to 0.99. Mean within- and between-run CVs were 8.0% and 11%, respectively. Recoveries of suPAR, sTREM-1, and MIF calibrators were 108%, 88%, and 51%, respectively. In plasma collected from 10 patients with bacterial sepsis confirmed by blood culture, the assay detected significantly increased concentrations of all 8 analytes compared with healthy controls.

Conclusions: A commercially available xMAP panel can be expanded with markers of interest. The combined multiplex assay can measure the 8 analytes with high reproducibility. The xMAP technology is an appealing tool for assaying conventional cytokines in combination with new markers.




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