Development and validation of a multiplex add-on assay for sepsis biomarkers using xMAP technology.
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-1beta (IL-1beta), 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.
Kofoed
Kristian
K
Clinical Research Unit, Copenhagen University Hospital Hvidovre, Hvidovre, Denmark. kristian.kofoed@hh.hosp.dk
Schneider
Uffe Vest
UV
Scheel
Troels
T
Andersen
Ove
O
Eugen-Olsen
Jesper
J
D015415:Biomarkers / Q000097:blood; D002630:Chemotactic Factors / Q000097:blood*; D003429:Cross Reactions; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000097:blood; D006801:Humans; D007118:Immunoassay; D007375:Interleukin-1 / Q000097:blood; D015850:Interleukin-6 / Q000097:blood; D016209:Interleukin-8 / Q000097:blood; D008264:Macrophages; D008562:Membrane Glycoproteins / Q000097:blood*; D011956:Receptors, Cell Surface / Q000097:blood*; D011971:Receptors, Immunologic / Q000097:blood*; D055293:Receptors, Urokinase Plasminogen Activator; D018805:Sepsis / Q000175:diagnosis*; D000073871:Triggering Receptor Expressed on Myeloid Cells-1; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis
D015415:Biomarkers; D002630:Chemotactic Factors; D007375:Interleukin-1; D015850:Interleukin-6; D016209:Interleukin-8; D008562:Membrane Glycoproteins; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D055293:Receptors, Urokinase Plasminogen Activator; C408842:TREM1 protein, human; D000073871:Triggering Receptor Expressed on Myeloid Cells-1; D014409:Tumor Necrosis Factor-alpha; C053609:macrophage migration enhancement factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor