An evaluation of multiplex bead-based analysis of cytokines and soluble proteins in archived lithium heparin plasma, EDTA plasma and serum samples.
OBJECTIVE
To assess the usability of archived plasma and serum by multiplex (Luminex) analysis of circulating proteins (analytes) by evaluating the day to day variation, the effect of several freeze-thaw cycles, and the influence of the media and choice of anticoagulant.


METHODS
Nineteen analytes in plasma and serum from 86 head and neck cancer patients and 33 controls were evaluated: EGFR, leptin, OPN, VEGFR-1, VEGFR-2, IL-2, IL-13, PDGF-bb, TNF, PAI-1, SDF-1a, IL-4, IL-6, IL-8, eotaxin, G-CSF, VEGF, GRO-a, and HGF.


RESULTS
The correlation between measurements of the same samples analyzed on different dates was reasonable. However, samples run on different dates could exhibit different absolute values. The 75th percentile of the fold differences for samples run on different dates was 2.2. No significant difference was found between one and four freeze-thaw cycles (except for HGF), and the correlation was high. We found significant differences in mean concentrations of the majority of analytes in different media and with different anticoagulants. Only the following analytes did not show difference in mean concentrations: EDTA plasma vs. serum: leptin and VEGFR-2, LH plasma vs. serum: IL-2, IL-13, and VEGF, LH plasma levels vs. EDTA plasma: IL-2 and IL-4.


CONCLUSIONS
Stored serum, LH plasma, and EDTA plasma from clinical trials can be used for analysis of circulating cytokines and proteins. Variations in measurements occur, but are within reasonable ranges. The optimal type of media depends on the analytes, as different analytes have low number of measurements below the lower limit of quantification and higher dynamic ranges in different media.
a Department of Experimental Clinical Oncology , Aarhus University Hospital , Aarhus , Denmark.
Line
L
Brøndum
a Department of Experimental Clinical Oncology , Aarhus University Hospital , Aarhus , Denmark.
Brita Singers
BS
Sørensen
b Department of Oncology , Odense University Hospital , Odense , Denmark.
Jesper Grau
JG
Eriksen
c Department of Oncology , Aarhus University Hospital , Aarhus , Denmark.
Lise Saksø
LS
Mortensen
d Department of Public Health, Section for Sport Science , Aarhus University , Aarhus , Denmark.
Simon
S
Lønbro
a Department of Experimental Clinical Oncology , Aarhus University Hospital , Aarhus , Denmark.
Jens
J
Overgaard
a Department of Experimental Clinical Oncology , Aarhus University Hospital , Aarhus , Denmark.
Jan
J
Alsner
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000925:Anticoagulants / Q000737:chemistry*; D016022:Case-Control Studies; D015925:Cryopreservation; D016207:Cytokines / Q000097:blood; D004492:Edetic Acid / Q000737:chemistry*; D066246:ErbB Receptors / Q000097:blood; D005260:Female; D006258:Head and Neck Neoplasms / Q000097:blood*; D006493:Heparin / Q000737:chemistry*; D017228:Hepatocyte Growth Factor / Q000097:blood; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008297:Male; D008875:Middle Aged; D015588:Observer Variation; D011786:Quality Control; D015203:Reproducibility of Results; D013048:Specimen Handling / Q000379:methods*; D042461:Vascular Endothelial Growth Factor A / Q000097:blood; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000097:blood; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000097:blood
D000925:Anticoagulants; D016207:Cytokines; C066855:HGF protein, human; D042461:Vascular Endothelial Growth Factor A; D017228:Hepatocyte Growth Factor; D006493:Heparin; D004492:Edetic Acid; C512478:EGFR protein, human; D066246:ErbB Receptors; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2
Blood; cytokines; multiplex analysis; quality control; specimen handling