A novel multiplex-protein array for serum diagnostics of colorectal cancer: impact of pre-analytical storage conditions.
BACKGROUND
Biomarker discovery studies seldom report on pre-analytical effects. We used a novel multiplex protein biochip for colorectal cancer screening to investigate effects of different storage temperatures and repeated freeze-thaw cycles.


METHODS
This biochip, composed of CEA, IL-8, VEGF, M-CSF, S100A11, C3adesArg, CD26, and CRP, was applied to twenty highly standardized preserved serum samples.


RESULTS
Aliquot comparison of long-term storage at -80°C (n=20) versus -170°C (n=20) did not show significant differences for any of the eight markers. In contrast, three freeze-thaw cycles (3 × 20 aliquots) detected changes in the serum level for all markers (p<0.05) but S100A11 and CD26: levels of CEA, IL-8, C3adesArg, and CRP increased, while VEGF and M-CSF levels decreased. However, applying diagnostic thresholds for CEA, IL-8, and CRP revealed that freeze-thaw cycles did not affect diagnostic performance. In contrast, analysis of samples stored at -80°C compared to -170°C failed to detect one out of three detectable malignancies.


CONCLUSIONS
We conclude that three freeze-thaw cycles modulated serum marker levels significantly, but do not compromise biochip diagnostic performance. For our marker panel, serum preservation at -80°C seems comparable to -170°C; however, storage at -80°C could lead to misdiagnosis. Our findings emphasize the need for standardized sample collection, processing, storage, and reporting.
1 Section for Translational Surgical Oncology and Biobanking, Department of Surgery, University of Lübeck , Lübeck, Germany .
Stefanie
S
Bünger
Katja
K
Klempt-Giessing
Vicki
V
Toner
Maria
M
Kelly
Stephen P
SP
FitzGerald
Hermann
H
Brenner
Ferdinand
F
von Eggeling
Jens K
JK
Habermann
D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000097:blood*; D015179:Colorectal Neoplasms / Q000097:blood* / Q000175:diagnosis; D005260:Female; D005615:Freezing; D057166:High-Throughput Screening Assays; D006801:Humans; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D040081:Protein Array Analysis / Q000379:methods*; D013048:Specimen Handling / Q000379:methods* / Q000592:standards; D013997:Time Factors
D015415:Biomarkers