A novel multiplex-protein array for serum diagnostics of colorectal Cancer: Impact of pre-analytical storage conditions

Introduction: 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. Conclusion: 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.