Development of blood-based biomarker tests for early detection of colorectal neoplasia: Influence of blood collection timing and handling procedures.
Author(s): Lech Pedersen N, Mertz Petersen M, Ladd JJ, Lampe PD, Bresalier RS, Davis GJ, Demuth C, Jensen SØ, Andersen CL, Ferm L, Jarle Christensen I, Nielsen HJ
Publication: Clin Chim Acta, 2020, Vol. , Page
PubMed ID: 32272156 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to investigate the effects of number of centrifugation steps and stage of colonoscopy bowel preparation (pre versus post) on levels of 14 biomarkers and cfDNA recovered from plasma and galectin-3 ligand recovered from serum.
Conclusion of Paper
Levels of the eight biomarkers assayed using the ARCHITECT automated immunoassay were generally strongly to very strongly correlated between pre and post bowel preparation, but correlations in the levels of the five biomarkers measured by the Luminex assay showed more variability and were lower for specimens centrifuged once than twice. Levels of von Willebrand factor (vWF) and CD44 were lower and Interleukin 6 Signal Transducer (IL6ST) higher in specimens centrifuged twice than those centrifuged once (P≤0.0001, P=0.016, and P=0.014, respectively). A non-significant 7% reduction in cfDNA yield was observed when plasma was centrifuged twice rather than once. CyFra21-1, BAG family molecular chaperone regulator 4 (BAG4), and ferritin decreased 29%, 12% and 18%, respectively after bowel preparation, but only the differences in CyFra21-1 and BAG4 were significant (P≤0.0001, and P=0.04, respectively). In contrast cfDNA concentrations increased 28% after bowel preparation (P<0.0001).
Studies
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Study Purpose
The purpose of this paper was to investigate the effects of number of centrifugation steps and stage of colonoscopy bowel preparation (pre versus post) on levels of 14 biomarkers and cfDNA recovered from plasma and galectin-3 ligand recovered from serum. Blood was collected from 125 patients with symptoms of colorectal cancer (5 later diagnosed with colorectal carcinoma and 46 with adenoma) at the pre-procedural visit and again after bowel preparation but before sedation into serum and EDTA vacutainer tubes. Serum tubes were stored at room temperature for 20-30 min while EDTA tubes were immediately centrifuged at 3000 x g for 10 min. The supernatant from the EDTA tubes was pipetted into two storage tubes and one tube was re-centrifuged along with the original serum tube at 3000 x g for 10 min at 21°C. All plasma and serum were placed in new storage tubes and frozen at -80°C with electronic surveillance before distribution to collaborators in the Netherlands and United States. Levels of alpha-fetoprotein (AFP), cancer antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA), high-sensitivity C-reactive protein (hs-CRP), CyFra21-1 (a fragment of cytokeratin 19), ferritin, galectin-3, and tissue inhibitors of matrix metalloproteinases (TIMP-1) were measured in EDTA plasma using an ARCHITECT automated immunoassay at Abbott Center of Excellence at Vrije University Medical Center (Amsterdam, Netherlands). Levels of BAG4, IL6ST, vWF, CD44, and epidermal growth factor receptor (EGFR) were quantified in EDTA plasma using a Luminex assay at Fred Hutchinson Cancer Research Center (Seattle, WA, USA). Galactin-3 ligand was measured in serum of 63 patients using a Luminex platform. Cell-free DNA was extracted from EDTA plasma using the QIAsymphony DSP Virus/Pathogen Midi Kit and quantified using a duplex droplet digital PCR for regions on chromosome 3 and 7.
Summary of Findings:
Levels of the eight biomarkers assayed using the ARCHITECT automated immunoassay were generally strongly to very strongly correlated between pre- and post-bowel preparation, regardless of number of centrifugation steps (0.82-0.99 except CyFra21 -1 which was 0.61-0.66), but correlations in the levels of the five biomarkers measured by the Luminex assay showed more variability (0.16-0.67) and were lower for specimens centrifuged once than twice (0.16-0.28 and 0.29-0.63, respectively). Levels of vWF and CD44 were lower and IL6ST higher in specimens centrifuged twice than those centrifuged once (P≤0.0001, P=0.016, and P=0.014; respectively). A non-significant, 7% reduction in cfDNA yield was observed when plasma was centrifuged twice rather than once. CyFra21-1, BAG4, and ferritin decreased 29%, 12%, and 18%; respectively after bowel preparation, but only the differences in CyFra21-1 and BAG4 were significant (P≤0.0001 and P=0.04, respectively). In contrast, cfDNA concentrations increased 28% after bowel preparation (P<0.0001).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Not specified
- Neoplastic - Benign
Platform:
Analyte Technology Platform DNA Digital PCR Peptide Clinical chemistry/auto analyzer Glycoprotein Clinical chemistry/auto analyzer Protein Immunoassay Glycoprotein Immunoassay Protein Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Time of biospecimen collection Initial visit
After bowel preparation
Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared