K3edta vacuette tubes

Venous blood was collected into two 9mL K3EDTA Vacuette tubes (Greiner Bio-One, Frickenhausen, Germany) from subjects either prior to them being sedated for colonoscopy but after consumption of bowel preparation solution, or prior to preparation for surgery but following colonoscopic diagnosis. A second sample was obtained from 26 CRC cases one month or more after surgery. Blood tubes were kept at 4 °C until commencing plasma processing. Plasma was prepared within 4 hours of blood collection by centrifugation at 1,500 g for 10 minutes at 4 °C (no braking), followed by retrieval of the plasma fraction and a repeat centrifugation. The resulting plasma was stored at -80 °C. Frozen plasma samples were shipped on dry ice to CGT and stored at -80 °C until testing.
No study-wide control of colonoscopy or pathology procedures or quality was undertaken as the study aimed to assess marker performance relative to outcomes determined in usual clinical practice. All procedures were performed by hospital-accredited specialists and so met site-specific standards for sedation, monitoring, imaging, and equipment. Histopathology and staging of neoplasia used routine procedures at each clinical site. Cases were excluded if any data crucial to clinical diagnosis was not obtainable, e.g. if colonoscopy was incomplete.

Venous blood was collected into two 9 mL K3EDTA Vacuette tubes (Greiner Bio‐One, Frickenhausen, Germany) at clinic visits. Blood collection tubes were kept at 4°C prior to plasma processing (not more than 4 h from blood collection). Plasma was prepared by centrifugation at 1500g for 10 min at 4°C (deceleration at lowest setting), followed by retrieval of the plasma fraction and a repeat centrifugation. The resulting plasma was stored at −80°C. Frozen plasma samples were shipped on dry ice to Clinical Genomics Technologies (North Ryde, NSW, Australia) and stored at −80°C until testing. Cases were excluded from analysis if these conditions were not met. No study‐wide control of radiological imaging or pathology procedures or quality was undertaken as the study aimed to assess marker performance relative to outcomes determined in usual clinical practice. All procedures were performed by hospital‐accredited specialists and so met site‐specific standards for venipuncture, monitoring, imaging, and equipment.

The plasma component from whole blood collected in K₃EDTA Vacuette tubes (Greiner Bio-One, Frickenhausen, Germany) was isolated by centrifugation at 1,500g for 10 minutes at 4°C (brakes disabled on centrifuge). The top-layer plasma fraction was retrieved and centrifugation was repeated. The resulting ‘two-spin’ plasma was stored at -80°C until further use. The total time from blood draw to freezing of plasma was no more than four hours at all recruitment sites.

Whole blood was collected from our healthy donors in K3 EDTA Vacuette tubes (Greiner #455036) via venipuncture and processed within one hour of collection based on density centrifugation at 400 g for 30 min at room temperature (RT) using Ficoll–Paque Plus solution (Cytiva #17-1440-03). Undiluted plasma was collected and stored at −80 °C. After lysing red blood cells, PBMCs were frozen in freezing media (Neat fetal bovine serum with 10% Dimethyl sulfoxide), stored at −80 °C, and subsequently stored in liquid nitrogen before analyses.