Mx plate reader

Blood was collected for study purposes on the morning of the second postoperative day; this was piggybacked onto clinical blood draws when possible. During phlebotomy, mechanical disruption was minimized to prevent platelet activation or hemolysis. Blood was stored on ice in heparinized tubes until processing. During processing, low speed centrifugation (1500 g for 15 minutes at 4°C) was used to separate plasma from cellular material, and plasma was stored at −80°C until analyzed. CRP was measured using a high-sensitivity enzyme linked immunosorbent assay (ELISA) kit from R&D Biosystems, with all standards and samples run in duplicate. The coefficient of variation of duplicate measures were generally <5%. If any CV was >10%, that plasma sample was repeated. An internal calibrator sample was present on every 96-well ELISA plate. The cross-plate variation was consistently below the range of 5–10% based on the internal calibrator value. ELISA plates were read using a BioTek MX plate reader at Optical Density (OD) = 450. A 4-parameter logistic curve was used for final calculations [34 ].

CRP on POD2 was measured in the entire SAGES sample using a high-sensitivity enzyme linked immunosorbent assay (ELISA) kit from R&D Systems, with all standards and samples run in duplicate (previously described [38 (link),42 ]), and coefficients of variation confirmed at ≤5%. ELISA plates were read using a BioTek MX plate reader at Optical Density 450. Since only community-based high-risk cutpoints for CRP have been identified (e.g., [43 ]) and are not relevant for patients 2 days post-surgery, a cutpoint for ‘high’ CRP on POD2 (i.e., our definition of a heightened stress response) was defined based on the CRP levels observed in the highest quartile of our sample (Q1 ≤116.31 mg/L, Q2 116.32-158.85 mg/L, Q3 158.86-245.83 mg/L, Q4, ≥234.12 mg/L). Additionally, we considered the possible dose-response effect of higher CRP levels by examining each sample based quartile of CRP (Q2-Q4) relative to Q1.