Bao A. V. Nguyen, MRCS, Francesca Fiorentino, PhD, Barnaby C

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Mini Bypass and Proinflammatory Leukocyte Activation: A Randomized Controlled Trial Bao A.V. Nguyen, MRCS, Francesca Fiorentino, PhD, Barnaby C. Reeves, DPhil, Kamran Baig, FRCS, Thanos Athanasiou, FRCS, Jon R. Anderson, FRCS, Dorian O. Haskard, FMedSci, Gianni D. Angelini, FRCS, Paul C. Evans, PhD The Annals of Thoracic Surgery Volume 101, Issue 4, Pages 1454-1463 (April 2016) DOI: 10.1016/j.athoracsur.2015.09.029 Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Summary of blood and blister fluid sampling times. Blood and cantharidin-induced skin blister fluid were sampled in patients exposed to conventional cardiopulmonary bypass (cCPB) and those who underwent miniaturized cardiopulmonary bypass (mCPB). A timeline is presented to summarize sampling times. The Annals of Thoracic Surgery 2016 101, 1454-1463DOI: (10.1016/j.athoracsur.2015.09.029) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Overview of participant selection. Trial participants were recruited and assessed for exclusion criteria. Computer randomization allocated participants to either miniaturized cardiopulmonary bypass (mCPB) (investigative group) or conventional cardiopulmonary bypass (cCPB) (control group). (CABG = coronary artery bypass grafting; LV = left ventricular.) The Annals of Thoracic Surgery 2016 101, 1454-1463DOI: (10.1016/j.athoracsur.2015.09.029) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Effect of cardiopulmonary bypass (CPB) on reactive oxygen species (ROS) induction and phosphorylation of p38-mitogen-activated protein kinase (MAPK) and phosphorylation of nuclear factor (NF)-κB p65. Coronary artery bypass grafting (CABG) was performed with conventional CPB (cCPB) or miniaturized CPB (mCPB). Peripheral blood samples were collected before the operation, after induction (PostInd), immediately before CPB (0 minutes), and at varying times after CPB initiation. (A) Samples were stained using either reactive oxygen species (ROS)-sensitive dye (3′-[p-aminophenyl] fluorescein). (B) Antibodies that recognize phosphorylated p38-MAPK were seen. (C) Antibodies that recognize Ser529 phosphorylated NF-κB were seen. Fluorescence of leukocyte cell subpopulations was quantified by flow cytometry after subtracting values from isotype-control antibodies. Data were pooled and log-transformed. Mean fluorescence intensity (MFI) values ± standard deviation are shown with arbitrary units (AU). (PE = phycoerythrin.) The Annals of Thoracic Surgery 2016 101, 1454-1463DOI: (10.1016/j.athoracsur.2015.09.029) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Modeled data on log-transformed measurements of reactive oxygen species (ROS), p38- mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB p65. CABG was performed with conventional cardiopulmonary bypass (cCPB) or miniaturized CBP (mCPB). Peripheral blood samples were collected before the operation, after induction, immediately before CPB (0 minutes), and at varying times after CPB initiation. (A) ROS induction, (B) phosphorylation of p38-MAPK, and (C) phosphorylation at Ser529 of nuclear factor (NF)-κB p65 subunits were measured. Data were log-transformed, and the effects of CPB were estimated by fitting mixed regression models. Mean fluorescence intensity (MFI) values ± standard deviation are shown with arbitrary units (AU). (FITC = fluorescein isothiocyanate; Ln = natural logarithms; PE = phycoerythrin.) The Annals of Thoracic Surgery 2016 101, 1454-1463DOI: (10.1016/j.athoracsur.2015.09.029) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Extravasation of leukocytes into cantharidin skin blisters. Coronary artery bypass grafting (CABG) was performed with conventional cardiopulmonary bypass (cCPB) or miniaturized CPB (mCPB). Blister fluid samples were collected before operation (Baseline) and at 5 hours after CPB initiation (Postoperative). Cells were collected, stained using Diff-Quick and visualized by microscopy. Representative images are shown at ×40 optical magnification. The Annals of Thoracic Surgery 2016 101, 1454-1463DOI: (10.1016/j.athoracsur.2015.09.029) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions

Fig 6 Summary of perioperative changes in C-reactive protein (CRP), white cell count, and creatinine and hemoglobin clinical measurements. Coronary artery bypass grafting (CABG) was performed with conventional cardiopulmonary bypass (cCPB) or miniaturized CPB (mCPB). Blood was sampled before operation (Baseline) and on the first and fourth days after operation. Log-transformed data for (A) CRP, (B) white cell counts, (C) serum creatinine, and (D) hemoglobin are presented ± standard deviation. (Ln = natural logarithms.) The Annals of Thoracic Surgery 2016 101, 1454-1463DOI: (10.1016/j.athoracsur.2015.09.029) Copyright © 2016 The Society of Thoracic Surgeons Terms and Conditions