Clinical Trial to Evaluate the Use of an Adsorption Membrane (oXiris®) During Cardiopulmonary Bypass Surgery

The oXiris® set connection to CPB

• The PrismafleX eXeed™ II (Baxter) system control unit will be used to deliver CRRT and thus connected to the CPB circuit through the oXiris® set lines.
• Priming. For oXiris® set priming use 2 L of normal saline according to the software instructions.
• Connection. Using a three-way stopcock, oXiris® set must be connected to the arterial line just after the oxygenator to derive blood into the oXiris® set and then returned into the venous reservoir through the return line. No additional vascular access will be required to deliver treatments.
• Modality. The Slow Continuous Ultrafiltration modality (SCUF modality) must be selected with no pre-set ultrafiltration rate (ultrafiltration rate: 0 ml/hr). A blood flow between 200-300 mL/min was set in the PrismafleX eXeed™ II (blood was derived through the arterial positive pressure line just after the oxygenator) to be delivered into the oXiris® set and then returned into the venous reservoir through the return line.
• All treatments should be delivered after reaching full-flow CPB.
• CRRT flow, pressure and alarms will be controlled in the PrismafleX eXeed™ II screen, independently from CPB circuit.
• If CPB is stopped for any reason, then PrismafleX eXeed™ II should be stopped temporarily to avoid alarm warning and/or any harm to the patient.
• No additional anticoagulation method will be needed besides the systemic anticoagulation required for the cardiopulmonary bypass pumping.
• The return line clamp (blue clamp) should be adjusted to reach a slight positive return pressure in order to avoid low-pressure alarm warning.

PrismafleX eXeed™ II weaning instructions. 5-10 min before the end of CPB, start the blood-return procedure into the venous reservoir of the CPB using 1 L of normal saline. Connect the solution into the three-way stopcock. Subsequently, set PrismafleX eXeed™ II flow in 80-100 ml/min. Select STOP in the PrismafleX eXeed™ II screen. Adjust the three-way stopcock to allow saline to flow into the oXiris® set arterial line an restart PrismafleX eXeed™ II to return blood into the venous reservoir.

• Register all manoeuvres, changes and adjustments in software settings and any technical problem during the procedure.

The oXiris® membrane will be only employed for adsorption (neither convection nor diffusion will be performed) during all CPB time. Using CPB flow, patient will not require venous catheter access neither changes in CPB normal settings.

The oXiris® membrane set is composed of a 1.5 m2 copolymer of acrylonitrile and sodium methylsulfonate (AN 69) with polyethylenimine treated surface and adhered heparin during set´s fabrication [oXiris® (Baxter Gambro)].

All blood samples collected during study will be obtained from CPB circuit or central venous catheters (standard cardiac surgery protocol).

During all CPB time besides the ordinary cardiac surgery assistance team, a supplementary CRRT expert team will be present in those patients randomized to CPB-oXiris®.

Blood samples and all variables will be equally collected and registered in the control group (CPB-Standard) with no modifications from CPB standard practice.

Adverse events will be reported (in less than 24 hours if severe) to the sponsor centre to be properly evaluated. If the severe adverse event (SAE) is finally evaluated by the study board as related to the intervention arm, urgent notification to health authorities must proceed and study should be interrupted until further decision.

Investigators will warrant a correct protocol application. Study data will be reviewed by an external monitoring committee from the clinical assay research central unit (UCICEC - IDIBELL). Monitors will contrast registered data from the collection data form (CDF) with data from patients' medical record. All patients' medical records will be indefinitely saved in electronic format to be reviewed if necessary.

Efficacy Assessments:

1. Registered variables and follow-up. Renal function related parameters Plasma creatinine and urine output will be registered at baseline, prior to CPB, at ICU admission, 6 hours ,12 hours, 24h postCBP and every 24h during the first week after cardiac surgery in order to evaluate CS-AKI based on KDIGO AKI criteria (KDIGO criteria Kidney inter., Suppl. 2012; 2: 1-138.) Renal function will be checked at hospital discharge. Hemolysis parameters Cell-free serum hemoglobin levels will be determined in plasma. These determinations will be measured at times T1 (at the end of CPB), and T3 (24 hours after ICU admission). Inflammatory response parameters Interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-18 (IL-18), complement 5a (C5a), high mobility binding protein 1 (HMBP-1), RANTES, GRO-alpha, leukocytes count, lactate, and D-dimer levels will be determined in plasma.These determinations will be measured baseline (0 hours), and at times T1 (at the end of CPB), T2 (ICU admission) and T3 (24 hours after ICU admission). Ordinary laboratory parameters Plasmatic urea, potassium, albumin, magnesium, phosphate, glucose, sodium, chloride, AST, ALT, blood cell count and coagulation times will be determined following the ordinary cardiac surgery protocol (every 8 hours during the first 24h). Clinical and respiratory parameters Hemodynamic and respiratory parameters will be registered every hour. Arterial and venous blood gases with arterial lactate will be determined every 8 hours during the first 24 hours. Temperature, heart rate, arterial blood pressure (systolic, diastolic, and mean), central venous pressure, vasopressor and/or inotrope dose will be registered during surgery and at ICU according to standard protocol. Demographic and clinical record data Age, gender, weight, height, relevant clinical records, previous or chronic medication, type of cardiac surgery, surgery times, time on CPB, surgical complications, blood transfusions during and after surgery, prognostic scores (SOFA, APACHE II and SAPS II), ICU stay, hospital stay. Clinical outcome (survival at discharge, 28 and 90 days) and renal outcome (renal function, and RRT requirements).
2. Techniques Cell-free serum hemoglobin analysis Cell-free serum hemoglobin concentration will be measured using the Cayman's Hemoglobin Colorimetric Assay Kit (Cayman Chemical, Ann Arbor, Michigan). Blood samples will be obtained at T1 and T3 into VACUETTE® Serum tubes (Greiner Bio One GmbH, Kremsmünster, Austria). After clotting at room temperature, serum will be separated by centrifugation at 2000 g for 15 minutes and stored at -80°C in 1 mL aliquots until use. Cell-free serum hemoglobin concentrations will be determined using manufacturer's protocols with provided calibration standards. All samples will be processed in duplicate. Changes in absorbance at 560 nm will be measured using a 96-well plate reader. Cytokines analysis IL-1, IL-6, IL-8, IL-10, IL-18, TNFα, MCP-1, HMBP-1, RANTES, GROα quantification will be performed with multiplex new technology. MILLIPLEX (Millipore) magnetic balls system will be used.
3. Study Limitations SIRS harmful effects which are initiated during CPB may persist after surgery and this is an important limitation as our study only evaluates the use of increased adsorption membranes during CPB. However, investigators hypothesize that the important effect on cytokine levels during CPB will be observed. Furthermore, patients requiring non emergent cardiac surgery represent a non absolutely homogeneous cohort and this could introduce potential bias in main and secondary objectives. To avoid this potential bias, temporary sequential randomization has been performed and advanced chronic renal failure patients have been excluded.
4. Statistical analysis

Missing data will try to be avoided by an exhaustive patient´s follow up by study investigators. Data registry has been created to include all variables with written individual data collection forms (DCF). Data will be bedside registered by the study members but final software database registration will be done by the statistics outside investigator who has no contact with patients situation. Cytokines levels will be introduced in DCF when measured.

Statistical analysis will be done by the statistics investigator who wont have any role in patient´s selection, randomization, or follow up. SPSS v. 20.0 for statistical analysis will be used. Variable distribution will be studied and logarithmic transformation will be used on those variables that don't present normal distribution, presumably cytokine levels. Univariate analysis comparing clinical, demographic, biochemical, metabolic, hemodynamic and respiratory baseline variables between both arms (CPB-OXIRIS® and CPB-Standard), will be done with two-tailed t test for continuous variables and chi-square test for categorical variables. Variables determined several times (T0, T1, T2, T3) will be analysed using a one-way repeated measures ANOVA test in order to demonstrate differences between both arms. Multivariate analysis will be completed to control those clinically relevant confounding variables as well as to discover baseline differences. Arm intervention (CPB-OXIRIS®, CPB-Standard) will be considered as the main independent variable adding other control independent variables. Intervention safety will be measured by the presence of any adverse effects not related to the standard technique. This effects will be recorded during CPB, after CPB, and during the first 24 hours postsurgery.

As the study is measuring cytokine levels in four different moments (T0, T1, T2, T3), in order to maximize statistical power and reduce control variables number, the area under the curve (AUC) we´ll be determined for every cytokine during the first 24 hours. Due to this statistical maneuver investigators will obtain a continuous variable that represents each cytokine level during the biochemical study period (24 hours). To evaluate if the arm intervention improvement in terms of efficacy and safety could be related to cytokine levels during the first 24 hours, a mediation complementary analysis will be done considering cytokine (represented by AUC) as a mediator between the independent variable (intervention arm) and the effect we study.