The effect ofetanercept on lung leukocyte margination and fibrin deposition following

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1 The effect ofetanercept on lung leukocyte margination and fibrin deposition following cardiac surgery Barry Dixon, Roger Smith, Duncan J. Campbell, Antony Tobin, Andrew E. Newcomb, Alexander Rosalion, Kenneth Opeskin, Helen Carter, John D. Santamaria ONLINE DATA SUPPLEMENT

2 Online Data Supplement Methods Study population: Patients undergoing coronary artery bypass grafting with CPB. Exclusion criteria included previous coronary artery bypass grafting (CABG), a planned surgical intervention in addition to CABG, a creatinine >250 mol/l, age <18 or >85, emergency surgery, use of an interleukin-1 (1L-1) antagonist, septic arthritis in last 12 months, the presence of any infection, pregnancy, multiple sclerosis or allergy to etanercept. The study was approved by the St. Vincent's Hospital Human Research Ethics Committee and all patients gave written informed consent before participation. Trial number ACTR Allocation concealment and randomisation: A computer-generated block randomisation sequence was prepared and held by a medical practitioner not involved in patient screening or data collection. Preparation and administration of the study drug:to facilitate intravenous infusion, the etanercept (Pfizer Pty Ltd, Australia) was added to0.9%sodium Chloride (Baxter Healthcare Pty Ltd, Australia) so the final volume was 100 ml. The placebo was 100 ml of 0.9% Sodium Chloride. The active drug and the placebo were identical in appearance. The study drug was given by intravenous infusion over 30 minutes, commencing after insertion of central venous access and the infusion was completed before the surgical incision. Anti-fibrinolytic and anticoagulation management:all patients were administered tranexamic acid IV following anesthetic induction (~5 grams). Prior to CPB patients were administered ~300 U/kg heparin to maintain an activated clotting time above 480 seconds. Protamine (~300 1

3 mg) was administered after discontinuation of CPB. Data collection: The alveolar dead space fraction, the partial pressure of arterial oxygen to the fraction of inspired oxygen (P a O 2 /F i O 2 ) ratio, and hemodynamic variables were measured following anesthetic induction, following sternotomy, at the end of CPB (time = 0) and at 2 and 4 hours post-cpb. At the same time points, and also at 30 minutes following the onset of CPB arterial blood was taken for hemoglobin, neutrophil, platelet, thrombin anti-thrombin (TAT) complex (Siemens, Enzygnost TAT micro), tissue plasminogen activator (Trinity Biotech TriniLIZE tpa Antigen), plasminogen activator inhibitor I antigen (Technozym, PAI-1 Actibind), D- Dimer (Vidas DDimer, BioMerieux) and interleukin-8 (IL-8), IL-1β, IL-6, IL-10, TNF and IL-12p70 levels (BD, CBA Human Inflammatory Cytokines Kit). PAI-1 levels were not available for the 12.5 mg dose of etanercept.troponin I levels were measured at 4 hours after CPB. The troponin assay used changed after enrolment of the 12.5 and 25 mg groups from the Siemens Advia Centaur TnI Ultra to the Abbott Architect Stat Troponin-I.C-reactive protein (CRP), the international normalised ratio (INR) and the activated partial thromboplastin time (APTT) were measured on the first post-operative day. The pre-operative blood samples were collected before commencement of the study drug infusion, while the pre-operative lung and cardiac function tests were performed following anesthetic induction, approximately 15 minutes after the study drug infusion was completed. The ventilator and ventilator settings were standardised for all measurements. The alveolar dead space fraction was measured using the Cosmo Plus Respironics monitor (Novametrix Medical Systems, CT, USA), as previously described.(1) 2

4 Other outcomes included mortality at 90 days, hospital, intensive careand mechanical ventilation duration and post-operative bleeding complications. Septic complications, including all positive microbiological cultures were recorded while in hospital. Patients were interviewed by telephone at day 90 regarding the need for antibiotics for wound discharge or other infections or any history of delayed wound healing. A lung biopsy was an optional part of the study and 55 patients consented to this procedure. The number of lung biopsies obtained in each group were placebo 20, 12.5 mg 10, 25 mg 7, 50 mg 8 and 100 mg 10. Around 15 minutes after protamine administration following disconnection from CPB and around 45 minutes after reperfusion of the lungs, an open wedge biopsy (usually left middle lobe) was performed using a TA re-loadable 4.8 mm stapler (Autosuture, Tyco, Norwalk, CT, USA). The lung tissue was immediately fixed in 10% neutral buffered formalin, followed by embedding in paraffin. Four m sections were cut and immunostained with polyclonal rabbit anti human fibrin antibody (Dako. 1:100,000) and counterstained with hematoxylin. The sections were digitally captured using the Aperio ScanScope image analysis system (Aperio, CA, USA). Histological assessment was blinded and undertaken by one of the authors (B.D.). Assessment of capillary leucocyte margination. For each slide, 20 random non-overlapping fields of 0.25 mm 2 were analyzed. Within each field non-alveolar tissue and vessels larger than 25 m in diameter were excluded. The positive pixel count analysis tool (Aperio)was used to measure the total area of nuclei in each field. This area was divided by an estimate of the average area of a single nucleus (13 um 2 ), to calculate the number of nuclei.cell nuclei comprised leukocytes, endothelial cells and pneumocytes.the positive pixel count analysis 3

5 tool (Aperio) was also used to estimate the area of alveolar tissue (excluding nuclei).the results were expressed as the number of cell nuclei/mm 2 of alveolar tissue. Assessment of capillary fibrin deposition.within each of the 20 fields non-alveolar tissue immunostained capillaryfibrin deposits were counted. Deposits were defined as focal areas of fibrin in vessels less than 25 m in diameter. Capillaryfibrin deposits were expressed as the number of capillaryfibrin deposits/mm 2 of alveolar tissue. Assessment of leukocyte margination and fibrin deposition in larger vessels. The whole slide was examined to identify larger vessels(veins, venules, arterioles and arteries). Venules and veinswere differentiated from pulmonary arteries and arterioles primarily by the appearance of continuous layers of thick smooth muscle (as opposed to discontinuous thin bundles in veins and venules) and the close proximity of arteries or arterioles to bronchi and bronchioles.(2)leukocyte margination was defined as 2 or more leukocytes either in direct contact with the endothelium or attached to the endothelium by fibrin. Focal deposits of fibrin in these vessels were identified by the immunostaining. The results were expressed as the percentage of venules or arterioles with margination and the percentage of venules or arterioles with fibrin deposition. Statistical analysis: On the basis of data from a previous publication, the study was powered to demonstrate a 30% difference in the alveolar dead space fraction, with 80% power at a significance level of 0.05.(1)One way analysis of variance (ANOVA) was used to compare baseline, operative and post-operative group differences. Comparison of the effect of etanercept with placebo was analyzed by two-way repeated measures ANOVAfor all time points. Comparison of the effect of etanercept with placebo for a single time point was 4

6 analyzed using Student s t test, Welch ANOVA or Wilcoxon rank sum test, where appropriate. Categorical variables were compared using χ 2 tests or Fisher s exact tests, where appropriate.as the 12.5 mg, 25 mg and 50 mg doses were not expected to have significant biological effectsonly the 100mg dose was analysedfor efficacy.continuous variables are reported as mean ± SD or median (interquartile range). All reported P values are two-sided. A P value of <0.05 was considered to indicatestatistical significance. Analyses were conducted with JMP software (SAS, Cary, NC). Results: The placebo and etanercept groups were similar with respect to baseline operative and post-operative characteristics. The alveolar dead space fraction was higher at baseline and a history of diabetes was more common for the 100 mg etanercept group. 5

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8 Panel A. Histological assessment of the number of cell nuclei / mm 2 of alveolar tissue. Compared with placebo levels were reduced with etanercept 100 mg (Student s t-test). Panel B. Histological assessment of the percentage of pulmonary venules with leukocyte margination. Compared with placebo levels were reduced with etanercept 100 mg (Student s t-test). Panel C. Histological assessment of the percentage of pulmonary arterioles with leukocyte margination.etanercept did not influencelevels. Cell nuclei comprised leukocytes, endothelial cells and pneumocytes. The lung biopsy was taken about 15 minutes after the end of cardiopulmonary bypass. The number of lung biopsies obtained per group: Placebo 20, 12.5 mg 10, 25 mg 7, 50 mg 8 and 100 mg 10. A pulmonary arteriole could be not identified in every biopsy. The number of biopsies in which an arteriole was identified: Placebo 17, 12.5 mg 10, 25 mg 7, 50 mg 7 and 100 mg 9. The bar represents the mean value for each group. 7

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10 Panel A.Histological assessment of the number of capillary deposits of fibrin / mm 2 of alveolar tissue. Compared with placebo etanercept 100 mg did not influencelevels.panel B. Histological assessment of the percentage of pulmonary venules with fibrin deposition. Compared with placebo etanercept 100 mg did not influencelevels.panel C. Histological assessment of the percentage of pulmonary arterioles with fibrin deposition. Compared with placebo levels were increased with etanercept 100 mg. The lung biopsy was taken about 15 minutes after the end of cardiopulmonary bypass. The number of lung biopsies obtained per group: Placebo 20, 12.5 mg 10, 25 mg 7, 50 mg 8 and 100 mg 10. A pulmonary arteriole could be not identified in every biopsy. The number of biopsies in which an arteriole was identified: Placebo 17, 12.5 mg 10, 25 mg 7, 50 mg 7 and 100 mg 9. The bar represents the mean value for each group. 9

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12 Interleukin (IL) levels. Over the entire study period there were no differences between etanercept groups and placebo when analyzed by two way repeated measures ANOVA. Post hoc analysis found IL-1, 6, 8 and 12 levels were lower at 2 hours for 100 mg etanercept vs placebo (*P < 0.05). Comparisons by t-test or Mann Whitney where appropriate. Stern (sternotomy). CPB (cardiopulmonary bypass). Mean and standard error values are shown. Bibliography 1. Dixon B, Campbell DJ, Santamaria JD. Elevated pulmonary dead space and coagulation abnormalities suggest lung microvascular thrombosis in patients undergoing cardiac surgery. Intensive Care Medicine 2008;34: Wang PM, Kachel DL, Cesta MF, Martin WJ, 2nd. Direct leukocyte migration across pulmonary arterioles and venules into the perivascular interstitium of murine lungs during bleomycin injury and repair. Am J Pathol 2011;178: