Appendix II: ECHOCARDIOGRAPHY ANALYSIS

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1 Appendix II: ECHOCARDIOGRAPHY ANALYSIS Two-Dimensional (2D) imaging was performed using the Vivid 7 Advantage cardiovascular ultrasound system (GE Medical Systems, Milwaukee) with a frame rate of 400 frames per second. A complete structural heart evaluation was performed to exclude congenital hearts defects. Serial echocardiography evaluations were performed at three specific time-points (baseline, after the loading dose and at steady state) by the research sonographer. a. Pulmonary Hemodynamics (i)pulmonary artery pressure (PAP) Pulmonary artery pressure was calculated as follows:. 1- A standard apical or subcostal 4-chamber view of the heart was used to obtain a clear image of the tricuspid valve. Color flow Doppler was applied to identify any regurgitant flow. A continuous wave (CW) Doppler gate was then placed in the region of the regurgitant jet and the flow pattern recorded. The peak velocity of tricuspid regurgitation was measured on 3 occasions and the average recorded. We then use a validated method for estimation of right ventricular systolic pressure (P) based on the Bernouilli equation (P = 4V 2 ), where V = velocity. According to this equation right ventricular systolic pressure = RAP + 4 (TR jet Vmax) 2, where RAP = estimated right atrial pressure. In healthy normal babies RAP is close to zero however for sick ventilated babies an allowance of 5-10 mmhg was used. 2- Interventricular septal wall motion was also evaluated and documented as normal, flattened or paradoxical. (ii) Right Ventricular Afterload ratio A short axis view, at the level of the aortic valve, was obtained and using color flow Doppler the pulmonary artery identified. Filters were set for maximum ranges for volumes and velocity settings with a medium degree of motion discrimination. The diameter of the pulmonary artery (PAD) was measured at

2 the hinge-point of the pulmonary valve leaflets. The pulsed Doppler gate was then positioned proximal to the pulmonary valve leaflets and aligned with an angle of insonation below 20 to maximize laminar flow. The pulmonary artery acceleration time (PAAT), right ventricular ejection time (RVET), peak pulmonary artery velocity (PA V max ) of the pulmonary Doppler profile were measured. PAAT is defined by the time of onset of systolic flow to peak pulmonary outflow velocity. RVET is the time from onset of systolic flow to completion of systolic pulmonary flow. The inverse ratio of PAAT: RVET has been shown to be a reliable surrogate of RV afterload and pulmonary vascular resistance. A normal PAAT: RVET is > (iii) Right ventricular output (RVO) was calculated according to the formula: RVO = PA VTI x HR x CSA [Π (PAD/2) 2 ], where PAD = pulmonary artery diameter, CSA = cross sectional area, HR = heart rate, VTI = velocity time integral. The VTI of the pulmonary artery Doppler profile was obtained by tracing the outer edge of the pulmonary outflow Doppler profile. b. Assessment of Myocardial Performance (i) LV systolic function M-Mode measurements: 1. Fractional Shortening (FS) The left ventricular end-diastolic dimension (LVEDD) and end-systolic dimension (LVESD) are measured in the parasternal long axis view at the level of the mitral valve, perpendicular to the interventricular septum, from the posterior edge of the interventricular septum to the anterior edge of the left ventricular free wall. FS = LVEDD LVESD/LVEDD normal value: 26-44%

3 2. Mean Velocity of Circumferential Fibre Shortening (mvcf) was calculated from a parasternal long axis view at the level of aortic leaflets and is a heart rate corrected modification of fractional shorterning. mvcf = LVDD-LVSD/LVDD x LVET where LVET = left ventricular ejection time, normal value: 1.5 +/ circ/s mvcfc= LVDD-LVSD / LVDD x (LVET/ R-R) corrected time interval, normal value: /- 0.07circ/s (ii) LV diastolic performance E/A ratio This represents the pattern and velocity of flow throughthe left atrioventricular (AV) valve. The E wave occurs early in diastole, and is caused by ventricular relaxation. The A wave is the result of AV gradient created by atrial contraction late in diastole. Pulse wave Doppler trace of the LV inflow and outflow was obtained from a long-axis or 5-chamber view of the heart. E/A ratio increases progressively reaching a unity at term. Echocardiographic measurements that influence or are influenced by the primary endpoint. a. Assessment of the ductus arteriosus Left atrial/aortic ratio (LA/Ao): The LA/Ao ratio was measured in the parasternal long axis view at the level of the aortic valve. Three measurements were averaged. Ductus arteriosus diameter and flow: The presence or absence of a PDA was determined by direct ductal imaging, using a high left parasternal short axis view or using a suprasternal approach with the transducer in the left or right supraclavicular region. Color doppler was also used to identify small PDAs with the lowest velocity variance setting, and

4 filters set for maximum ranges for volumes and velocity settings with a medium degree of motion discrimination. Color gain was set to obtain an optimal color flow image within the course of the ductus and to eliminate peripheral color interference. If a PDA was present the minimal diameter (i.e. point of maximum constriction) within the course of the ductus was measured using frame-by-frame analysis. Three of the best quality end-systolic frames with the sharpest image of the ductus were averaged to obtain the ductal diameter. The ductus arteriosus was considered closed when a color image of shunt flow is no longer detected. b. Assessment of cardiac output and input 1-Cardiac Output Aortic velocity time integral (Ao VTI) was measured by tracing the aortic flow from a continuous wave tracing obtained using a pencil probe placed in the suprasternal notch. An average of three readings was obtained. Aortic cross sectional area was estimated using a measurement of the aortic root diameter from a parasternal long axis view. Cardiac output (CO) (mls/kg/min) = Heart Rate x Ao VTI x Ao CSA indexed to body weight. 2- Cardiac Input From a subcostal view the SVC flow was identified by angling the beam anteriorly until the flow into the right atrium from SVC is seen using color Doppler. The pulsed Doppler was placed at junction of SVC and right atrium (RA). The SVC flow pattern is pulsatile with two peaks; the first associated with ventricular systole (S wave) and the other with early ventricular diastole (D wave). The mean velocity of blood flow was calculated from the integral of the Doppler velocity tracings [SVC VTI] and the average from 5 consecutives cardiac cycles. The heart rate was derived from the interval between cycles. The SVC was viewed from a parasternal long axis view with the beam in a true sagittal plane and angled to the right of the ascending aorta. On m-mode imaging the maximal and minimum internal diameters [SVC diameter] were measured at the SVC/RA junction. An average of three readings is obtained.

5 The SVC flow was calculated using the following formula: SVC flow (ml/kg/min) = [velocity flow integral x π x (mean SVC diameter2 /4) x heart rate] / body wt