114 Chapter 6 or CMD.15,16 In both patients and healthy controls, MCE was conducted to quantify MBV and MBF. Measurements were taken at baseline, after two hours of HE-clamp, and during intravenous dobutamine infusion. Myocardial contrast echocardiography MCE was performed during constant intravenous infusion of gas-filled contrast microbubbles (1.5 mL Luminity, Lantheus Medical Imaging, Newbury, United Kingdom) with an infusion rate of 110 to 160 mL/h. The infusion rate was adjusted to achieve optimal opacification of the myocardium without inducing basal attenuation due to contrast in the left ventricle cavity. Ultrasound images were captured in real-time using a low detection mechanical index (MI) of 0.10–0.15 and a frame rate of 27–30 Hz. After achieving a steady-state, microbubbles were locally destroyed using a high mechanical index ultrasound pulse of 1.3. Video intensity was monitored and recorded for ten seconds until the microbubble concentration in the coronary microcirculation returned to steadystate, indicating myocardial replenishment. Recordings were made in duplicate in apical 4-chamber, 2-chamber and 3-chamber views. This procedure was conducted under three conditions: at rest (baseline), during HE-clamp and during dobutamine-induced stress to evaluate their impact on MBV and MBF. Quantification of myocardial perfusion All MCE-images were stored for offline analysis using the Region of Interest (ROI) plug-in from QLAB Cardiac Analysis Software (Philips Healthcare). Two separate, experienced readers (CV and EJ), blinded to the group designations, conducted the echocardiographic analyses. Analyses were performed on end-systolic frames. According to the standard 16-segment myocardial perfusion territory model,17 ROIs of approximately 50mm2 were drawn within the myocardium and in the adjacent left ventricular cavity (Figure 1). Time-intensity curves of each segment were fitted to a mono-exponential function [y = A (1 − e−βt)], where y is acoustic intensity at pulsing interval of t, A is the plateau signal intensity representing microvascular blood volume (MBV, unitless), and β is the rate of signal intensity increase that represents the myocardial blood flow velocity (s − 1).6 Because A may vary within and between myocardial regions due to various factors such as therapeutic interventions, biologic variables (such as age, gender, body mass index (BMI) and cardiovascular risk factors), and technical factors like attenuation6, 7, MBV was corrected for blood contrast density, and possible signal changes due to biological variances or positioning of the heart. This correction was performed following Vogel et al., calculating MBV as the ratio of plateau signal intensity in the myocardium (A) to that in the adjacent left ventricle cavity (ALV). This approach ensures the most reliable estimate of MBV.5 MBV was calculated for the total myocardium by averaging all myocardial ROI’s, and for the individual myocardial perfusion territories as defined by the American Heart Association 16-segment model (Supplemental Figure 1).
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