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Behm CA, et al. (2007/744037-R)
ONLINE SUPPLEMENTAL DATA
Validation of Targeted Imaging Data
The signal intensity from retained (Ir) microbubbles 10 min after targeted agent injection is determined by the product of the number of microbubbles entering into the tissue of interest and their retention fraction (f). The number of microbubbles entering into tissue is determined by the relative blood flow. Hence, in situations where blood flow (and microbubble influx) is not constant, f is a better indicator of the degree of targeted ligand expression than Ir. In this study, Ir was normalized to relative blood flow in the region-of-interest.as an index of f. True validation of this method was not possible because of lack of methods capable of quantifying target molecule expression in locations available to targeted microbubbles that are confined to the vascular compartment. Accordingly, we compared flow-normalized data to f values calculated by mathematical modeling of time-intensity curves to deconvolve the functions describing signal from two populations: (1) those that freely transit through the tissue, and (2) microbubbles that are retained during transit.32 In other words, when microbubbles behave as free-flowing tracers, their signal within the muscle after a bolus injection can be described by a -variate function:
/ (1)where A is a scaling factor, t is time, and /2 is the mean transit rate. If all microbubbles entering into the muscle were to adhere and remain stable, then their concentration can be described by the integral of the equation above:
/ (2)If only a fraction ( f ) of the microbubbles persist and the remaining (1- f ) pass through unencumbered, then the concentration of microbubbles within the myocardium can be described by the function :
/ (3)which can be simplified to
Data for this analysis were obtained from 6 separate wild-type animals using VCAM-1-targeted microbubbles 3-10 days after iliac occlusion. Low-power CEU at an MI of 0.18 was performed to record microbubble signal without destruction during continuous imaging at 20 Hz for the first 10 seconds after microbubble injection, then images were obtained every 10 s for 10 min. An example of a time-intensity curve obtained after injection of VCAM-1-targeted microbubbbles is illustrated in Supplemental Figure I where persistent enhancement occurs late after injection from microbubble persistence in tissue. For each injection, the f values were compared to flow-normalized values of signal intensity obtained with high MI imaging as described that was performed after the 10 min low-MI image acquisition period. Illustrated by supplemental Figure II, a linear relation was found between microbubble retention fraction obtained during low-MI imaging, and the flow normalized signal enhancement obtained with high-power imaging.