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Purpose: Sensitivity of contrast-enhanced ultrasound (CEUS) to microvascular flow modifications can be limited by intra-injection variability (injected dose, rate, volume).Procedures: To evaluate the effect of injection variability on microvascular flow evaluation, CEUS was compared between controlled and manual injections where enhancement was assessed in vitro within a flow phantom, in normal murine kidney (N = 12) and in murine ectopic tumors (N = 10).Results: For both in vitro and in vivo measurements in the renal cortex, controlled injections significantly improved reproducibility of functional parameter estimation. Their coefficient of variation (CV) in the renal cortex ranged from 4 to 19 % for controlled injection vs. 5 to 43 % for manual injections. For measurements in tumors, controlled injection only decreased the CV significantly for the mean transit time. In tumors, multiple injections of contrast agent with a 15-min delay between each were shown to strongly modify contrast uptake by facilitating penetration of microbubbles.Conclusion: Improved reproducibility of CEUS assessments in murine models should provide more robust quantification of flow parameters and more sensitive evaluation of tumor modifications in therapeutic models. [ABSTRACT FROM AUTHOR]