Contrast-enhanced ultrasound (CEU) limb perfusion imaging is a promising approach for evaluating peripheral artery disease (PAD). However, low signal enhancement in skeletal muscle has necessitated high-power intermittent imaging algorithms, which are not clinically feasible. We hypothesized that CEU using a combination of intermediate power and a contrast agent resistant to inertial cavitation would allow real-time limb stress perfusion imaging.
In normal volunteers, CEU of the calf skeletal muscle was performed on separate days with Sonazoid, Optison, or Definity. Progressive reduction in the ultrasound pulsing interval was used to assess the balance between signal enhancement and agent destruction at escalating mechanical indices (MI, 0.1–0.4). Real-time perfusion imaging at MI 0.1–0.4 using postdestructive replenishment kinetics was performed at rest and during 25 W plantar flexion contractile exercise.
For Optison, limb perfusion imaging was unreliable at rest due to very low signal enhancement generated at all MIs and was possible during exercise-induced hyperemia only at MI 0.1 due to agent destruction at higher MIs. For Definity, signal intensity progressively increased with MI but was offset by microbubble destruction, which resulted in modest signal enhancement during CEU perfusion imaging and distortion of replenishment curves at MI ≥ 0.2. For Sonazoid, there strong signal enhancement at MI ≥ 0.2, with little destruction detected only at MI 0.4. Accordingly, high signal intensity and nondistorted perfusion imaging was possible at MI 0.2–0.3 and detected an 8.0- ± 5.7-fold flow reserve.
Rest-stress limb perfusion imaging in humans with real-time CEU, which requires only seconds to perform, is possible using microbubbles with viscoelastic properties that produce strong nonlinear signal generation without destruction at intermediate acoustic pressures.
There is increasing interest in applying stress-rest perfusion imaging protocols similar to those used in the heart to evaluate patients with peripheral artery disease (PAD). Limb blood flow assessment requires quantitative methods since regional heterogeneity cannot be used to diagnose PAD. Quantitative contrast-enhanced ultrasound (CEU) perfusion imaging has been demonstrated to yield information on PAD severity beyond that provided by ankle-brachial index. The main challenge for CEU is the relatively low signal-to-noise produced during conventional low-power real-time CEU imaging, which is attributable to low skeletal muscle blood flow (0.05–0.3 mL/min/g). High acoustic power CEU imaging results in greater signal, however, it also results in microbubble destruction through inertial cavitation, thereby requiring more time-consuming and technically difficult image acquisition protocols.
During low-power real-time CEU, the signal produced by microbubble stable cavitation, defined as nonlinear oscillation without destruction, increases with the acoustic power. However, there is an upper limit where stable cavitation converts to inertial cavitation. This limit is dependent largely on composition-related viscoelastic properties of microbubbles. It has been shown in small and large animal models that Sonazoid, which is an acoustically durable lipid microbubble agent that has a more than twofold higher bulk modulus than other lipid-shelled microbubbles, produces almost five-fold higher signal during real-time CEU limb perfusion imaging when used in conjunction with intermediate-power CEU. This advantage was due to this agent’s ability to undergo nonlinear oscillation without destruction at mechanical indices (MIs) of 0.3–0.4. In this study, we hypothesized that Sonazoid when used for limb perfusion imaging in humans would convey a benefit of higher signal-to-noise compared with more fragile microbubble agents that are destroyed by inertial cavitation during intermediate-power CEU.
The study was approved by the Investigational Review Board at Oregon Health and Sciences University. The design was a prospective open-label nonrandomized study performed in 14 healthy normal volunteers. Subjects ≥19 years of age without any major medical problems were recruited. Subjects were excluded for history or symptoms of coronary or PAD, heart failure, muscle disease, vasculitis, known or suspected right-to-left shunt, pregnancy, or abnormalities in peripheral pulse examination. Subjects were also excluded for allergy to ultrasound contrast agents, eggs, or blood products.
CEU limb perfusion imaging protocols were performed on two separate days separated by <14 days using Sonazoid (GE Healthcare, Amersham, United Kingdom) on one day and either Definity (Lantheus Medical Imaging, N. Billerica, MA) or Optison (GE Healthcare; n = 7 for each) on the other day. Subject vital signs and oxygen saturation were continuously measured during the protocol. Twelve subjects returned within 6 weeks to evaluate test-retest variability of both rest and stress CEU perfusion assessment with Sonazoid.
The use of Sonazoid was performed under an approved Investigational New Drug application from the United States Food and Drug Administration (IND 125975). Microbubble dilutions in 0.9% saline and intravenous infusion rates were varied for the three agents to produce similar dosing rates of microbubbles based on manufacturer information of microbubble concentration (see Figure 1 ). Manual agitation and rotation of the handheld infusion pump was used to maintain a uniform microbubble suspension. Halfway through each imaging protocol, microbubble suspensions were sampled to measure actual microbubble concentration, diameter, and volume by electrozone sensing (Multisizer III, Beckman Coulter, Brea, CA).
Transaxial imaging of the calf bilaterally one-third the distance from the popliteal fold to the ankle was performed using a phased-array transducer coupled to an ultrasound system (IE-33, Philips Ultrasound, Andover, MA). A multipulse contrast-specific imaging algorithm using amplitude modulation was performed at 1.8 MHz. Images were acquired at end diastole by gating to the electrocardiogram (ECG). Gain settings at each MI and acoustic focus were optimized prior to contrast administration to levels below that which produced tissue speckle and were kept constant between agents.
CEU imaging was performed at MIs of 0.1, 0.2, 0.3, and 0.4 using pulsing intervals (PI) that were progressively shortened from every 10 to one cardiac cycles. With this protocol, the acoustic lability of microbubbles was defined by the progressive decline in signal enhancement with shorter PIs, whereas the maximal signal enhancement was measured at a PI of 10, which provides sufficient time for complete ultrasound sector volume replenishment.
To evaluate the influence of microbubble destruction and signal-to-noise during real-time quantitative CEU perfusion imaging, destruction-replenishment kinetics were measured. Microbubbles within the volume of tissue in the ultrasound sector were destroyed with a high-power (MI > 0.9) five-frame pulse sequence, after which real-time replenishment kinetics were assessed at an MI of 0.1, 0.2, 0.3, or 0.4. Frames were acquired at end diastole gated to the ECG in order to minimize signal from intramuscular arteries and in order to not expose microbubbles unnecessarily to potentially destructive ultrasound energy created by imaging frames that are not used for analysis. Background-subtracted intensity from a region of interest placed over the soleus was measured using the immediate postdestruction frame as background, and time-intensity data were fit to the function
y = A ( 1 − e − β t ) ,
Unilateral calf muscle perfusion imaging at MIs of 0.1 and 0.3 was repeated during exercise. Subjects performed exercise with a 60° plantar flexion arc on a calibrated pedal ergometer at 25 W of power. Plantar flexion was performed every 5 seconds for 1 minute. CEU acquisition was initiated within 2 seconds of exercise termination.
Microbubble Cavitation Response
In vivo cavitation of Sonazoid during ultrasound exposure at MIs of 0.1–0.4 was assessed by passive cavitation detection (PCD). A spherically focused broadband (10 KHz to 20 MHz) hydrophone (Y-107, Sonic Concepts, Inc., Bothell, WA) with a focal depth of 20 mm and a focal width of 0.4 mm was confocally positioned with the ultrasound transducer on the calf. Received signals were digitized (100 MHz) and saved in a 4-channel oscilloscope (Waverunner, Teledyne LeCroy, Chestnut Ridge, NY) using sequence mode. Data analysis was performed with the Matlab (MathWorks, Natick, MA) using data averaged for 10 acquisitions.
Statistical analysis was performed with Prism (V.6.02, GraphPad, La Jolla, CA). For multiple comparisons, a one-way analysis of variance (ANOVA) with post hoc Student’s t -test (paired for comparisons between agents and acoustic powers) and Bonferroni correction were used for normally distributed data based on the D’Agostino-Pearson omnibus test. For nonnormally distributed data, a Kruskal-Wallis test was used for assessing changes in A or β according to MI or agent. When applicable, post hoc Wilcoxon rank sum test (paired data) or Mann-Whitney test (for nonpaired data) was used. Tests for differences in CEU data according to MI and post hoc tests for linear trends were performed by repeated-measures ANOVA. Linear regression analysis was used for assessment of test-retest variability. For all tests, P < .05 was considered statistically significant.
Patient Characteristics and Microbubble Infusions
The median age of the subjects studied was 36 ± 11 (range, 22–49) years, and the majority of the subjects were female ( Table 1 ). Baseline vital signs and oxygen saturation were normal in all subjects and did not change during plantar flexion exercise. Microbubble infusions were well tolerated in all subjects, and there was only one report of mild dyspnea 3–4 hours after study completion in a single subject receiving Definity. Microbubble size, size distribution, dosing information, and measured concentration after dilution are presented in Figure 1 . Mean microbubble size and size distribution varied between agents. Despite efforts to produce equipoise in dosing using manufacturer information, the microbubble administration rates based on actual measurements after dilution differed. The highest microbubble dose rate was for Definity and the lowest for Sonazoid. Because of differences in microbubble size distribution, the microbubble gas volume rate was also calculated and was similar for Definity and Sonazoid but higher for Optison.