At low MI, symmetrical oscillations occur, and the frequency of the scattered signals is the same as the transmitted pulse. Microbubbles resonate equally and symmetrically (linear response) with the high and low pressures generated by the incident ultrasound wave with minimal destruction. They act as very efficient signal scatterers, which is attributed to the difference in their compressibility and density compared to the surrounding tissues (Fig. 57.2). Scattering efficiency increases as a function of microbubble radius to the sixth power, and therefore larger bubbles display higher backscatter coefficients [8]. Furthermore, increasing shell stiffness or gas density in turn decreases the microbubble response. Under low MI imaging, the intensity of the scattered signal is linearly related to that of the incident ultrasound beam [12]. Low MI is useful in conventional Doppler applications and real-time contrast imaging.

The amplitude of the microbubble oscillations in an ultrasound field increases as the MI is increased. As the amplitude increases, the microbubble oscillations become asynchronous with the ultrasound wave (Fig. 57.3). By manipulating the local acoustic power, the microbubbles begin to echo a harmonic response at frequencies that differ from that of the incident wave (fundamental frequency). The highest intensity response is that of the second harmonic response, which is found at twice the fundamental frequency. Those responses found at higher frequencies are called higher harmonics. Specific types of harmonics called ultra harmonics can be obtained at specific frequencies such as those at 1.5 or 3.5 times the fundamental frequency [8]. Intermediate MI imaging possesses two main advantages. First, the technique enables avoidance of microbubble destruction while eliciting a good harmonic contrast signal. Second, the harmonics elicited from the surrounding tissues are reduced. As the advantage of contrast agents is their harmonic echo, any tissue harmonics become the background “noise.” Tissue is less compressible and denser than the microbubbles, therefore requiring a higher MI for a harmonic response. Intermediate MI scanning allows for a higher contrast-to-tissue ratio compared to a high MI by limiting the tissue harmonic response and thus eases the removal of the background “noise” from the contrast signal [20].

The destruction of microbubbles occurs at higher acoustic powers. The encapsulating shell is broken and the gas diffuses into the surrounding fluid. The rupture of the microbubble causes an intense echo, very rich in nonlinear (harmonic) components (Figs. 57.4 and 57.5). Although the destruction of contrast agents is often viewed as a limitation, it allows for improved sensitivity in microbubble detection. When color and power Doppler are used, the change in the echo of two consecutive pulses is seen as a particular color pixel. These color signals thus correlate with the distribution of the microbubbles with little affect from blood flow characteristics [8].

Contrast agents can be employed when using conventional B-mode imaging and Doppler imaging modalities, including color, power, and spectral imaging. They can be used to opacify fluid-containing cavities such as the bladder, uterus, cardiac chambers (Fig. 57.9), and some large venous structures. Echovist (Schering AG, Berlin, Germany) has been used for sonosalpingography [30], and Levovist (Schering) has been used to detect vesicoureteric reflux [31]. This allows equally sensitive and specific imaging alternatives to conventional radiographs while avoiding ionizing radiation exposure. The limitation of contrast agents with B-mode imaging is that there is no added enhancement in solid structures. Doppler imaging provides excellent detection of microbubbles, but it is limited by color blooming and oversaturation artifacts [8].

This microbubble destructive modality involves an initial wave of ultrasound at a high mechanical index, which ruptures the microbubbles. Signal responses are recorded before and after this destruction, and the loss of correlation between the two signals maps the distribution of the contrast agent [10]. As mentioned above, some organ-specific contrast agents accumulate only in normal liver tissue. This characteristic has been utilized to detect liver metastases using Levovist. Metastases appear as signal defects [32] (Figs. 57.10 and 57.11).

Conventional Doppler imaging can be used with multiple pulse techniques. After the ideal bandwidth is determined, consecutive pulses are emitted. The flow signals are isolated from static tissue signals by amplifying their intercorrelation at different times, and the static tissue signals are removed. This technique, called advanced dynamic flow, allows better spatial delineation of the microvasculature of superficial structures compared to conventional Doppler modes. However, its sensitivity is limited by the depth of the structure of interest. The utilization of contrast agents with this technique expands its uses to the visualization of deep structures, hypoperfused structures, and even real-time perfusion imaging using lower MIs to preserve microbubbles [8].

This is a nonlinear imaging modality that can detect microbubble harmonic responses at a higher sensitivity than the response from the surrounding tissues. The weaker nonharmonic echoes from the multiple responses from the body wall (as with obese patients) are filtered from the final image as well, improving the signal-to-noise ratio [12]. This sensitivity is derived from the fact that the microbubbles produce a unique nonlinear, harmonic response that can be separated from the tissue echoes and large vessel blood flow. Currently, there are commercially available transducers that can both emit ultrasound energy and detect the microbubble harmonic response. These transducers are tuned in to detect the resonant frequency of microbubbles, which range in diameter from 1 to 9 μm, in the 1–9 MHz range [33, 34]. Several techniques can be used with harmonic imaging to refine the detection of the nonlinear, harmonic response of the contrast agents including conventional imaging, subtraction techniques using single (coherent imaging mode) or multiple pulses (pulse or phase inversion), and a combination of the latter for multiframe subtraction techniques. Conventional imaging uses a monopulse technique in which a single pulse of ultrasound is emitted and the second harmonic of the microbubble echo is isolated using filters. The microbubble signal returns at 10–15 dB higher than the signal from the surrounding clutter. Harmonic imaging can provide grayscale and Doppler information; however, it does have its limitations. First, the filtering process to isolate the second harmonic from the fundamental frequency affects the image quality. Also, the increased attenuation of the harmonic frequency compared to the fundamental frequency limits the depth of the imaging capabilities [8].

Pulse or phase inversion involves the emission of two consecutive pulses in inverted phases and the detection of the difference of the unique resonance of microbubbles in each phase [34]. Since microbubbles have a unique harmonic response to the consecutive pulses, the difference in the echo signals in the two phases results in an increased sensitivity in the detection of the microbubbles. Power pulse inversion utilizes these same principles using a multipulse technique. The multipulse technique adds to the sensitivity of the modality and improves the differentiation between liquids and solids, as well as limiting motion artifacts [14]. Intermittent imaging and scanning are employed when using a high MI imaging modality for microbubble destruction [8]. This approach allows for replenishment of the contrast agent between images. The frame rate is reduced to about one frame per second compared to the conventional 30 frames per second. The frame rate can also be synchronized with the cardiac cycle so that the microbubble can be carried into the area of interest where the microbubbles have been destroyed. Depending on the imaging delay time after the microbubbles have been destroyed, regions with either increased or decreased blood volumes can be determined. Intermittent imaging is thus limited by its inability to provide real-time imaging. Another limitation is motion artifact between image acquisitions.

Flash echo imaging is a technique that involves using a low MI between intermittent high MI destructive phases and following the target to be imaged during the entire study. A real-time anatomic image and a contrast-enhanced image during bubble destruction can be viewed simultaneously on the image screen [8] (Toshiba Medical Systems, Tokyo, Japan) (Fig. 57.12).

Another imaging sequence involves real-time digital subtraction of a bubble destruction phase image from a consecutively acquired background image taken milliseconds apart. This technique limits motion artifact and improves the visibility of the microbubbles in the resulting image [8] (Fig. 57.13).

Imaging of vascular structures using CEUS allows simultaneous assessment of the vascular lumen (using the CEUS signal) and the vascular wall (using the classical B-mode signal). Thus, vascular anatomy and vessel patency can be evaluated, allowing reliable detection of several vascular abnormalities [35]. Since CEUS can easily be used at the bedside and during interventions, it is also suitable for guiding and monitoring vascular interventions and following them up [36]. CEUS can also be used to image vascular plaques. Location and extension as well as ulcerations can be visualized.

Diagnosis of renal artery stenosis, determination of microperfusion and extent of infarcts, as well as delineation of solid or cystic masses are all applications of CEUS that are being studied. It has been shown that using contrast agents with ultrasound evaluation improved the efficacy of the scan by enhancing the operator’s ability to visualize the renal arteries and by increasing the number of definitive examinations. Although angiography has been considered the “gold standard” in the evaluation of renal artery stenosis, its invasiveness and patient exposure to radiation and nephrotoxic contrast agents restrict its use as a screening modality. Conventional Doppler ultrasound is routinely used for renal artery stenosis evaluation; however, studies have shown that there is great variability from center to center in its correlation to angiographic findings. In addition, the ability to obtain technically successful examinations was also shown to vary among the studies. Avasthi et al. reported good correlation between the studies when evaluating 52 renal arteries with both Doppler ultrasound and angiogram [37]. They reported 89% sensitivity and 73% specificity for Doppler ultrasound in detecting lesions with a greater than 50% lumen reduction. Technically adequate examinations were performed in approximately 84% of the patients. Similarly, Norris et al. performed successful examinations in 90% of the patients studied, with Doppler ultrasound having 73% sensitivity and 97% specificity when compared to angiography [38]. In contrast, Berland et al. obtained adequate examinations in only 58% of their patients, while being unable to identify any of the seven patients who had renal artery stenosis on angiogram. They reported only 37% specificity, as patients without renal artery stenosis were detected on only 7 out of 19 ultrasound examinations [39]. Desberg et al. reported success in only 51% of ultrasound examinations [40]. Such variability in the success of Doppler ultrasound in the evaluation of renal artery stenosis is reflective of the technical difficulty of performing an adequate examination in a wide variety of patients. Patient body habitus and variable renal artery anatomy also complicate examinations. CEUS is proving to be the imaging modality that can overcome these limitations. Claudon et al. reported the results of a randomized crossover study of 198 patients from 14 European centers who were referred for renal arterial angiography because they were suspected of having renal arterial stenosis [15]. They reported a 63.9–83.8% increase in successful examinations when adding contrast to the ultrasound scan, including in obese patients and those with renal dysfunction. When comparing Doppler ultrasound to angiography, CEUS results correlated with angiographic results in the diagnosis or exclusion of renal artery stenosis more often than conventional Doppler ultrasound (p = 0.001). In addition, Lacourciere et al. reported results from 78 patients involved in a Canadian multicenter controlled pilot study who were undergoing evaluation for renal artery stenosis comparing captopril-enhanced scintigraphy and unenhanced and enhanced ultrasound [41]. Results revealed that CEUS examination yielded a diagnosis in more patients than unenhanced ultrasound or scintigraphy—99%, 82%, and 81%, respectively (p = 0.002). The proportion of technically successful ultrasound examinations increased significantly with the addition of contrast. CEUS has also been used to evaluate the microperfusion of the kidney and to define areas of ischemia or infarct. Renal perfusion has been quantified by using a high MI to destroy microbubbles and then using low MI intermittent harmonic imaging at various pulse intervals to plot pulse interval versus video intensity to derive microbubble velocity and renal blood volume fraction [42] (Fig. 57.14). Wei et al. showed that measuring the rate of microbubble replenishment after destruction reflected microbubble velocity (MV). When the tissue is completely replenished with contrast, the signal reflects the tissue blood volume fraction (BVF). The product of MV and BVF correlates with tissue nutrient blood flow (NBF) [43]. Total renal blood flow (RBF) is estimated by cortical MV since >90% of total RBF supplies the renal cortex. Wei et al. showed that CEUS provides an assessment of RBF and tissue nutrient blood flow that correlates with blood flow measurements obtained with a conventional Doppler flow probe [42]. The added assessment of NBF with CEUS allows the evaluation of renal pathologies that affect NBF with little effect on RBF and vice versa. Thus, CEUS provides improved information on the perfusion pattern of the kidneys in conditions such as pyelonephritis, embolism to the kidney, and posttransplant assessment of a transplanted kidney [44]. In addition to the imaging capabilities of CEUS, recent reports have demonstrated nonvector gene transfer into the kidney using microbubbles and ultrasound as a delivery vehicle. For example, Lan et al. studied the use of ultrasound and microbubbles to transfer a doxycycline-regulated Smad7 gene into the kidney as a potential therapy for renal fibrosis. They found that compared with non-ultrasound treatment, the combination of ultrasound microbubble-mediated delivery largely increased Smad7 transgene expression up to a 1000-fold in all kidney tissues [45]. CEUS is an imaging and therapeutic modality that will hopefully provide a safe, noninvasive, and easily applicable alternative in the management of renal disease.

Left ventricular cavity opacification (LVO) is clinically important in the evaluation of cardiac structure and ventricular function in resting and stress echocardiography. In the United States, contrast agents are FDA approved for ventricular opacification and enhancement of endocardial border definition. CEUS is used in those patients with a technically suboptimal echocardiogram. Contrast has been shown to increase the diagnostic capability of echocardiograms in numerous studies. In one study with 200 patients, the patient cohort was selected on the basis of suboptimal baseline echocardiograms with nonvisualization of at least two of six segments in the apical four-chamber view. CEUS in these patients converted a nondiagnostic study to a diagnostic echocardiogram in 75% of those patients examined. This added improvement in image quality resulted in a greater ability to answer the primary referral question in as many as 50% of patients [46]. Similar findings have been observed after left ventricular (LV) opacification produced by intravenous injections of investigational contrast agents [19, 47].

Administration of an intravenous contrast agent has also been shown to enable more accurate measurement of LV volume and ejection fraction in humans [48]. It has also been demonstrated clinically that with the use of contrast agents, harmonic imaging produces improvements over fundamental imaging in LVO, endocardial border definition, and reviewer confidence in the assessment of systolic function [14, 15, 20, 21, 34, 38, 44, 49]. The use of contrast agents has been demonstrated in other cardiac pathologies. The diagnosis of complications of myocardial infarction, such as wall rupture and left ventricle pseudoaneurysm formation, has been facilitated by the use of contrast agents. CEUS has also been used to improve the accuracy of transesophageal echocardiography in ascending aortic dissection by discriminating the true and false lumen. Intracardiac masses, such as tumors or thrombi, have been easier to identify with the use of ultrasonic contrast.

The significance of inflammation in relation to plaque stability has been well established in animal models of disease. The inflammatory response includes the recruitment of monocyte-derived macrophages and leukocytes to atherosclerotic endothelium leading to plaque propagation, increased susceptibility of rupture, and vascular remodeling [50]. Identifying these inflamed, vulnerable plaques would be valuable in determining which patients would require an immediate intervention to prevent ischemic complications from a ruptured plaque. Those microbubbles with shells composed of albumin or lipids bind to activated leukocytes in response to ischemic injury or exposure to proinflammatory mediators. The extent of leukocyte binding by these microbubbles correlates with the amount of inflammation, allowing for quantification of the extent of the inflammatory process [19, 51]. The albumin and lipid shells of microbubbles bind to these activated leukocytes via different mechanisms, although they both require the upregulated expression of adhesion molecules stimulated by activated leukocytes in response to inflammation. Albumin is bound via the leukocyte β2-integrin Mac-1 to endothelial ICAM-1, while lipid shells become coated with complement proteins that are recognized by complement receptors on the leukocyte surface [21, 22, 27, 51]. Once the microbubble is bound to the leukocyte, it is phagocytosed whole by neutrophils and monocytes. Within these cells the microbubble remains capable of oscillating enough to create an echo response [23, 51]. Another strategy for the targeting of inflamed plaques involves engineering the microbubbles to incorporate ligands to extracellular adhesion molecules (ECAMs) into the encapsulating shell. Targeting of ECAMs allows for tissue-specific binding since they are expressed on the surfaces of plaques, in neovessels of plaques, and in adventitial vessels and are absent in normal vessels [51, 52] (Fig. 57.15). ECAMs known to be associated with atherosclerosis include ICAM-1, vascular cell adhesion molecule (VCAM)-1, selectins, and the integrin αvβ3 [51, 52]. Several investigators have exploited the expression of ECAMs to target regions of inflammation. Villaneueva et al. targeted activated endothelial cells in a flow chamber system by conjugating a monoclonal antibody to ICAM-1 on the surface of a microbubble encapsulated in a lipid shell [53]. Demos et al. employed this same strategy using acoustically active liposomes instead of microbubbles. They demonstrated in vivo targeting of these antibody-carrying liposomes to inflamed atherosclerotic plaques within the carotid arteries of pigs [54]. Linder et al. also used antibodies to P-selectin to target inflamed venules as a means of identifying ischemia-reperfusion injury [21].