Developments in ultrasound contrast media

Ultrasound microbubble contrast agents are effective and safe echo enhancers. An ingenious array of methods are employed to achieve stability and provide a clinically useful enhancement period. Microbubbles enhance ultrasound signals by up to 25 dB (greater than 300-fold increase) due to resonant behaviour. This is used to rescue failed Doppler studies and may be extended to image the microcirculation of tumours and the myocardium using non-linear modes. Functional studies open up a whole range of applications by using a variety of active and passive quantitation techniques to derive indices from the transit of contrast through a tissue of interest. This has been especially successful in the detection of liver metastases and cirrhosis and shows great promise as a clinical tool. It also has great potential in measuring microcirculatory flow velocity. The demonstration that some microbubbles are not just pure blood pool agents but have a hepatosplenic specific phase has extended the versatility of ultrasound. Imaging of this stationary phase with non-linear modes such as phase inversion and stimulated acoustic emission, has improved the sensitivity and specificity of ultrasound in the detection and characterisation of focal liver lesions to rival that of CT and MR. Received: 23 June 2000 Revised: 18 July 2000 Accepted: 24 July 2000     

Ultrasound microbubbles for molecular diagnosis, therapy, and theranostics

Ultrasound imaging is clinically established for routine screening examinations of breast, abdomen, neck, and other soft tissues, as well as for therapy monitoring. Microbubbles as vascular contrast agents improve the detection and characterization of cancerous lesions, inflammatory processes, and cardiovascular pathologies. Taking advantage of the excellent sensitivity and specificity of ultrasound for microbubble detection, molecular imaging can be realized by binding antibodies, peptides, and other targeting moieties to microbubble surfaces. Molecular microbubbles directed against various targets such as vascular endothelial growth factor receptor-2, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, selectins, and integrins were developed and were shown in preclinical studies to be able to selectively bind to tumor blood vessels and atherosclerotic plaques. Currently, the first microbubble formulations targeted to angiogenic vessels in prostate cancers are being evaluated clinically. However, microbubbles can be used for more than diagnosis: disintegrating microbubbles emit acoustic forces that are strong enough to induce thrombolysis, and they can also be used for facilitating drug and gene delivery across biologic barriers. This review on the use of microbubbles for ultrasound-based molecular imaging, therapy, and theranostics addresses innovative concepts and identifies areas in which clinical translation is foreseeable in the near future.     

Assessment of tissue perfusion by contrast-enhanced ultrasound

Contrast-enhanced ultrasound (CEUS) with microbubble contrast agents is a new imaging technique for quantifying tissue perfusion. CEUS presents several advantages over other imaging techniques in assessing tissue perfusion, including the use of microbubbles as blood-pool agents, portability, availability and absence of exposure to radiation or nuclear tracers. Dedicated software packages are necessary to quantify the echo-signal intensity and allow the calculation of the degree of tissue contrast enhancement based on the accurate distinction between microbubble backscatter signals and native tissue background. The measurement of organ transit time after microbubble injection and the analysis of tissue reperfusion kinetics represent the two fundamental methods for the assessment of tissue perfusion by CEUS. Transit time measurement has been shown to be feasible and has started to become accepted as a clinical tool, especially in the liver. The loudness of audio signals from spectral Doppler analysis is used to generate time-intensity curves to follow the wash-in and wash-out of the microbubble bolus. Tissue perfusion may be quantified also by analysing the replenishment kinetics of the volume of microbubbles after their destruction in the imaged slice. This allows to obtain semiquantitative parameters related to local tissue perfusion, especially in the heart, brain, and kidneys.     

Contrast-specific ultrasound techniques

The advent of microbubble contrast agents has determined an important evolution of ultrasound (US) technology due to the introduction of contrast-specific US techniques. This was due to the fact that neither colour or power Doppler are suitable for correct management of the signals produced by microbubble insonation, as they are limited by the heavy presence of artefacts. Microbubbles may be insonated by a characteristic frequency named resonance or fundamental frequency (f (0)) by using a high or low transmit power. If insonated by a high transmit power, microbubbles produce a wideband harmonic signal due to microbubble destruction. If insonated by a low transmit power, microbubbles produce harmonic frequencies (2f, 3f, 4f) due to their nonlinear physical behaviour. Contrast-specific US techniques have recently undergone an important technical development with the introduction of innovative algorithms able to register selectively the harmonic signals produced by microbubbles and to suppress the signal produced by stationary tissues. The different contrast-specific US techniques may be distinguished by their basic principle into pseudo-Doppler, harmonic, phase-modulation, amplitude-modulation and phase-and amplitude-modulation techniques.     

Impact of European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB) guidelines on the use of contrast agents in liver ultrasound

The introduction of microbubble contrast agents and the development of contrast-specific techniques have opened new prospects in liver ultrasound. Over the past few years several reports have shown that contrast ultrasound can substantially improve detection and characterization of focal liver lesions with respect to baseline studies. The advent of second-generation agents and low mechanical index real-time scanning techniques has been instrumental in improving the easiness and the reproducibility of the examination. With the publication of the guidelines for the use of contrast agents in liver ultrasound by the European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB), contrast ultrasound enters into clinical practice. The guidelines define the indications and recommendations for the use of contrast ultrasound in focal liver lesion detection, characterization, and follow-up after tumor ablation procedures. We discuss the impact of EFSUMB guidelines on diagnostic protocols currently adopted in liver imaging.     

Microbubble contrast agents: targeted ultrasound imaging and ultrasound-assisted drug-delivery applications

The use of microbubble contrast agents for general tissue delineation and perfusion enjoys steady interest in ultrasound imaging. Microbubbles as contrast materials require a small dosage and show excellent detection sensitivity. Targeting ligands on the surface of microbubbles permit the selective accumulation of these particles in the areas of interest, which show an up-regulated level of receptor molecules on vascular endothelium. Selective contrast imaging of inflammation, ischemia-reperfusion injury, angiogenesis, and thrombosis has been achieved in animal models. Ultrasound-assisted drug delivery and activation, performed by combining microbubble agent containing drug substances or coadministered with pharmaceutical agents (including plasmid DNA for transfection), has been achieved in multiple model systems in vitro and in vivo. Ultrasound and microbubbles-based targeted acceleration of the thrombolytic enzyme action already have reached clinical trials. Overall, microbubble targeting and ultrasound-assisted microbubble-based drug-delivery systems will offer a step toward the application of targeted personalized diagnostics and therapy.     

Ultrasound of focal liver lesions

This paper gives a comprehensive overview of ultrasound of focal liver lesions. Technical aspects such as examination technique and the use of Doppler modes as well as recent developments such as tissue harmonic imaging and microbubble contrast agents are discussed. The clinical significance and sonographic features of various liver lesions such as haemangioma, focal nodular hyperplasia, adenoma, regenerative nodule, metastasis, hepatocellular carcinoma and various types of focal infections are described. With the exception of cysts and typical haemangiomas, definitive characterisation of a liver lesion is often not possible on conventional ultrasound. This situation has changed with the recent advent of ultrasound contrast agents, which permit definitive diagnosis of most lesions. Contrast-enhanced sonography using recently developed contrast-specific imaging modes dramatically extends the role of liver ultrasound by improving its specificity in the detection and characterisation of focal lesions to rival CT and MRI.     

Therapeutic applications of microbubbles

Microbubbles, currently used as contrast agents have potential therapeutic applications. Microbubbles, upon insonation of sufficiently intense ultrasound will cavitate. Cavitation with microbubbles can be used to dissolve blood clots or deliver drugs. Targeting ligands and drugs can be incorporated into microbubbles to make highly specific diagnostic and therapeutic agents for activation with ultrasound. In this paper I will review some of these potential applications and experimental results using such agents for thrombolysis, drug and gene delivery.     

Quantification of blood flow

Traditionally, Doppler ultrasound has been used to estimate blood flow as the mean velocity multiplied by the vessel area, but this is subject to significant errors and may be difficult to perform accurately. Microbubbles, developed as contrast agents for ultrasound, were initially envisaged as useful for increasing the intensity of echoes and thus rescuing Doppler studies that were technical failures because of attenuated signals or very slow flow. However, they can act as tracers and, by analogy with isotope techniques, can be used to measure blood flow with transit-time methods which exploit both arterial and venous time-intensity data. An acceptable compromise is to acquire both a tissue intensity curve and one from the feeding artery. The transit of microbubbles across an organ or tissue can be used to estimate haemodynamic alterations, e.g. the arterialisation of the supply to the liver in malignancies and cirrhosis and the delayed arterio-venous transit in the transplant kidney during rejection. The fragility of microbubbles can be turned to advantage by being exploited to create a negative bolus by exposing a tissue slice to a high power beam. The rate of refilling of this slice by circulating microbubbles can then be followed with a low-intensity monitoring beam and the resulting rising exponential curve analysed to extract indices of both the reperfusion rate (the slope) and the fractional vascular volume (the asymptote). The product of these is a measure of true tissue perfusion.     

Detection of individual microbubbles of ultrasound contrast agents: imaging of free-floating and targeted bubbles

RATIONALE AND OBJECTIVES: During echo examinations with microbubble contrast, individual "dots" of ultrasound reflection can be visualized. To address the question whether these signals represent individual microbubbles, very dilute suspensions of ultrasound contrast agents or individual microbubbles attached to Petri dishes were prepared and studied by ultrasound imaging. METHODS: Microbubble suspensions were diluted in saline and evaluated by a clinical ultrasound imaging system. Microbubble concentration was verified by Coulter counter. Single microbubble preparation on a Petri dish was established by streptavidin-biotin interaction under microscopy control and subjected to ultrasound imaging. RESULTS: Ultrasound of dilute microbubble dispersions demonstrated distinct white foci; concentration of these sites was consistent with signals from individual microbubbles as determined by Coulter. Individual microbubbles immobilized on polystyrene were also visualized by ultrasound. CONCLUSION: Ultrasound medical systems can resolve backscatter signals from individual microbubbles of ultrasound contrast, both in solution and in the targeted immobilized state, implying picogram sensitivity.     

Microbubbles as ultrasound contrast agents for molecular imaging: preparation and application

OBJECTIVE: The purpose of this review is to describe trends in microbubble application in molecular imaging. CONCLUSION: Microbubbles are used for contrast ultrasound imaging as blood-pool agents in cardiology and radiology. Their promise as targeted agents for molecular imaging is now being recognized. Microbubbles can be functionalized with ligand molecules that bind to molecular markers of disease. Potential clinical applications of molecular imaging with microbubble-based ultrasound contrast agents are in the monitoring of the biomarker status of vascular endothelium, visualizing tumor vasculature, and imaging inflammation and ischemia-reperfusion injury zones and thrombi.     

Evaluation of cerebral microemboli during radiofrequency ablation of lung tumors in a canine model with use of impedance-controlled devices

PURPOSE: To evaluate the incidence of cerebral microemboli during radiofrequency (RF) ablation of lung tumors in a canine model and evaluate the adverse effects of these microemboli on the brain parenchyma with use of magnetic resonance (MR) imaging and histopathologic examination. MATERIALS AND METHODS: Percutaneous RF ablation of 12 lung tumors in 12 dogs was performed under computed tomography (CT) guidance with use of impedance-controlled devices. The common carotid artery was continuously monitored in each animal during RF ablation with duplex Doppler ultrasonography. All animals underwent brain MR imaging shortly after RF ablation. Delayed brain MR imaging (5-8 days after RF ablation) was performed in eight animals. The MR examinations included diffusion-weighted echo-planar imaging. The animals were euthanized 3-11 days after RF ablation. The brain was harvested from each animal and examined by an experienced veterinary pathologist for evidence of ischemia. RESULTS: RF ablation was technically successful in all animals. Microbubbles were detected in the carotid artery in two animals (17%). Acute and delayed MR studies demonstrated no evidence of ischemic brain injury in any of the animals. Gross and histopathologic assessment of brain tissue also demonstrated no ischemic changes. CONCLUSIONS: During RF ablation of lung tumors, microbubbles are detected in the carotid arteries in a small number of cases. These microbubbles are too few and too small to be detected by CT imaging of the brain and do not cause ischemic brain injury.     

In vivo study of microbubbles as an MR susceptibility contrast agent.

The potential application of gas microbubbles as a unique intravascular susceptibility contrast agent for MRI has not been fully explored. In this study, the MR susceptibility effect of an ultrasound microbubble contrast agent, Optison, was studied with rat liver imaging at 7 T. Optison suspension in two different doses (0.15 mL/kg and 0.4 mL/kg) was injected into rats, and induced transverse relaxation rate increases (deltaR2*) of 29.1 +/- 1.6 s(-1) (N = 2) and 61.5 +/- 12.9 s(-1) (N = 6), respectively, in liver tissue. Liver uptake of intact albumin microbubbles was observed 10 min after injection. Eight of the 16 rats studied showed no susceptibility enhancement. This is probably attributable to the intravascular microbubble growth due to transmural CO2 supersaturation in the cecum and colon in small animals that causes microbubble aggregation and trapping in the inferior vena cava (IVC). In vitro deltaR2* measurements of Optison suspension at different concentrations are also reported.     

Pulse inversion imaging of liver blood flow: improved method for characterizing focal masses with microbubble contrast

RATIONALE AND OBJECTIVES: To create a microbubble contrast image of vessels that lie below the resolution of an ultrasound system, a technique is required that detects preferentially the agent echo, rejecting that from tissue. Harmonic imaging exploits the nonlinear behavior of microbubbles but forces a compromise between image sensitivity and axial resolution. The authors describe and evaluate a new method that overcomes this compromise and improves contrast imaging performance: pulse inversion imaging. METHODS: Sequences of pulses of alternate phase are transmitted into tissue and their echoes summed. A prototype scanner equipped with pulse inversion was used to image phantoms and 16 patients with focal liver masses. RESULTS: Pulse inversion images show contrast sensitivity and resolution superior to that of harmonic images. Vessels can be imaged at an incident power sufficiently low to avoid destroying the agent, allowing unique visualization of tumor vasculature. Distinct patterns were seen in hemangiomas, metastases, and hepatocellular carcinomas. CONCLUSIONS: Pulse inversion imaging is an improved bubble-specific imaging method that extends the potential of contrast ultrasonography.     

Focal liver lesions: role of contrast-enhanced ultrasound

The introduction of microbubble contrast agents and the development of contrast-specific techniques have opened new possibilities in liver imaging. Initially, only intermittent imaging with Doppler detection was available. Second-generation contrast agents and low mechanical index real-time scanning techniques are decisive advances that enable convenient liver examinations with high sensitivity and specificity. Hepatic lesions usually show typical perfusion and enhancement patterns through the various contrast phases, which help their characterization. Several published studies and the daily clinical routine show that, as opposed to conventional ultrasound (US), contrast-enhanced US can substantially improve detection and differentiation of focal liver lesions. Today, contrast-enhanced US is the dynamic imaging modality of choice for differentiation of focal liver lesions. Contrast uptake patterns of the most relevant liver lesions, as well as important clinical indications are presented and discussed.     

Advances in ultrasound

Ultrasound (US) has undergone dramatic changes since its inception three decades ago; the original cumbersome B-mode gantry system has evolved into a high resolution real-time imaging system. This review describes both recent advances in ultrasound and contrast media and likely future developments. Technological advances in electronics and computing have revolutionized ultrasound practice with ever expanding applications. Developments in transducer materials and array designs have resulted in greater bandwidths with improvements in spatial and contrast resolution. Developments in digital signal processing have produced innovations in beam forming, image display and archiving. Technological advances have resulted in novel imaging modes which exploit the non-linear behaviour of tissue and microbubble contrast agents. Microbubble contrast agents have dramatically extended the clinical and research applications of ultrasound. Not only can Doppler studies be enhanced but also novel non-linear modes allow vessels down to the level of the microcirculation to be imaged. Functional and quantitative studies allow interrogation of a wide spectrum of tissue beds. The advent of tissue-specific agents promises to improve the sensitivity and specificity of ultrasound in the detection and characterization of focal liver lesions to rival that of computed tomography (CT) and magnetic resonance imaging (MRI). Ultrasound has recently moved into therapeutic applications with high intensity focused ultrasound (HIFU) and microbubble assisted delivery of drugs and genes showing great promise.     

Effect of microbubble ligation to cells on ultrasound signal enhancement: implications for targeted imaging

OBJECTIVES: Molecular imaging with contrast-enhanced ultrasound (CEU) relies on the detection of microbubbles retained in regions of disease. The aim of this study was to determine whether microbubble attachment to cells influences their acoustic signal generation and stability. MATERIALS AND METHODS: Biotinylated microbubbles were attached to streptavidin-coated plates to derive density versus intensity relations during low- and high-power imaging. To assess damping from microbubble attachment to solid or cell surfaces, in vitro imaging was performed for microbubbles charge-coupled to methacrylate spheres and for vascular cell adhesion molecule-1-targeted microbubbles attached to endothelial cells. RESULTS: Signal enhancement on plates increased according to acoustic power and microbubble site density up to 300 mm. Microbubble signal was reduced by attachment to solid spheres during high- and low-power imaging but was minimally reduced by attachment to endothelial cells and only at low power. CONCLUSION: Attachment of targeted microbubbles to rigid surfaces results in damping and a reduction of their acoustic signal, which is not seen when microbubbles are attached to cells. A reliable concentration versus intensity relationship can be expected from microbubble attachment to 2-dimensional surfaces until a very high site density is reached.     

Prevalence and clinical significance of pleural microbubbles in computed tomography of thoracic empyema

AIM: To determine the prevalence and clinical significance of pleural microbubbles in thoracic empyema. MATERIALS AND METHODS: The charts of 71 consecutive patients with empyema were retrospectively reviewed for relevant demographic, laboratory, microbiological, therapeutic and outcome data. Computed tomography (CT) images were reviewed for various signs of empyema as well as pleural microbubbles. Two patient groups, with and without microbubbles were compared. RESULTS: Mean patient age was 49 years and 72% were males. Microbubbles were detected in 58% of patients. There were no significant differences between patients with and without microbubbles in regard to pleural fluid chemistry. A causative organism was identified in about 75% of cases in both. There was no difference in the rates of pleural thickening and enhancement, increased extra-pleural fat attenuation, air-fluid levels or loculations. Microbubbles were diagnosed after a mean of 7.8 days from admission. Thoracentesis before CT was performed in 90 and 57% of patients with and without microbubbles (p=0.0015), respectively. Patients with microbubbles were more likely to require repeated drainage (65.9 versus 36.7%, p=0.015) and surgical decortication (31.7 versus 6.7%, p=0.011). Mortalities were 9.8 and 6.6% respectively (p=0.53). CONCLUSION: Pleural microbubbles are commonly encountered in CT imaging of empyema but have not been systematically studied to date. Microbubbles may be associated with adverse outcome such as repeated drainage or surgical decortication. The sensitivity and specificity of this finding and its prognostic implications need further assessment.     

Microbubbles assist goat liver ablation by high intensity focused ultrasound

High intensity focused ultrasound (HIFU) has been introduced to treat cancers. However, this therapy is a time-consuming procedure; destructing a deeper volume is also difficult as ultrasonic energy attenuates exponentially with increasing depth in tissues. The aim of the present study was to investigate the effects of introducing microbubbles on liver HIFU ablation. Seventeen goats were divided into groups A (n=8) and B (n=9). The livers in both groups were ablated using HIFU (1.0 MHz, 22,593 W/cm²) performed in the manner of a clinical regime using a clinical device. A microbubble agent was bolus-injected intravenously before HIFU exposure in group B. All animals in group A and seven goats in group B were euthanased to evaluate the ablation efficiency 24 h after HIFU. The necrosis rate (mm³/s), which was the volume of necrosis tissue per second of HIFU exposure, was used to judge the ablation efficiency. Pathological examinations were performed to determine whether there were residual intact tissues within the exposed volume. The other two goats in group B were used to determine the delayed pathological changes 7 days after ultrasonic ablation. The necrosis rate (mm³/s) was increased in group B (14.4647±4.1960 versus 33.5302±12.4484, P=0.0059). Pathological examinations confirmed that there were no residual unaffected tissue focuses within the exposed volume. Two remarkable changes occurred in the other two goats in group B 7 days after HIFU: there were ghost-cell islands at the periphery of the ablated tissues, and surrounding adjacent tissues outside the reactive zone necrotized. These findings showed that microbubbles could be used to assist liver HIFU ablation. T. Yu and X. Fan contributed equally to this paper.     

Mechanism of hepatic parenchyma-specific contrast of microbubble-based contrast agent for ultrasonography: microscopic studies in rat liver

OBJECTIVE: The objective of this study was to elucidate the mechanism of hepatic parenchyma-specific contrast of Sonazoid (microbubble contrast agent) using microscopic techniques. MATERIALS AND METHODS: Sonazoid was intravenously injected into rats to investigate the microbubble dynamics and distribution within hepatic microcirculation in exteriorized liver using intravital microscopy and to observe dose dependency of ultrasound hepatic contrast effect. In vitro and in vivo uptake of microbubbles by Kupffer cells was examined using confocal laser scanning microscopy. RESULTS: Intravital observation demonstrated freely flowing microbubbles in the sinusoid and some microbubbles co-localized with Kupffer cells. The microbubbles internalized in Kupffer cells were identified with reflected light by confocal laser scanning microscopy. The percentage of Kupffer cells taking up microbubbles was about 1% at clinical dose at which the homogeneous hepatic contrast was observed. CONCLUSIONS: The hepatic parenchyma-specific contrast by Sonazoid is due to distribution of the microbubbles in Kupffer cells.