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Contrast echocardiography: new agents
Ultrasound in Med. & Biol., Vol. 30, No. 4, pp. 425– 434, 2004 Copyright © 2004 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/04/$–see front matter
doi:10.1016/j.ultrasmedbio.2003.12.002
● Review Article CONTRAST ECHOCARDIOGRAPHY: NEW AGENTS ANDREW P. MILLER and NAVIN C. NANDA* Division of Cardiovascular Diseases, University of Alabama at Birmingham, Birmingham, AL, USA (Received 5 June 2003; revised 23 November 2003; in final form 9 December 2003)
Abstract—In this report, we review the history, rationale, current status and future directions of contrast agents in echocardiography. First, we discuss the historic development of contrast agents through a review of important physical principles of microbubbles in ultrasonography. Second, we identify attributes of an ideal contrast agent and review those that are currently available or in the “pipeline” for clinical use. Third, we review indications for contrast echocardiography, including endocardial border detection, perfusion quantification and reperfusion assessment, and validate these observations by comparisons with other imaging modalities. Then, we briefly review different methodologies of performing a contrast study, including interrupted, real-time and a hybrid modality. Finally, we identify novel future applications of the newest contrast agents. These newer concepts in contrast echocardiography should form a foundation for nearly limitless application of echocardiography in improved anatomical assessment, perfusion imaging and even special applications, such as detection of vascular inflammation and site-specific drug delivery. (E-mail: [email protected]) © 2004 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound, Blood flow, Contrast agent, Inflammation, Myocardial contrast echocardiography, History, Quantification.
INTRODUCTION
PHYSICAL PRINCIPLES OF MICROBUBBLES IN ULTRASONOGRAPHY
The search for a comprehensive imaging modality that best evaluates cardiac function, perfusion and structure continues in the practice of cardiology. In recent years, the diagnostic application of cardiac ultrasound (US) imaging has greatly expanded with the advent of contrast echocardiography (CE). Through the injection of microbubbles and microbubble contrast agents, CE is already a valuable tool to delineate endocardial borders, direct invasive procedures, detect intracardiac shunts, assess myocardial perfusion and viability, and quantify coronary flow reserve and blood volumes. Further, the future potential of this new technique seems to be limitless, as it currently reaches beyond diagnostic uses and into therapeutic delivery of growth factors, genes and antithrombotic agents. In this report, we discuss the history, rationale, current status and future directions of contrast agents in echocardiography.
History Contrast echocardiography has a long history. CE began with the observation by Joyner and publication by Gramiak and Shah (1968) that injection of saline into the ascending aorta and, thus, the production of microbubbles during echocardiographic recording resulted in better echo signals in the lumen of the aorta and heart chambers. In the early 1970s, saline CE was used to delineate intracardiac shunts, to define congenital heart disease and to examine the right-sided valves as an adjunct to the normal transthoracic echocardiography (TTE) exam (Nanda et al. 1975, 1976, 1997; Gramiak et al. 1972; Rothbard and Nanda 1981, 1983). The commercial development of contrast agents began in the 1980s with observations that led to the stabilization and miniaturization of the microbubble. Carroll et al. (1980) demonstrated that gelatin-encapsulated nitrogen bubbles were stable enough to be used for US enhancement. Then, Feinstein et al. (1984a) showed that microbubbles sonicated from human serum albumin were small and stable enough to traverse the pulmonary circulation and to opacify the left ventricle. These findings led to signif-
Address correspondence to: Navin C. Nanda, M.D., University of Alabama at Birmingham, Heart Station, SW/S102, 619 South 19th Street, Birmingham, AL 35249 USA. E-mail: [email protected] 425
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icant interest in the development of a commercially available US contrast agent by many large and small pharmaceutical companies. Several programs were initiated to develop an easy to use and clinically useful contrast agent for ultrasound. Schering AG (Berlin, Germany) was the first company to be successful. Their first agent was Echovist威 (1982), which enabled right side enhancement only, followed by Levovist威 (1985), which achieved enhancement of the left ventricle. Schering’s agents were approved in Europe, Japan and Canada. Molecular Biosystems Inc. (MBI; San Diego, CA) developed Albunex威, the first agent approved by the US Food and Drug Administration (FDA) in 1994 for USA distribution. The agent was based on technology utilizing sonicated human serum albumin to encapsulate and stabilize the air microbubble. Both Albunex威 and Levovist威 utilize air as the gas component of the microbubble. This resulted in reduced longevity in vivo and limited the commercial success of these agents. The use of various types of perfluorocarbon (PFC) gas to increase the persistence of the microbubble was first introduced in the early 1990s. Many agents under development incorporated PFC technology into their formulations. These products include reformulation of the Albunex威 technology utilizing a PFC gas. This product, Optison威 (MBI), was approved by the FDA for commercial use in 1997. Additional agents have been developed that utilize PFC gas and vary the constituents in the shell that encapsulate the gas. The two such agents currently approved by the FDA are Definity威 (2001) distributed by Bristol Myers Squibb (New York, NY) and Imagent威 (2002), distributed by IMCOR (IMCOR, San Diego, CA), a division of Photogen Technologies Inc. Another agent, SonoVue威 (Bracco Diagnostics Inc., Princeton, NJ), utilizes sulfur hexafluoride, a low-solubility fluorinated gas, and is currently approved only in Europe (2001). These development activities have resulted in smaller, more stable microbubble agents, which, when coupled with improvements in US equipment technology, have resulted in the extension of CE from opacification to perfusion.
change that results in CE signal. The inherent properties of the bubble shell and the gas inside determine the output signature of each microbubble. The mechanical index (MI) of the US beam is used to modulate this output and to incite different bubble responses. MI is defined as the peak rarefactional pressure (i.e., the peak negative pressure) divided by the square root of the US frequency (McCullough et al. 2000). In other words, MI is the amount of negative acoustic pressure within a US field. In clinical practice, the range for MI varies from 0.05 to 1.9. By changing MI, the ultrasonographer can create three phenomena of CE (Becher and Burns 2000). First, at a low MI (0.1), the microbubbles undergo linear oscillation with compression and rarefaction that are equal in amplitude. No special CE signal is created at this stage using fundamental imaging. When the MI is increased (0.2 to 0.7), nonlinear oscillation occurs preferentially with the bubbles undergoing rarefaction that is greater than compression, and ultrasonic waves are created at harmonics of the delivered frequency. These CE signals are bubble-specific. Finally, although microbubble destruction occurs to some degree at all mechanical indices, microbubble destruction is extensive at the highest MI (0.8 to 1.9), creating a brief, but high, output signal that is unique to the contrast agent.
CE physics The mechanism of microbubble CE is based on the physical principles of rarefaction and compression created by a US wave insonating a gas-filled microbubble along with the mechanical index of the ultrasonic beam. Rarefaction occurs with the reduction in pressure of the gas when struck by the negative portion of an acoustic wave, and compression refers to the rise in pressure of a medium struck by the positive portion of the acoustic cycle (Frinking et al. 2000). Compression and rarefaction lead to volume pulsations of microbubbles, and it is this
where R ⫽ bubble radius, d ⫽ gas density, DIFS ⫽ gas diffusivity and constsat ⫽ saturation constant (Raisinghani and DeMaria 2002). Thus, a shell with low gas diffusivity and a gas with a low saturation constant and high density would result in longer persistence. The search for a gas with these properties led to the perfluorocarbons. The second great advance in CE was the ability reliably to produce microbubbles small enough to traverse the pulmonary circulation. The ideal agent for left ventricular opacification and, eventually, for perfusion
Contrast agent principles The first major advancement in CE came with development of more stable microbubbles. When agitated saline containing air bubbles was injected in the early studies, the air would quickly leak from the thin bubble shell into the blood, where it dissolved. The smaller bubbles that were capable of traversing the capillary bed did not survive long enough for imaging because the small amount of air quickly dissipated into the blood. So, persistence was sought in the form of a more stable shell and a higher density gas. Persistence is defined by the equation: 共R ⫻ d兲/共2DIFS ⫻ constsat兲
New agents in contrast echocardiography ● A. P. MILLER
studies, is an agent that could pass this capillary bed. This meant developing agents for which the predominant population of the microbubbles approximated the size of red blood cells (6 to 8 m in diameter) while minimizing the presence of larger-sized microbubbles that could plug microvascular flow. Most of the currently available products have been shown to transit the microcirculation successfully. Finally, recent research with contrast agents has focused on investigating the unique properties that permit targeting of tissues, or even cells. Some agents currently in development have demonstrated the ability to deposit in myocardial tissues, attach to endothelial cells, or even absorb into leukocytes. These distinctive features permit improved tissue contrast, potential targeting of therapies and inflammation or endothelial health assessment. ATTRIBUTES OF AN IDEAL CONTRAST AGENT The explosion of CE has led to the development of many new contrast agents, as well as improvements in the equipment available to image the agents. Optimization of the agents, as well as the equipment, continues. Several physical properties of microbubbles decide their utility, some in conflict with others. For example, the ideal agent would be resilient enough to freely transit the microcirculation and persist in the bloodstream, but fragile enough to be disrupted by US energy. We will explore the attributes of the ideal agent individually. First, as discussed above, the imaging properties of an agent are established by the shell and the gas. The shell must sufficiently stabilize the microbubble to provide adequate persistence, permitting the CE agent to reach the left ventricle or myocardium for imaging. Persistence is also defined by the density and diffusibility of the gas within the shell, and the ideal agent would contain a gas of high density and low diffusibility. However, these properties must be balanced with the ultimate goal of CE, which is to disrupt or even explode the microbubble by insonating with US energy. In other words, a balance of persistence and fragility is required. Additionally, the ideal agent would be neutrally charged to freely transit the microcirculation without interacting with the blood components, be imaged at low MI and be disrupted at higher MI (Fisher et al. 2002). Second, imaging artefacts, the plague of echocardiography, must be limited. The reflected energy of CE agents is often very high, resulting in attenuation that may obscure structures distal to the imaging agent. Attenuation of the image can be impacted by the size range of the microbubbles, with larger microbubbles causing greater attenuation. The concentration of the micro-
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bubbles within the imaging window can also affect the degree of attenuation observed. For example, contrast enhancement of the anterior wall may lead to an artificial “cold spot” in the inferior wall. The ideal agent would produce enough output to provide enhancement without shadowing distal structures to the point of inhibiting interpretation. Third, additional special properties are required for the CE agents under development for targeting of leukocytes or endothelial cells. For example, some agents have been shown to be taken up by endothelial cells. If a therapeutic agent could be incorporated into the shell or the gas of these agents, targeted disruption could provide a unique delivery system to the endothelium. Further, inflammation as a mechanism of atherosclerosis and of myocardial injury is currently a topic of intense study. CE agents that have been shown to target leukocytes may provide exceptional markers for local inflammation, and are currently under investigation to study this phenomenon. Finally, to reach from the bench to the bedside, CE agents must be safe, be easily used by physicians and nurses, and be of low cost. The early experience with saline contrast taught us that CE could lead to a potentially significant air embolus. With the current microbubble technology, the small size of the commercially available CE agents reduces this concern; however, some complications have been observed (Skyba et al. 1998; Van Der Wouw et al. 2000). CONTRAST AGENTS APPROVED AND “IN THE PIPELINE” FOR CLINICAL USE In the USA, three CE agents for left ventricular opacification and endocardial border detection are commercially available: Optison威, Definity威 and Imagent威. Several others are on the horizon: Levovist威 and Sonovue威 are already approved in Europe, and investigational agents continue to emerge. Table 1 lists these CE agents, their development status and their properties. As the agents have become smaller, more stable, and more uniform, more exciting applications for these agents, such as microvasculature assessment, have become possible. Albunex威 (MBI), not listed in Table 1 because it is now unavailable, was the first generation agent that utilized sonicated human albumin to create a 4-m air-filled microbubble for left ventricular opacification. This agent, developed by Feinstein et al. (1984b), did pass to the left ventricle and enhance endocardial border detection, but it poorly demonstrated microvasculature perfusion. Second-generation agents were developed that incorporated high-density gases with low diffusion and saturation constants (i.e., perfluorocarbons) into the microbubble (Figs. 1 and 2). These agents were
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Table 1. Echo Contrast Agents Agent Research Name
Company
Shell
Charge1
Gas
Microbubble Count/size
Storage
Prep
Vial volume
Galactose/Palmitic acid
Negative
Air
Varies with concentration2 95%⬍10m
Room temp 15–30°C
Reconstitute with 5 to 17 mL water
Optison威9 FS069
Amersham Health
Human Serum Albumin N-acetyltryptophan, Caprylic acid
Slight Negative
Octafluoropropane
5–8⫻108/mL 93%⬍10m
Refrigerate 2–8°C
Hand agitate
Definity威10 DMP 115
Bristol-Myers Squibb Medical Imaging, Inc.
Lipids:DPPA, DPPC, MPEG5000 DPPE
Negative
Octafluoropropane
1.2⫻1010/mL 98%⬍10m
Refrigerate 2–8°C
Activate through Vialmix agitation
2 mL
Imagent威11 AF0150
Lipid: DMPC
Neutral
Perfluorohexane/ Nitrogen
1.4⫻109/mL 99.8%⬍10m
10 mL
Lipids: Macrogol 4000, DSPC, DPPG, Palmitic acid
Negative
Sulfurhexafluoride
1–5⫻108/mL 99%⬍11m
Room Temp 15–30°C Room Temp 15–30°C
Reconstitute with 10 mL water
Sonovue威12 BR1
IMCOR Pharmaceuticals, Inc. Bracco Diagnostics, Inc.
Reconstitue with 5 mL saline
5 mL
CardioSphere威 PB 127 AI-700
Point Biomedical Corp. Acusphere, Inc.
Slight Negative Negative
Nitrogen3
Sonazoid威 NC100100 BR14
Amersham Health
Polylactide/ Albumin3 Poly-L-lactide co glycolide4 Lipid Stabilized (not disclosed)6 not disclosed
Bracco Diagnostics, Inc.
Negative
Perfluorocarbon4 (not disclosed) Perfluorobutane6
Negative
Perfluorobutane7
Reconstitute with water Reconstitute with 2 mL water5
Varies with conc. 6.5 to 19.5 mL 3 mL
EU-approved LVO/Doppler Canada-approved LVO/Doppler USA and EU approved LVO/EBD/Doppler Canada-approved LVO/EBD Investigational MCE USA-approved LVO/EBD Canada-approved LVO/EBD Investigational MCE USA-approved LVO/EBD Investigational MCE EU-Approved LVO/EBD, micro and macrovascular Doppler (non-cardiac) Investigational MCE Investigational MCE Investigational MCE
2 mL
Development in USA and EU suspended13 Investigational MCE
Abbreviations: DPPA ⫽ Dipalmatoylphosphatidic acid; DPPC ⫽ Dipalmatoylphosphatidylcholine MPEG5000; DPPE ⫽ Dipalmatoylphosphatidalethanolamine; DPPG ⫽ Dipalmitoylphosphatidylglycerol Sodium; DSPC ⫽ Distearoylphosphatidylcholine; DMPC ⫽ Dimyristoylphosphatidalcholine; EBD ⫽ endocardial border detection; EU ⫽ European Union; LVO ⫽ left ventricular opacification; MCE ⫽ myocardial contrast echocardiography; USA ⫽ United States of America; 1 ⫽ Fisher NG, Christiansen JP, Klibanov A, et al. Influence of microbubble surface charge on capillary transit and myocardial contrast enhancement. J Am Coll Cardiol 2002;40:811– 819; 2 ⫽ Website: http://home.intekom.com/pharm/schering/levovist.html; 3 ⫽ Website: http://www.heartandlungresearch.com/protocols/echocard.html, Cheririf et al: Protocol CSP 127-005 A Familiarization and Safety Study of Myocardial Perfusion Contrast Echocardiography with PB127 & Ultrasound Contrast Agent in Patient with Suspected Obstructive Coronary Artery Disease; 4 ⫽ Website: http://www.acusphere.com/700.html AI-700: Our Ultrasound Contrast Agent; 5 ⫽ Lepper W, Hoffmann R, Kamp O, et al: Assessment of myocardial reperfusion by intravenous myocardial contrast echocardiography and coronary flow reserve after primary percutaneous transluminal coronary angiography in patients with acute myocardial infarction. Circulation 2000;101:2368 –2374; 6 ⫽ Hvattum E, Normann PT, Oulie I, et al: Determination of perfluorobutane in rat blood by automatic headspace capillary gas chromatography and selected ion monitoring mass spectrometry. JPBA 2001;24:487– 494; 7 ⫽ Seidel G, Meyer K. Algermissen C, Broillet A: Harmonic imaging of the brain parenchyma using a perfluorobutane-containing ultrasound contrast agent. Ultrasound in Med & Biol 2001;27:915–918; 8 –13 ⫽ Approval Documents: Package Inserts, Summary Basis for Approval, Product Monographs, News Releases.
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Levovist威8 SHU 508A
Status
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Fig. 2. Schematic of a Definity威 “bubble,” showing the phospholipid shell enclosing the octafluoropropane gas. Reproduced with permission from Bristol-Myers Squibb Medical Imaging, Inc., N. Billerica, MA, USA.
more stable, more uniform and, hence, capable of opacifying the myocardium, giving birth to myocardial contrast echocardiography (MCE). These agents have proven to be excellent for perfusion studies along with left ventricular opacification and endocardial border delineation. INDICATIONS FOR CONTRAST ECHOCARDIOGRAPHY
Fig. 1. (a) Scanning electron microscopy image of Imagent威 as a dry powder. The Imagent威 microspheres are produced in a predetermined size range as hollow spheres with the shells comprised predominately of starch, salts and phospholipid. The shells contain pores that allow integration of the perfluorocarbon/nitrogen gas mixture into the hollow microsphere. (b) Light microscopy image of Imagent威 microbubbles following reconstitution. On adding sterile water to the Imagent威 vial, the starch and salts in the shell dissolve to prepare an isotonic suspension, and the phospholipid orients around the gas bubble to produce a stable flexible microbubble. (c) (left) Video micrograph of Cardiosphere bubble containing nitrogen (black) surrounded by two shells consisting of albumin (outer) and polymer (inner); (right) After exposure to diagnostic US, the shells fragment, liberating nitrogen. (a) and (b) reproduced with permission from IMCOR Pharmaceuticals, Inc., San Diego,
Contrast echocardiography is proving to be a useful tool in many facets of cardiology. Hand agitation of saline injected into a peripheral vein is used to opacify the right atrium and ventricle, detect intra-atrial and intraventricular shunts, diagnose hepatopulmonary syndrome and intrapulmonary shunts, enhance Doppler measurements of pulmonary artery pressure, and visualize left-to-right shunts by negative enhancement of contrast-free blood. Further, agitated saline is useful for
CA, USA; and (c) with permission from POINT Biomedical Corporation, San Carlos, CA, USA. Reprinted from J Am Soc Echocardiogr, Vol 15, Leong-Poi H, Song J, Rim SJ, Christiansen J, Kalu S, Lindner JR, Influence of Microbubble Shell Properties on Ultrasound Signal: Implications for Low-Power Perfusion Imaging, pp. 1269 –1276, Copyright (2002), with permission from “The American Society of Echocardiography”.
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providing guidance in pericardiocentesis when performed with US monitoring. Commercially available CE agents have expanded applications in the left side of the heart (Fig. 3). The first-generation agents permitted endocardial border delineation, useful during stress echocardiography or in subjects with poor acoustic windows, as well as Doppler enhancement for color Doppler mapping of mitral regurgitation and continuous-wave Doppler aortic stenosis assessment. Several studies have documented good agreement between CE and angiography or magnetic resonance imaging (MRI) for left ventricular ejection fraction and regional wall motion detection at rest and with stress with both first- and second-generation CE agents (Zotz et al. 1996; Hundley et al. 1998; Falcone et al. 1995; Rainbird et al. 2001). More importantly, MCE with the second-generation agents permits assessment of myocardial perfusion and viability (Fig. 4). With stress and rest imaging, MCE can be used to better assess myocardial tissue ischemia (fixed vs. reversible perfusion defects), coronary artery stenosis, (coronary flow reserve and physiological importance of obstruction), blood volume in the coronary arteries and myocardial capillaries, myocardial stunning and hibernation, myocardium at risk in an acute coronary syndrome (ACS), and “no-reflow” after a revascularization procedure (Fig. 5). Perfusion patterns When coupled with the functional data from the usual TTE examination, MCE is capable of accurately assessing myocardial ischemia, necrosis, stunning and hibernation. In the evaluation of chest pain, a stress and resting MCE could identify: 1. a fixed defect (rest and stress perfusion defects) coupled with akinetic wall motion that would be indicative of necrosis, or 2. a reversible defect (stress defect with normal resting perfusion) coupled with inducible wall motion abnormalities indicative of ischemia. Myocardial stunning would be defined by a resting TTE/MCE study that revealed wall motion abnormalities in a myocardial territory with normal microvasculature perfusion after a recent insult (ACS with reperfusion or severe stress with resolved ischemia), and hibernation would be represented by hypokinetic and hypoperfused myocardium. Coronary artery stenosis and flow reserve Despite significant narrowing, most coronary artery lesions do not result in myocardial perfusion or wall motion changes due to compensatory vasodilation and collateral flow. One way to quantify the physiological significance of a stenotic lesion is to evaluate coronary flow reserve. Coronary flow reserve is defined as the maximal
Fig. 3. Technically difficult echocardiogram performed on a 53-year-old woman referred for history of angina and palpitation. The administration of Imagent威 improved visualization of the lateral wall and septum, and enabled detection of wall thickness, resulting in a normal interpretation. Reproduced with permission from Nanda et al. (2003).
change in flow that can be induced by maximal vasodilatation of a single coronary artery. This measurement is obtained by comparing resting and maximal stress flow using exercise, inotropic agents or chemical vasodilators. In MCE, Lindner et al. (1999) first described a technique to quantify myocardial blood volume and flow velocity. By administering the CE agent at a constant rate and concentration, a steady state is achieved with equal concentrations of CE agent in the blood pool and the myocardium. Using a single high MI pulse, microbubbles are destroyed in the area of interest in the myocardium. The CE agent then replenishes microbubbles to the myocardial capillaries. US pulses are then repeatedly delivered at longer time intervals until maximal video intensity of the CE agent is achieved. A time-intensity curve is calculated, with the rate of microbubble replenishment being consistent with coronary flow velocity, and the
New agents in contrast echocardiography ● A. P. MILLER
Fig. 4. Assessment of myocardial perfusion using power contrast imaging. (top) 4.0-mL bolus injection of SonoVue威 was administered IV while the apical four-chamber view was recorded. Note the presence of apical perfusion defect. (bottom left) six regions-of-interest are displayed on the digital clip, and the resultant time-intensity curves are plotted (right). Contrast first appears in the left ventricle and then in the basal and mid septum and the lateral wall. The apex remains flat, consistent with the patient’s apical filling defect. The rapid decrease of contrast in the lateral wall is consistent with a US dropout artefact. (bottom right) green ⫽ basal septum; dark blue ⫽ mid septum; magenta ⫽ apex; yellow ⫽ anterolateral wall; light blue ⫽ lateral wall; red ⫽ LV cavity. Reproduced with permission from Nanda et al. (2000), Figs. 1A and 1E.
total intensity is used as a measure of myocardial blood volume. Myocardial blood flow is the product of blood volume and velocity. Stress (usually adenosine) and rest measurements of this parameter can be used to calculate severity of coronary artery stenosis. MCE in acute coronary syndromes (ACS) As mentioned above, MCE can identify several abnormal perfusion patterns that, when coupled with the functional data from the TTE examination, yield diagnoses of myocardial ischemia, necrosis, stunning and hibernation. In ACS, these data have important ramifications for interventions and prognosis. Due to its ability to provide spatial and temporal resolution, real-time imaging and accessibility for patient care in all areas of the hospital, MCE should expand from its limited research role to a first-line diagnostic modality for patients with ACS. In patients with acute myocardial infarction, MCE will initially reveal only a small amount of contrast in the area of involved myocardium, identifying myocardium at risk.
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Fig. 5. Assessment of myocardial perfusion using harmonic power Doppler and a continuous IV infusion of Cardiosphere威 0.175 mg/kg body weight. Apical three-chamber views acquired after (left) four beats of refill at rest and (right) after one beat at stress using dipyridamole infusion show a reversible defect in the left ventricular inferior wall. Reproduced with permission from POINT Biomedical Corporation, San Carlos, CA, USA.
With revascularization or reperfusion, contrast will be seen in the area of viable myocardium with intact microvasculature integrity, and this portion will demonstrate a pattern consistent with stunned myocardium and usually recover function. “No-reflow” areas will be identified by persistent poor contrast enhancement and will usually become scar. Several reports have validated this technique and its prognostic implications (Villanueva et al. 1993; Ito et al. 1992, 1996; Rocchi et al. 2001).
Fig. 6. (a) Low MI real-time imaging with full myocardial opacification. (b) A higher MI pulse creates a “flash” effect and destroys contrast in the myocardium. (c) The myocardium is cleared of contrast agent; and (D) seconds later contrast has been replenished in myocardium. Reproduced with permission from “A compendium of advanced technologies for echocardiography” by ATL, A Philips Medical Systems Co., 2000, Bothell, WA, USA.
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COMPARISON OF CONTRAST ECHOCARDIOGRAPHY WITH OTHER NONINVASIVE IMAGING MODALITIES Currently, myocardial perfusion imaging with radiolabeled tracers is the most commonly used method for evaluating myocardial perfusion and viability. This technique is limited, though, by its restriction to licensed centers, lack of portability, expense, inability to perform real-time imaging, low resolution, attenuation artefacts, use of ionizing radiation and requirements for radioactive waste disposal. On the other hand, MCE is more widely available, relatively inexpensive in terms of examination and equipment cost, portable, provides precise real-time anatomical and perfusion/ viability data and uses no ionizing radiation. Comparisons of these two imaging modalities continues favorably to demonstrate the assets of MCE (Kaul et al. 1997; Marwick et al. 1998; Heinle et al. 2000; Rocchi et al. 2001; Lepper et al. 2003). Kaul et al. (1997) first demonstrated the effectiveness of a second-generation CE agent for perfusion imaging. In a study of 30 participants with suspected or known coronary artery disease (CAD), MCE with Optison威 was compared to 99mTc-sestamibi single-photon emission computed tomography (SPECT) for concordance of 10 segmental perfusion scores and for the determination of the presence or absence of CAD. Concordance for segmental scores was 92% and agreement for the detection of CAD was 86%. However, when tested in a larger population, a different contrast agent, Sonazoid威, did not fare as well with the technology of the late 1990s. Marwick et al. (1998) compared fundamental or harmonic MCE with continuous or triggered imaging with 99mTc-sestamibi SPECT in 203 participants with known CAD. Although the specificity for detection of extensive (⬎ 12% of the left ventricle) perfusion defects was good (63% to 100% depending on the technique used), sensitivity was limited with MCE alone (13% to 48%). When coupled with wall motion data, sensitivity did improve to 46% to 55%. Heinle et al. (2000) defined what has become the Achilles’ heel of MCE, falsely abnormal lateral wall perfusion defects. In a study of 123 participants referred for SPECT imaging for known or suspected CAD, MCE was performed using harmonic power Doppler imaging and Optison威 at rest and during adenosine stress as well. Overall concordance for MCE and SPECT was 81%, but was only 72% for the left circumflex artery distribution. Notably, 33% of participants demonstrated fixed defects of the lateral wall by MCE, but only 14% did by SPECT. The further refinement of harmonic power Doppler imaging has led to improved accuracy of MCE, as more recent evidence compares MCE and SPECT to catheterbased assessments of coronary perfusion. Rocchi et al.
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(2001) documented 82% sensitivity and 95% specificity for MCE-identification of infarct-related artery perfusion defects in 30 patients with conservatively treated acute myocardial infarctions when compared with a coronary angiogram “gold standard.” Accuracies for MCE and SPECT were similar in this study. More recently, Lepper et al. (2003) compared MCE and SPECT with pressurewire–measured fractional flow reserve in 21 participants who presented for diagnostic coronary angiography in evaluation of chest pain. Agreement for MCE was 90%, but agreement for SPECT was only 71%. METHODOLOGY OF CONTRAST ECHOCARDIOGRAPHY The evolution of the equipment “hardware” for MCE has probably yielded the most innovations in this field. Instrument settings have grown from fundamental B-mode imaging to harmonic B-mode imaging and now to harmonic power Doppler imaging. Interrupted or ECG-triggered imaging was followed by pulse-inversion imaging and now by real-time power Doppler pulseinversion imaging at low MI. Three methods are predominantly used for perfusion imaging today: 1. interrupted or ECG-gated imaging in conjunction with harmonic power Doppler mode; 2. realtime power Doppler pulse inversion imaging at low MI combined with high MI bubble destruction; and 3. a combination of the previous two methods with endsystolic, ECG-gated, real-time imaging of every beat. Triggered imaging at a normal MI allows systolic gating for imaging of the myocardium at its thickest portion, which creates the best resolution for detecting perfusion patterns. With this method, a time-intensity curve can also be created with quantification of myocardial blood volume and coronary artery flow, as previously described. Limitations of this technique include movement artefacts, corruption of the image and the fact that imaging is not real-time. Real-time imaging at a low MI permits evaluation of instantaneous perfusion changes and offers improved application to stress imaging or assessment of reperfusion (Fig. 6). This technique suffers from a lower signalto-noise ratio and also from poor imaging of the lateral left ventricular wall. A combination technique of ECG-gating to obtain the thickest myocardial segment in systole and near real-time imaging with capture of signal from each beat is an appealing solution to the above limitations. With the improved signal-generating contrast agents currently available and refined instrumentation, this method should allow portable real-time perfusion imaging.
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FUTURE APPLICATIONS OF CONTRAST ECHOCARDIOGRAPHY Several unique properties of CE agents have been documented that may yield new diagnostic and therapeutic possibilities. We will discuss: 1. assessing endothelial function; 2. detecting vascular inflammation; and 3. delivering gene/drug therapies. Endothelial function Endothelial dysfunction is an early step in the development of symptomatic coronary artery disease and is currently a topic of intense research. Current US technologies crudely determine endothelial health by evaluating macrovascular flow patterns or responses to vasodilators. However, microbubbles may offer a more direct approach. Intercellular adhesion molecule-1 (ICAM-1) and other adhesion molecules are overexpressed in activated endothelial cells in probably the first step that leads to atherosclerosis (Ross 1999). Villanueva et al. (1998) placed monoclonal antibodies to human ICAM-1 on the shell of a microbubble. In vitro, anti-ICAM-1 bubbles bind to endothelial cells overexpressing ICAM-1, which could potentially lead to clinical use as direct evidence of early atherogenesis. Inflammation Taking this research tool one step further down the evolution of an atherosclerotic plaque, microbubbles have also been shown to attach to activated leukocytes. Originally, the hypothesis that microbubbles marked inflammation was postulated from observations that airfilled albumin CE agents administered by intracoronary injection “hung up” in regions of myocardial injury (Lindner et al. 1998). Further research has yielded direct observation of albumin-coated microbubbles bound to and phagocytosed by leukocytes in inflamed tissue (Lindner et al. 2000). As a marker of vascular inflammation, CE agents may provide evidence of plaque instability, reperfusion injury extent and restenosis likelihood in the not-so-distant future. Gene/drug therapy delivery CE agents offer a unique mechanism for delivery of therapeutic agents to specific anatomical and histologic targets. Microbubbles could be engineered with an adenovirus vector or pharmaceutical agent attached to the shell and injected IV. Then, destruction of microbubbles at high MI could be targeted to the sonicated area of a specific organ, with a high concentration delivered to that area. Further, other shell additives, such as adhesion molecule antibodies, could guide the microbubble to the target cell type.
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Initial work in this field has already yielded promising results. Shohet et al. (2000) incorporated recombinant adenovirus containing -galactosidase into the albumin surface of perfluoropropane-filled microbubbles and successfully delivered the functioning vector to the myocardium by US-mediated microbubble destruction. Drug and gene delivery should be a blossoming subspecialty of CE in the very near future. CONCLUSION The application of echocardiography is expanding with the development of contrast agents that permit improved anatomical assessment, perfusion imaging and special applications in vascular inflammation and drug delivery. Further refinement of the “hardware” (mechanical index, triggered and real-time imaging) and new developments in the “software” (CE agents) of CE should continue to revolutionize this nearly limitless field of echocardiography. Acknowledgments—The authors acknowledge with thanks help from IMCOR Pharmaceuticals, Inc., Bristol-Myers Squibb, Philips Ultrasound and POINT Biomedical Corporation.
REFERENCES Becher H, Burns PN. Handbook of contrast echocardiography: Left ventricular function and myocardial perfusion. Heidelberg: Springer Verlag, 2000. Carroll BA, Turner RJ, Tickner EG, et al. Gelatin encapsulated nitrogen microbubbles as ultrasonic contrast agents. Invest Radiol 1980; 15:260–266. Falcone RA, Marcovitz PA, Perez JE, et al. Intravenous Albunex during dobutamine stress echocardiography: Enhanced localization of left ventricular endocardial borders. Am Heart J 1995;130:254– 258. Feinstein SB, Shah PM, Bing RJ, et al. Microbubble dynamics visualized in the intact capillary circulation. J Am Coll Cardiol 1984a;4: 595–600. Feinstein SB, Ten Cate FJ, Zwehl W, et al. Two-dimensional constrast echocardiography. I. In vitro development and quantitative analysis of echo contrast agents. J Am Coll Cardiol 1984b;3:14–20. Fisher NG, Christiansen JP, Klibanov A, et al. Influence of microbubble surface charge on capillary transit and myocardial contrast enhancement. J Am Coll Cardiol 2002;40:811–819. Frinking PJA, Bouakaz A, Kirkhorn J, et al. Ultrasound contrast imaging: Current and new potential methods. Ultrasound Med Biol 2000;26:965–975. Gramiak R, Shah PM. Echocardiography of the aortic root. Invest Radiol 1968;3:356–366. Gramiak R, Nanda NC, Shah PM. Echocardiographic detection of the pulmonary valve. Radiology 1972;102:153–157. Heinle SK, Noblin J, Goree-Best P, et al. Assessment of myocardial perfusion by harmonic power Doppler imaging at rest and during adenosine stress: Comparison with 99mTc-sestamibi SPECT imaging. Circulation 2000;102:55–60. Hundley WG, Kizilbash AM, Afridi I, et al. Adminstration of an intravenous perflurorocarbon contrast agent improves echocardiographic determination of left ventricular volumes and ejection fraction: Comparison with cine magnetic resonance imaging. J Am Coll Cardiol 1998;32:1426–1432. Ito H, Maruyama A, Iwakura K, et al. Clinical implications of the “no reflow” phenomenon: A predictor of complications and left ven-
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tricular remodeling in reperfused anterior wall myocardial infarction. Circulation 1996;93:223–228. Ito H, Tomooka T, Sakai N, et al. Lack of myocardial perfusion immediately after successful thrombolysis: A prediction of poor recovery of left ventricular function in anterior myocardial infarction. Circulation 1992;85:1699–1705. Kaul S, Senior R, Dittrich H, et al. Detection of coronary artery disease with myocardial contrast echocardiography: Comparison with 99m Tc-sestamibi single-photon emission computed tomography. Circulation 1997;96:785–792. Lepper W, Nowak B, Franke A, et al. Myocardial contrast echocardiography, single-photon emission computed tomography, and regional function analysis for coronary stenosis description during vasodilator stress. Am J Cardiol 2003;91:445–448. Linder JR, Dayton PA, Coggins MP, et al. Non-invasive imaging of inflammation by ultrasound detection of phagocytosed microbubbles. Circulation 2000;102:531–538. Lindner JR, Ismail S, Spotnitz WD, et al. Albumin microbubble persistence during myocardial contrast echocardiography is associated with microvascular endothelial glycocalyx damage. Circulation 1998;98:2187–2194. Lindner JR, Wei K, Kaul S. Imaging of myocardial perfusion with Sonovue in patients with a prior myocardial infarction. Echocardiography 1999;16:753–760. Marwick TH, Brunken R, Meland N, et al. Accuracy and feasibility of contrast echocardiography for detection of perfusion defects in routine practice: Comparison with wall motion and technetium99m sestamibi single-photon emission computed tomography. J Am Coll Cardiol 1998;32:1260–1269. McCullough M, Gresser C, Moos S, et al. Ultrasound contrast physics. J Am Soc Echocardiogr 2000;13:959–967. Nanda NC, et al. Assessment of myocardial perfusion by power contrast imaging using a new echo contrast agent. Echocardiography 2000;17:457–461. Nanda NC, et al. Imagent improves endocardial border delineation, inter-reader agreement, and the accuracy of segmental wall motion assessment. Echocardiography 2003;20:151–161. Nanda NC, Schlief R, Goldberg BB, eds. Advances in echo imaging using contrast enhancement, 2nd ed. London: Kluwer Academic, 1997.
Volume 30, Number 4, 2004 Nanda NC, Shah PM, Gramiak R. Echocardiographic evaluation of tricuspid valve incompetence by contrast injections (Abstr.). Clin Res 1976;24:233A. Nanda NC, Steward S, Gramiak R, Manning JA. Echocardiography of the intra-atrial baffle in dextro-transposition of the great vessels. Circulation 1975;51:1130–1135. Rainbird AJ, Mulvagh SL, Oh JK, et al. Contrast dobutamine stress echocardiography: Clinical practice assessment in 300 consecutive patients. J Am Soc Echocardiogr 2001;14:378–385. Raisinghani A, DeMaria AN. Physical principles of microbubble ultrasound contrast agents. Am J Cardiol 2002;90(Suppl.):3J–7J. Rocchi G, Kasprzak JD, Galema TW, et al. Usefulness of power Doppler contrast echocardiography to identify reperfusion after acute myocardial infarction. Am J Cardiol 2001;87:278–282. Ross R. Atherosclerosis—An inflammatory disease. N Engl J Med 1999;340:115–126. Rothbard RL, Nanda NC. Contrast echocardiography. Semin Ultrasound 1981;2:167–172. Rothbard RL, Nanda NC. How to apply contrast echocardiographic techniques. J Cardiovasc Med 1983;736 –744. Shohet RV, Chen S, Zhou YT, et al. Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium. Circulation 2000;101:2554–2556. Skyba DM, Price RJ, Linka AZ, et al. Direct in vivo visualization of microvessel rupture and tissue injury caused by intravascular destruction of microbubbles by ultrasound. Circulation 1998;98: 1279–1285. Van Der Wouw PA, Brauns AC, Bailey SE, et al. Premature ventricular contractions during triggered imaging with ultrasound contrast. J Am Soc Echocardiogr 2000;13:288–294. Villanueva FS, Glasheen WP, Sklenar J, Kaul S. Characterization of spatial patterns of flow within the reperfused myocardium by myocardial contrast echocardiography: Implications in determining extent of myocardial salvage. Circulation 1993;88:2596–2606. Villanueva FS, Jankowski RJ, Klibanov S, et al. Microbubbles targeted to intercellular adhesion molecule-1 bind to activated coronary artery endothelial cells: A novel approach to assessing endothelial function using myocardial contrast echocardiography. Circulation 1998;98:1–5. Zotz RJ, Genth S, Waaler A, et al. Left ventricular volume determination using Albunex. J Am Soc Echocardiogr 1996;9:1–8.
doi:10.1016/j.ultrasmedbio.2003.12.002
● Review Article CONTRAST ECHOCARDIOGRAPHY: NEW AGENTS ANDREW P. MILLER and NAVIN C. NANDA* Division of Cardiovascular Diseases, University of Alabama at Birmingham, Birmingham, AL, USA (Received 5 June 2003; revised 23 November 2003; in final form 9 December 2003)
Abstract—In this report, we review the history, rationale, current status and future directions of contrast agents in echocardiography. First, we discuss the historic development of contrast agents through a review of important physical principles of microbubbles in ultrasonography. Second, we identify attributes of an ideal contrast agent and review those that are currently available or in the “pipeline” for clinical use. Third, we review indications for contrast echocardiography, including endocardial border detection, perfusion quantification and reperfusion assessment, and validate these observations by comparisons with other imaging modalities. Then, we briefly review different methodologies of performing a contrast study, including interrupted, real-time and a hybrid modality. Finally, we identify novel future applications of the newest contrast agents. These newer concepts in contrast echocardiography should form a foundation for nearly limitless application of echocardiography in improved anatomical assessment, perfusion imaging and even special applications, such as detection of vascular inflammation and site-specific drug delivery. (E-mail: [email protected]) © 2004 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound, Blood flow, Contrast agent, Inflammation, Myocardial contrast echocardiography, History, Quantification.
INTRODUCTION
PHYSICAL PRINCIPLES OF MICROBUBBLES IN ULTRASONOGRAPHY
The search for a comprehensive imaging modality that best evaluates cardiac function, perfusion and structure continues in the practice of cardiology. In recent years, the diagnostic application of cardiac ultrasound (US) imaging has greatly expanded with the advent of contrast echocardiography (CE). Through the injection of microbubbles and microbubble contrast agents, CE is already a valuable tool to delineate endocardial borders, direct invasive procedures, detect intracardiac shunts, assess myocardial perfusion and viability, and quantify coronary flow reserve and blood volumes. Further, the future potential of this new technique seems to be limitless, as it currently reaches beyond diagnostic uses and into therapeutic delivery of growth factors, genes and antithrombotic agents. In this report, we discuss the history, rationale, current status and future directions of contrast agents in echocardiography.
History Contrast echocardiography has a long history. CE began with the observation by Joyner and publication by Gramiak and Shah (1968) that injection of saline into the ascending aorta and, thus, the production of microbubbles during echocardiographic recording resulted in better echo signals in the lumen of the aorta and heart chambers. In the early 1970s, saline CE was used to delineate intracardiac shunts, to define congenital heart disease and to examine the right-sided valves as an adjunct to the normal transthoracic echocardiography (TTE) exam (Nanda et al. 1975, 1976, 1997; Gramiak et al. 1972; Rothbard and Nanda 1981, 1983). The commercial development of contrast agents began in the 1980s with observations that led to the stabilization and miniaturization of the microbubble. Carroll et al. (1980) demonstrated that gelatin-encapsulated nitrogen bubbles were stable enough to be used for US enhancement. Then, Feinstein et al. (1984a) showed that microbubbles sonicated from human serum albumin were small and stable enough to traverse the pulmonary circulation and to opacify the left ventricle. These findings led to signif-
Address correspondence to: Navin C. Nanda, M.D., University of Alabama at Birmingham, Heart Station, SW/S102, 619 South 19th Street, Birmingham, AL 35249 USA. E-mail: [email protected] 425
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icant interest in the development of a commercially available US contrast agent by many large and small pharmaceutical companies. Several programs were initiated to develop an easy to use and clinically useful contrast agent for ultrasound. Schering AG (Berlin, Germany) was the first company to be successful. Their first agent was Echovist威 (1982), which enabled right side enhancement only, followed by Levovist威 (1985), which achieved enhancement of the left ventricle. Schering’s agents were approved in Europe, Japan and Canada. Molecular Biosystems Inc. (MBI; San Diego, CA) developed Albunex威, the first agent approved by the US Food and Drug Administration (FDA) in 1994 for USA distribution. The agent was based on technology utilizing sonicated human serum albumin to encapsulate and stabilize the air microbubble. Both Albunex威 and Levovist威 utilize air as the gas component of the microbubble. This resulted in reduced longevity in vivo and limited the commercial success of these agents. The use of various types of perfluorocarbon (PFC) gas to increase the persistence of the microbubble was first introduced in the early 1990s. Many agents under development incorporated PFC technology into their formulations. These products include reformulation of the Albunex威 technology utilizing a PFC gas. This product, Optison威 (MBI), was approved by the FDA for commercial use in 1997. Additional agents have been developed that utilize PFC gas and vary the constituents in the shell that encapsulate the gas. The two such agents currently approved by the FDA are Definity威 (2001) distributed by Bristol Myers Squibb (New York, NY) and Imagent威 (2002), distributed by IMCOR (IMCOR, San Diego, CA), a division of Photogen Technologies Inc. Another agent, SonoVue威 (Bracco Diagnostics Inc., Princeton, NJ), utilizes sulfur hexafluoride, a low-solubility fluorinated gas, and is currently approved only in Europe (2001). These development activities have resulted in smaller, more stable microbubble agents, which, when coupled with improvements in US equipment technology, have resulted in the extension of CE from opacification to perfusion.
change that results in CE signal. The inherent properties of the bubble shell and the gas inside determine the output signature of each microbubble. The mechanical index (MI) of the US beam is used to modulate this output and to incite different bubble responses. MI is defined as the peak rarefactional pressure (i.e., the peak negative pressure) divided by the square root of the US frequency (McCullough et al. 2000). In other words, MI is the amount of negative acoustic pressure within a US field. In clinical practice, the range for MI varies from 0.05 to 1.9. By changing MI, the ultrasonographer can create three phenomena of CE (Becher and Burns 2000). First, at a low MI (0.1), the microbubbles undergo linear oscillation with compression and rarefaction that are equal in amplitude. No special CE signal is created at this stage using fundamental imaging. When the MI is increased (0.2 to 0.7), nonlinear oscillation occurs preferentially with the bubbles undergoing rarefaction that is greater than compression, and ultrasonic waves are created at harmonics of the delivered frequency. These CE signals are bubble-specific. Finally, although microbubble destruction occurs to some degree at all mechanical indices, microbubble destruction is extensive at the highest MI (0.8 to 1.9), creating a brief, but high, output signal that is unique to the contrast agent.
CE physics The mechanism of microbubble CE is based on the physical principles of rarefaction and compression created by a US wave insonating a gas-filled microbubble along with the mechanical index of the ultrasonic beam. Rarefaction occurs with the reduction in pressure of the gas when struck by the negative portion of an acoustic wave, and compression refers to the rise in pressure of a medium struck by the positive portion of the acoustic cycle (Frinking et al. 2000). Compression and rarefaction lead to volume pulsations of microbubbles, and it is this
where R ⫽ bubble radius, d ⫽ gas density, DIFS ⫽ gas diffusivity and constsat ⫽ saturation constant (Raisinghani and DeMaria 2002). Thus, a shell with low gas diffusivity and a gas with a low saturation constant and high density would result in longer persistence. The search for a gas with these properties led to the perfluorocarbons. The second great advance in CE was the ability reliably to produce microbubbles small enough to traverse the pulmonary circulation. The ideal agent for left ventricular opacification and, eventually, for perfusion
Contrast agent principles The first major advancement in CE came with development of more stable microbubbles. When agitated saline containing air bubbles was injected in the early studies, the air would quickly leak from the thin bubble shell into the blood, where it dissolved. The smaller bubbles that were capable of traversing the capillary bed did not survive long enough for imaging because the small amount of air quickly dissipated into the blood. So, persistence was sought in the form of a more stable shell and a higher density gas. Persistence is defined by the equation: 共R ⫻ d兲/共2DIFS ⫻ constsat兲
New agents in contrast echocardiography ● A. P. MILLER
studies, is an agent that could pass this capillary bed. This meant developing agents for which the predominant population of the microbubbles approximated the size of red blood cells (6 to 8 m in diameter) while minimizing the presence of larger-sized microbubbles that could plug microvascular flow. Most of the currently available products have been shown to transit the microcirculation successfully. Finally, recent research with contrast agents has focused on investigating the unique properties that permit targeting of tissues, or even cells. Some agents currently in development have demonstrated the ability to deposit in myocardial tissues, attach to endothelial cells, or even absorb into leukocytes. These distinctive features permit improved tissue contrast, potential targeting of therapies and inflammation or endothelial health assessment. ATTRIBUTES OF AN IDEAL CONTRAST AGENT The explosion of CE has led to the development of many new contrast agents, as well as improvements in the equipment available to image the agents. Optimization of the agents, as well as the equipment, continues. Several physical properties of microbubbles decide their utility, some in conflict with others. For example, the ideal agent would be resilient enough to freely transit the microcirculation and persist in the bloodstream, but fragile enough to be disrupted by US energy. We will explore the attributes of the ideal agent individually. First, as discussed above, the imaging properties of an agent are established by the shell and the gas. The shell must sufficiently stabilize the microbubble to provide adequate persistence, permitting the CE agent to reach the left ventricle or myocardium for imaging. Persistence is also defined by the density and diffusibility of the gas within the shell, and the ideal agent would contain a gas of high density and low diffusibility. However, these properties must be balanced with the ultimate goal of CE, which is to disrupt or even explode the microbubble by insonating with US energy. In other words, a balance of persistence and fragility is required. Additionally, the ideal agent would be neutrally charged to freely transit the microcirculation without interacting with the blood components, be imaged at low MI and be disrupted at higher MI (Fisher et al. 2002). Second, imaging artefacts, the plague of echocardiography, must be limited. The reflected energy of CE agents is often very high, resulting in attenuation that may obscure structures distal to the imaging agent. Attenuation of the image can be impacted by the size range of the microbubbles, with larger microbubbles causing greater attenuation. The concentration of the micro-
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bubbles within the imaging window can also affect the degree of attenuation observed. For example, contrast enhancement of the anterior wall may lead to an artificial “cold spot” in the inferior wall. The ideal agent would produce enough output to provide enhancement without shadowing distal structures to the point of inhibiting interpretation. Third, additional special properties are required for the CE agents under development for targeting of leukocytes or endothelial cells. For example, some agents have been shown to be taken up by endothelial cells. If a therapeutic agent could be incorporated into the shell or the gas of these agents, targeted disruption could provide a unique delivery system to the endothelium. Further, inflammation as a mechanism of atherosclerosis and of myocardial injury is currently a topic of intense study. CE agents that have been shown to target leukocytes may provide exceptional markers for local inflammation, and are currently under investigation to study this phenomenon. Finally, to reach from the bench to the bedside, CE agents must be safe, be easily used by physicians and nurses, and be of low cost. The early experience with saline contrast taught us that CE could lead to a potentially significant air embolus. With the current microbubble technology, the small size of the commercially available CE agents reduces this concern; however, some complications have been observed (Skyba et al. 1998; Van Der Wouw et al. 2000). CONTRAST AGENTS APPROVED AND “IN THE PIPELINE” FOR CLINICAL USE In the USA, three CE agents for left ventricular opacification and endocardial border detection are commercially available: Optison威, Definity威 and Imagent威. Several others are on the horizon: Levovist威 and Sonovue威 are already approved in Europe, and investigational agents continue to emerge. Table 1 lists these CE agents, their development status and their properties. As the agents have become smaller, more stable, and more uniform, more exciting applications for these agents, such as microvasculature assessment, have become possible. Albunex威 (MBI), not listed in Table 1 because it is now unavailable, was the first generation agent that utilized sonicated human albumin to create a 4-m air-filled microbubble for left ventricular opacification. This agent, developed by Feinstein et al. (1984b), did pass to the left ventricle and enhance endocardial border detection, but it poorly demonstrated microvasculature perfusion. Second-generation agents were developed that incorporated high-density gases with low diffusion and saturation constants (i.e., perfluorocarbons) into the microbubble (Figs. 1 and 2). These agents were
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Table 1. Echo Contrast Agents Agent Research Name
Company
Shell
Charge1
Gas
Microbubble Count/size
Storage
Prep
Vial volume
Galactose/Palmitic acid
Negative
Air
Varies with concentration2 95%⬍10m
Room temp 15–30°C
Reconstitute with 5 to 17 mL water
Optison威9 FS069
Amersham Health
Human Serum Albumin N-acetyltryptophan, Caprylic acid
Slight Negative
Octafluoropropane
5–8⫻108/mL 93%⬍10m
Refrigerate 2–8°C
Hand agitate
Definity威10 DMP 115
Bristol-Myers Squibb Medical Imaging, Inc.
Lipids:DPPA, DPPC, MPEG5000 DPPE
Negative
Octafluoropropane
1.2⫻1010/mL 98%⬍10m
Refrigerate 2–8°C
Activate through Vialmix agitation
2 mL
Imagent威11 AF0150
Lipid: DMPC
Neutral
Perfluorohexane/ Nitrogen
1.4⫻109/mL 99.8%⬍10m
10 mL
Lipids: Macrogol 4000, DSPC, DPPG, Palmitic acid
Negative
Sulfurhexafluoride
1–5⫻108/mL 99%⬍11m
Room Temp 15–30°C Room Temp 15–30°C
Reconstitute with 10 mL water
Sonovue威12 BR1
IMCOR Pharmaceuticals, Inc. Bracco Diagnostics, Inc.
Reconstitue with 5 mL saline
5 mL
CardioSphere威 PB 127 AI-700
Point Biomedical Corp. Acusphere, Inc.
Slight Negative Negative
Nitrogen3
Sonazoid威 NC100100 BR14
Amersham Health
Polylactide/ Albumin3 Poly-L-lactide co glycolide4 Lipid Stabilized (not disclosed)6 not disclosed
Bracco Diagnostics, Inc.
Negative
Perfluorocarbon4 (not disclosed) Perfluorobutane6
Negative
Perfluorobutane7
Reconstitute with water Reconstitute with 2 mL water5
Varies with conc. 6.5 to 19.5 mL 3 mL
EU-approved LVO/Doppler Canada-approved LVO/Doppler USA and EU approved LVO/EBD/Doppler Canada-approved LVO/EBD Investigational MCE USA-approved LVO/EBD Canada-approved LVO/EBD Investigational MCE USA-approved LVO/EBD Investigational MCE EU-Approved LVO/EBD, micro and macrovascular Doppler (non-cardiac) Investigational MCE Investigational MCE Investigational MCE
2 mL
Development in USA and EU suspended13 Investigational MCE
Abbreviations: DPPA ⫽ Dipalmatoylphosphatidic acid; DPPC ⫽ Dipalmatoylphosphatidylcholine MPEG5000; DPPE ⫽ Dipalmatoylphosphatidalethanolamine; DPPG ⫽ Dipalmitoylphosphatidylglycerol Sodium; DSPC ⫽ Distearoylphosphatidylcholine; DMPC ⫽ Dimyristoylphosphatidalcholine; EBD ⫽ endocardial border detection; EU ⫽ European Union; LVO ⫽ left ventricular opacification; MCE ⫽ myocardial contrast echocardiography; USA ⫽ United States of America; 1 ⫽ Fisher NG, Christiansen JP, Klibanov A, et al. Influence of microbubble surface charge on capillary transit and myocardial contrast enhancement. J Am Coll Cardiol 2002;40:811– 819; 2 ⫽ Website: http://home.intekom.com/pharm/schering/levovist.html; 3 ⫽ Website: http://www.heartandlungresearch.com/protocols/echocard.html, Cheririf et al: Protocol CSP 127-005 A Familiarization and Safety Study of Myocardial Perfusion Contrast Echocardiography with PB127 & Ultrasound Contrast Agent in Patient with Suspected Obstructive Coronary Artery Disease; 4 ⫽ Website: http://www.acusphere.com/700.html AI-700: Our Ultrasound Contrast Agent; 5 ⫽ Lepper W, Hoffmann R, Kamp O, et al: Assessment of myocardial reperfusion by intravenous myocardial contrast echocardiography and coronary flow reserve after primary percutaneous transluminal coronary angiography in patients with acute myocardial infarction. Circulation 2000;101:2368 –2374; 6 ⫽ Hvattum E, Normann PT, Oulie I, et al: Determination of perfluorobutane in rat blood by automatic headspace capillary gas chromatography and selected ion monitoring mass spectrometry. JPBA 2001;24:487– 494; 7 ⫽ Seidel G, Meyer K. Algermissen C, Broillet A: Harmonic imaging of the brain parenchyma using a perfluorobutane-containing ultrasound contrast agent. Ultrasound in Med & Biol 2001;27:915–918; 8 –13 ⫽ Approval Documents: Package Inserts, Summary Basis for Approval, Product Monographs, News Releases.
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Levovist威8 SHU 508A
Status
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Fig. 2. Schematic of a Definity威 “bubble,” showing the phospholipid shell enclosing the octafluoropropane gas. Reproduced with permission from Bristol-Myers Squibb Medical Imaging, Inc., N. Billerica, MA, USA.
more stable, more uniform and, hence, capable of opacifying the myocardium, giving birth to myocardial contrast echocardiography (MCE). These agents have proven to be excellent for perfusion studies along with left ventricular opacification and endocardial border delineation. INDICATIONS FOR CONTRAST ECHOCARDIOGRAPHY
Fig. 1. (a) Scanning electron microscopy image of Imagent威 as a dry powder. The Imagent威 microspheres are produced in a predetermined size range as hollow spheres with the shells comprised predominately of starch, salts and phospholipid. The shells contain pores that allow integration of the perfluorocarbon/nitrogen gas mixture into the hollow microsphere. (b) Light microscopy image of Imagent威 microbubbles following reconstitution. On adding sterile water to the Imagent威 vial, the starch and salts in the shell dissolve to prepare an isotonic suspension, and the phospholipid orients around the gas bubble to produce a stable flexible microbubble. (c) (left) Video micrograph of Cardiosphere bubble containing nitrogen (black) surrounded by two shells consisting of albumin (outer) and polymer (inner); (right) After exposure to diagnostic US, the shells fragment, liberating nitrogen. (a) and (b) reproduced with permission from IMCOR Pharmaceuticals, Inc., San Diego,
Contrast echocardiography is proving to be a useful tool in many facets of cardiology. Hand agitation of saline injected into a peripheral vein is used to opacify the right atrium and ventricle, detect intra-atrial and intraventricular shunts, diagnose hepatopulmonary syndrome and intrapulmonary shunts, enhance Doppler measurements of pulmonary artery pressure, and visualize left-to-right shunts by negative enhancement of contrast-free blood. Further, agitated saline is useful for
CA, USA; and (c) with permission from POINT Biomedical Corporation, San Carlos, CA, USA. Reprinted from J Am Soc Echocardiogr, Vol 15, Leong-Poi H, Song J, Rim SJ, Christiansen J, Kalu S, Lindner JR, Influence of Microbubble Shell Properties on Ultrasound Signal: Implications for Low-Power Perfusion Imaging, pp. 1269 –1276, Copyright (2002), with permission from “The American Society of Echocardiography”.
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providing guidance in pericardiocentesis when performed with US monitoring. Commercially available CE agents have expanded applications in the left side of the heart (Fig. 3). The first-generation agents permitted endocardial border delineation, useful during stress echocardiography or in subjects with poor acoustic windows, as well as Doppler enhancement for color Doppler mapping of mitral regurgitation and continuous-wave Doppler aortic stenosis assessment. Several studies have documented good agreement between CE and angiography or magnetic resonance imaging (MRI) for left ventricular ejection fraction and regional wall motion detection at rest and with stress with both first- and second-generation CE agents (Zotz et al. 1996; Hundley et al. 1998; Falcone et al. 1995; Rainbird et al. 2001). More importantly, MCE with the second-generation agents permits assessment of myocardial perfusion and viability (Fig. 4). With stress and rest imaging, MCE can be used to better assess myocardial tissue ischemia (fixed vs. reversible perfusion defects), coronary artery stenosis, (coronary flow reserve and physiological importance of obstruction), blood volume in the coronary arteries and myocardial capillaries, myocardial stunning and hibernation, myocardium at risk in an acute coronary syndrome (ACS), and “no-reflow” after a revascularization procedure (Fig. 5). Perfusion patterns When coupled with the functional data from the usual TTE examination, MCE is capable of accurately assessing myocardial ischemia, necrosis, stunning and hibernation. In the evaluation of chest pain, a stress and resting MCE could identify: 1. a fixed defect (rest and stress perfusion defects) coupled with akinetic wall motion that would be indicative of necrosis, or 2. a reversible defect (stress defect with normal resting perfusion) coupled with inducible wall motion abnormalities indicative of ischemia. Myocardial stunning would be defined by a resting TTE/MCE study that revealed wall motion abnormalities in a myocardial territory with normal microvasculature perfusion after a recent insult (ACS with reperfusion or severe stress with resolved ischemia), and hibernation would be represented by hypokinetic and hypoperfused myocardium. Coronary artery stenosis and flow reserve Despite significant narrowing, most coronary artery lesions do not result in myocardial perfusion or wall motion changes due to compensatory vasodilation and collateral flow. One way to quantify the physiological significance of a stenotic lesion is to evaluate coronary flow reserve. Coronary flow reserve is defined as the maximal
Fig. 3. Technically difficult echocardiogram performed on a 53-year-old woman referred for history of angina and palpitation. The administration of Imagent威 improved visualization of the lateral wall and septum, and enabled detection of wall thickness, resulting in a normal interpretation. Reproduced with permission from Nanda et al. (2003).
change in flow that can be induced by maximal vasodilatation of a single coronary artery. This measurement is obtained by comparing resting and maximal stress flow using exercise, inotropic agents or chemical vasodilators. In MCE, Lindner et al. (1999) first described a technique to quantify myocardial blood volume and flow velocity. By administering the CE agent at a constant rate and concentration, a steady state is achieved with equal concentrations of CE agent in the blood pool and the myocardium. Using a single high MI pulse, microbubbles are destroyed in the area of interest in the myocardium. The CE agent then replenishes microbubbles to the myocardial capillaries. US pulses are then repeatedly delivered at longer time intervals until maximal video intensity of the CE agent is achieved. A time-intensity curve is calculated, with the rate of microbubble replenishment being consistent with coronary flow velocity, and the
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Fig. 4. Assessment of myocardial perfusion using power contrast imaging. (top) 4.0-mL bolus injection of SonoVue威 was administered IV while the apical four-chamber view was recorded. Note the presence of apical perfusion defect. (bottom left) six regions-of-interest are displayed on the digital clip, and the resultant time-intensity curves are plotted (right). Contrast first appears in the left ventricle and then in the basal and mid septum and the lateral wall. The apex remains flat, consistent with the patient’s apical filling defect. The rapid decrease of contrast in the lateral wall is consistent with a US dropout artefact. (bottom right) green ⫽ basal septum; dark blue ⫽ mid septum; magenta ⫽ apex; yellow ⫽ anterolateral wall; light blue ⫽ lateral wall; red ⫽ LV cavity. Reproduced with permission from Nanda et al. (2000), Figs. 1A and 1E.
total intensity is used as a measure of myocardial blood volume. Myocardial blood flow is the product of blood volume and velocity. Stress (usually adenosine) and rest measurements of this parameter can be used to calculate severity of coronary artery stenosis. MCE in acute coronary syndromes (ACS) As mentioned above, MCE can identify several abnormal perfusion patterns that, when coupled with the functional data from the TTE examination, yield diagnoses of myocardial ischemia, necrosis, stunning and hibernation. In ACS, these data have important ramifications for interventions and prognosis. Due to its ability to provide spatial and temporal resolution, real-time imaging and accessibility for patient care in all areas of the hospital, MCE should expand from its limited research role to a first-line diagnostic modality for patients with ACS. In patients with acute myocardial infarction, MCE will initially reveal only a small amount of contrast in the area of involved myocardium, identifying myocardium at risk.
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Fig. 5. Assessment of myocardial perfusion using harmonic power Doppler and a continuous IV infusion of Cardiosphere威 0.175 mg/kg body weight. Apical three-chamber views acquired after (left) four beats of refill at rest and (right) after one beat at stress using dipyridamole infusion show a reversible defect in the left ventricular inferior wall. Reproduced with permission from POINT Biomedical Corporation, San Carlos, CA, USA.
With revascularization or reperfusion, contrast will be seen in the area of viable myocardium with intact microvasculature integrity, and this portion will demonstrate a pattern consistent with stunned myocardium and usually recover function. “No-reflow” areas will be identified by persistent poor contrast enhancement and will usually become scar. Several reports have validated this technique and its prognostic implications (Villanueva et al. 1993; Ito et al. 1992, 1996; Rocchi et al. 2001).
Fig. 6. (a) Low MI real-time imaging with full myocardial opacification. (b) A higher MI pulse creates a “flash” effect and destroys contrast in the myocardium. (c) The myocardium is cleared of contrast agent; and (D) seconds later contrast has been replenished in myocardium. Reproduced with permission from “A compendium of advanced technologies for echocardiography” by ATL, A Philips Medical Systems Co., 2000, Bothell, WA, USA.
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COMPARISON OF CONTRAST ECHOCARDIOGRAPHY WITH OTHER NONINVASIVE IMAGING MODALITIES Currently, myocardial perfusion imaging with radiolabeled tracers is the most commonly used method for evaluating myocardial perfusion and viability. This technique is limited, though, by its restriction to licensed centers, lack of portability, expense, inability to perform real-time imaging, low resolution, attenuation artefacts, use of ionizing radiation and requirements for radioactive waste disposal. On the other hand, MCE is more widely available, relatively inexpensive in terms of examination and equipment cost, portable, provides precise real-time anatomical and perfusion/ viability data and uses no ionizing radiation. Comparisons of these two imaging modalities continues favorably to demonstrate the assets of MCE (Kaul et al. 1997; Marwick et al. 1998; Heinle et al. 2000; Rocchi et al. 2001; Lepper et al. 2003). Kaul et al. (1997) first demonstrated the effectiveness of a second-generation CE agent for perfusion imaging. In a study of 30 participants with suspected or known coronary artery disease (CAD), MCE with Optison威 was compared to 99mTc-sestamibi single-photon emission computed tomography (SPECT) for concordance of 10 segmental perfusion scores and for the determination of the presence or absence of CAD. Concordance for segmental scores was 92% and agreement for the detection of CAD was 86%. However, when tested in a larger population, a different contrast agent, Sonazoid威, did not fare as well with the technology of the late 1990s. Marwick et al. (1998) compared fundamental or harmonic MCE with continuous or triggered imaging with 99mTc-sestamibi SPECT in 203 participants with known CAD. Although the specificity for detection of extensive (⬎ 12% of the left ventricle) perfusion defects was good (63% to 100% depending on the technique used), sensitivity was limited with MCE alone (13% to 48%). When coupled with wall motion data, sensitivity did improve to 46% to 55%. Heinle et al. (2000) defined what has become the Achilles’ heel of MCE, falsely abnormal lateral wall perfusion defects. In a study of 123 participants referred for SPECT imaging for known or suspected CAD, MCE was performed using harmonic power Doppler imaging and Optison威 at rest and during adenosine stress as well. Overall concordance for MCE and SPECT was 81%, but was only 72% for the left circumflex artery distribution. Notably, 33% of participants demonstrated fixed defects of the lateral wall by MCE, but only 14% did by SPECT. The further refinement of harmonic power Doppler imaging has led to improved accuracy of MCE, as more recent evidence compares MCE and SPECT to catheterbased assessments of coronary perfusion. Rocchi et al.
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(2001) documented 82% sensitivity and 95% specificity for MCE-identification of infarct-related artery perfusion defects in 30 patients with conservatively treated acute myocardial infarctions when compared with a coronary angiogram “gold standard.” Accuracies for MCE and SPECT were similar in this study. More recently, Lepper et al. (2003) compared MCE and SPECT with pressurewire–measured fractional flow reserve in 21 participants who presented for diagnostic coronary angiography in evaluation of chest pain. Agreement for MCE was 90%, but agreement for SPECT was only 71%. METHODOLOGY OF CONTRAST ECHOCARDIOGRAPHY The evolution of the equipment “hardware” for MCE has probably yielded the most innovations in this field. Instrument settings have grown from fundamental B-mode imaging to harmonic B-mode imaging and now to harmonic power Doppler imaging. Interrupted or ECG-triggered imaging was followed by pulse-inversion imaging and now by real-time power Doppler pulseinversion imaging at low MI. Three methods are predominantly used for perfusion imaging today: 1. interrupted or ECG-gated imaging in conjunction with harmonic power Doppler mode; 2. realtime power Doppler pulse inversion imaging at low MI combined with high MI bubble destruction; and 3. a combination of the previous two methods with endsystolic, ECG-gated, real-time imaging of every beat. Triggered imaging at a normal MI allows systolic gating for imaging of the myocardium at its thickest portion, which creates the best resolution for detecting perfusion patterns. With this method, a time-intensity curve can also be created with quantification of myocardial blood volume and coronary artery flow, as previously described. Limitations of this technique include movement artefacts, corruption of the image and the fact that imaging is not real-time. Real-time imaging at a low MI permits evaluation of instantaneous perfusion changes and offers improved application to stress imaging or assessment of reperfusion (Fig. 6). This technique suffers from a lower signalto-noise ratio and also from poor imaging of the lateral left ventricular wall. A combination technique of ECG-gating to obtain the thickest myocardial segment in systole and near real-time imaging with capture of signal from each beat is an appealing solution to the above limitations. With the improved signal-generating contrast agents currently available and refined instrumentation, this method should allow portable real-time perfusion imaging.
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FUTURE APPLICATIONS OF CONTRAST ECHOCARDIOGRAPHY Several unique properties of CE agents have been documented that may yield new diagnostic and therapeutic possibilities. We will discuss: 1. assessing endothelial function; 2. detecting vascular inflammation; and 3. delivering gene/drug therapies. Endothelial function Endothelial dysfunction is an early step in the development of symptomatic coronary artery disease and is currently a topic of intense research. Current US technologies crudely determine endothelial health by evaluating macrovascular flow patterns or responses to vasodilators. However, microbubbles may offer a more direct approach. Intercellular adhesion molecule-1 (ICAM-1) and other adhesion molecules are overexpressed in activated endothelial cells in probably the first step that leads to atherosclerosis (Ross 1999). Villanueva et al. (1998) placed monoclonal antibodies to human ICAM-1 on the shell of a microbubble. In vitro, anti-ICAM-1 bubbles bind to endothelial cells overexpressing ICAM-1, which could potentially lead to clinical use as direct evidence of early atherogenesis. Inflammation Taking this research tool one step further down the evolution of an atherosclerotic plaque, microbubbles have also been shown to attach to activated leukocytes. Originally, the hypothesis that microbubbles marked inflammation was postulated from observations that airfilled albumin CE agents administered by intracoronary injection “hung up” in regions of myocardial injury (Lindner et al. 1998). Further research has yielded direct observation of albumin-coated microbubbles bound to and phagocytosed by leukocytes in inflamed tissue (Lindner et al. 2000). As a marker of vascular inflammation, CE agents may provide evidence of plaque instability, reperfusion injury extent and restenosis likelihood in the not-so-distant future. Gene/drug therapy delivery CE agents offer a unique mechanism for delivery of therapeutic agents to specific anatomical and histologic targets. Microbubbles could be engineered with an adenovirus vector or pharmaceutical agent attached to the shell and injected IV. Then, destruction of microbubbles at high MI could be targeted to the sonicated area of a specific organ, with a high concentration delivered to that area. Further, other shell additives, such as adhesion molecule antibodies, could guide the microbubble to the target cell type.
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Initial work in this field has already yielded promising results. Shohet et al. (2000) incorporated recombinant adenovirus containing -galactosidase into the albumin surface of perfluoropropane-filled microbubbles and successfully delivered the functioning vector to the myocardium by US-mediated microbubble destruction. Drug and gene delivery should be a blossoming subspecialty of CE in the very near future. CONCLUSION The application of echocardiography is expanding with the development of contrast agents that permit improved anatomical assessment, perfusion imaging and special applications in vascular inflammation and drug delivery. Further refinement of the “hardware” (mechanical index, triggered and real-time imaging) and new developments in the “software” (CE agents) of CE should continue to revolutionize this nearly limitless field of echocardiography. Acknowledgments—The authors acknowledge with thanks help from IMCOR Pharmaceuticals, Inc., Bristol-Myers Squibb, Philips Ultrasound and POINT Biomedical Corporation.
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