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77 6 CHAPTER Ultrasonic contrast agents HISTORY AND DEVELOPMENT 77 MICROBUBBLE DYNAMICS 78 TYPES OF CONTRAST AGENTS 78 Lipid-stabilised contrast microbubbles 79 SonoVue 79 Definity 79 Sonazoid 80 Imavist 80 Albumin-coated microbubbles 80 Optison 80 PESDA 80 Polymer-coated microbubbles 80 CARDIOsphere 80 Imagify (AI-700) 81 Other types of agents 81 Levovist 81 IMAGING OF CONTRAST MICROBUBBLES 81 Fundamental imaging 81 Second harmonic imaging 81 Low MI techniques 82 Pulse inversion imaging/phase inversion imaging (PI) 82 Amplitude modulation/power modulation (PM/AM) 82 Power-modulated pulse inversion (PIAM) 82 High MI techniques 82 Flash contrast imaging; triggered imaging; destruction-replenishment 82 Newer imaging techniques 84 Subharmonic imaging 84 Coded excitation 84 Radial modulation imaging 84 CLINICAL APPLICATIONS OF CONTRAST IMAGING 84 Radiology applications 84 Liver 84 Renal 84 Spleen and pancreas 85 Transcranial 85 Urology 85 Cardiology 85 OTHER POTENTIAL USES OF CONTRAST MICROBUBBLES 86 Targeted contrast microbubbles 86 Drug and gene delivery 87 SAFETY OF CONTRAST MICROBUBBLES 87 IMAGING ARTEFACTS 88 Propagation artefacts 88 Doppler artefact 88 Carmel M. Moran HISTORY AND DEVELOPMENT Ultrasound signal enhancement using a contrast agent was first reported in the 1960s and initial experiments were performed by Joyner (unpublished) using physiological saline as an ultrasound contrast agent in the anatomical identification of the mitral valve echo. The same phenomena were subsequently observed using dextrose and indeed the patient’s own blood in the assessment of cardiac anatomy and function. 1 Over the next 20 years contrast agents were used mainly to determine the presence of cardiac shunts and to validate cardiac anatomy. Indeed the use of agi- tated saline is still routinely used in cardiac studies today for the assessment of a patent foramen ovale (PFO) (Fig. 6.1). Such free (unencapsulated) bubble contrast agents were initially made by agitating the carrier liquids; however, the range of bubble size was large. By a process called ‘sonication’ in which a low- frequency sonic field (20 kHz) is applied to liquids, small cavita- tion bubbles of a more limited size range (<10 µm) were created. 2 Various in-vivo experiments using early contrast agents including colloidal suspensions, aqueous solutions, emulsions and encapsulated microbubbles have been reviewed 3 and it is evident from this review and subsequent work that agents working on the principle of scattering from microbubbles were most likely to be successful in clinical applications. In the mid-1980s clinical interest re-emerged with rapid commercial development of a large range of contrast agents, many of which were the precursors of those now commercially available. By 1987 bubbles smaller than 5 µm were produced by sonicating albumin at high intensity and low frequency. 4 The encapsulating shell consisted of the denatured albumin protein. Another practical approach to making small encapsulated microbubbles was to support the small bubbles in a saccharide crystal structure with the addition of palmitic acid as a surfactant to stabilise the bubbles. 5 These breakthroughs in the production of stable encap- sulated bubbles which could be injected intravenously and survive passage through the pulmonary circulation opened up the possibility of a wide range of clinical applications. In addition, there existed an increasing clinical demand for the development of an ultrasonic technique that was capable of measuring myocardial perfusion. A longer-term interest in microbubbles is related to their potential use as carriers of pharmaceuticals or genes to targeted sites where the microbubbles would be destroyed, once attached, by an ultrasound beam, releasing the contents of the microbubbles in situ. Some types of contrast agents are discussed in this chapter; they should be considered as examples of different formulations and not an exhaustive list. In addition, although different methods of imaging contrast agents are discussed, this is an area of continuing commercial sensitivity for ultrasonic scanner manufacturers so only an overview of different approaches to contrast imaging details are given.

Transcript of Ultrasonic Agents

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6CHAPTER

Ultrasonic contrast agents

HISTORY AND DEVELOPMENT 77

MICROBUBBLE DYNAMICS 78

TYPES OF CONTRAST AGENTS 78Lipid-stabilised contrast microbubbles 79

SonoVue  79Definity  79Sonazoid  80Imavist  80

Albumin-coated microbubbles 80Optison  80PESDA  80

Polymer-coated microbubbles 80CARDIOsphere  80Imagify (AI-700)  81

Other types of agents 81Levovist  81

IMAGING OF CONTRAST MICROBUBBLES 81Fundamental imaging 81Second harmonic imaging 81Low MI techniques 82

Pulse inversion imaging/phase inversion imaging (PI)  82Amplitude modulation/power modulation (PM/AM)  82Power-modulated pulse inversion (PIAM)  82

High MI techniques 82Flash contrast imaging; triggered imaging; destruction-replenishment  82

Newer imaging techniques 84Subharmonic imaging  84Coded excitation  84Radial modulation imaging  84

CLINICAL APPLICATIONS OF CONTRAST IMAGING 84Radiology applications 84Liver 84Renal 84Spleen and pancreas 85Transcranial 85Urology 85Cardiology 85

OTHER POTENTIAL USES OF CONTRAST MICROBUBBLES 86Targeted contrast microbubbles 86Drug and gene delivery 87

SAFETY OF CONTRAST MICROBUBBLES 87

IMAGING ARTEFACTS 88Propagation artefacts 88Doppler artefact 88

Carmel M. Moran

HISTORY AND DEVELOPMENT

Ultrasound signal enhancement using a contrast agent was first reported in the 1960s and initial experiments were performed by Joyner (unpublished) using physiological saline as an ultrasound contrast agent in the anatomical identification of the mitral valve echo. The same phenomena were subsequently observed using dextrose and indeed the patient’s own blood in the assessment of cardiac anatomy and function.1 Over the next 20 years contrast agents were used mainly to determine the presence of cardiac shunts and to validate cardiac anatomy. Indeed the use of agi-tated saline is still routinely used in cardiac studies today for the assessment of a patent foramen ovale (PFO) (Fig. 6.1). Such free (unencapsulated) bubble contrast agents were initially made by agitating the carrier liquids; however, the range of bubble size was large. By a process called ‘sonication’ in which a low- frequency sonic field (20 kHz) is applied to liquids, small cavita-tion bubbles of a more limited size range (<10 µm) were created.2 Various in-vivo experiments using early contrast agents including colloidal suspensions, aqueous solutions, emulsions and encapsulated microbubbles have been reviewed3 and it is evident from this review and subsequent work that agents working on the principle of scattering from microbubbles were most likely to be successful in clinical applications. In the mid-1980s clinical interest re-emerged with rapid commercial

development of a large range of contrast agents, many of which were the precursors of those now commercially available. By 1987 bubbles smaller than 5 µm were produced by sonicating albumin at high intensity and low frequency.4 The encapsulating shell consisted of the denatured albumin protein. Another practical approach to making small encapsulated microbubbles was to support the small bubbles in a saccharide crystal structure with the addition of palmitic acid as a surfactant to stabilise the bubbles.5 These breakthroughs in the production of stable encap-sulated bubbles which could be injected intravenously and survive passage through the pulmonary circulation opened up the possibility of a wide range of clinical applications. In addition, there existed an increasing clinical demand for the development of an ultrasonic technique that was capable of measuring myocardial perfusion. A longer-term interest in microbubbles is related to their potential use as carriers of pharmaceuticals or genes to targeted sites where the microbubbles would be destroyed, once attached, by an ultrasound beam, releasing the contents of the microbubbles in situ. Some types of contrast agents are discussed in this chapter; they should be considered as examples of different formulations and not an exhaustive list. In addition, although different methods of imaging contrast agents are discussed, this is an area of continuing commercial sensitivity for ultrasonic scanner manufacturers so only an overview of different approaches to contrast imaging details are given.

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acoustic pressure increases the bubble cannot contract as much as it can expand, due to the gas within the microbubble. This results in asymmetric motion of the microbubbles and the resultant scat-tered wave is consequently distorted and contains frequency com-ponents that are multiples (harmonics) of the incident (fundamental) frequency. As the acoustic pressure increases further, the micro-bubble can expand and then violently implode. Such an event is known as inertial (transient) cavitation and can produce extremely large, highly localised temperatures, free radicals and high-velocity jets. The manner in which the microbubbles collapse is dependent on a range of insonating conditions including pressure amplitude, pulsing regime and ultrasound frequency.11 Also important are microbubble parameters such as composition of the encapsulating shell, size of the microbubbles and proximity of the microbubbles to other structures (including other microbub-bles and cell walls). The shells of the microbubbles have been observed to buckle, fragment and crack, allowing the gas to leak from the shells over successive incident ultrasound pulses.12 The range of phenomena that can be induced at different acoustic pres-sures are discussed elsewhere.13

On commercial scanners, an indication of the acoustic pressure is given by the mechanical index (MI), defined as:

  MI ve= −P f  

where P−ve is the peak derated negative (rarefaction) acoustic pres-sure and f is the ultrasonic frequency (see Chapter 4). This param-eter is an on-screen index designed to give an indication of the likelihood of mechanical bioeffects. Although defined and used as a safety index, the mechanical index is often used as an indicator of microbubble behaviour for a particular scanner set-up and contrast agent. In general for low MI imaging, the number of contrast micro-bubbles collapsing as a result of the insonating pressure pulse will be minimal. However, as the MI increases, the likelihood of inertial cavitation increases as the contrast microbubbles can be forced to collapse releasing free gas bubbles. Clinical contrast imaging tech-niques utilise both low MI and high MI applications.

TYPES OF CONTRAST AGENTS

Commercially available ultrasonic contrast agents are gas-filled microbubbles which are generally coated with a lipid, protein or polymer shell. Although free gas bubbles scatter ultrasound much more effectively than encapsulated microbubbles, they dissolve quickly in the blood, generally in the order of milliseconds, due to surface tension and diffusion effects. The encapsulation is required to lengthen the lifetime of the bubbles within the blood, by provid-ing an elastic membrane surrounding a gaseous interior. The gas contained within commercially available clinical microbubbles is generally a perfluorocarbon as these gases diffuse less readily than air within blood. Microbubble diameters generally lie within the 1–5 µm range although production of smaller microbubbles of a

MICROBUBBLE DYNAMICS

The equations which govern the dynamics of the motion of gas bubbles in an acoustic field were derived by Rayleigh6 and Plesset7 and consider the response of a spherical, unencapsulated bubble to a time-varying pressure field in an incompressible fluid. This model was extended to consider the effect of an elastic thin layer encap-sulating a gas-filled microbubble.8 It was demonstrated that the addition of a thin shell significantly increased the damping of the bubble oscillations and reduced the acoustic response of the micro-bubble in comparison to free microbubbles. However, the resonant oscillation was shown to still add significantly to the magnitude of the backscattered intensity.

It is serendipity that microbubbles of a size similar to red blood cells resonate at low megahertz frequencies, similar to those used routinely in diagnostic clinical ultrasound. Theoretical and experi-mental determination of the resonance frequency of free and encap-sulated gas bubbles is an active area of research.9 The equation below describes a derived approximation of the resonance fre-quency, f0, of a free air bubble in water:

  f R0 03 3= .  

where R0 is the bubble radius in microns. Substituting appropriate values into this formula shows that free bubbles with radii between 1 and 3 µm have resonances which lie approximately in the range of 5 to 1 MHz. Addition of an outer shell, such as albumin, shifts this resonant frequency higher. A more detailed review of the theory which governs the interaction of ultrasound with both free and encapsulated microbubbles in water may be found elsewhere.10 However, although such theory is useful to describe the resonant behaviour of the microbubbles, the interaction of encapsulated microbubbles with an insonating acoustic field in a biological system is complex. This is particularly true as the acoustic pressure incident on the microbubbles increases. At low acoustic pressures, theory predicts that the acoustic wave incident on a contrast micro-bubble with an elastic shell causes the microbubble to oscillate, expanding when the pressure is negative (rarefaction) and contract-ing when the pressure is positive (compression). This results in minimal destruction of the microbubbles but may cause micro-streaming in the fluid in the vicinity of the microbubbles. As the

Figure 6.1 Four-chamber view of the heart after injection of agitated saline in the clinical assessment of PFO. Right atrium and right ventricle are filled with agitated saline. There is no evidence of bubbles in the left atrium or the left ventricle indicating that there is no PFO. (Image courtesy of Audrey White, Western General Hospital, Edinburgh, Scotland.)

Microbubble dynamics

• Microbubbles oscillate in response to the ultrasound beam.• It is the compressibility of the gas within the microbubbles that

makes them such efficient scatterers.• The resonant frequency of commercial microbubbles lies in the

diagnostic frequency range of ultrasound scanners.• The ultrasound beam may force the microbubbles to oscillate in a

non-linear manner, resulting in the generation of harmonics.• At high acoustic pressures, the microbubbles can be forced to

collapse or fragment, releasing free gas bubbles which diffuse rapidly in the blood.

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Lipid-stabilised contrast microbubbles

The majority of commercially available contrast agents fall into this category with a mono- or bi-lipid layer providing the stabilising layer required for the microbubbles to pass into the systemic system. Phospholipid layers tend to vary in thickness from 10 to 200 nm. These microbubbles are distinct from echogenic multi-lamellar liposomes which have been developed principally for research applications and for which the echogenicity is achieved by encapsulation of gas within the lamellar structure.15

SonoVueSonoVue (Bracco) consists of sulphur hexafluoride gas bubbles encapsulated in a lipid monolayer. The size distribution of the microbubbles is such that 99% of the microbubbles are less than 11 µm in diameter. A typical concentration is 2 × 108 mL−1. The agent is presented in an integral kit which enables 5 mL of saline to be injected into 25 mg of the dry lyophilised powder in an atmos-phere of sulphur hexafluoride (Fig. 6.2). The agent does not require refrigeration and is currently licensed in the European Union (EU) for cardiac applications (including endocardial border enhance-ment (EBD) in the chambers of the heart and left ventricular opaci-fication (LVO). It is also used for Doppler exams of both cardiac and non-cardiac microvasculature. This agent can be administered both as a bolus and as an infusion.

DefinityDefinity (acquired in 2008 from Bristol-Myers Squibb by Lantheus Imaging) is also known as Luminity in the majority of EU countries.

well-defined radius (mono-disperse) and usually polymer- encapsulated is becoming more common. The number of microbub-bles in commercial products tends to range from 108 to 1010 mL-1 with typical injection doses ranging from 0.1 mL to 1 mL, although larger or multiple doses may be administered dependent on specific manufacturers’ recommendations. Contrast agent injections are given intravenously, generally as a bolus injection, but infusions are possible and in some instances necessary for clinical studies such as in the assessment of cardiac perfusion.

When reviewed historically, ultrasonic contrast agents are often subdivided into ‘generations’. First-generation contrast agents are non-transpulmonary contrast agents including free gas bubbles and Echovist, an agent developed by Schering and still used for hysterosalpingo-contrast-sonography for the assessment of Fallo-pian tube patency. Second-generation agents are those that are transpulmonary but have a relatively short half-life (several minutes) and can be seen in the vascular bed. These microbubbles generally encapsulate air within their structure. Third-generation agents are also transpulmonary vascular agents but tend to have a longer half-life and encapsulate fluorocarbons. One exception to this is SonoVue, which incorporates sulphur hexafluoride.

Table 6.1 gives a listing of different types of contrast microbub-bles that have been developed over recent years – some of these are commercially available, others are currently undergoing clinical trials. The majority of the commercially available agents are licensed for left ventricular opacification (LVO) and endocardial border defi-nition (EBD). In certain countries, the use of contrast agents for the assessment of focal liver lesions is also included. The use of contrast agents in the assessment of myocardial perfusion has not yet been licensed but at least one manufacturer has an agent currently sub-mitted to the FDA in the USA for approval for this application. A more extensive list of contrast agent properties can be found elsewhere.14

Table 6.1 Examples of commercially developed ultrasonic contrast agents. Early-generation agents that encapsulated air have been omitted

Contrast agent Manufacturer

Capsule/Shell Gas

Bubble diameter Charge Status

CARDIOsphere (PB 127)

Point Biomedical Corp

Polylactide/Albumin

Nitrogen 4 µm Slight negative

Not available

Imagify (AI 700)

Acusphere Inc Poly(L-lactide-co-glycolide)

Decafluorobutane NA Negative Awaiting approval in USA

Definity (USA/Canada)Luminity (EU)

Lantheus Medical Imaging

Lipids: DPPA, DPPC, MPEG5000, DPPE

Octafluoropropane 98% <10 µm

Negative USA approvedCA approvedLVO, EBD

Imavist/AF0150

Imcor Pharmaceuticals Inc

Lipid: DMPC Perfluorohexane/Nitrogen

99.8% <10 µm

Neutral USA approved LVO, EBD

Sonazoid/NC100100

GE Healthcare Lipid: HEPS Perfluorobutane Median 2.6 µm99.9% <7 µm

Negative Japan approved,focal liver lesions

SonoVue (BR1) Bracco Lipids: Macrogol 4000, DSPC, DPPG, palmitic acid

SF6 99% <11 µm

Negative EU approved LVO, EBDDoppler (non-cardiac studies)

Optison/FS069 GE Healthcare/Amersham

Albumin Octafluoropropane 93% <10 µm

Slight negative

EU, USA approved, EBD, LVO

HEPS, hydrogenated egg phosphatidyl serine; DMPC, dimyristoylphosphatidylcholine; DPPC, dipalmitoyl glycerophosphocholine; DPPA, dipalmitoyl glycerophosphate; DPPE, dipalmitoyl glycerophosphoethanolamine; DSPC, distearoyl glycerophosphocholine; DPPG, dipalmitoyl glycerophosphoglycerol.

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distribution of the microbubbles is such that 99.8% are less than 10 µm in diameter. The agent is approved in the USA for EBD and LVO.

Albumin-coated microbubbles

OptisonOptison (GE Healthcare) microbubbles are a development from the first-generation contrast agent Albunex, which was one of the first commercially available agents to demonstrate EBD. In Optison, octafluoropropane has replaced the air that was encapsulated in Albunex. The shell of Optison microbubbles is of denatured human serum albumin and the size distribution is such that 93% are less than 10 µm in diameter. A typical concentration is 5 × 108 mL−1. The agent is supplied in vials that must be refrigerated prior to use. The agent is hand agitated to produce a milky solution prior to with-drawal of the agent from the vial. Optison is licensed for EBD and LVO in the USA and EU.

PESDAPESDA (perfluorocarbon exposed dextrose albumin) comprises sonicated dextrose albumin microbubbles containing decafluorobu-tane gas. It has been developed by Porter et al. exclusively for research use.17 The mean size of the microbubbles is 5.1 µm and the concentration is 3.1 × 109 mL−1.

Polymer-coated microbubbles

Polymer-coated microbubbles have a much stiffer shell than lipid- or albumin-encapsulated microbubbles. As a result these microbub-bles do not oscillate (expand and contract) in the ultrasound field at low acoustic pressures.18 However, as the acoustic pressure increases, the polymer shell can buckle and crack, releasing free gas (Fig. 6.4).

CARDIOsphereCARDIOsphere microbubbles (POINT Biomedical Corp) are manu-factured using a technique described as BiSphere technology in

Figure 6.3 Vialmix with inset of Definity/Luminity vial.

Figure 6.4 Schematic representation of lipid (left column) and polymer (right column) microbubble interaction with ultrasound of increasing intensity (top to bottom). (Reprinted from Advanced Drug Delivery Reviews, Vol 60, Hernot S, Klibanov AL. Microbubbles in ultrasound-triggered drug and gene delivery. 1153–1166.Copyright (2008) with permission of Elsevier.)

No US

Lipid microbubble Polymer microbubble

Very low intensity US

Low intensity US

High intensity US

Figure 6.2 Preparation kit for SonoVue.

It is a lipid bi-layer microbubble encapsulating octafluoropropane. The size distribution of the microbubbles is such that 98% are less than 10 µm in diameter. A typical concentration is 109 mL−1. The agent is supplied in vials as a clear liquid with a head-space of octafluoropropane. To form the bubbles, the vial must be agitated in a mechanical shaker (Vialmix) for 45 seconds prior to withdrawal of the milky solution (Fig. 6.3). The vial is vented using a needle prior to withdrawal of the solution. The agent must be refrigerated prior to use. Luminity is licensed in the EU but is not currently marketed there. In the USA and Canada the agent is licensed for LVO and EBD.

SonazoidSonazoid (GE Healthcare) is a lipid (hydrogenated egg phosphati-dyl serine) encapsulated perfluorobutane microbubble. The agent is reconstituted using 2 mL of water and manually mixed for 1 min to produce a milky solution. A typical concentration is 1.2 × 109 mL−1 with a median size of 2.6 µm.16 This agent is currently not available in the EU and USA but is licensed in Japan for the assessment of focal liver lesions.

ImavistImavist (IMCOR Pharmaceuticals Inc) is a lipid-encapsulated, per-fluorohexane and nitrogen encapsulated microbubble. The size

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scattered wave to provide sensitive contrast imaging techniques. Current contrast imaging techniques can be divided into either low MI imaging techniques which minimise destruction of the micro-bubbles or high MI techniques which maximise microbubble destruction.

Fundamental imaging

Initial clinical imaging of the first commercial ultrasound contrast agents was performed in the fundamental imaging mode: ultra-sound transducers emitting and receiving the ultrasound signal over the same bandwidth of the transducer. In these early years, the use of the term contrast agent was clearly a misnomer as although the agent provided transient enhancement of the blood pool, the relative backscatter of tissue to blood was reduced. In some cases, the quantity of contrast agent injected in order to achieve enhancement was such that significant and long-lasting acoustical shadowing in distal regions of the image made diagnosis difficult. It was in ‘rescuing’ Doppler studies that the first clear indications of benefit were seen using contrast in studies where the blood signal was too small to be picked up (Fig. 6.5). Fundamental imaging of contrast agents has now been completely replaced by contrast-specific imaging techniques.

Second harmonic imaging

When the radius of a microbubble changes its size in direct propor-tion to the pressure variations of the ultrasonic wave it is said to be responding in a linear fashion. In this case the frequency of the scattered wave is the same as that of the incident wave. However, for larger pressure fluctuations the change in radius is not in pro-portion and the bubble is said to be responding in a non-linear way. When a bubble responds in a non-linear manner, the scattered ultrasound wave is distorted and contains additional frequency components above and below the frequency of the incident wave. These additional components are called harmonics and there is particular interest in the second harmonic which can be as strong as the ultrasound scattered from the microbubbles at the incident frequency, i.e. the first harmonic (fundamental frequency). Second harmonic imaging relies upon detecting and displaying only this second harmonic signal from the scattered ultrasound. Although at low acoustic pressures (low MI), second harmonic imaging significantly improves the contrast-to-tissue ratio (CTR), at higher

which microbubbles are produced comprising an outer layer of a biocompatible material (albumin) and an inner polymer layer. The microbubbles are filled with air and have been developed for myo-cardial perfusion studies. Their mean diameter is 4.0 µm. Using BiSphere technology, smaller sub-micron microbubbles have also been manufactured for lymphatic imaging.

Imagify (AI-700)Imagify (Acusphere Inc) consists of a synthetic polymer poly(L-lactide co-glycolide) shell and a phospholipid layer encapsulating a perfluorobutane gas. The agent is made using a spray drying technique to produce microbubbles. The mean size of the microbub-bles is 2.0 µm. FDA approval is currently being sought for this agent for myocardial contrast perfusion imaging.

Other types of agents

LevovistLevovist (Schering, Germany) is a first-generation agent and one of the first agents to undergo clinical trials and be commercially avail-able. Unlike the majority of commercial agents, Levovist is an air-filled microbubble. The microbubbles are formed within a galactose microstructure which controls the size of microbubbles formed and the microbubbles are stabilised by the addition of palmitic acid. The diameter of the microbubbles lies between 3 and 5 µm. The agent is supplied in vials containing 4 g of dry galactose granules and 0.1% palmitic acid. Sterile water is injected into the vials, the quan-tity of water determining the concentration of the agent. Recom-mended concentrations are 200 mg mL−1 (17 mL injection of water into vial), 300 mg mL−1 (11 mL injection of water into vial) or 400 mg mL−1 (8 mL injection of water into vial). Once the water is injected, the granules are vigorously shaken for 5–10 seconds and then allowed to stand for a further 2 minutes prior to injection into the patient. By this stage the solution is milky white. Care must be taken to avoid excessive increases in temperature caused by holding the vials tightly in hand or strong negative pressure when drawing up the solution.

IMAGING OF CONTRAST MICROBUBBLES

The development of imaging techniques to detect contrast micro-bubbles continues to be an exciting and active area of research. Such techniques aim to isolate and differentiate the acoustic signatures from tissue and contrast. Current approaches adopted by commer-cial ultrasound scanner manufacturers include utilising the differ-ent frequency components of the scattered ultrasound signal (fundamental, harmonics, subharmonics), and the different acoustic amplitude responses of the microbubbles. Indeed several manufac-turers utilise both frequency and amplitude components of the

Contrast agents

• Ultrasonic contrast agents are micron-sized encapsulated bubbles.

• The shells of the microbubbles are generally either lipid or polymer.

• The gases within current contrast microbubbles are perfluorocarbons.

• There are approximately 109 microbubbles/mL of contrast agent solution.

• Contrast agents are generally injected into the femoral vein as either a bolus or infusion. Figure 6.5 A: Suspected middle cerebral artery (MCA) stenosis on

unenhanced transcranial colour-coded duplex sonography. In the Doppler frequency spectrum only suspicious low-frequency bidirectional signals can be obtained. In colour mode the MCA is barely visible. B: Improvement of the Doppler frequency spectrum and the colour mode depiction of the MCA after application of an ultrasound contrast agent. Flow velocities >300 cm/s ascertain the presence of a high-grade MCA stenosis. (Reprinted from Seminars in Cerebrovascular Diseases and Stroke, Vol 5, Stolz EP and Kaps M, Ultrasound contrast agents and imaging of cerebrovascular disease 111–131.Copyright (2005) with permission from Elsevier.)

A B

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therefore the difference in the received pulses is due to the non-linear response of the target (Fig. 6.8).

Power-modulated pulse inversion (PIAM)This technique combines the two techniques described previously. In this case the two pulses that are sent down each scan line vary in both amplitude and phase. This results in a more sensitive detec-tion of non-linear signals.20

High MI techniques

Flash contrast imaging; triggered imaging; destruction-replenishmentThe contrast-specific imaging modes that have been discussed to date are predominantly used at low acoustic outputs where the

acoustic pressures (high MI) non-linear propagation through tissue and the generation of tissue harmonics23 results in a reduction of CTR compared to low MI. There is thus an inherent limitation to the potential increase in the CTR using second harmonic imaging. In addition, second harmonic imaging requires broad bandwidth transducers to enable separate transmit frequencies and receive frequencies (at twice the transmit frequency). This requirement in the transmitted and received bandwidths can result in a reduction in axial resolution (Fig. 6.6).

The advent of second harmonic imaging marked the beginning of an exciting period of development in both contrast agent manu-facture and contrast-specific imaging techniques to further capital-ise on the non-linear properties.

Low MI techniques

Pulse inversion imaging/phase inversion imaging (PI)The specific algorithms and bandwidth filters used within com-mercial scanners to separate microbubble and tissue echoes are not known, but pulse/phase inversion techniques have the potential to overcome the bandwidth limitations of second harmonic imaging.19 In this technique, two consecutive pulses are sent down each trans-mit line such that the second pulse is identical but inverted with respect to the first pulse (180° out of phase). The detection and summation of the two scattered echoes from linear scatterers will equate to zero. However, scattering from non-linear targets, such as microbubbles, will result in non-identical pulses. Summation of the two pulses will predominantly cancel out the fundamental part of the signal. The image is formed from the remaining signal (Fig. 6.7).

Pulse/Phase inversion is a low MI imaging technique which does not destroy the microbubbles in situ. One limiting factor of this technique is a reduction in the frame rate since it is necessary to send two pulses down each transmit line rather than one.

Amplitude modulation/power modulation (PM/AM)In amplitude/power modulation two, or more usually three, pulses of identical phase but different magnitude are sent along each trans-mit line. The amplitude of the pulses is often in a sequence where the first and third pulses are half the magnitude of the second pulse. Upon reception the signals are combined such that the sum of the response from the half amplitude transmit pulses is subtracted from the response from the full amplitude pulse. Lower-amplitude trans-mit pulses will generate less harmonics than high-amplitude pulses,

Figure 6.6 Schematic diagram illustrating the overlap in the transmit and receive bandwidths with second harmonic imaging, where f0 is the central transmit frequency.

Ampl

itude

Transducer bandwidth

Transmitbandwidth

Receivebandwidth

Frequencyf0 2f0

Figure 6.7 Schematic diagram illustrating the principle of the pulse inversion imaging technique. Two pulses are transmitted down each line, the second pulse the inverse of the first. The sum of the scattered pulses from linear scatterers will sum to zero, while the sum from non-linear scatterers will not equal zero.

Transmitted pulses Linear scatterers Non-linear scatterers

Pulse 1

Pulse 2

Pulse 1 + Pulse 2

Figure 6.8 Schematic diagram illustrating the principle of amplitude modulation imaging techniques. In this technique, three pulses are emitted, all of them in phase with one another. The first and third pulses are half the amplitude of the second pulse. Upon reception, summation of the scattered signals from linear scatterers will sum to zero, those from non-linear scatterers will not.

Transmitted pulses Linear scatterers Non-linear scatterers

Pulse 1

Pulse 2

Pulse 3

Pulse 1 – Pulse 2+ Pulse 3

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moving bubbles to trace out the vascular structure.24 In these tech-niques, a high MI pulse is applied to the organ to destroy the microbubbles, and then a low MI pulse inversion technique is used to image the organ as new individual microbubbles enter the field of view. The images are processed so that the echoes from the

Figure 6.9 A ‘flash frame’ acquired during a perfusion study. A high mechanical index frame disrupts SonoVue microbubbles within the myocardium and ventricle.

majority of the microbubbles are not destroyed. In some instances, however, destruction of the microbubbles in situ by one or several high pressure frames (flash frames) can be used to assess blood flow within an organ. Formerly for an assessment of flow within the myocardium, this technique involved varying the interval between successive high MI frames triggered from the ECG and not scan-ning between the high MI frames.21 More recently this technique has been developed further such that low MI imaging is performed between the high MI destruction frames. In such cases the micro-bubbles can be viewed initially using a low MI imaging technique. Once a constant enhancement has been established by infusion, application of a single, or several, high MI frames causes rupture of the microbubble shells. Upon rupture the microbubbles release free gas bubbles which rapidly diffuse into the blood and are there-fore generally only visualised in one frame (Fig. 6.9). On return to continuous low MI imaging, new contrast microbubbles may be visualised entering the scan plane and replenishing the destroyed contrast. The time taken for the contrast to refill the scan-plane is an indicator of myocardial blood flow22 (Fig. 6.10) and can be studied using refill kinetics theory. In this approach it is assumed that the enhancement of the ultrasound backscatter signal within the scan-plane is proportional to the microbubble concentration in the tissue, the infusion rate of the contrast and the pulse interval between successive high MI destruction pulses. When intensity measurements are made with increasing pulse intervals, a graph can be plotted of intensity, I(t), as a function of t, the time pulse interval between successive high MI destruction pulses (Fig. 6.11). The ultrasound intensity, I(t), is given by:

  I t I e t( ) = −( )−0 1 β  

where I0 is the ultrasound intensity of the plateau and β is a time constant. From this graph, β and I0 can be calculated. The former is related to the blood flow velocity, and the latter to the vascular volume.

High MI destruction techniques are also used in other organs, e.g. liver and spleen.23 Early techniques called loss-of-correlation imaging (LOC) or stimulated acoustic emission (SAE) imaging used colour Doppler to display the rapid collapse of the microbubbles as a chaotic colour Doppler region within the image. These imaging techniques have largely been replaced by contrast-specific imaging techniques such as maximum intensity projection or microbubble trace imaging, which are similar techniques that use the interplay of high and low MI techniques to study the path of

Figure 6.10 Contrast perfusion study using infusion of SonoVue with graphical display illustrating variation in backscatter signal in selected ROI within the myocardium. A pulse inversion imaging technique is used to image reperfusion of the myocardium following application of a high MI pulse. (Image courtesy of Dr Stephen Glen, Stirling Royal Infirmary, Scotland.)

Figure 6.11 Schematic example of intensity–time curve. I0 is the plateau intensity (related to vascular volume) and β is a time constant (related to the blood flow velocity).

Ultr

asou

nd in

tens

ity (I

)I0

Pulse interval (ms)

β

Imaging techniques

• Contrast microbubbles can be imaged using a variety of techniques.

• Harmonic imaging filters out the harmonic component of the signal.

• Pulse inversion separates the fundamental and harmonic components by subtraction technique rather than filtering so can utilise wider bandwidths.

• Pulse inversion and amplitude modulation imaging techniques are low MI imaging techniques and take advantage of the non-linear properties of contrast agents.

• At MI values of 0.1 or less, contrast microbubbles are not significantly destroyed.

• Destruction-replenishment imaging involves switching between high MI and low MI techniques is often used in cardiology.

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pulse dependent upon their current oscillatory state. By insonating the microbubbles with a low-amplitude, low-frequency pulse (modulation pulse), the microbubbles are initially forced to oscil-late. While the bubbles are oscillating, they are then insonated with two consecutive higher-frequency imaging pulses. Because of the lower-frequency modulation pulse, the bubbles will be in different stages of their expansion and contraction cycle when they are insonated by these higher-frequency imaging pulses and conse-quently the response of the microbubbles will be different. In com-parison the response from the tissue should not vary.27 The potential benefits of using this technique have yet to be proven but initial in-vitro results look very promising.

CLINICAL APPLICATIONS OF CONTRAST IMAGING

One of the most significant advantages of the use of contrast agents is the real-time nature of contrast enhancement and the resultant real-time diagnostic potential. In addition, the ability provided by some commercial ultrasound manufacturers to view the B-mode image and the contrast image simultaneously using a split-screen function (Fig. 6.12) enables visualisation of the organ of interest on one half of the screen, while simultaneously visualising the uptake of contrast on the other half. The conventional B-mode image is acquired at low MI to avoid disruption of the microbubbles.

The number of applications for which contrast agents are now used has increased significantly over the past 5 years and has been reviewed by the European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB).28 In this review the guidelines for each contrast procedure and recommendations for good clinical practice are presented. Some of these applications are not currently licensed (e.g. myocardial perfusion, renal applications) but the diagnostic benefits that clinicians have observed using contrast agents for applications other than those for which the agent is licensed (off-label applications) merit a mention in this chapter and elsewhere in this book.

Radiology applications

The initial use of ultrasonic contrast agents was primarily focused on ‘rescuing’ colour Doppler studies where the signal from blood was too small to be detected. Injection of contrast microbubbles increased these low signals so that they could be detected and displayed (Fig. 6.5).35

Liver

The organ in which contrast agents have provided substantial diag-nostic impact is in the liver for the characterisation of focal liver lesions. There is now substantial evidence to suggest that ultrasonic contrast agent imaging using low MI techniques (during the arte-rial, portal venous and late phases) is an effective diagnostic tool for the characterisation of focal liver lesions29 (Fig. 6.13). Maximum diagnostic potential from these studies is achieved when the pre-contrast image is such that there is little tissue structure visible and only major anatomical markers such as the diaphragm are presented. This relies upon interplay of both the gain and MI controls.

Renal

No contrast agents have currently been licensed for the assessment of renal lesions. However, there has been substantial documenta-tion of the benefits of using contrast microbubbles in the assessment of complex cystic lesions (Fig. 6.14), vascular diseases and trans-planted kidneys.30 For the assessment of renal lesions, contrast is injected as a bolus and low MI imaging techniques are used.

Figure 6.12 Image of a benign splenic lesion showing the pulse inversion harmonic image on the right side (about 15 seconds after injection of SonoVue), and the microbubble trace image on the left side. (Courtesy of Christoph Dietrich.)

microbubbles are summated over time, thus showing the path of the microbubbles through the vascular bed. In addition, the echoes are weighted such that the intensity decreases with elapsed time so that the direction of movement of the microbubbles can be studied (Fig. 6.12). Early studies have indicated that these techniques can illustrate arterial structure but the diagnostic benefit is as yet undetermined.

Newer imaging techniques

Subharmonic imagingSubharmonic imaging is based upon the principle that contrast microbubbles when scattering non-linearly not only generate har-monics higher than the fundamental but also generate subharmon-ics. In addition, tissue does not generate subharmonics and therefore at subharmonic frequencies the contrast-to-tissue ratio is generally higher than that achieved at second harmonic frequencies. Much of the work associated with subharmonic imaging has been performed at frequencies normally associated with intravascular ultrasound imaging (30 MHz). An improvement of contrast-to-tissue ratio of 30 dB at the subharmonic (15 MHz) in comparison to the funda-mental (30 MHz) has previously been achieved, highlighting the potential of using subharmonic imaging for vasa vasorum imaging.25

Coded excitationCoded excitation involves the transmission of a high energy long transmit pulse with limited peak amplitude and is well known as a means of improving signal-to-noise ratio in ultrasound B-mode imaging. Recovery of axial resolution is achieved by the receive electronics examining the signals and picking out the echoes of similar shape to the transmitted pulse. Use of coded excitation in combination with phase and amplitude modulation techniques has been shown to improve sensitivity to non-linear signals from micro-bubbles at relatively low signal to noise but with limited benefit at high signal-to-noise levels. A review of coded excitation techniques is given elsewhere.26

Radial modulation imagingRadial modulation imaging is based upon the premise that contrast microbubbles respond differently to an incident acoustic imaging

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Urology

Contrast microbubbles can also be injected into body cavities rather than intravenously. Vesicoureteric reflux can be demonstrated after contrast has been placed in the bladder. The kidneys and bladder can then be scanned and refluxing microbubbles observed.32

Cardiology

The majority of commercially available contrast agents have been licensed for cardiac applications and in particular for enhancement of endocardial border and left ventricular opacification. The ability to be able to identify the endocardial border is of importance in cardiac patients undergoing either a pharmacological or physical

Spleen and pancreas

No current contrast agents have been licensed for the assessment of splenic and pancreatic lesions. However, contrast agents have been used to depict and characterise lesions (Fig. 6.15).

Transcranial

No contrast agents have currently been licensed for the assessment of perfusion in transcranial studies. For the assessment of cerebral arteries, contrast is injected as a bolus to enhance either duplex sonography or colour Doppler imaging. It is used to enhance the Doppler signals in regions where the skull significantly attenuates the Doppler signal or the blood volume is too low.31

Figure 6.13 A: Image of an echogenic liver metastasis (between callipers) acquired in fundamental imaging. B: Image acquired 1 minute and 24 seconds after bolus injection of SonoVue (portal-venous stage). Low reflective area in the contrast-enhanced image is indicative of malignancy. (Images courtesy of Dr Paul Sidhu, King’s College Hospital, London, UK.)

A B

Figure 6.14 A: Image acquired in fundamental mode and suggestive of a complex renal cyst. B: Image acquired after a bolus injection of SonoVue using a low MI imaging technique. No septation enhancement evident, therefore lesion unlikely to be malignant. (Images courtesy of Dr Paul Sidhu, King’s College Hospital, London, UK.)

A B

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Ultrasonic contrast agents can also be used to assess other abnor-malities such as LV apical thrombus many of which are difficult to image without the use of contrast microbubbles (Fig. 6.17). A com-prehensive review of clinical applications of ultrasonic contrast agents in echocardiography is given in a recent American Society of Echocardiography Statement.34

OTHER POTENTIAL USES OF CONTRAST MICROBUBBLES

Targeted contrast microbubbles

One of the most exciting areas of progress in contrast agent devel-opment has been that of selective targeting of the microbubbles to

diagnostic stress test, where regions of the myocardium are indi-vidually assessed for movement under rest, stress and recovery conditions. Such studies are normally undertaken at low MI, ensur-ing that the complete ventricular endocardial border can be visual-ised without microbubble disruption and associated shadowing effects (Fig. 6.16).33

There are currently no ultrasonic contrast agents that are licensed to aid in the assessment of myocardial flow and hence perfusion. As described previously, when microbubbles are administered as an infusion and a consistent level of enhancement is achieved, it is possible to destroy the microbubbles in the scan-plane using a single or several high MI pulses. By then returning to low MI imaging, the time required for segments within the myocardium to become enhanced can be measured. Since this value is related to myocardial blood flow, areas of the myocardium that have a reduced blood flow take longer to enhance.

Figure 6.15 A: Image acquired in fundamental mode and suggestive of a possible splenic cyst. B: Image acquired after a bolus injection of SonoVue and using a low MI technique to image the spleen. Honeycomb pattern suggestive of a spleen abscess. (Images courtesy of Dr Paul Sidhu, King’s College Hospital, London, UK.)

A B

Figure 6.16 Cardiac parasternal short-axis view of the heart during a pharmacologically stressed cardiac stress-echo study. A low MI imaging technique is used. Images acquired at baseline (A), low dose stress (B), high dose stress (C) and recovery (D). (Images courtesy of Dr Stephen Glen, Stirling Royal Infirmary, Scotland.)

A B

C D

Figure 6.17 Contrast-enhanced images of a cardiac thrombus obtained in three imaging planes. (Images courtesy of Dr Stephen Glen, Stirling Royal Infirmary, Scotland.)

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microbubble to fuse with the cell membrane. However, from the literature it appears that many factors affect the size and perma-nency of the opening in the cell wall not least the relative closeness of the microbubble to the cell wall.42

Once sonoporation has occurred, extracellular molecules, such as drugs or genes, either incorporated within the microbubbles or injected near the site of sonoporation, may then enter the cells during this period of permeability thus enhancing the payload of the drugs or genes to the cell.43 Comprehensive reviews of this topic are available.44

SAFETY OF CONTRAST MICROBUBBLES

The main area of concern over the safety of ultrasonic contrast agents is based around the likelihood of acoustic cavitation taking place under routine clinical scanning conditions. The potential hazards and risks of diagnostic ultrasound with and without con-trast agents have recently been addressed.45 In summary, since acoustic cavitation is extremely unlikely to occur in a conventional scan due to the lack of pre-existing gas bubbles within the body, the probability of harm is extremely low. If contrast is introduced the risk of acoustic cavitation and the severity of harm induced by it are considered higher.

The safety of two contrast agents (Definity and Optison) has recently been assessed retrospectively in a large multicentre clinical study. The conclusions of the study indicated that the agents had a good safety profile in both cardiac and abdominal ultrasound appli-cations and the incidence of severe adverse reactions is less than that associated with other contrast agents used with other imaging modalities.46

The World Federation for Ultrasound in Medicine and Biology (WFUMB) published a series of articles as a result of a safety sym-posium dedicated to the use of ultrasonic contrast agents in diag-nostic applications. This series of articles comprehensively addresses clinical applications and safety concerns for contrast agents,47 in-vitro bioeffects,48 in-vivo bioeffects,49 exposures from diagnostic ultrasound equipment50 and mechanisms for the interaction of ultrasound.51 As a result of this symposium, recommendations for reducing the likelihood of bioeffects when using ultrasound con-trast agents included: (i) scanning at low MI, (ii) scanning at higher

Figure 6.18 Methods to attach targeting ligands to microbubble shell. A: Non-covalent method, where avidin is embedded in the shell during bubble formation. B: Biotinylated bubble is coated with streptavidin and then biotinylated ligand. C: Ligand is covalently attached to the bubble shell by a peptide bond. (Reprinted from Klibanov AL. Ultrasound molecular imaging with targeted microbubble contrast agents. J Nucl Cardiol 2007;14:876–884.)

A B C

HN–C=O HN–C=O

HN–C=OHN–C=O

HN–C=O

COO–

COO–C

OO

COO–

Gas insidethe bubble

Gas insidethe bubble

Gas insidethe bubble

Albumin Avidin Biotin Biotinylatedtargeting ligand

Spacer arm Streptavidin

particular sites within the body, enabling acoustic visualisation of localised biological markers expressed in disease processes.

However, the proportion of microbubbles that bind to the surface is dependent on many factors including the number of receptor sites available for binding and the shear stress exerted on the micro-bubbles at the receptor sites.35 Initial in-vitro studies demonstrated encouraging results under well-controlled flow dynamics36 and a review of this field is given elsewhere.37 Figure 6.18 illustrates sche-matically the potential methods of attaching ligands onto the surface of lipid- or albumin-coated microbubbles. Most commonly the binding for liposomal agents is achieved by formulating the liposome using a biotinylated lipid and using streptavidin as the linking mechanism between the biotinylated liposome and a bioti-nylated ligand. The antibody-loaded microbubble when injected into the body is then targeted to biological markers in which the receptor antigens are expressed. Increases in attachment can also be achieved using the radiation force of an ultrasound beam to push the targeted microbubbles towards the cell receptors by applying the ultrasound beam perpendicularly to blood flow.38 Although this has been shown to be useful for in-vitro applications, it is difficult to assess how controllable this would be in clinical situations.

Early results in this area have shown promise with applications that include targeting of microbubbles to atherosclerosis, intra vascular thrombi39 and sites of angiogenesis and tissue inflammation.40

Drug and gene delivery

Although targeting mechanisms enable the microbubbles to become attached to particular biological markers, it is the ability of ultra-sound in the presence of the microbubbles to cause transient pora-tion of the cell wall that enables the microbubbles, their contents or other localised extracellular materials to pass into the cell. This transient permeability of the cell wall is known as sonoporation. To date, it is unclear from the literature the exact range of phenomena that are responsible for sonoporation. Although early research sug-gested that inertial cavitation was the source, more recent work using high speed cameras suggests that micro-streaming associated with non-inertial cavitation may also be associated with sonopora-tion.41 Alternatively, the generation of extremely high temperatures associated with inertial cavitation may result in an increase in the fluidity of the phospholipid membranes thus allowing the

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image cannot be directly related to individual bubbles. The echo signals from bubbles in this situation interfere to form a speckle pattern, just as in the familiar case of echoes from the scattering centres in the parenchyma of organs. It is worth noting that the motion of the speckle pattern does not necessarily relate directly to the motion of the bubbles.

Where strong scatterers are present, such as gas bubbles, the echo signals may not travel directly back to the transducer; instead the path may involve multiple scatters. The multiple scattering delays the return time of an echo and hence it is depicted deeper than the actual position of the original source of the echo. The distal aspect of the region containing bubbles may therefore be displayed beyond its true boundary.

Doppler artefact

After the injection of a bolus of contrast agent, an artefact known as colour blooming may be observed during colour Doppler studies. When contrast agent enters a vessel the magnitude of the signal scattered from within the vessel increases, giving a corresponding increase in the Doppler signal. This effectively broadens the width of the scanning beam, allowing flow to be detected in the weaker regions of the beam. The resulting degradation of the lateral resolu-tion causes the colour-coded flow region in the image to expand, i.e. ‘bloom’. The effect is similar to having the Doppler gain too high and may be compensated by reducing the gain.

REFERENCES1. Gramiak R, Shah P, Kramer D. Ultrasound cardiography: contrast

study in anatomy and function. Radiology 1969;92:939–948.2. Feinstein SB, ten Cate FJ, Zwehl W, et al. Two-dimensional contrast

echocardiography. I. In vitro development and quantitative analysis of echo contrast agents. J Am Coll Cardiol 1984;3(1):14–20.

3. Ophir J, Parker KJ. Contrast agents in diagnostic ultrasound. Ultrasound Med Biol 1989;15(4):319–333.

4. Keller M, Feinstein SB, Watson DD. Successful left ventricular opacification following peripheral venous injection of sonicated contrast agent: an experimental evaluation. Am Heart J 1987;114: 570–575.

5. Schlief R, Schurmann R, Balzer T, et al. Saccharide based contrast agents. In: Nanda C, Schlief R, eds. Advances in echo imaging using contrast enhancement. Dordrecht: Kluwer Academic Publishers; 1993.

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frequencies, (iii) reducing total acoustic exposure time, (iv) reduc-ing contrast agent dose and (v) adjusting the timing of cardiac triggering to avoid systole where most ventricular arrhythmias have been shown to occur.52 The safety of ultrasound is discussed more fully in Chapter 4.

IMAGING ARTEFACTS

There are several artefacts that must be taken into consideration when making clinical ultrasound measurements after the injection of a bolus of contrast. These may be subdivided into propagation artefacts and Doppler artefacts.

Propagation artefacts

When contrast is injected into the body, although it scatters ultra-sound strongly it also strongly attenuates and consequently as the bolus of contrast passes through a vessel or through the heart, organs distal to the vessels will temporarily disappear from the screen until the bolus clears through the system. This is readily observed in cardiac scanning where a large amount of the contrast agent can build up within the ventricles and obscure the posterior walls (Fig. 6.19). Shadow artefacts are commonly helpful in making a diagnosis in ultrasonic imaging; however, those created by con-trast agents have not been used in that way.

Although a single bubble can be detected as a single spot in an image, when there are a large number of bubbles the spots in the

Figure 6.19 Example of acoustic shadowing during a cardiac study after a bolus injection of contrast. A: complete shadowing of left ventricle by contrast in right ventricle. B: contrast begins to clear from right ventricle, shadowing reduces. C: complete left ventricular opacification is now visualised.

CBA

Clinical applications and safety

• Ultrasound contrast microbubbles can be targeted to sites within the body.

• They are currently being investigated as drug delivery agents.• WFUMB recommendations for the safe use of ultrasound include:

• scanning at low MI• scanning at higher frequencies• reducing total acoustic exposure time• reducing contrast dose• adjusting cardiac triggering to avoid end-systole triggering.

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