Bioeffects Considerations forDiagnostic UltrasoundContrast Agents

Douglas L. Miller, PhD, Michalakis A. Averkiou, PhD,Andrew A. Brayman, PhD, E. Carr Everbach, PhD,Christy K. Holland, PhD, James H. Wible, Jr, PhD, Junru Wu, PhD

iagnostic ultrasound contrast agents have beendeveloped for enhancing the echogenicity of bloodand for delineating other structures of the body.Approved agents are suspensions of gas bodies (sta-

bilized microbubbles), which have been designed for persis-tence in the circulation and strong echo return for imaging. Theinteraction of ultrasound pulses with these gas bodies is a formof acoustic cavitation, and they also may act as inertial cavitationnuclei. This interaction produces mechanical perturbation and apotential for bioeffects on nearby cells or tissues. In vitro, sono-poration and cell death occur at mechanical index (MI) valuesless than the inertial cavitation threshold. In vivo, bioeffectsreported for MI values greater than 0.4 include microvascularleakage, petechiae, cardiomyocyte death, inflammatory cell infil-tration, and premature ventricular contractions and are accompa-nied by gas body destruction within the capillary bed. Bioeffectsfor MIs of 1.9 or less have been reported in skeletal muscle, fat,myocardium, kidney, liver, and intestine. Therapeutic applica-tions that rely on these bioeffects include targeted drug deliveryto the interstitium and DNA transfer into cells for gene therapy.Bioeffects of contrast-aided diagnostic ultrasound happen on amicroscopic scale, and their importance in the clinical settingremains uncertain. Key words: acoustic cavitation; contrastagent adverse effects; echocardiography; mechanical index.

Received April 12, 2007, from the Department ofRadiology, University of Michigan, Ann Arbor,Michigan USA (D.L.M.); Philips Medical Systems,Bothell, Washington USA (M.A.A.); AppliedPhysics Laboratory, University of Washington,Seattle, Washington USA (A.A.B.); Department ofEngineering, Swarthmore College, Swarthmore,Pennsylvania USA (E.C.E.); Department of BiomedicalEngineering, University of Cincinnati, Cincinnati,Ohio USA (C.K.H.); Tyco Healthcare/Mallinckrodt Inc,St Louis, Missouri USA (J.H.W.); and Department ofPhysics, University of Vermont, Burlington, VermontUSA (J.W.). Revision requested April 18, 2007. Revisedmanuscript accepted for publication December 4,2007.

We thank the members of the BioeffectsCommittee of the American Institute of Ultrasoundin Medicine for valuable suggestions for revision andimprovement of this report.

Address correspondence to Douglas L. Miller, PhD,University of Michigan, 3315 Kresge III, 200 ZinaPitcher Pl, Ann Arbor, MI 48109-0553 USA.

E-mail: douglm@umich.edu

AbbreviationsCAT, chloramphenicol acetyltransferase; CK, creatinekinase; ECG, electrocardiographic; FDA, Food and DrugAdministration; MCE, myocardial contrast echocardiog-raphy; MI, mechanical index; PESDA, perfluorocarbon-exposed sonicated dextrose albumin; PRPA, peakrarefactional pressure amplitude; PVC, premature ven-tricular contraction

1. Introduction

The ability to “see” inside the body represents oneof the most potent diagnostic tools of modernmedicine. Ultrasound imaging is particularly attrac-tive owing to the portability of imaging machines andthe inherent safety of low-power acoustic interroga-tion of tissue. The desire for means to enhance imageinformation has led to the development of contrastagents for pulse-echo diagnostic ultrasound, as for

© 2008 by the American Institute of Ultrasound in Medicine • J Ultrasound Med 2008; 27:611–632 • 0278-4297/08/$3.50

D

AIU

M C

on

sensu

s Rep

ort o

n Po

tential B

ioeffects o

f Diag

no

stic Ultraso

un

d

Article includes CME testCME

other imaging modalities (ie, radiography andmagnetic resonance imaging). Although similarin concept to radiologic contrast agents, ultra-sound contrast agents have unique propertiesdesigned for echogenic interaction with theultrasonic pulses, which make them specialcases for safety assurance in diagnosis.

The primary purpose of ultrasound contrastagents is to enhance the echogenicity of blood.Although modern Doppler imaging equipmentis very good at showing the location and motionof rapidly flowing blood, it fails in other impor-tant diagnostic problems such as capillary per-fusion. What could reasonably be added toblood to enhance its echogenicity? The answer ismicrobubbles. Over the past 2 decades, contrastagents consisting of suspensions of gas bodies(specially stabilized microbubbles) have beendeveloped that can pass through the lungs afterintravenous injection and persist in circulationfor useful periods.1–3 Ultrasound contrast agentscan enhance B-mode and Doppler images, andspecial imaging methods can show blood distri-bution at the capillary level to reveal tissue perfu-sion.4 Owing to the promise of safe and morecost-effective vascular diagnosis than might bepossible with contrast-enhanced radiography ormagnetic resonance imaging, a substantialresearch and development effort has been pur-sued to achieve approval and bring ultrasoundcontrast agents to the clinic.

The interaction of ultrasound with gas bodiesfor enhanced echogenicity also introduces apotential for bioeffects. Ultrasonic cavitation isdefined as the interaction of ultrasound with abody of gas and is a potent mechanism for bio-logical effects of ultrasound.5,6 Cavitation involv-ing microbubbles (microcavitation) has longbeen recognized as the most likely potentialmechanism for nonthermal bioeffects of diag-nostic ultrasound.7,8 At the megahertz frequen-cies used for diagnostic ultrasound, the mostefficiently echogenic (ie, resonant) gas bodiesare a few (≈1–5) micrometers in diameter. Atlow ultrasonic pressure amplitudes, less thanatmospheric pressure (0.1 MPa), cavitationmicrobubbles pulsate linearly in response to thetime-varying acoustic pressures of the ultra-sound field. At higher pressure amplitudes, thepulsation becomes nonlinear, and expansion

during the rarefactional pressure phase maybecome much greater than the initial microbub-ble radius. The large expansions are followedby violent collapse of the microbubble, in whichthe collapse is dominated by the inertia of theinrushing fluid surrounding the microbubble.For this reason, this special case of ultrasoniccavitation is called inertial cavitation. The mini-mum threshold for inertial cavitation was calcu-lated for the diagnostically relevant frequencyrange,9 and this theory served as the basis for themechanical index (MI) used for display on ultra-sound imaging machines.10

Gas bodies suitable for nucleation of ultrasoniccavitation are normally practically absent fromthe body, minimizing the possibility of cavitation-al bioeffects for diagnostic ultrasound withoutcontrast agents.6 The fortuitous coincidence thatthe microbubble sizes most strongly activated bydiagnostic ultrasound pulses to yield highechogenicity are small enough to pass throughthe circulatory system has allowed the creation ofultrasound contrast agents. However, the use ofgas body contrast agents introduces a potentialfor microcavitation bioeffects into the clinicaldiagnostic setting. At low MIs, the stabilizedmicrobubbles scatter ultrasound effectively, butthe amplitudes of oscillation are too small to havesignificant effects on nearby cells. At higher MIs,the contrast agent microbubbles may be destabi-lized, allowing loss of the gas or nucleation ofmicrocavitation activity.11 This destabilizationprocess can be useful for certain diagnostic pro-cedures but can also damage nearby cells. Thebioeffects possible with contrast agents and diag-nostic ultrasound are sufficiently robust that sev-eral therapeutic applications are under study.12

The subject considered in this report is thepotential for contrast-enhanced diagnostic ultra-sound to induce cavitational bioeffects by ultra-sound interaction with the gas bodies. Thispotential has been the subject of authoritativereviews previously.5,6 In addition, a symposiumon the safe use of ultrasound contrast agents wasconducted recently by the World Federation forUltrasound in Medicine and Biology.13 Thisreport arose from the 2005 American Institute ofUltrasound in Medicine Bioeffects ConsensusConference. Aspects of this subject consideredbelow include (1) the nature of present contrast

612 J Ultrasound Med 2008; 27:611–632

Bioeffects Considerations for Diagnostic Ultrasound Contrast Agents

AIU

M C

on

sen

sus

Rep

ort

on

Po

ten

tial

Bio

effe

cts

of

Dia

gn

ost

ic U

ltra

sou

nd

agents, including diagnostic applications andultrasound imaging modes; (2) the basic in vitrobioeffects of contrast gas body activation; and (3)reported in vivo bioeffects, including the thera-peutic potential of contrast-enhanced diagnosticultrasound. The text will only briefly revisit infor-mation included in the earlier conference report5

and will concentrate on bioeffects data relevant todiagnostic imaging, which have become availablebetween 1998 and the 2005 conference. Summaryconclusions are then presented with recommen-dations intended to optimize the safety profile ofcontrast-aided diagnostic ultrasound.

2. Contrast Agents for DiagnosticUltrasound

2.1. Ultrasound Contrast AgentsFirst-generation contrast agents used air as thecore gas. These air bubbles were surrounded by afatty acid, lipid, or protein shell. The shell sur-rounding the air bubble increased the stability ofthe microbubbles both in the vial and in thebody. Commercially available first-generationagents included Echovist (SH U 454; Schering AG,Berlin, Germany), Levovist (SH U 508A;, ScheringAG), both of which were only available in Europe,and Albunex (Molecular Biosystems, Inc, SanDiego, CA), which was the first agent approved bythe US Food and Drug Administration (FDA) foruse in the United States.

One of the problems with the first-generationmicrobubbles was the short duration of efficacyafter intravenous injection. These first-genera-tion agents used air as the active component.However, because of an inherent unsaturation ofdissolved gases (primarily oxygen, nitrogen, andcarbon dioxide) in blood, the air contained with-in these contrast agents readily diffused out ofthe microspheres or microbubbles.14 With thisloss of air, the first-generation contrast agentsquickly lost the ability to produce ultrasoundcontrast. The next advancement made in ultra-sound contrast agents (the second generation)was the inclusion of gases having decreasedsolubility and diffusivity. These microspheresor microbubbles retain their gas for a longerperiod of time; thus, the durations of contrastand Doppler enhancement increase from sever-al seconds to several minutes.15

Optison (FS-069, perflutren protein type Amicrospheres for injection; GE Healthcare,Princeton, NJ) was the first second-generationultrasound contrast agent approved by the FDA.This agent consists of a protenaceous shell sur-rounding a gas bubble of octafluoropropane gas.16

This relatively insoluble gas is inert and eliminat-ed through normal gas exchange in the lung.Optison opacifies the cardiac ventricular cham-bers at doses much smaller than Albunex (0.2 mLcompared with 15–20 mL, respectively) andopacifies the chamber for a much longer period oftime than Albunex (>5 minutes versus 30–45 sec-onds).17 Currently, Optison is indicated to opacifythe left ventricular chamber and to improve thedelineation of the endocardial border.

Definity (MRX-115, perflutren lipid micro-spheres; Bristol-Myers Squibb Medical Imaging,North Billerica, MA) is another agent that encap-sulates octafluoropropane, but this agent usesphospholipids to coat the microbubbles. Beforeinjection, Definity must be activated by agitationin a “dental shaker” for approximately 45 seconds.Small volumes of these microbubbles opacify theleft ventricular chamber and enhance the Dopplersignal from the peripheral vasculature for pro-longed periods of time.18,19 The microvascular rhe-ology of Definity microspheres has been shown tobe similar to that of erythrocytes in the circula-tion.20 Currently in the United States, Definity isindicated for opacification of the left ventricularchamber and to improve the delineation the endo-cardial border. In Canada, Definity is indicated forboth cardiology and radiology applications.

Imagent (AFO150, perflexane lipid micro-spheres; IMCOR Pharmaceuticals, San Diego, CA)is another ultrasound contrast agent that uses aperfluorocarbon to increase in vivo stability. Thisagent is composed of phospholipids, phosphatebuffers, sodium chloride, and a blend of perfluo-rohexane and nitrogen.21 As with the other twoapproved agents, Imagent is indicated for opacifi-cation of the left ventricular chamber and toimprove delineation of the endocardial borders.

Although not approved for sale in the UnitedStates, SonoVue (BR-1, sulfur hexafluoride; BraccoInternational BV, Amsterdam, the Netherlands) isalso a second-generation ultrasound contrastagent approved for use in Europe. This agent iscomposed of phospholipids, ethylene glycol,

J Ultrasound Med 2008; 27:611–632 613

Miller et al

AIU

M C

on

sensu

s Rep

ort o

n Po

tential B

ioeffects o

f Diag

no

stic Ultraso

un

d

and sulfur hexafluoride gas.22 Originally,SonoVue was approved in March 2001 toincrease the echogenicity of the blood, opacifythe cardiac chambers, improve delineation of theendocardial borders, improve the Doppler signal-to-noise ratio in the cerebral, extracranial carotid,and peripheral arteries, and improve the visual-ization of the vascularity of liver and breastlesions (SonoVue package insert). In May 2004,the European Medicines Agency restricted theuse of SonoVue to noncardiac imaging proce-dures.23 This restriction was later removed, and aprecautionary statement was issued stating thatextra caution should be exercised in patients withconditions such as severe hypotension, bradycar-dia, cardiac arrest, and myocardial infarction.

The second-generation ultrasound contrastagents relied on core gases that possessed lowsolubility in blood to prolong their duration inthe circulation. A third generation of contrastuses engineered changes in the microsphereshell to impart unique features to the contrastmaterials. Although these agents are still indevelopment, two are in the later stages of theirinitial clinical development.

CARDIOsphere (PB127; Point Biomedical Corp,San Carlos, CA) is composed of a bilayer shell madeup of polylactide and albumin. The polylactideinner layer is a biodegradable polymer that pro-vides specific physical characteristics, which con-trol acoustic properties of the contrast agent. Theouter layer, composed of human albumin, func-tions as the biological interface and provides bio-compatibility. These microspheres use nitrogen asthe gas core. They were designed to collapse undervery specific ultrasound conditions.24 Once themicrospheres are destroyed, the encapsulatednitrogen is released, and the gas quickly dissolvesinto the surrounding blood. This rapid loss of gasproduces an intense signal using harmonic powerDoppler imaging techniques.25 Another third-gen-eration agent, AI-700 (Acusphere, Inc, Watertown,MA), uses a synthetic porous microparticle to trapan insoluble gas. These microparticles are com-posed of D,L-lactide co-glycolide, a biodegradablepolymer. These microparticles appear to be moreresistant to the destructive effects of ultrasound.26,27

Several companies continue to develop newultrasound contrast agents with unique proper-ties and niche applications. Their developmental

status, physical characteristics, and imaging pro-files are less well known. A description of thesevarious materials must wait until they move fur-ther along the developmental pathway. Inaddition, several other agents (eg, Ecogen,[Sonos Pharmaceuticals, Bothell, WA], Sonazoid[Nycomed, Zurich, Switzerland], Sonovist[Schering AG], and Quantison [Andaris Ltd,Nottingham, England]) failed to make it throughthe entire developmental process and gain regula-tory approval for one reason or another. Althoughthese agents did not make it to the clinical market,the information derived from their use both pre-clinically and clinically has contributed to a betteroverall understanding of ultrasound contrastagents, their effects and their applications.

2.2. Diagnostic Applications of UltrasoundContrast Agents

Echocardiographic applications for Optison,Definity, and Imagent approved for use in theUnited States include left ventricular opacificationand border delineation. Contrast echocardiogra-phy can also be used (but is not yet approved inthe United States) for measurement of myocardialperfusion.28 Levovist and SonoVue are used inother countries for cardiac, microvascular, andtranscranial indications. Possibly the most promi-nent clinical application of contrast agents inCanada, Europe, and Asia is the detection andcharacterization of liver lesions. There are manyother potential applications for imaging, includ-ing tissue perfusion, inflammation, and tumors, inany tissue accessible to ultrasound interrogation,including liver, kidney, breast, spleen, and oth-ers.29–31 Ultrasound contrast agents are useful alsofor imaging body structure and function, such asfor the diagnosis of vesicoureteral reflux32,33 andfallopian tubal patency.34,35

2.3. Diagnostic Ultrasound Modes Used WithContrast Agents

Ultrasound imaging enhanced by the use ofultrasound contrast agents has been the subjectof several recent reviews.36–38 Ultrasound con-trast agents can be used most simply by bolusinjection or infusion to enhance the echogenici-ty of blood in B-mode or Doppler images. High MIvalues, such as those used for harmonic imagingwithout contrast agents, result in the destruction

614 J Ultrasound Med 2008; 27:611–632

Bioeffects Considerations for Diagnostic Ultrasound Contrast Agents

AIU

M C

on

sen

sus

Rep

ort

on

Po

ten

tial

Bio

effe

cts

of

Dia

gn

ost

ic U

ltra

sou

nd

of the contrast agent and loss of contrast. Thisproperty of contrast agents has been used by inter-mittent imaging with agent infusion, which allowsthe tissue vasculature to refill with the contrastagent before the next image frame is processed (eg,see Porter et al39 and Kuersten et al40). This proce-dure can be used to image perfusion by varying theinterframe trigger timing, as for myocardial con-trast echocardiography (MCE).41 Doppler tech-niques are well suited to image microbubbledestruction. Harmonic power Doppler imagingcombined with some form of triggering has beenused extensively to image microbubbles in themacrocirculation and microcirculation.

The increased sensitivity provided by newerimaging techniques makes it possible to imagecontrast microbubbles relatively nondestructivelyin real time at very low acoustic pressures. Low-MIimaging is important for two reasons. First, at alow MI, bubble destruction is avoided. Althoughmicrobubbles differ in their shell composition,work completed to date indicates that at an MI ofabout 0.15, the microbubbles examined are notsignificantly destroyed yet give a good harmonic(nonlinear) contrast signal.37 The second majorreason for low-MI scanning is the reduction of theharmonic component in the tissue echoes relativeto bubble echoes. While tissue harmonics havebenefited routine diagnostic scanning, it is thebackground “noise” signal that the contrast signalmust rise above. Because tissue is less nonlinearthan bubbles, it requires a higher MI than the con-trast microbubbles for a certain harmonicresponse. Therefore, at a low MI, the contrast-to-tissue ratio is higher than at a high MI, helpingremove the tissue signal and leave only the con-trast. For quantification purposes, both high andlow MIs may be combined, in which a high-MIpulse is sent to destroy the contrast microbubblesin a scan plane, and then a low MI is used after-ward to monitor the contrast replenishment.

3. Ultrasound Interaction With ContrastAgent Gas Bodies: Physical Theory

3.1. PulsationThe physical interaction between ultrasoundpulses and contrast agent gas bodies producesmicrobubble pulsations, which are responsiblefor their high echogenicity but which also increase

the potential for local bioeffects. Theory has beendeveloped for the interaction of ultrasound withencapsulated gas bubbles in contrast agents suchas Optison.42,43 The theory is similar to that for afree cavitation microbubble but with added elas-ticity and viscosity-related damping parametersto account for the shell. This theory has beenused to describe the scattering properties of sus-pensions of the agents, leading to empirical val-ues for the shell parameters.44 The theoriesappear to be reasonable models of the gas bodybehavior for low levels of excitation. In addition,the presence of blood cells around the contrastagent gas bodies has a relatively small effect on thetheoretical dynamics of the pulsation.45

3.2. Influence of Contrast Agents on TissueHeating

Although thermal effects of ultrasound are treat-ed in other articles,46,47 the presence of contrastagents may have an effect on tissue heating. Thetheory for this effect has been advanced48 andtested in nonbiological materials such as tissue-mimicking phantoms.49 Because of the ability ofbubbles to oscillate nonlinearly and producehigher-frequency components than are presentin the insonifying beam, acoustic energy can bedissipated much more effectively as heat.Moreover, the effects of fluid viscosity near oscil-lating bubbles are enhanced because of the largecomponent of acoustic radiation (especially forbubbles above the resonance size50). Numericalmodeling of the theory agrees reasonably wellwith in vitro experiments.51–53 In addition, Strideand Saffari54 have noted that the viscous proper-ties of the stabilizing shells could enhance localheating near the gas bodies.

Measurements of excess heating of tissue dueto the presence of echo contrast agents fall most-ly in the insonification regimen of therapeuticultrasound. Fujishiro et al55 insonified beef sam-ples using 1.5-MHz continuous ultrasound at 0.9W/cm2 for 3 minutes and found an equivalenttemperature rise to doubling the intensity with-out contrast agents present. Wu56 found excesstemperature elevation of several degrees Celsiusdue to the presence of contrast agents in suspen-sions insonified at 3.5 W/cm2 and 1 MHz. Sokka etal57 monitored lesion formation in rabbit thighsafter bubbles were detected following a 7-W con-

J Ultrasound Med 2008; 27:611–632 615

Miller et al

AIU

M C

on

sensu

s Rep

ort o

n Po

tential B

ioeffects o

f Diag

no

stic Ultraso

un

d

tinuous insonation at 1.7 MHz for 20 seconds.The lesions were larger in volume by up to a fac-tor of 3 for the bubble-enhanced exposures.

3.3. Cavitation NucleationAt low pressure amplitudes, the contrast agentgas bodies may remain stable; that is, theirshells may remain intact as the gas body oscil-lates. However, destabilization of gas bodiesappears to occur at modest peak rarefactionalpressure amplitudes (PRPAs), particularly forshell-encapsulated designs. At relatively highpressure amplitudes, the stabilized gas bodiesare destroyed but can serve as cavitation nuclei.As the incident ultrasound pressure amplitudeincreases from 0, the stabilizing shell experi-ences oscillating stresses. Above rather modestexcitation PRPAs, the theoretical stresses maybe sufficient to expect shell failure.58 From invitro bioeffects research on contrast agents, it isevident that bioeffects are often associated withgas body destabilization, and that observationof the loss of gas bodies appears to agree withthe theory for expected shell failure as a func-tion of frequency.59 Uncertainty remains as tohow the theories can be used to describe thedestabilization of the gas bodies and the com-plex behavior at higher amplitudes. The fate ofdestabilized gas bodies can include gradualshrinkage or rapid fragmentation, dependingon physical conditions.60,61

Nucleation of cavitation involves a transitionfrom restricted pulsation of gas bodies to the freepulsation of cavitation microbubbles. Cavitationnucleation by ultrasound contrast agents isimportant with regard to the bioeffects potentialin vivo because there normally are few, if any,cavitation nuclei in the body suitable for directactivation by diagnostic ultrasound pulses.62

Ultrasound contrast agents can supply suchnuclei. For example, direct evidence of cavita-tional activity has been obtained by detection ofbroadband noise emissions from the myocardi-um during contrast echocardiography.63

At relatively modest PRPAs, the pulsation of freemicrobubbles results in collapse driven by theinertia of the surrounding liquid during the com-pression phase of the oscillation. This phe-nomenon defines inertial cavitation, which isstrongly associated with many bioeffects. These

bioeffects are caused by fluid jets, extreme heat-ing, and free radicals generated on collapse.5 Thethreshold for inertial cavitation derived for opti-mum-sized nuclei was the basis for the MI. Byassuming the presence of these optimally sizednuclei, the inertial cavitation threshold p forblood at frequency f was found to fit the formula9

The square root of this formula approximates theform of the MI (ie, the PRPA in MPa divided bythe square root of the frequency in megahertz).The minimum inertial cavitation threshold inblood can therefore be expressed approximatelyin terms of the MI as MIt = 0.4. As noted below insection 5, several in vivo bioeffects associatedwith contrast-aided diagnostic ultrasound havebeen reported at and above this MI value,although the frequency dependence of ultra-sound bioeffects thresholds in the presence of anecho contrast agent may differ from that suggest-ed by the MI (see Figure 1).

3.4. Shear Stress Theory for BioeffectsBioeffects induced by ultrasound interactionwith contrast agents are amenable to theoreticalconsideration. However, detailed theory for esti-mating the amount of biological perturbationexpected from a given exposure situation is notpresently available for use in medical applicationsof contrast agents. Even for moderate pressureamplitudes, a number of different mechanismsmay plausibly have a role in bioeffects.64 Largeoscillatory and steady fluid shear stresses occur forgas body pulsation near solid surfaces. The con-trast agent gas bodies can destabilize and nucleateinertial cavitation. Cavitation, in the absence ofcontrast agents, is rare in most tissues and is dis-cussed in other conference reports.65,66 However,the use of ultrasound contrast agents introducesthe potential for cavitational bioeffects into therisk/benefit equation for diagnostic ultrasound.

For low PRPAs and simple in vitro conditions,cellular bioeffects have been modeled theoreti-cally with some success. Fluid shear stress gener-ated in the vicinity of a pulsating gas body canproduce mechanical membrane damage.67,68 Themicrostreaming shear stress model has provenuseful in describing several bioeffects situations

13. .067.1

=f

p

616 J Ultrasound Med 2008; 27:611–632

Bioeffects Considerations for Diagnostic Ultrasound Contrast Agents

AIU

M C

on

sen

sus

Rep

ort

on

Po

ten

tial

Bio

effe

cts

of

Dia

gn

ost

ic U

ltra

sou

nd

involving gas body activation in terms of the dam-age of cell membranes by shear stress in acousticmicrostreaming fluid flow near the oscillating gasbody.69 Approximate theory is available for thenonoscillatory steady shear stress generated innear-boundary acoustic microstreaming and canbe used to estimate the pressure amplitudesrequired to exceed the critical shear stress for bio-logical membranes. The average steady stress canalso persist with a time-average value given by thepeak stress times the fractional duty cycle. Miller70

considered this theoretical model with respect toultrasound contrast agents. Significant shear stresswas found to be possible for relatively low pressureamplitudes, particularly for the case of freemicrobubbles. The pressure amplitude for whichthe shear stress was expected to exceed critical lev-els increased approximately in proportion to fre-quency for encapsulated gas bodies. Wu71 used themicrostreaming shear stress theory to model theeffects of contrast agent gas bodies attached tocells when exposed to ultrasound. Shear stressesgenerated by 1- to 2-MHz ultrasound were foundto be sufficient for sonoporation (transient perme-abilization with resealing) of cells at a pressureamplitude of only 0.12 MPa. Destabilization withliberation of free bubbles was shown to produce amuch higher shear stress with potential cell killing.The shear stress model was applied to a specificexperimental system for which contrast agent gasbodies were exposed to pulsed ultrasound whileattached to monolayer cells.72,73 The observedPRPA thresholds p for destabilization at differentultrasound frequencies f correspond to approxi-mately constant relative pulsation amplitudes(amplitude divided by the initial radius). The con-stant relative amplitudes theoretically yieldapproximately constant shell stresses, which weresufficient to induce destabilization. The observedcell death thresholds corresponded theoretically toapproximately constant radial velocity ampli-tudes. The constant velocity amplitudes theoreti-cally yield slowly increasing microstreaming shearstress for 1.8- to 0.2-microsecond pulse durationsin the 1- to 10-MHz range. For this model system,contrast agent gas body destabilization and pulsa-tion-induced cell membrane damage was expect-ed to occur for similar critical values of theparameter p/f. The comparison to in vitro bioef-fects is noted below in section 4.2.

4. Basic In Vitro Studies of BioeffectsPotential

In vitro experimental models can provideinsights into the fundamental processes andminimum ultrasound exposures needed for bio-effects induced by the interaction of ultrasoundwith contrast agents. There are two qualitativelydifferent situations for cultured cells: suspen-sions and monolayers. Selected PRPA values forthresholds of in vitro bioeffects are plotted as afunction of frequency in Figure 1.

4.1. Effects Produced in Cell SuspensionsSonolysis of red blood cell suspensions (orhemolysis) containing contrast agent gas bodieshas been studied extensively.5 The frequencydependence of thresholds for hemolysis inwhole blood with added Albunex in a stationarychamber was determined by Miller et al74 forpulsed (10-microsecond pulses) ultrasound(Figure 1). The threshold at 2.4 MHz withOptison was found to be somewhat lower,presumably due to the greater presence of themicrobubbles.75

A correlation between contrast-aided-ultra-sound induced hemolysis in a rotating cham-ber and the amount of passively detectedinertial cavitation activity (an inertial cavita-tion “dose”) has been established.78–81 This cor-relation is so robust that hemolysis can be usedas a cavitation dosimeter.81,82 In blood contain-ing ultrasound contrast agent gas bodies, expo-sure to 1-MHz ultrasound with a 2.0-MPa PRPAproduces detectable hemolysis and inertial cav-itation dose with pulses as brief as 2 cycles.80

The inertial cavitation dose and hemolysisevolve and reach limiting values very rapidly invitro as microbubbles are destroyed. Both endpoints are influenced strongly by the PRPA,with thresholds at 1 MHz of approximately 0.5to 1 MPa.81 Hemolysis induced by inclusion ofcontrast agents in ultrasound-exposed sampleshas a very strong frequency dependence atsuprathreshold PRPAs.79,82,83 That is, the PRPA toproduce a given level of hemolysis increases withfrequency much faster than the f 0.5 frequencydependence of the MI. The MI, therefore, haspoor predictive value for the magnitude ofobserved hemolysis above the threshold.

J Ultrasound Med 2008; 27:611–632 617

Miller et al

AIU

M C

on

sensu

s Rep

ort o

n Po

tential B

ioeffects o

f Diag

no

stic Ultraso

un

d

Suspended phagocytic cells, which have bound orphagocytosed contrast agent gas bodies, are lysedwhen exposed to megahertz-frequency acousticpressures within the capabilities of diagnosticimaging machines.84,85 When the expansion phaseof the phagocytosed bubble oscillation exceededthe cell’s ability to expand, cell membrane damagewas observed. For 3-µm-diameter gas bodies and a2.25-MHz frequency, a 0.9-MPa pulse did notappear to damage the membrane, while a 1.6-MPapulse resulted in membrane rupture.

Exposure of whole blood to 3.5-MHz ultra-sound produced by a diagnostic ultrasoundscanner (MI = 1.9) induced platelet activation ata high gas body concentration of Levovist.86

Killing of lymphocytes cosuspended with con-trast agent gas bodies in vitro results from expo-sure to low-amplitude (0.2-MPa PRPA, 20/180cycles on/off) 2-MHz ultrasound in the rotatingtube system.87

4.2. In Vitro Studies of Bioeffects on CellMonolayers

Miller and Bao88 studied cell killing in Chinesehamster ovary monolayers insonified in the pres-ence of Albunex. Monolayers were exposed to 10-microsecond pulses of 3.3-MHz ultrasoundwhile “inverted” (ie, the monolayer was locatedat the top of the vessel during exposure so thatthe gas bodies would rise to become adjacent tothe monolayer). This model system was designedto maximize the potential for cellular effects andmay simulate the concentration of gas bodies ata distal vessel wall by acoustic radiation forces.89

A second transducer was used to detect subhar-monic emissions associated with oscillating bub-bles. Cell killing and bubble acoustic emissionswere correlated with the PRPA, with a thresholdof 0.56 MPa. Similar experiments using Optisonand 3.5-MHz diagnostic ultrasound showed epi-dermoid cell sonoporation to be strongly depen-dent on gas body concentration.90 Sonoporationwas detected at PRPAs as low as 0.23 MPa in thepulsed Doppler mode and 0.39 MPa in the B-mode. Experiments to characterize gene transferand cell killing were conducted with similarinverted epidermoid cell monolayers and used adiagnostic scanner operating in the harmonicmode (1.5-MHz transmit frequency) to producethe ultrasound exposures. With 2% Optison, thePRPA threshold for cell killing was less than 0.7MPa, and gene transfer was detected above 1.7MPa.91

Brayman et al76 modeled the endothelial layerof blood vessels with V79 fibroblast monolayerswhose orientation was varied to simulate eitherthe proximal or distal walls of blood vesselsscanned by ultrasound. Contrast agentsincreased damage to both distal and proximalmonolayers at 1.0-, 2.1-, and 3.5-MHz frequen-cies with a sharp frequency dependence (Figure1). Distal monolayers were damaged more thanproximal monolayers. Kudo et al92 used a high-speed camera to directly observe ultrasound-induced (1 MHz, 0.6-MPa PRPA) oscillations of

618 J Ultrasound Med 2008; 27:611–632

Bioeffects Considerations for Diagnostic Ultrasound Contrast Agents

AIU

M C

on

sen

sus

Rep

ort

on

Po

ten

tial

Bio

effe

cts

of

Dia

gn

ost

ic U

ltra

sou

nd

Figure 1. Critical (apparent threshold) measurements of thePRPA for cell damage by pulsed ultrasound interaction with con-trast agent gas bodies in in vitro test systems. Circles74 are fittedby line A for hemolysis in a whole-blood suspension withAlbunex. Triangles are for hemolysis with an Albunex (top) orOptison (bottom) suspension in whole blood.75 Line B is for ero-sion of endothelial-like cell monolayers by pulsed ultrasoundinteraction with Albunex suspended in the medium.76 Filled dia-monds72 are fitted by line C for killing of phagocytic monolayercells with Optison gas bodies attached to the cells. Open dia-monds represent apparent thresholds for cell killing (top) andsonoporation (bottom) of epidermoid cells with Optison gasbodies allowed to rise and contact the monolayer cells.77

Reproduced with permission from Miller D. Overview of experi-mental studies of biological effects of medical ultrasound causedby gas body activation and inertial cavitation. Prog Biophys MolBiol 2007; 93:314–330.

contrast agent gas bodies adjacent to bovineendothelial cell monolayers. Obvious cell shapedistortions and killing were associated with non-spherical bubble collapse.

Phagocytic cell monolayers (RAW-264.7) prein-cubated with Optison and then washed toremove unbound gas bodies were killed by expo-sure to ultrasound produced by a diagnosticultrasound machine operated in the spectralDoppler mode, with a PRPA threshold of approx-imately 0.2 MPa.77 Similar experiments usingOptison showed the PRPA threshold to be 0.8MPa for exposures consisting of a single 2-cyclepulse.72 Using 2-cycle ultrasound pulses, thePRPA thresholds for killing RAW-264.7 cellspreloaded with Optison showed a linear correla-tion (r2 = 0.982) with frequencies over the 1- to10-MHz range (Figure 1), increasing with a slopeof approximately 0.06 MPa/MHz.73 The authorsnote that these pressure thresholds are lowerthan those for nucleation of inertial cavitationand have a markedly different frequency depen-dence. As noted above in section 3, the frequen-cy dependence of gas body destabilization andcellular bioeffects observed for this in vitro sys-tem can be modeled by the theory for shellstresses and acoustic microstreaming shearstress on cells.59 The theory substantiated theobserved linear dependence of thresholds onfrequency. Owing to the design of this modelmonolayer system for maximum sensitivity forcellular bioeffects, the thresholds observed mayapproximate the lowest PRPAs for which biolog-ically significant bioeffects (ie, cell killing) can beexpected for contrast-aided pulsed ultrasound.

5. In Vivo Studies of Bioeffects

In this section, available reports on in vivo bioef-fects associated with contrast ultrasound will bereviewed. Some reports of bioeffects at highpulse amplitudes, which did not involve actualdiagnostic ultrasound or pulsed ultrasoundintended to simulate diagnostic ultrasound, arebriefly noted to indicate the types of bioeffectsthat might occur above the guideline upper lim-its for diagnostic ultrasound. Results of clinicalresearch conducted during the approval processare generally not available for review. In addition,research on possible pharmacologic side effects

(ie, without ultrasound interaction) of the con-trast agents are not reviewed here. No epidemio-logic studies are available on possible adverse (orfavorable) health effects of the use of ultrasoundcontrast agents. Most in vivo research has beenconducted using mice or rats. Reports have cen-tered mostly on skeletal or cardiac muscle, aslisted in Table 1. Several studies on other tissuesare listed in Table 2. Research on the possibletherapeutic use of diagnostic ultrasound aidedby gas bodies is also reviewed briefly.

5.1. Skeletal Muscle and MyocardiumThe behavior and resulting bioeffects of contrastagent gas body destruction by diagnostic ultra-sound were observed by intravital microscopy ofthe spinotrapezious muscle in rats.93 The musclewas positioned in a custom-built chamber filledwith Ringer’s solution containing adenosine forvascular dilation and propidium iodide to stainnuclei of dead cells. A phased array diagnosticultrasound system was used in the harmonicmode at 2.3 MHz to image the muscle. Optisonwith fluorescently labeled gas bodies wasinfused into the femoral vein for 1 minute beforeobtaining a single image frame at a specific MI,with MIs of 0.4, 0.5, 0.7, and 1.0 used for each ani-mal. After exposure, the muscle was examinedfor microvessel rupture and dead (stained) cells.The number of capillary rupture sites andstained cells was near 0 at an MI of about 0.4 andincreased rapidly for the higher MI values.

The induction of petechiae by contrast-aideddiagnostic ultrasound was confirmed for skeletalmuscle by Miller and Quddus.94 A 2.5-MHzprobe was directed at the abdomens of anes-thetized mice mounted in a water bath to pro-vide for free-field exposure conditions. Atissue-mimicking phantom was used to simulateintervening tissue. Evans blue dye, used to indi-cate microvascular leakage, and Optison wereintroduced by retro-orbital injection. After imag-ing, the abdominal muscle and intestines wereexamined for microvascular injury. For 10 peri-ods with 10 seconds on and 10 seconds off with5-mL/kg Optison, petechiae counts in the tissuewere significantly elevated relative to shams atPRPAs above 0.64 MPa (measured equivalent MI= 0.4) and were proportional to the square of thePRPA. A single image frame was sufficient to pro-

J Ultrasound Med 2008; 27:611–632 619

Miller et al

AIU

M C

on

sensu

s Rep

ort o

n Po

tential B

ioeffects o

f Diag

no

stic Ultraso

un

d

duce petechiae. The petechiae number wasapproximately proportional to the contrast agentdose. Evans blue leakage was evident, and excessdye could be extracted from tissue within the

scan plane relative to sham samples. Miller andQuddus94 also reported capillary rupture in fat,small intestine, and Peyer patches (intestinallymph nodes).

Bioeffects Considerations for Diagnostic Ultrasound Contrast Agents

AIU

M C

on

sen

sus

Rep

ort

on

Po

ten

tial

Bio

effe

cts

of

Dia

gn

ost

ic U

ltra

sou

nd

Table 2. Bioeffects of Diagnostic or Pulsed Ultrasound With Contrast Agents in Various Tissues Other Than Muscle

Frequency, Reference MHz Mode Agent Animal Tissue Effect Critical MI

Miller and Gies106,107 1.09 10-µs pulses Albunex, Levovist Mouse intestine Petechiae 0.812.3 Optison, PESDA 1.2

Kobayashi et al108 1.8 Harmonic Levovist Intravital rat Endothelial cell killing ≈0.61 onlyB-mode mesentery

Schlachetzki et al109 2.0–3.5 Transcranial Levovist, Optison Human brain Negative for magnetic <1.9color duplex resonance contrast leakage

Wible et al110 1.8 Continuous or Optison, MP1950 Rat kidney Glomerulus, hemorrhage 0.944.0 triggered B-mode MP2211 1.0

Kobayashi et al101 1.8 Harmonic B-mode Definity, Levovist Intravital rat Endothelial cell killing, ≈0.1mesentery hemorrhage

Shigeta et al86 8 B-mode Levovist Rat liver Platelet aggregation, ≈1.8 only12 endothelial injury ≈0.7 only

O’Brien et al111 3.1 1.2-µs pulses Optison Rat lung Negative for enhanced 1.6, 3.3hemorrhage

Miller and Dou112 1.5 Harmonic B-mode Definity Mouse tumor Negative for enhanced 1.9 onlymetastasis

*The different MI values correspond to the different frequencies.

Table 1. Bioeffects Induced by Contrast-Aided Diagnostic Ultrasound in Skeletal and Cardiac Muscle

Frequency, Reference MHz Mode Agent Animal Tissue Effect Critical MI

Skyba et al93 2.3 B-mode Optison Rat spinotrapezius Microvessel rupture, ≈0.4cell killing

Miller and Quddus94 2.5 B-mode Optison Mouse abdominal Petechiae, capillary 0.4leakage

van der Wouw et al95 1.66 Triggered B-mode AIP101 Human heart PVCs 1.1–1.5Ay et al96 1.8 Triggered B-mode PESDA, Rabbit isolated Function, lactate, 1.0

Sonazoid, heart capillary ruptureOptison, Levovist

Chen et al97 1.3 Triggered B-mode Optison, Rat heart Troponin T elevation, ≈1.2Definity negative histologic

findingsBorges et al98 1.7–1.9 Triggered B-mode Optison Human heart Negative for PVCs, 1.4–1.7 only

troponin I, CK, CK-MB

Raisinghani et al99 NS Triggered Doppler PB127 Human heart Negative for PVCs <1.0Li et al100 1.7 Triggered B-mode Optison Rat heart PVCs 0.77

Petechiae, leakage 0.41Kobayashi et al101 1.8 Harmonic B-mode Definity Rat heart Negative for petechiae ≈0.61 only

Miller et al102 1.7 Triggered B-mode Optison Rat heart Microvascular leakage 1.5 only<20 min

Li et al103 1.7 Triggered B-mode Optison, Rat heart PVCs 0.77Imagent, Petechiae 0.31Definity

Miller et al104 1.5 Triggered B-mode Optison Rat heart Histologic microlesions 1.7 onlyChapman et al105 1.7 Triggered B-mode PESDA Human heart PVCs, arrhythmia <0.8

NS, not specified.

620 J Ultrasound Med 2008; 27:611–632

Increased numbers of premature ventricularcontractions (PVCs) were reported in humansundergoing contrast echocardiography by vander Wouw et al.95 The contrast agent AIP101(not commercially available) was infused intra-venously into healthy volunteers. Cardiacscans were conducted at 1.66 MHz with inter-mittent image frames, which allowed refill ofthe tissue with the contrast agent betweenframes, and MI values of 1.1 and 1.5. A signifi-cant increase in PVCs to about 1 per minutewas seen for end-systolic triggering at an MI of1.5 but not at an MI of 1.1.

The microvascular effects of contrast echocar-diography were examined in isolated rabbithearts by Ay et al.96 A cardiac ultrasound systemwas operated at 1.8 MHz with 1-Hz triggering ofimage frames using the machine display MI as ameasure of the exposure. The perfusate con-tained the laboratory-made perfluorocarbon-exposed sonicated dextrose albumin (PESDA)contrast agent (effects were also confirmed withcommercial agents); however, it is difficult torelate the dosage to an in vivo intravenous dose.Scanning at an MI of 1.6 led to a transientdecrease in left ventricular pressure. Lactaterelease was significant at MIs of 1.0 and higher.Light microcopy revealed capillary damage anderythrocyte extravasation.

Potential injurious effects associated with theuse of Optison or Definity with diagnostic ultra-sound exposure were examined for in vivo rathearts.97 Imaging was performed at 1.3 MHz withelectrocardiographic (ECG) triggering at every 4cardiac cycles. The contrast dosage was 0.1 mL insaline delivered by infusion into the jugular vein.Left ventricular function was not perturbed(maximum MI = 1.6). Elevations in troponin T inblood plasma, indicating myocardial damage,were detected after 30 minutes for MIs of 1.2 and1.6. The elevation was significant at an MI of 1.6for both agents, declining to normal by day 4.Histologic examination of scanned tissueobtained on day 7 did not show evidence ofnecrosis, vascular damage, or inflammation.

A clinical study was reported for humansundergoing MCE by Borges et al.98 A dose of 3 mLof Optison in saline was injected as a bolus intothe cubital vein over 3 minutes followed by asaline flush. A diagnostic scanner was used in the

harmonic imaging plus power Doppler imagingduplex mode at 1.7 to 1.9 MHz for MIs of 1.4 to1.7 and with end-systolic triggering at every 1 to3 beats. Blood samples were taken before and upto 24 hours after scanning and were analyzed formyoglobin, troponin I, creatine kinase (CK), andCK isoenzyme MB. One patient had a transientincrease in troponin I after the examination, butthere were no consistent changes in measuredparameters that could be related to the ultra-sound examinations.

A large group (135) of humans was studied withregard to induction of PVCs during MCE using anew contrast agent, PB127.99 Several cardiacultrasound machines were used in the dual-frame triggering mode with MI settings of 0.9 to1.0. One group of patients also had dipyridamoleinfusion for a stress test. Premature ventricularcontractions were observed in the patient groupbut were not associated with the frame triggers.There was no significant increase in PVC fre-quency during or after imaging. The negativeresult was reassuring, but higher MI valueswould have been needed for direct comparisonwith the results of van der Wouw et al.95

An in vivo rat model of MCE was used to exam-ine microvascular permeabilization and PVCswith respect to the method of imaging, ultra-sound exposure, and agent dose.100 A 1.7-MHzdiagnostic ultrasound system was used to scanthe rats in a water bath for 3 minutes. Evans bluedye, a marker for microvascular leakage, and abolus of Optison were injected intravenously.Neither PVC nor microvascular leakage was seenin controls, rats imaged without the injectedcontrast agent, or rats with the injected contrastagent but not imaged. Triggering 1:4 at end sys-tole produced the most PVCs, petechiae, andmicrovascular leakage, followed by end systole1:1 triggering, continuous scanning, and enddiastole 1:1 triggering. All effects increased withincreasing Optison doses over the range of 25 to500 µL/kg. Threshold PRPAs above which effectswere significant were 1.0 MPa for PVCs and 0.54MPa for microvascular leakage.

The possible occurrence of microvascularinjury to rat hearts was also examined byKobayashi et al.101 A phased array ultrasoundsystem was used at 1.8 MHz and a 4-cm focuswith frame rates and on-screen MI combina-

J Ultrasound Med 2008; 27:611–632 621

Miller et al

AIU

M C

on

sensu

s Rep

ort o

n Po

tential B

ioeffects o

f Diag

no

stic Ultraso

un

d

tions of 1.6 and 1 Hz, 0.2 and 30 Hz, and 1.6 and30 Hz. After scanning of the rat mesentery,described below, the probe was moved to thechest wall for 3 minutes of exposure whileDefinity at 0.1 or 1.0 mL/kg was administered.This arrangement may have placed the rat heartin the near field of the array (ie, at lower PRPAsthan implied by the on-screen MI), but the heartimages were monitored on the ultrasoundscreen. After scanning, the hearts were fixed forhistologic examination. No hemorrhages werefound in the rat heart sections.

The timing and influence of vasoactive drugs onmicrovascular leakage induced by MCE in ratswere investigated by Miller et al.102 Hairless ratswere anesthetized and transthoracically scannedwith a diagnostic ultrasound system (GE VingMedSystem V) at 1.7 MHz with 1:4 triggered frames atend systole in a water bath with the heart at thefocal position. Optison, vasoactive medications,and Evans blue dye were injected via the tail vein.Effects were similar to those in the previousstudy.100 Propranolol and isoproterenol had littleeffect on the microvascular leakage, which sug-gests that the microvascular leakage was primarilya mechanical effect rather than a physiologicresponse. Capillary leakage occurred during andafter exposure but diminished for Evans blue injec-tions administered 20 minutes after scanning.

The effects of PVCs, petechiae, and microvascu-lar leakage reported by Li et al100 were comparedfor Optison, Definity, and Imagent.103 On the basisof the volume dose, MCE using Definity producedmore microvascular leakage. An example of thepetechial hemorrhages and microvascular leakageseen in rat hearts after Definity MCE is shown inFigure 2. However, when expressed in terms of thenumber of gas bodies, there was no apparent dif-ference between the three agents’ microvasculardamage potential, which increased linearly withthe gas body dose at low doses, as shown in Figure3. Myocardial contrast echocardiography usingDefinity resulted in fewer PVCs than the otheragents. The effects increased strongly with thePRPA, with calculated thresholds for petechiae atabout 0.4 MPa (MI = 0.31) and for PVCs at about 1.0MPa (MI = 0.77). The was no apparent threshold forthe visual detection of an Evans blue leakage areaon the heart surface, which was significant for thelowest exposure of 0.54 MPa (MI = 0.41).

Histologically defined microlesions withinflammatory cell infiltration induced by MCEwere reported by Miller et al.104 Myocardial con-trast echocardiography with 1:4 end-systolictriggering was performed in rats at 1.5 MHz andan MI of 1.7 in a short-axis view of the left ventri-cle in rats. Two high doses (500 µL/kg) of Optisonwere given 5 minutes apart during 10 minutes ofechocardiography. In rats killed 10 minutes afterMCE, microvascular leakage and petechiae wereevident. After 24 hours, microlesions with inflam-matory infiltrates were scattered primarily overthe anterior half of the sections. Lesion areas inthe anterior wall were scored from photomicro-graphs, and there was inflammatory cell infiltra-tion in areas of 0.5% ± 0.8% (SD) for shams and7.4% ± 5.0% for MCE (P < .02). For rats killed 1and 6 weeks after MCE, the microlesions healedto form small fibrous regions interspersed withnormal myocytes.

Dalecki et al113 examined the induction of pre-mature contractions in mice by 1.2-MHz pulsedultrasound with Albunex or Optison in the circu-lation. Pulses were triggered during diastolefrom the ECG. Using 5-millisecond pulses, thethreshold for premature contractions was low-

622 J Ultrasound Med 2008; 27:611–632

Bioeffects Considerations for Diagnostic Ultrasound Contrast Agents

AIU

M C

on

sen

sus

Rep

ort

on

Po

ten

tial

Bio

effe

cts

of

Dia

gn

ost

ic U

ltra

sou

nd

Figure 2. Microvascular leakage of Evans blue dye and petechialhemorrhages in a rat heart model of MCE.103 Diagnostic B-modeultrasound at 1.7 MHz with an in situ PRPA of 1.9 MPa (MI ≈ 1.5)was used to image the heart using frame triggering at end-sys-tole at each fourth heartbeat. A bolus of Definity at the recom-mended dose of 10 µL/kg was injected into the tail vein withscanning continuing for 5 minutes after contrast appeared in theheart. Scale bar indicates 2 mm. Reproduced with permissionfrom Miller D. Overview of experimental studies of biologicaleffects of medical ultrasound caused by gas body activation andinertial cavitation. Prog Biophys Mol Biol 2007; 93:314–330.

ered by a factor of 10 below a previously deter-mined threshold without the contrast agent toabout 0.2 MPa, which suggests that cavitationwas responsible for the bioeffect. For 10-microsecond pulses, the threshold was about 1MPa. The thresholds were similar for the two dif-ferent agents.

The occurrence of arrhythmias, such as PVCs,was examined and compared for physical thera-py ultrasound and for triggered diagnostic ultra-sound in humans.105 The therapeutic modeinvolved an unfocused 1-MHz ultrasound beamwith continuous or burst mode operation atmeasured PRPAs up to only 0.39 MPa. The diag-nostic ultrasound machine was operated at 1.7MHz with frames triggered at every 4 cardiaccycles and had a measured PRPA of 1.0 MPa for a1.3-MI setting on the machine. The therapydevice produced significantly more arrhythmiathan the diagnostic imager for the sametransthoracic exposure windows for the heart,which indicates that factors other than PRPA,such as a continuous versus pulsed mode, areimportant for this bioeffect. The low numbers ofPVCs induced by the diagnostic ultrasound witha moderate PRPA,

was consistent with other observations inhumans95 and also in rats.103

5.2. Bioeffects on Other TissuesThe intestine has been of interest with regard topotential nonthermal bioeffects, owing to its nat-ural content of free gas bubbles. Miller andGies106,107 used pulsed ultrasound to simulatediagnostic ultrasound (10-microsecond pulsesand a 1-kHz pulse repetition frequency for 100seconds) to expose mouse intestines and searchfor petechiae in the intestinal wall. Contrastagents were introduced by retro-orbital injection.Peak rarefactional pressure amplitude thresholdsfor induction of petechiae in the presence of 10-mL/kg Albunex increased from 0.85 MPa at 1.09MHz to 2.3 MPa at 2.4 MHz. Levovist, Optison,and PESDA all yielded more petechiae thanAlbunex at 2.3 MHz, and thresholds were as low as1.8 MPa (MI ≈ 1.2) for Levovist. Owing to the con-tinuous exposure in the near field of unfocusedtransducers, the apparent thresholds are not com-parable to intermittent diagnostic scanning.

The occurrence of microvascular injury to therat mesentery was examined by Kobyashi et al108

using diagnostic ultrasound in an intravitalpreparation. A phased array ultrasound systemwas used at 1.8 MHz and a 4-cm focus withframe rates and MI combinations of 1.6 and 1Hz, 0.2 and 30 Hz, and 1.6 and 30 Hz. Field mea-surements indicated a PRPA of 0.82 MPa (MI ≈0.61). Levovist was injected via the femoral vein.Propidium iodide was used to stain venule andcapillary endothelial cells killed by contrast-aided scanning. Capillary ruptures were rare.This same system was used to compare Levovistwith Definity for induction of endothelial cellinjury and microvessel bleeding.101 The systemwas adjusted for a measured PRPA value of either0.14 MPa (MI ≈ 0.1) or 0.82 MPa (MI ≈ 0.61). Nomicrovessel injury was seen for either ultrasoundalone or the contrast agent alone. Microvesselbleeding was rare and only seen for the 30-Hzframe rate at the higher exposure. Endothelialcell damage was observed for all conditions atthe higher exposure. Significant cell killing wasproduced using the low exposure and 30-Hz

8.0<f

p,

J Ultrasound Med 2008; 27:611–632 623

Miller et al

AIU

M C

on

sensu

s Rep

ort o

n Po

tential B

ioeffects o

f Diag

no

stic Ultraso

un

d

Figure 3. Comparison of the counts of petechiae seen on the ratheart surface after MCE at 1.7 MHz, 1:4 end-systolic triggering,and a PRPA of 1.9 MPa. The doses of the three agents are com-pared on the basis of the numbers of gas bodies in the bolusinjections. The curves are simple exponential functions with lin-ear dependence at low doses and saturation at higher doses.Reproduced with permission from Li P, Armstrong WR, Miller DL.Impact of myocardial contrast echocardiography on vascularpermeability: comparison of three different contrast agents.Ultrasound Med Biol 2004; 30:83–91.

frame rate with 1.0-mL/kg Definity in capillariesand venules (but not arterioles).

The possible alteration of the blood-brainbarrier by contrast-aided ultrasound was inves-tigated by Schlachetzki et al.109 Transcranialcolor-coded sonography was performed onhuman volunteers with a 2- to 3.5-MHz phasedarray probe with maximal output settings.Frames were triggered from the ECG at eachheartbeat, with speckling in the color Dopplerimages indicating microbubble destruction.Levovist and Optison were used for the con-trast ultrasound, and the magnetic resonancecontrast agent Magnevist (Bayer HealthCarePharmaceuticals, Leverkusen, Germany) wasalso injected intravenously. Evidence ofmicrovascular leakage was sought using mag-netic resonance imaging of the brain. There wereno indications of focal signal enhancementattributable to extravasation of the Magnevist.

Wible et al110 studied renal capillary hemor-rhage induced by contrast ultrasound in rats. Adiagnostic ultrasound probe was placed 1 cmfrom the kidney with one kidney exposed at a 30-Hz frame rate and the other at a 1-Hz frame rate.Frequencies of 1.8, 4, and 6 MHz were used atdisplayed MI values of 0.4 to 1.6, with the actualPRPA values measured at 1.5 cm. The contrastagents MP1950, MP2211, and Optison wereadministered at a dose of 40 million microbub-bles/kg. Contrast-aided ultrasound caused areasof small hemorrhages visible on the kidney sur-face within the scanned plane. The small hemor-rhages involved escape of red blood cells fromthe glomerular tuft into the Bowman capsule andproximal convoluted tubules. Intermittent expo-sure was more effective at producing the smallhemorrhages than continuous scanning andgave a significant increase in renal hemorrhagesfor a PRPA of 1.26 (MI = 0.94). The severitydecreased with increasing ultrasound frequen-cies but was significant at a PRPA of 2.02 at 4 MHz(MI = 1.0). At 6 MHz, no significant hemorrhagiceffect was seen, but the maximum measuredPRPA was only 1.6 MPa (MI = 0.65).

The liver is often the subject of ultrasoundexaminations, and these can be improved bycontrast agents. Effects of Levovist-aided ultra-sound on rat liver were investigated by Shigeta etal.114 The on-screen MI values were 1.8 at 8 MHz

and 0.7 at 12 MHz, and both were used on eachrat. The transducers were moved to expose theentire liver. Electron microscopy was performedon the rat livers with control, sham, and ultra-sound-only groups and two groups with ultra-sound plus contrast, 1 killed immediately and theother killed 5 hours later. Qualitative observationof the specimens revealed platelet aggregation inthe liver sinusoids for ultrasound-only and ultra-sound-plus-contrast groups. Endothelial celldamage was seen in the ultrasound-plus-con-trast group with the 5-hour delay.

Pulsed ultrasound has been shown to inducelung hemorrhage under some conditions, butthe exact mechanism is not clear. O’Brien et al111

tested the hypothesis that cavitation was themechanism by using saline or Optison injectionwith pulsed ultrasound exposure. Ten-secondexposures were performed on rats at 3.1 MHzwith 1.2-microsecond pulses and a 1-kHz pulserepetition frequency and in situ PRPAs of 2.74and 5.86 MPa (equivalent to in situ MI values of1.6 and 3.3, respectively). The contrast agentgroups did not have an increase in lung lesionoccurrence or size relative to the saline groups,which suggests that microbubble cavitation wasnot the mechanism for the lung hemorrhage.This finding confirms an earlier test usingAlbunex.115

Contrast-aided ultrasound scanning of varioustissues can assist in the identification of malignanttumors but might cause microvascular perturba-tions. Melanoma tumors growing on the thighs ofmice, which undergo metastatic spread to thelungs, were scanned with 1.5-MHz diagnosticultrasound during or after Definity injection.112

Image frames were triggered at a 1-Hz rate, andfour 10-µL/kg retro-orbital injections of the con-trast agent were made over a 100-second expo-sure. Sham exposure involved scanning for 100seconds followed by Definity injection with theultrasound off. For ultrasound plus the contrastagent, observation of a brightening of the tumorimage confirmed the interaction of ultrasoundwith the contrast agent within the tumor. One dayafter scanning, the primary tumor was surgicallyremoved, and the possible lung metastasis wasallowed to develop for 28 days. No significantincrease in lung metastases was seen in the lungsfor the contrast-aided ultrasound group.

624 J Ultrasound Med 2008; 27:611–632

Bioeffects Considerations for Diagnostic Ultrasound Contrast Agents

AIU

M C

on

sen

sus

Rep

ort

on

Po

ten

tial

Bio

effe

cts

of

Dia

gn

ost

ic U

ltra

sou

nd

5.3. Bioeffects Above the Diagnostic LimitSeveral researchers have studied bioeffects ofultrasound contrast agents with pulsed ultra-sound (for which cavitation might not normallyoccur) that had pressure amplitudes greater thanthe US FDA guideline upper limit for diagnosticultrasound. Miller and Gies116 injected hairlessmice with Albunex and exposed the abdominalregion to lithotripter shock waves. An increase inmortality was found after exposure to severalhundred shock waves, with increasing mortalityfor increasing Albunex doses. Hynynen et al117

found that 1.63-MHz focused ultrasound with10-microsecond pulses at 6.3 MPa produced his-tologically observable tissue damage in rabbitbrains. For this work, a window was created inthe skull for exposure, and a bolus of Optisonwas injected 10 seconds before exposure. Hwanget al118 examined effects of pulsed 1-MHz ultra-sound on rabbit ear veins with Optison in the cir-culation. Vessel wall damage with Evans blueextravasation was increased by the contrastagent at 6.5 MPa, and a small percentage of theendothelial surface was damaged at 3.35 MPawith the contrast agent. Hemolysis in suspen-sions has been studied for many years as an indi-cator of cavitation bioeffects. Dalecki et al119

detected hemolysis in vivo in mice with Albunexin the circulation. For a 10-microsecond pulsedexposure of the heart, thresholds were 3.0 MPa(peak positive or 1.9-MPa negative pressureamplitude) at 1.15 MHz but in excess of 10 MPa(peak positive) at 2.35 MHz.

The capillary hemorrhage effect was studied inmice injected with 0.1 mL of Albunex using pos-itive or negative pulses from an endoscopiclithotripter (≈0.4 MHz).120 One hundred pulses of3.6 or –3.6 MPa in amplitude were delivered tothe mouse abdomen. The negative pulses weresignificantly more effective than the positivepulses in producing hemorrhage in various tis-sues, including kidney, intestine, muscle, andstomach, which showed that the hemorrhageresulted from inertial cavitation.

Premature complexes (ECG signals represent-ing ventricular electrical activity) were seen for10 of 20 rats exposed to 3.1-MHz ultrasound with1.3-microsecond pulses at 15.9 MPa and Optisonin the circulation.121 However, myocardialdegeneration was identified by histologic stain-

ing in 16 rats, which suggests that the presence ofmyocardial degeneration alone was not a suffi-cient explanation of the premature complexes.

5.4. Potential Therapeutic ApplicationsContrast-aided ultrasound is capable of induc-ing a variety of in vivo bioeffects, and some ofthese effects may have useful clinical applica-tions for therapy. There have been many reportsof high-power contrast-aided ultrasound use forgene therapy, thrombolysis, and surgical appli-cations, which will not be considered here. Inthis document, only reports of in vivo therapeu-tic applications that have involved actual diag-nostic ultrasound systems for treatment arenoted. These reports do not directly address theproblem of bioeffects risks in diagnosis and ofteninvolved special gas body agents (they are notlisted in Tables 1 and 2), but they have a bearingon the perceived significance of the possibleeffects.

The contrast ultrasound-induced effect of vas-cular permeabilization has been suggested as ameans of drug delivery from the blood pool tothe interstitium.122 The method can accommo-date small particles as well as molecular drugs.Drug delivery was aided by Optison and wastargeted to skeletal muscle by 2.3-MHz diagnos-tic ultrasound, which served both as an imageguidance device and as the ultrasound exposuresystem.

Sonoporation, which is defined as transientultrasound-induced enhancement of cell mem-brane permeability, has been used in applica-tions of gene and drug delivery. Pislaru et al123

used a phased array transducer of a commercialultrasound imaging system (GE VingMed SystemV) in in vitro and in vivo experiments. For in vivoexperiments, Sprague-Dawley male rats wereexposed with a 1.7-MHz diagnostic ultrasoundsystem and PESDA to transfect skeletal musclewith the luciferase marker plasmid. Tissue sam-ples were also taken from remote, noninjectedmuscle or from the liver, kidney, lung, andheart. Luciferase activities were about 10-foldhigher than with intramuscular injections of theplasmid alone. Cationic lipid-DNA complexesincorporated into microbubbles have also beentested for DNA transfer in skeletal muscle.124

Diagnostic ultrasound at 1.75 MHz was used in

J Ultrasound Med 2008; 27:611–632 625

Miller et al

AIU

M C

on

sensu

s Rep

ort o

n Po

tential B

ioeffects o

f Diag

no

stic Ultraso

un

d

the B-mode with in situ PRPA values of 1.04 to1.14 MPa. The luciferase marker gene was used,and gene expression was assessed after 4 days.Intramuscular injection of the plasmid aloneproduced strong gene expression, which wasmatched by the intra-arterial treatment withplasmid-loaded microbubbles and ultrasound.No luciferase expression was seen outside theultrasound-treated region in liver or lung tissueor in muscle treated with ultrasound and theplasmid intra-arterially but without enhance-ment of cavitation by added microbubbles.

Gene delivery to the myocardium of rats wasenhanced by treatment with harmonic modediagnostic ultrasound, a microbubble contrastagent, and a viral β-galactosidase vector.125 Thecontrast agent was prepared in the laboratoryand was processed with the vector to attach thevirus particles to the microbubbles. Threeframes from a 1.3-MHz transducer destroyed themicrobubbles evident in the second-harmonicimage, and 3 frame bursts were triggered inter-mittently to allow refill of the tissue betweenscans. The hearts of all animals that received thecombined ultrasound plus microbubble treat-ment showed expression of the transgene.Vannan et al126 used cationic microbubble-linked plasmids and diagnostic ultrasoundexposure to enhance transfer of the chloram-phenicol acetyltransferase (CAT) marker gene indog hearts. Diagnostic ultrasound was deliveredinto anesthetized closed-chest dogs at 1.3 MHzand the highest power settings (MI = 1.5–1.7).Multiple frames were triggered at every 4 to 6cardiac cycles in an apical 4-chamber view. Thespecially prepared microbubbles were injectedinto a cephalic vein. For ultrasound with theplasmid-loaded microbubbles, CAT expressionwas found in several regions of the heart, with303 ± 188 ng/g in the myocardium for 4 dogs.However, for the ultrasound treatment of theheart only, CAT expression was found in distanttissues of the lungs, liver, kidney, and skeletalmuscle.

Ultrasound-enhanced gene transfer to cardiactissue was also shown using albumin and lipidmicrobubbles containing a luciferase plas-mid.127 The agents were infused for 20 minutesthrough the jugular vein of anesthetized rats,and the hearts were scanned with a 1.3-MHz

cardiac ultrasound machine at an MI of 1.5 with4 frames triggered from the ECG at every 4 car-diac cycles. Luciferase expression after 4 dayswas primarily detected in the heart, with somegene expression evident in the liver for the albu-min microbubbles and in the pancreas for thelipid microbubbles. The echocardiographictreatment parameters were varied to find theoptimum treatment conditions for the aden-ovirus- or plasmid-modified contrast agentmicrobubbles.128 Cardiac scanning was per-formed in anesthetized rats to transfer theluciferase plasmid. Triggered imaging at 1.3MHz was more effective than continuous imag-ing for gene transfer to the heart. An increase ofthe MI from the normal maximum of 1.6 for thediagnostic machine used in the study to 2.0 pro-duced significantly greater gene transfer (theFDA guideline upper limit for diagnostic ultra-sound is MI = 1.9).

Myocardial infarction might be treated byangiogenic gene therapies. Zhigang et al129 usedultrasound to enhance DNA transfer of a genevector coding for vascular endothelial growthfactor in a rat model of myocardial infarction.An albumin-based contrast agent was mixedwith a plasmid and incubated to attach theplasmid to the microbubbles. Three days afterinfarction, the plasmid vehicle was injected viathe tail vein and targeted to the heart by 1.8-MHz echocardiography at the maximum MIwith ECG triggering at every 6 to 8 beats. A sta-tistically significant increase in the microvascu-lar density in the ischemic myocardium wasfound in the ultrasound-plus-plasmid group.The rat model of myocardial infarction was alsoused by Kondo et al130 to test the efficacy ofgene therapy by hepatocyte growth factor. Thenaked plasmid coding for hepatocyte growthfactor was injected through a catheter insertedinto the left ventricle, while the femoral veinwas used to infuse Optison microbubbles. Thetreatment involved 1.3-MHz ultrasound trig-gered in 3 frame bursts 1:8 at end systole at aPRPA of 2.16 MPa. The capillary density in thearea around the infarct was 50% greater in thecontrast-plus-plasmid group than in controlgroups, and staining for scar formation showed asignificantly smaller scar area.

626 J Ultrasound Med 2008; 27:611–632

Bioeffects Considerations for Diagnostic Ultrasound Contrast Agents

AIU

M C

on

sen

sus

Rep

ort

on

Po

ten

tial

Bio

effe

cts

of

Dia

gn

ost

ic U

ltra

sou

nd

6. Conclusions

6.1. General ConclusionsDiagnostic ultrasound exposure can destabilizecontrast agent gas bodies (microspheres, or sta-bilized microbubbles). In practice, the use ofhigh MI values (>0.8) involves rapid gas bodydestruction, while use of low MI values (<0.2)involves minimal gas body destruction. Physicalmodels of gas body behavior indicate that iner-tial cavitation potentially can occur with expo-sure conditions corresponding to MI valuesgreater than approximately 0.4, which thereforerepresents a theoretical boundary between non-inertial and inertial activity regimens. The com-plex relationship between contrast agentdestabilization and inertial cavitation remainsthe subject of active research.

In suspensions of nonphagocytic cells, thedominant mechanism by which extensive celllysis is produced by ultrasound exposure withcontrast agents appears to be the occurrence ofinertial cavitation, and cavitation acoustic emis-sions can be used as cavitation “dosimeters.” Invitro studies have shown that diagnostic ultra-sound exposures of very modest PRPAs can killattached monolayer cells when in contact withcontrast agent gas bodies. The cell injury canoccur below the inertial cavitation threshold,apparently by a microstreaming shear stressmechanism. Because contrast agent gas bodiesattach to phagocytic cells, these cells, whichconstitute the mononuclear phagocytic systemin the body, may be particularly vulnerable.However, conditions in the body are differentfrom specialized in vitro conditions, and transla-tion of this basic research finding to in vivo con-ditions is not possible at this time.

Studies of bioeffects induced by contrast-aideddiagnostic ultrasound in vivo, primarily in smallanimals, have shown biologically significantmicroscale effects, such as petechial hemor-rhage with ultrasound exposures correspondingto MI values above 0.4. This value agrees with thetheoretical threshold for inertial cavitation inblood, which contains potential cavitationnuclei. Additional information confirms that anMI of 0.4 is an important boundary, as stated inthe American Institute of Ultrasound inMedicine statement Bioeffects of Diagnostic

Ultrasound with Gas Body Contrast Agents.131

Above an MI of 0.4, bioeffects appear to increaserapidly in magnitude, possibly as the square orhigh-order exponent of the PRPA. The magni-tude of effects appears to be proportional to thecontrast agent dose in terms of the number ofgas bodies for low doses. Reported bioeffects ofcontrast-aided diagnostic ultrasound includesonoporation, microvascular leakage, capillaryrupture (petechial hemorrhage), microlesionswith inflammatory cell infiltration, and PVCsduring ultrasound scanning. The bioeffects areprimarily in the form of scattered microscopicinjuries, which would not be expected to be clin-ically detectable in the short term. An exceptionis the induction of PVCs during contrastechocardiography, which is clinically observablebut ceases on cessation of the ultrasound. Thelonger-term medical significance of the reportedbioeffects is uncertain. Intentional bioeffects fortherapeutic purposes can be produced orenhanced with diagnostic ultrasound exposureof contrast agents, such as in gene therapy. Noepidemiologic studies are available on possibleadverse (or favorable) human health effects ofthe clinical use of ultrasound contrast agents.

6.2. Specific Conclusions

1. Studies of bioeffects induced by contrast-aided diagnostic ultrasound in vivo, primari-ly in small animals, have shown bioeffects,such as PVCs and petechial hemorrhage withultrasound exposures corresponding to MIvalues above 0.4. This value agrees with thetheoretical threshold for inertial cavitation inblood, which contains cavitation nuclei.

2. Above an MI of 0.4 bioeffects appear toincrease rapidly in magnitude as the squareor higher exponent of the PRPA. The magni-tude of effects appears to be proportional tothe concentration of gas bodies for concen-trations at or below the manufacturers’ rec-ommended dose.

3. In vitro studies have shown that diagnosticultrasound exposures can induce death ofattached monolayer cells when in contactwith contrast agent gas bodies. Cell injurycan occur below the inertial cavitationthreshold; the minimum reported thresh-

J Ultrasound Med 2008; 27:611–632 627

Miller et al

AIU

M C

on

sensu

s Rep

ort o

n Po

tential B

ioeffects o

f Diag

no

stic Ultraso

un

d

olds for cell death were approximately p/f =0.06 MPa/MHz.

4. Contrast agent gas bodies can be bound andinternalized by phagocytic cells, makingthem particularly vulnerable to injury fromultrasound exposure. The clinical implica-tions, if any, of these results are unknown.

5. Inertial cavitation is the dominant mecha-nism of cell lysis in whole blood exposed invitro or in vivo to diagnostic ultrasound in thepresence of contrast agents at suprathresh-old pressure amplitudes. The clinical impli-cations, if any, of these results are unknown.

6. Interaction of diagnostic ultrasound withcontrast agents is under investigation fortherapeutic applications.

7. Recommendations

1. For imaging with contrast agents at MIsgreater than 0.4, practitioners should use theminimal agent dose, MI, and exposure timeconsistent with acquisition of diagnosticinformation.

2. Practitioners of contrast-aided echocar-diography should note that use of high MIvalues (>0.8) involves rapid gas bodydestruction with a potential for bioeffects(eg, PVCs), whereas bioeffects have notbeen observed at low values of

(<0.2), which involve minimal gas body destruction.

3. The ECG should be monitored duringhigh-MI contrast cardiac-gated perfusionechocardiography, particularly in patientswith a history of myocardial infarction orunstable cardiovascular disease.*

4. Output display indices represent importantinformation and should be documented aspart of the permanent record of the exami-nation to enable future research.

5. The initial setting of the MI at mode selec-tion for contrast-aided ultrasound imagingshould be 0.4 or less.

6. Contrast-specific exposure indices shouldbe developed that reflect the destructionthresholds and more accurately account forthe in situ exposure values (derating factor).

7. Studies in laboratory animals are needed toinvestigate the potential for ultrasound-induced adverse fetal effects in the presenceof ultrasound contrast agents or otherexogenously introduced bubbles.

References

1. Deng CX, Lizzi FL. A review of physical phenomena associ-ated with ultrasonic contrast agents and illustrative clinicalapplications. Ultrasound Med Biol 2002; 28:277–285.

2. Frinking PJ, Bouakaz A, Kirkhorn J, Ten Cate FJ, de Jong N.Ultrasound contrast imaging: current and new potentialmethods. Ultrasound Med Biol 2000; 26:965–975.

3. Mulvagh SL, DeMaria AN, Feinstein SB, et al. Contrastechocardiography: current and future applications. J AmSoc Echocardiogr 2000; 13:331–342.

4. Dawson D, Vincent MA, Barrett EJ, et al. Vascular recruit-ment in skeletal muscle during exercise and hyperinsuline-mia assessed by contrast ultrasound. Am J PhysiolEndocrinol Metab 2002; 282:E714–720.

5. American Institute of Ultrasound in Medicine. MechanicalBioeffects from Diagnostic Ultrasound: AIUM ConsensusStatements. J Ultrasound Med 2000; 19:67–168.

6. National Council on Radiation Protection andMeasurements. Exposure Criteria for Medical DiagnosticUltrasound, II: Criteria Based on All Known Mechanisms.Bethesda, MD: National Council on Radiation Protectionand Measurements; 2002. Report 140.

7. Apfel RE. Acoustic cavitation: a possible consequence ofbiomedical uses of ultrasound. Br J Cancer 1982; 45(suppl):140–146.

8. Flynn HG. Generation of transient cavitation in liquids bymicrosecond pulses of ultrasound. J Acoust Soc Am 1982;72:1926–1932.

9. Apfel RE, Holland CK. Gauging the likelihood of cavitationfrom short-pulse, low-duty cycle diagnostic ultrasound.Ultrasound Med Biol 1991; 17:179–185.

10. American Institute of Ultrasound in Medicine, NationalElectrical Manufacturers Association. Acoustic OutputMeasurement and Labeling Standard for DiagnosticUltrasound Equipment. Rockville, MD: American Institute

f

p

628 J Ultrasound Med 2008; 27:611–632

Bioeffects Considerations for Diagnostic Ultrasound Contrast Agents

AIU

M C

on

sen

sus

Rep

ort

on

Po

ten

tial

Bio

effe

cts

of

Dia

gn

ost

ic U

ltra

sou

nd

*The US FDA issued a Healthcare Provider Advisory in October2007 stating that rare but serious cardiopulmonary complica-tions, including fatalities, have occurred during or within 30minutes after use of some approved ultrasound contrastagents (Definity and Optison). Changes to the package insertadded at that time state that the ECG should be monitored inall patients, and monitoring should be continued for at least30 minutes after administration of these agents. At the timeof preparation of this document, the data associated with theFDA recommendations were not available to the BioeffectsCommittee for review.

of Ultrasound in Medicine; Rosslyn, VA: National ElectricalManufacturers Association; 1992.

11. Holland CK, Apfel RE. Thresholds for transient cavitationproduced by pulsed ultrasound in a controlled nuclei envi-ronment. J Acoust Soc Am 1990; 88:2059–2069.

12. Price RJ, Kaul S. Contrast ultrasound targeted drug andgene delivery: an update on a new therapeutic modality. JCardiovasc Pharmacol Ther 2002; 7:171–180.

13. World Federation for Ultrasound in Medicine and Biology.WFUMB symposium on the safe use of echo-contrastagents. Ultrasound Med Biol 2007; 33:171–234.

14. Van Liew HD, Burkard ME. Bubbles in circulating blood:stabilization and simulations of cyclic changes of size andcontent J Appl Physiol 1995; 79:1379–1385.

15. Van Liew HD, Burkard ME. Behavior of bubbles of slowlypermeating gas used for ultrasonic imaging contrast.Invest Radiol 1995; 30:315–321.

16. Meza M, Greener Y, Hunt R, et al. Myocardial contrastechocardiography: reliable, safe, and efficacious myocar-dial perfusion assessment after intravenous injections of anew echocardiographic contrast agent. Am Heart J 1996;132:871–881.

17. Dittrich HC, Kuvelas T, Dadd K, et al. Safety and efficacy ofthe ultrasound contrast agent FS069 in normal humans:results of a phase one trial. Circulation 1995; 92:I–464.

18. Kitzman DW, Goldman ME, Gillam LD, Cohen JLAurimemma GP, Gottdiener JS. Efficacy and safety of thenovel ultrasound contrast agent perflutren (Definity) inpatients with suboptimal baseline left ventricular echocar-diographic images. Am J Cardiol 2000; 86:669–674.

19. Metzger-Rose C, Wright WH, Baker MR, et al. Effects ofphospholipid-coated microbubbles (MRX-115) on thedetection of testicular ischemia in dogs. Acad Radiol 1996;3:S314–S316.

20. Lindner JR, Song J, Jayaweera AR, Sklenar J, Kaul S.Microvascular rheology of Definity microbubbles afterintra-arterial and intravenous administration. J Am SocEchocardiogr 2002; 15:396–403.

21. Mulvagh SL, Foley DA, Aeschbacher BC, Klarich KK,Seward JE. Second harmonic imaging of an intravenouslyadministered echocardiographic contrast agent: visualiza-tion of coronary arteries and measurement to coronaryblood flow. J Am Coll Cardiol 1996; 27:1519–1525.

22. Schneider M, Arditi M, Barrau MB, et al. BR1: a new ultra-sonographic contrast agent based on sulfur hexafluoride-filled microbubbles. Invest Radiol 1995; 30:451–457.

23. European Medicines Agency. Evaluation of Medicines forHuman Use. Public Statement on SonoVue (SulphurHexafluoride): New Contraindication in Patients WithHeart Disease—Restriction of Use to Non-Cardiac Imaging.London, England: European Medicines Agency; 2004.Publication EMEA/CPMP/212/04:2004.

24. Leong-Poi H, Song J, Rim SJ, Christiansen J, Kaul S, LindnerJR. Influence of microbubble shell properties on ultrasoundsignal: implications for low-power perfusion imaging. J AmSoc Echocardiogr 2002; 15:1269–1276.

25. Masugata H, Cotter B, Peters B, Ohmori K, Mizushige K,DeMaria AN. Assessment of coronary stenosis severity andtransmural perfusion gradient by myocardial contrastechocardiography: comparison of grayscale and B-modewith power Doppler imaging. Circulation 2000; 102:1427–1433.

26. Church CC. The acoustical response of AI-700 in vitro[abstract]. J Am Soc Echocardiogr 2000; 13:457.

27. Straub JA, Chickering DE, Church CC, Shah B, Hanlon T,Bernstein H. Porous PLGA microparticles: AI-700, an intra-venously administered ultrasound contrast agent for use inechocardiography. J Control Release 2005; 108:21–32.

28. Lindner JR, Wei K. Contrast echocardiography. Curr ProblCardiol 2002; 27:454–519.

29. Goldberg BB, Raichlen JS, Forsberg F (eds). UltrasoundContrast Agents. 2nd ed. London, England: Martin DunitzLtd; 2001.

30. Leen E, Moug SJ, Horgan P. Potential impact and utilizationof ultrasound contrast media. Eur Radiol 2004; 14(suppl8):16–24.

31. Abramowicz JS. Ultrasound contrast media: has the timecome in obstetrics and gynecology? J Ultrasound Med2005; 24:517–531.

32. Blomley MJ, Cooke JC, Unger EC, Monaghan MJ,Cosgrove DO. Microbubble contrast agents: a new era inultrasound. BMJ 2001; 322:1222–1225.

33. Darge K, Riedmiller H. Current status of vesicoureteralreflux diagnosis. World J Urol 2004; 22:88–95.

34. Strandell A, Bourne T, Bergh C, Granberg S, Asztely M,Thorburn J. The assessment of endometrial pathology andtubal patency: a comparison between the use of ultra-sonography and X-ray hysterosalpingography for theinvestigation of infertility patients. Ultrasound ObstetGynecol 1999; 14:200–204.

35. Jakobsen JA, Correas JM. Ultrasound contrast agents andtheir use in urogenital radiology: status and prospects. EurRadiol 2001; 11:2082–2091.

36. Burns PN. Instrumentation for contrast echocardiography.Echocardiography 2002; 19:241–258.

37. Averkiou M, Powers J, Skyba D, Bruce M, Jensen S.Ultrasound contrast imaging research. Ultrasound Q 2003;19:27–37.

38. Albrecht T, Blomley M, Bolondi L, et al. Guidelines for theuse of contrast agents in ultrasound: January 2004.Ultraschall Med 2004; 25:249-256.

39. Porter TR, Xie F, Kricsfeld D, Armbruster RW. Improvedmyocardial contrast with second harmonic transient ultra-sound response imaging in humans using intravenous perfluorocarbon-exposed sonicated dextrose albumin. J Am Coll Cardiol 1996; 27:1497–1501.

40. Kuersten B, Murthy TH, Li P, et al. Ultraharmonic myocar-dial contrast imaging: in vivo experimental and clinical datafrom a novel technique. J Am Soc Echocardiogr 2001;14:910–916.

J Ultrasound Med 2008; 27:611–632 629

Miller et al

AIU

M C

on

sensu

s Rep

ort o

n Po

tential B

ioeffects o

f Diag

no

stic Ultraso

un

d

41. Wei K, Jayaweera AR, Firoozan S, Linka A, Skyba DM, KaulS. Quantification of myocardial blood flow with ultra-sound-induced destruction of microbubbles administeredas a constant venous infusion. Circulation 1998;97:473–483.

42. de Jong N, Cornet R, Lancee CT. Higher harmonics ofvibrating gas-filled microspheres, part one: simulations.Ultrasonics 1994; 32:447–453.

43. Church CC. The effects of an elastic solid surface layer onthe radial pulsations of gas bubbles. J Acoust Soc Am1995; 97:1510–1521.

44. Marsh JN, Hughes MS, Hall CS, et al. Broadband through-transmission signal loss measurements of Albunex suspen-sions at concentrations approaching in vivo doses. J AcoustSoc Am 1998; 104:1654–1666.

45. Stride E, Saffari N. Theoretical and experimental investiga-tion of the behaviour of ultrasound contrast agent particlesin whole blood. Ultrasound Med Biol 2004; 30:1495–1509.

46. O’Brien WD Jr, Deng CX, Harris GR, et al. The risk of expo-sure to diagnostic ultrasound in postnatal subjects: thermaleffects. J Ultrasound Med 2008; 27:517–535.

47. Abramowicz JS, Barnett S, Duck F, Edmonds P, Hynynen K,Ziskin M. Fetal thermal effects of diagnostic ultrasound. J Ultrasound Med 2008; 27:541–559.

48. Chavrier F, Chapelon JY. Modeling of high-intensityfocused ultrasound-induced lesions in the presence of cav-itation bubbles. J Acoust Soc Am 2000; 108:432–440.

49. Edson PL. The Role of Acoustic Cavitation in EnhancedUltrasound-Induced Heating in a Tissue-MimickingPhantom [dissertation]. Boston, MA: Boston University;2001.

50. Prosperetti A. The thermal behavior of oscillating gas-bub-bles. J Fluid Mech 1991; 222:587–616.

51. Holt RG, Roy RA. Measurements of bubble-enhanced heat-ing from focused, MHz-frequency ultrasound in a tissue-mimicking material. Ultrasound Med Biol 2001;27:1399–1412.

52. Hilgenfeldt S, Lohse D, Zomack M. Response of bubbles todiagnostic ultrasound: a unifying theoretical approach. EurPhys J B 1998; 4:247–255.

53. Hilgenfeldt S, Lohse D, Zomack M. Sound scattering andlocalized heat deposition of pulse-driven microbubbles. J Acoust Soc Am 2000; 107:3530–3539.

54. Stride E, Saffari N. The potential for thermal damage posedby microbubble ultrasound contrast agents. Ultrasonics2004; 42:907–913.

55. Fujishiro S, Mitsumori M, Nishimura Y, et al. Increased heat-ing efficiency of hyperthermia using an ultrasound contrastagent: a phantom study. Int J Hyperthermia 1998;14:495–502.

56. Wu J. Temperature rise generated by ultrasound in thepresence of contrast agent. Ultrasound Med Biol 1998;24:267–274.

57. Sokka SD, King R, Kynynen K. MRI-guided gas bubbleenhanced ultrasound heating in in vivo rabbit thigh. PhysMed Biol 2003; 48:223–241.

58. Stride E, Saffari N. On the destruction of microbubble ultra-sound contrast agents, Ultrasound Med Biol 2003;29:563–573.

59. Miller DL, Dou C. Theoretical gas body pulsation in relation toempirical gas body destabilization and to cell membranedamage thresholds. J Acoust Soc Am 2004; 116:3742–3749.

60. Chomas JE, Dayton P, May D, Ferrara K. Threshold of frag-mentation for ultrasonic contrast agents. J Biomed Opt2001; 6:141–150.

61. Chomas JE, Dayton P, Allen J, Morgan K, Ferrara K.Mechanisms of contrast agent destruction. IEEE TransUltrason Ferroelectr Freq Control 2001; 48:232–248.

62. Carstensen EL, Gracewski S, Dalecki D. The search for cav-itation in vivo. Ultrasound Med Biol 2000; 26:1377–1385.

63. Porter TR, Everbach C, Kricsfeld D, Xie F. Myocardial cavita-tional activity during continuous infusion and bolus intra-venous injections of perfluorocarbon-containing microbub-bles. J Am Soc Echocardiogr 2001; 14:618–625.

64. Miller MW, Miller DL, Brayman AA. A review of in vitro bio-effects of inertial ultrasonic cavitation from a mechanisticperspective. Ultrasound Med Biol 1996; 22:1131–1154.

65. Stratmeyer M, Greenleaf J, Dalecki D, Salvesen, K. Fetalultrasound: mechanical effects. J Ultrasound Med 2008;27:597–605.

66. Church CC, Carstensen EL, Nyborg WL, Carson PL, FrizzellLA, Bailey MR. The risk of exposure to diagnostic ultra-sound in postnatal subjects: nonthermal mechanisms. J Ultrasound Med 2008; 27:565–592.

67. Rooney JA. Shear as a mechanism for sonically induced bio-logical effects. J Acoust Soc Am 1972; 52:1718–1724.

68. Nyborg WL. Physical Mechanisms for Biological Effects ofUltrasound. Rockville, MD: Bureau of Radiological Health;1997.

69. Miller DL. A review of the ultrasonic bioeffects ofmicrosonation, gas-body activation, and related cavitation-like phenomena. Ultrasound Med Biol 1987; 13:443–470.

70. Miller DL. Frequency relationships for ultrasonic activationof free microbubbles, encapsulated microbubbles, and gas-filled micropores. J Acoust Soc Am 1998; 104:2498–2505.

71. Wu, J Theoretical study on shear stress generated bymicrostreaming surrounding contrast agents attached toliving cells. Ultrasound Med Biol 2002; 28:125–129.

72. Miller DL, Dou C. Membrane damage thresholds for pulsedor continuous ultrasound in phagocytic cells loaded withcontrast agent gas bodies. Ultrasound Med Biol 2004;30:405–411.

73. Miller DL, Dou C. Membrane damage thresholds for 1- to10-MHz pulsed ultrasound exposure of phagocytic cellsloaded with contrast agent gas bodies in vitro. UltrasoundMed Biol 2004; 30:973–977.

630 J Ultrasound Med 2008; 27:611–632

Bioeffects Considerations for Diagnostic Ultrasound Contrast Agents

AIU

M C

on

sen

sus

Rep

ort

on

Po

ten

tial

Bio

effe

cts

of

Dia

gn

ost

ic U

ltra

sou

nd

74. Miller DL, Gies RA, Chrisler WB. Ultrasonically inducedhemolysis at high cell and gas body concentrations in athin-disc exposure chamber. Ultrasound Med Biol 1997;23:625–633.

75. Miller DL, Gies RA. Enhancement of ultrasonically-inducedhemolysis by perfluorocarbon-based compared to air-based echo-contrast agents. Ultrasound Med Biol 1998;24:285–292.

76. Brayman AA, Lizotte LM, Miller MW. Erosion of artificialendothelia in vitro by pulsed ultrasound: acoustic pressure,frequency, membrane orientation and microbubble con-trast agent dependence. Ultrasound Med Biol 1999;25:1305–1320.

77. Miller DL, Quddus J. Lysis and sonoporation of epidermoidand phagocytic monolayer cells by diagnostic ultrasoundactivation of contrast agent gas bodies. Ultrasound MedBiol 2001; 27:1107–1113.

78. Everbach EC, Makin IR, Azadniv M, Meltzer RS. Correlationof ultrasound-induced hemolysis with cavitation detectoroutput in vitro. Ultrasound Med Biol 1997; 23:619–624.

79. Miller MW, Everbach EC, Cox C, Knapp RR, Brayman AA,Sherman TA. A comparison of the hemolytic potential ofOptison and Albunex in whole human blood in vitro:acoustic pressure, ultrasound frequency, donor and passivecavitation detection considerations. Ultrasound Med Biol2001; 27:709–721.

80. Chen WS, Brayman AA, Matula TJ, Crum LA, Miller MW.The pulse length dependence of inertial cavitation doseand hemolysis. Ultrasound Med Biol 2003; 29:739–746.

81. Chen W-S, Brayman AA, Matula TJ, Crum LA. Inertial cav-itation dose and hemolysis produced in vitro with or with-out Optison. Ultrasound Med Biol 2003; 29:725–737.

82. Miller MW, Everbach EC, Miller WM, Battaglia LF. Biologicaland environmental factors affecting ultrasound-inducedhemolysis in vitro, 2: medium dissolved gas (pO2) content.Ultrasound Med Biol 2003; 29:93–102.

83. Brayman AA, Strickler PL, Luan H, et al. Hemolysis of 40%hematocrit, Albunex-supplemented human erythrocytesby pulsed ultrasound: frequency, acoustic pressure andpulse length dependence. Ultrasound Med Biol 1997;23:1237–1250.

84. Lindner JR, Dayton PA, Coggins MP, et al. Noninvasive imag-ing of inflammation by ultrasound detection of phagocy-tosed microbubbles. Biophys J 2000; 80:1547–1556.

85. Dayton PA, Chomas JE, Lum AFH, et al. Optical and acous-tical dynamics of microbubble contrast agents inside neu-trophils. Biophys J 2001; 80:1587–1586.

86. Shigeta K, Taniguchi N, Omoto K, et al. In vitro platelet acti-vation by an echo contrast agent. J Ultrasound Med 2003;22:365–373.

87. Ward M, Wu J, Chiu JF. Experimental study of the effectsof Optison concentration on sonoporation in vitro.Ultrasound Med Biol 2000; 26:1169–1175.

88. Miller DL, Bao S. The relationship of scattered subharmon-ic, 3.3-MHz fundamental and second harmonic signals to

damage of monolayer cells by ultrasonically activatedAlbunex. J Acoust Soc Am 1998; 103:1183–1189.

89. Dayton P, Klibanov A, Brandenburger G, Ferrara K.Acoustic radiation force in vivo: a mechanism to assist tar-geting of microbubbles. Ultrasound Med Biol 1999;25:1195–1201.

90. Miller DL, Quddus J. Sonoporation of monolayer cells bydiagnostic ultrasound activation of contrast-agent gasbodies. Ultrasound Med Biol 2000; 26:661–667.

91. Miller DL, Dou C, Song J. DNA transfer and cell killing inepidermoid cells by diagnostic ultrasound activation ofcontrast agent gas bodies in vitro. Ultrasound Med Biol2003; 29:601–607.

92. Kudo N, Miyaoka T, Okada K, Niwa K. Study on mecha-nism of cell damage caused by microbubbles exposed toultrasound. Proc IEEE Ultrason Symp 2002; 1351–1354.

93. Skyba DM, Price RJ, Linka AZ, Skalak TC, Kaul S. Direct invivo visualization of intravascular destruction of microbub-bles by ultrasound and its local effects on tissue.Circulation 1998; 98:290–293.

94. Miller DL, Quddus J. Diagnostic ultrasound activation ofcontrast agent gas bodies induces capillary rupture in mice.Proc Natl Acad Sci USA 2000; 97:10179–10184.

95. van der Wouw P, Brauns AC, Bailey SE, Powers JE, WildeAA. Premature ventricular contractions during triggeredimaging with ultrasound contrast. J Am Soc Echocardiogr2000; 13:288–294.

96. Ay T, Havauz X, Van Camp G, et al. Destruction of contrastmicrobubbles by ultrasound effects on myocardial func-tion, coronary perfusion pressure and microvascularintegrity. Circulation 2001; 104:461–466.

97. Chen S, Kroll MH, Shohet RV, Frenkel P, Mayer SA,Grayburn PA. Bioeffects of myocardial contrast microbub-ble destruction by echocardiography. Echocardiography2002; 19:495–500.

98. Borges AC, Walde T, Reibis RK, et al. Does contrastechocardiography with Optison induce myocardial necrosisin humans? J Am Soc Echocardiogr 2002; 15:1080–1086.

99. Raisinghani A, Wei KS, Crouse L, et al. Myocardial contrastechocardiography (MCE) with triggered ultrasound doesnot cause premature ventricular complexes: evidence fromPB127 MCE studies. J Am Soc Echocardiogr 2003;16:1037–1042.

100. Li P, Cao LQ, Dou CY, Armstrong WR, Miller DL. Impact ofmyocardial contrast echocardiography on vascular perme-ability: an in vivo dose response study of delivery mode,ultrasound power and contrast dose. Ultrasound Med Biol2003; 29:1341–1349.

101. Kobayashi N, Yasu T, Yamada S, et al. Influence of contrastultrasonography with perflutren lipid microspheres onmicrovessel injury. Circ J 2003; 67:630–636.

102. Miller DL, Li P, Armstrong WF. The effect of time and ofvasoactive drugs on capillary leakage induced duringmyocardial contrast echocardiography. Echocardiography2004; 21:125–132.

J Ultrasound Med 2008; 27:611–632 631

Miller et al

AIU

M C

on

sensu

s Rep

ort o

n Po

tential B

ioeffects o

f Diag

no

stic Ultraso

un

d

103. Li P, Armstrong WR, Miller DL. Impact of myocardial con-trast echocardiography on vascular permeability: compari-son of three different contrast agents. Ultrasound Med Biol2004; 30:83–91.

104. Miller DL, Li P, Gordon D, Armstrong WF. Histological char-acterization of microlesions induced by myocardial contrastechocardiography. Echocardiography 2005; 22:25–34.

105. Chapman S, Windle J, Xie F, McGrain A, Porter TR.Incidence of cardiac arrhythmias with therapeutic versusdiagnostic ultrasound and intravenous microbubbles. J Ultrasound Med 2005; 24:1099–1107.

106. Miller DL, Gies RA. Gas-body-based contrast agent enhancesvascular bioeffects of 1.09 MHz ultrasound on mouse intes-tine. Ultrasound Med Biol 1998; 24:1201–1208.

107. Miller DL, Gies RA. The influence of ultrasound frequencyand gas-body composition on the contrast agent-medicat-ed enhancement of vascular bioeffects in mouse intestine.Ultrasound Med Biol 2000; 26:307–313.

108. Kobayashi N, Yasu T, Yamada S, et al. Endothelial cell injuryin venule and capillary induced by contrast ultrasonogra-phy. Ultrasound Med Biol 2002; 28:949–956.

109. Schlachetzki F, Holscher T, Koch HJ, et al. Observation onthe integrity of the blood-brain barrier after microbubbledestruction by diagnostic transcranial color-coded sonogra-phy. J Ultrasound Med 2002; 21:419–429.

110. Wible JH Jr, Galen KP, Wojdyla JK, Hughes MS, Klibanov AL,Brandenburger GH. Microbubbles induce renal hemor-rhage when exposed to diagnostic ultrasound in anes-thetized rats. Ultrasound Med Biol 2002; 28:1535–1546.

111. O’Brien WD Jr, Simpson DG, Frizzell LA, Zachary JF. Effectof contrast agent on the incidence and magnitude of ultra-sound-induced lung hemorrhage in rats. Echocardiography2004; 21:417–422.

112. Miller DL, Dou C. Contrast-aided diagnostic ultrasounddoes not enhance lung metastasis in a mouse melanomatumor model. J Ultrasound Med 2005; 24:349–354.

113. Dalecki D, Rota C, Raeman CH, Child SZ. Premature cardiaccontractions produced by ultrasound and microbubblecontrast agents in mice. Acoust Res Lett Online 2005;6:221–226.

114. Shigeta K, Itoh K, Ookawara S, Taniguchi N, Omoto K.Endothelial cell injury and platelet aggregation induced bycontrast ultrasonography in the rat hepatic sinusoid. J Ultrasound Med 2004; 23:29–36.

115. Raeman CH, Dalecki D, Child SZ, Meltzer RS, CarstensenEL. Albunex does not increase the sensitivity of the lung topulsed ultrasound. Echocardiography 1997; 14:553–558.

116. Miller DL, Gies RA. Consequences of lithotripter shockwaveinteraction with gas body contrast agent in mouse intes-tine. J Urol 1999; 162:606–609.

117. Hynynen K, McDannold N, Martin H, Jolesz FA,Vykhodtseva N. The threshold for brain damage in rabbitsinduced by bursts of ultrasound in the presence of an ultra-sound contrast agent (Optison). Ultrasound Med Biol 2003;29:473–481.

118. Hwang JH, Brayman AA, Reidy MA, Matula TJ, KimmeyMB, Crum LA. Vascular effects induced by combined 1-MHz ultrasound and microbubble contrast agent treat-ments in vivo. Ultrasound Med Biol 2005; 31:553–564.

119. Dalecki D, Raeman CH, Child SZ, et al. Hemolysis in vivofrom exposure to pulsed ultrasound. Ultrasound Med Biol1997; 23:307–313.

120. Dalecki D, Child SZ, Raeman CH, Xing C, Gracewski S,Carstensen EL. Bioeffects of positive and negative acousticpressures in mice infused with microbubbles. UltrasoundMed Biol 2000; 26:1327–1332.

121. Zachary JF, Hartleben SA, Frizzell LA, O’Brien WD Jr.Arrhythmias in rat hearts exposed to pulsed ultrasoundafter intravenous injection of a contrast agent. J UltrasoundMed 2002; 21:1347–1356.

122. Price RJ, Skyba DM, Kaul S, Skalak TC. Delivery of colloidalparticles and red blood cells to tissue through microvesselruptures created by targeted microbubble destruction withultrasound. Circulation 1998; 98:1264–1267.

123. Pislaru SV, Pislaru C, Kinnick RR, et al. Optimization of ultra-sound-mediated gene transfer: comparison of contrast agentsand ultrasound modalities. Eur Heart J 2003; 24:1690–1698.

124. Christiansen JP, French BA, Klibanov AL, Kaul S, Lindner JR.Targeted tissue transfection with ultrasound destruction ofplasmid-bearing cationic microbubbles. Ultrasound MedBiol 2003; 29:1759–1767.

125. Shohet RV, Chen S, Zhou Y, et al. Echocardiographicdestruction of albumin microbubbles directs gene deliveryto the myocardium. Circulation 2000; 101:2554–2556.

126. Vannan M, McCreery T, Li P, et al. Ultrasound-mediatedtransfection of canine myocardium by intravenous admin-istration of cationic microbubble-linked plasmid DNA. J AmSoc Echocardiogr 2002; 15:214–218.

127. Bekeredjian R, Chen S, Frenkel PA, Grayburn PA, ShohetRV. Ultrasound-targeted microbubble destruction canrepeatedly direct highly specific plasmid expression to theheart. Circulation 2003; 108:1022–1026.

128. Chen S, Shohet RV, Bekeredjian R, Frenkel P, Grayburn PA.Optimization of ultrasound parameters for cardiac genedelivery of adenoviral or plasmid deoxyribonucleic acid byultrasound-targeted microbubble destruction. J Am CollCardiol 2003; 42:301–308.

129. Zhigang W, Zhiyu L, Haitao R, et al. Ultrasound-mediatedmicrobubble destruction enhances VEGF gene delivery tothe infarcted myocardium in rats. Clin Imaging 2004;28:395–398.

130. Kondo I, Ohmori K, Oshita A, et al. Treatment of acutemyocardial infarction by hepatocyte growth factor genetransfer: the first demonstration of myocardial transfer of a“functional” gene using ultrasonic microbubble destruc-tion. J Am Coll Cardiol 2004; 44:644–653.

131. American Institute of Ultrasound in Medicine. Bioeffects ofDiagnostic Ultrasound with Gas Body Contrast Agents.Laurel, MD: American Institute of Ultrasound in Medicine;2002. Available at: http://www.aium.org/publications/state-ments/_statementSelected.asp?statement=25.

632 J Ultrasound Med 2008; 27:611–632

Bioeffects Considerations for Diagnostic Ultrasound Contrast Agents

AIU

M C

on

sen

sus

Rep

ort

on

Po

ten

tial

Bio

effe

cts

of

Dia

gn

ost

ic U

ltra

sou

nd