1) The study investigated the effects of an overpressure of 90 mmHg on the cavitation threshold and echogenicity of Definity®, an ultrasound contrast agent. 2) Stable and inertial cavitation thresholds as well as bubble activity were found to be approximately equal for overpressures of 0 and 90 mmHg. 3) The onset of echogenicity loss for Definity® insonified with pulsed Doppler was found to occur at a higher mechanical index for an overpressure of 0 mmHg than 90 mmHg.

1. THE EFFECT OF AN OVERPRESSURE OF 90 mmHg ON THE
CAVITATION THRESHOLD AND ECHOGENICITY OF
DEFINITY®
Tyler Fosnight, Kirthi Radhakrishnan, and Kevin Haworth
School of Energy, Environmental, Biological
and Medical Engineering, University of Cincinnati, Cincinnati, OH
Abstract- Cavitation of ultrasound contrast agents is currently being investigated as an
ultrasound mediated therapeutic mechanism. In this in vitro study, cavitation thresholds and
echogenicity change thresholds were measured for the commercial ultrasound contrast agent
Definity® insonified by a clinical transducer operated in pulsed Doppler (center frequency 6
MHz, pulse repetition frequency 1250 Hz, mechanical indexes ranging from 0.0 to 0.56 (i.e. peak
rarefactional pressure (PRP) 0 to 1.43 MPa)). Definity® was diluted in 0.5% (w/v) bovine serum
albumin and flowed through a latex tube at a flow rate of 5 ml/min with the overpressures, with
respect to atmospheric, set to 0 mmHg or 90 mmHg (0 kPa and 12 kPa, respectively).
Subharmonic and broadband emissions acquired using a 10 MHz spherically focused passive
cavitation detector (PCD) were used to define the stable and inertial cavitation thresholds,
respectively. Images of Definity® were acquired by operating the clinical transducer in B-mode.
Single frames before insonification with pulsed Doppler and after insonification with pulsed
Doppler were acquired. Mean grayscale values from captured single frames were used to define
an echogenicity loss threshold. For the UCA insonified with 6 MHz pulsed Doppler, the onset of
echogenicity loss was at a higher MI with an overpressure of 0 mmHg than 90 mmHg. The stable
and inertial cavitation thresholds and bubble activity were approximately equal, for both
overpressures of 0 and 90 mmHg. The independence of cavitation thresholds and the cavitation
emission energy on the overpressure suggests that these thresholds may be considered applicable
to in vivo environments as well.
Introduction
The use of ultrasound to enhance drug and gene delivery has many potential benefits in
the treatment of diseases like ischemic stroke (Datta, 2008; Smith, 2010), myocardial infarctions
(Christiansen, 2002), and deep vein thrombosis (Datta, 2008). Several studies have demonstrated
that ultrasound mediated enhancement of drug and gene delivery are correlated with some form
of bubble activity, as measured by the power in certain frequency bands of the signal, in the
presence of ultrasound. Cavitation can be defined as the formation and/or oscillation of a gas
phase (i.e. bubble) in a liquid phase due to changes in pressure. For therapeutic applications,
stable or inertial cavitation are the two areas most studied. During stable cavitation, the bubble
experiences nonlinear oscillations resulting in sub- and ultra-harmonic acoustic emissions
(Leighton, 1997). Inertial cavitation takes place when sufficient acoustical pressure is reached
causing the bubble to reach a critical radius thus resulting in the violent collapse of the bubble
indicated by the presence of broadband emissions from the bubble (Holland & Apfel, 1989).
Datta et al. (2008) showed that stable cavitation activity correlated well with enhanced lysis of
1

2. 2
clots. On the other hand Lai et al. (2006) showed that delivery of genes into cells were
concomitant with inertial cavitation.
Datta et al. (2008) showed that the presence of pre-existing nuclei in the form of a
contrast agent, such as Definity® lowered the thresholds of cavitation and enhanced clot mass
loss. Definity®, is a lipid-shelled ultrasound contrast agent (UCA) containing octafluoropropane
gas which produces enhanced echogenicity or contrast because of the difference in the acoustical
impedance between the encapsulated gas and the surrounding blood pool and tissue (Kremkau,
2006). Intravenous administration of Definity® is currently clinically approved for use only to
improve delineation of the endocardial border in previously suboptimal echocardiograms
(Kremkau, 2006). However, in order to use Definity® and other UCAs as cavitation nucleating
agents as adjuvants in ultrasound mediated therapy in clinical settings, a rigorous study of their
cavitation thresholds under physiologic conditions is required primarily because overpressures
above atmospheric levels have been shown to suppress subharmonic emissions and cavitation
from contrast agents. Adam (2001), observed this phenomenon for Optison®, a suspension of
human serum-albumin coated microspheres filled with octafluoropropane. Specifically, Adam
(2001) reported a subharmonic amplitude that decreased over time for overpressures of 70, 140,
and 210 mmHg (9, 19, and 28 kPa, respectively) in an in vitro system while insonifing at 2 MHz.
Processing cavitation signals to identify different types of cavitation can be a
computationally intensive process. Also, cavitation monitoring requires additional equipment
such as hydrophones, preamplifiers, and oscilloscopes. Therefore, it would be desirable to
demonstrate a correlation between cavitation activity and changes in loss of echogenicity on
standard ultrasound B-mode images. This would allow for a more clinically relevant approach to
monitor cavitation during therapy (Porter, 2006; Smith, 2007).
The objective of this study was to determine the effects of an overpressure of 90 mmHg
on the cavitation threshold and echogenicity of Definity®. The hypotheses tested in this study
are (a) the presence of a mean overpressure, with respect to atmospheric, of 90 mmHg would
result in the delayed onset of both stable and inertial cavitation activity for a sample volume of
Definity® being exposed to 6 MHz pulsed Doppler with a pulse duration of 5.83 µs and pulse
repetition frequency of 1250 Hz, and (b) the change of onset in echogenicity of Definity® under
these conditions correlates with the change of onset in stable cavitation emissions.
Materials and Methods
Flow phantom
The flow phantom, figure 1, consisted of a latex tube with an inner diameter of 3 mm and
a wall thickness of 400 microns. A peristaltic pump (Rainin Instrument Rabbit Peristatic Pump,
Oakland, CA, USA), was used to maintain a flow rate of 5 ml/min within the tube. A mean
overpressure, with respect to atmospheric, of 0 mmHg or 90 mmHg was introduced using a
gravity-mediated afterload reservoir. The overpressures were measured using a pressure
transducer and pressure monitor (Hugo Sachs Elektronik 73-0045, March-Hugstetten, Germany).
Pressure measurements were recorded every 0.016 s and the files were saved to a personnel

3. 3
computer. The latex tubing was immersed in a Lucite tank filled with degassed water, maintained
at 37˚C, with a dissolved oxygen content at 40% saturation as measured by a dissolved oxygen
meter (Oakton DO100, Vernon Hills, IL, USA). The UCA was pumped from the UCA reservoir
maintained at room temperature.
Fig. 1. Experimental setup for 6 MHz pulsed Doppler exposure of a flow phantom containing Definity® and bovine
albumin serum (BSA). A focused 10 MHz PCD, aligned confocally with and orthogonally to the acoustical axis of
the L12-5 transducer Doppler sample volume.
Contrast agent preparation
According to manufacturer instructions, Definity® vials were activated at room
temperature using the Vialmix ™ (Bristol-Myers Squibb Medical Imaging, MA, USA). After the
activation period of 45 s the vial was left on the bench to return to room temperature. The agent
was drawn from the vial using a 20 gage needle while allowing venting to the atmosphere using
a second 20 gage needle with a 0.2 µm filter. A 0.05 mL volume of the agent was drawn and
then diluted into 0.5% (w/v) bovine serum albumin (BSA) in deionized water to a concentration
of 0.31 µm/mL at room temperature. Fifty milliliters of this dilution was transferred into the
UCA reservoir seen in figure 1.
Insonation parameters and echogencity measurements
The L12-5, an imaging transducer used with the Philips HDI-5000 clinical ultrasound
scanner (Philips HDI 5000, Bothell, WA, USA), was used to both record B-mode images for
echogenicity measurements and to insonate the Definity® using a 6 MHz pulsed Doppler signal.
“2-D Live” mode was used for this study as it allowed the use of pulsed Doppler or B-mode
separately. To acquire images the B-mode was turned on for 1 s to acquire an image of the UCA
with the flow turned off (i.e. 0 mL/min) as a representative image of Definity® prior to exposure
to Doppler pulses. The transducer was then switched to Doppler mode at the appropriate
mechanical index (MI), and flow was turned on. As the Definity® flowed through the pulsed
Doppler sample volume located at the center of the B-mode image, as shown in figure 2, they
were exposed to Doppler pulses, cavitated, and subsequently lost echogenicity. To acquire an
image of the Definity® exposed to Doppler pulses, both the transducer and flow were turned off

4. 4
and the transducer was set again in B-mode to acquire an image. The B-mode MI was set to 0.04
(95 kPa) and was left on for no more than 1 s in order to minimize any destruction that may be
caused by B-mode pulses.
Fig. 2
A representative image captured after Definity® exposure to Doppler pulses at an MI of 0.56 (1.43 MPa). The image
depicts the regions of interest (ROIs) that were used for obtaining the echogenicity.
The pulsed Doppler sample volume was centered in the azimuthal direction of the B-
mode imaging plane and placed at a depth of 1 cm. Previous calibrations showed that this
placement results in a 0.8 mm -3 dB lateral beamwidth. For the experiments described in this
study, the L12-5 transducer in pulsed Doppler mode produced a pulse with a 5.83 µs pulse
duration at a 1250 Hz pulse repetition frequency.
Cavitation detection
The passive cavitation detector (PCD) (Valpey Fisher, Hopkinton, MA , USA) was a
circular, spherically focused, single-element, 10 MHz transducer with a -3 dB axial beamwidth
of 2 cm, lateral beamwidth of 1 mm, and a focal distance of 2 cm. An illustration of the
experimental set up and alignment is shown in figure 1. The PCD was placed 90° to the acoustic
axis of the L12-5 transducer and confocally aligned with the pulsed Doppler sample volume
using two micrometer three-axis translation stages (Newport 423, Irvine, CA, USA). To obtain
this alignment, the latex tube was filled with degassed water and then insonified with the pulsed
Doppler signal. The PCD was moved until the signal was maximized indicating that the PCD
was aligned to both the top of the tube and confocal with the clinical transducer along the axis of
the tube. Subsequently, the PCD was driven with a pulser-receiver (Panametrics, Squarewave
Pulser/Receiver 5077PR, Waltham, MA, USA) at 10-MHz and a pulse repetition frequency of
100 Hz. The PCD was then moved down by 1.5 mm, half the inner diameter of the latex tube, so
that it was aligned to the center of the latex tube and the echoes from the pulser-receiver were
maximized and corresponded to the correct inner diameter of the latex tube.
After alignment, either degassed water or Definity® was flowed through the system and
insonified at varying pressures (Table 1) at the desired overpressure of either 0 mmHg or 90
mmHg. The cavitation emissions were detected with the PCD, amplified 46 dB by a power
amplifier (Amplifier Research 50A15), and stored using a digital oscilloscope (LeCroy
Waverunner LTS84, Chestnut Ridge, NY, USA). The oscilloscope was operated in sequence

5. 5
mode with a sampling rate of 50 MHz for each sequence. Each segment contained 250 traces
corresponding to 250 consecutive Doppler firings. A total of 1000 time domain traces were
recorded. Files from the oscilloscope were then transferred to a personnel computer for later post
processing in MATLAB (The MathWorks Inc., Natick, MA, USA).
Table 1
Peak Rarefraction Pressure [MPa]
0.00 0.09 0.11 0.13 0.16 0.18 0.20 0.22 0.25 0.28 0.31 0.35 0.39 0.45 0.50 0.55 0.62 0.72 0.80 0.91 1.03 1.15 1.27 1.43
Mechanical Index (MI)
0 0.04 0.05 0.06 0.07 0.08 0.09 0.1 0.11 0.13 0.14 0.16 0.18 0.2 0.22 0.24 0.27 0.3 0.33 0.37 0.41 0.46 0.5 0.56
Peak rarefactional pressures (and corresponding MIs) used to insonate both degassed water and Definity® with over
pressures of 0 mmHg or 90 mmHg.
Image and cavitation analysis
For each MI setting, B-mode images of Definity® were saved before and after exposure
to Doppler as described above. On each image two regions of interest were defined upstream and
downstream of the Doppler sample volume as shown in figure 2. The ROIs had a height equal to
the tubing image inner diameter and a length that was approximately one quarter of the tubing
image length. The mean gray scale value (MGSV) of the ROI was computed. The average
MGSV was then calculated over eight trials at each MI to determine echogenicity thresholds.
At each MI, the average time-domain degassed water cases were subtracted from the
individual Definity® time domain traces to remove the coherent scattering from the tubing. A
Tukey window (r = 0.5) was applied to the subtracted time-domain signal to reduce artifactual
signal components. These windowed time-domain traces were converted into power spectra by
applying a 2048-point fast Fourier transform in MATLAB. Averaged power spectra were
computed from the 1000 traces captured at each MI. The mean subharmonic power was
computed as the mean power in the subharmonic band centered at 3 MHz with a 1 MHz
bandwidth. This bandwidth is the same as the bandwidth of the main lobe at 6 MHz. The mean
subharmonic power is indicative of stable cavitation activity. A comb filter was used to extract
the broadband components from the frequency ranges 9.5-10.5 MHz, 13.5-14.5 MHz and 15.5-
16.5 MHz, which lie outside the harmonic and ultraharmonic frequency bands. To determine the
cavitation threshold, the logarithms of the subharmonic and broadband powers were computed
from four trials and were then plotted against the peak rarefactional pressures (PRP).
Results
Echogenicity
The MGSV versus the peak rarefactional pressure for 0 mmHg and 90 mmHg are shown,
respectively, in figures 3 and 4. Both figures show the MGSV for the upstream and downstream
ROIs both before and after pulsed Doppler insonation. One does not see any significant changes
in echogenicity for either ROI in the before insonation cases (figures 3a and 4a). This is expected
and serves as a check that the echogenicity is on average uniform across experimental runs. This
suggests that the statistical measures of the bubble populations themselves remain stationary.
Each data point is an average of eight samples, and the error bars represent the standard
deviation of the measurements. In comparision, the echogenicity changes were seen in the

6. 6
downstream ROIs for Definity® exposed to 6 MHz pulsed Doppler (figure 3b and 4b) at higher
MIs.
(a)
50
45
40
35
30 Down stream
MGSV
25
Up stream
20
15
10
5
0
0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600
Peak rarefactional pressure (MPa)
(b)
50
45
40
35
30
Down Stream
MGSV
25
Up stream
20
15
10
5
0
0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600
Peak rarefactional pressure (MPa)
Fig. 3. Echogenicity, as measured by MGSV, as a function of peak rarefactional pressure under an overpressure of 0
mmHg for (a) ROIs downstream and upstream of the pulsed Doppler sample volume before pulsed Doppler
insonation was turned on, and (b) ROIs downstream and upstream of the pulsed Doppler sample volume after pulsed
Doppler insonation was turned on. A sample size of eight was used for each set of MIs, and error bars represent the
standard deviation of the measurements.

7. 7
(a)
50
45
40
35 Down stream
30
Up stream
MGSV
25
20
15
10
5
0
0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600
Peak rarefactional pressure (MPa)
(b)
50
45
40
35
30 Down Stream
MGSV
25
Up Stream
20
15
10
5
0
0.000 0.200 0.400 0.600 0.800 1.000 1.200 1.400 1.600
Peak rarefactional pressure (MPa)
Fig. 4. Change in echogenicity, as measured as the MGSV, as a function of peak rarefactional pressure under an
overpressure of 90 mmHg for (a) ROIs downstream and upstream of the pulsed Doppler sample volume with the
pulsed Doppler insonation off; and (b) ROIs downstream and upstream of the pulsed Doppler sample volume with
the pulsed Doppler insonation on. A sample size of eight was used for each set of MIs, and error bars represent the
standard deviation of the measurements.

8. 8
Cavitation
The logarithm of the mean subharmonic power and the broadband power plotted against
the peak rarefactional pressure correlating to stable and inertial cavitation activity are shown in
figure 5a and b respectively. Each data point is the average of the power from four samples, and
the error bars are the standard deviation of the measurements. For the stable cavitation activity, it
is evident that both thresholds were equal for both overpressures of 0 and 90 mmHg.
Furthermore, the stable cavitation bubble activity, represented by the subharmonic power, is also
approximately the same for both cases. For inertial cavitation, both overpressures show a
threshold that is approximately equal. The inertial bubble activity also remains approximately
equal for both overpressures.
(a)
-2.50
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
Log(normalized subharmonic power)
-3.00
Def inity at 0 mmHg
-3.50
Degassed Water at 0
mmHg
-4.00
Def inity at 90 mmHg
Degassed H2O at 90
-4.50 mmHg
-5.00
-5.50
-6.00
-6.50
Peak rarefactional pressure (MPa)

9. 9
(b)
-3.00
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
Log(mean broadband power)
-3.50
Definity at 0 mmHg
Degassed Water 0
-4.00 mmHg
Definity at 90 mmHg
Degassed Water at
90 mmHg
-4.50
-5.00
-5.50
Peak Rarefactional Pressure (MPa)
Fig. 5. The logarithm of the mean (a) subharmonic and (b) broadband power as a function of the peak rarefactional
pressure (MPa) for Definity® and degassed water at 0 mmHg, and Definity® and degassed water at 90 mmHg.
Discussion
The effect of an overpressure above atmospheric on cavitation and echogenicity
thresholds were explored in this study. This study also looked to see if the cavitation activity
could be correlated with the echogenicity loss. Correlation of echogenicity changes and
cavitation activity could help to simplify the monitoring of drug and gene delivery in the clinical
setting. Correlation of the thresholds for cavitation onset and echogenicity loss did not appear to
be concomitant.
For echogenicity loss, the threshold for change appears to decrease slightly (though a
robust statistical method was not used to define a precise threshold) at the higher overpressure.
This may be due to the additional pressure on the bubble making it more susceptible to
dissolution. It is interesting to note, the pressure at which echogenicity loss occurred for
Definity®, is close to the value seen by Porter et al. (2006) for Optison®. Porter et al. attributed
this loss to a process termed acoustically driven diffusion. While the experimental protocols are
different between the work of Porter et al. and the one described in this study, it may be that a
similar process is causing echogenicity loss as was observed here.
Further, the results indicate that the cavitation thresholds for Definity® are independent
of overpressures at 0 mmHg and 90 mmHg above atmospheric for both stable and inertial
cavitation. Earlier, Adam (2001) observed the amplitude of the first, second, and sub-harmonic
for the sterile suspension of human serum-albumin coated microspheres filled with
octafluoropropane UCA, (Optison®), decreased over time for overpressures of 70, 140, 210
mmHg (9, 19, and 28 kPa, respectively) in an in vitro system while insonifing at 2 MHz. Along
the same lines, Anderson et al. (2010) compared the energy ratio of the subharmonic and
fundamental to overpressures of 0 to 187 mmHg (0 and 25 kPa, respectively) for the UCA

10. 10
SonoVue®, a phospholipid membrane containing sulfur hexafluoride. The UCA was insonified
at 4 MHz in a static chamber while constantly mixing the UCA with a magnetic stir bar. They
reported a decrease in the energy ratio as a function of the overpressure. Shi et al. (1999)
demonstrated in vitro, for overpressures of 0 to186 mmHg (0 and 25 kPa, respectively), that the
UCA Levovist®, a glactose/palamitic acid shell encapsulating air, insonated at 2 MHz had a
reduction of amplitude of the first, second, and sub-harmonics by 2.4, 1.8, and 9.6 dB,
respectively, for a constant acoustic pressure of 0.39 MPa. Another study (Bailey, 2001), that
focused on reducing distortion of the shape and location of high intensity focused ultrasound
(HIFU) lesions in tissue by introducing an overpressure (5.6 MPa) to suppress gas or vapor water
bubbles resulting from heating, saw similar results in reducing bubble activity. These studies
have demonstrated the effect of an overpressure on bubble activity on various UCAs. The
novelty of this study is in investigating the cavitation thresholds and bubble activity for
Definity® under physiologically relevant overpressures, which hasn't been done in previous
studies. Further, in sharp contrast to the results reported in earlier studies we found that an
overpressure of 90 mmHg did not affect the cavitation threshold or bubble activity for stable or
inertial cavitation in Definity®. Further investigation of this result needs to be carried out to
determine the reason. It may be due to Definity® having a different shell composition than the
other UCAs, or the dependence of cavitation activity on the flow (i.e. with flow, without flow, or
constant agitation in a static chamber).
Conclusions
Echogenicity loss and subharmonic emissions can be used to monitor and measure
cavitation activity, so a clear understanding of the dependence of cavitation thresholds and
bubble activity with respect to overpressures is needed to move these methods into in vivo
systems. In this study, no loss of echogenicity was observed due to overpressures alone. While
differences in the onset of echogenicity loss for the overpressures of 0 and 90 mmHg could be a
result of dissolution of the UCA. Stable and inertial cavitation thresholds and bubble activity for
both 0 and 90 mmHg overpressures were approximately equal thus supporting the use of these
cavitation thresholds from this in vitro study for future in vivo studies.
Future Work
Future work could include repeating the protocol described in this study with other
UCAs, such as Optison®, that were used in the before mentioned reports to see if their results
can be repeated. This would allow the exploration of the dependence of cavitation activity and
echogenicity on the UCA composition. Other future work could be concentrated on studying the
effect of varying pulse repetition frequency (PRF) and pulse duration (PD) on cavitation activity
and echogenicity for Definity®. Data might be collected for the parameter sets listed in Table 2
(numbers in nonshaded cells indicate the maximum MI available). Within this study a statistical
method to derive a threshold for echogenicity should be investigated in order to more
quantitatively compare echogenicity changes with cavitation activity.

11. 11
Table 2. Suggested combinations of PRF and PD. The numbers in non-shaded cells indicate the maximum MI for
the Philips HDI-5000 clinical ultrasound scanner for the corresponding PRF/PD combinations.
Pulse Duration (PD) [µs]
1.67 3.33 5.83 8.33 16.7
P 1250 0.9 0.7 0.56 0.47 0.31
R 2500 0.53
F 5000 0.39
[H 8333 0.32
z] 0.27
11905
Acknowledgements- I gratefully acknowledge the assistance of Kirthi Radhakrishnan and Kevin
Haworth for their help in completing this study, specifically the analysis of the cavitation data by
Kirthi, and for the valuable discussions. I also gratefully acknowledge all other valuable
discussions with the rest of the members in the Holland Lab.
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