Ultrasound in Med. & Biol., Vol. 26, No. 5, pp.

807– 818, 2000

Copyright © 2000 World Federation for Ultrasound in Medicine & Biology
Printed in the USA. All rights reserved
0301-5629/00/$–see front matter

PII S0301-5629(00)00215-5

● Original Contribution

AN IN VITRO COMPARISON OF ULTRASONIC CONTRAST AGENTS IN

SOLUTIONS WITH VARYING AIR LEVELS

V. SBOROS, C. M. MORAN, T. ANDERSON and W. N. MCDICKEN

Department of Medical Physics and Medical Engineering, University of Edinburgh, Edinburgh, UK

(Received 27 August 1999; in final form 29 February 2000)

Abstract—The performance, in particular, the stability of ultrasound (US) contrast agents has yet to be assessed.
An in vitro system has been set up to investigate the properties of ultrasonic contrast agents under different
suspension conditions. This is designed to contribute to the optimal use of agents in clinical practice. In this study,
the contrast agents were introduced into solutions of different oxygen concentration levels, as might be
encountered in blood, and their relative performance was assessed in terms of decay in the solution environment.
The partial pressures of oxygen in those solutions ranged between 1.5 and 26 kPa. Three IV and one arterial
contrast agents were used: Levovist威, DMP115, Quantison™ and Myomap™. Levovist威 showed the highest
sensitivity to oxygen concentration in the solution, and the other three proved tolerant for the above values of
oxygen concentrations. © 2000 World Federation for Ultrasound in Medicine & Biology.

Key Words: Ultrasound, RF data, Contrast agents, Physical properties, Stability, Levovist威, DMP115, Quanti-
son™, Myomap™.

INTRODUCTION pi ⫽ pl ⫹ p␴ , (1)
Ultrasound (US) contrast agents have been widely recog-
nised not only to improve the outcome of investigations where pl is the pressure in the liquid and p␴ is the surface
using various US imaging modalities, but also to create a tension. The internal pressure also equals:
new perspective in ultrasonic imaging. Quantitation of
perfusion is one of the major goals of US contrast im- pi ⫽ pg ⫹ p␯ , (2)
aging, and its requirements have already been high-
lighted in the literature (Wiencek et al. 1993). One im- where pg is the pressure of the gas in the bubble and p␯
portant requirement for US contrast agents is that they is the pressure of the liquid vapour. Combining eqns (1)
must not decay at the time of the examination or, at least, and (2), we get:
decay in a predictable manner. de Jong et al. (1993)
demonstrated that the first commercial ultrasonic con- pg ⫽ pl ⫺ p␯ ⫹ p␴ , (3)
trast agent Albunex威 could not sustain pulmonary pas-
sage and the echo enhancement of the left ventricle was
which means that the gas pressure in the bubble is greater
severely reduced. Wilson et al. (1993) produced similar
than pl ⫺ p␯. Consequently, even in a saturated solution,
results. Wiencek et al. (1993) observed, in their labora-
the gas pressure inside the bubble is greater than that in
tory, that a degassed saline solution diminished the
the liquid and, therefore, the bubble will tend to dissolve
acoustic enhancement of Albunex威 microbubbles.
according to Henry’s law. Epstein and Plesset (1950)
The pressure within a free bubble at rest is greater
calculated the dissolution times of air bubbles in water
than the pressure of the liquid surrounding the bubble
and also their change in size. Their calculations showed
because of the surface tension forces (Leighton 1994).
that, if surface tension is neglected, then a bubble would
Therefore, the internal pressure of a bubble pi equals:
not dissolve in a saturated solution, taking the surface
tension into account would result in a finite dissolution
time for the bubble. According to their calculation, a
Address correspondence to: Vassilis Sboros, Department of
Medical Physics, Royal Infirmary of Edinburgh, 1 Lauriston Place, 10-␮m air bubble would dissolve in 1.17 s in a degassed
EH3 9YW, UK. E-mail: Vassilis.Sboros@ed.ac.uk water solution, and in 6.63 s in an air-saturated water

807
808 Ultrasound in Medicine and Biology
solution. Using these calculations, de Jong et al. (1991)
extended this theory to show that larger bubbles disap-
pear slower than the smaller ones. Dissolution of a bub-
ble means shrinkage and, therefore, decrease of scatter-
ing cross-section (resonance is not accounted for).
A simulation of exchanges of multiple gases in vivo
showed the complexity of the problem. Burkard and Van
Liew (1994) have shown that gases interact with each
other and, after an injection of contrast, the bubble may
take in several gases that are present in vivo. Calculations
based on this model showed that exchanges of O2, CO2
and N2 between the bubble and the blood might affect the
size of the bubble (Van Liew and Burkard 1995a). Ka-
balnov et al. (1998b) used the same approach to calculate
the dissolution times of “sparingly water-soluble gases”
used in modern contrast agents (like fluorocarbons), tak-
ing into account that a gas may condense into a liquid.
The results of the above studies showed an initial swell-
ing of the fluorocarbon bubbles, followed by diffusion
and probable condensation. Air bubbles, however, would
diffuse from the moment of introduction to the blood-
stream. Experimental work on rabbits showed little
agreement with the above theoretical predictions (Ka-
balnov et al. 1998a). The persistence of the bubbles was
more prolonged than predicted and did not relate to the
gas consistency. Such a finding illustrated the importance
of the role of the shell in the stabilisation process. It is
definite, however, that nonsoluble gases make better
contrast agents (Kabalnov et al. 1998a, Seidel et al.
1998).
The case of stabilised microbubbles using surface
films or surfactant monolayers to cancel out the surface
tension, responsible for the diffusion of the bubbles, was
examined in a model (Van Liew and Burkard 1995b).
The permeability was, however, considered high enough
to allow the exchange of gases. The complexity of shell
modelling lies not only in the accurate prediction of
pressures exerted, but also in the general mechanical and
chemical properties of the shell material.
Increase in hydrostatic pressure in the liquid would
also increase the gas pressure in the bubble according to
eqn (3), and this would proportionally reduce its volume
according to Boyle’s law. Application of pulsatile pres-
sure has shown that backscatter was partially recover-
able, which implies that the bubble size changes fol-
lowed the pressure changes (Padial et al. 1995). Schnei-
der et al. (1992) applied 150 mmHg pressure to air-filled
albumin particles and observed a decay in the backscatter
signal over time. de Jong et al. (1993) applied 160
mmHg hydrostatic pressure to Albunex威 microbubbles
and, apart from compression, they microscopically no-
ticed that the bubbles deform and disappear. This is in
agreement with Vuille et al. (1994), who noticed that the
application of increasing hydrostatic pressure would pro-
Volume 26, Number 5, 2000
portionally reduce the reflectivity of the bubbles in an
irreversible way (i.e., the release of pressure application
would not recover the reflectivity of the bubbles). This
result suggests that compression is not the sole mecha-
nism for the decay of the agents. Vuille et al. (1994), in
attempting to explain this irreversibility, suggested that
diffusion of the bubbles accelerated by the application of
pressure was more likely to be responsible, which also
would be the outcome from eqn (3).
Decompression sickness can occur in deep-sea
divers when the ascent is rapid. In that situation, expo-
sure to lower pressure oversaturates nitrogen in the
blood, resulting in bubble formation. The opposite hap-
pens in a rapid descent (West 1985). A gas-saturated
solution can become undersaturated under the applica-
tion of pressure; the gas concentration gradient is in-
creased across the wall of the bubble and, according to
Henry’s law, may cause accelerated diffusion, which
would, therefore, decrease the dissolution time. Changes
in hydrostatic pressure would, therefore, alter the gas
concentration gradient across the bubble wall in two
different ways, not only due to changes in the partial
pressure of gases inside a bubble, but also to partial
pressure changes of the dissolved gases in blood. Padial
et al. (1995) applied pulsatile pressure to hand-agitated
Angiovist威 (Berlex Laboratories, Wayne, NJ), which
showed a cyclic variation as well as an overall decrease
in backscatter, but the same treatment to Albunex威 so-
lutions provided a nonrecoverable decay. This paper
concluded that increases in hydrostatic pressure or in
pulse rate accelerated the diffusion of all types of bub-
bles. Shi et al. (1999) have demonstrated an excellent
negative correlation between subharmonic backscatter
signal amplitude and hydrostatic pressure, displaying a
different aspect in the influence of the hydrostatic pres-
sure changes on contrast agents.
Experimental in vitro investigations have been car-
ried out to assess the impact of the hydrostatic pressure
on the stability of the agents, but little has been carried
out experimentally on the role of the gas content of the
suspending solution. The variation of gas content in the
blood might affect significantly the stability of the con-
trast bubbles. In this paper, four agents are tested in terms
of relative decay in solutions with different oxygen con-
centrations. Traditional methods of degassing, such as
boiling, were not used and, instead, the introduction of
helium gas to the dilutant was employed, which is the
routine approach for degassing in chromatography and is
undoubtedly reproducible (Williams and Miller 1962;
Sboros et al. 2000). The results give information on the
tolerance of the agents in the arterial and venous blood
environments.
 In vitro contrast agents ● V. SBOROS et al.
Table 1. Characteristics of contrast agents
Levovist
Nature galactose ⫹ fatty acid
Gas air
Mean diameter (␮m) 3 2.5
Thickness of shell not available
Concentration (bubbles/mL) 4 ⫻ 107
MATERIALS AND METHODS
Contrast agents
Levovist威 (Schering AG Berlin, Germany),
DMP115 (DuPont Pharmaceutical Co, Waltham, MA),
Quantison™ (Quadrant Healthcare Ltd, Nottingham,
UK), and Myomap™ (Quadrant Healthcare Ltd, Notting-
ham, UK) were studied in these series of experiments.
Their characteristics are shown in Table 1. All the agents
in this study are intravenous, apart from Myomap™,
which is an intraarterial agent, because it is of larger
average bubble size (larger than capillaries), as seen in
Table 1. The different gas content, as well as the nature
of the coating, make these agents different from each
other and representative of a range of common contrast
agents. The manufacturer’s recommended preparation
method was followed in each case. During the experi-
ments, the vials were constantly agitated with a mixer.
Suspending solutions
Sterile water was used as the suspending medium
for the agents. Different air concentrations were achieved
by placing the sterile water in a 3-L total parenteral
nutrition plastic bag (KabiBag™, Kabi Pharmacia, Mil-
ton Keynes, UK), and introducing either helium or air.
The technique is described in a previous communication
(Sboros et al. 2000). Helium was introduced into 3 L of
sterile water for 20 min to achieve an oxygen partial
pressures (pO2) of 1.5 ⫾ 0.36 kPa. Air was also intro-
duced into 3 L of sterile water for 15 min; the partial
pressure achieved with this preparation was 24.7 ⫾ 1.0
kPa. The pO2 is a good indicator of how degassed the
water is. Nitrogen is less soluble than oxygen, and he-
lium is a nonsoluble gas and, therefore, they are all less
likely to be in bigger quantities than oxygen. Therefore,
the measurement of the partial pressure of the most
soluble gas (O2) is a good indicator of how degassed a
solution is.
Acquisition of data
The experimental setup, in addition to the data-
acquisition system, is presented and evaluated in Sboros
et al. (2000). A total of 200 mL of suspension was
introduced in an anechoic tank, and was scanned at
809
DMP115 Quantison™ Myomap™
liposomes human serum albumin human serum albumin
perfluoropropane air air
3.2 10
15 nm 200–300 nm 1 ␮m
109 1.5 ⫻ 109 1.5 ⫻ 107
3-MHz frequency using an ATL Ultramark 9 (UM9)
ultrasonic scanner. The data-acquisition system collected
complete RF sector frames.
The average radiofrequency (RF) backscatter inten-
sity of a region of interest of all the frames was referred
to the same region of interest of the acoustic test grey-
scale object (Cardiff grey-scale test object, Diagnostic
Sonar, Livingston, UK) under the same machine settings,
and the resulting normalised backscatter was calculated
(Sboros et al. 2000). The normalised backscatter (N) is a
pure number from which the integrated backscatter (IB)
is derived using the formula (Moran et al. 1994; Sboros
et al. 2000):
IB ⫽ 10 ⫻ Log10共N兲. (4)
The transmit power was set at 2.24% of maximum on the
scanner console, which corresponds to 0.27 MPa peak
negative pressure at 3 cm from the probe, with the focus
located at 4.25 cm from the transducer. The latter was
also the distance of the probe from the bottom of the
tank. Gain and time gain compensation (TGC) were kept
constant throughout the whole experiment.
Experimental protocol
Backscatter vs. bubble concentration. In this study,
200 mL of sterile water was introduced into the tank, and
different volumes of agent were injected into the tank.
They were allowed to mix for 28 s with the help of a
magnetic stirrer, and then were insonated for 2 s, of
which the last image frame of digitised RF echo data was
collected. Table 2 gives full details of the concentrations
of injection for each agent. For DMP115, the injections
ranged from 0.0005 mL to 0.005 mL, with a 0.0005-mL
step (over all 10 injections). For Quantison™, the injec-
tions ranged from 0.05 mL to 0.65 mL, with a 0.05-mL
step (over all 13 injections). For Myomap™, the injec-
tions ranged from 0.3 mL to 2.7 mL, with a 0.3-mL step
(over all 9 injections). For Levovist威, the injections
ranged from 0.2 mL to 1.6 mL, with a 0.2-mL step (over
all 8 injections).
Lifetime in the vial. In this experiment, the echoge-
nicity of the agents was tested over a long period of time
810 Ultrasound in Medicine and Biology Volume 26, Number 5, 2000

Table 2. Concentration range of contrast agents

Levovist DMP115 Quantison™ Myomap™

Minimum concentration* 0.2/40,000 0.0005/2500 0.05/375,000 0.3/22,500

Maximum concentration* 1.6/320,000 0.005/25,000 0.65/4,875,000 2.7/202,500
Step* 0.2/40,000 0.0005/2500 0.05/375,000 0.3/22,500
Amount of injections 8 10 13 9

*mL of contrast agent per 200 mL of suspension/number of bubbles per mL of suspension.

to assess the period of time that the contrast agents are
stable in the vial and, therefore, suitable for experiments.
Table 3 illustrates the protocol followed. DMP115,
Quantison™ and Myomap™ were tested for 5 h, starting
with a measurement 10 min after preparation. A mea-
surement was repeated for those agents every 20 min,
and the bubble concentrations were chosen to be in the
linear part of the “backscatter vs. concentration” plots
(Table 4). Levovist威 has a shorter lifetime in the vial
and, therefore, was tested for 30 min, starting 2 min after
its preparation, as advised by the manufacturer.
Variation of the pO2 in the suspension. In this ex-
periment, the agents underwent two sets of measure-
ments, a long term and a short term.
1. Long-term experiment: After the production of the
suspending medium in the bag, the agent was injected
into the bag, and stirred for 5 min. The concentrations
that were selected for the different agents were deter-
mined after the completion of the experiment (back-
scatter vs. concentration), and were chosen so that
they would lie in the linear range of the resulting
curves (Fig. 1). A 200-mL sample was taken from the
bag and inserted into the measuring tank. The solution
was stirred for 29.5 s with a magnetic stirrer and then
insonated for 0.5 s. The last frame was acquired. This
period of insonation was determined in a pilot study,
and showed that it had a negligible effect on the decay
of the agents. After 10 min, a new 200-mL sample
was inserted into the tank, and the same procedure
was repeated. That way, a frame was acquired every
10 min until 15 frames were acquired. This experi-
ment was repeated for two concentrations of oxygen
(i.e., two independent values of pO2 in the bag).
Levovist威 did not undergo this set of measurements
because it is not designed to remain echogenic for the
above time ranges.
2. Short-term experiment: Instead of injecting the agent
into the bag, 200-mL of the suspending medium of
the bag was inserted in the tank and then the agent
was injected in the tank. The new solution was con-
tinuously mixed and every 29.5 s there was 0.5 s of
insonation and the last frame of the insonation period
was acquired. This lasted until 15 frames were ac-
quired. The experiment was repeated as previously
for the two concentrations of oxygen, and it was also
repeated 3 times for every concentration of oxygen, to
assess the variability of the measurements. For Levo-
vist威, the protocol for the short-term experiment was
altered. After the introduction of the 200 mL to the
tank, Levovist威 was injected into the tank and stirred
for 9.5 s and then insonated for 0.5 s, and the last
frame of that was acquired. The same was repeated
until 15 frames were acquired. The experiment was
repeated 6 times, for each of the above two oxygen
levels, not only for reproducibility purposes, but also
because Levovist威 is a shorter-lived agent compared
to the other three. The concentrations of contrast used

Table 3. Protocol for assessing the lifetime of contrast agents

Table 4. Linear range of the backscatter vs. concentration
in the vial
curve
Levovist DMP115 Quantison™ Myomap™
Bubble concentration
(bubbles/mL of suspension) a b r2
Concentration
(bubbles/mL) 160,000 12,500 2,250,000 22,500
Levovist 160,000 8 ⫻ 10⫺5 9.98 0.90
First measurement
DMP115 12,500 0.0011 ⫺0.042 0.96
(min after
Quantison™ 2,250,000 3 ⫻ 10⫺8 0.018 0.98
preparation) 2 10 10 10
Myomap™ 75,000 6 ⫻ 10⫺6 0.073 0.96
Last measurement
(min after
The values of bubble concentrations correspond to those pointed by
preparation) 30 290 290 290
the arrows in Fig. 1, a is the slope of the corresponding linear fit to the
Time between
data up to the above concentrations, b is the intercept of the fit, and r2
measurements
is the regression coefficient. For Myomap™, the linear fit refers to the
(min) 2 20 20 20
whole plot.
 In vitro contrast agents ● V. SBOROS et al. 811

Fig. 1. All contrast agents display a linear increase of normalised backscatter with the bubble concentration. For the IV
agents, this leads to a plateau due to self-attenuation. The saturation in backscatter is not apparent in the case of
Myomap™ because the concentrations used were not as high. The concentrations of bubbles are displayed in Table 2.
The arrows point the highest concentration that provided a linearly related normalised backscatter (except for
Myomap™).

in this experiment are stated in the first part of the
Results section.
For every set of 15 frames of the short-term experiment,
an exponential fit was applied and the decay constant and
the intercept of it were derived. The mean value and
standard deviation of these were calculated for each pO2,
and a paired t-test was performed.
RESULTS
Backscatter vs. bubble concentration.
The graphs of results are plotted in Fig. 1. All the
agents have a distinct linear part for the lower values
of concentrations. For Levovist威, DMP115 and Quan-
tison™, there is a distinct shoulder in the curve for
higher values of concentrations, which implies self-
attenuation effects for those values (de Jong and Hoff
1993). Table 4 shows the maximum bubble concen-
trations for this linear part of each curve, the coeffi-
cients of the least square linear fit Y ⫽ a*X ⫹ b (where
X is the bubble concentration, Y is the normalised
backscatter, a is the slope of the linear fit, and b is the
intercept of the fit), and the regression coefficient that
corresponds to the linear fit. The following experi-
ments use bubble concentrations that belong to the
linear part of these curves. The concentrations used for
Myomap™ were not high enough to show where this
shoulder starts, due to limited availability of the agent.
Thus, the bubble concentration (arrow) in Table 4
refer to the one used in the following experiments, and
the regression coefficient r2, to the linear fit that con-
tains all the values of the plot.
Lifetime in the vial
Over 5 h, DMP115, Quantison™ and Myomap™
showed no decay in the vial (Fig. 2a). Levovist威 has a
shorter lifetime, but shows considerable stability over 30
min (Fig. 2b).
Variation of pO2 in the suspension
Long-term experiment. After the completion of
the experiment “backscatter vs. bubble concentration,”
the concentration of the three contrast agents that were
to be used in subsequent experiments was determined.
Table 5 shows the concentrations that were used in
this experiment. Myomap™ and Quantison™ showed
similar behaviour when injected into the bag with
different pO2. Both agents showed very low correla-
tion between the normalised backscatter and time for
both pO2 (Fig. 3a and b), which is due to the lack of
decay in the solution environment. The increased vari-
ability shown by Quantison™ was caused by occa-
sional bright scatterers in several frames. DMP115, on
812 Ultrasound in Medicine and Biology Volume 26, Number 5, 2000

Fig. 2. (a) Lifetime in the vial for DMP115, Quantison™ and Myomap™. (b) Lifetime in the vial for Levovist威. The
protocol of both experiments appears in Table 3.

the contrary, showed high correlation with the expo-
nential decay (Fig. 3c), but no sign of difference in
decay between the two environments. After 145 min in
two different solutions, the backscatter from DMP115
was almost indistinguishable from the noise level.
Even if the assumption that the US field caused neg-
ligible destruction to the DMP115 bubbles is not en-
tirely correct, the decay constant in this experiment is
accurately assessing the decay in the bag because
exposure to US was the same for every sample, as
explained in the experimental protocol.
Short-term experiment. As mentioned in the third
section of the experimental protocol, the concentrations
for this part of the experiments are taken from the “back-
scatter vs. bubble concentration” experiment, and are
shown in Table 6. Some examples of curves produced in
these experiments are shown in Fig. 4. For DMP115,
Myomap™ and Quantison™, for each value of pO2, the
 In vitro contrast agents ● V. SBOROS et al. 813

Table 5. Summary of the results of the long-term experiment

DMP115 Quantison™ Myomap™

Concentration (microbubbles/mL) 1.67 ⫻ 104 2 ⫻ 106 5 ⫻ 104

Low pO2 pO2 (start in kPa) 1.8 1.5 1.7
decay (min⫺1) 0.037 ⫾ 0.001 0.0007 ⫾ 0.0003 0.0012 ⫾ 0.0005
r ⫺0.99 ⫺0.34 ⫺0.54
pO2 (end in kPa) 2.5 2 1.9
High pO2 pO2 (start in kPa) 25.5 26.2 24.7
decay (min⫺1) 0.045 ⫾ 0.003 ⫺0.0007 ⫾ 0.0025 ⫺0.0011 ⫾ 0.0013
r ⫺0.98 0.25 0.24
pO2 (end in kPa) 25.1 25.5 24.7

For all three agents, the pO2 in the start (0 min) and in the end (150 min) of the measurements, the decay constants, and also the correlation
coefficient to the exponential decay, are stated. Negative values in the decay constants correspond to an increase in backscatter. Decay and intercept ⫾
their standard deviation of the mean, calculated by the regression analysis performed on the logged data, are shown.

measurements were repeated 3 times and, for Levovist威,
6 times. Quantison™ was the only agent that did not
have good correlation with the exponential fit, mainly
due to the lack of decay (Fig. 4a, Table 6). The increased
variability shown by Quantison™ was caused again by
occasional bright scatterers in several frames. It is not
certain whether they belong to impurities in the suspen-
sion or are due to the contrast material. The best corre-
lation with the exponential fit is exhibited by the
DMP115, with values very close to one (Fig. 4c, Table
6). Myomap™ and Levovist威 also showed good corre-
lation in all measurements (Fig. 4b and d, respectively,
Table 6). Quantison™, Myomap™ and DMP115 exhib-
ited no difference in the decay constants, and intercepts
deduced from the exponential fits, for the two pO2 values
of the solution. Levovist威 was the only agent for which
these two parameters were significantly different for the
two solutions. The first point of all the curves for Levo-
vist威 was an outlier from the exponential model. It was
found that, indeed, the air-saturated solution gave a
higher normalised backscatter value for these outliers.
However, performing a paired t-test for the mean outlier
value for each pO2 in the solution, there is no significant
difference between the two solutions (p ⫽ 0.057). Per-
forming a t-test for the decays and intercepts, excluding
the outliers, both these characteristics of the curves are
highly significantly different for the two solutions (p ⬍
0.001).
DISCUSSION
The first two experiments that are presented are
considered necessary, not only for the later experiments
in this paper, but also for in vitro or in vivo investigations
that aim at the quantitation of contrast enhancement.
The linear portion of the graph in Fig. 1, that all
agents showed in the lower range of the bubble concen-
trations, is simply pointing out that, at this range of
concentrations, the normalised backscatter is propor-
tional to the number of bubbles that are insonated, be-
cause it is proportional to the scattering cross-section of
each bubble. Similarly to de Jong and Hoff (1993), the
attenuation affects significantly the normalised backscat-
ter above certain concentrations. The linear range of
normalised backscatter vs. concentration allowed com-
parisons between agents because we assume that they all
are exposed to the same ultrasonic field, and that negli-
gible destruction of contrast occurs in that field. Table 4
stated that the slope, a, of the linear part of the curve in
Fig. 1 for each agent, is approximately proportional to
the average scattering cross-section of each agent when
the intercept of the fit, b, is close to zero. Only Quanti-
son™ and Levovist威 displayed a significant intercept.
For Quantison™, this was probably due to the very low
values of normalised backscatter that were close to the
background signals. The significance of the intercept for
Levovist威, however, suggested that the slope, a, was not
an accurate estimate of the relationship between the
normalised backscatter and microbubble concentration.
Levovist威 is the most unstable of all four agents and it is
difficult to determine an accurate estimate of the number
of microbubbles in suspension at the time of measure-
ment. Furthermore, for the same reason, it was impossi-
ble reproducibly to perform measurements at lower con-
centrations with the same protocol to increase the num-
ber of points for the linear fit.
In Fig. 1, DMP115 and Levovist威 occupy a similar
range of normalised backscatter values and provided
significantly higher backscatter levels than Quantison™
and Myomap™. The comparison illustrated in Fig. 1 is
highlighted by the comparison of slopes in Table 4
(Levovist威 is not included in this comparison). DMP115
and Levovist威 have much thinner shells (Table 1) and,
thus, provide less damping in the oscillatory motion
triggered by the incident US pulse. Frinking and de Jong
(1998) showed that Quantison™ demonstrated low back-
scatter at low acoustic pressures due to its “high stiffness
814 Ultrasound in Medicine and Biology Volume 26, Number 5, 2000

Fig. 3. Backscattering performance of three contrast agent solutions. (a) Quantison™, (b) Myomap™ and (c) DMP115,
in two solutions: one degassed (pO2 ⬇ 2 kPa), and one air-saturated (pO2 ⬇ 25 kPa). The plots for Quantison™ and
Myomap™ show no decay in either of the two solutions, and the DMP115 plots show an almost identical decay for both
solutions.

and friction parameter of the shell.” Myomap™ provided
a considerably steeper slope, a, than Quantison™, due to
the larger average size of bubbles, also in agreement with
Frinking and de Jong (1998).
For Quantison™ and Myomap™, there was no
indication of decay in the long experiment, which
suggests great tolerance of the agents under the spe-
cific in vitro environment (Fig. 3a and b) and demon-
strates that the shells of these agents did not permit
diffusion of the gas. The DMP115 did decay, but there
was no sign of difference in decay between the two
pO2 levels (Fig. 3c). The agent was not stable as long
as it was in the vial (Fig. 2a), but it exceeded the
predicted survival time for similar gas materials (Ka-
 In vitro contrast agents ● V. SBOROS et al. 815

Fig. 3. Continued

balnov et al. 1998b), which demonstrates the impor-
tance of the shell in the stability of the bubbles.
However, the decay was higher in comparison to
Quantison™ and Myomap™ (Fig. 3), even though
they contain air, which is a more soluble gas than
perfluoropropane. This is suggestive that the DMP115
shell was more permeable compared to the other two
agents, and allowed gas exchange. The lack of signif-
icance between the decay constants at different pO2
levels showed that there was nothing to suggest a

Table 6. Summary of the results of the short-term experiment

Levovist DMP115 Quantison™ Myomap™

Concentration (microbubbles/mL) 1.6 ⫻ 10 5

1.25 ⫻ 10 4
2.25 ⫻ 10 6
7.5 ⫻ 104
Low pO2 pO2 (start in kPa) 1.3 ⫾ 0.2 1.5 ⫾ 0.3 1.7 ⫾ 0.1 1.6 ⫾ 0.3
r ⬍ 0.7 0 0 3 0
r ⬎ 0.9 6 3 0 2
pO2 (end in kPa) 6.4 ⫾ 0.7 8.5 ⫾ 0.5 8.7 ⫾ 0.6 8.3 ⫾ 0.6
Intercept 10.55 ⫾ 5.64 15.61 ⫾ 1.73 0.16 ⫾ 0.01 0.15 ⫾ 0.01
Decay constant
(min⫺1) 1.228 ⫾ 0.250 0.065 ⫾ 0.015 0.012 ⫾ 0.019 0.059 ⫾ 0.002
High pO2 pO2 (start in kPa) 25.1 ⫾ 1.0 25.4 ⫾ 0.9 23.9 ⫾ 0.3 24.8 ⫾ 0.7
r ⬍ 0.7 0 0 2 0
r ⬎ 0.9 3 2 0 3
Intercept 25.51 ⫾ 5.41 13.57 ⫾ 1.53 0.14 ⫾ 0.03 0.15 ⫾ 0.02
Decay constant
(min⫺1) 0.308 ⫾ 0.068 0.058 ⫾ 0.005 0.030 ⫾ 0.027 0.059 ⫾ 0.004
Comparison degrees of freedom 10 4 4 4
Intercepts t 4.69 1.53 1.075 ⫺0.089
p 0.0008 0.201 0.343 0.933
Decays t ⫺8.69 ⫺0.754 0.932 0.262
p 5.6 E–06 0.493 0.404 0.806

For all four agents, the pO2 at the start of the experiments was measured. The measurement was repeated only for degassed suspensions at the end
of the experiments, to assess whether it still remained degassed. Also, the decay constants and intercepts of the exponential fit are summarised in terms
of mean ⫾ one standard deviation. The correlation coefficients of the above fit were classified as high (⬎ 0.9) or low (⬍ 0.7), and the number of
models that belong to either category are stated. The results of the t-test comparing the decay constants and intercepts between the two suspension
environments are also stated for each agent.
816 Ultrasound in Medicine and Biology Volume 26, Number 5, 2000

Fig. 4. Normalised backscatter plotted against time (short experiment). (a) Quantison™ does not exhibit any sign of
decay; (b) Myomap™ exhibits some decay, but there is no significant difference in decay between the two solutions;
(c) DMP115 is similar to Myomap™; and (d) Levovist威 not only decays, but there is a significant difference between
the degassed and air-saturated solutions. These measurements were repeated 3 times for all agents, apart from Levovist威;
these were repeated 6 times.

swelling of the bubbles due to initial introduction of the decay in the degassed suspension would be con-
air that was present in the solution and missing from siderably faster.
the bubble (Van Liew and Burkard 1995b; Kabalnov High shell permeability might apply for Levovist威
et al. 1998b). In a case of initial swelling of the agent, too, but here the diffusion rate of the gas was much faster
 In vitro contrast agents ● V. SBOROS et al. 817

Fig. 4. Continued

than the one demonstrated by DMP115. This explanation
is further confirmed by the accelerated diffusion of the
Levovist威 microbubbles when subjected to a degassed
suspension, compared to the air-saturated suspension,
which demonstrated that the thin coating provided by the
palmitic acid does not stop the exchange of gases be-
tween the suspension and the microbubbles. It is also
evident from Fig. 4d that a large population of bubbles
dissolves within less than 20 s in suspension and the rest
dissolves at a much slower rate. It can be speculated that
this could reflect different levels of shell permeability
among bubbles. Similar findings were demonstrated in
an experiment where Albunex威 bubbles were insonated
at 1MPa peak negative pressure (Wu and Tong 1998).
That study was a different experiment, but showed, on a
different time scale, that, after an initial drop in the
number of scatterers after insonation, a subsequent less
pronounced decrease of the remaining number followed.
The authors suggested that there were two different pop-
ulations of bubbles. One “weak” population was easily
818 Ultrasound in Medicine and Biology
destroyed by the insonation, and a “stronger” one was
more resistant to the particular acoustic pressures. The
recirculation of Levovist威 demonstrated in a dog model
by Schwarz et al. (1996) does not contradict the above
results. The decelerated decay demonstrated by Levo-
vist威 after the initial 20 s in suspension perhaps belongs
to the population of bubbles that were able to recirculate
in the above paper.
Myomap™ and DMP115 showed higher decay
rates in the short-term experiment (Table 6) compared to
the long experiment (Table 5), which is evidence that the
acoustic field (0.27 MPa peak negative pressure) was
responsible for the decay of the agents. Quantison™ did
not display a significant decay in any experiment. The
design of these experiments did not aim at understanding
the contribution of the ultrasonic beam in the decay
process of the agents, and any conclusion or comparison
with work of others can only be speculative.
The above results are only suggestive of the behav-
iour of the four agents in an in vivo situation. Venous
blood has 6 –9 kPa pO2, and arterial blood 11–14 kPa
pO2. These values vary further in different pathological
situations. The pO2 in the present study was arranged to
take values above and below the physiological range of
blood pO2. In spite of the fact that the results cannot be
conclusive of the behaviour of the agents in vivo, this
study illustrated the potential effect of the gas content of
different vascular spaces on different agents. Further
investigation is needed to outline the combined effect of
different physical environments in addition to that of the
US beam. This can be provided with few additions to the
setup used in the present study.
SUMMARY
Four agents were compared in this study at low
acoustic pressures. The ones with thinner coating
(DMP115 and Levovist威) demonstrated an enhanced
backscatter compared to agents with thicker coating
(Quantison™ and Myomap™). The role of oxygen con-
tent of a suspension was tested in the destruction of four
different ultrasonic contrast agents. All agents, apart
from Levovist威, proved tolerant in suspensions with low
pO2. The behaviour of all four agents in vitro is expected
to be a good indicator of their behaviour in vivo. The
stability of contrast agents needs to be known to perform
quantitative studies.
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