Ultrasound in Med. & Biol., Vol. 27, No. 10, pp.

1367–1377, 2001
Copyright © 2001 World Federation for Ultrasound in Medicine & Biology
Printed in the USA. All rights reserved
0301-5629/01/$–see front matter

PII: S0301-5629(01)00440-9

● Original Contribution

CONTRAST AGENT STABILITY: A CONTINUOUS B-MODE IMAGING

APPROACH

V. SBOROS, C. M. MORAN, S. D. PYE, and W. N. MCDICKEN

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

(Received 15 January 2001; in final form 11 July 2001)

Abstract—The stability of contrast agents in suspensions with various dissolved gas levels has not been reported
in the literature. An in vitro investigation has been carried out that studied the combined effect of varying the
acoustic pressure along with degassing the suspension environment. In this study, the contrast agents were
introduced into suspensions with different oxygen concentration levels, and their relative performance was
assessed in terms of decay rate of their backscatter echoes. The partial pressures of oxygen in those solutions
ranged between 1.5 and 26 kPa. Two IV and one arterial contrast agents were used: Definity, Quantison™, and
Myomap™. It was found that Quantison™ and Myomap™ released free bubbles at high acoustic pressure that
also dissolved faster in degassed suspensions. The backscatter decay for Definity did not depend on the air content
of the suspensions. The destruction of bubbles was dependent on acoustic pressure. Different backscatter
performance was observed by different populations of bubbles of the last two agents. The physical quantity of
“overall backscatter” (OB) was defined as the integral of the decay rate over time of the backscatter of the
contrast suspensions, and improved significantly the understanding of the behaviour of the agents. A quantitative
analysis of the backscatter properties of contrast agents using a continuous imaging approach was difficult to
achieve. This is due to the fact that the backscatter in the field of view is representative of a bubble population
affected by the ultrasound (US) field, but this bubble population is not representative of the contrast suspension
in the whole tank. Single frame insonation is suggested to avoid the effects of decay due to the ultrasonic field,
and to measure a tank-representative backscatter. The definition of OB was useful, however, in understanding
the behaviour of the agents. (E-mail: Vassilis.Sboros@ed.ac.uk) © 2001 World Federation for Ultrasound in
Medicine & Biology.

Key Words: Ultrasound, Contrast agents, Stability, Free bubble, Degassing, Definity, Quantison™, Myomap™.

INTRODUCTION tissue (Christopher 1997; Averkiou et al. 1997). On the

Ultrasound (US) contrast agents are microbubble-based
scatterers that undergo a range of interactions with US to
achieve echo enhancement in diagnostic US. This en-
hancement is based on the impedance mismatch between
the liquid that surrounds a contrast bubble and the bubble
itself, as well as the motion of the bubble caused by the
US wave. Exposure even to a low-amplitude US field
provokes nonlinear bubble oscillations that create har-
monic components in the scattered wave (Schrope et al.
1992; Schrope and Newhouse 1993). From this, a new
imaging mode called second harmonic imaging emerged
(Burns 1996; Tiemann et al. 1997) and, furthermore, an
imaging modality that did not involve contrast was cre-
ated, based on the detection of harmonic signals from
Address correspondence to: Vassilis Sboros, Department of
Medical Physics, Royal Infirmary of Edinburgh, 1 Lauriston Place,
EH3 9YW, UK. E-mail: Vassilis.Sboros@ed.ac.uk
other hand, it was found that increase in the amplitude of
incident US destroyed the contrast bubbles as a result of
the acceleration of the diffusion of the encapsulated gas
(Porter and Xie 1995), which introduced the use of
intermittent imaging with contrast agents. The idea lies
in the fact that a much lower frame rate would allow time
for the introduction of a fresh agent into the field of view.
Another interesting modality, called LOC (loss of corre-
lation) imaging, also emerged in colour Doppler US
(Uhlendorf and Hoffman 1994; Tiemann et al. 1997).
Ongoing research offers novel ideas for the use of US
contrast agents. Subharmonic signals from contrast
agents showed good correlation with hydrostatic pres-
sure (Shi et al. 1999). In this context, a systematic
investigation of the properties of contrast microbubbles
is necessary.
It has been impossible so far to model satisfactorily
the oscillatory motion of microbubbles, or to fully com-

1367
1368 Ultrasound in Medicine and Biology
Table 1. Characteristics of contrast agents
Definity
Shell composition Liposome
Gas Perfluoropropane
Mean diameter (␮m) 2.5
Thickness of shell 15 nm
Concentration in vial (bubbles/mL) 109
Concentration in suspension (bubbles/mL) 1.5 ⫻ 104
prehend their stability in suspension. Even before the
development of a wide spectrum of US imaging modal-
ities, due to the introduction of contrast agents, the
requirement of microbubble stability, or at least of its
predictable decay, was understood (Wiencek et al. 1993).
A short review of the literature on the stability of contrast
introduced a previous communication (Sboros et al.
2000a). The main reason for contrast decay lies in the
dissolution of the gas of the bubble into the liquid me-
dium. The rate of dissolution results mainly from the gas
concentration or pressure imbalance across the bubble
wall and the contribution of the stabilising effect of the
bubble coating. Although the behaviour of free gas bub-
bles in a liquid environment has been well predicted
(Epstein and Plesset 1950; Leighton 1994; de Jong et al.
1991), models that include the stabilising effect of the
coating (Van Liew and Burkard 1995a) or sparingly
soluble gases (Van Liew and Burkard 1995b; Kabalnov
et al. 1998b) have not matched the experimental data
(Kabalnov et al. 1998a; Basude et al. 2000). The com-
plexity 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
(Basude et al. 2000).
This paper does not propose a new model but,
instead, reports that the combined effects of suspension
environment and exposure to US add further complexity
to the problem. In vitro work showed that Albunex威
(Molecular Biosystems Inc., San Diego, CA) and Levo-
vist (Schering AG, Berlin, Germany) were sensitive to
degassed liquid suspensions (Wiencek et al. 1993; Sbo-
ros et al. 2000a), but newer contrast agents were not, for
example, Quantison™ (Quadrant Healthcare Ltd, Not-
tingham, UK), Myomap™ (Quadrant Healthcare) and
Definity (DuPont Pharmaceutical Co, Waltham, MA)
(Sboros et al. 2000a). The experiments for the latter three
were carried out using a continuous imaging insonation
setting at 0.27 MPa peak negative pressure (PNP). Myo-
map™ and Definity decayed during exposure to this
field, but Quantison™ did not. Seven different acoustic
pressures were used in this study to clarify the effect of
the US field on the stability of these agents and, further-
more, if a degassed medium would have a different effect
on contrast bubbles subjected to different acoustic pres-
Volume 27, Number 10, 2001
Quantison™ Myomap™
Human serum albumin Human serum albumin
Air Air
3.2 10
200–300 nm 1 ␮m
1.5 ⫻ 109 1.5 ⫻ 107
2.25 ⫻ 106 7.5 ⫻ 104
sures. The previous study (Sboros et al. 2000a) assumed
that the contrast agent in suspension was such that its
backscatter was linearly related to the concentration of
contrast bubbles in the whole suspension. This assumed,
for continuous imaging insonation, that, if the acoustic
field caused destruction of some microbubbles, the num-
ber of destroyed bubbles would be very small compared
to the total number of bubbles in the field of view. Thus,
the concentration of bubbles in the field of view would
be almost identical to that in the tank. If this is not the
case, then the frames captured in continuous imaging do
not have contrast backscatter that belongs to a concen-
tration identical to that of the whole suspension. In such
a case, the backscatter of a frame is not the same as that
of the bubble concentration in suspension. This assump-
tion is also discussed in the present study.
MATERIALS AND METHODS
The setup and the acquisition of data
A description of the experimental setup and its
calibration is given by Sboros et al. (2000b). A 200-mL
suspension was introduced into an anechoic tank and
scanned at 3-MHz centre frequency using an Ultramark
9 (UM9) ultrasonic scanner (Advanced Technology Lab-
oratories, Bothell, WA). The nominal transmit power
ranged from 2.24% to 100% of maximum output power.
This corresponded to a range of 0.27 to 1.52 MPa PNP at
3 cm from the probe. The centre of the focal region was
located at 4.25 cm from the transducer, which was also
the distance of the probe from the bottom of the tank.
The data-acquisition system collected complete RF sec-
tor frames at 12-MHz digitisation rate. The average
backscatter intensity of a region-of-interest (ROI) at 0.5
cm above the bottom of the tank was used in the analysis
(distance from bottom of ROI). The ROI was 2.9 ⫾ 0.1
cm wide and 1.4 ⫾ 0.1 cm deep.
Contrast agents and suspending solutions
Three contrast agents were used in this study:
Definity, Quantison™ and Myomap™. Their character-
istics are shown in Table 1. The different gas content, as
well as the nature of the shell, make these agents differ-
ent from each other and representative of a range of
 Contrast agent stability ● V. SBOROS et al.
available contrast agents. The manufacturer’s recom-
mended preparation method was followed in each case.
During the experiments, the vials were constantly agi-
tated with a mixer.
The preparation of the suspensions is described by
Sboros et al. (2000b). Sterile water was placed into a 3-L
total parenteral nutrition plastic bag (KabiBag™, Kabi
Pharmacia, Milton Keynes, UK), and either helium for
20 min or air for 15 min was introduced to produce either
a degassed or an air-saturated suspension.
The pO2 for the degassed suspension was 1.4 ⫾ 0.1
kPa, with maximum and minimum values of 1.6 and 1.3
kPa, respectively. The content of each bag was used for
approximately 2 h, and random checks proved pO2 stable
to within 0.2 kPa. The pO2 is a good indicator of how
degassed the water is. Nitrogen is less soluble than
oxygen, and helium is classed as a nonsoluble gas.
Experimental protocol
The bubble concentrations in the different suspen-
sions are given in Table 1. Continuous mixing was
applied in the tank for 28 s after the introduction of
contrast, and insonation (61-Hz frame rate) was applied
for 2 s as mixing continued. The RF data of the last frame
of the insonation period were acquired. This routine was
repeated until 15 frames were acquired. This procedure
was repeated, for each agent, for each suspension (de-
gassed and air-saturated), and for seven different acous-
tic pressure settings ranging from 0.27 to 1.52 MPa PNP
at the focus. Each experiment was repeated 3 times to
assess its variability. Overall, 126 sets of 15 frames were
collected.
A separate experiment was performed for all three
agents in air-saturated suspension at 1.37 MPa. The
insonation time was increased to 10 s and the time
between insonations was 30 s. This experiment was
performed to achieve total dissolution of the gas of the
contrast bubbles and, consequently, to reduce the back-
scatter level to a negligible amount. The RF data of the
last frame of the insonation period were acquired and the
procedure was repeated until 15 frames were acquired.
Each set of measurements was repeated 3 times. In
addition to the RF data being collected, all measurements
were recorded on SVHS videotape for subsequent in-
spection.
Statistical analysis
The analysis was based on normalising the back-
scatter intensity of a ROI of the contrast agent suspen-
sions (Sboros et al. 2000a). Only the data from the final
experiment at 1.37 MPa were not subjected to normal-
isation because comparison with other settings was not
intended. In the normalisation, the average backscatter
intensity of the agents, at the specific ROI, was referred
1369
to a suspension whose composition was similar to the
blood-mimicking fluid (BMF) described by Ramnarine
et al. (1998). In this suspension, the ingredients dextran
and glycerol were omitted and replaced by water, so as to
match the viscosity of the water-based contrast suspen-
sion. The backscatter intensity of this suspension is
shown to be linear with transmit acoustic intensity at that
ROI (Sboros et al. 2000b).
An exponential fit was applied to each 15-frame
normalised data set. The linear correlation coefficient,
the decay constant and the intercept on the normalised
backscatter axis were calculated. The decay constant
represents the decay rate of the bubbles in suspension in
terms of backscatter, and the intercept is the normalised
backscatter of the contrast suspension when no in-
sonation has occured.
A criterion was used to assess if an exponential
decay was followed. Correlation coefficients lower than
0.7 were considered as poor correlation to the exponen-
tial curve, and correlation coefficients higher than 0.9
were considered as satisfactory correlation to the expo-
nential decay. Each combination of contrast agent, pO2,
and acoustic pressure was repeated 3 times. Both decay
constants and intercepts were averaged and a t-test as-
suming equal variances was performed to assess the
difference between the degassed and air-saturated sus-
pensions.
RESULTS
All the agents showed decay when exposed to US at
almost all different acoustic pressures. Figure 1 shows a
representative data set with acoustic pressure at maxi-
mum for (a) Definity, (b) Quantison™ and (c) Myo-
map™ at air-saturated suspensions. The vast majority of
the data sets for Definity are highly correlated with the
exponential fit (r ⬎ 0.9) and, for Quantison™, the cor-
relation is considered poor (r ⬍ 0.7) (Table 2). Almost
half of the data sets (24) correlate highly with the expo-
nential decay for Myomap™, but the rest of the sets
provide a lower correlation coefficient, although not as
poor as for Quantison™. No tendency to higher or lower
correlation coefficients was observed for either different
pO2 or acoustic pressures.
The appearances of the captured frames at maxi-
mum acoustic pressures was similar to those obtained at
1.37 MPa (Fig. 2a.2, b.2, c.2). In Fig. 2a.2, Definity
appeared as a homogeneous suspension of scatterers.
This was also supported by the videotape and explains
the high correlation to the exponential decay (Fig. 1a),
because backscatter homogeneity in a ROI would pro-
vide less backscatter variability between frames. Thus,
ROI-average backscatter values at different times, calcu-
lated by a narrow distribution of backscatter amplitudes,
1370 Ultrasound in Medicine and Biology
Fig. 1. Backscatter intensity vs. time for (a) Definity, (b)
Quantison™ and (c) Myomap™. Note the difference in regres-
sion coefficients and the scattered values of the plots around the
trends, as well as the decay constants and the intercepts. The
units of the average backscatter intensity are arbitrary, and
result from the digitisation.
would lie closer to the curve that describes the general
behaviour of the backscatter with time. This explains the
closeness to the exponential model. The videotape
showed that Definity microbubbles, when suspended in
much lower concentrations (e.g., in the 15th insonation
interval, when most of the bubbles were destroyed and
individual scattering events were observed), needed
more than one frame to disappear, because the same
scatterers were easy to trace in consecutive frames.
Looking at the VCR recordings of all the data, the
suspensions of Definity appeared visually homogeneous
Volume 27, Number 10, 2001
Table 2. Behaviour of contrast agents when insonated at
different acoustic pressures and po2.
DMP-115 Quantison™ Myomap™
Number of acoustic pressures 7 7 7
Number of pO2 levels 2 2 2
r ⬍ 0.7 0 of 42 24 of 42 1 of 42
r ⬎ 0.9 40 of 42 2 of 42 24 of 42
Number of nonsignificant 5 of 7 5 of 7 1 of 7
intercepts
Number of nonsignificant 6 of 7 6 of 7 3 of 7
decays
Three data sets were collected for each (of seven) acoustic pressure,
and each (of two) pO2 levels. Definity and Quantison™ did not have
significantly different decays (both 5 of 7 nonsignificantly different
decay constants) and intercepts (both 6 of 7 nonsignificantly different
intercepts) between the 2 pO2 levels. The relationship of normalised
backscatter vs. time for Quantison™ had a rather poor correlation with
the exponential decay (24 of 42 fits had a correlation coefficient lower
than 0.7), and Definity had a very high correlation (40 of 42 fits had a
correlation coefficient higher than 0.9). Myomap™ had a good corre-
lation with the exponential decay, but the six highest values of acoustic
pressures provided significantly different intercepts. The same applied
to 4 of 7 decay constants.
and, when most bubbles were destroyed, the frames
provided almost identical backscatter intensity to the
background (pure water).
The Quantison™ frames, however, were different
(Fig. 2b.2). The images showed that there were two
species of scatterers in the suspension, one whose back-
scatter was very large and another with relatively lower
backscatter. The appearance of two distinct species of
scatterers occurs at 0.83 MPa and all the higher acoustic
pressures. The images of Myomap™ did not differ sig-
nificantly from Quantison™, except that the lower back-
scattering species of contrast provided much higher
backscatter compared to the low-backscattering species
of Quantison™ (Fig. 2c.2). Videotapes with suspensions
of Quantison™ showed that almost all the bright species
of scatterers disappeared completely after one frame, and
the rest continued to form a uniform and low backscat-
tering suspension. Similar to that were the videotapes
recorded with Myomap™ suspensions. The variability
about the exponential fit was smaller for both Quanti-
son™ and Myomap™ at the two lowest acoustic pres-
sure settings, but the correlation to the exponential decay
was poor because there was negligible decay. All frames
were relatively homogeneous at the lowest acoustic pres-
sure (Fig. 2a.1, 2b.1, 2c.1). The video images further
confirm the uniform mixing of all the scatterers in the
suspension.
To compare the effect of the degassed and air-
saturated suspensions, a t-test with equal variance was
performed at each acoustic pressure for each agent. The
t-tests showed that the decays and intercepts were not
significantly different for Definity and Quantison™ for
 Contrast agent stability ● V. SBOROS et al.
Fig. 2. All the images refer to frames captured after 2 s of
insonation. Images (1) refer to the lowest acoustic pressure
applied to the agents (0.27 MPa), and (2) refer to the second
highest acoustic pressure (1.37 MPa). Images (a) refer to
Definity, (b) to Quantison™, and (c) to Myomap™.
the vast majority of acoustic pressures (Table 2). Myo-
map™ had significantly different intercepts for the six
highest acoustic pressures, and four significantly differ-
ent decays. Figure 3 illustrates the characteristics of the
data sets vs. the applied acoustic pressure. The solid lines
correspond to the air-saturated suspensions, and the
dashed lines to the degassed suspensions. The decay
constants are plotted in the left side (1) and the intercepts
in the right side (2) of Fig. 3. Both parameters were, in
general, lower at lower values of pO2. For all agents,
there was an increase in the decay constant with increas-
ing acoustic pressure and increasing pO2. The intercepts
increased with acoustic pressure only for Quantison™
and Myomap™ and, for Definity, there was a small
decrease.
For the captured data sets at 1.37 MPa, where the
insonation time was increased to 10 s, the average curve,
from three data sets for each agent, was produced to
smooth out the variability of individual data sets (Fig. 4).
An investigation followed to assess the variability within
each average curve. An exponential model was fitted to
1371
Fig. 3. Applying an exponential trend to each set of data
(normalised backscatter), the decay constant and the intercepts
were calculated (sets of graphs. 1 and 2, respectively). Decay
constants are measured in min⫺1 and intercepts, in normalised
backscatter. The points are connected with lines to make the
effect of different pO2 values and the relationship of the above
parameters with PNP more distinguishable. Solid lines-air-
saturated suspensions, dotted lines-degassed suspensions. In-
terpolation between points was performed only so that the
tendencies of each curve can be observed, especially when the
plots overlap. Definity, Quantison™, and Myomap™, are (a),
(b), and (c) pairs of graphs, respectively. The error bars dis-
play ⫾ SD of three experiments.
the initial part of the curve (first 6 –10 points), to the
remainder of the curve and, finally, to the whole curve.
The initial part of the curve was chosen to be the one that
fitted a significantly different exponential model from the
remainder of the curve. For Definity, the exponential
trend for insonation time up to 80 s (solid line in Fig. 4a)
provided a similar regression coefficient to that of the
trend of the whole curve (dotted line in the same figure).
Even though the trend for the first 80 s overestimated
both intercept and decay constant, the difference was not
significant.
Figure 4b and c shows the different behaviour of
Quantison™ and Myomap™. In addition to giving a
significant underestimation of the intercept and decay
constant, the exponential fit of the whole curve (dashed
line) in Fig. 4b also gave a poorer regression coefficient,
when compared with the trend of the first 80 s. The
exponential curve provided a satisfactory description of
1372 Ultrasound in Medicine and Biology
Fig. 4. Curve of backscatter intensity against total insonation
time produced by averaging three data sets at 1.37 MPa for the
10-s insonation time. (a) Definity, (b) Quantison™ and (c)
Myomap™. Solid line exponential trend of the initial 80 s of
insonation for (a) and (b) and for 60 s for (c); also, forecasting
the backscatter of the following time of insonation, is shown to
lie at lower values than those measured for Quantison™ and
Myomap™. Its equation and regression coefficient is in the
middle of each graph. Light grey lines-exponential trend for the
whole curve, its equation and regression coefficient is in the left
bottom corner of each graph. See text for further detail.
the behaviour of the agent for the first 80 s of insonation.
The same did not apply for the remainder of the in-
sonation time because the forecast of the exponential
trend (of the 80 s) for the rest of the data points lay at
lower values than actually measured. In this case, the
agent showed a departure from its initial behaviour. This
departure happened earlier for Myomap™ (Fig. 4c). The
agent seemed to alter its initial behaviour after the first
60 s of insonation.
Volume 27, Number 10, 2001
The definition of the overall backscatter (OB)
The behaviour of the decay constant and the inter-
cept of the decay curves provides useful information
about the behaviour of the agents at different acoustic
pressures. However, these parameters are not useful in
assessing the impact of the degassed suspensions on the
stability of the microbubbles. Table 2 showed that Myo-
map™, in a degassed suspension, has generally lower
backscatter levels than in the air-saturated ones. This
result contradicts the faster decay of air-saturated sus-
pensions. Quantison™ does not demonstrate signifi-
cantly different parameters between the two suspensions,
but Figs. 3b.1 and b.2 shows a similar tendency for lower
values for both parameters at degassed suspensions. Poor
correlation to exponential decay for the Quantison™
suspensions (Table 2) is responsible for the large error
bars shown in Fig. 3, and could probably be responsible
for nonsignificant differences between the degassed and
air-saturated suspensions. A new parameter that would
better demonstrate the difference between the degassed
and air-saturated suspensions should probably take into
account both the decay and intercept. The normalised
backscatter N(t) is assumed to follow the exponential
model as a function of time. Hence:
N共t兲 ⫽ ae ⫺bt, (1)
where a is the intercept and b is the decay constant
(min⫺1). The integral of normalised backscatter over
time (OB for overall backscatter) that represents the
normalised backscatter intensity capacity of a particular
suspension of scatterers would be:
OB ⫽ 冕0
⬁
N共t兲dt ⫽ 冕
0
⬁
a
ae ⫺btdt ⫽ ,
b
(2)
which is the ratio of the intercept to the decay constant.
Figure 5 plots this integral against PNP. Errors were not
plotted in Fig. 5, to make reading of the graphs easier.
There are several observations that can be made from
this figure. 1. The variability of each OB curve is signif-
icantly reduced compared to the variability of the decay
and intercept in Fig. 3.2. The comparative effect of decay
and backscatter of the agents on continuous imaging is
demonstrated. 3. The curves that corresponded to the two
different suspensions seemed to follow a similar pattern
for all agents.
A paired t-test was performed to evaluate differ-
ences in OB between the degassed and the air-saturated
suspension (dotted and solid lines, respectively, in Fig.
5). There was no difference at 5% significance level for
any of the pairs of lines. However, if the first two points
 Contrast agent stability ● V. SBOROS et al.
Fig. 5. The ratio intercept/decay plotted against PNP. Equation
(2) shows that it represents the OB. Dotted lines-low pO2
values (degassed suspension); solid ones lines high pO2 values
(air-saturated suspension). The ratios are not significantly dif-
ferent for the two pO2 levels for Definity, but they become
higher above 0.83 MPa for the high pO2 values for the other
two agents. (‚) ⫽ Definity; (586) ⫽ Myomap™; (x) ⫽ Quan-
tison™.
were omitted (i.e., the ratios that refer to the two lowest
acoustic pressures), the paired t-test showed that both
Myomap™ and Quantison™ had significantly lower
OBs for the degassed suspension (p⬃0.014 in both
cases). Definity demonstrated insignificant difference be-
tween the two pO2 values ( p ⫽ 0.1). A plot of the
difference of the OB, between the two suspensions,
against acoustic pressure was more illuminating (Fig. 6).
This difference correlated highly with acoustic pressure
above 0.83 MPa PNP, and was almost identical for both
agents.
DISCUSSION
Destruction of bubbles
The images in Fig. 2 gave some information on the
nature of these agents, but the observation of sequences
of frames recorded on videotape was valuable in under-
standing their behaviour. In Fig. 2, all the (l) images
referred to the lowest acoustic pressure, where the scat-
terers were homogeneously distributed. The differences
of backscatter of these agents at low acoustic pressure
have been discussed by Sboros et al (2000a), and will not
be included in this discussion.
Above 0.83 MPa PNP, it is suggested that the lower
backscattering species of scatterers, that set the “back-
ground” of the images, were the encapsulated bubbles
and remained so after insonation, and the Myomap™
bubbles are expected to have higher backscatter than the
smaller Quantison™ ones. Both materials are reported to
have robust shells, and the number of microbubbles
before and after insonation has been reported to be iden-
1373
Fig. 6. The difference of OB refers to the subtraction of the OB
of the air-saturated suspension from that of the degassed one.
The differences of OBs are plotted against peak negative pres-
sure for the Quantison™ and Myomap™. The two suspensions
provided significantly different OBs at 0.83 MPa and higher
acoustic pressures for the two agents. As shown in the graphs,
the differences of OBs at those pressures correlated highly with
acoustic pressure. Also, the slope and intercept of the resulting
regression were less than 10% different between the two
agents.
tical (Johnson 1997; Frinking et al. 1999), which implies
that the gas leaks or diffuses out of the shell when the
microbubbles are subjected to an US pulse. This robust-
ness restricts shell motion, which strongly suggests that
the lower backscattering species of scatterers for both
these agents has a behaviour close to that of linear
scatterers. For the same reason, it also seems reasonable,
at high acoustic pressures, that the bright species for both
agents were free bubbles that leaked from their coating.
The belief that these bright scatterers were free bubbles
is further supported by their disappearance after one
frame of insonation, which implies that they dissolve
fast, which is likely for a highly soluble free air bubble
(61 Hz frame rate; i.e., dissolution time ⬍ 16 ms).
Theoretical calculations and experiment agree with the
above, and have shown that Quantison™ bubbles have a
dissolution time of about 11 ms when exposed to 2-MHz
frequency (Frinking et al. 1999). Moreover, Frinking et
al. (1999) calculated that only 1% of the entire popula-
1374 Ultrasound in Medicine and Biology
tion of bubbles is releasing free bubbles that are able to
scatter US strongly and, therefore, also suggested the
existence of two different populations of bubbles.
The behaviour of Definity in the present work is in
agreement with theoretical calculations, as well as with
observations of other studies. Dayton et al. (1999) ob-
served that octafluoropropane remained a free bubble for
280 ⫾ 180 ms, and similar order dissolution times have
been measured for sparingly soluble bubbles by Ka-
balnov et al. (1998b) in vivo. Dayton and colleagues also
witnessed expansion (6% from bubble at rest) and con-
traction (9% from bubble at rest) for a phospholipid-
coated bubble (similar to the coating of Definity), which
did not occur for the albumin-coated Optison威 (Mallin-
crodt, St Louis, MO). The behaviour of Definity is uni-
form across the range of acoustic pressures used in this
study, and this is suggested to be related to the elasticity
of the coating that, perhaps, is slightly damping the bubble
oscillation. This does not rule out the possibility of free
bubble release at higher acoustic pressures, where the coat-
ing might not be able to withstand the oscillation amplitude
or might be directly destroyed by the ultrasonic field.
Figure 4 further illuminates the destruction mecha-
nism of the agents. Extended insonation time showed
that Definity bubbles have a uniform behaviour and the
exponential decay of the agent is consistent until the
backscatter reaches the background levels. This unifor-
mity is not followed by the other two agents (Fig. 4b and
c). The backscatter of both Quantison™ and Myomap™
did not reach the background levels in the course of the
experiment, which also expresses a departure of the
initial (80 s and 60 s, respectively) exponential decay
behaviour. The late slower-decaying batch of contrast
also included bright scatterers that disappeared in one
frame. This suggested that a proportion of the bubble
population that provided low backscatter levels released
the encapsulated gas after more than a single frame
exposure to US. This indicates that the coating of those
agents provided leaking sites or cracks for gas release
after being hit several times by the US wave. The two
exponential decays fitted in different parts of the data for
Quantison™ and Myomap™ belong to the different de-
cay behaviours of the two populations of bubbles present
in suspension. The decay constant of the initial part of
the curve is high and belongs to the bubbles, perhaps
initially with shell deficiency, that release their gas in one
frame. The decay constant of the latter part is small and
belongs to bubbles that have a firmer shell that requires
several hits from an US wave to provide the necessary
leaking site for release of a free bubble.
The overall backscatter (OB)
Vigorous mixing would increase the number of
fresh bubbles not previously in the field of view and, as
Volume 27, Number 10, 2001
a consequence, the number of bubbles likely to release a
free bubble. Morgan et al. (1998) have encountered a
similar situation with a flow phantom, and found that the
increase of flow rate increased the backscatter intensity
of contrast. Likewise, a lower frame rate would also
increase backscatter intensity. Therefore, the frame rate
and the mixing procedure determined the number of
particles that gave free bubbles for Quantison™ and
Myomap™. Taking into account that the mixing is not
perfectly homogeneous, the number of bubbles intro-
duced in the field-of-view (FOV) would vary signifi-
cantly from frame to frame. This, and the fact that the
backscatter of the bright scatterers was significantly
higher than that of the low backscattering ones, ex-
plained the poor correlation of Quantison™ with the
exponential decay (Fig. 1b and Table 2). The same
reasoning is justified by the only slightly improved cor-
relation of the Myomap™ decay curves (Fig. 1c and
Table 2), where the low backscattering scatterers have an
enhanced backscatter when compared to the Quantison™
ones.
Because the slice width of the ultrasonic field is not
infinitely small, the mixing is inadequate in keeping the
concentration of potentially free bubbles in the ultrasonic
field equal to that of the rest of the suspension. This is
explained by the fact that already insonated micro-
bubbles can appear in consecutive frames, and their
proportion in the FOV is different than the one in the rest
of the tank. It was apparent for the case of Quantison™
that the time between frames was sufficient to dissolve
the high backscattering bubbles (free bubbles as dis-
cussed above) in the scan plane, and that the scatterers
that would appear in the next frame were those intro-
duced into the scan plane by the mixing between two
frames (16 ms). At a specific mixing speed, larger time
intervals between frames (i.e., lower frame rate) would
allow more fresh microbubbles into the volume of the
FOV. A near inverse relationship, therefore, exists be-
tween the frame rate and the magnitude of the backscat-
ter intensity in each frame, and would be responsible for
an underestimation of the intercept (Fig. 3). Therefore, a
higher frame rate would decrease the amount of bright
scatterers and the intercept, which suggests also that the
intercept is no longer representative of the bubble con-
centration in suspension.
The above explanation is compatible with the be-
haviour of the decay constant, which is generally de-
creased for the degassed suspensions (Fig. 3b.1 and c.1).
Because the magnitude of the backscatter intensity is
decreased, and no longer is representative of the back-
scatter behaviour of the tank, it is expected subsequently
to affect the slope of the decay curve. The decay constant
is no longer representative of the decay of the agent. At
the six highest acoustic pressures used, the backscatter
 Contrast agent stability ● V. SBOROS et al.
intensities of the decay curves lie at significantly lower
values for the degassed suspensions, when compared
with the air-saturated ones, resulting to a less steep slope
for the curves of the degassed suspensions. The decay
constant is, therefore, smaller and, subsequently, mis-
leading for the behaviour of the agents.
Although Definity seemed different compared to the
other two agents, it is rather unlikely to be correct,
especially when the decay becomes larger, to assume that
the concentration of scatterers in the ultrasonic field was
equal to that of the rest of the suspension. Furthermore,
increasing acoustic pressure, which increases the decay,
would lead to an increasing underestimation of the above
intercept, which was probably the reason for the decreas-
ing intercept in the case of Definity (Fig. 3a.2). Hence,
the intercept was affected by the decay process, and the
decay constant was probably not accurately describing
the decay process of the contrast agents. This may be the
reason why, for both Quantison™ and Myomap™, both
parameter graphs followed similar relative behaviour
(i.e., both intercept and decay constant remained lower
for the degassed suspension) (Fig. 3b and c). For Myo-
map™, this was a significant effect for all apart from the
lowest acoustic pressure. The definition of the OB re-
solved these problems. The OB represents the nor-
malised backscatter intensity capacity of a particular
suspension of scatterers, and is proportional to the inter-
cept and inversely proportional to the decay constant,
eqn (2). A degassed suspension demonstrated a signifi-
cantly reduced OB above 0.83 MPa, for Quantison™ and
Myomap™. The reduced variability and similar behav-
iour of the pairs of the curves of OB against acoustic
pressure for each agent further justified its choice, and
shows that the decay constant and the intercept were not
independent of each other and were not the physical
quantities that assess the decay and the representative
backscatter for the tank, respectively.
Figure 5 was much more conclusive of the behav-
iour of the different agents, compared with Fig. 3. The
decay of Quantison™ at very low acoustic pressures was
negligible and, therefore, the OB would be large. The
increase of acoustic pressure increased dramatically the
decay of Quantison™ (Fig. 3b.1), which results in the
decrease of the OB. However, the largely enhanced nor-
malised backscatter of the Quantison™ (Fig. 3b.2) with
increasing acoustic pressure (i.e., appearance of free
bubbles) was the reason for the increase of the OB after
0.83 MPa PNP seen in Fig. 5. For Myomap™, this
enhancement was less pronounced (Fig. 5) and the gra-
dient of OB increase (vs. acoustic pressure) was lower.
As discussed above, the intercept is expected to be un-
derestimated and was probably the reason that Definity
exhibits a decrease in the OB with the increase of acous-
tic pressure.
1375
The calculation of OB for the different parts of Fig.
4 for Quantison™ and Myomap™ can also provide
interesting information. The ratios of OB of the later part
of the curve to the initial one were 0.48 and 0.19,
respectively. Knowing that the bubbles of these agents
dissolve in one frame, these ratios can stand as a crude
comparison of the populations of bubbles that corre-
spond to the different parts of the curves. Hence, the
bubbles’ shells that take longer to break are approxi-
mately half those that release a free bubble in one frame
for Quantison™. For Myomap™, the bubbles that re-
lease a free bubble in one frame are 5 times more than the
ones that need more than a frame to be released. The
assessment of different populations of bubbles with the
above approach is limited, due to the background popu-
lation of lower backscattering bubbles that add to the OB
and distort the ratio. Therefore, the above results have
only a qualitative sense. These results, however, do not
contradict those of Frinking et al. (1999), who calculated
the free bubble population to be about 1% of the entire
population of bubbles. It is speculated that a large pro-
portion of the Quantison™ and Myomap™ bubbles are
not containing gas even in the vial environment, and do
not provide the potential of free bubble formation.
Acoustic pressure dependence of backscatter
Definity demonstrated a linear increase in backscat-
ter intensity with increasing incident intensity that was
illustrated by a negligible decrease of normalised back-
scatter with acoustic pressure (Fig. 3a.2). This near in-
dependent behaviour of the normalised backscatter with
acoustic pressure suggests that Definity scatterers have a
scattering cross-section independent of acoustic pressure.
This is linear scatterer behaviour, like the ones used for
normalising, and is also encountered with contrast agents
at very low acoustic pressure fields (Chang et al. 1996).
As discussed above, the intercept did not provide an
accurate measure of the backscatter of each agent. Strong
suggestion of an increasing underestimation of the inter-
cept with the increase of acoustic pressure is produced by
the increase of decay. A nonlinear increase is, perhaps,
expected for the backscatter intensity of Definity with the
increase of transmitted intensity.
The appearance of free bubbles for Quantison™ at
0.83 MPa PNP coincided with an increase of both inter-
cept and OB with acoustic pressure (Figs. 3b.2 and 5).
Myomap™ showed similar behaviour, but less pro-
nounced (Figs. 3c.2 and 5). Both agents demonstrated
increasing decay with acoustic pressure, but the increase
of OB above 0.83 MPa PNP with acoustic pressure
suggested a higher-order nonlinear dependence than
Definity (speculated order ⬎ 1) of both agents’ scattering
cross-sections. Frinking and de Jong (1997) speculated
that the release of free bubbles is the cause of the
1376 Ultrasound in Medicine and Biology
transient effect unique to Quantison™ and Myomap™. It
is, however, somehow strange that gas is only allowed
out of the shell due to some rupture or leakage at high
acoustic pressures. It might be the case that a rupture in
a shell is not a structural imperfection, but damage
caused by exposure to US. Defects of the albumin shell
of Optison威 that was followed by a free gas bubble
release has been demonstrated microscopically when the
bubbles were subjected to US at 2.25-MHz transmit
frequency (Dayton et al. 1999). If this occurs, then the
frequency of ruptures among the population of bubbles
would probably be dependent on the amplitude of the
incident wave. It seems logical to hypothesise that in-
crease of acoustic pressure would increase the proportion
of free bubbles in a Quantison™ or a Myomap™ sus-
pension. That way, the enhancement was not only due to
the free bubbles, but also to the increasing population of
them with the increase of acoustic pressure. However,
although our observations are consistent with this theory,
there is no direct evidence of such an occurrence. Further
investigation is required to prove such a property for the
two agents.
Stability in degassed and air-saturated suspensions
Previous studies have demonstrated the high solu-
bility of air-based contrast agents compared with per-
fluorocarbon-based ones (Sboros et al. 2000a; Basude et
al 2000). It has been difficult, however, to isolate the
contribution of the shell to the dissolution of the gas of
contrast bubbles. Quantison™ and Myomap™, although
air-based agents, have demonstrated nonsignificant dif-
ferences in backscatter decay of degassed suspensions
compared with air-saturated ones, which can be attrib-
uted to the thick and probably unpermeable shell (Sboros
et al. 2000a).
The air from Myomap™ and Quantison™ dissolved
more readily in the degassed suspension triggered by the
ultrasonic field above 0.83 MPa PNP. The difference of
OBs between the two suspensions, plotted in Fig. 6
against acoustic pressure, correlated significantly with
acoustic pressure above 0.83 MPa PNP (r ⫽ 0.99 for
both agents). The propagated error was high due to the
small sample size, but the differences also had almost
identical linear fits for the two agents (⬍ 10% difference
in slope or intercept in the graph). This similarity sug-
gests that Quantison™ and Myomap™ had identical
dissolution behaviour. It is further suggested that the
dissolution time reduced with increasing acoustic pres-
sure. However, the difference in OB for the two agents
occurred only when free bubbles were present. If the
above hypothesis of free-bubble concentration depen-
dence on acoustic pressure is correct, then the dissolution
time could be independent of acoustic pressure. Hence,
the dependence of the difference of OB between the two
Volume 27, Number 10, 2001
suspensions on acoustic pressure could be related to the
dependence of the number of free bubbles on acoustic
pressure.
SUMMARY
The present study concluded that continuous imag-
ing is of value in assessing the properties of contrast
agents. Even though the decay constant and associated
intercept were proven not to have a solid physical mean-
ing in the experimental setup described herein, the def-
inition of the integral of normalised backscatter over
time (OB) showed potential in assessing subtle proper-
ties of the different agents using continuous imaging.
The contributions of the dissolved gas concentration in
suspension and the acoustic pressure on the stability of
the agents were clarified. It was shown that Quantison™
and Myomap™ release free bubbles at high acoustic
pressure, that also dissolve faster in degassed suspen-
sions. The destruction of bubbles was dependent on
acoustic pressure. The scattering cross-sections of all
agents were also related to acoustic pressure, but differ-
ences between Definity and the other two agents were
strongly suggestive of a dependence between free-bubble
formation and acoustic pressure for Quantison™ and
Myomap™. Further in vitro investigation should be per-
formed at the microscopic level to assess this. It was
shown that a degassed suspension accelerated the disso-
lution of free bubbles in a reproducible manner.
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