Ultrasound Imaging
(Contrast Agents and Therapy)
MEng in Bioinstrumentation
Why Ultrasound?
Over half a century old technique!
Arguably the most widely used imaging technologies in
medicine.
Portable, free of radiation risk, and relatively inexpensive
compared to MRI, CT and PET
Tomographic, i.e., offering a “cross-sectional” view of
anatomical structures.
“Real time,”- providing visual guidance for interventional
procedures
Do you expect any
similarities?
Most amazing is that sound can actually help us to see what is
hidden, just like the way bats 'see'.
Bats always have the night shift. They go hunting for things to eat at
night where food isn't well lit.
Fortunately, bats are gifted with a system of locating things with
sound. First they emit sound.
• The human ear cannot hear below 20 Hz.
• Elephants can use infra sound.
• The human ear cannot hear above 20,000 Hz.
• Bats use ultrasound to locate food.
• Dolphins use it to communicate.
Ultrasound used in medical imaging operate at frequencies way
above human hearing: about 2 million Hz - 20 million Hz (2-20 MHz).
Sound travels in waves. Ultrasound physics has to do
with the higher frequencies of sound.
Human hearing is from about 20 cycles per second or
20HZ (a low hum) to about 20,000 cycles per second
or 20KHZ.
A grasshopper sends out sound
waves at 40KHZ. A dog can hear at
about 30KHZ and bats send chirps
and listens for the echoes at 100KHZ.
Properties of Sound Waves
Amplitude
Crest
Trough
wavelength
•
•
•
•
Frequency
Velocity
Wavelength
Amplitude
Pressure
High Frequency Wave
• Units to describe frequency:
Hertz= 1 cycle in one sec
kHz= 1000 Hz= 1000 cycles per sec
MHz= 1000000 Hertz
US imaging frequency range: 2-12
MHz
Time
Pressure
Period
Low Frequency Wave
Time
Period
• The number of cycles occurring in one sec of time (cycles per sec)
• The high frequency wave sounds higher than the low freq wave
http://www.genesis-ultrasound.com/Ultrasound-physics-2.html
Wavelength
• Length of space over which one cycle occurs (distance)
wavelength
Distance
wavelength
Distance
• Given a constant velocity, as frequency increases
wavelength decreases (V=  x f)
• Common US frequencies and wavelengths
-2.25MHz = 0.6 microns
-5.0 MHz = 0.31 microns
-10.0 MHz = 0.15 microns
Ultrasound Wavelength and Frequency
• High frequency US waves High axial resolution  More
attenuation  Superficial structure
• Low frequency US waves Lower resolution  Less degree
attenuation  Deeper penetration
• High frequency transducers (10-15
MHz) to image superficial structures
(e.g. stellate ganglion blocks)
• Low frequency transducers (2-5
MHz) to image the lumbar neuraxial
structure
Higher frequency waves are more highly attenuated than lower
frequency waves at a given distance
Velocity
• Average speed of US in the human body is 1540 m/sec
• Directly related to the stiffness of media
• Inversely related to the density of media
• Slowest in air/gasses
• fastest in solids
c=×f
=c/f
Medium Velocity (m/sec)
-------------------------------------------Air
330
Fat
1450
Water
1480
Soft tissue
1540
Blood
1570
Muscle
1580
Bone
4080
Amplitude
• The strength/intensity of the sound wave at any given
point in time
• Represented by the height of the wave
• Amplitude/intensity decreases with increasing depth
• Magnitude of the pressure changes along the sound
wave
• Power: rate at which energy is transferred from a sound beamproportional to the amplitude squared
• Intensity (Watts/cm2) is the concentration of energy in a sound beam
Attenuation Coefficient
8 MHz
10MHz
12MHz
The ultrasound amplitude decreases in certain media as a
function of ultrasound frequency (attenuation coefficient)
ScN-Sciatic nerve, PA - Popliteal artery.
Practical consequence of attenuation: the penetration decreases as frequency increases
8 MHz
10MHz
12MHz
A 0.5-mmdiameter
object
• Ultrasound frequency affects the resolution of the imaged
object.
• Resolution can be improved by increasing frequency and
reducing the beam width by focusing.
For a constant acoustic velocity, higher frequency US can detect smaller objects and provide
a better resolution image.
Spatial Resolution
Axial and Lateral. Axial resolution is the minimum
separation of above-below planes along the beam
axis.
It is determined by spatial pulse length, which is equal
to the product of wavelength and the number of
cycles within a pulse.
Axial resolution = wavelength (λ) × number of cycle per
pulse (n) ÷ 2
Common Frequencies for Clinical US
Dystrophic calcification of the choroids
Portal Vein Ultrasound
Ablative therapy
MRI of a large tumor in the left kidney (L) and 12 days following HIFU treatment (R).
Color Doppler imaging shows a thrombus in upper
PV moderately dilated (14.5 mm) with
splenomegaly: Cirrhosis with PV thrombosis.
Dr. Karl Theo Dussik, an Austrian neurologist, was the first to apply
US to image the brain.
Ultrasound in Med. & Biol., Vol. 30, No. 12, pp. 1565 - 1644, 2004
T1: ultrasonic generator, Q1: transmitter, Q2: receiver, T2:
converter amplifier, W: water bath, L: light, P: photographic/
heat-sensitive paper
Ideal Characteristics of an Ultrasound Probe
• High echogenicity
• Low attenuation
• Low blood solubility
• Low diffusivity
• Ability to traverse
pulmonary system
• Lack of biological effects in
repeat exposures
Contrast Agents and Their Distribution
Relative to the Vascular Endothelial Cells
Diagram illustrating development stage of microbubbles, nanobubbles, and nanodroplets
for diagnostic and therapeutic purposes. HIFU = high-intensity focused ultrasound; KDR =
kinase domain receptor.
Microbubbles Response to US
Microbubbles volumetrically oscillate due to continuous changes in the external
pressure caused by an acoustic sound wave.
At high pressure amplitudes, microbubbles collapse violently, producing water
jetting and shock waves, and other inertial phenomena.
Lindner JR. Microbubles in medical imaging: current applications and future directions. Nat Rev Drug Discov. 2004 Jun;3(6):527-32.
A slow motion capture of interial
cavitations of micro bubbles
Contrast harmonic image of liver in vivo
Right frame: Image using both tissue and contrast agent
fundamental frequency backscatter
Left frame: the fundamental tissue signal has been filtered leaving
the higher harmonic signal corresponding only to the contrast agent
and, subsequently, the blood flow of the liver.
Acoustic Properties of Microbubbles
Lindner JR. Microbubles in medical imaging: current applications and future directions. Nat Rev Drug Discov. 2004 Jun;3(6):527-32.
Non Linear Response of the Microbubble
Microbubble Contrast Agents
Name
Manufacturer
Shell
Gas
Mean Size
(µm)
Albunex
Molecular
Biosystems
Albumin
Air
4.3
Optison
Mallinckrodt/
Amersham
Albumin
Octafluoropropane
2-4.5
Definity
Bristol-Myers
Squibb
Lipid/surfactant
Octafluoropropane
1.1-3.3
Imagent
Imacor
Lipid/surfactant
N2 /
perfluorohexane
6.0
Sonovue
Bracco
Diagnostics
Lipid
Sulphur hexafluoride
2-3
Levovist
Schering AG
Lipid/Galactose
Air
2-4
Ultrasonic Molecular Imaging of Tumor
Angiogenesis in Breast Cancer
Orthotopically implanted human
Bubbles coalescing in the breast adenocarcinoma xenograft
extravascular space become (MDA-MB-231 cells) in nude mouse
following
intravenous
detectable by US due to their imaged
7 VEGFR2administration
of
5×10
micron sized collections
targeted probe. Scale bar =3 mm
Albunex
• Air-filled sonicated albumin MBs (MBI, San Diego,
California)
• 1994: First ultrasound CA approved for use in US
• Albumin shell designed to prevent outward diffusion
of air from MBs
• Substantial loss of gas volume occurred during transit
to systemic circulation following intravenous injection
• Markedly decreased contrast enhancement and short
duration of clinically useful contrast
Newer Agents
• Newer agents designed to improve intravascular
stability
• Modifications in shell and gas content
• “Air-tight” polymer shells or lipid-galactose stabilized
shells designed to minimize outward diffusion of gas
• Use of gases less prone to outward diffusion than air
• Inert high-molecular-mass gases with low diffusion
coefficients and low solubility in water (low Ostwald
coefficient); result in prolonged lifespan of MBs
OptisonTM
• Perflutren Protein-Type A
Injectable Microspheres
• GE Healthcare,
Buckinhamshire, UK
Structural Formula
• Octafluoropropane
• Manufacturer has voluntarily
suspended marketing since
2005
http://www.amershamhealth-us.com/optison/monograph/om03-02.html
Optison with RBCs
Definity
• FDA approval in 2001
• Bristol-Myers Squibb Medical Imaging, Billerica, MA
• $65 million in sales in 2006
• More than 2 million patients dosed
How Do We Make These Bubbles?
SEM photograph of MBs (50 000×)
Subcutaneous human ovarian adenocarcinoma xenograft tumor
(arrows) imaged with targeted contrast-enhanced ultrasound
imaging after intravenous injection of integrin-targeted (A) and MB(control) (B). Imaging signal obtained after MB-targeted injection
was substantially higher than that obtained after control injection.
MBs Loaded with Chemotherapeutics
Docetaxel
The prepared nanoparticles in vitro released the drug
steadily for 72 hours without occurrence of burst release.
Influence of ultrasound exposure on tumor
temperature
After ultrasound exposure, the tumor temperature
increased steadily in a time dependent manner.
The growth curve of hepatocellular carcinoma xenografts.The tumor volume increased
persistently in model group, while in all the docetaxel treated groups, the tumor volume
increment was inhibited, especially in the GDN+UE group.
Laplace Pressure
The Laplace pressure is the pressure difference between the inside and
the outside of a curved surface. The pressure difference is caused by
the surface tension of the interface between liquid and gas.
The Laplace pressure is determined from the Young–Laplace
equation given as
where R1
tension.
and R2
are the radii of curvature and  is the surface
Bubble diameter
(2r) (µm)
(Pa)
(atm)
1000
288
0.00284
3.0
96000
0.947
0.3
960000
9.474
Phase-change contrast agents (PCCAs)
Consist of perfluorocarbons (PFCs) that are initially in
liquid form, but can then be vaporized with acoustic
energy.
• Exposing pre-formed PFC micro bubbles to decreased ambient temperature and
increased ambient pressure results in condensation of the gaseous core.
• The decreased size results in an increased Laplace pressure, which serves to preserve the
particle in the
Phase-change contrast agents (PCCAs)
Exposing pre-formed PFC micro bubbles to decreased ambient temperature
and increased ambient pressure results in condensation of the gaseous core.
The decreased size results in an increased Laplace pressure, which serves to
preserve the particle in the
PCCAs take advantage of the volumetric increase that occurs during
vaporization.
Microscale droplets limited to flow in the vascular space have been proposed
for a number of techniques, including temporary occlusion of blood vessels
and enhancing cavitation activity.
PCCAs manufactured with diameters in the 100-nm range may be able to
diffuse out by EPR before phase-transition
Contrast-enhanced extravascular imaging, cell-specific targeting, drug delivery,
and therapy via ultrasound.
A microscale droplet of MBs exposed to a 0.25 µs
pulse at approximately 0.55 MPa vaporizes to form
a gas bubble approximately 5-fold larger
Bright field (a, c) and fluorescence (b, d) microscopy illustrating that the shell is preserved
when DiI-labeled DFB microbubbles (a, b) are condensed to the liquid state (c, d), the shell
is preserved through the change in volume.
Therapy with PCCA
Schematic illustration of drug transfer from nanodroplets through
microbubbles into cells under the action of ultrasound.
PFP/PEG-PLLA microbubbles in a plasma clot before (A) and after sonication for 1 min by 1MHz, 3.4 W/cm2 (B) and 90-kHz, 2.8 W/cm2 ultrasound (C) at room temperature.
Fig. 2A. PEG-PCL nanodroplets inserted in PBS through a 18 G needle (left) or 26 G needle
(right). Bubbles formed when nanoemulsion is injected through a thin needle are visualized
as bright spots (indicated by arrows in the right panel); bubbles rise to the sample surface
while droplets precipitate to the bottom of a test tube.
Fig. 2B. PEG-PCL nanodroplets injected in the agarose gel through a 18 G (left) or 26 G
(right) needle. Injection through the thin needle results in immediate formation of very
bright bubbles whose size and brightness increase with time; the brightness of the droplets
also increases with time suggesting a post-injection droplet-to-bubble transition.
Photographs of a mouse bearing two ovarian carcinoma tumors (A) - immediately before
and (B) - three weeks after the treatment; a mouse was treated by four systemic injections
of nanodroplet-encapsulated PTX, nbGEN (20 mg/kg as PTX) given twice weekly; the right
tumor was sonicated by 1-MHz CW ultrasound (nominal output power density 3.4 W/cm2,
exposure duration 1 min) delivered 4 hours after the injection of the drug formulation.
Ultrasound was delivered through a water bag coupled to a transducer and mouse skin by
Aquasonic coupling gel.
Effective regression of the ovarian carcinoma tumor treated by nbGEN/ultrasound
combination as described in Materials and Methods. The first photograph was taken before
the start of the treatment, the second – two weeks later, i.e. immediately after the last
treatment of the first treatment round. The third photograph was taken one week after the
completion of the first treatment round.
Fig. 6B. Normalized tumor growth/regression curve for the mouse presented in Figure 6A.
Bubble-mediated Gene Delivery
US and Bubble-mediated Drug Delivery
Through BBB
Ultrasound and pH Dually Responsive
Polymer Vesicles for Anticancer Drug Delivery
Upon ultrasound radiation, the disruption and re-self-assembly lead to smaller vesicle. Upon decreasing the solution pH,
the shrinkage of vesicles by the recrystallization of PTMA chains surpasses the swelling of vesicles by the partial
protonation of DEA, leading to smaller vesicles. Further decreasing the solution pH will lead to the complete protonation
of DEA and finally the disassembly of vesicles. Both ultrasound radiation and decreasing pH can lead to faster drug
release.
A) Recanalization was observed in angiograms taken post-HIFU treatment. 2/4 (50%) of
animals treated with 415 W power, exhibited recanalization when compared to poststroke angiograms. There was no damage or leakage from the vessel apparent on the
angiogram. B) Post-treatment H&E showed no evidence of tissue damage or hemorrhage
due to HIFU in the region of sonication or in the surrounding brain tissue. Scale bar = 500
µm. C) Prussian blue staining was used to detect small fragments of the disrupted ironloaded blood clots. doi:10.1371/journal.pone.0042311.g003
Intravital fluorescence
images of subcutaneous
pancreatic tumors before
and after FUS treatment
(A) and (B) – two coronal slices in the 19F MR images superimposed on the low resolution proton anatomic images of a
pancreatic tumor bearing mouse. A 2% PFCE nanoemulsion stabilized with PEG-PCL copolymer was systemically injected
every two hours, 200 μl each. Four injections were given, for a total of the PFCE dose of 2 mmol/kg. Images were recorded
an hour after the last nanodroplet injection (seven hours after the start). (C)- Multiple liver metastases (arrows) are
revealed at the necropsy of the mouse (indicated by long thing arrows). Transparent large tumor is indicated by a thick
arrow. Organs could be displaced at necropsy.
Dramatic regression of a breast cancer MDA MB231 tumor
treated by four systemic injections of paclitaxel loaded 1%
PFCE/0.25% PEG-PCL nanoemulsion and focused 1-MHz
CW ultrasound applied for 60 s
FDA “Black Box” Warning
• Issued on October 10, 2007
• Post-marketing reports of 11 deaths 1-12 hours following
administration of perflutren-based contrast agents
• 10 patient deaths following Definity injection and 1 death
following Optison injection
• 4 patient deaths temporally related to contrast injection
• Perflutren-based compounds contraindicated for use in patients
with:
1. Acute coronary syndromes
2. Acute myocardial infarction
3. Worsening or clinically unstable heart failure
http://www.fda.gov/cder/drug/InfoSheets/HCP/microbubbleHCP.htm
Safety Data
Adverse
Experience
Placebo
(n=42)
5 ul/kg
(n=85)
10ul/kg
(n=85)
All Perflutren
(n=169)
Headache
3 (7%)
4 (5%)
5 (6%)
9 (5%)
Dizziness
1 (2%)
2 (2%)
1 (1%)
3 (2%)
Back Pain
0
0
3 (4%)
3 (2%)
Nausea
0
2 (2%)
1 (1%)
3 (2%)
Flushing
0
0
2 (2%)
2 (1%)
Chest pain
0
0
2 (2%)
2 (1%)
Pruritis
0
1 (1%)
1 (1%)
2 (1%)
Rash
0
1 (1%)
1 (1%)
2 (1%)
Sweating
0
1 (1%)
1 (1%)
2 (1%)
IV Site Pain
1 (2%)
1 (1%)
0
1 (1%)
Fatigue
1 (2%)
1 (1%)
0
1 (1%)
Kitzman DW, Goldman ME, Gilliam LD, Cohen JL, Aurigemma GP, Gottdiener JS. Efficacy and safety of the novel ultrasound contrast agent perflutren
(definity) in patients with suboptimal baseline left ventricular echocardiographic images. Am J Cardiol. 2000 Sep 15;86(6):669-74.
Safety Data
• No clinically significant change in physical examination, vital signs,
electrocardiographic tracings, or chemistry or hematology
laboratory values
• Adverse event rates similar across treatment groups
• 30 of 169 patients (18%) in the combined perflutren-treated group
(15% in the 5 ml/kg group and 20% in the 10 ml/kg group)
• 6 of 42 placebo-treated patients (14%)
• Headache was most frequently reported adverse event (9 of 169
patients who received perflutren (5%) and 3 of 42 patients who
received placebo (7%)
Kitzman DW, Goldman ME, Gilliam LD, Cohen JL, Aurigemma GP, Gottdiener JS. Efficacy and safety of the novel ultrasound contrast agent perflutren
(definity) in patients with suboptimal baseline left ventricular echocardiographic images. Am J Cardiol. 2000 Sep 15;86(6):669-74.
“Pseudocomplication”
• Main ML, Goldman JH, and Grayburn PA. Thinking outside the
“Box”—the ultrasound contrast controversy. J Am Coll Cardiol
2007; 50 (25): 2434-2437.
• Complications occurring after a medical procedure may be due to
either the procedure itself or due to progression of the underlying
disease state
• Major cardiovascular events are more likely to occur in patients
who are “ill enough” to require diagnostic testing
• Echocardiography often the test of choice (or the only test
available) for critically ill patients (shock, hypotension, tamponade,
etc.)
• Association of adverse events following contrast administration
does not establish causality