• Non ci sono risultati.

UNIVERSITY OF PISA FACULTY OF ENGINEERING

N/A
N/A
Protected

Academic year: 2021

Condividi "UNIVERSITY OF PISA FACULTY OF ENGINEERING"

Copied!
28
0
0

Testo completo

(1)

UNIVERSITY OF PISA

FACULTY OF ENGINEERING

DEPARTMENT OF ELECTRICAL SYSTEMS AND AUTOMATION

RESEARCH DOCTORATE IN

AUTOMATION, ROBOTICS AND BIOENGINEERING

DOCTORAL THESIS

A

COUSTIC

C

HARACTERIZATION OF

U

LTRASOUND

C

ONTRAST

A

GENTS FOR

A

DVANCED

B

IOMEDICAL

I

MAGING

TUTORS:

Chiar.mo Prof. LUIGI LANDINI

Dr. Ing. SERGIO CASCIARO

CANDIDATE:

Ing. FRANCESCO CONVERSANO

(2)
(3)

To My Family

and

To My Best Friends

(4)
(5)

Contents

Acknowledgements ix

Symbols and abbreviations xi

Abstract xiii

1. Introduction.. . . 1

1.1 Medical ultrasound imaging and contrast agents.. . . 1

1.2 Historical development of contrast echography . . . 2

1.3 Current and future applications of ultrasound contrast agents. . 5

1.3.1 Cardiac imaging. . . 5

1.3.2 Vascular imaging.. . . 6

1.3.3 Solid-organ imaging... . . . 7

1.3.4 Molecular imaging and therapeutic applications. . . 8

1.4 Aims of this study... . . . ... . 8

1.5 Thesis organization.. . . 10

PART I: ULTRASOUND IMAGING AND CONTRAST AGENTS 2. Ultrasound imaging principles . . . 15

2.1 Ultrasound generation and propagation... . . .. . 15

2.2 Echographic image reconstruction. . . 19

2.2.1 A-mode analysis ... . . 19

2.2.2 B-mode analysis and transducer technologies . . . 20

2.2.3 Image resolution.. . . 21

3. Contrast agent principles....... . . 23

3.1 Physical phenomena involved in ultrasound-contrast agent interaction . . . 23

3.2 Theoretical modelling of microbubble behavior... . . . 24

3.2.1 Introduction.... . . 24

(6)

Contents

3.2.2 The scattering approach . . . 25

3.2.3 The bubble dynamics approach.. . . 27

3.2.4 Scattering, absorption and attenuation. . . 31

3.3 Echographic modes for contrast agent detection... . . . . ... 34

3.3.1 Fundamental B-mode imaging. . . .. 35

3.3.2 Harmonic B-mode imaging... . . . 35

3.3.3 Harmonic power Doppler imaging.... . . 36

3.3.4 Pulse-inversion imaging.. . . 37

3.3.5 Transient imaging techniques.... . . 37

3.3.6 Subharmonic imaging... . . . 39

PART II: PHANTOM DESIGN AND PRODUCTION 4. Tissue-mimicking material characterization..... . . 43

Abstract... . . 43

4.1 Introduction.... . . 44

4.2 Hydrogel synthesis and characterization... . . . 45

4.2.1 Hydrogel sample preparation. . . 45

4.2.2 Pig liver sample preparation.... . . 46

4.2.3 Ultrasound analysis and hydrogel selection.. . . 46

4.3 Nonlinear behavior investigation... . . . 47

4.3.1 Experimental set-up and measurement procedure . . . 47

4.3.2 Effect of mechanical index... . . 49

4.3.3 Effect of penetration depth.. . . 51

4.4 Conclusions.... . . 53

5. Sound-absorbent material selection.. . . 55

Abstract... . . 55

5.1 Introduction.... . . 56

5.2 Materials and methods... . . . 56

5.2.1 Data acquisition.. . . 56

5.2.2 Off-line analysis. . . 57

5.3 Results.... . . 59

5.4 Conclusions.... . . 60

6. Phantom production and flow circuit construction . . . 61

Abstract... . . . 61

6.1 Preliminary phantom fabrication and testing... . . 61

6.2 Final phantom production... . . 62

6.3 Pump selection and flow circuit optimization... . . . 64

PART III: MICROBUBBLES IN FLOWING CONDITIONS 7. Effect of mechanical index on backscattered spectrum. . . 69

Abstract... . . . 69 vi

(7)

Contents

7.1 Introduction.... . . 69

7.2 Materials and methods... . . . 70

7.2.1 Contrast agent.... . . 70

7.2.2 Experimental set-up and data acquisition.... . . 71

7.2.3 Off-line analysis. . . 71

7.3 Results.... . . 73

7.4 Discussion... . . 75

7.5 Conclusions.... . . 76

8. Effect of contrast agent concentration.. . . 79

Abstract... . . . 79

8.1 Introduction.... . . 80

8.2 Materials and methods .... . . 80

8.2.1 Contrast agent preparation and data acquisition... . . . 80

8.2.2 Off-line analysis. . . 81

8.3 Results.... . . 82

8.4 Discussion... . . 83

8.4.1 Linearity between incident power and backscatter amplitude. . . 83

8.4.2 Contrast agent concentration effect.. . . 84

8.5 Conclusions.... . . 85

PART IV: MICROBUBBLES IN STATIC CONDITIONS 9. Optimizing imaging parameters... . . 89

Abstract... . . . 89

9.1 Introduction.... . . 90

9.2 Materials and methods... . . . 90

9.2.1 Experimental set-up. . . 90

9.2.2 Acquisition system. . . 91

9.2.3 Measurement procedure and data acquisition... . . . 93

9.2.4 Off-line analysis. . . 93

9.3 Results and discussion... . . . 95

10. Microbubble nonlinear oscillation threshold....... . . 97

Abstract... . . . 97

10.1 Introduction.. . . 97

10.2 Materials and methods.. . . 98

10.2.1 Experimental set-up. . . 98

10.2.2 Contrast agent preparation . . . 99

10.2.3 Granulometric measurement procedure. . . 100

10.2.4 Ultrasound parameter settings and attenuation measurements.. .101

10.2.5 Data analysis... . . . 102

10.3 Results and discussion. . . 102

10.4 Conclusions.. . . 105

(8)

Contents

11. Microbubble destruction mechanisms.. . . 107

Abstract... . . . 107

11.1 Introduction.. . . 108

11.2 Materials and methods.. . . 108

11.3 Results... . . . 109

11.4 Discussion... . . . 111

11.5 Modelling acoustical behavior of BR14 microbubbles . . . 115

11.6 Conclusions.. . . 117

12. General conclusions and future perspectives....... . . 119

12.1 Summary and conclusions. . . 119

12.2 Future developments related to this thesis... . . 120

References... . . 123

List of publications..... . . 143

(9)

Acknowledgements

I would like to thank my supervisors: Prof. Luigi Landini, for the support and the advices he has provided me throughout the duration of my Ph.D. course, and Dr. Ing. Sergio Casciaro, for his irreplaceable daily guidance and encouragement, starting from first definitions about ultrasound signals until last revisions of this thesis.

I am also deeply grateful to Prof. Alessandro Distante, for introducing me to this exciting research field, and to all his collaborators that welcomed me, both at ISBEM (Euro-Mediterranean Scientific Biomedical Institute, Brindisi) and at the Institute of Clinical Physiology (IFC-CNR, Lecce).

In this context, a particular thanks goes to all the members of the Bioengineering Division, directed by Sergio Casciaro, for their helpful collaboration and generous friendship. I would like to acknowledge in a special way the other Ph.D. students of the Ultrasound Laboratory (Rosa Palmizio Errico, Christian Demitri and Graziano Palma), with whom I have worked over the past three years, and some undergraduate engineering students (Luigi Ostuni, Serena Lettera and Angelo Mesagne), that contributed to this work during their Bachelor theses. I am grateful to ISBEM and IFC-CNR Lecce also for their funding and I wish to show my appreciation to the Doctorate Council and to its Coordinator, Prof. Mario Innocenti, for giving me the opportunity of doing my research activities in Puglia. Nevertheless, I had several occasions to meet my colleagues of the Electrical Systems and Automation Department in Pisa: I would like to thank all of them for the time they spent with me, and in particular I am very grateful to Roberto Mati, who has become a close friend of mine.

Then, I would like to express my gratitude to all those persons with whom I had very useful discussions about single topics treated in this thesis: Prof. Alfonso Maffezzoli and Dr. Alessandro Sannino, for providing valuable suggestions and guidance in the hydrogel synthesis and preparation; Giuseppe Loliva, for his help concerning the employment of ESAOTE ultrasound transducers; Bracco Research SA

(10)

Acknowledgements

(Geneva, Switzerland) for providing the contrast agent vials and, in particular, Dr. Marcel Arditi and Dr. Eric Allemann for their fruitful comments and support; Rodolfo Facchini and Erica Magrini, for their kindness in teaching me the basic concepts about the use of FEMMINA platform and FortezzaTM software; Dr. Lars Hoff, for his helpful suggestions in the field of ultrasound signal processing; Prof. Nico de Jong, who has welcomed me in three editions of the European Symposium on Ultrasound Contrast Imaging he organized in Rotterdam, and also showed me several interesting perspectives involving my research work.

Again a very special thanks goes to Prof. Distante and to Sergio Casciaro for the important chance they gave me in October 2005, when I had the honour of representing our research group at the “20th Advances in Contrast Ultrasound Conference” (Chicago, IL), where we had four posters that were awarded with the First Prize Poster Context. Finally, last but never least, my deepest thanks is for those persons that have always believed in me, being my stable reference points and my energy source: my parents, Antonio and Gemma, that have constantly provided me with every kind of support and encouragement; my grandmother Giulia and my brothers Roberto, Stefano and Gabriele, who in particular has been living with me in Lecce during the past three years, so being close to me in many unforgettable moments; all my best friends, with whom I shared these important years, facing various difficulties but also having a lot of fun and enjoyable time.

(11)

Symbols and abbreviations

Symbols

A Acoustic wave amplitude

b Effective damping constant of the bubble-liquid system

c Speed of sound

d Distance

dSe Shell thickness at rest

e Base of natural logarithms (e ≈ 2.718)

f Acoustic wave frequency

f0 Bubble resonance frequency

GS Shell shear modulus

H1 First harmonic amplitude

H2 Second harmonic amplitude

i Imaginary unit (i= −1)

I Acoustic wave intensity

I0 Acoustic wave intensity for z=0

Ii Intensity of an incident acoustic wave

Ir Intensity of a reflected acoustic wave

It Intensity of an acoustic wave after its passage through a discontinuity surface K Compressibility

k Acoustic wave number (k=2π/λ)

ks Effective stiffness of the bubble-liquid system

m Effective mass of the bubble-liquid system n Number of bubbles per unit volume

p Acoustic pressure

pa Ambient pressure

(12)

Symbols and abbreviations

pg0 Initial gas pressure inside the bubble

pneg Negative peak of acoustic pressure

pv Vapour pressure

Pa Absorbed acoustic power

Pe Extinct acoustic power

Ps Scattered acoustic power

R Bubble radius at time t

R& First time derivative of bubble radius

R&& Second time derivative of bubble radius

R0 Bubble radius at t=0

R1 Inner bubble radius

R2 Outer bubble radius

Re Reynolds number Sf Shell friction parameter

Sp Shell stiffness parameter

t Time

Time interval between ultrasound pulse transmission

T and echo detection Tl Pulse temporal length

VS Microbubble shell volume

z Distance along wave propagation direction

Z Acoustic impedance

α Attenuation coefficient

Γ Polytropic exponent of the gas

γ Gas adiabatic constant

δ Total damping coefficient

δrad Damping term from acoustic radiation

δsh Damping term from internal friction inside the shell

δth Damping term from thermal conduction

δvis Damping term from liquid viscosity

θi Incidence angle

θr Reflection angle

θt Transmission angle

λ Wavelength

µL Liquid shear viscosity

µS Shell shear viscosity

ξ Bubble radial displacement (ξ=R-R0)

(13)

Symbols and abbreviations

ρ Density

ρg Density of the gas inside the microbubble

ρL Density of the liquid surrounding the microbubble

ρS Density of the shell material

σ Surface tension coefficient

σ1 Surface tension coefficient at the gas-shell interface

σ2 Surface tension coefficient at the shell-liquid interface

σa Microbubble absorption cross-section

σe Microbubble extinction cross-section

σs Microbubble scattering cross-section

<σe> Average extinction cross-section per microbubble

Abbreviations

A-mode Amplitude mode B-mode Brightness mode

CA Contrast agent

CMCNa Carboxymethylcellulose sodium salt DVS Divinylsulphone

EEP Echograph electrical power ET Emitting transducer EVA© Ethylene Vinyl Acetate

FEMMINA Fast Echographic Multiparameter Multi Image Novel Apparatus

FFT Fast Fourier Transform FG Function generator HEC Hydroxyethilcellulose

MI Mechanical index PC Personal computer PFB Perfluorobutane

PRF Pulse repetition frequency RF Radiofrequency

RFA Radiofrequency amplifier ROI Region of interest

RT Receiving transducer TGC Time Gain Compensation

(14)

Symbols and abbreviations TS Trigger signal US Ultrasound VI Virtual instrument xiv

(15)

Abstract

This thesis introduces the use of a series of new experimental systems, and the corresponding signal processing techniques, to investigate the acoustic properties of ultrasound contrast agents, both in flowing and static conditions. Considered properties include backscatter, attenuation, resonance phenomena, nonlinear oscillation and destruction mechanisms as a function of the applied ultrasound parameters. These properties have been investigated by acoustic and granulometric measurements conducted on a last generation microbubble experimental contrast agent (BR14, Bracco Research SA, Geneva, Switzerland) for frequencies in the medical diagnostic range.

In the first part of this work, a new tissue-mimicking phantom was designed, manufactured and employed to evaluate microbubble behavior in very similar conditions to those encountered inside the human body. During these studies microbubbles were insonified by means of a common commercial echograph while flowing through the phantom, that had been inserted into an ad hoc assembled flow circuit, aimed to simulate human microcirculation conditions. In addition, in order to explore possible improvements in signal processing techniques, the echograph was connected to a prototypal platform for acquisition and independent processing of the raw unfiltered radiofrequency signals. In the second part, whose focus was on the determination of microbubble intrinsic properties, microbubbles were studied in static conditions, employing single-element ultrasound probes driven by laboratory devices, offering higher flexibility in terms of generated signal parameters, and linked to high-performance data acquisition boards.

In particular, a new methodology has been developed to study microbubble destruction mechanisms and three main patterns have been identified in the microbubble acoustic behavior, also establishing the boundary conditions for the onset of the corresponding underlying phenomena. These findings could represent interesting bases for

(16)

Abstract

theoretical modeling of microbubble acoustic behavior from linear oscillation to complete shell disruption.

The outcome of each performed study has been also analyzed and interpreted to exploit possible implications for the improvement of current clinical techniques or for the potential development of new ones. A global characterization of the acoustic behavior of the studied contrast agent is finally provided, together with many indications for its effective employment in further in vivo testing and clinical trials.

(17)

Chapter 1

Introduction

1.1 Medical ultrasound imaging and contrast enhancement

Ultrasonography is the most widely used clinical imaging technique in the world: it has been estimated that 100 million ultrasound scans of the heart, vascular system and abdominal organs are conducted worldwide each year [1]. Ultrasound imaging is so attractive for a number of reasons: it is versatile, not invasive, portable, easy to use, has low costs and produces real-time images. However, unlike the other major medical imaging modalities, such as X-ray radiology and magnetic resonance imaging, until a few years ago ultrasound techniques have suffered the lack of effective contrast agents.

A contrast agent, by definition, alters image contrast in a meaningful way that helps the diagnostician to distinguish between normal and abnormal conditions. Visualization of blood and blood flow within an organ is then essential for helping to differentiate normal tissues from injured ones [1].

Medical ultrasound imaging is based on the echoes generated from inhomogeneities in the investigated tissues. These echoes are elaborated in the echographic scanner and used to compose images of the interior of the body. Ultrasound scatter from blood is much weaker than scatter from other tissues, typically 30 to 60 dB weaker [2], so the lumen of small vessels is not easily distinguishable from surrounding tissues. Hence the aim of introducing contrast agents is to increase the scatter of sound from blood. This is used to increase the information content in ultrasound images and to obtain diagnostic information not available without contrast agent (e.g. more accurate measurements of heart functional parameters, visualization of blood flow in small vessels, evaluation of tissue perfusion in various tissues and organs).

It has been widely showed [2,3] that very strong ultrasound scattering can be achieved by using extremely compressible particles, since they introduce a marked difference in acoustic impedance with respect to the surrounding blood. These particles have to pass successfully the

(18)

Introduction

pulmonary and coronary microcirculation and their longevity within the systemic circulation must be sufficient for an effective clinical examination to be performed. For these reasons, the most common and efficacious ultrasound contrast agents are in the form of gas-filled encapsulated microbubbles having diameters in the range 1-7 µm. Currently this is by far the most common approach to produce an ultrasound contrast agent [2], as confirmed by the fact that there are several microbubble contrast agents in clinical trials and a few are already commercially available [1].

Contrast agents can provide real-time images of blood flowing through the heart chambers, vessels and capillary beds. Some agents are then slowed in the circulation of the liver and spleen or are taken up by phagocytic cells, specifically enhancing these organs [4]. Some other microbubbles can be designed to specifically target a receptor system, opening new exciting perspectives for molecular imaging and therapeutic applications of ultrasound contrast agents, potentially employable as carriers of drugs or genes.

Successful introduction of mentioned applications in clinical routine should dramatically increase the diagnostic performance of ultrasound imaging, while decreasing the time and cost for diagnosis together with the invasivity of the required examination. These considerations determined the widespread interest recently achieved by ultrasound contrast imaging, pushing research efforts in this field towards a deeper understanding of the dynamics of microbubble-ultrasound interactions, in order to effectively develop innovative microbubble detection strategies and to properly design new contrast agents particularly suited for each given application.

Next paragraphs provide a brief synthesis of the historical development of contrast echography, followed by an overview of current and future applications of ultrasound contrast agents. Specific aims of this study are then illustrated and, finally, the contents of the subsequent chapters are summarized.

1.2 Historical development of contrast echography

Ultrasound contrast agents were pioneered in the late 1960s by Gramiak and Shah [5,6], who observed that strong short-lived echoes were generated in aorta and heart after agitated saline solutions containing free air bubbles had been injected into patients through an intra-aortic catheter. Shortly thereafter it was discovered that other fluids containing gas bubbles produced similar results.

Contrast effects discussed in those early experiences involved only free gas bubbles, with inherent shortcomings due to the bubble 2

(19)

Introduction

generation method itself, such as indeterminate size, short life-time and inability to traverse the lung circulation.

However, this exciting new tool in echocardiography was explored by a large number of investigators in the period 1970-1980, as documented in the review by Meltzer and Roelandt [7]. All the research investigations performed in this period were aimed to determine in what manner this new method could enhance the diagnostic potentials of echocardiography.

Apart from the injection of the contrast material directly into the left side of the circulation by means of a catheter, left sided applications of contrast bubbles after intravenous injection were not possible in those years because of the blocking action of the lungs.

An important step forward in this area was the introduction of encapsulated microbubbles, whose first employments for remote sensing of local blood pressure [8] and for indicator dilution measurements [9] were reported in the late 1970s. Anyway, there was still the need for substantial improvements in the microbubble production processes before reasonably aiming to the introduction in clinical routine.

In the period 1980-1990 many clinical experiments were carried out in order to make contrast echocardiography an established technique. In the beginning of the 1980s researchers were forced to use home-made contrast agents: their studies were focused on determining myocardial perfusion and they were able to show that areas of normal and insufficient perfusion could be visualized [10].

In 1984 Feinstein et al [11] reported the study of a sonication technique which produced small and stable microbubbles, whose transpulmonary effect was investigated by Ten Cate et al [12]. This was the first successfully reproducible method of producing microbubbles. In the same year a videodensitometric method for quantifying myocardial perfusion studies was also presented [13], stimulating an increasing interest for computer applications involving contrast ultrasound.

The first ultrasound contrast agents launched as commercial products in the early 1990s were Echovist (Schering AG, Germany) and Albunex (Molecular Biosystems Inc., USA). Echovist consisted of microcrystalline galactose particles that acted as a template providing nidation sites within which air bubbles formed when suspended in water [14], while Albunex consisted of air microbubbles inside heat-denatured human albumin shells [15]. These particles were, however, not stable enough to pass the pulmonary capillary bed and reach the left side of the heart when injected intravenously. Their survival within the systemic circulation could be measured in seconds, and thus provided limited utility.

Schering subsequently developed Levovist, which also consisted of microcrystalline galactose microparticles, but was complemented with 3

(20)

Introduction

palmitic acid, which coated the microbubbles that formed after dissolution in water and vigorous agitation, providing increased stability [16,17].

Molecular Biosystems, on the other hand, improved the intravascular stability of Albunex by replacing air with perfluoropropane. The new product, Optison, consisted of a ready-to-use suspension of microcapsules of heat-denatured human albumin filled with perfluoropropane [18-24]. This agent, which requires refrigerated storage, was introduced on the market in 1998.

Optison belongs to the so-called “second-generation ultrasound contrast agents” [25]. The most significant change in physical characteristics of microbubbles addressed by second generation agents was the gas content. In almost all agents, air was substituted by high-molecular-weight perfluorocarbons, which are poorly soluble in water and blood, having longevity proportional to the length of their carbon chain [26,27].

Another change took place in the composition of microbubble shell: semi-rigid shells of denatured albumin have progressively given way to lipid shells, that are more flexible and so require less acoustic energy to oscillate in such a manner to produce an effective ultrasound scattering. Differences in shell composition also resulted in different methods of producing microbubbles: while sonication is generally employed for the production of albumin shell microbubbles, mechanical or hand agitation is effective for producing microbubbles with lipid shells [25].

The concentration of most microbubble contrast agents is in the range 0.2-1.5 × 109

microbubbles/mL [28]. Research studies and clinical trials in humans indicated that doses of agent as low as 0.1 mL are sufficient for myocardial opacification, while even less is necessary for left ventricular opacification and delineation [29,30].

The toxicity and safety of second generation microbubble contrast agents have been also widely investigated [29-32], showing that such agents produce no significant effects on cardiac or systemic hemodynamic function, myocardial blood flow, left ventricular wall thickening and pulmonary gas exchange.

Several second generation microbubble contrast agents are already available on the market since a few years [1], while further interesting experimental preparations are currently being evaluated, including, in particular, perfluorobutane-exposed sonicated dextrose albumin (PESDA) microbubbles [33], perfluorobutane microbubbles with phospholipidic shell, like BR14 (Bracco Research SA, Geneva, Switzerland) [34], and AI-700 (Acusphere, USA), consisting of perfluorocarbon-containing microbubbles enclosed in a biodegradable polymeric shell [35].

(21)

Introduction

1.3 Current and future applications of ultrasound contrast

agents

Although the field of contrast ultrasound was born in the 1960s and was introduced into clinical application during the late 1980s and the 1990s, it did not achieve widespread interest until very recent times. Three critical breakthroughs, all achieved in the last few years, propelled the field into wider clinical practice and gave new energy to further research efforts: 1) with the introduction of second generation contrast agents, microbubble survival within systemic circulation was extended from seconds to minutes, thus making their use more attractive for many clinical purposes; 2) the advent of completely digital ultrasound equipments and the adoption of broadband transducers simplified the implementation of novel pulsing schemes and signal processing techniques; 3) the introduction of harmonic and pulse-inversion imaging techniques allowed real-time suppression of tissue signals without affecting bubble signals, so dramatically increasing image contrast.

Microbubble contrast agents are designed to remain within the vascular space to image the blood pool. This characteristic is ideal for both contrast echocardiography and vascular imaging, particularly when combined with bubble-specific imaging modes such as harmonic imaging and pulse-inversion. Restriction of microbubbles to the vascular space, together with their capability of being destroyed by higher intensity ultrasound pulses, makes them very useful also for imaging solid organs such as liver and kidneys.

Applications of ultrasound contrast agents to cardiac, vascular and solid-organ imaging are shortly overviewed below, giving also indications on the potential further developments of contrast-enhanced echographic techniques in the various mentioned fields, including the new exciting perspectives of molecular imaging and therapeutic applications.

1.3.1 Cardiac imaging

The development of effective ultrasound contrast agents consisting of

injectable micrometer-sized gas bubbles that highlight the endocardial border has made contrast echocardiography a common clinical reality [36-39].

Around 20% of conventional echographic examinations of the heart, in fact, does not provide images of adequate quality to allow visualization of inner border of the heart (endocardial border) for accurate diagnosis of ventricular dysfunction [1]. Thus, before the advent of ultrasound contrast agents, additional non-ultrasound testing procedures were

(22)

Introduction

required, which were often more invasive, always more expensive, and sometimes presented also higher risk for the patient.

The use of contrast agents provided a clear delineation of the endocardial wall and improved definition of wall motion and thickening [40]. Enhanced endocardial border definition enabled a better evaluation of cardiac structure and function, which has lead to substantial improvements in diagnostic and prognostic accuracy and confidence. In particular, effective delineation of ventricular border helps to assess global heart function by accurately measuring the ejection fraction (the percentage of the blood in the left ventricle that is ejected during a heart beat).

New methods to achieve an accurate assessment of myocardial perfusion through microbubble contrast agents are currently in clinical trials, as well as innovative contrast ultrasound techniques to improve detection of coronary insufficiency.

Contrast-specific ultrasound imaging of the myocardium has in fact the potential to offer results equivalent to perfusion imaging with radiopharmaceuticals, both at rest and with exercise, with the advantage of providing real-time information on both heart function and perfusion [24], accompanied by the elimination of all the problems inherent the radioactive materials.

1.3.2 Vascular imaging

The purposes of vascular imaging are [1]: 1) to verify that supply vessels are not occluded; 2) to assess the presence of arterial wall abnormalities such as atherosclerotic plaques and to evaluate their effect on flow; 3) to determine whether the direction of flow and velocity profile through the cardiac cycle are normal; 4) to acquire a visualization of the vascular tree within organs.

Standard Doppler imaging provides flow velocity data, but it is not reliable at demonstrating structural changes in vessels. Filling the vessel with the signal from contrast media has been already shown to be effective at detecting disease with X-ray angiography, X-ray-computed tomography and magnetic resonance imaging, and it is now extending also to ultrasound imaging.

Contrast agent employment in vascular imaging with ultrasound is important in addition for thrombus detection, since a blood clot typically assumes a similar appearance to blood, which allows the vessel to look normal when actually it may be totally occluded. Filling the vein with an ultrasound contrast medium allows for clear visualization of clots and even of the tiny channels that form as a clot begins to recanalize [41]. These channels are recognizable in consequence of the ability of ultrasound to detect single bubbles as they negotiate their way through the clot. This capability is particularly useful in the assessment of 6

(23)

Introduction

abdominal and pelvic veins, which cannot be assessed by the application of an external compression over the vein.

When imaging the extracranial carotid arteries, standard echography cannot consistently depict the noncalcified atherosclerotic plaques. Filling the artery with an ultrasound contrast agent can accurately display the arterial inner wall, which helps to observe plaques in detail [42,43]. When tested in patients undergoing standard carotid X-ray angiography, contrast-enhanced ultrasonography was as accurate as X-ray angiography in directly measuring the percentage of diameter reduction [43].

Further goals of contrast sonography now appear to be within reach in the field of vascular imaging. They include in particular providing more simple and more effective methods for detection and classification of plaques in the carotid arteries or abdominal or pelvic veins, based on innovative quantitative analysis of the corresponding contrast-enhanced echographic images.

1.3.3 Solid-organ imaging

The ability to recognize perfused tissues and the pattern of perfusion is critical for the detection of diseased tissues. The use of an ultrasound contrast agent to fill all the tissue vascular spaces enables the clear depiction of regions not perfused, thus allowing the detection of vascular occlusion immediately after it occurs. Observing the pattern of perfusion as microbubbles fill the organ in real time also allows the detection of abnormal perfusion patterns, as is observed in tumors [44].

Conventional echography is capable of detecting tumors within organs as they typically alter the appearance of normal tissue architecture. However, detecting small tumors (<2 cm) remains difficult, and even more difficult is to distinguish between benign and malignant tumors. Tumor detection rate for conventional echography of the liver, the most commonly involved solid organ, ranges from 40% to 70%, depending on the operator skill [1].

By observing the filling pattern and the arterial tree within an organ in real time, contrast-specific ultrasound imaging can not only enable the detection of abnormal regions, but also improve the ability to characterize diseases [45].

The introduction of contrast echography has already allowed the quantification of tumor microcirculation and the assessment of angiogenesis consequent to tumor growth [46]. Significant improvement in disease detection and characterization has been achieved in recent years both in animal models and in humans [45,47,48]. Clinical investigations of prostate vascularity have also indicated that contrast agents may improve the echographic detection of prostate cancer [47]

(24)

Introduction

and that renal cortical blood flow and ischemia can be assessed as well [49].

Furthermore, microbubbles have demonstrated to be useful for imaging the liver after they have been taken up by the reticulo-endotelial system and to aid the visualization of liver lesions, including carcinoma and small metastases [50,51].

The full clinical potential of ultrasound contrast agents for solid-organ imaging is still far from exploited and its development appears extremely promising. Currently the main research goals in this field are optimal quantification of regional ischemia or infarction, enhancement of the accuracy of cancer detection, more accurate characterization and staging of tumors in organs such as liver, breast and prostate. Research is also underway to look at brain perfusion [52].

1.3.4 Molecular imaging and therapeutic applications

The increased diagnostic accuracy and reliability gained by combining second generation ultrasound contrast agents with improvements in the software instruments, together with the low cost and portability of this imaging technique relative to most other modern diagnostic modalities, are likely to further expand the range of ultrasound imaging applications. Further medical advances involving microbubbles, in particular, are emerging in fields such as specific tissue targeting and drug or gene delivery.

Microbubbles can be outfitted with a targeting device and used to seek and mark specific targets, which then become visible during ultrasound imaging. Such detection of the specific molecular signature of a given pathology is referred to as “molecular imaging” [53]. Privileged target tissues include thrombi, atheroma plaques, areas of inflammation and tumors [54-56].

Microbubbles subjected to ultrasound have also a potential as therapeutic tools. For example, in vivo arterial thrombolysis has been achieved in rabbits without a thrombolytic drug, but simply using microbubbles and externally applying ultrasound energy [57,58].

Finally, the combination of bubble targeting and ultrasound-triggered bubble disruption in the targeted tissue offers further opportunities. A drug substance can in fact be incorporated into the shell of a bubble or attached to its surface. Bubble/drug circulation and distribution can then be monitored by ultrasound imaging. Therefore, ultrasound-mediated bubble disruption and local release of the active substances can be triggered and focused when the microspheres reach the intended target. This strategy could help to protect surrounding normal tissue from toxic drugs.

(25)

Introduction

1.4 Aims of this study

The key to further developing the above mentioned clinical diagnostic procedures lies in a deeper understanding of the influence of ultrasound transmission parameters on contrast microbubble behavior.

Assessing the efficacy of ultrasound contrast agents on the basis of their physical characteristics is a complex issue. The characterization of the contrast media behavior, in fact, requires in vitro attenuation and scattering experiments to be performed on a wide range of parameter settings and so, during past years, microbubble dynamics have been widely investigated [59-65]. In particular, it is known from literature [66] that the dependency of bubble volume pulsation on the incident pressure amplitude can be divided into three regimes: linear oscillation at low pressure, nonlinear oscillation with harmonic emission at intermediate pressure and bubble rupture associated with a strong transient backscatter enhancement at higher pressure.

This thesis introduces a series of new experimental systems aimed to provide a global characterization of the acoustical behaviour of a last generation experimental contrast agent (BR14, Bracco Research SA, Geneva, Switzerland), both in flowing and static conditions. Main microbubble properties have been then considered and are related to backscatter, attenuation, resonance phenomena, nonlinear oscillation and destruction mechanisms as a function of the applied ultrasound parameters.

Experimental results can be also exploited in terms of possible implications for effective clinical employment of the studied agent and potential improvement of current microbubble detection strategies. In addition, some previous studies [66-76] specifically investigated nonlinear properties of contrast microbubbles, aiming to improve existing contrast-assisted imaging modalities and to develop new ones. Our effort has been addressed to the characterization of a new experimental contrast agent, that was not previously covered by any of those studies.

Moreover, improved knowledge of microbubble destruction phenomena could allow to choose the contrast agent better suited for a given application. Just to cite two important cases, we could refer to reperfusion imaging [77-79], made possible by the knowledge of acoustic pressure thresholds for microbubble destruction, and therapeutic applications [80-83], which require the use of thick-shelled microbubbles, robust enough to carry drug or gene and then to be destroyed at higher acoustic pressures.

A further goal of this thesis was, then, the implementation of new techniques to improve the theoretical and experimental knowledge of microbubble behavior by means of attenuation measurements, with the 9

(26)

Introduction

specific aim of determining pressure thresholds for the onset of nonlinearities and different destruction mechanisms for the investigated microbubbles. In order to do this, results of detailed granulometric analyses performed on microbubble solutions have been exploited and closely correlated with acoustic measurement outcome, giving new perspectives in the interpretation of microbubble population behavior.

1.5 Thesis organization

This thesis consists of twelve chapters and it is conceptually divided into four parts. After this introductory chapter, the first part, Ultrasound

imaging and contrast agents, summarizes basic principles of

echographic imaging and microbubble contrast agents and comprises chapters 2 and 3:

• Chapter 2 briefly reviews physical principles related to generation and propagation of ultrasound signals. Fundamental steps of echographic image reconstruction are also explained. • Chapter 3 is focused on ultrasound-microbubble interaction,

giving details about involved physical phenomena, main available theoretical models and contrast imaging methods that are currently used or under investigation.

After the illustration of the theoretical background underlying the experimental studies reported in this thesis, the second part, entitled

Phantom design and production and comprising chapters 4-6, describes

the procedures adopted for manufacturing the tissue-mimicking “phantom” (which has to be intended as a simplified vascular model of a target human organ) and the assembly of an appropriate flow circuit:

• Chapter 4 illustrates the synthesis and characterization processes of a new tissue-mimicking hydrogel, able to accurately simulate linear and nonlinear acoustic properties of liver tissue, that had been chosen as the target tissue for this study.

• Chapter 5 shows the experimental procedure adopted to select a sound-absorbent material to cover the phantom bottom, in order to minimize artefacts due to environmental boundary conditions. • Chapter 6 depicts the progressive steps of phantom fabrication

and testing, until the final phantom production. Details of the assembled flow circuit are also provided.

(27)

Introduction

Then, the third part, Microbubbles in flowing conditions, comprising chapters 7-8 and aimed to evaluate microbubble behavior in very similar conditions to those encountered inside the human body, describes the main studies conducted on flowing microbubbles employing the experimental set-up introduced in previous chapters:

• Chapter 7 discusses the effect of incident ultrasound amplitude on microbubble backscattered signal.

• Chapter 8 addresses the effect of contrast agent concentration on the amplitude of ultrasound signals backscattered by microbubbles. Some considerations are also made on variable effectiveness of different ways of indicating contrast agent concentration itself.

In the fourth part, entitled Microbubbles in static conditions and comprising chapters 9-11, focus is moved on the determination of microbubble intrinsic properties through the employment of different experimental set-ups and innovative methodologies of data analysis:

• Chapter 9 describes the employment of a new hydrogel-containing set-up, just designed to estimate optimal acoustic pressures for effective microbubble imaging in different echographic modalities.

• Chapter 10 introduces the last experimental set-up developed within this thesis: a new system for acoustic attenuation measurements in “through-transmission” mode. Its application in estimating microbubble nonlinear oscillation threshold is also shown.

• Chapter 11 presents the assessment of microbubble destruction mechanisms, which is achieved through a new methodology based on the combination of acoustic and granulometric measurements. Main obtained results are grouped into three main patterns, representing a proposed model of BR14 microbubble acoustical behavior.

Finally, in chapter 12, the most important conclusions of this work are summarized and some speculations are given on the most promising future developments of this research topic.

(28)

Riferimenti

Documenti correlati

Aims The objectives of the present study were to describe epidemiology and outcomes in ambulatory heart failure (HF) patients stratified by left ventricular ejection fraction (LVEF)

Usefulness of a combination of systolic function by left ventricular ejection fraction and diastolic function by E/E’ to predict prognosis in patients with heart

N o changes in plasma catecholamine concentration occurr ed C AD: cor onar y arter y disease; LVEF: left ventricular ejection fraction; HF: heart failure; NY AH: Ne w Y ork

Abbreviations: MR-proANP, mid-regional pro-atrial natriuretic peptide; HF, heart fail- ure; LVEF, left ventricular ejection fraction; BMI, body mass index; NT-proBNP,

Methods: Twenty patients (68 ± 7 years old, 20% females) with stable chronic heart failure due to reduced left ventricular ejection fraction (31 ± 8 %) participated in a

When comparing to placebo, heart rate and blood lactate when taking the combination of supplements, was similar to that of caffeine alone which shows that probably there

Data from various randomised trials show that both biphasic insulin lispro and insulin aspart provide more effective postprandial control of blood glucose than