Introduction: Haemodialysis (HD) is a treatment in which blood from the patient is lead through a tubing system into a dialysis device in a extracorporeal circuit. This circuit contains semipermeable membranes (dialyzer). Blood with uraemic toxins flows on one side, and a salt solution flows on the other side. The salt solution flushes away waste products that have passed the membrane by diffusion or convection through small pores. From there the blood returns to the patient through a tubing system that contains an air-trap and a sensor to avoid air contamination in the blood. Besides air contamination, this treatment is burdened with safety problems such as biocompatibility, electrical safety and mechanical safety. The aim of this thesis was to investigate the safety issues in haemodialysis devices regarding leakage current and air contamination during standard procedures and simulated fault conditions. Does the dialysis device constitute a risk for the patient? Methods: To determine the extent of leakage current in HD machines, measurements at the filter-coupling site were performed in vitro according to the safety standard, IEC 601-1, in 5 types of dialysis machines. To determine, in vitro, to what extent blood and priming fluid allowed leakage current to pass to the patient, leakage current were also measured in the blood lines. The blood line was filled with blood from donors or priming fluid in eight different runs. To determine if leakage current could influence biocompatibility, a Fresenius 2008C dialysis machine and 8 hemophan dialyzers were used. Blood lines contained about 400 ml heparinized blood from each of 8 different donors (in vitro). C3d was measured, in vitro, before start of a simulated dialysis and at 15, 30, 45 and 60 min. during standard dialysis procedure. Then 1.5 mA current was switched on and additional samples were drawn at 75 and 90 min. Some patients need a central dialysis catheter (CDC) for access, placed close to or within the heart. To analyze if leakage current during standard HD would influence the ECG, patients with CDC or with AV-fistula as access were investigated. To analyse if air contamination could occur without activating security alarms in the dialysis device, various modes of in vitro dialysis settings were studied, some using a dextran solution to mimic blood viscosity. Besides visual inspection an ultrasound detector for microemboli and microbubbles was also used. Results: The data showed leakage current at the filter coupling site that was significantly higher for some devices than for others. The leakage current could pass through blood and priming fluid. It exceeded the cardiac floating (CF)-safety limit (<50μA) at the top of the CDC using the test mains on applied part for saline (median 1008μA), for blood (median 610μA) and for a single fault condition using saline (median 68 μA) or blood (47 μA). The leakage current experiments showed that complement activation worsened as the leakage current increased. During standard dialysis arrhythmia could occur. Microbubbles were visible at the bottom of the air-trap and bubbles could pass the air-trap towards the venous line without triggering the alarm. During recirculation, several ml of air could be collected in an intermediate bag after the venous line. Ultrasound showed the presence of bubbles of sizes 2.5-50 μm as well as more than 50 μm silently passing to the venous line in all runs performed. In conclusion, the data showed that a leakage current in HD devices can be high enough to be a safety risk for the patient. This risk is greater if a single fault arises in the dialysis machine or another device connected to the same patient, or during mains contact to the patient. Then the current flow may be high enough to cause arrhythmia for the patient, especially when using a CDC. There is also reason for concern that micro bubble transmission may occur without inducing an alarm. These factors need to be looked over to improve safety regulations and optimize HD treatment and service schedules.

This thesis presented a microfluidic preparation of bubbles-templated micro-size materials. In particular, this thesis focused on the microfluidic formation and dissolution of CO2 bubbles. First, this thesis described pH-regulated behaviours of CO2 bubbles in the microfluidic channel. This method opened a new way to generate small (<10 µm in diameter) with a narrow size distribution (CV<5%). Second, the microfluidic dissolution of CO2 bubbles possessed the important feature: the local change of pH on the bubble surface. This allowed us to encapsulate the bubbles with various colloidal particles. The bubbles coated with particles showed a high stability against coalescences and Ostwald ripening. The dimensions and shapes of bubbles with a shell of colloidal particle were manipulated by the hydrodynamic and chemical means, respectively. Third, we proposed a microfluidic method for the generation of small and stable bubbles coated with a lysozyme-alginate shell. The local pH decrease at the periphery of CO2 bubbles led to the electrostatic attraction between lysozyme on the bubble surface and alginate in the continuous phase. This produced the bubbles with a shell of biopolymers, which gave a long-term stability (up to a month, at least) against the dissolution and coalescence. Fourth, we presented a single-step method to functionalize bubbles with a variety of nanoparticles. The bubbles showed the corresponding properties of nanoparticles on their surface. Further, we explored the potential applications of these bubbles as contrast agents in ultrasound and magnetic resonance imaging.

In vivo real-time data of ultrasound and microbubble assisted blood-brain barrier disruption is centrally based on low-resolution magnetic resonance images. Additional information can be gained using online microscopic monitoring. This study presents the first ever in vivo two-photon microscopy, four-dimensional data sets of ultrasound and microbubble assisted blood-brain barrier disruption. It characterized the threshold pressures and mechanical index needed to disrupt the vasculature with 800 kHz ultrasound, and found three different leakage constants from the compromised vasculature. Furthermore, using numerical models, an ultrasound array was designed and optimized to perform specifically with our two-photon microscope. It was fabricated, fully characterized, and its performance met both the required pressure field profile and the pressure values needed for our in vivo two-photon microscopy experiments. This array is an important step in microscopically characterizing ultrasound and microbubble assisted blood-brain barrier disruption.

Microbubble ultrasound (US) contrast agents have been used to monitor the progression of anti-angiogenic chemotherapies. However, US backscatter measurements used in contrast imaging are inherently variable, given the presence of many microbubbles of random position and size. A model was developed to investigate the influence of US scanner and microbubble characteristics on these variable measurements. The Coefficient of Variation was used to measure variability. It was found that an optimum excitation frequency exists that minimizes this variability. In the case of DefinityTM, a 2.25 MHz centre-frequency pulse yielded a less variable measurement than at 5 MHz. Conversely, decreasing microbubbble concentration was found to significantly increase variability. Evidence suggests that microbubbles are no longer Rayleigh scatterers at sufficient low concentrations. Post-processing was found to aid in reducing measurement variability by averaging samples where microbubble positions are uncorrelated. As well, reduction can be achieved by averaging about a region-of-interest of uniform perfusion.

In vivo real-time data of ultrasound and microbubble assisted blood-brain barrier disruption is centrally based on low-resolution magnetic resonance images. Additional information can be gained using online microscopic monitoring. This study presents the first ever in vivo two-photon microscopy, four-dimensional data sets of ultrasound and microbubble assisted blood-brain barrier disruption. It characterized the threshold pressures and mechanical index needed to disrupt the vasculature with 800 kHz ultrasound, and found three different leakage constants from the compromised vasculature. Furthermore, using numerical models, an ultrasound array was designed and optimized to perform specifically with our two-photon microscope. It was fabricated, fully characterized, and its performance met both the required pressure field profile and the pressure values needed for our in vivo two-photon microscopy experiments. This array is an important step in microscopically characterizing ultrasound and microbubble assisted blood-brain barrier disruption.

This thesis presented a microfluidic preparation of bubbles-templated micro-size materials. In particular, this thesis focused on the microfluidic formation and dissolution of CO2 bubbles. First, this thesis described pH-regulated behaviours of CO2 bubbles in the microfluidic channel. This method opened a new way to generate small (<10 µm in diameter) with a narrow size distribution (CV<5%). Second, the microfluidic dissolution of CO2 bubbles possessed the important feature: the local change of pH on the bubble surface. This allowed us to encapsulate the bubbles with various colloidal particles. The bubbles coated with particles showed a high stability against coalescences and Ostwald ripening. The dimensions and shapes of bubbles with a shell of colloidal particle were manipulated by the hydrodynamic and chemical means, respectively. Third, we proposed a microfluidic method for the generation of small and stable bubbles coated with a lysozyme-alginate shell. The local pH decrease at the periphery of CO2 bubbles led to the electrostatic attraction between lysozyme on the bubble surface and alginate in the continuous phase. This produced the bubbles with a shell of biopolymers, which gave a long-term stability (up to a month, at least) against the dissolution and coalescence. Fourth, we presented a single-step method to functionalize bubbles with a variety of nanoparticles. The bubbles showed the corresponding properties of nanoparticles on their surface. Further, we explored the potential applications of these bubbles as contrast agents in ultrasound and magnetic resonance imaging.

A Lagrangian formalism presented by Hay, Ilinskii, Zabolotskaya, and Hamilton [J. Acoust. Soc. Am. 132, 124--137 (2012)] to calculate the pulsation of a spherical bubble, immersed in liquid and near one or two viscoelastic layers, is extended here to include bubble translation. The method presented here is simplified from that given by Hay et al. in that only a single interface between a liquid and a viscoelastic half-space is considered. In the present approach the force on the bubble due to the presence of the liquid-solid interface is calculated using a Green's function that takes into account elastic waves and viscosity in the layer, and the viscous boundary layer within the liquid near the interface. Previous models and experiments have shown that the direction of bubble translation near a viscoelastic layer is correlated with the direction of a liquid jet often produced by the bubble during collapse. In this dissertation an attempt is made to model the pulsation and translation of a spherical bubble near a liquid-solid interface to infer the direction of bubble translation in reference to material parameters of the liquid and viscoelastic medium, and the standoff distance of the bubble from the interface. The analysis is simplified by demonstrating that the direction of bubble translation can be inferred from the phase of the component of the Green's function associated with the reverberant pressure gradient. For linear bubble pulsation it is shown that the domain of material properties of the viscoelastic medium which generally corresponds to bubble translation away from the interface occurs when the effective stiffness of the viscoelastic medium is greater than the effective damping for both itself and the liquid. The analysis is performed assuming the viscoelastic medium is similar to soft tissue, and its dynamics are described by a Voigt, Kelvin, or Maxwell model. The simulations are compared with existing experimental data. Effects of high-amplitude bubble pulsation are explored in terms of how the simulations differ as the pulsation amplitude increases. At higher pulsation amplitudes, it is shown that bubble translation is still described qualitatively by analyzing the phase of the reverberant pressure gradient. / text

Diagnostic ultrasound (US) is safer, quicker and cheaper than other diagnostic imaging modalities. Over the past two decades, the applications of US imaging has been widened due to the development of injectable, compressible and encapsulated microbubbles (MBs) that provide an opportunity to improve conventional echocardiographic imaging, blood flow assessment and molecular imaging. The encapsulating material is manufactured by different biocompatible materials such as proteins, lipids or polymers. In current research, researchers modify the encapsulated shell with the help of advanced molecular chemistry techniques to load them with dyes (for fluorescent imaging), nanoparticles and radioisotopes (for multimodal imaging) or functional ligands or therapeutic gases (for local drug delivery). The echogenicity and the radial oscillation of MBs is the result of their compressibility, which undoubtedly varies with the encapsulated shell characteristics such as rigidity or elasticity. In this thesis, we present acoustic properties of novel type of polyvinyl alcohol (PVA)-shelled microbubble (PVA-MB) that was further modified with superparamagnetic iron oxide nanoparticles (SPIONs) to work as a dual-modal contrast agent for magnetic resonance (MR) imaging along with US imaging. Apparently, the shell modification changes their mechanical characteristics, which affects their acoustic properties. The overall objective of the thesis is to investigate the acoustic properties of modified and unmodified PVA-MBs at different ultrasound parameters. The acoustic and mechanical characterization of SPIONs modified PVA-MBs revealed that the acoustical response depends on the SPION inclusion strategy. However they retain the same structural characteristics after the modification. The modified MBs with SPIONs included on the surface of the PVA shell exhibit a soft-shelled behavior and produce a higher echogenicity than the MBs with the SPIONs inside the PVA shell. The fracturing mechanism of the unmodified PVA-MBs was identified to be different from the other fracturing mechanisms of conventional MBs. With the interaction of high-pressure bursts, the air gas core is squeezed out through small punctures in the PVA shell. During the fracturing, the PVA-MBs exhibit asymmetric (other modes) oscillations, resulting in sub- and ultra-harmonic generation. Exploiting the US imaging at the other modes of the oscillation of the PVA-MBs would provide an opportunity to visualize very low concentrations of (down to single) PVA-MBs. We further introduced the PVA-MBs along with particles mimicking red blood cells in an acoustic standing-wave field to observe the acoustic radiation force effect. We observed that the compressible PVA-MBs drawn toward pressure antinode while the solid blood phantoms moved toward the pressure node. This acoustic separation method (acoustophoresis) could be an efficient tool for studying the bioclearance of the PVA-MBs in the body, either by collecting blood samples (in-vitro) or by using the extracorporeal medical procedure (ex-vivo) at different organs. Overall, this work contributes significant feedback for chemists (to optimize the nanoparticle inclusion) and imaging groups (to develop new imaging sequences), and the positive findings pave new paths and provide triggers to engage in further research. / <p>QC 20150827</p> / 3MiCRON

Microbubble contrast agents for ultrasound are widely used in numerous medical applications, both diagnostic and therapeutic. Due to their size, similar to that of red blood cells, microbubbles are able to traverse the entire vascular bed, enabling their utilization for applications such as tumour diagnosis. Vaporizable submicron droplets of liquid perfluoro- carbon potentially represent a new generation of extravascular contrast agents for ultrasound. Droplets of a few hundred nanometers in diameter have the ability to extravasate selectively in regions of tumour growth while staying intravascular in healthy tissues. Upon extravasation, these droplets may be vaporized with ultrasound and converted into gas bubbles. In this thesis we argue that vaporized submicron perfluorocarbon droplets possess the necessary stability and acoustic characteristics to be potentially applicable as a new gener- ation of extravascular ultrasound contrast agents. We examine, separately, the ultrasound conditions necessary for vaporization of the droplets into microbubbles, the size and stability of these bubbles following vaporization, on timescales ranging from nanoseconds to minutes, and the bubbles’ acoustic response to incident diagnostic ultrasound. We show that submicron droplets may be vaporized into bubbles of a few microns in diameter using single ultrasound pulse within the diagnostic range. The efficiency of conversion is shown to be on the order of at least 10% of the exposed droplets converting into stable microbubbles. The bubbles are shown to be stabilized by the original coating material encapsulating the droplet precursors, and be stable for at least minutes following vaporization. Finally, vaporized droplets are shown to be echogenic, with acoustic characteristics comparable to these of the commercially available ultrasound contrast agents. The results presented here show that vaporized droplets possess the necessary stability properties and echogenicity required for successful application as contrast agents, suggesting potential for their future translation into clinical practice.

Microbubble contrast agents for ultrasound are widely used in numerous medical applications, both diagnostic and therapeutic. Due to their size, similar to that of red blood cells, microbubbles are able to traverse the entire vascular bed, enabling their utilization for applications such as tumour diagnosis. Vaporizable submicron droplets of liquid perfluoro- carbon potentially represent a new generation of extravascular contrast agents for ultrasound. Droplets of a few hundred nanometers in diameter have the ability to extravasate selectively in regions of tumour growth while staying intravascular in healthy tissues. Upon extravasation, these droplets may be vaporized with ultrasound and converted into gas bubbles. In this thesis we argue that vaporized submicron perfluorocarbon droplets possess the necessary stability and acoustic characteristics to be potentially applicable as a new gener- ation of extravascular ultrasound contrast agents. We examine, separately, the ultrasound conditions necessary for vaporization of the droplets into microbubbles, the size and stability of these bubbles following vaporization, on timescales ranging from nanoseconds to minutes, and the bubbles’ acoustic response to incident diagnostic ultrasound. We show that submicron droplets may be vaporized into bubbles of a few microns in diameter using single ultrasound pulse within the diagnostic range. The efficiency of conversion is shown to be on the order of at least 10% of the exposed droplets converting into stable microbubbles. The bubbles are shown to be stabilized by the original coating material encapsulating the droplet precursors, and be stable for at least minutes following vaporization. Finally, vaporized droplets are shown to be echogenic, with acoustic characteristics comparable to these of the commercially available ultrasound contrast agents. The results presented here show that vaporized droplets possess the necessary stability properties and echogenicity required for successful application as contrast agents, suggesting potential for their future translation into clinical practice.