QUANTIFICATION OF FLOW PARAMETERS IN COMPLEX VASCULATURE FLOW PHANTOMS USING CONTRAST-ENHANCED ULTRASOUND METHOD ASAWARI PAWAR Bachelors of Technology in Biomedical Engineering Padmashree Dr. D. Y. Patil University June, 2012 Submitted in partial fulfillment of requirements for the degree of MASTER OF SCIENCE IN BIOMEDICAL ENGINEERING at the CLEVELAND STATE UNIVERSITY August, 2015 We hereby approve this thesis of Asawari Pawar Candidate for the Master of Science in Biomedical Engineering degree for the Department of Chemical and Biomedical Engineering and the CLEVELAND STATE UNIVERSITY College of Graduate Studies _________________________________________________________________ Thesis Chairperson, Dr. Gregory T. Clement _________________________________________ Department & Date _________________________________________________________________ Thesis Committee Member, Dr. Joanne M. Belovich _________________________________________ Department & Date _________________________________________________________________ Thesis Committee Member, Dr. Chandra Kothapalli _________________________________________ Department & Date Student’s Date of Defense: 8/4/2015 ii This thesis is dedicated to my parents Asha Pawar & Prakash Pawar, my sister Amita Pawar and my best friend Sidharth Oberoi iii ACKNOWLEDGEMENT First and foremost, I would like to thank God the almighty for providing me this opportunity and granting me the capability to do this thesis project. I owe a debt of gratitude to my thesis advisor and principal investigator at Cleveland Clinic, Dr. Gregory Clement, he was the first to welcome me into the clinic and encourage my participation in clinical ultrasound lab. I am grateful for his help laying out a fascinating project that I am proud to have been a part. But most of all, I will remember his unconditional and genuine support and quick wit. I would like to offer my sincere thanks to my thesis committee members, Dr. Kothapalli and Dr. Belovich for always being encouraging throughout my study and research. In addition, I express my appreciation to Dr. Kothapalli and Dr. Belovich for giving me valuable suggestions, helpful ideas and constant support. Also I would like to thank the clinic team members for all their help especially; Mark Howell for his untiring support, without him this thesis would not be possible. I would like to thank my family and my sister for all the trust and support they showed me. Last but not the least; I would like to thank Sidharth Oberoi, the most reliable friend and a beautiful soul one could have, who I absolutely adore. I would have never learned the lessons of life and grown into a stronger person had I never made the decision to be with you. He played a crucial role in my evolution as a person today I am. Thank you for standing tall by me, appreciating me in whatever I did and choosing me to be a part of your life . I will be grateful to him forever. iv QUANTIFICATION OF FLOW PARAMETERS IN COMPLEX VASCULATURE FLOW PHANTOMS USING CONTRAST-ENHANCED ULTRASOUND METHOD ASAWARI PAWAR ABSTRACT Currently, there are few simple-to-construct in vitro, wall-less phantoms that have accurate acoustic properties while mimicking the complex normal and neoplastic geometries of the vascular network. The purpose of this study was to develop agar-based tissue-mimicking phantoms (TMP) to model such networks and to quantify the mean intensity and the slope measurements of the ultrasound flow. Three types of vascular networks were developed; (1) single vessel, (2) multi-vessel with artery bifurcations and (3) multi-vessel with artery bifurcations and structural abnormalities typical of diseased (tumor) vascular networks. Bolus injections of ultrasound contrast agents (UCAs) were performed under varying flow conditions relevant to our ongoing work in developing techniques to simultaneously quantify both the total volume and flow measurements within a tumor phantom The correlation coefficient between the mean slope measurements and the least squares fitted line was all higher than 0.95, indicating a good linear relationship between the mean slope and the flow-rates. For all single-vessel flow phantom data, the mean proportional difference of the slope measurements and flow-rate was 0.695 ± 0.18 (mean ± SD). It was found that the correlation coefficient between the mean slope v measurements and the least squares fitted line is 0.90, indicating a good linear correlation between the single-vessel and Multi-vessel flow phantoms. However, the parameters obtained from tumor region 1, 2 and 3 did not show any significant flow pattern and correlation after contrast injection of UCAs. Finally, the slope measurements of intensity at the tumor regions in-flow and out-flow demonstrated the nonlinear and undefined relationship between the measured intensity and varied flow-rates. Ideally, most information could be obtained when intensities at both the input and the output of the tumor periphery are measured. For further studies, alternative modeling of the problem is required because in physiology, microvasculature flow system has multiple input vessels and multiple output vessels. Developing new time-intensity-based techniques is the focus of future research. vi TABLE OF CONTENTS ABSTRACT……………………………..……………………………………………………..……..……………....v List of Tables……………………………………………...……..…………..………………………………...…..xi List of Figures……………….……….…………………………………...……………………………...……..xiii CHAPTER I. Introduction………………………………….………………...…………………….…………….....1 II. Background………………………………………………………………………………………..….6 2.1 The Circulatory System……………………………………………………………...6 2.1.1 General Description……………………………………………………..6 2.1.2 Normal Vasculature……………………………………………………..8 2.2 Diseased Microcirculation………………………………………………………….9 2.2.1 General Description……………………………………………………..9 2.2.2 Angiogenesis………………………………………………………….…..10 2.2.3 Tumor vascular……………………………………………………….…11 2.3 Techniques to measure Microcirculation Blood Flow………………..12 2.4 Principles of Ultrasound Imaging……………………………………………..13 2.4.1 DCE-US - Contrast Enhanced Ultrasound……………………..17 vii 2.4.2 DCE CT - Dynamic Contrast Enhanced Computed Tomography……………………………….......................................................19 2.5 Contrast agents…………………………………….…………………………………20 2.5.1 Microbubble Material Preparation……………………………...23 2.5.2 Detection of Microbubbles UCAs…………………………………25 2.5.2.1. Non-linear behavior of Microbubbles…25 2.5.2.2. Relevant Studies…………………………………27 2.5.2.3. Principles for Evaluating the Microvasculature with Contrast Agents……………………………………………..…28 2.5.2.4. Evaluating the Microvasculature with Contrast Agents…………………………………………………….....29 2.6 Flow Phantoms models……………………………………………………………31 2.6.1 Single-vessel flow phantom model…………………………...35 2.6.2. Multi-vessel Flow Model………………………………………….37 2.6.3. Tumor Vessel Flow Model………………………………………37 III. MATERIALS & METHODS……………………………………………………………………42 viii 3.1 Preparation of Ultrasound Contrast Agents (UCAs) Microbubbles………………………………………………………………………………..42 3.1.1. Selection of Internal Gas: Octafluoropropane………........42 3.1.2. Selection of Outer Lipid Shell………...……………………….…..43 3.1.3. Preparation of Stock Solution………………………………...…45 3.1.4. Observation of Microbubbles under Microscope………49 3.1.5. Analysis of Microbubbles using Invitrogen Counting Chamber………………………………………………..………………..49 3.2 Preparation of Tissue-mimicking vascular flow phantoms……..….50 3.2.1 Single vessel flow Phantom………………………………………...50 3.2.2 Multi-vessel Flow phantom…………………………………….......56 3.2.3 Tumor-vasculature flow phantom………………………………60 IV. RESULTS & DISCUSSION……………………………………………………………………..68 4.1 RESULTS…………………………………………………………………………………………..68 4.1.1. Ultrasound Contrast agents………………………….………………….68 4.1.2. Detection by Ultrasound………………………………………….71 4.1.3. Tissue-mimicking vascular flow phantom experiments…………………………………………………………….73 ix 4.1.3.1. Single Vessel Flow Phantom…………………………………………….77 4.1.3.2. Multi-Vessel Flow Phantom………….……..79 4.1.3.3. Slopes vs. Flow-rate for Single vessel and multi-vessel flow phantoms………………86 4.1.4. Measurement of Intensity (Maximum Intensity, Imax) at Different flow-rat….......................................................................89 4.1.5. Ultrasound based velocity measurements…………..…….89 4.1.6. Tumor vasculature flow phantoms……………………….…..92 4.1.7. Computed Tomography (CT): Comparative study of Vessel diameter measurement…………………………………97 4.1.7.1. Data analysis…………………………………...…97 4.2. Discussion…………………………………………………………………………….103 V. Conclusions & Recommendations……………………………………………………….107 5.1 Conclusions………………………………………………………………..…………107 5.1Recommendations…………..…………………..…………………………………119 BIBLIOGRAPHY…………………………………………………………………….………………………….111 APPENDIX………………………………………………………………………………………………………..134 x LIST OF TABLES Table Page I. The B-mode Ultrasound imaging parameters……….…………………………………...55 II. The B-mode ultrasound imaging-experimental parameters for single-vessel and multi-vessel phantom………………………………………………………………………..56 III. Summary of the derived slopes of the time-intensity curves with different flow-rates and imaging planes within a single-vessel flow phantom………….78 IV. Summary of the derived slopes of the time-intensity curves with different flow-rates and imaging planes within a single-vessel flow phantom…………..80 V. The maximum intensity vs. Flow-rates in single vessel flow Phantom………..83 VI. The maximum intensity vs. Flow-rates in a multi-vessel flow Phantom…………………………………………………………………………………………………85 VII. The summary of the ultrasound based velocity measurements derived from the distance