Clinical trial NCT02489045
Conditions: Liver Diseases Portal Hypertension
Sponsor: Thomas Jefferson University
Start date: 2015-04-06; Completion date: 2019-03-31
Enrollment: 178 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This is an open-label, non-randomized trial that will be conducted at two clinical sites, Thomas Jefferson University (TJU) and the Hospital of the University of Pennsylvania (HUP). Enrolled patients undergoing trans-jugular liver biopsy with hepatic vein pressure gradient (HVPG) measurements will receive a continuous infusion of Sonazoid® (GE Healthcare, Oslo, Norway) co-infused with 0.9% NaCl solution over a 5-10 minute time period. Ultrasound imaging will be performed using a Logiq 9 scanner with a 4C transducer (GE Healthcare, Milwaukee, WI) and the novel SHAPE (subharmonic aided pressure estimation) algorithm will be used to measure pressure values in the hepatic and portal veins. Data will be stored on a PC and compared to pressure-catheter measurements, Subjects identified in the initial examination as having portal hypertension (by HVPG results) will be monitored by SHAPE for up to 18 months. These subjects typically have surveillance Computed tomography (CT) or magnetic resonance imaging (MRI) scans every 6 months to screen for liver cancer, and at those times a repeat SHAPE examination will be performed (ideally within 1 month of their clinically indicated imaging follow up appointment). In patients who undergo more frequent screening (generally 3 month intervals), SHAPE exams will be performed at 6 month intervals. Any repeat trans-jugular liver biopsies performed in this population will also trigger a repeat SHAPE study. Results of blood test evaluations (performed every 3 months in this population), medication, concomitant imaging study or procedure (including endoscopies) will be noted (all blood tests and imaging are clinically indicated only and are not required by this protocol). The end point for this part of the study will be any one new complication (e.g., liver cancer) or a marked worsening in any complication, liver transplantation, death, or the end of this clinical trial (after 3 years). The investigators expect these patients will be monitored three times during the course of this clinical trial. The time to reach the end point will be noted if a new complication or a marked worsening in any complication occurs.

Detailed description:

Arms: arm 1: 48 µl of Sonazoid microbubbles (GE Healthcare, Oslo, Norway) will be co-infused at a rate of 0.024 µl/kg body weight/minute together with a 0.9% NaCl solution infused at a rate of at least 2 ml/min.
Interventions: intervention 1: SHAPE measurement (Sonazoid ultrasoud contrast agent)
Intervention descriptions: intervention 1: Three vials with 48 µl of Sonazoid (GE Healthcare, Oslo, Norway) microbubbles (6 ml) will be prepared and drawn into a 10 ml syringe, placed in a syringe pump. Sonazoid will be co-infused at a rate of 0.024 µl/kg body weight/minute (suspension infusion rate of 0.18 ml/kg/hour) together with a 0.9% NaCl solution infused at a rate of at least 2 ml/min.

Protocol documents:
Prot_SAP_000.pdf: 
 
NONINVASIVE SUBHARMONIC AIDED PRESSURE ESTIMATION OF PORTAL 
HYPERTENSION 
 
Version 3.1 – July 21, 2014; submitted April 19, 2021 
 
NCT02489045 


 
1
PROTOCOL 
 
TITLE: NONINVASIVE SUBHARMONIC AIDED PRESSURE ESTIMATION OF PORTAL 
HYPERTENSION 
 
VERSION & DATE: Version 3.1 – July 21, 2014 
 
PRINCIPAL INVESTIGATOR:  
Flemming Forsberg, PhD 
Professor, Department of Radiology 
Thomas Jefferson University  
132 South 10th Street, 7th Floor 
Philadelphia, PA 19107 
Email address: Flemming.Forsberg@jefferson.edu 
Tel: 215-955-4870 
Fax: 215-955-8549 
 
 
KEY PERSONNEL: 
Jonathan Fenkel, MD  
Colette Shaw, MD  
 
John Eisenbrey, PhD 
Professor of Medicine, Pharmacology  
Assistant Professor  
 
Assistant Professor  
And Experimental Therapeutics  
of Radiology  
 
 
of Radiology 
Div. of Gastroenterology and Hepatology  
Department of Radiology 
 
Dept of Radiology 
Thomas Jefferson Univ 
Thomas Jefferson Univ 
 
Thomas Jefferson Univ  
 
 
Michael Soulen, MD  
Chandra Sehgal, PhD 
Professor of Radiology & Surgery 
Professor of Radiology 
Department of Radiology 
Dept of Radiology  
Hospital of the University of Pennsylvania Univ of Pennsylvania 
 
 
Medical Monitor: 
TBN 
 
Research Coordinator: TBN 
 
 
Sonographer:  TBN 
 
 
LOCATION OF STUDY: 
Thomas Jefferson University 
 
 
Hospital of the University of Pennsylvania 
132 South 10th Street, 7th Floor  
 
3400 Spruce Street 
Philadelphia, PA 19107  
 
 
Philadelphia, PA 19104 
 
STUDY DURATION: 3 years 
 
CONFIDENTIALITY STATEMENT 
This document is the confidential property of Thomas Jefferson University.  No part of it may be 
transmitted, reproduced, published, or used by other persons without the permission of Thomas Jefferson 
University. 
 
 


 
2
Contents 
SYNOPSIS .................................................................................................................................................................... 3 
1. INTRODUCTION ..................................................................................................................................................... 9 
1.1 Background .......................................................................................................................................................... 9 
1.2  Rationale ........................................................................................................................................................... 13 
2.  TRIAL OBJECTIVES ............................................................................................................................................ 14 
3.  TRIAL DESIGN ..................................................................................................................................................... 14 
3.1  Trial Duration ................................................................................................................................................... 15 
4.  TRIAL POPULATION .......................................................................................................................................... 16 
4.1  Inclusion Criteria .............................................................................................................................................. 17 
4.2  Exclusion Criteria ............................................................................................................................................. 17 
5.  MEDICATIONS ..................................................................................................................................................... 18 
5.1  Contraindications .............................................................................................................................................. 20 
5.2  Randomization .................................................................................................................................................. 21 
5.3  Blinding and Unblinding Methods ................................................................................................................... 21 
5.4  Storage .............................................................................................................................................................. 21 
6.  TRIAL PROCEDURES.......................................................................................................................................... 21 
6.1  Screening Assessments ..................................................................................................................................... 21 
6.2  Treatment Administration ................................................................................................................................. 22 
6.3  Trial Assessments ............................................................................................................................................. 23 
6.3.1  Medical History ......................................................................................................................................... 23 
6.3.2 Ultrasound Imaging .................................................................................................................................... 23 
6.4  Efficacy Assessments ....................................................................................................................................... 24 
6.5  Safety Assessments ........................................................................................................................................... 24 
6.5.1  Risks/Benefits Assessment. ....................................................................................................................... 24 
6.6.2  Adverse Events (AE) ................................................................................................................................. 25 
6.7  End-of-Treatment and End-of-Trial Evaluations .............................................................................................. 28 
6.7.1  Discontinuation of Subjects ....................................................................................................................... 28 
7.  DATA MANAGEMENT AND STATISTICAL ANALYSES .............................................................................. 29 
7.1  Data Management ............................................................................................................................................. 29 
7.2  Statistical Analyses ........................................................................................................................................... 29 
7.2.1  Hypotheses ................................................................................................................................................. 29 
7.2.2  Analysis of Results .................................................................................................................................... 30 
7.2.3  Sample Size Calculation ............................................................................................................................ 30 
7.2.4 Disposition of Software .............................................................................................................................. 32 
8.  REFERENCES ....................................................................................................................................................... 33 
APPENDIX A - INVESTIGATOR OBLIGATIONS ................................................................................................. 34 
APPENDIX B - GE Provided Study Specific Protocol Supplement on Sonazoid Suspension and Infusion .............. 37 
 
 


 
3
SYNOPSIS 
 
Protocol Title: Noninvasive Subharmonic Aided Pressure Estimation of Portal Hypertension 
 
Trial Objectives: The primary objectives of this trial are: 
 
• 
Evaluate the use of in vivo subharmonic aided pressure estimation (SHAPE) as a first qualitative 
screening modality for determining the presence of portal hypertension in patients undergoing a 
transjugular liver biopsy compared to catheter based pressure measurements (as the reference 
standard) 
• 
Determine if SHAPE measurements can provide a quantitative, noninvasive measurement of 
hepatic venous pressure gradient (HVPG) to monitor disease progression or treatment response in 
patients identified with portal hypertension by comparing the results to repeat biopsies and/or 
clinical outcomes 
 
Trial Design: This is an open-label, non-randomized trial that will be conducted at two clinical sites, 
Thomas Jefferson University (TJU) and the Hospital of the University of Pennsylvania (HUP).  Enrolled 
patients undergoing trans-jugular liver biopsy with hepatic vein pressure gradient (HVPG) measurements 
will receive a continuous infusion of Sonazoid® (GE Healthcare, Oslo, Norway) co-infused with 0.9% 
NaCl solution over a 5-10 minute time period using the setup and infusion rates described in GE 
Healthcare (GEHC) Protocol Supplement provided in Appendix B.  Ultrasound imaging will be 
performed using a Logiq 9 scanner with a 4C transducer (GE Healthcare, Milwaukee, WI) and the 
SHAPE algorithm will be used to measure pressure values in the hepatic and portal veins.  Data will be 
stored on a PC and compared to pressure-catheter measurements,  Subjects identified in the initial 
examination as having portal hypertension (by HVPG results) will be monitored by SHAPE for up to 18 
months.  These subjects typically have surveillance CT or MRI scans every 6 months to screen for liver 
cancer, and at those times a repeat SHAPE examination will be performed (ideally within 1 month of their 
clinically indicated imaging follow up appointment).  In patients who undergo more frequent screening 
(generally 3 month intervals), SHAPE exams will be performed at 6 month intervals. Any repeat trans-
jugular liver biopsies performed in this population will also trigger a repeat SHAPE study.  Results of 
blood test evaluations (performed every 3 months in this population), medication, concomitant imaging 
study or procedure (including endoscopies) will be noted (all blood tests and imaging are clinically 
indicated only and are not required by this protocol).  The end point for this part of the study will be any 
one new complication (e.g., liver cancer) or a marked worsening in any complication, liver 


 
4
transplantation, death, or the end of this clinical trial (after 3 years).  We expect these patients will be 
monitored three times during the course of this clinical trial.  The time to reach the end point will be noted 
if a new complication or a marked worsening in any complication occurs. 
 
 
Trial Population: This trial will consist of up to 300 adults (21 years of age or older) undergoing trans-
jugular liver biopsy with HVPG measurement at TJU or at HUP. 
 
Trial Procedures:  Subjects eligible for trial enrollment will be identified by the co-investigators, Dr. 
Fenkel (at TJU) and Dr. Soulen (at HUP) from their patient population of subjects scheduled for a trans-
jugular liver biopsy.  The research coordinators for this study will explain the study to the patients at the 
respective clinical sites.  The patient will be given time to consider the risks and benefits of the study and 
ask questions about participation.  The coordinator will review the consent form with the patient and then 
the patient will be given the form to review.  The patient, coordinator, and a study investigator will all 
sign the consent form.  The patient will be given a copy of the signed consent form for their records. 
 
After consenting to participate in this study, a full demographic profile, known drug allergies or 
intolerances, and review of the subject’s medical/surgical history will be recorded.  Patients with known 
cardiac shunts will be excluded from the study.  If the subject is a woman of childbearing potential, she 
will have a urine pregnancy test (the results of which will be made available to the subject prior to study 
initiation). 
 
Whenever a subject undergoes a trans-jugular liver biopsy with transcatheter free and wedged hepatic 
venous pressure measurements as part of their standard clinical care and agrees to participate in the study, 
we will perform the corresponding SHAPE measurement within the 3 hours of post-recovery observation 
to ease transportation for patients and ensure that hydration status and other variables, which could 
potentially affect portal pressures, are equivalent.  Administration of Sonazoid will be performed under 


 
5
direct supervision of a medical doctor.  Additionally, resuscitation equipment will be in immediate 
proximity to the patient during Sonazoid infusion.  Three vials (6 ml) of Sonazoid microbubbles (16 μl 
per vial when reconstituted) will be prepared for each subject by resuspending each vial in 2 ml of water 
for injection, supplied by GEHC, as described in the instructions provided by GEHC (Appendix B).  
Infusions will be performed as described in Appendix B.  All materials and supplies used for the infusion 
procedure will be identical to those described in GE Healthcare’s current Sonazoid IND.  Briefly, 
anintravenous  cannula will be placed in a vein in the subject’s arm.  A 0.9% NaCl solution will be started 
and used to fill up the connecting tubes before being connected to a 3-way stopcock.  The stopcock will 
then be connected to the extension tubing leading to the cannula.  All three vials of suspended Sonazoid 
will be drawn into a 10 ml syringe, placed in a syringe pump at the same level or below the patient, and 
connected directly to the stopcock.  After the stopcock to the 0.9% NaCl solution has been opened, the 
saline solution will be infused at a rate of at least 2 ml/min.  Then, Sonazoid will be co-infused at the 
target rate of 0.024 µl/kg body weight/minute (suspension infusion rate of 0.18 ml/kg/hour), but the rate 
may be reduced if necessary to achieve optimal levels of contrast.  This infusion rate was selected based 
on our group’s previous experiences with Sonazoid infusion in human subjects in general [Halpern et al. 
2002; Landmark et al. 2008] and in SHAPE portal hypertension subjects in particular [Eisenbrey et al. 
2013].  This dosing scheme is designed to avoid (or limit) the number of cases that has to be discarded 
due to insignificant enhancement in the hepatic veins (as in our pilot study; Eisenbrey et al. 2013). The 
dosage is based off the GEHC provided, study specific resuspension and infusion instructions (Appendix 
B) and the total Sonazoid dose given over a maximum of 15 minutes (0.36 µl/kg) will be well below the 
maximal tested dose which was still found safe in Phase I clinical trials (2.7 µl of microbubbles/kg in PBI 
0001 as detailed in the Sonazoid Investigator’s Brochure).  The duration of contrast agent infusion will 
range from 5 to 10 minutes. 
 
After beginning the contrast infusion, a modified Logiq 9 scanner (GE Healthcare) with a broad 
bandwidth curvi-linear array (model 4C) will be used to obtain RF data (for SHAPE measurements) 
within the hepatic and portal veins.  Both the experimental machine and software will be labeled as an 


 
6
investigational device with a GRC provided label prior to beginning any clinical experiments (21 CRF 
812.5).  Once the infusion of Sonazoid has started, ultrasound imaging will be performed with the 4C 
curved linear array to guide SHAPE ROI placement into the portal vein (while also visualizing a hepatic 
vein).  The SHAPE optimization algorithm will be activated ([Dave et al. 2013]) and the acoustic power 
will be adjusted to produce the maximum change in subharmonic amplitudes (i.e., maximizing the 
sensitivity of SHAPE).  Then the ROI will be increased to visualize the hepatic and the portal veins, and 
the corresponding subharmonic data will be acquired at the optimal acoustic power setting in 5 s segments 
during the infusion of the Sonazoid suspension.  Ultrasound imaging will be performed at a transmit 
frequency of 2.5 MHz and the subharmonic obtained at 1.25 MHz.  All measurements will be repeated 
three times.  The depths, sizes, and locations of the portal and hepatic veins will be determined by the 
sonographer and saved in a reference image. 
 
The proposed agent for the study, Sonazoid (GE Healthcare,), is a sterile non-pyrogenic suspension of 
lipid stabilized perfluorobutane microbubbles for contrast-enhancement, with a median diameter between 
2.4 and 3.5 µm (Forsberg et al. 2008).  Previous in vitro results have shown Sonazoid to be superior for 
SHAPE measurements relative to other ultrasound contrast agents (Halldorsdottir et al. 2011).  The 
United States Food and Drug Administration (FDA) has yet to approve Sonazoid for human use in this 
country.  Consequently, we intend to apply to the FDA for an investigator initiated IND (investigational 
new drug) to cover the use of Sonazoid in patients undergoing a trans-jugular liver biopsy.  It should be 
pointed out that the safety of Sonazoid has already been established in numerous animal studies and in 
human studies for the evaluation of the liver and heart both in this country and in Europe (Landmark et al. 
2008).  Sonazoid has been approved for use in patients with liver lesions in Japan and has been marketed 
in that country since early 2007, and has been approved more recently in South Korea (Bouakaz et al. 
2007; Jang et al. 2013.  Finally, no adverse reactions attributable to contrast agent infusions were 
observed in our pilot study (Eisenbrey et al. 2013). 
 
All examinations will be recorded.  During the examination, the subharmonic pressure estimates will be 


 
7
displayed on the Logiq 9 scanner, these estimates will be noted down and the data will be stored on a PC.  
Data will be compared to the transcatheter portal pressure measurements, which comprise the free and 
wedged hepatic venous pressure (FHVP and WHVP, respectively) and the hepatic vein pressure gradient 
(HVPG; measured as the difference between the WHVP and FHVP.  Emergency services (e.g., a crash 
cart) will be available within the hospital in case of any acute adverse reactions.  Subjects will be 
monitored for adverse reactions 2 hours post biopsy (roughly 1 hour post Sonazoid injection), again as a 
standard of care procedure.  Patients will be monitored for a minimum of one hour post Sonazoid 
administration.  During this time a trained CPR personnel and resuscitation equipment will be available.  
SHAPE data will later be compared to all clinical variables obtained from the patient to determine 
potential correlations to liver dysfunction. 
 
Statistical Methodology:  This project is a large clinical trial to further develop SHAPE and to employ 
this method to conduct a large scale human clinical trial to conclusively establish the accuracy of SHAPE 
for the assessment of portal hypertension.  Our statistical analysis will address following major questions: 
1. Can patients with HVPG of 10 mmHg or greater be identified using SHAPE? The fundamental 
hypothesis is that a sensitivity of 89% and a specificity of 81 % can be achieved by using 
SHAPE measurements to identify patients with HVPGs of 10 mmHg or greater. 
2. Can portal vein pressures (in the form of HVPGs) in patients with liver disease be quantified 
accurately using SHAPE?  The fundamental hypothesis is that a correlation coefficient of 0.82 
can be achieved between in vivo SHAPE and HVPG measurements. 
3. Can SHAPE accurately monitor disease progression or treatment response in patients identified 
with portal hypertension?  The fundamental hypothesis is that correlation coefficients above 0.90 
can be achieved between SHAPE and repeat HVPG results and that SHAPE will significantly 
differentiate responders from non-responders (using clinical outcomes as the reference standard). 
 
The findings of SHAPE will be correlated to the invasive catheter based measurements of portal vein 
blood pressure (i.e., HVPG) using least squares, multiple linear regression analysis with robust standard 
errors.  Comparisons of sensitivities, specificities and accuracies for diagnosing portal hypertension with 
SHAPE will be conducted with an unpaired Student’s t-test (using HVPG measurements as the reference 
standard).  Receiver operating characteristic (ROC) curves will be computed using a continuous scale 


 
8
with HVPG cutoff levels of 10 and 12 mmHg.  The optimal operating point (OOP) will be determined as 
the point closest to ‘0,1’ on the ROC curve (representing 100% sensitivity and 100% specificity).  All 
clinical variables (e.g., concomitant imaging results, presence of cancer or ascites, blood tests, fibrosis 
and MELD scores etc.) will also be compared to the SHAPE values.  When both types of variables are 
ordinal or continuous, correlations will be calculated.  When one type of variable is nominal and one 
continuous non-parametric rank order tests such as Mann-Whitney U-tests or Kruskal-Wallis tests will be 
performed [Rosner 1990].  All of the statistical analyses proposed for the human clinical trial will be 
repeated split by racial and ethnic groups to determine if clinically important race/ethnicity differences 
exist in the ability of SHAPE to diagnose portal hypertension. 
 
All analyses and computations will be performed using NCSS/PASS 2008 and Stata 12.0 (Stata 
Corporation, College Station, TX), while the study database will be designed and implemented in 
Filemaker Pro 10.0 (Filemaker Inc, Santa Clara, CA).  This database will contain all patient information 
(except names and other identifiers), including results of biopsies, HVPG and SHAPE measurements as 
well as the clinical variables. 


 
9
1. INTRODUCTION 
 
There is clearly a well-established clinical need to accurately measure the hepatic vein pressure gradient 
(HVPG) (Garcia-Tsao 1985).  Unfortunately, current techniques are either invasive, such as catheter 
based pressure transducers, which have to be introduced directly into the hepatic vasculature, or 
noninvasive but indirect and qualitative, such as using CT, MRI or ultrasound imaging to diagnose liver 
cirrhosis (Kudo et al. 2008).  However, the diagnostic accuracy of CT, MRI and ultrasound for grading 
cirrhosis into 3 categories (chronic hepatitis, cirrhosis and pre-cirrhosis) was only between 64 and 70% in 
a recent multi-center study (Kudo et al. 2008) and this is still only a very indirect measure of HVPG. 
 
Our group has proposed and patented a novel and innovative technique called subharmonic-aided 
pressure estimation (SHAPE) for noninvasive pressure measurements (Forsberg et al. 2005; Shi et al. 
1999; 2001).  We have shown hydrostatic pressure can be measured through variations of the 
subharmonic amplitude of ultrasound contrast agents during insonation.  Our group has just completed a 
first-in-humans clinical trial of SHAPE in 45 patients with chronic liver disease supported by RC1 
DK087365 [Eisenbrey et al. 2013].  The SHAPE gradient between the portal and hepatic veins was in 
good overall agreement with the HVPG (R = 0.82) indicating SHAPE may be a useful tool for the 
diagnosis of portal hypertension.  This project will serve as a large clinical trial to further develop SHAPE 
and to employ this method to conduct a large scale human clinical trial to conclusively establish the 
accuracy of SHAPE for the assessment of portal hypertension. 
 
1.1 Background 
 
Portal hypertension is defined as an increase in the pressure gradient between the portal vein and hepatic 
veins or the inferior vena cava (IVC) exceeding 5 mmHg (Sanyal et al. 2008).  Cirrhosis (prevalence of 2-
10%) accounts for at least 90% of cases of portal hypertension in the developed world.  From a 
pathophysiological perspective, the increase in portal venous pressure results from a sequence of events 
that begins with an increase in resistance to portal outflow, into the liver (Navarro et al. 2007).  This is the 
result of two factors; architectural distortion of the liver parenchyma, as occurs in the accumulation of 


 
10 
fibrosis, and vasoconstriction of the hepatic microcirculation.  The rise in portal pressure is largely 
asymptomatic until the body mounts a homeostatic response to systemic arterial vasodilatation.  
Specifically, systemic vasodilatation yields predictable physiological consequences, including systemic 
arterial hypotension, retention of salt and water and the formation of ascites, and renal insufficiency.  In 
addition, up to 90% of the portal flow can be diverted into the systemic circulation through portosystemic 
collaterals (Sanyal et al. 2008). 
 
Cirrhosis due to most causes, without the clinical manifestations of portal hypertension, has a small effect 
on mortality.  However, it is the manifestations of portal hypertension which greatly impact survival.  
Common complications of portal hypertension include gastroesophageal varices, ascites, and 
portosystemic encephalopathy.  Patients who develop ascites have a 50% two year mortality and those 
who develop spontaneous infection of the ascites fluid carry a 70% one year mortality.  Patients with 
cirrhosis have a 5-10% yearly incidence of gastroesophageal variceal formation, and a 4-15% yearly 
incidence of bleeding, with larger varices having a greater risk for bleeding (D’Amico et al. 2006).  
Although the mortality associated with each bleeding event has fallen significantly due to endoscopic, 
pharmacological, and clinical care interventions, each bleeding episode continues to carry up to a 20% 
risk of death (D’Amico et al. 2006; Navarro et al. 2007; Sanyal et al. 2008). 
 
In patients with chronic liver disease, transcatheter portal pressure measurement represents the “gold 
standard”, albeit indirect, for assessment of portal hypertension.  The wedge hepatic venous pressure 
(WHVP) reflects values of portal venous pressure in cirrhosis (Sanyal et al. 2008).  Another useful 
parameter is the gradient between the portal vein and the hepatic vein pressure, the hepatic vein pressure 
gradient (HVPG).  The value of HVPG in assessing and monitoring several of the complications of portal 
hypertension is well established (D’Amico et al. 2006; Sanyal et al. 2008).  It has also been found that an 
HVPG above 10 mmHg predicts the presence of varices, while an HVPG of 12 mmHg or more is 
required for varices to bleed (Garcia-Tsao 1985). 


 
11 
 
Variceal hemorrhage represents one of the most dramatic and immediately life threatening complications 
of portal hypertension and while the mortality rate has declined over the last decade it still ranges from 15 
to 20% (Sanyal et al. 2008).  Clinical management depends upon preventing an initial bleed when varices 
are present, intervening to stop active bleeding, and providing measures to prevent repeat bleeding.  A 
sustained reduction in the HVPG to below 12 mmHg is the best predictor of successful reduction in the 
risk of bleeding with non-selective β-blockers.  Likewise, a reduction in the risk of re-bleeding from 
varices is associated with a reduction in the HVPG of more than 20% from baseline or to less than 12 
mmHg.  Finally, the HVPG has been evaluated as a prognostic marker for disease progression, survival 
and mortality (Kalambokis et al. 2007).  Most studies found that the HVPG, when added to other 
measures such as the Child Pugh or MELD scores, increased the ability to predict survival. 
 
Consequently, our group has proposed and patented a novel and innovative technique called 
subharmonic-aided pressure estimation (SHAPE) for noninvasive pressure measurements (Forsberg et al. 
2005; Shi et al. 1999; 2001).  An accurate marker for portal hypertension would impact literally millions 
of Americans with liver disease.  Such a marker could be used to predict the presence of portal 
hypertension and its complications (in particular varices) and might also be used to predict morbidity and 
mortality.  Moreover, the value of HVPG measurements in the management of hepatocellular cancer 
(HCC) has been demonstrated.  The noninvasive and accurate measurement of HVPG would also be 
clinically useful for trans-jugular intrahepatic portosystemic shunt (TIPS) procedures.  TIPS are 
frequently performed for patients with intractable ascites not responding to medications or variceal 
bleeding which does not respond to pharmacological or endoscopic means.  Patients with suspected 
variceal bleeding routinely undergo ultrasound examination to evaluate patency of the portal venous 
branches and determine whether TIPS placement is feasible.  Getting a reliable estimate of HVPG in 
these patients with SHAPE would expedite therapy.  SHAPE may also assist in the evaluation of 
ultrasound findings at TIPS follow-up studies, which can be technically challenging. 


 
12 
The proposed agent for the current study, Sonazoid® (GE Healthcare, Oslo, Norway), is a sterile non-
pyrogenic suspension of lipid stabilized perfluorobutane microbubbles for contrast-enhancement, with a 
median diameter between 2.4 and 3.5 µm (Forsberg et al. 2008).  Previous in vitro results have shown that 
Sonazoid is superior for SHAPE measurements relative to other ultrasound contrast agents (Halldorsdottir 
et al. 2011).  The FDA has yet to approve Sonazoid for human use in this country.  Consequently, we 
intend to apply to the FDA for an investigator initiated IND to cover the use of Sonazoid in patients 
undergoing a trans-jugular liver biopsy.  It should be pointed out that the safety of Sonazoid has already 
been established in numerous animal studies and in human studies for the evaluation of the liver and heart 
both in this country and in Europe (Landmark et al. 2008).  Sonazoid has been approved for use in 
patients with liver lesions in Japan and has been marketed in that country since early 2007 and more 
recently in South Korea (Bouakaz et al. 2007; Jang et al. 2013).  Finally, no adverse reactions attributable 
to contrast agent infusions were observed in our pilot study (Eisenbrey et al. 2013). 
 
 
Sonazoid Clinical Safety 
 
Sonazoid was administered in clinical trials in 1699 patients.  In these patients 404 (23.8%) reported at 
least one adverse event, while 12 of 62 (19.4%) of patients receiving a placebo reported adverse events. 
 
Forty-three (2.5 %, 43/1699) subjects experienced serious adverse events, including 13 deaths.  None of 
these events were considered by the investigators to be related to Sonazoid.  All the serious adverse 
events were considered to be caused by the underlying disease or related treatment.  In addition, there 
were no clinically significant trends in laboratory tests, vital signs, ECGs, or physical examination 
findings. 
 
The most commonly noted adverse events were headache (3.6%, 62/1699), chest pain (2.3%, 39/1699), 
abdominal pain (1.5%, 25/1699), diarrhea (1.5%, 25/1699), and nausea (1.6%, 28/1699). The majority of 
adverse events were mild to moderate in severity (92.6%, 652/704). 


 
13 
Table 1. 
Selected Adverse Events Reported in ≥ 0.5% of the Subjects who Received Sonazoid in Controlled 
Clinical Studies (% of 1699) 
 
No. of Patients Exposed to Sonazoid 
 
1699 
 
 
No. of Patients Reporting an Adverse Event 
404  
(23.8%) 
Application Site Disorders 
9 
(0.5%) 
Body as a Whole 
91  
(5.4%) 
 
Back Pain 
10  
(0.6%) 
 
Chest Pain 
39  
(2.3%) 
Cardiovascular System, General 
45 
(2.6%) 
 
ECG Abnormal Specific 
15  
(0.9%) 
 
Hypotention 
9  
(0.5%) 
Nervous 
94 
(5.5%) 
 
Dizziness 
18          (1.1%) 
 
Headache 
62          (3.6%)  
Gastrointestinal 
126 
(7.4%) 
 
Abdominal Pain 
25          (1.5%) 
 
Diarrhea 
25          (1.5%) 
 
Nausea 
28           (1.6%) 
 
Vomiting 
21           (1.2%) 
Hear Rate & Rhythm 
55           (3.2%) 
Liver and Billiary System 
22           (1.3%) 
 
SGOT/AST increased 
9 
(0.5%) 
Metabolic & Nutritional 
42 
(2.5%) 
 
Increased LDH 
17 
(1.0%) 
Musculo-Skeletal System 
18 
(1.1%) 
Myo-, Endo-, Pericardial & Valve 
29 
(1.7%) 
 
Angina Pectoris 
14 
(0.8%) 
Platelet, Bleeding & Clotting 
21 
(1.2%) 
Psychiatric 
19 
(1.1%) 
Respiratory System 
58 
(3.4%) 
 
Dyspnoea 
19 
(1.1%) 
Skin & Appendages 
18 
(1.1%) 
Urinary System 
31 
(1.8%) 
 
Albuminuria 
17 
(1.0%) 
White Cell & RES 
9 
(0.5%) 
  
Additional information concerning pre-clinical and clinical experience with Sonazoid, including the 
dosing levels and reported subject complaints, can be found in the Sonazoid investigational drug brochure 
(Version 7, October 4th 2002). 
 
1.2  Rationale 
 


 
14 
The quantitative aspect of this project is based on the assumption that the magnitude of subharmonic 
signals from insonated contrast agents depend on the hydrostatic pressure of the surrounding media.  
SHAPE estimates internal pressure variations by transmitting at one frequency, receiving at its 
subharmonic frequency and then monitoring the subharmonic amplitude variations from the contrast 
bubbles.  The optimal sensitivity for SHAPE was determined in vitro to be at a transmit frequency of 2.5 
MHz (i.e., receiving at 1.25 MHz) at a 0.35 MPa acoustic pressure using the contrast agent Sonazoid (GE 
Healthcare, Oslo, Norway), which declined approximately 14.4 dB over a 0 to 186 mmHg pressure range. 
 
We propose a large clinical trial at two sites to further develop SHAPE and to employ this method to 
conduct a large scale human clinical trial to conclusively establish the accuracy of SHAPE for the 
assessment of portal hypertension.  The purpose of this study is to evaluate if SHAPE measurements may 
be used as a first qualitative screening modality for determining the presence of portal hypertension in 
patients undergoing a transjugular liver biopsy and determine if SHAPE measurements may be used to 
monitor disease progression or treatment response for patients diagnosed with portal hypertension.  
 
 
2.  TRIAL OBJECTIVES 
 
 
Trial Objectives: The primary objectives of this trial are: 
 
• 
Evaluate the use of in vivo subharmonic aided pressure estimation (SHAPE) as a first qualitative 
screening modality for determining the presence of portal hypertension in patients undergoing a 
transjugular liver biopsy compared to catheter based pressure measurements (as the reference 
standard) 
• 
Determine if SHAPE measurements can provide a quantitative, noninvasive measurement of 
hepatic venous pressure gradient (HVPG) to monitor disease progression or treatment response in 
patients identified with portal hypertension by comparing the results to repeat biopsies and/or 
clinical outcomes. 
 
 
3.  TRIAL DESIGN 
 
This is an open-label, non-randomized trial that will be conducted at two clinical sites, Thomas Jefferson 


 
15 
University (TJU) and the Hospital of the University of Pennsylvania (HUP).  Enrolled patients 
undergoing trans-jugular liver biopsy will receive a continuous infusion of Sonazoid® (GE Healthcare, 
Oslo, Norway) co-infused with 0.9% NaCl solution over a 5-10 minute time period using the setup and 
infusion rates described in GE Healthcare (GEHC) Protocol Supplement provided in Appendix B.  
Ultrasound imaging will be performed using a Logiq 9 scanner with a 4C transducer (GE Healthcare, 
Milwaukee, WI) and the SHAPE algorithm will be used to measure pressure values in the hepatic and 
portal veins.  Data will be stored on a PC and compared to pressure-catheter measurements, which are 
taken as part of the standard of care for all patients undergoing a trans-jugular biopsy.  Subjects identified 
in the initial examination as having portal hypertension (by HVPG results) will be monitored by SHAPE 
for up to 18 months.  These subjects typically have surveillance CT or MRI scans every 6 months to 
check for liver cancer and at those times a repeat SHAPE examination will be performed (ideally within 2 
weeks).  Any repeat transjugular biopsies performed in this population will also trigger a repeat SHAPE 
study. Results of blood test evaluations (performed every 3 months in this population), medication, 
concomitant imaging study or procedure (including endoscopies) done as standard of care will be noted.  
The end point for this part of the study will be any one new complication (e.g., liver cancer) or a marked 
worsening in any complication, liver transplantation, death, or the end of the clinical trial participation (up 
to 18 months).  On an average, these patients will be monitored three times during the course of this 
clinical trial. 
A full demographic profile, known drug allergies or intolerances, and review of the subject’s 
medical/surgical history will be recorded.  If the subject is a woman of childbearing potential, she will 
have a urine pregnancy test (the results of which will be made available to the subject prior to study 
initiation). 
 
 
3.1  Trial Duration 
 
Individual participation in this trial will be limited to ultrasound imaging studies within the two hour 
period following a trans-jugular liver biopsy (during which their hepatic wedge pressure will be measured 


 
16 
with a pressure catheter as part of the standard of care at TJU and HUP).  Patients are monitored for two 
hours (1 hour post Sonazoid injection).  The entire ultrasound imaging protocol will require 
approximately one hour.  Subjects identified in the initial examination as having portal hypertension (by 
HVPG results) will be monitored by SHAPE for up to 18 months.  These subjects typically have 
surveillance CT or MRI scans every 6 months to check for liver cancer and at those times a repeat 
SHAPE examination will be performed.  Any repeat trans-jugular biopsies performed in this population 
will also trigger a repeat SHAPE study.  Moreover, these patients will have a complete blood test every 3 
months and these clinical variables (platelets, albumin, MELD, etc.) as well as any medication will be 
recorded.  Finally, most subjects with portal hypertension will undergo an endoscopy examination every 1 
to 2 years [de Franchis 2005; Merkel et al. 2004].  Results of any concomitant imaging study or procedure 
(including endoscopies) will be noted.  The end point for this part of the study will be any one new 
complication (e.g., liver cancer) or a marked worsening in any complication (e.g., MELD scores), liver 
transplantation, death, or the end of this clinical trial (after 3 years).  On an average, these patients will be 
monitored three times during the course of this clinical trial.  The time to reach the end point will be noted 
if a new complication or a marked worsening in any complication occurs. 
 
Subject recruitment is expected to last 3 years (April, 2014 – January, 2017). This includes monitoring 
disease progression and/or treatment response.  Analysis and publication of results are expected to take an 
additional 2 months (January– March, 2017).  Volunteer Registry Database forms will be submitted to the 
National Institute of Health’s Office of Regulatory Compliance and Quality at the completion of the 
research study. 
 
4.  TRIAL POPULATION 
 
Trial Population: This trial will consist of up to 300 adults (21 years of age or older) undergoing trans-
jugular liver biopsy with HVPG measurement at TJU or at HUP. 
 


 
17 
Subjects eligible for trial enrollment will be identified by the co-investigators, Dr. Fenkel (at TJU) and 
Dr. Soulen (at HUP) from their patient population of subjects scheduled for a trans-jugular liver biopsy.  
The research coordinators for this study will explain the study to the patients at the respective clinical 
sites.  The patient will be given time to consider the risks and benefits of the study and ask questions 
about participation.  The coordinator will review the consent form with the patient and then the patient 
will be given the form to review.  The patient, coordinator, and a study investigator will all sign the 
consent form.  The patient will be given a copy of the signed consent form for their records. 
 
4.1  Inclusion Criteria 
All subjects accepted for this trial must fulfill all the following criteria: 
• 
Be scheduled for trans-jugular liver biopsy the day of the ultrasound procedure. 
• 
Be at least 21 years of age. 
• 
Be medically stable. 
• 
If a female of child-bearing potential, must have a negative pregnancy test. 
• 
Be conscious and able to comply with study procedures. 
• 
Have read and signed the IRB-approved Informed Consent form for participating in the study. 
 
4.2  Exclusion Criteria 
 
Subjects who fulfill any of the following conditions or who have had the following procedures will be 
excluded from this trial: 
• 
Females who are pregnant or nursing. 
• 
Patients not scheduled for trans-jugular liver biopsy 
• 
Patients who have received an investigational drug in the 30 days before study drug 
administration, or will receive one within 72 h afterwards,. 
• 
Patients with known or suspected right-to-left, bi-directional, or transient right-to-left cardiac 
shunts 
• 
Patients with pulmonary hypertension or unstable cardiopulmonary conditions 
• 
Patients currently on chemotherapy or with other primary cancers requiring systemic or hepatic 
loco-regional treatment. 
• 
Patients who are medically unstable, patients who are seriously or terminally ill, and patients 
whose clinical course is unpredictable.  For example: 
• 
Patients on life support or in a critical care unit. 
• 
Patients with unstable occlusive disease (e.g., crescendo angina) 
• 
Patients with clinically unstable cardiac arrhythmias, such as recurrent ventricular 
tachycardia. 
• 
Patients with uncontrolled congestive heart failure (NYHA Class IV) 
• 
Patients with recent cerebral hemorrhage. 
• 
Patients who have undergone surgery within 24 hours prior to the study sonographic 
examination. 
• 
Patients with a history of anaphylactic allergy to eggs or egg products, manifested by one or more 


 
18 
of the following symptoms: generalized urticaria, difficulty in breathing, swelling of the mouth 
and throat, hypotension, or shock.  (Subjects with nonanaphylactic allergies to eggs or egg 
products may be enrolled in the study, but must be watched carefully for 1 h following the 
administration of SONAZOID). 
• 
Patients with congenital heart defects. 
• 
Patients with severe emphysema, pulmonary vasculitis, or a history of pulmonary emboli. 
• 
Patients with respiratory distress syndrome  
• 
Patients with thrombosis within the hepatic, portal, or mesenteric veins. 
 
Subject identification will be maintained with a study specific alphanumeric code including the institution 
(TJU or HUP), patient number (001 and onwards at both the sites) and the patient’s initials.   
 
5.  MEDICATIONS 
 
Sonazoid will be provided by GEHC (Oslo, Norway).  An FDA Sponsor-Investigator IND application 
will be submitted to the FDA and studies will not commence until regulatory approval has been obtained 
from the FDA as well as the IRB committee for TJU and HUP. 
 
Sonazoid is a sterile non-pyrogenic suspension of microspheres of lipid stabilized perfluorobutane (PFB) 
for contrast-enhancement, with a median diameter between 2.4 and 3.5 µm and less than 0.1 % larger than 
7 µm.  Sonazoid is formulated as a powder for injection consisting of lyophilized sucrose entrapping 
hydrogenated egg phosphatidyl serine (HEPS) PFB microspheres under a PFB headspace.  Each milliliter 
of Sonazoid contains roughly 1.2 billion microspheres. 
 
Sonazoid is supplied as a dry powder within 10 ml sealed vials.  The headspace of the vials contains PFB.   
Three vials with 48 µl of Sonazoid microbubbles (6 ml) will be prepared for each subject by resuspending 
each vial in 2 ml of injection grade water supplied by GEHC, as described in Appendix B.  Infusions will 
be performed as described in the Instructions for Use protocol supplement provided by GEHC (Appendix 
B). All materials and supplies used for the infusion procedure will be identical to those described in GE 
Healthcare’s current Sonazoid IND. A diagram of this infusion setup is shown below in Figure 1. 
 


 
19 
 
Figure 1: Setup used for Sonazoid infusion for all patients during SHAPE imaging. This setup is 
discussed briefly in section 5 and in detail in the attached GEHC provided infusion protocol (Appendix 
B). 
 
Briefly, a 20-22 gauge cannula will be placed in a vein in the subject’s right or left arm. A 0.9%NaCl 
solution will be started and used to fill up the connecting tubes before being connected to a 3-way 
stopcock.  The stopcock will then be connected to the extension tubing leading to the cannula.  All three 
Extension set 
100cm 
Infusion of  
NaCl 
(2 mL/min.) 
Syringe pump 
Reconstituted 
SONAZOID 
3-way 
stopcock 
SONAZOID 
infusion 
Clip 
Venflo


 
20 
vials of suspended Sonazoid will be drawn into a 10 ml syringe, placed in a syringe pump at the same 
level or below the patient, and connected directly to the stopcock.  After the stopcock has been opened, 
the NaCl solution will be infused at a rate of at least 2 ml/min, and Sonazoid will be infused at the desired 
rate of 0.024 µl/kg body weight/minute (suspension infusion rate of 0.18 ml/kg/hour), but may be reduced 
if necessary to achieve optimal levels of contrast.  This infusion rate was selected based on our group’s 
previous experiences with Sonazoid infusion in human subjects in general [Halpern et al. 2002; Landmark 
et al. 2008] and in SHAPE portal hypertension subjects in particular [Eisenbrey et al. 2013].  This dosing 
scheme is designed to avoid (or limit) the number of cases that has to be discarded due to insignificant 
enhancement in the hepatic veins (as in our pilot study; Eisenbrey et al. 2013).  This dosage is based on 
GEHC’s previous experience of dosing Sonazoid by infusion.  The maximum amount of microbubbles 
infused over 15 minutes (0.36 µl/kg) is well below the maximal tested dose which was still found safe in 
Phase I clinical trials (2.7 µl of microbubbles/kg in PBI 0001 as detailed in the Sonazoid Investigator’s 
Brochure). 
Sonazoid will be stored in a secure cabinet, with only the study investigators and research personnel 
having access.  New vials must be prepared for each subject's contrast administration.  Unused drug and 
empty vials will be properly disposed of after reconciling in the log of study drug. 
 
5.1  Contraindications 
Sonazoid should not be administered to patients with known or suspected hypersensistivity to egg 
phosphatidyl serine.  Patients with a history of anaphylactic allergy to eggs or egg products will be 
excluded from the study.  
 
The safety of ultrasound contrast agents in patients with cardiac shunts has recently been called into 
question.  Therefore, patients with known or suspected right-to-left, bi-directional, or transient right-to-
left cardiac shunts will be excluded from this study. 
 


 
21 
The safety of Sonazoid in patients with 1) severe emphysema, pulmonary vasculitis or a history of 
pulmonary emboli; and 2) respiratory distress syndrome has not been studied.  Therefore, patients with 
any of these conditions will be excluded from participation.  Additional exclusion criteria are listed in 
section 4.2. 
 
5.2  Randomization 
 
This is a non-randomized trial; therefore, no randomization procedure is required. 
 
5.3  Blinding and Unblinding Methods 
 
This is an open-label trial; therefore, no blinding or unblinding procedures for the trial drug are required. 
 
5.4  Storage 
 
Sonazoid vials will be stored in a secure cabinet, with only the study investigators and research personnel 
having access.  The study researchers will be responsible for drug reconstitution and inventory control. 
Sonazoid is stable for 8 hours after reconstitution.  If the agent is suspended and not used within 8 hours it 
will be discarded. 
 
 
6.  TRIAL PROCEDURES 
 
6.1  Screening Assessments 
Screening assessments will be performed within 4 weeks prior to the administration of Sonazoid.  All 
subjects will receive a written consent form and a verbal explanation of the trial by the investigator and/or 
the research study coordinator and will be asked to sign the written informed consent.  Trial participants 
will have the presence of inclusion criteria and absence of exclusion criteria verified by providing a 
comprehensive medical history, which will include screening assessments for their scheduled trans-
jugular liver biopsy.  Patients with cardiac shunts will be excluded from the study. 
 


 
22 
6.2  Treatment Administration 
 
Administration of Sonazoid will be performed under direct supervision of a medical doctor.  Emergency 
services (e.g., a crash cart) will be available within the hospital in case of any acute adverse reactions. 
Additionally, resuscitation equipment will be in immediate proximity to the patient during Sonazoid 
infusion.  Patients will be monitored for AEs post Sonazoid treatment for a minimum of one hour. 
Trained CPR personnel and resuscitation equipment will be in attendance during this monitoring period.  
A dose of three vials with 48 µl of Sonazoid microbubbles (6 ml total after resuspension) will be prepared 
for each subject by resuspending each vial in 2 ml of injection grade water supplied by GEHC.  Infusions 
will be performed as described in the GEHC protocol supplement (Appendix B), and shown in Figure 1. 
All materials and supplies used for the infusion procedure will be identical to those described in GE 
Healthcare’s current Sonazoid IND. Briefly, an IV cannula will be placed in a vein in the subject’s right 
arm. A 0.9%NaCl solution will be started and used to fill up the connecting tubes before being connected 
to a 3-way stopcock. The stopcock will then be connected to the extension tubing leading to the stopcock 
the cannula. All three vials of suspended Sonazoid will be drawn into a 10 ml syringe, placed in a syringe 
pump at the same level or below the patient, and connected directly to the stopcock. After the stopcock 
has been opened, the NaCl solution will be infused at a rate of at least 2 ml/min, and Sonazoid will be 
infused at the desired rate of 0.024 µl/ kg body weight/ minute (suspension infusion rate of 0.18 
ml/kg/hour), but may be reduced if necessary to achieve optimal levels of contrast . This infusion rate was 
selected based on our group’s previous experiences with Sonazoid infusion in human subjects in general 
[Halpern et al. 2002; Landmark et al. 2008] and in SHAPE portal hypertension subjects in particular 
[Eisenbrey et al. 2013].  This dosing scheme is designed to avoid (or limit) the number of cases that has to 
be discarded due to insignificant enhancement in the hepatic veins (as in our pilot study; Eisenbrey et al. 
2013). This dosage is based off the GEHC instructions for storage and well below the maximal tested 
dose which was still found safe in Phase I clinical trials (2.7 µl of microbubbles/kg in PBI 0001 as 
detailed in the Sonazoid Investigator’s Brochure). 


 
23 
 
6.3  Trial Assessments 
 
6.3.1  Medical History 
 
A full demographic profile, known drug allergies or intolerances, and a review of the subject’s 
medical/surgical history will be recorded.  If the subject is a woman of childbearing potential, she will 
have a urine pregnancy test (the results of which will be made available to the subject prior to study 
initiation).  Emergency services (e.g., a crash cart) will be available within the hospital in case of any 
acute adverse reactions.  Adverse events will be monitored during the entire procedure. 
 
6.3.2 Ultrasound Imaging 
 
The ultrasound examinations will be performed by a qualified sonographer.  Procedures and equipment 
for this trial will be used in accordance with typical clinical procedures.  All trial procedures will be 
conducted in accordance with Good Clinical Practice.  A modified Logiq 9 scanner (GE Healthcare, 
Milwaukee, WI) with a broad bandwidth curvi-linear array will be used in to obtain RF data (for SHAPE 
measurements) within the hepatic and portal veins.  Both the experimental machine and software will be 
labeled as an investigational device with a GE provided label prior to beginning any clinical experiments 
(21 CRF 812.5).  Ultrasound imaging will be performed at a transmit frequency of 2.5 MHz and the 
subharmonic obtained at 1.25 MHz.  Using this setup, acoustic pressure amplitudes will all be below 1.21 
MPa peak negative pressure, 2.19 MPa peak positive pressure.  The SHAPE algorithm will be activated  
and the acoustic power will be adjusted to produce the maximum change in subharmonic amplitudes (i.e., 
maximizing the sensitivity of SHAPE).  Subharmonic data from the microbubbles (i.e., SHAPE) will be 
acquired in 5 s segments during the infusion of the Sonazoid suspension.  All measurements will be 
repeated three times. 
 
During the examination, the subharmonic pressure estimates will be displayed on the Logiq 9 scanner, 
these estimates will be noted and the data will be stored on a PC.  Data will be compared to the 


 
24 
transcatheter portal pressure measurements, which comprise the free and wedged hepatic venous pressure 
(FHVP and WHVP, respectively) and the hepatic vein pressure gradient (HVPG; measured as the 
difference between the WHVP and FHVP), which are acquired as part of standard of care for all patients 
undergoing trans-jugular liver biopsy at TJU and HUP. 
 
6.4  Efficacy Assessments 
 
The findings of SHAPE will be correlated to the invasive catheter based measurements of portal vein 
blood pressure (i.e., HVPG) using least squares, multiple linear regression analysis with robust standard 
errors.  Comparisons of sensitivities, specificities and accuracies for diagnosing portal hypertension with 
SHAPE will be conducted with an unpaired Student’s t-test (using HVPG measurements as the reference 
standard).  Receiver operating characteristic (ROC) curves will be computed using a continuous scale 
with HVPG cutoff levels of 10 and 12 mmHg.  The optimal operating point (OOP) will be determined as 
the point closest to ‘0,1’ on the ROC curve (representing 100% sensitivity and 100% specificity).  All 
clinical variables (e.g., concomitant imaging results, presence of cancer or ascites, blood tests, fibrosis 
and MELD scores etc.) will also be compared to the SHAPE values. 
 
6.5  Safety Assessments 
Emergency services (e.g., a crash cart) will be available within the hospital in case of any acute adverse 
reactions.  Adverse events will be monitored during the entire procedure.  Specifically, the patient will be 
monitored with non-leading questions to monitor the patient for the occurrence of transient side effects 
that are described below. 
 
6.5.1  Risks/Benefits Assessment. 
 
The known risks from the administration of Sonazoid are minimal.  Transient side effects that have been 
described as possibly related to Sonazoid administration include headache (3.6%, 62/1699), chest pain 
(2.3%, 39/1699), abdominal pain (1.5%, 25/1699), diarrhea (1.5%, 25/1699), and nausea (1.6%, 28/1699). 
The majority of adverse events were mild to moderate in severity (92.6%, 652/704). 


 
25 
The use of an intravenous needle and the fluids given through the needle may cause minor discomfort, 
bleeding under the skin (bruise), and possible infection at the site of needle insertion. 
 
Clinically significant adverse effects from the administration of Sonazoid are unlikely.  The use of 
contrast with the new ultrasound imaging techniques is expected to provide significantly more 
information than conventional ultrasound techniques.  This may lead to a non-invasive method for 
measurement of hepatic pressure.  
 
To minimize and/or eliminate risks a nurse will be present during the entire procedure. Emergency 
services (e.g., a crash cart) will be available within the hospital in case of any acute adverse reactions. 
Adverse events will be monitored during the entire procedure.   
 
The risk benefit ratio is low.  Based on the available nonclinical and clinical safety data and the 
anticipated dose levels of Sonazoid that will be used in this study, safety concerns are minimal.  The 
potential side effects related to Sonazoid administration are described above and listed in greater detail in 
the investigator’s brochure. 
 
6.6.2  Adverse Events (AE) 
 
An AE includes any condition that was not present prior to trial treatment, but appeared following 
initiation of trial medication; any condition that was present prior to trial treatment, but worsened during 
trial medication; or any condition, of which the subject has a history, that was not present prior to trial 
medication initiation but reappeared following administration of Sonazoid.  This would include 
conditions that are likely to be associated with an underlying or intermittent disease (e.g., angina, flu, 
etc.). 
 
The subjects will be monitored for AEs during the entire procedure.  All AEs, including observed or 


 
26 
volunteered problems, complaints, signs or symptoms, and diagnoses, occurring from the initiation of 
Sonazoid dosing until the completion of the Sonazoid administration will be recorded on a serious or non-
serious AE data form, whether or not associated with the use of the trial medication. GEHC will be 
informed of all AEs. In addition all unexpected and serious adverse events (SAEs) are reported to the TJU 
or HUP IRB (as applicable) and to the FDA. The investigator is required to submit all unexpected and 
serious adverse events to the TJU or HUP IRB (as applicable) within 48 hours, and to GEHC. Fatal 
adverse events related to treatment which are unexpected must be reported within 24 hours to the TJU or 
HUP IRB (as applicable) and to GEHC. Fatalities not related to the study drug/device must be reported 
within 5 days. The PI of the study has previous experience running ultrasound clinical trials and will serve 
as the study sponsor. He will be responsible for ensuring all FDA requirements are met and all AE are 
properly reported. 
 
The AE forms will include: subject identification number and initials; subject’s date of birth, gender, and 
ethnicity; name of the institution, date of Sonazoid administration; signs/symptoms and severity; date of 
onset; date of resolution or death; relationship to the study drug; action taken; concomitant medication(s) 
including dose, and route and duration of treatment.   
 
Whenever possible, the AE will be evaluated and reported as a diagnosis rather than individual signs and 
symptoms.  If a definitive diagnosis is not possible, the individual signs and symptoms will be recorded.  
The investigator(s) will evaluate and note the duration, intensity, and relationship to (association with) the 
Sonazoid administration, the action taken, and the determination of seriousness for each AE. 
 
INTENSITY OF AEs 
The intensity of the AE will be characterized as mild, moderate, or severe. 
Mild AEs are usually transient, require no special treatment, and do not interfere with the subject's daily 
activities. 
Moderate AEs traditionally introduce a low level of inconvenience or concern to the subject and may 


 
27 
interfere with daily activities but are usually ameliorated by simple therapeutic measures. 
Severe AEs interrupt a subject's usual daily activity and typically require systemic drug therapy or other 
treatment. 
When the intensity of the AE changes over time, the maximum intensity will be recorded. 
 
RELATIONSHIP TO SONAZOID ADMINISTRATION 
 
The relationship or association of the AE to the Sonazoid administration will be characterized as 
"unlikely," "possible," or "probable."  A relationship assessment will be performed by the investigator to 
determine if an AE is attributable to Sonazoid and will be recorded on a data form.  The investigator will 
refer to the Sonazoid investigator brochure for assistance in determining AE relationship. 
 
An "unlikely" relationship indicates that there is little or no chance that Sonazoid caused the reported AE; 
other conditions, including concurrent illnesses, progression or expression of the disease state, or a 
reaction to a concurrent medication, appear to explain the reported AE. 
 
A "possible" relationship indicates that the association of the AE with Sonazoid is unknown.  However, 
the AE is not reasonably attributed to any other condition. 
 
A "probable" relationship indicates that a reasonable temporal association exists between the AE and 
Sonazoid administration and, based upon the investigator's clinical experience, the association of the 
event with the trial medication seems likely. 
 
SERIOUS ADVERSE EVENTS 
 
A "serious" AE (SAE) is defined as a significant clinical hazard, contraindication, or precaution that: 
 
Results in death 
 
Is life-threatening (In the opinion of the investigator, there is an immediate risk of death from 
the AE as it occurred. This does not include an AE that had it occurred in a more serious form 
may have caused death.) 


 
28 
 
Results in a persistent or significant temporary disability/incapacity defined as a substantial 
disruption of a person's ability to conduct normal life functions 
 
Results in or prolongs an existing in-patient hospitalization (an overnight stay in the hospital, 
regardless of length) [Note: A hospitalization for an elective procedure or treatment which is 
not associated with an AE, hospitalization for a pre-existing condition which did not worsen, 
and hospitalization for reasons of convenience or observation, do not constitute an SAE.] 
 
Is a congenital anomaly/birth defect (in offspring of a subject taking the trial medication, 
regardless of time to diagnosis) 
 
Is an important medical event that may not result in death, be life-threatening, or require 
hospitalization but based upon the appropriate medical judgment, the event may jeopardize 
the subject and may require medical or surgical intervention to prevent one of the outcomes 
listed for the definition of a serious adverse experience. 
 
The designated medical monitor will review all serious and unexpected adverse events associated with the 
protocol and provide an unbiased written report of the event within 10 calendar days of the initial report.  
At a minimum, the medical monitor will comment on the outcomes of the adverse event and relationship 
of the event to the Sonazoid administration.  The medical monitor will also indicate whether he concurs 
with the details of the report provided by the principal investigator. 
 
A copy of the SAE will be retained on file with the respective subject's data forms.  
 
 
6.7  End-of-Treatment and End-of-Trial Evaluations 
 
6.7.1  Discontinuation of Subjects 
Subjects will be free to discontinue trial participation at any time.  The investigator will also discontinue 
any subject from the trial if, in the investigator's opinion, it is not safe for the subject to continue.  The 
date the subject is withdrawn from a treatment and/or from the trial and the reason for discontinuation 
will be recorded on the CRF.  For portal hypertension patients that will be monitored by SHAPE for up to 
18 months, the end point will be any one new complication (e.g., liver cancer) or a marked worsening in 
any complication (e.g., MELD scores) or the end of their clinical trial participation (up to 18 months).  
The time to reach the end point will be noted if a new complication or a marked worsening in any 
complication occurs.  The time to reach the end point will be noted. 
 
Trial participation will be considered completed if the subject has met all of the following trial 


 
29 
requirements: 
 
 
Has received at least one infusion of Sonazoid 
 
Has undergone the complete ultrasound imaging study as described in this protocol 
 
If a subject's participation in the trial is interrupted for any reason (e.g., because of an AE or if the subject 
is lost to follow-up) and the subject has met the criteria described above for completing the trial, the 
subject's trial participation will be considered completed.  If a subject's trial participation is interrupted for 
any reason by the subject's or investigator's choice and the subject has not met all of the criteria listed 
above, then the subject will be considered a discontinued subject. 
 
 
7.  DATA MANAGEMENT AND STATISTICAL ANALYSES 
 
7.1  Data Management 
 
Data forms will be completed for all subjects enrolled in the trial.  The patient study files will be stored in 
a secure file cabinet and maintained by the research study coordinators at each institution (TJU and HUP).  
Patient study files will be kept for 7 years after the completion of the study.  The PI of the study has 
previous experience running ultrasound clinical trials and will serve as the study sponsor.  He will be 
responsible for ensuring all FDA requirements are met and all AE are properly reported. 
 
The final data will be entered into a database.  The investigator will be responsible for management of the 
database.  The database will be maintained within an organized and secure directory system. 
 
7.2  Statistical Analyses 
 
7.2.1  Hypotheses 
 
H1:  Can patients with HVPG of 10 mmHg or greater be identified using SHAPE? The fundamental 
hypothesis is that a sensitivity of 89% and a specificity of 81 % can be achieved by using SHAPE 
measurements to identify patients with HVPGs of 10 mmHg or greater. 


 
30 
H2: A correlation coefficient of 0.82 will be achieved between in vivo SHAPE and HVPG measurements.  
H3: A correlation coefficients above 0.90 will be achieved between SHAPE and repeat HVPG results.  
H4: SHAPE will significantly differentiate responders from non-responders using clinical outcomes as the 
reference standard. 
 
7.2.2  Analysis of Results 
The findings of SHAPE will be correlated to the invasive catheter based measurements of portal vein 
blood pressure (i.e., HVPG) using least squares, multiple linear regression analysis with robust standard 
errors.  Receiver operating characteristic (ROC) curves will be computed using a continuous scale with 
HVPG cutoff levels of 10 and 12 mmHg.  The optimal operating point (OOP) will be determined as the 
point closest to ‘0,1’ on the ROC curve (representing 100% sensitivity and 100% specificity).  All clinical 
variables (e.g., concomitant imaging results, presence of cancer or ascites, blood tests, fibrosis and MELD 
scores etc.) will also be compared to the SHAPE values.  When both types of variables are ordinal or 
continuous, correlations will be calculated.  When one type of variable is nominal and one continuous 
non-parametric rank order tests such as Mann-Whitney U-tests or Kruskal-Wallis tests will be performed 
[Rosner 1990].  All of the statistical analyses proposed for the human clinical trial will be repeated split 
by racial and ethnic groups to determine if clinically important race/ethnicity differences exist in the 
ability of SHAPE to diagnose portal hypertension.   
All analyses and computations will be performed using NCSS/PASS 2008 and Stata 12.0 (Stata 
Corporation, College Station, TX), while the study database will be designed and implemented in 
Filemaker Pro 10.0 (Filemaker Inc, Santa Clara, CA).  This database will contain all patient information 
(except names and other identifiers), including results of biopsies, HVPG and SHAPE measurements as 
well as the clinical variables. 
 
7.2.3  Sample Size Calculation 
 
As the most clinically important aim, the sample size analysis is based on specific aim 1 where each 
patient represents an independent entity.  Based on our pilot study, the sensitivity and specificity of 


 
31 
SHAPE for the diagnosis of portal hypertension (i.e., HVPG > 10 mmHg) is 89% and 88%, respectively 
[Eisenbrey et al. 2013].  However, the confidence intervals achieved in the pilot study were (due to the 
limited sample size) very wide (worst case scenario: 52–100%).  Hence, a power analysis was performed 
using NCSS/PASS 2008 (NCSS, East Kaysville, UT) to estimate the number of cases required to produce 
clinically acceptable 95% two-sided confidence intervals (for one correlation) [Newcombe 1998].  Table 
2 below illustrates sample size calculations for the number of independent observations needed in the 
study population based upon 95% two-sided confidence intervals ranging from 0.06 to 0.10 for one 
correlation. A sample size between 259 and 278 subjects will provide 95% confidence intervals from 0.84 
to 0.92 (i.e., a width of 0.08).  A similar analysis was conducted based on the correlation between SHAPE 
and HVPG of 0.82 obtained in our pilot study [Eisenbrey et al. 2012a].  In that case to demonstrate that 
SHAPE can accurately measure HVPG values with a correlation coefficient of 0.82 requires between 264 
and 343 patients (for confidence interval widths of 0.08 and 0.07, respectively).  Hence, we selected a 
sample size for this study of 300 subjects. 
 
 
 
 
Table 2: Sample size calculations for different 95% confidence intervals in the human clinical trial. 
Target width 
Proportion 
(R) 
Lower limit 
Upper 
limit 
Sample size 
(N) 
0.06 
0.88 
0.848 
0.908 
483 
0.06 
0.89 
0.857 
0.917 
450 
0.07 
0.88 
0.842 
0.912 
359 
0.07 
0.89 
0.851 
0.921 
335 
0.08 
0.88 
0.836 
0.916 
278 
0.08 
0.89 
0.845 
0.925 
259 
0.09 
0.88 
0.830 
0.920 
222 
0.09 
0.89 
0.839 
0.929 
207 
0.10 
0.88 
0.824 
0.923 
181 
0.10 
0.89 
0.833 
0.933 
170 
 


 
32 
The Interventional Radiology Divisions at Thomas Jefferson University (TJU) and the Hospital of the 
University of Pennsylvania (HUP) performs approximately 85 and 100 transjugular liver biopsy 
procedures with pressure measurements per year, respectively.  The patient population of this project will 
reflect the population demographics found at major American urban academic health centers.  The overall 
hospital demographics for TJU and HUP include 66% Caucasian, 30% African American, and 4% Asian, 
with 10% representing Hispanic patients.  The goal is to enroll 300 patients over the last 3 years of this 
project, which means we are aiming for a recruitment rate of approximately 54%. 
 
7.2.4 Disposition of Software 
 
Investigational software containing the modified SHAPE algorithm will be removed from the Logiq 9 
scanner upon completion of the study to avoid the investigational device being used for any unapproved 
investigations. At the conclusion of the study, the developed software will be saved on CD for future 
experimentation. These investigations may ultimately further the development of SHAPE technology as a 
pressure monitoring system. However, under no circumstances with the software be used in a clinical 
environment (i.e., for human studies) without GE’s consent. This will be ensured by the use of a GE 
controlled password for installation of experimental software which is currently in use and will expire at 
the conclusion of the study. 
 
 


 
33 
8.  REFERENCES 
 
Bouakaz A, de Jong N. WFUMB Safety Symposium on Echo-Contrast Agents: nature and types of 
ultrasound contrast agents. Ultrasound Med Biol. 33:187-196, 2007. 
D'Amico G, Garcia-Tsao G, Pagliaro L. Natural history and prognostic indicators of survival in cirrhosis: 
a systematic review of 118 studies. J Hepatol, 44:217-231, 2006. 
Dave JK, Halldordottir VG, Eisenbrey JR, et al.. On the implementation of an automated acoustic output 
optimization algorithm for subharmonic aided pressure estimation. Ultrasonics, 53: 880-888, 2013 
de Franchis R.  Evolving consensus in portal hypertension. Report of the Baveno IV consensus workshop 
on methodology of diagnosis and therapy in portal hypertension.  J Hepatol 2005; 43:167-176 
Eisenbrey JR, Dave JK, Halldorsdottir VG, et al.  Noninvasive subharmonic aided pressure estimation of 
hepatic venous pressure gradient.  Radiology, 2013. (published online; in press) 
Forsberg F, Liu JB, Shi WT, Furuse J, Shimizu M, Goldberg BB. In vivo pressure estimation using 
subharmonic contrast microbubble signals: proof of concept. IEEE Trans Ultrason Ferroelec Freq 
Contr, 52:581-583, 2005. 
Forsberg F, Dave J, Halldorsdottir VG, Leodore LM, Lin F, Hall AL, Thomenius K. Applying real-time 
noninvasive pressure estimation obtained from subharmonic contrast microbubble signals. Proc 
IEEE Ultrason Symp, 1694–1697, 2008. 
Garcia-Tsao G, Groszmann J, Fisher RL, Conn HO, Atterbury CE, Glickman M. Portal pressure, presence 
of gastroesophageal varicies and variceal bleeding. Hepatology, 5(3):419-424, 1985. 
Halldorsdottir VG, Dave JK, Leodore LM, Eisenbrey JR, Park S, Hall AL, Thomenius KE, Forsberg F. 
Subharmonic contrast microbubble signals for noninvasive pressure estimation under static and 
dynamic flow conditions. Ultrasonic Imaging, 33:153-164, 2011 
Halpern EJ, McCue PA, Aksnes AK, Hagen EK, Frauscher F, Gomella LG. Contrast-enhanced US of the 
prostate with Sonazoid: comparison with whole-mount prostatectomy specimens in 12 patients. 
Radiology, 2002; 222:361-366. 
Jang JY, Kim MY, Joeng SW et al. Current consensus and guidelines of contrast enhanced ultrasound for 
the characterization of focal liver lesions. Clinical and Molecular Heptalogy. 19:1-16, 2013 
Kalambokis G, Manousou P, Vibhakorn S, et al. Transjugular liver biopsy--indications, adequacy, quality 
of specimens, and complications--a systematic review. J Hepatol 47:284-294, 2007  
Kudo M, Zheng RQ, Kim SR, et al. Diagnostic accuracy of imaging for liver cirrhosis compared to 
histologically proven liver cirrhosis. A multicenter collaborative study. Intervirology, 51(Suppl 
1):17-26, 2008.  
Landmark KE, Johansen PW, Johnson JA, et al. Pharmacokinetics of Perfluorobutane following 
intravenous bolus injection and continuous infusion of Sonazoidin in healthy volunteers and in 
patients with reduced pulmonary diffusing capacity. Ultrasound Med Biol, 34:494-501, 2008. 
Merkel C, Marin R, Angeli P, et al.  A placebo-controlled clinical trial of nadolol in the prophylaxis of 
growth of small esophageal varices in cirrhosis.  Gastroenterology 2004; 127:476-484. 
Navarro V, Rossi S, Herrine S. Hepatic Cirrhosis. In Pharmacology and Therapeutics: Principles to 
Practice, 2007. 
Rosner B.  Fundamentals of Biostatistics (3rd Ed.).  Boston, MA: PWS-Kent Publishing Co., 1990 
Sanyal 
AJ, 
Bosch 
J, 
Blei 
A, 
Arroyo 
V. 
Portal 
hypertension 
and 
its 
complications. 
Gastroenterology;134:1715-1728, 2008.  
Shi WT, Forsberg F, Goldberg BB, Raichlen S. Method and system for pressure estimation using 
subharmonic signals from microbubble-based ultrasound contrast agents. U.S. Patent No. 
6,302,845, 2001.  
Shi WT, Forsberg F, Raichlen JS, Needleman L, Goldberg BB. Pressure dependence of subharmonic 
signals from contrast microbubbles. Ultrasound Med. Biol. 25:275-283, 1999.. 


 
34 
APPENDIX A - INVESTIGATOR OBLIGATIONS 
 
 
A. 
Institutional Review Board (IRB) and Human Subjects Research Review Board (HSRRB) 
Review/Approval 
 
The protocol and informed consent for this study must be reviewed and approved by an appropriate IRB 
and HSRRB prior to enrollment of participants in the study.  It is the responsibility of the investigator to 
assure that all aspects of the ethical review are conducted in accordance with FDA Regulations 21 CFR 
Part 56.  A letter documenting the IRB and HSRRB approval which specifically identifies the 
study/protocol must be obtained by the investigator prior to initiation of the study.  Amendments to the 
protocol will be subject to the same requirements as the original protocol.  The HSRRB must review and 
approve each modification to the study prior to implementation. 
 
A progress report with a request for re-evaluation and re-approval will be submitted by the investigator to 
the IRB and HSRRB at intervals required by the IRB, and not less than annually.  
 
After completion or termination of the study, the investigator will submit a final report to the IRB. This 
report should include: deviations from the protocol, the number and types of participants evaluated, the 
number of participants who discontinued (with reasons), results of the study, if known, and all AEs, 
including deaths. 
 
 
B. 
Informed Consent 
 
Signed, written informed consent which conforms to FDA Regulation 21 CFR Part 50, must be obtained 
from each participant prior to entering the study.  Each participant will be provided a written consent form 
and verbal information in an understandable manner which describes the nature and duration of the study.  
The research study coordinator or the investigator will conduct the informed consent interview in a 
private examination room.  The potential subject will be allowed to discuss the study with the 
investigator, research study coordinator, or any persons who may have accompanied the potential subject.  
Additionally, the participant must be allowed adequate time to consider the potential risks and benefits 
associated with his participation in the study.  The research study coordinator will sign the informed 
consent as the person conducting the consent interview.   
 
 
C. 
Data Reporting and Data Forms  
 
Data reflecting participant's experiences with the study will be recorded on CRFs by the investigator.  
 
 
D. 
Records Retention 
 
All records pertaining to the conduct of the clinical study, including CRFs, informed consent forms, 
source documents, and other study documentation must be retained for seven (7) years after the end of the 
study. 
 
Other study documentation includes all protocols and amendments, drug supply receipt, dispensing and 
final disposition records, IRB correspondence and approvals, signed consent forms, a blank copy of study 
consent forms, Form 1572, curriculum vitae or biosketches of members of the research team including the 
medical monitor, HSRRB correspondence and approval, and Statement of Investigator forms. 


 
35 
 
Source documents include all original records of observations, results, and activities necessary to 
reconstruct and evaluate the study.  Source documents include but are not limited to laboratory reports, 
electrocardiogram tracings, X-ray films, ultrasound images, subject diaries, subject progress notes, 
hospital charts, appointment books, radiologic reports or pharmacy records, and any other records or 
reports of procedures performed during the study.  Source documents also may include copies of the CRF 
or sponsor supplied worksheets when original information is recorded directly onto these forms. 
 
Whenever possible, an original recording of an observation should be retained as the source document.  
However, a photocopy of a record is acceptable provided it is legible and is a verified copy of the original 
document. 
 
 
E. 
Deviation from the Protocol 
 
The investigator will not deviate from the protocol without prior written approval from the IRB and the 
HSRRB.  In medical emergencies, the investigator will use medical judgment and remove the participant 
from immediate hazard.  The HSRRB and the IRB will be notified regarding the type of emergency and 
course of action taken.  Any other changes to or deviations from the protocol will be made as an 
amendment to the protocol.  The amendment must be submitted for review and approval to the local IRB 
and the HSRRB for review and approval. 
 
 
F. 
Roles and Responsibilities of Study Personnel 
 
Flemming Forsberg, Ph.D., Professor of Radiology and Director of Ultrasound Physics, will serve as 
Principal Investigator on this project.  He will be responsible for the scientific goals of the project.  Dr. 
Forsberg will oversee patient recruitment, informed consent, ultrasound studies, and the data entry and 
statistical analyses.  He will also supervise the SHI data acquisition from patients.  Dr. Forsberg will also 
prepare any manuscript(s) resulting from this grant.   
 
Jonathan Fenkel, M.D., Professor of Medicine, Pharmacology And Experimental Therapeutics Division 
of Gastroenterology and Hepatology will assist with the patient recruitment, interpret hepatic pressure 
results and advise on clinical issues. 
 
Collete Shaw, M.D., Research Professor of Radiology will assist with the patient recruitment, interpret 
ultrasound images and advise on clinical issues. 
 
John Eisenbrey, Ph.D., Assistant Professor of Radiology will aid in patient recruitment, informed consent, 
ultrasound studies, and the data entry and statistical analyses. 
 
Michael Soulen, M.D., Professor of Radiology at the Hospital of the University of Pennsylvania (site #2), 
Principal Investigator, will oversee all aspects of project at site #2. 
 
Chandra Sehgal, Ph.D., Professor and Director of Ultrasound Research, Site #2 Investigator, will oversee 
all Ultrasound activities. 
 
Stephen Hunt, M.D., Ph.D., Radiology Clinical Instructor, Site #2 Investigator, who will assist the PI with 
clinical procedures and project oversight. 
 
Susan Schultz, R.D.M.S., Radiology research sonographer who will perform ultrasound studies. 


 
36 
 
Suzanne Sweeney, B.S.N., R.N., Investigator/nurse manager, will perform and manage IV infusions. 
 
Bradford Dungan, B.S.N., R.N., Investigator/nurse will perform and manage IV infusions under Suzanne 
Sweeney. 
 
Evelyn Stainthorpe, B.S., C.C.R.C., will coordinate all aspects of the project under Dr. Soulen including 
IRB submission and reporting, recruitment, consent and data collection and submission. 
 
 
 
 
Signature of PI:  
 
 
 
 
 
 
 
 
Flemming Forsberg, PhD 
 
 
 
 


 
37 
APPENDIX B - GE PROVIDED STUDY SPECIFIC PROTOCOL SUPPLEMENT ON 
SONAZOID SUSPENSION AND INFUSION 
 
INSTRUCTION FOR STORAGE AND USE OF SONAZOIDTM POWDER FOR INJECTION, 16 
µL MICROBUBBLES PER VIAL 
 
Emergency drugs and equipment should be present in or nearby the examination room for immediate 
treatment, should a serious adverse event occur. 
 
1 
CONTENTS OF PACKAGE 
• 
SONAZOIDTM Powder for Injection, 16 µL microbubbles per vial;   
• 
Sterile water for reconstitution of SonazoidTM  
• 
Sterile NaCl 0.9 % for i.v. infusion, Baxter 
• 
1 Plastic syringe 10 mL for reconstituted product, Becton Dickinson  
• 
5 Plastic syringes 2 mL with hypodermic needles 
• 
Venflon i.v. cannula 22G (Luer-Lock) 
• 
CODAN Chemospike (contains 0.20 µm air filter and 5 µm liquid filter), 5 spikes  
• 
Connecta Plus3, 3-way stopcock ,  Becton Dickinson  
• 
Original Perfusor-Leitung MR, B. Braun, 150 cm  
• 
Administration set, R87 plus, Ohmeda GmbH & Co, 180cm  
 
2 
STORAGE OF SONAZOIDTM  
2 - 30°C / 35.6 - 86°F protected from light.   
 


 
38 
3 
RECONSTITUTION OF PRODUCT 
An illustration of the reconstitution procedure is shown in Figure 1.  
3.1 
Perforate the stopper of the SONAZOIDTM vial with the provided CODAN Chemospike. 
3.2 
Remove protective cap from the syringe port of the Chemospike. 
3.3 
Using a 2-mL syringe, add 2 mL sterile water through the Chemospike.  
3.4 
With the syringe remaining attached to the Chemospike, immediately shake the product for 1 minute to 
ensure a homogeneous product. 
3.5 
Withdraw the product into the syringe and re-inject the product back into the vial again. This is to avoid 
dilution of the product due to the dead-space volume in the Chemospike. 
3.6 
Remove the syringe from the syringe port and reattach the protective cap. The concentration of the 
reconstituted product is 8 µL microbubbles/mL. 
3.7 
Repeat the reconstitution procedure for the necessary number of vials as stated in Table 1. 
 
 
Figure 1 Reconstitution procedure for Sonazoid™ 


 
39 
4 
APPEARANCE OF RECONSTITUTED PRODUCT 
The reconstituted product appears as a milky white dispersion.  
 
5 
STORAGE OF RECONSTITUTED PRODUCT 
If the reconstituted product is not used immediately, leave the vial at rest in an upright position.  Store between 15-
25°C.  
 
NOTE - The infusion procedure must be started within 1 hour from reconstitution of the product. 
NOTE - Always shake the product for 10-15 seconds before withdrawal from the vial.  
6 
ADMINISTRATION PROCEDURE 
6.1 
Infusion set up 
The infusion set up and procedure is illustrated in Figures 2 and 3. 
 
6.1 Place the cannula in a vein in the subjects right upper limb (preferably), as the venous circulation of 
the left arm might be compromised during imaging procedures if the subject is placed in the left 
decubitus position. 
 
7.1 Connect the 3-way stopcock to the 150 cm extension tube, and connect it to the administration set.  
 
NOTE - The stopper of the third port on the 3-way stopcock should not be removed yet.  
8.1 Put the 3-way stopcock in the right position (Figure 3A) and start the infusion of 0.9% NaCl, fill up 
the tubes and connect the outlet to the Venflon cannula.  Set the Infusion rate:  NLT 2.0 mL/min 
(=  NLT 40 drops/min with the submitted infusion set). 
 
9.1 Close the stopcock as shown in Figure 3B in order to temporarily stop the infusion. 
 
10.1 Place the syringe pump in a vertical position so that the syringe is at the same level or below 
the patient. This is important in order to maintain a homogenous product during infusion. 
 


 
40 
6.2 
Infusion procedure:  
For each subject, reconstitute the number of vials of Sonazoid given in Table 1. 
 
6.2.1 
Shake the reconstituted product immediately before product withdrawal. 
  
 
6.2.2 
Just before start of infusion, draw reconstituted Sonazoid from all three vials into the 10 mL 
syringe via the CODAN Chemospike.  
 
6.2.3 
Remove air from the 3-way stopcock by removing the stopper of the third port (Figure 3C).  
Attach the 10 mL syringe containing the homogenous contrast agent to the 3-way stopcock 
according to Figure 1 and Figure 3D. 
6.2.4     Open the 3-way stopcock (Figure 3E) so that all ports are open and check the saline infusion rate 
(NLT 40 drops/min).   
6.2.5     Place the syringe in the vertically positioned pump in an upright position (syringe outlet 
up), and start infusion immediately (as described below). 
NOTE - Make sure that the 3-way stopcock is open. 
NOTE - Check that the correct syringe is recognized by the pump (10 mL BD Plastipak). 
NOTE - Ensure that the tubing has no bends/kinks that may cause obstruction. 
6.2.6    Enter the syringe pump rate according to the subject’s b.w., see Table 1.  
6.2.7    START PUMP.   
NOTE - Maximum infusion time is 15 minutes.  The syringe contains a calculated overage of product in 
order to ensure homogeneity. The overage should be discarded after infusion is finished. 
NOTE - The infusion rate of NaCl should always be NLT 2 mL/min (= 40 drops/min) during 
Sonazoid infusion, to ensure a homogeneous infusion of Sonazoid. 
 
 
 
 
 
 
 
 


 
41 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2 The infusion set up 
 
Extension tube 
150cm 
Infusion of  
NaCl 0.9% 
(2 mL/min.) 
Reconstituted 
SONAZOID 
3-way  
stopcock 
SONAZOID 
 infusion 
 
Venflon 
Vertically positioned 
Syringe pump 


 
42 
 
Figure 3 Positions of the 3-way stopcock as described in the text 
  
  
 
 
 
NaCl
NaCl
Closed
Closed
NaCl
Closed
Open to 
remove air
Closed
NaCl
Attach 
syringe
syringe
NaCl
Closed
Start syringe 
pump
NaCl
A
B
C
D
E
F
NaCl
NaCl
NaCl
Closed
Closed
NaCl
Closed
Closed
NaCl
Closed
Open to 
remove air
NaCl
Closed
Open to 
remove air
Closed
NaCl
Attach 
syringe
NaCl
Attach 
syringe
syringe
NaCl
syringe
NaCl
Closed
Start syringe 
pump
NaCl
A
B
C
D
E
F


 
43 
Table 1 
Infusion rate of reconstituted SonazoidTM 8 µL microbubbles/mL.   
BODY WEIGHT 
 
INFUSION RATE = 
SYRINGE PUMP RATE (mL/hour) 
 
Kg 
Lbs. 
Number of Sonazoid  
vials to be withdrawn into 
the 10 mL syringe1 
40 
88 
3 
3.6 mL/hour 
45 
99 
3 
4.1 mL/hour 
50 
110 
3 
4.5 mL/hour 
55 
121 
3 
5.0 mL/hour 
60 
132 
3 
5.4 mL/hour 
65 
143 
3 
5.9 mL/hour 
70 
154 
3 
6.3 mL/hour 
75 
165 
3 
6.8 mL/hour 
80 
176 
3 
7.2 mL/hour 
85 
187 
3 
7.7 mL/hour 
90 
198 
3 
8.1 mL/hour 
95 
209 
3 
8.6 mL/hour 
100 
220 
3 
9.0 mL/hour 
105 
231 
3 
9.5 mL/hour 
110 
242 
3 
9.9 mL/hour 
115 
253 
3 
10.4 mL/hour 
120 
264 
3 
10.8 mL/hour 
125 
275 
3 
11.3 mL/hour 
130 
286 
3 
11.8 mL/hour 
135 
297 
3 
12.2 mL/hour 
140 
308 
3 
12.6 mL/hour 
145 
319 
3 
13.1 mL/hour 
150 
330 
3 
13.5 mL/hour 
 
Calculation of infusion rate:  
Table 1 lists the infusion rate per hour, which corresponds to the syringe pump rate for a body 
weight range of 40-130 kg.  
Dose of 0.012 µL microbubbles/kg bw/minute corresponds to 0.72 µL microbubbles/kg bw/hour: 
 
 
 
 
Infusion rate (mL/hour) = 0.090 x body weight in kg  
1The calculation of number of vials includes a necessary overage of minimum 2mL. The maximum 
extractable volume from each vial should be drawn into the 10 mL syringe.