Conditions: Cardiac Disease
Keywords: Left Ventricular Ejection Fraction DEFINITY® Enhanced Echocardiogram Cardiac Magnetic Resonance
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 160
Healthy volunteers: no

Brief summary:
This is a Phase 3, prospective, open-label, multicenter study to evaluate Left Ventricular Ejection Fraction (LVEF) measurement accuracy and reproducibility of DEFINITY® contrast-enhanced and unenhanced echocardiography as compared with non-contrast cardiac magnetic resonance imaging (CMR) used as the truth standard.

Detailed description:

Arms:
arm 1: Each patient will undergo an unenhanced ultrasound examination and a DEFINITY® contrast-enhanced ultrasound

Interventions: intervention 1: DEFINITY®
Intervention descriptions: intervention 1: All subjects will receive a single dose of DEFINITY®

Protocol document:
Prot_000.pdf: 
Clinical Study Protocol:  DEF-315 
Study Title: 
A Phase III, Open-Label, Multicenter Trial to Evaluate Ejection 
Fraction, End-Diastolic and End-Systolic Volumes, by Unenhanced 
and DEFINITY®-enhanced 2D-Echo and Magnetic Resonance 
Imaging 
Study Number: 
DEF-315 
Study Phase: 
Phase 3 
Product Name: 
DEFINITY® 
IND Number: 
048626 
Indication: 
DEFINITY® is indicated to improve the accuracy of left ventricular 
ejection fraction measurements with echocardiography. 
Investigators: 
Multicenter 
Sponsor: 
Lantheus Medical Imaging, Inc. 
Sponsor 
Contact: 
331 Treble Cove Road 
North Billerica, MA 01862 
+1 (978)-667-9531 
Medical 
Monitor: 
Cesare Orlandi, MD 
 
 
 
Date 
Original Protocol 
07 February 2018  
Amendment 1 
02 May 2018 
Amendment 2 
12 June 2018 
Amendment 3 
15 August 2018 
 
Confidentiality Statement 
This document is the confidential property of Lantheus Medical Imaging (LMI). No part of it 
may be transmitted, reproduced, published, or used by other persons without the permission 
of LMI. 
 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
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Page 2 of 49 
SYNOPSIS 
Sponsor:  Lantheus Medical Imaging, Inc. 
Study Title: 
A Phase III, Open-Label, Multicenter Trial to Evaluate Ejection Fraction, End-Diastolic 
and End-Systolic Volumes, by Unenhanced and DEFINITY®-enhanced 2D-Echo and 
Magnetic Resonance Imaging 
Study Number:  DEF-315 
Study Phase:  Phase 3 
Primary Objective(s):  
The primary objective of this study is to demonstrate improvement in accuracy in left 
ventricular ejection fraction (LVEF) assessment using DEFINITY® contrast-enhanced over 
unenhanced echocardiography.  
Secondary Objective(s): 
The secondary objectives of this study are to: 
1. Demonstrate improvement in accuracy in left ventricular ejection fraction (LVEF) 
assessment using DEFINITY® contrast-enhanced over unenhanced 
echocardiography in subjects with suboptimal echocardiograms. 
2. Demonstrate a reduction in inter-reader variability for the assessment of LVEF 
using DEFINITY® contrast-enhanced versus unenhanced echocardiography. 
3. Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography. 
4. Demonstrate a reduction in inter-reader variability for the assessment of LVEF 
using DEFINITY® contrast-enhanced versus unenhanced echocardiography in 
subjects with suboptimal echocardiograms. 
5. Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms. 
 


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Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
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Study Design: 
This is a Phase 3, prospective, open-label, multicenter study to evaluate LVEF 
measurement accuracy and reproducibility of DEFINITY® contrast-enhanced and 
unenhanced echocardiography as compared with non-contrast cardiac magnetic resonance 
imaging (CMR) used as the truth standard.   
Subjects will be enrolled over approximately 10 months at approximately 10 centers 
located in the United States.  Subjects will be screened based on LVEF measurements 
obtained via 2D echo with or without contrast or other methods (e.g. CMR, MUGA scan) 
obtained within 6 months of enrollment (Day 0) to achieve an approximately even 
distribution within four pre-defined LVEF groups (>50, 41-50, 30–40, <30%).  Subjects 
with optimal and sub-optimal echocardiograms will be enrolled.  
Each patient will undergo an unenhanced ultrasound examination and a DEFINITY® 
contrast-enhanced examination on the same day at Day 0.  Subjects will remain at the 
clinical site for at least 30 minutes of observation after DEFINITY® administration.  The 
site investigator will determine whether an echocardiogram is optimal or sub-optimal 
based on the unenchanced echocardiogram performed on Day 0.   
A non-contrast CMR will be used as the truth standard.  CMR studies will be performed 
within ±30 days of the echocardiographic assessments at Day 0. Final assignment of each 
subject to an LVEF group will be determined by CMR. 
In addition, a safety follow-up phone call will be conducted at 72±24 hours after 
DEFINITY® administration.   
All echocardiography images will be interpreted by 3 experienced independent blinded 
readers at a central imaging core laboratory.  A single, independent, blinded 
echocardiologist will read each subject’s unenhanced echocardiogram as either optimal or 
suboptimal.  This will be independent of the site investigator determination.  All CMR 
images will be read centrally by a single reader.  Echocardiographic imaging data will be 
compiled, analyzed, and stored without knowledge of the CMR findings. 
 
Study Population:  
Approximately one hundred and fifty (150) evaluable subjects will be enrolled at 
approximately 10 US centers.  Subjects who have had a 2D echo with or without contrast 
or other methods (e.g. CMR, MUGA scan) to evaluate screening LVEF within 6 months 
prior to enrollment (Day 0) may be enrolled.  The study population will consist of males 
and non-pregnant and non-lactating females 18 years-of-age or older. 
Duration of Treatment:  
Subjects will receive DEFINITY® treatment once as a part of a single day imaging session.   


DEFINITY® 
 
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Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
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Study Treatment:  
DEFINITY® will be administered as a diluted bolus injection.  1.3 mL of activated 
DEFINITY® will be diluted with 8.7 mL of preservative-free saline to evenly distribute 
microspheres.  An initial injection of up to 3 mL of diluted DEFINITY® will be 
administered with subsequent injections of 1 to 2 mL, as needed. 
Efficacy Assessments: 
The primary endpoint is to demonstrate an improvement in LVEF accuracy from 
unenhanced imaging to imaging with DEFINITY® contrast enhancement using CMR as 
the truth standard.   
The secondary endpoints are to: 
1. Demonstrate improvement in accuracy in left ventricular ejection fraction (LVEF) 
assessment using DEFINITY® contrast-enhanced over unenhanced echocardiography 
in subjects with suboptimal echocardiograms. 
2. Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography. 
3. Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography. 
4. Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography in subjects with 
suboptimal echocardiograms. 
5. Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms. 
 
Safety Assessments: 
Enrolled subjects will be followed for adverse events (AEs), serious adverse events (SAEs) 
and changes in concomitant medications from the time the Informed Consent (IC) is 
signed through 72±24 hours after completion of DEFINITY® administration.  All AEs will 
be recorded on the electronic case report form (eCRF).  The National Cancer Institute 
Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 4.0 will be 
utilized for Adverse Event (AE) reporting of severity. 
Statistical Methods for Primary Objective:  
The primary objective of the study is to demonstrate improvement in accuracy in left 
ventricular ejection fraction (LVEF) assessment using DEFINITY® contrast-enhanced 
over unenhanced echocardiography, using CMR as the truth standard.   
The following analyses will be carried out for each of the three blinded readers.  For each 
subject, the absolute value of the difference of DEFINITY® LVEF minus CMR LVEF 
will be calculated.  Similarly, the absolute value of the difference of unenhanced imaging 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 5 of 49 
LVEF minus CMR LVEF will be calculated for each subject.  The primary analysis is to 
assess the significance of the difference between DEFINITY® and unenhanced echo with 
respect to the mean “absolute value of the difference vs. CMR”.  These means will be 
compared with a paired t-test at a two-sided 0.05 level of significance.  Specifically, the 
null and alternative hypotheses are: 
H0: µD = µU vs. H1: µD ≠ µU 
Where µD is the mean of the absolute value of the differences between the DEFINITY®-
enhanced echo LVEF and the CMR LVEF. Similarly, µU is the mean of the absolute value 
of the differences between the unenhanced echo LVEF and the CMR LVEF.  A sample of 
size of 150 enrolled subjects has 90% power to reject the null hypothesis in favor of the 
alternative if the true difference µD - µU is at least 2.75 with a standard deviation of 10 or 
less and allows for approximately 5% premature withdrawal.  If the null hypothesis is 
rejected in favor of DEFINITY® for at least two of three blinded readers, the primary 
analysis will be considered a success. 
The following are additional analyses to be carried out separately for each blinded reader 
to achieve the primary objective: 
Bias is the mean of the actual (not absolute value) per-subject differences between the 
imaged LVEF and CMR LVEF.  The bias, the two-sided 95% confidence interval of the 
bias, the precision (standard deviation of the per-subject differences) and the root mean 
square error (RMSE) will be calculated for DEFINITY®.  The RMSE is the square root of 
the bias-squared + precision-squared and is considered a measure of overall accuracy.  
These analyses will be repeated for unenhanced echocardiography vs. CMR.   It is 
anticipated that the RMSE will be smaller for DEFINITY®-enhanced LVEF than for 
unenhanced LVEF. 
DEFINITY®-enhanced and unenhanced echocardiography LVEF will each be assessed for 
measurement accuracy against the reference CMR using Bland-Altman analysis and 
Deming regression analysis.  For the Bland-Altman analysis, a plot of per-subject actual 
difference between DEFINITY®-enhanced echocardiography and CMR LVEF will be 
plotted vs. the per-subject sum of the two measurements. Limits of agreement (defined as 
the mean of the DEFINITY® vs. CMR difference) +/- 2 SD will be shown on the plot.  
Similar analyses will be carried out for the unenhanced echocardiography LVEF again 
using CMR as the reference standard. 
Deming regression plots of LVEF vs. CMR LVEF will be generated for each of 
DEFINITY®-enhanced and unenhanced echocardiography.  Unweighted Deming 
regression will be employed to estimate the regression slope and intercept with two-sided 
95% confidence intervals of each, assuming the measurement error is the same for CMR 
and each of the echocardiography error techniques. 


DEFINITY® 
 
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Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
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The intra-class correlation coefficient (ICC) and its two-sided 95% confidence interval will 
be calculated for DEFINITY®-enhanced echo vs. CMR and for unenhanced echo vs. 
CMR.    
Statistical Methods for Secondary Objectives: 
A secondary objective of this study is to demonstrate improvement in accuracy in left 
ventricular ejection fraction (LVEF) assessment using DEFINITY® contrast-enhanced over 
unenhanced echocardiography in subjects with suboptimal echocardiograms.  For this, the 
above analyses for the primary objective will be repeated on subjects with suboptimal 
echocardiograms. 
An additional secondary objective is to demonstrate a reduction in inter-reader variability 
for the assessment of LVEF and End-Diastolic/Systolic volumes using DEFINITY® 
contrast-enhanced versus unenhanced echocardiography.  The inter-observer variability 
among each pair of readers within each imaging modality will be estimated using an intra-
class correlation coefficient (ICC); descriptive comparisons of the ICC between 
DEFINITY® and unenhanced echocardiography will be carried out.  Similar analyses will 
be conducted for the final secondary objective of assessing inter-reader variability within 
subjects with suboptimal echocardiograms.   
 
 


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Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
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TABLE OF CONTENTS 
SYNOPSIS .................................................................................................................................2 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ..........................................11 
1 
INTRODUCTION ...........................................................................................................14 
1.1 
Background ............................................................................................................14 
2 
STUDY OBJECTIVES ....................................................................................................15 
2.1 
Primary Objective ..................................................................................................15 
2.2 
Secondary Objectives.............................................................................................15 
3 
INVESTIGATIONAL PLAN ..........................................................................................16 
3.1 
Overall Study Design and Plan ..............................................................................16 
3.2 
Rationale for Study Design ....................................................................................17 
3.3 
Study Duration .......................................................................................................18 
4 
STUDY POPULATION ..................................................................................................19 
4.1 
Inclusion Criteria ...................................................................................................19 
4.2 
Exclusion Criteria ..................................................................................................19 
5 
STUDY TREATMENT(S) ..............................................................................................21 
5.1 
Description of Treatment(s) ...................................................................................21 
5.1.1 
Investigational Product ................................................................................21 
5.2 
Treatments Administered .......................................................................................21 
5.2.1 
Administering Study Drug ...........................................................................21 
5.3 
Selection and Timing of Dose for Each Patient .....................................................21 
5.4 
Method of Assigning Subjects to Treatment Groups .............................................21 
5.5 
Blinding..................................................................................................................22 
5.6 
Concomitant Therapy.............................................................................................22 
5.7 
Treatment Compliance ...........................................................................................22 
5.8 
Packaging and Labeling .........................................................................................22 
5.9 
Storage and Accountability ....................................................................................22 
5.10 
Investigational Product Retention at Study Site ....................................................22 
6 
STUDY PROCEDURES .................................................................................................23 
6.1 
Screening................................................................................................................23 
6.2 
Informed Consent...................................................................................................23 
6.3 
Medical History .....................................................................................................23 
6.4 
Physical Examination.............................................................................................24 
6.5 
Vital Signs ..............................................................................................................24 


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Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
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6.6 
Pregnancy Test .......................................................................................................24 
6.7 
Cardiac Magnetic Resonance Imaging ..................................................................24 
6.7.1 
Image Acquisition ........................................................................................24 
6.7.2 
Imaging Core Laboratory .............................................................................25 
6.8 
Echocardiography Imaging Procedures .................................................................25 
6.8.1 
Image Acquisition ........................................................................................25 
6.8.2 
Imaging Core Laboratory .............................................................................26 
6.9 
Efficacy Assessments.............................................................................................26 
6.9.1 
Primary Efficacy Assessments .....................................................................26 
6.9.2 
Secondary Efficacy Assessments .................................................................26 
6.10 
Adverse Event Assessments ..................................................................................26 
6.10.1 
Performing Adverse Event Assessments .....................................................26 
6.10.2 
Timing ..........................................................................................................28 
6.10.3 
Severity ........................................................................................................28 
6.10.4 
Relationship .................................................................................................29 
6.10.5 
Clinical Significance ....................................................................................29 
6.10.6 
Serious Adverse Events ...............................................................................29 
6.10.6.1 
Definition ...........................................................................................29 
6.10.6.2 
Reporting Serious Adverse Events ....................................................30 
6.11 
Removal of Subjects from the Trial or Investigational Product ............................31 
7 
QUALITY CONTROL AND ASSURANCE .................................................................32 
8 
PLANNED STATISTICAL METHODS ........................................................................33 
8.1 
Statistical Analyses ................................................................................................33 
8.1.1 
General Considerations ................................................................................33 
8.1.2 
Analysis Populations ....................................................................................33 
8.1.2.1 
Safety Population ...............................................................................33 
8.1.2.2 
Intent-to-Treat ....................................................................................33 
8.1.2.3 
Modified Intent-to-Treat Population ..................................................33 
8.1.2.4 
Per-Protocol Population .....................................................................33 
8.2 
Efficacy Endpoint(s) ..............................................................................................34 
8.2.1 
Primary Efficacy Endpoint ..........................................................................34 
8.2.2 
Other Primary Endpoint(s) ...........................................................................36 
8.3 
Secondary Endpoint(s) ...........................................................................................36 
8.3.1 
Secondary Efficacy Endpoint(s) ..................................................................36 
8.3.1.1 
Improvement in accuracy in left ventricular ejection fraction 
(LVEF) assessment using DEFINITY® contrast-enhanced over 


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Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
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unenhanced echocardiography in subjects with suboptimal 
echocardiograms ................................................................................36 
8.3.1.2 
Reduction in inter-reader variability for the assessment of LVEF 
using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography ...............................................................................36 
8.3.1.3 
Reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced 
versus unenhanced echocardiography ................................................37 
8.3.1.4 
Reduction in inter-reader variability for the assessment of LVEF 
using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms ......37 
8.3.1.5 
Reduction in inter-reader variability for the assessment of End-
Diastolic/Systolic volumes using DEFINITY® contrast-enhanced 
versus unenhanced echocardiography in subjects with suboptimal 
echocardiograms ................................................................................37 
8.3.2 
Other Secondary Endpoint(s) .......................................................................37 
8.4 
Safety Endpoint(s) .................................................................................................37 
8.4.1 
Primary Safety Endpoint(s)..........................................................................37 
8.4.2 
Secondary Safety Endpoint(s) ......................................................................37 
8.5 
Demographics and Baseline Characteristics ..........................................................38 
8.6 
Interim Analysis .....................................................................................................38 
8.7 
Determination of Sample Size ...............................................................................40 
9 
ADMINISTRATIVE CONSIDERATIONS ....................................................................41 
9.1 
Investigators and Study Administrative Structure .................................................41 
9.2 
IRB or IEC Approval .............................................................................................41 
9.3 
Ethical Conduct of the Study .................................................................................41 
9.4 
Patient Information and Consent ...........................................................................42 
9.5 
Patient Confidentiality ...........................................................................................42 
9.6 
Study Monitoring ...................................................................................................43 
9.7 
Case Report Forms and Study Records .................................................................43 
9.8 
Data Monitoring Committee ..................................................................................44 
9.9 
Protocol Violations/Deviations ..............................................................................44 
9.10 
Access to Source Documentation ..........................................................................44 
9.11 
Data Generation and Analysis ...............................................................................45 
9.12 
Retention of Data ...................................................................................................45 
9.13 
Financial Disclosure...............................................................................................45 
9.14 
Publication and Disclosure Policy .........................................................................46 


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Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
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10 REFERENCES ................................................................................................................47 
 
LIST OF IN-TEXT FIGURES 
Figure 3-1 
Study Event Overview ..................................................................................17 
 
LIST OF APPENDICES 
Appendix 1 Schedule of Procedures .................................................................................48 
Appendix 2 Dose Preparation and Administration Guide ................................................49 
 
 


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Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
Abbreviation 
Definition 
A2C 
apical 2-chamber 
A3C 
apical 3-chamber 
A4C 
apical 4-chamber 
AE 
adverse event 
BMI 
body mass index 
CABG 
coronary artery bypass graft 
CAD 
coronary artery disease 
CFR 
Code of Federal Regulations 
CIOMS 
Council for International Organizations of Medical Sciences 
cm 
centimeter 
CMR 
cardiac magnetic resonance 
CRO 
Contract Research Organization 
DMC 
Data Monitoring Committee 
ECG 
electrocardiogram 
eCRF 
electronic case report form 
ED 
effective dose 
Echo 
Echocardiogram 
EEG 
electroencephalography 
EF 
ejection fraction 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
GSM 
Group sequential method 
HEENT 
head, ears, eyes, nose and throat  
IB 
Investigator’s Brochure 


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Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
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Abbreviation 
Definition 
IC 
informed consent 
ICC 
intra-class correlation coefficient 
ICF 
informed consent form 
ICH 
International Conference on Harmonization 
IEC 
Institutional Ethics Committee 
IRB 
Institutional Review Board 
ITT 
intent-to-treat 
LC 
liquid chromatography 
LCX 
left circumflex 
LMI 
Lantheus Medical Imaging 
LV 
left ventricular 
LVEF 
left ventricular ejection fraction 
MedDRA 
Medical Dictionary for Regulatory Activities 
MI 
myocardial infarction 
mITT 
modified intent-to-treat 
MUGA 
Multiple-gated acquisition 
g 
microgram 
mg 
milligram 
MI 
mechanical index 
min 
minute 
mL 
milliliter 
mmHg 
millimeters of mercury 
NCI-CTCAE 
National Cancer Institute Common Terminology Criteria for Adverse 
Events 
O2 Sat 
oxygen saturation 


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Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
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Abbreviation 
Definition 
pH 
potential of hydrogen 
PFP 
phospholipids-encapsulated perfluoropropane 
PT 
preferred term 
QC 
quality control 
QT [interval] 
interval on the ECG from the onset of the QRS complex to the end of the 
T-wave 
QTcB 
Bazett-Corrected QT interval 
RCA 
right coronary artery  
RMSE 
root mean square error 
RR 
respiratory rate 
SAE 
serious adverse event 
SAF 
Safety Evaluable Population 
SAS® 
Statistical Analysis System 
sec 
second(s) 
SOC 
system organ class 
SP 
safety population 
TE 
echo time 
TEAE 
treatment emergent adverse event 
TIA 
transient ischemic attack 
TR 
repetition time 
US 
United States 
WOCBP 
women of childbearing potential 
 


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Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
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1 
INTRODUCTION 
1.1 
Background 
Left ventricular (LV) volumes and ejection fraction (EF) are important clinical parameters 
for the diagnosis, management, and prognosis of subjects with cardiac diseases. Important 
treatment decisions and the evaluation of therapeutic effects are based on these parameters.  
Several techniques have been used for the analysis of LV volumes and EF, among them cine 
ventriculography, echocardiography, cardiac magnetic resonance (CMR) and computed 
tomography (Foley 2012).1 CMR has evolved into the preferred reference technique because 
of its high spatial resolution and ability to obtain complete volumetric data sets, allowing 
very accurate determinations of regional and global LV function.  Although the most 
frequently used modality in clinical practice, echocardiography has been hampered by its 
moderate reproducibility and accuracy to define LVEF. Poor acoustic windows and 
inadequate discrimination of the endocardial border are the main reasons for the poor 
reproducibility and accuracy of the test, besides geometric assumptions resulting from the 
two-dimensional approach (Foley 2012).1 In a few studies, contrast echocardiography has 
been shown to allow improved assessment of LV volumes and EF, especially in subjects with 
difficult imaging conditions (Hoffman 2005, 2014,).2,3  Recent innovations in contrast-
specific ultrasound techniques have further enabled improvements in visualization of the LV 
endocardial border above the level already shown in previous trials with the use of contrast-
enhanced ultrasound imaging. 
DEFINITY® (perflutren injectable suspension) is a sterile, non-pyrogenic suspension of 
phospholipids-encapsulated perfluoropropane microbubbles that is activated by shaking with 
the aid of the Vialmix™.  In subjects with suboptimal echocardiograms, the addition of 
DEFINITY® improves the assessments of cardiac structure (ventricular chambers and 
endocardial borders) and function (regional wall motion). 
The objective of this multicenter study is to determine the accuracy and inter-reader 
agreement of DEFINITY® contrast-enhanced and unenhanced 2D echocardiography for the 
assessment of LV volumes and EF in comparison with non-contrast CMR. 
This trial will be conducted strictly according to the protocol, Good Clinical Practice (GCP), 
the sites’ Independent Ethics Committees (IECs)/Institutional Review Boards (IRBs), and 
other related laws and regulations as applicable. 


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Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
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2 
STUDY OBJECTIVES 
2.1 
Primary Objective  
The primary objective of the study is to demonstrate improvement in accuracy in left 
ventricular ejection fraction (LVEF) assessment using DEFINITY® contrast-enhanced over 
unenhanced echocardiography. 
2.2 
Secondary Objectives 
The secondary objectives of the study are to: 
1. Demonstrate improvement in accuracy in left ventricular ejection fraction (LVEF) 
assessment using DEFINITY® contrast-enhanced over unenhanced echocardiography in 
subjects with suboptimal echocardiograms. 
2. Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography. 
3. Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography. 
4. Demonstrate a reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography in subjects with 
suboptimal echocardiograms. 
5. Demonstrate a reduction in inter-reader variability for the assessment of end-
diastolic/systolic volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms. 
 


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Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
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3 
INVESTIGATIONAL PLAN 
3.1 
Overall Study Design and Plan 
This is a Phase 3, prospective, open-label, multicenter study to evaluate LVEF measurement 
accuracy and reproducibility of DEFINITY® contrast-enhanced and unenhanced 
echocardiography as compared with non-contrast cardiac magnetic resonance imaging 
(CMR) used as the truth standard.  Approximately one-hundred fifty (150) evaluable subjects 
will be enrolled and will undergo unenhanced and DEFINITY®-enhanced echocardiograms 
and CMR.   
Subjects will be screened based on LVEF measurements obtained via 2D echo with or 
without contrast or other methods (e.g. CMR, MUGA scan) obtained within 6 months prior 
to enrollment.    Subjects will be stratified to achieve an approximately even distribution 
within four pre-defined LVEF groups (>50, 41-50, 30–40, <30%) as measured by CMR.  
Subjects with optimal and suboptimal echocardiograms will be enrolled.   
Each patient will undergo an unenhanced ultrasound examination and a DEFINITY® 
contrast-enhanced examination on the same day.  A minimum of 360 seconds of images will 
be collected during both the unenhanced and the DEFINITY® contrast-enhanced 
examinations.   
The site investigator will determine whether an echocardiogram is optimal or sub-optimal 
based on the unenchanced echocardiogram performed on Day 0.  An echocardiogram is 
considered suboptimal if 2 or more segments of the ventricular border are classified as not 
adequately visualized in any one of the 3 apical views.   
Subjects will remain at the clinical site for at least 30 minutes of observation after the end of 
the administration of DEFINITY®.  A safety follow-up telephone call will be conducted for 
all subjects at approximately 72±24 hours after completion of the imaging sessions.   
Enrolled subjects will be followed for AEs, SAEs, and changes in concomitant medications 
from the time the Informed Consent is signed through the safety follow-up telephone call.   
Unenhanced and DEFINITY®-enhanced echocardiograms will be performed with standard 
apical 2 chamber, apical 3 chamber, and apical 4 chamber, (A2C, A3C, and A4C, 
respectively) views.  DEFINITY®-enhanced echocardiograms will be performed using low or 
very low mechanical index (MI) imaging.  Images will be recorded in standard digital format, 
masked to patient identifiers, and sent to a central echocardiography facility for analysis.  
There, 3 experienced independent blinded readers will interpret the results according to the 
Image Review Charter; additional analyses will also be performed as described in the Image 
Review Charter.  Each reader’s independent interpretation will be recorded in a database. 


DEFINITY® 
 
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CMR images will also be collected and independently read at a central laboratory by a single 
reader. 
The schedule of events is provided in Appendix 1. 
Figure 3-1 displays an overview of study events. 
Figure 3-1 
Study Event Overview 
 
 
3.2 
Rationale for Study Design 
The study is intended to demonstrate the utility of DEFINITY® contrast-enhanced 
echocardiography in providing accurate and reproducible measurements of LVEF and LV 
volumes as compared with unenhanced 2D echocardiography using non-contrast CMR as the 
truth standard. 
LVEF measurement obtained via 2D Echo or other 
methods (e.g. CMR, MUGA scan) within 6 months prior 
to enrollment Day 0  
Screening 
Days -7 to Day 0 
Echocardiograms & DEFINITY® 
administration  
Day 0 
 
Safety telephone 
follow-up 
72 ± 24 hours 
CMR within ±30 days of Day 0 


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A broad-based population with known or suspected cardiac disease with either optimal or 
suboptimal 2D echocardiograms or other imaging methods from which LVEF can be derived 
(e.g. CMR, MUGA scan) will be included.   
The primary endpoint of improved accuracy in LVEF measurement will be assessed from the 
results of an independent assessment and subsequent analysis conducted by the central 
imaging laboratory.  Secondary endpoints will also be assessed, including accuracy in the 
suboptimal echocardiogram subset and inter-reader variability in the overall population and 
the suboptimal echocardiogram subset. 
Interpretation of images will be conducted with a blinded read by three experienced 
physicians at a central imaging laboratory who are not associated with patient recruitment.  
In addition, a single, independent, blinded echocardiologist will read each subject’s 
unenhanced echocardiogram as either optimal or suboptimal.  This will be independent of the 
site investigator determination performed at Day 0.  The independent readers will employ a 
prospectively defined and standardized methodology described in the Image Review Charter, 
which will reduce the potential for variation in the criteria used for image reads. 
CMR images will also be collected and read independently at a central imaging laboratory, 
using a methodology that will also be described in the Image Review Charter. 
3.3 
Study Duration 
All screening assessments will occur within 7 days prior to enrollment/DEFINITY® 
administration (Day 0).  Non-contrast CMR studies will occur within ± 30 days of 
DEFINITY® administration.  Safety monitoring will begin with the signing of the informed 
consent and continue up to 72±24 hours post-DEFINITY® administration.  The expected 
duration of subject participation is not more than 41 days.  Subjects are expected be enrolled 
over a 10 month period.   


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4 
STUDY POPULATION 
Approximately one hundred and fifty (150) evaluable subjects who have previously 
undergone a 2D echocardiogram with or without contrast or other assessment (CMR, MUGA 
scan) will be enrolled in this trial.  The study population will consist of male and female 
subjects 18 years of age or older.   
Enrollment will be stratified according to CMR LVEF to achieve an approximately even 
distribution across four subgroups (LVEF >50, 41-50, 30–40, <30%).  LVEF for screening 
purposes will be determined by the investigator and based on measurements with 2D echo or 
other methods (e.g. CMR, MUGA scan) obtained within 6 months prior to enrollment (Day 
0). Subjects with optimal and sub-optimal echocardiograms will be enrolled.  Suboptimal 
echocardiograms will be based on the site investigator’s evaluation of the unenhanced 
echocardiography images obtained on Day 0.  An echocardiogram is considered suboptimal 
if 2 or more segments of the ventricular border are classified as not adequately visualized in 
any one of the 3 apical views. 
4.1 
 Inclusion Criteria 
Potential study subjects must meet the following inclusion criteria to be enrolled in this 
study. 
1. Men and women ≥ 18 years of age in sinus rhythm 
2. Able to communicate effectively with trial personnel 
3. LVEF measurements obtained via 2D Echo with or without contrast or other methods 
(e.g. CMR, MUGA scan) obtained within 6 months prior to enrollment (Day 0) 
4. Has provided signed informed consent after receiving a verbal and written explanation of 
this clinical trial 
 
4.2 
Exclusion Criteria 
Potential study subjects who meet any of the following criteria will be excluded from the 
study: 
1. Female subjects who are pregnant or lactating.  All women of child bearing potential 
[WOCBP] must have a negative urine pregnancy test at screening regardless of 
contraceptive use history. 
2. Women of child-bearing potential are excluded unless they: 
a. are post-menopausal defined as amenorrhea ≥ 12 consecutive months, OR 
b. have undergone successful surgical sterilization (hysterectomy, bilateral tubal ligation 
or bilateral oophorectomy), OR  


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c. have been using an adequate and medically approved method of contraception to 
avoid pregnancy for at least 1 month prior to DEFINITY® dose administration and be 
willing to continue using the same method for the duration of the study. 
3. Current illness or pathology that would prevent undergoing investigational product 
administration due to a significant safety risk to the patient.  
4. Uncontrolled arterial hypertension (defined as systolic blood pressure ≥ 200 mmHg or 
diastolic blood pressure ≥ 110 mmHg) or arterial hypotension (defined as systolic blood 
pressure ≤ 90 mmHg). 
5. Unstable cardiovascular status defined as: 
a. myocardial infarction or unstable angina pectoris within 6 months prior to 
enrollment/DEFINITY® dose administration day  
b. transient ischemic attack or stroke within 3 months prior to DEFINITY® dose 
administration  
c. symptomatic valvular heart disease or moderate to severe stenotic valvular heart 
disease  
d. clinically significant congenital heart defects 
e. current uncontrolled cardiac arrhythmias causing symptoms or hemodynamic 
compromise  
f. acute pulmonary embolus or pulmonary infarction  
g. acute myocarditis or pericarditis  
h. acute aortic dissection   
i. atrial fibrillation 
6. any major surgery within 4 weeks prior to screening 
7. known contraindications to undergoing CMR (e.g. implanted pacemakers, cardioverter, 
defibrillators) or claustrophobia 
8. participation in any investigational drug, device, or placebo study within 30 days prior to 
screening  
9. known hypersensitivity to perflutren, or any of the excipients in DEFINITY® 
10. prisoners or those who are subject to compulsory detention or involuntary incarceration 
for treatment of either a psychiatric or physical illness (e.g., infectious disease) 
 


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5 
STUDY TREATMENT(S) 
5.1 
Description of Treatment(s) 
5.1.1 
Investigational Product 
DEFINITY® is an ultrasound contrast agent consisting of phospholipids-encapsulated 
perfluoropropane (PFP), also known as perflutren, microspheres, which is designed to 
enhance echocardiographic ultrasound images.  DEFINITY® (perflutren injectable 
suspension) will be provided in a 2-mL clear vial containing a 1.5 mL fill volume. 
DEFINITY® contains no preservative.  Bacterial contamination with the risk of 
post-administration septicemia can occur following the puncture of the elastomeric septum.  
It is essential to follow directions for preparation of DEFINITY® carefully and to adhere to 
strict aseptic procedures during preparation. 
The chemical, and pharmacologic properties and formulation of DEFINITY® are described in 
detail in the Investigator’s Brochure (IB).   
5.2 
Treatments Administered 
All treatments will be administered as described in the Dose Preparation and Administration 
Guide.  Appendix 2. 
5.2.1 
Administering Study Drug  
DEFINITY® will be administered as a diluted bolus injection.  1.3 mL of activated 
DEFINITY® will be diluted with 8.7 mL of preservative-free saline to evenly distribute 
microspheres.  An initial injection of up to 3 mL of diluted DEFINITY® will be administered 
with subsequent injections of 1 to 2 mL, as needed.  Full details are available in the Dose 
Preparation and Administration Guide – Appendix 2. 
5.3 
Selection and Timing of Dose for Each Patient 
All subjects will receive administration of DEFINITY® as described above and in the Dose 
Preparation and Administration Guide – Appendix 2. 
5.4 
Method of Assigning Subjects to Treatment Groups 
There is only one treatment group in this study. 


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5.5 
Blinding 
This is an open-label study; therefore, no blinding procedures are required for investigators 
and subjects.   
5.6 
Concomitant Therapy 
Concomitant medications will be collected from the screening visit to the 72±24 hour safety 
follow-up phone call, and will be recorded on the eCRF.   
5.7 
Treatment Compliance 
The investigator will comply with the protocol and all applicable regulatory requirements in 
assuring that the study drug is dispensed to subjects by authorized personnel only. 
5.8 
Packaging and Labeling 
The labeling of the study drug will meet local regulatory requirements, but at a minimum will 
provide the investigational product name, expiry, lot number, manufacturer name and 
address, storage conditions, and caution warnings for investigational product use.   
5.9 
Storage and Accountability 
The investigator is responsible for ensuring that deliveries of study drug are safely and 
properly received, recorded, handled, and stored in accordance with the national and/or local 
laws and are used in accordance with this protocol. 
DEFINITY® should be stored in a refrigerator (2-8° C) upon receipt of the vials.  Prior to 
activation, the vial should be warmed to room temperature.  Following activation, 
DEFINITY® can be stored at room temperature (do not store above 30°C) and should be used 
within 12 hours of preparation.  Refer to DEFINITY® Dose Preparation and Administration 
Guide (Appendix 2) for full storage details. 
5.10 Investigational Product Retention at Study Site 
The site will document the administration of the investigational product for each subject. All 
investigational product, used and unused, will be accounted for by a study monitor.   
All unused or residual study drug must be properly destroyed at the study site in accordance 
with national and/or local laws and institutional policies and properly documented. 


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6 
STUDY PROCEDURES 
6.1 
Screening 
The timing of study procedures is provided in Appendix 1.  All Screening/Baseline 
assessments will occur within 7 days prior to administration of study drug (Day 0). 
6.2 
Informed Consent 
Informed consent (IC) must be obtained from each patient prior to the initiation of any study 
related procedures unless those procedures are considered standard of care for the diagnosis 
and/or treatment of the underlying disease process.  The IC must meet the requirements as 
defined by International Conference on Harmonization (ICH) - Good Clinical Practice (GCP) 
guidelines, US Federal Regulations and/or conform to national or regional regulatory agency 
requirements, or local laws, whichever provides the greatest level of protection.  Each patient 
must be provided, in an understandable manner, a written informed consent form (ICF) that 
describes the nature and duration of the study.  Additionally, the patient must be allowed 
adequate time to consider the potential risks and benefits associated with his/her participation 
in the study.  The IC must also contain language that allows the Sponsor (or designated 
representative), regulatory authorities, and the Institutional Review Board (IRB)/ Institutional 
Ethics Committee (IEC) direct access to the patient’s medical records for the purpose of 
review. 
In situations where study subjects are not legally competent to provide consent (i.e., 
cognitively incapacitated patient), written consent must be obtained from an authorized 
guardian or representative.  In these situations, the ICF must be signed and dated by a witness 
and, if possible, assent must be obtained from the patient.  The ICF must also be signed and 
dated by the individual obtaining the IC. 
The Investigator, as part of the study documentation, must retain the original signed and 
dated ICF and must provide a signed copy to each patient or authorized representative.  The 
Investigator will not undertake any measures specifically required only for the clinical study until 
valid consent has been obtained. 
6.3 
Medical History 
A general medical history for each patient will be obtained by interview and through a 
review of available medical records.  Data to be collected and recorded on the eCRF will 
include demographics (e.g. age, gender, race/ethnicity) and an overview of significant past or 
present illnesses, including previous administration of DEFINITY®.  Known drug allergies, 
other allergies, or history of hypersensitivity reactions will also be recorded on the eCRF.  


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Information obtained via interview should be supplemented with data from hospital records 
or clinic charts as available. 
All concomitant medications will be recorded on the eCRF.  This information will be 
obtained by interview and through a review of available medical records. 
6.4 
Physical Examination 
A physical examination will be performed at screening and will include height, weight, 
general appearance, and assessment of appearance (head, ears, eyes, nose and throat 
[HEENT]); neck; cardiovascular; lungs; abdomen; lymph nodes; extremities; neurological; 
skin; musculoskeletal; and other).  All physical examination abnormalities will be recorded 
on the eCRF. 
6.5 
Vital Signs 
All vital sign measurements should be obtained with the patient lying supine, having rested 
quietly for at least 2 minutes, as feasible.  Vital signs (including heart rate, respiratory rate, 
and systolic and diastolic blood pressure) will be collected at the Screening visit only. 
6.6 
Pregnancy Test 
A urine sample for pregnancy testing will be obtained for all women of childbearing potential 
at screening and within 24 hours prior to dosing the study drug. 
6.7 
Cardiac Magnetic Resonance Imaging 
6.7.1 
Image Acquisition 
Subjects will receive a non-contrast enhanced CMR examination. MRI examinations will be 
performed by a qualified technologist supervised by a physician experienced with cardiac 
MRI. Steady-state free precession (SSFP) MRI will be performed using a 1.5 T (or above) 
imaging system and a standardized and harmonized pre-specified method in accordance with 
practice guidelines for images that are to be used for LVEF evaluation. Cardiac slices relative 
to single apical 2- and 4-chamber views as well as multi-slice short-axis images running from 
the apex to the base will be obtained for each patient. The apical 2-chamber view should be 
parallel to the interventricular septum and intersect the cardiac apex and mid-mitral valve; 
whereas, the apical 4-chamber view should be perpendicular to the interventricular septum, 
maximizing ventricular size, and should intersect the apex and mid-mitral valve. The left 
ventricular outflow tract and aorta must not be visualized in this view. Parasternal short-axis 
images of the left ventricle will be acquired, perpendicular to the long axis of the ventricle 
spanning apex to base.   


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Consecutive breath held short-axis cine images will be obtained with full ventricular 
coverage.   
All images will then be transferred to an Imaging Core Laboratory for further analysis. No 
site analysis will be performed. Instructions for MRI data transfer and storage at the central 
imaging laboratory will be provided in the study Imaging Manual.   
6.7.2 
Imaging Core Laboratory 
To identify the LV end-diastolic and end-systolic images, the full stack of short axis images 
will be evaluated.  The LV end-diastolic image will be chosen as the phase with the largest 
LV blood volume.  The LV end-systolic image will be chosen as the phase with the smallest 
LV blood volume.   The Simpson’s method of summation of discs will then then applied 
(sum over all slices of cross sectional area multiplied by the sum of the slice thickness and 
the inter-slice gap) to determine LV end-diastolic (LVEDV) and LV end-systolic (LVESV) 
volumes for entire LV. Quantification of LVEF will then be calculated based on volumes 
using the formula: LVEF = (LVEDV − LVESV)/LVEDV.4,5 
A single central MR reader will verify and correct if necessary the identification of the LV 
end-systolic and diastolic images provided by a trained and blinded technologist to determine 
the LV end diastolic and end-systolic volumes. Similarly a trained and blinded technologist 
will provide initial ventricular contours which will be verified and corrected if necessary by 
the central MR reader prior to final calculation of the volumes. 
6.8 
Echocardiography Imaging Procedures 
6.8.1 
Image Acquisition 
A bedside resting transthoracic echocardiogram will be performed by personnel trained in 
study procedures according to standardized instructions provided here and in the study 
Imaging Manual.  Unenhanced echocardiography will be performed in each of the standard 
A2C, A3C, and A4C views after optimization of these views.  At least 3 consecutive cardiac 
cycles should be recorded in standard digital format in each view.  After unenhanced imaging 
has been conducted, study drug will be administered as described above and in the Dose 
Preparation and Administration Guide.  DEFINITY®-enhanced images will be obtained with 
low or very low MI imaging using a standardized and harmonized method consistent with 
practice guidelines.  After study drug administration and subsequent image optimization, the 
same A2C, A3C, and A4C views should be obtained as with unenhanced imaging over at 
least 3 consecutive cardiac cycles. 
Subjects will remain at the site for observation until at least 30 minutes after the end of the 
administration of study drug.   


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All images will then be transferred to an Imaging Core Laboratory for further analysis. No 
site analysis will be performed. Instructions for echo data transfer and storage at the central 
imaging laboratory will be provided in the study Imaging Manual.   
6.8.2 
Imaging Core Laboratory 
Left ventricular end-diastolic and end-systolic volumes will be measured using the modified 
biplane Simpson's method in the apical 4 chamber and apical 2 chamber views. End-diastole 
will be identified as the frame in which the left ventricular cavity size is visually 
largest.  End-systole will be identified as the frame in which the left ventricular cavity size is 
visually smallest, just before mitral valve opening. The inner contour of the LV cavity 
(interface between left ventricular cavity and compacted myocardium) will be traced 
according to the recommendations of the American Society of Echocardiography (ASE), 
leaving the papillary muscles and trabeculations within the cavity.  Left ventricular ejection 
fraction will be calculated from the biplane volumes using the formula: 
LVEF = (LVEDV − LVESV)/LVEDV.6 
A blinded echocardiography technologist at the Imaging Core Laboratory will provide an 
initial contour of the LV cavity for the blinded read.  The blinded echocardiography readers 
will adjust contours based on their judgement and will provide independent LV 
measurements.  The same method for LVEF calculation will be used for all echocardiograms, 
including suboptimal and non-suboptimal and both enhanced and non-enhanced.   
6.9 
Efficacy Assessments 
Three blinded independent readers will perform all of the efficacy assessments following the 
methodology described in the Imaging Review Charter. 
6.9.1 
Primary Efficacy Assessments 
The primary efficacy assessment will be the measurement of LVEF. 
6.9.2 
Secondary Efficacy Assessments 
Secondary efficacy assessments will be the end-systolic and end-diastolic LV volumes. 
6.10 Adverse Event Assessments 
6.10.1 
Performing Adverse Event Assessments 
In this clinical trial, an AE is defined as any new untoward medical occurrence or worsening 
in severity or frequency of a pre-existing medical condition in a study patient, which does not 
necessarily have a causal relationship with investigational product.  AEs occurring from the 


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time the informed consent is signed until the 72±24 hour telephone follow-up will be 
reported.  Treatment-emergent adverse events (TEAEs) are a safety endpoint and are 
discussed in Section 8.4.1.  The NCI-CTCAE version 4.0 will be utilized for Adverse Event 
(AE) reporting of severity by grade. 
An AE can, therefore, be any unfavorable and unintended sign (including an abnormal 
laboratory finding, for example), symptom or disease temporally associated with the use of a 
medicinal (investigational or marketed) product, whether or not considered related to the 
medicinal (investigational or marketed) product. 
Note:  For this clinical trial, observations and findings (previously unknown) discovered 
during the procedures and medically assessed as most-likely pre-existing (e.g., myocardial 
wall hypokinesis, thrombus, cardiac valve deficits, and so on) will not be considered AEs and 
will not be reported as such in the eCRF.  However, these observations and any 
hospitalizations/treatments required to treat the new findings will be reported elsewhere in 
the eCRF.  Any complications resulting from the study procedures (e.g., intravenous 
extravasations) will be recorded as an AE.   
A medication error is defined as any event related to the administration of a medicinal or 
investigational product that may cause or lead to inappropriate use or patient harm.  
Medication errors can be actual or potential errors and may include misuse, administration 
error, overdose, under-dose, or abuse.  For this clinical trial, any medication errors (e.g., 
administration error, overdose, etc., that occur at the time of study product administration) 
must be recorded as an AE.  
All identified AEs must be recorded and described on the appropriate AE section of the 
eCRF by the investigator (or designee). If known, a diagnosis should be recorded, rather than 
multiple individual symptoms.  In addition to the event term, the following information 
should be captured for all AEs: 
• Date (and time) of onset and resolution  
• Severity of the event (see definitions below)  
• Investigator’s opinion of the causal relationship to study drug or comparator, procedure, 
or underlying disease (see definitions below)  
• Treatment action taken  
• Information regarding resolution/outcome 
 


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6.10.2 
Timing 
Timely and complete reporting of safety information helps to identify any untoward medical 
occurrence, thereby allowing: 
• Protection of safety of study subjects, 
• Greater understanding of the overall safety profile of the investigational product, 
• Appropriate modification of study protocols, and 
• Adherence to worldwide regulatory requirements. 
 
Adverse event monitoring will be initiated at the time the informed consent is signed and will 
continue until completion of all echo assessments and patient discharge.  All subjects will be 
followed up by telephone assessments for AEs at 72±24 hours following DEFINITY® 
administration. 
6.10.3 
Severity 
The Investigator (or designee) will assess the severity of the AE using the National Cancer 
Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 4.0 will 
be utilized for Adverse Event (AE) reporting.  The NCI-CTCAE definitions of severity are 
below.   
Grade 
Degree of Severity 
1 
Mild, with mild or no symptoms; no interventions required 
2 
Moderate; minimal intervention indicated; some limitation of activities 
3 
Severe but not life threatening; hospitalization required; limitation of patient's ability 
to care for him/herself 
4 
Life threatening; urgent intervention required 
5 
Death related to adverse event 
 
This assessment is subjective and the Investigator should use good clinical judgment to 
compare the reported AE to similar type experiences observed in clinical practice. 


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6.10.4 
Relationship 
The Investigator will assess the relationship of the AE to DEFINITY®. The following 
categories and definitions of causal relationship should be used for this study: 
• Not related: indicates that there is little or no chance that the study treatment or 
procedure caused the reported AE; other conditions, including concomitant illnesses, 
progression or expression of the disease state, or a reaction to a concomitant medication, 
appear to explain the reported AE.  For the purposes of determining expedited 
reportability, AEs categorized as unlikely related to investigational product will not be 
considered associated with the investigational product. 
• Possibly related: indicates that the association of the AE with the study treatment or 
procedure is unknown; however, the AE is not reasonably attributed to any other 
condition 
• Related: indicates that a reasonable temporal association exists between the AE and 
study treatment or procedure and, based upon the investigator’s clinical experience, the 
association of the event with the study treatment seems likely 
 
6.10.5 
Clinical Significance 
Any abnormality in physical examinations or vital signs not present at Baseline should be 
judged for clinical significance by the Investigator. 
6.10.6 
Serious Adverse Events 
6.10.6.1 
Definition 
A serious adverse event (SAE) is defined by regulation as any AE occurring at any dose that 
results in any of the following outcomes:  
• Death 
• Life-threatening AE 
• In-patient hospitalization or prolongation of existing hospitalization 
• Persistent or significant disability/incapacity, or  
• Congenital anomaly/birth defect. 
 
Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered an SAE when, based upon appropriate medical judgment, 
they may jeopardize the patient and may require medical or surgical intervention to prevent 
one of the outcomes listed in this definition.  Examples of such medical events include 


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allergic bronchospasm requiring intensive treatment in an emergency room or at home, and 
blood dyscrasias or convulsions that do not result in inpatient hospitalization. 
Note: Hospitalization for scheduled elective procedures or hospitalization from clinically 
indicated patient management decisions based on pre-study conditions should not be 
classified as SAEs. Examples of this would include a scheduled coronary intervention or 
CABG procedure within the SAE reporting period.  Additionally, a treatment procedure 
scheduled due to diagnosis during the investigational procedure would not be classified as an 
SAE.  An example of this would be angioplasty to treat a patient’s underlying CAD.  Events 
resulting from the investigational procedure or a worsening of a pre-study condition which 
satisfy the definition of a SAE must be reported.  
6.10.6.2 
Reporting Serious Adverse Events 
All SAEs, whether related or unrelated to the investigational product, must be reported 
within 1 calendar day to LMI Drug Safety by confirmed facsimile transmission, email or 
phone.  Overnight express mail may also be used in lieu of a facsimile transmission.  If only 
limited information is initially available, follow-up reports are required.  The original SAE 
form must be kept on file at the study site. 
Collection of complete information concerning SAEs is extremely important.  Thus, 
follow-up information that becomes available as the SAE evolves, as well as supporting 
documentation (e.g., hospital discharge summaries and autopsy reports), should be collected 
subsequently, if not available at the time of the initial report, and immediately sent using the 
same procedure as the initial SAE report. 
The contact information for reporting SAEs and follow-up SAE information is: 
Contact 
Telephone Number 
Fax Number 
Email Address 
LMI Safety 
1-800-343-7851 
+ 978-677-9531 
FAX: 1-978-436-7296 
lantheussafety@lantheus.com 
 
 
If the investigator needs to discuss an SAE or any other aspect of the protocol with the 
Medical Monitor, s/he can be reached at the following numbers:  
LMI Medical Monitor 
24-Hour phone number:  +1-240-481-8818 
Fax:  +1-978-436-7296  
 


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In the event of AEs that are serious, unexpected, and possibly related or related to 
DEFINITY® administration, LMI will contact the Investigator and will provide a MedWatch 
and/or Council for International Organizations of Medical Sciences (CIOMS) narrative of the 
case, which will include an Analysis of Similar Events.  The Investigator is responsible for 
notifying the appropriate IRB in accordance with institutional requirements.  The Investigator 
and IRB will determine if the IC requires revision.  The Investigator should also comply with 
the IRB procedures for reporting any other safety information. 
6.11 Removal of Subjects from the Trial or Investigational Product 
The investigator may withdraw a patient from the study for any of the following reasons: 
• Patient withdraws consent 
• Patient is lost to follow up 
• Patient has an AE that, in the opinion of the Investigator, requires the patient’s 
discontinuation 
• Discretion of the Investigator  
• The Sponsor or Investigator terminates the study  
 
All events that result in discontinuation of study treatment will be appropriately recorded and 
reported.  In addition, for all subjects who discontinue prematurely, an evaluation which 
reflects the status of the patient at premature termination, along with a final assessment and 
the reasons for termination, will be recorded on the eCRF.   


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7 
QUALITY CONTROL AND ASSURANCE 
The sponsor has implemented and maintains Quality Assurance and Quality Control systems 
with written procedural documents (e.g. SOPs, work instructions, monitoring plans, etc.) to 
ensure that the study is conducted, and data are generated, recorded, and reported in 
compliance with the protocol, ICH-GCP, and all applicable regulatory requirements. 
These quality systems include monitoring (see Section 9.6); data collection, review and entry 
(see Section 9.11); training; and audit by the sponsor’s Quality group or representative.  
Audits are independent of routine monitoring and quality control functions, and are 
conducted to evaluate trial conduct and compliance with the protocol, procedural documents, 
ICH-GCP and applicable regulatory requirements. 


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8 
PLANNED STATISTICAL METHODS 
8.1 
Statistical Analyses 
8.1.1 
General Considerations 
The contract research organization (CRO) will be responsible for data management and 
statistical analysis in this trial.  All statistical analyses will be performed using SAS (SAS 
Institute, Inc., Cary, NC) version 9.4 or higher.  Patient data listings and tabular presentations 
of results will be provided.  Presentation of summary statistics for continuous variables will 
include N, mean, median, and standard deviation, as well as the minimum and maximum 
values.  For categorical variables, the number and percent of each category within a 
parameter for non-missing data will be calculated.  All statistical tests will be two-sided 
employing a significance level of 5% unless otherwise specified.  Further details of the 
criteria and conduct of the statistical analyses are below and will be included in the Statistical 
Analysis Plan for this study. 
8.1.2 
Analysis Populations 
8.1.2.1 
Safety Population 
The Safety Population (SP) will include all subjects who have signed informed consent and 
who have received any amount of DEFINITY® in the study. This is the primary analysis 
population for the safety analysis.  
8.1.2.2 
Intent-to-Treat 
The Intent-to-Treat (ITT) population will include all subjects who have signed informed 
consent. 
8.1.2.3 
Modified Intent-to-Treat Population 
The Modified Intent-to-Treat (mITT) population will include all ITT subjects who complete 
unenhanced imaging, DEFINITY® -enhanced imaging, and cardiac MR studies.  This is the 
primary analysis population for the efficacy endpoints. 
8.1.2.4 
Per-Protocol Population 
The Per-Protocol (PP) population will include all subjects in the mITT population who a) did 
not violate inclusion and exclusion criteria that would likely have an effect on the primary 
outcome, b) do not have major protocol violations; c) have LVEF data on both unenhanced 
and DEFINITY®- enhanced for at least one reader; (d) have CMR LVEF.  


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Efficacy analyses will be conducted for both mITT and PP population sets.  Differences in 
results using the two populations will be carefully examined and the results of that evaluation 
presented in the study report. 
8.2 
Efficacy Endpoint(s) 
8.2.1 
Primary Efficacy Endpoint  
The primary analysis is to demonstrate an improvement in LVEF accuracy from unenhanced 
imaging to imaging with DEFINITY® contrast enhancement using CMR as the truth 
standard.  For each patient, the absolute value of the difference of DEFINITY® LVEF minus 
CMR LVEF will be calculated.  Similarly, the absolute value of the difference of unenhanced 
imaging LVEF minus CMR LVEF will be calculated for each patient.  The primary analysis 
is to assess the significance of the difference between DEFINITY® and unenhanced echo 
with respect to the mean “absolute value of the difference vs. CMR”.  These means will be 
compared with a paired t-test at a two-sided 0.05 level of significance.  Specifically, the null 
and alternative hypotheses are: 
H0: µD = µU vs. H1: µD ≠ µU 
where µD and µU are the mean of the DEFINITY® and unenhanced echo absolute value of 
the difference vs. CMRs, respectively.  A sample of size 150 enrolled subjects has 90% 
power to reject the null hypothesis in favor of the alternative if the true difference µD - µU is 
at least 2.75 (in favor of DEFINITY®) with a standard deviation of 10 or less and allows for 
approximately 5% premature withdrawal. 
The analyses will be conducted separately for each of the three blinded readers of the 
DEFINITY® enhanced and unenhanced images; the criterion for success is that the null 
hypothesis is rejected in favor of DEFINITY® for at least 2 of the 3 blinded readers.  CMR 
LVEF is interpreted by a single reader; this is the CMR LVEF that will be used as the 
comparator for each of the three blinded readers. 
The primary analysis will be performed on mITT subjects with non-missing LVEF for 
DEFINITY®, unenhanced echocardiography, and the reference standard.  A supportive 
analysis will be run where a missing echocardiography or CMR LVEF is multiply imputed 
using the fully conditional specification (FCS) multiple regression, as outlined below and as 
will be further detailed in the Statistical Analysis Plan. 
The following are additional analyses to be carried out separately for each blinded reader to 
achieve the primary objective: 
Bias is the mean of the actual (not absolute value) per-subject differences between the 
imaged LVEF and CMR LVEF.  The bias, the two-sided 95% confidence interval of the bias, 


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the precision (standard deviation of the per-subject differences) and the root mean square 
error (RMSE) will be calculated for DEFINITY®.  The RMSE is the square root of the bias-
squared + precision-squared and is considered a measure of overall accuracy.  These analyses 
will be repeated for unenhanced echocardiography vs. CMR.   It is anticipated that the RMSE 
will be smaller for DEFINITY®-enhanced LVEF than for unenhanced LVEF. 
DEFINITY®-enhanced and unenhanced echocardiography LVEF will each be assessed for 
measurement accuracy against the reference CMR using Bland-Altman analysis and Deming 
regression analysis.  For the Bland-Altman analysis, a plot of the per-subject difference 
between DEFINITY®-enhanced echocardiography and CMR LVEF will be plotted vs. the 
per-subject sum of the two measurements. Limits of agreement (defined as the mean of the 
DEFINITY® vs. CMR difference) +/- 2 SD will be shown on the plot.  The same analyses 
will be performed for unenhanced echocardiography using CMR as the reference standard. 
Deming regression plots of LVEF vs. CMR LVEF will be generated for each of 
DEFINITY®-enhanced and unenhanced echocardiography.  Unweighted Deming regression 
will be employed to estimate the regression slope and intercept with two-sided 95% 
confidence intervals of each assuming the measurement error is the same for CMR and each 
of the echocardiography error techniques. 
The intra-class correlation coefficient (ICC) and its two-sided 95% confidence interval will 
be calculated for DEFINITY®-enhanced echo vs. CMR and for unenhanced echo vs. CMR.  
The ICCs and their two-sided confidence intervals will be calculated using between and 
within mean squares from an ANOVA model with method (echocardiography, CMR) and 
subject as the main effects and LVEF as the dependent variable.  The ICC will be calculated 
using the SAS macro developed by Hamer (1990).7 
Assessment Across Study Centers:  The mean “absolute value of the difference vs. CMR” 
for each DEFINITY® and unenhanced echocardiography, as well as the difference between 
the two treatments with respect to this mean, will be presented for each study center and 
blinded reader.  Within each blinded reader, an assessment of study center effect on the mean 
treatment difference will be carried out using one-way analysis of variance. For each blinded 
reader, a site difference that is not significant at the 0.15 level of significance, or a site 
difference that is significant but where for every site the mean treatment difference is more 
favorable for DEFINITY® than enhanced echocardiography, will support pooling subjects 
across sites for the primary analysis for that reader. 
Missing Data:  Within each blinded reader, the primary analysis will be performed on 
subjects with non-missing LVEF for DEFINITY®, unenhanced echocardiography, and the 
reference standard.  A supportive analysis will be run where, within each blinded reader, 
missing LVEF is multiply imputed using the fully conditional specification (FCS) multiple 
regression.  The covariates in the imputation model will be fully specified in the formal 


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statistical analysis plan and will include baseline and demographic variables and will also 
include imaging method (DEFINITY®, unenhanced, or CMR reference standard).  A total of 
50 imputations will be generated; the paired t-test comparing mean bias between 
DEFINITY® and unenhanced will be performed separately on each of the 50 imputed 
datasets, and the t-test results will be combined across datasets using the usual multiple 
imputation techniques to create one overall paired t-test results on imputed data. 
8.2.2 
Other Primary Endpoint(s) 
None 
8.3 
Secondary Endpoint(s) 
8.3.1 
Secondary Efficacy Endpoint(s) 
8.3.1.1 
Improvement in accuracy in left ventricular ejection fraction (LVEF) assessment 
using DEFINITY® contrast-enhanced over unenhanced echocardiography in 
subjects with suboptimal echocardiograms  
The above primary analyses will be repeated here, but on the subgroup of subjects with 
suboptimal echocardiograms.  There will be no imputation of missing data for this analysis. 
8.3.1.2 
Reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography 
The inter-observer variability among each pair of readers within each imaging modality will 
be estimated using an intra-class correlation coefficient (ICC).  The ICC assesses rating 
reliability by comparing the variability of different ratings of the same subject with the total 
variation across all ratings and all subjects. The inter-observer variability in the assessment of 
LVEF between two readers will be determined by percentage of error.  The percentage of 
error will be calculated using the formula: 
Percentage of error = SD between 2 measurements/ mean of the 2 measurements x 100 
The mean percentage of error and its 95% confidence interval will be calculated for each pair 
of readers within each imaging modality.  
There will be no imputation of missing data for this analysis. 


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8.3.1.3 
Reduction in inter-reader variability for the assessment of end-diastolic/systolic 
volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography 
The above analyses used to assess inter-reader variability on LVEF will be carried out, but on 
end-diastolic/systolic volumes.  There will be no imputation of missing data for this analysis. 
8.3.1.4 
Reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography in 
subjects with suboptimal echocardiograms 
The above analyses used to assess inter-reader variability on LVEF will be carried out, but on 
the subgroup of subjects with suboptimal echocardiograms.  There will be no imputation of 
missing data for this analysis. 
8.3.1.5 
Reduction in inter-reader variability for the assessment of End-Diastolic/Systolic 
volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms 
The above analyses used to assess inter-reader variability on LVEF in subjects with 
suboptimal echocardiograms will be carried out, but on end-diastolic/systolic volumes.  
There will be no imputation of missing data for this analysis. 
8.3.2 
Other Secondary Endpoint(s) 
None. 
8.4 
Safety Endpoint(s) 
8.4.1 
Primary Safety Endpoint(s) 
Safety analyses will include tabulations of the incidence (number and percent of subjects) of 
treatment-emergent adverse events (TEAEs) and procedure-related AEs by MedDRA system 
organ class (SOC) and preferred term (PT).  This will be repeated for serious adverse events, 
for adverse events leading to study withdrawal and for adverse events at least possibly related 
to study procedures.  This will be performed on the safety population. 
8.4.2 
Secondary Safety Endpoint(s) 
None. 


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8.5 
Demographics and Baseline Characteristics 
Demographic profiles including age, gender, race, ethnicity, height, weight and BMI will be 
summarized for all Safety evaluable subjects.  Descriptive statistics (n, mean, median, 
standard deviation, minimum and maximum) will be provided for each quantitative variable, 
and frequencies and percentages will be provided for each categorical variable. 
8.6 
Interim Analysis 
This study will utilize an adaptive design that allows one interim sample size re-calculation 
after a minimum of 75 subjects have been enrolled and followed. The pre-specified 
maximum allowable adjusted sample size following re-estimation will be 300 enrolled, or 
two times the initial planned sample size of 150 enrolled. The re-estimation of sample size 
will be conducted by an independent biostatistician following a pre-specified plan using the 
method of by Mehta and Pocock (2011).1 Specifically, at the interim stage the conditional 
power (CP) for obtaining a significant beneficial effect of DEFINITY® over unenhanced 
imaging with respect to the primary endpoint will be calculated for each blinded reader, 
using the protocol-specified planned sample size of 150 enrolled subjects (approximately 143 
evaluable subjects assuming 5% premature withdrawal). This conditional power will be 
calculated under the assumption that the interim estimate of DEFINITY® -unenhanced 
difference is the true population difference. Specifically, the calculation of conditional power 
will be as follows: 
Let µD be the true mean of the absolute value of the LVEF differences between the 
DEFINITY®-enhanced echo LVEF and the CMR LVEF, and let µU be the true mean of the 
absolute value of the LVEF differences between the unenhanced echo LVEF and the CMR 
LVEF.  After 75 patients are enrolled and followed (which should lead to approximately 71 
evaluable patients), an unblinded interim analysis will be conducted to determine whether 
sample size should be increased to maintain adequate conditional power of up to 90% for 
each reader. Conditional Power (CP) for rejecting the null hypothesis in favor of 
DEFINITY® by the planned final sample size of 143 evaluable patients is calculated as 
follows for each reader: 
CP = 𝑃(𝑍>
𝐶2√𝑖2−𝑡1√𝐼1−(𝐼2−𝐼1)∆
√𝐼2−𝐼1
) 
where  
a. Z is a random standard random variate 
b. c2 is the t-critical value to be used in the final analysis = 1.97681 (one-sided 0.025 level 
of significance; , with 142 df assuming the final evaluable sample size is 143, which is 
the number of evaluable subjects expected for the final analysis with 150 enrolled 
subjects with 5% premature withdrawal) 


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c. Δ is the assumption of the true difference “µU minus µD” where µD is the mean of the 
absolute value of the differences between the DEFINITY®-enhanced echo LVEF and the 
CMR LVEF, and µU is the mean of the absolute value of the differences between the 
unenhanced echo LVEF and the CMR LVEF; Δ will be set to the value observed at the 
interim (the calculation of Δ is described below; it is not correct to calculate it as the 
interim point estimate of µU minus the interim point estimate of µD). 
d. I1 is the observed “information” at the interim analysis based on the interim observed 
standard deviation of µD minus µE; its calculation is described below. 
e. I2 is the anticipated “information” at the final analysis; its calculation is described below. 
f. t1 is the interim paired t-statistic testing the null hypothesis, calculated from the data 
observed at the interim 
 
Further information on the above parameters is as follows: 
Assuming the Definity-CMR raw LVEF difference (not absolute value) is normally 
distributed with mean μ and sd σ, then its absolute value, D, follows the folded normal 
distribution with true mean and sd as follows: 
𝜇𝐷= √2
𝜋𝜎𝑒−𝜇2
𝜎2 + 𝜇[1 −2Φ (−𝜇
𝜎)]         𝜎𝐷= √𝜇2 + 𝜎2 −𝜇𝐷
2 
where ϕ is the standard normal cumulative distribution function. A similar algorithm can be 
applied to the unenhanced-CMR LVEF raw difference and its absolute value U to determine 
µU  and σU.  In the above CP formula, Δ will be set to the estimate of µD - µU, where the 
estimates of µU and µD  are each calculated from the above equation using point estimates of 
the relevant μ and σ.  The standard deviation of the difference between the absolute value of 
D minus the absolute value of U is  
√𝜎𝐷
2 + 𝜎𝑈
2 −2 ∗𝐶𝑜𝑣(𝐷, 𝑈) where 2 ∗𝐶𝑜𝑣(𝐷, 𝑈) = 𝜌∗ 𝜎𝐷∗𝜎𝑈  
 
where ρ is the correlation between D and U. This standard will be used in the calculation of 
the “information”, as further described below.  
So, in the CP formula for this example, 
a. Δ will be set to the estimate of µD - µU 
b. I1  = 
1
𝑠2/𝑛1 where s = the interim estimate of the above standard deviation between the 
absolute values D and U 
c. I2  = 
1
𝑠2/𝑛2 where s - the interim estimate of the above standard deviation between the 
absolute values D and U and n2 is the planned final sample size = 143.   


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•  
The study will not be stopped for overwhelming efficacy or futility at this interim stage. 
Instead, based on Table 1 of Mehta and Pocock (2011),1 the following algorithm will be 
carried out:  (1) if the CP ≤ 36% for at least two of the three blinded readers based on the 
final sample size, the study will continue to its original planned sample size of 150 enrolled 
subjects; otherwise (2) if the CP ≥ 90% for at least two of the three blinded readers based on 
the final sample size, the study will continue to its original planned sample size of 150 
enrolled subjects; (3) if 36% < CP <90% (the Mehta and Pocock “promising zone”) for at 
least two of the three readers based on the final sample size, then the sample size will be 
increased so that the CP is at least 90% for every reader whose CP fell in the promising zone.  
However, if there is a reader who does not fall in the promising zone, then that reader’s 
primary results will be based only on the patients enrolled under the original protocol-
specified sample size.  Further, for every reader for whom the sample size is increased, that 
reader’s results will only be based on the first X images, where X is the number of images 
that yielded CP of 90%.  The sample size may be increased  up to 300 enrolled subjects 
(twice the protocol-planned sample size) for a given reader in order to obtain 90% CP.  If for 
a given reader that falls into the promising zone, the maximum increase to 300 enrolled 
subjects still does not yield 90% CP, the sample size may still be increased to 300 for this 
reader to maximize conditional power, at the discretion of the independent statistician).  If 
(1), (2), and (3) above are not satisfied, the study will continue to its original planned sample 
size of 150 enrolled subjects. This approach to sample size increase does not inflate the 
overall Type I error for a given reader. 
The sponsor and investigators will remain blinded to the interim results for the duration of 
the ongoing study.  After the interim analysis is carried out, the recommendation made to the 
sponsor will only be to keep sample size as is or to increase enrolled sample size to a given 
value; the reason for the recommendation will not be given to the sponsor. 
8.7 
Determination of Sample Size 
See Section 8.2.1 on the primary efficacy endpoint for the determination of sample size. 


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9 
ADMINISTRATIVE CONSIDERATIONS 
9.1 
Investigators and Study Administrative Structure 
Each Investigator is responsible for conducting the study in accordance with the protocol, all 
applicable laws, regulations and ICH-GCP.   
Lantheus Medical Imaging is the Sponsor of this study and is responsible for overseeing its 
conduct.  Study-specific information regarding the imaging procedures will be described in 
the Imaging Manual. 
9.2 
IRB or IEC Approval 
It is the Investigator’s responsibility to ensure that all aspects of the ethics review are 
conducted in accordance with the current ICH and GCP guidelines, and/or in accordance 
with local laws, whichever provides the greatest level of protection for the study subjects.  
The protocol and any information supplied to the patient to obtain informed consent, 
including written ICF(s), patient recruitment procedures (e.g., advertisements) and written 
information (e.g., information leaflets), must be reviewed and approved by a qualified 
IRB/IEC prior to enrolling subjects in the study.  Before study initiation, the Sponsor must 
receive documentation of the IRB/IEC approval, which specifically identifies the 
study/protocol, and the assurance number or a list of the committee members. 
Amendments to the protocol and revisions to the IC must also be submitted to and approved 
by the IRB/IEC. 
At intervals required by the IRB/IEC, but not less than annually, the Investigator must submit 
to the IRB/IEC a progress report with a request for re-evaluation and re-approval of the 
study.  A copy of the progress report and re-approval of the study must be sent to the 
Sponsor. 
When the Sponsor provides the Investigator with a Safety Report, the Investigator must 
promptly forward a copy to the IRB/IEC. 
After completion or termination of the study, the Investigator must submit a final report to 
the IRB/IEC and forward a copy to the Sponsor.  As part of the record retention requirements 
for the study, the Investigator must maintain documentation of all submissions, 
correspondence and approvals to and from the IRB/IEC. 
9.3 
Ethical Conduct of the Study 
This study will be conducted in conformance with all applicable country or local 
requirements regarding ethical committee review and informed consent using the guidelines 


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for the protection of the rights and welfare of subjects participating in biomedical research, as 
described in the following documents: 
• US Code of Federal Regulations (CFR) dealing with clinical studies (21 CFR including 
parts 50 and 56 concerning informed consent and IRB US regulations) 
• Good Clinical Practice:  Consolidated Guidance (ICH-E6 R2) 
 
By signing the protocol, the investigator(s) will agree to adhere to the instructions and 
procedures described in it and thereby to adhere to the principles of GCPs to which it 
conforms. 
9.4 
Patient Information and Consent 
Informed consent must be obtained from each patient prior to entering the study, and must 
meet the requirements as defined by the ICH GCP guideline, US Federal Regulations and/or 
conform to local laws, whichever provides the greatest level of protection.  Each patient must 
be provided, in an understandable manner, written and verbal information that describes the 
nature and duration of the study.  Additionally, the patient must be allowed adequate time to 
consider the potential risks and benefits associated with his/her participation in the study.  
The IC must also contain language that allows the Sponsor (or designated representative), 
regulatory authorities, and the IRB/IEC direct access to the patient’s medical records for the 
purpose of review and copying. 
The ICF must be signed and dated by the study patient.  In situations where a study patient is 
not legally competent to provide consent (i.e., a mentally incapacitated patient), written 
consent must be obtained from an authorized guardian or representative.  In these situations, 
the consent form must be signed and dated by a witness and, if possible, assent must be 
obtained from the patient.  The ICF must also be signed and dated by the individual obtaining 
the IC. 
The Investigator, as part of the study documentation, must retain the original signed and 
dated ICF and must provide a signed copy to each patient or authorized representative.  The 
Investigator will not undertake any measures specifically required only for the clinical study until 
valid consent has been obtained. 
9.5 
Patient Confidentiality 
The Sponsor affirms and upholds the principle of the patient’s right to protection against 
invasion of privacy.  


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9.6 
Study Monitoring 
Each study will be monitored by qualified representatives of the Sponsor.  Monitoring visits 
provide the Sponsor with the opportunity to: evaluate the progress of the study; verify the 
accuracy and completeness of eCRFs; assure that all protocol requirements, applicable laws 
and/or regulations and Investigator obligations are being fulfilled; and resolve any 
inconsistencies in the study records.  The Investigator must allow the Sponsor’s 
representatives direct access to periodically review, at mutually convenient times during the 
study and after the study has been completed, all eCRFs and office, hospital, and laboratory 
records supporting the participation of each patient in the study.  In the case where medical 
records are stored electronically, direct access to the relevant records relating to the clinical 
trial including past medical history must be provided to the study monitor, auditors or other 
representatives of the sponsor.  eCRFs and supporting documentation of the study conduct 
must be kept up-to-date and available for each monitoring visit. 
Deviations from the protocol with regard to patient enrollment, study conduct or in any other 
category will be recorded.  The Investigator is responsible for abiding by the IRB/EC’s rules 
and regulations for reporting protocol deviations.  The Sponsor may adjust the monitoring 
schedule for more frequent visits to allow more prompt collection of the data; provide 
continued training to the study center personnel on an as needed basis; and/or to follow up on 
any issues identified at the study center. 
Details of the monitoring activities will be described in the Clinical Monitoring Plan.   
9.7 
Case Report Forms and Study Records 
Data reflecting the patient’s participation in the study and experiences with the study 
treatment must be reported by the Investigator to the Sponsor.  These data must be recorded 
on eCRFs or other media approved by the Sponsor.  For rules regarding completion and 
correction of eCRFs, see the eCRF completion guidelines that accompany the eCRFs.  
eCRFs must be reviewed, electronically signed and dated by the Investigator or Sub-
investigator and must be submitted in a timely manner to the Sponsor.  Significant delays in 
submission of study data to the sponsor may result in a site being placed on enrollment hold. 
Study documentation includes all eCRFs; Safety Reports received from the Sponsor; SAE 
reports sent to the Sponsor; data correction forms; source documents; monitoring logs; 
Sponsor-Investigator correspondence; protocols and amendments; clinical supplies receipts; 
dispensing and final disposition records; IRB/IEC correspondence and approvals; signed 
consent forms; and Statement of Investigator forms. 
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 


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photographs, patient diaries, patient progress notes, hospital charts, appointment books, 
radiology reports or pharmacy records, and any other records or reports of procedures 
performed during the study.  Source documents also may include copies of the eCRF or 
Sponsor supplied worksheets when original information is recorded directly onto these 
forms. 
Whenever possible, an original recording of an observation must 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. 
9.8 
Data Monitoring Committee 
No DMC will be formed for this study. 
9.9 
Protocol Violations/Deviations 
The Investigator will not intentionally deviate from the protocol without prior written 
agreement from the Sponsor and the IRB/IEC except when necessary to eliminate an 
immediate hazard(s) to study subjects.  The investigator must take all possible steps to avoid 
unintentional deviations.  
All deviations, whether intentional or unintentional, must be documented and submitted to 
the appropriate parties (Sponsor, IRB/IEC, etc.) as required by applicable regulation or local 
practice.  
9.10 Access to Source Documentation 
In compliance with international and federal (US) regulations relative to monitoring clinical 
studies and the fulfillment of its obligation to verify compliance with this protocol, the 
Sponsor will require the investigator to permit the Sponsor’s representatives and, when 
necessary, representatives of the FDA and other regulatory authorities, to review and/or copy 
any or all portions of the patient medical records relevant to this study.  The potential for 
disclosure of patient’s confidential medical information will be included in the informed 
consent form signed by all subjects in the study. 
At some point during the study, individuals from the Sponsor’s Quality group or their 
authorized representative may visit the Investigator’s site to conduct an audit of the study.  
The purpose of this visit will be to determine the Investigator’s adherence to the protocol, 
applicable regulations and the Sponsor’s procedures, in addition to assessing the accuracy of 
the study data.  Before initiating this audit, the Investigator will be contacted by the Sponsor 
to arrange a convenient time for this visit.  The Investigator and staff are expected to 
cooperate with the auditors and allow access to all patient records supporting the eCRFs and 
other study-related documents. 


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9.11 Data Generation and Analysis 
All database and data management activities will be handled by a CRO contracted by LMI 
using qualified and validated database systems.  At scheduled monitoring visits, eCRFs will 
be verified against source documentation and submitted as final data.  Any subsequent 
changes to the eCRFs must be performed in accordance with the CRO’s standard operating 
procedures for editing and clarifying eCRFs.  Data entry will be performed by the sites using 
an electronic data capture (EDC) system.  Comment fields on the eCRFs will be used as a 
means of clarification and communication between the investigator and the sponsor; 
however, comments entered in these fields will not be edited or clarified. 
9.12 Retention of Data 
The Investigator must ensure that all records pertaining to the conduct of the clinical study, 
including signed eCRFs, informed consent forms, drug accountability records, source 
documents and other study documentation are adequately stored for the required time period 
to allow for review and reconstruction of the study.  This documentation must be retained 
for: 
• 2 years following approval of the last marketing application that the data was used to 
support; or 
• 2 years after formal discontinuation of the clinical development program; or 
• a record retention period mandated by any national and/or local laws, regulations, and 
customs. 
 
The Investigator must not destroy any records associated with the study without receiving 
written approval from the Sponsor.  The Investigator must notify the Sponsor in the event of 
accidental loss or destruction of any study records.  If the Investigator leaves the institution 
where the study was conducted, the Sponsor must be contacted to arrange alternative record 
storage options. 
9.13 Financial Disclosure 
All Clinical Investigators participating in clinical studies subject to FDA Regulation 21 CFR 
Part 54 – Financial Disclosure by Clinical Investigators are required before study initiation to 
submit a completed Clinical Investigator Financial Certification/Disclosure Request Form 
that sufficiently details any financial interests and arrangements that apply.  For the purpose 
of this regulation, Clinical Investigator is defined as any Investigator or Sub-investigator who 
is directly involved in the treatment or evaluation of research subjects, including the spouse 
and any dependent child of each Investigator and Sub-investigator.  These requirements 
apply to both US and foreign Clinical Investigators conducting covered clinical studies. 


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Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 46 of 49 
The Clinical Investigators will also be reminded that they must report any changes in their 
financial information regarding significant equity interests and significant payments for the 
duration of the trial, and for a period of 1 year after completion of their participation in the 
covered clinical study.  At the conclusion of the study, the Clinical Investigators may, again, 
be required to submit a completed Clinical Investigator Financial Certification/Disclosure 
Request Form to document any changes in financial interests. 
9.14 Publication and Disclosure Policy 
All information collected during this study will be regarded as confidential.  Investigators 
must obtain written permission from the Sponsor prior to disclosure or publication of data 
obtained during the conduct of this study.  Manuscripts prepared for publication will be 
submitted to the Sponsor for review and comment prior to submission to a publisher, in 
accordance with policies set forth in the Clinical Trail Agreement signed by the Principal 
Investigator.  


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 47 of 49 
10 REFERENCES 
1. Foley TA, Mankad SV, Anavekar NS, Bonnichsen CR, Morris MF, Miller TD, et al.  
Measuring left ventricular ejection fraction – techniques and potential pitfalls.  Eur 
Cardiol. 2012;8(2):108114. 
2. Hoffman R, von Bardeleben S, ten Cate F, Borges AC, Kasprzak J, Firschke C, et al.  
Assessment of systolic left ventricular function: A multi-centre comparison of 
cineventriculography, cardiac magnetic resonance imaging, unenhanced and contrast-
enhanced echocardiography.  Eur Heart J. 2005;26:607-616. 
3. Hoffman R, Barletta G, von Bardeleben S, Vanoverschelde JL, Kasprzak J, Greis C, et al.  
Analysis of left ventricular volumes and function:  A multicenter comparison of cardiac 
magnetic resonance imaging, cine ventriculography, and unenhanced and contrast-
enhanced two-dimensional and three-dimensional echocardiography.  J Am Soc 
Echocardiogr. 2014;27:292-301. 
4. ACCF/ACR/AHA/NASCI/SCMR 2010 Expert Consensus Document on CMR. 
Circulation. 2010;121(22):2461-2508. 
5. Schulz-Menger J, Bluemke DA, Bremerich J, Flamm SD, Fogel MA, Friedrich MG, et al.  
Standardized image interpretation and post processing in cardiovascular magnetic 
resonance: Society for Cardiovascular Magnetic Resonance (SCMR) board of trustees 
task force on standardized post processing.  J Cardiovasc Magn Reson. 2013 May 
1;15:35.  
6. Lang RM, Badano LP, Mor-Avi V, Afilalo J, Armstrong A, Ernande L, et al. 
Recommendations for cardiac chamber quantification by echocardiography in adults: an 
update from the American Society of Echocardiography and the European Association of 
Cardiovascular Imaging. J Am Soc Echocardiogr. 2015;28(1):1-39.e14. 
7. Hamer RM.  INTRACC.SAS Macro to Calculate Reliabilities for Intraclass Correlations 
[Internet].  SAS Institute;1990 [cited 15 January 2018].  Available from  
https://github.com/friendly/SAS-macros/blob/master/intracc.sas. 
8. Mehta CR and Pocock SJ.  Adaptive increase in sample size when interim results are 
promising:  A practical guide with examples.  Statist Med. 2011;30:3267-3284. 
 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 48 of 49 
Appendix 1 
Schedule of Procedures 
Study Procedures 
Screening/Baseline1 
Echo Imaging Session 
Day 0 
Telephone Follow-up 
CMR Imaging Session 
Informed consent 
X 
 
 
 
Inclusion/Exclusion 
X 
 
 
 
Medical history 
X 
 
 
 
Physical exam 
X 
 
 
 
Body weight and height 
X 
 
 
 
Vital Signs 
X 
 
 
 
Urine pregnancy test2 
X 
X 
 
 
Resting unenhanced echocardiogram 
 
X 
 
 
DEFINITY® administration and resting 
echocardiogram3 
 
X 
72 ± 24 hours after imaging 
session completion 
Within ±30 days of imaging 
session 
Concomitant medications 
X 
X 
X 
 
Safety assessments (AEs, SAEs)4 
 
 
1Procedures may be conducted up to 7 days prior to administration of study drug (Day 0). 
2A urine pregnancy test will be performed at screening and within 24 hours prior to dosing study drug. 
3Patient will remain at the clinical site for observation until at least 30 minutes after the end of study drug administration. 
4AEs will be recorded from at the time the ICF is signed until the 72 ± 24 hour telephone follow-up. 
 
 


DEFINITY® 
 
Lantheus Medical Imaging, Inc. 
Clinical Study Protocol:  DEF-315 Amendment 3 
 
15 August 2018 
 
 
 
Confidential 
Page 49 of 49 
Appendix 2 
Dose Preparation and Administration Guide 
 

SAP_001.pdf: 
 
 
 
 
 
This document is confidential. 
SAP Version: Final Version 1.0, 31-May-2019 
Controlled Document ID: 3903A.01, Effective Date 29-Oct-2018 
Filing requirements: TMF 
Page 1 of 31 
 
 
Statistical Analysis Plan  
 
 
Sponsor Name: Lantheus Medical Imaging, Inc. 
 
 
 
Protocol Number: DEF-315 
Protocol Title: A Phase III, Open-Label, Multicenter Trial to Evaluate Ejection Fraction, End-Diagnostic 
and End-Systolic Volumes, by Unenhanced and DEFINITY®-enhanced 2D-Echo and Magnetic 
Resonance Imaging 
 
 
 
Protocol Version and Date: Amendment 3, 15-Aug-2018 
 
Statistical Analysis Plan Version and Date: Version 1.0, 31-May-2019 
 
Syneos Health Project Code: 1011321 
 
Author: Heather L Murphy, Principal Biostatistician 
 
 
 
 
 
 
 
 
 
 
 
 
 
Notice of Confidential and Proprietary Information: 
The information contained in this document is confidential belonging to Lantheus Medical Imaging, Inc. 
Acceptance of this document constitutes agreement by the recipient that no information contained herein 
will be published or disclosed without prior written authorization from an official of Lantheus Medical 
Imaging, Inc..  However, this document may be disclosed to appropriate Institutional Review Board and 
Ethics Committees or duly authorized representatives of a national regulatory authority under the 
condition that they are requested to keep it confidential. In the event of an actual or suspected breach of 
this obligation, Syneos Health should be notified promptly. 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
This document is confidential. 
SAP Version: Final Version 1.0, 31-May-2019 
Controlled Document ID: 3903A.01, Effective Date 29-Oct-2018 
Filing requirements: TMF 
Page 2 of 31 
Revision History 
Version # 
Date 
(DD-Mmm-YYYY) 
Document Owner 
Revision Summary 
V0.01 
02-Apr-2019 
Syneos Health 
Initial Release Version 
V1.0 
31-May-2019 
Syneos Health 
Version 1.0 
 
 
 
 


 
 
 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
This document is confidential. 
SAP Version: Final Version 1.0, 31-May-2019 
Controlled Document ID: 3903A.01, Effective Date 29-Oct-2018 
Filing requirements: TMF 
Page 4 of 31 
Table of Contents 
Revision History .......................................................................................................................... 2 
Approvals ................................................................................... Error! Bookmark not defined. 
 
Glossary of Abbreviations ................................................................................................... 7 
1.
 
Purpose .............................................................................................................................. 9 
2.
2.1. 
Responsibilities ...................................................................................................... 9 
2.2. 
Timings of Analyses ............................................................................................... 9 
 
Study Objectives .............................................................................................................. 10 
3.
3.1. 
Primary Objective .................................................................................................10 
3.2. 
Secondary Objective(s) .........................................................................................10 
3.3. 
Brief Description ...................................................................................................10 
3.4. 
Subject Selection ..................................................................................................12 
3.4.1. 
Inclusion Criteria .................................................................................................. 13 
3.4.2. 
Exclusion Criteria ................................................................................................. 13 
3.5. 
Determination of Sample Size ...............................................................................13 
3.6. 
Treatment Assignment & Blinding .........................................................................14 
3.7. 
Administration of Study Medication .......................................................................14 
3.8. 
Study Procedures and Flowchart ..........................................................................14 
3.8.1. 
Screening ............................................................................................................. 14 
3.8.2. 
Echocardiography Imaging Session .................................................................... 15 
3.8.3. 
Telephone Assessment ....................................................................................... 15 
3.8.4. 
Cardiac Magnetic Resonance Imaging ................................................................ 15 
 
Endpoints ......................................................................................................................... 16 
4.
4.1. 
Primary Efficacy Endpoint .....................................................................................16 
4.2. 
Secondary Efficacy Endpoints ..............................................................................16 
4.3. 
Safety Endpoints ...................................................................................................16 
 
Analysis Sets .................................................................................................................... 17 
5.
5.1. 
Screened Population.............................................................................................17 
5.2. 
Safety Population ..................................................................................................17 
5.3. 
Intent-to-Treat Population .....................................................................................17 
5.4. 
Modified Intent-to-Treat Population .......................................................................17 
5.5. 
Per-Protocol Population ........................................................................................17 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
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5.6. 
Protocol Deviations ...............................................................................................17 
 
General Aspects for Statistical Analysis ........................................................................... 18 
6.
6.1. 
General Methods ..................................................................................................18 
6.2. 
Missing Data .........................................................................................................18 
6.2.1. 
Primary Supportive Analysis ................................................................................ 18 
6.2.2. 
Concomitant Medications ..................................................................................... 19 
6.2.3. 
Adverse Events .................................................................................................... 19 
 
Demographic, Other Baseline Characteristics and Medication ......................................... 20 
7.
7.1. 
Subject Disposition ...............................................................................................20 
7.2. 
Demographic and Other Baseline Characteristics .................................................20 
7.3. 
Medical History .....................................................................................................20 
7.4. 
Medication ............................................................................................................20 
7.4.1. 
Prior Medication ................................................................................................... 20 
7.4.2. 
Concomitant Medication ...................................................................................... 20 
 
Efficacy ............................................................................................................................ 21 
8.
8.1. 
Primary Efficacy Endpoint and Analysis ................................................................21 
8.1.1. 
Other Analyses for the Primary Endpoint ............................................................ 21 
8.2. 
Secondary Efficacy Endpoint(s) and Analyses ......................................................22 
8.2.1. 
Improvement in accuracy in LVEF assessment using DEFINITY® contrast-
enhanced over unenhanced echocardiography in subjects with suboptimal 
echocardiograms.................................................................................................. 22 
8.2.2. 
Reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography .............. 22 
8.2.3. 
Reduction in inter-reader variability for the assessment of end-diastolic/systolic 
volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography ................................................................................................ 23 
8.2.4. 
Reduction in inter-reader variability for the assessment of LVEF using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography in subjects 
with suboptimal echocardiograms........................................................................ 23 
8.2.5. 
Reduction in inter-reader variability for the assessment of end-diastolic/systolic 
volumes using DEFINITY® contrast-enhanced versus unenhanced 
echocardiography in subjects with suboptimal echocardiograms ........................ 23 
 
Safety ............................................................................................................................... 24 
9.
9.1. 
Exposure ..............................................................................................................24 
9.2. 
Treatment Compliance ..........................................................................................24 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
This document is confidential. 
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9.3. 
Adverse Events .....................................................................................................24 
9.4. 
Laboratory Evaluations .........................................................................................25 
9.5. 
Vital Signs .............................................................................................................25 
9.6. 
Physical Examination ............................................................................................26 
9.7. 
Study Center Effects .............................................................................................26 
 
Interim Analyses ............................................................................................................... 27 
10.
 
Changes from Planned Analysis  ...................................................................................... 29 
11.
11.1. 
Changes from Analysis Planned in Protocol .........................................................29 
11.2. 
Changes from Analysis Planned in FDA Approved Statistical Analysis Plan 
(06Oct2018) ..........................................................................................................29 
 
Reference List .................................................................................................................. 30 
12.
 
Appendices ...................................................................................................................... 31 
13.
13.1. 
Schedule of Procedures ........................................................................................31 
 
 
List of In-Text Figures 
Figure 1.  Schedule of Events ................................................................................................................ 12 
 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
This document is confidential. 
SAP Version: Final Version 1.0, 31-May-2019 
Controlled Document ID: 3903A.01, Effective Date 29-Oct-2018 
Filing requirements: TMF 
Page 7 of 31 
 
Glossary of Abbreviations 
1.
Abbreviation 
Description 
A2C 
Apical 2-chamber 
A3C 
Apical 3-chamber 
A4C 
Apical 4-chamber 
AE 
Adverse Event 
ANOVA 
Analysis of Variance model 
BMI 
Body Mass index 
BP 
Blood Pressure 
CI 
Confidence Interval 
cm 
Centimeters 
CP 
Conditional Power 
CMR 
Cardiac Magnetic Resonance Imaging 
FCS 
Fully Conditional Specification 
IA 
Interim Analysis  
ICC 
Intra-class Correlation Coefficient 
ITT 
Intent-to-Treat 
kg 
Kilogram 
LVEF 
Left Ventricular Ejection Fraction 
MedDRA 
Medical Dictionary for Regulatory Activities 
m 
Meter 
mL 
Milliliter 
mITT 
Modified Intent-to-Treat 
MRI 
Magnetic Resonance Imaging 
MUGA scan 
Multiple-Gated Acquisition scan 
PP 
Per-Protocol 
PT 
Preferred Term 
RMSE 
Root Mean Square Error 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SAS 
Statistical Analysis System 
SD 
Standard Deviation 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
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Abbreviation 
Description 
SOC 
System Organ Class 
SP 
Safety Population 
TEAE 
Treatment Emergent Adverse Event 
TLFs 
Tables, Listings, and Figures 
WHO 
World Health Organization 
 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
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Purpose 
2.
The purpose of this statistical analysis plan (SAP) is to ensure that the data listings, summary tables and 
figures (TLFs) which will be produced, and the statistical methodologies that will be used, are complete 
and appropriate to allow valid conclusions regarding the study objectives. 
The SAP for this study will provide a framework in which answers to the protocols’ objectives may be 
achieved in a statistically rigorous fashion, without bias or analytical deficiencies. Specifically, the SAP 
has the following purpose: To prospectively outline the analyses and presentations of data that will form 
the basis for conclusions to be reached that will answer the studies’ objectives outlined in the protocol, 
and to explain in detail how the data will be handled and analyzed, adhering to commonly accepted 
standards and practices of biostatistical analysis in the clinical trial industry.  
This analysis plan is based on the protocol dated 15-Aug-2018, amendment 3, and the original SAP, 
dated 06-Oct-2018, approved by the FDA along with the protocol. As Syneos Health was commissioned 
to perform the statistical analysis, they were also asked to write a SAP following enrollment in the study. 
 
Responsibilities 
2.1.
Syneos Health will perform the statistical analyses and is responsible for the production and quality 
control of all TLFs.   
 
Timings of Analyses 
2.2.
The primary analysis of safety and efficacy will take place after all subjects complete the final study visit, 
or terminate early from the study, and the database has been locked. 
An interim analysis to evaluate sample size assumptions will be conducted when a minimum of 75 
subjects have been enrolled and imaged.  An unblinded team from Syneos Health biostatistics will 
perform the analyses as described in Section 10.0 of this SAP to maintain the blinding of the study. 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
This document is confidential. 
SAP Version: Final Version 1.0, 31-May-2019 
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Page 10 of 31 
 
Study Objectives 
3.
 
Primary Objective 
3.1.
Demonstrate improvement in accuracy in left ventricular ejection fraction (LVEF) assessment using 
DEFINITY® contrast-enhanced over unenhanced echocardiography. 
 
Secondary Objective(s) 
3.2.
• 
Demonstrate improvement in accuracy in LVEF assessment using DEFINITY® contrast-enhanced 
over unenhanced echocardiography in subjects with suboptimal echocardiograms 
• 
Demonstrate a reduction in inter-reader variability for the assessment of LVEF using DEFINITY® 
contrast-enhanced versus unenhanced echocardiography 
• 
Demonstrate a reduction in inter-reader variability for the assessment of end-diastolic/systolic 
volumes using DEFINITY® contrast-enhanced versus unenhanced echocardiography 
• 
Demonstrate a reduction in inter-reader variability for the assessment of LVEF using DEFINITY® 
contrast-enhanced versus unenhanced echocardiography in subjects with suboptimal 
echocardiograms 
• 
Demonstrate a reduction in inter-reader variability for the assessment of end-diastolic/systolic 
volumes using DEFINITY® contrast-enhanced versus unenhanced echocardiography in subjects 
with suboptimal echocardiograms 
 
Brief Description 
3.3.
This is a Phase 3, prospective, open-label, multicenter study to evaluate LVEF measurement accuracy 
and reproducibility of DEFINITY® contrast-enhanced and unenhanced echocardiography as compared 
with non-contrast cardiac magnetic resonance imaging (CMR) used as the truth standard. Approximately 
one-hundred fifty (150) subjects will be enrolled over approximately 10 months at approximately 10 
centers located in the United States. The study population will consist of male and female subjects 18 
years of age of older. 
Subjects will be screened based on LVEF measurements obtained via 2D echo with or without contrast or 
other methods (e.g. CMR, MUGA scan) obtained within 6 months of enrollment (Day 0). Subjects will be 
stratified to achieve an approximately even distribution within four pre-defined LVEF groups (>50, 41-50, 
30–40, <30%). LVEF for screening purposes will be determined by the investigator and based on 
measurements with 2D echo or other methods (e.g. CMR, MUGA scan) obtained within 6 months prior to 
enrollment (Day 0). The site investigator will determine whether an echocardiogram is optimal or sub-
optimal based on the unenhanced echocardiogram performed on Day 0. Subjects with optimal and sub-
optimal echocardiograms (based on investigator opinion) will be enrolled. An echocardiogram is 
considered sub-optimal if 2 or more segments of the ventricular border are classified as not adequately 
visualized. 
Each subject will undergo an unenhanced ultrasound examination and a DEFINITY® contrast-enhanced 
examination on the same day at Day 0. A minimum of 360 seconds of images will be collected during 
both the unenhanced and the DEFINITY® contrast-enhanced examinations. Subjects will remain at the 
clinical site for at least 30 minutes of observation after DEFINITY® administration. A safety follow-up 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
This document is confidential. 
SAP Version: Final Version 1.0, 31-May-2019 
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Page 11 of 31 
phone call will be conducted for all subjects at approximately 72±24 hours after completion of the imaging 
sessions, including DEFINITY® administration.   
Enrolled subjects will be followed for adverse events (AEs), serious adverse events (SAEs), and changes 
in concomitant medications from the time the Informed Consent is signed through the safety follow-up 
telephone call. 
A non-contrast CMR will be used as the truth standard. CMR studies will be performed within ±30 days of 
the echocardiographic assessments at Day 0. Final assignment of each subject to an LVEF group will be 
determined by CMR.  
Unenhanced and DEFINITY®-enhanced echocardiograms will be performed with standard apical 2 
chamber, apical 3 chamber, and apical 4 chamber (A2C, A3C, and A4C, respectively) views using 
harmonic imaging. Images will be recorded in standard digital format, masked to subject identifiers, and 
sent to a central imaging core laboratory for analysis.  
At the central imaging core laboratory, 3 experienced independent blinded readers will interpret the 
results according to the Image Review Charter. A single, independent, blinded echocardiologist will read 
each subject’s unenhanced echocardiogram as either optimal or suboptimal. This will be independent of 
the site investigator determination. All CMR images will be read centrally by 1 experienced independent 
blinded reader.  
Echocardiographic imaging data will be compiled, analyzed, and stored without knowledge of the CMR 
findings. 
All screening assessments will occur within 7 days prior to enrollment/DEFINITY® administration (Day 0). 
CMR studies will occur within ±30 days of DEFINITY® administration. Safety monitoring will continue up to 
72±24 hours post-DEFINITY® administration. The expected duration of subject participation is not more 
than 41 days (see Figure 1). 
 
 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
This document is confidential. 
SAP Version: Final Version 1.0, 31-May-2019 
Controlled Document ID: 3903A.01, Effective Date 29-Oct-2018 
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Figure 1.  Schedule of Events 
 
 
 
Subject Selection 
3.4.
Approximately one hundred and fifty (150) evaluable subjects who have previously undergone a 2D 
echocardiogram with or without contrast or other assessment (CMR, MUGA scan) will be enrolled in this 
trial. The study population will consist of male and female subjects 18 years of age or older. 
Enrollment will be stratified according to CMR LVEF to achieve an approximately even distribution across 
four subgroups (LVEF >50, 41-50, 30–40, <30%). LVEF for screening purposes will be determined by the 
investigator and based on measurements with 2D echo or other methods (e.g. CMR, MUGA scan) 
obtained within 6 months prior to enrollment (Day 0). Subjects with optimal and sub-optimal 
echocardiograms will be enrolled. Suboptimal echocardiograms will be based on the site investigator’s 
evaluation of the unenhanced echocardiography images obtained on Day 0. An echocardiogram is 
considered suboptimal if 2 or more segments of the ventricular border are classified as not adequately 
visualized in any one of the 3 apical views. 
 
 
LVEF measurement obtained via 2D Echo or other methods 
(e.g. CMR, MUGA scan) within 6 months prior to enrollment 
Day 0  
Screening 
Days -7 to Day 0 
Echocardiograms & DEFINITY® 
administration  
Day 0 
Safety telephone 
follow-up 
72 ± 24 hours 
CMR within ±30 days of Day 0 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
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Page 13 of 31 
 
Inclusion Criteria 
3.4.1.
Potential study subjects must meet the following inclusion criteria to be enrolled in this study. 
1. Men and women ≥ 18 years of age in sinus rhythm 
2. Able to communicate effectively with trial personnel 
3. LVEF measurements obtained via 2D Echo with or without contrast or other methods (e.g. CMR, 
MUGA scan) obtained within 6 months prior to enrollment (Day 0) 
4. Has provided signed informed consent after receiving a verbal and written explanation of this clinical 
trial 
 
Exclusion Criteria 
3.4.2.
Potential study subjects who meet any of the following criteria will be excluded from the study. 
1. Female subjects who are pregnant or lactating.  All women of child bearing potential [WOCBP] must 
have a negative urine pregnancy test at screening regardless of contraceptive use history. 
2. Women of child-bearing potential are excluded unless they: 
a. are post-menopausal defined as amenorrhea ≥ 12 consecutive months, OR 
b. have undergone successful surgical sterilization (hysterectomy, bilateral tubal ligation or bilateral 
oophorectomy), OR  
c. have been using an adequate and medically approved method of contraception to avoid 
pregnancy for at least 1 month prior to DEFINITY® dose administration and be willing to continue 
using the same method for the duration of the study. 
3. Current illness or pathology that would prevent undergoing investigational product administration due 
to a significant safety risk to the subject.  
4. Uncontrolled arterial hypertension (defined as systolic blood pressure ≥ 200 mmHg or diastolic blood 
pressure ≥ 110 mmHg) or arterial hypotension (defined as systolic blood pressure ≤ 90 mmHg). 
5. Unstable cardiovascular status defined as: 
a. myocardial infarction or unstable angina pectoris within 6 months prior to enrollment/DEFINITY® 
dose administration day  
b. transient ischemic attack or stroke within 3 months prior to DEFINITY® dose administration  
c. symptomatic valvular heart disease or moderate to severe stenotic valvular heart disease  
d. clinically significant congenital heart defects 
e. current uncontrolled cardiac arrhythmias causing symptoms or hemodynamic compromise  
f. 
acute pulmonary embolus or pulmonary infarction  
g. acute myocarditis or pericarditis  
h. acute aortic dissection   
i. 
atrial fibrillation 
6. Any major surgery within 4 weeks prior to screening 
7. Known contraindications to undergoing CMR (e.g. implanted pacemakers, cardioverter, defibrillators) 
or claustrophobia 
8. Participation in any investigational drug, device, or placebo study within 30 days prior to screening  
9. Known hypersensitivity to perflutren, or any of the excipients in DEFINITY® 
10. Prisoners or those who are subject to compulsory detention or involuntary incarceration for treatment 
of either a psychiatric or physical illness (e.g., infectious disease) 
 
Determination of Sample Size 
3.5.
The primary analysis is to demonstrate an improvement in LVEF accuracy from unenhanced imaging to 
imaging with DEFINITY® contrast enhancement using CMR as the truth standard. 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
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For each subject, the absolute value of the difference of DEFINITY® LVEF minus CMR LVEF will be 
calculated. Similarly, the absolute value of the difference of unenhanced imaging LVEF minus CMR LVEF 
will be calculated for each subject. The primary analysis is to assess the significance of the difference 
between DEFINITY® and unenhanced echo with respect to the mean “absolute value of the difference 
versus CMR”. These means will be compared with a paired t-test at a two-sided 0.05 level of significance. 
Specifically, the null and alternative hypotheses are: H0: µD = µU versus H1: µD ≠ µU, where µD and µU 
are the mean of the DEFINITY® unenhanced echo absolute value of the difference versus CMRs, 
retrospectively. A sample size of 150 enrolled subjects has 90% power to reject the null hypothesis in 
favor of the alternative if the true difference µD - µU is at least 2.75 (in favor of DEFINITY®) with a 
standard deviation of 10 or less and allows for approximately 5% premature withdrawal. Power was 
calculated using the PASS 15 software (NCSS,LLC).  
 
Treatment Assignment & Blinding 
3.6.
There is only one treatment group in this open-label study. 
To allow the statistical team to be able to work on the interim analysis (IA), without unblinding to the IA 
results, the statistical team will remain blinded to the reader and CMR reported results until all subjects 
complete the final study visit, or terminate early from the study, and the database has been locked. A 
separate unblinded statistical team will create the unblinded results to be reviewed during the IA. 
 
Administration of Study Medication 
3.7.
DEFINITY® will be administered as a diluted bolus injection. 1.3 mL of activated DEFINITY® will be diluted 
with 8.7 mL of preservative-free saline to evenly distribute microspheres. An initial injection of up to 3 mL 
of diluted DEFINITY® will be administered with subsequent injections of 1 to 2 mL, as needed.  Full details 
are available in the Dose Preparation and Administration Guide in the protocol.  
 
Study Procedures and Flowchart 
3.8.
The schedule of events for this study is shown in Figure 1. A schedule of procedures is displayed in 
Appendix 13.1. Below is a description, by visit, of all activities. 
 
Screening 
3.8.1.
All Screening/Baseline assessments will occur within 7 days prior to administration of study drug (Day 0). 
• 
Determine eligibility according to inclusion/exclusion criteria 
• 
Informed consent completed 
• 
General medical history information collected 
• 
Concomitant medications collected 
• 
Physical examination conducted 
• 
Vital signs collected 
• 
Urine pregnancy test (as applicable) 
• 
Adverse event collection 
 
 
 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
This document is confidential. 
SAP Version: Final Version 1.0, 31-May-2019 
Controlled Document ID: 3903A.01, Effective Date 29-Oct-2018 
Filing requirements: TMF 
Page 15 of 31 
 
Echocardiography Imaging Session 
3.8.2.
Subjects providing informed consent and meeting the inclusion/not meeting the exclusion criteria will be 
enrolled into the study and undergo the following assessments on Day 0: 
• 
Urine pregnancy test   
• 
Concomitant medications collected 
• 
Bedside resting transthoracic echocardiogram  
• 
Following the unenhanced imaging scan, the study drug (DEFINITY®) will be administered  
• 
Following study drug administration and subsequent image optimization, the same A2C, A3C, 
and A4C views will be obtained 
• 
After completion of all assessments, subjects will remain on site for at least 30 minutes of 
observation 
• 
Adverse event collection 
 
Telephone Assessment 
3.8.3.
A safety follow-up call will be conducted for all subjects at approximately 72±24 hours after completion of 
the imaging sessions. Concomitant medications and adverse events will be collected during this call. 
 
Cardiac Magnetic Resonance Imaging 
3.8.4.
Within ±30 days of the Day 0 imaging session, subjects will receive a non-contrast enhanced CMR 
examination as described in the Study Imaging Manual.  


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Endpoints 
4.
 
Primary Efficacy Endpoint 
4.1.
The primary endpoint of this study is improvement in accuracy in left ventricular ejection fraction (LVEF) 
assessment using DEFINITY® contrast-enhanced over unenhanced echocardiography. The primary 
analysis is to compare LVEF accuracy from unenhanced imaging to imaging with DEFINITY® contrast 
enhancement using cardiac magnetic resonance imaging (CMR) as the truth standard. 
 
Secondary Efficacy Endpoints 
4.2.
The secondary endpoints are as follow: 
• 
Improvement in accuracy in LVEF assessment using DEFINITY® contrast-enhanced over 
unenhanced echocardiography in subjects with suboptimal echocardiograms 
• 
Reduction in inter-reader variability for the assessment of LVEF using DEFINITY® contrast-
enhanced versus unenhanced echocardiography 
• 
Reduction in inter-reader variability for the assessment of end-diastolic/systolic volumes using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography 
• 
Reduction in inter-reader variability for the assessment of LVEF using DEFINITY® contrast-
enhanced versus unenhanced echocardiography in subjects with suboptimal echocardiograms 
• 
Reduction in inter-reader variability for the assessment of end-diastolic/systolic volumes using 
DEFINITY® contrast-enhanced versus unenhanced echocardiography in subjects with suboptimal 
echocardiograms 
 
Safety Endpoints 
4.3.
Enrolled subjects will be followed for AEs, SAEs, and changes in concomitant medications from the time 
the informed consent is signed through 72±24 hours after completion of DEFINITY® administration. 
 


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Analysis Sets 
5.
There will be 5 populations for this study. They are listed below. 
 
Screened Population 
5.1.
The Screened Set will include all subjects screened in the study. Unless specified otherwise, this set will 
be used for subject listings and summaries of subject disposition.  
 
Safety Population 
5.2.
The Safety population (SP) will include all subjects who have signed an informed consent and who have 
received any amount of DEFINITY® in the study. This is the primary analysis population for the safety 
analysis. Unless otherwise stated, the SP will be used for all analyses of safety endpoints and for the 
presentation of subjects in all subject listings.  
 
Intent-to-Treat Population  
5.3.
The Intent-to-Treat (ITT) population will include all subjects who have signed an informed consent.  
 
Modified Intent-to-Treat Population 
5.4.
The Modified Intent-to-Treat (mITT) population will include all ITT subjects who complete unenhanced 
imaging, DEFINITY®-enhanced imaging, and cardiac MRI assessments. This is the primary analysis 
population for the efficacy endpoints. 
 
Per-Protocol Population  
5.5.
The Per-Protocol (PP) population will include all subjects in the mITT population who 
a) Did not violate inclusion and/or exclusion criteria that would likely have an effect on the primary 
outcome;  
b) Do not have major protocol violations;  
c) Have LVEF data on both unenhanced and DEFINITY®-enhanced for at least one reader; and  
d) Have CMR LVEF. 
Efficacy analyses will be conducted for both the mITT and PP populations. Differences in results between 
the 2 populations will be carefully examined. 
 
Protocol Deviations 
5.6.
Deviations from the protocol, as defined in the protocol, will be documented and monitored on an ongoing 
basis by the Sponsor, study monitors, and project manager throughout the study. 
Prior to database lock, the study team will forward all relevant protocol violation documentation in a pre-
specified format to the study statistician. These violations will be used to determine eligibility for the PP 
population and will be listed in a separate report to be included in the clinical study report. 


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General Aspects for Statistical Analysis 
6.
 
General Methods 
6.1.
Syneos Health will be responsible for data management and statistical analysis. All statistical analyses 
will be performed using SAS version 9.4 or higher. Subject data listings and tabular presentations will be 
provided. Summary statistics for continuous variables will include, unless otherwise stated, the following 
values: N, mean, median, standard deviation, minimum, and maximum. For categorical variables, the 
number and percent of subjects in each category will be calculated, while the number of subjects with 
missing data will be presented as a ‘Missing’ category. Unless otherwise stated, subjects with missing 
values will be included in the denominator count when computing percentages.  
Results will be presented to 1 decimal place when applicable. All statistical tests will be two-sided 
employing a significance level of 5% unless otherwise specified. Further details of each analysis will be 
described in the appropriate section. 
Demographic and efficacy analyses will be conducted on the mITT population as the main analysis 
population. Efficacy analyses will also be conducted on the PP population. The safety analyses will be 
conducted on the SP. 
The primary analysis will be performed on subjects with non-missing LVEF for DEFINITY®-enhanced 
echocardiography imaging, unenhanced imaging, and the truth standand CMR. A supportive analysis will 
be conducted where missing LVEF is multiply imputed using the fully conditional specification (FCS) 
multiple regression, as detailed in section 6.2. Otherwise, there will be no imputation of missing efficacy 
data. 
 
Missing Data 
6.2.
There will be no imputations for missing efficacy data except the supportive primary endpoint paired t-
test. For all other analyses, the number of subjects with missing data will be presented under a ‘Missing’ 
category. Unless otherwise stated, subjects with missing values will be included in the denominator count 
when computing percentages. When continuous data are being summarized, only the non-missing values 
will be evaluated for computing summary statistics. 
 
Primary Supportive Analysis 
6.2.1.
A supportive analysis will be run where, within each blinded reader, missing LVEF is multiply imputed 
using the FCS multiple regression prior to carrying out the primary endpoint analysis. The covariates in 
the imputation model will be study center, age, gender, body weight (kg), race, and ethnicity. This 
procedure will be done separately for each imaging method (DEFINITY® enhanced, unenhanced, and 
CMR truth standard). A total of 50 imputations will be generated. The paired t-test comparing mean bias 
between DEFINITY® and unenhanced (see primary endpoint null and alternative hypotheses in section 
3.5) will be performed separately on each of the 50 imputed datasets, and the t-test results will be 
combined across datasets using the usual multiple imputation techniques to create one overall paired t-
test results on imputed data.  
 
 


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Concomitant Medications 
6.2.2.
In cases of a missing start or end date, the following methods will be implemented: 
Missing start date: 
o 
Missing entire date: estimate the date of DEFINITY® administration  
o 
Missing day and month: 
 
If the year is the same as the year of the DEFINITY® administration date, it will be 
estimated by the date of DEFINITY® administration 
 
If the year is different to the year of the DEFINITY® administration date, it will be 
estimated as 1st January of that year 
o 
Missing day only: 
 
If the month/year are the same as the month/year of the DEFINITY® administration 
date, it will be estimated by the date of DEFINITY® administration 
 
If the month/year is different to the month/year of the DEFINITY® administration date, 
it will be estimated as the first day of the month 
Missing end date: 
o 
Missing entire date: estimate the End of Study date 
o 
Missing day and month: 
 
If the year is the same as the year of the End of Study date, is will be estimated by 
End of Study date 
 
If the year is prior to the year of End of Study date, it will be estimated by as 31st 
December of that year 
o 
Missing day only: 
 
If the month/year are the same as the month/year of End of Study date, it will be 
estimated by the End of Study date 
 
If the year or month is different from the year or month of End of Study date, it will be 
estimated by the last day of the month 
 
Adverse Events 
6.2.3.
If any part of the start date is missing for an AE, and it cannot be determined whether it occurred prior to 
the start of DEFINITY® administration, a conservative approach will be taken and the AE start date will be 
estimated to be the day of DEFINITY® administration. 


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Demographic, Other Baseline Characteristics and Medication 
7.
 
Subject Disposition  
7.1.
Subject disposition will include the number and percentage of ITT subjects who were enrolled in the trial, 
who received DEFINITY®, who completed the trial, and who discontinued from the trial. The number and 
percentage of ITT subjects who discontinued from the trial will also be presented by reason of 
discontinuation. The number and percentage of subjects in each analysis population will be presented. All 
percentages will be based on the total number of ITT subjects.  
 
Demographic and Other Baseline Characteristics 
7.2.
All baseline and demographic characteristics will be summarized overall on the mITT. This will include 
age at screening, age group at screening, gender, ethnicity, race, height (cm), weight (kg), and body 
mass index (BMI). Descriptive statistics will be provided for each continuous variable; whereas, 
frequencies and percentages will be provided for categorical variables. Age, Age Group, and BMI 
calculations are described below: 
 
Age Group = two age groups will be created: (1) < 65 years, (2) ≥ 65 years 
 
BMI = weight(kg)/height2(m) 
 
Medical History  
7.3.
Medical History will be summarized overall on the mITT population. Medical history data will be collected 
by body system for all enrolled subjects. The details of history by body system are collected as open text. 
Medical history will be coded according to Medical Dictionary for Regulatory Activities (MedDRA) version 
21.0 or higher. The number and percentage of subjects within each system organ class (SOC) and 
preferred term (PT) will be presented. A subject experiencing a medical history within more than one SOC 
and PT will be counted only once within that SOC and PT, respectively. 
Active medical history is defined as histories marked as ongoing at time of screening. Past medical 
histories are defined as histories marked as resolved at time of screening. A summary table will be 
provided for both active and past medical history. A listing of combined medical history will also be 
provided. 
 
Medication 
7.4.
Medications will be coded using the World Health Organization (WHO) Drug dictionary, version Q3-2016. 
The number and percentage of subjects will be presented overall, by WHO therapeutic area and WHO 
preferred drug name. Both a table and listing will be created independently for the prior and concomitant 
medications for the SP. Methods for handling and categorizing missing medication data are reported in 
section 6.2.2. 
 
Prior Medication 
7.4.1.
Prior medications are those that started and stopped before exposure to DEFINITY®.  
 
Concomitant Medication 
7.4.2.
Concomitant medications are all medications taken during the study period, including those which started 
before administration, but were reported ongoing at the first administration. 


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Efficacy 
8.
All efficacy parameters will be summarized and presented in tables based on the mITT and PP 
populations. Note, approximately 10% of imaging scans will be re-read by each reader. Unless otherwise 
specified, the first/original read is what will be used for all reporting and statistical analyses. 
 
Primary Efficacy Endpoint and Analysis 
8.1.
The primary endpoint is LVEF accuracy. The primary analysis is to demonstrate an improvement in LVEF 
accuracy from unenhanced imaging to that derived from imaging with DEFINITY® contrast enhancement 
using CMR as the truth standard.  
For each subject, the absolute value of the difference of DEFINITY® LVEF minus CMR LVEF will be 
calculated. Similarly, the absolute value of the difference of unenhanced imaging LVEF minus CMR LVEF 
will be calculated for each subject. The primary analysis is to assess the significance of the difference 
between DEFINITY® and unenhanced echo with respect to the mean “absolute value of the difference 
versus CMR”. 
The null and alternative hypotheses for this study are: 
H0: µD = µU versus H1: µD ≠ µU; or, that is 
H0: µU - µD = 0 versus H1: µU - µD ≠ 0 
where µD and the µU are the mean of the DEFINITY® and unenhanced echo absolute value of the 
difference versus CMR, retrospectively. The null hypothesis will be tested at a two-sided 0.05 level of 
significance using a paired t-test derived from the sample point estimates of the mean and standard 
deviation of the difference between unenhanced echo’s and DEFINITY’s® “absolute value of the 
difference versus CMR”. 
The analyses will be conducted separately for each of the three blinded readers of the DEFINITY® 
enhanced and unenhanced images. The criterion for success is that the null hypothesis is rejected in 
favor of DEFINITY® for at least 2 of 3 blinded readers for all subjects. CMR LVEF will be used as the 
comparator for each of the 3 blinded readers, and is interpreted by a single reader. 
The primary analysis will be performed on mITT subjects with non-missing LVEF for DEFINITY®, 
unenhanced echocardiography, and the truth standard. A supportive analysis will be run where a missing 
echocardiography or CMR LVEF is multiply imputed using the fully conditional specification (FCS) 
multiple regression as described in section 6.2.1. 
 
Other Analyses for the Primary Endpoint 
8.1.1.
The following will be carried out separately for each of the 3 blinded readers without imputation of missing 
LVEF. 
Bias is the mean of the actual (not absolute value) per-subject differences between the imaged LVEF and 
CMR LVEF. The bias, the two-sided 95% confidence interval (CI) of the bias, the precision (standard 
deviation of the per-subject differences) and the root mean square error (RMSE) will be calculated for the 
LVEF derived from the DEFINITY®-enhanced images. The RMSE is the square root of the bias-squared + 
precision-squared and is considered a measure of overall accuracy. These analyses will be repeated for 


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LVEF values derived from the unenhanced echocardiography versus those from CMR. It is anticipated 
that the RMSE will be smaller for DEFINITY®-enhanced LVEF than for unenhanced LVEF. 
DEFINITY®-enhanced and unenhanced echocardiography LVEF will each be assessed for measurement 
accuracy against the reference CMR using Bland-Altman analysis and Deming regression analysis. For 
the Bland-Altman analysis, a plot of the per-subject actual difference between DEFINITY®-enhanced 
echocardiography and CMR LVEF will be plotted versus the per-subject sum of the two measurements. 
Limits of agreement (defined as the mean of the DEFINITY® versus CMR difference) ±2 standard 
deviations will be shown on the plot. The same analyses will be performed for unenhanced 
echocardiography using CMR as the reference standard.  
Deming regression plots of LVEF versus CMR LVEF will be generated for each of DEFINITY®-enhanced 
and unenhanced echocardiography. Unweighted Deming regression will be employed to estimate the 
regression slope and intercept with two-sided 95% CI of each assuming the measurement error is the 
same for CMR and each of the echocardiography error techniques. 
The intra-class correlation coefficient (ICC) and its two-sided 95% CI will be calculated for DEFINTIY®-
enhanced echo versus CMR and for unenhanced echo versus CMR. The ICCs and their two-sided CIs 
will be calculated using between and within mean squares from an ANOVA model with method 
(echocardiography, CMR) and subject as the main effects and LVEF as the dependent variable. The ICC 
will be calculated using the SAS macro developed by Hamer (1990). 
 
Secondary Efficacy Endpoint(s) and Analyses 
8.2.
 
Improvement in accuracy in LVEF assessment using DEFINITY® contrast-enhanced over 
8.2.1.
unenhanced echocardiography in subjects with suboptimal echocardiograms 
The primary endpoint analysis will be repeated in the subset of subjects with suboptimal 
echocardiograms. This will include all primary analyses described in sections 8.1 and 8.1.1, excluding the 
supportive analyses at the end of section 8.1. The study is not powered to reject the primary endpoint null 
hypothesis, so the focus is more on the estimate of µD - µU, on Bland-Altman plots and on the Deming 
regression for each blinded reader. There will be no imputation of missing LVEF data for this analysis. 
 
Reduction in inter-reader variability for the assessment of LVEF using DEFINITY® contrast-
8.2.2.
enhanced versus unenhanced echocardiography 
The inter-reader variability among each pair of readers within each imaging modality will be estimated 
using an intra-class correlation (ICC) and its two-sided 95% CI. The ICC assesses rating reliability by 
comparing the variability of different ratings of the same subject with the total variation across all ratings 
and all subjects. The inter-observer variability in the assessment of LVEF between two readers will be 
determined by percentage of error. The percentage of error will be calculated using the following formula:  
 
Percentage of error = (SD between 2 measurements / mean of the 2 measurements) x 100 
The mean percentage of error and its 95% CI will be calculated for each pair of readers within each 
imaging modality. The pairwise ICCs and percentages of error differences will be compared descriptively 
between contrast-enhanced and unenhanced echocardiography.   
In addition, specific to only section 8.2.2, there will also be a separate ICC analysis to assess intra-reader 
variability as approximately 10% of all echo results will be re-read by each reader. 


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Reduction in inter-reader variability for the assessment of end-diastolic/systolic volumes 
8.2.3.
using DEFINITY® contrast-enhanced versus unenhanced echocardiography 
The same analysis used to assess inter-reader variability on LVEF (section 8.2.2) will be conducted for 
end-diastolic/systolic volumes. 
 
Reduction in inter-reader variability for the assessment of LVEF using DEFINITY® contrast-
8.2.4.
enhanced versus unenhanced echocardiography in subjects with suboptimal 
echocardiograms 
The same analysis used to assess inter-reader variability on LVEF (section 8.2.2) will be conducted for 
the subgroup of subjects with suboptimal echocardiograms. 
 
Reduction in inter-reader variability for the assessment of end-diastolic/systolic volumes 
8.2.5.
using DEFINITY® contrast-enhanced versus unenhanced echocardiography in subjects with 
suboptimal echocardiograms 
The same analysis used to assess inter-reader variability on LVEF (section 8.2.4) for the subgroup of 
subjects with suboptimal echocardiograms will be conducted for end-diastolic/systolic volumes. 


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Safety 
9.
The population used for safety analyses will be the Safety population (SP).  Safety will be assessed on 
the basis of adverse event (AE) or serious adverse event (SAE) reports, physical examinations, and vital 
signs.  
 
Exposure 
9.1.
DEFINITY® will be administered as a diluted bolus injection. 1.3 mL of activated DEFINITY® will be diluted 
with 8.7 mL of preservative-free saline to evenly distribute microspheres. An initial injection of up to 3 mL 
of diluted DEFINITY® will be administered with subsequent injections of 1 to 2 mL, as needed. Information 
about the administration of DEFINITY® is collected on the case report form. Exposure to DEFINITY® 
study drug will be summarized. The number and percentage of ITT subjects who are administered 
DEFINITY® will be presented. Summary statistics of the dose administered (mL) will be summarized for 
the safety population. 
 
Treatment Compliance  
9.2.
Not applicable 
 
Adverse Events  
9.3.
A treatment-emergent adverse event (TEAE) is an AE that started or worsened in severity following the 
start of administration of DEFINITY®. A table of AEs will be summarized for the following: 
 
Number of subjects having experienced at least one TEAE 
 
Number of subjects having experienced at least one TEAE related to DEFINITY® 
 
Number of subjects having experienced at least one TEAE related to study procedure 
 
Number of subjects having experienced at least one severe TEAE 
 
Number of subjects with study procedure interrupted due to a TEAE 
 
Number of subjects with study procedure interrupted due to a TEAE related to DEFINITY® 
 
Number of subjects with study procedure interrupted due to a TEAE related to the study 
procedure 
 
Number of subjects having experienced at least one serious TEAE 
 
Number of subjects having experienced at least one serious TEAE related to DEFINITY® 
 
Number of subjects having experienced at least one serious TEAE related to a study procedure 
 
Number of subjects having experienced at least one severe serious TEAE 
 
Number of subjects with study procedure interrupted due to a serious TEAE 
 
Number of subjects with study procedure interrupted due to a serious TEAE related to 
DEFINITY® 
 
Number of subjects with study procedure interrupted due to a serious TEAE related to the study 
procedure 
 
Number of subjects having experienced a fatal TEAE 
 
Number of subjects having experienced a fatal TEAE related to DEFINITY® 
 
Number of subjects having experienced a fatal TEAE related to the study procedure 
If an AE is described as being ‘related to DEFINITY®, unless otherwise stated, this will be defined as a 
relationship to DEFINITY® administration where the relationship is defined as either possibly related or 
related. If an AE is described as being ‘related to study procedure’, unless otherwise stated, this will be 
defined as a relationship to DEFINITY® enhanced echo and/or DEFINITY® administration and/or 
unenhanced echo and/or CMR where the relationship is defined as either possibly related or related. 


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A summary table of TEAEs (number and percentage of subjects who experienced an AE and number of 
events) grouped by primary SOC and PT will be presented for the following categories of events: 
 
All TEAEs 
 
All TEAEs related to DEFINITY® 
 
All TEAEs related to study procedure 
 
All serious TEAEs 
 
All serious TEAEs related to DEFINITY® 
 
All serious TEAEs related to study procedure 
 
All severe TEAEs 
 
All TEAEs leading to study discontinuation 
A summary table of TEAEs by SOC and PT will also be presented by maximum severity. 
A subject with more than one occurrence of the same AE in a particular SOC and PT will be counted only 
once in the total of subjects experiencing AEs in that particular SOC and PT, respectively. If a subject 
experiences the same AE at more than one severity, or with more than one relationship category, the 
most severe rating or the stronger causal relationship will be reported. 
Any missing severity or relationship of an AE should be replaced by the worst case as follows: 
 
If severity is missing, then the AE will be included in the “severe” category 
 
If relationship is missing, then the AE will be included as “related to DEFINITY®” 
Time to onset and duration of events in days will also be listed where: 
 
Time to onset is defined as (AE start date – date of DEFINITY® administration) 
 
Duration of event is defined as (AE stop date – AE start date + 1) 
AEs with missing start dates will be included in the count of events, but a time will not be calculated. AEs 
will be coded using MedDRA version 21.0 or higher. 
 
Laboratory Evaluations 
9.4.
A urine pregnancy test will be performed at screening and Day 0 as applicable. Results will be listed. 
 
Vital Signs  
9.5.
The vital sign measurements are collected only at the screening/baseline visit. The following vitals will be 
summarized at baseline using descriptive statistics: systolic/diastolic blood pressure (BP), heart rate, and 
respiratory rate. In addition, all vital signs will be listed by subject. 
 
 


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The number and percentage of subjects with potentially clinically significant vital sign values at baseline 
will be tabulated. Potentially clinically significant vital sign values are detailed below and should be used 
to determine what is ‘normal’ and ‘potentially clinically significant’: 
Parameter 
Unit 
Normal 
Range 
Potentially Clinically 
Significant 
Heart Rate 
Beats per  
    minute 
60 – 100  
1) <50 bpm  
2) >100 bpm  
Systolic BP 
mmHg 
90 – 139  
1) >190 mmHg  
2) <80 mmHg 
 
Diastolic BP 
mmHg 
60 – 89  
1) >110 mmHg  
2) <50 mmHg 
 
 
Physical Examination 
9.6.
Physical examination body system inspection results are collected at baseline and will be listed.  
 
Study Center Effects 
9.7.
The mean “absolute value of the difference versus CMR” for each of DEFINITY®, and unenhanced 
echocardiography, as well as the difference between the two treatments with respect to this mean, will be 
presented for each study center and each blinded reader. Within each blinded reader, an assessment of 
study center effect on the mean treatment difference will be assessed using a one-way ANOVA. For each 
blinded reader, a site difference that is not significant at the 0.15 level of significance, or a site difference 
that is significant but where for every site the mean treatment difference is more favorable for DEFINITY® 
than unenhanced echocardiography, will support pooling subjects across sites for the primary analysis for 
that reader. 


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 Interim Analyses 
10.
This study will utilize an adaptive design that allows one interim sample size re-calculation after a 
minimum of 75 subjects have been enrolled and followed. The pre-specified maximum allowable adjusted 
sample size following re-estimation will be 300 enrolled, or two times the initial planned sample size of 
150 enrolled. The re-estimation of sample size will be conducted by an independent, unblinded 
biostatistician following a pre-specified plan using the method Mehta and Pocock (2011). Specifically, at 
the interim stage, the conditional power (CP) for obtaining a significant beneficial effect of DEFINITY® 
over unenhanced imaging with respect to the primary endpoint will be calculated for each blinded reader, 
using the protocol-specified planned sample size of 150 enrolled subjects (approximately 143 evaluable 
subjects assuming 5% premature withdrawal). This conditional power will be calculated under the 
assumption that the interim estimate of the mean unenhanced-DEFINITY® difference in LVEF accuracy is 
the true population mean difference. Specifically, the calculation of conditional power and sample size 
increase will be as follows: 
Let µD be the true mean of the absolute value of the differences between the DEFINITY®-enhanced echo 
LVEF and the CMR LVEF, and let µU be the true mean of the absolute value of the differences between 
the unenhanced echo LVEF and the CMR LVEF. After a minimum of 75 subjects are enrolled and 
followed (which should lead to approximately 71 evaluable subjects), an unblinded interim analysis will be 
conducted to determine whether sample size should be increased to maintain adequate conditional power 
of up to 90% for each reader. Conditional Power (CP) for rejecting the null hypothesis in favor of 
DEFINITY® by the planned final sample size of 143 evaluable subjects is calculated as follows for each 
reader: 
CP = 𝑃൬𝑍>
𝐶2ඥ𝑖2−𝑡1ඥ𝐼1−(𝐼2−𝐼1)∆
ඥ𝐼2−𝐼1
൰ 
 
where,  
a. Z is a random standard random variate 
b. c2 is the t-critical value to be used in the final analysis = 1.97681 (one-sided 0.025 level of 
significance; with 142 degrees of freedom assuming the final evaluable sample size is 143, which is 
the number of evaluable subjects expected for the final analysis with 150 enrolled subjects with 5% 
premature withdrawal). 
c. Δ is the assumption of the true difference “µU minus µD” where µD is the mean of the absolute value 
of the differences between the DEFINITY®-enhanced echo LVEF and the CMR LVEF, and µU is the 
mean of the absolute value of the differences between the unenhanced echo LVEF and the CMR 
LVEF; Δ will be set to the interim sample’s point estimate of µU minus µD; i.e., Δ will be set to x̅U - x̅D, 
where x̅U and x̅D are the sample mean point estimates of µU and µD, respectively. 
d. I1 is the observed “information” at the interim analysis; specifically, I1 =  
1
𝑠2
𝑛1
ൗ where s = the interim 
sample point estimate of the standard deviation of the difference between the unenhanced and 
DEFINITY® echo’s “absolute value of the difference versus CMR” and n1 is the interim sample size. 
e. I2 is the anticipated “information” at the final analysis; specifically, I2 =  
1
𝑠2
𝑛2
ൗ  where s is as defined in 
item ‘d’ directly above and n2 is the planned final evaluable sample size = 143. 
f. t1 is the interim paired t-statistic testing the null hypothesis, derived from the interim sample point 
estimates of the mean and standard deviation of the difference between the unenhanced and 
DEFINITY® echo’s “absolute value of the difference versus CMR”. 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
This document is confidential. 
SAP Version: Final Version 1.0, 31-May-2019 
Controlled Document ID: 3903A.01, Effective Date 29-Oct-2018 
Filing requirements: TMF 
Page 28 of 31 
 
The study will not be stopped for overwhelming efficacy or futility at this interim stage. Instead, based on 
Table 1 of Mehta and Pocock (2011), the following algorithm will be carried out:   
• 
If the CP ≤ 36% for at least two of the three blinded readers based on the final sample size, the 
study will continue to its original planned sample size of 150 enrolled subjects.  
• 
If the CP ≥ 90% for at least two of the three blinded readers based on the final sample size, the 
study will continue to its original planned sample size of 150 enrolled subjects.  
• 
If 36% < CP <90% for exactly two out of the three readers, let n be the total protocol-specified 
enrolled sample size. Assume na is the total enrolled sample size for reader 1 to reach 90% CP, 
nb is the total enrolled sample size for reader 2 to reach 90% CP, and nc is the total enrolled 
sample size for reader 3 to reach 90% CP. Without loss of generality, assume both reader 1 and 
2 had a CP within the range of 36% < CP <90%, reader 3 did not. Using this example, the final 
total number of enrolled subjects will be the sample size with which both readers have more than 
90% CP but not exceeding 2n; i.e., min(max(na, nb), 2n). All 3 readers’ final analysis will be based 
on this min(max(na, nb), 2n) enrolled subjects.  
• 
If 36% < CP <90% for all three readers, the final total number of enrolled subjects will be the 
sample size with which all readers have more than 90% CP but not exceeding 2n; i.e., 
min(max(na, nb, nc), 2n). Each reader’s final analysis will be based on min(max(na, nb, nc), 2n) 
enrolled subjects. 
The number of enrolled subjects is not changed from the final protocol-specified sample size in any other 
cases. That is, in all such other cases, each reader’s final analysis will be based on the n enrolled 
subjects. 
The sample size may be increased up to 300 enrolled subjects (twice the protocol-planned sample size) 
for a given reader in order to obtain 90% CP. As stated above, if different sample size increases are 
specified for different readers, only the largest sample size increase will be shared with those blinded to 
the results. Thus, all readers will review the same number of scans and each reader’s final analysis will 
be based on the number of subjects enrolled in the study.  
The statisticians, sponsor, and investigators will remain blinded to the interim results for the duration of 
the ongoing study. After the interim analysis is carried out, the recommendation made to the sponsor will 
be either to keep sample size as is or to increase enrolled sample size to a given value not greater than 
300. No other information derived from this interim analysis, including the reason for the recommendation, 
will be provided to the sponsor and other blinded personnel. 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
This document is confidential. 
SAP Version: Final Version 1.0, 31-May-2019 
Controlled Document ID: 3903A.01, Effective Date 29-Oct-2018 
Filing requirements: TMF 
Page 29 of 31 
 Changes from Planned Analysis  
11.
 
Changes from Analysis Planned in Protocol 
11.1.
There have been no changes in analyses from those defined in the protocol. 
 
Changes from Analysis Planned in FDA Approved Statistical Analysis Plan (06Oct2018) 
11.2.
The variable ‘Age’ has been amended from being a derived value to being a reported value. No date of 
birth is collected for subjects in this study. 
In section 3.6, a section was added to clarify that the study statistical team will remain blinded to the 
reader and CMR reported results until all subjects complete the final study, or terminate early from the 
study, and the database has been locked. A separate unblinded statistical team will create the unblinded 
results to be reviewed during the IA. The reason for this amended text is to ensure the study statistical 
team will also remain blinded to the IA study results along with the sponsor and investigators. 
In section 8, it is noted that approximately 10% of the imaging scans will be re-read by each reader. 
Unless otherwise specified, the original read is what will be used for all reporting and statistical analyses. 
Further, in section 8.2.2, a separate ICC analysis was added to assess intra-reader variability. Any 
additional information regarding the re-reading of scans can be found in the Imaging Charter for this 
study. 
In the vital signs table, section 9.5, the following updates have been made: 
• 
For Heart Rate, the phrases ‘and ≥25% decrease (increase) from baseline’ were removed 
• 
For Systolic blood pressure, the phrases ‘(entry is <160 mmHg’ and ‘decrease from baseline 
>30mmHg’ were removed 
• 
For Diastolic blood pressure, the phases ‘(entry is <90 mmHg)’ and ‘change from baseline of 20 
mmHg (increase or decrease)’ were removed; and a greater than sign was added in front of the 
Potentially Clinically Significant value of 110 mmHg as it was missing in the previous FDA 
approved SAP 
Since the vitals signs are only collected once, at the screening/baseline visit, there will be no way to 
determine increases/decreases from baseline.  
 
In section 10 (interim analysis), the two bullets to clarify how to proceed with the study in cases of CP ≤ 
36% or CP ≥ 90% have been added for clarification, along with additional text regarding how to evaluate 
the final study results. 
 
 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
This document is confidential. 
SAP Version: Final Version 1.0, 31-May-2019 
Controlled Document ID: 3903A.01, Effective Date 29-Oct-2018 
Filing requirements: TMF 
Page 30 of 31 
 Reference List 
12.
1. Hamer RM.  INTRACC.SAS Macro to Calculate Reliabilities for Intraclass Correlations [Internet].  
SAS Institute;1990 [cited 15 January 2018].  Available from  https://github.com/friendly/SAS-
macros/blob/master/intracc.sas. 
2. Mehta CR and Pocock SJ.  Adaptive increase in sample size when interim results are promising:  A 
practical guide with examples.  Statist Med. 2011;30:3267-3284. 
 
 


Statistical Analysis Plan for Interventional Studies 
Sponsor: Lantheus Medical Imaging, Inc.; Protocol No.: DEF-315 
 
 
This document is confidential. 
SAP Version: Final Version 1.0, 31-May-2019 
Controlled Document ID: 3903A.01, Effective Date 29-Oct-2018 
Filing requirements: TMF 
Page 31 of 31 
 Appendices 
13.
 
Schedule of Procedures 
13.1.
Study Procedures 
Screening/ 
Baseline1 
Echo Imaging Session 
Day 0 
Telephone Follow-
up 
CMR Imaging 
Session 
Informed consent 
X 
 
 
 
Inclusion/Exclusion 
X 
 
 
 
Medical history 
X 
 
 
 
Physical exam 
X 
 
 
 
Body weight and height 
X 
 
 
 
Vital Signs 
X 
 
 
 
Urine pregnancy test2 
X 
X 
 
 
Resting unenhanced 
echocardiogram 
 
X 
 
 
DEFINITY® administration and 
resting echocardiogram3 
 
X 
72 ± 24 hours after 
imaging session 
completion 
Within ±30 days of 
imaging session 
Concomitant medications 
X 
X 
X 
 
Safety assessments (AEs, SAEs)4 
 
 
1Procedures may be conducted up to 7 days prior to administration of study drug (Day 0). 
2A urine pregnancy test will be performed at screening and within 24 hours prior to dosing study drug. 
3Subject will remain at the clinical site for observation until at least 30 minutes after the end of study drug administration. 
4AEs will be recorded from at the time the ICF is signed until the 72 ± 24 hour telephone follow-up.