Conditions: Transthoracic Echocardiography Suspected or Known Structural or Functional Cardiac Abnormality
Keywords: Contrast-enhanced echocardiography (CE-ECHO) Transthoracic echocardiogram Child Left ventricular opacification (LVO) Left ventricular ejection fraction (LVEF) Left ventricular endocardial border delineation (LV EBD)
Allocation: NON_RANDOMIZED | Intervention model: SINGLE_GROUP
Enrollment: 39
Healthy volunteers: no

Brief summary:
Study to determine the safety and appropriate dosage of OPTISON in pediatric participants since OPTISON has been tested in adult participants only during the clinical development

Detailed description:

Arms:
arm 1: Participants with body weight greater than or equal to (\>=) 20 to less than or equal to (\<=) 28 kilograms (kg) received OPTISON 0.1 milliliter (mL; dose level 1) or 0.2 mL (dose level 2); and participants with body weight greater than (\>) 28 to \<=40 kg received OPTISON 0.2 mL (dose level 1) or 0.3 mL (dose level 2) intravenous (IV) injection, 10 minutes apart on Day 1. The maximum allowed cumulative dose for body weight \>=20 to \<=28 kg and \>28 to \<=40 kg was 1.0 mL and 1.5 mL per dose level, respectively.

The treated subjects received both dose levels. Per protocol, up to 3 injections per dose level were allowed for acquiring different imaging views. A maximum of 2 injections was recorded in the treated population. arm 2: Participants with body weight \>40 kg received OPTISON 0.2 (dose level 1) or 0.4 mL (dose level 2) IV injection, 10 minutes apart on Day 1. The maximum allowed cumulative dose was 1.8 mL per dose level.

The treated subjects received both dose levels. Per protocol, up to 3 injections per dose level were allowed for acquiring different imaging views. A maximum of 2 injections was recorded in the treated population.

Interventions: intervention 1: Optison
Intervention descriptions: intervention 1: Optison is administered intravenously

Protocol document:
Prot_000.pdf: 
GE Confidential 
Page 1 of 58 
Clinical Study Protocol 
GE Healthcare 
GE-191-008 
Title: A Phase 4, Open-Label, Non-randomized, Multicenter Study to Evaluate Safety 
and Efficacy of Intravenous Administration of OPTISON™ for Contrast-
Enhanced Echocardiography in Pediatric Patients 
REVISED TO INCORPORATE AMENDMENT A01 
Sponsor 
GE Healthcare Ltd. and its Affiliates (hereinafter referred to as the “sponsor”) 
Medical Director 
 
 
 
GE Healthcare Life Sciences 
Pollards Wood, Nightingales Lane 
Chalfont St Giles, Bucks HP8 4SP 
United Kingdom 
T: 
 
Confidentiality Statement 
This protocol is provided for conducting a clinical research study.  The information contained 
in this document is confidential and, except to the extent necessary to obtain informed consent 
or IEC/IRB approval, cannot be disclosed unless required by governmental regulation.  Persons 
to whom any portion of the contents of this document is disclosed must be informed that the 
information is confidential and may not be further disclosed by them. 
GE-191-008 Clinical Protocol Amendment A01
Version 2.0 Approved  04 Oct 2022
ClinicalTrials.gov ID: NCT03740997


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Investigator’s Signature Page 
I have read this protocol and all associated case report forms and agree to conduct this study in 
full accordance with the stipulations of the protocol described herein. 
 
 
 
Signature 
 
 
 
Date 
Print Name 
 
 
 
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1 
SYNOPSIS 
Name of Sponsor/Company: 
GE Healthcare Ltd. and its 
Affiliates 
Individual Study Table 
Referring to Part of Dossier in 
Which the Individual Study or 
Study Table is Presented: 
 
Volume: 
 
Reference: 
 
(For National Authority Use 
Only) 
 
Name of Finished Product: 
OPTISONTM 
Name of Active Ingredient: 
Perflutren Protein – Type A 
Microspheres Injectable 
Suspension, USP 
Title of Study:  
A Phase 4, Open-Label, Non-randomized, Multicenter Study to Evaluate Safety and Efficacy of Intravenous 
Administration of OPTISONTM for Contrast-Enhanced Echocardiography in Pediatric Patients 
Protocol Number: GE-191-008  
Investigators and Study Centers: Up to 12 centers in North America and Europe.  
Phase of Development: Phase 4 
Objectives:  
Primary: 
• 
To determine a suitable dosage of OPTISON for contrast-enhanced echocardiography (CE-ECHO) in 
pediatric patients. 
Secondary: 
• 
To assess the safety profile of intravenous administrations of OPTISON in the pediatric population. 
• 
To compare diagnostic confidence of left ventricular endocardial border delineation (LV EBD) and 
wall motion between non-contrast and OPTISON-enhanced echocardiography. 
• 
To compare the diagnostic confidence in the evaluation of left ventricular ejection fraction (LVEF) 
between non-contrast and OPTISON-enhanced echocardiography. 
Study Design:  
This will be a Phase 4, open-label, non-randomized, multi-center, multi-dose study in North America and 
Europe.  The study is designed to evaluate overall efficacy and safety of intravenous administration of 
OPTISON at various doses in pediatric patients undergoing transthoracic echocardiography.   
Approximately 50 pediatric patients between ≥9 and <18 years of age weighing ≥20 kg who are clinically 
indicated for a transthoracic echocardiogram and are likely to have a limited transthoracic echocardiographic 
window based on body habitus and/or previous cardiac operation or have a previous suboptimal non-contrast 
echocardiogram will be enrolled into the study.  OPTISON will be administered according to pediatric body 
weight (≥20 to ≤28 kg, >28 to ≤40 kg, and >40 kg).  Each subject will receive administrations of 2 different 
dose levels.  Injections should be at least 10 minutes apart between the 2 dose levels to allow for clearance of 
the previous dose. 
For echocardiographic imaging, standard apical 4-chamber and 2-chamber views will be obtained at baseline 
using tissue harmonic imaging, thereafter with contrast using a contrast-imaging mode.  Additional views 
may be obtained at the clinician’s discretion.  All image views will be obtained prior to and after contrast 
administration until 10 minutes post injection.  All images will be recorded on disk and sent to image core 
lab for preparation of blinded image evaluation (BIE).   
Efficacy assessment will be based on the results of the BIE.  Clinical safety data will be collected throughout 
the study, beginning at baseline throughout 72 hours post OPTISON administration.   
Primary Endpoint: 
• 
Visualization of the 12 segments of the left ventricle wall in standard apical 4-chamber and 2-chamber 
views measured by the qualitative EBD visualization scale. 
Secondary Endpoints: 
• 
Overall safety profile in terms of occurrence of adverse events (AEs) and changes in vital signs, arterial 
oxygen saturation (SaO2), physical examinations and 12-lead electrocardiograms (ECGs) following 
administration of different OPTISON doses in pediatric patients. 
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Name of Sponsor/Company: 
GE Healthcare Ltd. and its 
Affiliates 
Individual Study Table 
Referring to Part of Dossier in 
Which the Individual Study or 
Study Table is Presented: 
 
Volume: 
 
Reference: 
 
(For National Authority Use 
Only) 
 
Name of Finished Product: 
OPTISONTM 
Name of Active Ingredient: 
Perflutren Protein – Type A 
Microspheres Injectable 
Suspension, USP 
• 
Left ventricular opacification (LVO) assessed by visual peak contrast intensity and peak left ventricular 
(LV) contrast filling, and duration of contrast enhancement following intravenous administration of 
OPTISON at various doses. 
• 
Comparison of diagnostic confidence of LV EBD and wall motion between non-contrast and 
OPTISON-enhanced echocardiography at various doses.  
• 
Comparison of diagnostic confidence in the evaluation of LVEF between non-contrast and OPTISON-
enhanced echocardiography. 
Selection of Subjects:  
Inclusion Criteria: 
(1) 
The subject is between ≥9 and <18 years of age and weighs ≥20 kg. 
(2) 
The subject is clinically indicated to undergo a transthoracic echocardiogram. 
(3) 
The subject has a suboptimal non-contrast echocardiogram defined as ≥2 contiguous segments in any 
given view that cannot be visualized. 
(4) 
The subject is able to comply with study procedures.  
(5) 
A parent or legal guardian of the subject has signed and dated an informed consent form, and age-
appropriate pediatric assent has been obtained where appropriate. 
(6) 
Post-menarchal female subjects must have a negative urine pregnancy test at screening and at pre-dose 
on the day of OPTISON administration. 
(7) 
Post-menarchal female subjects must be practicing abstinence, or be using an effective form of birth 
control (e.g., intrauterine device, oral contraceptives, contraceptive implants or injections, diaphragm 
with spermicide, cervical cap, or consort use of condom) for at least 30 days before being enrolled in 
the study. 
Exclusion Criteria 
(1) 
The subject was previously enrolled in this study.  
(2) 
The subject has received an investigational medicinal product within 30 days before or is scheduled to 
receive one from time of entry into this study until completion of the follow-up period proposed for this 
study. 
(3) 
The subject has a known or suspected hypersensitivity to any of the components of OPTISON, blood, 
blood products, or albumin. 
(4) 
The subject has pulmonary hypertension or unstable cardiopulmonary conditions.  
(5) 
The subject has severe liver disease based on medical history. 
(6) 
The subject had a recent (<6 months) neurological event. 
(7) 
The subject presents any clinically active, serious, life-threatening disease, with a life expectancy of 
less than 1 month or where study participation may compromise the management of the subject or other 
reason that in the judgment of the investigator makes the subject unsuitable for participation in the 
study. 
(8) 
The subject is a pregnant or lactating female, or is a female of childbearing potential not using an 
acceptable form of birth control (negative urine pregnancy test also required). 
Number of Subjects/Centers Planned: Approximately 50 subjects at up to 12 centers in North America and 
Europe. 
Treatment of Subjects:  
Investigational Medicinal Product:  
OPTISON (Perflutren Protein – Type A Microspheres Injectable Suspension [USP]) is a sterile non-
pyrogenic suspension of perflutren-containing microspheres (5.0 to 8.0 x 108/mL with a mean diameter range 
of 3.0 to 4.5 µm) of heat-treated human serum albumin with perflutren for contrast enhancement during the 
indicated ultrasound imaging procedures.  The approximate amount of perflutren gas in each milliliter of 
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Name of Sponsor/Company: 
GE Healthcare Ltd. and its 
Affiliates 
Individual Study Table 
Referring to Part of Dossier in 
Which the Individual Study or 
Study Table is Presented: 
 
Volume: 
 
Reference: 
 
(For National Authority Use 
Only) 
 
Name of Finished Product: 
OPTISONTM 
Name of Active Ingredient: 
Perflutren Protein – Type A 
Microspheres Injectable 
Suspension, USP 
OPTISON is 0.19 mg corresponding to 7 µL.  The vial contains a clear liquid lower layer and a white upper 
layer that, after resuspension by gentle mixing, provides a homogeneous, opaque, milky-white suspension for 
intravenous injection. 
In adults, the recommended dose of OPTISON ranges from 0.5 to 3.0 mL injected into a peripheral vein with 
the maximum total dose not exceeding 8.7 mL in any one patient.  However, there is no established dosage 
for children.  For this study, the dose information is based on 2 publications using OPTISON in pediatric 
echocardiography.  Each patient will receive 2 different doses of OPTISON administration for their 
CE-ECHO. 
OPTISON will be administered according to pediatric body weight of ≥20 to ≤28 kg, >28 to ≤40 kg and 
>40 kg.  According to the low volume of OPTISON required for these children, the volume of OPTISON 
will be diluted within the same volume of saline to facilitate the injection.  In children weighing ≥20 to 
≤28 kg, 0.1 or 0.2 mL of OPTISON per injection will be given in ascending order.  The cumulative dose will 
not exceed 1.0 mL.  If children weigh >28 to ≤40 kg, 0.2 or 0.3 mL of OPTISON per injection will be 
administered in ascending order with total dose not to exceed 1.5 mL.  For children whose weight is >40 kg, 
0.2 or 0.4  mL of OPTISON per injection will be administered in ascending order with total dose not to 
exceed 1.8 mL.  The contrast will be administered as a slow bolus at a steady rate of approximately 0.05 mL 
per second for contrast-enhanced echocardiographic imaging followed by a 5.0 mL slow injection of saline.   
Duration of Treatment: 
Subject involvement in this study will be up to 3 weeks from time of screening to the completion of safety 
evaluation at 72 hours after OPTISON administration. 
Efficacy and Safety Evaluation 
Efficacy Evaluation: 
Images will be evaluated by 3 independent blinded readers unaffiliated with enrolling centers.   
Evaluation of EBD on 4- and 2-chamber views with non-contrast and OPTISON-enhanced 
echocardiography.  A qualitative score will be assigned to each LV segment:  
0 = no visualization of the LV endocardial border 
1 = poor visualization 
2 = fair visualization 
3 = good/optimal visualization 
The number of left ventricle segments visualized on 4- and 2-chamber views with non-contrast and 
OPTISON-enhanced echocardiography will be derived from the EBD score (segments rated as “fair 
visualization” or “good/optimal visualization”). 
Evaluation of LVO: 
• 
Peak contrast intensity in the LV chamber is determined using categorical scales: none, low, medium, 
high, and blooming for different dose levels. 
• 
Peak LV contrast filling will be categorized as: 
0 = none (0% filling) 
1 = faint (around 33% filling) 
2 = intermediate (around 67% filling) 
3 = full (100% filling) 
• 
Contrast enhancement duration is determined from the time the contrast appears in the LV to the time 
the contrast almost dissipates from the left chamber. 
Evaluation of image quality: a visual evaluation of the overall image quality will be categorized as one of the 
following: 
• 
Excellent/good: homogeneous contrast enhancement with neither blooming nor shadowing artifacts. 
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Name of Sponsor/Company: 
GE Healthcare Ltd. and its 
Affiliates 
Individual Study Table 
Referring to Part of Dossier in 
Which the Individual Study or 
Study Table is Presented: 
 
Volume: 
 
Reference: 
 
(For National Authority Use 
Only) 
 
Name of Finished Product: 
OPTISONTM 
Name of Active Ingredient: 
Perflutren Protein – Type A 
Microspheres Injectable 
Suspension, USP 
• 
Adequate: sufficient contrast enhancement (i.e., enough to visualize EBD) but less than ideal 
enhancement and/or short enhancement duration and/or with minimal blooming or shadowing artifacts. 
• 
Poor: no or insufficient contrast enhancement/contrast duration or too much contrast enhancement 
leading to significant blooming and shadowing artifacts to assess EBD. 
If any images are deemed as poor quality, an explanation is to be given as to the reason(s). 
The diagnostic usefulness will be evaluated as follows: 
• 
Non-diagnostic: the image quality is not sufficient to confidently visualize the endocardial border. 
• 
Diagnostic: sufficient or excellent image quality for EBD assessments. 
If any images are deemed non-diagnostic, an explanation is to be given as to the reason(s).   
Diagnostic confidence for the assessment of LV EBD and wall motion will be scored for non-contrast and 
OPTISON-enhanced echocardiographic acquisitions separately using the following scale: 
 0 = no confidence 
 1 = low confidence 
 2 = moderate confidence 
 3 = high confidence 
And the change in diagnostic confidence will be derived by comparing non-contrast and OPTISON-enhanced 
diagnostic confidence scores as follows (contrast-enhanced minus unenhanced): 
≤-1 = confidence is lower with contrast-enhanced images 
 0 = confidence is the same between unenhanced and contrast-enhanced images 
 1 = confidence is slightly improved with contrast-enhanced images 
 2 = confidence is moderately improved with contrast-enhanced images 
 3 = confidence is greatly improved with contrast-enhanced images 
Diagnostic confidence for the evaluation of LVEF (LVEF will be calculated using the formula: (end diastole 
volume – end systole volume)/end diastole volume) will be scored for non-contrast and OPTISON-enhanced 
echocardiographic acquisitions separately using the following scale: 
 0 = no confidence 
 1 = low confidence 
 2 = moderate confidence 
 3 = high confidence 
And the change in diagnostic confidence will be derived by comparing non-contrast and OPTISON-enhanced 
diagnostic confidence scores as follows (contrast-enhanced minus unenhanced): 
≤-1 = confidence is lower with contrast-enhanced images 
 0 = confidence is the same between unenhanced and contrast-enhanced images 
 1 = confidence is slightly improved with contrast-enhanced images 
 2 = confidence is moderately improved with contrast-enhanced images 
 3 = confidence is greatly improved with contrast-enhanced images 
Further details of the independent assessment are given in the study’s Independent Review Charter. 
Safety Evaluation: 
Safety parameters will include:  
• 
Treatment-emergent adverse events (TEAEs) and serious adverse events (SAEs) 
• 
Vital signs: heart rate, respiratory rate and blood pressure 
• 
Arterial oxygen saturation (SaO2) by pulse oximetry 
• 
Physical examination 
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Name of Sponsor/Company: 
GE Healthcare Ltd. and its 
Affiliates 
Individual Study Table 
Referring to Part of Dossier in 
Which the Individual Study or 
Study Table is Presented: 
 
Volume: 
 
Reference: 
 
(For National Authority Use 
Only) 
 
Name of Finished Product: 
OPTISONTM 
Name of Active Ingredient: 
Perflutren Protein – Type A 
Microspheres Injectable 
Suspension, USP 
• 
Continuous ECG monitoring 
• 
12-lead ECGs 
Statistical Methods and Planned Analysis:  
All subjects who receive any study drug will be included in the safety population for safety analyses.  
Efficacy analyses will be performed on the intent-to-treat (ITT) and per-protocol populations.  The ITT 
population includes all subjects enrolled in the study who have available non-contrast harmonic images and 
OPTISON-enhanced echocardiographic images, irrespective of quality of their CE-ECHO.  The per-protocol 
population includes all subjects whose non-contrast harmonic images are available and whose OPTISON 
echocardiography images have adequate quality (sufficient contrast enhancement to visualize EBD). 
Demographic variables and patient characteristics will be summarized descriptively by treatment assignment 
and overall, independent of investigational medicinal product assignment.  Demographic variables will 
include age, weight, height, gender, body mass index and race/ethnicity. 
Efficacy: Since this is a dose-finding study, no hypotheses are being tested.  Continuous variables will be 
summarized using means, standard deviations, medians, maxima, minima and sample size. Categorical data 
will be summarized using counts and percentages.  
The suitable dosage of OPTISON for CE-ECHO in pediatric patients will be primarily determined by the 
improvement in the number of segments adequately visualized over non-contrast images, supplemented by 
LVO peak contrast intensity assessed as medium or better, peak LV contrast filling to around 67% or better, 
and duration of contrast enhancement ≥1.5 minutes.  Image quality and diagnostic confidence, as well as 
overall safety of OPTISON in the pediatric population will also be taken into consideration for the dose 
selection. 
Safety: Summary and descriptive statistics will be employed for safety analysis.  TEAEs as reported on the 
case report forms (CRFs) will be mapped to System Organ Class (SOC) and preferred terms using the 
Medical Dictionary for Regulatory Activities (MedDRA; current version or the immediately preceding 
version).  The numbers and percentages of patients reporting each event will be summarized as follow: 
• 
Overall number (frequency) of TEAEs (from dosing through 72±8 hours after the administration of the 
contrast agent). 
• 
TEAEs by maximum severity, relationship to study drug and seriousness. 
• 
TEAEs causing discontinuation from the study. 
For vital signs, observed values and changes from baseline will be summarized with descriptive statistics, 
and a shift table analysis will be performed.   
For ECGs, the overall ECG interpretation will be summarized.  The observed values and changes from 
baseline in ECG intervals will be summarized with descriptive statistics, and a shift analysis will be 
performed for each ECG parameter.  In addition, a summary will be made of clinically notable observed 
values and changes from baseline in QTc intervals.   
Concomitant medications will be summarized using the World Health Organization (WHO) Drug Dictionary 
Anatomical-Therapeutic-Chemical (ATC) classification. 
Physical examination results will be listed but not summarized. 
Sample Size 
The maximum sample size for this clinical investigation will be approximately 50 patients, which will 
provide adequate data to determine OPTISON efficacy in terms of LV EBD, LVO characteristics, wall 
motion assessment, diagnostic confidence for the evaluation of LVEF, and safety profile following 
intravenous administration in pediatric patients.  The data will support dosing information in children. 
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2 
TABLE OF CONTENTS 
2.1 
Overall Table of Contents 
Page Number 
1 
SYNOPSIS ..................................................................................................................... 3 
2 
TABLE OF CONTENTS ............................................................................................... 8 
2.1 
Overall Table of Contents .................................................................................. 8 
2.2 
List of Tables .................................................................................................... 10 
2.3 
List of Figures ................................................................................................... 11 
3 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS .............................. 12 
4 
BACKGROUND INFORMATION ............................................................................. 13 
5 
STUDY OBJECTIVES AND PURPOSE .................................................................... 14 
6 
STUDY DESIGN ......................................................................................................... 15 
6.1 
Overall Study Design and Plan ......................................................................... 15 
6.2 
Study Rationale ................................................................................................ 16 
6.2.1 
Justification of Dose Selection and Efficacy Analysis ................... 17 
6.2.2 
Justification of Safety Monitoring Plan .......................................... 17 
6.3 
Study Timeframe .............................................................................................. 17 
6.4 
Risks and Benefits to Subjects.......................................................................... 18 
7 
SELECTION AND WITHDRAWAL OF SUBJECTS................................................ 19 
7.1 
Procedures for Enrolment ................................................................................. 19 
7.2 
Inclusion Criteria .............................................................................................. 19 
7.3 
Exclusion Criteria ............................................................................................. 19 
7.4 
Withdrawal and Termination Criteria............................................................... 20 
7.4.1 
Subject Withdrawal ........................................................................ 20 
7.4.2 
Study or Site Termination .............................................................. 21 
8 
TREATMENT OF SUBJECTS .................................................................................... 22 
8.1 
Investigational Medicinal Product .................................................................... 22 
8.1.1 
OPTISON ....................................................................................... 22 
8.1.2 
Comparator ..................................................................................... 23 
8.1.3 
Registration of Investigational Medicinal Product Complaints ..... 23 
8.2 
Method of Numbering Subjects and Assigning Subjects to Treatment Groups23 
8.3 
Blinding ............................................................................................................ 23 
8.4 
Prior and Concomitant Therapy ....................................................................... 23 
8.5 
Contraception and Pregnancy Avoidance Procedure ....................................... 24 
8.6 
Treatment Compliance ..................................................................................... 25 
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9 
STUDY PROCEDURES .............................................................................................. 26 
9.1 
Screening Period ............................................................................................... 26 
9.2 
Baseline Period ................................................................................................. 27 
9.3 
Treatment Period .............................................................................................. 27 
9.4 
Post-Treatment Period ...................................................................................... 28 
10 
EFFICACY, SAFETY, AND OTHER VARIABLES ................................................. 29 
10.1 
Study Endpoints ................................................................................................ 29 
10.1.1 
Primary Endpoint ............................................................................ 29 
10.1.2 
Secondary Endpoints ...................................................................... 29 
10.2 
Efficacy Assessment ......................................................................................... 29 
10.2.1 
Efficacy Variables and Method of Measurement ........................... 30 
10.2.2 
Image Acquisition .......................................................................... 32 
10.2.3 
Comparator or Reference Imaging ................................................. 32 
10.2.4 
Image Interpretation ....................................................................... 32 
10.3 
Safety Assessments .......................................................................................... 33 
10.3.1 
Vital Signs ...................................................................................... 33 
10.3.2 
Electrocardiograms ......................................................................... 33 
10.3.3 
Prior and Concomitant Medication................................................. 34 
10.3.4 
Physical Examination ..................................................................... 34 
10.3.5 
Adverse Events ............................................................................... 34 
10.3.6 
Serious Adverse Events (SAEs) ..................................................... 35 
10.3.7 
Other Significant Adverse Events .................................................. 36 
10.3.8 
Adverse Event and Serious Adverse Event Reporting ................... 36 
10.3.9 
Urgent Safety Measures.................................................................. 37 
10.3.10 
Pregnancy Reporting ...................................................................... 38 
10.4 
Other Variables ................................................................................................. 39 
10.5 
Appropriateness of Measurements ................................................................... 39 
11 
DATA HANDLING AND QUALITY ASSURANCE ................................................ 40 
11.1 
Completing and Signing Case Report Forms ................................................... 40 
11.2 
Clinical Data Management ............................................................................... 40 
11.3 
Data Quality Assurance .................................................................................... 40 
11.4 
Record Retention .............................................................................................. 41 
12 
STATISTICAL METHODS AND PLANNED ANALYSIS ...................................... 42 
12.1 
General Statistical Considerations .................................................................... 42 
12.2 
Subject Characteristics ..................................................................................... 42 
12.2.1 
Determination of Safety and Efficacy Population .......................... 42 
12.3 
Efficacy Analysis .............................................................................................. 43 
12.3.1 
Primary Efficacy Analysis .............................................................. 43 
12.3.2 
Secondary Efficacy Analysis .......................................................... 43 
12.3.3 
Handling of Uninterpretable Images .............................................. 44 
12.3.4 
Reader Differences ......................................................................... 44 
12.4 
Safety Analysis ................................................................................................. 45 
12.4.1 
Vital Signs ...................................................................................... 45 
12.4.2 
Electrocardiograms ......................................................................... 45 
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12.4.3 
Prior and Concomitant Medications ............................................... 46 
12.4.4 
Physical Examination ..................................................................... 46 
12.4.5 
Adverse Events ............................................................................... 46 
12.5 
Sample Size Determination .............................................................................. 47 
12.6 
Significance Level ............................................................................................ 47 
12.7 
Procedures for Missing, Unused and Spurious Data ........................................ 47 
12.8 
Rules for Excluding Subjects from Analysis .................................................... 48 
12.9 
Procedures for Reporting Deviations from Original Statistical Plan ............... 48 
13 
SPECIAL REQUIREMENTS AND PROCEDURES ................................................. 49 
13.1 
Regulatory and Ethical Considerations ............................................................ 49 
13.2 
Investigator’s Responsibilities .......................................................................... 50 
13.2.1 
Overall Responsibilities .................................................................. 50 
13.2.2 
Parental Permission/Subject Informed Assent ............................... 50 
13.2.3 
Direct Access to Source Data/Documents ...................................... 50 
13.2.4 
Confidentiality Regarding Study Subjects ..................................... 51 
13.2.5 
Data Protection ............................................................................... 51 
13.3 
Protocol Deviations .......................................................................................... 51 
13.4 
Study Monitoring .............................................................................................. 51 
13.5 
Audit and Inspection ......................................................................................... 52 
13.6 
Financial Disclosure ......................................................................................... 52 
13.7 
Insurance ........................................................................................................... 52 
13.8 
Publication Policy ............................................................................................. 52 
14 
REFERENCES ............................................................................................................. 53 
15 
APPENDICES .............................................................................................................. 55 
15.1 
Information on Investigational and Registered Products ................................. 55 
15.2 
Equipment Parameters ...................................................................................... 55 
15.3 
Pediatric Normal Ranges .................................................................................. 55 
16 
CLINICAL PROTOCOL AMENDMENT SUMMARIES .......................................... 57 
16.1 
Amendment A01 .............................................................................................. 57 
16.1.1 
Reasons for Amendment ................................................................ 57 
16.1.2 
Description of Changes .................................................................. 57 
2.2 
List of Tables 
Table 1 
OPTISON Dosing Table ........................................................................................... 22 
Table 2 
Study Schedule of Events ......................................................................................... 26 
Table 3 
Efficacy Variables and Method of Measurement ..................................................... 31 
Table 4 
Power Calculation .................................................................................................... 47 
Table 5 
Pediatric Vital Signs Normal Ranges ....................................................................... 55 
Table 6 
Pediatric ECG Normal Ranges ................................................................................. 55 
Table 7 
Mean and Percentiles of ECG Cardiac Measures Among Pubertal Children .......... 56 
 
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2.3 
List of Figures 
Figure 1 
Study Diagram .......................................................................................................... 16 
Figure 2 
Segmentation schemes for segmental analysis of LV EBD ( adapted from [Lang 
et al. 2015]) ............................................................................................................... 30 
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3 
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS 
AE 
Adverse event 
ATC 
Anatomical-Therapeutic-Chemical 
BIE 
Blinded image evaluation 
CE-ECHO 
Contrast-enhanced echocardiography 
CRF 
Case report form 
CRO 
Contract research organization 
EBD 
Endocardial border delineation 
ECG 
Electrocardiogram 
GCP 
Good Clinical Practice 
ICH 
International Council for Harmonisation 
IEC 
Independent Ethics Committee 
IMP 
Investigational medicinal product 
IRB 
Institutional/Independent Review Board 
ITT 
Intent-to-treat 
LV 
Left ventricular 
LV EBD 
Left ventricular endocardial border delineation 
LVEF 
Left ventricular ejection fraction 
LVO 
Left ventricular opacification 
MedDRA 
Medical Dictionary for Regulatory Activities 
PMC 
Post-marketing commitment 
SAE 
Serious adverse event 
SOC 
System organ class 
SOPs 
Standard operating procedures 
TEAE 
Treatment-emergent adverse event 
 
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4 
BACKGROUND INFORMATION 
Transthoracic echocardiography is an ideal tool for cardiac assessment, as it is noninvasive, 
portable, and efficacious in providing detailed anatomic, hemodynamic, and physiologic 
information.  Unfortunately, up to 30% of adult studies are deemed technically difficult due to 
poor image quality, making these studies very challenging to interpret.  Patients with large 
body habitus, chest wall deformities, and severe chronic lung disease are at higher risk of 
having suboptimal image quality ([Mulvagh et al. 2000], [Geleijnse et al. 1997], 
[Weissman et al. 2000]).  Intravenous contrast-enhanced echocardiography (CE-ECHO) has 
been demonstrated to be a useful tool in optimizing endocardial border delineation (EBD) in 
adult patients ([Candido et al. 2003], [Clark and Dittrich. 2000], [Kornblith et al. 2000]) and in 
3 studies only in pediatric patients ([Zilberman et al. 2003], [McMahon et al. 2005], [Kutty et 
al. 2016]).   
Echocardiography plays a role in the diagnosis and assessment of congenital and acquired heart 
disease in infants and children.  Despite the relatively high quality of echocardiographic 
imaging in children, echocardiographic images are not always diagnostic in some patients as a 
result of body habitus, previous cardiac operation, or both.  However, there is limited 
experience with contrast agents in children to date ([Zilberman et al. 2003], 
[McMahon et al. 2005], [Kutty et al. 2016]).   
OPTISON™ (Perflutren Protein-Type A Microspheres Injectable Suspension, USP) is a sterile 
non-pyrogenic suspension of microspheres of human serum albumin approved for use in adult 
patients with suboptimal echocardiograms to opacify the left ventricle and to improve the 
delineation of the left ventricular (LV) endocardial borders.  
At the time of original new drug application (NDA) approval, a post-marketing commitment 
(PMC) was agreed to study the efficacy and safety of OPTISON in pediatric patients: 
• To determine suitable dosage and administration instructions and any technical 
differences in instrumentation or settings that may affect OPTISON performance and 
safety in pediatric patients compared to adult patients. 
The current study is therefore to fulfill the PMC requirement on dosage and safety assessment 
of OPTISON for the pediatric patients. 
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5 
STUDY OBJECTIVES AND PURPOSE 
The primary and secondary objectives of the study are as follows: 
Primary: 
• 
To determine a suitable dosage of OPTISON for CE-ECHO in pediatric patients. 
Secondary: 
• 
To assess the safety profile of intravenous administrations of OPTISON in the pediatric 
population. 
• 
To compare diagnostic confidence of left ventricular endocardial border delineation 
(LV EBD) and wall motion between non-contrast and OPTISON-enhanced 
echocardiography. 
• 
To compare the diagnostic confidence in the evaluation of left ventricular ejection 
fraction (LVEF) between non-contrast and OPTISON-enhanced echocardiography. 
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6 
STUDY DESIGN 
6.1 
Overall Study Design and Plan 
This will be a Phase 4, open-label, non-randomized, multi-center, multi-dose study in North 
America and Europe.  The study is designed to evaluate efficacy and safety of intravenous 
administration of OPTISON at various doses in pediatric patients undergoing transthoracic 
echocardiography.   
Approximately 50 pediatric patients between ≥9 and <18 years of age weighing ≥20 kg who 
are clinically indicated for a transthoracic echocardiogram and are likely to have a limited 
transthoracic echocardiographic window based on body habitus and/or previous cardiac 
operation or have a previous suboptimal non-contrast echocardiogram will be enrolled into the 
study. 
Each patient will undergo an echocardiographic procedure first without contrast administration 
then following intravenous bolus injection of OPTISON at various doses.  The dose of 
OPTISON for each subject will be determined according to pediatric body weight group and 
2 different dose levels will be tested in each subject.  The details on OPTISON administration 
are presented in Section 8.1 of this protocol. 
For echocardiographic imaging, standard apical 4-chamber and 2-chamber views will be 
obtained at baseline using tissue harmonic imaging, thereafter with contrast using a contrast-
imaging mode.  Additional views may be obtained at the clinician’s discretion.  All image 
views will be obtained prior to and after contrast administration until 10 minutes post injection.  
All images will be recorded on disk and sent to sponsor’s image core lab for preparation of 
blinded image evaluation (BIE).   
Efficacy of echocardiographic images will be evaluated by independent blinded readers based 
on characteristics of left ventricular opacification (LVO), diagnostic confidence of LV EBD 
and wall motion, and diagnostic confidence of LVEF.  Clinical safety data will be collected 
throughout the study, beginning from dosing through 72 hours post OPTISON administration 
to determine the safety profile of intravenous administration of OPTISON at various doses in 
the pediatric population. 
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Figure 1 Study Diagram 
 
6.2 
Study Rationale 
The study is to determine a suitable dosage and administration instructions for CE-ECHO with 
OPTISON in pediatric patients.  
OPTISON is a registered product.  It has been approved since 1997 for LVO and EBD in adult 
patients with non-diagnostic echocardiogram.  Currently, several injections of 0.5 mL may be 
injected, up to a cumulative maximum of 8.7 mL.  The current study will help to determine an 
appropriate dose for pediatric echocardiography.  
Screening (≤2 weeks) 
Informed consent, study criteria, pregnancy test 
(post-menarchal females), demographic information, 
medical/surgical history 
Baseline (from-1 ± 1 hour prior to OPTISON) 
Entry criteria re-check, pregnancy test (post-menarchal females), vital signs, 
pulse oximetry, physical examination, ECG, prior and concomitant 
medications, AEs (from time of informed consent), unenhanced scan 
Dosing/Imaging (0) 
OPTISON administration(s), CE-ECHO, pulse oximetry, 
continuous ECG monitoring (0 to 30 min), AE monitoring 
Post Last Injection (+10 ±5 min & 1 hour ±5 min) 
Vital signs, pulse oximetry, ECG (+10 ±5 min & 30±5 min), AE monitoring, 
concomitant medication, physical examination (1 hour ±5 min only) 
Post Last Injection (+24 ±4 hours phone) 
Concomitant medication, AE reporting 
Image Transfer to Core Lab 
Post Last Injection (72 ±8 hour phone) 
Concomitant medication, AE reporting 
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6.2.1 
Justification of Dose Selection and Efficacy Analysis 
In the adult controlled clinical trials for the LVO and EBD indication, the duration of the useful 
imaging time for LV chamber opacification was 2.5 to 4.5 minutes for a dose of 0.5 to 3.0 mL 
[OPTISON PI].   
McMahon et al conducted rest non-contrast and CE-ECHO in 20 children (9 to 18 years) with 
limited transthoracic echo windows [McMahon et al. 2005].  OPTISON was administered at a 
dose of 0.3 mL per injection for children <20 kg and 0.5 mL per injection for those ≥20 kg.  
The EBD was significantly improved in all patients with CE-ECHO and LVEF was measurable 
in all 20 patients using contrast echo versus only 11 with non-contrast echocardiography.  
Zilberman et al performed contrast stress echocardiography with OPTISON administration in 
22 children (8 months to 19 years; mean age 9.3 ± 3.9 years) [Zilberman et al. 2003].  A dose 
of 0.1 mL was given for patients ≤25 kg, and 0.2 mL for patients >25 kg.  These doses 
provided adequate opacification of the LV cavity for up to 60 seconds. 
Similar to the two publications, OPTISON dose in this study will be administered based on 
body weight.  According to the Centers for Disease Control and Prevention Pediatric Growth 
Charts [CDC Growth Charts], children >9 years of age usually weigh >28 kg and 
children >12 years of age usually weigh >40 kg for both boys and girls in the United States, but 
weight varies greatly for a specific age.  Two dose levels will be tested in each weight cohort: 
0.1 or 0.2 mL per injection to children weighing ≥20 to ≤28 kg, 0.2 or 0.3 mL per injection to 
those who weigh >28 to ≤40 kg, and 0.2 or 0.4 mL per injection to those who weigh >40 kg.  It 
is expected that one or more of these dose levels will be able to provide adequate LVO and 
EBD in these groups of pediatric patients with a limited transthoracic echocardiographic 
window. 
6.2.2 
Justification of Safety Monitoring Plan 
This study’s safety monitoring plan is justifiable and adequate from a safety standpoint: 
• 
The design of the safety plan permits a comparison of the safety response to OPTISON 
at baseline and post-investigational medicinal product (IMP) administration conditions 
in the same subject. 
• 
Consideration of a 3-day safety monitoring follow-up permits the evaluation of 
late-appearing adverse effects that may emerge or progress after the administration of 
OPTISON. 
• 
The measures used to assess safety are well-defined and reliable, and the proposed 
safety analyses are adequate to assess the effects of the administration of OPTISON. 
6.3 
Study Timeframe 
The duration of each subject’s participation is expected to be up to 3 weeks, including 
screening (2 weeks), procedure, and safety follow-up (24 hours and 72 hours post OPTISON 
administration). 
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The entire subject recruitment phase is expected to last up to 2 years. 
6.4 
Risks and Benefits to Subjects 
Study participants may help future patients by contributing to a knowledge base for further 
evaluation of the use of OPTISON in pediatric patients with cardiac disease.  For some patients 
participating in the study, the benefit might be detection or more precise evaluation of their 
cardiac disease. 
OPTISON is well tolerated in adults.  Adverse reactions to OPTISON are rare and usually of a 
non-serious nature.  In general, the administration of human albumin has been associated with 
transient altered taste, nausea, flushing, rash, headache, vomiting, chills and fever.  The shell of 
the OPTISON bubble is composed of albumin.  Rare anaphylactic reactions have been 
associated with the administration of human albumin products.  The reported adverse events 
(AEs) following the use of OPTISON in Phase 3 human clinical studies have been mild to 
moderate with subsequent full recovery. 
For further information please refer to the currently approved OPTISON Package Insert 
[OPTISON PI]. 
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7 
SELECTION AND WITHDRAWAL OF SUBJECTS 
7.1 
Procedures for Enrolment 
Investigators are responsible for subject recruitment at their sites.  A signed and dated (with 
time) informed consent form will be obtained from a parent or legal guardian of each study 
subject prior to study entry or any study-specific procedures.  Age-appropriate pediatric assent 
will be obtained where appropriate.  Subjects will be screened for entry through the use of 
clinical examination and historic assessment.  A unique study-related subject number will be 
assigned at baseline before the unenhanced echocardiography procedure. 
7.2 
Inclusion Criteria 
Subjects may be included in the study if they meet all of the following criteria: 
(1) 
The subject is between ≥9 and <18 years of age and weighs ≥20 kg. 
(2) 
The subject is clinically indicated to undergo a transthoracic echocardiogram. 
(3) 
The subject has a suboptimal non-contrast echocardiogram defined as ≥2 contiguous 
segments in any given view that cannot be visualized.  
(4) 
The subject is able to comply with study procedures.  
(5) 
A parent or legal guardian of the subject has signed and dated an informed consent 
form, and age-appropriate pediatric assent has been obtained where appropriate. 
(6) 
Post-menarchal female subjects must have a negative urine pregnancy test at screening 
and at pre-dose on the day of OPTISON administration. 
(7) 
Post-menarchal female subjects must be practicing abstinence, or be using an effective 
form of birth control (e.g., intrauterine device, oral contraceptives, contraceptive 
implants or injections, diaphragm with spermicide, cervical cap, or consort use of 
condom) for at least 30 days before being enrolled in the study. 
7.3 
Exclusion Criteria 
Subjects must be excluded from participating in this study if they meet any of the following 
criteria: 
(1) 
The subject was previously enrolled in this study.  
(2) 
The subject has received an IMP within 30 days before or is scheduled to receive one 
from time of entry into this study until completion of the follow-up period proposed for 
this study. 
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(3) 
The subject has a known or suspected hypersensitivity to any of the components of 
OPTISON, blood, blood products, or albumin. 
(4) 
The subject has pulmonary hypertension or unstable cardiopulmonary conditions.  
(5) 
The subject has severe liver disease based on medical history. 
(6) 
The subject had a recent (<6 months) neurological event. 
(7) 
The subject presents any clinically active, serious, life-threatening disease, with a life 
expectancy of less than 1 month or where study participation may compromise the 
management of the subject or other reason that in the judgment of the investigator 
makes the subject unsuitable for participation in the study. 
(8) 
The subject is a pregnant or lactating female, or is a female of childbearing potential not 
using an acceptable form of birth control (negative urine pregnancy test also required). 
7.4 
Withdrawal and Termination Criteria 
7.4.1 
Subject Withdrawal 
During the conduct of the study, the sponsor will review the safety data for trends and signals 
that would indicate the need for withdrawal of a subject.  If a subject undergoes an intervention 
or has a significant change in disease status between the administration of OPTISON and the 
completion of study follow-up, the subject will be withdrawn from the study. 
In accordance with the Declaration of Helsinki, each subject is free to withdraw from the study 
at any time.  Investigator(s) also have the right to withdraw subjects from the study in the event 
of illness, AEs, or other reasons concerning the health or well-being of the subject, or in the 
case of lack of co-operation. 
Should a subject decide to withdraw after administration of OPTISON, or should the 
investigator(s) decide to withdraw the subject, all efforts will be made to complete and report 
the observations up to the time of withdrawal as thoroughly as possible.  A complete final 
evaluation at the time of the subject’s withdrawal should be made and an explanation given of 
why the subject is withdrawing or being withdrawn from the study.   
The reason for withdrawal must be noted in the Case Report Form (CRF).  If the reason for 
withdrawal is a serious adverse event (SAE), monitoring will continue until the outcome is 
evident.  The specific event or test result(s) must be recorded in the CRF.   
If a subject is withdrawn before completion of CE-ECHO, the subject will be replaced.  The 
replaced subject will be assigned a new subject number. 
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7.4.2 
Study or Site Termination 
GE Healthcare is committed to complete subject enrolment in approximately 2 years.  
However, if after one year, <10% of the 50 subjects have been enrolled at 12 sites, additional 
study modifications or study termination will be considered.  The sponsor reserves the right to 
terminate the study at any time.  
Should termination be necessary, both the sponsor and the investigators will ensure that 
adequate consideration is given to the protection of the subjects’ interest.  Termination of the 
study will be considered in the event of a significant safety finding occurring at any time 
during the conduct of the study. 
Investigators have the responsibility to comply with International Council for Harmonisation 
(ICH) E6-Good Clinical Practice (GCP) guidance.  The sponsor, the Institutional Review 
Board (IRB)/Independent Ethics Committee (IEC), or the health authorities may terminate a 
center for the following (but not limited to) reasons: 
(1) 
Failure of the investigator to comply with pertinent ICH E6-GCP guidelines and 
regulations. 
(2) 
Occurrence of serious protocol violations. 
(3) 
Submission of knowingly false information from the research facility to the sponsor, 
clinical monitor, or other party involved in the study. 
(4) 
Failure of the investigator to enroll subjects into the study at an acceptable rate as 
agreed with the sponsor. 
(5) 
Repeated failure to have imaging data transferred to the sponsor or the CRF completed 
and ready for review by the sponsor in the agreed time frame. 
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8 
TREATMENT OF SUBJECTS 
Each investigator is responsible for ensuring that deliveries of IMP and other study materials 
from the sponsor are correctly received, recorded, handled, and stored safely and properly in 
accordance with all applicable regulatory guidelines, and used in accordance with this protocol. 
All IMP containers (opened, unopened, or empty) must be returned to the sponsor or destroyed 
on site after the study and overall drug accountability have been completed by the sponsor or 
representative.  A list of IMP(s) and other materials that were returned, or destroyed, must be 
prepared and signed by the principal investigator or designee.  If there are any discrepancies, 
an explanation for these should also be provided. 
8.1 
Investigational Medicinal Product 
8.1.1 
OPTISON 
OPTISON (Perflutren Protein-Type A Microspheres Injectable Suspension, [USP]) is a sterile 
non-pyrogenic suspension of perflutren-containing microspheres (5.0 to 8.0 108/mL with a 
mean diameter range of 3.0 to 4.5 m) of heat-treated human serum albumin with perflutren 
for contrast enhancement during the indicated ultrasound imaging procedures.  The 
approximate amount of perflutren gas in each milliliter of OPTISON is 0.19 mg corresponding 
to 7 µL.  The vial contains a clear liquid lower layer and a white upper layer that, after 
resuspension by gentle mixing, provides a homogeneous, opaque, milky-white suspension for 
intravenous injection.  OPTISON will be provided in 3-mL vials for single use.  OPTISON 
should be stored refrigerated at 2°C to 8°C (36°F to 46°F).  Investigators must follow the 
package insert instruction to prepare and administer OPTISON. 
In adults, the recommended dose of OPTISON ranges from 0.5 to 3.0 mL injected into a 
peripheral vein with the maximum total dose not exceeding 8.7 mL in any one patient.  
However, there is no established dosage for children.  For this study, the dose information is 
based on 2 publications using OPTISON in pediatric echocardiography (see Section 6.2.1).  
Each patient will receive 2 different doses of OPTISON administration for CE-ECHO; the 
actual doses of OPTISON will be based on pediatric body weight of ≥20 to ≤28 kg, >28 to 
≤40 kg, and >40 kg and up to 3 injections per dose level are allowed for acquiring different 
imaging views (Table 1).  According to the low volume of OPTISON required for these 
children, the volume of OPTISON will be diluted within the same volume of saline to facilitate 
the injection.   
Table 1 
OPTISON Dosing Table 
Body weight (kg) 
Dose/injection (mL) 
Dose level 1 
Dose/injection (mL) 
Dose level 2 
Total dose (mL) not to exceed 
(3 injections/dose level) 
≥20 to ≤28 
0.1  
0.2 
1.0 
>28 to ≤40 
0.2 
0.3 
1.5 
>40 
0.2 
0.4 
1.8 
 
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The contrast will be administered as a slow bolus at a steady rate of approximately 0.05 mL per 
second for contrast-enhanced echocardiographic imaging followed by a 5.0 mL slow injection 
of saline.  Injections should be at least 10 minutes apart to allow for clearance of the previous 
dose.   
8.1.2 
Comparator 
Unenhanced grey-scale echocardiography will be the comparator. 
8.1.3 
Registration of Investigational Medicinal Product Complaints 
In the event of an IMP complaint (e.g., breakage, leakage, particulate matter, discoloration), the 
investigator or recipient of the IMP is requested to report the problem on the IMP shipping 
documentation (e.g., ‘Delivery Note for Product’, Drug Shipping and Receiving Form, or 
equivalent form).  This should be promptly forwarded to the person indicated on the shipping 
documentation.  Once received, the Clinical Supplies Manager will register the complaint and 
determine if the complaint is minor or significant according to sponsor procedures.  All 
complaints will be followed-up and the appropriate action will be implemented according to 
sponsor procedures. 
8.2 
Method of Numbering Subjects and Assigning Subjects to 
Treatment Groups 
No treatment will be assigned to a subject before it is ascertained that the subject has met all of 
the inclusion criteria and none of the exclusion criterion and that written informed consent (and 
age-appropriate pediatric assent where appropriate) has been obtained.  An allocation number 
will be assigned to each consented subject so that all of the subjects from a center will be given 
allocation numbers in successive order of inclusion.  The subject’s allocation number and vial 
number are separate and independent. 
8.3 
Blinding 
This is an open-label study.  No blinding will be conducted for the subject allocation or 
application of the IMP in this study. 
For determination of efficacy and optimal dose level, 3 qualified independent blinded readers 
will conduct BIE of echocardiographic images.  Non-contrast and OPTISON-enhanced images 
will be presented in random order to the 3 readers without dosing information.  
8.4 
Prior and Concomitant Therapy 
Any prior, concomitant, or procedural medications or therapy given to or taken by the subject 
within 24 hours before and up to the end of the observation period (3 days post OPTISON 
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administration) will be recorded in the CRF along with the indication.  Either the generic or the 
trade name may be recorded.  The sponsor will encode all therapy and medication according to 
a current well-recognized dictionary of medical codes. 
8.5 
Contraception and Pregnancy Avoidance Procedure 
Females of child-bearing potential are “fertile, following menarche and until becoming post-
menopausal, unless permanently sterile.  Permanent sterilization methods include 
hysterectomy, bilateral salpingectomy and bilateral oophorectomy” [CTFG Guidance 2014].   
A man is considered fertile after puberty unless permanently sterile by bilateral orchidectomy 
or vasectomized with confirmation of success.  
Females of child-bearing potential who are sexually active with a non-sterile male partner must 
be using an acceptable method of contraception (as defined below) from at least 30 days before 
being enrolled in the study.  
Acceptable methods of contraception considered as highly effective are those with no higher 
than a 1% failure rate.  Such methods include [CTFG Guidance 2014]:  
• 
Combined (estrogen and progestogen containing) hormonal contraception associated 
with inhibition of ovulation, provided there is no concern about interaction with the 
IMP: 
• 
oral 
• 
intravaginal 
• 
transdermal 
• 
Progestogen-only hormonal contraception associated with inhibition of ovulation, 
provided there is no concern about interaction with the IMP: 
• 
oral 
• 
injectable 
• 
implantable 
• 
Intrauterine device 
• 
Intrauterine hormone-releasing system, provided there is no concern about interaction 
with the IMP 
• 
Bilateral tubal occlusion 
• 
Vasectomized partner (provided that the vasectomized partner is the sole sexual partner 
of the female of child-bearing potential and that the vasectomized partner has received 
medical confirmation of the surgical success) 
• 
Sexual abstinence (only if the subject refrains from heterosexual intercourse during the 
entire period of risk associated with the study treatments.  The reliability of sexual 
abstinence needs to be evaluated in relation to the duration of the clinical trial and the 
preferred and usual lifestyle of the subject). 
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Acceptable, but not highly effective, birth control methods that result in a failure rate of more 
than 1% per year include [CTFG Guidance 2014]:  
• 
progestogen-only oral hormonal contraception, where inhibition of ovulation is not the 
primary mode of action 
• 
male or female condom with or without spermicide 
• 
cap, diaphragm or sponge with spermicide 
Subjects will be provided with information on acceptable methods of contraception as part of 
the subject informed consent process.  Females of child-bearing potential must have a negative 
result for a urine human chorionic gonadotropin pregnancy test at screening and at pre-dose 
(according to the Study Schedule of Events, Table 2).   
8.6 
Treatment Compliance 
Subjects will receive OPTISON administration under direct supervision of study personnel.  
Each administration volume will be checked and the volume per administration will be 
recorded in each subject’s CRF. 
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9 
STUDY PROCEDURES 
All efficacy and safety measurements obtained during the course of the study are summarized 
in the Study Schedule of Events (Table 2). 
Table 2 
Study Schedule of Events 
Variables 
Pre-procedure 
Dosing/ 
Imaging 
(0) 
Post-last OPTISON Administration 
Screening 
(≤2 weeks) 
Baseline 
(from -1 
± 1 h) 
10 min 
(±5 min) 
30 min  
(±5 min) 
1 h  
(±5 min) 
24 h  
(±4 h) 
72 h  
(±8 h) 
Informed Consent 
X 
 
 
 
 
 
 
 
Entry Criteria 
< ------------ > 
 
 
 
 
 
 
Demographic 
Information 
X 
 
 
 
 
 
 
 
Medical/Surgical 
History 
X 
 
 
 
 
 
 
 
Pregnancy test a 
X 
X 
 
 
 
 
 
 
Physical 
Examination 
 
X 
 
 
 
X 
 
 
Vital Signs 
 
X 
 
X 
 
X 
 
 
SaO2 by Pulse 
Oximetry 
 
X 
X 
X 
 
X 
 
 
12-Lead ECG 
Recording 
 
X 
 
X 
X 
 
 
 
Prior and 
concomitant 
Medications b 
 
< -------------------------------------------------------------------------------------------------------- > 
Continuous ECG 
Monitoring c 
 
 
< ---------------------------------------- > 
 
 
 
OPTISON 
Administration 
 
 
X 
 
 
 
 
 
Image Acquisition  
 
X 
X 
 
 
 
 
 
Phone Calls 
 
 
 
 
 
 
X 
X 
AEs and SAEs 
Monitoring 
X 
<------------------------------------------------------------------------------------------------------ > 
AE = Adverse event; ECG = Electrocardiogram; SAE = Serious adverse event; SaO2 = Oxygen saturation 
a Urine β-hCG pregnancy test at screening and at pre-dose; post-menarchal females only. 
b Any prior or concomitant medication taken by the subject within 24 hours before OPTISON injection and up to 
the end of the 72-hour follow-up period. 
c Continuous ECG monitoring is from time of first OPTISON injection until 30 minutes after the last OPTISON 
injection 
9.1 
Screening Period 
The screening evaluations may occur within 2 weeks prior to baseline day 0.  The subject’s 
study participation must be documented in the institution’s files including subject’s 
identification, date of inclusion and the study number.   
The screening evaluations include the following: 
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• 
Signed, dated, and timed informed consent must be obtained from parents or legal 
guardian of all subjects prior to their entering the study or any study specific procedure.  
Age-appropriate pediatric assent must be obtained where appropriate.   
• 
Assessment of inclusion and exclusion criteria.  All subjects must satisfy all the 
inclusion criteria and none of the exclusion criteria listed in Sections 7.2 and 7.3.  
• 
Collection of demographic information including age and gender.  
• 
Obtaining medical/surgical history. 
• 
Urine β-hCG pregnancy test (post-menarchal females). 
• 
Recording of AEs (from time of consent). 
9.2 
Baseline Period 
The subjects will enter the baseline period on the day of the echocardiography.  The baseline 
period will begin at 1  1 hour prior to OPTISON administration and continue until time of 
first injection of OPTISON.  
The following procedures must be completed before OPTISON administration: 
• 
Re-assessment of inclusion and exclusion criteria. 
• 
Urine β-hCG pregnancy test (post-menarchal females) 
• 
Perform a physical examination. 
• 
Vital signs (heart rate, blood pressure and respiratory rate) will be measured and 
recorded. 
• 
Monitoring and recording pulse oximetry. 
• 
12-lead electrocardiograms (ECGs) will be performed with the subject in the supine 
position. 
• 
Recording prior and concomitant medication taken within 24 hours prior to procedure. 
• 
Record any AEs. 
• 
Acquiring unenhanced echocardiography images. 
9.3 
Treatment Period 
The treatment period includes OPTISON administration, imaging procedure and safety follow-
up.  The following procedures will be performed: 
• 
OPTISON must be prepared and administered according to the package insert 
instruction (also see Section 8.1.1). 
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• 
The volume per OPTISON administration, number of bolus injections, and total volume 
administered will be recorded.   
• 
CE-ECHO images will be acquired according to instruction in Section 10.2.2.  
• 
Continuous ECG monitoring from time of first OPTISON injection until 30 minutes 
after the last OPTISON injection. 
• 
Monitoring and recording pulse oximetry. 
• 
Subjects will be monitored for the occurrence of AEs and SAEs. 
9.4 
Post-Treatment Period 
After completion of contrast administration and imaging acquisition, the subjects will be 
monitored at the following time points for safety assessment: 
• 
At 10 ± 5 minutes and 30 ± 5 minutes after the last OPTISON administration, 12-lead 
ECG will be recorded.  Investigators may also record 12-lead ECG at additional time 
points if thought to be clinically relevant; however, only 12-lead ECG recordings at 
10 ± 5 minutes and 30 ± 5 minutes will be considered as study data.   
• 
At 10 ± 5 minutes and 1 hour ± 5 minutes after the last OPTISON administration, vital 
signs and pulse oximetry will be obtained.   
• 
A physical examination will be performed at 1 hour ± 5 minutes.  The occurrence of 
AEs and SAEs will be monitored and recorded. 
• 
At 24 ± 4 hours and 72 ± 8 hours after the last OPTISON administration, the subject’s 
parents or legal guardian will be contacted by telephone for assessing AEs/SAEs and 
recording concomitant medications. 
• 
The subject’s parent/guardian will be provided with an emergency phone number and 
instructed to contact study site personnel with any questions or concerns.  The subject’s 
parent/guardian may be called at any time if considered necessary by study personnel. 
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10 
EFFICACY, SAFETY, AND OTHER VARIABLES 
10.1 
Study Endpoints 
10.1.1 
Primary Endpoint 
• 
Visualization of the 12 segments of the left ventricle wall in standard apical 4-chamber 
and 2-chamber views measured by the qualitative EBD visualization scale. 
10.1.2 
Secondary Endpoints 
The secondary endpoints will be the following: 
• 
Overall safety profile in terms of occurrence of AEs and changes in vital signs, arterial 
oxygen saturation (SaO2), physical examinations and 12-lead ECGs following 
administration of different OPTISON doses in pediatric patients. 
• 
LVO assessed by visual peak contrast intensity and peak LV contrast filling, and 
duration of contrast enhancement following intravenous administration of OPTISON at 
various doses.  
• 
Comparison of diagnostic confidence of LV EBD and wall motion between non-
contrast and OPTISON-enhanced echocardiography at various doses. 
• 
Comparison of diagnostic confidence in the evaluation of LVEF between non-contrast 
and OPTISON-enhanced echocardiography. 
10.2 
Efficacy Assessment 
Efficacy will be assessed by 3 independent blinded readers unaffiliated with enrolling centers 
and based on characteristics of LV EBD, LVO, diagnostic confidence for the assessment of LV 
EBD and wall motion, and for the evaluation of LVEF for both non-contrast and CE-ECHO.   
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10.2.1 
Efficacy Variables and Method of Measurement 
Figure 2 Segmentation schemes for segmental analysis of LV EBD ( adapted from [Lang 
et al. 2015]) 
 
Basal segments  
Mid segments 
 
Apical segments 
2 = Basal anterior 
  8 = Mid anterior  
13 = Apical anterior 
3 = Basal anterolateral 
  9 = Mid anterolateral 
14 = Apical lateral 
5 = Basal inferior  
11 = Mid inferior  
15 = Apical inferior 
6 = Basal inferoseptal 
12 = Mid inferoseptal 
16 = Apical septal 
 
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Table 3 
Efficacy Variables and Method of Measurement 
Efficacy Variable 
Method of Measurement 
EBD 
Evaluation of EBD on 4- and 2-chamber views with non-contrast and OPTISON-enhanced 
echocardiography.  A qualitative score will be assigned to each ventricular segment:   
0 = no visualization of the LV endocardial border 
1 = poor visualization 
2 = fair visualization 
3 = good/optimal visualization 
The number of left ventricle segments visualized on 4- and 2-chamber views with non-
contrast and OPTISON-enhanced echocardiography will be derived from the EBD score 
(segments rated as “fair visualization” or “good/optimal visualization”). 
LVO  
• 
Peak contrast intensity in the LV chamber is determined by blinded readers using 
categorical scales: none, low, medium, high, and blooming for different dose levels. 
• 
Peak LV contrast filling will be categorized as: 
0 = none (0% filling) 
1 = faint (around 33% filling) 
2 = intermediate (around 67% filling) 
3 = full (100% filling) 
• 
Contrast enhancement duration is determined from the time the contrast appears in the 
LV to the time the contrast almost dissipates from the left chamber. 
Image quality 
Independent blinded readers assess CE-ECHO images for overall image quality as 
excellent/good, adequate, or poor. 
• 
Excellent/good: homogeneous contrast enhancement with neither blooming nor 
shadowing artifacts. 
• 
Adequate: sufficient contrast enhancement (i.e., enough to visualize EBD) but less than 
ideal enhancement and/or short enhancement duration and/or with minimal blooming 
or shadowing artifacts. 
• 
Poor: no or insufficient contrast enhancement/contrast duration or too much contrast 
enhancement leading to significant blooming and shadowing artifacts to assess EBD. 
If any images are deemed as poor quality, an explanation is to be given as to the reason(s). 
The diagnostic usefulness will be evaluated as follows: 
• 
Non-diagnostic: the image quality is not sufficient to confidently visualize the 
endocardial border. 
• 
Diagnostic: sufficient or excellent image quality for EBD assessments. 
If any images are deemed non-diagnostic, an explanation is to be given as to the reason(s)  
Diagnostic confidence 
for the assessment of 
LV EBD and wall 
motion 
Diagnostic confidence for the assessment of LV EBD and wall motion will be scored for 
non-contrast and OPTISON-enhanced echocardiographic acquisitions separately using the 
following scale: 
 0 = no confidence 
 1 = low confidence 
 2 = moderate confidence 
 3 = high confidence 
And the change in diagnostic confidence will be derived by comparing non-contrast and 
OPTISON-enhanced diagnostic confidence scores as follows (contrast-enhanced minus 
unenhanced): 
≤-1 = confidence is lower with contrast-enhanced images 
 0 = confidence is the same between unenhanced and contrast-enhanced images 
 1 = confidence is slightly improved with contrast-enhanced images 
 2 = confidence is moderately improved with contrast-enhanced images 
 3 = confidence is greatly improved with contrast-enhanced images 
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Table 3 
Efficacy Variables and Method of Measurement 
Efficacy Variable 
Method of Measurement 
Diagnostic confidence 
for the evaluation of 
LVEF 
Diagnostic confidence for the evaluation of LVEF (LVEF will be calculated using the 
formula: (end diastole volume – end systole volume)/end diastole volume) will be scored 
for non-contrast and OPTISON-enhanced echocardiographic acquisitions separately using 
the following scale: 
 0 = no confidence 
 1 = low confidence 
 2 = moderate confidence 
 3 = high confidence 
And the change in diagnostic confidence will be derived by comparing non-contrast and 
OPTISON-enhanced diagnostic confidence scores as follows (contrast-enhanced minus 
unenhanced): 
≤-1 = confidence is lower with contrast-enhanced images 
 0 = confidence is the same between unenhanced and contrast-enhanced images 
 1 = confidence is slightly improved with contrast-enhanced images 
 2 = confidence is moderately improved with contrast-enhanced images 
 3 = confidence is greatly improved with contrast-enhanced images 
 
The suitable dosage of OPTISON for CE-ECHO in pediatric patients will be primarily 
determined by the improvement in the number of segments adequately visualized over non-
contrast images, supplemented by LVO peak contrast intensity assessed as medium or better, 
peak LV contrast filling to around 67% or better, and duration of contrast enhancement to ≥1.5 
minutes.  Image quality and diagnostic confidence, as well as overall safety of OPTISON in the 
pediatric population, will also be taken into consideration for the dose selection. 
10.2.2 
Image Acquisition 
For each echocardiographic imaging, standard apical 4-chamber and 2-chamber views will be 
obtained at baseline using tissue harmonic imaging, thereafter with contrast using a contrast 
imaging mode.  Additional views may be obtained either prior to or after contrast 
administration under clinician’s discretion.   
For details, please refer to the Imaging Manual provided as a separate document. 
10.2.3 
Comparator or Reference Imaging 
Non-contrast harmonic images will be used as comparator in this study. 
10.2.4 
Image Interpretation  
All echocardiographic images will be evaluated by 3 independent blinded readers for 
characteristics of LV EBD, LVO, diagnostic confidence for the assessment of LV EBD and 
wall motion, and for the evaluation of LVEF.  The BIE process is described in detail in the 
Independent Review Charter. 
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10.3 
Safety Assessments 
The investigator(s) and the sponsor will review the safety data.  The following safety data will 
be collected and evaluated (see Table 2): 
• 
Vital signs: systolic/diastolic blood pressure, heart rate, respiratory rate, and continuous 
pulse oximetry. 
• 
Continuous ECG monitoring. 
• 
12-lead ECG recording. 
• 
Physical examination. 
• 
Post-treatment events (treatment-emergent AEs [TEAEs] and SAEs). 
Pre-specified normal limits for vital signs and ECG intervals are provided in Section 12.4.   
10.3.1 
Vital Signs 
Vital signs will be measured at the various pre- and post-treatment time point ranges described 
in Table 2.  Vital sign parameters include measurements of heart rate, respiratory rate, systolic 
and diastolic blood pressures, and pulse oximetry.  Before vital signs are measured, subject 
should be resting for at least 5 minutes (if possible).  The same position will be used each time 
vital signs are measured for a given subject and blood pressure will be measured from the arm 
contra-lateral to the site of OPTISON administration whenever possible. 
Any finding that is classified by the investigator as a clinically significant (i.e., that resulted in 
a change in subject management) worsening compared to a previous finding will be considered 
an AE or TEAE as applicable and will be documented on the subject’s CRF, and followed until 
the outcome is known. 
10.3.2 
Electrocardiograms 
Continuous ECG monitoring will begin from time of first OPTISON injection and continue 
until 30 minutes after the last dose of OPTISON.  12-lead ECGs will be recorded at baseline 
prior to contrast administration and 10 minutes and 30 minutes after the last OPTISON 
administration.  
All ECG recordings will be read at the investigational site.  Any finding that is classified by the 
investigator as a clinically significant worsening compared to a previous finding will be 
considered an AE or TEAE as applicable and will be documented on the subject’s CRF and 
followed until the outcome is known. 
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10.3.3 
Prior and Concomitant Medication 
Prior and concomitant medication will be recorded during the study procedures and at follow-
up appointments throughout the study period. 
10.3.4 
Physical Examination 
A licensed physician or qualified designee will conduct the physical examinations at the 
baseline visit and 1 hour after the last OPTISON administration.  The same individual should 
conduct the physical examination at both required time points.  The physical examination will 
include recording an assessment for the presence of abnormalities of the following: general 
appearance, respiratory and cardiovascular system.  Neurological examination will include 
level of consciousness, motor function, and sensory function. 
Any finding that is classified by the investigator as a clinically significant worsening compared 
to a previous finding will be considered an AE or TEAE as applicable and will be documented 
on the subject’s CRF, and followed until the outcome is known. 
10.3.5 
Adverse Events 
The AE profile of OPTISON has not yet been established in pediatric patients.  Study 
personnel must remain vigilant for the occurrence of AEs, particularly those that may be life 
threatening.  Personnel who are trained in the management of acute anaphylaxis and other 
emergencies and who have access to appropriate clinical supplies must be immediately 
available for at least 30 minutes to observe for possible anaphylactoid reactions after dosing.  
Treatment of SAEs should be primarily supportive of vital functions. 
An AE is defined as any untoward medical occurrence in a patient or clinical investigation 
subject which does not necessarily have to have a causal relationship with the study treatment.  
AEs will be asked for from time of informed consent through 72 ±8 hours after the last 
administration of contrast agent.   
A TEAE is any unfavorable and unintended sign, symptom, or disease temporally associated 
with the use of OPTISON, whether or not considered related to that product.  Only 
symptoms/signs that begin or worsen in severity and/or frequency after OPTISON 
administration/use will be recorded as TEAEs in the CRF.   
The subjects will be closely observed and questioned for any kind of AE during the study 
procedures and at follow-up appointments throughout the study period with non-leading 
questioning (e.g., “How do you feel?”).  The subjects will be instructed to immediately report 
any symptoms and signs to the study staff (i.e., between formal observations). 
See section 10.3.8 for documentation of SAEs in the CRF. 
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10.3.6 
Serious Adverse Events (SAEs) 
An SAE is defined as any untoward medical occurrence that: 
a) Results in death. 
b) Is life threatening. 
• The term ‘life-threatening’ in the definition of ‘serious’ refers to an event in which the 
subject was at risk of death at the time of the event.  It does not refer to an event, which 
hypothetically might have caused death, if it were more severe. 
c) Requires in-patient hospitalization or prolongation of existing hospitalization. 
• In general, hospitalization signifies that the subject has been detained (usually involving 
at least an overnight stay) at the hospital or emergency ward for observation and/or 
treatment that would not have been appropriate in the physician’s office or outpatient 
setting.  Complications that occur during hospitalization are AEs.  If a complication 
prolongs hospitalization or fulfills any other serious criteria, the event is serious.  When 
in doubt as to whether “hospitalization” occurred or was necessary, the AE should be 
considered serious. 
• Hospitalization for elective treatment of a pre-existing condition that did not worsen 
from baseline is not considered an AE. 
d) Results in persistent or significant disability or incapacity. 
• The term disability means a substantial disruption of a person’s ability to conduct 
normal life functions. 
• This definition is not intended to include experiences of relatively minor medical 
significance such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, 
and accidental trauma (e.g., sprained ankle) which may interfere with or prevent 
everyday life functions but do not constitute a substantial disruption. 
e) Is a congenital anomaly or birth defect. 
f) Is another important medical event. 
• Medical or scientific judgment should be exercised in deciding whether SAE reporting 
is appropriate in other situations such as important medical events that may not be 
immediately life-threatening or result in death or hospitalization but may jeopardize the 
subject or may require medical or surgical intervention to prevent one of the other 
outcomes listed in the above definition. These events should usually be considered 
serious. 
• Examples of such events include invasive or malignant cancers, intensive treatment in 
an emergency room or at home for allergic bronchospasm, blood dyscrasias or 
convulsions that do not result in hospitalization, or development of drug dependency or 
drug abuse. 
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Subjects (or, when appropriate, a caregiver, surrogate, or the subject’s legally authorized 
representative) will report AEs to the investigator and/or qualified designee. 
The investigator and any qualified designees are responsible for detecting, documenting, and 
recording events that meet the definition of an AE or SAE and remain responsible for 
following up AEs that are serious, considered related to the IMP or study procedures, or that 
cause the subject to discontinue the study. 
10.3.7 
Other Significant Adverse Events 
Any events that lead to an intervention (including premature discontinuation of OPTISON, 
dose reduction or significant additional concomitant therapy), other than those reported as 
SAEs, will be reported and evaluated as other significant AEs. 
10.3.8 
Adverse Event and Serious Adverse Event Reporting 
All AEs should be recorded using acceptable diagnoses, if possible.  If an AE has already been 
reported it is not necessary to report each individual sign and symptom of that AE as a separate 
AE.  For example, if myocardial infarction is reported as an AE, there is no need to report 
elevated creatine phosphokinase and abnormal ECG, or other related signs, symptoms, or 
laboratory values as separate AEs.  However, if both occurred in isolation and myocardial 
infarction was not diagnosed, then each event would be reported as an AE. 
The intensity of all AEs will be graded as mild, moderate, or severe using the following 
definitions: 
Mild: 
Tolerable. 
Moderate: 
Interferes with normal activity. 
Severe: 
Incapacitating (causes inability to perform usual activity or work). 
The investigator will be instructed to closely monitor each subject who experiences an AE 
(whether ascribed to the IMP or not) until the outcome of the AE has been determined. 
Both the investigator and GE Healthcare will perform a causality assessment on any AE, to 
assess whether or not there is a reasonable possibility (evidence to suggest) that the IMP 
caused the event.   
SAEs will be recorded in the CRF and on the SAE reporting form if they occurred as follows: 
• 
After a subject first signed informed consent and throughout the subject’s follow-up 
period*, whether or not considered related to the OPTISON, and 
• 
After the subject’s follow-up period, and for which a causal relationship to the 
OPTISON is a reasonable possibility (evidence to suggest) that the OPTISON caused 
the event.  Causality evaluation for any AE shall be documented. 
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(*Follow-up period is defined as the protocol-stipulated period of 72±8 hours after 
administration of the IMP or, for subjects prematurely withdrawn from a study, the duration of 
a subject’s participation.) 
All serious and non-serious AEs must be followed for a final outcome until the end of the 
follow-up period.  An outcome of “unknown” is not considered to be an acceptable final 
outcome.  An outcome of “not yet resolved” is an acceptable final outcome for non-serious 
AEs at the end of a subject’s participation in a study, and for SAEs at database lock. 
The sponsor will report all SAEs to local health authorities, IECs/IRBs and investigators as 
required by local regulations and sponsor standard operating procedures (SOPs).   
In this study, pediatric patients with known or suspected heart disease may be enrolled.  
Therefore, it is anticipated that echocardiography (with or without OPTISON) may detect 
abnormalities that clearly must have been pre-existing, and that some of these may result in 
new/prolonged hospitalization, surgery, etc.  In such cases, the newly detected pre-existing 
abnormality need not be reported as an AE or SAE.  
Study centers are instructed to report all SAEs to the sponsor within 24 hours.   
Notification of SAEs (using the standard SAE form provided by the sponsor) must be sent by 
email to the attention of “Covance DDSS” at SAEIntake@Covance.com immediately (within 
24 hours) of becoming aware of an SAE.  Covance will forward the SAE form to GE 
Healthcare within 24 hours to GPV.DrugSafety@GE.com 
For any protocol or safety-related questions please contact the Medical Director: 
 
 
 
GE Healthcare Life Sciences 
Pollards Wood, Nightingales Lane 
Chalfont St Giles, Bucks HP8 4SP 
United Kingdom 
T: 
 
 
10.3.9 
Urgent Safety Measures 
In accordance with the principles of GCP as laid out in ICH E6, the investigator(s) has/have 
primary responsibility for assuring subject safety throughout the performance of study 
procedures.  An urgent safety measure is defined as any measure which an investigator may 
need to implement which is a deviation from, or a change in, the protocol to eliminate an 
immediate hazard(s) to trial subjects without prior IEC/IRB approval/favorable opinion. 
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The investigator may take appropriate urgent safety measures to protect the subjects of a 
clinical trial against any immediate hazards to their health or safety.  However, the investigator 
must inform the sponsor within 24 hours of having taken such measures. 
The sponsor in turn shall immediately, and in any event no later than 3 days from the date the 
measures are taken, give written notice to the licensing authority and the relevant IEC/IRB of 
the measures taken and the circumstances giving rise to those measures. 
All urgent safety measures must be reported to the Medical Director using the contact numbers 
listed in Section 10.3.8 within 24 hours of having to take such a measure(s).  Such reports can 
be initiated by telephone but must be officially documented by the investigator (by email or 
fax) and must include details of what measures were taken and the circumstances giving rise to 
those measures. 
10.3.10 
Pregnancy Reporting 
This process is aimed at ensuring the appropriate monitoring of the potential risk related to 
IMP exposure of pregnant women and/or fetuses regarding congenital abnormalities or birth 
defects in their offspring.  It also ensures compliance with applicable international and local 
regulations. 
The requirements are applicable to all subjects following exposure to IMP. 
Female trial subjects: The trial subject must be advised by the investigator to inform him/her 
immediately if she suspects she may be pregnant and believes that conception occurred within 
30 days after administration of OPTISON. 
When a female trial subject reports a pregnancy (post-IMP administration) to the investigator, a 
pregnancy test should be arranged for the trial subject by the investigator within 7 days of the 
pregnancy being reported. 
Notification of pregnancy (using the standard pregnancy form provided by the sponsor) must 
be sent by email to the attention of “Covance DDSS” at SAEIntake@Covance.com 
immediately (within 24 hours) of obtaining a positive pregnancy test.  Covance will forward 
the pregnancy form to GE Healthcare within 24 hours to GPV.DrugSafety@GE.com.  The 
investigator should include an estimated date of conception when communicating with the 
sponsor/CRO. 
A reported pregnancy will be followed to determine the outcome.  The investigator will collect 
follow-up information on the participant and the neonate and the information will be forwarded 
to the sponsor.  Generally, follow-up will not be required for longer than 6 to 8 weeks beyond 
the estimated delivery date.  Any termination of pregnancy will be reported, regardless of fetal 
status (presence or absence of anomalies) or indication for the procedure. 
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10.4 
Other Variables 
Other study relevant information, such as demographic data, medical and surgical history, and 
prior and concomitant medication will be collected at screening and during the study period as 
appropriate in the CRFs. 
10.5 
Appropriateness of Measurements 
All assessments and measurements are appropriate and generally regarded as standard medical 
practice.   
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11 
DATA HANDLING AND QUALITY ASSURANCE 
11.1 
Completing and Signing Case Report Forms 
For electronic CRFs, data will be entered by trained site personnel with reasons given for any 
missing data.  Any errors should be corrected within the electronic system.  The audit trail will 
record all changes made, the date and time of the correction, and the person correcting the 
error.  The appropriate electronic signature will be provided. 
Any data recorded directly in the CRF, for which no other written or electronic record will be 
maintained in the subject’s medical record, will be considered source data and should be signed 
by the investigator(s) (e.g., results of physical examinations, vital signs, or the IMP 
administration procedure). 
11.2 
Clinical Data Management 
The sponsor or Contract Research Organization (CRO) will be responsible for the processing 
and quality control of the data.  Data management will be carried out by the sponsor or CRO.  
The handling of data, including data quality control, will comply with all applicable regulatory 
guidelines. 
11.3 
Data Quality Assurance 
All subject data relating to the study will be recorded on electronic CRFs unless transmitted to 
the sponsor or designee electronically (e.g., laboratory data).  The investigator is responsible 
for verifying that data entries are accurate and correct by electronically signing the CRF. 
The investigator must maintain accurate documentation (source data) that supports the 
information entered in the CRF. 
The investigator must permit study-related monitoring, audits, IRB/IEC review, and regulatory 
agency inspections and provide direct access to source data documents. 
Monitoring details describing strategy (e.g., risk-based initiatives in operations and quality 
such as Risk Management and Mitigation Strategies and Analytical Risk-Based Monitoring), 
methods, responsibilities and requirements, including handling of noncompliance issues and 
monitoring techniques (central, remote, or on-site monitoring) are provided in the Monitoring 
Plan. 
The sponsor or designee is responsible for the data management of this study including quality 
checking of the data. 
The sponsor assumes accountability for actions delegated to other individuals (e.g., CROs). 
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Study monitors will perform ongoing source data verification to confirm that data entered into 
the CRF by authorized site personnel are accurate, complete, and verifiable from source 
documents; that the safety and rights of subjects are being protected; and that the study is being 
conducted in accordance with the currently approved protocol and any other study agreements, 
ICH GCP, and all applicable regulatory requirements. 
11.4 
Record Retention 
All study documentation at the investigator site and sponsor site will be archived in accordance 
with ICH E6-GCP and the sponsor’s quality standards and SOPs. 
All study documentation at the Investigator site and sponsor site will be retained for a 
minimum of 15 years following completion or discontinuation of the study, unless notified 
otherwise by the sponsor or a longer period is required by local legislation.  The Investigator 
must request written agreement from the sponsor before destruction of archived study 
documentation.  No records may be transferred to another location or party without written 
notification to the sponsor. 
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12 
STATISTICAL METHODS AND PLANNED ANALYSIS 
12.1 
General Statistical Considerations 
Tabulations of summary statistics, graphical presentations, and statistical analyses will be 
performed using SAS software.  All summary tables and data listings will be separated by 
dose levels.  The last pre-administration observation will be used as the baseline value for 
calculating post-administration changes from baseline.  All data obtained on the CRF and 
entered into the database will be provided in separate data listings showing individual subject 
values.  The planning and reporting of statistical analysis will be carried out as described in the 
sponsor’s SOPs governing clinical studies. 
The relevant variables will be presented using the appropriate descriptive statistics, categorical 
variables will be summarized by counts and percentages and continuous variables by means, 
standard deviations, medians, maxima, minima and sample size. No specific clinical data will 
be imputed beyond the point of discontinuation for subjects who withdraw prematurely from 
the study. Demographic variables and other baseline patient characteristics will be summarized 
descriptively by dose level and overall, independent of IMP assignment.  Demographic 
variables will include age, weight, height, gender, body mass index and race/ethnicity. 
12.2 
Subject Characteristics 
A table will be provided with the following information: 
• 
Number of subjects enrolled. 
• 
Number of subjects included in the intent-to-treat (ITT) analysis. 
• 
Number of subjects included in the per-protocol analysis.  
• 
Number of subjects included in the safety analysis. 
• 
Number of subjects withdrawn from the study and the reason for withdrawal. 
Demographic information (age, height, weight, and body mass index) will be summarized 
using descriptive statistics.  Gender and race/ethnicity will be summarized by counts and 
percentages. 
Medical/surgical history data will be summarized by counts and percentages.   
12.2.1 
Determination of Safety and Efficacy Population 
The study analysis populations are defined as follows: 
Safety population 
All subjects enrolled in the study who receive administration of OPTISON. 
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ITT population 
All subjects enrolled in the study who have available non-contrast harmonic images and 
OPTISON-enhanced echocardiographic images, irrespective of the quality of CE-ECHO.  
Per-protocol population 
All subjects whose non-contrast harmonic images are available and whose OPTISON 
echocardiography images have adequate quality (sufficient contrast enhancement to visualize 
EBD).   
12.3 
Efficacy Analysis 
Since this is a dose-finding study, no hypotheses are being tested.  All efficacy results will be 
summarized for the ITT and per-protocol populations. 
12.3.1 
Primary Efficacy Analysis 
Visualization of the 12 segments of the left ventricle wall in standard apical 4-chamber and 2-
chamber views will be measured by the qualitative EBD visualization scale.  Results will be 
summarized for non-contrast and OPTISON dose levels.  Additionally, the mean number of 
segments visualized will be derived from the EBD score and summarized (segments rated as 
“fair visualization” or “good/optimal visualization” will be considered visualized). 
12.3.2 
Secondary Efficacy Analysis 
The following secondary endpoints will be summarized: 
• 
The LV peak contrast intensity, determined as none, low, medium, high or blooming, 
will be summarized as a categorical variable by OPTISON dose level.  
• 
The peak LV contrast filling, determined as none, faint, intermediate or full, will be 
summarized as a categorical variable by OPTISON dose level.  
• 
The contrast enhancement duration in the LV chamber determined from the time 
contrast appears in the LV to the time it almost dissipates from the chamber as 
determined by investigators, will be summarized with descriptive statistics by 
OPTISON dose level. 
• 
Image quality, categorized as excellent/good, adequate or poor will be summarized as a 
categorical variable for OPTISON dose levels.  
• 
Diagnostic usefulness, evaluated as non-diagnostic or diagnostic, will be summarized as 
a categorical variable for non-contrast and OPTISON dose levels.   
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• 
The diagnostic confidence of the assessment of LV EBD and wall motion, assessed 
using a 5-point scale in comparison to non-contrast echocardiographic images 
(confidence was lower with contrast-enhanced images, same between unenhanced and 
contrast-enhanced images, slightly improved with contrast-enhanced images, 
moderately improved, greatly improved), will be summarized as a categorical variable 
by OPTISON dose level. 
• 
The diagnostic confidence of the evaluation of LVEF, assessed using a 5-point scale in 
comparison to non-contrast echocardiographic images (confidence was lower with 
contrast-enhanced images, same between unenhanced and contrast-enhanced images, 
slightly improved with contrast-enhanced images, moderately improved, greatly 
improved), will be summarized as a categorical variable by OPTISON dose level. 
• 
LV function assessed by LVEFs, calculated using the formula: (end diastole volume – 
end systole volume)/end diastole volume for each ventricle, will be summarized with 
descriptive statistics by OPTISON dose levels and compared with non-contrast images. 
A composite assessment of dose suitability will be made at the subject level and results will be 
tabulated by OPTISON dose level. A dose will be considered suitable of it meets the following 
criteria: 
(1) 
There is an increase in the number of segments adequately visualized over non-
contrast images  
(2) 
LVO peak contrast intensity assessed as medium or better 
(3) 
Peak LV contrast filling to around 67% or better 
(4) 
Duration of contrast enhancement ≥1.5 minutes 
12.3.3 
Handling of Uninterpretable Images 
In the ITT analysis of the primary endpoint, the un-interpretable images will be categorized as 
none for contrast intensity and 0 visualization of EBD.  In the secondary analyses, the un-
interpretable images will be considered as 0 enhancement duration, poor for image quality, and 
non-diagnostic for diagnostic usefulness.  Additionally, the number of un-interpretable images 
will be summarized by non-contrast and dose level.  Uninterpretable images will be excluded 
from the per-protocol population analysis. 
12.3.4 
Reader Differences 
Summaries of the image readings by reader will be presented. 
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12.4 
Safety Analysis 
The safety of the intravenous administration of OPTISON in the pediatric population will be 
measured by the incidence of AEs, vital signs, SaO2 by pulse oximetry, physical examination 
and ECG results.   
12.4.1 
Vital Signs 
Vital signs will be measured at the various pre- and post-treatment time point ranges described 
in Table 2.  Vital sign parameters include measurements of systolic and diastolic blood 
pressure, heart rate, respiratory rate, and pulse oximetry.  Normal ranges for the observed 
values of heart rate, respiratory rate, systolic blood pressure and weight by age group are 
defined in Table 5.   
The presentation of vital signs focuses on the descriptive summaries at baseline, at 10 minutes, 
and at 1 hour post-OPTISON administration, and the change from baseline to the 10-minute 
and 1-hour post-OPTISON assessments.  All subjects in the safety population who have a 
baseline and a post-baseline assessment of vital signs will be included in the presentation of the 
results. 
In addition to the change from baseline results, the shifts from baseline to the post baseline 
visit will be summarized in tabular form for heart rate and systolic blood pressure.  Subjects 
will be classified at baseline and the post-baseline visit in one of the following categories based 
on a comparison of the vital sign result to the reference range for that vital sign parameter: 
below normal, normal, or above normal.  A shift table will be constructed in which each 
subject is cross-classified based upon the reference range categories.  The number and 
percentage of subjects in each shift table cell will be displayed.  
12.4.2 
Electrocardiograms 
A 12-lead ECG will be measured at the various pre- and post-treatment time point ranges 
described in Table 2.  The ECGs will be characterized as normal or abnormal, and, if abnormal, 
whether the abnormality is clinically significant.  These overall readings will be summarized.  
Descriptive statistics will be used to describe the observed values and change from baseline for 
ECG intervals (heart rate, PR, RR, QRS, QT, QTcB [Bazett], QTcF [Fridericia]).  Summaries 
will be presented by dose level, at each post-baseline time point.  QTcB and QTcF will be 
defined as follows, 
QTcB = QT/RR 
QTcF = QT/3RR  
Normal ranges for the observed values and change from baseline by age group are defined in 
Table 6 and Table 7.  
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In addition to the observed values and change from baseline results, the shifts from baseline to 
the post-baseline visit will be summarized in tabular form for each ECG parameter.  Subjects 
will be classified at baseline and the post-baseline visit in one of the following categories based 
on a comparison of the ECG result to the reference range for that ECG parameter: below 
normal, normal, or above normal.  A shift table will be constructed in which each subject is 
cross-classified based upon the reference range categories.  The number and percentage of 
subjects in each shift table cell will be displayed.  In addition, observed values and changes in 
QTc consistent with the [ICH E14] guidance will be specified in the statistical analysis plan, 
and a categorical analysis of study QTc values based on these pre-specified values will be 
performed.  The results will be summarized in a table. 
12.4.3 
Prior and Concomitant Medications 
Prior and concomitant medications will be recorded and coded using the Anatomical-
Therapeutic-Chemical (ATC) classification text from the World Health Organization (WHO) 
Drug Dictionary.  Medications will be presented by ATC Class 1 and preferred term. 
12.4.4 
Physical Examination 
Physical examination findings at baseline and after OPTISON administration will be 
summarized. 
12.4.5 
Adverse Events 
An AE will be considered as treatment-emergent if it starts or worsens after OPTISON 
administration.  Any AE with an onset on the day of IMP administration for which the time of 
onset is missing will be assumed to be treatment-emergent. 
All summaries of AEs will be based on TEAEs and will be prepared by dose level and overall.  
AEs will be mapped to System Organ Class (SOC) and preferred terms using the Medical 
Dictionary for Regulatory Activities (MedDRA; current version or the immediately preceding 
version).  The number and percentage of subjects experiencing AEs will be summarized by 
SOC and preferred term.  Summaries by maximum severity, and summaries and tabular listings 
by relationship to study treatment will also be provided.  SAEs and AEs leading to 
discontinuation of study treatment will be presented by SOC and preferred term.   
The sponsor will discuss other significant AEs, defined as laboratory abnormalities that qualify 
as AEs (other than those meeting the definition for serious) and any events that led to an 
intervention (including premature discontinuation from the study, dose reduction, or significant 
additional concomitant therapy), in addition to those reported as SAEs. 
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12.5 
Sample Size Determination 
Although this is a dose-finding study and no hypotheses are being tested, it was thought useful 
to estimate sample size based on one of the study endpoints as a guide.  Since there is no 
historical literature about LV EBD results for OPTISON, the power estimation is based on the 
endpoint of contrast enhancement duration.  
A paired t-test was used for this power analysis with a one-sided significance level of 0.025.  
The null hypothesis is that the mean value of (contrast enhancement duration for the high dose 
– contrast enhancement duration for the low dose within the same patient ≤0 minutes), and the 
alternative hypothesis is that the mean value of (contrast enhancement duration for the high 
dose – contrast enhancement duration for the low dose within the same patient) >0 minutes.  
Based on the results from [Cohen et al. 1998], we assume that the estimated mean value of 
(contrast enhancement duration for the high dose – contrast enhancement duration for the low 
dose within the same patient) equals 0.7 minutes, with a standard deviation of 1.5 minutes. 
Table 4 displays the power calculation for the hypothesis testing with different sample sizes.   
Table 4 
Power Calculation 
Total sample size  
Power 
30 
70% 
40 
82% 
50 
90% 
60 
94% 
70 
97% 
80 
98% 
 
Therefore, the sample size for this clinical investigation will be approximately 50 subjects, 
which will provide at least 90% power to detect an efficacy difference of 0.7 minutes between 
the high and low doses, with the greater duration for the high dose.  In addition, a sample size 
of 50 is considered to be adequate to address the other efficacy endpoints (LV EBD, LVO 
characteristics, wall motion assessment, and diagnostic confidence for the evaluation of LVEF) 
and OPTISON’s safety profile following intravenous administration in pediatric subjects.  The 
data will support dosing information in children. 
12.6 
Significance Level 
Since no hypotheses are being tested in this study, this section is not applicable. 
12.7 
Procedures for Missing, Unused and Spurious Data 
Missing values will not be substituted by estimated values, but treated as missing in the 
statistical evaluation.  All data from all subjects dosed in the study will be included in all 
listings, plots, summary tables, and statistical analyses when appropriate. 
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12.8 
Rules for Excluding Subjects from Analysis 
All dosed subjects will be included in the analyses unless otherwise specified.  The sponsor 
will make any decisions regarding whether any subjects or any individual values belonging to a 
subject will be excluded from the evaluations when the protocol violation is considered to have 
a negative impact on the scientific aspects and interpretation of the study results.  If the subject 
has received any IMP, all available safety data will be used.  The reason(s) for any exclusion 
will be described in the report. 
12.9 
Procedures for Reporting Deviations from Original Statistical Plan 
Any deviations from the statistical analysis outlined in this protocol will be described, and 
reasons for the deviations listed, in the final Clinical Study Report. 
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13 
SPECIAL REQUIREMENTS AND PROCEDURES 
13.1 
Regulatory and Ethical Considerations 
This study will be conducted in accordance with the protocol and with the following: 
• 
Consensus ethical principles derived from international guidelines including the 
Declaration of Helsinki and Council for International Organizations of Medical 
Sciences (CIOMS) International Ethical Guidelines 
• 
Applicable ICH GCP Guidelines 
• 
Applicable laws and regulations 
Before starting this study, the protocol (authorized by the sponsor), Informed Consent Form, 
Investigator Brochure, and other relevant documents (e.g., advertisements) must be submitted 
to an IEC/IRB for evaluation and reviewed and approved by the IEC/IRB.  The protocol will 
also be submitted to regulatory bodies/local health authorities in accordance with local 
regulations as required.  The study will not start before the IEC/IRB gives written approval or a 
favorable opinion in accordance with ICH E6-GCP and all applicable regulatory bodies/local 
health authorities give approval or a favorable opinion as required. 
No changes from the final approved (authorized) protocol will be initiated without the 
IEC’s/IRB’s prior written approval or favorable opinion of a written amendment, except when 
necessary to eliminate immediate hazards to the subjects or when the change involves only 
logistics or administration.  The sponsor will authorize and the principal investigator(s) will 
sign the protocol amendment prior to submission to the IEC/IRB.  Protocol amendments should 
be submitted to the IEC/IRB without delay.  Any significant deviation from the protocol when 
no approved amendment exists will be regarded as a protocol violation, and will be addressed 
as such during the reporting of the study. 
The investigator will be responsible for the following: 
• 
Providing written summaries of the status of the study to the IEC/IRB annually or more 
frequently in accordance with the requirements, policies, and procedures established by 
the IEC/IRB 
• 
Notifying the IEC/IRB of SAEs or other significant safety findings as required by 
IEC/IRB procedures 
• 
Providing oversight of the conduct of the study at the site and adherence to 
requirements of 21 CFR, ICH guidelines, the IEC/IRB, European regulation 536/2014 
for clinical studies (if applicable), and all other applicable local regulations. 
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13.2 
Investigator’s Responsibilities 
13.2.1 
Overall Responsibilities 
The investigator(s) is/are responsible for conducting the study in full accordance with the 
Protocol and the Declaration of Helsinki, the Good Clinical Practice: Consolidated Guideline, 
approved by the ICH, and any applicable national and local laws and regulations.  Information 
regarding any investigational centers participating in this study that cannot comply with these 
standards will be documented. 
13.2.2 
Parental Permission/Subject Informed Assent 
Written and oral information about the study in an understandable language will be given to the 
subject and subject’s parent/legal guardian.  Each subject’s willingness to participate in the 
study will be documented in a signed and dated informed assent form and parental informed 
permission form before any procedures or assessments are performed and after the aims, 
methods, anticipated benefits, potential hazards, and insurance arrangements in force are 
explained.  Age-appropriate pediatric assent will be obtained where required.   
It will also be explained to the subject and the subject’s parent/ legal guardian that they are free 
to refuse to participate in the study and free to withdraw from the study at any time without 
prejudice to future treatment.   
The informed consent/assent process will be documented in the subject's medical record and 
the investigator will sign, date and time the informed consent form after the subject’s 
parent/legal guardian have signed, dated and recorded the time.   
The investigator(s) will keep the original consent forms and copies will be given to the subjects 
or the subject’s parent/legal guardian. 
13.2.3 
Direct Access to Source Data/Documents 
Source documents provide evidence for the existence of the subject and substantiate the 
integrity of the data collected.  Source documents are filed at the investigator’s site. 
Data reported on the CRF or entered in the electronic CRF that are transcribed from source 
documents must be consistent with the source documents or the discrepancies must be 
explained.  The investigator may need to request previous medical records or transfer records, 
depending on the study.  Also, current medical records must be available. 
The monitor(s), auditor(s), authorized personnel of the sponsor, health authority inspector(s) or 
their agents, and authorized members of IECs/IRBs will be given direct access to source data 
and documentation (e.g., medical charts/records, laboratory results, printouts, videotapes, etc.) 
for source data verification, provided that subject confidentiality is maintained in accordance 
with local requirements. 
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13.2.4 
Confidentiality Regarding Study Subjects 
The investigator(s) must assure that the privacy of the subjects, including their personal 
identity and all other personal medical information, will be maintained at all times.  In CRFs 
and other documents or image material (including materials from all examinations, 
e.g., ultrasound examinations) submitted to the sponsor, subjects will not be identified by their 
names, but by an identification code (e.g., initials and study subject number). 
Personal medical information may be scrutinized for the purpose of verifying data recorded in 
the CRF.  This may be done by the monitor(s), properly authorized persons on behalf of the 
sponsor, the quality assurance unit, or regulatory authorities.  Personal medical information 
will always be treated as confidential. 
13.2.5 
Data Protection 
Subjects will be assigned a unique identifier by the sponsor.  Any subject records or datasets 
that are transferred to the sponsor will contain the identifier only; subject names or any 
information which would make the subject identifiable will not be transferred. 
The subject and subject’s parent/legal guardian must be informed that his/her personal study-
related data will be used by the sponsor in accordance with local data protection law.  The level 
of disclosure must also be explained to the subject and subject’s parent/legal guardian who will 
be required to give consent for their data to be used as described in the informed consent. 
The subject and subject’s parent/legal guardian must be informed that his/her medical records 
may be examined by Clinical Quality Assurance auditors or other authorized personnel 
appointed by the sponsor, by appropriate IEC/IRB members, and by inspectors from regulatory 
authorities. 
13.3 
Protocol Deviations 
Any deviation from the protocol when no approved amendment exists must be documented as 
a protocol deviation and reported according to local requirements.  If appropriate, corrective 
and preventative action must be implemented to avoid repetition.  Protocol deviations and any 
potential impact on the study results will be discussed during the reporting of the study. 
Waivers or protocol exceptions will not be granted prospectively by the sponsor under any 
circumstances. 
13.4 
Study Monitoring 
Study monitoring will be performed in accordance with ICH E6-GCP, the sponsor SOPs, the 
protocol, and applicable local regulations. 
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13.5 
Audit and Inspection 
According to ICH E6-GCP, the sponsor or regulatory authorities may audit the investigational 
site.  The sponsor’s Quality Assurance Unit, independent of the Clinical Research and 
Development Department, is responsible for auditing the study. 
The investigator(s) must accept that regulatory authorities may conduct an inspection to verify 
compliance of the study with GCP. 
13.6 
Financial Disclosure 
According to 21 CFR, Part 54, the sponsor is required to completely and accurately disclose or 
certify information concerning the financial interests of a clinical investigator (or investigating 
institution) who is not a full-time or part-time employee to the FDA.  Therefore, the 
investigator(s) (or investigating institution) must either provide the sponsor with sufficient, 
accurate financial certification that none of the following financial arrangements (further 
defined in 21CFR Part 54.2) exist with the sponsor or fully disclose the nature of the 
arrangement.  This financial disclosure also applies to any financial arrangements that exist 
between the sponsor and the investigator’s spouse(s) or dependent children: 
• 
Compensation for participation in the study is affected by the outcome of the study. 
• 
Significant equity (greater than $50,000) interest in the sponsor’s company. 
• 
Proprietary interest in the tested product. 
• 
Significant payments of other sorts, exceeding a monetary value of $25,000. 
13.7 
Insurance 
This study is covered under the sponsor’s Liability Insurance Policy (under General Electric 
Insurance Company and/or a company designated by the study sponsor).  A Certificate of 
Insurance and/or an information leaflet containing essential information about the insurance 
coverage can be provided upon request. 
13.8 
Publication Policy 
The investigator and/or Institution shall have the right to publish the results of their work 
conducted under this protocol, subject to providing the sponsor with the opportunity to review 
the contents of any proposed abstract or publication concerning the work, including any results 
of the study, in advance of publication and if necessary to delay publication for a limited time 
not to exceed 60 days in order to protect the confidentiality or proprietary nature of any 
information contained therein.  The sponsor will make every reasonable effort to consider and 
release each proposed publication within 30 days, or proposed abstract within 15 days, of 
submission. 
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14 
REFERENCES 
[Candido et al. 2003] 
Candido A, Coucelo J, Galvao J, et al.  Contrast echocardiography in segmental analysis and 
intraventricular gradient quantification in hypertrophic cardiomyopathy.  Rev Port Cardiol 
2003;22:789-798 
[CDC Growth Charts] 
2000 CDC Growth Charts for the United States: Methods and Development.  Vital and health 
statistics.  2002;11:246. ( accessed on 13 Jun 2012 at http://www.cdc.gov/growthcharts) 
[Clark and Dittrich. 2000] 
Clark LN, Dittrich HC.  Cardiac imaging using Optison.  Am J Cardiol. 2000;86:14G-18G 
[Cohen et al. 1998] 
Cohen JL, Cheirif J, Segar DS, et al.  Improved left ventricular endocardial border delineation 
and opacification with OPTISON (FS069), a new echocardiographic contrast agent.   
JACC 1998;32:746-752 
[CTFG Guidance 2014] 
Clinical Trial Facilitation Group (CTFG) (2014) Recommendations related to contraception 
and pregnancy testing in clinical trials. 
http://www.hma.eu/fileadmin/dateien/Human_Medicines/01-
About_HMA/Working_Groups/CTFG/2014_09_HMA_CTFG_Contraception.pdf.  Accessed 
08 October 2019. 
[Geleijnse et al. 1997] 
Geleijnse ML, Fioretti PM, Roelandt JR.  Methodology, feasibility, safety and diagnostic 
accuracy of dobutamine stress echocardiography.  J Am Coll Cardiol.  1997;30:595-606 
[ICH E14] 
International conference on harmonisation of technical requirements for registration of 
pharmaceuticals for human use.  The clinical evaluation of QT/QTC interval prolongation and 
proarrhythmic potential for non-antiarrhythmic drugs--E14.  Step 4 version; May, 2005 
[Kornblith et al. 2000] 
Kornblith M, Liang DH, Brown P, et al. Contrast echocardiography is superior to tissue 
harmonics for assessment of left ventricular function in mechanically ventilated patients.  Am 
Heart J.  2000;140:291-296 
[Kutty et al. 2016] 
Kutty S, Xiao Y, Olson J, et al. Safety and Efficacy of Cardiac Ultrasound Contrast in Children 
and Adolescents for Resting and Stress Echocardiography. J Am Soc Echocardiogr. 
2016;29:655-62 
[Lang et al. 2015] 
Lang RM, Badano LP, Mor-Avi V, et al. Recommendations for cardiac chamber quantification 
by echocardiography in adults: an update from the American Society of Echocardiography and 
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the European Association of Cardiovascular Imaging.  Eur Heart J Cardiovasc Imaging. 
2015;16(3):233-70 
[McMahon et al. 2005] 
McMahon CJ, Ayres NA, Bezold LI, et al. Safety and Efficacy of Intravenous Contrast 
Imaging in Pediatric Echocardiography.  Pediatr Cardiol 2005:26:413–417 
[Mulvagh et al. 2000] 
Mulvagh SL, DeMaria AN, Feinstein SB, et al. Contrast echocardiography: current and future 
applications.  J Am Soc Echocardiogr. 2000;13:331-42 
[OPTISON PI] 
GE Healthcare, OPTISON – Package Insert, Currently approved version.  
[Weissman et al. 2000] 
Weissman NJ, Cohen MC, Hack TC, et al. Infusion versus bolus contrast echocardiography: a 
multicenter, open-label, crossover trial.  Am Heart J.  2000;139:399-404 
[Zilberman et al. 2003] 
Zilberman MV, Witt SA, Kimball TR.  Is there a role for intravenous transpulmonary contrast 
imaging in pediatric stress echocardiography?  J Am Soc Echocardiogr. 2003;16:9-14 
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15 
APPENDICES 
15.1 
Information on Investigational and Registered Products 
The currently approved OPTISON package insert or Summary of Product Characteristics will 
be provided to all investigators as a separate document.  The package insert/Summary of 
Product Characteristics provides information on the efficacy and safety of the OPTISON, and 
is used for assessing expectedness of Serious Adverse Drug Reactions, in order to determine 
regulatory reportability.  An unexpected Adverse Drug Reaction is a reaction, for which the 
nature, seriousness, severity or outcome is not consistent with the applicable product 
information presented in the package insert/Summary of Product Characteristics.   
15.2 
Equipment Parameters 
Please refer to the Imaging Manual. 
15.3 
Pediatric Normal Ranges 
Table 5 
Pediatric Vital Signs Normal Ranges 
Age Group 
Respiratory 
Rate 
Heart Rate 
Systolic Blood 
Pressure 
Weight in 
kilos 
Weight in 
pounds 
Newborn 
30 – 50 
120 – 160 
50 – 70 
2 – 3 
4.5 – 7 
Infant (1-12 months) 
20 – 30 
80 – 140 
70 – 100 
4 – 10 
9 – 22 
Infant (1-12 months) 
20 – 30 
80 – 140 
70 – 100 
4 – 10 
9 – 22 
Preschooler (3-5 yrs.) 
20 – 30 
80 – 120 
80 – 110 
14 – 18 
31 – 40 
School Age (6-12 yrs.) 
20 – 30 
70 – 110 
80 – 120 
20 – 42 
41 – 92 
REF: Source: http://www.rnceus.com/psvt/psvtvs.html 
 
Table 6 
Pediatric ECG Normal Ranges 
 
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Table 7 
Mean and Percentiles of ECG Cardiac Measures Among Pubertal Children 
Source: Child and Adolescent Psychiatry and Mental Health 2007;1:11 
 
 
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16 
CLINICAL PROTOCOL AMENDMENT SUMMARIES 
16.1 
Amendment A01 
16.1.1 
Reasons for Amendment 
• 
The requirement for a minimum of 10 subjects to be enrolled in each body weight 
group is removed. 
• 
The requirement to record the vial code in the CRF is removed. 
16.1.2 
Description of Changes 
Where appropriate, the changes documented below are also made in the synopsis. 
Section 6.1, Overall Study Design and Plan, Third Paragraph 
Previously read: 
Each patient will undergo an echocardiographic procedure first without contrast administration 
then following intravenous bolus injection of OPTISON at various doses.  The dose of 
OPTISON for each subject will be determined according to pediatric body weight group and 
2 different dose levels will be tested in each subject.  A minimum of 10 subjects will be 
enrolled in each body weight group.  The details on OPTISON administration are presented in 
Section 8.1 of this protocol. 
Now reads: 
Each patient will undergo an echocardiographic procedure first without contrast administration 
then following intravenous bolus injection of OPTISON at various doses.  The dose of 
OPTISON for each subject will be determined according to pediatric body weight group and 
2 different dose levels will be tested in each subject.  The details on OPTISON administration 
are presented in Section 8.1 of this protocol. 
 
Section 8.6, Treatment Compliance 
Previously read: 
Subjects will receive OPTISON administration under direct supervision of study personnel.  
Each administration volume will be checked and the vial code and volume per administration 
will be recorded in each subject’s CRF. 
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Now reads: 
Subjects will receive OPTISON administration under direct supervision of study personnel.  
Each administration volume will be checked and the volume per administration will be 
recorded in each subject’s CRF. 
 
GE-191-008 Clinical Protocol Amendment A01
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Date of signature: 04-Oct-2022 08:33:15 GMT+0000
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SAP_001.pdf: 
Page 1 of 30 
STATISTICAL ANALYSIS PLAN 
GE HealthCare 
GE-191-008 
Title: 
A Phase 4, Open-Label, Non-randomized, Multicenter Study to Evaluate 
Safety and Efficacy of Intravenous Administration of OPTISONTM for 
Contrast Enhanced Echocardiography in Pediatric Patients 
Date of Creation: 
06Mar2023 
Version: 
1.0 
This Statistical Analysis Plan was prepared by the following personnel: 
Name 
 
Title 
 
Company 
 
Reviewed by: 
Name 
 
Title 
 
Company 
 
Sponsor reviewed by: 
Name 
 
Title 
 
Company 
 
GE-191-008 Statistical Analysis Plan
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ClinicalTrials.gov ID: NCT03740997


 
Page 2 of 30 
 
Table of Contents 
1 
INTRODUCTION ................................................................................................ 5 
2 
STUDY OBJECTIVES, DESIGN AND PROCEDURES ................................... 5 
2.1 
Objectives ................................................................................................. 5 
2.2 
Study Design............................................................................................. 6 
2.3 
Sample Size .............................................................................................. 9 
2.4 
Interim Analysis ..................................................................................... 10 
2.5 
Randomization and Blinding .................................................................. 10 
3 
STUDY ENDPOINTS ........................................................................................ 11 
3.1 
Primary Endpoint .................................................................................... 11 
3.2 
Secondary Endpoints .............................................................................. 11 
4 
ANALYSIS POPULATIONS ............................................................................ 12 
4.1 
Definition for Analysis Populations ....................................................... 12 
4.1.1 
Safety Population .................................................................. 12 
4.1.2 
Full Analysis Set (FAS) Population ..................................... 12 
4.1.3 
Per-Protocol (PP) Population................................................ 12 
5 
ANALYSIS CONVENTIONS ........................................................................... 12 
6 
STATISTICAL ANALYSES ............................................................................. 15 
6.1 
Demographics, Baseline Characteristics, Medical History, and 
Prior/Concomitant Medications .............................................................. 15 
6.1.1 
Demographic and Baseline Characteristics .......................... 15 
6.1.2 
Medical History .................................................................... 16 
6.1.3 
Prior and Concomitant Medications ..................................... 16 
6.2 
Subject Disposition ................................................................................. 16 
6.3 
Study Drug Exposure and Compliance .................................................. 17 
6.4 
Efficacy Analysis .................................................................................... 17 
6.4.1 
General Considerations......................................................... 17 
6.4.2 
Primary Efficacy Analysis .................................................... 17 
6.4.3 
Secondary Efficacy Analyses ............................................... 18 
6.4.4 
Handling of Uninterpretable Images .................................... 20 
6.4.5 
Reader Differences ............................................................... 20 
6.5 
Safety Analysis ....................................................................................... 21 
6.5.1 
General Considerations......................................................... 21 
6.5.2 
Analysis of Adverse Events .................................................. 21 
6.5.2.1 
Treatment-Emergent Adverse Events ................. 21 
6.5.2.2 
SAEs (Including Deaths) and Adverse Events 
Leading to Study Drug Discontinuation ............. 22 
6.5.3 
Analysis of Vital Signs and Weight ..................................... 22 
6.5.3.1 
Variables and Criteria Defining Abnormality .... 22 
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6.5.3.2 
Statistical Methods ............................................. 22 
6.5.4 
Analysis of ECG Parameters ................................................ 23 
6.5.4.1 
Variables and Criteria Defining Abnormality .... 23 
6.5.4.2 
Statistical Models ............................................... 24 
6.5.5 
Analysis of Physical Examination ........................................ 25 
7 
REFERENCES ................................................................................................... 25 
8 
LIST OF TABLES, FIGURES AND DATA LISTINGS THAT ARE TO BE 
PROGRAMMED................................................................................................ 26 
9 
SUMMARY OF CHANGES.............................................................................. 29 
10 
APPENDIX ........................................................................................................ 30 
 
List of Tables 
Table 1 
OPTISON Dosing Table ......................................................................................... 6 
Table 2 
Study Schedule of Events ....................................................................................... 9 
Table 3 
Power Calculation................................................................................................. 10 
Table 4 
Pediatric Vital Signs Normal Ranges ..................................................................... 22 
Table 5 
Mean and Percentiles of ECG Cardiac Measures Among Pre-pubertal Children .. 23 
Table 6 
Mean and Percentiles of ECG Cardiac Measures Among Pubertal Children ........ 24 
Table 7 
Mean and Percentiles of ECG Cardiac Measures Among Post-pubertal Children 24 
 
List of Figures 
Figure 1 Study Diagram ........................................................................................................ 8 
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Abbreviations 
AE 
Adverse event 
ATC 
Anatomical-Therapeutic-Chemical 
BIE 
Blinded image evaluation 
CE-ECHO 
Contrast-enhanced echocardiography 
CHMP 
Committee for Medicinal Products for Human Use 
EBD 
Endocardial border delineation 
ECG 
Electrocardiogram 
eCRF 
Electronic Case Report Form 
ICH 
International Council for Harmonisation 
IMP 
Investigational medicinal product 
ITT 
Intent-to-treat 
LV 
Left ventricular 
LV EBD 
Left ventricular endocardial border delineation 
LVEF 
Left ventricular ejection fraction 
LVO 
Left ventricular opacification 
MedDRA 
Medical Dictionary for Regulatory Activities 
SAE 
Serious adverse event 
SAP 
Statistical Analysis Plan 
SOC 
System organ class 
TEAE 
Treatment-emergent adverse event 
WHO-DD 
World Health Organization Drug-Dictionary 
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1 
INTRODUCTION 
The statistical analysis plan (SAP) defines the methods and analyses that GE HealthCare 
plans to use to analyze the data from Protocol GE-191-008. This SAP is based on the 
final study protocol (Amendment A01 Version 2.0) dated 04 October, 2022. This SAP 
complies with guidance promulgated by the International Conference on Harmonization 
(ICH) and the US Food and Drug Administration (FDA). If the protocol is subsequently 
amended, this SAP may be amended as well. Should the SAP and the protocol be 
inconsistent with respect to the planned analyses, the language of the SAP prevails. 
2 
STUDY OBJECTIVES, DESIGN AND PROCEDURES 
2.1 
Objectives 
The primary and secondary objectives of the study are as follows: 
Primary: 
• To determine a suitable dosage of OPTISON for contrast-enhanced 
echocardiography (CE-ECHO) in pediatric patients. 
Secondary: 
• To assess the safety profile of intravenous administrations of OPTISON in the 
pediatric population. 
• To compare diagnostic confidence of left ventricular endocardial border 
delineation (LV EBD) and wall motion between non-contrast and OPTISON-
enhanced echocardiography. 
• To compare the diagnostic confidence in the evaluation of left ventricular ejection 
fraction (LVEF) between non-contrast and OPTISON-enhanced 
echocardiography. 
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2.2 
Study Design 
This is a Phase 4, open-label, non-randomized, multi-center, multi-dose study in North 
America and Europe.  The study is designed to evaluate overall efficacy and safety of 
intravenous administration of OPTISON at various doses in pediatric patients undergoing 
transthoracic echocardiography. 
Approximately 50 pediatric patients between ≥9 and <18 years of age weighing ≥20 kg 
who are clinically indicated for a transthoracic echocardiogram and are likely to have a 
limited transthoracic echocardiographic window based on body habitus and/or previous 
cardiac operation or have a previous suboptimal non-contrast echo cardiogram will be 
enrolled into the study. Per FDA Advice/information request dated 2023, February 16th, a 
reduction of the sample size to 30 evaluable subjects was agreed. 
OPTISON will be administered according to pediatric body weight (≥20 to ≤28 kg, >28 
to ≤40 kg, and >40 kg).  Each patient will receive administrations of 2 different dose 
levels. Up to 3 injections per dose level are allowed to be administrated if significant 
technical issue(s) have occurred e.g., patient related needs, scanner malfunction, or drug 
injection error, with the total injected dose not to exceed the value defined in Table 1. 
Injections should be at least 10 minutes apart between the 2 dose levels to allow for 
clearance of the previous dose. 
Table 1 
OPTISON Dosing Table 
Body weight (kg) 
Dose/injection (mL) 
Dose level 1 
Dose/injection (mL) 
Dose level 2 
Total dose (mL) not to exceed (3 
injections/dose level) 
≥20 to ≤28 
0.1  
0.2 
1.0 
>28 to ≤40 
0.2 
0.3 
1.5 
>40 
0.2 
0.4 
1.8 
 
For echocardiographic imaging, standard apical 4-chamber and 2-chamber views will be 
obtained at baseline using tissue harmonic imaging, thereafter with contrast using a 
contrast-imaging mode.  Additional views may be obtained at the clinician’s discretion.  
All image views will be obtained prior to and after contrast administration until 10 
minutes post injection.  All images will be recorded on disk and sent to sponsor’s image 
core lab for preparation of blinded image evaluation (BIE). 
Efficacy of echocardiographic images will be evaluated by independent blinded readers 
based on characteristics of left ventricular opacification (LVO), diagnostic confidence of 
LV EBD and wall motion, and diagnostic confidence of LVEF.  Echocardiographic 
Analyses as per independent review charter includes:  
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1. Evaluation of Left ventricular (LV) Endocardial Border Delineation (EBD) 
2. Evaluation of diagnostic confidence for the assessment of LV EBD and wall motion 
3. Evaluation of Left Ventricular Opacification (LVO) 
4. Evaluation of Image Quality and Diagnostic Usefulness 
5. Evaluation of Left Ventricular Function 
6. Evaluation of diagnostic confidence for the assessment of LVEF 
7. Evaluation of Duration of Contrast Enhancement. 
Clinical safety data will be collected throughout the study, beginning from dosing 
through 72 hours post OPTISON administration to determine the safety profile of 
intravenous administration of OPTISON at various doses in pediatric population. 
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Figure 1 Study Diagram 
 
All efficacy and safety measurements obtained during the course of the study are 
summarized in the Study Schedule of Events (Table 2). 
Screening (≤2 weeks) 
Informed consent, study criteria, pregnancy test 
(post-menarchal females), demographic information, 
medical/surgical history 
Baseline (from-1 ± 1 hour prior to OPTISON) 
Entry criteria re-check, pregnancy test (post-menarchal females), vital signs, 
pulse oximetry, physical examination, ECG, prior and concomitant 
medications, AEs (from time of informed consent), unenhanced scan 
Dosing/Imaging (0) 
OPTISON administration(s), CE-ECHO, pulse oximetry, 
continuous ECG monitoring (0 to 30 min), AE monitoring 
Post Last Injection (+10 ±5 min & 1 hour ±5 min) 
Vital signs, pulse oximetry, ECG (+10 ±5 min & 30±5 min), AE monitoring, 
concomitant medication, physical examination (1 hour ±5 min only) 
Post Last Injection (+24 ±4 hours phone) 
Concomitant medication, AE reporting 
Image Transfer to Core Lab 
Post Last Injection (72 ±8 hours phone) 
Concomitant medication, AE reporting 
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Table 2 
Study Schedule of Events 
Variables 
Pre-procedure 
Dosing/ 
Imaging 
(0) 
Post-last OPTISON Administration 
Screening 
(≤2 weeks) 
Baseline 
(from -1 
± 1 h) 
10 min 
(±5 min) 
30 min  
(±5 min) 
1 h  
(±5 min) 
24 h  
(±4 h) 
72 h  
(±8 h) 
Informed Consent 
X 
 
 
 
 
 
 
 
Entry Criteria 
< ------------ > 
 
 
 
 
 
 
Demographic 
Information 
X 
 
 
 
 
 
 
 
Medical/Surgical 
History 
X 
 
 
 
 
 
 
 
Pregnancy test a 
X 
X 
 
 
 
 
 
 
Physical 
Examination 
 
X 
 
 
 
X 
 
 
Vital Signs 
 
X 
 
X 
 
X 
 
 
SaO2 by Pulse 
Oximetry 
 
X 
X 
X 
 
X 
 
 
12-Lead ECG 
Recording 
 
X 
 
X 
X 
 
 
 
Prior and 
concomitant 
Medications b 
 
< ------------------------------------------------------------------------------------------------ > 
Continuous ECG 
Monitoring c 
 
 
< ---------------------------------------- > 
 
 
 
OPTISON 
Administration 
 
 
X 
 
 
 
 
 
Image 
Acquisition  
 
X 
X 
 
 
 
 
 
Phone Calls 
 
 
 
 
 
 
X 
X 
AEs and SAEs 
Monitoring 
X 
<------------------------------------------------------------------------------------------------------> 
AE = Adverse event; ECG = Electrocardiogram; SAE = Serious adverse event; SaO2 = Oxygen saturation 
a Urine β-hCG pregnancy test at screening and at pre-dose; post-menarchal females only. 
b Any prior or concomitant medication taken by the subject within 24 hours before OPTISON injection and 
up to the end of the 72-hour follow-up period. 
c Continuous ECG monitoring is from time of first OPTISON injection until 30 minutes after the last 
OPTISON injection 
2.3 
Sample Size 
Although this is a dose-finding study and no hypotheses are being tested, it was thought 
useful to estimate sample size based on one of the study endpoints as a guide.  Since there 
is no historical literature about LV EBD results for OPTISON, the power estimation is 
based on the endpoint of contrast enhancement duration. 
A paired t-test was used for this power analysis with a one-sided significance level of 
0.025.  The null hypothesis is that the mean value of (contrast enhancement duration for 
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the high dose – contrast enhancement duration for the low dose within the same patient) 
≤0 minutes, and the alternative hypothesis is that the mean value of (contrast 
enhancement duration for the high dose – contrast enhancement duration for the low dose 
within the same patient) >0 minutes.  Based on the results from [Cohen et al. 1998], we 
assume that the estimated mean value of (contrast enhancement duration for the high 
dose – contrast enhancement duration for the low dose within the same patient) equals 
0.7 minutes, with a standard deviation of 1.5 minutes.  Table 3 displays the power 
calculation for the hypothesis testing with different sample sizes. 
Table 3 
Power Calculation 
Total sample size  
Power 
30 
70% 
40 
82% 
50 
90% 
60 
94% 
70 
97% 
80 
98% 
 
Therefore, the sample size for this clinical investigation was approximately 50 subjects, 
which would provide at least 90% power to detect an efficacy difference of 0.7 minutes 
between the high and low doses, with the greater duration for the high dose.  In addition, 
a sample size of 50 was considered to be adequate to address the other efficacy endpoints 
(LV EBD, LVO characteristics, wall motion assessment, and diagnostic confidence for 
the evaluation of LVEF) and OPTISON’s safety profile following intravenous 
administration in pediatric subjects.  The data will support dosing information in children.  
Per FDA Advice/information request dated 2023, February 16th, a reduction of the 
sample size to 30 evaluable subjects was agreed. 
2.4 
Interim Analysis 
There is no interim analysis planned for this study. 
2.5 
Randomization and Blinding 
This is an open-label study.  No blinding will be conducted for the subject allocation or 
application of the investigational medicinal product (IMP) in this study. 
For determination of efficacy and optimal dose level, 3 qualified independent blinded 
readers will conduct BIE of echocardiographic images.  Non-contrast and OPTISON-
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enhanced images will be presented in random order to the 3 readers without dosing 
information. 
3 
STUDY ENDPOINTS 
3.1 
Primary Endpoint 
• Visualization of the 12 segments of the left ventricle wall in standard apical 4-
chamber and 2-chamber views measured by the qualitative EBD visualization 
scale. 
3.2 
Secondary Endpoints 
The secondary endpoints will be the following: 
• Overall safety profile in terms of occurrence of AEs and changes in vital signs, 
arterial oxygen saturation (SaO2), physical examinations, and 12-lead ECGs 
following administration of different OPTISON doses in pediatric patients. 
• LVO assessed by visual peak contrast intensity and peak LV contrast filling, and 
duration of contrast enhancement following intravenous administration of 
OPTISON at various doses. 
• Comparison of diagnostic confidence of LV EBD and wall motion between non-
contrast and OPTISON-enhanced echocardiography at various doses. 
• Comparison of diagnostic confidence in the evaluation of LVEF between non-
contrast and OPTISON-enhanced echocardiography. 
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4 
ANALYSIS POPULATIONS 
4.1 
Definition for Analysis Populations 
Enrolled subjects are subjects who signed the informed consent.  
4.1.1 
Safety Population 
The Safety Population will include all subjects enrolled in the study who receive ≥1 
administration of OPTISON. 
4.1.2 
Full Analysis Set (FAS) Population 
The FAS population will include all subjects enrolled in the study who have available 
non-contrast harmonic images and OPTISON-enhanced echocardiographic images, 
irrespective of the quality of CE-ECHO. 
4.1.3 
Per-Protocol (PP) Population 
The PP population will include all subjects in the FAS population who do not have 
important protocol deviation impacting the study primary endpoint. 
5 
ANALYSIS CONVENTIONS 
Post-text tables and listings will be prepared in accordance with the current International 
Council for Harmonisation (ICH) Guidelines.  The information and explanatory notes to 
be provided in the “footer” or bottom of each table and listing will include the following 
information: 
1. Date and time of output generation 
2. SAS® program name, including the path that generates the output 
3. Any other output specific details that require further elaboration 
In general, tables will be formatted with a column displaying findings recorded on the 
electronic case report form (eCRF) for all subjects combined.  Row entries in tables are 
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made only if data exists for at least one subject (i.e., a row with all zeros will not appear).  
The only exception to this rule applies to tables that list the termination status of subjects 
(e.g., reasons for not completing the study).  In this case, zeros will appear for study 
termination reasons that no subject satisfied.  The summary tables clearly indicate the 
number of subjects to which the data apply and unknown or not performed are 
distinguished from missing data. 
Supportive individual Subject Data Listings, as a minimum, will be sorted and presented 
by weight group, subject number, visit date, and dose level if applicable.  Listings will 
also include days relative to the first dose of study drug, if applicable. 
Specific algorithms are discussed for imputing missing or partially missing dates, if 
deemed appropriate, under specific data topics.  Imputed or derived data are flagged in 
the individual subject data listings.  Imputed data are not incorporated into any raw or 
primary datasets.  These data are retained in derived (or analysis) datasets. 
This section details general conventions to be used for the statistical analyses.  The 
following conventions will be applied to all data presentations and analyses. 
• Summary statistics for categorical variables will consist of the number and 
percentage of responses in each level.  The number and percentage of responses 
will be presented in the form XX (XX.X%). 
• Summary statistics for continuous variables will consist of the sample size (n), 
mean, standard deviation (SD), median (1st and 3rd quartile), minimum, and 
maximum values. 
• All mean and median values, as well as Q1 and Q3, will be formatted to one more 
decimal place than the measured value.  Standard deviation values will be 
formatted to two more decimal places than the measured value.  Minimum and 
maximum values will be presented with the same number of decimal places as the 
measured value. 
• All p-values, if applicable, will be rounded to 4 decimal places. All p-values that 
round to 0.0000 will be presented as ‘<0.0001’ and p-values that round to 1.0000 
will be presented as ‘>0.9999’. Two-sided P-values < 0.05 will be considered to 
be statistically significant unless otherwise specified. 
• All summary tables will include the analysis population sample size (i.e., number 
of subjects). 
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• Study Day 1 is defined as the date at which the subject receives the first dose of 
study medication.  All study days are determined relative to Day 1.   
• Study days prior to Day 1 will be calculated as: 
o Study Day = Assessment Date – First Injection Date 
• Study days after Day 1 will be calculated as: 
o Study Day = Assessment Date – First Injection Date + 1. 
• Baseline values will be defined as the last non-missing value recorded prior to 
first dose of study drug. 
• Change from baseline will be calculated as follows: Change = Post-baseline value 
- baseline value. 
• All pre- and post- investigational product assessments including unscheduled or 
repeat assessments will be included in the data listings.  For unscheduled or repeat 
pre-dose assessments, the last assessment taken for a time point will be used in 
the data summaries (summary tables, figures, and statistical analysis); for all post-
dose time points, the initial assessment for any given time point will be used in 
the safety data summaries (summary tables, figures, and statistical analysis, 
except the summary tables of clinically notable values and shift tables where all 
values will be included). For efficacy summaries, the site records in the eCRF 
which assessment was considered for the image interpretation. The corresponding 
assessment will be used in the efficacy summaries. 
• Date variables will be formatted as DDMMMYYYY for presentation. 
• Tables, figures, and listings will be presented in landscape orientation. 
• SAS® Version 9.4 or higher will be the statistical software package used for all 
data analyses. 
Definition of Analysis Windows 
There are no visit windows for this study.  For the statistical analyses, data will be 
analyzed by the nominal visit that was collected on the electronic case report form 
(eCRF). 
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Unscheduled visits will not be used in the by-visit analysis, but will be used for the 
following where appropriate: 1) derivations of baseline/last on-treatment measurements; 
2) derivations of the maximum/minimum on-treatment values and maximum/minimum 
changes from baseline values for safety analyses; 3) data listings. 
Definition of Missing Data Imputation 
Missing values will not be substituted by estimated values but treated as missing in the 
statistical evaluation.  Data for all enrolled participants will be presented in the data 
listings.  A participant who is enrolled but who did not receive any amount of study drug 
will be included in those data listings for which they have data but will be excluded from 
all data summaries. 
Missing or partial dates for AE or for medications may be imputed for determination of 
treatment emergent AE (TEAE) or concomitant medication, as follows.  
• If the AE start date is completely missing, then the AE will be considered 
treatment emergent unless it can be determined that the AE end date/time 
occurred prior to IMP injection. If this is the case, the AE will not be considered 
treatment emergent.   
• If the AE start date/time is partially missing and the partial date is not sufficient to 
determine if the event occurred after the IMP injection, then the AE will be 
considered treatment emergent unless it can be determined that the AE end 
date/time occurred prior to the injection. 
6 
STATISTICAL ANALYSES 
6.1 
Demographics, Baseline Characteristics, Medical History, and 
Prior/Concomitant Medications 
6.1.1 
Demographic and Baseline Characteristics 
Age (years between birth and time informed consent form was signed), sex, race, 
ethnicity, and baseline height, weight, weight group (≥20 to ≤28, >28 to ≤40, >40 kg; as 
well as ≤40, >40 kg), and body mass index (BMI) will be summarized overall, 
independent of IMP assignment, based on Safety, FAS, and PP populations. 
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Categorical data (sex, weight group, race, and ethnicity) will be summarized using 
frequencies and percentages.  The number of subjects with missing information will also 
be summarized. 
Continuous data (age, baseline height, weight and BMI) will be summarized with the 
number of non-missing observations by mean, standard deviation, median, Q1, Q3, 
minimum and maximum values. 
6.1.2 
Medical History 
Medical/surgical history data will be coded using the Medical Dictionary for Regulatory 
Activities (MedDRA) version 24.0.  It will be summarized and presented using system 
organ class (SOC) and preferred term (PT).  The SOC will be presented in alphabetical 
order and the PT will be presented in alphabetical order within each SOC. The number 
and percentage of subjects with a particular condition/diagnosis will be summarized 
overall and by weight group (≤40, >40 kg) based on the Safety population.  Subjects 
reporting more than one condition/diagnosis within a body system will be counted only 
once for that body system. 
6.1.3 
Prior and Concomitant Medications 
Prior and concomitant medications will be recorded and coded using the Anatomical-
Therapeutic-Chemical (ATC) classification text from the World Health Organization 
Drug Dictionary (WHO-DD) version, March 1, 2021.  They will be summarized overall 
and by weight group (≤40, >40 kg) based on the Safety population.  A prior medication is 
defined as any medication taken prior to the first dose of study drug.  A concomitant 
medication is defined as any medication that started prior to the first dose of study drug 
and continued to be taken after the first dose of study drug or any medication that started 
after the first dose of study drug, but not after the last dose of study drug.  Medications 
with unknown or incomplete end date will be considered concomitant unless there is 
clear evidence that the medication was stopped prior to the first dose of study drug. The 
number and percentage of subjects who have taken medications will be summarized by 
ATC Class 3 and generic drug name, separately for prior and concomitant medications.   
6.2 
Subject Disposition 
The number of subjects for each of the following categories will be summarized, overall 
and by weight group (≤40, >40 kg): 
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● Enrolled subjects 
● Subjects who received at least one dose of study drug (i.e., the safety 
population). 
● Subjects included in the FAS population 
● Subject included in the PP population 
● Subjects who completed the study 
● Subjects who withdrawn from the study and the reason for withdrawal 
All protocol deviations will be listed. Deviations will be classified as Important or Non-
important. Important deviations excluding from the PP population will be identified. 
6.3 
Study Drug Exposure and Compliance 
The total treatment exposure (OPTISON injection in mL as well as number of 
administrations) received at two dose levels will be summarized by descriptive statistics 
overall and by weight group (≤40, >40 kg) based on Safety population.  A summary of 
each subject’s dosing information will be presented in a listing.  The injection used for 
image interpretation will be identified in this listing. Reasons why OPTISON dose was 
repeated will also be listed. 
6.4 
Efficacy Analysis 
6.4.1 
General Considerations 
Since this is a dose-finding study, no hypotheses are being tested.  All efficacy results 
will be summarized for the FAS and PP populations. 
Assessments to be used for image interpretation are identified by the site and recorded in 
the eCRF. These identified assessments will be used for efficacy analyses.  
Efficacy summaries will be presented for each reader. 
6.4.2 
Primary Efficacy Analysis 
Visualization of the 12 segments of the left ventricle wall in standard apical 4-chamber 
and 2-chamber views will be measured by the qualitative EBD visualization scale: 
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0 = no visualization of the LV endocardial border  
1 = poor visualization  
2 = fair visualization  
3 = good/optimal visualization 
Results for each segment will be summarized for non-contrast and OPTISON dose levels 
overall and by weight group (≤40, >40 kg). A category “Visualized”, corresponding to 
scores 2 or 3, will also be presented.  Additionally, the number of segments visualized 
(segments rated as “fair visualization” or “good/optimal visualization”) will be derived 
from the EBD score and summarized for non-contrast and OPTISON dose levels, overall 
and by weight group. 95% CI will be presented. A Paired t-test will be used to compare 
the number of segments visualized between non-contrast and each OPTISON dose level. 
The number and percentage of subjects with an increase in the number of segments 
adequately visualized over non-contrast images will also be tabulated overall and by 
weight group for each OPTISON dose levels. 
The Total LV EBD score, calculated as the sum of the individual scores assigned to each 
of the segment (range 0 to 36), will be tabulated overall and by weight group for non-
contrast and each OPTISON dose level. The change from non-contrast to each OPTISON 
dose level will also be presented. 95% CI will be presented. A Paired t-test will be used 
to compare Total LV EBD scores between non-contrast and each OPTISON dose level. 
A suboptimal echocardiogram is defined as ≥ 2 contiguous segments in any given view 
that cannot be visualized. The number and percentage of subjects with suboptimal 
echocardiogram will be presented for non-contrast and each OPTISON dose level, overall 
and by weight group. 
6.4.3 
Secondary Efficacy Analyses 
The following secondary endpoints will be summarized, overall and by weight group 
(≤40, >40 kg): 
• 
The LV peak contrast intensity, determined as None, Low, Medium, High or 
Blooming, will be summarized as a categorical variable for each OPTISON dose 
level.  
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• 
The peak LV contrast filling, categorized as None (0% filling), Faint (around 33% 
filling), Intermediate (around 67% filling) or Full (100% filling), will be 
summarized as a categorical variable for each OPTISON dose level.  
• 
The contrast enhancement duration in the LV chamber determined from the time 
contrast appears in the LV to the time it almost dissipates from the chamber as 
determined by investigators, will be summarized with descriptive statistics for 
each OPTISON dose level. 
• 
Image quality, categorized as Excellent/good, Adequate or Poor will be 
summarized as a categorical variable for each OPTISON dose levels.  
• 
Diagnostic usefulness, evaluated as Non-diagnostic (the image quality is not 
sufficient to confidently visualize the endocardial border) or Diagnostic 
(sufficient or excellent image quality for EBD assessments), will be summarized 
as a categorical variable for non-contrast and OPTISON dose levels.   
• 
The diagnostic confidence of the assessment of LV EBD and wall motion, 
assessed using a 4-point scale (0 = no confidence, 1 = low confidence, 2 = 
moderate confidence, 3 = high confidence) will be described for non-contrast and 
OPTISON dose levels. 
The change in diagnostic confidence will be derived by comparing non-contrast 
and OPTISON-enhanced diagnostic confidence scores as follows (contrast-
enhanced minus unenhanced): 
• ≤-1 = confidence is lower with contrast-enhanced images  
• 0 = confidence is the same between unenhanced and contrast-enhanced 
images  
• 1 = confidence is slightly improved with contrast-enhanced images  
• 2 = confidence is moderately improved with contrast-enhanced images  
• 3 = confidence is greatly improved with contrast-enhanced images. 
Diagnostic confidence in comparison to non-contrast echocardiographic images 
will be summarized as a categorical variable by OPTISON dose level. 
• 
The diagnostic confidence of the evaluation of LVEF, assessed using a 4-point 
scale (0 = no confidence, 1 = low confidence, 2 = moderate confidence, 3 = high 
confidence) will be described for non-contrast and OPTISON dose levels. 
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The change in diagnostic confidence will be derived using the same method as for 
change diagnostic confidence of the assessment of LV EBD and wall motion and 
summarized as a categorical variable by OPTISON dose level. 
• 
LV function assessed by LVEFs, calculated using the formula: (end diastolic 
volume – end systolic volume)/end diastolic volume for each ventricle, will be 
summarized with descriptive statistics for non-contrast and OPTISON dose levels  
• 
A composite assessment of dose suitability will be made at the subject level and 
results will be tabulated by OPTISON dose level. A dose will be considered 
suitable of it meets the following criteria: 
(1) 
There is an increase in the number of segments adequately visualized over 
non-contrast images  
(2) 
LVO peak contrast intensity assessed as medium or better 
(3) 
Peak LV contrast filling to around 67% or better 
(4) 
Duration of contrast enhancement ≥1.5 minutes. 
6.4.4 
Handling of Uninterpretable Images 
In the FAS analysis of the primary endpoint, the uninterpretable images will be 
categorized as none for contrast intensity and 0 visualization of EBD.  In the secondary 
analyses, the uninterpretable images will be considered as 0 enhancement duration, poor 
for image quality, and non-diagnostic for diagnostic usefulness.  Additionally, the 
number of uninterpretable images will be summarized by non-contrast and dose level.   
6.4.5 
Reader Differences 
All efficacy summaries described in Section 6.4 will be presented by reader.   
Data from re-read will be listed. 
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6.5 
Safety Analysis 
6.5.1 
General Considerations 
The safety of the intravenous administration of OPTISON in the pediatric population will 
be measured by the incidence of AEs, vital signs, SaO2 by pulse oximetry, physical 
examination and ECG results. Safety data will be summarized overall and for each 
weight group (≤40, >40 kg). 
6.5.2 
Analysis of Adverse Events 
6.5.2.1 
Treatment-Emergent Adverse Events 
An AE will be considered as treatment-emergent if it starts or worsens after OPTISON 
administration.  Any AE with an onset on the day of IMP administration for which the 
time of onset is missing will be assumed to be treatment-emergent. 
All summaries of AEs will be based on TEAEs and will be prepared by weight group 
(≤40, >40 kg) and overall for the Safety Population.  AEs will be mapped to System 
Organ Class (SOC) and preferred terms using the MedDRA version 24.0. All AEs will be 
listed.  
An overall summary of TEAEs will present the number (%) of subjects and number of 
events for the following events: 
- 
TEAE 
- 
Severe TEAE 
- 
Serious TEAE 
- 
TEAE considered related to IMP 
- 
Serious TEAE considered related to IMP  
- 
TEAE leading to discontinuation of study drug 
- 
TEAE leading to death 
 
The number and percentage of subjects experiencing TEAEs, and the number of events 
will be summarized by SOC and preferred term in alphabetical order.  Summaries of 
TEAEs by maximum severity and TEAEs considered related to the study drug will also 
be presented by SOC and preferred terms.   
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6.5.2.2 
SAEs (Including Deaths) and Adverse Events Leading to Study Drug 
Discontinuation 
The number and percentage of subjects, with the corresponding number of events, 
experiencing treatment-emergent SAEs (including deaths) and adverse events leading to 
discontinuation of study drug will be tabulated  by SOC and  preferred term, overall and 
by weight group. Deaths, SAEs and TEAEs leading to discontinuation of study treatment 
will be listed. 
6.5.3 
Analysis of Vital Signs and Weight 
6.5.3.1 
Variables and Criteria Defining Abnormality 
Vital sign variables are systolic blood pressure, diastolic blood pressure, heart rate, 
respiratory rate, and pulse oximetry.  These will be measured at pre- and post-treatment 
time points as specified in Table 2. 
Table 4 presents the criteria for vital sign normal limits in a pediatric population by age 
group for respiratory rate, heart rate, systolic blood pressure, and weight. Besides, pulse 
oximetry < 95% will be considered as abnormal in this study. 
Table 4 
Pediatric Vital Signs Normal Ranges 
Age Group 
Respiratory 
Rate 
Heart Rate 
Systolic Blood 
Pressure 
Weight in kilos 
Weight 
in 
pounds 
School Age (6-12 yrs.) 
20 – 30 
70 – 110 
80 – 120 
20 – 42 
41 – 92 
Adolescent (13+ yrs.) 
12 – 20 
55 – 105 
110 – 120 
>50 
>110 
REF: Source: http://www.rnceus.com/psvt/psvtvs.html 
6.5.3.2 
Statistical Methods 
The presentation of vital signs focuses on the descriptive summaries at baseline, at 10 
minutes, and at 1 hour post-OPTISON administration, and the change from baseline to 
the 10-minute and 1-hour post-OPTISON assessments.  All subjects in the safety 
population who have a baseline and a post-baseline assessment of vital signs will be 
included in the presentation of the results.  Results will be presented by weight group 
(≤40, >40 kg) and overall. 
In addition to the change from baseline results, the shifts from baseline to the post 
baseline visit will be summarized in tabular form for heart rate and systolic blood 
pressure.  Subjects will be classified at baseline and the post-baseline visit in one of the 
following categories based on a comparison of the vital sign result to the reference range 
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for that vital sign parameter: below normal, normal, or above normal.  A shift table will 
be constructed in which each subject is cross-classified based upon the reference range 
categories.  The number and percentage of subjects in each shift table cell will be 
displayed.  The number of subjects in the safety population missing either baseline or 
post-baseline vital sign information will also be presented.  Results will be presented by 
weight group and overall. All vital signs results will be listed. Classification (below 
normal, normal, or above normal) will be presented in the listing for respiratory rate, 
heart rate, systolic blood pressure, and pulse oximetry. 
6.5.4 
Analysis of ECG Parameters 
6.5.4.1 
Variables and Criteria Defining Abnormality 
A 12-lead ECG will be measured at the various pre- and post-treatment time point ranges 
described in Table 2.  The ECGs will be characterized as normal or abnormal, and, if 
abnormal, whether the abnormality is clinically significant.  These overall readings will 
be an interpretation based on ECG intervals (heart rate, PR, RR, QRS, QT, QTcF 
[Fridericia]).  QTcF will be defined as follows: 
● Fridericia’s correction (QTcF):   
3 RR
QT
F
QTC
=
 
● Bazett’s correction (QTcB):   
RR
QT
B
QTC
=
 
Normal ranges for the observed values and change from baseline by age group are 
defined in Table 5, Table 6 and Table 7. 
Table 5 
Mean and Percentiles of ECG Cardiac Measures Among Pre-pubertal 
Children 
ECG Variable 
Mean (sd) 
1st %ile 
5th %ile 
Median 
95th %ile 
99th %ile 
Heart Rate (bpm) 
83.0 (12.1) 
59 
65 
82 
104 
114 
RR Interval (ms) 
738.5 (107.8) 
526.3 
576.9 
731.7 
923.1 
1040 
PR Interval (ms) 
132.8 (17.3) 
100 
108 
130 
162 
180 
QRS Interval (ms) 
80.0 (9.0) 
64 
70 
80 
96 
102 
QT Interval (ms) 
354.7 (24.3) 
300 
318 
352 
396 
416 
QTc Bazett (ms) 
414.0 (19.6) 
367 
383 
414 
443 
460 
QTc Data-derived (ms) 
399 (17.3) 
357.4 
372 
398.7 
424.1 
439.6 
QTc Fridericia (ms) 
393.2 (17.1) 
353.3 
366.3 
392.9 
419 
433 
Note: Pre-pubertal group (N = 1537) defined as females whose age was ≤ 8 years (n = 224) and males whose age was ≤ 9 
years (n = 1313). Source: Child and Adolescent Psychiatry and Mental Health 2007;1:11 
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Table 6 
Mean and Percentiles of ECG Cardiac Measures Among Pubertal 
Children 
ECG Variable 
Mean (sd) 
1st %ile 
5th %ile 
Median 
95th %ile 
99th %ile 
Heart Rate (bpm) 
77.4 (11.8) 
54 
60 
76 
98 
109 
RR Interval (ms) 
792.9 (121.1) 
555.6 
612.2 
780 
1000 
1132 
PR Interval (ms) 
136.8 (18.5) 
100 
112 
136 
170 
190 
QRS Interval (ms) 
83.0 (9.1) 
68 
70 
80 
96 
106 
QT Interval (ms) 
368.8 (27.0.) 
310 
330 
368 
412 
440 
QTc Bazett (ms) 
415.6 (21.3) 
362 
381 
416 
448 
468 
QTc Data-derived (ms) 
403.9 (19.1) 
357 
373.3 
404 
433.1 
450.1 
QTc Fridericia (ms) 
399.4 (19.0) 
354.5 
369.4 
399.6 
429 
443.9 
Note: Pubertal group (N = 3656) defined as females whose age was > 8 and ≤ 13 years (n = 881) and males whose age was 
> 9 and ≤ 14 years (n = 2775). Source: Child and Adolescent Psychiatry and Mental Health 2007;1:11 
Table 7 
Mean and Percentiles of ECG Cardiac Measures Among Post-pubertal 
Children 
ECG Variable 
Mean (sd) 
1st %ile 
5th %ile 
Median 
95th %ile 
99th %ile 
Heart Rate (bpm) 
70.8 (11.4) 
48 
52 
70 
91 
102 
RR Interval (ms) 
870.3 (144.3) 
588.2 
659.3 
857.1 
1153.8 
1250 
PR Interval (ms) 
140.8 (18.9) 
104 
116 
140 
176 
192 
QRS Interval (ms) 
87.8 (9.9) 
70 
72 
88 
104 
112 
QT Interval (ms) 
382.9 (29.0) 
328 
340 
380 
432 
470 
QTc Bazett (ms) 
412.2 (20.7) 
350 
376 
413 
444 
457 
QTc Data-derived (ms) 
405.0 (18.5) 
357.2 
371.6 
406.5 
433 
447.5 
QTc Fridericia (ms) 
402.1 (18.5) 
354.5 
371.1 
402.6 
430.9 
446.5 
Note: Post-Pubertal group (N = 737) defined as females whose age was >13 yrs (n = 240) and males whose age was > 14 
yrs (n = 497). Source: Child and Adolescent Psychiatry and Mental Health 2007;1:11 
6.5.4.2 
Statistical Models 
All subjects in the safety population who have a baseline and a post-baseline assessment 
of ECG data will be included in the presentation of the results.  Overall ECG investigator 
interpretation will be categorized as normal, abnormal (Not Clinically Significant), and 
abnormal (Clinically Significant).  Number and percentage of subjects in each category at 
each timepoint will be presented. 
Consistent with ICH E14 guidance, ECG data will be presented both as analyses of 
central tendency (e.g., means, medians) and categorical analyses. ECG data will be 
summarized overall and by weight group (≤40, >40 kg). 
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Twelve-lead ECG data (observed values and changes from baseline) will be presented 
using descriptive statistics by weight group (≤40, >40 kg) and overall for each time point 
(baseline, 10 minutes, and 30 minutes post-dose).   
In addition to the observed values and change from baseline results, the shifts from 
baseline to post-baseline time points will be summarized in tabular form for each ECG 
parameter and the overall interpretation.  The overall interpretation will be based on the 
categories of the interpretation listed previously, while the ECG parameters will be listed 
based on being below normal (<1st percentile), normal, or above normal (>99th percentile) 
compared to the reference ranges in Table 5, Table 6 and Table 7.  The number and 
percentage of subjects in each shift table cell will be displayed.  The number of subjects 
who are missing either baseline or post-baseline ECG data will also be provided for each 
timepoint.   
All ECG results will be listed including overall interpretation and status compared to 
normal ranges (below normal / normal / above normal). 
In addition, changes from baseline in the QTc interval will be displayed according to the 
Committee for Medicinal Products for Human Use (CHMP) criteria (absolute QTc 
interval prolongation, of >450, >480, >500 ms and change from baseline in QTc 
interval >30 and >60 ms).  
6.5.5 
Analysis of Physical Examination 
Physical examination findings at baseline and after OPTISON administration will be 
summarized for the safety population in the form of a shift table.  Findings for each body 
system will be categorized as normal, abnormal (non-clinically significant), and abnormal 
(clinically significant).  The number and percentage of subjects in each shift table cell 
will be displayed.  The number of subjects who are missing either baseline or post-
baseline Physical examination data will also be provided for each timepoint.  Data will be 
presented by weight group (≤40, >40 kg) and overall. 
7 
REFERENCES 
[ICH E14] 
International conference on harmonisation of technical requirements for registration of 
pharmaceuticals for human use.  The clinical evaluation of QT/QTC interval prolongation 
and proarrhythmic potential for non-antiarrhythmic drugs–E14.  Step 4 version; May, 
2005 
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[Cohen et al. 1998] 
Cohen JL, Cheirif J, Segar DS, et al.  Improved left ventricular endocardial border 
delineation and opacification with OPTISON (FS069), a new echocardiographic contrast 
agent.   
JACC 1998;32:746-752 
8 
LIST OF TABLES, FIGURES AND DATA LISTINGS THAT 
ARE TO BE PROGRAMMED 
Whenever applicable, tables listed below will present results overall and by weight group 
and will be repeated by reader. 
TABLE 
NUMBER 
TABLE TITLE 
POPULATION 
FOR 
ANALYSIS 
14.1.1 
Summary of Subject Disposition 
All Subjects 
14.1.2.x 
Summary of Subject Demographics 
Safety, FAS, PP 
14.1.3 
Summary of Medical History 
Safety 
14.1.4.1 
Summary of Prior Medications 
Safety 
14.1.4.2 
Summary of Concomitant Medications 
Safety 
14.1.5 
Summary of Exposure to OPTISON 
Safety 
14.2.1.1a 
Summary of Visualization of LV Wall by Segments 
ITT 
14.2.1.1b 
Summary of Visualization of LV Wall by Segments 
PP 
14.2.1.2a 
Summary of Visualization of LV Wall Overall 
ITT 
14.2.1.2b 
Summary of Visualization of LV Wall Overall 
PP 
14.2.2a 
Summary of Peak Contrast Intensity in LV 
ITT 
14.2.2b 
Summary of Peak Contrast Intensity in LV 
PP 
14.2.3a 
Summary of Peak Contrast Filling in LV 
ITT 
14.2.3b 
Summary of Peak Contrast Filling in LV 
PP 
14.2.4a 
Summary of Contrast Enhancement Duration in LV 
ITT 
14.2.4b 
Summary of Contrast Enhancement Duration in LV 
PP 
14.2.5a 
Summary of Image Quality 
ITT 
14.2.5b 
Summary of Image Quality 
PP 
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TABLE 
NUMBER 
TABLE TITLE 
POPULATION 
FOR 
ANALYSIS 
14.2.6a 
Summary of Diagnostic Usefulness 
ITT 
14.2.6b 
Summary of Diagnostic Usefulness 
PP 
14.2.7a 
Summary of Diagnostic Confidence of LV EBD and Wall Motion 
Between Contrast and Non-Contrast Enhanced Images 
ITT 
14.2.7b 
Summary of Diagnostic Confidence of LV EBD and Wall Motion 
Between Contrast and Non-Contrast Enhanced Images 
PP 
14.2.8a 
Summary of Diagnostic Confidence of LVEF Between Contrast and 
Non-Contrast Enhanced Images 
ITT 
14.2.8b 
Summary of Diagnostic Confidence of LVEF Between Contrast and 
Non-Contrast Enhanced Images 
PP 
14.2.9a 
Summary of LV Function Assessed by LVEF 
ITT 
14.2.9b 
Summary of LV Function Assessed by LVEF 
PP 
14.2.10a 
Summary of Dose Suitability 
ITT 
14.2.10b 
Summary of Dose Suitability 
PP 
14.3.1 
Overall summary of Treatment Emergent Adverse Events 
Safety 
14.3.1.1 
Summary of Treatment Emergent Adverse Events by System Organ 
Class and Preferred Term 
Safety 
14.3.1.2 
Summary of Treatment Emergent Adverse Events by maximum 
severity by System Organ Class and Preferred Term  
Safety 
14.3.1.3 
Summary of Treatment Emergent Adverse Events considered related 
to IMP by System Organ Class, Preferred Term 
Safety 
14.3.2.1 
Summary of Serious Treatment Emergent Adverse Events by System 
Organ Class and Preferred Term 
Safety 
14.3.2.4 
Summary of Treatment Emergent Adverse Events Leading to Study 
drug Discontinuation by System Organ Class and Preferred Term 
Safety 
14.3.3.1 
Summary and Change from Baseline – Vital Signs 
Safety 
14.3.3.2 
Shifts from Baseline to Post-Administration Timepoints – Vital Signs 
Safety 
14.3.4.1 
Summary and Change from Baseline – Electrocardiograms 
Safety 
14.3.4.2 
Shifts from Baseline to Post-Administration Timepoints – 
Electrocardiograms 
Safety 
14.3.4.3 
Summary of Overall Interpretation by Timepoint - 
Electrocardiograms 
Safety 
14.3.4.4 
Shift from Baseline to Post-Administration Timepoints of Overall 
Interpretation - Electrocardiograms 
Safety 
14.3.4.5 
Summary of QTc Interval Outlier Categories 
Safety 
14.3.5 
Shift from Baseline to Post-Administration Timepoint – Physical 
Examination 
Safety 
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LISTING NUMBER 
LISTING TITLE 
POPULATION 
FOR 
ANALYSIS 
16.2.1 
Subject Disposition 
All Subjects 
16.2.2 
Protocol Deviations 
All Subjects 
16.2.3 
Inclusion/Exclusion Criterion 
All Subjects 
16.2.4.1 
Demographic Information 
Safety 
16.2.4.2 
Medical History 
Safety 
16.2.4.3 
Prior and Concomitant Medications 
Safety 
16.2.5 
Exposure to OPTISON 
Safety 
16.2.6.1 
Visualization of Segments 
ITT 
16.2.6.2 
BIE Interpretations 
ITT 
16.2.6.3 
Non-Contrast and Contrast Enhanced Comparisons 
ITT 
16.2.6.4 
Dose Suitability 
ITT 
16.2.7.1 
Adverse Events 
Safety 
16.2.7.2 
Deaths 
Safety 
16.2.7.3 
Serious Adverse Events 
Safety 
16.2.7.4 
TEAEs leading to discontinuation of study treatment 
Safety 
16.2.8.1 
Vital Signs 
Safety 
16.2.8.2 
Electrocardiogram – Reader Results 
Safety 
16.2.8.3 
Electrocardiogram – Overall Interpretation 
Safety 
16.2.8.4 
Physical Examination 
Safety 
 
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9 
SUMMARY OF CHANGES 
Changes from the statistical analyses planned in the protocol are summarized in the 
following table: 
Protocol Amendment A01 Version 2.0 
Changes 
ITT population  
All subjects enrolled in the study who 
have available non-contrast harmonic 
images and OPTISON-enhanced 
echocardiographic images, irrespective 
of the quality of CE-ECHO. 
 
PP Population 
The PP population will include all 
subjects in the FAS population whose 
OPTISON-enhanced 
echocardiography images have 
adequate quality (sufficient contrast 
enhancement to visualize EBD). 
For consistency with ICH E9 guidance, the 
efficacy population has been renamed from ITT 
population to Full Analysis Set. 
 
The PP Population definition has been updated 
to all subjects in the FAS population who do not 
have important protocol deviation impacting the 
study primary endpoint. A lack of contrast 
enhancement is not considered as a protocol 
deviation. Nevertheless, any important 
deviation from the Imaging Manual (e.g. wrong 
settings for echocardiographic imaging, 
standard apical 4-chamber and 2-chamber views 
not acquired...) will be reviewed during the 
Data Review Meeting and exclusion from the 
PP Population will be assessed. 
All summary tables and data listings 
will be separated by dose levels. 
Whenever relevant, summaries and listings will 
be presented by Weight group (≤40, >40 kg) 
and dose level. 
Medications will be presented by ATC 
Class 1 and preferred term 
Prior and concomitant medications will be 
tabulated by ATC Class 3 and preferred term 
Primary endpoint analysis 
Supportive analyses added in Section 6.4.2 
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Reference ranges for Vital signs 
Adolescent (13+ yrs.) category added in 
Reference ranges table  
Table with reference ranges for Post Pubertal 
Children added 
Reference ranges for ECG 
Table with reference ranges for Post Pubertal 
Children added 
 
Any deviations from the statistical analysis outlined in the SAP will be described, and 
reasons for the deviations listed, in the final Clinical Study Report. 
10 
APPENDIX 
Not applicable 
 
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