Conditions: Hypoxic-Ischemic Encephalopathy Brain Ischemia Hypoxia
Keywords: Ultrasound Contrast-enhanced ultrasound
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 1
Healthy volunteers: no

Brief summary:
Neonates presenting with neurologic symptoms require rapid, non-invasive imaging with high spatial resolution and tissue contrast. The purpose of this study is to evaluate brain perfusion using contrast-enhanced ultrasound CEUS in bedside monitoring of neonates and infants with hypoxic ischemic injury (HII). Investigational CEUS scan will be performed separately from clinically indicated conventional US, in the ICU. Subjects will be scanned with CEUS at two different time-points (at the time HII is first suspected or diagnosed and at time of MRI scan), separately from clinically indicated ultrasound. The CEUS scan will be interpreted by the sponsor-investigator.

The study will be conducted at one site, The Children's Hospital of Philadelphia. It is expected that up to 100 subjects will be enrolled per year, for up to two years, for a total enrollment of up to 200 subjects.

Detailed description:
Contrast-enhanced ultrasonography (CEUS) is a novel imaging technique in which gas-filled microbubbles, smaller than red blood cells, generate increased signal due to the acoustic impedance mismatch. Injection of ultrasound contrast agents into blood increase the echogenicity, allowing enhanced visualization of a blood vessel. Ultrasound contrast agents have been approved for use in Europe for almost two decades. In the case of SonoVue (now called LumasonTM), a second-generation lipid/sulfur hexafluoride US contrast agent (Bracco, Milan, Italy), European Union approved its intravenous use in adults in 2001. The FDA in the United States just recently approved the use of LumasonTM for evaluation of focal hepatic lesions in pediatric population in 2016, and very recently (Jan 2017) approved its use in children for the evaluation of the urinary tract in pediatric patients with known or suspected vesicoureteral reflux. For the remainder of clinical applications, the ultrasound contrast agents are being used off-label in both Europe and the United States.

There is already good evidence in the literature that CEUS may be very valuable in children, and that is why it is routinely performed off-label for this purpose in the USA. However, there are ways in which it could be improved, and more studies are needed to definitively establish its diagnostic performance.

Sulfur hexafluoride lipid-type A microspheres (LumasonTM, Bracco Inc) is an FDA-approved ultrasound contrast agent. The study duration per subject will be approximately 15 minutes including the time to prepare LumasonTM contrast agent and perform the CEUS, as well as the 60 minute monitoring period after the first and second injection of LumasonTM. CEUS will be performed at the time HII is first suspected or diagnosed and within 24 hours of clinically indicated MRI (which occurs approximately on day 5 of life) for a total of two CEUS exams of 1 hour and 15-minute duration each. Study participation will be complete when the 60 minute monitoring period after the second CEUS is complete.

Injection of LumasonTM contrast agent will be performed via the existing peripheral intravenous line or central line using the FDA-recommended dose of up to 0.03 mg/kg. Contrast-agent injection will be performed twice per CEUS scan to ensure image quality and test reproducibility. In the case of more stable patients without an IV line, a peripheral IV line will be started to conduct the investigational CEUS. Two bolus injections will performed to evaluate for dynamic brain perfusion and several 2-minute cine clips as well as static images will be acquired during the exam.

Qualitative analysis with visual assessment and quantitative analysis of the acquired CEUS scans will be performed and interpreted by the PI. The scans will be assessed for diagnostic quality of images, artifacts encountered, and the presence of additional contributory diagnostic information.

Arms:
arm 1: Intravenous administration of contrast agent Sulfur hexafluoride lipid-type A microspheres before performing contrast-enhanced ultrasound (CEUS). In pediatric patients, after reconstitution 0.03 mL per kg is administered intravenously. The weight-based dose of 0.03 mL per kg will be repeated one time during a single examination. Following each injection, an intravenous flush of 0.9% Sodium Chloride is injected. The study duration per subject will be approximately 15 minutes including the time to prepare the contrast agent and perform the CEUS, as well as the 60 minute monitoring period after the first and second injection of the contrast agent.

Interventions: intervention 1: Sulfur hexafluoride lipid-type A microspheres
Intervention descriptions: intervention 1: Injection of Sulfur hexafluoride lipid-type A microspheres (Lumason) contrast agent will be performed via the existing peripheral intravenous line using the FDA-recommended dose of 0.03 mg/kg.

Protocol document:
Prot_SAP_000.pdf: 
Title: 
Improved Diagnosis and Prognostication of Hypoxic Ischemic 
Injury in Neonates and Infants Using Contrast-Enhanced 
Ultrasound 
Short Title 
Contrast-Enhanced Ultrasound Evaluation of Hypoxic 
Ischemic Injury 
 
 
 
 
Drug Name(s): 
Sulfur hexafluoride lipid-type A microspheres (LumasonTM) 
FDA IND 
137921 
Regulatory Sponsor: 
Misun Hwang, MD 
eIRB Number 
IRB 18-014912 
Protocol Date: 
January 10, 2018 
 
Amendment 1 Date: 27 April 2018 
Amendment 3 Date: 11 March 2019 
Amendment 2 Date: 20 November 2018 
Amendment 4 Date: 10 October 2019 
Amendment 5 Date: 13 April 2020 
Amendment 6 Date: 11 January 2021  
Amendment 7 Date: 7 February 2022          Amendment 8 Date: 11 April 2022 
 
 
 
 
Sponsor-Investigator 
Misun Hwang, MD 
The Children’s Hospital of Philadelphia 
Department of Radiology 
Philadelphia, PA, 19104 
Phone 267.425.7110 
email: Hwangm@chop.edu  
 
 


2 
 
 
 
 
TABLE OF CONTENTS 
 
Abbreviations and Definitions of Terms .............................................................................. 4 
 
BACKGROUND INFORMATION AND RATIONALE ..................................................................... 11 
 
INTRODUCTION ........................................................................................................................................... 11 
 
NAME AND DESCRIPTION OF INVESTIGATIONAL PRODUCT OR INTERVENTION ........................................... 19 
 
COMPLIANCE STATEMENT .......................................................................................................................... 19 
 
STUDY OBJECTIVES ............................................................................................................................ 19 
 
PRIMARY OBJECTIVE (OR AIM) ................................................................................................................... 19 
 
SECONDARY OBJECTIVE (OR AIM) .............................................................................................................. 20 
 
INVESTIGATIONAL PLAN.................................................................................................................. 20 
 
GENERAL SCHEMA OF STUDY DESIGN ........................................................................................................ 20 
3.1.1 
Screening Phase ..................................................................................................................................... 20 
3.1.2 
Study Treatment Phase .......................................................................................................................... 20 
 
ALLOCATION TO TREATMENT GROUPS AND BLINDING ............................................................................... 21 
 
DURATION OF STUDY PARTICIPATION ........................................................................................................ 21 
 
TOTAL NUMBER OF STUDY SITES/TOTAL NUMBER OF SUBJECTS PROJECTED ............................................ 21 
 
STUDY POPULATION ................................................................................................................................... 21 
3.5.1 
Inclusion Criteria ................................................................................................................................... 21 
3.5.2 
Exclusion Criteria .................................................................................................................................. 21 
 
STUDY PROCEDURES ......................................................................................................................... 22 
 
SCREENING ................................................................................................................................................. 22 
 
STUDY TREATMENT PHASE ......................................................................................................................... 22 
4.2.1 
CEUS #1 ................................................................................................................................................ 24 
4.2.2 
CEUS #2 ................................................................................................................................................ 24 
 
CONCOMITANT MEDICATION ...................................................................................................................... 25 
 
RESCUE MEDICATION ADMINISTRATION .................................................................................................... 25 
 
SUBJECT COMPLETION/WITHDRAWAL ........................................................................................................ 25 
4.5.1 
Early Termination Study Visit ................................................................................................................ 25 
4.5.2 
Review of medical records from EPIC and/or other sources ................................................................. 25 
4.5.3 
Review of diagnostic images from the PACS (iSite Radiology or iSite Enterprise) ............................... 26 
 
STUDY EVALUATIONS AND MEASUREMENTS ........................................................................... 26 
 
STATISTICAL CONSIDERATIONS ................................................................................................... 26 
 
PRIMARY ENDPOINT ................................................................................................................................... 27 
 
SECONDARY ENDPOINTS ............................................................................................................................. 27 
 
STATISTICAL METHODS .............................................................................................................................. 27 
6.3.1 
Baseline Data ......................................................................................................................................... 27 
6.3.2 
Safety Analysis ....................................................................................................................................... 28 
 
SAMPLE SIZE AND POWER ........................................................................................................................... 28 
 
STUDY DRUG ......................................................................................................................................... 28 
 
DESCRIPTION .............................................................................................................................................. 28 
 
DOSING ....................................................................................................................................................... 29 


3 
 
 
INVESTIGATIONAL DRUG HANDLING AND ACCOUNTABILITY ..................................................................... 30 
 
SAFETY MANAGEMENT ..................................................................................................................... 30 
 
CLINICAL ADVERSE EVENTS ....................................................................................................................... 30 
 
ADVERSE EVENT REPORTING ..................................................................................................................... 31 
 
DEFINITION OF AN ADVERSE EVENT ........................................................................................................... 31 
 
DEFINITION OF A SERIOUS ADVERSE EVENT (SAE) .................................................................................... 31 
8.4.1 
Relationship of SAE to study drug or other intervention ....................................................................... 32 
 
IRB/IEC NOTIFICATION OF SAES AND OTHER UNANTICIPATED PROBLEMS .............................................. 32 
8.5.1 
Follow-up report .................................................................................................................................... 32 
 
NOTIFICATIONS OF SAES/IND SAFETY REPORTS TO THE FDA .................................................................. 33 
 
MEDICAL EMERGENCIES ............................................................................................................................. 33 
 
STUDY STOPPING RULES ............................................................................................................................. 33 
 
STUDY ADMINISTRATION ................................................................................................................. 34 
 
DATA COLLECTION AND MANAGEMENT ..................................................................................................... 34 
 
CONFIDENTIALITY ...................................................................................................................................... 34 
 
DATA AND SAFETY MONITORING PLAN ...................................................................................................... 35 
 
REGULATORY AND ETHICAL CONSIDERATIONS .......................................................................................... 35 
9.4.1 
Risk Assessment ..................................................................................................................................... 35 
9.4.2 
Potential Benefits of Trial Participation ................................................................................................ 36 
9.4.3 
Risk-Benefit Assessment ......................................................................................................................... 37 
 
RECRUITMENT STRATEGY ........................................................................................................................... 37 
 
INFORMED CONSENT AND HIPAA AUTHORIZATION ................................................................................... 37 
 
PAYMENT TO SUBJECTS/FAMILIES .............................................................................................................. 37 
 
PUBLICATION ....................................................................................................................................... 37 
 
REFERENCES ......................................................................................................................................... 37 
 
 
 


4 
 
ABBREVIATIONS AND DEFINITIONS OF TERMS 
 
 
 
 
 
 
AE 
Category B 
Drug 
 
CEUS 
CFR 
CHOP 
CT 
DSMP 
DWI 
 
Echo-PIV 
FDA 
HII 
HIPAA 
ICU 
IDS 
IND 
IV 
IRB 
LumasonTM 
LVT 
MRI 
ORC 
PET 
PI 
PIV 
PHI 
PTV 
RMS 
SAE 
TCEUS 
US 
PACS 
 
 
Adverse Event 
Animal reproduction studies have failed to demonstrate a risk to the fetus 
and there are no adequate and well-controlled studies in pregnant women 
Contrast-Enhanced Ultrasound 
Code of Federal Regulations 
Children’s Hospital of Philadelphia 
Computed Tomography 
Data Safety Monitoring Plan 
Diffusion-weighted magnetic resonance imaging (MRI DWI) is the use of 
specific MRI sequences as well as software that generates images from 
the resulting data 
Echocardiographic Particle Image Velocimetry 
Food and Drug Administration 
Hypoxic Ischemic Injury 
Health Insurance Portability and Accountability Act 
Intensive Care Unit 
CHOP Investigational Drug Service 
Investigational New Drug 
Intravenous 
Institutional Review Board 
FDA-approved for contrast-enhanced ultrasound; the investigational drug 
Left Ventricular Thrombus 
Magnetic Resonance Imaging 
The CHOP Office of Research Compliance 
Positron Emission Tomography 
Principal Investigator 
Particle Image Velocimetry 
Protected Health Information 
Particle Tracking Velocimetry 
Root Mean Square 
Serious Adverse Event 
Transfontanellar Contrast Enhanced Ultrasound 
Conventional Ultrasound Scan 
Picture Archiving and Communication System 
 
 
 
 
 
 
 
 


5 
 
 
ABSTRACT 
Context:   
Neonates presenting with neurologic symptoms require rapid, non-invasive imaging with 
high spatial resolution and tissue contrast. The purpose of this study is to evaluate brain 
perfusion using contrast-enhanced ultrasound CEUS in bedside monitoring of neonates 
and infants with hypoxic ischemic injury.  
Objectives: Two possible methods have been proposed to rapidly and accurately detect 
perfusion abnormalities:  
• Primary: Identify regions of interest in gray versus white matter to detect ratio of 
perfusion changes, in cases of diffuse white matter ischemia.   
• Secondary: Generate a microbubble velocity mapping technique to detect 
regions of ischemia.   
Study Design:  
Single site, open-label clinical study. 
Setting/Participants: 
Subjects in the neonatal or pediatric intensive care unit aged 1.5 years of age or 
younger with open fontanelles and suspected or diagnosed hypoxic ischemic injury. 
The study will be performed at Children’s Hospital of Philadelphia (CHOP). CHOP will 
be the only site of the study. 
Study Interventions and Measures:  
Contrast-enhanced ultrasound scan with a duration of approximately 20 minutes. 
Qualitative analysis with visual assessment and quantitative analysis of the acquired 
scans will be performed by the sponsor-investigator and reviewed by the second 
radiologist (co-investigator). The scans will be assessed for diagnostic quality of 
images, artifacts encountered, and the presence of additional contributory diagnostic 
information.  
 


6 
 
Protocol Synopsis 
 
Study Title 
Improved Diagnosis and Prognostication of Hypoxic Ischemic 
Injury in Neonates and Infants Using Contrast-Enhanced 
Ultrasound 
Funder 
Children’s Hospital of Philadelphia 
Clinical Phase 
Phase II 
Study Rationale 
Neonates presenting with neurologic symptoms require rapid, 
non-invasive imaging with high special resolution and tissue 
contrast. Magnetic resonance imaging (MRI) is currently the 
most sensitive and specific neuroimaging modality for 
evaluation of neonatal neurological diseases. This modality 
does come with several challenges in the neonatal 
population, namely the need to transport a possibly critically 
sick neonate to the MRI suite and the necessity of the 
neonate to remain still for a significant length of time, 
occasionally requiring sedation. Cranial ultrasound has 
provided neuroradiologists and clinicians with an invaluable 
neuroimaging modality that allows a rapid, bedside point of 
care evaluation without ionizing radiation. The major 
drawback of cranial ultrasound is its lower sensitivity and 
specificity for subtle/early lesions. Contrast enhanced 
ultrasound (CEUS) has the potential to improve sensitivity 
and specificity for a variety of neonatal neurological diseases 
and expand the indications for cranial ultrasound. 
 
There is no current diagnostic tool with high soft tissue 
contrast that can assess brain perfusion of neonates and 
infants at the bedside. Critically ill neonates cannot be 
transported easily down to the magnetic resonance suite, and 
even when transported, ultrafast sequences are performed 
(which lack perfusion imaging) due to their clinical condition. 
In the case of neonatal hypoxic ischemic injury, our 
preliminary evidence has shown that contrast-enhanced 
ultrasound (CEUS) of the brain delineates areas of injury, as 
the perfusion to the injured regions of the brain become 
altered. Extensive literature exists in regard to the value of 
perfusion imaging in brain injury, especially in terms of 
prognostication. Since the CEUS of the brain can be 
performed at bedside, serially if needed over the course of 
injury evolution, the technique can be of significant clinical 
value in caring for neonates and infants with brain injury. It will 
be important to validate the initial evidence from early CEUS 


7 
 
brain scanning studies, and establish standardized 
quantitative techniques to reduce interpretation variations and 
errors in the clinical research setting. 
 
There is a dire need to introduce better imaging tools such as 
contrast-enhanced ultrasound to the clinical setting that can 
detect HII at an early stage and prompt therapeutic 
implementation. In this regard, contrast-enhanced ultrasound 
(CEUS) enables safe, serial monitoring of dynamic 
quantification of brain perfusion at the bedside.  
 
Safety of intravenous use of Sulfur hexafluoride lipid-type A 
microspheres was based on evaluation of published literature 
involving use of LumasonTM in over 900 pediatric patients, as 
noted on the 2016 FDA product label. Non-fatal anaphylaxis 
was reported in one pediatric patient, but none in a neonate. 
Animal data of daily intravenous administration of Sulfur 
hexafluoride lipid-type A microspheres to rats (administered 
up to 10 times the recommended maximum human dose) and 
rabbits (administered up to 20 times the recommended 
maximum human dose) for 30 consecutive days and 14 
consecutive days, respectively, resulted in no toxicity to the 
fetus in animal studies, as noted on the 2016 FDA product 
label. Specifically pertaining to the use of Sulfur hexafluoride 
lipid-type A microspheres in brain imaging in pediatric 
patients, we expect a similar risk of adverse events. For the 
proposed study, the same pediatric dosage, route of 
administration, safety monitoring guidelines, and low 
mechanical index used for contrast ultrasound settings will be 
used. 
 
Study Objective(s) 
Primary  
• 
The primary objective of this study is to identify regions of 
interest in gray versus white matter to detect the ratio of 
perfusion changes, in cases of diffuse white matter 
ischemia.    
Secondary 
• 
The secondary objective of this study is to generate a 
microbubble velocity mapping technique to detect regions 
of ischemia. 
 
Test Article(s) 
(If Applicable) 
Sulfur hexafluoride lipid-type A microspheres (LumasonTM, 
Bracco Inc) is an FDA-approved ultrasound contrast agent 
which consists of active ingredients including Sulfur 


8 
 
hexafluoride (strength 60.7 mg in 1 mg), 
Distearoylphosphatidylcholine, DL- (strength 0.19 mg in 1 
mg), 1,2-Dipalmitoyl-Sn-Glycero-3-Phospho-(1’-Rac-
Glycerol), Sodium Salt (0.19 mg in 1 mg). Inactive ingredients 
include Polyethylene Glycol 4000 (strength 24.56 mg in 1 mg) 
and Palmitic Acid (0.04 mg in 1 mg). The Sulfur hexafluoride 
lipid microspheres are composed of SF6 (molecular weight 
145.9) gas in the core surrounded by an outer shell 
monolayer of phospholipids consisting of 1,2-Distearoyl-sn-
glycero-3-phosphocholine (DSPC) and 1,2-Dipalmitoyl-sn-
glycero-3-phosphoglycerol, sodium salt (DPPG-Na) with 
palmitic acid as stabilizer. Sulfur hexafluoride lipid-type A 
microspheres fall under Category B, that is, animal 
reproduction studies have failed to demonstrate a risk to the 
fetus and there are no adequate and well-controlled studies in 
pregnant women.1,2-Distearoyl-sn-glycero-3-phosphocholine 
(DSPC), with empirical formula C44H88NO8P, has a 
molecular weight of 790.6. 1,2-Dipalmitoyl-sn-glycero-3-
phospho-rac-glycerol sodium (DPPG-Na), with empirical 
formula C38H74 NaO10P, has a molecular weight of 745. 
 
Study Design 
 
Single site, open-label clinical study. 
Subject Population 
key criteria for 
Inclusion and 
Exclusion: 
Inclusion Criteria 
1. Males and females aged 1.5 years or younger with 
open fontanelles and known or suspected hypoxic 
ischemic injury 
2. Post menstrual age of 34 weeks or older  
3. Patient in the CHOP NICU or PICU 
4. Parental permission 
Exclusion Criteria 
1. Medical history of Lumason hypersensitivity 
2. Hemodynamic instability as defined by rapid escalation 
of cardiopulmonary support in the past 12-24 hours, as 
defined by the clinical care team including >1 intensive 
care physician not part of the study team 


9 
 
3. Pulmonary insufficiency as defined by FiO2 
requirements of >40% and/or subjects with pulmonary 
hypertension requiring nitric oxide 
 
Number Of Subjects  
 
200; CHOP will be the only site of the study. 
Study Duration 
The study duration per subject will be approximately 20 
minutes including the time to prepare LumasonTM contrast 
agent and perform the pre-contrast imaging and the CEUS, 
as well as the 60 minute monitoring period after the first and 
second injection of LumasonTM.  
 
CEUS will be performed at the time HII is first suspected or 
diagnosed. A second CEUS may be performed at short-term 
follow-up (approximately within 1-2 weeks from the first scan) 
for a total of two CEUS exams of 1 hour and 15-minute 
duration each.  
 
Study participation will be complete when the 60 minute 
monitoring period of the last CEUS performed is complete 
(after the first CEUS in patients who undergo one exam, or 
after the second CEUS is complete in patients who undergo 
two exams). 
 
Study Treatment 
 
CEUS has a total duration of 1 hour and 20 minutes: CEUS 
has a duration of approximately 20 minutes for the CEUS and 
60 minutes for post-examination monitoring of potential 
adverse events. 
 
Efficacy Evaluations 
There are no efficacy evaluations for this diagnostic study. 
The endpoints of this study are: 
 
Primary Endpoint 
• To qualitatively evaluate perfusion abnormalities in 
correlation with clinical MRI results. 
Secondary Endpoint 
• To quantitatively evaluate perfusion abnormalities in 
correlation with clinical MRI results. 
 


10 
 
Pharmacokinetic 
Evaluations 
There are no pharmacokinetic evaluations. 
Safety Evaluations 
All subjects entered into the study and receiving at least one 
injection of investigational drug will be included in the safety 
analysis. The frequencies of AEs by type, severity, and 
temporal relationship to the CEUS scan will be summarized. 
SAEs (if any) will be described in detail. 
Statistical And 
Analytic Plan 
Baseline and demographic characteristics will be summarized 
by standard descriptive summaries. All subjects entered into 
the study and receiving at least one injection of investigational 
drug will be included in the safety analysis. The frequencies of 
AEs by type, severity, and temporal relationship to the CEUS 
scan will be summarized. SAEs (if any) will be described in 
detail. Details of sample size and power calculations for this 
study are described in Section 6 of the protocol. 
DATA AND SAFETY 
MONITORING PLAN 
The safety monitoring for this study is the primary 
responsibility of the sponsor-investigator. Monitoring the 
safety outcomes following IV administration of the 
investigational drug will be conducted primarily by the 
Principal Investigator and/or specifically designated study 
personnel. An independent safety monitor will be designated 
to oversee the safety reports, and help adjudicate attribution 
of serious adverse events, should they occur. Regular 
meetings to discuss the outcomes of the study, and of the 
safety events, will be conducted by the study team. The 
occurrence of adverse events, serious adverse events and 
unanticipated events will be reported by the study team in 
accordance with federal and institutional guidelines, as 
outlined in Section 8 of this clinical study. 
 
Prior to study initiation, the Office of Research Compliance 
(ORC) will conduct a pre-trial monitoring visit, to assess trial 
readiness of the study staff. Once the pre-trial monitoring visit 
has been successfully completed, the ORC will also monitor 
the IND study on at least an annual basis. 
 


11 
 
 
Background Information and Rationale 
 
 Introduction 
 
Contrast-enhanced ultrasonography (CEUS) is a novel imaging technique in which gas-
filled microbubbles, smaller than red blood cells, generate increased signal due to the 
acoustic impedance mismatch. Injection of ultrasound contrast agents into blood 
increase the echogenicity, allowing enhanced visualization of a blood vessel. 
Ultrasound contrast agents have been approved for use in Europe for almost two 
decades. In the case of SonoVue (now called LumasonTM), a second-generation 
lipid/sulfur hexafluoride US contrast agent (Bracco, Milan, Italy), European Union 
approved its intravenous use in adults in 2001. The FDA in the United States just 
recently approved the use of LumasonTM for evaluation of focal hepatic lesions in 
pediatric population in 2016, and very recently (Jan 2017) approved its use in children 
for the evaluation of the urinary tract in pediatric patients with known or suspected 
vesicoureteral reflux. For the remainder of clinical applications, the ultrasound contrast 
agents are being used off-label in both Europe and the United States. All patients in this 
study will already be scheduled to have a CEUS study for clinical indications. This 
protocol focuses on imaging of hypoxic ischemic injury in neonates and infants. 
 
Unlike CT or MRI contrast agents, ultrasound contrast agents have no associated renal 
toxicity, and there is no need for accompanying ionizing radiation or sedation. The risk 
of adverse events is the lowest of all contrast agents available, with CT contrast being 
the highest (0.6%), followed by MRI contrast (0.0088%) and ultrasound contrast 
(0.0086%) [1]. Several studies detail the safety profile of ultrasound contrast agents in 
children and have shown minor adverse events including altered taste, tinnitus, light-
headedness, nausea [2-4]. One rare severe reaction in a child documented symptoms 
of generalized pruritus, nausea, hypotension with tachycardia initially then bradycardia 
[5]. Management in this instance consisted of oxygen, intravenous epinephrine, and 
fluids (0.9% normal saline) with resolution of symptoms in two hours. Treatment of both 
minor, mild, and severe adverse reactions post LumasonTM administration are the same 
as that of CT or MRI contrast agents. In comparison to CT or MRI contrast agents, 
however, ultrasound contrast agents have proven to be much safer in children over 
decades of its use to date (contrasting to approximately 15-20 adverse events per 2000 
children if CT contrast agent were to be used). 
 
Neonates presenting with neurologic symptoms require rapid, non-invasive imaging with 
high spatial resolution and tissue contrast. Magnetic resonance imaging (MRI) is 
currently the most sensitive and specific imaging modality for evaluation of neurological 
pathology. This modality does come with several challenges in the neonatal population, 
namely the need to transport a possibly critically sick neonate to the MRI suite and the 
necessity of the neonate to remain still for a significant length of time, occasionally 
requiring sedation. Cranial ultrasound has provided radiologists and clinicians with an 
invaluable imaging modality that allows rapid, bedside point of care evaluation without 
ionizing radiation. The major drawback of cranial ultrasound is its lower sensitivity and 
specificity for subtle/early lesions. Contrast enhanced ultrasound (CEUS) has the 


12 
 
potential to improve sensitivity and specificity for a variety of neuropathologies and 
expand the indications for cranial ultrasound. 
 
CEUS has several advantages over traditional ultrasonographic grey scale and color-
coded Doppler images. CEUS utilizes ultrasonographic contrast agents to allow for 
precise visualization of vasculature, determination of relative blood flow, and enhanced 
visualization of solid organs and lesions [6, 7]. Ultrasound contrast agents are gas-filled 
microbubbles that are injected into the systemic vasculature that appear echogenic on 
grey scale ultrasound [8, 9]. With respect to neuroimaging, CEUS not only provides a 
more accurate depiction of the cerebral macrovascular circulation but also has the 
ability to define regional cerebral blood flow, a surrogate marker of cerebral 
microcirculation. Early animal models of neonatal hypoxia in piglets have demonstrated 
that transcranial CEUS can be used to qualitatively track regional cerebral perfusion 
changes. Furthermore, CEUS measures of cerebral perfusion correlated with MRI [10]. 
There is, however, a paucity of published data on the subject of brain imaging using 
CEUS. This is due to a combination of lack of FDA approval and lack of familiarity with 
the technique amongst neonatal intensive care providers. Despite the favorable safety 
profile of ultrasound contrast agents, the institutional and governmental oversight as 
well as cumbersome consent procedures required for the off-label application of CEUS 
in the evaluation of neonatal brain conditions has limited its usage in this particular 
clinical setting throughout the United States. 
 
Few studies have examined imaging findings in transfontanellar contrast enhanced 
ultrasound (TCEUS). Kastler and colleagues performed one of the first studies of 
TCEUS in 2013 [11]. Kastler examined several neonates with suspected neurological 
conditions using TCEUS. Specifically, the authors looked for abnormal parenchymal 
enhancement patterns on arterial, venous, and delayed phases to define regions of 
abnormal perfusion and masses/lesions. TCEUS findings were then correlated with 
brain MRI [11]. Twelve neonates underwent TCEUS and MRI. Diagnostic accuracy 
between TCEUS and MRI was graded based on the following scale: No correlation for 
discordant findings; Good correlation for accurate diagnosis with an underestimate of 
the extent of the lesion; Excellent correlation for accurate diagnosis and satisfactory 
estimation of the extent of the lesion. Ten out of 12 neonates exhibited enhancement 
abnormalities on TCEUS. TCEUS successfully defined brain perfusion abnormalities 
(9/10 neonates) and several hypovascular (4/10 neonates) and avascular lesions (5/10 
neonates) which were felt to represent ischemic pathology [11].  
 
When compared with MRI findings, TCEUS findings suggesting regional ischemia were 
accurate in the majority of cases, with either good or excellent correlation (10/12 
neonates). In cases that were deemed to be discordant, TCEUS underestimated the 
extent of ischemic or hemorrhagic injury [11]. In this paper, calculated sensitivity and 
specificity for detecting pathological brain lesions in neonates was 88.9% and 66.6% 
respectively. For comparison, in larger samples of adults presenting after acute stroke, 
transcranial ultrasound has demonstrated a sensitivity and specificity for localizing 
areas of infarction ranging from 86-100% and 96-100% respectively [12, 13]. These 
figures are particularly impressive given the fact that adult examinations are limited by 


13 
 
significant acoustic impedance from the skull [11-13]. Thus, it is reasonable to postulate 
that sensitivity and specificity for detecting ischemic regions in the neonate may end up 
being more favorable than previously reported given the lack of acoustic impedance 
through the fontanelle. Further studies with larger samples sizes will of course be 
required to accurately determine sensitivity and specificity for a variety of neurological 
pathologies. No immediate or late adverse events were reported after TCEUS [11].  
 
An area of deep interest is the application of cranial CEUS to investigate brain perfusion 
characteristics in neonatal hypoxic ischemic injury, specifically, elucidating quantitative 
parameters and qualitative perfusion patterns in neonates with a variety of hypoxic 
injuries. With contrast quantification software it is possible to determine contrast 
enhancement kinetics. When a region of interest is identified a time intensity curve is 
generated allowing for the calculation of the wash in (time from injection to peak 
intensity of contrast), peak intensity (maximum value of contrast on the time intensity 
curve), half washout (time between peak intensity and time when half the enhancement 
has disappeared), and washout (the time from peak enhancement to when 
enhancement has completely disappeared) can be calculated. 
 
The area under the curve is calculated from the beginning of contrast enhancement to 
the end of washout which can be thought of as a surrogate for perfusion within that 
region of interest [14, 15]. Two quantitative methods are being developed by our group 
to rapidly and accurately detect perfusion abnormalities: 1) drawing regions of interest in 
gray versus the white matter to detect ratio of perfusion changes, in cases of diffuse 
white matter ischemia 2) generating a microbubble velocity mapping technique to detect 
regions of ischemia (unpublished data). With both qualitative and quantitative data, we 
believe that cranial CEUS can detect subtle ischemic injuries that cannot be detected 
with non-contrast cranial US that correlate well with gold standard MRI [14]. At Johns 
Hopkins, we initially started applying TCEUS for the evaluation of neonatal hypoxic 
ischemic injury. For extremely injured brain cases, nuclear scan (for diagnosis of brain 
death) instead of MRI may be performed as part of gold standard. Using both qualitative 
and quantitative parameters we have been able to characterize global perfusion 
abnormalities in neonates with different etiologies of hypoxic ischemic injury (see 
Preliminary Results section, below).  
 
The temporal evolution of perfusion following brain ischemia has been well studied in 
pediatric and adult patients [16-21]. A previous study on infants with hypoxic ischemic 
injury applied Doppler US to perform serial cerebral blood flow measurements and 
showed initial marked increase followed by decrease at 21 days in comparison to 
controls [18]. Another group evaluated infants with hypoxic ischemic injury with MR 
perfusion and also revealed similar results by noting lower cerebral blood flow in the 
basal ganglia and thalamus by the second week of life, likely reflective of decreased 
metabolic state of the central gray matter structures following irreversible brain injury 
[19]. 
 
Previous study with PET performed in infants with hypoxic ischemic encephalopathy 
showed decreased cerebral uptake at 2 weeks correlated with poor outcome at 2 years 


14 
 
[20]. Using PET, low cerebral blood flow has been shown during the first few days of life 
in two infants with basal ganglia injury [22]. The discrepant findings may be of clinical 
significance, as some infants with inadequate reperfusion in the acute post-ischemic 
phase may be correlated with increased neuronal cell death and poor long-term clinical 
outcome. Not only the hypoperfusion, but also continued or exaggerated post-ischemic 
hyperperfusion may cause brain injury: post-ischemic regional hyperperfusion to the 
basal ganglia and thalamus has been suggested to increase the vulnerability of 
metabolically active regions [22, 23]. An accurate delineation of perfusion changes 
following ischemia is important and may provide an additional therapeutic window of 
opportunity in infants with reduced or increased brain perfusion. 
 
Prior CEUS study on post-ischemic reperfusion in a piglet showed hyperperfusion 
during hypoxia and early resuscitation state, and revealed the dynamic perfusion 
changes during the first hours of injury [10]. In the histologically injured brains, 
increased CEUS quantification parameters including time to peak and area under the 
curve was seen in both the basal ganglia and whole brain at the time of injury and 
resuscitation to a greater degree compared to 7 hours after injury. These findings 
emphasize the need for monitoring dynamic perfusion changes during the first few 
hours following ischemia, and show the convenience and practicality of CEUS in 
studying temporal perfusion changes of neonatal brain ischemia. This study also 
revealed compromised cerebral perfusion with 100% oxygen treatment, with a decrease 
of peak intensity and area under the curve during and shortly after resuscitation, and 
suggested a suspected pathomechanism of oxygen induced reduction of perivascular 
nitric oxide production and resultant vasoconstriction resulting in reduced cerebral blood 
flow [24].  
 
In the near future, the investigative team anticipates that widespread use of contrast 
enhanced ultrasound will complement some of the conventional imaging modalities in 
specific patient populations.  Contrast enhanced ultrasound will be an important imaging 
modality among the various tools in the arsenal of pediatric diagnostic imaging. 
 
Preliminary Results: High Diagnostic Sensitivity of Contrast-Enhanced 
Ultrasound in Detection of Brain Injury 
 
All patients were scanned at Johns Hopkins for clinically indicated reasons which in 
these cases was suspected brain injury. Contrast-enhanced ultrasound and 
elastography were performed as part of the standard of care brain ultrasound 
examinations. The studies were obtained due to the inconvenience or inability of 
transporting the patients to the MRI scanner at the time due to the nature of their illness, 
support staff or support devices limitations. Figure 1 demonstrates that from the 
qualitative evaluation of CEUS exams the injury patterns as validated on MRI can be 
inferred. 


15 
 
Figure 1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fig 1. Top rows denote coronal brain CEUS exams of neonates with bottom rows 
representing corresponding coronal brain diffusion-weighted MRI (MRI DWI) images 
obtained hours after the CEUS exams. Figure 1A is of a 2-month-old male born at 35 
weeks gestational age, with trisomy 21 and associated laryngomalacia and 
tracheomalacia status post supraglottoplasty scanned for respiratory distress and 
suspected brain injury. The CEUS scan depicts a coronal slice through the brain at the 
level of the frontotemporal lobes and basal ganglia showing homogeneous perfusion 
without focal or diffuse perfusion abnormality. Figure 1B shows the corresponding coronal 
MRI DWI slice obtained hours after the CEUS exam confirming the absence of brain 
injury. Figure 1C is a coronal brain CEUS of a 2-week-old male born at 40 weeks 
gestational age, delivered via caesarian section due to arrest of descent. The newborn 
presented with hypoglycemia and seizures with multifocal perfusion abnormalities in the 
parietooccipital regions bilaterally confirmed on the corresponding coronal MRI DWI of the 
brain (Figure 1D) to have multifocal injuries in the parietooccipital regions. Figure 1E is of a 
3 day old male born at 35 weeks gestational age, with uncomplicated birth history who 
initially presented with seizures. Brain CEUS scan on Day 14, and brain MRI on Day 15, 
confirmed diffuse white matter injuries. Coronal CEUS brain images of the frontotemporal 
lobes showed generalized hyperperfusion. Comparison of coronal MRI DWI image in 
Figure 1F confirmed diffuse white matter injury. Figure 1G is a coronal CEUS scan of a 6-
month-old male 3 hours after he suffered a prolonged cardiac arrest of unknown etiology. 
The scan shows no significant perfusion to the brain except for few intracranial vessels. Of 
note, wash-out of had not occurred 30 minutes after contrast administration in the post 
cardiac arrest patient signifying extremely poor cerebral circulation. Unfortunately, the 6-
month-old died before any further imaging could be obtained or formal brain death 
evaluation was conducted. 
Passed away 30 
minutes after the 
CEUS exam 


16 
 
 
 
Figure 2. Figure 2A shows a coronal DWI brain image (top left) and corresponding 
CEUS image (top right) with region of interest (dotted red circle) placed in the left 
occipital lobe based on which microbubble wash-in graph (second row) and graph 
(bottom row) are derived. Note the irregular, inhomogeneous perfusion to the region. 
Figure 2B shows a coronal DWI brain image (top left) and corresponding CEUS image 
(top right) with region of interest (dotted red circle) placed in the right temporal lobe 
based on which microbubble wash-in graph (second row) and graph (bottom row) are 
derived. Note the double peak wash-in phase with a more regular, homogeneous 
perfusion to the region without significant variability in intensity as on Figure 2A. 
Notable differences include significantly quicker wash-in and higher area under the 
curve representative of total microbubble or blood volume in Figure 2B as compared to 
Figure 2A [15]. 


17 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3. Hypothetical Microbubble 
Velocity Map of the Brain. Cine (movie) 
clips of contrast scans will be analyzed to 
track the movement and velocity of an 
individual microbubble across hundreds of 
frames. The data will be used to generate 
a super-resolution velocity maps of the 
brain. Above is an example of how an  
individual microbubble can be tracked 
across three successive frames; the same 
microbubble as denoted by a dotted red 
circle is traced (arrows) from the left to mid 
to right temporally sequential images, 
each obtained within milliseconds of each 
other. The acquired information across 
hundreds of successive frames will be 
compiled to generate an image detailing 
regional velocity information, with the high 
speed denoting normal velocity (red) and 
low speed denoting abnormal velocity as 
in ischemia (blue). [Hwang et al, J 
Neuroimaging 2017, in press] 
A 
B 
C 
Figure 4: (a) Sample original clinical 
echocardiographic images containing 
ultrasound 
contrast 
agent; 
(b) 
Enhanced image of the bubble traces of 
the same image; and (c) Vectors sowing 
the corresponding velocity distribution 
and flow direction calculated using the 
new echo-PIV/PTV procedures. The 
color map shows the vorticity (angular 
velocity) distribution (Sampath et al., 
submitted for publication).  
Figure 5: (a) A sample instantaneous brain CEUS showing clear 
bubble traces; and (b) Distribution of RMS values of contrast over 
time. Bright (red) regions represent regions with rapid motions and 
dark (blue) regions represent stagnant zones with weak seeding 
and/or circulation.  


18 
 
Microbubble Velocity Mapping Technique 
As a further advancement to the existing CEUS quantification tools, we developed a 
novel microbubble velocity mapping technique for quantifying the instantaneous 
distributions of brain perfusion abnormalities that may serve as an alternative to 
diffusion-weighted sequence of MRI. 
 
Echocardiographic particle image velocimetry (echo-PIV) has been introduced in recent 
years to obtain quantitative velocity distributions from CEUS data [25, 26]. However, 
images acquired from routine clinical CEUS are prone to noise as well as spatially and 
temporally non-uniform distribution of tracers, adversely affecting the quality of data 
obtained using standard PIV algorithms. Consequently, we have developed an 
optimized procedure that integrates the image enhancement, PIV, and particle tracking 
velocimetry (PTV), which tracks individual tracers in time, to process clinical ultrasound 
images and obtain reliable, time-resolved, two-dimensional velocity distributions from 
non-uniformly distributed bubbles [27]. So far, to demonstrate the clinical value of the 
optimized procedures, the new tools have been used for analyzing cardiac contrast 
images acquired from four patients with left ventricular thrombus (LVT). A sample 
original contrast echo image is presented in Figure 4a, the enhanced traces are shown 
in Figure 4b, and the corresponding velocity and vorticity distributions calculated using 
the new PIV/PTV procedure is presented in Figure 4c. Time series of such velocity 
maps enable us to follow the formation, decay and fragmentation of the left-ventricular 
vortex (large swirling structure in Figure 4c) as it migrates towards the LV apex. The 
flow induced by this vortex is believed to play a key role in apical washing, an important 
factor in determining susceptibility to LVT formation. Furthermore, the velocity 
distribution enables direct quantification of the local washing around the LVT, which 
might be useful for assessing risk of LVT. A sample of a recently acquired brain CEUS 
is presented in Figure 5a. The bubble traces are clear and can be readily followed in 
time. In some areas, they move at high speed while in others, they are slow or even 
stagnant. As a preliminary indication of the advantages offered by mapping the velocity, 
Figure 5b shows the root mean square (RMS) of the contrast intensity over time. Bright 
areas indicate high speed flow regions, and dark regions indicate sites with weak or no 
flow. Our objective is to integrate the new procedures into routine CEUS imaging, 
providing bedside real time data on the velocity distribution, flow directions, and 
clearance rates of injured brains.  
 
The goal of the study is to perform contrast enhanced ultrasound (CEUS) for diagnosis 
and monitoring of brain injury in neonates and infants. The CEUS technique offers several 
advantages over MRI in that the technique can be performed at bedside, at lower cost, 
and without the need for sedation, and thus may serve as a valuable alternative to MRI in 
critically ill neonates and infants. Our preliminary evidence has shown that CEUS can 
delineate regions of brain injury due to associated altered perfusion. Extensive literature 
demonstrates the value of monitoring injury associated perfusion response in 
prognostication and therapy. This study will enroll neonates and infants in the neonatal 
intensive care unit or pediatric intensive care unit at Children’s Hospital of Philadelphia 
who are already scheduled to receive a standard of care grayscale brain ultrasound scan.  
 
A 
B 


19 
 
  Name and Description of Investigational Product or Intervention 
 
Sulfur hexafluoride lipid-type A microspheres (LumasonTM, Bracco Inc) is FDA-approved 
ultrasound contrast agent which consist of active ingredients including Sulfur 
hexafluoride (strength 60.7 mg in 1 mg), Distearoylphosphatidylcholine, DL- (strength 
0.19 mg in 1 mg), 1,2-Dipalmitoyl-Sn-Glycero-3-Phospho-(1’-Rac-Glycerol), Sodium 
Salt (0.19 mg in 1 mg). Inactive ingredients include Polyethylene Glycol 4000 (strength 
24.56 mg in 1 mg) and Palmitic Acid (0.04 mg in 1 mg). The sulfur hexafluoride lipid 
microspheres are composed of SF6 (molecular weight 145.9) gas in the core 
surrounded by an outer shell monolayer of phospholipids consisting of 1,2-Distearoyl-
sn-glycero-3-phosphocholine (DSPC) and 1,2-Dipalmitoyl-sn-glycero-3-
phosphoglycerol, sodium salt (DPPG-Na) with palmitic acid as stabilizer. Sulfur 
hexafluoride lipid-type A microspheres fall under Category B, that is, animal 
reproduction studies have failed to demonstrate a risk to the fetus and there are no 
adequate and well-controlled studies in pregnant women.1,2-Distearoyl-sn-glycero-3-
phosphocholine (DSPC), with empirical formula C44H88NO8P, has a molecular weight 
of 790.6. 1,2-Dipalmitoyl-sn-glycero-3-phospho-rac-glycerol sodium (DPPG-Na), with 
empirical formula C38H74 NaO10P, has a molecular weight of 745. In pediatric 
patients, after reconstitution 0.03 mL per kg is administered intravenously. The weight-
based dose of 0.03 mL per kg will be repeated twice during a single examination. 
Following each injection, an intravenous flush of 0.9% Sodium Chloride is injected.  
 
  Compliance Statement 
 
This study will be conducted in full accordance of all applicable Children’s Hospital of 
Philadelphia Research Policies and Procedures and all applicable federal and state 
laws and regulations including 45 CFR 46. All episodes of noncompliance will be 
documented. Research will also be conducted in full accordance with Food and Drug 
Administration (FDA) regulations 21 CFR 50 (Protection of Human Subjects), 21 CFR 
56 (Institutional Review Boards) and 21 CFR 312 (Investigational New Drug). 
 
The investigators will perform the study in accordance with this protocol, will obtain 
consent, and will report unanticipated problems involving risks to subjects or others in 
accordance with The Children’s Hospital of Philadelphia IRB Policies and Procedures 
and all federal requirements. Collection, recording, and reporting of data will be 
accurate and will ensure the privacy, health, and welfare of research subjects during 
and after the study.  
 
STUDY OBJECTIVES 
 
The purpose of this study is to evaluate brain perfusion using contrast enhanced 
ultrasound CEUS in neonates and infants with hypoxic ischemic injury. 
 
  Primary Objective (or Aim) 
 
The primary objective is to identify regions of interest in gray versus white matter to 
detect the ratio of perfusion changes, in cases of diffuse white matter ischemia. 


20 
 
 
  Secondary Objective (or Aim) 
 
The secondary objective is to generate a microbubble velocity mapping technique to 
detect regions of ischemia. 
 
INVESTIGATIONAL PLAN 
 
  General Schema of Study Design 
 
Patients scheduled for routine cranial ultrasound as part of clinical care for screening or 
monitoring of hypoxic ischemic injury will be recruited for the study. Following parental 
consent, the subject will undergo an investigational CEUS exam, which will be 
performed separately from any clinically indicated conventional ultrasound. The CEUS 
exam includes a pre-contrast ultrasound evaluation with FDA-approved technologies 
(e.g. gray-scale ultrasound, Doppler ultrasound, microvascular imaging). Pre-contrast 
ultrasound and CEUS duration is of approximately 20 minutes, followed by 60 minutes 
of monitoring.  
 
3.1.1 Screening Phase 
 
Potential subjects will be identified by referral from neonatologists of patients during the 
neonatal or pediatric intensive care unit stay for suspected, at risk of or diagnosed 
hypoxic ischemic injury and review of orders for cooling blankets, brain imaging and/or 
EEG exams in neonates.  
 
Before discussing participation in the study, we will confirm eligibility by reviewing the 
subjects’ medical records. Participation will be discussed between the PI and the 
referring neonatologist. Participation will be discussed with the parents/guardian by the 
neonatologist and/or radiologist after identification and confirmation of eligibility. 
Consent of the parents/guardian will be obtained prior to the exam by the PI, co-
investigator, or neonatologist co-investigator in a private setting. Questions will be 
answered by the PI, co-investigator, or referring neonatologist co-investigator.  
 
3.1.2 Study Treatment Phase 
 
Investigational CEUS scan will be performed separately from clinically indicated 
conventional US, in the ICU. CEUS will be performed at the time HII is first suspected or 
diagnosed. A second CEUS may be performed at short-term follow-up (approximately 
within 1-2 weeks from the first scan) for a total of two CEUS exams of 1 hour and 20-
minute duration each.  
Injection of LumasonTM contrast agent will be performed via the existing peripheral 
intravenous line or central line using the FDA-recommended dose of up to 0.03 mg/kg. 
Contrast-agent injection will be performed twice per CEUS scan to ensure image quality 
and test reproducibility. In the case of more stable patients without an IV line, a 
peripheral IV line will be started to conduct the investigational CEUS. Two bolus 


21 
 
injections will performed to evaluate for dynamic brain perfusion and several 2-minute 
cine clips as well as static images will be acquired during the exam. 
 
  Allocation to Treatment Groups and Blinding 
 
Not applicable. The CEUS scan will be interpreted by the sponsor-investigator only.  
 
 Duration of Study Participation 
 
The study duration per subject will be approximately 20 minutes including the time to 
prepare LumasonTM contrast agent, perform the pre-contrast imaging, and perform the 
CEUS, as well as the 60 minute monitoring period after the first and second injection of 
LumasonTM.  
 
CEUS will be performed at the time HII is first suspected or diagnosed. A second CEUS 
may be performed at short-term follow-up (approximately within 1-2 weeks from the first 
scan) for a total of two CEUS exams of 1 hour and 20-minute duration each.  
 
Study participation will be complete when the 60 minute monitoring period of the last 
CEUS to be performed (after the first CEUS in patients who undergo one exam, or after 
the second CEUS is complete in patients who undergo two exams). 
 
 
 Total Number of Study Sites/Total Number of Subjects Projected 
 
The study will be conducted at one site, The Children’s Hospital of Philadelphia. It is 
expected 200 subjects will be enrolled to produce 100 evaluable subjects. 
 
 Study Population 
 
3.5.1 Inclusion Criteria 
1. Males and females aged 1.5 years or younger with open fontanelles and known 
or suspected hypoxic ischemic injury 
2. Post menstrual age of 34 weeks or older  
3. Patient in the CHOP NICU or PICU 
4. Parental permission 
3.5.2 Exclusion Criteria 
1. Medical history of Lumason hypersensitivity 


22 
 
2. Hemodynamic instability as defined by rapid escalation of cardiopulmonary 
support in the past 12-24 hours, as defined by the clinical care team including >1 
intensive care physician not part of the study team 
3. Pulmonary insufficiency as defined by FiO2 requirements of >40% and/or 
subjects with pulmonary hypertension requiring nitric oxide 
In this regard, there is a published report of infusion of the investigational drug in twelve 
neonates, ranging from 26.9 to 41 weeks gestational age, including four premature 
infants from 26.9 to 29.5 weeks gestational age [11] as well as 3 additional premature 
subjects from unpublished data of the sponsor. The infusion of the investigational drug 
in these neonates and premature infants was safe, and imaging results of good quality. 
 
Brain CEUS has been performed successfully in intubated subjects without alteration of 
image quality or microbubble pharmacokinetics, as illustrated by the extensive evidence 
of intrasurgical brain CEUS after bone flap removal and/or transtemporal and 
transforaminal bone windows [28-33]. The specific ventilator settings and nitric oxide 
administration for pulmonary hypertension were chosen as exclusion criteria because 
they are indicative of unstable pulmonary status. These settings were determined 
according to clinical practice as communicated by Dr. John Flibotte (Co-medical 
Director of the Neonatal Neurocritical Care Program at CHOP), attending neonatologist 
with extensive clinical experience of HII patient care and supervision of clinical trials in 
this population.  
 
Subjects who do not meet all of the enrollment criteria may not be enrolled. Subjects will 
be excluded from the study, if in the judgement of the primary clinical team, they are too 
unstable to tolerate the procedure.  
 
Any violations of these criteria must be reported in accordance with IRB policies and 
procedures. 
 
STUDY PROCEDURES 
 
 Screening 
 
• 
Identify patients with suspected or diagnosed hypoxic ischemic injury 
• 
Review of medical records (for inclusion and exclusion criteria) 
• 
Discussion of the case with neonatologists and study team to determine 
eligibility 
• 
If subject is considered eligible, written consent can be obtained at this stage 
by PI, co-investigator, or neonatologist co-investigator 
• 
Coordinate cranial ultrasound schedule 
 
 Study Treatment Phase 
 


23 
 
Subjects enrolled to this clinical trial are anticipated to receive the study drug and have 
CEUS imaging conducted in the ICU setting. In this case, the study drug and US 
imaging unit will be brought to the ICU for study drug administration and imaging. Any 
clinically indicated non-contrast cranial ultrasounds be performed prior to contrast-
enhanced cranial ultrasound so as not to delay clinical care.  
 
Pre-injection documentation 
Prior to injection, Vital signs and baseline assessment will be recorded and documented 
from the medical record and clinically in-place monitoring. Baseline assessment will 
consist on neurological status as described in the clinical evaluations recorded in the 
medical chart prior to intervention. 
 
Pre-injection evaluation 
The exam will include a pre-contrast injection evaluation with FDA-approved 
technologies (e.g. gray-scale ultrasound, Doppler ultrasound, microvascular imaging) to 
assess anatomical structures and guide contrast evaluation. This will last approximately 
5 minutes.   
 
CEUS scan 
CEUS duration is of approximately 15 minutes.  
 
CEUS will be performed at the time HII is first suspected or diagnosed. A second CEUS 
may be performed at short-term follow-up (approximately within 1-2 weeks from the first 
scan) for a total of two CEUS exams of 1 hour and 20-minute duration each.  
 
Study participation will be complete when the 60-minute monitoring period of the last 
CEUS to be performed (after the first CEUS in patients who undergo one exam, or after 
the second CEUS is complete in patients who undergo two exams). 
 
For those neonates/infants not undergoing hypothermia therapy, the first CEUS scan 
will be performed at the time HII is first suspected or diagnosed and the second CEUS 
scan will be performed within 24 hours of clinically indicated MRI. It is part of standard 
of care to obtain a clinically indicated MRI as follow-up of diagnosed or suspected 
hypoxic ischemic injury. 
 
60-minute monitoring period 
The study team, who are composed of personnel trained in recognizing signs of infusion 
reaction, will conduct the 60 minute monitoring period and record any untoward reaction 
that may be related to the infusion of the contrast drug. The 60 minute monitoring post-
CEUS will be conducted by study team members. 
 
Vital signs will be recorded and documented from the medical record and clinically in-
place monitoring at 1) 30 minutes post-scanning, and 2) 60 minutes post-scanning. 
During monitoring, subjects will be assessed for rash, allergic reactions, anaphylaxis, 
and abrupt deviations from the subject's baseline hemodynamic parameters trend not 
related to medical intervention. 


24 
 
 
Adverse event assessment and documentation 
Adverse events will be recorded at 1) 60 minutes post-scanning when the monitoring 
period is completed, and 2) through 48 hours post-scanning, with documentation at 48 
hours post-scanning if no adverse event presents until this point. During the 48-hour AE 
assessment period, the following AEs of special interest will also be assessed: 
a. 
worsening cardiopulmonary status,  
b. 
worsening pulmonary hypertension, which may be suggested by new 
requirement of nitric oxide use, elevated pulmonary artery pressures on 
echocardiography, or differential limb pulse oximetry measurements,  
c. 
worsening neurological status, and 
d. 
serious gastrointestinal complications 
 
4.2.1 CEUS #1 
 
• 
Obtain consent prior to scheduled CEUS exam (if not previously obtained by, 
PI, co-investigator, or neonatologist co-investigator) 
• 
Record vital signs and baseline assessment prior to injection of contrast 
• 
Perform pre-contrast injection evaluation. 
• 
CEUS scan performed at the time HII is first suspected or diagnosed 
• 
Monitor subject for 60 minutes following the CEUS scan for documentation 
and treatment of potential adverse events 
• 
Documentation of any adverse events through 48 hours post-scanning 
 
4.2.2 CEUS #2 
 
A second CEUS may be performed at short-term follow-up (approximately within 1-2 
weeks from the first scan) for a total of two CEUS exams of 1 hour and 20-minute 
duration each.  
 
• 
Record vital signs and baseline assessment prior to injection of contrast 
• 
Perform pre-contrast injection evaluation. 
• 
CEUS scan performed on the same day of MRI exam  
• 
Monitor patient for 60 minutes following the scan for documentation and 
treatment of potential adverse events 
Documentation of any adverse events through 48 hours post-scanning 
Study participation will be complete when the 60-minute monitoring period of the last 
CEUS to be performed (after the first CEUS in patients who undergo one exam, or after 
the second CEUS is complete in patients who undergo two exams). 
 
The study team will not use sedation or general anesthesia to conduct the research 
CEUS scans. The lowest mechanical index (MI) possible will be implemented in both 
CEUS, with a maximum MI value <0.2. 
 
The results of the CEUS imaging will be collected for research purposes only. The 
results of the CEUS will not be used to direct clinical care decisions, without 


25 
 
confirmation of diagnosis by another medically established diagnostic product or 
procedure 
 
 Concomitant Medication 
 
No concomitant medications will be recorded, with the exception of rescue medications, 
as noted below. 
 
 Rescue Medication Administration 
 
All the rapid response equipment and resuscitation staff are readily available 24/7 in the 
intensive care unit setting at Children’s Hospital of Philadelphia. In the rare event that a 
significant allergic reaction occurs, or anaphylaxis results following injection of 
LumasonTM, standard clinical medication to treat the subject will be administered. For 
presumed allergic reactions, medications may include intravenous diphenhydramine 
and bolus corticosteroids (prednisolone), based on clinical care. For anaphylaxis, 
medications may include epinephrine as well as fluid and oxygen administration for 
emergency treatment, based on clinical care decision making and on the severity of 
symptoms. Any severe allergic or anaphylactic reaction will be reported to both the IRB 
and the FDA. 
 
 Subject Completion/Withdrawal 
 
Subjects’ families may withdraw their child from the study at any time without prejudice 
to their child’s care. A study investigator may withdraw a subject to protect the subject 
for reasons of safety or for administrative reasons. It will be documented whether or not 
each subject completes the clinical study. If the Investigator becomes aware of any 
serious, related adverse events after the subject completes or withdraws from the study, 
the adverse events will be recorded and reported. 
 
4.5.1 Early Termination Study Visit 
 
A subject may be withdrawn by a parent prior to or during a CEUS scan, provided the 
60 minute monitoring is completed after injection of the investigational drug.  The CEUS 
examination will be terminated if there is a deterioration in the subject’s clinical status 
during imaging. 
 
4.5.2 Review of medical records from EPIC and/or other sources 
 
• 
Date of birth 
• 
Weight 
• 
Clinical diagnosis 
• 
Treatment history (medications, chemotherapy, antibiotics, steroids)  
• 
Surgical history  
• 
Pathology report 
 


26 
 
4.5.3 Review of diagnostic images from the PACS (iSite Radiology or iSite 
Enterprise) 
 
• 
Review of cranial ultrasound and CEUS images (iSite) 
• 
Review of available MRI images 
 
STUDY EVALUATIONS AND MEASUREMENTS 
 
The results of the pre-contrast injection and CEUS imaging will be collected for research 
purposes only. The results of the CEUS will not be used to direct clinical care decisions, 
without confirmation of diagnosis by another medically established diagnostic product or 
procedure. 
 
Qualitative analysis 
Visual rating by 2 teams consisting of primary investigator and second radiologist (co-
investigator). Each scan will be rated for diagnostic quality and qualitative rating of 
cranial perfusion. The visual rating scale used will be: 0 (absent flow), 1 (decreased 
flow), 2 (normal flow), 3 (increased flow).  
 
Quantitative analysis 
Qontrast (Bracco Diag Inc., 510K regulatory status granted in 2004) contrast 
quantification software or similar software will be used to analyze the obtained CEUS 
scans. For each scan, wash-in and wash-out curves will be generated to quantify the 
rate of wash-in, time to peak intensity, peak intensity, and area under the curve. 
 
The CEUS scans will be interpreted by the sponsor-investigator, and a second 
interpretation by a “second reader” in Radiology, who is part of a group of radiologists 
with sufficient training and expertise to read CEUS scans. 
 
Monitoring After Investigational Drug Administration: 
 
The monitoring post-administration will encompass 60 minutes, during which time the 
subject will be observed for the occurrence of infusion reactions. Any concern by the 
investigative team or attending staff for severe allergic reaction or anaphylaxis will be 
managed as all similar reactions are managed as part of clinical care, with close 
observation and treatment as clinically indicated, which may include diphenhydramine, 
corticosteroids, fluids, oxygen and epinephrine. Post-infusion reactions will be recorded 
and reported following established IRB and FDA reporting guidelines. 
 
STATISTICAL CONSIDERATIONS 
 
Up to 100 evaluable patients should be adequate for assessment of clinical feasibility 
and optimization of protocol. Qualitative analysis will be used as detailed above. 
Quantitative analysis will be performed by drawing a region of interest in brain segments 
and compared to control subjects (those with suspected or at risk of HII but turns out to 
be normal on imaging and clinical evaluation) using non-parametric Wilcoxon testing. 
 


27 
 
The study will optimize current techniques in the application of contrast ultrasound. For 
instance in brain imaging, the optimal plane in which to obtain the contrast perfusion 
kinetics curve is suggested but not established. The region of interest placement for 
perfusion kinetics quantification is also suggested by few publications, but not 
established as the standard practice. All this is part of the optimization process that is 
pivotal to improving the current contrast ultrasound techniques.  
 
Specific analysis plan consists of both qualitative and quantitative assessment in which 
description of focal perfusion deficit or lesion, for instance, is reported in correlation with 
clinical information. Perfusion abnormalities will be qualitatively evaluated (absent – 0, 
mild hypoperfusion – 1, normal – 2, hyperperfusion – 3) and quantitatively (region of 
interest placed on brain regions for acquisition of wash-in curves using QLab or 
Qontrast or other similar software). These abnormalities will be correlated to MRI 
findings as gold standard, non-contrast ultrasound, and clinical information (neurologic 
exam, hemodynamics, behavioral outcomes) between serial exams. For extremely 
injured brain cases, nuclear scan (for diagnosis of brain death) instead of MRI may be 
performed as part of gold standard 
 
The sample size will be based on the available cases, and based on the current number 
of patients presenting to Children’s Hospital of Philadelphia neonatal or pediatric 
intensive care unit with clinical suspicion for hypoxic ischemic injury. It is estimated that 
the study will achieve a sample size of up to 50 neonates and infants per year for up to 
two years.   
 
Baseline and demographic characteristics will be summarized using descriptive 
measures. Statistical analysis will consist of comparing diagnostic tests, in this case 
conventional grayscale ultrasound and contrast ultrasound, on each patient using the 
McNemar’s test. Differences between perfusion before and after resuscitation (in the 
setting of hypoxic ischemic injury) will be evaluated with the Wilcoxon signal rank test 
for related samples. 
 
 Primary Endpoint 
 
Qualitative analysis of perfusion abnormalities in correlation with clinical MRI results. 
 
 Secondary Endpoints 
 
Quantitative analysis of perfusion abnormalities in correlation with clinical MRI results. 
This will be achieved by quantitative analysis, identifying regions of interest and 
generating time intensity curves.  
 
 Statistical Methods 
 
6.3.1 Baseline Data 
 


28 
 
Baseline and demographic characteristics will be summarized by standard descriptive 
summaries. 
 
6.3.2 Safety Analysis 
 
All subjects entered into the study and receiving at least one injection of investigational 
drug will be included in the safety analysis. The frequencies of AEs by type, severity, 
and temporal relationship to the CEUS scan will be summarized. SAEs (if any) will be 
described in detail. 
 
Adverse events will be recorded at 1) 60 minutes post-scanning when the monitoring 
period is completed, and 2) through 48 hours post-scanning, with documentation at 48 
hours post-scanning if no adverse event presents until this point. 
 
 Sample Size and Power 
 
Power analyses indicate that the study is sufficiently powered to detect group perfusion 
differences. For example, given a basal ganglia PE mean value in the control group of 
1.37 (SD = 0.23) and in the patient group of 0.96 (0.09), with an alpha p = 0.05, power 
to detect group differences with a sample of 25 per group approaches 1.00. As another 
example, given a peak cortical perfusion mean value in the control group of 2.46 (SD = 
1.96) and in the patient group of 0.69 (0.20), with an alpha p = 0.05, power to detect 
group differences with a sample of 50 per group approaches 0.84. 
 
Given a sample of about 100 imaged patients, pilot data suggest that approximately 
one-half will show abnormal perfusion, thus providing a final sample of approximately 50 
patients with abnormal perfusion and 50 patients with normal perfusion. The above 
sample sizes were used to compute the power to detect group differences in the ratio of 
basal ganglia to cortex perfusion, using means and standard deviations obtained from 
the pilot sample (see Preliminary Data).  
  
Power analyses indicate that the study is sufficiently powered to detect group perfusion 
differences. For example, given a basal ganglia PE mean value in the control group of 
1.37 (SD = 0.23) and in the patient group of 0.96 (0.09), with an alpha p = 0.05, power 
to detect group differences with a sample of 50 per group approaches 1.00. As another 
example, given a peak cortical perfusion mean value in the control group of 2.46 (SD = 
1.96) and in the patient group of 0.69 (0.20), with an alpha p = 0.05, power to detect 
group differences with a sample of 50 per group approaches 0.85. 
 
An interim statistical analysis may be considered to review image quality and 
comparisons between the results of each subject’s CEUS scans and available clinical 
US exams, to better inform the number of subjects required for statistical significance.   
 
STUDY DRUG 
 
 Description 


29 
 
 
LumasonTM is currently FDA approved for use in the pediatric population for 
echocardiography and evaluation of focal hepatic lesions, and very recently (Jan 2017) 
approved for use in children for the evaluation of the urinary tract in pediatric patients 
with known or suspected vesicoureteral reflux. Previously, the presence of cardiac 
shunts was a contraindication for its use, but this was recently cancelled by the FDA as 
of December 2016. However, for this clinical study, the FDA has recommended that 
subjects with significant PDA and right-to-left cardiac shunts be excluded from study 
participation. Ultrasound contrast agents have been approved for use in Europe for 
almost two decades. In the case of SonoVue (now called LumasonTM), a second-
generation lipid/sulfur hexafluoride US contrast agent (Bracco, Milan, Italy), the 
European Union approved its intravenous use in adults in 2001. The FDA in the United 
States just recently approved the use of LumasonTM for evaluation of focal hepatic 
lesions in the pediatric population in 2016. Through decades of clinical utilization of 
ultrasound contrast agents, there are established recommended doses for the 
intravenous route of administration. Recommended intravenous dose for LumasonTM is 
weight-based, 0.03mL/kg as an intravenous injection, up to a maximum of 2.4mL per 
injection. As an example, for the intended study population it may be roughly estimated 
that the maximum weight of an infant 1 year of age may be up to approximately 10 kg, 
for a maximum dose of 0.30 mL. Two injections per exam will be performed. 
 
 Dosing 
 
Through decades of clinical utilization of ultrasound contrast agents, there are 
established recommended doses for the intravenous route of administration. 
Recommended intravenous dose for LumasonTM is weight-based, 0.03mL/kg as an 
intravenous injection, up to a maximum of 2.4mL per injection. As an example, for the 
intended study population it may be roughly estimated that the maximum weight of an 
infant 1 year of age may be up to approximately 10 kg, for a maximum dose of 0.30 mL. 
Two injections per exam will be performed. Since investigational findings are subject to 
pre-analytic variability, performing two contrast-agent injections facilitates validation of 
findings if reproducibility is found. No increased risk was found at initial experience at 
Johns Hopkins Hospital, where 5 of 10 patients required double injection of contrast-
agent. Clinical cases at Johns Hopkins Hospital and The Children’s Hospital of 
Philadelphia have required double injection due to variable factors such as microbubble 
trapping within intravenous tubing. Adverse effects are not dose-dependent, thus risk is 
not increased by modifying the timing of interventions or increasing the number of 
contrast-agent injections. 
 
Even though the initial dosing of investigational drug for this study is 0.03 mL/kg, it is 
possible that the optimal dose for CEUS imaging of hypoxic ischemic injury may be less 
than 0.03 mL/kg. Since there would be no apparent safety concern regarding the 
administration of a lower dose of the investigational drug, the study team would proceed 
with a lower dose administration, if initial imaging studies suggest that a dose less than 
0.03 mL/kg may provide more optimal imaging results. Therefore, the study team 
proposes that a dose range of 0.01-0.03 mL/kg be considered for dose optimization of 


30 
 
the initial subjects, as indicated. Based on the published report of safe infusion of the 
investigational drug in four premature infants from 26.9 to 29.5 weeks gestational age 
[11] as well as 3 additional premature subjects from unpublished data of the sponsor, 
this dose range seems appropriate. For each subject, the dose per injection, as well as 
the total dose delivered, will be recorded in the study file for each administration of 
study drug. The expectation is that the intravenous injection of this ultrasound contrast 
agent will permit noninvasive, non-ionizing delineation of physiology and 
pathophysiology with higher resolution and accuracy than conventional ultrasound 
techniques. Since preliminary studies at Hopkins showed that half of neonates required 
two injections of contrast agent to achieve evaluable CEUS images, two injections will 
be performed per CEUS scan in all subjects to ensure adequate image quality and 
reproducibility. 
 
 Investigational Drug Handling and Accountability 
 
Bulk supplies of drug will be directly shipped to IDS, by drug supplier. On an as-needed 
basis, only 1 box of either 5 or 20 vials, will be ordered from IDS by the principal 
investigator. The 1 box supply will be located in a storage unit, inside the Ultrasound 
Suite, with access limited to study personnel and dedicated Ultrasound personnel. 
 
The investigational imaging drug, LumasonTM, will be maintained as a separate supply 
from the Radiology Departments and central supply’s LumasonTM that is used for clinical 
care purposes. Specifically, the investigational drug LumasonTM for this IND will be 
physically segregated from the clinical use LumasonTM, and stored in a location that 
identifies the study drug as “Investigational Drug LumasonTM, For IND Research Only” 
so that all Radiology personnel know to limit the use of the investigational drug 
LumasonTM for study in this particular IND research exclusively.   
 
The investigational drug LumasonTM will be labeled according to FDA regulations, and 
identified as for IND research use only. The study team will use the investigational drug 
exclusively for the purposes of this IND study, and will not distribute or administer the 
investigational drug to persons not participating in the clinical study.  
 
The study team will ensure that each LumasonTM vial used in the IND clinical trial is 
entered into an investigational drug log that contains at least the following details of 
each vial: Lot #number and expiration date. For each subject, the LumasonTM lot 
#number, expiration date, # of vials used, disposition of unused LumasonTM, and 
discard procedure, will be recorded as part of the study record. Expired lots of 
LumasonTM will not be used in the clinical trial, and will be discarded. 
 
SAFETY MANAGEMENT 
 
 Clinical Adverse Events 
 
Clinical adverse events (AEs) will be monitored throughout the study. Subjects enrolled 
to this clinical trial are anticipated to receive the study drug and have CEUS imaging 
conducted in the ICU setting. In this case, the study drug and US imaging unit will be 


31 
 
brought to the ICU for study drug administration and imaging. CEUS has a duration of 
approximately 20 minutes. The 60 minute monitoring post-CEUS will be conducted by 
the study team members. The study team, who are composed of personnel trained in 
recognizing signs of infusion reaction (rash, allergic reactions, anaphylaxis) will conduct 
the 60 minute monitoring period and record any untoward reaction that may be related 
to the infusion of the contrast drug. Adverse events will be recorded at 1) 60 minutes 
post-scanning when the monitoring period is completed, and 2) through 48 hours post-
scanning, with documentation at 48 hours post-scanning if no adverse event presents 
until this point. All adverse events suspected of being related to study drug infusion will 
be reported to the regulatory authorities. 
 
In addition to assessing each patient after dosing, after 10 subjects, a complete safety 
analysis to determine the safety of continuing the study will be performed. 
 
 Adverse Event Reporting 
 
Unanticipated problems related to the research involving risks to subjects or others that 
occur during the course of this study (including SAEs) will be reported to the IRB in 
accordance with CHOP IRB SOP 408: Unanticipated Problems Involving Risks to 
Subjects. AEs that are not serious but that are notable and could involve risks to 
subjects will be summarized in narrative or other format and submitted to the IRB at the 
time of continuing review.  
 
AEs will be recorded and graded per the International Neonatal Consortium (INC) 
Neonatal AE Terminology. 
 
 
 Definition of an Adverse Event 
 
An adverse event is any untoward medical occurrence in a subject who has received an 
intervention (drug, biologic, or other intervention).  The occurrence does not necessarily 
have to have a causal relationship with the treatment.  An AE can therefore be any 
unfavorable or unintended sign (including an abnormal laboratory finding, for example), 
symptom, or disease temporally associated with the use of a medicinal product, whether 
or not considered related to the medicinal product. 
 
All AEs (including serious AEs) will be noted in the study records and on the case report 
form with a full description including the nature, date and time of onset, determination of 
non-serious versus serious, intensity (mild, moderate, severe), duration, causality, and 
outcome of the event. Adverse events will be recorded at 1) 60 minutes post-scanning 
when the monitoring period is completed, and 2) through 48 hours post-scanning, with 
documentation at 48 hours post-scanning if no adverse event presents until this point. 
 
 Definition of a Serious Adverse Event (SAE) 
 


32 
 
An SAE is any adverse drug experience occurring at any dose that results in any of the 
following outcomes: death, a life-threatening event (at risk of death at the time of the 
event), requires inpatient hospitalization or prolongation of existing hospitalization, a 
persistent or significant disability/incapacity, or a congenital anomaly/birth defect in the 
offspring of a subject.   
 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug event when, based upon 
appropriate medical judgment, they may jeopardize the subject and may require 
medical or surgical intervention to prevent one of the outcomes listed in this definition. A 
distinction should be drawn between serious and severe AEs.  A severe AE is a major 
event of its type.  A severe AE does not necessarily need to be considered serious.  For 
example, nausea which persists for several hours may be considered severe nausea, 
but would not be an SAE.  On the other hand, a stroke that results in only a limited 
degree of disability may be considered a mild stroke, but would be an SAE.  
 
8.4.1 Relationship of SAE to study drug or other intervention 
 
The relationship of each SAE to the study intervention should be characterized using 
one of the following terms in accordance with CHOP IRB Guidelines: definitely, 
probably, possibly, unlikely or unrelated.  
 
 IRB/IEC Notification of SAEs and Other Unanticipated Problems 
 
The Investigator will promptly notify the IRB of all on-site unanticipated, serious Adverse 
Events that are related to the research activity. Other unanticipated problems related to 
the research involving risk to subjects or others will also be reported promptly. Written 
reports will be filed using the eIRB system and in accordance with the timeline below. 
External SAEs that are both unexpected and related to the study intervention will be 
reported promptly after the investigator receives the report. 
 
Type of Unanticipated 
Problem 
Initial Notification  
(Phone, Email, Fax) 
Written Report 
Internal (on-site) SAEs 
Death or Life Threatening  
24 hours 
Within 2 calendar days 
Internal (on-site) SAEs 
All other SAEs 
7 days 
Within 7 business days 
Unanticipated Problems 
Related to Research 
7 days  
Within 7 business days 
All other AEs 
N/A 
Brief Summary of 
important AEs may be 
reported at time of 
continuing review 
 
8.5.1 Follow-up report 
 


33 
 
If an SAE has not resolved at the time of the initial report and new information arises 
that changes the investigator’s assessment of the event, a follow-up report including all 
relevant new or reassessed information (e.g., concomitant medication, medical history) 
should be submitted to the IRB. The investigator is responsible for ensuring that all SAE 
are followed until either resolved or stable.  
 
 Notifications of SAEs/IND Safety Reports to the FDA 
 
Unexpected fatal or life-threatening adverse events that are related to the study drug, 
will be reported to FDA as soon as possible but no later than 7 calendar days following 
the sponsor’s initial receipt of the information. 
 
Unexpected serious adverse events that are related to the study drug but not fatal or 
life-threatening, will be reported to FDA as soon as possible but no later than within 15 
calendar days following the sponsor’s initial receipt of the information. 
 
Follow-up reporting: Any relevant additional information obtained by the sponsor that 
pertains to a previously submitted IND safety report will be submitted as a Follow-up 
IND Safety Report. Such report will be submitted as soon as the information is 
available, but no later than 15 calendar days after the sponsor receives the information. 
 
All other adverse events, will be reported to the FDA at or by the time of the Annual 
Report. 
 
 Medical Emergencies 
 
Any medical emergencies that develop following injection of the investigational drug will 
be managed according to clinical care. See protocol Section 4.4 for a more complete 
description of clinical care management for serious adverse events following infusion of 
the investigational drug.   
 
 Study Stopping Rules 
 
The study will be stopped for image futility, if non-diagnostic imaging is obtained in the 
first 3 subjects. 
 
Affected neonates and infants with hypoxic ischemic injury have substantially increased 
mortality and long-term neurological sequelae. There are some circumstances which 
potentially may arise, requiring temporary study stop. The FDA requires that the study 
be stopped for all patients (to allow for review of the protocol and procedures based on 
study related events) after one episode of anaphylaxis or death or other serious adverse 
event, regardless of relation to study drug. Therefore, anaphylaxis or death or another 
SAE will prompt a temporary stop to formally discuss the event with the FDA. As such 
an event (if deemed unrelated) may not meet the prompt reporting criteria for the IRB, 
the IRB will be notified as applicable (in accordance with CHOP IRB SOP 408). The 


34 
 
study will proceed only with documented concurrence of the FDA (and the IRB, as 
applicable).  
 
The occurrence of two non-fatal SAEs directly related to use of the study drug or one 
death directly attributed to use of the study drug will stop the study. 
 
STUDY ADMINISTRATION 
 
 Data Collection and Management 
 
Confidentiality: All subjects will be assigned a number unrelated to their medical record 
number and this will be kept in a master list. All of the data collected will be recorded 
using the respective “research number” to maintain anonymity. Only the master list will 
contain patient identifiers and a link to the research-specific code, and the data 
collection sheet will not contain patient identifiers.  
 
Security:  All files (master list and data collection sheet) will be password protected and 
will be stored on a password protected computer at CHOP on the secure storage 
network on the secure Hospital server. A password protected excel spreadsheets will be 
used for data collection. The paper scoresheets will be identifiable only by the coded # 
of each subject and will be stored in a binder in a locked cabinet located in the study 
coordinator's office. The only way we will use to transfer information between co-
investigators will be [send secure] emails using the study members @email.chop.edu 
account. These e-mails will be only accessed from CHOP network computers and will 
be erased after the download of the password-protected excel sheet. All computers will 
meet CHOP IT Policy A-3-6: Acceptable Use of Technology Resources. 
 
Anonymization, de-identification, or destruction: After the study is finalized (results 
published in a scientific journal), the master list containing the reference to PHI will be 
archived in accordance with FDA and CHOP requirements. De-identified scoresheets 
and data gathered from the study will be archived in a password-protected folder on the 
primary computer of the P.I. These data will be destroyed only after a period of time 
compliant with federal and institutional guidelines.  
 
 Confidentiality 
 
All data and records generated during this study will be kept confidential in accordance 
with Institutional policies and HIPAA on subject privacy and that the Investigator and 
other site personnel will not use such data and records for any purpose other than 
conducting the study. Patient names will be removed from images for use in the 
educational setting. 
   
No identifiable data will be used for future study without first obtaining IRB approval. 
The investigator will obtain a data use agreement between the provider (the PI) of the 
data and any recipient researchers (including others at CHOP) before sharing a limited 
dataset (PHI limited to dates and zip codes).  
 


35 
 
 Data and Safety Monitoring Plan 
 
The safety monitoring for this study is the primary responsibility of the sponsor-
investigator. Monitoring the safety outcomes following IV administration of the 
investigational drug will be conducted primarily by the Principal Investigator. An 
independent safety monitor will be designated to oversee the safety reports, and help 
adjudicate attribution of adverse events, should they occur. Regular meetings to discuss 
the outcomes of the study, and of the safety events, will be conducted by the study 
team. The occurrence of adverse events, serious adverse events and unanticipated 
events will be reported by the study team in accordance with federal and institutional 
guidelines, as outlined in Section 8 of this clinical study. 
 
Prior to study initiation, the Office of Research Compliance (ORC) will conduct a pre-
trial monitoring visit, to assess trial readiness of the study staff. Once the pre-trial 
monitoring visit has been successfully completed, the ORC will also monitor the IND 
study on an annual basis.    
 
 Regulatory and Ethical Considerations 
 
9.4.1 Risk Assessment 
 
The pre-contrast scan is non-invasive and poses risk no greater than minimal. The 
potential risks associated with contrast-enhanced ultrasonography have been 
extensively studied, and the risk associated with the technique is less than that of CT or 
MRI contrast agents. The risk of adverse events is the lowest of all contrast agents 
available, with CT contrast being the highest (0.6%), followed by MR contrast (0.0088%) 
and ultrasound contrast (0.0086%) [1]. Further studies detail the safety profile of 
ultrasound contrast agents in children and have shown minor adverse events including 
nausea, tinnitus, lightheadedness, altered taste sensation [3-5]. Serious 
cardiopulmonary reactions, including fatalities, have occurred uncommonly in adults that 
had complex comorbidities. Risk for these reactions may be increased among patients 
with unstable cardiopulmonary conditions. 
 
One documented severe reaction in a child documented symptoms of generalized 
pruritus, nausea, hypotension with tachycardia initially then bradycardia [6]. 
Management in this instance consisted of oxygen, intravenous epinephrine, and fluids 
(0.9% normal saline) with resolution of symptoms in two hours. Treatment of both minor, 
mild, and severe adverse reactions post LumasonTM administration are the same as that 
of CT or MRI contrast agents. In comparison to CT or MRI contrast agents, however, 
ultrasound contrast agents have proven to be much safer in children with only one 
serious adverse event over decades of its use to date (contrasting to approximately 15-
20 adverse events per 2000 children if CT contrast agent were to be used). No serious 
adverse event has been reported in a neonate since its clinical use in this population. 
Animal studies on its toxicity profile also validate no fetal toxicity and the ultrasound 
contrast agent belongs to category B.  
 


36 
 
In the case of SonoVue (now called LumasonTM), a second-generation lipid/sulfur 
hexafluoride US contrast agent (Bracco, Milan, Italy), the European Union approved its 
intravenous use in adults in 2001. The FDA in the United States just recently approved 
the use of LumasonTM for evaluation of focal hepatic lesions and vesicoureteral reflux in 
pediatric population in 2016. For the remainder of clinical applications, the ultrasound 
contrast agents are being used off-label in both Europe and the United States. 
 
Risks of the administration of the study drug are considered a minor increase above 
minimal risk, without the prospect of direct benefit. Adverse effects are not dose-
dependent, thus risk is not increased by modifying the timing of interventions or 
increasing the number of contrast-agent injections. 
 
Another risk of the study includes the insertion of a peripheral IV line. This is a no 
greater than minimal risk procedure, with the main risks of discomfort, bruising, and 
infection which are generally self-limited. There is a no greater than minimal risk of 
breach of confidentiality, which is minimized by having all of study personnel undergo 
HIPAA training.  
 
Interference of CEUS with MRI is not expected since CEUS contrast-agent Lumason 
clears within minutes after injection. Elimination of Lumason (Sulfur Hexafluoride Lipid-
Type A Microspheres) occurs via the lungs in the first minutes following contrast-agent 
injection (please see Package Insert's section 12.3 Pharmacokinetics for more detail, 
attached in Application's section 12.02 (3.0)). Additionally, Misun Hwang, the sponsor-
investigator, has also previously performed brain CEUS before MRI in neonatal patients 
without adverse events. 
 
Steps Taken to Minimize Risks 
 
Parents and/or legal guardians of participants will be asked about contraindications to 
contrast enhanced ultrasonography examinations, as listed in the exclusion criteria. In 
order to appropriately treat potential rare adverse events, patients will be monitored by 
the study team for 60 minutes following contrast administration. 
 
Vital signs will be recorded from the medical chart and in-place monitoring and 
documented at 1) 30 minutes post-scanning, and 2) 60 minutes post-scanning. During 
monitoring, subjects will be assessed for rash, allergic reactions, anaphylaxis, and 
abrupt deviations from the subject's baseline hemodynamic parameters trend not 
related to medical intervention. 
 
Adverse events will be recorded at 1) 60 minutes post-scanning when the monitoring 
period is completed, and 2) through 48 hours post-scanning, with documentation at 48 
hours post-scanning if no adverse event presents until this point. 
 
 
9.4.2 Potential Benefits of Trial Participation 
 


37 
 
The patient will not receive a direct benefit as a result of participating in the study. 
Indirect benefits may include improvement in current diagnostic algorithm for detection 
and monitoring of HII and downstream reduction of high mortality and morbidity 
associated with HII. 
 
9.4.3 Risk-Benefit Assessment 
 
The benefit to society outweighs the risks of this study.   
 
 Recruitment Strategy 
 
Potential subjects will be identified by reviewing the subjects’ medical chart and referral 
from neonatologists of patients during the neonatal or pediatric intensive care unit stay 
for suspected, at risk of or diagnosed hypoxic ischemic injury and review of orders for 
cooling blankets, brain imaging and/or EEG exams in neonates. Potential participation 
will be discussed by the PI with the referring neonatologist (co-investigator). 
Parents/legal guardians may be approached over phone/e-mail by a member of the 
study team to determine if they are interested in receiving more information about the 
study. Participation will be discussed with the parents/guardian by the neonatologist 
and/or radiologist. Parental/guardian permission (informed consent) will be obtained.  
 
Additionally, the CHOP Research Discovery Finder, e-mails, and a tearpad flyer will be 
used in the recruitment strategy 
 
 Informed Consent and HIPAA Authorization 
 
Approved members of the study team will obtain parental/guardian consent prior to the 
proposed study in a private setting. The investigators will assure that parents/guardian 
comprehend the nature of the study, the study procedures and the risks and benefits of 
participation, steps that will be taken to avoid coercion and documentation of consent. A 
combined HIPAA consent-authorization document will be used.  
 
 Payment to Subjects/Families 
 
Families will be offered a gift card of 50 USD value for participation in the study. 
 PUBLICATION 
 
The investigative team plans to publish the data collected in a scientific journal.  Data 
may also be presented as abstract, podium presentation, or poster presentations at 
scientific meetings and conventions.  No patient identifying information will be used in 
publications. 
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