A Phase II Multi-site Study of Autologous Cord Blood Cells for Hypoxic (HIE)
Phase: PHASE2
Conditions: Moderate or Severe Hypoxic-ischemic Encephalopathy in Newborns
Sponsor: Michael Cotten

A Phase II Multi-site Study of Autologous Cord Blood Cells for Hypoxic Ischemic 
Encephalopathy (HIE)   IND 14753  (BABYBAC II) 
 
February 6, 2018                       Version 8.0 
 
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NCT02612155 
 
 
Principal Investigator 
Joanne Kurtzberg, MD 
Duke University Medical Center 
Durham, NC  27710 
Phone:  919-668-1100 
 
Co-Principal Investigator 
Michael Cotten, MD, MHS 
Duke University Medical Center 
Durham, NC 27710 
Phone:  919-681-4844 
 
Statistician 
Abhik, Das, PhD 
RTI International  
Research Triangle Park, NC 
Phone: 301-770-8214 
 
 
 
 

A Phase II Multi-site Study of Autologous Cord Blood Cells for Hypoxic Ischemic 
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Contents 
INVESTIGATOR SIGNATURE PAGE ........................................................................................ 4 
1. 
Abbreviations........................................................................................................................... 5 
2. 
Protocol Summary ................................................................................................................... 6 
3. 
Introduction ............................................................................................................................. 7 
4. 
Study Objectives ...................................................................................................................... 7 
5. 
Background and Pilot Study Results ....................................................................................... 8 
5.1. 
Animal Studies ..................................................................................................................... 8 
5.2. 
Human Clinical Data using Umbilical Cord Blood in Infants with HIE ............................. 9 
6. 
Study Design.......................................................................................................................... 10 
6.1. 
Study Flow Chart ............................................................................................................... 11 
6.2. 
Study Population and Site selection ................................................................................... 11 
7. 
Inclusion and exclusion criteria ............................................................................................. 12 
7.1. 
Inclusion criteria ................................................................................................................ 12 
7.1.1. 
Exclusion criteria............................................................................................................ 14 
7.1.2. 
Screening and Enrollment .............................................................................................. 14 
8. 
Study product and Placebo .................................................................................................... 15 
8.1. 
Study product ..................................................................................................................... 15 
8.2. 
Placebo ............................................................................................................................... 15 
8.3. 
shipment of remaining cord blood products to Duke for cyropreservation ....................... 16 
8.4. 
Infusions ............................................................................................................................. 16 
8.5. 
Monitoring During and After Infusion .............................................................................. 16 
8.5.1. 
Vital Signs ...................................................................................................................... 16 
8.5.2. 
Metabolic Status ............................................................................................................. 16 
8.5.3. 
Respiratory Status .......................................................................................................... 17 
8.5.4. 
Neurologic Status ........................................................................................................... 17 
8.5.5. 
Hematologic Status ........................................................................................................ 17 
8.5.6. 
Follow up visits .............................................................................................................. 17 
9. 
Schedule of events ................................................................................................................. 18 

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10. 
Other Treatments/Tests/Concurrent Medications .............................................................. 19 
11. 
Primary Outcome Measures ............................................................................................... 19 
11.1. 
Bayley Scales of Infant and Toddler Development- III ................................................. 19 
11.2. 
MRI ................................................................................................................................ 20 
11.3. 
NICU discharge .............................................................................................................. 20 
11.4. 
Short term/hospital outcomes/Study Endpoint ............................................................... 20 
11.5. 
Long term outcomes/Primary Outcome/Study Endpoint ............................................... 20 
12. 
Risks and Benefits.............................................................................................................. 20 
12.1. 
Risk and benefits of study .............................................................................................. 20 
13. 
Safety and Adverse Event Reporting ................................................................................. 21 
13.1. 
Adverse Events (AE) ...................................................................................................... 21 
13.1.1. 
Adverse Events (AE) .................................................................................................. 22 
13.1.2. 
Serious Adverse Events (SAE) ................................................................................... 22 
13.1.3. 
Grade/Severity ............................................................................................................ 22 
13.1.4. 
Suspected Adverse Reaction ...................................................................................... 22 
13.1.5. 
Causality ..................................................................................................................... 22 
13.2. 
Adverse Event Reporting ............................................................................................... 22 
13.3. 
Unblinding ...................................................................................................................... 23 
13.4. 
Interim monitoring for Safety Outcomes ....................................................................... 23 
13.5. 
Interim monitoring for Efficacy Outcome ..................................................................... 25 
13.6. 
Interim Estimation of Treatment Efficacy ..................................................................... 25 
13.7. 
Interim Estimation of Treatment Futility ....................................................................... 26 
14. 
Data Safety Monitoring Committee ................................................................................... 26 
15. 
Statistical Consideration .................................................................................................... 27 
15.1. 
Study Design .................................................................................................................. 27 
15.2. 
Estimates of available and required sample size ............................................................ 27 
16. 
References .......................................................................................................................... 29 
 
 
 

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INVESTIGATOR SIGNATURE PAGE 
A Phase II Multi- site Study of Autologous Cord Blood Cells for Hypoxic Ischemic 
Encephalopathy (HIE) 
 
I agree to conduct and supervise this clinical study in accordance with the design and 
specific provisions of this protocol; modifications to the study or protocol are acceptable 
only with a mutually agreed upon protocol amendment except when necessary to protect 
the safety of subjects.  I agree to await IRB approval for the protocol and informed 
consent before initiating the study, to obtain informed consent from subjects’ parent or 
guardian prior to their enrollment in the study.  I agree to report to responsible regulatory 
agencies and IRB (when necessary) adverse events that occur in the course of this 
investigation.  I agree to maintain accurate and adequate records in the case report forms 
as required by this protocol and maintain those records for the period of time required.   I 
will make the study documentation available for safety oversight committee review 
and/or for other inspections as required.  I agree to maintain study documentation for the 
period of time required.  I agree to comply with all other requirements regarding the 
obligations of clinical investigators according to FDA regulations and guidance.  I agree 
to ensure that all people assisting in the conduct of this study are informed in meeting the 
above commitments. 
 
 
________________________________ 
(Investigator’s printed name) 
 
 
______________________________ 
(Investigator’s signed name) 
 
 

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1. ABBREVIATIONS 
AE 
Adverse Event 
BDNF 
Brain-derived neurotropic factor 
BSID 
Bayley Scales of Infant and Toddler Development III 
CBU 
Cord Blood Unit 
CMV 
Cytomegalovirus 
CPD 
Citrate Phosphate Dextrose 
CRF 
Case report form 
CTCAE 
Common Terminology Criteria for Adverse Events 
DMSO 
Dimethyl sulfoxide 
DSMC 
Data Safety Monitoring Committee 
ECMO 
Extracorporeal membrane oxygenation 
FDA 
Food and Drug Administration 
GDNF 
Glial cell-derived neurotropic factor 
HIE 
Hypoxic Ischemic Encephalopathy 
HIV 
Human Immunodeficiency Virus 
HTLV 
human T-cell lymphotropic virus 
ICU 
Intensive Care Unit 
IND 
Investigational New Drug 
IRB 
Internal Review Board 
IV 
intravenous 
MOP 
Manual of Procedures 
MRI 
Magnetic Resonance Imaging 
NAT 
Nucleic Acid Testing 
NGF 
Nerve Growth Factor 
NICHD 
The National Institute of Child Health and Human Development 
NICU 
Neonatal Intensive Care Unit 
NRN 
Neonatal Research Network 
PRBC 
Packed Red Blood Cells 
PT/PTT 
Prothrombin time/Partial thromboplastin time 
RBC 
Red Blood Cells 
RPR 
Rapid Plasma Reagin 
SAE 
Serious Adverse Event 
TNCC 
Total Nucleated Cell Count 
UCB 
Umbilical Cord Blood 
 
 

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2. PROTOCOL SUMMARY  
 
Title: 
 
A Phase II Study of Autologous Cord Blood Cells for 
Hypoxic Ischemic Encephalopathy (HIE) 
 
Study Phase: 
 
Phase II  
Study Sites: 
Duke University and up to 14 additional sites 
 
Study description:  
 
The study will include two groups, randomized to receive 
up to two doses of cells (with a dose range of 2-5 x 107 
cells/kg per dose) or up to two doses of placebo. The 
randomization will be stratified by severity of 
encephalopathy, as defined in the NRN’s Optimizing 
Cooling trial. 
 
Objectives: 
Primary objective:  Evaluate the efficacy of up to two 
infusions of autologous umbilical cord blood in a 
prospective, randomized, double-blind, placebo controlled 
multi-center study.  Efficacy will be measured by one year 
survival and on Bayley III scores in all three domains > 
85.   
 
Research Participant Population: 
The study population includes infants with HIE who meet 
the NICHD cooling study enrollment criteria, (1) and have 
cord blood collected and available for infusion(s) in the 
first 48 postnatal hours 
 
Study Design: 
 
Prospective, randomized, double blind, placebo controlled, 
multi-center Phase II trial in up to 160 infants 
 
 
 

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3. INTRODUCTION 
Treatment of babies with moderate to severe Hypoxic Ischemic Encephalopathy (HIE) with 
hypothermia results in normal outcomes in approximately 50% of the treated neonates 1-4. 
Cooling targets inflammatory and excitatory neurotransmitter-related cell injury and early death 
5-8. Animal studies indicate that providing human cord blood cells after hypoxic ischemic injury 
also reduces the initial inflammatory response and enhances production of neurotrophic 
compounds and facilitates ongoing repair 9-12. 
Our phase I, single center, open label study of infusion of autologous cord blood cells in infants 
treated with hypothermia for moderate to severe HIE has demonstrated that infusion of cells in 
the first 48 postnatal hours was safe.  At one year, cell recipients were more likely to have 
survived with neurodevelopmental assessment scores in the normal range than infants with HIE 
concomitantly treated in our center with hypothermia alone 13, 25. 
The accumulating animal studies, and the results of our phase I study justify a need for further 
study of safety and efficacy of autologous cord blood cells in a multi-center, double-blind, 
placebo controlled randomized trial in infants cooled for moderate to severe HIE. We 
hypothesize that umbilical cord blood will improve the outcome of neonates with HIE 
associated with evidence of hypoxic-ischemic injury undergoing hypothermia at 12 months 
compared to neonates treated with hypothermia alone. Our long-term goal is to optimize the 
use of human neonatal umbilical cord blood by understanding the mechanism by which 
umbilical cord blood interrupts the pathophysiologic cascade that is unleashed following 
hypoxic-ischemic injury. This study will move us toward our long-term goal by determining if 
administration of autologous umbilical cord blood will improve the outcomes of neonates with 
HIE who are undergoing therapeutic hypothermia.   
4. STUDY OBJECTIVES 
The primary objective is to assess the safety and efficacy of up to two intravenous infusions of 
autologous umbilical cord blood cells as compared with placebo in neonates with HIE 
undergoing hypothermia treatment. Efficacy will be measured by one year survival and score on 
Bayley III scores in all three domains > 85.   
 
 

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5. 
BACKGROUND AND PILOT STUDY RESULTS 
The composite results of whole body hypothermia studies indicates a significant reduction in risk 
of death or impairment when hypothermia is initiated in the first 6 postnatal hours and continued 
for 72 hours; however, in the four whole-body hypothermia studies, 44 – 51% of infants died or 
survived with disabilities, 24 - 38% of babies with HIE and were cooled died, and 13 – 28% of 
the survivors were later diagnosed with cerebral palsy. While cooling is helpful, the results of 
these trials provide strong incentive for development of adjunct therapies. Investigators are now 
testing multiple interventions, including inhaled xenon and erythropoietin, in pre-clinical and 
phase I and now phase III clinical trials 14. Cell-based interventions have also shown promise 15.   
5.1.   ANIMAL STUDIES 
The first study of human cord blood cells infused in a neonatal animal with a hypoxic-ischemic 
brain injury used the Rice-Vannucci model of unilateral carotid artery ligation followed by 
global hypoxia via exposure to very low oxygen concentration environment 16,17. Neonatal rats 
were infused intraperitoneally with 1 x 107 human umbilical cord blood nucleated cells 24 hours 
after injury. Human cells localized to the injured hemisphere, but did not proliferate in situ. 
Tissue injury was reduced in cell recipients, and spastic paresis was alleviated 9. Subsequently, 
another group infused cells and cells plus mannitol intraperitoneally 7 days after injury and 
found dendritic densities among cell recipients to be 75% of controls, and 80% of controls for 
those receiving cells plus mannitol 14 days after cell infusion. Dendritic densities for animals 
that received the vehicle solution or mannitol alone were approximately 50% of controls. These 
investigators also noted better neurobehavioral outcomes for cells and cells plus mannitol 
recipients, and higher brain tissue neurotrophic factor levels (BDNF, NGF, and GDNF). These 
results were obtained with a lower dose than that used in the first study (1.5 x 104  vs 1 x 107 
human umbilical cord blood nucleated cells) 11.  
In collaboration with Dr. Sid Tan, a series of experiments in a rabbit model of intrauterine brain 
injury were performed 18, 19, 24. In these studies, pregnant rabbits had an intravascular catheter 
with balloon inserted in the uterine artery at day 22 of the usual 31 day pregnancy. The balloon 
was inflated for 40 minutes, restricting intrauterine blood flow, which results in rabbit pups born 
with a hypertonic-cerebral palsy-like phenotype. In our studies, human umbilical cord blood cells 
were intravenously infused within 4 hours after birth in animals with severe injury phenotypes. 
In the first experiment, we used a dose of 5 x 106 human cord blood cells in one milliliter of 
saline, and the infusion was delivered over one minute. Animals born with a severe phenotype 
that received cells and survived the neonatal period demonstrated improvement in motor 
function, but mortality was higher (although not statistically significantly higher) among cell 
recipients. In a subsequent replication study using the same infusion volume but half the cell 
amount (2.5 x106), the mortality was very similar for cell recipients and those receiving saline 
but the motor outcomes for cell recipients were improved compared to saline recipients. Studies 
with MRI demonstrated very sparse presence of human cord blood cells in the days after 

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infusion, but brain tissue samples obtained closer to the time of infusion demonstrate human 
DNA markers within the homogenized tissue samples 24.   This indicated that cells homed to the 
ischemic area but engraftment was transient.  Despite this fact, a clinical effect was observed, 
likely due to paracrine signaling from the cord blood cells. 
5.2. HUMAN CLINICAL DATA USING UMBILICAL CORD BLOOD IN INFANTS 
WITH HIE 
We initiated a phase 1 study based on the emerging animal data and over a decade of experience 
at Duke University in performing allogeneic cord blood transplants for inherited metabolic 
disease.  Our clinical experience with these allogeneic transplants noted surprising neurologic 
results, which included post-transplant myelination in the presence of disease that should have 
caused de-myelination 19.  We initiated a phase I open- label feasibility and preliminary safety 
study of use of autologous cord blood cells for infants with HIE in 2009 13. 
Our phase I study has enrolled 45 subjects. The initial intent was to provide up to 4 infusions of 
2-5 x 107cells/kg each in the first 72 postnatal hours. After release of guidance for public cord 
blood banking in 2011, the FDA implemented a requirement for regulation of all non-
homologous uses of umbilical cord blood. Accordingly, we submitted an IND application for the 
phase I study. In July 2011, FDA indicated that our IND was safe to proceed, and our study 
moved forward under an IND with a protocol modification allowing a maximum of 2 infusions 
of fresh cells in the first 48 postnatal hours; FDA requested the number of infusions limited until 
there was sufficient evidence to ensure reliable viability of unfrozen cells beyond 48 hours.   
Study operations included collection of cord blood, usually with verbal assent for collection for 
potential use in the study, unless the family had previously provided consent for collection for 
public banking of cord blood. If blood was collected, and the infant met cooling criteria, and the 
parents provided consent for enrollment in the phase I study, cells were volume and red blood 
cell reduced on the Sepax device (Biosafe, International), or manually if cord blood volumes 
were below 30 milliliters. For the majority of enrollees (after 2011), two infusions were provided 
in the first 48 postnatal hours.  
The improved survival and one-year neurologic outcomes of subjects who received cells and 
concurrent cooling compared to cooled infants who did not receive cells provide impetus to 
move forward to a multi-center phase II placebo controlled study.  Since publication of the phase 
I paper in May 2014, we have accumulated and reported additional data on infants with HIE and 
one year outcomes 25. Thirty-nine (39) infants have been cooled and received cells (16 since our 
initial report) and 146 have been cooled for HIE without receiving cells. For those receiving 
cells, median collection and infusion volumes were 33 mL and 5 mL.   None of the cell 
recipients died prior to discharge while 16 (11%) of cooled non-cell recipients died prior to 
hospital discharge (p = 0.03). Ten (26%) cell recipients and 31 (21%) cooled-only infants were 
discharged on seizure medications (p = 0.56). Of the 25 cell recipients with known one year 

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outcomes, 16 (64%) survived with Bayley III scores ≥ 85 in all three domains, and of the 63 
cooled-only infants with known one year outcomes, 25 (40%) survived with one year Bayley III 
scores ≥ 85 in all three domains (p = 0.04). While the results for the cell recipients are better than 
for infants who were cooled, important facts must be acknowledged. More of the cell recipients 
were inborn, and more of the cooled only group were not seen at one year follow-up.  
There were no serious adverse event directly related to study therapy.  No significant infusion 
reactions were noted.  Heart rate, mean arterial pressure, and oxygen saturation did not vary 
significantly before and after infusions for the first two infusions.  Mean oxygen saturation was 
lower after the third and fourth infusion.  One infant, born at 35 weeks gestation, had a cord 
blood pH of 6.72 and a spontaneous ileal perforation prior to the fourth infusion.  One infant had 
a positive umbilical cord blood (UCB) culture for E. coli, which was also present in the mother’s 
blood culture.  The infant’s own postnatal blood culture was negative and prophylactic 
antibiotics were started.  For one subject, an infusion was stopped due to an occluded iv line but 
subsequent infusions were successfully completed.  It was concluded the safety profile of the 
Phase 1 study warranted further study.  We believe that the accumulating evidence in pre-
clinical studies and the initial signals of safety and feasibility in the phase I study are 
sufficient evidence to justify a phase II study to further assess potential efficacy of 
autologous cord blood cells in the first postnatal days for infants treated with hypothermia 
for moderate to severe HIE. 
6. STUDY DESIGN 
This is a prospective, randomized, double-blind, placebo controlled multi-center Phase 2 study in 
up to 160 infants who meet the NICHD cooling study enrollment criteria and have cord blood 
collected and available for up to two infusions in the first 48 postnatal hours.  We will evaluate 
the efficacy of the infusions of autologous umbilical cord blood.  We project accrual of 60-80 
subjects per year.  The study will include two groups (up to 80 subjects per group), randomized 
to receive up to two doses of cells (with a dose range of 2-5 x 107 cells/kg per dose) or up to two 
doses of placebo. The randomization will be stratified by severity of encephalopathy, as defined 
in the NRN’s Optimizing Cooling trial 19.  Investigators and families will be blinded to enrolled 
study group.   
 
 

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6.1. STUDY FLOW CHART 
< 24 Hours Postnatal
< 48 Hours Postnatal
12 month follow up visit
Collect UCB
Consent (assent)
Determine Eligibility
Randomization
Bayley III Assessment
Infusion 1 (cord 
blood or placebo)
Infusion 2 (cord 
blood or placebo)
Infusions of cord blood or placebo must occur by 48 hours of life.  
Number of doses is dependent on # of cells and timing of 
infusions (must be at least 8 hours apart).  
 
6.2. STUDY POPULATION AND SITE SELECTION 
The study population will include up to 160 infants who meet the NICHD cooling study 
enrollment criteria19, and have cord blood collected and available for infusion(s) in the first 48 
postnatal hours. 
Study sites will include Duke University and up to fourteen additional sites that are Level 3 
neonatal ICUs with a standardized hypothermia protocol.   
 
 
 

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7. 
INCLUSION AND EXCLUSION CRITERIA 
7.1. INCLUSION CRITERIA 
Enrolled subjects must meet all of the following criteria: 
 
1. Cord blood must be available for volume and red blood cell reduction before 45 hours of 
age.  Please follow local IRB rules regarding consent/assent for cord blood collection.   
2. The infant must be able to receive at least one dose of autologous cord blood before 48 
hours of age 
3. All infants must have signs of encephalopathy within 6 hours of age utilizing the two step 
(A and B) approach used in the Network’s Optimizing Hypothermia Study 20. 
The eligibility criteria: 
 
 Infants will be evaluated in two steps; evaluation by clinical and biochemical 
criteria (Step A, which is either A1 or A2, depending on available information and 
severity of blood gas abnormalities), followed by a neurological exam (Step B)  
 Once infant meets either A1 or A2, proceed to neurologic examination. (See part 
B) 
 The presence of moderate/severe encephalopathy (a “2” or a “3”) defined as 
seizures OR presence of signs in 3 of 6 categories in the table below. For the 
categories with more than one item, such as PRIMITIVE REFLEXES, the item 
(SUCK, MORO) with the highest score determines the level of encephalopathy 
assigned for that category 
 The neurologic examination will be performed by a physician examiner 
 
Steps A1 and A2. 
All infants will be evaluated for the following:  
1. 
Is the baby ≥ 36 weeks gestation? 
2.         Is there a history of an acute perinatal event (abruptio placenta, cord prolapse, 
severe FHR abnormality: variable or late decelerations)? 
3. 
Is the Apgar score <5 at 10 minutes or is there a continued need for ventilation 
initiated at birth and continued for at least 10 minutes? 
4. 
What is the cord pH or first postnatal blood gas pH at <1 hour? 
5. 
What is the base deficit on cord gas or first postnatal blood gas at <1 hour?  
 
If infant meets criteria A1 or A2 and criteria B and does not meet exclusion criteria, the 
infant is eligible and is therefore eligible for study enrollment if cells are available 
 
 

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IF BLOOD GAS IS AVAILABLE: 
IF BLOOD GAS IS  
NOT AVAILABLE 
OR pH between 7.0 and 7.15,  
OR  
BASE DEFICIT 10 to 15.9mEq/L 
A1 
A2 
Answer to #1 is ‘YES’ AND 
Answer to #1 AND  
 
 
Acute perinatal event (#2) and either 
 
Cord pH or first postnatal blood gas 
within 1 hour with pH  <7.0 (#4) 
 
An Apgar score <5 at 10 minutes 
(#3) 
OR 
 OR 
 
Base deficit on cord gas or first 
postnatal blood gas within 1 hour at >16 
mEq/L (#5) 
 
Continued need for ventilation 
initiated at birth and continued for at least 10 
minutes (#3) 
 
Part B: Neurologic Assessment 
 CATEGORY  
SIGNS OF HIE IN EACH LEVEL 
                                                                         
NORMAL/MILD HIE  
MODERATE HIE  
SEVERE HIE  
1. LEVEL OF 
CONSCIOUSNESS  
1 
2 = Lethargic  
3 = Stupor/coma  
2. SPONTANEOUS 
ACTIVITY  
1 
2 = Decreased activity  
3 = No activity  
3. POSTURE  
1 
2 = Distal flexion, 
complete extension  
3 = Decerebrate  
4. TONE  
1 
2a = Hypotonia (focal or 
general)  
2b = Hypertonia  
3a = Flaccid  
3b = Rigid  
5. PRIMITIVE REFLEXES  
           Suck  
1 
2 = Weak or has bite  
3 = Absent  
           Moro  
1 
2 = Incomplete  
3 = Absent  
6. AUTONOMIC SYSTEM  
          Pupils  
1 
2 = Constricted  
3 = Deviation/dilated/non-
reactive to light  
         Heart rate  
1 
2 = Bradycardia  
3 = Variable HR  
         Respiration  
1 
2 = Periodic breathing  
3 = Apnea or requires 
ventilator  
3a=on vent with spont breaths  
3b=on vent without spont 
breaths  
 
 

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7.1.1. 
EXCLUSION CRITERIA 
Enrolled subjects must not meet any of the following criteria: 
1. Major congenital or chromosomal abnormalities 
2. Severe growth restriction (birth weight <1800 g) 
3. Opinion by attending neonatologist that the study may interfere with treatment or safety 
of subject 
4. Moribund neonates for whom no further treatment is planned 
5. Infants born to mothers are known to be HIV, Hepatitis B, Hepatitis C or who have active 
syphilis or CMV infection in pregnancy 
6. Infants suspected of overwhelming sepsis 
7. ECMO initiated or likely in the first 48 hours of life 
8. Mother suspected to have chorioamnionitis at time of birth. Refer to MOP-01, section 4.3 
for definition.  
9. ALL blood gases (cord and postnatal) done within the first 60 minutes had a pH >7.15 
AND a base deficit < 10 mEq/L (source can be arterial, venous or capillary) 
10. Mother with documented Zika infection during this pregnancy 
 
 
7.1.2. 
SCREENING AND ENROLLMENT  
Centers will screen for potential subjects among all deliveries of infants > 35 6/7th weeks who 
appear to be at risk for delivery of an infant with encephalopathy. From our single site 
experience, the obstetric team collected cord blood at 5 – 6 deliveries of potentially 
encephalopathic infants for every one infant who met enrollment criteria in the Phase 1 study.  If 
an infant whose cord blood was collected for potential enrollment in the study is subsequently 
not enrolled in the study, the collected cord blood will be discarded as medical waste at the study 
site (unless arrangements are made by the family for private banking).   
7.1.3. RANDOMIZATION AND STRATIFICATION   
After informed consent is obtained, eligible infants will be randomized to one of two arms (cells 
or placebo). Neonates randomized to receive cells will receive up to two doses of cells (with a 
dose range of 2-5 x 107 cells/kg per dose), while those randomized to the placebo arm will 
receive up to two doses of placebo.      
Randomization will be conducted using permuted block design and stratified by stage of 
encephalopathy (moderate or severe). A central web based randomization system available 24 
hours a day, 7 days a week, will be developed and maintained by the Data Coordinating Center 
at RTI International, Research Triangle Park, NC.  

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8. STUDY PRODUCT AND PLACEBO 
8.1.  STUDY PRODUCT 
Autologous cord blood will be collected in utero during the 3rd stage of labor or shortly after 
delivery of the placenta from neonates at risk for the development of HIE. Sites must follow 
local IRB rules regarding consent/assent for cord blood collection for autologous use. . The cord 
blood will be collected into Pall Medical Sterile CB Collection Unit Code 791-08 containing 35 
mL of citrate phosphate dextrose (CPD) anticoagulant.  It is important to collect the maximum 
volume of cord blood.   The cord blood unit (CBU) will be transported to the processing 
laboratory where it will be red blood cell (RBC) and volume reduced and up to 2 study doses 
prepared.  The infusion volume will be ≤3.0 cc/kg with an infusion time of 10-20 minutes.  A 
manual of procedures will be provided for all sites to assure consistency of processing.    
If randomized to treatment arm, cell preparation will include volume- and RBC reduction after 
20-30 minute incubation with 6% Hespan (hetastarch, Hospira, Lake Forest, Illinois, USA ) 
using the Sepax automated processing system (Biosafe, Geneva, Switzerland) for units with > 
30mL of umbilical cord blood (UCB).  A manual method will be used if the collected cord blood 
volume is < 30mL using procedures in the manual of procedures. A sample of processed cord 
blood is used to determine the cell count and decide if one or two doses are aliquotted into 
individual dose syringes. A sample is also removed for the gram stain. 
A gram stain from the study product and placebo will be performed and resulted prior to infusion 
of the study product or placebo to indicate sterility.  If the gram stain is positive, then the cells or 
placebo will not be infused and the cord blood is discarded.   If sterility cultures, which will be 
performed on the collected cord blood at Duke University Stem Cell Laboratory (STCL), 
subsequently become positive, the study team will notify the clinical team of the nature of the 
positive culture and a follow up strategy will be developed by the clinical and research teams.  In 
general, babies with HIE are placed on empirical antibiotic therapy shortly after birth.  If the 
baby is on concurrent antibiotic therapy and if the antibiotic covers the organism grown in the 
culture obtained from the cord blood, the baby will complete a course of therapy.  If, on the other 
hand, the baby is no longer on antibiotics or the organism identified in the culture is not sensitive 
to the antibiotics the baby is being treated with and is not considered a contaminant, a course of 
antibiotic therapy covering the organism isolated will be administered. 
8.2.  PLACEBO 
Placebo will be prepared from a small volume of unprocessed cord blood diluted in saline.  This 
formulation will be described in a manual of procedures.  Placebo will have a similar appearance 
to the study product. A gram stain from the CBU must be performed and resulted prior to release 
of placebo (see section 8.1). Only products with negative gram stain will be released for infusion.   

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8.3.  SHIPMENT OF REMAINING CORD BLOOD PRODUCTS TO DUKE FOR 
CYROPRESERVATION 
The entire cord blood unit or cord blood remaining after processing and removal of cells for 
dosing and the remaining material in the PRBC bags and plasma bags from remote sites will be 
shipped to the Stem Cell Transplant Laboratory at Duke University for cryopreservation and 
testing for potential future autologous use by the study subject. Each infant’s cord blood unit’s 
characteristics will be recorded, including total nucleated cell counts pre- and post-processing, 
post-processing viable CD34+ cell content, colony forming units, sterility, and viability. The 
manual of procedures describes packing and shipping to STCL at Duke University.   
Cryopreservation is dependent on receipt of cord blood or processed cord blood within 96 hours 
of collection at STCL at Duke University. It is best to ship as soon as possible to preserve the 
viability and potency of the cord blood cells. 
8.4. INFUSIONS  
For babies meeting entry criteria, the first dose of cells/placebo will be infused intravenously as 
soon as possible. Infants will be pretreated with hydrocortisone, 1 mg/kg IV 30-60 minutes prior 
to each infusion if the subject was not on hydrocortisone for clinical purposes. The placebo 
group is receiving hydrocortisone to minimize the risk of infusion-related and/or allergic reaction 
to the placebo - the same risk minimization as for the active stem cell recipients. Cells/placebo 
will be infused over 10-20 minutes, followed by a saline flush (≤ 5.0mL) to clear the 
intravascular line.  The study allows up to two doses of cells or placebo if the cell number is 
adequate from the cord blood unit.  There must be a minimum of 8 hours between doses and both 
doses need to be completed by 48 hours post delivery.     Babies receiving placebo infusions will 
be treated in an identical fashion to those receiving cells.  The infusion product will be blinded to 
the hospital staff administering the cells and evaluating the babies’ outcomes.as well as the 
family.  
8.5. MONITORING DURING AND AFTER INFUSION 
8.5.1. 
VITAL SIGNS 
Vital signs will be assessed pre- infusion and for 15 minutes post infusion and per hospital 
routine thereafter. 
8.5.2. 
METABOLIC STATUS 
Daily chemistries are routinely obtained in infants with moderate to severe HIE when they are 
treated with hypothermia.  Serum electrolytes, CBC, BUN, and creatinine, and frequently liver 
function tests are monitored at baseline, and then daily.  We will record results of metabolic 
laboratories collected for clinical purposes during the cooling period and the first 24 hours post 
re-warming in the case report forms.   

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8.5.3. 
 RESPIRATORY STATUS 
A daily blood gas is standard in infants with moderate to severe HIE.  Results from daily blood 
gases obtained for clinical purposes during cooling and for the first 24 hours after re-warming 
will be collected in the case report forms.   
8.5.4. 
NEUROLOGIC STATUS 
A neurological assessment will be performed at baseline, daily during cooling, and at discharge. 
This will be performed by a trained examiner. 
8.5.5. 
HEMATOLOGIC STATUS 
Monitoring of coagulation studies is considered routine for infants with HIE.  PT/PTT results 
obtained for clinical purposes during cooling and for the first 24 hours after re-warming will be 
recorded in the case report forms. 
8.5.6. 
FOLLOW UP VISITS  
Subjects will return at one year (12 mo - 16 mo) corrected age.  The following assessments will 
be recorded:  History and physical exam, Bayley Scales of Infant Development III (BSID), 
neurological exam, adverse events and concomitant medications.  Infants not testable on the 
BSID due to Cerebral Palsy, blindness or deafness  will be assigned scores of 54, 46 and 46 
respectively on the cognitive, language and motor of the BSID scale.    
 
 
 

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9. 
SCHEDULE OF EVENTS 
 
 
 
 
 
First Dose<40 
hours 
postnatal if 
two doses are 
feasible 
<48 hours if 
only one dose 
is feasible 
Second 
dose if 
feasible 
< 48 hours 
postnatal 
Rewarming 
through 
postnatal day 
21 
Visit 1 
12-16months 
Screening 
X 
 
 
 
 Maternal 
consent or 
assent 
X 
 
 
 
Collect UCB 
X 
 
 
 
 
 
 
 
 
Labs1 
X 
 
 
 
Blood Gas2 
X 
 
 
 
Neurological 
Assessment3 
X 
X 
X 
X 
Infusion and 
vitals4 
X 
X (if cells 
are 
available) 
 
 
History and 
Physical 
Exam 
X 
 
 
X 
Adverse 
events and 
concomitant 
meds 
X 
X 
X 
X 
MRI 
 
 
X 
 
PT/PTT5 
X 
 
X 
 
 
1 Serum Electrolytes, BUN, CBC, Creatinine, liver function obtained as part of 
routine care for HIE (section 8.5.2) 
2Collected daily (section 8.5.3) 
3 Collected at baseline, daily during cooling and prior to discharge (section 8.5.4) 
4 Assessed pre-infusion and for 15min post infusion and per hospital SOC 
(section 8.5.1) 
5 Collected during cooling and 24 hours after re-warming if obtained as part of 
routine care (section 8.5.5) 
 

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10. OTHER TREATMENTS/TESTS/CONCURRENT MEDICATIONS 
Anti-seizure medication and sedation ideally will not be administered prior to hypothermia or 
study infusions, unless clinically indicated. All medications will be listed in concomitant 
medications in the case report forms.  
11. PRIMARY OUTCOME MEASURES 
11.1.BAYLEY SCALES OF INFANT AND TODDLER DEVELOPMENT- III 
The Bayley Scales of Infant and Toddler Development-III (BSID) 23 will be administered with all 
children at 12-16. Months corrected age. The Bayley-III is an individually-administered 
examination that assesses the current developmental functioning of infants and young children 
from birth to 42 months of age. This instrument has been shown to have adequate reliability and 
validity, and is considered the gold standard of infant and toddler assessment tools. The Bayley 
is a standardized, norm-referenced measure that assesses development in Cognitive, Language 
and Motor domains. Composite standard scores can be derived that have a mean of 100 and a 
standard deviation of 15.  
All Bayley examiners for this study will be trained and certified to a criterion of 80% accuracy 
for administration and scoring. Certification will be completed via submission of a DVD of 
administration of the Bayley-III Scales with a non-study child of the target age for the study. The 
DVD will be reviewed and the accuracy of administration and scoring will be determined by the 
study gold standard examiner within two weeks of submission of the DVD. Additional DVDs 
will be submitted by the examiner until the criterion of 80% accuracy is reached for both 
administration and scoring. Each examiner must be certified once per year. No study participants 
may be assessed by an examiner who has not been certified.  
For children with sensory impairments, an attempt should be made to administer Bayley items 
and a decision about validity of assessment will be made subsequently with consultation with the 
gold standard Bayley examiner. However, some children with sensory impairment cannot be 
reliably assessed with the Bayley-III. This includes children who are: 1) legally blind (< 20/200 
with glasses). 2) Hearing impaired and have no amplification/hearing aids. If a child does have 
hearing aids, the Bayley must be completed with the hearing aids in place.  If the child has 
profound hearing loss and is expected to undergo a cochlear implant, the child will not be able to 
be tested. 3) Child is both vision and hearing impaired (Deaf/Blind). These children cannot be 
tested with the Bayley. Infants not testable on BSID due to CP, blindness or deafness be assigned 
respective scores of 54, 46 and 46 on the cognitive, language and motor components of the BSID 
III.  

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Non-English language learners will be assessed by a bilingual examiner who has been certified 
or with the assistance of a qualified interpreter. Interpreters must be instructed to translate the 
Bayley instructions to the child verbatim and to not repeat instructions unless instructed to do so 
by the examiner. Language scores will not be derived for non-English language learners as the 
normative developmental progression of language milestones in other languages may not be 
accurately reflected on the Bayley Language Scales. 
11.2.MRI 
An MRI is routinely obtained on HIE infants between 7 – 28 postnatal days.  Ideally the MRI 
will be done optimally at 10 days of life (but with an acceptable window between 7 days and 21 
days of life as suggested by the 2014 revised guidelines for infants with HIE, to assess extent of 
injury) 21.  Results from the standard of care MRI will recorded for the study. 
11.3.NICU DISCHARGE   
Tracking information will be recorded at discharge, and will include phone numbers and home 
address.  All surviving infants will be followed for one year for the in-person assessment.  
An attempt will be made at each site to obtain an autopsy in case of death both prior to and 
following NICU discharge. Autopsy results, if available, will be included in the case report 
forms. 
11.4. SHORT TERM/HOSPITAL OUTCOMES/STUDY ENDPOINT 
Short term outcomes will be considered secondary outcomes for the study. We will compare 
adverse outcomes, including mortality, seizures, need for nitric oxide and need for ECMO, need 
for G-tube feeding at discharge, and discharge on anti-epileptic medications. This information 
will be collected in case report forms. 
11.5.LONG TERM OUTCOMES/PRIMARY OUTCOME/STUDY ENDPOINT  
The Primary outcome of the study will be survival at one year with Bayley III scores in all three 
domains > 85.  
12. RISKS AND BENEFITS 
12.1.RISK AND BENEFITS OF STUDY 
Infusion of a neonate’s own cord blood cells during routine cooling is hoped to improve survival 
and neurological outcome as compared to infants that are only cooled and receive red blood 
cells.  Autologous cells should eliminate any risk of graft vs host reaction.  If cord blood cells are 
collected and not infused, cord blood cells will be cryopreserved for later autologous use for the 
subject.  Complications of cord blood infusions could include infection, volume overload, 
hypertension, allergic or anaphylactic reactions, hyperviscosity or pulmonary edema and will be 

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monitored during and after infusion.  The medication hydrocortisone given prior to the infusions 
may increase blood sugar, blood pressure and fluid retention.   
 
13. 
SAFETY AND ADVERSE EVENT REPORTING 
Adverse Events (AE)  Adverse events and serious adverse events parameters are listed below. 
The parameters for events are derived from prior studies of thawed/washed cord blood cells used 
for allogeneic transplant, in combination with expected physiologic responses to therapeutic 
hypothermia, which by definition and inclusion criteria, will be concurrently underway when the 
study infusions are administered. The physiologic response to hypothermia leads to decreased 
heart rate during cooling (the mean Heart Rate during cooling in the NICHD NRN pivotal trial 
was 109); therefore a lower level to define significant bradycardia is used (Shankaran 2005). 
Other AE/SAE definitions are taken from the NICHD Optimizing cooling trial, to account for the 
concurrent use of therapeutic hypothermia, and the AE/SAE category definitions developed for 
the NICHD Pediatric Trials Network (Shankaran 2014 ;England 2016).  Definitions for each AE 
parameter are listed in MOP-01 Section 10.1. 
SAEs may include: 
 Acidosis 
 Allergic reaction/hypersensitivity 
 GI Pathology 
 Hemoglobinuria 
 Hyperglycemia 
 Hypoglycemia 
 Hypertension 
 Hypotension, and fever in the absence of neutropenia 
 Hypoxia 
 Infectious Disease 
 Polycythemia 
 Pulmonary Hypertension, within the first 24 hours 
 Sinus bradycardia 
 Sinus tachycardia 
 Dyspnea 
 Any other cardiac rhythm disturbances, 
 Any other reported SAE, including death 
 

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13.1.1. 
ADVERSE EVENTS (AE) 
Adverse event means any untoward medical occurrence associated with the use of the 
investigational product, whether or not considered related to the investigational product.    
13.1.2. 
SERIOUS ADVERSE EVENTS (SAE) 
An adverse event or suspected adverse reaction is considered “serious” if, in the view of 
either the investigator or sponsor, it results in any of the following outcomes: death, a 
life-threatening adverse event, inpatient hospitalization or prolongation of existing 
hospitalization, a persistent or significant incapacity or substantial disruption of the 
ability to conduct normal life functions, or a congenital anomaly/birth defect. Important 
medical events that may not result in death, be life-threatening, or require hospitalization 
may be considered serious when, based upon appropriate medical judgment, they may 
jeopardize the patient or subject and may require medical or surgical intervention to 
prevent one of the outcomes listed in this definition. 
13.1.3. 
GRADE/SEVERITY 
Grade/Severity will be performed per CTCAE guidelines. 
13.1.4. 
SUSPECTED ADVERSE REACTION 
Suspected adverse reaction means any adverse event for which there is a reasonable 
possibility that the investigational product caused the adverse event. ‘Reasonable 
possibility’ means there is evidence to suggest a causal relationship between the 
investigational product and the adverse event. 
13.1.5. 
CAUSALITY 
The Investigator will use the following question when assessing causality of an adverse 
event to the investigational product:  Is there a reasonable possibility that the drug caused 
the event?  An affirmative answer designates the event as a suspected adverse reaction. 
 
13.2.ADVERSE EVENT REPORTING 
All adverse events reported or observed during the study beginning at the time of the autologous 
cord blood infusion must be recorded and maintained in the study participant’s paper files or 
electronic case report forms. Information to be reported includes when the site became aware of 
the event, investigator-specified assessment of severity and relationship to study therapy, 
whether there is an alternative etiology, seriousness, as well as any required treatment or 
evaluations, and outcome.  In general, investigators should report adverse events as diseases or 
syndromes whenever possible, instead of reporting individual component symptoms, signs, 
laboratory abnormalities, and sequelae. 

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Severe adverse reactions (fatal or life threatening) that are unexpected and related will be 
reported to the FDA by Duke University within seven calendar days of receipt of the information 
by telephone, mail or fax, following FDA guidelines.  All non-life threatening serious, related, 
and unexpected adverse events will be reported to the FDA via a written report within 15 days of 
receipt of the information (21 CFR 312.32) by Duke University.  If the principal investigator 
assesses the event to be unrelated to the study, then the event will not require expedited reporting 
but will be included in a summary report.  
Investigators are requested to notify the Principle Investigators at Duke University within 24 
hours of all SAEs.  The Principal Investigator or Sub-Investigator at each site is responsible for 
informing their Institutional Review Board (IRB). Copies of SAE correspondence with all 
Principal Investigators or Sub-Investigators, governing authorities, ethics committees, and the 
sponsor must be submitted to Duke University for filing. 
 
 
13.3.  UNBLINDING 
 
Safety or other clinical or legal considerations including requests from a child’s family may, on 
rare occasions, require unblinding of the infant with regard to the actual intervention received. If 
such events make it necessary, the lab staff, responsible for preparation of study product at each 
site will be able to unmask the study drug product given to an individual infant.  Before 
unblinding the lab staff must contact the Center’s Investigator and the overall study Principal 
Investigators prior to releasing this information, and a protocol violation form will be completed. 
 
 
13.4.INTERIM MONITORING FOR SAFETY OUTCOMES 
The intervention for this trial will be conducted during the first 48 hours of life for the study 
subjects, while the primary outcome will be assessed at 12 (12 – 16) months of age. Thus, the 
primary focus of interim monitoring for this trial will be based on monitoring adverse events 
within 24 hours of cord blood infusion. Adverse events monitored will include the following, 
plus a composite (any of the listed events), on which statistical comparisons between the 
treatment groups will be based: 
 Acidosis 
 Allergic reaction/hypersensitivity 
 GI Pathology 
 Hemoglobinuria 
 Hyperglycemia 

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 Hypoglycemia 
 Hypertension 
 Hypotension, and fever in the absence of neutropenia 
 Hypoxia 
 Infectious Disease 
 Polycythemia 
 Pulmonary Hypertension, within the first 24 hours 
 Sinus bradycardia 
 Sinus tachycardia 
 Dyspnea 
 Any other cardiac rhythm disturbances, 
 Any other reported SAE, including death 
In keeping with the Bayesian nature of the trial design, we will use a Bayesian interim 
monitoring approach to determine whether systematic differences are emergent among the two 
treatment groups in the incidence of any of the adverse events listed above. Specifically, after 
every 40 babies enrolled in the trial have spent 24 hours post their final study transfusion, we 
will calculate the posterior probability that one group has higher incidence of any adverse events 
than another. In statistical terms, the treatment will be considered harmful (i.e., the DSMC may 
consider termination of the trial) if for a pre-specified threshold , the posterior probability of 
treatment harm (in terms of the above adverse events) is greater than  ; in other words if the 
predictive probability 





X
P
|
1
, where  denotes the relative risk favoring the treatment 
group and X is the data available. In order to allow for a liberal safety monitoring regime to 
ensure patient safety, we propose to take = 80%. However, we will also present the 95% 
credible interval for, as well as the entire posterior distribution for  in a graphical fashion to 
the DSMC so that they have a full appreciation for the range of possible values of . All interim 
analyses will adjust for baseline level of encephalopathy, the stratification variable for this trial, 
whenever computationally feasible. 
The choice of a prior distribution is essential and controversial in Bayesian analyses. Thus, we 
can compute the posterior probability of treatment harm and the posterior distribution of the 
associated relative risk under two sets of priors – (a) a non-informative prior that does not 
assume any substantive prior information about treatment differences in terms of the adverse 
events listed above, and (b) an informative prior based on treatment differences for these adverse 
events observed in the Phase I trial. However, we realize that the standard Bayesian approach of 
running the analysis with different priors provides only a partial solution, as the results on 
stopping are often inconclusive, especially when few data have accumulated. Instead of worrying 
about the selection of a single correct prior, we will also explore the use of a robust Bayesian 
approach which replaces a prior distribution with a class of priors and calculates the 
corresponding posterior probabilities for decision making26.  

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13.5. INTERIM MONITORING FOR EFFICACY OUTCOME  
 
The relatively rare condition that this trial addresses means that it may take considerable time for 
this trial to enroll the requisite number of babies. Thus, even though the primary outcome is only 
available at 12 months corrected age, the enrollment period for the trial may make some interim 
efficacy data on the primary outcome available for monitoring by the DSMC. Note that because 
of the Bayesian nature of the trial design, interim efficacy looks at the data will not have any 
adverse Type I error consequences that are normally the case for a conventionally designed (or, 
‘frequentist’) trial.  
 
We propose to conduct one formal interim analysis of the accruing primary outcome data, 
examining both efficacy and futility, at a third of primary outcome accrual for this trial, provided 
that recruitment and intervention have not been completed by that point in time. The analysis for 
both efficacy and futility will adjust for baseline level of encephalopathy, the stratification 
variable for this trial, if computationally feasible 
 
13.6. INTERIM ESTIMATION OF TREATMENT EFFICACY  
This interim efficacy analysis will first evaluate whether the intervention is beneficial, 
equivalent, or harmful relative to usual care. A Bayesian logistic regression model will be fit to 
the data to obtain adjusted estimates of interim treatment efficacy, adjusting for the trial 
stratification variables.  
 
A prior distribution is chosen in Bayesian analyses to provide an estimate of initial beliefs 
concerning the size of the potential treatment benefit. Thus, if one hopes that the results from the 
trial will influence the treatment of future patients, the prior distribution should represent the 
level of skepticism that is expressed by those clinicians that one seeks to influence. There are 
three principal types of prior we could use: (i) the uninformative, or reference prior, (ii) the 
skeptical prior and (iii) the enthusiastic prior. The uninformative prior represents a lack of 
clinical opinion as to the likely treatment difference, and in that sense contains no information 
about prior beliefs or other prior knowledge (eg. a relative risk for treatment benefit set at 1.0). A 
skeptical prior would be that overall effect of cord blood is actually harmful in this setting (i.e., a 
relative risk set higher than 1). An enthusiastic prior would be that the treatment is beneficial, as 
in the Phase I trial (relative risk set lower than 1). 
 
It is generally useful to present the results of Bayesian analyses under several alternative prior 
distributions, so that the impact of differing levels of prior belief on the observed data may be 
reviewed. The uninformative prior has the advantage of corresponding roughly to the classical 
frequentist approach, and so this together with the skeptical and enthusiastic priors gives a broad 
and useful overview of the implications of terminating a clinical trial27. Accordingly, we will use 

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all three prior distributions to present adjusted mean posterior relative risk (RR) estimates of 
treatment efficacy relative to survival free of cognitive impairment, along with corresponding 
95% credible intervals for the same. These priors will be set at RR = 1.1 (skeptical prior), 1.0 
(uninformative prior) and 0.625 (enthusiastic prior). 
 
The DSMC may consider proposing termination of the trial for benefit if the upper 95% credible 
limit for the mean posterior RR of treatment benefit, calculated using a skeptical prior, is less 
than 1.0. We propose to use a skeptical prior for this consideration to provide a more 
conservative basis for trial termination, and also to convince even those who strongly believed at 
the outset that the intervention will not succeed in improving survival free of cognitive 
impairment. In addition, we note that early stopping of a trial based on treatment benefit 
generally produces a biased and sometimes misleading estimate of the treatment effect 28, 29. 
 
13.7. INTERIM ESTIMATION OF TREATMENT FUTILITY 
In addition to providing estimates of interim treatment efficacy, we will also conduct interim 
futility analyses for treatment efficacy. For this purpose, at each interim safety look, we will use 
the available data to calculate the posterior probability of concluding that the probability of 
survival free of cognitive impairment in the cord blood group would be higher than in the 
placebo group (i.e., the mean posterior RR for treatment benefit is greater than 1.0), if the trial 
continued until 120 patients were randomized and had their primary outcomes evaluated. In 
order to provide a more conservative basis for this estimate, and also to convince even those who 
strongly believed in this intervention at the outset, we will primarily use an optimistic prior for 
this calculation, with the prior belief set at a level of treatment benefit observed in the Phase I 
trial. However, in order to provide a broad overview and to explore the robustness of this 
estimate with respect to prior assumptions, this probability will also be calculated and presented 
for the two other prior distributions (skeptical and uninformative). 
 
The DSMC may consider proposing termination of the trial for futility if this posterior 
probability of observing any treatment benefit, if the trial continued to full primary outcome 
accrual, is less than 10%.  
 
14. DATA SAFETY MONITORING COMMITTEE 
A DSMC will be responsible for monitoring the safety of the trial.  The DSMC will have a 
minimum of three members including a biostatistician, a neonatologist, and an expert in cord 
blood stem cell transplant. The DSMC members will be independent from any enrolling center 
and guided by a DSMC charter.   

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15. STATISTICAL CONSIDERATION  
15.1.STUDY DESIGN 
This study is a prospective Phase II prospective, randomized, double-blind, placebo controlled, multi-
center trial designed to assess the safety and efficacy of two infusions of autologous UCB cells or placebo 
in newborns born in distress.   
15.2.ESTIMATES OF AVAILABLE AND REQUIRED SAMPLE SIZE 
In our phase I study to date, 16 of 25 (64%) cell recipients with known one year outcomes have 
survived with all three Bayley III assessments (Cognitive, Motor, and Language) > 85, and 25 of 
63 (40%) of the cooled-only infants with known one year outcomes has survived with all three 
Bayley III domain scores > 85. (13) We acknowledge the limitations of a one year Bayley score, 
but would note that our rate of survival with Bayley scores > 85 among cooled-only infants is 
consistent with the previously reported Whole Body Hypothermia studies rates of survival at 12– 
24 months without moderate or severe impairment (1 -4).  
For the phase II study, rather basing the sample size on effect size, we would propose a Bayesian 
approach as used in the NRN Late Hypothermia study. Because this is phase II where our aim is 
to detect a trend favoring cell recipients, and to accumulate data that will inform design and 
sample size calculations for a phase III study of efficacy, we anticipate that two years of 
enrollment would provide approximately 120-160 infants, 60-80 in each group.  
Control  
Cell Recipients 
95% Confidence Interval 
Power 
0.6 
0.30 
0.45 
0.92 
0.6 
0.35 
0.48 
0.81 
0.6 
0.40 
0.51 
0.62 
0.6 
0.45 
0.54 
0.38 
0.6 
0.60 
0.54 
0.056 
 
The Bayesian analysis will permit calculation of a probability of treatment benefit. Within the 
range of an active group proportion of 0.3 to 0.45, the average 95% credible interval width 
ranges from 0.45 to 0.54 when the control group has a proportion having the primary outcome 
(death or Bayley III scores < 85) of 0.6. If the decision rule for declaring a win is the posterior 
95% credible interval must exclude 1, then the design has power ranging from 0.92 to 0.38 with 
a Type 1 error of about 0.056. 
15.3. STATISTICAL ANALYSIS PLAN 
All statistical analyses, including interim monitoring, will be conducted using the intent to treat 
principle, unless explicitly specified otherwise for certain secondary and/or post-hoc analyses.  A 
Bayesian approach will be used for conducting the primary analysis in this trial. We propose to 
apply a log-binomial regression model and a neutral prior that will be centered at RR=1.0 to final 

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efficacy and safety data, where final data is defined as data contained in the study database after 
database lock. Database lock will occur either after 160 infants have completed the study or the 
study is terminated early for efficacy, futility, or safety. In addition to using a neutral prior, final 
analyses on locked data and some interim analyses will develop posterior distributions using 
skeptical (centered at RR = 1.1) priors and/or enthusiastic (centered at RR = 0.625) priors as 
well. In all analyses, MCMC methods will be used to sample from the Posterior distribution. 
All analyses will involve estimating a Posterior distribution based on a log-binomial regression 
model with covariates and MCMC methods. In addition to estimating the treatment effect on the 
log relative risk scale, the log-binomial regression will adjust for level of encephalopathy. For 
the intercept and the encephalopathy terms, Normal (mean = 0, sd=100) priors will be used.  For 
the term representing the log relative risk, skeptical, neutral, and enthusiastic priors will be used 
to estimate posterior distributions for the relative risk. 
With respect to the primary efficacy endpoint, the estimated Posterior distribution of relative risk 
based on the data and the neutral prior will be used to determine posterior probabilities of >0, 
>10 and >20% decrease in death or moderate/severe disability, as well as graphical displays of 
the Posterior distribution and appropriate 95% credible intervals for the relative risk.  In addition 
to adjusting for level of encephalopathy and center in the log-binomial model, the final analysis 
of efficacy will also perform sensitivity analyses concerning possible treatment effect 
confounders present at randomization.  All additional covariates added to the model will utilize 
Normal (mean=0, sd=100) priors. 
The lack of preexisting data suggests that the neutral prior distribution may be most appropriate 
for presentation of final results; however, generation of final results using the skeptical (RR of 
1.10) and enthusiastic priors (RR of 0.625) will also be produced to give a broad overview of the 
implications of the trial’s efficacy results. 
In addition to the analyses specified above, we will also conduct equivalent frequentist analyses 
using robust Poisson regression to obtain relative risk estimates of the treatment effect adjusted 
for level of encephalopathy at baseline. Moreover, we will also conduct secondary analyses 
stratified by inborn or outborn status to estimate the treatment effect separately within each of 
these two groups. 
 
 

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