Transfusion of Prematures (TOP) Trial: Does a Liberal Red Blood Cell Transfusion Strategy Improve Neurologically-Intact Survival of Extremely-Low-Birth-Weight Infants as Compared to a Restrictive Strategy?
Phase: NA
Conditions: Infant, Newborn, Diseases, Infant, Extremely Low Birth Weight, Infant, Small for Gestational Age, Bronchopulmonary Dysplasia (BPD), Anemia
Sponsor: NICHD Neonatal Research Network

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Transfusion of Prematures (TOP) Trial:  
Does a Liberal Red Blood Cell Transfusion 
Strategy Improve Neurologically-Intact Survival 
of Extremely-Low-Birth-Weight Infants as 
Compared to a Restrictive Strategy? 
 
Haresh Kirpalani, BM, MSc  chair, Ed Bell, MD v. chair, Carl, D’Angio, MD, Susan Hintz, MD, 
Kathleen Kennedy, MD, Robin Ohls, MD, Brenda Poindexter MD, MS, Kurt Schibler, MD, 
Barbara Schmidt, MD, Betty Vohr, MD, Jack Widness, MD, Abhik Das, PhD, Rose Higgins, MD, 
John Zupancic, MD, ScD, Robin Roberts, MSc Robin Whyte MD, MD, Aasma Chaudhary, BS,  
Karen Johnson, BSN 
 
 
Version 5.0 
Final: October 8, 2012 
Revised: August 27, 2013 
Revised: April 9, 2014 
Revised: June 15, 2015 
Revised: April 7, 2016

 
 
 
 
ii 
Table of Contents 
Chapter 
Page 
TABLE OF CONTENTS 
II 
LIST OF TABLES AND FIGURES 
IV 
1.0 SYNOPSIS 
1 
2.0 BACKGROUND AND SIGNIFICANCE 
2 
2.1 
Increasing Survival of Newborn Preterms Less Than 1000 g BW ............................................ 2 
2.2 
Poor Outcomes of Survivors Born at Less Than 1000 g BW ...................................................... 2 
2.3 
High Frequency of Blood Transfusions in Neonatal Units........................................................... 2 
2.4 
Failure of Other Strategies to Prevent Allogeneic Red Blood Cell Transfusions ................. 3 
2.5 
Contradictions of Data on Risks and Benefits of Transfusion from Observational Data – 
only weak evidence based recommendations ................................................................................... 3 
3.0 INNOVATION 
6 
3.1 
Research Design and Milieu ................................................................................................................... 6 
3.2 
Economic Outcomes ................................................................................................................................. 6 
3.3 
Differing Blood Bank Practices ............................................................................................................ 7 
3.3.1 
Red Blood Cell Storage Lesions and Age of Transfused RBCs .................................. 7 
3.3.2 
Storage Media for Red Blood Cells (RBCs) ...................................................................... 7 
3.3.3 
Leukoreduction ............................................................................................................................ 8 
3.3.4 
Ensuring Cytomegalovirus-Negative Blood Products for Transfusion...................... 8 
3.3.5 
Irradiation of Blood .................................................................................................................... 8 
3.3.6 
How this Trial will Deal with These Possible Confounders ......................................... 9 
4.0 SPECIFIC AIMS 
10 
4.1 
Specific Aim 1 .......................................................................................................................................... 10 
4.2 
Specific  Aim 2 ......................................................................................................................................... 10 
5.0 APPROACH 
12 
5.1 
Preliminary Studies ................................................................................................................................. 12 
5.1.1 
Follow-up of Randomized Cohorts .................................................................................... 13 

 
 
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6.0 STUDY DESIGN 
15 
6.1 
Study Population, Inclusion and Exclusion Criteria, Withdrawals .......................................... 15 
6.2 
Intervention Maneuver -- How We Developed the Algorithm Triggering Transfusions .. 15 
6.3 
Stratification, Random Allocation, and Enrollment Procedure ................................................. 17 
6.4 
Prevention of Bias in an Unblinded Trial ......................................................................................... 17 
6.5 
Maintaining Adherence to Algorithm and Minimizing Deviation from Protocol .............. 17 
6.6 
Choice of Primary Outcome ................................................................................................................. 18 
6.7 
Main secondary Outcomes .................................................................................................................... 19 
6.8 
Potential Confounders and Strategy to Limit their Impact ......................................................... 19 
6.9 
Statistical Design Considerations ....................................................................................................... 20 
6.10 
Analysis Plan ............................................................................................................................................. 20 
6.11 
Data and Information Management ................................................................................................... 21 
7.0 METHODS 
22 
7.1 
Randomization Method .......................................................................................................................... 22 
7.2 
Recruitment and Enrollment Plans ..................................................................................................... 22 
8.0 DATA SAFETY AND MONITORING 
24 
9.0 STUDY ORGANIZATION AND ADMINISTRATION 
25 
9.1 
Study Sites.................................................................................................................................................. 25 
9.2 
Regulatory Compliance and Monitoring .......................................................................................... 25 
9.3 
Recruitment Pace ..................................................................................................................................... 25 
10.0 ETHICS 
26 
10.1 
Risk Category, Privacy .......................................................................................................................... 26 
10.2 
Consent and Oversight from NRN and RTI, and Interaction with Local IRBs ................... 27 
10.3 
Involvement of Human Subjects ......................................................................................................... 28 
REFERENCES 
29 
APPENDIX A:  DATA AND SAFETY MONITORING PLAN 
A-1 

 
 
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iv 
List of Tables and Figures 
Table 1 
Population Comparison between Trials ............................................................................................ 12 
Table 2 
Intervention Comparison between Trials ......................................................................................... 12 
Table 3 
Primary Outcomes ................................................................................................................................... 13 
Table 4 
Secondary Outcomes .............................................................................................................................. 13 
Table 5 
PINT-OS 18 to 24-Month Outcomes ................................................................................................. 13 
Table 6 
Transfusion Thresholds (Hemoglobin g/dl) .................................................................................... 16 
Table 7 
Transfusion Thresholds (Hematocrit)................................................................................................ 16 
Table 8 
Enrollment Timeline for the TOP Trial ............................................................................................ 22 
 
Figure 1. 
PINT-OS Outcomes Using the post-hoc Outcome of MDI<85 (mean minus 1 SD) ......... 14 
 
 

 
 
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1.0 
Synopsis 
Long-term outcomes of extremely low birth weight (ELBW) preterm infants, those 
weighing less than 1000 g at birth, are poor and pose a major health care burden. Virtually all of 
these infants are transfused, but at inconsistent hemoglobin (Hgb) thresholds. This results from 
conflicting data on when these infants should be transfused, preventing an evidence-based 
recommendation. However, the Prematures In Need of Transfusion (PINT) study1 suggests that 
higher Hgb thresholds for transfusion may be beneficial to long-term neurocognitive outcomes 
at 18-22 months.2 If a definitive adequately powered trial verifies this, the potential impact is 
high. An economic analysis of the PINT study outcomes showed that the increased costs of 
transfusion in the liberally transfused group would be more than offset by benefits of reduction in 
poor outcomes at 24 months.3  On the other hand, the Iowa RCT showed that short-term 
potential neurological benefits of more liberal blood transfusion4 were offset by poorer outcomes 
in brain structure and function at 7-10 year follow-up.5,6  This study however had a significant 
attrition making it difficult to understand its implications.We propose in TOP to randomize infants 
≤1000 g BW and < 29 weeks GA to receive red blood cell (RBC) transfusions according to one 
of two strategies of Hgb thresholds, either a high Hgb (liberal transfusion) or a low Hgb 
(restrictive transfusion) algorithm. It is currently unknown which transfusion strategy is superior. 
TOP is powered to demonstrate which strategy reduces the primary outcome of death or 
neurodisability in survivors at 22-26 months.  
We therefore propose an open, parallel-group multicenter randomized controlled trial 
(RCT) analyzed by intention to treat. TOP randomizes extremely-low-birth-weight infants (birth 
weight less than or equal to 1000 g) and gestational age at least 22 weeks and <29 weeks) to 
either a liberal or a restricted red cell transfusion regimen according to hemoglobin thresholds. 
Following consent of eligible infants, central randomization will be performed within strata of 
birth weight (<750 g versus 750-1000 g) and study center, with variable block sizes. Compared 
to larger preterm infants, ELBW infants are at greater risk of death and neurodevelopmental 
sequelae. Two previous trials - the PINT trial 1  and its follow-up PINT-Outcome Study (PINT-
OS) 2,, and the Iowa 4 trial and its follow up study 5 - raise important but contradictory 
implications for CNS injury and development. These can only be resolved in a new large trial. 
The objective of the TOP trial is to determine whether higher hemoglobin thresholds for 
transfusing ELBW infants resulting in higher hemoglobin levels lead to improvement in the 
primary outcome of survival and rates of neurodevelopmental impairment (NDI) at 22-26 
months. 
 

 
 
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2.0 
Background and Significance 
2.1 
Increasing Survival of Newborn Preterms Less Than 1000 g BW 
Preterm birth at any gestational age is a serious and large burden for individual families, 
the health care system, and society. “In an average week in the United States: 10,056 babies 
are born preterm (less than 37 completed weeks of pregnancy); 1,604 babies are born very 
preterm (less than 32 completed weeks); 6,511 babies are born with a low birth weight (less 
than 2500 grams birth weight); 1,188 babies are born at very low birth weight” (less than 1500 g 
BW) .7 The societal economic cost (medical, educational and lost productivity) is estimated for 
2005 as in excess of $26 billion.8 Most of the health burden stems from the most vulnerable 
infants in this group, those <1000 g BW, termed extremely-low-birth-weight (ELBW) infants. 
2.2 
Poor Outcomes of Survivors Born at Less Than 1000 g BW 
As survival has improved in ELBW infants, focus increases on the degree of how ‘intact’ 
the survival is. Intact survival is an end-point that is meaningful to parents of surviving children, 
society overall, and health care providers. Recent ELBW outcomes are not reassuring. Among 
infants born at less than 25 weeks and <1000 grams in 2002-2004 in the NICHD Neonatal 
Research Network, 51% of survivors had BSID-II Mental Development Index (MDI) scores <70 
at 18-22 months corrected age.9 Survivors at school age have even more concerning outcomes. 
For example, ELBW survivors at 6 years of age were approximately 40% more likely than 
matched classmates born at term to have cognitive delay, defined as >2 SD below test mean 
scores.10 Follow-up to adulthood adds concerns of a lack of age-appropriate sociability, which 
ranges from potentially beneficial effects of less delinquent behavior 11 to more worrying effects 
including infertility.12 It is true that a small study of selected adults born with ELBW show high 
self-perceived health-related quality of life scores.13 However, this may reflect an 
accommodation and adjustment to a lower functional ability.14,15 
2.3 
High Frequency of Blood Transfusions in Neonatal Units 
ELBW infants all become anemic in early life, and approximately 90% receive one or 
more blood transfusions.16 Preterm infants require transfusions for several reasons. Relative to 
term infants, preterm infants at birth have lower hemoglobin levels.17 Repeated iatrogenic losses 
for laboratory sampling exacerbate the problem18,19 and reflect prolonged intensive care. The 
anemia of prematurity is characterized by a reduced red cell life span, low levels of endogenous 
erythropoietin and a hyporegenerative bone marrow. This results in a universal and generally 
severe anemia in this population. The most powerful predictors of transfusion are low 
gestational age, low initial hemoglobin, large volume of iatrogenic loss, and low endogenous 
erythropoietin.20 In the 1980’s reported rates of transfusion were 80 to 90% for infants <1.5 kg 
BW, and 100% for infants <1000 g BW.21 However, transfusion practices are changing.22,23 
Comparing 1982 and 1993 transfusion data21, found that the number of transfusions per infant 
dropped significantly (from 7.0 ± 7.4 to 2.3 ± 2.7). This decline was associated with a decrease 
in pre-transfusion hematocrit (33.6 +/- 2.8% in 1982, 29.8 +/- 5.1% in 1993). In the past decade, 
RBC transfusion rates remained high in all studies. From 2000 to 2005 in the University of 
Iowa’s NICU, the mean (±SD) of RBC transfusions given to ELBW infant’s prior discharge was 
5.4 ± 0.9 (unpublished).  

 
 
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2.4 
Failure of Other Strategies to Prevent Allogeneic Red Blood Cell Transfusions 
Unfortunately, strategies to limit blood transfusions have had only limited success. Several 
methods have been proposed to “keep the blood in the baby”.24,25 Average phlebotomy losses 
are high, from 40 to 80 ml/kg.26,27 Spinning down blood samples to re-infuse red cells, is 
logistically awkward and time consuming.28 “Point-of-care” testing in an RCT29 did not reduce 
transfusion. Initial enthusiasm for aggressive human recombinant erythropoietin to boost bone 
marrow RBC production has waned. A systematic review by Vamvakas and Strauss30 found 
only modest effects of erythropoietin on transfusion need. In infants, a Cochrane review 
suggests that erythropoietin may elevate the risk of retinopathy.31 A large, well designed trial 
found erythropoietin therapy did not significantly reduce transfusions in infants and conferred no 
benefits in survival, chronic lung disease, retinopathy of prematurity or intraventricular 
hemorrhage.32 On follow-up to 22 months, these infants had no benefit in neurodevelopmental 
outcome33, except in a small highly selected post-hoc subset.34 In the TOP trial, most 
participating sites do not use erythropoietin except for children of parents with religious 
objections to blood transfusion. Increasing the red cell mass by delaying cord clamping may 
also boost blood volume, and seven trials in preterm infants are summarized in the Cochrane 
review.35 With delay in cord clamping of up to 120 seconds, reductions were seen in 
transfusions (3 trials, 111 infants; relative risk (RR) 2.01, 95% CI 1.24, 3.27); and hypotension 
(2 trials, 58 infants; RR 2.58, 95% CI 1.17, 5.67); and intraventricular hemorrhage (five trials, 
225 infants; RR 1.74, 95% CI 1.08, 2.81). Two later systematic reviews find a total of fifteen 
studies enrolling in total 734 preterms <37 weeks.36,37 Unfortunately, few infants below 1000 g 
have been enrolled in RCTs of either delayed cord clamping or cord milking. A meta-analysis by 
this group found only a total of 63 infants below 1000 g. 38  
 
2.5 
Contradictions of Data on Risks and Benefits of Transfusion from 
Observational Data – only weak evidence based recommendations 
Blood product use has risen sharply over recent years, but sound indications for use are 
lacking.39 Whether higher or lower transfusion thresholds are optimal in preterm infants remains 
controversial. Summarizing the data, a Cochrane review of infant studies shows no clear 
advantage of higher or lower hemoglobin transfusion thresholds for preterm infants and justifies 
clinical and scientific equipoise.40 Historically, both higher and lower transfusion thresholds were 
proposed on physiological grounds. But data have been contradictory, resulting in variable 
practices. This is still the case as shown by an international survey conducted by the PIs – 
where neonatologists chose to transfuse a sample infant over a very large range of hemoglobin 
or hematocrit.41 The controversy arises because of the dearth of high quality data, impeding the 
development of robust evidence-based guidelines. Many NICUs base their transfusion 
guidelines on those used in a 1995 trial of recombinant human erythropoietin to reduce 
transfusions.42 Trials show that strict criteria can reduce transfusions.43 However number of 
transfusions may not be the appropriate end point to study. 
In the absence of firm evidence, guidelines for transfusion threshold have trended 
downward, including those of the Fetus and Newborn Committee of the Canadian Paediatric 
Society.44 Current reports advocating lower transfusion thresholds for preterm infants are 
predominantly based on observational or retrospective study designs.45 Authors of such reports 
often cite adult trials, such as the TRIC trial.46 But extrapolating from these trials to the infant 
population carries obvious serious limitations. One biological challenge is that the preterm brain 
(in contrast to the adult brain) is developing and may have greater potential for damage from 
hypoxemia. Moreover, the adult data have had methodological limitations. In particular, the 
major cited finding of the TRICC trial was that younger and less sick patients were at higher risk 
of mortality. 46 However, this was a subgroup analysis not confirmed in a systematic review of 

 
 
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4 
multiple studies.47 Contradictory findings have prompted calls for a new large adult randomized 
trial.48 
We review here the arguments for lower and higher transfusion thresholds. First, data 
arguing for lower thresholds are discussed. A retrospective study in Brazil found an association 
between blood transfusions and neonatal mortality.49 The authors found an increased relative 
risk of death of 1.49 (1.17-1.78) in infants who received at least one RBC transfusion compared 
with non-transfused infants. However, methodological difficulties include confounding analyses 
in such a data set – sick infants are more likely to be transfused. Moreover, in the largest 
neonatal RCT of high versus low hemoglobin threshold, the RR favored higher thresholds, but 
this effect was not statistically significant.1 These methodological issues are discussed in a 
commentary editorial on dos Santos.50 Frequent transfusions can result in iron overload, which 
was found at autopsy to correlate with number of transfusions given to preterm infants.51 Excess 
tissue iron acts as an oxidizing agent. This has implications for some of the major and serious 
complications affecting ELBW infants, namely ROP, BPD and NEC.52,53,54,55,56 Several recent 
studies report increased risk of NEC following blood transfusions.57,58,59,60,61,62,63 One such report 
found that blood from male donors was more likely to be associated with NEC.64 Unfortunately, 
these are mainly retrospective observations, and all demonstrate, at best, association and not 
causality. 
Finally, liberal transfusion policies must consider infection risks. Assessing the risk-
benefit ratio of transfusion requires consideration of the unknown but real potential for 
transmission of infective agents that are undetected or even unknown.65 With new standards of 
screening for the HIV virus, this agent is less prominent. However, other blood-borne agents 
previously not considered as potential vectors (including babesiosis and Creutzfeldt-Jakob 
disease variant viruses) have recently been implicated in transmission of infection.66,67 
Cytomegalovirus (CMV) is still a potential concern despite efforts to mitigate its transmission in 
donated blood products.68 Despite leukoreduction of donor blood or the use of CMV-negative 
donors, rates of CMV break-through infection continue at 1-3% of transfused subjects.69 At 
current low rates of transmission, we acknowledge that our proposal is unlikely to be large 
enough to address these concerns directly. 
Those who favor higher transfusion thresholds, on the other hand, cite evidence that 
transfusing at higher hemoglobin thresholds reduces apnea of prematurity and fosters weight 
gain70 , increases oxygenation in ventilated infants71, and reduces compensatory increases in 
cardiac output.72,73 However, apnea is reduced by simple volume infusion as well as RBC 
transfusion.74 Moreover, the two RCTs that examined the impact of transfusion threshold on 
apnea differed in their findings: the Iowa trial found less frequent apnea with higher transfusion 
thresholds4, whereas the PINT study found no difference.1 The beneficial impact of higher 
hemoglobin thresholds on weight gain was not supported by PINT or other studies.75 Improved 
echocardiographic findings of cardiac parameters with higher hemoglobins71 may be simply 
surrogate physiological adaptations. Claims of increases in general body oxygenation are also 
challenged by measures of near infrared spectroscopy in peripheral forearm tissue.76,77 There 
was too large an overlap between symptomatic and asymptomatic infants, which precluded 
distinguishing on the basis of peripheral oxygen extraction.78 Nonetheless, the same technology 
for assessing brain oxygen saturation appears to confirm that infants with lower hemoglobins 
tend to have lower regional brain oxygen saturation.79,80,81 This may be relevant to the 
observation that lower rates of IVH were associated with higher hemoglobins.82,83 Even those 
who advocate lower hemoglobin ranges for transfusion often recommend higher hemoglobin 
thresholds in the first week of life in the hope that this will help to protect against brain 
hemorrhage.45 However, once again, the data are contradictory. A large observational study of 

 
 
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417 newborns found that a simple grade I germinal matrix hemorrhage was more likely to 
evolve into a grade 3 or 4 IVH if there had been a preceding RBC transfusion (OR 2.92; 95% CI 
2.19, 3.90).84 If it is true that higher hemoglobins confer neuroprotection from IVH, this implies a 
physiological mechanism to explain the observation in PINT-OS of better developmental 
outcome at higher hemoglobin thresholds.2 
To conclude:  The contradictory findings of previous studies make evidence-based 
recommendations nearly impossible. The TOP trial would produce substantial evidence on 
which to base recommendations to guide transfusion practice for ELBW infants. 
 

 
 
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6 
3.0 
Innovation 
Summary:  Many neonatologists have shifted toward using lower hemoglobin thresholds 
for transfusion, based largely on retrospective data. For example Valieva et al stated: “In our 
study of all ELBW infants treated at UW NICU during 2006, we did not identify any clinical 
benefits of transfusing PRBCs with the guidelines in place at the time. As a result of this study, 
the transfusion guidelines at the UWMC NICU were made more restrictive“.45 In contrast, the 
TOP study proposes a higher-level study design, namely a randomized controlled trial. In 
addition, the need to assess long-term outcomes of randomized trials is compelling in the 
newborn, where developmental outcomes are of paramount interest to both parents and 
society.85 A randomized comparison will enable comparison of health care costs of the two 
strategies in weighing competing risks and benefits. Our group has analyzed the short and long-
term costs of the two transfusion strategies used in the PINT study.3 Finally, in designing the 
current study, we have designed a strategy that accounts for differing blood bank practices and 
differences in dispensed products across multiple study sites. An ideal solution would be to 
establish tight guidelines for blood bank practices, but this would be difficult to implement and 
would limit the generalizability of the results. Instead, we will first conduct a study to survey 
individual blood bank practices and assess the correlation of hematocrit with volume and weight 
of dispensed cells. This will allow an a-priori adjustment for potential variation in hematocrit by 
dispensed RBCs by site. 
3.1 
Research Design and Milieu 
This prospective randomized unmasked Phase III trial defines a clinically significant 
long-term outcome that will be assessed by examiners blinded to group assignment. It is led by 
experienced clinical trialists and biostatisticians who are familiar with the relevant 
methodological issues. Three of the four key investigators for the two paired applications (HK, 
EB, DB) have had specific experience with RCTs comparing transfusion strategies. The two 
major trials pertinent to this study question were designed and performed independently by the 
two clinical PIs. They were published within 6 months of each other. In reality, TOP is the result 
of a new partnership created by merging the two rival groups who had conducted the Iowa and 
PINT trials. These two studies, taken together, resulted in new hypotheses about the impact of 
hemoglobin level (or hematocrit) on the developing brain. These form the basis for the proposed 
TOP trial, which will be built on the very experienced, productive framework of the NICHD 
Neonatal Research Network. 
3.2 
Economic Outcomes 
We will conduct a formal, prospective economic evaluation with patient-level cost and 
efficacy data alongside the parent TOP randomized controlled trial, using standard accepted 
methodology86, as previously demonstrated by our group in similar multi-center trials.87,88,89,90,91 
The analysis will take a comprehensive societal perspective and a similar 24-month timeline to 
the parent trial. Direct medical resource utilization during the initial birth hospitalization will be 
ascertained through collection of itemized billing records and UB-04 forms, a uniform billing 
statement recommended by the National Uniform Billing Committee and utilized for reporting of 
hospital expenditures by all third party payers including the Centers for Medicare and Medicaid 
Services (CMS). We will convert hospital-reported charges to costs by applying the appropriate 
Center for Medicare and Medicaid Services cost-center specific ratio of costs to charges.92,93,94 
Physician professional fees for the initial hospitalization will be based on CMS reimbursement 

 
 
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7 
levels for each day of stay and non-bundled procedure, using information in the TOP case 
report forms to assign a level of illness acuity and a billing code according to the Resource-
Based Relative Value Scale.95,96 Family out-of-pocket expenses and work-related productivity 
losses, as well as post-discharge health-care utilization will be ascertained by questionnaire 
administered during the initial hospitalization and at follow-up visit. 
Sample size will be based on power considerations for the parent clinical trial.  All 
analyses will utilize an intention-to-treat approach. Data confirmed to be missing at random will 
be addressed as appropriate using multiple imputation.97 We will first directly compare mean 
cost between the study groups using generalized linear modeling to account for the typically 
right skewed cost data distribution.98,99  We will then calculate the incremental cost-effectiveness 
ratio, defined as the difference in mean cost per patient in the high and low transfusion 
threshold arms divided by the difference in the mean effect between the study arms. The 
outcome will thus be expressed as the cost per survivor without neurodevelopmental 
impairment. To assess statistical uncertainty in the joint distribution of costs and effects, we will 
use nonparametric bootstrapping and report results as cost-effectiveness acceptability 
curves.100,101,102,103 Finally, we will assess uncertainty in parameter values by using sensitivity 
analysis, in which we will recalculate the cost-effectiveness after varying the input values for 
certain variables through a plausible range. 
3.3 
Differing Blood Bank Practices 
After collection, donor blood must be safeguarded against red cell hemolysis and 
infection. However, donor blood is stored with differing anticoagulants and varying conditions 
and for varying periods of time in different centers. Some such factors could act as co-
interventions. A recent multinational study “confirmed the great variability in the hemoglobin 
levels in the RBCs transfused to patients”.104 However this is a generalizable and pragmatic 
trial. We will record these differences, but not attempt to control for them. A-priori secondary 
analyses will take account of these factors.  
3.3.1 
Red Blood Cell Storage Lesions and Age of Transfused RBCs 
In the U.S., the Food and Drug Administration allows RBCs to be refrigerated for up to 
42 days .105 Stored blood is favored over fresh blood because it lowers administrative and 
collection costs, minimizes waste, and assures an adequate and readily available supply of 
blood. Although longer storage might be detrimental106,107, human data are conflicting as to the 
benefits of “newer” (fresher) blood. In adults undergoing coronary artery bypass surgery, receipt 
of blood stored <14 days, was associated with improvement in all measured outcomes 
compared with those receiving older blood, including in-hospital mortality.108  However, this 
retrospective study had an imbalance of massively transfused patients between the groups: 
more than half of the patients who died had received more than 6 units and were 
disproportionately more likely to have received older blood.106 The question of the safety and 
efficacy of fresh versus stored blood is still open. An ongoing adult randomized trial109and a trial 
in preterm infants (Clinicaltrials.gov NCT00326924: The Age of Red Blood Cells in Premature 
Infants Study (ARIPI) are currently examining optimal length of RBC storage, with regard to 
clinically relevant outcomes including mortality. 
3.3.2 
Storage Media for Red Blood Cells (RBCs) 
RBCs are stored so as to remain sterile, metabolically active (usually mandating 
dextrose solution) and intact, with specified targets for cell survival such that acute hemolysis of 
transfused RBCs is less than 1% and 24-hour survival of the RBCs in the recipient is at least 

 
 
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75%.110 Anticoagulation is achieved with citrate, and phosphate is used as a buffer. Thus, most 
solutions contain citrate, phosphate, and dextrose (CPD). Additions of mannitol and adenine are 
intended to stabalize the RBC membrane and maintain 2,3-diphosphoglycerate and ATP levels 
within the RBC. The use of adenine has boosted the storage life of RBCs from 21 days for CPD 
to 35 days for citrate-phosphate-dextrose-adenine (CPDA-1), and up to 42 days with even 
newer formulations (Adsol, Optisol, and Nutricell). Reports suggest that prolonged storage may 
result in metabolic abnormalities.111 Washing RBCs may limit exposure of the heavily transfused 
neonate to these storage compounds. There is inadequate information on the safety of these 
compounds, and the efficacy of washing RBCs is also unknown. Based on the quantities of 
anticoagulant preservatives solutions, during large volume transfusions to neonates, metabolic 
abnormalities are anticipated and documented in a few small studies.111 
3.3.3 
Leukoreduction 
RBC transfusion may down-regulate immune status, a phenomenon termed transfusion-
related immunomodulation or TRIM.112,113,30 White cells (WBCs) in donor blood may be the 
source of both cytokines and histamine mediators as they deteriorate in storage, which may 
result in detrimental effects. Randomized trials have compared white cell reduction by buffy coat 
depletion against more targeted specific WBC reduction. Benefits of aggressive leukoreduction 
may include reducing hospital-acquired infections.114,115 Other trials suggest leukoreduction may 
decrease absolute total postsurgical mortality by 4.2%.116 In 1999, the Canadian national blood 
service adopted universal leukoreduction. Following this, a large retrospective study examined 
mortality effects. Both unadjusted and adjusted absolute hospital mortality rates were 
approximately 1% lower with leukoreduction (adjusted OR, 0.87; 95% CI, 0.75, 0.99; P=0.04).117 
A study of leukoreduction in premature infants found no change in the primary outcome of 
infection, but on secondary analysis, the composite rates of BPD, IVH, ROP and NEC were 
significantly reduced (OR 0.31 (95% CI, 0.17, 0.56).118 Nonetheless, methodological problems 
preclude firm recommendations. Meta-analysis of trials in oncology supports skepticism about 
accepting these findings.119,112,120 
3.3.4 
Ensuring Cytomegalovirus-Negative Blood Products for Transfusion 
Newborns are vulnerable to transfusion acquired cytomegalovirus (CMV). CMV is 
frequently present in asymptomatic adults, with some 70% of the population being 
seropositive.121,122 Seropositive donors present a risk of transmission, even when not actively 
viremic. Some consider that CMV-seronegative blood should be given to preterm infants at risk 
of frequent transfusion. 121 However, this complicates testing the blood supply and assuring an 
adequate stock. Since the virus is thought to be latent in neutrophils, alternatives include 
leukocyte reduction or storage methods to reduce viral survival such as freezing in glycerol. 
Finally, 3rd-generation leukocyte-depletion filters prevent primary CMV in neonates123 and in 
immunologically deficient adult bone-marrow transplant patients. However, there is no 
consensus on how best to prevent transfusion-acquired CMV infection. 
3.3.5 
Irradiation of Blood 
Irradiation of blood does not prevent CMV transmission from RBC transfusion, as 
irradiation affects primarily lymphocytes. Thus, irradiation of blood is a center-specific measure 
with unproven benefit in reducing risk of CMV transmission to transfused newborns.124 The 
relevance for newborns is unclear since the risk of graft versus host disease is unknown in 
ELBW infants. 

 
 
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3.3.6 
How this Trial will Deal with These Possible Confounders 
We will randomize 1824 infants and examine the primary outcome of death or 
neurodevelopmental impairment at 22-26 months. Given our large, generalizable, pragmatic trial 
design, we cannot mandate local laboratory practices to adopt uniform blood banking practices. 
Instead we have adopted the following strategy: 
i. 
We have appointed a ‘TOP Trial Blood Banking Committee’ led by Dr. Naomi Luban 
as Chair Members were selected for expertise in blood banking practices. They will 
conduct a survey of all the blood banks by site to ensure that practices fall within an  
acceptable range for standards of care. Before the study begins, we will conduct a 
survey on pertinent blood bank practices in the 18 participating sites of the NRN. 
This will be the subject of a separate report and will inform the TOP trial of the likely 
magnitude of variation among the sites.  
ii. 
Patients will be stratified by center, and analyses will adjust for center. 
 

 
 
TOP Protocol 
 
10 
4.0 
Specific Aims 
4.1 
Specific Aim 1 
To examine whether the clinically relevant composite primary outcome of death or 
significant neurodevelopmental impairment in survivors at 22-26 months of corrected age is less 
common among preterm infants who, by transfusion practice, are maintained at higher 
hemoglobins. Neurodevelopmental assessments will be performed by trained and annually 
recertified examiners, who are masked to treatment allocation, using standardized tools: the 
Bayley Scales of Infant Development-III (BSID-III) and the Gross Motor Function Classification 
Scale (GMFCS).125,126 The primary outcome consists of any one of the following: (i) Cognitive 
delay where delay is defined as BSID-III cognitive score < 85 (>1 standard deviation below the 
mean). (ii) Cerebral Palsy if the child has a non-progressive motor impairment characterized by 
abnormal muscle tone and impaired range or control of movements. Moderate cerebral palsy 
(CP) is defined as level II or III on the GMFCS. Severe or non-ambulatory CP consists of 
GMFCS IV or V. (iii) Severe vision impairment, defined as corrected visual acuity in the better 
eye less than 20/200, determined by pediatric ophthalmologists. (iv) Severe hearing impairment, 
defined as bilateral hearing loss requiring amplification or cochlear implant. (v) Death before 22-
26 month follow-up assessment. The primary outcome is deemed present if one or more of the 
individual components of the composite outcome are present, or absent if no component was 
present. If no component is present, one or more missing components render the primary 
outcome to be deemed missing. 
4.2 
Specific  Aim 2 
We will examine in which arm the following secondary outcomes are more common: 
Short term, to NICU discharge: 
 
(a) Survival to discharge without severe morbidity, defined as any of the following: 
bronchopulmonary dysplasia, retinopathy of prematurity (stage >3 or requiring 
treatment), or serious brain abnormality (grade 3 or 4 intraventricular hemorrhage, 
periventricular leukomalacia, or ventriculomegaly). 
(b) Weight, length, and head circumference at 36 weeks postmenstrual age or at 
discharge from NICU, whichever occurs first. 
(c) Serious abnormality on cranial ultrasound examination: grade 3 or 4 intraventricular 
hemorrhage, periventricular leukomalacia, or ventriculomegaly; 
(d) Age at final tracheal extubation; age at final caffeine dose 
(e) Number of transfusions, numbers of donor exposures by RBC donors or other blood 
product 
(g) For survivors, length of hospital stay in the level 3 NICU referral site, or in the level 3 
area of the referral site; 
(h) Episodes of necrotizing enterocolitis of Bell stage 2 or higher, and time to full feeds; 
 
Long term (survivors only), at 22-26 months corrected age: 
 
(i) The incidence of ambulatory and non-ambulatory CP defined by GMFCS; 
(j) Hydrocephalus shunt, microcephaly, or seizure disorder; 

 
 
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11
(k) The presence of respiratory disease necessitating readmission before 22-26 months 
follow-up. 
(l) All individual components of the composite outcome of NDI or death, including 
cognitive outcomes at follow-up at 1 SD cut-off on the BSID III standardized scales 
(m) BSID III cognitive, language and motor scores at 2 SD cut-offs (<70) at follow-up. 
(n) Economic cost-benefit analysis to time of discharge and to 22-26 month follow-up. 
 
Potential Impact: This trial is powered to address an important unresolved clinical 
problem. Current trials have shown the hypothesis to be reasonable, and needs a definitive trial 
to prove or disprove. This trial will change clinical practice, whether or not the higher arm 
improves NDI. If it does improve, important further research should target how neurological 
pathways are improved by transfusion. 
 

 
 
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12
5.0 
Approach 
This trial directly builds on and benefits from the two major prior trials – led by the PIs of 
the TOP study - and their follow-up studies.1,2,4,5,6 
5.1 
Preliminary Studies 
Historical trials are of limited help in today’s clinical environment of infants <1000 g.127,128 
In the ELBW population, four trials are relevant. One trial examined differing thresholds for 
transfusion but with the adjunctive therapy of erythropoietin, and no significant differences were 
found.129 One trial is too small (n=36) to be really informative130, and its inclusion in the 
Cochrane review on neonatal transfusions did not alter the conclusions.2 The Iowa and PINT 
trials are directly comparable, and erythropoietin was used in neither study.1,4 These trials 
asked: “In high-risk preterm infants, does a low or a high hemoglobin or hematocrit transfusion 
threshold strategy lead to a clinically detectable benefit?” Subjects in both trials were high risk, 
but the birth weight criterion for entry was lower in PINT trial (Table 1). PINT ran in Canada, 
U.S. and Australia. 
The intervention algorithm for transfusions also varied, but both trials adopted the 
principle of higher target hemoglobins for infants requiring oxygen and or respiratory support 
(Table 2). The main difference was in the hemoglobin separation between the restrictive and 
liberal groups, which was 2.7 g/dl in the Iowa trial as opposed to 1.1 g/dl in PINT. The 
transfusion thresholds were arrived at in differing ways. The Iowa group had extrapolated above 
and below current clinical ranges, and the PINT group stayed within the confines of 
recommendations of the Fetus and Newborn Committee of Canada131 – to which participating 
units adhered. 
Table 1 
Population Comparison between Trials  
 
Iowa Trial 
PINT Trial 
 
Restrictive 
Liberal 
Restrictive 
Liberal 
Participating centers 
1 
10 
No. of subjects 
100 
451 
Treatment allocation 
Randomized 
Randomized 
Stratification 
Birth weight 
Birth weight, center 
Mean BW (g) 
954 
958 
771 
769 
Mean GA (wk) 
28 
28 
26 
26 
Table 2 
Intervention Comparison between Trials 
 
The primary outcomes selected were different (Table 3). Iowa used a reduction in the number of 
 
Iowa Trial 
PINT Trial 
 
Restrictive 
Liberal 
Restrictive 
Liberal 
Transfusion thresholds 
    Hemoglobin, g/dl 
    Highest 
11.3 
15.3 
11.5 
13.5 
    Lowest 
7.3 
10.0 
7.5 
8.5 
Mean hemoglobin 
8.3 
11.0 
10.1 
11.2 
Mean hemoglobin difference 
2.7 
1.1 

 
 
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13
transfusions, which was significantly different. PINT constructed a composite outcome of death 
or any one of: BPD, ROP stage 3 or higher, or brain Injury (PVL, IVH grade 4, or 
ventriculomegaly). The primary outcome in PINT was not significantly different between the 
restrictive and liberal transfusion groups. The PINT trial also showed a reduction in transfusions. 
In an unplanned secondary analysis, the Iowa trial found excess severe IVH and PVL in the 
restrictive group (Table 4). 
Table 3 
Primary Outcomes 
 
Iowa Trial 
PINT Trial 
 
Primary outcome 
 
Number of transfusions 
Composite outcome of death 
or any of the following:  BPD, 
ROP stage >3, or brain injury 
(PVL, IVH grade 4, or 
ventriculomegaly) 
Result 
Restrictive 
Liberal 
Restrictive 
Liberal 
 
3.3 
5.2 
74% 
70% 
 
P=0.025 
Not significant 
Table 4 
Secondary Outcomes 
 
Iowa Trial 
PINT Trial 
 
Restrictive 
Liberal 
Restrictive 
Liberal 
IVH grade 3 or 4 
10% 
16% 
 
 
PVL 
14%* 
0 
2.7% 
2.3% 
PVL or IVH grade 4 
12% 
0 
 
 
Death 
4% 
2% 
21.5% 
17.5% 
* Of those with late head ultrasound examinations 
 
5.1.1 
Follow-up of Randomized Cohorts 
The PINT study reported follow-up of survivors at 18-24 months age, in the outcome 
study - PINT-OS.2 The a-priori primary outcome was a composite of death or survival with one 
or more neurodevelopmental impairments: cognitive delay (Bayley II MDI <70); cerebral palsy; 
blindness; or deafness requiring amplification. The follow-up rate was 93%. For the primary 
outcome, the OR for low vs high transfusion thresholds was 1.45 (95% CI 0.94, 2.21) (p=0.091). 
The rate of death or impairment was 45.2 % in the low group vs 38.5% in the high group. 
However, in a secondary a-priori outcome assessment, the component parts of the primary 
outcome cluster were examined separately (Table 5). 
Table 5 
PINT-OS 18 to 24-Month Outcomes 
 
Odds Ratio 
95% CI 
Death 
1.18 
0.72,1.93 
Cerebral Palsy 
1.32 
0.53, 3.27 
Cognitive delay, Bayley MDI <70 
(2 SD below mean) 
1.74 
0.98, 3.11 
Blindness 
2.16 
0.19, 24.1 
Deafness 
1.45 
0.32,6.58 
 
All point estimates favored the high threshold group, but none was statistically 
significant. However, the nearly significant difference in the risk of cognitive delay, strongly 

 
 
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14
suggested a true effect of statistical significance was possible. We therefore conducted a post-
hoc analysis using Bayley MDI score <85 (1 SD below the mean) to determine prevalence of 
cognitive impairment. This result is summarized below (Figure 1).  As can be seen, the 
composite outcome is significant using the MDI cutoff of 85. The data do not allow firm 
conclusions, but a benefit to higher hemoglobin transfusion thresholds is plausible in reduced 
death (OR 1.38, 95% CI 0.84, 2.27) and/or reduced neurocognitive deficit in survivors. We 
believe these benefits of higher transfusion thresholds are sufficiently important to justify a new 
adequately powered study. 
 
Figure 1. PINT-OS Outcomes Using the post-hoc Outcome of MDI<85 (mean minus 1 SD)  
 
 
OR
95% CI
p
1.71
1.12, 2.61
0.013
1.18
0.72 , 1.93 
0.52
1.32
0.53 , 3.27 
0.55
1.81
1.12,2.93
0.016
2.16
0.19 , 24.1 
0.53
1.45
0.32 , 6.58 
0.63
USING A CUT-OFF OF MDI<85

 
 
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15 
6.0 
Study Design 
6.1 
Study Population, Inclusion and Exclusion Criteria, Withdrawals 
Inclusion criteria:  Infants – inborn or outborn – who are admitted to participating 
NICUs and who fulfill the following inclusion criteria are eligible for enrollment:  (a) Birth weight 
≤1000 g and gestational age at least 22 weeks but less than 29 weeks; and (b) Less than 48 
hours of age. 
Exclusion criteria:  Infants meeting any of the following criteria will not be eligible: (a) 
Considered nonviable by the attending neonatologist. (b) Cyanotic congenital heart disease. (c) 
Parents opposed to the transfusion of blood. (d) Parents with hemoglobinopathy or congenital 
anemia. (e) In-utero fetal transfusion. (f) Twin-to-twin transfusion syndrome. (g) Isoimmune 
hemolytic disease. (h) Lack of parental consent. (i) Prior blood transfusion on clinical grounds 
beyond the first 6 hours of life. (j) High probability that the family is unable to return for the  
follow-up assessment at 22-26 months. (k) Infant has received erythropoietin prior to 
randomization, or is intended to receive erythropoietin through the neonatal course. (l) 
Congenital condition, other than premature birth, that adversely affects life expectancy or 
neurodevelopment.   
Algorithm Deviations:  Infants who by either late diagnosis or clinical deterioration are 
found to have conditions listed in the exclusions will be transfused according to the preference 
of their medical team rather than by study protocol. However, their outcomes will be measured 
and analyzed according to original allocation by intention-to-treat principles. 
6.2 
Intervention Maneuver -- How We Developed the Algorithm Triggering 
Transfusions 
Previous trials using a titrated therapy were criticized as departing from standard 
practice.132,133 Therefore, the ethically correct approach to define the Hgb thresholds is to remain 
within the range of current clinical standards of practice.134,135 Threshold Hgbs used in practice 
vary from center to center, may depend on the neonatologist and are often unclear. We base 
our proposed transfusion thresholds on: 
i. 
The range of hemoglobin thresholds used clinically to guide transfusion decisions in 
the participating NICUs of the NICHD Neonatal Research Network; 
ii. 
A poll of the range of hemoglobin thresholds that would be acceptable to each 
neonatologist in an NRN site within the context of an RCT. 
The low threshold values reflect more common practice, so this is considered the ‘usual 
treatment’ group. In this group, the transfusion thresholds are similar to those used for the 
restrictive group in both the PINT and Iowa trials. The highest threshold for the liberal 
transfusion group was the highest acceptable to neonatologists at the majority of NRN centers. 
We identified two potential clinical modifiers of triggers to transfusion, postnatal age and 
respiratory support. The use of postnatal age to define transfusion thresholds is based on the 
physiologic fall in hemoglobin seen in infancy. Respiratory support is defined as mechanical 
ventilation, continuous positive airway pressure, FiO2 in excess of 0.35, or oxygen by nasal 
cannula ≥ 1 liter per min (please note: room air nasal cannula 1 lpm or greater is considered 
respiratory support). The final intervention algorithm reflects substantial dialogue with all NRN 

 
 
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16
site members. It will be applied to the enrolled population up to the postmenstrual age of 36 
weeks or until the infant is discharged from the NICU, whichever occurs first. 
The transfusion thresholds for the two groups are listed in Table 6 below, as hemoglobin 
in g/dl. 
Table 6 
Transfusion Thresholds (Hemoglobin g/dl)  
 
High threshold 
Low threshold 
 
 
Resp support 
No support 
 
Resp support 
No support 
Period: 
 
 
 
 
 
 
  Week 1 
 
13.0 
12.0 
 
11.0 
10.0 
  Week 2 
 
12.5 
11.0 
 
10.0 
8.5 
  Weeks ≥3 
 
11.0 
10.0 
 
8.5 
7.0 
 
The values given for Week 3 and later, will be used as transfusion thresholds until 
postmenstrual age 36 weeks or hospital discharge, whichever occurs first.  
Since some NICUs prefer to use hematocrit, we provide the table by HCT also,  using  
the conversion formula: Hematocrit (%) = 2.941 x Hgb (g/dl) 
(http://www.heartpumper.com/hematocrit.htm). – to provide the table below. 
The transfusion thresholds for the two groups are listed in Table 7 below, as hematocrit. 
Table 7 
Transfusion Thresholds (Hematocrit)  
 
High threshold 
Low threshold 
 
 
Resp support 
No support 
 
Resp support 
No support 
Period: 
 
 
 
 
 
 
  Week 1 
 
38 
35 
 
32 
29 
  Week 2 
 
37 
32 
 
29 
25 
  Weeks ≥3 
 
32 
29 
 
25 
21 
 
Our proposed algorithm results in a difference in hemoglobin threshold of 2-3 g/dl in all 
age and respiratory categories. Moreover, these thresholds conform to the ranges within which 
all sites have confirmed equipoise. 
Volumes of transfusions and hemoglobin measures:  We will use a standard 
transfusion volume of 15 ml/kg, expecting to raise Hgb by about 4 g/dl. A fixed transfusion 
volume will yield higher compliance and a consistent difference in Hgb levels between the two 
arms. Centers are free to obtain blood samples for Hgb determination from venous, arterial, or 
prewarmed heel capillary blood according to vascular access and their usual practice. We 
recognize that values from large veins and arteries are systematically lower than capillary 
samples by about 8.5%.17 However for a pragmatic trial, these small differences are not 
problematic and do not require adjustment. We will not mandate Hgb checks, but by polling we 
have ensured that most units obtain these at least weekly in the first month. We will record all 
Hgb values obtained. 

 
 
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17
Non-algorithm, or clinically driven transfusions:  The algorithm will determine 
transfusion for routine or “top-up” transfusions, but additional transfusions may be given for 
infants with urgent need for transfusion because of bleeding or anticipated bleeding, e.g. during 
and after surgery. All blood transfusions will be recorded and categorized as “threshold 
reached” or “other indication” (e.g., shock, severe sepsis with coagulopathy; or perioperative). 
All subsequent events will be analyzed by intention-to-treat principles, irrespective of additional 
transfusions, trial dropouts, or protocol violations. 
Transfusions and blood banking:  All RBCs transfused will be tested and screened 
according to the policies of the blood banks serving each hospital participating in the trial. These 
practices will be recorded throughout the trial, with notification to the DCC of any policy 
changes. The blood transfused will be ABO and Rh compatible with the infants’ type. Individual 
unit practices related to blood transfusion vary among the participating sites. Because 
randomization will be stratified by site, variation in local blood banking practices will not result in 
imbalances between treatment groups and will allow greater generalizability of the study results. 
The details of blood bank practice to be recorded for each site will include screening for CMV, 
storage buffer, maximum storage age, policies for RBC washing and leukoreduction, and 
methods of storing and concentrating RBCs. The age of donor blood will be recorded for each 
transfusion, but there is no restriction on age beyond individual site practice.  
6.3 
Stratification, Random Allocation, and Enrollment Procedure 
Parents of eligible infants who conform to the inclusion and exclusion criteria will be 
approached by the clinical study team (either a research nurse coordinator or a physician 
investigator) within 48 hours of birth in order to offer study participation. A log will be maintained 
of all screened infants indicating which are eligible and which were enrolled. On receipt of 
consent, treatment assignment (high or low transfusion threshold group) will be made using a 
telephone system of randomization by the Data Coordinating Center (RTI International). 
Randomization will be performed within the appropriate birthweight and center stratum. Detailed 
instructions for treatment allocation and implementation and monitoring of the study protocol will 
be given to the research nurse and to on-call physicians in each participating center.  
6.4 
Prevention of Bias in an Unblinded Trial 
All eligible infants of ≤1000 g birth weight and gestational age 22 0/7 through 28 6/7 
weeks will be screened and logged, reducing selection bias. Randomization will be concealed, 
preventing allocation bias. It is impossible practically or ethically to blind either the caregivers or 
parents to the assigned hemoglobin threshold. Interim results will be presented to the Data 
Safety and Monitoring Committee (DSMC) with treatment assignments labeled only as group 
“A” or “B”. We will record all hemoglobin measures and transfusions given in order to assess 
protocol compliance. Site investigators will review compliance with clinicians at the onset of the 
trial and thereafter monthly as appropriate. Examiners at the 22-26 month examination will be 
unaware of treatment assignment, as will the radiologists interpreting the brain ultrasounds. 
Within NRN centers, local site interpretations were highly accurate and inter-rater reliability was 
excellent for the diagnosis of grade 3 or 4 IVH.136 
6.5 
Maintaining Adherence to Algorithm and Minimizing Deviation from Protocol 
As noted above the algorithm will drive the majority of the transfusions. In exceptional 
circumstances, as defined below, the algorithm may be set aside until the infant is deemed 
stable to allow resumption of transfusion by study algorithm. Examples of such situations are 
infants with shock, severe sepsis with coagulopathy; or need for perioperative transfusion, 

 
 
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18
where the critical clinical indication for transfusion will override the threshold. In the PINT 
multicenter trial, the rate of deviation from protocol was acceptably low (15.9% in the low 
threshold arm and 6.7% in the high threshold arm). We assess adherence and separation of 
arms by recording mean Hgb at transfusion and at discharge. No Hgb determinations are 
mandated by study, but only as clinically ordered. 
6.6 
Choice of Primary Outcome 
We will use a composite outcome of death or significant neurodevelopmental impairment 
in survivors at 22-26 months of corrected age. Children will be assessed at 22-26 months of 
corrected age by staff unaware of assignment. Centers will schedule visits as early as possible 
within this window. Outcome assessments will be conducted by physicians or nurses 
specializing in neonatal follow-up examination and certified psychologists. Neurodevelopmental 
Impairment will be defined in this study, as one or more of the following: cognitive delay, 
moderate or severe cerebral palsy, or severe visual or hearing impairment, as defined below. 
Cognitive delay will be assessed by certified psychologists will assess infants using the 
Bayley Scales of Infant Development III (BSID-III). Cognitive delay is defined as BSID-III 
cognitive score < 85 (>1 standard deviation below the mean). 
Cerebral Palsy is diagnosed if the child has a non-progressive motor impairment 
characterized by abnormal muscle tone and impaired range or control of movements. Medical 
history, physical examination and neurological examinations will be used to determine the 
presence of moderate or severe cerebral palsy (CP). Moderate CP is defined as level II or III on 
the Gross Motor Function Classification Scale (GMFCS).125,126 Severe or non-ambulatory CP 
consists of GMFCS IV or V. 
Severe vision impairment; is defined as corrected visual acuity in the better eye less 
than 20/200 determined by pediatric ophthalmologists. 
Severe hearing impairment, is defined as bilateral hearing loss requiring amplification 
or the insertion of a cochlear implant as determined by a sound field hearing assessment or 
auditory brainstem responses. 
The primary outcome will be deemed present if one or more of the individual 
components of the composite outcome are known to be present, or absent if no component was 
present. If no component was present, one or more missing components will cause the primary 
outcome to be deemed missing.  
Cognitive delay is defined using the cutoff of 85 in Cognitive score because the Bayley 
III cognitive score below 70 identifies a smaller group than the Bayley II MDI below 70. This is 
because the Bayley III administered at 18-22 months corrected age among children born 
extremely preterm results in higher scores than the Bayley II.137 One of two alternative 
explanations explains this difference. Either we collectively have previously underestimated 
cognitive development, or current testing overestimates cognitive outcomes. We believe a 
conservative approach to sample size will account for potential measurement error at 22-26 
months. Although the more common composite primary outcome uses a cognitive score cutoff 
of 70 (i.e., 2 SD below the mean), we have chosen a cutoff score of 85, to answer possible 
effects on neurodevelopment raised by PINT, and to allow a conservative approach regarding 
the sample size. 

 
 
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19
At the 22-26 month follow-up, each of the composite parts of the Primary Outcome will 
be examined also as a secondary outcome. All secondary outcomes at this time point are listed, 
but they will include: behavioral, emotional and social problems as evaluated by the Child 
Behavior Checklist 1.5 to 5.  This is a 99 item instrument that has been widely used in follow up 
studies. It is being utilized by the Neonatal Research Network in the Follow Up Study for 
extremely low birth weight infants, as well as in the follow up components of clinical other trials.  
6.7 
Main secondary Outcomes 
Secondary outcomes are measured at two time points: 
 
(A) During initial hospitalization:  (i) Ultrasound grade 3 or 4 IVH, cystic PVL, or 
ventriculomegaly; (ii) Bell stage 2 or 3 NEC; (iii) Apnea mandating either caffeine or 
respiratory support; (iv) Age at final tracheal extubation; (v) Number of donor 
exposures by RBC donors or other blood product; (vi) Number of transfusions; (vii) 
ROP stage 3 or greater or requiring treatment; (viii) Time to regain birth weight; (ix) 
Time to full feedings; (x) Death before discharge; (xi) BPD at 36 weeks 
postmenstrual age based on need for oxygen supplementation following an oxygen 
reduction test; (xii) Decreased height, weight, or head circumference at 36 weeks 
postmenstrual age; (xiii) length of hospital stay at Level 3 care; and (xiv) Costs of 
hospitalization. 
 
(B) At 22-26 months corrected age, each of the composite outcomes individually: (i) 
Moderate or severe cerebral palsy; (ii) Severe vision impairment; (iii) Severe 
hearing impairment; (iv) BSID-III Cognitive score < 85; (v) BSID-III cognitive, 
language, or motor score <70; (vi) Gross Motor Function level ≥ II; (vii) CBCL 
Internalizing, Externalizing, and Total Problems aggregate T scores of >= 64 
(clinical range) and 60-63 (borderline range) will be evaluated. (viii) Death before 
22-26 month follow-up; (ix) Decreased height, weight, or head circumference at 22-
26 month assessment; and (x) Cost-benefit analysis of primary outcome at 22-26 
months. 
 
Analyses of all primary and secondary outcomes will be adjusted for the design effects of 
stratification by center and birth weight group, and correlation between multiple births. 
6.8 
Potential Confounders and Strategy to Limit their Impact 
(i) Severity of initial illness:  Randomization should achieve balance at baseline. Adjustment 
for the SNAPPE score138 on admission to NICU will be performed post-hoc if imbalances 
arise. 
(ii) Blood bank practices:  Center stratification will ensure balance across study.  
(iii) Gender of blood donor: A retrospective cohort study of infants with birth weight <1500 g 
suggested that transfusion from male donors is associated with an increase in rate of 
NEC.64 Thus, the proportion or total volume of transfused blood from male donors will be 
covariates in the model to predict NEC. This will be collected while preserving HIPPA 
anonymity, after the main trial is complete. This will be done by only using the blood unit 
ID number. This will be relayed to the issuing blood banks, who would provide only the 
gender of the donor to the investigators.   
(iv) Sociological determinants:  Home environmental influences and potential enhanced 
neurodevelopmental programs will be recorded as highest maternal educational 
levels, at enrollment. Adjustment for these will be conducted in secondary analyses. 
 

 
 
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20
6.9 
Statistical Design Considerations 
The primary objective of the TOP trial is to assess the difference in the primary outcome of 
survival without significant NDI (or, equivalently, death or NDI) at 22-26 months adjusted age in 
the two treatment arms. This outcome is similar to that used in the PINT trial, which found rates 
of death or neurosensory impairment at 18 months of 45.2% in the low trigger group and 38.5% 
in the high trigger group. Based on these findings, we believe that an absolute difference of 7% 
is the minimal clinically significant difference we would wish to detect in the rate of death or NDI. 
We considered several other factors in estimating the sample size for this study. First, we 
calculated the rate of death or NDI among infants born at NRN centers in 2005-2008 with GA 
22-26 weeks and birth weight <1000 g who survived to 12 hours, which was 52%. However, we 
recognized that this population differed somewhat from that of the TOP trial, which proposes to 
enroll infants less than 29 weeks GA. In addition, the current NRN definition of NDI incorporates 
only the cognitive composite score of the Bayley III, while future definitions will likely also 
involve the Bayley III motor scales which have been collected in the Network only since January 
2010. Thus, we recognized the limitations of estimating our sample size based only on current 
NRN data. At the same time, however, we were aware that the most conservative sample size 
estimate would result from an event rate of 50%, which current NRN data indicated was not 
implausible in our population. This led us to choose a conservative estimate based on assumed 
outcome rates of 53.5% and 46.5% (i.e., centered around an overall event rate of 50%) in the 
two treatment groups. With a two-tailed alpha of 0.05 and 80% power, the initial sample size 
estimate was 1,658 infants. The sample size was additionally inflated by 10% to account for loss 
to follow up at 22-26 months, yielding a final sample size estimate of 1,824. 
Although the sample size calculations described above were based on the primary 
outcome, we will have 80% power with a two-tailed alpha of 0.05 to detect the following 
differences in important secondary outcomes (rates in the low threshold group are based on 
findings of the PINT trial): any transfusion 89.0% in the low threshold group (LOW) vs. 92.9% in 
the high group (HI); NEC 8.5% in LOW vs. 5.0% in HI; brain injury (PVL, echodense lesions or 
ventriculomegaly) on latest ultrasound (US) or neuroimaging closest to 36 weeks 18.5% in LOW 
vs. 24.6% in HI; death before discharge 21.5% in LOW vs. 16.1% in HI; combined death, severe 
ROP, BPD, or US brain injury 74.0% in LOW vs. 67.7% in HI.  
6.10 
Analysis Plan 
Main Study:  All analyses will be conducted on an intention-to-treat basis, with the caveat that 
infants surviving to discharge, but lost to follow-up at 22-26 months, will be excluded from the 
primary analysis and considered ignorably missing. We will, however, assess the impact of 
outcomes lost to follow-up on our results by performing sensitivity analyses that assume all 
positive or all negative outcomes for those with missing data. We will also conduct bivariate 
analyses to compare the in-hospital characteristics of children lost to follow-up versus those with 
assessment of the primary outcome. Note that, based on previous NRN trials in similar 
populations, we expect the follow-up rate among survivors to be above 90%. 
All analyses will be adjusted for design effects: stratification by center and birth weight 
group. The primary outcome of death or NDI at 22-26 months adjusted age will be analyzed 
using robust Poisson regression. Adjusted relative risks and 95% confidence intervals will be 
reported. Secondary analyses of the primary outcome may adjust for additional baseline 
covariates such as SNAPPE scores at NICU admission and socioeconomic status..  
The consistency of the treatment effect over birth weight strata and gender, and 
treatment heterogeneity across the different centers, will be investigated. 

 
 
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21
Secondary outcomes including comorbidities such as NEC, ROP, BPD, and NDI among 
survivors at follow up, as well as other binary outcomes, will be analyzed using robust Poisson 
regression to produce adjusted relative risk estimates for the treatment effect, as described 
above. Models may also be adjusted for covariates known to be associated with the secondary 
outcomes in this population. For example, both gender and socioeconomic status have been 
implicated as crucial confounders for cognitive outcomes among survivors at follow up 
(Nopoulos P 2011); thus, it may be appropriate to include them as covariates in models for such 
outcomes. Number of transfusions will be analyzed using Poisson regression. Other secondary 
outcomes, such as time to regain birth weight and time to full feeds, will be analyzed using Cox 
proportional hazards survival regression. Although the basic Cox model assumes proportional 
hazards between groups, if this assumption is found to be violated in our data, time-varying 
covariates can be added to the models to allow for changes in the proportion over time. Weekly 
measures of growth (weight, length, head circumference) will be analyzed using longitudinal 
models to test whether growth trajectories differ between treatment groups. All models 
incorporating the treatment effect will be adjusted for the stratification variables (center and birth 
weight group). All secondary outcomes analyses will be considered exploratory because the trial 
is not primarily powered to detect these associations.  
  
Economic Evaluation: We will conduct a formal, prospective economic evaluation with 
patient-level cost and efficacy data, using standard methods used in similar multicenter trials 
(56-60). Medical expenditure data from hospitals and families will be collected. The analysis will 
first directly compare mean costs between the two treatment groups using generalized linear 
modeling to account for the typically right-skewed cost data distribution. We will then calculate 
the incremental cost-effectiveness ratio, defined as the difference in mean cost per patient in the 
two groups, divided by the difference in the mean effect between the study arms. The outcome 
will thus be expressed as the cost per survivor free of significant NDI. To assess statistical 
uncertainty in the joint distribution of costs and effects, we will use nonparametric bootstrapping 
and report results as cost-effectiveness accepTOPlity curves. Finally, we will assess uncertainty 
in parameter values by using sensitivity analysis, in which we will recalculate the cost-
effectiveness after varying the input values for certain variables through a plausible range. 
Further details are provided in the fuller protocol.  
6.11 
Data and Information Management  
The data management system (DMS) recommended for the TOP study is a general 
purpose distributed data entry and management system developed to enable clinics to enter 
data from medical record extractions or other sources. This table-driven system is designed in a 
way that allows quick implementation of new studies into the existing system. Versions of this 
system, continually updated, have been successfully used for data collection for all NRN studies 
since 1998. An expanded version of this section is contained in the ‘Data Safety and Monitoring 
Plan’(Appendix A).  
 

 
 
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22 
7.0 
Methods 
7.1 
Randomization Method  
Eligible infants whose parents consent to enrollment will be randomized to the high or 
low threshold group. Permuted block randomization with randomly chosen block sizes of two 
and four will be performed. The allocation ratio will be 1:1 and infants will be stratified by study 
center and birth weight (<750 g versus 750-999 g). Multiple births, which are common in this 
premature population (comprising 26% of infants <29 weeks gestational age in the 2009 NRN 
Generic Data Base), will be randomized independently. The study allocation (low or high 
threshold) will be randomly assigned to each patient number (to be assigned to enrolled patients 
sequentially within each center), in advance, using a computer-based random number 
generator. Study personnel at each center will telephone a secure automated toll-free line at 
RTI to obtain the randomized assignment for each enrolled patient. Back-up personnel at RTI 
will be in place in case of failure of the automated system, to ensure 24/7/365 randomization. 
Details of interaction of RTI with study sites, and clinical case record forms (CRFs) are provided 
in the Manual of Procedures (MOP).  
7.2 
Recruitment and Enrollment Plans 
The NRN, with its 18 large academic medical centers, several with large satellite sites of 
their own, is uniquely positioned to successfully complete this rather large and ambitous 
neonatal trial. The trial poses an important and unresolved question in neonatal medicine, and 
both the PINT and Iowa trials indicate collective equipoise and high consent rates (>70%). 
Moreover, NRN centers have demonstrated the ability to recruit and randomize patients at all 
hours in previous studies where time was an important factor, and have an impressive track 
record in following infants through 18-22 months of age.. The NRN has achieved > 90% 
outcome assessment at 18-22 months follow-up in each of its previous trials. The most recent 
examples are the Phototherapy and Hypothermia trials, where >91% subjects had primary 
outcomes at 18-22 months and 6-7 years of age, respectively (47,50).  In Table 8, below we 
provide different scenarios on the recruitment timeline for the TOP trial, based on most recent 
numbers from the NRN Generic Database. It demonstrates that this trial can indeed be 
successfully completed by the NRN centers within the requested funding period.  
Table 8 
Enrollment Timeline for the TOP Trial 
Predicted 
GDB 
Enrollment 
Per Month 
Percent 
Recruited 
Per Month 
(per center) 
Estimated 
Total Time 
to Enroll 
Consent 
Rate 
Unable to 
Recruit 
for Other 
Reasons 
152 
70 
10 
91 (5.7) 
20 months 
152 
60 
10 
76 (4.7) 
24 months 
152 
50 
10 
60 (3.8) 
30 months 
 
Similar to other NRN trials, recruitment will be monitored closely by the DCC through 
regular enrollment reports, and low enrollment at a center will prompt the CCC, DCC, NIH, and 

 
 
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23
other members of the trial subcommittee to hold conference calls with such centers to identify 
problems and develop solutions. 
 
Although the acute enrollment is envisaged to be for 30 months, the follow-up phase will be 
longer. Our window for the follow-up is centered on 24 months, and extends from 22 to 26 
months.  

 
 
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24 
8.0 
Data Safety and Monitoring 
Data Safety Monitoring Committee: The NRN has a standing, independent Data 
Safety Monitoring Committee (DSMC) which monitors all Network trials. Because the proposed 
study will be conducted within the NRN, and because the procedures of this DSMC are 
consistent with NHLBI policies for Monitoring Boards for Data and Safety, the NICHD DSMC will 
monitor patient safety for this protocol. The committee includes experts in neonatology, 
maternal and fetal medicine, neurodevelopment, epidemiology, biostatistics, bioethics, high-risk 
infant follow-up, and clinical trials. A neonatal transfusion specialist will be added to the DSMC 
for the purposes of this study. An expanded version of this section is contained in the ‘Data 
Safety and Monitoring Plan’ (Appendix A).  
 

 
 
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25 
9.0 
Study Organization and Administration 
9.1 
Study Sites  
The study organization is guided by the procedures of the Neonatal Research Network. 
The Network is a collaboration among eighteen clinical centers appointed after competitive 
review, the DCC, and the NICHD. The Network has a Steering Committee consisting of the 
eighteen principal investigators (PIs) of the clinical centers, the NICHD Program Officer Dr. 
Rosemary Higgins, and an independent chairman. The Committee, which meets quarterly, sets 
policy and makes decisions.  
9.2 
Regulatory Compliance and Monitoring  
The TOP DCC will operate under guidelines to ensure data accuracy, integrity, 
confidentiality, and security. DCC activities are governed formally by RTI Standard Operating 
Procedures or SOPs or less formally through technical operating procedures and guidance 
documents, which also help ensure regulatory compliance at the clinical centers.  
We will continuously monitor data quality using point-of-entry quality checks in the DMS 
and other cross-form or longitudinal checks indicated by the investigators. We will identify 
errors, missing forms/items, and post center-specific reports on the private portion of the NRN 
website so that clinical staff can follow up. We have used this approach successfully for the 
NRN and other studies, producing automated routine reports on enrollment, retention, missing 
visits/items, and other problems, that enable us to evaluate general center performance and 
quickly initiate corrective actions as needed.  
We will conduct targeted site visits using data audits on accrual, drop-outs, unresolved 
queries, and protocol violations to inform cost-efficient site monitoring decisions (54). 
Established checklists and reporting templates will be used to ensure that centers have the 
necessary resources; review research records and regulatory documents and observe study 
procedures, such as screening and recruitment. Following the site visit, the DCC will provide a 
written report to the center and NIH, and follow up on unresolved issues. 
9.3 
Recruitment Pace 
Similar to other NRN trials, recruitment will be monitored closely by the DCC through 
regular enrollment reports, and low enrollment at a center will prompt the CCC, DCC, NIH, and 
other members of the trial subcommittee to hold conference calls with such centers to identify 
problems and develop solutions.

 
 
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26 
 
10.0 
Ethics 
10.  
Ethics 
10.1 
Risk Category, Privacy  
We avoid breaching current practice boundaries of thresholds of hemoglobin that would 
trigger a transfusion by assuring broad buy-in of NRN clinicians and a transfusion algorithm that 
was developed by consensus. This approach avoids the dilemmas sometimes faced by other 
trials.134  
This open trial, allows clinicians to bypass the study transfusion algorithm in 
circumstances where an infant’s condition warrants acute transfusion, but then revert to the 
algorithm thereafter. The potential risks to subjects participating in this study or their parents are 
categorized in this study as ‘minimal risk’. The trial maneuvers – other than randomization – are 
all within the range of current clinical practice and well within the likely variation of normal 
clinical practice.  
We do not mandate any additional blood sampling nor specific route of sampling of 
blood, thus not departing from the standard practice of the NICU. The informed consent 
document and patient information materials will be thoroughly reviewed with the parents prior to 
their completing the written informed consent. Families will be given ample opportunity to ask 
questions regarding alternative options.  
The basic rights of study participants will be respected and maintained by the 
investigators and by all who are involved in the collection or processing of individually identified 
data. Our data collection and processing procedures are designed to protect individual rights 
and to comply with all applicable laws and ethical principles. All staff who conduct or support 
research involving human subjects are required to undergo training on the protection of human 
subjects in research. 
Minimizing risks from procedures used to collect data is the responsibility of the clinical 
centers and the DCC, although procedures to minimize risk will be described in the study 
protocols and include medical management common for this study population. Confidentiality 
procedures for subject data will be established by the trial investigators before data are 
transferred to RTI. All subject-identifying data will be kept solely at the clinical centers. The data 
collection forms will include unique study ID numbers only and basic demographic data as 
participant identifiers. Thus, the files maintained at RTI will contain limited identifying information 
and protect subject confidentiality. Safeguards are in place to greatly decrease the chances that 
characteristics of a case can be linked to the individual participating in the study.  
The DCC will receive subject data from the clinical centers identified by a study ID only 
and will never have contact with the subjects. All procedures related to DCC activities, including 
data transmission and data security procedures, are reviewed and approved by the RTI IRB 
prior to receipt of any study data at RTI.  

 
 
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27
10.2 
Consent and Oversight from NRN and RTI, and Interaction with Local IRBs 
All NRN clinical centers and RTI have IRB committees that convene on a fixed schedule 
every month to review protocols and associated informed consent forms and data collection 
procedures for all research to ensure that they are in compliance with all applicable human 
subject regulations. IRB approval must be granted prior to beginning any study, and study 
progress and procedures must be reviewed by the IRB at least annually.  
Potential subjects or their legal guardians must be fully informed about the details of any 
research study in which they are considering participation and what their involvement will entail. 
Specific consent forms are developed for each protocol and reviewed and approved by the IRBs 
at RTI and at each clinical center. RTI reviews forms used at the sites to ensure that essential 
elements of consent are presented and comply with federal law 
All NRN clinical centers and the DCC at RTI have Federal wide Assurance (FWA) by the 
Office for Human Research Protections (OHRP) of the Department of Health and Human 
Services (HHS). This FWA is an agreement between each center, RTI and the U.S. government 
that all research with human subjects will be conducted according to appropriate federal 
regulations and allows us to undertake its own Institutional Review Board (IRB) review and 
monitoring of research with human subjects. Each NRN institution participating in the TOP trial 
holds an FWA, which ensures that the institution’s human research activities, overseen by their 
regulatory authorities, comply with the requirements set forth in 45 CFR 46, as well as the terms 
of Assurance. The DCC at RTI is responsible for obtaining appropriate clearances at our 
institution with respect to HIPAA and Human Subjects Research regulations and verifying 
similar clearances at the clinical centers.  
TOP trial study procedures are subject to the approval of the IRBs at the participating 
clinical centers and the DCC. For this trial, the protocol, informed consent (IC), and other study 
documents used at each center must be reviewed and approved by the respective IRBs before 
the study is initiated. The IRBs monitor the research process to ensure that the procedures for 
protecting human subject rights are followed. Every protocol is reviewed by each IRB at least 
annually. Each IRB reviews AE reports and approves all proposed changes to a research 
protocol before any changes are implemented. If necessary, the IRBs will mandate changes 
needed to protect the rights and welfare of human subjects or suggest solutions for problems 
that arise during a project.  
After IRB approval, infants eligible for the TOP trial will be identified by the clinical center 
PI or study staff from among the infants who are cared for in the NRN Neonatal Intensive Care 
Units. The PI or their staff will explain to the parents (1) consent forms that have been 
specifically to address the nature, duration, and purpose of the study; (2) means by which it is to 
be conducted; (3) possible benefit or lack of benefits; (4) potential risks, hazards, and 
discomforts; and (5) possible alternative procedures. Specific decisions regarding the 
operational details of how consent will be sought (including the timing and the level of details 
presented regarding various aspects of the study) are made at the level of each clinical center 
under the guidelines of their local IRBs and practice policies and traditions.  
NICHD and NHLBI will defer to local IRB ruling for the conduct of the TOP trial at all the 
clinical centers. Should a local IRB determine safety issues and require actions, including any 
related to suggested changes to the consent form, the local center research staff 
(investigator/coordinator) will notify the NRN DCC (RTI), the NHLBI and NICHD Program 
Scientists via phone or email within 24 hours of discovery. NHLBI and NICHD will direct the 

 
 
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28
DCC in further action. Note that any changes to the protocol itself (directed either by any IRB or 
the NRN DSMC) will require discussion and approval by the TOP trial subcommittee, NHLBI 
and NICHD. The DCC will maintain all communications between the clinical center(s), NHLBI 
and NICHD through resolution of all these IRB issues.   
Conflicts of Interest 
We will ensure that no member appointed to the DSMC has a conflict of interest, and 
they   will be required to sign to that effect. This is in the DSMC charter. No one on the study 
team including the proposed steering committee of the trial, has any financial interests related to 
either blood products, or alternatives to standard blood based infusion fluids.  Nor do they act as 
consultants to any commercial blood product agencies. 
10.3 
Involvement of Human Subjects 
The primary goal of this study is to conduct a randomized clinical trial of varying 
transfusion thresholds in extremely low birth weight (≤1000 g) or ELBW newborns to determine 
the effects of relatively high or low transfusion triggers on long-term survival and 
neurodevelopmental outcomes in this population. The study will involve this population, with 
baseline and sociodemographic data, as well as informed consent obtained from parents or 
legal guardians.  
In addition to our internal safeguards, we have a Data Safety and Monitoring Committee 
(DSMC) and a Data Safety and Monitoring Plan (Appendix A), which will be implemented 
together with NICHD, the institute that oversees the Neonatal Research Network and will 
support this trial. 

 
 
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80. Dani C, Pratesi S, Fontanelli G, Barp J, Bertini G. Blood transfusions increase cerebral, 
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83. Mercer JS, Vohr BR, McGrath MM, Padbury JF, Wallach M, Oh W. Delayed cord clamping 
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84. Baer VL, Lambert DK, Henry E, Snow GL, Christensen RD. Red blood cell transfusion of 
preterm neonates with a Grade 1 intraventricular hemorrhage is associated with extension 
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85. Zhang B, Schmidt B. Do we measure the right end points? A systematic review of primary 
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87. McBride JA, Parad R, davis J, Allred L, Zupani J. Cost analysis on the use of recombinant 
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Retrospective economic evaluation of a controlled trial of indomethacin prophylaxis for 
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89. McBride JA, Parad RB, Davis JM, Zheng Z, Zupancic JA. Economic evaluation of 
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91. Dukhovny D, Lorch SA, Schmidt B, Doyle LW, Kok JH, Roberts RS, et al. Economic 
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94. Taira DA, Seto TB, Siegrist R, Cosgrove R, Berezin R, Cohen DJ. Comparison of analytic 
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95. RBRVS: What is it and how does it affect pediatrics?: American Academy of Pediatrics; 
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96. Hsiao WC, Braun P, Dunn D, Becker ER, DeNicola M, Ketcham TR. Results and policy 
implications of the resource-based relative-value study. N Engl J Med. 1988;319(13):881-
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97. Briggs A, Clark T, Wolstenholme J, Clarke P. Missing... presumed at random: cost-
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98. Manning WG, Basu A, Mullahy J. Generalized modeling approaches to risk adjustment of 
skewed outcomes data. J Health Econ. 2005;24(3):465-88. 
99. Manning WG, Mullahy J. Estimating log models: to transform or not to transform? J Health 
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100. Efron B, Tibshirani RJ. An introduction to the boostrap. New York: Chapman and 
Hall/CRC; 1993. 
101. Fenwick E, Claxton K, Sculpher M. Representing uncertainty: the role of cost-effectiveness 
accepTOPlity curves. Health Econ. 2001;10(8):779-87. 
102. Fenwick E, O'Brien BJ, Briggs A. Cost-effectiveness accepTOPlity curves--facts, fallacies 
and frequently asked questions. Health Econ. 2004;13(5):405-15. 
103. Polsky D, Glick HA, Willke R, Schulman K. Confidence intervals for cost-effectiveness 
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104. Reikvam H, van de Watering L, Prowse C, Devine D, Heddle NM, Hervig T; BEST 
collaborative.  Evaluation of noninvasive methods for the estimation of haemoglobin 
content in red blood cell concentrates. Transfus Med. 2011 Jun;21(3):145-9. 
105. Adamson JW. New blood, old blood, or no blood? N Engl J Med. 2008;358(12):1295-6. 
106. Zimrin AB, Hess JR. Current issues relating to the transfusion of stored red blood cells. 
Vox Sang. 2009;96(2):93-103. 
107. Marik PE, Sibbald WJ. Effect of stored-blood transfusion on oxygen delivery in patients 
with sepsis. JAMA. 1993;269(23):3024-9. 
108. Koch CG, Li L, Sessler DI, Figueroa P, Hoeltge GA, Mihaljevic T, et al. Duration of red-cell 
storage and complications after cardiac surgery. N Engl J Med. 2008;358(12):1229-39. 
109. Tinmouth A, Fergusson D, Yee IC, Hebert PC. Clinical consequences of red cell storage in 
the critically ill. Transfusion. 2006;46(11):2014-27. 
110. Roback J, Combs MR, MT(ASCP)SBB, Grossman B and Hillyer C. Technical Manual, 16th 
ed. Bethesda: American Association of Blood Banks (AABB); 2008. 
111. Fasano R, Luban NL. Blood component therapy. Pediatr Clin North Am. 2008;55(2):421-
45, ix. 
112. Vamvakas E, Moore SB. Perioperative blood transfusion and colorectal cancer recurrence: 
a qualitative statistical overview and meta-analysis. Transfusion. 1993;33(9):754-65. 
113. Blajchman MA. Allogeneic blood transfusions, immunomodulation, and postoperative 
bacterial infection: do we have the answers yet? Transfusion. 1997;37(2):121-5. 
114. Houbiers JG, Brand A, van de Watering LM, Hermans J, Verwey PJ, Bijnen AB, et al. 
Randomised controlled trial comparing transfusion of leucocyte-depleted or buffy-coat-
depleted blood in surgery for colorectal cancer. Lancet. 1994;344(8922):573-8. 

 
 
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115. Jensen LS, Kissmeyer-Nielsen P, Wolff B, Qvist N. Randomised comparison of leucocyte-
depleted versus buffy-coat-poor blood transfusion and complications after colorectal 
surgery. Lancet. 1996;348(9031):841-5. 
116. van de Watering LM, Hermans J, Houbiers JG, van den Broek PJ, Bouter H, Boer F, et al. 
Beneficial effects of leukocyte depletion of transfused blood on postoperative 
complications in patients undergoing cardiac surgery: a randomized clinical trial. 
Circulation. 1998;97(6):562-8. 
117. Hebert PC, Wells G, Blajchman MA, Marshall J, Martin C, Pagliarello G, et al. A 
multicenter, randomized, controlled clinical trial of transfusion requirements in critical care. 
Transfusion Requirements in Critical Care Investigators, Canadian Critical Care Trials 
Group. N Engl J Med. 1999;340(6):409-17. 
118. Fergusson D, Hebert PC, Lee SK, Walker CR, Barrington KJ, Joseph L, et al. Clinical 
outcomes following institution of universal leukoreduction of blood transfusions for 
premature infants. JAMA. 2003;289(15):1950-6. 
119. McAlister FA, Clark HD, Wells PS, Laupacis A. Perioperative allogeneic blood transfusion 
does not cause adverse sequelae in patients with cancer: a meta-analysis of 
unconfounded studies. Br J Surg. 1998;85(2):171-8. 
120. Thurer RL, Luban NL, AuBuchon JP, Silver H, McCarthy LJ, Dzik S, et al. Universal WBC 
reduction. Transfusion. 2000;40(6):751-2. 
121. Luban NL. Transfusion safety: Where are we today? Ann N Y Acad Sci. 2005;1054:325-
41. 
122. Glynn SA. Blood supply safety: an NHLBI perspective. Transfusion. 2008;48(8):1541-4. 
123. Gilbert GL, Hayes K, Hudson IL, James J. Prevention of transfusion-acquired 
cytomegalovirus infection in infants by blood filtration to remove leucocytes. Neonatal 
Cytomegalovirus Infection Study Group. Lancet. 1989;1(8649):1228-31. 
124. Strauss RG. Data-driven blood banking practices for neonatal RBC transfusions. 
Transfusion. 2000;40(12):1528-40. 
125. Rosenbaum PL, Walter SD, Hanna SE, Palisano RJ, Russell DJ, Raina P, et al. Prognosis 
for gross motor function in cerebral palsy: creation of motor development curves. JAMA. 
2002;288(11):1357-63. 
126. Palisano RJ, Cameron D, Rosenbaum PL, Walter SD, Russell D. STOPlity of the gross 
motor function classification system. Dev Med Child Neurol. 2006;48(6):424-8. 
127. Ransome OJ, Moosa EA, Mothebe FM, Spector I. Are regular 'top-up' transfusions 
necessary in otherwise well, growing premature infants? S Afr Med J. 1989;75(4):165-6. 
128. Blank JP, Sheagren TG, Vajaria J, Mangurten HH, Benawra RS, Puppala BL. The role of 
RBC transfusion in the premature infant. Am J Dis Child. 1984;138(9):831-3. 
129. Bifano EM, Bode MM, D’Eugenio DB. Prospective randomized trial of high vs low 
hematocrit I ELBW infants: one year growth and neurodevelopmental outcome. Pediatr 
Red. 2002:51:325A. 
130.  Chen HL, Tseng HI, Lu CC, Yang SN, Fan HC, Yang RC. Effect of blood transfusions on 
the outcome of very low body weight preterm infants under two different transfusion 
criteria. Pediatr Neonatol. 2009;50(3):110-6. 

 
 
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131. Guidelines for transfusion of erythrocytes to neonates and premature infants. Fetus and 
Newborn Committee, Canadian Paediatric Society. CMAJ. 1992;147(12):1781-92. PMCID: 
1336654. 
132. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute 
lung injury and the acute respiratory distress syndrome. The Acute Respiratory Distress 
Syndrome Network. N Engl J Med. 2000;342(18):1301-8. 
133. Mann H. Controversial choice of a control intervention in a trial of ventilator therapy in 
ARDS: standard of care arguments in a randomised controlled trial. J Med Ethics. 
2005;31(9):548-53. PMCID: 1734226. 
134. Deans KJ, Minneci PC, Suffredini AF, Danner RL, Hoffman WD, Ciu X, et al. 
Randomization in clinical trials of titrated therapies: unintended consequences of using 
fixed treatment protocols. Crit Care Med. 2007;35(6):1509-16. 
135. Minneci PC, Eichacker PQ, Danner RL, Banks SM, Natanson C, Deans KJ. The 
importance of usual care control groups for safety monitoring and validity during critical 
care research. Intensive Care Med. 2008;34(5):942-7. 
136. Hintz SR, Slovis T, Bulas D, Van Meurs KP, Perritt R, Stevenson DK, et al. Interobserver 
reliability and accuracy of cranial ultrasound scanning interpretation in premature infants. J 
Pediatr. 2007;150(6):592-6, 6 e1-5. PMCID: 2757063. 
137. Vohr BR, Stephens BE, Higgins RD, Bann CM, et al. Are outcomes of extremely preterm 
infants improving? Impact of Bayley assessment on outcomes. J Pediatr. 2012 
Aug;161(2):222-8.e3. 
138. Zupancic JA, Richardson DK, Horbar JD, Carpenter JH, Lee SK, Escobar GJ. Revalidation 
of the Score for Neonatal Acute Physiology in the Vermont Oxford Network. Pediatrics. 
2007;119(1):e156-63. 

 
 
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Appendix A:  DATA AND SAFETY MONITORING PLAN  
 
1. Composition of the Data and Safety Monitoring Board (DSMB) 
TOP is a study which is investigator-initiated and sponsored by the Eunice Kennedy Shriver 
NICHD Neonatal Research Network (NRN). Accordingly the standing NRN Data Safety 
Monitoring Committee (hereafter referred to as ‘DSMC’) provides the basis of study safety 
oversight.  
 
In order to ensure full co-oversight by the NHLBI, two representatives of the Blood division of 
NHLBI are appointed to the Board, which enables a joint oversight to be exercised.  Precedence 
for this model comes from the NRN SUPPORT trial (co-funded by the NHLBI) where additional 
specialists joined the NRN DSMC for monitoring that trial. The joint oversight is provided by the 
two representatives of the Blood division on the DSMC. These are firstly Dr. Traci Mondoro MD 
(Project Officer for the Blood division of NHLBI), and Dr. Catherine S. Manno, a pediatric 
transfusion expert.  
 
All members of the DSMC are completely independent of the study and the PIs and will be 
required to sign documentation to this effect.  The NRN DSMC conforms to the recommended 
standards of the NIH (http://www.fda.gov/RegulatoryInformation/Guidances/ucm127069.htm). 
 
We detail some specifics of DSMC functioning: 
a) The executive secretary is Dr. Marion Willinger, who is based at the Pregnancy and 
Perinatology Branch, Center for Developmental Biology and Perinatal Medicine NICHD, 
NIH.  
Dr. Willinger is not involved at any level with the study other than in her function as 
Executive Secretary of the DSMC. 
 
b) The DSMC is chaired by Dr. Christine Gleason (Specialty: Neonatology, Cerebral-
vascular Physiology; Department of Pediatrics; University of Washington). Both she and 
all other members are independent of the study.  Collectively the NRN DSMC includes 
experts in neonatology, maternal and fetal medicine, neurodevelopment, epidemiology, 
biostatistics, bioethics, high-risk infant follow-up, and clinical trials.  Both the individuals 
and their expertise are shown in Appendix.    
c) The Charter under which the DSMC operates for the NRN was drawn up in accordance 
with the NIH guidelines, and details the roles of the DSMC. It can be found at the 
password protected private gateway of the NRN as follows:  
https://neonatal.rti.org/private/pdf/Administration/PolicyNProcedures/DSMC_Charter.pdf 
The charter has been reviewed and approved by the DSMC. 
The DSMC has at least one annual in-person meeting and numerous other 
teleconferences, as necessary, organized by RTI, the Data Coordinating Center (DCC) 
for the NRN.  
 
In brief the Committee will: 
(i) 
Review the research protocol, review model informed consent documents, and 
plans for data and safety monitoring, including all proposed revisions;  
(ii) 
Review methodology used to help maintain the confidentiality of the study data 
and the results of monitoring by reviewing procedures put in place by 
investigators to ensure confidentiality; 

 
 
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A-2 
(iii) 
 Monitor study design, procedures and events that will maximize the safety of 
the study participants and minimize the risks; 
(iv) 
Evaluate the progress of the study, including periodic assessments of data 
quality and timeliness, participant recruitment, accrual and retention, 
participant risk versus benefit, performance of the study site(s), and other 
factors that may affect study outcome; 
(v) 
Consider factors external to the study when relevant information becomes 
available, such as scientific or therapeutic developments that may have an 
impact on the safety of the participants or the ethics of the studies; 
(vi) 
 Review serious adverse event documentation and safety reports and make 
recommendations regarding protection of the safety of the study participants; 
(See: 
http://www.nichd.nih.gov/funding/policies/upload/NICHD_Sample_IDMC_Ch
arter.pdf) 
 
d) The DSMC will be asked to critically assess the study protocol and study documents, in 
order to approve the study protocol, the data and safety monitoring plan, informed 
consent template, reporting templates for data to be presented to the board. The DSMC 
is fully empowered to require any other specific changes to the trial (pertaining to patient 
safety and trial feasibility issues) prior to any local IRB submissions and prior to any start 
to enrollment. 
 
e) The DSMC deliberations will be recorded as minutes to summarize topics discussed, 
recommendations made; and will be signed by the board chair.  All participating NRN 
sites and their site PIs will receive a sanitized summary of board recommendations, to 
be forwarded to the IRB of each participating site.  
 
f) Any potential and actual Conflicts of Interest (COI) for board members will be reviewed 
and managed appropriately 
 
2) Details of data acquisition and maintenance and protection of confidentiality 
a) Database and Study Records 
The data management system (DMS) recommended for the TOP study is a general purpose 
distributed data entry and management system developed to enable clinics to enter data from 
medical record extractions or other sources. This system allows quick implementation of new 
studies and has been successfully used for data collection for all NRN studies since 1998.  
As the NRN DCC, RTI has tailored our DMS for more than 40 primary and secondary 
NRN studies with as many as 15 different studies active at one time. It has been used to enter, 
edit, and manage the data for more than 650 forms and 30,000 data items, and includes point of 
entry quality checks, such as range, skip pattern, and consistency checks. It provides a user-
friendly data entry interface that closely resembles the paper study forms to ensure more 
accurate data entry.  
 
Features of the NRN DMS 

 
 
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A-3 
• Provides role-based security. Each user has an individual username and password with a 
specified data entry role. For example, only users with a certified user role can key study 
data. 
• Allows the user to enter data in any order. Users can select any form in the system to key 
and can select any question to answer. 
• Provides a comprehensive search capability. Users can search for a patient by entering 
various identifying information, such as a range of birth dates, gestational ages, birth 
weights. 
• Generates reports. The DMS generates several different types of reports: an Adjusted Age 
Calculator, Field Range Report, Incomplete Records List for each protocol, and the Keyed 
Forms Inventory for each protocol.  
• Generates data backups. Although study data are stored on two mirrored drives, the DMS 
provides an additional mechanism that allows the users to back up their study data files 
onto password protected CDs. 
• Enables medical record storage. The DMS provides a mechanism for users to store and 
transmit various medical data files such as oximeter data files, aEEG records, to the DCC. 
• Performs batch edits. In addition to range, consistency, and skip patterns checks, cross-
form edits are applied to the data. Edit reports are generated and electronically submitted 
to the sites for resolution. 
• Maintains audit trail. The DMS maintains an audit trail on any data changes made to 
previously completed data records. 
• Builds analysis files. Using the codebook that automatically generates the data entry 
screens and database table structures, database tables are converted to SAS files for use 
by DCC statisticians. 
• Provides a data transmission system. Study data are transmitted to RTI. Updates to the 
DMS that include new studies or version changes can also be transmitted to the research 
center computer via the transmission system. 
 
Research center staff key study data into the DMS on their NRN research computers 
and transmit these data to RTI at least weekly. Provided by RTI, these computers are already in 
place at all the clinical centers. At RTI, data from the different clinical centers are combined into 
one dataset and are then converted to SAS data files for use by RTI study statisticians for data 
analysis. Patient confidentiality and privacy is maintained within the system by assigning 
patients a study identification number. Neither patient names nor addresses are transmitted to 
RTI. No other personally identifiable information (PII) or protected heath information (PHI) other 
than dates (i.e., dates of birth, discharge, intervention and hospital outcomes) are collected or 
transmitted to RTI. Once a combined dataset has been created, the data will go through 
additional higher level data edits and resulting queries to research center staff are posted on the 
secure portal of the NRN website.  
The combined dataset is also utilized by the NRN capitation system to calculate monthly 
payments to the research centers to reimburse them for their study costs. The Steering 
Committee and RTI define the rules or data items that trigger payments. Additional data items 
will be added to the capitation system for this study. 
The TOP study will be easily added as a new study module to the existing NRN DMS. 
Once programmed, it will go through a rigorous review that includes testing of each data item, 
table, form, report, and all DMS features before it is distributed to the NRN research centers. 
The TOP study module will be transmitted to the research centers’ computer and automatically 
added to the NRN DMS on the center’s NRN computer. Users can begin keying data into the 
study immediately after a successful transmission.  

 
 
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A-4 
An additional module will be added to the NRN telephone randomization system for the 
TOP trial. This system allows clinical center staff to randomize patients at any time of the day, 
quickly and efficiently. Center coordinators dial a toll-free number, enter a password on the 
phone’s keypad, provide any stratifying information (e.g., days of life, gestational age), and 
receive a treatment assignment through the system. 
 
2b) Maintaining confidentiality of subject data 
All named study PIs at the NRN sites, and their research coordinators are CITI certified 
in conduct of clinical trials, as a requirement of their participation in any NRN study. The basic 
rights of study participants will be respected and maintained by the investigators and by all who 
are involved in the collection or processing of individually identified data. NRN data collection 
and processing procedures are designed to protect individual rights and to comply with all 
applicable laws and ethical principles including confidentiality. Among the rights that must be 
protected are:  
 
the right to informed consent, which requires that prospective participants in a research 
project and, if needed, their family members, be provided adequate information about the 
potential risks, benefits, and requirements of participation so that each can make an informed 
decision about participation 
 
the right to decline, which requires that prospective participants be fully informed that their 
participation is completely voluntary, that they may withdraw at any time, that access to 
adequate health care will be provided whether or not they participate in the research, and that 
they may refuse to answer any question  
 
the right to privacy, which requires guarantees of confidentiality of information and other 
specific protection as specified in the Privacy Act of 1974. 
 
2c) Data Quality Control  
2c i) Internal Audit 
We will continuously monitor data quality using point-of-entry quality checks in the DMS 
and other cross-form or longitudinal checks indicated by the investigators. We will identify 
errors, missing forms/items, and post center-specific reports on the private portion of the NRN 
website so that clinical staff can follow up. We have used this approach successfully for the 
NRN and other studies, producing automated routine reports on enrollment, retention, missing 
visits/items, and other problems, that enable us to evaluate general center performance and 
quickly initiate corrective actions as needed.  
 
2c ii) Quality Assurance 
The TOP DCC at RTI will operate under guidelines to ensure data accuracy, integrity, 
confidentiality, and security. DCC activities are governed formally by RTI Standard Operating 
Procedures or SOPs (see examples below) or less formally through technical operating 
procedures and guidance documents, which also help ensure regulatory compliance at the 
clinical centers.   
 
SOPs Guiding QA Activities for TOP 
DCC Role 
Associated SOPs 
Statisticians 
• Developing Statistical Analysis Plan 
• Creating, testing, and documenting all statistical products 
• Writing reports 

 
 
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A-5 
Computer 
Programmers 
• Designing, testing, and documenting all software developed 
• Developing and maintaining QC systems within DMS 
• Software development lifecycle 
Data Managers 
• Developing Data Management Plan  
− Checks and edits to ensure quality data entry 
− Audit trails 
− Data lock 
Project Management 
• Protocol development 
• Human subjects protection 
• Documenting study protocols 
• Clinical site monitoring and training 
• AE/SAE monitoring 
• Essential documents management 
 
 
 
2c iii) Participating Sites 
All NRN clinical centers and RTI have IRB committees that convene on a fixed schedule 
every month to review protocols and associated informed consent forms and data collection 
procedures for all research to ensure that they are in compliance with all applicable human 
subject regulations. IRB approval must be granted prior to beginning any study, and study 
progress and procedures must be reviewed by the IRB at least annually. All staff who conduct 
or support research involving human subjects are required to undergo training on the protection 
of human subjects in research. 
We will conduct targeted site visits using data audits on accrual, drop-outs, unresolved queries, 
and protocol violations to inform cost-efficient site monitoring decisions. Established checklists 
and reporting templates will be used to ensure that centers have the necessary resources; 
review research records and regulatory documents and observe study procedures, such as 
screening and recruitment. Following the site visit, the DCC will provide a written report to the 
center and NIH, and follow up on unresolved issues. 
 
3) Collection and reporting of Adverse Events 
a. We will use standard definitions as follows.  
 
3a i) Unanticipated Problems 
i. Unanticipated Problem – any incident, experience, or outcome that meets all 
of the following criteria: 
1. Is unexpected in terms of nature, severity, or frequency in relation to 
(a) the research risks that are described in the IRB-approved research 
protocol and informed consent document; Investigator’s Brochure or 
other study documents, and (b) the characteristics of the subject 
population being studied; and 
2. Is related or possibly related to participation in the research; and 
3. Places subjects or others at a greater risk of harm (including physical, 
psychological, economic, or social harm) than was previously know 
or recognized. 

 
 
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A-6 
 
 
 
3a ii) Adverse Event (AE) 
“Defined as any reaction, side effect, or untoward event that occurs during the course of 
the clinical trial associated with the intervention, whether or not the event is considered related 
to the treatment or clinically significant.  A new illness, symptom, sign or clinically significant 
laboratory abnormality or worsening of a pre-existing condition or abnormality is considered an 
Adverse Event”. 
 
AE will be monitored during the study to ensure timely detection of events that may affect the 
safety or continued participation of research subjects. In the TOP Trial this extremely high-risk 
and fragile population will each experience expected and unexpected adverse events. Adverse 
events and their relationship to study, severity, time of experience, expectation, actions taken to 
resolve the event and final outcome will be recorded as documented in the medical record, or if 
reported by the NICU team even before documentation. These event rates will be part of 
reporting the final results of the study, and for the DSMC safety monitoring. 
Secondary outcomes of interest in this trial that can be compared across the two treatment 
groups include death; Bell Stage 2-3 necrotizing enterocolitis; periventricular leukomalacia or 
echodense lesions or ventriculomegaly; apnea mandating either caffeine or respiratory support; 
bronchopulmonary dysplasia; retinopathy of prematurity requiring intervention; and each 
component of the primary outcome (available only after the 18- to 22-month follow-up visit). 
Rates of these events, historically observed among similar extremely low gestation/birth weight 
infants, will be provided to the DSMC for comparison. The set of common, serious neonatal 
morbidities as defined in the NRN Generic Data Base (GDB) will also be collected. These 
include data on in-hospital growth, the incidence and severity of intraventricular hemorrhage, 
seizures, patent ductus arteriosus (PDA) and its treatment, nosocomial sepsis (and organisms), 
hearing impairment, and pneumothorax. 
 
3a iii) Serious Adverse Events (SAE) 
Definitions in this study are listed below. Based on the premature infant population 
studied, the SAE categories of events that result in congenital anomaly/birth defect or require 
intervention to prevent permanent impairment or damage (Devices) do not apply when defining 
events. 
An adverse event or suspected adverse reaction is considered serious if in the review of 
the investigator or the sponsor, it results in any of the following: 
 
Death of Subject 
An event that results in the death of a subject. 
Life-Threatening 
An event that, in the opinion of the investigator, would have 
resulted in immediate fatality if medical intervention had not 
been taken. This does not include an event that would have 
been fatal if it had occurred in a more severe form. 

 
 
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Prolongation of 
Hospitalization 
An event that prolongs the subject's hospital stay.   
Results in persistent or 
significant 
disability/incapacity 
An adverse event that may result in a substantial disruption of 
the ability to conduct normal life functions, i.e., the adverse 
event resulted in a significant, persistent or permanent change, 
impairment, damage or disruption in the infant’s body 
function/structure, physical activities and/or quality of life. 
Important Medical Event 
Requiring Medical or 
Surgical Intervention to 
Prevent Serious 
Outcome 
 
An important medical event that may not be immediately life-
threatening or result in death or hospitalization, but based on 
medical judgment may jeopardize the subject and may require 
medical or surgical intervention to prevent any of the outcomes 
listed above (i.e., death of subject, life-threatening, prolongation 
of hospitalization). An example of such events would be an 
allergic bronchospasm requiring intensive treatment. 
 
Expedited Serious Adverse Events (SAE) 
The sponsor NICHD will determine if individual serious adverse events a) meet the 
criteria for expedited  reporting in accordance with 21CFR 312.32 IND safety reporting and b) 
are promptly reported to the  DSMBCand NHLBI. 
 
ii) 
Unexpected Adverse Event 
“Any adverse event, the specificity or severity of which is not listed in the study protocol, 
product inserts, or informed consent document” 
 
iii) 
Attribution – the determination of whether an adverse event is related to a medical 
treatment or procedure: 
This will be performed by the site PI and study team, according to the basis of the below 
classification:  
1. Definitely 
2. Probably 
3. Possibly 
4. Unlikely 
5. Unrelated 
This will then be shared with all the bodies listed below under 3 (b) 
 
3.b) Adverse Event Management and Reporting 
If an adverse event meets any of the criteria listed aboveof Serious (3a i), Unexpected (3 
a ii) and at least possibley attributable (3a iii) to the study therapy or intervention, it will be 
reported by study team at the recruiting site to the NICHD, the Data Coordinating Center (DCC) 

 
 
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RTI, and the local IRB (per site specific IRB procedures) as a serious adverse event via the 
Medwatch Safety Reporitng Form 3500A.  
These eventsis must be performed within 24 hours of the site being made aware of the 
SAE.  NICHD will report the event to the NHLBI.  A Medwatch form 3500A will be completed.   
Adverse Event /Serious Adverse Event forms will be completed by site research staff 
(investigators and/or coordinators) for each reportable (as defined by protocol ) adverse event 
that infants may experience from the time of randomization through the predefined intervention 
and/or events monitoring period.  Events will be captured documented by clinical site staff using 
protocl caase report forms (CRF), reviewed by the site investigators for accuracy and 
completeness then entered into the specified Data Management System (DMS) form(s) as 
instructed in the manual of procedures that will be developed for this trial.  Specific study 
defined events of interest, death and any other event that could be classified as serious, 
unexpected and potentially attributable to study are to be reported using FDA form 3500A 
(MedWatch) by fax or email to the RTI DCC Coordinator and NHLBI and NICHD Program 
Scientists within one business day of discovery. The Program Scientist at NICHD and/or the 
DCC will determine whether expedited DSMC reviews are necessary. The DSMC can 
recommend further action and the  the the DCC is responsible for notification to the local IRB, 
NICHD and NHLBI. NHLBI and NICHD may request the DCC notify required parties through 
established communication mechanisms via technical memos. 
Reporting to the Food and Drug Administration will not be required for this trial as it involves no 
new drug or device intervention mandating an IND/IDE. 
 
iv) 
Amendments to the study protocol: process of review and implementation 
a. It is not expected that we will encounter any major modifications to the protocol, as 
the most significant portions of this have been already pre-tested within the PINT 
trial. However it is always possible that these may be needed. In that event, the 
Principal Investigators of the trial (HK, EB, AD) will be jointly responsible for the 
coordination and development of all protocol amendments in collaboration with the 
study subcommittees, NICHD and NHLBI as necessary. Dissemination of this 
information to the participating centers will be managed by the NRN DCC.   
b. Any changes to the protocol or consent will be made in the form of an amendment 
and must be approved prior to implementation and this process will take the following 
shape:  
 
The initial assessment of the need to have a study design-protocol change will be first 
assessed by the PIs (HK, ED, AD). If a substantive need is felt, these three will convene a sub-
committee meeting. It will then be sent to the Steering Committee of the NRN. Following 
substantive discussion there, if the collective decision is that there needs amendment, this will 
be drafted and sent to the NRN DSMC for approval. Once the DSMC has approved the 
changes, they will be sent to each PI of the each site, who will be instructed to ask their local 
IRB to vet and pass the amendment. Only after each IRB has agreed to an amendment will the 
changes written there, be implemented to the study as it pertains to human subjects.   
 
v) 
Benefits and Risks of the Study to enrolled subjects 
We avoid stepping beyond current practice boundaries of thresholds of hemoglobin that 
would trigger a transfusion by assuring broad buy-in of NRN clinicians and a transfusion 
algorithm that was developed by consensus. This approach avoids the dilemmas 
sometimes faced by other trials (Deans KJ 2007). This open trial, allows clinicians to 
bypass the study transfusion algorithm in circumstances where an infant’s condition 

 
 
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warrants acute transfusion, but then revert to the algorithm thereafter. The potential risks 
to subjects participating in this study or their parents are categorized in this study as 
‘minimal risk’. The trial maneuvers – other than randomization – are all within the range 
of current clinical practice and well within the likely variation of normal clinical practice. 
We do not mandate any additional blood sampling nor specific route of sampling of 
blood, thus not departing from the standard practice of the NICU. The informed consent 
document and patient information materials will be thoroughly reviewed with the parents 
prior to their completing the written informed consent. Families will be given ample 
opportunity to ask questions regarding alternative options.  
 
6. Data analysis plan 
Main Study:  
 Design The primary objective of the TOP trial is to assess the difference in the primary 
outcome of survival without significant NDI (or, equivalently, death or NDI) at 22-26 months 
adjusted age in the two treatment arms. This outcome is similar to that used in the PINT trial, 
which found rates of death or neurosensory impairment at 18 months of 45.2% in the low trigger 
group and 38.5% in the high trigger group. Based on these findings, we believe that an absolute 
difference of 7% is the minimal clinically significant difference we would wish to detect in the 
rate of death or NDI. We considered several other factors in estimating the sample size for this 
study. First, we calculated the rate of death or NDI among infants born at NRN centers in 2005-
2008 with GA 22-26 weeks and birth weight <1000 g who survived to 12 hours, which was 52%. 
However, we recognized that this population differed somewhat from that of the TOP trial, which 
proposes to enroll infants less than 29 weeks GA. In addition, the current NRN definition of NDI 
incorporates only the cognitive composite score of the Bayley III, while future definitions will 
likely also involve the Bayley III motor scales, which have been collected in the Network only 
since January 2010. Thus, we recognized the limitations of estimating our sample size based 
only on current NRN data. At the same time, however, we were aware that the most 
conservative sample size estimate would result from an event rate of 50%, which current NRN 
data indicated was not implausible in our population. This led us to choose a conservative 
estimate based on assumed outcome rates of 53.5% and 46.5% (i.e., centered around an 
overall event rate of 50%) in the two treatment groups. With a two-tailed alpha of 0.05 and 80% 
power, the initial sample size estimate was 1,658 infants. The sample size was additionally 
inflated by 10% to account for loss to follow up at 2-26 months, yielding a final sample size 
estimate of 1,824. 
Although the sample size calculations described above were based on the primary 
outcome, we will have 80% power with a two-tailed alpha of 0.05 to detect the following 
differences in important secondary outcomes (rates in the low threshold group are based on 
findings of the PINT trial): any transfusion 89.0% in the low threshold group (LOW) vs. 92.9% in 
the high group (HI); NEC 8.5% in LOW vs. 5.0% in HI; brain injury (PVL, echodense lesions or 
ventriculomegaly) on latest ultrasound (US) to 36 weeks 18.5% in LOW vs. 24.6% in HI; death 
before discharge 21.5% in LOW vs. 16.1% in HI; combined death, severe ROP, BPD, or US 
brain injury 74.0% in LOW vs. 67.7% in HI.  
Analysis All analyses will be conducted on an intention-to-treat basis, with the caveat 
that infants surviving to discharge, but lost to follow-up at 22-26 months, will be excluded from 
the primary analysis and considered irrevocably missing. We will, however, assess the impact of 
outcomes lost to follow-up on our results by performing sensitivity analyses that assume all 
positive or all negative outcomes for those with missing data. We will also conduct bivariate 
analyses to compare the in-hospital characteristics of children lost to follow-up versus those with 

 
 
TOP Protocol 
 
A-10 
assessment of the primary outcome. Note that, based on previous NRN trials in similar 
populations, we expect the follow-up rate among survivors to be above 90%.   
All analyses will be adjusted for design effects: stratification by center and birth weight 
group. The primary outcome of death or NDI at 22-26 months adjusted age will be analyzed 
using robust Poisson regression. Adjusted relative risks and 95% confidence intervals will be 
reported. Secondary analyses of the primary outcome may adjust for additional baseline 
covariates such as SNAPPE scores at NICU admission and socioeconomic status. The 
consistency of the treatment effect over birth weight strata and gender, and treatment 
heterogeneity across the different centers, will be investigated. 
Secondary outcomes including comorbidities such as NEC, ROP, BPD, and NDI among 
survivors at follow up, as well as other binary outcomes, will be analyzed using robust Poisson 
regression, as described above. In addition to the stratification variables, models may also be 
adjusted for covariates known to be associated with the secondary outcomes in this population. 
For example, both gender and socioeconomic status have been implicated as crucial 
confounders for cognitive outcomes among survivors at follow up (53); thus, it may be 
appropriate to include them as covariates in models for such outcomes. Number of transfusions 
will be analyzed using Poisson regression. Other secondary outcomes, such as time to regain 
birth weight and time to full feeds, will be analyzed using Cox proportional hazards survival 
regression. Although the basic Cox model assumes proportional hazards between groups, if this 
assumption is found to be violated in our data, time-varying covariates can be added to the 
models to allow for changes in the proportion over time. Weekly measures of growth (weight, 
length, head circumference) will be analyzed using longitudinal models to test whether growth 
trajectories differ between treatment groups. All models incorporating the treatment effect will be 
adjusted for the stratification variables. All secondary outcomes analyses will be considered 
exploratory because the trial is not primarily powered to detect these associations.  
 
6b) Interim Analysis and 6c) Stopping Rules 
Interim analyses for safety will be conducted at four time points during the study, after  
250, 500, 1000 and 1500 babies enrolled have reached status, as defined in the NRN (death, 
discharge, transfer, or 120 days of age, whichever occurs earlier). Rates of death and other 
prespecified adverse events (AEs), including NEC and adverse head US findings, will be 
compared between the two treatment groups. Analyses of AEs that were not prespecified may 
be added during the course of the study as deemed appropriate by the DSMC or the Steering 
Committee, in accordance with any new findings reported in the literature that may be of 
relevance to this trial.  
The statistical significance of interim safety analyses will be based on O’Brien Fleming 
bounds calculated with a Lan-DeMets spending function to preserve an overall Type-I error rate 
of 0.05 for a composite safety outcome of death or NEC or adverse head US findings, and tests 
that exceed the boundary will be considered as evidence of a statistically significant difference 
between treatment groups. In the event that a test approaches significance using these stopping 
rules, the DCC will report to the DSMC Chair who can then take appropriate action. To ensure 
that Drs. Das and Brambilla remain blinded to evolving results, they will not be directly involved 
in these analyses. The results of the interim analyses will thus remain confidential, with only the 
trial statistician having access to unblinded data.  
We recommend that the DSMC meet formally to review safety and other aspects of 
accruing data (such as enrollment, data completeness and protocol compliance) three times 
during the study, at roughly 25% , 50% and 75% of subject accrual). Formal DSMC reports will 
be prepared by the DCC for these meetings. These reports will include the following information: 

 
 
TOP Protocol 
 
A-11 
• 
Brief summary of the trial design, including primary and secondary hypotheses and 
outcomes, study population, inclusion and exclusion criteria, recruitment, screening, 
randomization and study intervention procedures, and statistical considerations for 
trial design and analysis 
• 
Interim monitoring plan 
• 
Enrollment, including screening, consent, randomization and study exit 
• 
Completeness of data and edit queries. Baseline study population characteristics, 
overall and by treatment group 
• 
Treatment compliance  
• 
Primary efficacy outcomes by treatment group, if at least 25% available 
• 
Secondary efficacy outcomes by treatment group, if at least 25% available 
• 
Safety outcomes, including death, by treatment group 
• 
Protocol deviations and violations, overall and by treatment group. 
These DSMC reports will be blinded, with treatments labeled as group A and group B. As per 
the NRN DSMC Charter, the DSMC may, however, request to be unblinded to perform their 
duties. If the DSMC recommends modification or cessation of the study protocol due to safety 
concerns, NIH will make the final determination.  
 
Because the enrollment period for this study is less than 3 years and the NDI component of the 
primary outcome will not be obtained until 22-26 months adjusted age, we do not plan to 
conduct formal interim analyses for efficacy. However, when the DSMC meets for formal safety 
analyses, members will be presented with, and may choose to make recommendations on the 
basis of, data regarding recruitment and retention, protocol adherence (including compliance 
with the intervention algorithm), and other factors that relate to the scientific validity of the trial. 
We also anticipate that towards the end of trial recruitment, some primary outcome data may be 
available, and such data will be included in the DSMC report for every 500 babies with primary 
outcome accrual if it is available on at least 25% of the total target sample size of 1824. O’Brien 
Fleming stopping bounds for efficacy, calculated with a Lan-DeMets spending function to 
preserve an overall Type-I error rate of 0.05 for the primary outcome will be used.