Randomized Controlled Trial on the Use of EPO to Reduce Top-up Transfusions in Neonates With Red Blood Cell Alloimmunization Treated With Intrauterine Transfusions
Phase: PHASE2
Conditions: Erythroblastosis, Fetal, Erythroblastosis Fetalis, Rh Disease, Erythroblastosis Fetalis Due to RH Antibodies, Erythroblastosis Fetalis Due to Isoimmunization
Sponsor: Sanquin-LUMC J.J van Rood Center for Clinical Transfusion Research

 
 
Randomized controlled trial on the use of EPO 
to reduce top-up transfusions in neonates  
with red blood cell alloimmunization treated with intrauterine 
transfusions  
 
Short Title: EPO-4-Rhesus Study 
 
 
Masja de Haas, Anske van der Bom, Sanquin Research, Center for Clinical Transfusion 
Research, Leiden, The Netherlands, and Jon J van Rood Center for Clinical Transfusion 
Medicine, Leiden University Medical Center 
Remco Visser, Vivianne Smits-Wintjens, Enrico Lopriore, Department of Pediatrics, Division 
of Neonatology, Leiden University Medical Center  
 
Dick Oepkes, Department of Obstetrics, Division of Fetal Medicine, Leiden University 
Medical Center 
 
Linda van der Hulst, Department of Pharmacology, Leiden University Medical Center 
 
Sandra Juul, Department of Pediatrics, Division of Neonatology, Seattle Children’s Hospital 
 
Nan van Geloven, Department of medical Statistics, Leiden University Medical Center 
 
 

EPO-4-Rhesus Study (26-04-2018) 
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PROTOCOL TITLE: Randomized controlled trial on the use of EPO to reduce top-up 
transfusions in neonates with red blood cell alloimmunization treated with intrauterine 
transfusions. 
Short title: 
EPO-4-Rhesus Study 
EudraCT number:  
2017-000583-15 
Version: 
26-04-2018 
Trial manager: 
Drs. I.M.C. Ree 
Department of Pediatrics, Division of Neonatology 
Leiden University Medical Centre 
Albinusdreef 2, 2333 ZA Leiden 
Center for Clinical Transfusion Research 
Sanquin Research 
Plesmanlaan 1a, 2333 BZ Leiden 
Email: I.M.C.Ree@lumc.nl 
Principal investigators:  
Prof. dr. M. de Haas  
Center for Clinical Transfusion Research 
Sanquin Research 
Plesmanlaan 1a, 2333 BZ Leiden 
Email: m.de_haas@lumc.nl 
 
Prof. dr. E. Lopriore 
Department of Pediatrics, Division of Neonatology 
Leiden University Medical Centre 
Albinusdreef 2, 2333 ZA Leiden 
Email: E.Lopriore@lumc.nl 
Sponsor: 
Leiden University Medical Center 
Department of Pediatrics, Division of Neonatology 
Albinusdreef 2, 2333 ZA Leiden 
Independent expert: 
Dr. A.A.W. Roest  
Department of Pediatrics, Division of Pediatric Cardiology   
Leiden University Medical Centre 
Albinusdreef 2, 2333 ZA Leiden LUMC 
Email: A.Roest@lumc.nl 
Pharmacy:  
Department of Clinical Pharmacology and Toxicology  LUMC 

EPO-4-Rhesus Study (26-04-2018) 
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PROTOCOL SIGNATURE SHEET 
Name 
Signature 
Date 
Head of Department: 
Prof. dr. E. Lopriore 
Pediatrician-Neonatologist 
 
 
 
 
Trial manager: 
Drs. I.M.C. Ree 
Arts-onderzoeker, Sanquin/LUMC 
 
Principal Investigators: 
Prof. dr. M. de Haas 
Head of Immunohematology 
Diagnostic Services, Sanquin and 
professor in Immunohaematology, 
LUMC 
 
Prof. dr. E. Lopriore 
Pediatrician-Neonatologist and 
professor in Neonatology and fetal 
medicine, LUMC 
  
 
 
 
 
 

TABLE OF CONTENTS 
1. 
LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS 
5 
2. 
SUMMARY 
6 
3. 
INTRODUCTION AND RATIONALE 
8 
4. 
OBJECTIVES 
10 
5. 
MATERIAL AND METHODS 
11 
5.1 Study population 
11 
5.2 Study design 
11 
5.4 Sample size calculation 
13 
5.5 End points 
14 
5.6 Randomization, blinding and treatment allocation 
15 
5.7 Withdrawal of individual subjects 
15 
5.8 Replacement of individual subjects after withdrawal 
15 
5.9 Cost-effectiveness 
15 
5.10 Statistical analysis 
16 
6. 
REQUIRED CLINICAL EVALUATION 
17 
6.1 Maternal and fetal baseline characteristics 
17 
6.2 Neonatal baseline characteristics 
17 
6.3 Measurements during follow-up 
17 
7. 
INVESTIGATIONAL PRODUCT 
18 
7.1 Name and description of investigational product 
18 
7.2 Summary of findings from non-clinical studies 
18 
7.3 Summary of findings from clinical studies 
19 
7.4 Summary of known and potential risks and benefits 
20 
7.5 Description and justification of route of administration and dosage 
20 
7.6 Preparation and labelling of Investigational Medicinal Product 
20 
7.7 Drug accountability 
21 
8. 
SAFETY REPORTING 
22 

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8.1 Section 10 WMO event 
22 
8.2.1 Adverse and serious adverse events (SAEs) 
22 
8.2.2 Suspected unexpected serious adverse reactions (SUSARs) 
24 
8.3 Annual safety report 
25 
8.4 Follow-up of adverse events 
25 
8.5 Monitoring 
26 
8.6 Data safety monitoring board (DSMB) 
26 
ETHICAL CONSIDERATIONS 
27 
9.1 Regulation statement 
27 
9.2 Recruitment and consent 
27 
9.3 Benefits and risks assessment, group relatedness 
27 
9.4 Compensation for injury 
27 
9. 
ADMINISTRATIVE ASPECTS AND MONITORING 
29 
10.1 Handling and storage of data and documents 
29 
10.2 Monitoring and Quality Assurance 
29 
10.3 Amendments 
29 
10.4 Annual progress report 
30 
10.5 End of study report 
30 
10.6 Public disclosure and publication policy 
30 
10. STRUCTURED RISK ANALYSIS 
31 
11.1 Potential issues of concern 
31 
11.2 Synthesis 
32 
11. REFERENCES 
34 
 

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1. LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
 
ALAT 
Alanine aminotransferase 
ASAT 
Aspartate aminotransferase 
BSID-III 
Bayley scales of infant development, third edition 
DSMB 
Data safety monitoring board 
EPO 
Erythropoietin 
EudraCT European drug regulatory affairs clinical trials  
GCP 
Good clinical practice 
γGT 
Gamma-glutamyl transferase 
HDFN 
Hemolytic disease of the fetus and newborn 
IUT 
Intrauterine red cell transfusion 
LDH 
Lactate dehydrogenase 
LUMC 
Leiden University Medical Center 
MDI 
Mental developmental indexes 
METC  
Medical research ethics committee (MREC); in Dutch: medisch ethische toetsing 
commissie (METC) 
PDI 
Psychomotor development indexes 
(S)AE 
(Serious) adverse event  
SUSAR 
Suspected unexpected serious adverse reaction 
Wbp 
Personal data protection act; in Dutch: Wet bescherming persoonsgegevens 
WMO 
Medical research involving human subjects act; in Dutch: wet medisch-
wetenschappelijk onderzoek met mensen 
 

EPO-4-Rhesus Study (26-04-2018) 
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2. SUMMARY 
Rationale: neonates with hemolytic disease of the fetus and newborn (HDFN) due to red cell 
alloimmunization often require top-up (red blood cell) transfusions to treat late anemia during 
the first 3 months of life. Late anemia in neonates with HDFN may be due to depressed 
erythropoiesis (hyporegenerative anemia) and/or persisting (intra-marrow) destruction of 
erythrocytes by remaining antibodies, which is not necessarily associated with parameters of 
hemolysis. Hyporegenerative anemia occurs in particular in neonates treated with 
intrauterine red cell transfusions (IUT) due to bone marrow suppression. Approximately 80% 
of infants with rhesus hemolytic disease  treated with IUT require at least one top-up 
transfusion, with an average of two top-up transfusions per infant.  
Since more than a decade erythropoietin (EPO) has been applied in small studies and 
casuistic reports in children with HDFN. It is controversial whether neonates with HDFN 
treated with or without IUT benefit from this treatment to reduce the risk of delayed anemia 
and subsequently, the need for top-up transfusions. Due to the lack of evidence, routine use 
of EPO is currently not recommended. To determine a role for EPO in patients treated with 
IUT, a well-designed randomized controlled clinical trial of sufficient sample size is required.  
Objective: to evaluate the effect of darbepoetin alfa on the need for top-up transfusions in 
neonates with HDFN due to red cell alloimmunization treated with IUT. Secondary outcomes 
are number of days of admission for top-up transfusions, percentage of infants with high 
ferritin levels and high blood pressure. 
Study design: randomized controlled trial. 
Study population: all (near)-term neonates (gestational age ≥ 35 weeks) admitted to the 
Leiden University Medical Center (LUMC) with HDFN, treated with IUT. 
Intervention: treatment with darbepoetin alfa subcutaneously at a dosage of 10 µg/kg once 
a week for a period of 8 weeks (intervention), or “standard care”.  
Main study endpoints: number of top-up transfusions required per infant. 
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: all infants participating in either treatment arm of the study are subjected 

EPO-4-Rhesus Study (26-04-2018) 
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to routine neonatal care. Darbepoetin alfa is given subcutaneously, once a week for a period 
of 8 weeks on home visits. Administration of darbepoetin alfa may cause local skin reactions, 
increased blood pressure with rarely convulsions and hypersensitivity reactions. However, 
these risks are small and are observed in different populations. If effective, darbepoetin alfa 
prevents or reduces the amount of top-up transfusions. Additionally, darbepoetin alfa may 
improve neurodevelopmental outcome. No extra blood will be drawn from the infants in 
addition to the weekly routine measurements of complete blood counts. Blood pressure will 
be measured three times throughout treatment. 
HDFN is a condition occurring exclusively in infants and can therefore only be studied in this 
specific population.  

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3. INTRODUCTION AND RATIONALE 
 
The mainstay of antenatal treatment of fetal anemia due to red cell alloimmunization is 
(serial) intrauterine red cell transfusions (IUT).1,2 The mainstay of postnatal treatment in  
hemolytic disease of the fetus and newborn (HDFN) is (1) intensive phototherapy and 
exchange transfusion to treat hyperbilirubinemia and prevent kernicterus, and (2) top-up 
transfusions to treat anemia.3 Up to 80% of infants with HDFN treated with IUT require at 
least one top-up transfusion for late anemia during the first 3 months of life, with an average 
of two top-up transfusions and therefore two hospital admissions per infant.3-5  
Late anemia is attributed to two mechanisms: “hyporegenerative anemia” characterized by 
depressed erythropoiesis6 and “late anemia of hemolytic disease” caused by persisting 
antibodies, destroying newly produced erythrocytes.4,7 “Hyporegenerative anemia” occurs in 
particular in neonates treated with several IUTs due to bone marrow suppression.4,6 To 
distinct these two mechanisms, a persistence of a high antibody titer can be used.8  
Several risk factors for late anemia have been reported, including serial IUT (due to bone 
marrow suppression), severity of HDFN, reduced use of exchange transfusions during the 
neonatal period and reduced survival of transfused red blood cells.3 Finally, erythropoietin 
deficiency is also considered as a possible contributing factor to late anemia.9-14   
Erythropoietin (EPO) has been increasingly used in neonates to prevent or reduce neonatal 
anemia without short or long-term adverse effects.13,15-17 Several small studies and casuistic 
reports suggest that neonates with HDFN may benefit from treatment with EPO to reduce the 
risk of delayed anemia and subsequent top-up transfusions.9-14 However, other authors found 
that EPO may be less effective than expected.8,18 Due to the lack of evidence, routine use of 
EPO is currently not recommended.3 To determine a role for EPO in this group of patients, a 
well-designed randomized controlled clinical trial of sufficient sample size is required.14 
Potentially, EPO stabilizes the hemoglobin levels of these infants and thus prevents anemia, 
hospital admissions for top-up transfusions and potential transfusion reactions, creating a 
more stable and natural postnatal course for patients with HDFN. In this scenario, the current 

EPO-4-Rhesus Study (26-04-2018) 
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management of weekly out-patient visits and weekly blood draws for hemoglobin level 
measurements, may be reduced, further contributing to reduction of the burden for these 
infants. 
HDFN is a condition occurring exclusively in infants and can therefore only be studied in this 
specific population.  

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4. OBJECTIVES 
The primary objective of this study is to investigate whether darbepoetin alfa is effective in 
reducing the incidence of late anemia in infants with HDFN treated with IUT and therefore in 
decreasing the number of top-up transfusions per infant. As secondary objectives the 
percentage of infants that required a top-up transfusions will be assessed, as well as the time 
from birth to first top-up transfusion, number of days of admission for top-up transfusions, 
percentage of infants with a systolic blood pressure ≥ 2 SD above age adjusted mean 
systolic blood pressure and the percentage of infants with ferritin levels >200 μg/L. For 
exploratory purposes, the long-term neurodevelopmental outcome at 2 years of age will be 
assessed using the BSID-III test. 

EPO-4-Rhesus Study (26-04-2018) 
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5. MATERIAL AND METHODS  
 
5.1 Study population 
All (near) term neonates (gestational age ≥ 35 weeks) with HDFN (due to Rhesus-D, -C, -c, -
E, Kell or other red blood cell alloimmunization) treated with IUT and admitted to the Leiden 
University Medical Center (LUMC) between May 2017 and October 2020 are eligible for the 
study. The LUMC is the single national referral center in the Netherlands for pregnancies 
complicated by maternal red blood cell alloimmunization. Approximately 15 eligible patients 
are treated in the LUMC a year.   
 
Inclusion criteria: 
 
Gestational age ≥ 35 weeks. 
 
Treatment with IUT due to Rhesus-D, -C, -c, -E, Kell or other red blood cell 
alloimmunization. 
 
Birth at the LUMC. 
Exclusion criteria: 
 
 Gestational age <35 weeks. 
 
Treatment with IUT due to other causes than red blood cell 
alloimmunization. 
 
Early onset proven neonatal sepsis. 
 
 
 
5.2 Study design 
Randomized controlled trial. Included neonates will be randomized at birth to treatment with 
EPO (intervention group) or “standard of care”, 1:1 allocation. In the treatment group, EPO 
(darbepoetin alfa) is administered subcutaneously at a dosage of 10 µg/kg once a week, 
starting at day 7, for a period of 8 weeks. Treatment is administered during weekly home 
visits after discharge from the LUMC. Concomitant therapy with folate (0.25 mg/day) is given 

EPO-4-Rhesus Study (26-04-2018) 
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in both groups (standard practice). Concomitant iron therapy is given if ferritin level drops 
below 75 microg/l.19 Weekly routine measurements of complete blood counts (including 
hemoglobin level, hematocrit and reticulocyte count) will be performed in both groups 
(standard practice). EPO is discontinued if hemoglobin level is ≥ 13 g/dL after at least 4 
weeks of treatment with EPO. Blood pressure will be measured at the start of treatment, after 
four weeks and after eight weeks. Monthly measurements of liver enzymes (aspartate 
aminotransferase (ASAT), alanine aminotransferase (ALAT), gamma-glutamyl transferase 
(γGT) and lactate dehydrogenase (LDH)) will also be performed in all groups (standard 
practice). In addition, the neonatal EPO-level will be determined at the start of the treatment 
with EPO. The number of top-up transfusions received during the first 3 months of life and 
hemoglobin levels prior to the top-up transfusion are recorded. After initial discharge from the 
LUMC, top-up transfusions are performed when hemoglobin levels fall below 7.2 g/dL or 
when hemoglobin is between 7.2 and 8.8 g/dL if clinical symptoms of anemia (lethargy, 
feeding difficulties or failure to thrive) are present. 
 
At two years of age a physical and neurological examination and an assessment of cognitive 
and neurological development using the Dutch version of the Bayley Scales of Infant 
Development, third edition (BSID-III) will be performed (standard practice). BSID-III scores 
provide mental developmental indexes (MDI) and psychomotor development indexes (PDI). 
 

EPO-4-Rhesus Study (26-04-2018) 
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5.3 Flow chart 
 
5.4 Sample size calculation 
Based on the (scarce) results in the literature, we expect a 50% reduction in the median 
number of top-up transfusions per patient with EPO treatment, from a median of 2 to 1. For 
sample size calculation we hypothesized a shift in the distribution of number of transfusions 
per infant as depicted in Figure 1. The distribution in the ‘care as usual’ group are based on 
data from 2000-2014. Based on these expected frequencies, group sample sizes of 21 
achieve 81% power to detect a difference of 1,1 between the null hypothesis that both group 
means are 1,9 and the alternative hypothesis that the mean of the EPO group is 0,8 with 
estimated group standard deviations of 1,5 and 0,9 and with a significance level (alpha) of 
0,05 using a two-sided Mann-Whitney test. The drop-out percentage is estimated at 5%, 
 
Neonates GA ≥ 35 weeks 
with HDN due to red cell 
alloimmunization and IUT 
 
Informed consent 
 
Randomization 
 
Conventional therapy 
+ EPO treatment: 
 10 µg/kg 1x/wk sc 
 
Conventional therapy  

EPO-4-Rhesus Study (26-04-2018) 
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adding (42/0.95 = 44) 1 person to each group’s sample size. 
 
hypothesized distribution
number of transfusions per infant
percentage of infants
0
1
2
3
4
5
6
7
0
10
20
30
40
50
care as usual
epo
care as usual
epo
care as usual
epo
care as usual
epo
care as usual
epo
 
Figure 1. Hypothesized distribution of number of top-up transfusions 
 
5.5 End points 
Primary end points:  
 
Number of top-up transfusions required per infant up to 3 months of life. 
 
Secondary end points: 
 
 
The percentage of infants requiring a top-up transfusion up to 3 months of life; 
 
Time form birth to first top-up transfusion (days); 
 
Number of days of admission for top-up transfusions;  
 
The percentage of infants with a systolic blood pressure ≥ 2 SD above age adjusted 
mean systolic blood pressure during treatment; 
 
The percentage of infants with ferritin levels >200 μg/L during treatment;  

EPO-4-Rhesus Study (26-04-2018) 
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Exploratory end point: 
 
Long-term neurodevelopmental outcome at 2 years of age using the BSID-III test. 
 
5.6 Randomization, blinding and treatment allocation 
Prior to randomization, informed consent will be obtained from parents or caregivers. 
Information will be given antenatal if possible to allow sufficient time to consider participation. 
Informed consent can also be obtained after birth, but not after the first week of life as not to 
interfere with the possible darbepoetin treatment schedule. If parents or caregivers agree to 
participate, treatment will be allocated by the web based randomization service of GRP 
validated program CASTORto obtain an unique trial number and assignment of treatment 
policy. As the primary objectives are based on hemoglobin levels (need for top-up 
transfusion) that are set out in a transfusion protocol, blinding or the use of placebo is not 
necessary. Due to the subcutaneous administration route, the use of a placebo is considered 
unethical.   
 
5.7 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without any 
consequences. The attending physician can decide to withdraw a subject from the study for 
urgent medical reasons. 
 
5.8 Replacement of individual subjects after withdrawal 
When parents or caregivers want to withdraw their infant for any reason, another infant will 
be included in the study. 
 
5.9 Cost-effectiveness 
By calculating the costs of the intervention (EPO treatment), and taking into account 

EPO-4-Rhesus Study (26-04-2018) 
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caregiver time and other services needed and the costs of top-up transfusions, we will 
assess the cost-effectiveness of the intervention compared to the standard of care. The total 
costs of EPO treatment using darbepoetin alfa are approximately 32 Euro (total of 8 vials of 
darbepoetin) per child. The costs of a clinical admission at a day-care-center for one top-up 
transfusion are approximately 700 Euros. An effective treatment with EPO will reduce the 
average rate of top-up transfusions from an average of 2 top-up transfusions per child to an 
average of 1 top-up transfusion per child (absolute reduction of 50%), hereby reducing the 
costs of treatment by almost 50%, from 1400 Euros (without EPO treatment) to 732 Euros 
(with EPO). 
 
5.10 Statistical analysis 
Both intention to treat and per protocol analysis will be carried out to study the differences in 
outcome measures between the intervention and control group. Intention to treat analysis is 
our primary choice of analysis. A minimal treatment with 5 out of 8 darbepoetin injections is 
defined as acceptable protocol deviation. A p-value < 0.05 will be considered to indicate 
statistical significance. The number of top-up transfusions per infant and, the number of days 
of admission for top-up transfusions will be compared between both groups using a Mann-
Whitney U test. The time from birth to top-up transfusion will be compared between both 
groups using survival analysis and a log rank test. The percentage of infants requiring a top-
up transfusion up to 2 months of life, the percentage of infants with a high blood pressure 
and the percentage of infants with high ferritin levels will be compared between both groups 
using χ2 tests. If despite randomization the groups differ at baseline in type of 
alloimmunization, EPO-levels at birth or treatment with exchange transfusion, a multivariable 
model may be used in a secondary analysis to correct for these factors.  
 
 

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6. REQUIRED CLINICAL EVALUATION 
6.1 Maternal and fetal baseline characteristics 
The following variables will be recorded: type of red blood cell alloimmunization, course and 
level of antibody-titers and ADCC (antibody-dependent cell-mediated cytotoxicity test)20 
during pregnancy, number of IUTs, fetal hemoglobin values at start of each IUT, presence of 
severe fetal anemia (< 5 SD), presence of fetal hydrops. 
 
6.2 Neonatal baseline characteristics 
The following variables will be recorded: birth weight, gestational age at birth, gender, Apgar 
score at 5 min, respiratory distress syndrome, patent ductus arteriosus, necrotizing 
enterocolitis, sepsis and cerebral lesions on cranial ultrasound examination, number of days 
of phototherapy, number of exchange transfusions, and highest bilirubin level during 
admission. At birth, the following measurements will be performed in umbilical cord blood as 
part of standard care: hemoglobin level, hematocrit, reticulocyte count, nucleated red cells 
(erythroblasts), iron tests (including serum iron, total iron binding capacity, transferrin and 
ferritin), liver enzymes (ASAT, ALAT, γGT and LDH). Additionally, EPO-levels will be 
measured in umbilical cord blood.  
 
6.3 Measurements during follow-up 
Complete blood counts including hemoglobin level and reticulocyte count are determined 
weekly in both groups (standard practice). The number of top-up transfusions received 
during the first 3 months of life and hemoglobin levels prior to the top-up transfusion are 
recorded. Iron tests (serum iron, ferritin, transferrin and total iron binding capacity) and liver 
enzymes (ASAT, ALAT, γGT and LDH) will be determined monthly (standard practice). Blood 
pressure will be measured at the start of treatment, after four weeks and after eight weeks of 
treatment. 

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7. INVESTIGATIONAL PRODUCT  
7.1 Name and description of investigational product 
In this study, the investigational medicinal product is darbepoetin alfa (Aranesp). Darbepoetin 
alfa is a long-acting synthetic substitute for the endogenous hormone erythropoietin that 
stimulates erythropoiesis. A detailed description of darbepoetin alfa can be found in the 
summary of product characteristics (SPC) which is added to this protocol as a separate 
document. The authorized indications of darbepoetin alfa are listed in the SPC on page 5. In 
children, darbepoetin alfa is used as treatment for symptomatic anemia caused by chronic 
kidney disease. Relevant information on darbepoetin alfa to treat anemia specifically caused 
by red cell alloimmunization, is provided in this section. Information is provided on both the 
erythropoietic and neuroprotective aspects of darbepoetin alfa, as neurodevelopmental 
outcome will be assessed as a secondary end point. 
 
7.2 Summary of findings from non-clinical studies 
Erythropoietic: animal studies in rats and dogs showed a marked increase in hemoglobulin 
levels, hematocrit and reticulocytes after administration of darbepoetin alfa. Adverse effects 
at high dosages were related to an increased pharmacological effect (decreased tissue 
perfusion due to increased blood viscosity). Darbepoetin alfa showed no genotoxic or 
carcinogenic potential, nor had any effect on the proliferation of non-hematological cells in 
vitro or in vivo (SPC page 20).   
 
Neuroprotective: both in vitro and in vivo studies in animal models revealed neuroprotective 
effects of exogenous EPO administration. These studies showed overall an anti-inflammatory 
role of EPO and a marked decrease in apoptosis and an increase in neuroregeneration after 
EPO administration (both recombinant human EPO as well as the long-acting variant 
darboepoetin alfa) (SPC page 20). Animal studies showed EPO to be able to penetrate the 
blood brain barrier and showed neuroprotective effects in animal models for neonatal 

EPO-4-Rhesus Study (26-04-2018) 
19 
 
hypoxic-ischemia and neonatal strokes.21,22 A recent study in rats showed neuroprotective 
effects of EPO (administration of 5000 µg/kg) in induced hemolytic hyperbilirubinemia.23 
 
7.3 Summary of findings from clinical studies 
Erythropoietic: although HDFN is not a registered indication for darbepoetin treatment, off-
label use of medication is common in neonatology. The potential effect of EPO in neonates 
with HDFN was shown in small studies and casuistic reports, showing overall an increased 
hemoglobulin level and decreased transfusion need. No severe adverse effects were 
reported (dosages 200-400 µg/kg three times a week, recombinant EPO; correlating to a 
darbepoetin dosage of 5-10 µg/kg once a week).9-14,18 
 
Systematic reviews of both early (27 randomized controlled trials) and late administration (30 
randomized controlled trials) of EPO in preterm infants have shown an overall reduction in 
RBC transfusions. The rates for mortality and morbidities including intraventricular 
haemorrhage and necrotizing enterocolitis were not significantly changed, EPO 
administration showed a trend in increased risk for retinopathy of prematurity in this 
population.15,16 A meta-analysis specifically addressing this issue showed no increased risk 
for any stage of retinopathy of prematurity. 24,25 EPO has been shown to protect developing 
retinas in sheep models of endotoxin induced retinal injury and exogenous EPO was found to 
improve retinal neuronal function in the early stages of retinal ischemia and prevent 
neovascularization in an animal model of oxygen-induced retinopathy. The previous reported 
increased risk of retinopathy of prematurity after EPO administration was attributed to the 
effect of excessive levels of iron, which is toxic to tissues and can cause oxidative damage to 
lipids, proteins, and DNA.25 
Other potential adverse effects include local skin reactions (pain and erythema) due to 
subcutaneous administration, increased blood pressure with rarely convulsions and 
hypersensitivity reactions (SPC page 14).  

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Neuroprotective: the short-term and long-term neuroprotective effects of EPO and 
specifically of darbepoetin alfa are most widely studied in preterm neonates and term 
neonates with hypoxic-ischemic encephalopathy. Darbepoetin alfa showed promising results 
in these groups compared to placebo and appears to be safe (dosage 10 µg/kg once a 
week).21,26,27  
 
7.4 Summary of known and potential risks and benefits 
Darbepoetin alfa is registered as treatment of symptomatic anemia due to chronic kidney 
disease. As such, it is known that darbepoetin potentially causes local skin reactions, 
increased blood pressure with rarely convulsions and hypersensitivity reactions. Despite 
these potential risks, darbepoetin alfa can increase hemoglobulin levels and thereby prevent 
or reduce the amount of top-up transfusions. Potential adverse effects of transfusions would 
be avoided and the number of days of hospitalization could be reduced. EPO may have an 
additional beneficial effect on long-term neurodevelopment.   
 
7.5 Description and justification of route of administration and dosage 
Darbepoetin alfa is administered subcutaneously, as is common in the registered indications. 
A dosage of 10 µg/kg once a week for a consecutive period of 8 weeks will be given. The 
optimal dose of darbepoetin is currently unknown, but the chosen dosage of 10 µg/kg is in 
agreement with previous studies.14,17 A lower dosage is used in anemic patients with chronic 
kidney disease (SMPC), supporting the probable effectiveness of this dosage to enhance 
erythropoiesis. When used for its neuroprotective potential, higher dosages are common and 
are safely used to achieve adequate levels of darbepoetin to pass the blood-brain barrier.21  
 
7.6 Preparation and labelling of Investigational Medicinal Product 
Preparation and labelling will be done according to relevant GMP guidelines. 

EPO-4-Rhesus Study (26-04-2018) 
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Darbepoetin alfa will be provided by the Department of Clinical Pharmacology and 
Toxicology of the LUMC. 
 
7.7 Drug accountability 
Drug accountability will follow current GMP guidelines. The LUMC pharmacy will take full 
responsibility and supervision of the drug accountability process.  
 
 

EPO-4-Rhesus Study (26-04-2018) 
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8. SAFETY REPORTING 
 
8.1 Section 10 WMO event 
In accordance to section 10, subsection 1, of the WMO, the investigator will inform the 
parents of the subjects and the accredited METC if anything occurs, on the basis of which it 
appears that the disadvantages of participation may be significantly greater than was 
foreseen in the research proposal. The study will be suspended pending further review by 
the accredited METC, except insofar as suspension would jeopardize the subjects’ health. 
The investigator will take care that all parents of the subjects are kept informed. 
 
8.2 AEs, SAEs and SUSARs 
8.2.1 Adverse and serious adverse events (SAEs) 
Adverse events are defined as any undesirable experience occurring to a patient during a 
clinical trial, whether or not considered related to the investigational drug. All adverse events 
reported spontaneously by the patient’s parents or caregivers or observed by the investigator 
or his staff will be recorded. Serious adverse events are those which result in:  
 
Death 
 
A life-threatening event (i.e. an immediate risk of death) 
 
Hospitalization or prolongation of hospitalization; 
 
Severe/permanent disability. 
 
During protocol treatment, all deaths, all SAE’s that are life-threatening and any unexpected 
SAE must be reported to the study coordinator within 48 hours of the initial observation of the 
event. All details should be documented on the serious adverse event and death report and 
signed by the responsible investigator or one of the authorized staff members. In 
circumstances where it is not possible to submit a complete report an initial report may be 
made giving only the mandatory information.  

EPO-4-Rhesus Study (26-04-2018) 
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The investigator will decide whether the SAE is related to the treatment (i.e. unrelated, 
unlikely, possible, probable, definitely and not assessable) and the decision will be recorded 
on the serious adverse event form. The assessment of causality is made by the investigator 
using the following criteria: 
 
The investigator will report all SAEs to the sponsor without undue delay after obtaining 
knowledge of the events. The sponsor will report the SAEs through the web portal 
ToetsingOnline to the accredited METC that approved the protocol, within 7 of first 
knowledge for SAEs that result in death or are life threatening followed by a period of 
maximum 8 days to complete the initial preliminary report. All other SAEs will be reported 
within a period of maximum 15 days after the sponsor has first knowledge of the serious 
adverse events.  
Relationship 
Description 
Unrelated 
There is no evidence of any causal relationship 
Unlikely 
There is little evidence to suggest there is a causal relationship (e.g. the 
event did not occur within a reasonable time after administration of the trial 
medication). There is another reasonable explanation for the event. 
Possible 
There is some evidence to suggest a causal relationship (e.g. the event 
occurred within a reasonable time after administration of the trial medication), 
but other factors may have contributed to the event (e.g. the patients clinical 
condition, other concomitant treatments). 
Probable 
There is evidence to suggest a causal relationship and the influence of other 
factors is unlikely. 
Definitely 
There is clear evidence to suggest a causal relationship and other 
contributing factors can be ruled out. 
Not 
assessable 
There is insufficient or incomplete evidence to make a clinical judgment of the 
causal relationship. 

EPO-4-Rhesus Study (26-04-2018) 
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8.2.2 Suspected unexpected serious adverse reactions (SUSARs) 
Adverse reactions are all untoward and unintended responses to an investigational product 
related to any dose administered. 
 
Unexpected adverse reactions are SUSARs if the following three conditions are met: 
1. 
the event must be serious (see chapter 8.2.1); 
2. 
there must be a certain degree of probability that the event is a harmful and an 
undesirable reaction to the medicinal product under investigation, regardless of the 
administered dose; 
3. 
the adverse reaction must be unexpected, that is to say, the nature and severity of 
the adverse reaction are not in agreement with the product information as recorded in 
the Investigator’s Brochure for an unapproved IMP or Summary of Product 
Characteristics (SPC) for an authorized medicinal product. 
 
The sponsor will report expedited the following SUSARs through the web portal 
ToetsingOnline to the METC: 
 
SUSARs that have arisen in the clinical trial that was assessed by the METC; 
 
SUSARs that have arisen in other clinical trials of the same sponsor and with the 
same medicinal product, and that could have consequences for the safety of the 
subjects involved in the clinical trial that was assessed by the METC. 
 
The remaining SUSARs are recorded in an overview list (line-listing) that will be submitted 
once every half year to the METC. This line-listing provides an overview of all SUSARs from 
the study medicine, accompanied by a brief report highlighting the main points of concern.  
The expedited reporting of SUSARs through the web portal ToetsingOnline is sufficient as 
notification to the competent authority. 
 

EPO-4-Rhesus Study (26-04-2018) 
25 
 
The sponsor will report expedited all SUSARs to the competent authorities in other Member 
States, according to the requirements of the Member States.  
The expedited reporting will occur not later than 15 days after the sponsor has first 
knowledge of the adverse reactions. For fatal or life threatening cases the term will be 
maximal 7 days for a preliminary report with another 8 days for completion of the report.  
 
8.3 Annual safety report 
In addition to the expedited reporting of SUSARs, the sponsor will submit, once a year 
throughout the clinical trial, a safety report to the accredited METC, competent authority, and 
competent authorities of the concerned Member States. This safety report consists of: 
 
a list of all suspected (unexpected or expected) serious adverse reactions, along with 
an aggregated summary table of all reported serious adverse reactions, ordered by 
organ system, per study; 
 
a report concerning the safety of the subjects, consisting of a complete safety 
analysis and an evaluation of the balance between the efficacy and the harmfulness 
of the medicine under investigation. 
 
8.4 Follow-up of adverse events 
All AEs will be followed until they have abated, or until a stable situation has been reached. 
Depending on the event, follow up may require additional tests or medical procedures as 
indicated, and/or referral to the general physician or a medical specialist. 
 

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8.5 Monitoring 
An external monitor will monitor the study and will assess (1) whether the rights and health of 
the subjects are protected, (2) whether data is correct and verifiable from source documents 
and (3) whether the study is in agreement with GCP, the approved protocol and legal 
requirements. A detailed monitor plan is added to this protocol.  
 
8.6 Data safety monitoring board (DSMB) 
 
A DSMB is established to perform ongoing safety surveillance which means that (S)AEs will 
be monitored. The DSMB will have insight into the total number of (S)AEs and will, as 
needed, review individual records to be able to analyze potential associations between 
complications and the study protocol. The members will not have insight in the efficacy data. 
Accumulating data on (S)AEs will be send to the DSMB for safety review by the researchers.  
All members have independent positions from the trial study group. The composition and 
responsibilities of the DSMB are described in detail in the DSMB charter.   
 
The advice(s) of the DSMB will only be sent to the principal investigators of the study. Should 
they decide not to fully implement the advice of the DSMB, they will send the advice to the 
reviewing METC, including a note to substantiate why (part of) the advice of the DSMB will 
not be followed. 
 

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ETHICAL CONSIDERATIONS  
 
9.1 Regulation statement 
The study will be conducted according to the principles of the Declaration of Helsinki 
(seventh revision, October 2013) and in accordance with the Medical Research Involving 
Human Subjects Act (WMO). 
 
9.2 Recruitment and consent 
Parents or caregivers of infants with known red blood cell alloimmunization that were 
monitored throughout pregnancy for anemia and were treated with IUT(s) for anemia, will be 
informed (verbally and in writing) about the study by the supervising neonatologist or a 
member of this research team. Parents can consider their decision at least 24 hours and can 
ask additional information or questions to an independent doctor before they are asked for 
their written consent. See appendix A and B (Patient information and Consent form). 
 
9.3 Benefits and risks assessment, group relatedness 
HDFN is a condition limited to the neonatal population (group relatedness) Late anemia is a 
common complication of this condition during the first three months of life, currently treated 
with top-up transfusions. EPO could potentially be a less invasive alternative to top-up 
transfusions and be beneficial to our study subjects in terms of reducing the number of 
needed transfusions.  
 
9.4 Compensation for injury 
A liability insurance is obtained in accordance with article 7 of the WMO. This insurance 
covers: 
1. 
A maximum of €650.000,- for injury or death for each subject who participates in this 
study;  

EPO-4-Rhesus Study (26-04-2018) 
28 
 
2. 
A maximum of €5.000.000,- for injury or death for all subjects who participate in this 
study;  
3. 
A maximum of €7.500.000,- for the total damage for all subjects of studies by the same 
organization in each year of insurance coverage.  
This insurance applies to the damage that becomes apparent during the study or within 4 
years after the end of the study. 
 

EPO-4-Rhesus Study (26-04-2018) 
29 
 
9. ADMINISTRATIVE ASPECTS AND MONITORING  
10.1 Handling and storage of data and documents 
Data management will be implemented according to Good clinical practice (GCP)-guidelines 
and will comply with the Dutch personal data protection act (Wbp). Data will be handled 
confidentially and anonymously by coding. The key to the code will be safeguarded by one of 
the investigators. The coded patient data will be entered in CASTOR (www.castoredc.com), 
a GCP conform internet based electronic data capture tool secured with login codes. Data 
will only be transferred encrypted. Data will be stored for 15 years according to the WGBO. 
 
10.2 Monitoring and Quality Assurance  
The conduct of the study will be monitored, as is described in detail in the monitoring plan.  
 
10.3 Amendments  
A ‘substantial amendment’ is defined as an amendment to the terms of the METC 
application, or to the protocol or any other supporting documentation, that is likely to affect to 
a significant degree: 
 
the safety or physical or mental integrity of the subjects of the trial; 
 
the scientific value of the trial; 
 
the conduct or management of the trial; or 
 
the quality or safety of any intervention used in the trial. 
 
All substantial amendments will be notified to the METC and to the competent authority. 
Non-substantial amendments will not be notified to the accredited METC and the competent 
authority, but will be recorded and filed by the sponsor.  
 

EPO-4-Rhesus Study (26-04-2018) 
30 
 
10.4 Annual progress report 
The investigator will submit a summary of the progress of the trial to the accredited METC 
once a year. Information will be provided on the date of inclusion of the first subject, numbers 
of subjects included and numbers of subjects that have completed the trial, serious adverse 
events / serious adverse reactions, other problems, and amendments.  
 
10.5 End of study report 
The sponsor will notify the accredited METC and the competent authority of the end of the 
study within a period of 90 days. The end of the study is defined as the last patient’s last visit.  
In case the study is ended prematurely, the sponsor will notify the accredited METC and the 
competent authority within 15 days, including the reasons for the premature termination. 
Within one year after the end of the study, the investigator will submit a final study report with 
the results of the study, including any publications/abstracts of the study, to the accredited 
METC and the Competent Authority.  
 
10.6 Public disclosure and publication policy 
The trial will be registered with European drug regulatory affairs clinical trials (EudraCT). 
Additionally, the trial will be registered with www.clinicaltrials.gov. Results of the study will be 
submitted for publication to a peer-reviewed international medical journals.  

EPO-4-Rhesus Study (26-04-2018) 
31 
 
10. STRUCTURED RISK ANALYSIS 
 
11.1 Potential issues of concern 
a. Level of knowledge about mechanism of action 
The mechanism of action of EPO in its currents indication is well known and detailed in the 
SPC, page 16. The use of EPO in a neonatal population with HDFN is further described in 
chapter 7, but overall seems safe with promising clinical results. In this population, affected 
fetuses are treated with IUTs. Blood transfusions, including IUTs, are known to enhance 
bone marrow suppression and suppress erythropoiesis.4,6 EPO could in turn stimulate 
erythropoiesis and reduce or treat anemia after birth. 
 
b. Previous exposure of human beings with the test product(s) and/or products with a similar 
biological mechanism 
The effect of the exposure of human beings to darbepoetin alfa is well known as it is a 
registered product in the treatment of anemia in chronic kidney disease. In patients with 
HDFN, off-label and/or experimental use has been described with no serious adverse 
events.9-14,18 
 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo human 
cell material? 
The working mechanism of EPO and specifically of darbepoetin have been extensively 
studied in non-clinical settings.21,22 
 
d. Analysis of potential effect 
A lower dosage is used in anemic patients with chronic kidney disease (SMPC), supporting 
the probable effectiveness of this dosage to enhance erythropoiesis. A similar dosage has 
been administered before14,17 and much higher dosages have been safely used when the 
primary objective was neuroprotection, as EPO only partly crosses the blood-brain barrier.21  

EPO-4-Rhesus Study (26-04-2018) 
32 
 
 
e. Pharmacokinetic considerations 
The half-life of darbepoetin alfa is longer than of recombinant human erythropoietin, justifying 
a less frequent administering for an equal biologic response. In adults with chronic kidney 
disease, the half-life is about 73 hours with subcutaneous injections. Pharmacokinetic 
research in pediatric patients (age 2 to 16 year) with chronic kidney disease, showed similar 
values (SPC page 19,20).  
 
f. Study population 
The study population is by its nature a vulnerable group, consisting of infants with HDFN. 
Standard procedure in our hospital is to admit neonates with HDFN to the high care unit, 
where they are clinically and biochemically (bilirubin, hemoglobulin) closely monitored and 
phototherapy treatment is started. Infants randomized to darbepoetin treatment will start 
treatment after the first week of life for a consecutive period of 8 weeks. All infants with 
HDFN are followed up to 3 months of age in an out-clinic setting.  
 
g. Interaction with other products 
No known interaction is known of darbepoetin alfa with other products (SPC page 12).  
 
h. Predictability of effect 
To evaluate the effect of darbepoetin administration, hemoglobulin levels will be carefully 
followed. National transfusion guidelines are followed to determine the need for top-up 
transfusion bases on hemoglobulin cut-offs.  
 
11.2 Synthesis 
Good neonatal research is essential if to improve the care and outcome of newborns.  The 
study has been designed to allow parents time to consider participation by approaching them 
timely for consent. The parents are under no obligation to join the trial and participation is 

EPO-4-Rhesus Study (26-04-2018) 
33 
 
completely voluntary, as specifically stated in the parent information leaflet.  
 
The results of this study are important to further optimize treatment of HDFN and reduce the 
transfusion need of this population. Darbepoetin alfa is an approved drug to treat or prevent 
anemia and did not give rise to any major safety concerns thus far. The study population is a 
vulnerable group as the patients are newborns, but this research cannot be conducted in 
another population. Apart from 8 subcutaneous injections with the study medication for half 
of the study population and an additional visit to assess development of the patients at the 
age of 2 years for all participants, there is no extra burden for participating infants.  

EPO-4-Rhesus Study (26-04-2018) 
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11. REFERENCES 
 
1. van Kamp IL, Klumper FJ, Meerman RH, Oepkes D, Scherjon SA, Kanhai HH. Treatment of fetal anemia due 
to red-cell alloimmunization with intrauterine transfusions in the Netherlands, 1988-1999. Acta Obstet Gynecol 
Scand. 2004;83:731-737. 
2. Weiner CP, Williamson RA, Wenstrom KD, Sipes SL, Widness JA, Grant SS, et al. Management of fetal 
hemolytic disease by cordocentesis. II. Outcome of treatment. Am J Obstet Gynecol. 1991;165:1302-1307. 
3. Smits-Wintjens VE, Walther FJ, Lopriore E. Rhesus haemolytic disease of the newborn: Postnatal 
management, associated morbidity and long-term outcome. Semin Fetal Neonatal Med. 2008;13:265-271. 
4. De Boer IP, Zeestraten EC, Lopriore E, van Kamp IL, Kanhai HH, Walther FJ. Pediatric outcome in Rhesus 
hemolytic disease treated with and without intrauterine transfusion. Am J Obstet Gynecol. 2008;198:54. 
5. Rath ME, Smits-Wintjens VE, Lindenburg I, Brand A, Oepkes D, Walther FJ, et al. Top-up transfusions in 
neonates with Rh hemolytic disease in relation to exchange transfusions. Vox Sang. 2010;99:65-70. 
6. Thorp JA, O'Connor T, Callenbach J, Cohen GR, Yeast JD, Albin J, et al. Hyporegenerative anemia 
associated with intrauterine transfusion in rhesus hemolytic disease. Am J Obstet Gynecol. 1991;165:79-81. 
7. Koenig JM, Ashton RD, De Vore GR, Christensen RD. Late hyporegenerative anemia in Rh hemolytic 
disease. J Pediatr. 1989;115:315-318. 
8. Pessler F, Hart D. Hyporegenerative anemia associated with Rh hemolytic disease: treatment failure of 
recombinant erythropoietin. J Pediatr Hematol Oncol. 2002;24:689-693. 
9. Scaradavou A, Inglis S, Peterson P, Dunne J, Chervenak F, Bussel J. Suppression of erythropoiesis by 
intrauterine transfusions in hemolytic disease of the newborn: use of erythropoietin to treat the late anemia. J 
Pediatr. 1993;123:279-284. 
10. Ovali F, Samanci N, Dagoglu T. Management of late anemia in Rhesus hemolytic disease: use of recombinant 
human erythropoietin (a pilot study). Pediatr Res. 1996;39:831-834. 
11. Zuppa AA, Maragliano G, Scapillati ME, Florio MG, Girlando P, Noia G, et al. Recombinant erythropoietin in 
the prevention of late anaemia in intrauterine transfused neonates with Rh-haemolytic disease. Fetal Diagn 
Ther. 1999;14:270-274. 

EPO-4-Rhesus Study (26-04-2018) 
35 
 
12. Nicaise C, Gire C, Casha P, d'Ercole C, Chau C, Palix C. Erythropoietin as treatment for late 
hyporegenerative anemia in neonates with Rh hemolytic disease after in utero exchange transfusion. Fetal 
Diagn Ther. 2002;17:22-24. 
13. Ohls RK, Wirkus PE, Christensen RD. Recombinant erythropoietin as treatment for the late hyporegenerative 
anemia of Rh hemolytic disease. Pediatrics. 1992;90:678-680. 
14. Zuppa AA, Alighieri G, Calabrese V, Visintini F, Cota F, Carducci C, et al. Recombinant human erythropoietin 
in the prevention of late anemia in intrauterine transfused neonates with Rh-isoimmunization. J Pediatr 
Hematol Oncol. 2010;32:e95-101.  
15. Ohlsson A, Aher SM. Early erythropoietin for preventing red blood cell transfusion in preterm and/or low birth 
weight infants. Cochrane Database Syst Rev. 2006;3:CD004863. 
16. Aher S, Ohlsson A. Late erythropoietin for preventing red blood cell transfusion in preterm and/or low birth 
weight infants. Cochrane Database Syst Rev. 2006;3:CD004868. 
17. Ohls RK, Kamath-Rayne BD, Christensen RD, Wiedmeier SE, Rosenberg A, Fuller J, et al. Cognitive 
outcomes of preterm infants randomized to darbepoetin, erythropoietin, or placebo. Pediatrics. 
2014;133(6):1023-1030. 
18. Dallacasa P, Ancora G, Miniero R, Gavella B, Brondelli L, Conte R, et al. Erythropoietin course in newborns 
with Rh hemolytic disease transfused and not transfused in utero. Pediatr Res. 1996;40:357-360. 
19.  Armony-Sivan R, Eidelman AI, Lanir A, Sredni D, Yehuda S. Iron status and neurobehavioral development of 
premature infants. J Perinatol. 2004;24:757-762. 
20. Oepkes D, van Kamp IL, Simon MJ, Mesman J, Overbeeke MA, Kanhai HH. Clinical value of an antibody-
dependent cell-mediated cytotoxicity assay in the management of Rh D alloimmunization. Am J Obstet 
Gynecol. 2001;184:1015-1020. 
21. Juul SE, Pet GC. Erythropoietin and Neonatal Neuroprotection. EClin Perinatol. 2015;42(3):469-481. 
22. van der Kooij MA, Groenendaal F, Kavelaars A, Heijnen CJ, van Bel F. Neuroprotective properties and 
mechanisms of erythropoietin in in vitro and in vivo experimental models for hypoxia/ischemia. Brain Res Rev. 
2008;59(1):22-33. 

EPO-4-Rhesus Study (26-04-2018) 
36 
 
23. Memisoglu A, Kolgazi M, Yaman A, Bahadir E, Sirvanci S, Yeğen BÇ, et al. Neuroprotective Effect of 
Erythropoietin on Phenylhydrazine-Induced Hemolytic Hyperbilirubinemia in Neonatal Rats. Neurochem Res. 
2016 Dec 19. [Epub ahead of print]  
24. Xu XJ, Huang HY, Chen HL. Erythropoietin and retinopathy of prematurity: a meta-analysis. Eur J Pediatr. 
2014;173(10):1355-1364. 
25. Chou HH, Chung MY, Zhou XG, Lin HC. Early Erythropoietin Administration does not Increase the Risk of 
Retinopathy in Preterm Infants. Pediatr Neonatol. 2017;58(1):48-56. 
26. Ohls RK, Cannon DC, Phillips J, Caprihan A, Patel S, Winter S, et al. Preschool Assessment of Preterm 
Infants Treated With Darbepoetin and Erythropoietin. Pediatrics. 2016;137(3):e20153859. 
27. Patel S, Ohls RK. Darbepoetin Administration in Term and Preterm Neonates. Clin Perinatol. 2015;42(3):557-
566.