A Feasibility Study to Consider the Relationship Between Markers of Red Cell Damage, Inflammation and the Recovery Process of Newborns Requiring Extracorporeal Membrane Oxygenation (ECMO) for Persistent Pulmonary Hypertension of the Newborn (PPHN): Mi-ECMO
Conditions: Persistent Pulmonary Hypertension of the Newborn
Sponsor: University of Leicester

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CLINICAL STUDY PROTOCOL 
 
The Mi-ECMO Study 
NCT02940327 
A FEASIBILITY STUDY TO CONSIDER THE RELATIONSHIP BETWEEN MARKERS OF RED CELL 
DAMAGE, INFLAMMATION AND THE RECOVERY PROCESS OF NEWBORNS REQUIRING 
EXTRACORPOREAL MEMBRANE OXYGENATION (ECMO) FOR PERSISTENT PULMONARY 
HYPERTENSION OF THE NEWBORN (PPHN) 
 
 
Professor Gavin J Murphy 
Department of Cardiovascular Sciences 
Glenfield Hospital 
Groby Road 
Leicester 
LE3 9QP 
Tel: 0116 258 3054 
Email: gjm19@le.ac.uk 
Dr Simon Robinson 
Paediatric Intensivist and ECMO Consultant 
Glenfield Hospital 
Groby Road 
Leicester 
LE3 9QP 
Tel : 0116 258 2796 
E-mail: simon.robinson@uhl-tr.nhs.uk 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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A FEASIBILITY STUDY TO CONSIDER THE RELATIONSHIP BETWEEN MARKERS OF RED CELL 
DAMAGE, INFLAMMATION AND THE RECOVERY PROCESS OF NEWBORNS REQUIRING 
EXTRACORPOREAL MEMBRANE OXYGENATION (ECMO) FOR PERSISTENT PULMONARY 
HYPERTENSION OF THE NEWBORN (PPHN). THE MI-ECMO STUDY. 
 
Study references: 
 
Protocol number: 
UNOLE 0553 
 
REC number: 15/NE/0398 
IRAS Project ID:  
191592    
 
 
 
 
 
Protocol version: 
4.0 
 
 
Date: 
27/02/2017 
 
 
Details of Sponsor: 
 
University of Leicester  
Research Support Office 
University Road 
Leicester, LE1 7RH 
Tel: 0116 252 2759 
Fax: 0116 252 2028 
Email: uolsponsor@le.ac.uk 
 
Chief Investigator & Research Team Contact Details 
 
Prof Gavin Murphy  
British Heart Foundation Professor of Cardiac 
Surgery 
University of Leicester 
Glenfield Hospital 
Groby Road 
Leicester, LE3 9QP 
Tel:  0116 258 3054 
Email: gjm19@le.ac.uk 
 
Dr Simon Robinson 
Consultant in Paediatric Intensive Care and 
ECMO 
Glenfield Hospital 
Groby Road 
Leicester, LE3 9QP 
Tel: 0116 2582796 
Email: simon.robinson@uhl-tr.nhs.uk 
 
Dr Paolo Pais 
Clinical Research Fellow in ECMO 
University of Leicester 
Glenfield Hospital 
Clinical Sciences Wing 
Groby Road 
Leicester, LE3 9QP 
Tel: 0116 2582796 
Email: pp257@leicester.ac.uk 
 
Dr Silvia Mariani 
Clinical Research Fellow in Cardiac Surgery 
University of Leicester 
Glenfield Hospital 
Clinical Sciences Wing 
Groby Road 
Leicester, LE3 9QP 
Tel: 0116 2582796 
Email: sm780@leicester.ac.uk 
 
Dr Marcin Wozniak 
Research Scientist 
University of Leicester 
Glenfield Hospital 
Clinical Sciences Wing 
Groby Road 
Leicester, LE3 9QP 
Tel: 0116 258 3028 
e-mail: mw292@le.ac.uk 
Dr Attilio Lotto 
Consultant Congenital Cardiac Surgeon 
Glenfield Hospital 
Clinical Sciences Wing 
Groby Road 
Leicester, LE3 9QP 
Tel : 0116 258 3076 
e-mail attilio.lotto@uhl-tr.nhs.uk 

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Mrs Tracy Kumar 
Research Manager in Cardiac Surgery  
University of Leicester 
Glenfield Hospital 
Clinical Sciences Wing 
Groby Road 
Leicester, LE3 9QP 
Tel: 0116 250 3039 
e-mail:tk98@le.ac.uk 
Dr Lorenzo Grazioli 
Locum Consultant Anaesthetist 
Glenfield Hospital 
Clinical Sciences Wing 
Groby Road 
Leicester, LE3 9QP 
Tel: 0116 2582796 
Email: lorenzo.grazioli@uhl-tr.nhs.uk 
Dr Nikol Sullo  
Research Associate 
University of Leicester 
Glenfield Hospital 
Clinical Sciences Wing 
Groby Road 
Leicester, LE3 9QP 
Tel:  0116 250 2606 
e-mail: ns331@le.ac.uk 
Dr Claire Westrope 
Consultant in Paediatric Intensive Care 
Glenfield Hospital 
Groby Road 
Leicester, LE3 9QP 
Tel: 0116 2582796 
Email: Claire.Westrope@uhl-tr.nhs.uk 
Ms Maria Jn Tala 
Clinical Perfusionist 
Division of Acute Care 
University Hospitals Leicester NHS Trust 
Glenfield Hospital 
Leicester LE3 8QZ 
Tel : 0116 258 3026 
e-mail: mariajntala02@gmail.com 
Ms Gail Faulkner 
ECMO Co-ordinator 
Glenfield Hospital 
Groby Road 
Leicester, LE3 9QP 
Tel: 0116 250 2380 
Tel: 0116 250 2431 (PA – Carolyn Shutt) 
Email: gail.faulkner@uhl-tr.nhs.uk 
 
 
 
 
 

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Table of contents 
 
Table of contents ......................................................................................................................... 4 
Glossary / abbreviations .............................................................................................................. 6 
1. 
Study Synopsis ..................................................................................................................... 7 
2. 
Lay Summary ..................................................................................................................... 10 
3. 
Background ....................................................................................................................... 10 
3.1 Respiratory Failure in Newborns ......................................................................................... 10 
3.2 Extracorporeal membrane oxygenation .............................................................................. 10 
3.3 ECMO, haemoglobin metabolism, endothelial injury and outcomes. ................................ 11 
4. 
Aims and Objectives ........................................................................................................... 13 
5. 
Plan of Investigation .......................................................................................................... 14 
5.1 Study design ........................................................................................................................ 14 
5.2 Study population ................................................................................................................. 14 
5.3 Primary and secondary endpoints ....................................................................................... 14 
5.4 Clinical management of Study Patients ............................................................................... 16 
6. 
Study Tests and Procedures ................................................................................................ 19 
6.1 Blood sampling .................................................................................................................... 19 
6.2 Urine sampling ..................................................................................................................... 20 
6.3 Respiratory sampling ........................................................................................................... 20 
6.4 Sample storage .................................................................................................................... 20 
6.5 Blood and urine samples assays .......................................................................................... 21 
6.6 MV Analyses ........................................................................................................................ 22 
6.7 Bronchial aspirates analysis ................................................................................................. 22 
6.8 Summary of trial specific tests and procedures .................................................................. 22 
6.9 End of the trial ..................................................................................................................... 23 
6.10 Data collection ..................................................................................................................... 24 
6.11 Patient recruitment and consent ........................................................................................ 24 
6.12 Planned recruitment rate and duration of follow-up .......................................................... 29 
6.13 Likely rate of loss to follow-up, protocol violations and clinical data completeness .......... 29 
6.14 Expenses and benefits ......................................................................................................... 30 
7. 
Statistical Considerations ................................................................................................... 31 
7.1 Sample Size .......................................................................................................................... 31 
7.2 Measures taken to avoid bias .............................................................................................. 31 
7.3 Plan of analysis .................................................................................................................... 31 
7.4 Criteria for stopping the study early ................................................................................... 31 
8. 
Study Management ............................................................................................................ 31 
8.1 Day-to-day management ..................................................................................................... 31 
8.2 Trial steering committee and data safety & monitoring committee .................................. 31 
9. 
Safety Reporting ................................................................................................................ 32 
9.1 Definitions ........................................................................................................................... 32 
9.2 Adverse event reporting ...................................................................................................... 32 
9.3 Expected adverse events ..................................................................................................... 32 
9.4 Period for recording adverse events ................................................................................... 35 
9.5 Methods of recording and assessing adverse events .......................................................... 35 
10. Ethical Considerations ........................................................................................................ 36 
10.1 Review by a NHS Research Ethics Committee ..................................................................... 36 
10.2 Risks and anticipated benefits ............................................................................................. 36 
10.3 Informing potential study participants of possible benefits and known risks .................... 37 
10.4 Obtaining informed consent from participants ................................................................... 37 
11. Research Governance......................................................................................................... 37 
11.1 Sponsor approval ................................................................................................................. 37 
11.2 NHS approval ....................................................................................................................... 37 

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11.3 Investigators' responsibilities .............................................................................................. 37 
11.4 Monitoring by sponsor ........................................................................................................ 38 
11.5 Indemnity............................................................................................................................. 38 
11.6 Clinical Trial Authorisation ................................................................................................... 38 
12. Data Protection and Patient Confidentiality ........................................................................ 38 
12.1 Data protection.................................................................................................................... 38 
12.2 Data handling, storage and sharing ..................................................................................... 38 
13. Dissemination of Findings .................................................................................................. 39 
14. References ......................................................................................................................... 40 
15. Protocol Signature Page ..................................................................................................... 44 
16. Appendix 1 ........................................................................................................................ 45 
 
 
 

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Glossary / abbreviations 
 
AE 
AKI 
ARG1 
Adverse event 
Acute kidney injury 
Arginase 1 
CD 
CDH 
CO2 
CmH2O 
Cluster of differentiation 
Congenital diaphragmatic hernia 
Carbon dioxide 
Centimetre of water 
CPB 
Cardiopulmonary bypass 
CRF 
CRRT 
CSCTT 
Case report form 
Continuous renal replacement therapy 
Cardiac surgery clinical trials team  
DMSC 
Data monitoring and safety committee 
ECMO 
Extra-corporeal membrane oxygenation 
EDTA 
Ethylenediaminetetraacetic acid 
FACS 
FiO2 
Fluorescence activated cell sorting 
Fraction of inspired oxygen 
GFR 
Hb 
HFOV 
HMOX1 
Glomerular filtration rate 
Haemoglobin 
High frequency oscillatory ventilation 
Heme oxygenase 1 
ICU 
Intensive care unit 
ICAM 
Intercellular adhesion molecule 
IL 
INOS 
Interleukin 
Inducible nitric oxide synthase 
KDIGO 
The International Society of Nephrology: Kidney Diseases Improving Global 
Outcomes (KDIGO) definition of acute kidney injury (AKI) 
MAS 
Meconium aspiration syndrome 
MABP  
Mean arterial blood pressure 
MCP 
Monocyte chemoattractant protein 
MI 
Myocardial infarction 
MP 
MV 
Microparticles 
Microvesicles 
NGAL 
Neutrophil gelatinase associated lipocalin 
NO 
Nitric oxide 
pCO2 
Partial pressure of carbon dioxide 
PEEP 
Positive End Expiratory Pressure 
PICU 
Paediatric intensive care unit 
PIL 
PPHN 
RDS 
Patient information leaflet 
Persistent pulmonary hypertension of the newborn 
Respiratory distress syndrome 
REC 
RNA 
Research ethics committee 
Ribonucleic acid 
SAE 
SaO2 
Serious adverse event  
Oxygen saturation 
SIRS 
Systemic inflammatory response syndrome 
SOP 
Standard operating procedures 
SSAR 
Suspected serious adverse reaction 
SUSAR 
Suspected unexpected serious adverse reaction  
TNF-α 
Tumour necrosis factor - alpha 
TSC 
Trial steering committee 

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UHL 
University Hospital Leicester 
VAD 
VV-ECMO 
VA-ECMO 
Ventricular assist device 
Veno Venous-ECMO 
Veno Arterial-ECMO 
 
1. 
Study Synopsis 
 
Title of Study 
A feasibility study to consider the relationship between markers of red cell 
damage, inflammation and the recovery process of newborns requiring 
Extra-Corporeal Membrane Oxygenation (ECMO) for Persistent Pulmonary 
Hypertension of the Newborn (PPHN) 
Acronym 
Markers of Inflammation and Lung recovery in ECMO patients for PPHN: the 
MI-ECMO study 
Name of Sponsor 
University of Leicester 
Study Hypothesis 
Our primary hypothesis is that damage to red blood cells by the exposure to 
the ECMO circuit will result in inflammatory responses that mitigate against 
successful weaning from Extra-Corporeal Membrane Oxygenation (ECMO) 
for Persistent Pulmonary Hypertension of the Newborn (PPHN). 
 
Our secondary hypothesis are: 
1. Damage to red cells will result in platelet, leukocyte and endothelial 
activation.  
2. Markers of platelet, endothelial and leukocyte activation are 
indicators of lung inflammation and injury severity and hence lung 
recovery. 
3. Markers of platelet, endothelial and leukocyte activation are 
indicators of kidney injury severity and hence acute kidney injury. 
4. The level of oxidative stress will correlate with type shifts in 
pulmonary macrophages, tissue iron deposition and organ injury. 
5. Ability to raise anti-oxidative response, measured by Heme 
Oxigenase-1 (HMOX 1) expression, will correlate with shorter 
intubation times and less severe kidney and lung injury. 
6. Granulocyte and platelets activation are secondary to rising redox 
potential and the levels of activation will correlate with longer 
intubation times and more severe organ injury. 
7. Markers of anti-oxidative response, platelet, endothelial and 
leukocyte activation, as well as oxidative stress levels have diagnostic 
and prognostic utility for the prediction of key clinical events 
including delayed time to recovery, acute kidney injury in paediatric 
patients undergoing Extra-Corporeal Membrane Oxygenation 
(ECMO) for Persistent Pulmonary Hypertension of the Newborn 
(PPHN). 
 
Study Objectives 
This is a pilot feasibility study that will establish the following: 
1. Recruitment rates and patient flows for 24 patients specified as the 
target population for the feasibility study 
2. Withdrawal rate, and completeness of follow-up and data collection 
in a paediatric population at high risk for death and major morbidity   
3. The proportions (categorical data) and variance (continuous data) 
for the primary and secondary outcomes of interest. These will be 
used to model the sample sizes and outcomes that may be used in a 
definitive study 

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4. Perceptions of family members whose children participate in the 
study as to the appropriateness of the screening and consent process 
(Appendix 1) 
  
Study Design 
Prospective, single-centre observational feasibility study 
Setting 
The study will be carried out at a regional paediatric ECMO centre in the UK, 
the University Hospitals of Leicester NHS Trust 
Planned Sample 
Size 
24 children undergoing ECMO will be enrolled 
Recruitment Rate 
This unit performs over 60 neonatal and paediatric ECMO per year, of which 
at least 40 are expected to be caused by PPHN in infants  
Subject Selection 
Criteria 
Participant may enter study if ALL of the following apply: 
1. Patients with a diagnosis of PPHN 
2. Patients that require ECMO support as determined by the ECMO team 
3. Patients aged less than 30 days 
4. Emergency consent obtained within 12 hours from cannulation, and 
ultimately full consent 
Participant may not enter study if ANY of the following apply: 
 
1. PPHN is caused by a congenital heart pathology 
2. ECMO is required for a congenital heart disease 
3. Lack of consent  
Primary outcome of 
interest 
Markers of platelet, leukocyte and endothelial cell activation in arterial blood 
at baseline (12 hours after ECMO commencement) and then at 24, 48 and 72 
hours post commencement and at weaning (end of ECMO support: 24 hours 
after decannulation) or immediately prior to ECMO support being stopped in 
case of death or treatment withdrawal  
Secondary 
outcomes of 
interest 
1. Serum haemoglobin levels. 
2. Data on recruitment, sampling, and compliance rates. 
3. Data on demographics, clinical characteristics and medications of study 
subjects. 
4. Time to wean from ECMO. 
5. Pulmonary inflammation as determined qualitatively by analysis of 
bronchial aspirates lysates 
6. Percentage of patients weaned from ECMO within 7 days 
7. Acute kidney injury as defined by the KDIGO definition (Table 2). 
8. Heart injury as determined by serum troponin levels. 
9. Renal inflammation as determined by urine NGAL concentrations. 
10. Allogenic red cell transfusion volume. 
11. Non red cell transfusion volume. 
12. Time to discharge from Glenfield Hospital ICU. 
13. Time to Glenfield Hospital discharge. 
14. Measures of haemoglobin metabolism, for example: serum hepcidin, 
transferrin saturation, serum ferritin, serum haptoglobin, plasma labile 
iron concentrations, oxidative stress, HMOX 1 expression in 
inflammatory cells from blood and bronchial aspirate. 
15. Levels of inflammatory cytokines in serum.  
16. MV levels and activation of leukocytes and platelets in circulation 
17. Other adverse events not specified above. 
18. A questionnaire will assess parents/family/guardians experience of the 
screening, consent and study procedures. (Appendix 1) 
 

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Statistical 
Considerations 
A statistical analysis plan will be established prior to any analysis. Normally 
distributed data will be reported as means (standard deviations). Non 
normally distributed data will be presented as geometric means (95% 
confidence intervals). Dichotomous data will be presented as percentages. 
The analysis for the feasibility study will be descriptive, i.e. describing the 
relationship between continuous and dichotomous primary and secondary 
outcomes of interest to address the primary and secondary objectives above. 
These analyses will contribute to the design of a further prospective study 
that will consider the role of red cell derived inflammatory responses as early 
biomarkers of lung recovery and/or kidney injury in paediatric patients 
requiring ECMO for primary pulmonary hypertension of the newborn 
(PPHN). 
 
 
 

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2. 
Lay Summary 
Respiratory failure in newborns is common and has high rates of death. Where conventional intensive 
care strategies have failed, newborn children are referred to treatment with Extra- Corporeal 
Membrane Oxygenation (ECMO) (1). This involves connecting children via large bore cannulas placed 
in their heart and major blood vessels to an artificial lung that adds oxygen to their blood and removes 
waste gases (carbon dioxide). Although this treatment saves lives (2, 3), it still has some limitations. In 
particular, severe complications like bleeding, or damage to the kidneys can occur. These 
complications can lead to death in some cases and long-term disability in others. Based on our ongoing 
research in adults and children undergoing cardiac surgery we have identified a new process that may 
underlie some of the complications observed in ECMO. We have noted that when transfused blood is 
infused in an ECMO circuit, this results in the accelerated release of substances from the donor cells 
that cause organ damage; at least in adults. There are treatments that can reverse this process. Before 
we explore whether these treatments should be used in newborn children on ECMO, we must first 
demonstrate that we can measure the complex inflammatory processes that occur in these critically 
ill children. We therefore propose to conduct a feasibility study to identify the practical issues and 
challenges that would need to be overcome in order to perform a successful trial in this high-risk 
population. 
 
3. 
Background 
 
3.1 
Respiratory Failure in Newborns 
Persistent Pulmonary Hypertension of the Newborn (PPHN) is a life-threatening condition that causes 
respiratory failure and death in neonates. PPHN is characterised by the absence of the normal 
postnatal fall in pulmonary vascular resistance, which leads to persisting right to left shunts across the 
foetal channels and resultant hypoxia and right ventricular dilatation. The condition primarily affects 
full term and late preterm babies with an incidence of 1–2/1000 live births (4). In many cases PPHN 
responds to treatments such as inhaled nitric oxide therapy or high frequency oscillatory ventilation 
(HFOV), however, in children refractory to those treatments, prior to the development of ECMO 
mortality was as high as 80% (5).  
 
3.2 
Extracorporeal membrane oxygenation 
Randomised trials have shown that ECMO (6) can reduce mortality in children with respiratory failure 
refractory to conventional treatments, and ECMO has been shown to reduce mortality rates for 
neonates with PPHN (5) to as low as 10-20%. Despite this progress, challenges remain. ECMO is highly 
invasive, which in itself results in high levels of morbidity (7) including bleeding (cannulation site 
bleeding 7.6%, gastro-intestinal bleeding 1.6%, disseminated intravascular coagulation 2.8%), stroke 
(haemorrhagic 7% and ischaemic 7.4%), acute kidney injury 64% (8) and infection 6%. (9). In a study 
of neonates with primary PPHN, 74% developed a severe complication while on ECMO (10). This 
morbidity contributes to mortality, and to long-term health and developmental problems with long-
term disability in 45% of survivors (11).  
 
Aetiology of PPHN is also an important determinant of outcome. Meconium aspiration syndrome 
(MAS) is the most common cause of PPHN because it represents 42% affecting about 42-84/100.000 
live-births; MAS is also the most common indication for ECMO (4). Idiopathic PPHN, a process that is 
poorly understood (4, 12, 13, 14), is the second PPHN cause and affects 27-54/ 100.000 live births 
(27% of all the PPHN), but is the most common in the near-term (>34 weeks gestation) and term 
newborns. 
Congenital Diaphragmatic Hernia (CDH) complicates 1 of every 2000 to 4000 live births and represents 
the second most common ECMO indication (4). The Extracorporeal Life Support Registry (ELS) reports 
neonatal survival rates of 94% for MAS, 77% for Idiopathic PPHN, and 51% for CDH respectively (9). 
This compares to a mean survival in children treated with ECMO for cardiac failure of 43% overall (9), 
which reflects the reversible nature of PPHN in new-borns and the potential additional benefits of 

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refinements in care to the short and long-term outcomes in these children. Other factors that 
influence outcome are pre-ECMO status, and therapies. Risk factors for mortality include prematurity, 
a pre-ECMO pH <7.2 , a pre-ECMO SaO2<65%, initiation of ECMO support at or beyond 5th day of life, 
and a duration of ECMO greater than 7 days (10).  
 
Our understanding of how these clinical factors interact to determine which children recover or do 
not recover is poor, and there are no biochemical or laboratory measures that have been widely 
adopted as diagnostic or prognostic markers. Our ongoing research has identified important 
pathophysiological processes that are associated with organ failure and recovery in adults and 
children undergoing cardiac surgery with cardiopulmonary bypass. These processes, in particular the 
role of free haemoglobin within extracorporeal circuits have not been studied extensively in children 
treated with ECMO. We are proposing to conduct a study to explore the feasibility of measuring 
inflammatory processes in children in ECMO, thus enabling further studies that can provide insights 
into lung recovery during ECMO, provide diagnostic or prognostic markers that may refine treatment, 
or allow the transfer to children of developments and treatments for organ failure in adult patients. 
This has not been done previously.   
 
3.3 
ECMO, haemoglobin metabolism, endothelial injury and outcomes. 
ECMO involves connecting the patients via large-bore cannulas placed in their heart and major blood 
vessels to an external device. During ECMO, blood is drained from the native vascular system, 
circulated outside the body by a mechanical pump, and reinfused into the circulation. While outside 
the body, the blood passes through an oxygenator and heat exchanger. In the oxygenator, 
haemoglobin becomes fully saturated with oxygen, while carbon dioxide (CO2) is removed. 
Oxygenation is determined by flow rate, where elimination of CO2 can be controlled by adjusting the 
rate of countercurrent gas flow through the oxygenator. 
 
In cases of Veno-Arterial ECMO the circuit augments perfusion pressures and cardiac output, whereas 
in Veno-Venous ECMO the circuit provides gas transfer only. Systemic anticoagulation is required to 
prevent circuit thrombosis attributable to the large non-endothelialized surface area that also elicits 
profound activation of inflammatory proteases in the blood along with leukocytes and platelets (15-
19). These processes result in systemic inflammation, and this has parallels with the inflammatory 
response observed in patients undergoing cardiac surgery with cardiopulmonary bypass (20-24). 
Activation of inflammatory processes within the circuit coupled with the mechanical shear stress of 
the pump also leads to red cell damage and release of free haemoglobin into the plasma. This is 
considered a major problem in neonatal ECMO where severe haemolysis and subsequent rise in free 
haemoglobin are reported in over 10% of cases (25-30). Free haemoglobin is highly reactive and can 
oxidize multiple species including plasma proteins and membrane lipids causing inflammation, 
endothelial injury and organ dysfunction (31-38). In particular, free haemoglobin has been implicated 
in the progression of pulmonary hypertension (39-41), and this may be relevant to the recovery for 
neonates with PPHN.   
 
In the recent REDWASH study (42), we have characterised the effects of centrifugal pumps on red cells 
in adult patients undergoing cardiac surgery. We showed that patients receiving washed blood had 
increased free haemoglobin levels (fig. 1A), caused by exposure of stored donor red cells to a 
centrifugal pump (fig. 1B) during washing. This free haemoglobin was highly reactive and directly 
activated cultured endothelial cells in vitro. In trial patients higher free haemoglobin levels in patients 
that received washed blood resulted in oxidative stress (protein carbonylation), endothelial injury 
(CD144 positive microvesicles increased), renal inflammation (elevated NGAL) and acute kidney injury 
(fig 1C-G).  
 
To prevent haemolysis, red cells can be treated with a rejuvenating solution (Rejuvesol), which 
restores energy levels (fig. 1A, 2A,B) and increases red cells resistance to physical stress (fig. 2C).  

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Figure 1. Role of free haemoglobin in organ injury in REDWASH patients: N=60 patients receiving a 
mean of 3 units of blood randomised to  standard care or red cell washing using the Fresenius CATS 
device. Results showed that red cell washing increased circulating free haemoglobin levels (A), which 
was due to the haemolysis during washing (B). Free haemoglobin led to a mild oxidative stress, as 
indicated by elevated carbonyl modifications in plasma proteins (C). Our in vitro experiments indicate 
that free haemoglobin and oxidative stress lead to endothelial activation, as indicated by higher levels 
of MV production (D). Higher levels of endothelial MV (estimated with flow cytometry) are also 
observed in patients transfused with washed blood (E) and correlate with carbonylation levels. 
Endothelial dysfunction most likely affected kidney function as indicated by NGAL levels (F) and higher 
incidence of AKI (G) identified according to the KDIGO criteria. 
 
Washing of stored red cells also let to higher ATP content by increasing RBC metabolic rate when 
exposed to increased shear stress in extracorporeal circuits. This results in red cell activation 
manifested by MV shedding (fig. 2D) and accelerated ATP depletion. The released MV could further 
increase oxidative stress by directly delivering haem to endothelial cells (Camus et al., 2015) or causing 
vaso-occlusions in kidney (Camus et a., 2012). Given that virtually all neonatal ECMO patients are 
transfused with stored red cells, the parallels we have observed between adults and children 
undergoing cardiac surgery in terms of inflammatory responses, and the results of the REDWASH trial, 
we now suggest that activation of red cells by the centrifugal pump and shear stress within the ECMO 
circuit may contribute to increased serum hemoglobin levels and inflammatory red cell microparticles 
release. We hypothesise that levels of free hemoglobin and red cell derived microparticles will be 
associated with endothelial activation, increased markers of pulmonary leucocyte activation and 
platelet activation, acute kidney injury and potentially failure to wean from ECMO (43-53).  
 
 
 

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Figure 2. RBC biochemical properties and the role of RBC MV: ATP (A), 2,3-DPG (B) and osmotic 
fragility (C) levels in 20 days old RBC, before and after washing or rejuvenation. (D)RBC MV production 
in REDWASH patients. 
 
If our hypothesis is correct, this study will identify new processes involved in the pathogenesis of 
ECMO morbidity and potentially identify new biomarkers of recovery or delayed recovery. Finally, 
Rejuvesol is FDA licenced and may represent an effective therapy for the prevention of these 
processes. Before we can explore this in detail, we will first conduct a feasibility study to evaluate the 
practical challenges and key considerations for the successful completion of a research study that 
evaluates complex inflammatory pathways in critically ill children. 
 
4. 
Aims and Objectives 
 
Our primary hypothesis is that damage to red blood cells by the exposure to the ECMO circuit will 
result in inflammatory responses that mitigate against successful weaning from extracorporeal 
membrane oxygenation (ECMO) for persistent pulmonary hypertension of the newborn (PPHN).  
 
Our secondary hypotheses are: 
1. Damage to red cells will result in platelet, leukocyte and endothelial activation.  
2. Markers of platelet, endothelial and leukocyte activation are indicators of lung inflammation 
and injury severity and hence lung recovery. 
3. Markers of platelet, endothelial and leukocyte activation are indicators of kidney injury 
severity and hence acute kidney injury. 
4. The level of oxidative stress will correlate with type shifts in pulmonary macrophages, tissue 
iron deposition and organ injury. 
5. Ability to raise anti-oxidative response, measured by Heme Oxigenase-1 (HMOX 1) expression, 
will correlate with shorter intubation times and less severe kidney and lung injury. 
6. Granulocyte and platelets activation are secondary to rising redox potential and the levels of 
activation will correlate with longer intubation times and more severe organ injury. 
7. Markers of anti-oxidative response, platelet, endothelial and leukocyte activation, as well as 
oxidative stress levels have diagnostic and prognostic utility for the prediction of key clinical 
events including delayed time to recovery, acute kidney injury in paediatric patients on ECMO 
for PPHN. 
 

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To assist with the design of a prospective observational study that will test these hypotheses we 
propose to undertake a feasibility study in 24 new-borns. The objectives of this feasibility study are:  
 
1. Recruitment rates and patient flows for 24 patients specified as the target population for the 
feasibility study. 
2. Withdrawal rate and completeness of follow-up and data collection in a paediatric population 
at high risk for death and major morbidity.   
3. The proportions (categorical data) and variance (continuous data) for the primary and 
secondary outcomes of interest. These will be used to model the sample sizes and outcomes 
that may be used in a definitive study. 
4. Perceptions of family members, whose children participate in the study as to the 
appropriateness of the screening and consent process. (Appendix 1) 
 
5. 
Plan of Investigation 
 
5.1 
Study design 
This is a prospective, single-centre observational feasibility study that will measure changes in free 
haemoglobin and markers of cellular activation, and their relationship to lung recovery or acute kidney 
injury in newborns with PPHN requiring ECMO support. 
The initial feasibility study will enrol 24 patients and will assist with the design of a subsequent 
observational study that will test our hypotheses. 
 
5.2 
Study population 
The study will be conducted at a regional ECMO centre in the UK, the University Hospitals of Leicester 
NHS Trust. This unit performs over 60 neonatal and paediatric ECMO per year, of which at least 40 are 
expected to be performed for the treatment of PPHN in infants. 
 
 
5.2.1 
Inclusion Criteria 
 
 Participant may enter study if ALL of the following apply: 
1. Patients with a diagnosis of PPHN  
2. Patients that require ECMO support as determined by the ECMO team 
3. Patients aged less than 30 days 
4.  Emergency consent obtained within 12 hours from cannulation, and ultimately full consent 
 
 
5.2.2 
Exclusion criteria 
 
Participant may not enter study if ANY of the following apply: 
 
1. PPHN is caused by a congenital heart pathology 
2. ECMO is required for a congenital heart disease 
3. Lack of consent  
 
Participants will be 24 consecutive patients who match the study criteria and that are referred to 
Glenfield Hospital from different British centres according to the ECMO referral system. The Glenfield 
Hospital is a national paediatric referral centre and provides ECMO support across UK from phone 
consultation to onsite cannulation and transport on ECMO. Patient referred to Glenfield Hospital are 
transferred to the Paediatric Intensive Care Unit for further care. 
 
5.3 
Primary and secondary endpoints 
 

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5.3.1 
Primary outcome 
Markers of platelet, leukocyte and endothelial cell activation in arterial blood at baseline (12 
hours after ECMO commencement) and then at 24, 48 and 72 hours post commencement 
and at weaning (24 hours after decannulation) or immediately prior to ECMO support being 
stopped in case of death or treatment withdrawal. 
 
5.3.2 
Secondary outcomes 
1. Serum haemoglobin levels. 
2. Data on recruitment, sampling, and compliance rates. 
3. Data on demographics, clinical characteristics and medications of study subjects. 
4. Time to wean from ECMO. 
5. Pulmonary inflammation as determined qualitatively by analysis of bronchial aspirates 
lysates. 
6. Percentage of patients weaned from ECMO within 7 days 
7. Acute kidney injury as defined by the KDIGO definition (Table 1). 
8. Heart injury as determined by serum troponin levels. 
9. Renal inflammation as determined by urine NGAL concentrations. 
10. Allogenic red cell transfusion volume. 
11. Non red cell transfusion volume. 
12. Time to discharge from Glenfield Hospital ICU. 
13. Time to Glenfield Hospital discharge. 
14. Measures of haemoglobin metabolism, for example: serum hepcidin, serum haptoglobin, 
transferrin saturation, serum ferritin, plasma labile iron concentrations, oxidative stress, 
HMOX 1 expression in inflammatory cells from blood and bronchial aspirate.  
15. Levels of inflammatory cytokines in serum.  
16. MV levels and activation of leukocytes and platelets in circulation 
17. Other adverse events not specified above. 
18. A questionnaire will assess parents/ family/ guardians experience of the screening, consent 
and study procedures. (Appendix 1) 
 
 
 
 
Table 1. KDIGO staging system for acute kidney injury (55), with modified Stage 3, as per the pRIFLE 
definition (56) 
 
Stage  Serum creatinine (SCr) 
Urine output 
1 
SCr ≥ 26.5 μmol/L (0.3 mg/dL) increase or 
SCr ≥1.5- to 1.9-fold from baseline 
<0.5 
mL/kg/hr 
for 
> 
6 
consecutive hrs   
2 
SCr ≥ 2 to 2.9-fold from baseline 
<0.5 mL/kg/ hr for > 12 hrs 
3 
SCr ≥3-fold from baseline or 
SCr of more than or equal to 353.6 μmol/L (4 mg/dL) or 
renal replacement therapy irrespective of stage or, in 
patients <18 years, decrease in estimated creatinine 
clearance (eCrCl) to less than 35 mL/min/1.73 m2 using 
the Schwartz equation:  
eCrCl(mL/min/1.73 m2) = (36.2 × Height in cm) / 
Creatinine in µmol/L 
<0.3 mL/kg/ hr for > 24 hrs or 
anuria for > 12 hrs 
The above criteria include both an absolute and a relative change in creatinine to accommodate variations 
related to age, gender, and body mass index and to reduce the need for a baseline creatinine but do require at 
least two creatinine values within 48 hours. Only one criterion (creatinine or urine output) has to be fulfilled to 

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qualify for a stage. Given the wide variation in indications and timing of initiation of renal replacement therapy 
(RRT), individuals who receive RRT are considered to have met the criteria for stage 3 irrespective of the stage 
they are in at the time of RRT. The modified stage 3 criteria for children reflect the observation that infants with 
small body mass may be unable to generate high serum creatinine values. 
 
5.4 
Clinical management of Study Patients 
 
5.4.1 
Pre-ECMO care and ECMO indication 
 
Eligible patients receive standard care during the pre-ECMO period, according to the different 
respiratory failure aetiology and protocols from referral hospital. Patients requiring ECMO support are 
intubated and ventilated with conventional or High Frequency Oscillatory Ventilation (HFOV). 
 
5.4.2 
The ECMO circuit and cannulation 
 
The ECMO circuit consists of vascular access cannulas, polyvinyl chloride tubing for blood drainage 
and reinfusion, a centrifuge pump, an appropriate sized oxygenator and a heat exchanger.  
Cannulation in neonates is always performed using an open surgical approach. VA (veno-arterial) or 
VV (veno-venous) cannulation is chosen based on the clinical status of the patient and the availability 
of the correct material. VA-ECMO is the first choice for patients who need both respiratory and 
hemodynamic support, while the VV-ECMO is preferred if only respiratory support is required. 
Currently, the indication for VA-ECMO in newborns is extended also to patients with good cardiac 
function since double lumen cannulas for VV-ECMO are not available for the smallest neck vessels. If 
the vessels size is adequate, VV-ECMO is chosen when the patient requires only a respiratory support. 
The ECMO consultant decides the best approach, according to the clinical status and the neck vessels 
diameter. 
VA ECMO involves surgical cannulation of the right common carotid artery and internal jugular vein, 
with the tip of the venous cannula advanced into the right atrium and the arterial catheter positioned 
at the junction of the right common carotid artery and aortic arch. The right-sided vessels are used 
specifically for ease of advancement of the venous catheter into the right atrium.  
Conversely, VV ECMO uses a single, double-lumen cannula that is advanced to the right atrium, where 
blood is drained and reinfused into the same chamber, thus only requiring cannulation of the right 
internal jugular vein while sparing the carotid artery.  
 
5.4.3 
ECMO management 
 
Glenfield ECMO centre protocols will determine the conduct of ECMO support. In reference to VA-
ECMO the flow is typically maintained at about 100-120 ml/kg/min to achieve normal saturation (SaO2 
> 95%), with a controlled pCO2 to achieve a normal pH. Venous saturation is typically kept greater than 
60%. The target of saturation during VV ECMO is 85% with a venous saturation > 60% and low lactates 
with targeted flow rates of 80 ml/kg/min. Sweep gas is usually maintained at a FiO2 100% and 
regulated on the pCO2 basis; the usual ECMO flow/sweep gas ratio is 1:1.  
 
5.4.4 
Ventilator Management 
 
During ECMO, the patient is commonly ventilated in a “lung rest” setting. Rest setting is defined as 
respiratory rate of 10 b/min, PEEP (positive and expiratory pressure) of 10 cmH20 and pressure over 
PEEP 10 cmH20 with FiO2 <40%. The clinician can decide to change this setting or to change in a HFOV 
modality according to clinical status. 
 
5.4.5 
Haemodynamic support 
 

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The haemodynamic management during VA and VV-ECMO is different. During VA-ECMO the patient 
receives haemodynamic support over the respiratory support, and the usual inotropes requirement is 
reduced or null. VV-ECMO provides only a respiratory support, in this case the clinician can use 
inotropic or vasoactive drug to maintain an adequate perfusion and cardiac output. 
In general practice targets are MABP > 40 mmHg, lactate < 2 mmol/l, central venous saturation > 60%. 
 
5.4.6 
Anaesthesia induction 
 
The standard Glenfield anaesthetic technique will be used where possible. This typically includes the 
administration of Ketamine 2 mg/kg for sedation and Atracurium 1 mg/kg as a neuromuscular blocking 
agent. The underlying sedation might be modified according to the requirements of the patient. 
Deviations from this protocol will be recorded.  
 
5.4.7 
Fluid Management  
 
Target urine output for neonates is > 0.5 ml/kg/h. This may be maintained using fluid boluses or 
diuretics at the discretion of the attending clinician. In some circumstances the clinician can choose 
CRRT (continuous renal replacement therapy) according the local protocols and fluid balance. 
 
5.4.8 
Administration of non-RBC Blood Components  
 
Children receiving blood transfusions frequently receive non RBC components that can affect 
inflammatory responses. These will be administered according to standard unit protocols, with the 
indication, volume and timing of their administration recorded. 
 
5.4.9 
Red cell transfusion 
 
The Glenfield  ECMO red cell transfusion policy indicates transfusion with 10 ml/kg of allogenic red 
cells if the Hb < 13 g/dL. Red cells may be transfused outwith this protocol if indicated for bleeding. 
 
5.4.10 Concomitant Treatment 
 
Patients may receive medications and/or other therapies to treat adverse events as deemed necessary 
by the investigator or the patient’s physician. Concomitant medications and/or therapy that become 
necessary during the study and any changes in concomitant medication and/or therapy will be 
recorded on the CRFs. Details of concomitant medications and therapy will include generic drug name, 
dose, route, frequency, duration and indication. 
 
5.4.11 Weaning process 
 
Clinician and patient clinical status guide ECMO weaning process. VA ECMO is usually weaned with 
“retrograde flow”, in this case the flow in the cannula is inverted inside the system using the ECMO as 
a brake and not as an engine. With this kind of procedure, the observational weaning time is prolonged 
with less clotting risk for the circuit. In VV ECMO, the support blood flow is maintained but the sweep 
gas is unplugged, the patient is fully ventilated to assess the lung function. Failure to wean the patient 
leads to restart the ECMO support or to treatment withdrawal.    
  
5.4.12 Withdrawal criteria 
 
The criteria for withdrawal of participants will be the following: 
 
1. Parent/guardian’s request  
2. If the patient is found not to meet all the inclusion criteria 

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In the event of withdrawal, we will request that all patient’s data and tissues collected until that time 
are retained for analysis. In case of denial, all data will be removed and tissue disposed of. 
 
 
Mi-ECMO study Patient Flow and outcomes  
 
 
 
 
Inclusion Criteria 
1.
Patients with a diagnosis of PPHN  
2.
Patients that require ECMO support as determined by the ECMO team 
3.
Patients aged less than 30 days 
4.
Emergency consent obtained within 12 hours from cannulation, and ultimately full consent 
Exclusion criteria 
1.
PPHN is caused by a congenital heart pathology 
2.
ECMO is required for a congenital heart disease 
3.
Lack of consent 
Yes 
No 
Emergency + full consent  
Yes 
No 
Outcomes : 
Primary Outcome:  
•
Markers of platelet, leukocyte and endothelial cell activation in arterial blood 
Secondary Outcomes: 
1.
Serum haemoglobin levels. 
2.
Data on recruitment, sampling, and compliance rates. 
3.
Data on demographics, clinical characteristics and medications of study subjects. 
4.
Time to wean from ECMO. 
5.
Pulmonary inflammation as determined qualitatively by analysis of bronchial aspirates lysates. 
6.
Percentage of patients weaned from ECMO within 7 days 
7.
Acute kidney injury as defined by the KDIGO definition. 
8.
Heart injury as determined by serum troponin levels. 
9.
Renal inflammation as determined by urine NGAL concentrations. 
10. Allogenic red cell transfusion volume. 
11. Non red cell transfusion volume. 
12. Time to discharge from Glenfield Hospital ICU. 
13. Time to Glenfield Hospital discharge. 
14. Measures of haemoglobin metabolism, for example: serum hepcidin, serum haptoglobin, transferrin 
saturation, serum ferritin, plasma labile iron concentrations, oxidative stress, HMOX 1 expression in 
inflammatory cells from blood and bronchial aspirates. 
15. Levels of inflammatory cytokines in serum. 
16. MV levels and activation of leukocytes and platelets in circulation 
17. Other adverse events not specified above. 
18. A questionnaire will assess parents/family/guardians experience of the screening, consent and study 
procedures. 
N=24 Patients 
Data: Before, during and after ECMO support, follow-up (24 hours after decannulation) 
Blood, urine and respiratory samples: baseline, 24 hrs, 48 hrs, 72 hrs, after 
decannulation/withdrawal  
STOP 

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6. 
Study Tests and Procedures 
 
6.1 
Blood sampling 
Blood samples will be collected from all participants in the trial at the following time points: 
- 
During ECMO support: 12 hours, 24 hours, 48 hours, 72 hours after cannulation 
- 
End of ECMO support: 24 hours after decannulation or immediately prior to ECMO support 
being stopped in case of death or treatment withdrawal 
A total amount of 5 blood samples will be taken per patient.  
 
The sample volumes to be taken from each patient will be determined by 4 factors: 
 
1. Patient’s weight: maximum safe volume (57) depends on body weight : patients will donate a 
maximum of 3 ml per sample or 15 ml in total over the whole study period, as in the p-Mivaki 
study (63). 
 
2. Minimum volumes required to perform the assays: calculations are based on our existing 
studies in children, and are listed in Table 3. 
 
3. Minimum volume that can be collected in a sample tube: blood will be collected in 3 different 
types of collection tubes (see Table 2) at each sampling time point from all patients. Although 
individual assays might require smaller volumes, it is not possible to collect accurately these 
smaller volumes in the available sample tubes. Therefore, in some cases, excess blood will be 
taken beyond what is required, and the design of the blood sampling study has anticipated this.   
 
4. Tests to be undertaken (Table 2) 
We will only take blood required to complete the specified analysis (see below). We aim to 
remove a maximum of 15 ml in total over the 5 time points: 1.4 ml (Citrate) per time point for 
leukocyte, platelets and biochemical markers analyses, and 1.1 ml (clotting) per time point for 
troponin, and cytokine analyses and 0.5 ml (Hirudin) for MP analysis. 
 
Table 2. Blood Sample Distribution  
 
Test 
Tube 
Volume Required 
[ml] 
Storage 
Tubes  
 
Creatinine, troponin, MAGPIX tests,  
clotting 
0.6  
(serum) 
-80C 
 
Total clotting sample 
 
0.6 
(serum)  
 
1x 1.1 ml 
Leukocyte, platelets (flow), full blood 
count 
citrate 
0.4 (blood) 
Hot test 
 
Free Hgb, hepcidin, carbonylation, iron 
citrate 
0.5 
(plasma) 
-80C 
 
Total citrated sample 
 
 
 
1x 1.4 ml 
MV analysis 
hirudin 
0.5 
-80C 
 
Total hirudin sample 
 
 
 
1x 0.5ml 
Total/time-point 
 
 
 
3.0ml 
TOTAL 
 
 
 
15ml 
 
Blood samples will be taken by medical or nurse staff responsible for the care of participants through 
an arterial line, which is routinely placed before or during ECMO support.  The authorised ECMO 
fellow/trial coordinator will ‘track’ each patient and prompt staff to take the samples required at the 
designated times, and will be responsible for the secure storage and transfer of samples until they are 

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analysed in the central laboratory. Serum and plasma samples will batched and transferred for storage 
in the -80°C freezer located in the Cardiovascular Research Centre based at Glenfield Hospital until 
analysis. They will be labelled and identified by the trial acronym, patient’s study ID, initials, encrypted 
date of birth and the time point of the sample. 
 
6.2 
Urine sampling 
 
Urine samples will be collected from all participants in the trial at the following time points: 
- 
Baseline: 12 hours after cannulation 
- 
During ECMO support: 24 hours, 48 hours, 72 hours after cannulation 
- 
End of ECMO support: 24 hours after decannulation, or immediately prior to ECMO support 
being stopped in case of death or treatment withdrawal 
All available urine at the time-point will be collected for each patient. Urine sample collection will 
occur at the same time of blood sample collection. Urine samples for measuring renal function and 
injury will be taken through an in situ urinary catheter that is usually positioned before or soon after 
ECMO commencement.  
Samples for the research will be taken over a 24-hour period. The start and stop times of the collection 
period will be recorded and the total urinary output will be measured. 
Urine samples will be taken through an in situ urinary catheter by the authorised ECMO fellow/trial 
coordinator at the predefined time points. Urine samples will be batched and transferred for storage 
in the -80° freezer located in the Cardiovascular Research Centre based at Glenfield Hospital until 
analysis. They will be labelled and identified by the trial acronym, patient’s study ID, initials, encrypted 
date of birth, sample type and the time point of the sample. 
 
6.3 
Respiratory sampling 
 
Respiratory samples will be collected from all participants in the trial through a respiratory tract 
suction procedure at the following time points: 
- 
Baseline: 12 hours after cannulation 
- 
During ECMO support: 24 hours, 48 hours, 72 hours after cannulation 
- 
End of ECMO support: 24 hours after decannulation or immediately prior to ECMO support 
being stopped in case of death or treatment withdrawal 
 
A total amount of 5 respiratory samples will be taken per patient.  
Each intubated patient routinely receives a regular suction to evacuate the secretion from respiratory 
tract. The routine suction procedure is regulated by a hospital protocol and completed by the bed 
space nurse as required by clinical purposes. The collected sputum would be normally wasted. We will 
collect part of the sputum routinely aspirated and no extra procedure will be performed in order to 
complete the sample collection for the study.  After the suction is completed, the fluid will be collected 
for the analysis.  
The authorised ECMO fellow/trial coordinator will ‘track’ each patient and prompt staff to take the 
samples required at the designated times, and will be responsible for the secure storage and transfer 
of samples until they are analysed in the central laboratory. Sputum samples will be transferred to the 
lab and analysis will be performed. The remaining cells will be lysed as described below and stored at 
-80°C in a freezer located in the Cardiovascular Research Centre based at Glenfield Hospital until 
analysis. Samples will be labelled and identified by the trial acronym, patient’s study ID, initials, 
encrypted date of birth, sample type and the time point of the sample. 
 
 
6.4 
Sample storage 
 
The urine, blood and respiratory samples will be transferred to the academic laboratory of Glenfield 
Hospital where they will be stored until analysed.  After the samples have been analysed, they may be 

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stored with patient's permission for further REC and R&D approved researches. The 
patient/parents/guardians will be informed through the patient information leaflet about the possible 
storage of the samples for future research and they will be asked to sign a consent form if they agree. 
We may share anonymised tissue or data with external collaborators, with patient permission. 
Otherwise, the samples will be disposed in accordance with the Human Tissue Authority's Code of 
Practice and the institution’s standard operating procedure for clinical wastage. 
In the event that the neonate does not survive before obtaining full consent, parents/legal 
representatives will be approached to obtain permission to retain the samples. If permission is not 
obtained, the samples will be destroyed and this will be fully documented in the medical notes. 
 
Glenfield Hospital  
Groby Road 
Leicester, LE3 9QP 
Tel: 0116 259 3039  
e-mail: gjm19@le.ac.uk 
 
Detailed description of the sample collection, storage and transfer procedures will be provided in the 
study laboratory manual. 
 
6.5 
Blood and urine samples assays 
 
Serum creatinine will be measured using fluorometric creatinine kit (Abcam, UK) at 12, 24, 48, 72 
hours after cannulation, and 24 hours after decannulation or immediately prior to ECMO support 
being stopped in case of death or treatment withdrawal. Estimated creatinine clearance will also be 
used to define Stage 3 AKI, using the Schwartz equation (table 2).  
 
Cytokines and chemokines important in monocyte/macrophage differentiation and renal injury, e.g. 
IL-6, -8, TNFα, MCP-1 and MIP-1, as well as markers of endothelial injury (circulating ICAM or E-
selectin) may be measured from serum on the Luminex MAGPIX Analyser (Oosterhout, NL). 
 
Troponin will be measured in serum using Troponin I ELISA kit (ENZO Life Sciences, Exeter, UK). 
Hepcidin (key regulator of iron in circulation) and carbonylation levels reflecting oxidative stress will  
be measured in citrated plasma using commercially available ELISA kits from DRG International Inc. 
(Springfield, USA) and Abcam (Cambridge, UK). 
 
Total iron and non-transferrin-bound iron (NTBI) levels will be measured as previously described 
(Gosriwatana et al., 1999). Iron is detected by measuring absorbance at 525 nm in colorimetric 
reaction with bathophenanthroline disulfonic acid disodium salt hydrate (BPT). For NTBI samples will 
be treated with iron chelator (nitrilotriacetic acid, NTA), ultrafiltered and analysed with BPT. 
 
Oxidative stress will be measured in plasma using commercially available carbonylation kit (R&D 
Systems, Minneapolis MN).  
HMOX-1, iNOS and Arg-1 expression will be estimated in cells from bronchial aspirates by Western 
blotting with specific antibodies. 
 
Platelets and leukocyte activation, as well as platelets—leukocyte aggregates will be determined with 
specific antibodies (CD41, PAC-1 and CD62P for platelets, CD64, CD163 and CD11a for leukocytes and 
CD14, CD16, CD41 for the aggregates) and analysed by flow cytometry. Full blood count will be 
measured. 
 
Urine biomarkers of renal oxidative stress; NGAL (ELISA Bioporto Diagnostics A/S Denmark) and/will 
be measured on the DS2 automated ELISA platform (Dynex, through Alere, UK).  
 

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6.6 
MV Analyses 
 
Circulating blood will be anticoagulated with hirudin at the defined time points (Section 6.1).  
MV will be isolated by centrifugation (1,500 g, 15 min ×2 at 4°C). MV size will be evaluated using the 
Nanosight. Mean size and particle concentration values will be calculated using Nanoparticle Tracking 
Analysis software using the manufacturer’s instructions. Antigen expression on MV, indicative of the 
cell of origin will be determined using flow cytometry (Beckman Coulter MCL-XK or a Cyan ADP 9 
colour instrument) as previously described [39, 40].  
 
MV will be identified with Annexin V-APC and subpopulations will be identified by co-staining with 
antibodies against cellular marker proteins. A suggested range of markers is listed in table 3.  
 
Table 3. 
 
Probe 
Target molecule 
Target cells 
Annexin V 
Phosphatidylserine 
Microvesicles, apoptotic cells 
CD235-FITC 
Glycophorin A 
Red blood cells 
CD41-PE 
Integrin α-IIb 
Platelets 
CD144-PE 
VE-cadherin 
Adherens junctions 
CD162-PE 
PSGL-1 
Adhesion molecule 
CD29-PE 
Integrin-β1 
Platelets 
CD142-FITC 
Tissue factor  
Coagulation 
CD 62-PE 
E-selectin 
Endothelium 
CD284-Ax488 
TLR-4 
Monocytes , granulocytes 
Phalloidin-FITC 
F-actin 
Cell fragmentation 
 
 
6.7 
Bronchial aspirates analysis 
 
Bronchial aspirates (BA) analysis is a valuable tool to assess the degree of lung injury. Analysis of 
immune cells will allow us to extrapolate the results obtained from blood analysis and conclude on 
events leading to injury and more importantly to recovery from lung injury and consequently shorter 
ECMO time. 
BA samples will be spun and the pelleted cells washed with PBS. Iron deposition will be assessed 
using ferrocyanide method where BA smears are stained with Prussian Blue and analysed by light 
microscopy. 
Remaining cells will be lysed and frozen at -80C for WB analysis with HMOX-1 antibodies to estimate 
oxidative stress response and iNOS and Arginase (Arg-1) antibodies to assess macrophage/T-cells 
M1/Th1 and M2/Th2 responses in lungs. 
 
 
 
6.8 
Summary of trial specific tests and procedures 
 
 
Baseline (12h 
after ECMO 
start 
Day 1 
(24 hrs 
Day 2 
(48h 
Day 3 
(72h +/- 
12 hrs) 
24h after 
decannulation/ 

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+/- 6 hrs) 
+/- 12 
hrs) 
+/- 12 
hrs) 
 
prior to ECMO stop 
for death or 
withdrawal (+/- 12 
hrs) 
Eligibility 
 
 
 
 
 
Written emergency 
consent 
 
 
 
 
 
Trial Registration 
 
 
 
 
 
Clinical outcomes 
 
 
 
 
 
Serious adverse event 
monitoring 
 
 
 
 
 
Bloods: Full blood 
count 
  
  
  
  
  
Bloods: Cytokines, total 
iron, non-transferrin-
bound iron 
 
 
 
 
 
Bloods: Platelets and 
leukocyte activation 
 
 
 
 
 
Bloods: MV analysis 
 
 
 
 
 
Respiratory Sample: 
ferrocyanide, oxidative 
stress 
 
 
 
 
 
Urine sample & 
volume: NGAL 
 
 
 
 
 
Serious adverse event 
monitoring 
 
 
 
 
 
Questionnaire on 
perception of family 
members 
 
 
 
 
 
 
† Indicates samples taken as part of normal care 
  
 
 
6.9 
End of the trial 
 
For an individual participant, the end of the trial is defined as a successful weaning from ECMO, or 
death. We consider finished the follow-up after discharge from UHL. The participant’s parents 
/guardians are free to withdraw from the study at any time for any reason. In the event of withdrawal, 
the primary reason must be recorded in the subject’s medical record and on the withdrawal form in 
the CRF. Any comments (spontaneous or elicited) or complaints made by the patient or clinical care 
team, together with the reason for termination, date and time of withdrawal must be recorded in the 
CRF and source documents. 
 
Patients will be considered lost to follow-up only if there is insufficient information to determine the 
patient’s status at end of trial, whether because of transfer to another unit, no contact has been re-
established following 3 documented attempts by the research team.  
The definition of the end of the trial as a whole is the date when all participants have completed the 
follow-up or have been lost to follow-up. 
 

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6.10 
Data collection 
 
After cannulation, the authorised ECMO fellow/trial coordinator will collect data on pre-printed forms 
to characterise the patients who are approached about the trial (e.g. eligibility criteria, key 
demographic data, willingness to participate and, if not willing, main reason for refusal).  These data 
will be entered promptly in the master subject tracker in order for the trial registration number to be 
generated for a study participant.  
 
Other pre-ECMO data likely to influence the speed of post cannulation recovery, procedure details 
(cannulation, details of the ECMO circuit), morbidity, duration of ICU and hospital stay and other non-
biochemical secondary outcomes will also be collected prospectively on pre-printed forms, including 
dates and times of relevant outcomes. 
These data will be entered into a database immediately after the end of follow-up. In the event that a 
patient has been withdrawn from the study, we will continue to analyse any data already collected, 
unless the patients expresses a wish for their samples and any associated data to be destroyed. 
 
Source data will be collected prospectively on designated CRF’s. In addition, the patient’s medical 
notes will be a source for additional clinical data, including blood and urine results performed in the 
clinical setting. University laboratory databases will be the source for other trial specific biochemical 
markers analysed in the blood and urine. 
 
Protocol adherence will be evaluated through the monitoring of protocol violations defined as any 
change, deviation, or departure from the study design or procedures of research project that is not 
approved by the Sponsor and Ethics Committee prior to its initiation or implementation.   
Protocol violations will be categorized as major or minor violations, according to the following 
definitions:  
 
A major protocol violation: 
• a serious and/or continuous failure on the part of the study team to comply with the protocol, 
standard operating procedures, good clinical practice (GCP), trial regulations.   
• has significant negative impact to subject safety. It may or may not result in actual harm to 
the subjects.   
• Significantly damages the completeness, accuracy and reliability of the data collected for the 
study. 
  
A minor protocol violation/deviation/non-compliance: 
• Does NOT represent a serious or continuous failure on the part of the study team to comply 
with the protocol, standard operating procedures, GCP, trial regulations. 
• Does NOT significantly impact subject safety or substantially alter potential risks to subjects 
• Does NOT result in actual harm  
• Does NOT significantly damage the completeness, accuracy and reliability of the data collected 
for the study 
 
Clinical protocol non-adherence will be defined as the failure to obtain the specified clinical data, 
blood volume or urine samples required for analysis at the required time. Protocol violation 
concerning the time of sample collection will be defined as a deviation of more than 6 hours from the 
planned collection time for the baseline data, and 12 hours for the remaining time points. Laboratory 
protocol non-adherence will be defined as the failure to complete laboratory essay as described in the 
protocol. 
 
 
6.11 
Patient recruitment and consent 
 

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Patients who meet the inclusion criteria and have been referred for ECMO at the Glenfield Hospital 
will be considered eligible for the study. Parents/guardians will be required to provide full written 
informed consent. Patient’s willingness to participate cannot be assessed since all the participants are 
younger than 1 month, therefore, they will be considered as unable to provide consent or assent.  
According to the Children Act and the Children Act (Northern Ireland) Order, parents and those with 
parental responsibility are allowed to consent to medical treatment or research on their behalf. 
Consent of only one parent is required. Patients’ parents/guardians are expected to read and 
understand the parent/guardian information leaflet and (with the assistance of a member of the 
clinical team) understand the purposes and consequences of the study to the patient at a level suitable 
for them. 
Patients can be formally enrolled only if emergency consent (59, 60) is obtained within 12h from ECMO 
cannulation. Details of all patients approached for the trial and reason(s) for non-participation (e.g. 
reason for being ineligible or patient refusal) will be documented.  
Since the Glenfield Hospital is a national paediatric referral centre collecting patients from different 
parts of UK, we expect that a percentage of parents/guardians will not reach the Hospital within the 
first hours from cannulation and will not be promptly available onsite. Moreover, we expect that in 
rare cases the mother will not reach the hospital for days after ECMO start due to delivery’s 
complications or health reasons. On the other hand, it is important to establish baseline values in 
order to test our hypothesis. For that reason, the first sample has to be collected within 12 hours from 
the cannulation. The Regulations prescribe a hierarchy for determining who should be approached to 
give informed consent on behalf of a minor prior to their inclusion in the trial as shown in Table 4. The 
provisions for informed consent by a legal representative only apply if due to the emergency nature 
of the trial, no person with parental responsibility can be contacted prior to the proposed inclusion of 
the minor. 
For the reasons discussed above, the recruitment strategy will follow the scheme described in the 
following flowchart. 
 
 
 

CLINICAL STUDY PROTOCOL 
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Parents/Guardians 
with parental 
responsibility are 
present at Glenfield 
Hospital
when patient is 
cannulated
A member of the research team 
approaches parents/guardian with 
parental responsibility and explains in 
summary the project,  asks for 
emergency consent and delivers PIL            
(Documents: PIL and Emergency 
consent for parents/Guardians or 
personal legal representative)
As soon as possible, a member of the 
research team approaches 
parents/guardians with parental 
responsibility or legal representative, 
explains in details the project, answers 
questions and ask for full consent 
(Document: full consent form for 
Parents/Guardians/Legal 
representative)
After at 
least 12 
hours
Phase 1: emergency consent
A member of the research team 
approaches a personal legal 
representative ( IE: Father if parents 
are not married) and explains in 
summary the project, asks for 
emergency consent and delivers PIL 
(Documents: PIL and Emergency 
consent for parents/Guardians or 
personal legal representative)
NO
A member of the research team 
approaches a professional legal 
representative (IE: Doctor in charge for 
the patients if not involved in the 
study) and explains in summary the 
project, asks for emergency consent
and delivers PIL (Document: 
Emergency consent for professional 
representative)
If the personal legal representative is 
not present
Consent
given
Collect 
the 
samples 
YES
Continue the research project
Phase 2: full consent
ENROLLMENT 
FLOW CHART
Consent
given
Collect 
the 
samples 
YES
NO
STOP
NO
STOP
Consent given
As soon as possible, a member of the 
research team approaches 
parents/guardian with parental 
responsibility or legal representative, 
explains in summary the project, and 
delivers PIL (Document: PIL for 
Parents/Guardians/Legal 
representative)
After at 
least 12 
hours
STOP
YES
NO

CLINICAL STUDY PROTOCOL 
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Table 4 
Person who may give the 
consent 
Definition 
Commentary 
Parent 
A parent or person with 
parental responsibility 
Should always be approached if 
available 
Personal legal representative 
A person not connected with 
the conduct of the trial who is: 
(a) suitable to act as the legal 
representative by virtue of 
their relationship with the 
minor, and 
(b) available and willing to do 
so 
May be approached if no 
person 
with 
parental 
responsibility can be contacted 
prior to the proposed inclusion 
of the minor, by reason of the 
emergency nature of the trial 
Professional legal 
representative 
A person not connected with 
the conduct of the trial who is: 
(a) 
the 
doctor 
primarily 
responsible for the medical 
treatment of the minor, or 
(b) a person nominated by the 
relevant health care provider 
(e.g. an acute NHS Trust or 
Health Board). 
May be approached if no 
person suitable to act as a 
personal legal representative is 
available. Informed consent 
must be given before the minor 
is entered into the trial  
 
 
6.11.1 
Parents/guardians with parental responsibility available within 12 hours from cannulation 
 
If parents/guardians with parental responsibility are available at patient admission to the Glenfield 
Hospital, an authorised member of the research team will approach them in order to explain the study 
and ask for emergency consent. 
 
Parents/guardians 
with 
parental 
responsibility 
will 
be 
provided 
with 
an 
emergency 
parent/guardian/personal legal representative information leaflet at the time of their arrival at the 
Glenfield Hospital. At the same time, an authorised member of the research team will explain in 
summary the purpose of the study in person and will provide answers to any questions that might be 
raised. Due to the urgency of the situation, if parents/guardians are willing to take part in the study, 
they will be asked to provide a written emergency consent. 
 
After parents/guardians’ emergency consent, the samples will be collected following the protocol and 
the parents/guardians will be provided with a complete version of the parent/guardian/personal legal 
representative information leaflet. They should therefore have at least 12 hours to read and consider 
the information and to ask questions before being asked to decide whether they are willing to 
continue the study. An authorised member of the research team will approach again 
parents/guardians and will explain in details the research project, answer the questions and ask for 
full consent (59, 60).  
 
In the event that a child withdraws, we will request that all patient’s data and tissues collected until 
that time are retained for analysis. If this request is denied, all data will be removed and tissues 
disposed of. All the leaflets and forms will be approved by the local Research Ethics Committee (REC). 
 

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6.11.2 Parents/guardians with parental responsibility not available within 12 hours from 
cannulation 
 
If parents/guardians with parental responsibility are not available at patient admission to the Glenfield 
Hospital, an authorised member of the research team will approach a personal legal representative 
(defined as in Table 4) in order to explain the study and ask for emergency consent. 
As personal legal representative will be considered the following persons: 
• 
Putative father 
• 
Grandparents  
The personal legal representative will be provided with an emergency parent/guardian/personal legal 
information leaflet at the time of his/her arrival at the Glenfield Hospital. At the same time, an 
authorised member of the research team will explain in summary the purpose of the study in person 
and will provide answers to any questions that might be raised. Due to the urgency of the situation, if 
the personal legal representative is willing to take part in the study, he/she will be asked to provide a 
written emergency consent. 
After the personal legal representative’s consent, the samples will be collected following the protocol 
and the personal legal representative will be provided with a complete version of the 
parent/guardian/personal legal representative information leaflet. He/she should therefore have at 
least 12 hours to read and consider the information and to ask questions before being asked to decide 
whether he/she is willing to continue the study. An authorised member of the research team will 
approach again the personal legal representative and will explain in details the research project, 
answer the questions and ask for full consent (59, 60). 
In the event that a child withdraws, we will request that all patient’s data and tissues collected until 
that time are retained for analysis. If this request is denied, all data will be removed and tissue 
disposed of. All the leaflets and forms will be approved by the local Research Ethics Committee (REC). 
 
 
6.11.3 Neither parents/guardians with parental responsibility nor personal legal representative 
available within 12 hours from cannulation 
 
If parents/guardians with parental responsibility and a personal legal representative are not available 
at patient admission to the Glenfield Hospital, an authorised member of the research team will 
approach a professional legal representative (defined as in Table 4) in order to explain the study and 
ask for emergency consent. The professional legal representative have to be a Consultant in Paediatric 
Intensive Care and/or ECMO not connected with the conduct of the trial. Before the start of the 
recruitment phase, the study will be explained to the whole medical staff working in the Paediatric 
Intensive Care Unit and in the ECMO team through several meetings. This will allow them to 
understand the details of the study in advance, consider all the information and ask questions.  
The professional legal representative will be provided with an emergency professional legal 
representative information leaflet at the time of the child’s arrival at the Glenfield Hospital. At the 
same time, an authorised member of the research team will explain in summary the purpose of the 
study in person and will provide answers to any questions that might be raised. Due to the urgency of 
the situation, if the professional legal representative is willing to take part in the study, he/she will be 
asked to provide a written emergency consent. After the professional legal representative’s consent, 
the samples will be collected following the protocol. 
As soon as possible, a member of the research team will approach parents/guardians with parental 
responsibility or a personal legal representative, explain in summary the project, and provide them 
with a complete version of the parent/guardian/personal legal representative information leaflet. 
They should therefore have at least 12 hours to read and consider the information and to ask 
questions before being asked to decide whether he/she is willing to continue the study. An authorised 
member of the research team will approach again the parents/guardians with parental responsibility 
or a personal legal representative and will explain in details the research project, answer the questions 
and ask for full consent (59-60).  

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In the event that a child withdraws, we will request that all patient’s data and tissues collected until 
that time are retained for analysis. If this request is denied, all data will be removed and tissue 
disposed of. All the leaflets and forms will be approved by the local Research Ethics Committee (REC). 
 
6.11.4 Satisfaction questionnaire 
 
Perceptions of family members whose children participate in the study as to the appropriateness of 
the screening and consent process will be assessed by means of a 11 items questionnaire (Appendix 
1) adapted from previous studies (61, 62). The questionnaire will be administered at the end of the 
study prior to hospital discharge. 
 
 
 
6.12 
Planned recruitment rate and duration of follow-up 
 
On the basis of historical data we estimate that there will be a target population of 40-60 eligible 
patients over 12 months at Glenfield Hospital. We plan to recruit, 24 of all eligible patients. In a 
previous observational clinico-experimental study undertaken by this group [The p-MiVAKI study 
(63)], that also required serial blood sampling in children undergoing cardiac surgery, an 84.37% 
consent rate was achieved.   
Participants will be followed up until discharge from UHL.   
 
6.13 
Likely rate of loss to follow-up, protocol violations and clinical data completeness 
 
Until discharge from hospital, the likely losses to follow-up will be due to death or participant 
withdrawing, or more commonly incomplete collection of the samples. In a previous observational 
clinico-experimental study undertaken by this group [The p-MiVAKI study (63)],that also required 
serial blood sampling in children, clinical data showed a completeness of 84.09% as described in Table 
5. 
 
Table 5 – Data collection 
Variable 
Completeness 
(%) 
Overall 
84.09 
Clinical variables 
94.61 
Clinical laboratory data 
77.5 
Categorical variables 
100 
 
Total sample collection reached a percentage of 93.75% (Table 6-7). Only in one case, parents refused 
consent  for one sample donation because of children discomfort. All patients concluded the study 
and no major protocol violations were recorded.  
 
Table 6 – Blood sample collection 
Patients  
(n) 
Expected 
samples (n) 
Collected 
samples (n) 
Collected 
samples (%) 
Patient’s weight: 2-5 Kg 
 6 
18 
18 
100% 

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Patient’s weight: 5-20 Kg 
14 
42 
41 
95.2% 
Patient’s weight:  > 20 Kg 
4 
12 
9 
75% 
Total 
24 
72 
68 
94.4% 
 
 
 
Table 7 – Urine sample collection 
Patients  
(n) 
Expected 
samples (n) 
Collected 
samples (n) 
Collected 
samples (%) 
Patient’s weight: 2-5 Kg 
 6 
18 
18 
100% 
Patient’s weight: 5-20 Kg 
14 
42 
40 
97.9% 
Patient’s weight: > 20 Kg 
4 
12 
9 
75% 
Total 
24 
72 
67 
93.1 % 
 
6.14 
Expenses and benefits 
 
Participants will not receive payment for taking part in the trial.  As they will not be attending as 
outpatients above and beyond their normal care there will be no additional reimbursement for travel 
or other expenses.  

 
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7. 
Statistical Considerations 
 
7.1 
Sample Size 
 
This is a feasibility study and no formal power calculation has been performed. Our sample size is 
based on our observations from the 24 patients in the p-MiVAKI study (63), where we have 
demonstrated significant differences in markers of platelet, leukocyte and endothelial activation 
between patients who subsequently did or did not develop AKI, and between those that did or did not 
receive transfusion.  
 
7.2 
Measures taken to avoid bias 
 
All necessary steps will be taken to reduce the risk of bias. Detection bias will be minimised by blinding 
of laboratory staff analysing cytokines, biomarkers and inflammatory processes.  Specifically, urine, 
serum and sputum samples obtained exclusively for the trial will be identified only by a trial acronym, 
patients’ study ID, initials and date of birth and the time at which the sample was taken, ensuring that 
laboratory staff performing analyses are blinded.  Detection bias for the clinical outcomes will also be 
minimised by the use of objective outcome criteria. Other samples, that are also obtained as part of 
routine care will be analysed routinely in NHS laboratories by personnel who are unaware that the 
participant is in a trial. Clinical decision about patient care will be made by clinical staff on the basis of 
existing institutional protocols and will not be affected by enrolment in the study. 
 
7.3 
Plan of analysis 
 
A statistical analysis plan will be drafted prior to analysis. Normally distributed data will be presented 
as means (standard deviation). Non normally distributed data will be presented as geometric means 
(95% confidence intervals). Dichotomous data will be presented as percentages. This data will be used 
to quantify the number of patients required to design a further prospective observational analysis. 
 
7.4 
Criteria for stopping the study early 
 
The study may be terminated early if the results of another study supersedes the necessity for 
completion of this study. 
 
8. 
Study Management 
 
The study will be managed by the Cardiac Surgery Clinical Trials Team (CSCTT) at the University of 
Leicester. The CSCTT will prepare trial documentation and data collection forms, register patients and 
carry out trial procedures; the CSCTT will also develop and maintain the study database, check data 
quality as the trial progresses, and carry out trial analyses in collaboration the statistician. 
 
8.1 
Day-to-day management 
 
The trial will be managed by a Clinical Trial Manager, supported by a Clinical Trial Coordinator from 
the Cardiac Surgery Clinical Trials Team at the University of Leicester. An authorised ECMO 
fellow/coordinator will be responsible for managing the trial on a day-to-day basis.  They will be 
responsible for identifying potential trial participants, seeking informed patient consent, liaising with 
blood and laboratory services, collecting trial data and ensuring the trial protocol is adhered to.  
 
8.2 
Trial steering committee and data safety & monitoring committee 
 

 
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The trial will be monitored by the Cardiovascular Surgery Research Group Steering Committee. As this 
is an observational study, no Data Monitoring and Safety Committee (DMSC) will be convened. 
The Trial Steering Committee will provide overall supervision of the trial and ensure that the local, 
national and international research framework is adhered to. The Trial Steering Committee will agree 
trial amendments, as necessary, and provide relevant guidance on study design and conduct to 
participating investigators. Membership includes members of the Trial executive, principal 
investigators, independent experts, a service user representative and a representative of the study 
Sponsor. 
 
 
9. 
Safety Reporting 
 
9.1 
Definitions 
 
As defined by the ICH Guidelines for Good Clinical Practice (CPMP/ICH/135/95) the following 
definitions will be used:  
 
Adverse Event 
Any untoward medical occurrence in a patient or clinical trial subject, who has been administered a 
clinical intervention, procedure, treatment or medicinal product and which does not necessarily have 
a causal relationship with this medical intervention. An adverse event can therefore be any 
unfavourable and unintended sign (including an abnormal laboratory finding), symptom or disease 
temporally associated with the intervention, whether or not related to the medical intervention. 
 
Serious Adverse Event 
Any untoward medical occurrence, which 
• 
results in death 
• 
is life threatening 
• 
requires inpatient hospitalisation or prolongation of existing hospitalisation 
• 
results in persistent or significant disability or incapacity 
• 
consist of a congenital anomaly or birth defect.  
• 
other situations, such as significant medical events, which may not be immediately life 
threatening or result in death or hospitalisation, but which may jeopardise the subject or may 
require intervention to prevent one of the other serious outcomes listed in the definition 
above, should also be considered. 
 
9.2 
Adverse event reporting 
 
Adverse events will be recorded and reported in accordance with the University of Leicester’s and 
University Hospitals Leicester NHS Trust’s policies for reporting Research Related Adverse Events. 
 
In intensive care management and especially in ECMO practice, adverse events are not unexpected 
and are not infrequent.  The research team will only notify deaths and ‘unexpected’ non-fatal SAEs to 
the Trial Sponsor (University of Leicester Research Support Office).  Unexpected events are those not 
listed in the trial protocol or on the case report forms. The sponsor will inform the research team 
which SAEs should be reported to the REC. 
 
 
9.3 
Expected adverse events 
 
The following adverse events are ‘expected’: 
 

 
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Cardiovascular: 
• 
Cardiac arrest 
• 
Myocardial stunning 
• 
Cardiac arrhythmias and arrhythmias requiring temporary or permanent pacing 
• 
Myocardial infarction including: 
o New Q’s in 2 contiguous leads 
o Raised Troponin I >0.5μg/l 
o New ST depression >2mm in 2 leads 
• 
Cardiac tamponade 
• 
Pericardial effusion 
• 
Perforation of heart or vessels 
• 
Cardiopulmonary resuscitation including cardiac defibrillation/DC shock  
• 
Chest opening 
• 
External/internal cardiac massage 
• 
Low cardiac output 
• 
Haemodynamic support, including use of: 
• 
Any inotropes   
• 
Pulmonary artery catheter  
• 
Any vasodilators  
 
Pulmonary: 
• 
Prolonged invasive ventilation  
• 
Tracheostomy 
• 
Bronchomalacia 
• 
Tracheomalacia 
• 
Subglottic stenosis 
• 
Initiation of mask continuous positive airway pressure ventilation after weaning from 
ventilation 
• 
Acute Respiratory Distress Syndrome 
• 
Pneumothorax or effusion requiring drainage 
 
Thromboembolic complications, including: 
 
• 
Deep vein thrombosis 
• 
Pulmonary embolism  
• 
Clotting of the ECMO circuit 
• 
Stroke 
• 
Arterial thrombosis  
• 
Limb ischemia 
 
Hemorragic complications, including: 
• 
Disseminated intravascular coagulation 
• 
Haemothorax 
• 
Cannulation site bleeding 
• 
Gastrointestinal bleeding 
• 
Brain haemorrhage 
• 
Haemolysis 
• 
Airways bleeding 
 
Renal complications, including: 
• 
Acute kidney injury 

 
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• 
Haemofiltration/dialysis 
 
Infective complications, including: 
• 
Sepsis (defined as antibiotic treatment for suspected infection, and the presence of SIRS1 
within 24 hours prior to start of antibiotic treatment) - SIRS will be defined as ≥2 of the 
following conditions: temperature >38oC or <36oC; heart rate >90 beats/minute; 
respiratory rate >20 breaths/min or PaCO2<32 mm Hg or <4.3 kPa; white cells count 
>12,000/mm3 or <4,000/mm3.   
• 
Septic shock 
• 
Wound infection  
• 
Respiratory infection  
• 
Ventilator associated pneumonia 
• 
Urinary tract infection 
• 
Catheter related bloodstream infection 
  
GI complications, including: 
• 
Pancreatitis (amylase >1500iu) 
• 
NEC (necrotizing enterocolitis ) 
• 
Other (e.g. laparotomy) 
 
Neurological complications 
• 
Irreversible neurological deficit 
• 
Seizures 
• 
Peripheral nerve damage 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Figure 3: Serious adverse event reporting flow chart  
 
 
 
 
9.4 
Period for recording adverse events 
 
Data on adverse events will be collected from ECMO commencement to UHL discharge. 
 
 
9.5 
Methods of recording and assessing adverse events 
 
Prior to study registration, a detailed medical history and physical examination, including laboratory 
tests, will be taken for baseline reference. At all subsequent days of hospital admission and during the 
follow up, the occurrence of AEs will be recorded in the appropriate section of the CRF. Among these, 
all SAEs must also be recorded in the SAE report form and will be notified to the Trial Sponsor 
(University of Leicester Research Support Office) in case of death or ‘unexpected’ non-fatal SAEs.   
 
(a) Unexpected serious adverse events – i.e. those not listed in study protocol 
Causally related to study procedures? 
Yes 
No 
Event is classified as a 
SUSAR  
Treat as per expected 
event above 
Resulted in death or life threatening? 
Yes 
No 
Report event to REC and 
DMSC immediately (maximum 
7 days).  Follow-up report 
within further 8 days. 
Report event to REC and 
DMSC immediately 
(maximum 15 days). 
(b) Expected serious adverse events – i.e. those listed in study protocol 
Event is a SSAR. Report event to 
DMSC in quarterly report and to REC 
in annual safety report. 
Causally related to study procedures? 
Yes 
No 
Report event to 
DMSC in 
quarterly report 
Responsibility of: 
 
 
 
INVESTIGATOR 
 
 
 
 
 
 
 
 
INVESTIGATOR 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
INVESTIGATOR 

 
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The following aspects of AEs will be captured in the CRF: 
• 
Description of event in medical term 
• 
Date of onset 
• 
Date of recovery, if applicable 
• 
Intensity of the event: 
o 
Mild:  
Awareness of event, but easily tolerated 
o 
Moderate:  Discomfort enough to interfere with usual daily activity 
o 
Severe:  
Inability to carry out usual daily activity 
o 
 Life-threatening: Life-threatening or disabling 
o 
Fatal:  
Death related to the AE 
• 
Causal relationship to the study procedures as assessed by the Investigator. The decisive 
factor in documentation is the temporal relationship between the AE and study intervention. 
The following judgements of causality are to be used: 
o 
Not related:  The event is related to an aetiology other than study procedures 
o 
Remote:  
The event is unlikely to be related to the study procedures based on 
existing knowledge and likely to be related to other factors.  
o 
Possibly: 
Reasonable possibility that the AE was caused by the study 
procedures and there is a plausible mechanism of action, but there may also be an 
alternative aetiology such as the participant’s clinical status or underlying disease.  
o 
Definite:  
There is an association between the event and the administration of 
study procedures, a plausible mechanism for the event to be related to the study 
product and causes other than study product have been ruled out. 
• 
Action taken: None, Concomitant medication or therapy or other 
 
 
10. 
Ethical Considerations 
 
10.1 
Review by a NHS Research Ethics Committee 
 
Ethics review of the protocol for the trial and other trial related essential documents (e.g. patient 
information leaflet, consent form) will be carried out by a UK NHS Research Ethics Committee (REC). 
Any amendments to these documents, after a favourable opinion from the REC has been given, will 
be submitted to the REC for approval prior to implementation.  
 
10.2 
Risks and anticipated benefits 
 
The response of neonates to ECMO is still poorly understood in terms of inflammatory signalling and 
interaction between the body and the machine. However, the haematological response to the circuit 
in low weight patients is considered to play an important role in the recovery process and in morbidity 
and mortality. There is a clear need for the development of novel interventions that reduce the 
severity of inflammatory organ injury during ECMO support. This study may lead to further research 
that will help future ECMO patients however there are no direct benefits to the recruited patients 
from taking part. 
As this is a non-interventional study, the potential risks from participating in the study are those risks 
associated with the trial procedures. Specifically the withdrawal of significant volumes of blood from 
children subject to an ECMO where circulating volume is smaller than in adults is our chief concern.  
To minimise this risk, we have taken steps to reduce the volume of blood removed from the children 
during and after the extracorporeal support. The volumes and tests to be undertaken are determined 
by patients weight and the minimum volumes of blood required to complete the assays. The detailed 
consideration of these factors is described in detail in Section 6.1 and Table 2. Our experience with a 
previous study in paediatric cardiac surgery [The p-MiVAKI study (63)], has shown that these volumes 

 
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are safe. We therefore do not anticipate that any child will be exposed to increased risk by 
participation in the study. 
 
No further invasive procedure will be carried out only for research purposes. Since the airway 
suctioning is performed routinely for clinical purpose and in the interest of the patient, no ethical 
issues can be raised. 
 
In summary, the study investigations have a low risk profile, but there are no direct benefits to be 
gained from trial participation other than helping the researchers in gaining a better understanding 
about the inflammatory processes that are involved in neonatal ECMO. 
 
 
10.3 
Informing potential study participants of possible benefits and known risks 
 
Information about benefits and risks of participation will be described in a Patient Information Leaflet.  
This information sheet will be part of our application for research ethics approval to the REC. 
 
 
10.4 
Obtaining informed consent from participants 
 
All participants will be required to give written informed consent to participate in the study. The 
consent process, including the information about the trial given to patients in advance of recruitment, 
has been described previously. Members of the clinical research team will be responsible for the 
consent process. 
 
 
11. 
Research Governance 
 
This clinical trial will be sponsored by the University of Leicester and will be conducted in accordance 
with: 
• 
International Conference for Harmonisation of Good Clinical Practice (ICH GCP) guidelines 
• 
Research Governance Framework for Health and Social Care, the European Union Directive 
2001/20/EC on clinical trials  
• 
Data Protection Act 1998.  
• 
Human Tissue Act 2004 
• 
Protection of Children Act, 1999  
 
11.1 
Sponsor approval 
 
Any amendments to the trial documents must be approved by the sponsor prior to submission to the 
REC and/or implementation. 
 
 
11.2 
NHS approval 
 
Approval from University Hospitals Leicester NHS Trust will be obtained prior to the start of the trial.   
 
Any amendments to the trial documents approved the REC will be submitted to the Trust for 
information or approval as required.  
 
11.3 
Investigators' responsibilities 
 

 
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The investigators will be required to ensure that the local research approvals have been obtained and 
that any contractual agreements required have been signed off by all parties before recruiting any 
participants.  Investigators will be required to ensure compliance to the protocol and study manual 
and with completion of the CRFs.  Investigators will be required to allow access to study 
documentation or source data on request for monitoring visits and audits performed by the Sponsor 
or any regulatory authorities. 
 
Investigators will be required to read, acknowledge and inform their trial team of any amendments to 
the trial documents approved the REC that they receive and ensure that the changes are complied 
with.  
 
11.4 
Monitoring by sponsor 
 
The study will be monitored and audited in accordance with the University of Leicester policy which is 
consistent with the Research Governance Framework.  All trial related documents will be made 
available on request for monitoring and audit by University of Leicester, the relevant REC and for 
inspection by the Human Tissue Authority (HTA) or other licensing bodies. 
 
11.5 
Indemnity 
 
University of Leicester insurance will cover potential legal liability of the sponsor from harm to 
participants arising from the management and design of the research. NHS indemnity will cover 
potential legal liability of investigators/collaborators arising from harm to participants in the conduct 
of the research. 
 
11.6 
Clinical Trial Authorisation 
 
This is not a randomised trial of an Investigational Medicinal Product and as such a Clinical Trial 
Authorisation from the MHRA is not required.  
 
 
12. 
Data Protection and Patient Confidentiality 
 
12.1 
Data protection 
 
Data will be collected and retained in accordance with the Data Protection Act 1998. 
 
12.2 
Data handling, storage and sharing 
 
12.2.1 Data handling 
 
Data will be entered onto a purpose designed database and data validation and cleaning will be carried 
out throughout the trial. Standard operating procedures (SOPs) for database use, data validation and 
data cleaning will be available and regularly maintained.   
 
12.2.2 Data storage 
 
All study documentation will be retained in a secure location during the conduct of the study. All the 
personal data will be stored for 3 years and all the anonymised data will be stored for 15 years after 
the end of the study, when all paper records will be destroyed by confidential means. In compliance 
with the MRC Policy on Data Preservation, the pseudonymised dataset, a separate secure electronic 
‘key’ with a unique patient identifier, and relevant ‘meta’-data about the trial will be retained in 

 
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electronic form indefinitely because of the potential for the raw data to be used subsequently for 
secondary research. 
 
12.2.3 Data sharing 
 
Data will not be made available for sharing until after publication of the main results of the study.  
Thereafter, anonymised individual patient data will be made available for secondary research, 
conditional on assurance from the secondary researcher that the proposed use of the data is 
compliant with the MRC Policy on Data Preservation and Sharing regarding scientific quality, ethical 
requirements and value for money.  A minimum requirement with respect to scientific quality will be 
a publicly available pre-specified protocol describing the purpose, methods and analysis of the 
secondary research, e.g. a protocol for a Cochrane systematic review.  The second file containing 
patient identifiers would be made available for record linkage or a similar purpose, subject to 
confirmation that the secondary research protocol has been approved by a UK REC or other similar, 
approved ethics review body. 
 
 
13. 
Dissemination of Findings 
 
The findings will be disseminated by usual academic channels, i.e. presentation at international 
meetings, as well as by peer-reviewed publications and through patient organisations and newsletters 
to patients, where available. 
 
 
 
 
 
 

 
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14. 
References 
 
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3. Mugford M, Elbourne D, Field D. Extracorporeal membrane oxygenation for severe respiratory 
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5. Bahrami KR, Van Meurs KP. ECMO for neonatal respiratory failure. Semin Perinatol. 2005 
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6. UK Collaborative ECMO Trail Group. UK collaborative randomised trial of neonatal extracorporeal 
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7. Gail M. Annich, William R Lynch, Graeme MacLaren, Jay M. Wilson, Robert H. Bartlett. ECMO 
Extrapulmonary Cardiopulmonary support in Critical Care 4th Edition. 2012 
8. Zwiers AJ, de Wildt SN, Hop WC, Dorresteijn EM, Gischler SJ, Tibboel D, Cransberg K. Acute kidney 
injury is a frequent complication in critically ill neonates receiving extracorporeal membrane 
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9. Extracorporeal Life Support Organization. ECLS Registry Report International Summary January 
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13. Bendapudi P, Rao GG, Greenough A. Diagnosis and management of persistent pulmonary 
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Versmold HT. Leukocyte and endothelial activation in a laboratory model of extracorporeal 
membrane oxygenation (ECMO). Pediatr Res. 2000 Nov;48(5):679-84. 
19. Golej J, Winter P, Schöffmann G, Kahlbacher H, Stoll E, Boigner H, Trittenwein G. Impact of 
extracorporeal membrane oxygenation modality on cytokine release during rescue from infant 
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responses to cardiopulmonary bypass in children weighing less than 10 kilograms. Ann Thorac Surg. 
1996 Aug;62(2):538-42. 
21. Kozik DJ1, Tweddell JS. Characterizing the inflammatory response to cardiopulmonary bypass in 
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22. Day JR, Taylor KM. The systemic inflammatory response syndrome and cardiopulmonary bypass. 
Int J Surg. 2005;3(2):129-40 
23. Warren OJ1, Smith AJ, Alexiou C, Rogers PL, Jawad N, Vincent C, Darzi AW, Athanasiou T. The 
inflammatory response to cardiopulmonary bypass: part 1--mechanisms of pathogenesis. J 
Cardiothorac Vasc Anesth. 2009 Apr;23(2):223-31 
24. Warren OJ, Watret AL, de Wit KL, Alexiou C, Vincent C, Darzi AW, Athanasiou T. The 
inflammatory response to cardiopulmonary bypass: part 2--anti-inflammatory therapeutic strategies. 
J Cardiothorac Vasc Anesth. 2009 Jun;23(3):384-93 
25. Williams DC, Turi JL, Hornik CP, Bonadonna DK, Williford WL, Walczak RJ, Watt KM, Cheifetz IM. 
Circuit oxygenator contributes to extracorporeal membrane oxygenation-induced haemolysis. ASAIO 
J. 2015 Mar-Apr;61(2):190-5 
26. Omar HR, Mirsaeidi M, Socias S, Sprenker C, Caldeira C, Camporesi EM, Mangar D. Plasma Free 
Haemoglobin Is an Independent Predictor of Mortality among Patients on Extracorporeal Membrane 
Oxygenation Support. PLoS One. 2015 Apr 22;10(4):e0124034 
27. Lou S , MacLaren G, Best D, Delzoppo C, Butt W. Haemolysis in pediatric patients receiving 
centrifugal-pump extracorporeal membrane oxygenation: prevalence, risk factors, and outcomes. 
Crit Care Med. 2014 May;42(5):1213-20 
28. Lubnow M, Philipp A, Foltan M, Bull Enger T, Lunz D, Bein T, Haneya A, Schmid C, Riegger G, 
Müller T, Lehle K.Technical complications during veno-venous extracorporeal membrane 
oxygenation and their relevance predicting a system-exchange--retrospective analysis of 265 cases. 
PLoS One. 2014 Dec 2;9(12):e112316 
29. Maslach-Hubbard A, Bratton SL. Extracorporeal membrane oxygenation for pediatric respiratory 
failure: History, development and current status. World J Crit Care Med. 2013 Nov 4;2(4):29-39 
30. Toomasian JM, Bartlett RH. Haemolysis and ECMO pumps in the 21st Century. Perfusion. 2011 
Jan;26(1):5-6 
31.Smith A, McCulloh RJ. Hemopexin and haptoglobin: allies against heme toxicity from 
haemoglobin not contenders. Front Physiol. 2015 Jun 30;6:187 
32. Mamikonian LS, Mamo LB, Smith PB, Koo J, Lodge AJ, Turi JL. Cardiopulmonary bypass is 
associated with haemolysis and acute kidney injury in neonates, infants, and children. Pediatr Crit 
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33. Schaer DJ, Vinchi F, Ingoglia G, Tolosano E, Buehler PW. Haptoglobin, hemopexin, and related 
defense pathways-basic science, clinical perspectives, and drug development. Front Physiol. 2014 
Oct 28;5:415.  
34. Hanssen SJ, van de Poll MC, Houben AJ, Windsant IC, Snoeijs MG, Bekers O, Buurman WA, Jacobs 
MJ. Haemolysis compromises nitric oxide-dependent vasodilatory responses in patients undergoing 
major cardiovascular surgery. Thorac Cardiovasc Surg. 2012 Jun;60(4):255-61 
35. Rother RP, Bell L, Hillmen P, Gladwin MT. The clinical sequelae of intravascular haemolysis and 
extracellular plasma haemoglobin: a novel mechanism of human disease. JAMA. 2005 Apr 
6;293(13):1653-62. 
36. Vermeulen Windsant IC, de Wit NC, Sertorio JT, van Bijnen AA, Ganushchak YM, Heijmans JH, 
Tanus-Santos JE, Jacobs MJ, Maessen JG, Buurman WA. Haemolysis during cardiac surgery is 
associated with increased intravascular nitric oxide consumption and perioperative kidney and 
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37. Vermeulen Windsant IC, Hanssen SJ, Buurman WA, Jacobs MJ. Cardiovascular surgery and organ 
damage: time to reconsider the role of haemolysis. J Thorac Cardiovasc Surg. 2011 Jul;142(1):1-11. 
38. Haase M, Bellomo R, Haase-Fielitz A. Novel biomarkers, oxidative stress, and the role of labile 
iron toxicity in cardiopulmonary bypass-associated acute kidney injury. J Am Coll Cardiol. 2010 May 
11;55(19):2024-33. 
39. Irwin DC, Baek JH, Hassell K, Nuss R, Eigenberger P, Lisk C, Loomis Z, Maltzahn J, Stenmark KR, 
Nozik-Grayck E, Buehler PW. Haemoglobin-induced lung vascular oxidation, inflammation, and 
remodeling contribute to the progression of hypoxic pulmonary hypertension and is attenuated in 
rats with repeated-dose haptoglobin administration. Free Radic Biol Med. 2015 May;82:50-62 
40. Brittain EL, Janz DR, Austin ED, Bastarache JA, Wheeler LA, Ware LB, Hemnes AR. Elevation of 
plasma cell-free haemoglobin in pulmonary arterial hypertension. Chest. 2014 Dec;146(6):1478-85 
41. Buehler PW, Baek JH, Lisk C, Connor I, Sullivan T, Kominsky D, Majka S, Stenmark KR, Nozik-
Grayck E, Bonaventura J, Irwin DC. Free haemoglobin induction of pulmonary vascular disease: 
evidence for an inflammatory mechanism. Am J Physiol Lung Cell Mol Physiol. 2012 Aug 
15;303(4):L312-26 
42. REDWASH: a randomised controlled trial of red cell washing for the attenuation of transfusion 
associated organ injury in cardiac surgery. The REDWASH Trial: REC number 12/EM/0475 - ISRCTN 
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43. Meyer AD, Gelfond JA, Wiles AA, Freishtat RJ, Rais-Bahrami K. Platelet-derived microparticles 
generated by neonatal extracorporeal membrane oxygenation systems. ASAIO J. 2015 Jan-
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44. Nascimbene A, Hernandez R, George JK, Parker A, Bergeron AL, Pradhan S, Vijayan KV, Civitello A, 
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cell-derived microparticles and adverse events in patients with nonpulsatile left ventricular assist 
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45. Chung J, Suzuki H, Tabuchi N, Sato K, Shibamiya A, Koyama T. Identification of tissue factor and 
platelet-derived particles on leukocytes during cardiopulmonary bypass by flow cytometry and 
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46. Fu L, Hu XX, Lin ZB, Chang FJ, Ou ZJ, Wang ZP, Ou JS. Circulating microparticles from patients with 
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47. Nieuwland R, Berckmans RJ, Rotteveel-Eijkman RC, Maquelin KN, Roozendaal KJ, Jansen PG, ten 
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48. Fontaine D, Pradier O, Hacquebard M, Stefanidis C, Carpentier Y, de Canniere D, Fontaine J, 
Berkenboom G. Oxidative stress produced by circulating microparticles in on-pump but not in off-
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49. Biró E1, Sturk-Maquelin KN, Vogel GM, Meuleman DG, Smit MJ, Hack CE, Sturk A, Nieuwland R. 
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50. Larson MC, Hillery CA, Hogg N. Circulating membrane-derived microvesicles in redox biology. 
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51. Piccin A, Murphy WG, Smith OP. Circulating microparticles: pathophysiology and clinical 
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52. Lovren F, Verma S. Evolving role of microparticles in the pathophysiology of endothelial 
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53.Yong PJ, Koh CH, Shim WS. Endothelial microparticles: missing link in endothelial dysfunction? 
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54. Fanaroff AA. Meconium aspiration syndrome: historical aspects. J Perinatol. 2008 Dec;28 Suppl 
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55. Improving Global Outcomes (KDIGO) Acute Kidney Injury Work Group. KDIGO Clinical Practice 
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56. Akcan-Arikan A, Zappitelli M, Loftis LL, Washburn KK, Jefferson LS, Goldstein SL. Modified RIFLE 
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57. HowieBlood SRC. Blood sample volumes in child health research: review of safe limits Bull World 
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58. Extracorporeal Life Support Organization. ELSO Registry Report – International Summary. Ann 
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59. Modi N, Vohra J, Preston J, Elliott C, Van't Hoff W, Coad J, Gibson F, Partridge L, Brierley J, 
Larcher V, Greenough A; Working Party of the Royal College of Paediatrics and Child Health. 
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60. Brierley J, Larcher V. Emergency research in children: options for ethical recruitment. J Med 
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61. Marc-Aurele KL, Steinman SL, Ransom KM, Finer NN, Dunn LB. Evaluation of the content and 
process of informed consent discussions for neonatal research. J Empir Res Hum Res Ethics. 2012 
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62. Joffe S, Cook EF, Cleary PD, Clark JW, Weeks JC. Quality of informed consent: a new measure of 
understanding among research subjects. J Natl Cancer Inst. 2001 Jan 17;93(2):139-47 
63. Acute Kidney Injury Following Paediatric Cardiac Surgery, the p-MiVAKI Study: REC number 
14/EM/1136 
 
 
 
 

 
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15. 
Protocol Signature Page 
 
 
 
Study Title: 
 
 
Study Identifier: 
 
Protocol Version: 
 
Details of Sponsor: 
 
University of Leicester  
Research Support Office 
University Road 
Leicester, LE1 7RH 
 
Tel: 0116 252 2759 
Fax: 0116 252 2028 
Email: uolsponsor@le.ac.uk 
Chief Investigator: 
(Specify Name, Institution, 
Address) 
Professor Gavin Murphy 
Cardiovascular Sciences 
University of Leicester 
Glenfield Hospital 
Clinical Sciences Wing 
Groby Road 
Leicester, LE3 9QP 
 
 
 
 
Chief Investigator Agreement:  
I, the undersigned, have reviewed this protocol and agree to conduct the study in accordance with 
Good Clinical Practice, the ethical principles set forth in the Declaration of Helsinki, The Medicines for 
Human Use Regulations and the applicable Local Research Governance Policies. 
 
             Printed Name:  
 
         Signature:  
 
 
Date: 
 
 
_______________________               _______________________ 
_________________ 
 
 
Sponsor's Representative  Agreement:  
I, the undersigned, have reviewed this protocol and agree to conduct the study in accordance with 
Good Clinical Practice, the ethical principles set forth in the Declaration of Helsinki, The Medicines for 
Human Use Regulations and the applicable Local Research Governance Policies. 
 
             Printed Name:  
 
         Signature:  
 
 
Date: 
 
 
_______________________               _______________________ 
_________________ 
 
 
 
 

 
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16. 
Appendix 1 
Mi-ECMO QUESTIONNAIRE 
When you signed the consent form to allow your baby to participate in the study, how well 
did you understand the following aspects of the study? If you didn’t understand the item at 
all, please circle 1. If you understood it very well, please circle 5. If you understood it 
somewhat, please circle a number between 1 and 5 
 
I didn’t 
understand 
this at all 
 
I understood 
this very 
well 
The fact that your baby’s treatment involves research 
1 
2 
3 
4 
5 
What the researchers are trying to find out in the study 
1 
2 
3 
4 
5 
How long your baby will be in the study 
1 
2 
3 
4 
5 
The procedures your baby will undergo 
1 
2 
3 
4 
5 
The possible disadvantages of participating in the study 
1 
2 
3 
4 
5 
How participation in this study may benefit future 
babies 
1 
2 
3 
4 
5 
The effect of the study on the confidentiality of your 
baby’s medical records 
1 
2 
3 
4 
5 
Whom you should contact if you have questions or 
concerns about the study 
1 
2 
3 
4 
5 
The fact that participation in the study is voluntary 
1 
2 
3 
4 
5 
The fact that you can withdraw your baby from the 
study any moment 
1 
2 
3 
4 
5 
Overall, how well did you understand the study when 
you signed the consent form? 
1 
2 
3 
4 
5