A Multi-centre Randomised Double Blinded Phase III Trial of the Effect of Standard Issue Red Blood Cell Blood Units on Mortality Compared to Freshest Available Red Blood Cell Units
Phase: PHASE3
Conditions: Transfusion, Age of Blood
Sponsor: Australian and New Zealand Intensive Care Research Centre

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CONFIDENTIAL 
 
 
 
 
 
 
 
TRANSFUSE  
STandaRd Issue TrANsfusion versuS Fresher red blood 
cell Use in intenSive carE– a randomised controlled trial. 
 
A multi-centre randomised double blinded phase III trial of the effect of standard 
issue red blood cell blood units on mortality compared to freshest available red 
blood cell units  
 
 
Chief Investigator: 
 
Prof Jamie Cooper  
 
 
 
 
 
The Australian & New Zealand Intensive Care Research Centre   
Department of Epidemiology and Preventive Medicine 
School of Public Health and Preventive Medicine, Monash University  
The Alfred Centre, 99 Commercial Road,  
Melbourne, Victoria, 3004 
AUSTRALIA 
Phone: +61 3 9903 0343 
Fax: +61 3 9903 0071 
Jamie.cooper@monash.edu 
 
 
Coordinating Centre:  
 
The Australian & New Zealand Intensive Care Research Centre  
Department of Epidemiology and Preventive Medicine 
School of Public Health and Preventive Medicine, Monash University  
The Alfred Centre, 99 Commercial Road,  
Melbourne, Victoria, 3004 
AUSTRALIA 
Phone: +61 3 9903 0247 
Fax: +61 3 9903 0071 
anzicrc@med.monash.edu.au 
 
 

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TABLE OF CONTENTS 
ABBREVIATIONS ...................................................................................................................................................... 4 
STUDY ADMINISTRATION STRUCTURE ................................................................................................................... 6 
Coordinating Centre & Data Management Centre ............................................................................................. 6 
Responsibilities ......................................................................................................................................... 6 
Executive committee .......................................................................................................................................... 7 
Responsibilities ......................................................................................................................................... 7 
Members ................................................................................................................................................... 7 
Management Committee ................................................................................................................................... 7 
Responsibilities ......................................................................................................................................... 7 
Members ................................................................................................................................................... 7 
Steering Committee ............................................................................................................................................ 8 
Responsibilities ......................................................................................................................................... 8 
Members ................................................................................................................................................... 8 
Contact Details .................................................................................................................................................... 8 
Chief investigator ...................................................................................................................................... 8 
Coordinating centre .................................................................................................................................. 9 
Project manager ........................................................................................................................................ 9 
LAY DESCRIPTION.................................................................................................................................................. 12 
BACKGROUND & RATIONALE................................................................................................................................ 13 
Clinical and Scientific Rationale ........................................................................................................................ 13 
OBJECTIVES ........................................................................................................................................................... 15 
Aim .................................................................................................................................................................... 15 
Hypothesis ........................................................................................................................................................ 15 
STUDY OUTCOME MEASURES .............................................................................................................................. 15 
Primary outcome .............................................................................................................................................. 15 
Secondary outcomes ........................................................................................................................................ 15 
OVERALL STUDY DESIGN ....................................................................................................................................... 15 
Study design...................................................................................................................................................... 15 
Study treatments .............................................................................................................................................. 15 
Study population .............................................................................................................................................. 15 
Inclusion criteria ............................................................................................................................................... 16 
Exclusion criteria ............................................................................................................................................... 16 
Co-enrolment .................................................................................................................................................... 16 
STUDY PROCEDURES ............................................................................................................................................. 16 
Eligible patients ................................................................................................................................................ 16 
Enrolment ......................................................................................................................................................... 16 
Randomisation .................................................................................................................................................. 16 
Blinding ............................................................................................................................................................. 17 
ETHICS ................................................................................................................................................................... 18 

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Guiding principles ............................................................................................................................................. 18 
Ethical issues of the study ................................................................................................................................ 18 
Ethics Committee approval .............................................................................................................................. 18 
Confidentiality of patient data.......................................................................................................................... 18 
Consent ............................................................................................................................................................. 18 
DATA MANAGEMENT ........................................................................................................................................... 19 
Data collection methods ................................................................................................................................... 19 
Data variables collected .................................................................................................................................... 19 
Data management ............................................................................................................................................ 20 
Data quality & monitoring ................................................................................................................................ 20 
STATISTICAL CONSIDERaTIONS ............................................................................................................................. 21 
Sample size calculation ..................................................................................................................................... 21 
Statistical and analytical plan ........................................................................................................................... 21 
Subgroup analyses ............................................................................................................................................ 21 
Interim analysis ................................................................................................................................................. 21 
SAFETY .................................................................................................................................................................. 22 
Data Safety Management Committee .............................................................................................................. 22 
Adverse events ................................................................................................................................................. 22 
Serious adverse events ..................................................................................................................................... 22 
Reporting .......................................................................................................................................................... 22 
FUNDING............................................................................................................................................................... 23 
PUBLICATION ........................................................................................................................................................ 23 
RESEARCH TIMELINES ........................................................................................................................................... 24 
APPENDIX 1 – SCHEDULE OF EVENTS ................................................................................................................... 25 
APPENDIX 2 – EURO QUALITY OF LIFE (EQ 5D) ..................................................................................................... 27 
APPENDIX 3 – THE IRON TRANSFUSE SUB STUDY (THE IT STUDY) ....................................................................... 28 
APPENDIX 4 - The RENALTRANSFUSE SUBSTUDY ................................................................................................. 34 
REFERENCES .......................................................................................................................................................... 37 
 
 
 
 

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ABBREVIATIONS 
 
2,3-DPG 
2,3-diphosphoglycerate 
ANZ  
Australia and New Zealand  
ANZIC RC 
Australia & New Zealand Intensive Care Research Centre 
ANZICS CTG 
Australian & New Zealand Intensive Care Society Clinical Trials Group 
APACHE III 
Acute Physiology and Chronic Health Evaluation 
ARBCS  
Australian Red Blood Cross Service  
ATP 
Adenosine triphosphate  
CIDMU  
Clinical Informatics and Data Management Unit  
CPMP 
Committee for proprietary medicinal products 
CRF 
Case report form 
DEPM 
Department of Epidemiology & Preventive Medicine 
DSMC 
Data & Safety Monitoring Committee 
FNHTR 
Febrile Non-Haemolytic Transfusion Reaction 
GCP 
Good clinical practice 
ICH 
International conference on harmonisation 
ICU 
Intensive care unit  
LOS 
Length of stay 
MV 
Mechanical ventilation 
NHMRC 
National Health and Medical Research Council 
POD 
Persistent Organ Dysfunction 
RBC 
Red Blood Cells 
RC 
Research Coordinator 
RCT 
Randomised Controlled Trial 
RRT 
Renal Replacement Therapy 
SOFA 
Sequential Organ Failure Assessment 
TRALI 
Transfusion Related Acute Lung Injury 
 
 

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SYNOPSIS 
Background 
Red blood cell (RBC) transfusion is a very common and potentially life-saving 
treatment in intensive care units (ICUs). However, RBC transfusion has also 
been associated with an increased risk of morbidity and/or mortality in 
critically ill, surgical and trauma patients. Although this association is 
multifactorial, attention has increasingly focused on the possible adverse 
impact of transfusing RBC which have been stored for a prolonged time. 
Aim 
The primary aim of the trial is to determine if transfusion of the freshest 
available RBC in critically ill patients compared to standard care decreases 
patient mortality.  
 
Design 
Multi-centre, randomised, double blinded, phase III trial 
Patient population 
Intensive care patients who require a RBC transfusion 
Sample size 
5000 
Methods 
Eligible patients who require a blood transfusion will be randomised by the 
ICU team using a web based system. The study number generated at 
randomisation will be conveyed to the transfusion service by telephone and 
on the blood request slip. The hospital transfusion service will allocate the 
appropriate blood to the patient as designated by a randomisation schedule. 
Allocation will be either standard practice (the oldest compatible available 
blood, front of fridge) or freshest compatible available (back of fridge). The 
patient will continue to receive the randomised treatment arm for any 
additional blood transfusion episodes during their hospital stay. A telephone 
follow-up will be conducted at 90 and 180days. 
 
Outcomes 
Primary Endpoint:  
 
90 day mortality  
 
Secondary Endpoints:  
 
28 day mortality 
 
Persistent Organ dysfunction combined with death at day 28 
 
Days alive and free of mechanical ventilation (post randomisation) 
at day 28 
 
Days alive and free of RRT post randomisation at day 28 
 
Blood stream infection rate in ICU, post randomisation 
 
Length of stay in ICU and in hospital post randomisation 
 
Proportion of patients who suffer at least one febrile non-
haemolytic transfusion reaction in ICU  
 
Quality of life (EQ5D) at 180 days 
 
 
 

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STUDY ADMINISTRATION STRUCTURE 
 
Coordinating Centre & Data Management Centre 
Responsibilities 
 
 
overall management of the study including assistance with HREC applications 
 
management of study budget and liaison with funding bodies 
 
protocol and case report form (CRF) design and production 
 
database design and management 
 
protocol training of research coordinators and TRANSFUSE study team 
 
preparation and arrangement of investigator payments 
 
study set-up 
 
randomisation  
 
coordination of data entry and feedback of data enquiries 
 
monitoring and close-out site visits 
 
organisation of investigator meetings 
 
serious adverse event notification 
 
data analysis and collaboration on publications 

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Executive committee 
Responsibilities 
 
Day to Day study management 
 
Liaise with Management Committee and sites  
Members 
Ms 
Bridget 
Ady 
TRANSFUSE Project Manager 
ANZIC-RC, Monash University 
Dr 
Cecile 
Aubron 
Adjunct Senior Research Fellow 
ANZIC-RC, Monash University 
Prof 
Jamie  
Cooper 
Co-Director 
ANZIC-RC, Monash University 
Dr 
Craig 
French 
ICU Director 
Western Hospital, Intensive Care Unit 
Dr 
Dashiell 
Gantner 
Senior Research Fellow 
ANZIC-RC, Monash University 
Dr 
Maija 
Kaukonen 
Senior Research Fellow 
ANZIC-RC, Monash University 
Mrs 
Lynne 
Murray 
Research Manager 
ANZIC-RC, Monash University 
Dr 
Alistair 
Nichol 
Assoc/Professor 
ANZIC-RC, Monash University 
Dr 
Ville 
Pettila 
ICU Director 
Helsinki University Meilahti Hospital (Finland) 
Dr 
Zoe 
McQuilten 
Clinical Research Fellow 
Transfusion Outcome Research Collaborative, 
Monash University 
 
Management Committee 
Responsibilities 
Overseeing all aspects of the study management including  
 
liaison with coordinating centre staff 
 
liaison with steering committee 
 
liaison with ANZICS CTG  
 
overseeing funding applications 
 
overseeing disbursement & administration of funds 
 
ensuring fiscal responsibilities are maintained 
 
development and approval of final protocol & study materials 
 
development and approval of data collection tools and methods 
 
general study management issues 
Members 
 
Ms 
Bridget 
Ady 
TRANSFUSE Project Manager 
ANZIC-RC, Monash University 
Dr 
Cecile 
Aubron 
Adjunct Senior Research Fellow 
ANZIC-RC, Monash University 
Assoc/Prof 
Michael  
Bailey 
Senior Biostatistician 
ANZIC-RC, Monash University 
Prof 
Rinaldo 
Bellomo 
Co-Director 
ANZIC-RC, Monash University 
Mr 
Andrew 
Webb 
Senior Scientist  
The Alfred, Blood Bank 
Prof 
Jamie 
Cooper 
Co-Director 
ANZIC-RC, Monash University 
Dr 
Craig 
French 
ICU Director 
Western Hospital, Intensive Care Unit 
Dr 
Dashiell 
Gantner 
Senior Research Fellow 
ANZIC-RC, Monash University 

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Dr 
David 
Irving 
Executive Director, Research and 
Business Development 
Australian Red Cross Blood Service 
Dr 
Maija 
Kaukonen 
Senior Research Fellow 
ANZIC-RC, Monash University 
Dr 
Colin 
McArthur 
Clinical Director 
Auckland City Hospital NZ (Dept Critical 
Care Medicine) 
Dr 
Phillip 
Mondy 
Transfusion Medicine Specialist 
Australian Red Cross Blood Service 
Mrs 
Lynne 
Murray 
Research Manager 
ANZIC-RC, Monash University 
Dr 
Alistair 
Nichol 
Assoc/Professor 
ANZIC-RC, Monash University 
Dr 
Neil 
Orford 
ICU Director 
The Geelong Hospital, Barwon Health 
Dr 
Ville 
Pettila 
ICU Director 
Helsinki University Meilahti Hospital 
(Finland) 
Dr 
Louise 
Phillips 
Head, Transfusion Research Unit 
DEPM, Monash University 
Dr 
Zoe 
McQuilten 
Clinical Research Fellow 
Transfusion Outcomes Research 
Collaborative, Monash University 
Dr 
Jeff 
Presneill 
Deputy Director Campus Wide Adult ICU 
Mater Health Services 
Prof 
Michael  
Reade 
Defence Professor of Military Medicine & 
Surgery 
Royal Brisbane & Women's Hospital 
Ms 
Shirley 
Vallance 
Research Manager 
The Alfred, Intensive Care Research 
 
Steering Committee 
Responsibilities 
 
Oversight and advisory role 
 
Data analysis, collaboration and approval of study publications 
Members 
 
Management committee (as above) 
 
Associate site investigators (4 from each site – suggested 1 Intensive Care Physician, 1 Transfusion 
Medicine Specialist, 1 Research Coordinator, 1 Blood Bank Scientist) 
Contact Details 
Chief investigator 
 
Professor Jamie Cooper 
 
Director Research, Intensive Care Unit 
The Alfred 
Melbourne 
Victoria 3004, Australia 
Ph: 
+61 3 99030343 
Fax: 
+61 3 90763780 
J.cooper@alfred.org.au 
 

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Coordinating centre 
 
ANZIC RC 
DEPM, School of Public Health and Preventive Medicine  
Monash University, The Alfred, Commercial Road,  
Melbourne VIC 3004, Australia  
 
Project manager 
 
Bridget Ady 
Research Fellow 
ANZIC RC 
DEPM, School of Public Health and Preventive Medicine  
Monash University, The Alfred, Commercial Road,  
Melbourne VIC 3004, Australia  
 
Tel 
+61 3 9903 0035 
Fax 
+61 3 9903 0071 
Mobile  +61 400 504 164 
E-mail 
Bridget.ady@monash.edu 
 

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MANAGEMENT COMMITTEE AUTHORISATION PAGE 
We the management committee have read the attached protocol and authorize it as the official protocol for 
the study entitled “A multi-centre randomised double blinded phase III trial of the effect of standard issue red 
blood cell blood units on mortality compared to freshest available red blood cell units”  
 
Management Committee 
 
Date 
 
Bridget Ady 
 
 
Management Committee 
 
Date 
 
Cecile Aubron 
 
 
Management Committee 
 
Date 
 
Michael Bailey 
 
 
Management Committee 
 
Date 
 
Rinaldo Bellomo 
 
 
Management Committee 
 
Date 
 
Andrew Webb 
 
 
Chief Investigator 
 
Date 
 
Jamie Cooper 
 
 
Management Committee 
 
Date 
 
Craig French 
 
 
Management Committee 
 
Date 
 
Dashiell Gantner 
 
 
Management Committee 
 
Date 
 
David Irving 
 
 
Management Committee 
 
Date 
 
Maija Kaukonen 
 
 

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Management 
Committee 
 
Date 
 
Colin McArthur 
 
 
Management 
Committee 
 
Date 
 
Phillip Mondy 
 
 
Management 
Committee 
 
Date 
 
Lynne Murray 
 
 
Management 
Committee 
 
 
Date 
 
Alistair Nichol 
 
 
Management 
Committee 
 
Date 
 
Neil Orford 
 
 
Management 
Committee 
 
Date 
 
Ville Pettila 
 
 
Management 
Committee 
 
Date 
 
Louise Phillips 
 
 
Management 
Committee 
 
Date 
 
Zoe McQuilten 
 
 
Management 
Committee 
 
Date 
 
Jeff Presneil 
 
 
Management 
Committee 
 
Date 
 
Shirley Vallance 
 
 

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LAY DESCRIPTION 
In Australia, blood for transfusion has a “use by” date of 42 days after collection. The actual age of blood given 
to patients depends on what is available at the time and the rate of usage.  During the last decade, it has been 
reported that blood transfusion in patients admitted to intensive care was associated with an independent 
increase in mortality. Some research suggests that transfusion of fresher blood might help patients in the 
intensive care unit to reach a better recovery. This project will test whether patients who receive ‘fresher’ 
blood do better than patients who receive ‘standard issue’ blood. 
 

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BACKGROUND & RATIONALE 
Clinical and Scientific Rationale 
Anaemia is common in critically ill patients: up to 90% of patients will be anaemic by day three of their 
intensive care unit (ICU) stay [1]. The RBC transfusion rate in critically ill patients is reported between 20-40% 
in ICU [1-3] , with a mean of 2 to 4.8 RBC units transfused per patient [2, 4]. In Australia, a mean of 786,097 
RBC units per year were delivered by the Blood Service between 2007 and 2010 [5]. Approximately 18% of 
these (~140,000) are administered to critically ill patients. While potentially life-saving for individual patients 
RBC transfusion has also been associated with an increased risk of morbidity and/or mortality in critically ill, 
surgical and trauma populations [6, 7]. Literature reports have increasingly focused the attention on the 
possible harmful impact of RBC storage on the outcome of ICU patients [8-11]. 
 
Storage Lesion 
Storage of RBCs in a preservative medium is associated with metabolic, biochemical and molecular changes to 
erythrocytes commonly referred to as “storage lesion” [9, 12]. These biochemical and biomechanical changes 
include: depletion of adenosine triphosphate (ATP) and of 2,3-diphosphoglycerate (2,3-DPG), membrane 
phospholipid vesiculation, protein oxidation and lipid peroxidation of RBC membrane, and loss of deformability 
[9, 12, 13]. Bioreactive substances (lipids and cytokines) accumulate during storage [12]. Over time, RBC shape 
changes with increased osmotic fragility and loss of deformability [12, 14]. Decreased membrane flexibility 
may compromise microcirculatory flow and leads to increased red cell-endothelial cell interaction, with 
activation of inflammation. Other consequences of the “storage lesion” include: alteration of oxygen delivery 
(secondary to a decrease of 2,3-DPG and pH) and cell lysis [9]. In addition, haemolysis during RBC storage 
increases free iron, which is also bio-reactive [15]. Free haemoglobin also affects the bioactivity of nitric oxide 
(NO) leading to endothelial dysfunction, intravascular thrombosis, vasoconstriction and leukocyte adhesion 
[16]. Recently, a study pointed out the higher extravascular haemolysis after old blood transfusion (storage of 
40 to 42 days) compared to fresh blood (storage of 3 to 7 days) in healthy volunteer and the possible harmful 
effect of iron delivery [17]. White blood cells may participate in the storage lesion, but do not fully explain 
it[12].  
The severity of illness of a blood transfusion recipient may further increase their susceptibility to deleterious 
effects of RBC storage. Critically ill patients frequently have disease states leading to impaired microcirculatory 
blood flow. In addition, patient neutrophils may first be primed by an event (e.g. sepsis or trauma) and then 
subsequent exposure to bio-reactive substances in a RBC unit may initiate enhanced activation of adherent 
leucocytes. This hypothetical “two-hit” model was confirmed during transfusion of aged RBC to 
lipopolysaccharide-pre-treated rats. Lung injury was more severe when compared with transfusion with fresh 
RBC in another group of pre-treated animals or with transfusion of aged RBC in healthy rats [18]. These data 
support the hypothesis that critically ill patients may be particularly sensitive to the adverse effects of the 
storage lesion (i.e. older transfused RBC) and that the transfusion of fresher blood may decrease mortality.  
 
Clinical significance 
Studies in trauma, ICU and patients undergoing cardiac surgery have demonstrated adverse outcomes 
potentially attributable to storage lesion. In 1993, Marik et al. demonstrated an inverse association between 
change in gastric intramucosal pH and age of the transfused blood and concluded that patients receiving old 
RBC developed splanchnic ischemia[19]. More recently, a prospective 66 patient study showed that 
transfusion of blood stored for more than 19 days failed to increase brain oxygenation, whereas the freshest 
RBC were effective[20]. Six, single centre retrospective observational studies conducted in trauma patients, 
and one in critically ill patients reported clinical adverse events related to RBC storage. Adverse events 
included multiple organ failure, increased ICU and hospital length of stay, deep vein thrombosis, nosocomial 
infections and mortality [21-27]. However, these studies are of insufficient quality to confirm the impact of 
duration of RBC storage (currently recommended up to 42 days) on RBC adverse effects, or to change current 
transfusion practice.  

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In the last two years, 6 large observational studies have shown that older compared to fresher blood increased 
patient mortality and morbidity [28, 29] [30] [31] [32]. Koch et al. reported prolonged mechanical ventilation 
(9.7% versus 5.6%, p<0.001) and an increased incidence of sepsis (4% versus 2.8%, p=0.01) and mortality in 
6,002 cardiothoracic patients. Transfusion of older RBCs was also independently associated with an increased 
risk-adjusted rate of a composite of serious adverse events (25.9% versus 22.4%, p=0.001)[28]. A Canadian 
prospective observational study in 4,933 acute care cardiovascular patients reported an adjusted relative risk 
for death of 1.48 (95% CI 1.07 to 2.05) for patients in the highest quartile of maximum age of blood compared 
with those in the lowest quartile [29]. A retrospective observational study conducted in 1,478 post cardiac 
surgery patients showed an increase risk of infections when the patients were transfused with blood older 
than 14 days [32]. Two prospective paediatric ICU studies, in 455 and 296 patients respectively, showed an 
independent association between duration of blood storage and morbidity [30, 31]. In 296 paediatric ICU 
patients, the odds ratio for development of new or progressive multiple organ dysfunction syndrome in 
patients receiving older blood (stored for more than 14 days) was 1.87 (95% CI 1.04 to 3.27, p=0.03) after 
correction for confounding variables. Patients receiving older blood (≥14 days) had a longer ICU length of stay 
(9.9±8.3 versus 14.0±10.4 days, p<0.0001)[31]. Similar results were reported by Gauvin et al. Furthermore, 
these authors found that a RBC storage time of more than 21 days compared to less than 21 days was 
associated with higher mortality (9.2% versus 3.8%, p<0.025)[30]. In 2008 we conducted a prospective 
observational study to assess any association between age of transfused RBC and outcome in 757 ANZ ICU 
patients. After adjustment for confounding factors including APACHE III score, number of transfusions, pre-ICU 
transfusions, fresh frozen plasma and platelet transfusions, leucodepletion status, pre-transfusion 
haemoglobin concentration, clustering of study sites, and cardiac surgery, the administration of “fresher” RBC 
was independently associated with decreased mortality in a heterogeneous group of critically ill patients: odds 
ratio for hospital mortality of the older three quartiles compared to the freshest quartile 2.01% (95% CI, 1.07 
to 3.77) [3]. 
However some investigators have not demonstrated any association between RBC storage lesion and clinical 
outcomes [33-39]. Vlaar et al. recently reported that storage of RBC did not influence TRALI occurrence [40]. In 
contrast to other studies of cardiac surgical patients a recent large retrospective study did not demonstrate 
any association between storage lesion and mortality [36].  
Most of the clinical studies in this area are observational: they are mostly retrospective, small, and subject to 
bias. Only two small RCTs have been performed: they were very small and under-powered to answer the 
question [41, 42]. Accordingly, systematic reviews [8-10, 43] and a meta-analysis[44] have been inconclusive.  
The impact of storage lesion on clinical outcomes remains uncertain and no definitive conclusions may be 
made. The available literature supports further research to determine the effect of storage lesion on clinical 
outcome in critically ill patients. 
Our study design does not use an arbitrary cut-off age for defining the youngest RBC, as others have 
proposed[45] [41],[46], but instead uses the “freshest available” RBC versus standard care. This design best 
reflects the clinical practice change which would be practical to implement if fresher blood was beneficial in 
ICU patients. Importantly if we find no difference between the 2 treatment arms, such practice change would 
not be necessary.  
The pragmatic design of the 5,000 patient TRANSFUSE-RCT expands and complements two current 
international studies.  ABLE (n=2500), a Canadian multicentre RCT will evaluate transfusion of RBC units stored 
for less than eight days compared to standard care in critically ill patients [45]. This design is logistically 
demanding and cannot translate to current practice where freshest available blood varies in age. With 2500 
patients ABLE is likely to be underpowered unless a much larger than anticipated treatment effect is observed. 
RECESS is a United States multicentre RCT comparing cardiac surgery patients transfused with leucodepleted 
RBC stored for 10 days or less, versus 21 days or more. The very low mortality in cardiac surgery patients 
requires a surrogate outcome (change in multiple organ dysfunction score); this reduces the clinical 
significance of any study outcome [46].  
Only a large multicentre RCT can address whether the age of RBC transfusion affects mortality. Accordingly the 
TRANSFUSE study will have important health policy implications regardless of the outcome, in Australia and 
internationally. 

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OBJECTIVES 
Aim 
This study aims to determine whether, compared to standard care, transfusion of the freshest available 
allogeneic RBC decreases mortality of patients admitted to ICU.   
Hypothesis 
In critically ill patients who require a RBC transfusion, compared to standard practice, the administration of the 
freshest available compatible RBC reduces 90-day patient mortality. 
STUDY OUTCOME MEASURES 
Primary outcome 
90 day mortality 
Secondary outcomes 
1. 28 day mortality  
2. Persistent Organ Dysfunction combined with death at 28 [47]  
3. Days alive and free of mechanical ventilation at day 28 post randomisation   
4. Day alive and free of renal replacement therapy at day 28 post randomisation 
5. Blood stream infection in ICU (post randomisation) 
6. Length of stay in ICU and in hospital post randomisation 
7. Proportion of patients who suffer at least one febrile non-haemolytic transfusion reaction in ICU 
8. EQ-5D score at day 180 post randomisation [48] 
OVERALL STUDY DESIGN  
Study design 
TRANSFUSE-RCT is a multi-centre, randomised, double blind, controlled trial, testing the effect of the freshest 
available RBC compared to standard practice, on mortality in critically ill patients who require RBC transfusion.   
Study treatments  
 
Freshest available blood group: These patients will receive the freshest available group-specific 
compatible RBC unit in the transfusion service.   
 
Standard care group: These patients will receive standard practice, which is the oldest available 
group-specific compatible RBC unit in the transfusion service.  
Study population 
ICU patients or emergency department patients accepted for ICU admission in participating hospitals who 
meet all inclusion criteria and have no exclusion criteria. The study will enrol 5000 patients, in approximately 
50 centres in Australia and New Zealand (ANZ). 
Our observational study indicated that the recruitment of 40 patients per week is feasible and conservative 
[3]. At such a rate study recruitment would be completed in approximately 2.5 years. Allowing for study set up 
(12 months), 90 day outcome assessments, monitoring and analysis, a study time period of 4 years is 
anticipated. 

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Inclusion criteria 
ICU patients* with an anticipated ICU stay of at least 24 hours, in whom the decision has been made by 
medical staff to transfuse at least one RBC unit. 
*A patient can be included into the study in the Emergency Department if the decision to admit the patient 
into the ICU has been taken. 
Exclusion criteria 
 Age younger than 18 
 Previous RBC transfusion during the current hospital admission (including transfusion in another 
hospital for transferred patients)  
 Diagnosis of transplantation or hematologic malignancy  
 Pregnancy 
 Cardiac surgery during the present hospital admission  
 Expected to die imminently (<24hrs)  
 The treating physician believes it is not in the best interest of the patient to be randomised in this trial. 
 Known objection to the administration of human blood products 
 Participation in a competing study (see below) 
Co-enrolment 
 
Co-enrolment into any RBC transfusion studies is not permitted  
 
TRANSFUSE patients cannot be co-enrolled into the POLAR, ARISE, TRISS, and IRONMAN studies.  
 
Co-enrolment in the ADRENAL, EPO-TBI, PHARLAP, and SPICE studies is permitted by the TRANSFUSE 
management committee 
 
Co-enrolment in single site studies is permitted (excluding blood transfusion studies) 
STUDY PROCEDURES 
Eligible patients  
Patients will be screened in the ICU and emergency department for their potential eligibility by the ICU 
research coordinator (RC) and clinicians. The ICU RC will place a sticker on the patient’s chart to assist the ICU 
team with identifying eligible patients when a blood transfusion is ordered.  
Enrolment 
The inclusion and exclusion criteria will be checked when a clinical decision is made to transfuse. Transfusion 
indication, timing and number of the RBC units will be determined by the treating clinicians. However, we 
recommend following the current ANZ transfusion guidelines for ICU patients [49].  
Randomisation 
Randomisation will be performed by the ICU research staff using a Web-based computer system. 
The patient will be allocated a unique study identification number at randomisation to identify their study 
involvement for the first and each subsequent RBC transfusion during their hospital stay. This number will be 
recorded on a sticker placed in the patient’s chart and also on the RBC request form sent to the hospital 
transfusion service. The treatment allocation will be determined using variable block randomisation in a 1:1 
ratio, stratified by centre.  

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The ICU staff (the physician or the nursing staff caring for the patient, or the research coordinator) will 
telephone the transfusion service to: 1) order the RBC units, 2) inform them of the patient’s study inclusion 
and unique study identification number. The hospital transfusion service will be given the randomisation 
schedule for their site and the allocated study number will be identified on the schedule and the appropriate 
group (freshest or standard care) will be allocated to the patient. 
For the freshest blood group, the transfusion service scientist will take the required number of compatible RBC 
units from the area of the fridge containing the freshest blood. For the standard care group, the transfusion 
service scientist will select the RBC units in accordance with the current practice and will take the required 
number of compatible RBC units from the area of the fridge containing the oldest blood. 
The blood scientist will flag the patient’s allocation in the transfusion service computer system to ensure they 
remain in the same study group throughout their primary hospital admission. In addition to this measure, 
while the patient is in ICU clinical staff will remind the transfusion service of the patient’s study enrolment 
when ordering RBC. For any subsequent transfusion episodes in the operating theatre or on the ward, the 
blood scientists will be reminded by the computer flag.  
At the time of the patient’s primary hospital discharge the ICU RC will notify the blood bank to remove the 
computer flag. Patients who are readmitted to hospital and receive further transfusion will receive standard 
issue RBC.  
Blinding 
Study group allocation will be concealed from the treating medical and nursing staff and the research 
coordinator.  
Only transfusion service staff will have access to patient allocation. The transfusion service staff will treat the 
randomisation schedule confidentially and will not reveal the study group to the research or clinical staff. In 
keeping with normal hospital protocol and transfusion practices, the RBC unit details including collection and 
expiry dates will be checked; however in ICU this will be performed by 2 nurses not involved in the direct care 
of the patient. One of the 2 non-bedside nurses will put the RBC unit in a bag with opaque panels to conceal 
the collection and expiry dates before commencing the transfusion. For those patients receiving transfusion in 
the operating theatre or ward following ICU discharge, RBC checking will be performed according to usual 
practice by treating staff. For the purposes of later data analysis, RBC collection and expiry dates will be 
obtained directly and sequentially from the ARBCS. Due to considerable variation in stock levels in the 
transfusion service, knowledge of these dates will not identify the study group. 
Our pilot study showed that clinical staff remained blind to treatment group, confirming the feasibility of the 
blinding procedures [50].       

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ETHICS 
Guiding principles 
This study is to be performed in accordance with the ethical principles of the Declaration of Helsinki (June 1964 
and amended 1975, 1983, 1989, 1996, 2000 and Note of Clarification 2002, 2004, 2008)[51], ICH GCP Notes for 
Guidance on Good Clinical Practice (CPMP/ICH/135/95) annotated with Therapeutic Goods Administration 
comments[52] and NHMRC National Statement on Ethical Conduct in Research Involving Humans (March 
2007)[53]  
Ethical issues of the study 
Patients will not have capacity to consent for themselves.  
Ethics Committee approval 
In Australia, this protocol will be submitted to a Human Research and Ethics Committee constituted according 
to the NHMRC National Statement on Ethical Conduct in Research Involving Humans (March 2007)[53] for 
each institution.  In New Zealand, this protocol will be submitted to the appropriate Health and Disability 
Ethics Committee, accredited by the Health Research Council and constituted in accordance with the 
Operational Standard for Ethics Committees March 2002. Approval of the protocol and related documents will 
be obtained prior to the start of the study at each site.  
It is the principal investigator’s responsibility to ensure that all conditions for approval of the study are met 
and that amendments to the protocol or serious adverse events are also reported to the HREC (or equivalent) 
as required by that committee. 
Confidentiality of patient data 
Patient’s will be randomised via a secure website and will be allocated a unique study number. The Research 
Coordinator will compile an enrolment log including the patient’s name, date of birth, hospital identification 
number, unique study number and date and time of randomization. Subsequent data will be identified by the 
unique study number only. The enrolment log and study data will be kept separately. Follow up details of the 
patient and their family will be collected including name, address and contact telephone numbers to allow the 
site research coordinator to conduct the telephone interview at 90 days. Follow up contact details will not be 
sent off site. All data collected in the follow up assessments will be identified by the unique study number 
only. Study data will be entered into a secure website managed by the Clinical Informatics and Data 
Management Unit (CIDMU, Monash University). No identifying data will be entered into the website. All 
contact details and study data will be kept in a locked office at both the study site and in a locked office at 
Monash. 
Consent 
The NHMRC National Statement on the Ethical Conduct of Research in Humans (March 2007)[53] 
acknowledges in Chapter 4.4 that research involving patients who are heavily dependent on medical care, such 
as the patients in this study, is necessary to assess and improve the efficacy and safety of interventions used in 
their treatment. Critically ill patients requiring blood transfusion are unlikely to be able to provide informed 
consent. This project involves emergency care research; recruitment has to be achieved rapidly as the 
treatment (RBC transfusion) is urgent. The Statement also acknowledges (4.4.6) that where the research 
involves emergency treatment a waiver for consent may be provided. Accordingly a waiver for consent is 
sought. The Statement also outlines the conditions when such a waiver may be granted (2.3.6).The reasons we 
believe such a waiver may be granted to this project include: it carries no more than low risk as defined by the 
National Statement (2.1.6 and 2.1.7); the potential benefits to society, irrespective of the result of the study, 
are significant; it is impractical to obtain consent given the emergency nature of the treatment; the study 
involves no investigational drug, both arms of treatment are within current practice, and there is no known 
reason for thinking the participants would not have consented if asked. As soon as practicable following 

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recruitment the participant and/or their legally authorised representative will be informed of the participant’s 
inclusion in the research and of the option to withdraw without any reduction in quality of care. If they choose 
to withdraw the patient, permission will be asked to use the data collected up to that time (4.4.13).   
Any interaction between research staff, participants and their person responsible will take into consideration 
the stress or emotional factors associated with critical illness and ensure that the dependency of potential 
participants and their relatives on medical personnel providing treatment does not compromise the freedom 
of the decision to continue participation (4.4.11). 
 
DATA MANAGEMENT 
Data collection methods 
All data will be collected by trained staff at each study site using a paper source document developed by the 
coordinating centre. Data will then be entered into a web database designed by the CIDMU. Data queries will 
be automatically generated via the electronic data collection database. 
Randomised patients will be followed up to death or 180 days post-randomisation. Data collection will be 
restricted primarily to those variables necessary to define clinical patient characteristics including: baseline 
demographics, primary diagnoses, physiological parameters, diagnostic interventions, therapeutic 
interventions and documentation of deaths and other adverse events.   
Patients and/or their legal surrogate will be asked to provide three possible points of contact (home and close 
family contact details) to the research staff prior to discharge. For patients discharged alive from ICU, follow-
up will be restricted to information concerning duration of hospital stay and vital status at hospital discharge, 
90 days and 180 days post randomisation. In addition, patients (or a proxy – generally a close family member) 
that are alive at 180 days after randomization will be interviewed by the site RC using a simple standardized 
structured telephone questionnaire to measure quality of life EQ5D [48].  
Data variables collected 
 
Demographic data: 
o 
Identity (initials, study number, date of birth, gender, postcode) 
o 
Blood group: A, B, O, AB, Rhesus status (positive or negative) 
o 
Alloantibodies (if present) 
o 
Co-morbidities: heart disease including angina, myocardial infraction and previous CABG, 
immuno-compromised 
o 
Ethnicity (for patients enrolled in New Zealand only) 
 
Hospital data 
o 
Hospital admission and discharge dates 
o 
Hospital discharge destination 
 
ICU data 
o 
ICU admission and discharge dates 
o 
Acute Physiology and Chronic Health Evaluation III [APACHE III] score and risk of death at ICU 
admission  
o 
Acute Physiology and Chronic Health Evaluation III [APACHE III] diagnosis code at admission 
o 
Current “unstable or active” heart disease –(cardiac arrest, arrhythmia, cardiogenic shock, 
angina, acute myocardial infarction) 

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o 
Sequential Organ Failure Assessment [SOFA] score at randomisation and daily during ICU 
admission 
o 
Haemoglobin at ICU admission 
o 
Requirement of invasive mechanical ventilation at randomisation 
o 
Duration of mechanical ventilation  
o 
Requirement of renal replacement therapy at randomisation 
o 
Duration of renal replacement therapy 
o 
Requirement of catecholamines at randomisation 
o 
Duration of catecholamine administration 
o 
Blood stream infection (date and pathogen)  
 
Transfusion information 
o 
Haemoglobin pre transfusion episode 
o 
Date and time of each RBC unit transfused 
o 
RBC Blood group (A,B, O, AB) / rhesus status (positive or negative)  
o 
Donation number (=pack number)  
o 
Fresh frozen plasma (number of units post randomisation) 
o 
Platelets (number of units post randomisation) 
o 
Blinding status (ie: units checked by non-beside nurse) 
o 
Adverse events including febrile non haemolytic reaction  
 
Vital status at 
o 
ICU discharge 
o 
Hospital discharge 
o 
28 days post randomisation 
o 
90 days post randomisation 
o 
180 days post randomisation 
 
Quality of life 
o 
180 day EQ5D score [48]  
Data management 
Data entry and data management will be coordinated by the Project Manager and the ANZIC-RC, including 
programming and data management support. 
Data quality & monitoring 
Several procedures to ensure data quality and protocol standardisation will help to minimise bias and to 
optimise data quality. These include: 
 
Start-up meeting for all research coordinators and investigators will be held prior to study 
commencement to ensure consistency in procedures; 
 
A detailed data dictionary will define the data to be collected on the case report form; 
 
CIDMU & the Project Manager will perform timely validation of data, queries and corrections.  

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The study will be monitored by quality control reviews of data queries and protocol violations. On site 
monitoring of data quality will be performed on 10% of the data. Additional onsite monitoring will be 
performed on a case by case basis if quality control issues are flagged by electronic review of data.  
STATISTICAL CONSIDERATIONS 
All statistical analyses will be conducted under the supervision of Michael Bailey at Monash University 
Department of Epidemiology and Preventive Medicine.  
Sample size calculation 
We estimated the baseline mortality using the prospective ANZ observational study data [3]. Over 5 weeks in 
2008, 757 patients were recruited. The exclusion criteria of TRANSFUSE leaves 45% recruitable patients. These 
patients had a hospital mortality of 25% and we conservatively estimate 90-day mortality to be 28%. Our 
sample size calculation is based on a 15% relative decrease in 90-day mortality, or an absolute decrease of 
4.2% from 28% to 23.8%. This effect size would be clinically highly relevant (Number needed to treat to save 
one life [NNT]=24). This relative decrease is much less than observed in the Koch study (30% RRR) [28], and in 
our observational study where we found a mortality of 21.3% in the older three quartile versus 13.2% in the 
freshest quartile (38% RRR) [3]. With a type I error of 0.05 and a type II error of 0.1 (power 90%), the required 
patient number is 2332 per group. According to our previous studies [54, 55], loss to follow up should not 
exceed 5%, giving an accurate number of 4898 patients which has been rounded up to 5000 patients. 
Statistical and analytical plan 
This study will be analysed on an intention-to-treat basis. Analysis will primarily be conducted using SAS 
version 9.2. (SAS Institute Inc., Cary, NC USA). Baseline comparisons will be performed using chi-square tests 
for equal proportion, student t-tests for continuously normally distributed variables and Wilcoxon rank sum 
tests otherwise. The primary outcome, 90 day all-cause mortality will be analysed using a Chi-square test 
statistic, with results reported as frequencies and percentages per arm. Adjustment for strata (centre) will be 
made using logistic regression for binomial outcomes and Cox Proportional Hazards regression for time to 
event data with results reported as Odds Ratios (95% CI) or Hazard ratios (95% CI) respectively. In the unlikely 
event that baseline imbalances are found to exist between treatment groups, sensitivity analysis will be 
performed with imbalanced variables included as covariates. A two-sided p-value of 0.05 will be considered to 
be statistically significant. Additionally a per protocol analysis will be undertaken to account for patients with 
alloantibodies for whom freshest blood is unavailable. A detailed statistical analysis plan will be published prior 
to analysis being performed. 
Subgroup analyses 
A priori subgroup analyses will include: 
 
Group O versus non group O 
 
Illness severity according to APACHE III and SOFA scores 
 
Blood < 8 days old  
Interim analysis 
One planned interim safety analysis will be performed by the independent data and safety monitoring 
committee (DSMC) at 50% patient enrolment. It will examine safety and efficacy with stopping rules to be 
determined by the DSMC at their first meeting and to be published in the statistical analysis plan. Additionally 
an independent statistician will assess treatment separation by reviewing the average age of blood in the 
freshest and standard issue groups. 
 

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SAFETY 
Data Safety Management Committee 
An independent Data and Safety and Monitoring Committee (DSMC), comprising experts in clinical trials, 
biostatistics, transfusion medicine and intensive care medicine will be established before patient enrolment to 
review all trial protocols. The DSMC will conduct one planned interim safety analysis at half (2500) of the 
specified overall (5000) patient recruitment target, and will assess the differential proportion of all-cause 
mortality between the two treatment groups (a safety assessment of the trial primary outcome) as well as the 
differential cumulative “serious adverse events” reports. The DSMC may, in its absolute discretion, request 
assessment of any other trial data at any time. 
Adverse events 
Defined adverse events will be collected. Any undefined adverse events will be collected as free text bearing in 
mind that it is recognised that the patient population with critical illness and requiring RBC transfusions will 
experience a number of common aberrations in laboratory values, signs and symptoms due to the severity of 
the underlying disease and the impact of standard therapies. These will not necessarily constitute adverse 
events unless they are considered to be causally related to the study intervention or are otherwise considered 
to be of concern in the investigator’s judgement.  
Serious adverse events 
The baseline mortality of intensive care patients enrolled in trials will be high due to the critical illness that has 
necessitated their ICU admission. They will frequently develop life-threatening organ failure(s) unrelated to 
study interventions and despite optimal management. Therefore, consistent with the advice of Cook et al[56], 
events that are part of the natural history of the primary disease process or expected complications of critical 
illness will not be reported as serious adverse events in this trial. Additionally, events already defined and 
reported as study outcomes (total mortality, febrile non-haemolytic transfusion reactions, bacteraemia, need 
for mechanical ventilation or renal replacement therapy) will not be labelled and reported separately as 
adverse events or SAEs unless they are considered to be causally related to the study intervention or are 
otherwise of concern in the investigator’s judgement. 
Reporting 
SAEs will be recorded on separate case report forms. SAEs which occur from the time of commencement of 
study treatment to 90 days post randomisation will be reported to the coordinating centre (ANZIC-RC) by 
faxing the provided SAE form. SAEs should be reported to the ANZIC-RC within 24 hours of study staff 
becoming aware of the event. Minimum information to report will include: 
 
Patient initials and study number 
 
Nature of the event 
 
Commencement and cessation of the event 
 
An investigator’s opinion of the relationship between study involvement and the event (unrelated, 
possibly, probably or definitely related). 
 
Whether treatment was required for the event and what treatment was administered. 
 
SAEs must be reported on the fax form. Follow up reporting may be required. The SAE reports will then be 
forwarded to all sites for reporting to their HRECs in accordance with local requirements.  
 
Attention: 
Bridget Ady 
Transfuse Project Manager 
Fax number for SAE Reporting:+ 61 3 9903 0035 
Telephone Numbers:+61 3 9903 0071 

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FUNDING 
The TRANSFUSE RCT is funded by a project grant from the National Health and Medical Research Council 
(NHMRC#1020694) and by the Australian Red Cross Blood Service. The ANZIC-RC will supply infrastructure and 
administrative support. 
 
PUBLICATION 
The study will be conducted in the name of the TRANSFUSE investigators, the ANZIC RC and the ANZICS CTG.  
The central project coordination and data management will be provided by the ANZIC RC at Monash 
University, Melbourne. The principal publication from the study will be in the name of the TRANSFUSE 
Investigators with full credit assigned to all collaborating investigators, research coordinators and institutions.  
Where an individuals’ name is required for publication it will be that of the writing committee, with the chair 
of the writing committee listed first and subsequent authors listed alphabetically. 
Funding bodies will be acknowledged in the publication. 
 

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RESEARCH TIMELINES 
Time frame indicator 
Milestone 
February 2012 
Appoint Project Manager 
March 2012 
Protocol & HREC approval (Monash University) 
April –Aug 2012 
CRF & database development 
April – Sept 2012 
Staff training & ethical approval at collaborating sites 
Oct 2012 
Commence recruitment 
Jan 2015  
Complete 50% recruitment 
Mar 2015  
Interim analysis 
Aug 2016  
Complete 100% recruitment 
Nov 2016  
Complete 90 day follow up 
Jan 2017 
Query resolution & database lock of primary endpoint 
data 
Feb 2017  
Complete 180 day follow up 
Mar 2017  
Query resolution and database lock of secondary 
endpoint data 
April 2017  
Primary manuscript submitted 
 
 
 

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APPENDIX 1 – SCHEDULE OF EVENTS 
 
Assessments / Procedures 
ICU 
admission  
Pre-1st RBC 
transfusion 
Daily 
ICU 
Admission 
ICU 
Discharge 
Hospital 
Discharge 
Follow-up – 
28 
days 
post-
randomisati
on 
Follow-up – 
90 
days 
post-
randomisati
on 
Follow-up-  
180 
days 
post 
randomisati
on 
Screening log 
X 
 
 
 
 
 
 
 
Inclusion & Exclusion criteria 
 
X 
 
 
 
 
 
 
Demographic data 
 
X 
 
 
 
 
 
 
Primary diagnosis 
X 
 
 
 
 
 
 
 
Comorbidities 
 
X 
 
 
 
 
 
 
Physiological parameters 
X 
X 
 
 
 
 
 
 
APACHE III 
X 
 
 
 
 
 
 
 
SOFA 
 
X 
X 
 
 
 
 
 
Patient’s blood group (ABO, Rh) 
 
X 
 
 
 
 
 
 
Randomisation 
 
X 
 
 
 
 
 
 
RBC Transfusion (each 
unit) 
Haemoglobin pre-transfusion 
 
X 
X 
 
 
 
 
 
Time & date of transfusion 
 
 
X 
 
X 
 
 
 
RBC blood group (ABO, Rh) 
 
 
X 
 
X 
 
 
 
Pack number 
 
 
X 
 
X 
 
 
 
Collection and expiry dates 
 
 
X 
 
X 
 
 
 
Blinding status (units checked 
by non-bedside nurse) 
 
 
X 
 
 
 
 
 

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Assessments / Procedures 
ICU 
admission  
Pre-1st RBC 
transfusion 
Daily 
ICU 
Admission 
ICU 
Discharge 
Hospital 
Discharge 
Follow-up – 
28 
days 
post-
randomisati
on 
Follow-up – 
90 
days 
post-
randomisati
on 
Follow-up-  
180 
days 
post 
randomisati
on 
Adverse Events 
 
 
X 
 
 
 
 
 
Serious Adverse Events 
 
 
X 
 
 
 
 
 
Number of units (ml) FFP post-randomisation 
 
 
X 
 
 
 
 
 
Number of units platelets post-randomisation 
 
 
X 
 
 
 
 
 
Febrile non-haemolytic transfusion reaction 
 
 
X 
 
 
 
 
 
Requirement for catecholamine 
 
X 
X 
 
 
 
 
 
Requirement for Invasive Ventilation 
 
X 
X 
 
 
 
 
 
Requirement for Renal Replacement Therapy (RRT) 
 
X 
X 
X 
 
 
 
 
Blood stream infection (date(s), pathogen(s)) 
 
 
X 
 
 
 
 
 
Vital status & cause of death 
 
 
 
X 
X 
X 
X 
X 
EQ5D 
 
 
 
 
 
 
 
X 
 
 
 
 
 

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APPENDIX 2 – EURO QUALITY OF LIFE (EQ 5D) 
 
By placing a cross in one box in each group below please indicate which statement best describes your own 
health state today 
 
 
Mobility 
 
 
 
         I have no problems in walking around 
 
         I have some problems in walking around 
 
         I am confined to bed 
 
 
 
 
Personal care 
 
 
 
         I have no problems with personal care 
 
         I have some problems with personal care  
 
         I am unable to wash or dress myself 
 
 
Usual activities(eg: work, 
study, 
housework, 
leisure 
activities) 
 
 
         I have no problems with performing my usual activities 
 
         I have some problems with performing my usual activities 
 
         I am unable to perform my usual activities 
 
 
Pain/discomfort 
 
 
 
         I have no pain or discomfort 
 
         I have some pain or discomfort 
 
         I have extreme pain or discomfort 
 
 
Anxiety/depression 
 
 
 
         I am not anxious or depressed 
 
         I am moderately anxious or depressed 
 
         I am extremely anxious or depressed 
 
 

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APPENDIX 3 – THE IRON TRANSFUSE SUB STUDY (THE IT STUDY) 
 
Investigators (in alphabetical order) 
Dr Cecile Aubron, Dr Melinda Dean, Dr Robert Flower, Dr Chris Hogan, Dr Zoe McQuilten, Dr Sant-rayn 
Pasricha, Dr Rosemary Sparrow and A/Prof Erica Wood on behalf of the TRANSFUSE investigators. 
 
Aims: 
To investigate changes in iron metabolism, bacterial growth in plasma and inflammatory profile after red blood 
cell (RBC) transfusion, and the role played by the duration of RBC storage in these changes, in critically ill 
patients.  
 
Background: 
Age of blood, iron metabolism and the TRANSFUSE trial 
One third of patients hospitalised in intensive care unit (ICU) will be administered an average of 3 to 4 RBC 
units during their ICU stay [1, 2]. While RBC transfusion is considered lifesaving, their administration is 
independently and significantly associated with an increase in mortality and morbidity in this population [7]. 
Mechanisms for this potential harmful effect are not understood, however experimental studies suggest that 
blood storage duration may play a key role through RBC storage lesions, including accumulation of bioactive 
substances over the storage period.  
 
One of the main consequences of the RBC storage lesion is the increase in extra vascular haemolysis in both 
the RBC unit and recipient, which results in a large amount of iron delivered to the recipient. Studies in 
animals, healthy volunteers and non-critically ill patients have described that transfusion of older RBC, 
compared to fresher RBC, leads to an increase in extra vascular haemolysis, an increase in non-transferrin 
bound iron (NTBI) and to a pro-inflammatory cytokine storm within the first hours after transfusion [57, 58]. 
NTBI is also involved in oxidative damage [59] [60], expression of endothelial adhesion molecules [61] and 
bacterial growth [62] [60]. For instance, it was reported, in 33 preterm infants, that transfusion of older RBC 
units was associated with a significantly higher concentration of malondialdehyde (MDA) 4 hours after 
transfusion compared to fresh RBC [59], suggesting elevated oxidative stress. However, these studies are 
restricted to animals or a non ICU-adult population and are limited in their sample size and design. Underlying 
disease appears to be a determinant in the occurrence of post transfusion adverse events [63].  
A large prospective study on pathophysiological consequences of RBC transfusion and RBC storage duration in 
critically ill patients is required to: i) better understand how old RBC may promote inflammation, bacterial 
growth, oxidative damage and leucocyte adhesion, and ii) to define the patients most at risk of these adverse 
effects.  
The TRANSFUSE (sTandaRd Issue TrANsfusion versuS Fresher red blood cell Use in intenSive carE)-trial 
(ACTRN12612000453886) enrols heterogeneous critically ill patients who did not receive blood before 
intensive care unit (ICU) admission and aims to determine whether freshest available allogeneic RBC compared 
to standard practice is associated with a decrease in mortality. This randomised controlled trial (RCT), 
conducted in Australia, New-Zealand and Europe, is the largest ongoing RCT on age of blood in critically ill 
patients at this time.  
The TRANSFUSE-trial provides a unique opportunity to study how the RBC storage lesion may be harmful in 
critically ill patients. This study will provide valuable data on in vivo iron metabolism after RBC transfusion and 
other pathophysiological consequences of RBC storage lesions. These include: i) changes in bacterial growth in 
plasma before and after transfusion and between patients who received old and fresh RBC post-transfusion; ii) 
inflammatory profile post-RBC transfusion in patients receiving fresh vs. old RBC,  and iii) plasma induced 
changes to leukocyte-endothelial interaction and the difference in leucocyte adhesion in patients receiving 
fresh vs. old RBC. 

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Significance 
The project aims to address important and unresolved questions regarding potential mechanisms for any 
adverse effect from the storage age of RBC on patient outcomes. These data will provide detailed 
measurements of the potential pathways (iron, inflammation, propensity to infection) by which RBCs may 
produce adverse outcomes in patients. Importantly, by utilising the randomised design of the TRANSFUSE trial, 
this study will be able to investigate the effect of age of RBCs on these potential pathways. Identification of 
this mechanism could help future researchers develop specific approaches to preventing these adverse effects. 
The findings of this project will contribute to the development of strategies to address these effects and 
inform clinical practice, both in the intensive care and other clinical settings. They will also form the basis for a 
future potential NHMRC project grant to fund further studies into the mechanisms of adverse effects of 
transfusion. The collection of patient samples while the TRANSFUSE-trial is recruiting will be an invaluable 
resource for further research on the mechanisms of adverse effects from RBC transfusion in critically ill 
patients.  
 
Objectives 
Hypothesis  
Transfusion of older RBC compared with freshest available RBC leads to greater extravascular haemolysis and 
subsequent increase in NTBI resulting in an increase in bacterial growth and inflammation. 
 
Primary Outcome 
Indices of increased erythrocyte damage (extravascular haemolysis, iron indices including NTBI and labile 
plasma iron [LPI]) at 4-hour and 24-hours post the first RBC. 
Secondary Outcomes 
Bacterial growth in plasma at 4-hour and 24-hours post the first RBC. 
Inflammatory cytokines at 4-hour and 24-hours post the first RBC transfusion  
Leucocyte adhesion at 4-hour and 24-hours post the first RBC transfusion  
 
Study Design 
A sub study of patients enrolled in the TRANSFUSE randomised control trial.  
Recruiting Site 
The Alfred Hospital Melbourne, the Austin Hospital Melbourne and Monash Medical Centre Melbourne 
Inclusion criteria 
 
Patients randomised in the TRANSFUSE trial and who are going to receive their first RBC transfusion in 
the current admission in ICU  
Exclusion Criteria 
 
Current therapy with iron supplementation or iron chelation. 
 
Ethics and consent  
Appropriate Human Research and Ethics Committee (HREC) approval and Research Governance, including the 
use of opt-out consent has been obtained for the TRANSFUSE trial at each participating site.  Application for an 
amendment will be made to the HREC to approve the inclusion of this study. Prospective consent will be 
obtained from the patient or the person responsible in the first instance, and if this is not possible telephone 
consent will be sought. If no person responsible is contactable after reasonable attempts, patients will be 
enrolled under section 42T.  

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Researchers will ensure that the requirements for section 42T are met before proceeding – 
 
Patient will not regain capacity within a reasonable time 
 
The person responsible cannot be ascertained or contacted despite making reasonable 
efforts 
 
The medical research procedure is not contrary to the person’s best interests 
 
The medical research procedure is not against the wishes of the person 
 
The HREC has agreed that the procedure may proceed for a person where that person or 
their person responsible is unable to consent 
 
The purpose of the research is to test the effectiveness of the proposed therapy  
 
The procedure poses no greater risk than the risk inherent in the person’s condition and 
alternative treatment 
 
The researcher must tell the patient or their person responsible of the medical research 
procedure once the patient has regained capacity or the person responsible has been 
ascertained or located and that they are advised that they may withdraw from the 
treatment. 
A copy of the section 42T form will be submitted to the HREC and also to the Office of the Public Advocate. 
Patients and the person responsible (if applicable) will be given the plain language and information form 
including details about the study design, methodology and significance.  
 
Research plan and Methods 
One hundred and fifty patients will be included in this sub-study. Wherever possible eligible patients will be 
enrolled consecutively.  
After randomisation in the TRANSFUSE-trial, a sample of 18 ml (31/2 teaspoons) of blood will be taken before 
the first RBC unit, at 4 and 24 hours after the transfusion episode.  
Figure. Study procedures 
 
 
 
 

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Patient and transfusion data: will be routinely collected as part of the data collected for the TRANSFUSE-trial. 
Dates and times when samples are taken and results of the experiments performed in the study sites will be 
recorded in a separate case report form (CRF). 
Blood samples: Blood samples (2 x5 ml in lithium heparin tube, 1 x K2E 3ml EDTA (lilac top) tube and 1 x 5 ml in 
gel lithium heparin) will be sent immediately to the pathology lab of each hospital where plasma samples will 
be aliquoted and stored at - 80ᵒC before transfer to the Australian Red Cross Blood Service Melbourne 
Processing Centre (MPC), where they will be stored until recruitment is complete and experiments are 
performed. For further details refer to the section below “blood sample schedule.” 
Experiments:  
- 
Bilirubin, free haemoglobin, haptoglobin, ferritin, serum iron, transferrin and transferrin saturation 
will be performed in each pathology laboratory at the 3 participating centres.  
- 
The other experiments will be performed at a later date once all patients have been enrolled and 
samples collected. These experiments are outlined below: 
i) 
Non transferrin bound iron (NTBI) and labile plama iron (LPI, a fraction of NTBI that is redox active) 
will be measured by FeROS eLPI and LPI assays (Aferrix, Israel), and serum hepcidin will be 
measured using a commercially available ELISA (DRG, Germany).  
ii) Bacterial growth will be performed in plasma before and after transfusion without and with 
desferroxiamine as described elsewhere [64]. 
iii) Inflammatory biomarkers:  sCD106 (VCAM-1), sCD62L, sCD62P, ICAM-1, MCP-1, MIP-1a, MIP-1β, IL-
1α, IP-10, IL-6, IL-8, IL-10, IL-12, IL-4, IL-13, IL-17, IL-21, IFN-γ and IFN-α, will be assessed in plasma 
samples via multiplex Cytometric Bead Array (BD Biosciences). 
iv) Leucocyte adhesion  
 
 
Blood samples schedule 
Samples will be taken within 1 hour prior to transfusion (T0), 4 hours after transfusion initiation (T4) and 24 
hours after transfusion initiation. For example, if the first RBC transfusion starts at 10 am, the samples T0 will 
be taken between 9 am and 9.59 am, at 2 pm (samples T4) and at 10 am the following day (samples T24).  
In pathology, samples will be processed by the pathology laboratory staff. Supernatant will be removed and 
0.5ml of plasma will be aliquoted and stored in cryovials at -80 degrees Celsius. At least five tubes per patient 
for each collection time-point will be stored. They will include four cryovials of plasma with heparin and one 
cryovial of plasma with EDTA. The storage tubes will be pre-labelled with the study number, the date, time and 
type of collection tube used (i.e. EDTA or lithium heparin). Patient identifiers will not be used on the cryovials. 
Each week, a research assistant (Shauna French, PhD student) from the Blood Service will collect the samples 
from the 3 sites and transfer them with ice to the MPC. The patient samples will be kept at -80 degrees Celsius 
until the study recruitment has been completed, when the samples will then be transferred for analysis. 
 
 
 
Samples/volume collected 
Processing and storage 
Tests 
T0 
Sample 
Before 
transfusion 
of the first 
RBC unit 
1x5ml lithium heparin 
tube  
 
Processed in laboratory 
for immediate analysis 
as per laboratory 
protocol 
Iron studies: ferritin, 
transferrin, transferrin 
saturation, serum iron 
Haptoglobin 
 
1 x5ml gel lithium heparin 
tube 
 
Processed in laboratory 
for immediate analysis 
as per laboratory 
protocol 
Free haemoglobin 
1 x5ml lithium heparin  
^Centrifuge and aliquot 
for -80 degrees Celsius 
storage  into: 
Plasma in heparin, (5 
Non-transferrin bound 
iron 
Labile plasma iron 
Hepcidin 

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tubes of 0.5ml) 
Bacterial growth  
1 x3ml EDTA tube 
^Centrifuge and aliquot 
for -80 degrees Celsius 
storage  into: 
Plasma in EDTA, (2 tubes 
of 0.5ml) 
Inflammatory 
biomarkers (as listed 
above) 
T4 
Sample 
4 hours after 
transfusion 
initiation* 
1x5ml lithium heparin 
tube  
 
Processed in laboratory 
for immediate analysis 
as per laboratory 
protocol 
Iron studies: ferritin, 
transferrin, transferrin 
saturation, serum iron 
Haptoglobin 
1 x5ml gel lithium heparin 
tube 
 
Processed in laboratory 
for immediate analysis 
as per laboratory 
protocol 
Free haemoglobin 
1 x5ml lithium heparin  
 
 
^Centrifuge and aliquot 
for -80 degrees Celsius 
storage  into: 
Plasma in heparin, (5 
tubes of 0.5ml) 
Non-transferrin bound 
iron 
Labile plasma iron 
Hepcidin 
Bacterial growth 
1 x3ml EDTA tube 
^Centrifuge and aliquot 
for -80 degrees Celsius 
storage  into: 
Plasma in EDTA, (2 tubes 
of 0.5ml) 
Inflammatory 
biomarkers (as listed 
above) 
 
T24 
Sample 
24 hours 
after 
transfusion 
initiation* 
1x5ml lithium heparin 
tube  
 
Processed in laboratory 
for immediate analysis 
as per laboratory 
protocol 
Iron studies: ferritin, 
transferrin, transferrin 
saturation, serum iron 
Haptoglobin 
1 x5ml gel lithium heparin 
tube 
 
Processed in laboratory 
for immediate analysis 
as per laboratory 
protocol 
Free haemoglobin 
1 x5ml lithium heparin  
 
 
^Centrifuge and aliquot 
for -80 degrees Celsius 
storage  into: 
Plasma in heparin, (5 
tubes of 0.5ml) 
Non-transferrin bound 
iron 
Labile plasma iron 
Hepcidin 
Bacterial growth 
1 x3ml EDTA tube 
^Centrifuge and aliquot 
for -80 degrees Celsius 
storage  into: 
Plasma in EDTA, (2 tubes 
of 0.5ml) 
Inflammatory 
biomarkers (as listed 
above) 
 
*In the event that multiple RBC units are transfused in the transfusion episode, samples will still be taken 4 
hours after initiation of the first RBC transfusion 
^Centrifugation will be performed at 16 degrees for 10 minutes with a speed of 2898 G 
Data analysis and statistics 
Sample size: 150 patients. Enrolment of 150 patients will enable detection of a difference in NTBI at 4 hours of 
2.2uM with SD of 3.7 (similar to that shown in healthy volunteers in the study by Hod et al., Blood 2011 [62]), 

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between patients receiving fresh and older RBC units, accounting for a 20% incomplete data collection rate 
(which is expected due to the complex management requirements of patients enrolled in this study). 
Recruitment of 150 patients prior to the completion of the TRANSFUSE-trial (anticipated 18-months from 
commencement of the sub-study) is feasible based on the 3 centre’s current combined recruitment rate of 
approximately 8 patients per month within usual working hours.  
 
Statistical analysis: Data collected for the sub-study will be treated and analysed separately after the 
TRANFSUE RCT has been completed. Data will be transformed when skewed and analysis performed on log-
transformed values. Parameters before and after the transfusion episode will be compared for each patient 
using paired t-test. Biological parameters post-transfusion, as well as differences between pre- and post-
transfusion values, will be compared according to RBC storage duration category, which will be defined 
according to the RCT (i.e.: freshest available vs. standard issue) and also according to whether patients were 
transfused with only RBC younger than 14 days vs. patients transfused with RBC older than 14 days. A 
correlation between patient severity at randomisation and each parameter (including: post-transfusion 
extravascular haemolysis and NTBI) will also be performed. A sub group analysis will be performed on the most 
severe patients according to their SOFA score on the day of transfusion. 
 
Safety 
As per the TRANSFUSE trial. There are no additional risks posed by the IT TRANSFUSE sub-study. The results of 
the study will be forwarded to the Data and Safety Monitoring Committee (DSMC). 
 
Funding  
This study is collaboration with the Australian Red Cross Blood Service, who has provided funding for the costs 
for research staff time, sample collection, processing and testing in addition to in kind support provided by the 
Blood Service. 
 
Publication 
The study will be conducted in the name of Monash University on behalf of the TRANSFUSE investigators. 
Authorship will be under the names of the sub-study investigators on behalf of the TRANSFUSE investigators. 
Publication of results will be embargoed until publication of the main study results unless specific permission 
for early publication is given by the TRANSFUSE management committee. 
 
 
 
 
 
 
 
 
 

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APPENDIX 4 - THE RENALTRANSFUSE SUBSTUDY 
 
Investigators 
 Liz Moore, Cecile Aubron, Zoe McQuilten, Bridget Ady, Lynne Murray, Maija Kaukonen, Michael Bailey, 
Rinaldo Bellomo, Jamie Cooper of ANZIC Research Centre, School of Public Health and Preventive Medicine, 
Monash University 
 
Aim 
This study aims to determine whether, compared to standard care, transfusion of the freshest available 
allogeneic red blood cell (RBC) unit is associated with a decrease in the incidence of acute kidney injury (AKI) in 
patients admitted to the intensive care unit (ICU) who require RBC transfusion.   
 
Hypothesis 
In critically ill patients who require a RBC transfusion, compared to standard practice, the administration of the 
freshest available compatible RBC reduces the incidence of AKI and the need for ongoing renal replacement 
therapy after hospital discharge. 
 
Background 
Age of blood, storage lesion and acute kidney injury 
There is increasing evidence supporting an association between RBC transfusion and the development of AKI 
and RBC transfusion is reported to be an independent risk factor for AKI in critically ill patients[7, 65]. 
Moreover, in critically ill patients, the deterioration of RBC over time as a result of the ‘storage lesion’ could 
lead to organ dysfunction, including AKI, through iron toxicity and/or microcirculation dysfunction. Indeed, a 
human study demonstrated that RBC administration is associated with an increase in plasma iron 
concentration secondary to transfused RBC haemolysis with the observed changes proportional to the age of 
the RBC units[17].  
In vitro and animal studies have shown that circulating free iron is harmful and leads to glomerular damage 
and alteration in proximal tubule cell homeostasis[66, 67]. These findings taken together strongly suggest that 
older RBC may have greater toxicity than fresh RBC on the kidney. One of the hypotheses for this adverse 
effect is the deleterious effect of free iron secondary to haemolysis which is partly dependent on RBC storage 
duration. 
Age of blood and AKI: clinical evidence 
The clinical effects of the age of transfused RBC on kidney function have not been well investigated and 
therefore remain uncertain. Only a few observational studies have been performed reporting conflicting 
findings[28, 34, 68, 69]. In a large retrospective single centre study published in the New England Journal of 
Medicine, Koch et al reported that renal failure was more frequent in the 2872 patients receiving only old RBC 
(storage duration>14 days) compared to patients (n=3130) receiving only young RBC (storage duration less 
than 14 days)[28]. Similarly, in 1724 trauma patients, Weinberg et al reported an increase in the incidence of 
renal failure in patients receiving blood older than 2 weeks[69]. In contrast, a study of 670 patients undergoing 
cardiac surgery found that the age of RBC was not associated with any outcome, including renal failure[34]. 
The only study to directly investigate the association between age of RBC and development of AKI in a 
heterogeneous ICU population was observational and did not find an independent association between age of 
RBC and AKI. However, with 652 transfused patients this study was likely to be underpowered for this 

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outcome[70]. Indeed, they did find that the incidence of Stage 3 AKI was significantly greater in patients in the 
RBC age quartiles 2 to 4 (older) compared to quartile 1 (21.4% v. 11.2% respectively, p=0.006). All these studies 
have methodological limitations, including their design and/or the definition of AKI used, precluding any 
definitive conclusion. Furthermore, a meta-analysis has also been inconclusive regarding the impact of blood 
storage on the occurrence of AKI[11]. This question requires investigation with a randomised controlled trial 
(RCT) to achieve a more definitive result. 
The Canadian Age of Blood Evaluation (ABLE) RCT measured a composite outcome of multi organ failure only, 
without including a staged consensus definition of AKI in its study outcomes[45]. The TRANSFUSE RCT provides 
a unique, cost-effective opportunity to properly investigate the impact of RBC storage duration on the 
incidence of AKI and to better understand transfusion-associated AKI. 
 
Significance 
This Renal substudy of the TRANSFUSE trial provides a unique opportunity to evaluate the effect of age of RBC 
on the incidence, severity and duration of AKI in critically ill patients and may also elucidate mechanisms 
involved in the body’s response to RBC transfusion, thereby enhancing the parent study results. Leveraging the 
infrastructure and study design of the TRANSFUSE trial, this substudy provides a highly cost-efficient 
opportunity to test a clinically important hypothesis which cannot be answered outside such a large RCT and 
which would be unlikely to be funded as a separate study. 
 
Methods 
Renal function will be assessed in the TRANSFUSE trial using the KDIGO (Kidney Disease, Improving Global 
Outcomes) classification system[71] and an additional question at 90 day follow-up on the need for ongoing 
renal replacement therapy. There are no additional procedures required for this substudy. 
Inclusion criteria: The same as for the TRANSFUSE trial. All patients recruited to the trial at Australian, New 
Zealand and other overseas sites that agree to participate in this substudy will be included.  
Exclusion criteria: The same as for the TRANSFUSE trial with the following addition: patients with pre-existing 
end stage renal failure (receiving chronic dialysis) and those already undergoing renal replacement therapy at 
randomisation will be excluded. 
Measurements 
Renal function: AKI will be classified using the KDIGO classification system, the latest consensus classification 
system for AKI which has now been validated and used extensively[71, 72].  
Ongoing renal replacement therapy: A question has been added to 90 day follow-up to ascertain whether the 
patient is receiving ongoing renal replacement therapy.  
The only additional data to collect would be baseline creatinine defined as the most recent creatinine pre-
randomisation, then the highest daily creatinine thereafter when available during the ICU stay for a maximum 
of 28 days (the duration of the trial follow-up in ICU). Collection of routine available creatinine values will be 
performed for this period, with no extra dedicated blood collection required for the substudy. The use of renal 
replacement therapy while in hospital will also be recorded as part of the TRANSFUSE parent trial. 
 
Outcome measures 
Primary outcome:  
Cumulative proportion of patients with any degree of AKI  
Secondary outcomes:  
a) The proportion of patients who develop stage 3 AKI (KDIGO criteria)  
b) The proportion of patients requiring ongoing renal replacement therapy at 90 day follow-up 

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c) The severity of AKI measured as the change in serum creatinine from baseline (continuous variable)  
d) The duration of AKI measured as AKI free days (number of days not in stage 1-3 categories) for ICU stay 
e) Comparison of the mean creatinine levels across the first 28 days  
The fact that we are including several outcomes related to renal function will allow this study to provide a 
more comprehensive picture of the impact of RBC storage duration on renal function and will increase the 
possibility of detecting an effect if there is one. 
 
Power calculations and sample size 
This substudy will analyse data for 2000 patients. With 1000 patients per group, we will have an 80% power (2 
sided p-value of 0.05) to show a difference of 5.3% (19.5% vs 24.8%) of AKI any stage. This is taken from a 
recent article reporting an incidence of AKI (defined by the RIFLE criteria) of 24.8% in anaemic transfused ICU 
patients compared to 16.7 % in anaemic non transfused ICU patients (p<0.05)[73]. If the harmful effect of 
blood is mainly due to storage duration[73] a sample size of 550 patients per group would be enough to detect 
a difference of this magnitude (8.1%, 16.7% v. 24.8%). However, we don’t exclude that fresh RBC may also be 
deleterious to a degree, therefore, we have decided on a more conservative approach with a sample size of 
1000 patients per group.  
Alternatively, stage 3 AKI (KDIGO criteria) is the most severe stage of AKI and therefore the category in which 
we are most likely to detect a difference between groups. With 1000 patients per group we will have more 
than enough power to detect a difference of 10.2% (11.2% vs 21.4%) in stage 3 AKI[70]. 
 
Statistical Analysis  
Data will be assessed for normality and log-transformed where appropriate. Univariate analysis will be 
conducted using student t-tests, chi-square test for equal proportion or non-parametric tests where 
appropriate. Multivariate analysis will be performed adjusting for baseline imbalances and known covariates 
and confounders. Multiple logistic regression will be used for AKI as a binomial outcome. Linear regression will 
be used for analysis of continuous outcomes. The difference between groups for daily serum creatinine will be 
determined using generalised linear modelling fitting main effect for group and time and an interaction 
between group and time to determine if the two groups behave differently over time. A two-sided p-value of 
0.05 will be considered to be statistically significant. All data will be analysed using Intercooled Stata, version 9 
and above (Statacorp, College Station, Tx, USA). 
 
Publication 
The study will be conducted in the name of Monash University on behalf of the TRANSFUSE investigators. 
Authorship will be under the names of the sub-study investigators on behalf of the TRANSFUSE investigators. 
Publication of results will be embargoed until publication of the main study results unless specific permission 
for early publication is given by the TRANSFUSE management committee. 
 
 
 

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