Point-of-Care RBC Washing to Prevent Transfusion-Related Pulmonary Complications
Phase: PHASE1
Conditions: Blood Transfusion Complications
Sponsor: Mayo Clinic

 
 
 
 
 
 
 
 
POINT-OF-CARE RBC WASHING TO PREVENT TRANSFUSION-RELATED PULMONARY COMPLICATIONS 
 
 
 
 
 
 
NCT02094118 
 
 
 
 
 
 
PRIMARY INVESTIGATOR 
DARYL KOR, MD. 
 
VERSION 6.3 
 JANUARY 17, 2019 
 

 
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Contents                                                                                                                        
 
Table of Events ................................................................................................................................................................. 5 
A. Specific Aims .............................................................................................................................................................. 6 
AIM 1: ............................................................................................................................................................................... 6 
Aim 1a .......................................................................................................................................................................... 6 
Aim 1b .......................................................................................................................................................................... 6 
Aim 1c ........................................................................................................................................................................... 6 
AIM 2: ............................................................................................................................................................................... 6 
Aim 2a .......................................................................................................................................................................... 6 
Aim 2b .......................................................................................................................................................................... 7 
Aim 2c……………………………………………………………………………….………………………………………………….…………………………………7 
AIM 3: ............................................................................................................................................................................... 7 
Aim 3a:......................................................................................................................................................................... 7 
B.  RESEARCH STRATEGY ............................................................................................................................................. 7 
B1. SIGNIFICANCE ...................................................................................................................................................... 7 
B2.  INNOVATION ...................................................................................................................................................... 10 
Study procedures: ..................................................................................................................................................... 15 
Screening: ................................................................................................................................................................. 15 
Randomization: ....................................................................................................................................................... 15 
Study intervention: ............................................................................................................................................... 15 
Off-protocol transfusions: ................................................................................................................................... 16 
Blinding:..................................................................................................................................................................... 16 
Other aspects of care: .......................................................................................................................................... 16 
Outcome Measures: .............................................................................................................................................. 16 
Statistical Considerations ........................................................................................................................................ 19 
Sample Size Considerations: ............................................................................................................................. 20 
Additional statistical considerations: .............................................................................................................. 21 
Protocol addendum: .............................................................................................................................................. 21 
 
 
 
 
 

 
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ABBREVIATIONS 
 
ALI 
acute lung injury 
APACHE 
acute physiology and chronic health evaluation 
ARDS 
acute respiratory distress syndrome 
BNP 
brain natriuretic peptide  
BRM 
biological response modifiers 
CABG 
coronary artery bypass graft 
CATS 
continuous autotransfusion system 
CCL5 
chemokine ligand 5 
CFH 
cell free hemoglobin 
CHF 
congestive heart failure 
Co-I 
Co-Investigator 
CTMS 
Clinical Trials Management System 
DUMC 
Duke University Medical Center 
EDTA 
ethylenediamine tetraacetic acid 
EHR 
electronic health record 
FDA 
Food and Drug Administration 
FiO2 
fraction of inspired oxygen 
Hb 
hemoglobin 
Hct 
hematocrit 
ICU 
intensive care unit 
IgA 
Immunoglobulin A 
IL-6 
interleukin 6  
IL-8 
interleukin 8  
IQR 
interquartile range 
ITT 
intention to treat 
LC 
liquid chromatography 
LR-RBCs 
leukocyte reduced red blood cells 

 
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LVBT 
large-volume blood transfusion 
LVEF 
left ventricular ejection fraction 
MAP 
mean arterial pressure 
MAR 
missing at random 
MC 
Mayo Clinic 
MCAR 
missing completely at random 
MS 
mass spectrometry 
NO 
nitric oxide 
OR 
operating room 
PAI-1 
plasminogen activator inhibitor-1 
PaO2 
partial pressure of oxygen in arterial blood 
PCWP 
pulmonary capillary wedge pressure 
PEEP 
positive end expiratory pressure 
PI 
Principal Investigator 
RAGE 
receptor for advanced glycation end-products 
RANTES 
regulated on activation, normal T expressed and secreted 
RAP 
right atrial pressure 
RBC 
red blood cell 
RBC-MPs 
red blood cell microparticles 
sCD40L 
soluble CD40 ligand 
SD 
standard deviation 
SOFA 
sequential organ failure assessment  
SpO2 
peripheral capillary oxygen saturation 
SVR 
systemic vascular resistance 
TACO 
Transfusion-associated circulatory overload 
TRALI 
Transfusion-related acute lung injury 
USCIITG 
United States Critical Illness and Injury Trials Group 
VFD 
ventilator free days 
 
 
 

 
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Table of Events 
 
 
 
 
Adverse 
Event 
Monitoring 
Oxygen titration 
monitoring** 
Monitoring of 
blood products 
Daily 
Assessment for 
ARDS/TRALI / 
TACO 
Research Blood 
Draw #5 
Research Blood 
Draw #4 
Research Blood 
Draw #3 (6 
hours) 
Research Blood 
Draw #2 
Baseline 
Research Blood 
Draw 
Event 
 
X 
 
X 
 
X 
 
X 
 
 
 
X* 
 
 X* 
 
X 
Study Day 
1 
 
X 
 
X 
 
X 
 
X 
 
 
X 
 
 
 
Study Day 
2 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
 
Study Day 
3 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
 
Study Day 
4 
 
X 
 
X 
 
X 
 
X 
X 
 
 
 
 
Study Day 
5 
 
X 
 
X 
 
X 
 
 
 
 
 
 
Study Day 
6-28 
*May actually occur on the next study day if the patient is a late surgical case 
 
**Oxygen titration only occurs on extubated patients 

 
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A. Specific Aims   
 
With the long-term goal of reducing pulmonary morbidity following perioperative transfusion, the objective of 
this proposal is to test the feasibility, safety, efficacy, and clinical impact of point-of-care washing of allogeneic 
Leukocyte-Reduced (LR) Red Blood Cells (RBCs).  Specifically, we will evaluate the impact of this intervention 
on intermediate mechanistic and physiologically-relevant markers of transfusion-related acute lung injury 
(TRALI) and transfusion-associated circulatory overload (TACO).  To this end, we propose a multicenter, 
randomized, phase I/II clinical trial in adult cardiac surgery patients receiving LR-RBCs on the day of surgery. 
TRALI and TACO are common and important causes of morbidity following cardiac surgery and are the leading 
causes of transfusion-related death in the US.1 Although successful TRALI prevention interventions exist for 
non-RBC components (e.g. plasma/platelets), the lack of effective prevention interventions for RBC-
associated TRALI and TACO represent critical knowledge gaps. In this context, the mechanisms 
underlying these RBC-associated complications remain poorly defined.  Increasingly, soluble biological 
response modifiers (BRMs) within the RBC storage solution are suggested to play an important role.2-6  Point-
of-care RBC washing may remove these BRMs, thereby mitigating their influence on postoperative 
transfusion-related complications.7-10 Such measures bear significant clinical importance in cardiac surgery 
which consumes over one million RBC units each year in the US alone.11 While the pro-inflammatory stimulus 
of cardiopulmonary bypass and postoperative reduction in left ventricular compliance may partly contribute to 
respiratory dysfunction, infusion of BRMs residing within stored RBC supernatant may explain why RBC 
transfusion also represents a major risk factor for postoperative pulmonary complications in this surgical 
population.  Indeed, numerous BRMs in the RBC product [e.g. neutral lipids,6,12 soluble CD40 ligand 
(sCD40L),3 chemokine (C-C motif) ligand (CCL5),13,14 RBC-derived microparticles (RBC-MPs),15-17 cell-free 
hemoglobin (CFH) 18-20] have been associated with acute transfusion reactions. Accordingly, safe, efficacious, 
and logistically feasible interventions to reduce RBC-associated respiratory dysfunction are needed.  
Washing blood products prior to transfusion has been shown to reduce inflammatory markers in pediatric 
cardiac surgery patients21 and improve survival in patients with acute leukemia22 However, widespread 
adoption of this practice has been limited by ongoing equipoise regarding its clinical impact as well as the 
logistical limitations of washing RBCs in the blood bank prior to transfusion.8  Our preliminary data demonstrate 
that: A we can predict which patients will receive RBC transfusions, thereby facilitating the targeted enrollment 
of a high-risk cohort, and B we can effectively remove BRMs from allogeneic LR-RBC units with a practical, 
point-of-care, Autotransfusion device.  Therefore, to demonstrate the safety, efficacy, feasibility and clinical 
impact of this approach, we propose the following specific aims and testable hypotheses.   
AIM 1: To determine the feasibility, safety, and efficacy of point-of-care RBC washing using the Continuous 
Autotransfusion System (CATS) in adult cardiac surgery patients receiving allogeneic LR-RBC transfusion. 
Aim 1a - We hypothesize that point-of-care washing of allogeneic LR-RBCs will be feasible in the time-
sensitive environment of cardiac surgery with minimal or no protocol violations. 
Aim 1b - We hypothesize that when compared to standard-issue allogeneic LR-RBCs, washed allogeneic LR-
RBCs will be associated with a comparable rise in hemoglobin concentration and will not be associated with 
evidence of increased hemolysis (CFH, haptoglobin) or acute kidney injury in the recipient. 
Aim 1c - We hypothesize that RBC washing will reduce TRALI (neutral lipids, sCD40L, CCL5) and TACO 
(RBC-MPs, CFH) related BRMs in the RBC unit by greater than 80%.  
AIM 2:  To demonstrate the extent to which point-of-care washing of allogeneic LR-RBCs impacts the 
recipient response to RBC transfusion when compared to standard-issue allogeneic LR-RBCs. 
Aim 2a - We hypothesize that RBC washing will reduce post-transfusion concentrations of validated lung 
injury-associated biomarkers (IL-6, IL-8, vWF, ICAM-1, RAGE, SP-D, PAI-1) in the transfusion recipient. 

 
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Aim 2b - We hypothesize that RBC washing will mitigate adverse physiologic consequences of RBC 
transfusion that have been associated with TACO (elevated mean arterial pressure, systemic vascular 
resistance, and pulmonary capillary wedge pressure). 
Aim 2c - We hypothesize that lower levels of putative BRMs (neutral lipids, sCD40L, CCL5, RBC-MPs, CFH) 
in transfused RBC components (and in the RBC recipient) will be associated reduced levels of lung injury 
biomarkers and an attenuated cardiopulmonary response to RBC transfusion. 
AIM 3:  To evaluate the impact of point-of-care washing of allogeneic LR-RBCs on recipient clinical 
outcomes.   
Aim 3a: We hypothesize that compared to standard-issue RBCs, point-of-care washed RBCs are associated 
with improved clinical outcomes (shorter duration of mechanical ventilation, oxygen supplementation, and ICU 
stay; improved oxygenation; and improved organ function).   
Our large and accessible at-risk population, established clinical trial infrastructure, and multidisciplinary 
experience/expertise in translational, patient-centered transfusion research will assure this proposal’s success. 
B.  RESEARCH STRATEGY 
 
B1. SIGNIFICANCE 
 
Problem statement:  Transfusion-related pulmonary complications are the leading cause of serious transfusion-
related adverse events.  Transfusion-Related Acute Lung Injury (TRALI) remains the leading cause of 
transfusion-related death in the US and although seemingly less appreciated, Transfusion-Associated 
Circulatory Overload (TACO) has been the second leading cause of transfusion-related death over the past 5 
years.1 In addition to their associated mortality, both syndromes result in substantial resource utilization and 
associated health care cost.  The majority of patients who develop TRALI will require intensive care unit (ICU) 
admission and ventilator support, which typically lasts from 3 to 10 days.23,24 Clinical evidence also notes that 
up to 21% of TACO cases are life-threatening and associated with increased lengths of ICU and hospital 
stay.25-27 Although specific preventative strategies have dramatically reduced the incidence of plasma-
associated TRALI (e.g. male-only plasma donation), no prevention strategies exist for RBC-associated TRALI 
or TACO.  Indeed, the lack of safe and feasible strategies that can mitigate risk of RBC-associated 
TRALI and TACO represent critical knowledge gaps in transfusion medicine.   
TRALI mechanisms:  Although TRALI and TACO share a similar clinical phenotype (pulmonary edema and 
hypoxemic respiratory insufficiency), each is the result of distinct pathophysiologic processes.2,4,5,26,28 TRALI is 
believed the result of a two-hit process that results in leukocyte priming, sequestration, and activation followed 
by endothelial injury and inflammatory lung edema.  The first insult typically relates to recipient factors (e.g. 
surgery, trauma, or infection) and the second “hit” from the infusion of factors residing in the blood component.  
For high-plasma volume components (e.g. 
plasma, apheresis platelets), this is most 
often the result of anti-leukocyte antibodies 
within the donor product reacting with 
recipient cognate antigens.  In contrast, 
multiple lines of evidence suggest alternate 
mechanisms are at play with RBC-associated 
TRALI.2-4,6  Here, the second insult is 
generally believed the infusion of soluble 
biologic response modifiers (BRMs) residing 
in the RBC supernatant.  Evidence supporting 
this theory  
include: 1) identification of specific BRMs  
Figure 1.  Temporal changes in the concentration of soluble 
biologic mediators in RBC supernatant during storage.14  

 
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which can generate an acute inflammatory response [e.g. bioactive lipids6,12, soluble CD40 ligand3, and CCL5 
(also known as RANTES)13,29,30 and RBC-MPs17,31,32]; 2) association of prolonged storage duration with 
adverse respiratory outcomes (BRMs accumulate during the storage process, Figure 1)12,14,15,33,34; and 3) the 
potential for mitigating transfusion reactions by washing RBCs prior to transfusion.21,35-38 Notably, RBC washing 
has been associated with abrogation of adverse immunologic effects resulting from transfusion in trauma 
patients, and improved survival in transfusion recipients with acute leukemia.22,35  Although promising, washing 
stored RBCs has been largely discounted due to concerns related to cost/feasibility.38  
TACO mechanisms:  The primary mechanism underlying TACO has historically been believed fluid 
overload.25,26 However, several lines of recent evidence suggest the potential for additional and potentially 
synergistic pathophysiologic processes.  First, the supposition that excessive fluid volume is the primary 
mechanism underlying TACO has not been consistently supported by clinical data.   We have recently shown 
the median (IQR) transfusion volume in patients who develop TACO is only 3 (2-7) units.39  Indeed, a large 
proportion of reported TACO cases present after a single blood unit exposure. 40,41 Moreover, Roubinian et al. 
recently compared fluid and transfusion volumes in patients with TACO and TRALI. 42  These investigators 
reported no statistically significant differences in hourly fluid balance or the number of blood component units 
transfused in the 24-hour interval preceding the TACO or TRALI episode.   
Second, TACO is characteristically associated with a substantial elevation in the systemic blood 
pressure.16,29,43 This hypertensive response exceeds what would be expected from the volume challenge alone 
and suggests the existence of vasoconstricting substances in the transfused product.  To this point, Donadee 
et al. have recently shown significant differences in the blood pressure response to RBC transfusion as a 
function of storage duration.18  These responses were strongly correlated with time-dependent increases in 
nitric oxide (NO) consumption as well as with increased concentrations of RBC-derived microparticles (RBC-
MPs) and cell free hemoglobin (CFH), both of which are known to scavenge NO.  As a potent microvascular 
vasodilating substance, NO has substantial influence on endothelial function and systemic vascular resistance 
(SVR).  Indeed, the role of NO scavenging, RBC-MPs, and CFH in endothelial dysfunction and transfusion-
related complications has been increasingly described. 16,17,19,20,31,44,453 In regards to TACO, a sudden increase 
in the SVR has the clear potential to compromise left ventricular function (particularly in transfusion recipients 
with altered baseline cardiac function), resulting in elevated left atrial pressures and ultimately hydrostatic 
pulmonary edema (the hallmark of TACO).  Notably, recent evidence also suggests that the delivery of 
therapies specifically targeting NO-mediated pathways (e.g., inhaled NO44,46 and haptoglobin47) can mitigate 
these adverse responses.  In further support of a potential role for RBC-MPs, CFH, and NO scavenging as a 
mechanism for adverse cardiopulmonary response to allogeneic RBC transfusion, it should also be noted that 
the primary adverse effect identified with hemoglobin (Hb)-based RBC substitutes is profound hypertension 
associated with nitric oxide scavenging.48-51 
   
As a third line of evidence suggesting alternative TACO mechanisms, our own preliminary data failed to 
correlate central venous pressure (a measure of intravascular volume status) with post-transfusion hypoxemia 
in closely monitored, mechanically ventilated patients, receiving single unit RBC transfusion. In contrast, an 
acute increase in SVR was associated with worsening gas exchange after RBC transfusion (Figure 2).   
Figure 2. Relative contribution of post-
transfusion increase in central venous 
pressure (CVP) and systemic vascular 
resistance (SVR) in worsening gas 
exchange (change in PaO2/FIO2 after 
transfusion) in a prospective study of 48 
mechanically ventilated patients receiving 
an elective transfusion of one unit of stored 
RBCs. Post-transfusion increase in SVR 
was associated with worsening gas 
exchange (left) while CVP increase had no 
effect on oxygenation (right) 

 
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Finally, Blumberg et al. recently evaluated temporal changes in transfusion outcomes and identified a reduced 
rate of cardiopulmonary complications (including TACO) with implementation of universal leukoreduction.52  
More importantly, the investigation noted a complete absence of reported TRALI and/or TACO cases 
associated with washed allogeneic RBCs (n = 28,120 RBC units).  Again, these findings suggest 
alternative TACO mechanisms and further imply that these mechanisms may be modifiable with RBC washing. 
Transfusion-related complications following cardiac surgery:  Cardiac surgery is a high-risk endeavor under the 
best of circumstances.  In addition to this baseline risk, multiple studies have associated RBC transfusion with 
increased risk of adverse events in this population.53-60  Numerous studies also suggest that respiratory 
complications are chief among the risks related to transfusion in this setting.53,59-61  In light of this, as well as the 
fact that patients who undergo cardiac surgery receive a substantial proportion of the greater than 15,000,000 
units of allogeneic RBCs transfused in the US each year11, the significance of improving transfusion safety and 
mitigating transfusion-related respiratory complications in this population is clear.     
Point-of-care washing of allogeneic RBCs for the prevention of RBC-associated respiratory dysfunction:  
Standard issue, 120-micrometer transfusion set filters 
permit the transfusion of cellular debris, microparticles 
and soluble mediators present in the supernatant of an 
RBC unit.62  RBC washing can remove soluble 
contaminants in the RBC supernatant including 
chemokines, biologically active lipids, cellular 
debris/microaggregates and possibly microparticles.7-10  
Importantly, however, washing RBCs in the blood 
bank may not always be the best solution.  A typical 
device used in blood banks, the Cobe 2991 Cell 
Processor (Terumo BCT, Denver, CO), is an example of 
a device designed to remove all traces of plasma, 
essential for IgA-deficient RBC recipients.63 Importantly, 
such aggressive washing may damage RBCs.  Indeed, 
this is illustrated in Figure 3 [adapted from O’Leary et 
al.63] where progressive hemolysis of the resuspended, 
washed RBCs occurred in the post-wash storage period.  
Notably, post-wash hemolysis appears to be attenuated 
when using cell-salvage devices.  To this point, potassium release is a marker of hemolysis and has been 
proposed as a washed product quality marker.64 Recently, O’Leary et al. demonstrated reduced hemolysis 
when comparing the Continuous AutoTransfusion System (CATS; Fresenius Kabi, Bad Homberg, Germany) to 
the Cobe 2991 Cell Processor.63  
Perhaps even more limiting is the time required for standard blood bank RBC washing procedures (typically 
greater than 1 hour).8 This time requirement precludes the feasible implementation of “on-demand” RBC 
washing in time-sensitive environments such as the operating room and ICU.  Moreover, RBC washed units 
have a 24-hour outdate.8  Therefore, pre-washing RBCs prior to a known transfusion need risks wastage of 
this scarce and expensive resource.  In contrast, point-of-care bedside RBC washing with cell-salvage devices 
may provide a practical, and possibly preferable option. Indeed, use of cell salvage devices to reduce 
allogeneic RBC transfusion during cardiac surgery is recommended by published guidelines65 and is currently 
standard of care in most cardiac surgery practices.  If washing of allogeneic RBCs using such a device proves 
effective in mitigating RBC-associated respiratory dysfunction, all RBCs (cell-salvaged and allogeneic) could 
quite feasibly be washed with such a bedside device. 
Of note, cell-salvage devices are also not identical. CATS is based on continuous cell separation with constant 
application of a low centrifugal force (330-680g)6658 during separation or “skimming” of the red cells, whereas 
the more typical Latham bowl-based cell separation devices employ an interrupted/discontinuous, higher 
centrifugal force (1220-2000g).66,67 The CATS design is associated with less hemolysis63 and has proved 
superior to Latham bowl based cell-salvage devices in the removal of oil emulsions7, RBC microaggregates10 
and separation of plasma from RBCs.9 Indeed, Gruber et al. have recently shown elimination rates exceeding 
 
Figure 3.  Progressive hemolysis following 
standard AABB-approved RBC washing 
procedures (Cobe 2991).  

 
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90% (a limit used for quality control in cell salvage) when washing allogeneic RBCs with the CATS device.8 
Summary on Significance:  In light of:  1) the impact of transfusion-related pulmonary complications on patient-
important outcomes; 2) the lack of effective preventative strategies for RBC-associated TRALI and TACO; 3) 
the biologic plausibility for the role of soluble BRMs in RBC-associated respiratory dysfunction; and 4) the 
ability to remove these BRMs with point-of care RBC washing, we believe this proposal is highly significant.  
Furthermore, this proposal specifically addresses multiple highlighted topics in this funding opportunity 
announcement including: 1)  Non-infectious complications of blood transfusion including transfusion-related 
acute lung injury (TRALI); 2)  Immunomodulatory, inflammatory and vasoregulatory properties of transfused 
blood components; and 3)  Evaluation of the impact of introducing a new blood product preparation or 
transfusion strategies on transfusion-associated adverse events and recipients’ clinical outcomes.   
 
B2.  INNOVATION 
 
At present, there are no effective strategies that can mitigate the occurrence and/or impact of RBC-associated 
TRALI or TACO.  This proposal seeks to evaluate the novel repurposing of a cell-salvage device (CATS) in an 
effort to provide an innovative strategy to prevent allogeneic RBC-associated pulmonary complications.  We 
will also evaluate novel mechanisms underlying TRALI and TACO. While specifically focused on BRMs and 
their association with respiratory dysfunction, our findings will more broadly differentiate the role of 
soluble BRMs (removed with washing) versus the role of the red blood cell itself (not removed with 
RBC washing).  We believe this application is highly innovative for the following reasons. 
Testing the feasibility and efficacy of point-of-care allogeneic RBC washing:  Although washing of stored RBCs 
has shown promise in isolated settings (e.g. pediatric cardiac surgery21 and acute leukemia22), concerns 
related to feasibility have prevented larger scale testing of this novel approach to improving transfusion safety.  
Major limitations with current RBC washing protocols include cost and the time requirements (typically one to 
several hours).8  In time-sensitive environments such as cardiac surgery or the ICU, this time delay is not 
feasible.  To address this limitation, this proposal takes an FDA-approved autotransfusion device (CATS) and 
proposes to repurpose it for point-of-care washing of allogeneic RBCs; an approach that is clearly innovative.  
If both feasibility and efficacy are established, this would present a novel, feasible and cost effective approach 
to improving transfusion safety.  Importantly, additional costly equipment would not be required as 
autotransfusion is standard of care for many surgical populations.  
There are no current strategies which mitigate the risk of RBC-associated TRALI or TACO:  Despite their 
impact on patient-important outcomes, no effective prevention strategies exist for RBC-associated TRALI or 
TACO.  Development of new strategies that can mitigate the onset and/or severity of these respiratory 
complications would be novel and highly significant.  We proposed to test a promising intervention, point-of-
care washing of allogeneic RBCs, in a high-risk population undergoing cardiac surgery.  There is clear biologic 
plausibility for cause-effect relationships between the infusion of soluble BRMs and development of these life-
threatening transfusion-related respiratory complications.3,4,6,13,29,30 Moreover, recent evidence as well as our 
preliminary data (see below), suggest the washing procedures proposed in this application are effective at 
removing RBC supernatant (and the BRMs contained within).7-10 Therefore, we believe our proposed 
intervention provides an innovative, feasible approach to preventing RBC-associated respiratory complications. 
Evaluating new mechanisms for RBC-associated TRALI:  Though our understanding of the mechanisms 
underlying TRALI has improved in recent years, these advances have been primarily limited to 
antibody/antigen interactions.2,68 Importantly, these specific episodes typically result from high-plasma 
containing blood components (plasma, apheresis platelet concentrates, whole blood).  In contrast, our current 
understanding of the processes underlying non-antibody mediated TRALI (the primary mechanism believed 
responsible following RBC transfusion) remain quite limited.  Indeed, the great preponderance of data in this 
regard has been generated by Dr. Christopher Silliman, a consultant on this grant application.4-6,12,38 We 
believe our innovative approach (evaluating the concentration of potentially putative BRMs in the donor unit, 
their change with RBC washing, the relationship between their concentration in the transfusion recipients and 

 
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intermediate markers of lung injury) will provide essential new hypothesis-generating mechanistic insights into 
the associations between RBC transfusion and post-transfusion lung injury.    
Evaluating new mechanisms for RBC-associated TACO:  Despite its clear significance to transfusion recipients 
and the healthcare team, our understanding of the mechanisms underlying this life-threatening syndrome 
remain incomplete.   
Although volume overload likely plays a key role in many cases of TACO, a growing body of evidence 
suggests the potential for alternate and potentially synergistic mechanisms that may present important 
opportunities for TACO mitigation.  Increasingly, evidence suggests a role for NO consumption, via RBC-
MPs16,17 and CFH18-20,46,47,69 in the hemodynamic response to RBC transfusion.  However, our understanding of 
how these processes impact TACO pathogenesis remains incomplete.  This is the first significant attempt to 
improve our understanding of the pathophysiology underlying TACO.  The targeted study of RBC-MPs and 
CFH in the RBC unit (as well as the transfusion recipient), and the evaluation of their association with the 
recipient’s physiologic response to transfusion, is novel and with high scientific merit.  
APPROACH 
 
We propose a multicenter, phase I/II clinical trial evaluating the feasibility, safety, efficacy, and clinical impact 
of point-of-care washing for allogeneic LR- RBC units in patients undergoing cardiac surgery.  Our group has 
demonstrated experience and expertise in the translational study of transfusion therapies and patient-
important transfusion-related respiratory complications.  We also have significant experience with the 
design and conduct of clinical trials.  Pertinent preliminary data are summarized here. 
Adequacy of the study population to ensure feasibility of the protocol:  Cardiac surgery remains a 
leading consumer of RBC component therapies.11  At Mayo Clinic in Rochester, MN, there were 2,344 unique 
patients who underwent cardiac surgery in 2011.  A total of 1,059 of these (45%) were transfused on the day of 
their surgical procedure with 310 patients receiving 4 or more units.  In the cohort of 10,653 patients 
undergoing CABG or CABG/valve surgery at Duke University Medical Center between 2000 and 2010, 28% of 
patients received between 4 and 20 units of allogeneic RBC.  Last year, Duke University Medical Center 
performed 1,343 cardiac operations in total of which ~350 were isolated Aortic or Mitral Valve 
repair/replacement. The remaining 1,000 cases would be of equivalent or greater risk of transfusion than the 
CABG and CABG/valve cohort.  Therefore, at least 250 patients per year will be eligible for screening for this 
proposal.  Of note, approximately 75% of allogeneic transfusions in this population are administered on the day 
of the surgical procedure (based on our preliminary data 
from 2011). 
A prediction model for RBC requirements in patients 
undergoing cardiac surgery70:  Utilizing a 10-year study 
population of isolated CABG surgery patients at Duke 
University Medical Center (n = 5,887), our team (Co-PI: 
Welsby) developed a prediction model for estimating 
perioperative RBC requirements in this setting.70 The model 
performed well with derivation and validation data set c-
indices of 0.79 and 0.78, respectively.   Applying this model 
in more complex cardiac surgery populations (combined 
CABG/valve, aortic repair/replacement, etc.) suits our 
purposes well, as it will likely underestimate RBC needs for 
these more complex procedures. Entering the predictive 
variables (see Table 1) into an on-line calculator tool, will 
enable screeners to enroll patient expected to receive 4 or 
more units of allogeneic RBCs into the proposed clinical trial. 
Clinical trial experience in transfusion medicine and lung injury prevention71,72: Our team (Co-PI: Kor; 
Co-I: Gajic) has experience in the design and conduct of multicenter clinical trials evaluating the impact of 
storage duration on respiratory and immunologic outcomes in populations similar to those targeted in this 
Table 1.    Factors predictive of RBC transfusion in 
CABG surgery. 

 
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proposal. More specifically, we recently completed a two-center (Mayo Clinic and the University of California-
San Francisco) randomized, double-blind clinical trial which compared fresh (< 5 days storage) to standard-
issue single-unit RBC transfusion in adult, intubated and mechanically ventilated patients.71  Novel automated 
hemoglobin screening algorithms were employed to identify, enroll, and randomize fifty patients to receive 
fresh RBCs an additional 50 to standard-issue RBCs.  The primary outcome evaluated was the change in 
partial pressure of arterial oxygen to fraction of inspired oxygen concentration ratio (Δ PaO2/FIO2) with 
secondary outcomes evaluating changes in immunologic and coagulation status.  Although no significant 
differences were noted in the endpoints evaluated (Table 2), the limited sample size, restriction to single unit 
RBC transfusion, and short duration of follow-up (median of 1.9 hours) were notable limitations with this 
investigation.   
Table 2:  Pertinent outcomes in a randomized controlled trial evaluating single-unit fresh versus standard issue RBC 
transfusion.  
 
Using the multicenter, interdisciplinary infrastructure of the US Critical Illness and Injury Trials Group, our 
investigative team (Co-PIs: Kor, Welsby; Co-Is:  Gajic, Carter) has also helped to design and conduct the first 
multicenter clinical trial evaluating the safety and efficacy of a promising ALI prevention agent 
(ClinicalTrials.gov ID:  NCT01504867).72  Specifically, this trial is evaluating the safety and efficacy of aspirin 
as an ALI prevention agent in patients at high risk for ALI.  Sixteen geographically diverse institutions 
throughout the US are enrolling patients into this trial and enrollment is currently exceeding plan.   
Experience with the study of transfusion-associated circulatory overload 39 :  Our team (Co-PI: Kor; Co-I: 
Gajic) has significant experience with the study of TACO epidemiology and case recognition.  In a 
retrospective observational cohort study, we evaluated risk factors for TACO among 901 patients who were 
transfused in the medical ICU setting.39  Fifty-one (6%) of these patients developed TACO and 16 were 
associated with RBC transfusion.  Several baseline predictors (e.g. left ventricular dysfunction; plasma ordered 
for the reversal of anticoagulant therapy) and transfusion factors (Table 3) were identified as TACO risk 
predictors. RBC storage duration was longer in the TACO cohort, but this did not meet significance in the 
underpowered subgroup analyses. 
 
Table 3:  Transfusion characteristics of TACO cases and matched transfused controls. 
 
Recently, our team (Co-PI: Kor; Co-I: Gajic) also developed innovative electronic health record (EHR) 
surveillance algorithms which can be used to facilitate the detection of transfusion-related pulmonary 
complications.54  Using a study population acquired from a NIH-sponsored prospecrtive cohort investigation,55, 
we developed optimal TRALI and TACO case detection algorithms using data contained within the EHR.  

 
13 | P a g e  
 
 
 
 
These algorithms accurately identified TRALI cases with a sensitivity and specificity of 83.9% (95% confidence 
interval [CI], 74.4%-90.4%) and 89.7% (95% CI, 80.3%-95.2%), respectively.  For TACO, the sensitivity and 
specificity were 86.5% (95% CI, 73.6%- 94.0%) and 92.3% (95% CI, 83.4%-96.8%), respectively.  Notably, of 
true-positive cases identified using the screening algorithms (TRALI/transfused ALI, n = 78; TACO, n = 45), 
only 11 (14.1%) and five (11.1%) were reported to the blood bank by clinical service, respectively. 
The Continuous AutoTransfusion System (CATS) can eliminate potentially dangerous BRMs from 
stored allogeneic RBCs:  In addition to previous work demonstrating effective eliminiation of protien8, CFH8, 
and RBC microaggregates10, our preliminary data suggest effective elimination of additional BRMs as well.  
Specifically, 4 LR-RBC units were washed with 
the CATS device using a 4:1 dilution process as 
previously described.8  RBC-MPs, sCD40L, and 
CCL5 concentrations were assessed before 
and after the washing procedures.  The 
reduction in microparticle concentration 
exceeded 80 % in all cases evaluated with a 
median (IQR) percent change of 92.2% 
(82.2% – 98.7%).  Similar reductions were 
noted in CCL5 for all 4 units evaluated and in 
sCD40L for 2 of the 4 units.  The remaining 
two units had low levels of sCD40L in the pre-
wash sample.  A representative pre-, post-
wash sample evaluating RBC-derived 
microparticle counts is presented in figure 4.   
Biomarkers as intermediate outcomes for 
clinical lung injury (Grant #:K23HL112855-01): 
Our team has substantial experience in the 
study and use of biomarkers as intermediate 
surrogate outcomes for clinical lung injury.  Our 
current K23 award (PI:  Kor) specifically aims to 
evaluate the use of biomarkers to better 
understand the mechanisms underlying 
postoperative acute respiratory distress 
syndrome (ARDS).  As prelminary data for the 
present proposal, we evaluated the biomarker 
profiles of 12 patients who developed ARDS 
following cardiovascular surgery with 12 
matched controls who did not.  As can be seen 
in Figure 4, differing biomarker profiles were 
noted in those who developed postoperative 
ARDS when compared to those who did not.  In 
light of this preliminary data, we have refined 
our biomarker panel in this A1 resubmission to include IL-6, IL-8, PAI-1, and RAGE as the intermediate 
outcomes for Aims 2a/c of this proposal. 
 
 
 
 
 
 
 
 
Figure 4. RBC microparticle counts before (left) and after 
(right) washing with the CATS.   
Figure 4.  Lung injury biomarkers in patients who do and do not 
develop ARDS.   

 
14 | P a g e  
 
 
 
 
PHASE I/II CLINICAL TRIAL EVALUATING POINT-OF-CARE RBC WASHING TO PREVENT RBC-
ASSOCIATED RESPIRATORY COMPLICATIONS FOLLOWING CARDIAC SURGERY 
 
 
 
 
Study population:  Study procedures are summarized in Figure 5.   We will enroll a total 154 patients 
receiving at least one RBC transfusion on the operative day.  We anticipate this will require randomizing up to 
190 patients in this trial (95 per group).  Adult patients (age >18) who are planned to undergo cardiac surgery 
at one of the two participating institutions and who have a predicted RBC transfusion requirement of > 4 units 
will be screened for the exclusion criteria outlined in Table 4.  Predicted RBC transfusion requirements will be 
determined utilizing a recently published large-volume blood transfusion (LVBT) prediction model (>4 units of 
RBC).73 A cut off of 4 predicted units of RBC administration was chosen because:  1) this transfusion volume 
has been associated with increased risk of pulmonary complications following cardiac surgery58; 2) we have 
previously identified this transfusion volume as a common “dose” in patients who experience TRALI and/or 
TACO28, 39,42; and 3) this threshold would still identify a sizable cardiac surgery population, ensuring study 
feasibility.   
 
 

 
15 | P a g e  
 
 
 
 
Study procedures:   
Screening:  Study coordinators will screen all adult (age > 18) patients who are scheduled to undergo coronary 
artery bypass graft 
surgery with or without 
valve 
repair/replacement, 
complex cardiac valve 
surgery, pericardial 
resection, and/or 
ascending aortic surgery 
in one of the two 
participating institutions.  
Eligible patients with a 
predicted transfusion 
requirement of 4 or more 
units will be approached 
before their elective 
surgical procedure by a 
study coordinator for 
consent.  After 
confirming the patient’s 
inclusion and exclusion 
criteria, the study 
coordinator at the 
respective institution will 
obtain informed consent 
and assign a study ID 
number to the study participant.  Screening logs will be maintained at each site to allow generation of a 
CONSORT diagram.  
Randomization:  Randomization will occur at the time of consent and analyses will be conducted using a 
modified intention-to-treat principle (See statistical considerations below). Randomization to RBC washing or 
control group (standard-issue RBC) will be conducted by the study’s electronic data management system’s 
Balance (Medidata) algorithm. This software uses dynamic minimization stratified by clinical center.  The 
software will return a confirmation of the randomization indicating the study participant’s treatment allocation 
status.  A note will be placed into the EHR identifying the patient as a study participant and the transfusion 
medicine service will have unblinded, electronic access to the treatment assignment.  Randomization and 
screening will halt after the 154th study participant has undergone surgery and received at least one RBC 
transfusion on the operative day.  Patients randomized but awaiting surgery will continue study participation. 
Study intervention:  The intervention in this investigation will be implemented for all allogeneic RBCs 
administered on the day of sugery (intraoperative and postoperatively on Study Day 1).  The decision to 
administer an allogeneic RBC transfusion will be left to the responsible clinical service and will not be 
prespecified in the study protocol.  We have elected to leave this decision to the clinical service for the 
following reasons:  1) the target population frequently experiences major acute blood loss; in these 
circumstances, typical measures assessing the need for RBC transfusoin (e.g. Hb) are often associated with 
unacceptable time delays and frequently do not reflect true red blood cell mass nor need for RBC transfusion; 
2) this design facilitates a more meaningul understanding of the feasibility of RBC washing in clinical practice.  
Upon receiving an order for allogeneic RBC transfusion, the transfusion medicine service will prepare the RBC 
product according to the allocated treatment assignment (intervention versus control).  Of note, all allogeneic 
RBC units at the two participating institutions undergo pre-storage leukocyte reduction. 
-Intervention cohort:  For patients randomized to receive washed RBCs, all allogeneic RBCs administered 
on the day of surgery will be washed with the CATS device prior to transfusion. To account for the time 
Table 4.  Exclusion criteria.    
Exclusion Criteria 
Justification 
• 
Emergency surgery 
Inability to randomize/perform study procedures 
• 
IgA deficiency 
Not ethical to randomize to standard issue RBCs 
• 
History of severe recurrent transfusion 
reaction 
Not ethical to randomize to standard issue RBCs  
• 
Refusal to receive allogeneic RBCs 
Inability to administer intervention of interest 
• 
Refusal to provide informed consent 
Not ethical to enroll into trial 
• 
Prevalent  Acute lung injury prior to 
randomization 
Inability to adequately assess outcome 
• 
Prevalent   hydrostatic pulmonary 
edema prior to randomization 
Inability to adequately assess outcome 
• 
Expected hospital stay < 48 hours 
Incomplete study procedures and outcome data 
• 
Not anticipated to survive > 48 hours 
Incomplete study procedures and outcome data  
• 
Previously enrolled in this trial 
Violation of the independence assumption 
• 
 No intention to measure cardiac output 
parameters during surgical procedure 
Inability to assess key physiologic parameters 
outlined in the study protocol 
                 
• 
Use of home oxygen therapy 
Inability to assess  oxygen use outcome 
• 
Complex RBC antibody profiles 
Washing not feasible due to testing delays 
• 
Need for the use of irradiated RBCs 
Intervention contraindicated 
• 
Intention to place a cardiac mechanical 
assist device 
Inability to assess key physiologic parameters 
outlined in the study protocol 
                 

 
16 | P a g e  
 
 
 
 
required to complete the ordering, delivery, washing, and administration procedures, we will restrict RBC 
washing to those RBC units ordered prior to 23:30 on the day of surgery.  Any RBC unit ordered after 23:30 on 
the day of surgery will no longer be considered an “intervention unit” and will therefore be standard issue (non-
washed) as it anticipated that the time required to perform the activities mentioned above (ordering, delivery, 
washing, and administration) would otherwise result in the RBC component’s actual administration time to 
occur on the day after surgery.  When the need for transfusion is identified by the primary team, the RBC 
product will be prepared.   As previously described8 and confirmed in our preliminary data, pre-dilution of 
stored, allogeneic RBCs results in the most effective elimination of supernatant. Therefore, an approximate 4:1 
dilution will be added to the reservoir of the CATS by gravity drainage.  The “High Quality” wash mode option 
will be selected for processing; this takes approximately 7-10 minutes to complete.74  Washed RBCs will then 
be drained from the reinfusion reservoir into sterile transfer bags (Fenwal Inc, Lake Zurich, ILL) for transfusion. 
  
-Control cohort:  For patients randomized to the control group arm, all RBCs administered on the day of 
surgery will be standard-issue RBCs.   
Off-protocol transfusions:  In the setting of cardiac surgery, it is occasionally necessary to provide allogeneic 
RBCs in an emergent fashion (e.g. acute, life-threatening bleeding).  In this particular circumstance,  time-
delays relating to study-related activities may prove unsafe.  To address this potential scenario, our study 
protocol will allow the administration of emergency “off-protocol” allogeneic RBCs.  “Off-protocol” RBC 
transfusions will be administered as per standard institutional practice.  These RBC transfusion episodes will 
be specifically noted as “off-protocol” and will be summarized and analyzed to assist in assessing the feasibility 
of point-of-care RBC washing in patients underoing cardiac surgery (see statistical description below).  In 
addition, autotransfusion (“cell-saver”) is frequently used in this patient population.  Cell-salvage will be 
implemented at the discretion, and under the direction, of the clinical team.  If cell-salvage is employed, the 
device used for this procedure will be distinict and separate from the intervention CATS device. 
Blinding: In light of the time-sensitive, point-of-care nature of the intervention being proposed, the patient, 
clinical team, and members of the study team involved in the RBC washing procedure will not be blinded to the 
patient’s treatment allocation status.  Blinding will be ensured for Drs. Norris and Silliman who will be running 
the proposed biomarker analyses.    
Other aspects of care:  ARDSnet trials have demonstrated the benefit of standardizing clinical processes in 
ICU trials.75,76  In this study, clinical care that may affect development of respiratory dysfunction and associated 
outcomes will be standardized to the extent possible.  To this end, our protocol will specify optimal ventilator 
strategies for both the OR and ICU environments (tidal volume < 8 mL/kg predicted body weight; peak 
inspiratory pressures < 35 cm H20, PEEP ≥ 5 cm while ventilated in the ICU).  Similarly, although we will not 
pre-specify RBC transfusion thresholds in this protocol, we will advise restrictive transfusion practices in the 
postoperative period (hemoglobin target > 8 g/dL in the absence of acute bleeding and/or ischemia).  This 
transfusion threshold was chosen as it is the current standard of care at the two participating institutions.  
Standardization of best practices in at-risk patients will decrease the heterogeneity of the risk modifiers that 
may otherwise confound our associations of interest (RBC washing and intermediate markers of respiratory 
outcomes).  Additionally, each center has adopted protocols on daily spontaneous awakening and 
spontaneous breathing trials to facilitate standardized weaning from ventilators (see attached appendix for 
specific protocols).  Non-intubated patients will undergo standard titration of oxygen twice daily (at 0700 and 
1900, +/- 2 hours).  Patients saturating 92% or greater on room air will not receive supplemental oxygen, 
unless specifically requested by the primary service.  If the primary care service requests oxygen 
supplementation for a patient saturating greater than or equal to 92% on room air, the reason for the deviation 
from oxygen weaning will be documented.  Patients will continue to undergo evaluation for oxygen titration until 
liberation from oxygen therapy for 24 hours, hospital day 28, or hospital discharge, whichever comes first.     
Outcome Measures:  To help ensure the integrity and quality of the laboratory analyses, all assays will be 
performed by our collaborators, Drs. Norris and Silliman. The safety outcome analyses (aim 1b) will be an 
exception as these will be run in “real-time” at each enrolling institution. All clinical outcome variables will be 
assessed by trained study coordinators using standardized definitions and operating procedures.  To the 

 
17 | P a g e  
 
 
 
 
extent possible, these study coordinators will also be blinded to the study participant’s treatment allocation 
status. 
Specific Aim 1:  The primary feasibility outcome in aim 1a will be the number and proportion of “off-protocol” 
allogeneic RBC transfusions administered during the study intervention period (day of surgery).  A secondary 
feasibility outcome will be the time required for the RBC washing procedures (defined as the time from 
determination of RBC need by the clinical team to time of delivery of the RBC unit to the clinical team).  This 
time will be computed for all patients and all transfusions during the study intervention period (in both the 
intervention and control cohorts).  The primary safety outcome in (aim 1b) include the change in the RBC 
recipient’s Hb concentration from pre- to post-transfusion as well as the concentration of CFH and haptoglobin 
following RBC transfusion.  To assess the primary safety outcomes, samples for total Hb, CFH, and 
haptoglobin will be obtained prior to transfusion, as soon as possible after the first transfusion, but no later than 
30 minutes following the first intervention/control RBC transfusion, as well as 6 hours (+/- 30 minutes) and 18 
hours (+ / - 30 minutes) from the end of first study RBC transfusion for all study participants.  We will also 
assess for clinical evidence of acute kidney injury throughout the hospitalization.  Safety labs will also be drawn 
anytime on study day #5 if the patient is still in the hospital. The primary efficacy outcome in aim 1c will be 
the change in the concentration of neutral lipids [arachidonic acid, 5-hydroxyeicosatetranoic acid (HETE), 12-
HETE, 15-HETE], sCD40L, CCL5/RANTES, RBC-MPs, and CFH in the RBC component from the pre- to the 
post-wash phase.  These data will allow us to calculate the CATS-related elimination rates for these BRMs, 
which have previously been associated with transfusion-related respiratory dysfunction and hemodynamic 
alterations.  To balance assay costs while ensuring the scientific success of aim 1c, pre- and post-wash 
samples will be obtained/analyzed for the first 75 washed RBC units.  If efficacy is not clearly established, we 
will continue to obtain both pre- and post-wash samples for 75 additional units.   
Specific Aim 2:  The outcomes measures for aim 2a will be the change in concentration of multiple, validated 
lung injury biomarkers representing the primary pathways leading to development of lung injury (Table 5).   
Patients will have blood drawn as soon as possible after the decision to transfuse, as soon as possible after 
transfusion, but not later than 30 minutes after the end of first study RBC transfusion, 6 hours (+ / - 30 minutes) 
from the end of first study RBC transfusion, and 18 hours (+ / - 30 minutes) from the end of first study RBC 
transfusion.   
Table 5.  Lung injury biomarkers and exploratory potentially pathogenic biologic response modifiers for specific aim 2.  
Validated lung injury biomarkers (Aim 2a) 
Primary process evaluated 
Supporting Reference 
Interleukin-6 
Inflammation 
 
77-79 
Interleukin-8 
77-81 
Von Willebrand Factor 
Endothelial activation/injury 
 
77,80-8570,73-76 
Intercellular Adhesion Molecule-1 
77,80,86-89 
Receptor of Advanced glycation end-products 
Epithelial Injury 
 
80,93 
Surfactant Protein-D 
77,80,90,91 
Plasma activator inhibitor-1 
Dysregulated coagulation 
79,86,94-96 
Exploratory biomarkers (Aim 2c) 
Primary process evaluated 
Supporting Reference 
Neutral lipids 
Lung inflammation 
6,12 
sCD40L 
3 
CCL5/RANTES 
13,29,30 
RBC-derived microparticles 
NO scavenging 
16-18 
Free Hemoglobin 
18-20 
N-terminal Brain Natriuretic Peptide 
Ventricular stretch/volume-overload 
97-99 

 
18 | P a g e  
 
 
 
 
 
In addition to these biomarker analyses, we will proceed with detailed assessment of the cardiopulmonary 
responses to study RBC transfusions administered in the operating room (Aim 2b).  Specific variables to be 
assessed and recorded are listed in Table 6.   Systemic vascular resistance will be calculated at each time 
point using the following equation:  SVR (dyns/cm5) = [(MAP – RAP)/CO] x 80.  Each of these variables will be 
assessed and recorded immediately prior to the intervention RBC transfusion and again immediately after the 
RBC transfusion.  Standard 
operating procedures for these 
cardiopulmonary assessments will 
be defined prior to study onset 
according to previously established 
recommendations.100  These 
secondary outcomes will allow a 
more detailed assessment of the 
cardiopulmonary response to RBC 
transfusion and will be expected to provide important insight into the pathophysiology of TACO.  Of note, 
pulmonary artery catheter placement is standard of care for this patient population at both of the enrolling 
institutions.   
The exploratory putative BRMs in Table 5 have been selected to evaluate specific potential mechanistic 
pathways for TRALI and TACO (aim 2c).  N-terminal BNP has been included as a marker of volume 
overload/ventricular strain.  To better elucidate the relationship between the “dose” of these soluble BRMs in 
the RBC components, their subsequent concentration in the recipient, and their ultimate relationship to 
recipient’s cardiopulmonary response to transfusion, levels of these potential putative agents will be 
determined in the RBC components prior to transfusion (in both the washed and standard issue cohorts) as 
well as in the transfusion recipient.  As we are targeting surgical patients who are expected to receive > 4 units 
of allogeneic RBCs, we will obtain samples from the RBC component for all RBC units administered up to and 
including the fourth unit for each study participant.  This will be a post-wash sample in those randomized to the 
RBC washing arm.  Of note, we recognize the potential for incomplete capture of relevant information in those 
who receive larger volumes of RBC transfusion.  However, we believe the four-unit cutoff to be the best 
compromise between study feasibility and scientific validity.  In the recipient, samples for the putative BRM 
assessments will be obtained at the same time points outlined above for the lung injury biomarker samples.    
 
Specific Aim 3:  To facilitate the design and conduct of future clinical trials, we will also pursue a number of 
exploratory clinical outcomes.  The clinical outcome measure of interest for aim 3 is the duration of 
postoperative mechanical ventilation.  Study coordinators will collect data daily until endotracheal extubation, 
death or hospital discharge regarding the need for mechanical ventilation. The primary clinical outcome will 
then be determined by calculating the duration of mechanical ventilation in hours for each study participant, 
determined by subtracting the time of ICU admission from the time of endotracheal extubation.   If the study 
participant is extubated prior to ICU admission, the duration of mechanical ventilation will be assigned as 0 
hours.  Recognizing the potential for early death (intraoperative or early postoperative) biasing our primary 
clinical outcome, the number of ventilator free days (VFD) at postoperative day 28 will also be determined.  
Those who die prior to day 28 will be determined to have had 0 VFD.  Those who are discharged from the 
hospital alive prior to day 28 will be assumed to have had no additional days of mechanical ventilation following 
hospital discharge.  Additional secondary outcomes for Aim 3 will include evaluations of hypoxemia (SpO2, 
PaO2/FiO2), duration of oxygen supplementation, sequential organ failure assessment scores, and durations of 
ICU and hospital stay.  The clinical diagnoses of TRALI, possible TRALI, and/or TACO will be measured 
through study day 5, or hospital discharge, whichever comes first. 
Specimen Handling and Laboratory Assays:  Aim 1b: 5 ml of blood will be drawn from each study participant 
at baseline, following the first RBC unit administered, 6 hours after the end of the first RBC transfusion, 18 
hours after the end of first RBC transfusion, and on hospital day 5 if the patient remains hospitalized (see 
Specific Aim 1 and Specific Aim 2 for more details on the timing of blood draws).  These safety laboratory 
Table 6.  Physiologic parameters to be measured during the study 
intervention period. 
Respiratory Variables 
 
Hemodynamic Variables 
PaO2, SpO2, FiO2 
Mean arterial pressure 
Tidal Volume 
Heart Rate 
Peak inspiratory pressure 
Cardiac output 
Plateau airway pressure 
Central venous/right atrial pressure 
Positive end-expiratory pressure 
Pulmonary artery wedge pressure 

 
19 | P a g e  
 
 
 
 
assessments (total Hb, CFH, haptoglobin) will be analyzed locally at the enrolling sites using standard clinical 
assays.  Aim 1c:  A 6-ml sample will be taken pre- and post-wash from the already anticoagulated intervention 
RBC units.  Aim 2:  10 mls of blood will be drawn from each study participant at baseline, following the first 
RBC unit administered, at 6 hours after the end of first study RBC transfusion, and 18 hours after the end of 
first study RBC transfusion.  All samples will be centrifuged, alloquoted and frozen.  Samples will be batch 
shipped to Mayo Clinic Rochester and analyzed in Dr. Norris’ laboratory at Blood Systems Research Institute 
(BSRI), and a separate aliquot provided for Dr. Silliman’s laboratory (neutral lipids only).   
Biomarkers:  Thawed platelet poor plasma will be diluted with assay buffers and measured (aims 1c/2c: 
sCD40L, CCL5/RANTES; aims 2a/2c:  IL-6, IL-8, PAI-1;    Plasma will be used to perform ELISA-based 
measurements of RAGE (aim 2a/2c).  NT-proBNP (aim 2c) will be measured.RBC-derived microparticles 
(aims 1c/2c): Thawed platelet-poor plasma will be spun, then labeled in preparation for flow cytometric 
measurement   Vesicles will be lysed with NP-40 detergent and samples re-run to confirm results and allow 
setting of gates.  Free hemoglobin (aims 1c/2c): will be measured.  Neutral lipids (aims 1c/2c):  Following 
the addition of ice-cold methanol, proteins will be precipitated, and non-polar lipids will be extracted/analyzed 
using high-pressure liquid chromatography (LC) interfaced into the electrospray source of a triple quadrupole 
mass spectrometer (MS) (liquid chromatography coupled to electrospray ionization mass spectrometry 
[LC/MS/MS]).  Lipid concentrations will be estimated using ratios to an internal standard (2H8-5-HETE), as 
previously described.101,103   
Predictor variables other than the intervention:  Pertinent baseline demographics and clinical 
characteristics such as age, sex, race, and comorbidities will be recorded.  Additional predictor variables will 
include:  vital signs and laboratory values that are obtained during the course of routine care, APACHE IV 
score, administration of statins, ace-inhibitors, insulin, amiodarone, or steroids; blood product administration for 
the up to day 28 or hospital discharge, whichever comes first, daily fluid status, and vasopressor requirements. 
Quality Control and Data Management:  Mayo Clinic will serve as the DSCC to coordinate data collection, 
quality control and data analysis.  The principal investigators at each study site will be responsible for the 
quality control of data collection according to standard definitions that are provided in the protocol.  In addition, 
the strategies to achieve a high level of protocol adherence will include 1) refresher education sessions for 
study coordinators two weeks after the study onset, 2) periodic checks of protocol compliance by the research 
coordinators at the DSCC and 3) computerized identification of protocol violations in the database.   
Mayo Clinic has implemented an enterprise-wide Clinical Trials Management System (CTMS).  CTMS is a 21 
CFR Part 11 compliant data management infrastructure to support multi-center clinical trials and participant 
registries.  The core of the CTMS project is the Medidata RAVE product.  Briefly, Medidata RAVE will serve as 
the electronic data capture and randomization system for the study.  The system has comprehensive audit 
trails, user authentication, security and/or disaster plans, and standardized training for end users.  The system 
provides real-time, cross-form data integrity checks to maximize data integrity while lessening the need for on-
site source document verification (See Facilities and Resources).   
 
Statistical Considerations (prepared in collaboration with our statistical:  Aim 1 is centered on the 
clinical and technical feasibility of on-demand RBC washing (hypothesis 1a). Feasibility is defined as the 
administration of protocol RBCs instead of “off-protocol” standard-issue RBCs. Simon’s two-stage design will 
be used to determine if the protocol needs to be modified to prepare RBCs prior to the surgical procedure. The 
null hypothesis rate for feasibility is p<=0.75 versus the alternative that p>=0.90, where p is the probability of 
successfully preparing and administering the intervention RBCs. Stage 1 of the design will examine the first 16 
cases randomized to RBC washing. If 12 or fewer transfusions were deemed “feasible”, the protocol will be 
modified to pre-wash RBCs (see limitations). If 13 or more are feasible, an additional 32 cases will be 
evaluated. Should 39 or fewer (of the 48 studied) not be feasible, the protocol will be modified to pre-wash.  If 
40 or more cases meet the feasibility definition, the remainder of the study will go as originally planned. 
(Sample size/ design calculations details: PASS 2008, alpha=0.10, beta=0.10, Optimal design). The safety 
endpoint for aim 1 is the change in hemoglobin level (hypothesis 1b). The change in hemoglobin after the first 
transfused unit will be used as the primary safety measure.  Wilcoxon rank sum tests will be used to compare 
change in hemoglobin levels between randomized groups. The change in concentrations of soluble BRMs (e.g. 

 
20 | P a g e  
 
 
 
 
neutral lipids; hypothesis 1c) will be assessed using paired t-tests. There is > 80% power to detect a 0.4 SD 
difference pre- to post-washing with 78 washed RBC products. Should there be fewer (due to feasibility), 
power will decrease.  However, the magnitude of differences observed in preliminary data (92% reduction, 
Figure 4) is many times larger than an effect size of 0.4.  Therefore, power for this hypothesis should not be a 
concern. 
 
Aim 2 examines the changes in biomarkers levels measured over three time points (Baseline, no later than 30 
minutes after the end of the first RBC transfusion, 6 hours (+ / - 30 minutes) from the end of first study RBC 
transfusion, and 18 hours (+ / - 30 minutes) from the end of first study RBC transfusion. Mixed models will be 
fit to model the linear trajectory of these biomarkers. A model with a random slope and intercept will be 
considered initially, and the primary parameter of interest will be the treatment group by time interaction. For 
this analysis, a “modified” intention to treat (ITT) principle will be considered. Only patients randomized to RBC 
washing who receive at least one unit of the washed cells will be included from RBC washing group in the 
analysis. Those not receiving RBC washed cells will be in the considered the control group. The reason for this 
is this aim is focused on mechanistic action and demonstrating biologic plausibility prior to formal evaluation of 
clinical outcomes (which would be analyzed using traditional ITT considerations). Since the functional form of 
the changes in biomarkers over time is not known, a discrete (2 D.F. test) representation of time will also be 
used to gauge the linearity assumption as well as provide a sensitivity analysis to the primary regression 
model. Standard mixed modeling practices will be utilized (e.g., assessment of residuals, verification of 
variance components, nested modeling to simply variance components and covariance patterns, etc). This 
modeling scenario will be conducted for each biomarker of interest. Since prior research have noted that these 
outcomes are “clustered”, the methodology by Shi et al. will be used to adjust for multiple comparisons.104  We 
will also compute O’Brien’s nonparametric global test statistic to provide an overall measure of treatment effect 
between the two treatment groups. 
 
The effects of storage duration will also be assessed. The mean age of the transfused blood products will be 
determined. This value will be used to test for effect modification (interaction with treatment). Specifically, the 
main effect of RBC age, its interaction action with treatment, time and the treatment-by-time interaction term 
will be used to determine if the biomarker trajectories are affected by RBC age. It is hypothesized that the 
washing protocol will attenuate the effects of age resulting in more uniform trajectories over the range of RBC 
age where as the unwashed RBC will show increases in biomarker values as RBC age increases. 
 
Sample Size Considerations: The sample size for this clinical trial is based on the mechanistic outcomes 
detailed for Specific Aim 2a. Table 2 in Fremont et al. was used to provide estimates of the range of effect 
sizes for the biomarkers considered in this study.80Using an approximation of the standard deviation from the 
interquartile range (SD~=IRQ/1.35), the median effect size was found to be 0.4. This magnitude of change 
would be relevant, and appropriate to power this study on. With equal allocation between the groups, the 
sample size for this study is estimated to be n=78/group (alpha=0.10, two-sided; see additional statistical 
considerations below). Actual power is expected to be higher on account of the repeated measures design. To 
allow for drop out and “non-feasible” cases, we plan to enroll and randomize patients until 154 patients have 
undergone surgey and had at least one transfusion on the operative day.  We anticipate this may require 
randomizing up to 190 total patients. Any patients randomized and awaiting surgery when the 154th patient is 
achieved will also be included. The final sample size may be adjusted further based on the feasibility findings 
observed in the two-stage design. 
Aim 3 will utilize standard analytical measures for comparing randomized treatment groups with ITT. For data 
that may be skewed (e.g., length of ventilation, ICU stay), Wilcoxon rank sum tests will be used to compare 
groups.  Serial measurements (oxygen saturation) will be analyzed using longitudinal summary statistics (e.g., 
AUC, mean value). It should be noted that the intermediate clinical outcomes are not being powered for in this 
study. Estimates of precision (confidence intervals) along with range of responses will be used to guide 
subsequent trial designs (e.g. a larger phase II/III trial with clinical outcomes as the primary outcome of 
interest).  

 
21 | P a g e  
 
 
 
 
Additional statistical considerations: Consistent with early phase clinical trials, we have selected a level of 
significance higher than the traditional 0.05. This will facilitate advancement of the technique should it prove 
feasible and demonstrates potential efficacy. It is noted that multiple testing may increase the overall family 
wise error rate, so further research, particularly with clinical events, will be needed to quantify clinical efficacy 
of the approach. Missing data is expected to be minimal during the course of the study given the close 
surveillance the surgical and ICU environment provides. Missing specimens may occur in the event of patient 
discharge, death or administrative issues. Initial analyses will be conducted under the assumption of 
MCAR/MAR (ignorable missing). Sensitivity analyses using multiple imputation and pattern mixture models will 
be used to assess the robustness of the model assumptions.  The analysis of specific aim 2 proposes to use a 
modification of the ITT principle. Justification for this is that it is a mechanistic aim with blinded examiners. We 
are interested in quantifying the effects of washing as clearly (and perhaps ideally) this is an early phase trial.  
In Aim 3, the clinical outcomes will be assessed using the traditional ITT definition to provide a more realistic 
measure of the effect size for planning subsequent studies. 
 
 
 
Protocol Addendum: 
 
The Aim 1c RBC washing efficacy outcome analyses evaluating the first 75 washed RBC units, comparing the 
pre-washed soluble biologic response modifier (BRM) concentrations to the post-washed unit concentrations, 
have been completed in collaboration with Drs. Norris and Silliman.   The Aim 1c findings indicate a decrease 
in the RBC micro-particle and cytokine concentrations, indicating the CATS washing procedure was effective in 
removing the BRM’s that have previously been associated with transfusion-related cardiopulmonary 
dysfunction.  However, the analyses also noted increased concentrations of cell free hemoglobin (CFH).   
 
In an effort to further understand the potential relevance/clinical importance, if any, of the increase in CFH, we 
will obtain 20 evaluable hematocrit values between the two sites, both before and after the washing procedure.  
To accomplish this, a small sample of 2 mls. will be drawn from the RBC unit prior to performing the washing 
procedure and again at the conclusion of the washing procedure prior to administering the unit to the patient.  
This hematocrit analyses will be conducted locally at each site.   
 
As we have completed the Aim 1c washing efficacy analyses defined a priori, we will no longer be collecting 
the 6 ml sample from the RBC bag prior to washing.  Thus, the total amount of sample drawn from the RBC 
units will be less than it has been up to this point in the trial.  Therefore, we would not anticipate any additional 
risks to the recipients of these washed allogeneic RBC units.   
 
Our decision to test the hematocrit levels before and after washing in 20 RBC units is the result of our 
interactions with the lead statistician of this protocol who has estimated this to be a sufficient sample size to 
better understand the increase in cell free hemoglobin in relation to the total hemoglobin present in the 
allogeneic RBC units.     
 
 

 
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