Physiologic Effects of RBC Storage in Chronic Transfusion Recipients: Vasoreactivity, Exercise Capacity, and Oxygen Consumption
Phase: PHASE4
Conditions: Red Blood Cell Transfusion
Sponsor: Emory University

 
1 
 
 
STUDY PROTOCOL: 
 
IRB00064523 
 
NCT02566577 
 
 
Physiologic effects of RBC storage in chronic transfusion recipients: 
vasoreactivity, exercise capacity, and oxygen consumption. 
 
Principal Investigator: 
 
Arshed Quyyumi, MD 
 
Date 10/31/2014 
 
 

 
2 
 
 
 
 
 
 
 
STUDY PROTOCOL: 
 
 
 
Physiologic effects of RBC storage in chronic transfusion recipients: 
vasoreactivity, exercise capacity, and oxygen consumption. 
 
 
 
 
 
 
 
Principal Investigator: 
 
Arshed Quyyumi, MD 
 
 
Co Principal Investigators and Mentors:  
 
John Roback, MD, PhD. 
 
 
Co-Investigator: 
Robert Neuman, MD 
Salim Hayek, MD 
 
 
Sponsor:  
National Blood Foundation 
 
 
 
 
 
 
 

 
3 
 
SPECIFIC AIMS:  Transfusion of red blood cells (RBCs) is a common intervention aimed at preventing mortality 
and morbidity in anemic and bleeding patients. However, some RBC units may have functional defects that 
impair their efficacy and could actually harm patients. For example, a growing body of evidence suggests that 
transfusion of older stored RBCs (termed storage-aged RBCs [saRBCs]) is associated with worse clinical 
outcomes including multi-organ failure and mortality.1-4 A constellation of cellular changes, commonly termed 
‘the storage lesion’, may underlie these outcomes. Among the saRBC storage changes of interest are those that 
reduce nitric oxide (NO) bioavailability.5,6 NO is a crucial vasodilator in small arterioles and capillaries which 
actively adjust to increase blood flow (and O2 delivery) to local tissues with high oxygen demands (hypoxic 
vasodilation).7,8 Some common disorders, such as cardiovascular disease (CVD) and diabetes mellitus, are 
known to reduce NO bioavailability which leads to vascular dysfunction and related morbidities in affected 
patients9,10.  We, and others have likewise demonstrated that saRBCs reduce NO bioavailability in vascular 
models, and that transfusion of saRBCs reduces NO-mediated vasodilation in animal and human transfusion 
recipients11-13 (please see preliminary data). One important component of the proposed studies will be our 
investigations to determine whether the impairment of NO-mediated vasodilation seen in these previous studies 
produces significant physiologic consequences on exercise tolerance and oxygen consumption in transfused 
patients.        
Up to now, prospective mechanistic investigations of the effects of saRBC transfusions on human 
recipients have been limited to either hospitalized subjects or healthy volunteers14-17_ENREF_14(preliminary data). 
The use of inpatient subjects is complicated by the fact that that are often critically ill with complex and dynamic 
medical conditions, their transfusion needs are unpredictable and heterogeneous. In contrast, with healthy 
volunteers, the timing and volume of study transfusions can be controlled. However, healthy volunteers cannot 
ethically have their hematocrits reduced to levels seen in anemic hospitalized patients, nor do they typically have 
underlying endothelial dysfunction seen in hospitalized patients, which may synergize with the effects of saRBC 
transfusions to further reduce NO bioavailability.  Thus, while both groups have been used successfully to study 
the effects of fresh vs. saRBC transfusions, each study population has limitations.  Ideally, investigations of the 
vasoinhibitory effects of saRBCs would best be performed in anemic patients who have predictable, repetitive 
transfusion requirements such that a cross-over transfusion design can be used to control for inter-individual 
heterogeneity.  At Emory, we care for a large population of chronically anemic patients who require outpatient 
blood transfusions every 2 to 4 weeks for thalassemia, aplastic anemia, myelodysplastic syndrome and other 
disorders. Thus, the second important component of the proposed studies will be the recruitment of these 
patients as study subjects: they have relatively stable disease-related anemias and vascular dysfunctions, are 
transfusion-dependent and can be randomized into a cross-over study design to receive fresh transfusions 
during one visit, and saRBC transfusions during the next (or vice versa). The proposed studies will test our 
hypothesis that transfusion of saRBCs (day 21-42 of storage), but not fresh RBCs (< 10 days), will impair NO-
mediated vasodilation and also reduce exercise tolerance and oxygen carrying capacity in an ambulatory 
transfusion-dependent adult population.  The Specific Aims are:  
 
Aim 1: To assess the effects of fresh vs. saRBC transfusions on NO-dependent endothelial function in 
chronically anemic adults using: 1) flow mediated dilation (FMD) and 2) reactive hyperemic index (RHI) measured 
by pulsatile arterial tomography (PAT). The primary endpoint of this Aim is the change in FMD with fresh vs. 
saRBC transfusion. The secondary endpoint is change in RHI.  
 
Aim 2: To assess the effects of fresh vs. saRBC transfusions on functional exercise capacity and 
oxygen carrying capacity by cardiopulmonary exercise testing. The primary endpoint is the change in peak 
oxygen consumption (VO2Max) after transfusion.  
 
Secondary endpoints for the above Aims are changes in metabolic markers before and after 
transfusion/exercise: inflammation (IL-2, IL-6, hsCRP), oxidative stress (glutathione/glutathione disulfide and 
cysteine/cystine redox potentials), and nitric oxide metabolites (nitrite, nitrate, and nitrosylated hemoglobin and 
thiols).  
These studies are a continuation of research I performed during my postdoctoral fellowship and represent an 
interdisciplinary approach with mentoring expertise in transfusion medicine and cardiovascular function. These 
novel studies will be crucial for my continued research training, and will provide data to support future 
independent NIH funding to further dissect the mechanisms of the adverse effects of saRBC transfusions and to 
develop approaches to abrogate those effects.   
BACKGROUND AND SIGNIFICANCE 

 
4 
 
Clinical Significance: While more than 15 million units of RBCs are transfused annually in the U.S., 
RBC transfusion is in some cases associated with serious adverse outcomes.18 Although optimal utilization of 
donated RBC units is dependent on the ability to store RBCs at refrigerated temperatures for up to 42 days prior 
to transfusion, multiple perturbations in RBC structure and function occur with prolonged storage. Notable 
alterations include: 1) increased free hemoglobin, extracellular potassium, and lactate levels, 2) accumulation of 
shed microparticles (shedisomes) and soluble biological response modifiers, and 3) decreased pH, 2,3-
disphosphoglycerate (2,3-DPG) and S-nitrosyl hemoglobin (SNO-Hb).1,2,5,19-21 When infused, saRBCs with these 
defects may alter recipient physiology and contribute to poor outcomes. Physiologic processes that may be 
perturbed include the disruption of nitric oxide (NO)-mediated vasodilation. In the INOBA (Insufficient Nitric Oxide 
Bio-Availability) hypothesis,22 we postulated that an interplay of variables related to the RBC unit (e.g., storage 
age) and the transfusion recipient (e.g., endothelial dysfunction) together determined local NO levels in the 
vasculature and hence affected tissue blood flow.  Thus, when NO bioavailability declines below a critical 
threshold (precipitated by saRBC transfusions, for example), local blood flow and O2 delivery are insufficient to 
meet tissue demands, resulting in end-organ complications. We now present data that validates key tenets of 
the INOBA hypothesis (please see preliminary data). In particular, we have shown in both in vitro model 
systems13 and in human transfusion studies that saRBCs (but not fresh RBCs) significantly disrupt NO-mediated 
vasodilation.   
The role of RBC storage time in transfusion-associated mortality and morbidity:  Even when the 
well-recognized non-infectious hazards of transfusion are excluded from analysis, transfusion (particularly of 
RBCs) remains an independent predictor of morbidity and mortality.2,3,5,6,18,20,23-34 Tinmouth and Wang have 
performed systematic reviews of dozens of studies that investigated the relationship between blood storage 
interval and adverse transfusion events 4,18.  Meta-analyses showed worse recipient outcomes following 
transfusion of saRBC.  Since the largest clinical studies included in these reviews were retrospective, further 
elucidation of the possible adverse effects of saRBCs may be provided by prospective randomized trials.  The 
largest to be published to date, ARIPI 15, compared fresh blood (stored up to 7 days; median = 5 days) with 
standard of care (stored up to 42 days;  median = 13 days) in low-birthweight neonates. Although the outcomes 
showed no difference between study arms, it is important to note that only 25% of the units transfused in the 
standard issue group were stored for > 20 days, and thus the study does not address the safety of RBC units 
near the end of the 42 day storage period. In fact, our results using in vitro aortic ring assays (see preliminary 
data) suggest that the vasoinhibitory effects of saRBCs first manifest after 21 days of storage13.  The possibility 
of adverse clinical outcomes following transfusion of saRBCs near outdate (35-42 days) may be better addressed 
in the ongoing RECESS 35, ABLE 16, and Red Cell Storage Duration and Outcomes in Cardiac Surgery studies 
17.  
The role of endothelial- and RBC-derived nitric oxide (NO) in the control of vascular tone:  The 
endothelium plays a crucial role in actively regulating vascular tone. This effect depends on tightly regulated 
release of vasodilators (NO, prostacycline, and endothelium-derived hyperpolarizing factor [EDHF]) and 
vasoconstrictors (endothelin and thromboxane).36-38 NO is synthesized in the endothelium by NO synthases 
(NOS) and acts in a paracrine manner by diffusing to the underlying smooth muscle where it causes muscle 
relaxation, and thus increases vessel diameter and blood flow to local tissues.  
Intrinsic RBC mechanisms that regulate NO levels have also been postulated to play a role in controlling local 
blood flow in order to preferentially perfuse, and thus provide O2, for the most hypoxic tissues.1 RBCs may 
participate in this process of hypoxic vasodilation in order to match blood flow (and O2 delivery) with tissue 
needs.39 Evidence suggests that this “endocrine” form of NO signaling by RBCs may affect NO bioavailability via 
at least three potential pathways:  1) S-nitrosylation of Hb (SNO-Hb), 40,41 in which NO is captured by the cys β-
93 on the R (oxygenated) conformation of Hb creating a storage form of NO which can then be released as 
bioactive NO when pO2 is reduced and the Hb transitions to the T-state (deoxygenated). Thus, RBCs may serve 
as a storage pool for NO, forming SNO-Hb under oxygenated conditions for subsequent downstream release of 
NO to promote vasodilation when O2 tension is low 1,6,21,42,43 2) Nitrite, present in high concentrations in plasma, 
can be converted to NO by Hb in hypoxic conditions.44 In this model, deoxygenated Hb binds nitrite and a proton 
to generate NO and met-Hb. The NO can then leave the RBC to cause vasodilation or can bind a second deoxy-
Hb molecule to form iron-nitrosyl-Hb (HbFe2+-NO). With either mechanism, NO must escape from the RBC to 
act on smooth muscles and produce vasodilation, although the mechanisms of NO egress are unclear.45,46 3) By 
promoting O2 release and stabilizing deoxy-Hb, 2,3-DPG likely facilitates the production of NO through this latter 
pathway. Thus, stored RBCs with depleted 2,3-DPG may be deficient in NO production. Interestingly, the time 
course over which 2,3-DPG is completely depleted (after ~ 14 days of storage) correlates with studies showing 

 
5 
 
that RBCs stored >14 days are more likely to cause morbidity and mortality.3,5 With any of these three pathways, 
its reasonable to postulate that storage of RBCs prior to transfusion may disrupt these mechanisms of NO 
production/release, and thus transfusion of saRBCs may impair NO bioavailability in the transfusion recipient.   
Alternatively, transfused saRBCs could produce an inhibitory factor that interrupts normal NO signaling 
between endogenous RBCs, endothelium and the underlying smooth muscle thereby allowing a small volume 
of transfused saRBCs to broadly impair hypoxic vasodilation.  Disruption of NO signaling by saRBCs could be 
mediated by free hemoglobin (or other RBC constituents) released into the plasma as a consequence of RBC 
hemolysis, or by hemoglobin or other factors encapsulated in RBC-derived microparticles.45-47 Under normal 
circumstances, Hb is isolated from the endothelium by encapsulation within RBCs that tend to flow in center of 
the blood stream and away from the endothelial wall.45,46,48 Microparticles may be important because they can 
flow closer to the endothelium than intact RBCs, bringing hemoglobin close to the sites of NO synthesis, which 
may further accentuate NO scavenging after transfusion.11 There also appear to be other mechanisms by which 
saRBCs can inhibit NO signaling, For example, our studies have indicated that plasma supernatant constituents, 
including free hemoglobin and microparticles, were not the only cause of vasoconstrictive effects in an aortic ring 
model13 (see preliminary data section).  Removal of the supernatant from saRBCs, either through volume 
reduction or washing, did not improve NO-mediated vasodilation. While addition of day 42 supernatant alone to 
the aortic rings did inhibit NO-mediated vasodilation, the effect was only significant when compared to aortic 
rings with no supernatant added;  there was no difference in ring vasodilation when day 42 supernatant was 
compared to day 0 supernatant. Thus, while free Hb exerts some inhibitory effects on NO signaling in this model, 
it does not appear to account for the majority of the vasoinhibitory activity seen with stored RBC samples.  Rather, 
the saRBCs themselves appear capable of inhibiting NO-mediated vasodilation, as others have recently 
suggested 12.  This effect may not occur through NO scavenging, but instead through suppression of endothelial 
NO production, a possibility we are currently investigating.  Furthermore, we note that these in vitro vasoinhibitory 
effects are comparable to the inhibition of NO-mediated vasodilation we have observed in hospitalized patients 
transfused with saRBCs, but not fresh blood (manuscript in preparation;  see preliminary data).  Thus, there are 
several potential mechanisms by which saRBCs may impair endothelial NO signaling and the normal 
vasodilatory response to hypoxia. 
Endothelial function and dysfunction: The endothelial dysfunction associated with chronic illnesses 
such as CVD is primarily due to reduced NO bioavailability and correlates with the traditional cardiovascular risk 
factor “burden”, such as hypertension and hypercholesterolemia.9,49,50 The presence and magnitude of 
endothelial dysfunction has significant clinical implications as it is an independent predictor of adverse long-term 
cardiovascular outcomes even in patients without overt atherosclerosis.10,51-61  Furthermore, clinical interventions 
can alter endothelial dysfunction.62,63 Many of the subjects of the clinical studies showing adverse effects of RBC 
storage had coronary artery disease risk factors, or were critically ill, and likely had some degree of endothelial 
dysfunction. Non-invasive methods for measurement of NO mediated vasodilation, and endothelial dysfunction, 
include brachial artery flow mediated dilation (FMD) and pulsatile arterial tomography (PAT). FMD represents a 
well-validated, non-invasive, ultrasound based approach to assess conduit vessel endothelial function.64 PAT 
measures blood flow in the digital microvessels. The digital reactive hyperemia index (RHI) measured using PAT 
correlates with coronary and peripheral endothelial function, is largely NO-dependent, and is an independent 
predictor of cardiovascular events.65 Used in concert, FMD and PAT provide a sensitive, non-invasive 
assessment of endothelial function and thus NO bioavailability in both the conductance and resistance vessels.  
Physiologic effects of defective saRBCs on vascular function:  In addition to our in vitro and in vivo 
studies described above and discussed in more detail in the preliminary results section, other studies have also 
investigated physiological interactions between saRBCs and blood vessels. Using aortic ring assays and animal 
transfusions, saRBCs have been shown to reduce NO-mediated vasodilation as compared to fresh RBCs.5,6 In 
a recent study in patients undergoing hemodialysis, hyper-hemolysis was associated with impairment of FMD, 
suggesting a measurable role for NO scavenging by free hemoglobin.66 However, no change in RHI using PAT 
was reported with transfusion of fresh (3 day) vs. stored (40 day) autologous transfusions in healthy adults.14 It 
is quite possible, however, that healthy adults who lack pre-existing endothelial dysfunction are less susceptible 
to saRBC-induced reductions in NO bioavailability (as postulated in the INOBA hypothesis67).  For example, we 
have seen impairments in NO-mediated vasodilation in hospitalized patients transfused with saRBCs, but not 
fresh RBCs (see preliminary data section).     
Transfusion and Cardiopulmonary Exercise Testing: Hypoxic vasodilation results in matching O2 
supply with demand. At the level of the whole organism, cardiopulmonary exercise testing (CPX) measures 

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8 
 
Rationale: 
In our preliminary studies, we demonstrated that saRBCs (but not fresh RBCs) impair NO-mediated vasodilation 
when studied using in vitro models as well as in hospitalized transfusion recipients. Some limitations of studying 
transfusion outcomes in hospitalized patients include the heterogeneity of this population, unpredictable times 
of transfusions, urgent/emergent circumstances, and a lack of standardization of inpatient covariates including 
time of day, diet and other associated therapies. Vascular tests (FMD and PAT) and measures of circulating NO, 
oxidative stress and inflammation are sensitive to co-morbidities, diet and medications. Finally, dynamic changes 
in patients’ condition can also potentially confound experimental results. Conversely, transfusion studies can be 
performed in healthy volunteers.  However, these subjects rarely have underlying endothelial dysfunction (which 
may synergize with saRBCs and magnify their effects on NO bioavailability) and for ethical reasons cannot be 
rendered anemic or transfused with allogeneic blood.  Thus, transfusion effects in healthy volunteers may not 
necessarily be reflective of outcomes seen in sick patients.  
In order to overcome these limitations, and to investigate the relationships between blood storage, reduced NO 
bioavailability, impaired vasodilation, and physiologic outcomes of transfusion, we propose to investigate the 
effects of saRBC transfusions in a chronically transfusion-dependent but ambulatory population. This approach 
has the following advantages: 1) these patients are stable over the course of days to weeks, 2) studies are 
performed under conditions of controlled diet, circadian rhythms, medications, etc., 3) the same subject receives 
both fresh and saRBCs in a randomized cross-over study design (see schema in Fig 4), and 4) because these 
patients are ambulatory, they can undergo cardiopulmonary exercise testing. Successful completion of the 
proposed studies will advance our understanding of mechanisms underlying the adverse events and functional 
consequences of saRBC transfusion and validate a model for subsequently evaluating potential treatments and 
storage techniques to reverse saRBC-mediated impairments (the latter objectives are beyond the scope of the 
present investigations).   
Specific Aims: 
 
Aim 1: To assess the effects of fresh vs. saRBC transfusions on NO-dependent endothelial function in 
chronically anemic adults using: 1) flow mediated dilation (FMD) and 2) reactive hyperemic index (RHI) measured 
by pulsatile arterial tomography (PAT). The primary endpoint of this Aim is the change in FMD with fresh vs. 
saRBC transfusion;  The secondary endpoint is change in RHI.  
 
Aim 2: To assess the effects of fresh vs. saRBC transfusions on functional exercise capacity and oxygen 
carrying capacity by cardiopulmonary exercise testing. The primary endpoint is the change in peak oxygen 
consumption (VO2Max) after transfusion.  
 
Secondary endpoints for the above Aims are changes in metabolic markers before and after 
transfusion/exercise: inflammation (IL-2, IL-6, hsCRP), oxidative stress (glutathione/glutathione disulfide and 
cysteine/cystine redox potentials), and nitric oxide metabolites (nitrite, nitrate, SNO-Hb and SNO-thiols).  
Hypotheses:  In patients with chronic transfusion-dependent anemia, transfusion of saRBCs will impair vascular 
reactivity compared to transfusion of fresh RBCs (Aim 1). Impairment of NO-mediated vasodilation will be 
associated with decreased exercise tolerance and peak oxygen consumption (VO2Max) following saRBC 
transfusion as compared to fresh RBCs (Aim 2), and will be associated with increased systemic oxidative stress 
and inflammation, and decreased circulating NO metabolites (Secondary endpoints).   
Subject Population: Twenty-four subjects diagnosed with stable transfusion dependent anemia will be recruited 
and undergo a screening health questionnaire and physical examination to ensure eligibility. All subjects will 
provide informed consent prior to entry into the study. We will recruit adult subjects without preference to any 
race, gender or socioeconomic status. Dr. Quyyumi’s translational research group in the Department of 
Cardiology regularly enrolls patients into clinical studies, and has experience consenting patients, randomizing 
them to study arms, and performing study protocols including the use of these non-invasive assays.65,72-74 This 
clinical research team oversaw execution of the clinical study comparing fresh vs. saRBC transfusions in 
hospitalized patients described in the preliminary results,  Furthermore, we have also consulted with our 
Hematology colleagues, and have identified a large number of potential subjects who fit our criteria for this study.  
The proposed study has been approved by the Emory IRB. 
Eligibility Criteria: Male or female volunteers age 21-80 with any condition resulting in RBC transfusion-
dependent anemia will be eligible to participate. Participants may receive either 1- or 2-unit transfusions as 
required clinically so long as their transfusion requirement is stable throughout the study period.  

 
9 
 
Ineligibility Criteria: Age <21 or > 80 years, pregnancy, acute infection in previous 4 weeks, active 
substance abuse within the past year, inability to give informed consent, inability to return for follow-up or the 
presence of alloantibodies that would limit the blood bank’s ability to obtain correctly aged RBC units. Participants 
with contraindications to cardiopulmonary exercise testing may be allowed to participate in the other study aims.   
Study Design:  Studies will be performed in a temperature-controlled vascular laboratory in the outpatient unit 
of the Clinical Research Network, ACTSI, at Emory University Hospital after an overnight fast. After providing 
informed consent, subjects will be randomized to receive their next clinically-indicated outpatient transfusion with 
fresh (<10 days old) blood or storage-aged (>21 day old) blood.  Following their first transfusion, subjects will be 
crossed-over to receive the opposite aged product. Subjects and individuals conducting and interpreting 
experimental tests will be blinded to the age of RBC units. To maintain safety, clinical staff will not be blinded.  
All subjects will receive clinically indicated transfusions with cross-matched, leukoreduced AS1 units of packed 
RBCs from available blood bank inventory. The effects of RBC transfusion on blood flow and peripheral O2 
delivery will be directly tested in the 24 volunteers using non-invasive assessments of endothelial function 
including: brachial artery flow-mediated dilation (FMD) and pulsatile arterial tonometry (PAT). Symptoms of 
fatigue and anemia will be objectively assessed at every visit using the self-administered FACT-An questionnaire 
(Functional Assessment of Cancer Therapy – Anemia). Vascular function will be assessed prior to transfusion 
as well as 1 hour and 24 hours after blood transfusion. Concurrently, subjects will undergo cardiopulmonary 
following each transfusion.  Blood samples will be obtained at baseline, prior to and following each transfusion 
and from each unit transfused to perform metabolic marker testing. 
Study Schedule (Figure 4, below): Each subject will be studied over a total of 4 visits, as shown in the Figure 
and described below.  
 
Screening: Subjects will be screened with history and physical for inclusion and exclusion criteria and 
provide written consent. Subjects to meet inclusion and exclusion criteria, and give consent, will be randomized 
into either Group A or Group B. 
Visit #1 (Transfusion #1): Subjects will return fasting to receive a scheduled, clinically-indicated RBC 
transfusion.  Depending on their randomization to Group A or B, they will receive either fresh or saRBCs; an 
infusion pump will be used to administer the blood over a 1 hour period.  FMD and PAT and will be performed 
and blood samples will be obtained for measurement of oxidative stress and other metabolic markers prior to 
and 1 hour following RBC transfusion.  
Visit #2 (Post-transfusion #1): Subjects will return fasting the day following transfusion. FMD and PAT 
will be performed and blood samples will be obtained. CPX will be performed. The visit #3 (and #5) time points 
are important to this study since our preliminary data showed that impairment of NO-mediated vasodilation by 
saRBC transfusion was greatest 24 hours after transfusion.   
 
Visit #3 (Transfusion #2): Visit #3 will recapitulate Visit #1 except that subject will receive the opposite 
aged product, as shown in the cross-over study design schema (Fig 4).  
 
Visit #4 (Post-transfusion #2): Visit #4 will recapitulate Visit #2.  
Procedures: 

 
10 
 
Blood Transfusion: Participants will receive either 1 or 2 packed RBC units as ordered by their attending 
physician. Units will be infused intravenously via programmable electronic infusion pump (Baxter, Inc) over 1 
hour per unit. The subjects will receive only cross-matched leukoreduced units. No pre-medication will be 
permitted. Subjects will be monitored closely for any reactions during transfusion following standard hospital 
policy. In the event that blood units of the prespecified age are not available, patient will receive their clinically 
indicated transfusion with blood units of any age per hospital protocol. This would be considered a protocol 
deviation and those patients will then be excluded from the study 
Study Measurements: 
 
Endothelial function assessment -- Brachial artery flow-mediated vasodilation (FMD):  All studies will be 
performed in a temperature-controlled vascular laboratory after an overnight fast. The brachial artery of the non-
dominant arm will be imaged using a high-resolution 13 MHz ultrasound transducer (Acuson) as previously 
described in detail and as used for the studies shown in Fig 3 of the preliminary results section.10,75,76  Briefly, a 
blood pressure cuff on the forearm will be inflated to supra-systolic pressures to produce 5 minutes of ischemia.  
On cuff deflation, reactive hyperemia produces an acute increase in shear stress with an accompanying release 
of NO.  Imaging of the brachial artery is performed continuously for the next 120 seconds, providing the data 
necessary to calculate flow-mediated dilation.  After a 10 minute recovery interval, sublingual nitroglycerin will 
be given (0.4 mg) and the brachial artery will be re-imaged after 3 minutes.  Image analysis will be performed by 
an experienced FMD investigator who is blinded to the treatment arm.  Arterial diameter will be measured using 
a validated proprietary software program (Medical Instruments Inc.).  Brachial artery FMD will be calculated as 
[(postischemia diameter - baseline diameter) / baseline diameter] X 100. 
 Reproducibility: FMD is well-accepted technique64. In our laboratory, the mean difference in FMD (%) between 
2 consecutive assessments performed in 11 subjects an average of 8 days apart was 1.26% (±0.76%, r=0.75). 
The mean difference in the FMD (%) between 2 readings of the same 11 measurements was 0.82% (±0.48%, 
r=0.97). (unpublished data) 
 
Endothelial function assessment – Reactive Hyperemia Index (RHI) as measured using Pulsatile Arterial 
Tonometry (PAT, Itamar Medical Ltd.):  This assay uses a device that measures reactive hyperemia of the finger. 
The magnitude of hyperemia correlates with coronary microvascular endothelial function, peripheral vascular 
endothelial function measured as brachial reactivity, and can be inhibited by LNG monomethyl arginine, indicating 
that it is NO-dependent.  We are employing this technique currently in our catheterization lab population and 
have found it to be easy to use and reproducible.65   
Technique:  Subjects will be studied in the supine position in a temperature-controlled room.  Baseline blood 
pressure of both hands is measured and PAT probes are placed one on each hand at the same finger (fingers 
2, 3 or 4).  Following a 10 min. equilibration period, the blood pressure cuff is inflated to 60 mmHg above systolic 
pressure for 5 minutes followed by deflation of the cuff while the pulsatile recordings from both study and control 
fingers are measured.   
 
Cardiopulmonary Exercise Testing (CPX): Graded treadmill exercise testing will be performed following 
American Heart Association guidelines using the Modified Balke protocol.68 A treadmill with full metabolic cart 
(Carefusion INC, USA) will be used for CPX. Testing measurements will include 1) Maximal oxygen uptake 
(VO2Max):  This is the Primary Endpoint of Aim 2 and is defined as the value achieved when VO2 remains stable 
despite a progressive increase in the intensity of exercise. This is synonymous with peak aerobic capacity. 
Secondarily, CPX will report: 2) Respiratory exchange ratio (RER): Related but not equivalent to its cellular 
counterpart, the respiratory quotient, RER is defined as the ratio of VCO2 to VO2. 3) O2 pulse: The amount of O2 
consumed from the volume of blood delivered to tissues by each heartbeat; this index is calculated as: O2 pulse 
= VO2 / heart rate. 4) Peak VO2lean: the peak oxygen uptake adjusted for lean body mass. Reported as a lean 
body weight–adjustment parameter in mL/kg per minute. These data are then integrated with heart rate, blood 
pressure, work rate, electrocardiography findings, and the occurrence of any clinical symptoms for a 
comprehensive analysis of exercise tolerance before and after transfusion.  
 
Blood Testing: Biomarkers of oxidative stress (glutathione/glutathione disulfide and cysteine/cystine 
redox potentials) are measured in an Emory University Core Lab directed by Dr. Dean Jones, and available for 
use by all Emory investigators. Dr. Jones is an internationally recognized expert in the area of glutathione (GSH) 
and cysteine metabolism, and has substantial experience with measurements of reactive oxygen species.  He 
has optimized HPLC methods for quantitation of oxidative stress markers.  NO metabolites (nitrate, nitrite, and 
nitrosylated hemoglobin and thiol) levels will be measured in Dr. David Lefer’s laboratory at Emory (see support 

 
11 
 
letter). 2,3-DPG, plasma free hemoglobin and inflammatory marker levels will be measured in Dr. Roback’s 
laboratory.  
Expected outcomes:  
Specific Aim 1: FMD and RHI at 24 hours hour post-transfusion with saRBCs will be lower than following 
transfusion of fresh RBCs in the same patient.  
Specific Aim 2: Exercise tolerance and VO2Max will increase following fresh RBC transfusions;  in 
contrast, transfusion of saRBC will either lead to a lesser increase in these markers or will produce an actual 
decrease in these parameters.  
Secondary Endpoints: Markers of oxidative stress (glutathione and cysteine redox ratios) and 
inflammatory markers will show a greater increase following saRBC transfusion than after fresh transfusions. 
Potential Limitations and Alternative Techniques:  
Since we have previously observed significant differences in FMD following transfusion of saRBCs, but not fresh 
RBCs, in hospitalized subjects, we do not anticipate difficulty measuring vascular reactivity in response to 
transfusion in this ambulatory chronically-transfused patient population. In fact, the proposed design using 
outpatients allows for improved adherence to optimal FMD and PAT technique including control of study timing 
relative to transfusions, medications, diet and circadian rhythms64. Furthermore, a cross-over design allows for 
control of inter-individual variation which was a significant limitation of our prior mechanistic studies. It is possible 
that some of these patients do not have the level of preexisting endothelial dysfunction we observed in the 
hospitalized population. If this is the case, and it turns out to limit our ability to measure the physiologic effects 
of saRBC transfusions, we can narrow the study population to transfusion dependent-individuals with baseline 
cardiovascular disease. 
Given that this is a chronically ill population, we may have difficulty recruiting participants may who are able to 
complete the CPX procedure. However, as demonstrated by our preliminary data in patients with peripheral 
arterial disease, we have previously had success measuring exercise outcomes in other populations with 
functional physical limitations.  We are also prepared to employ other exercise protocols, if necessary, including 
the modified Balke or Gardner protocols for use with treadmill testing, or by utilizing a seated cycle ergometer 
as opposed to treadmill.  
Statistical considerations: 
Sample size justification for Aim 1: Subject numbers are based on the primary objective (Aim 1) of 
measuring change in FMD (%) with transfusion.  For sample size calculations, we use the paired t-test statistic 
and assume a two-sided test with α=0.05 and power = 0.8. Based on the FMD changes we observed in 
transfused hospitalized patients (see preliminary data) of 2.4% with saRBC transfusion and a standard deviation 
of FMD in our lab of 3.11%, with a sample size of 24 we have power to observe a 0.85% change in FMD with 
blood transfusion. This is a clinically meaningful change.10   
 
Sample size justification for Aim 2:  Subject numbers are based on the primary objective of measuring 
change in VO2Max with transfusion of fresh vs saRBCs (Aim 2). A sample size of 24 achieves 86% power to 
detect a VO2Max difference of 130 ml/min between fresh and saRBC transfusions. These calculations are based 
on a mean of 1100 and the alternative hypothesis mean of 1230 with an estimated standard deviation of 190.0 
and with a significance level (alpha) of 0.05 using a two-sided Wilcoxon test assuming that the actual distribution 
is normal. This calculation is based on published VO2Max responses to exercise in an anemic population.71 
 
Statistical analysis will be performed in consultation with faculty statisticians within the Emory Clinical 
Cardiovascular Research Institute. Descriptive statistics will be performed initially by calculating means, standard 
deviation, range, descriptive plots, and box-plot histograms for each variable.  This will assist in determining 
distributional properties of the outcome variable and the choice of appropriate subsequent analytical approaches 
(parametric vs non-parametric).  For most analyses, we will use a generalized linear mixed model to analyze 
these data provided that outcomes are normally distributed. Otherwise we will use transformation methods. 
Given that there are multiple hypotheses with multiple outcomes it is necessary to adjust for multiple 
comparisons.  With the proposed sample size, we will not adjust for multiple comparisons with different outcomes 
but will adjust for comparisons within each outcome. The sample size may be too small for certain outcomes 
given the heterogeneity of subjects: therefore we will interpret results carefully, particularly with secondary 
outcomes.  In such cases we will report the confidence intervals.   

 
12 
 
Time table:   
Subjects will be enrolled concurrently into Aims 1 and 2 over the first 18 months of the grant period. Data will be 
recorded continuously and analyzed over that same period with manuscript preparation in months 18-24.  
 
Year 1 
Year 2 
Subject enrollment -Aim 1 and 2 
 
 
 
Data analysis 
 
 
 
 
 
 
 
 
 
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