VO2 Max: In Vivo Model for Functional Red Cell Testing. Can RECESS be Explained?
Phase: PHASE4
Conditions: Healthy
Sponsor: Stony Brook University

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VO2 Max: In Vivo Model for Functional Red Cell Testing. Can RECESS be Explained? 
NCT02918851 
June 12, 2017 
 
 

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TITLE: VO2 Max: In Vivo Model for Functional Red Cell Testing. Can RECESS be Explained? 
STUDY PERSONNEL: 
Elliott Bennett-Guerrero, MD (PI) 
Professor and Vice Chair for Clinical Research & Innovation, 
Department of Anesthesiology 
 
Medical Director, Perioperative Quality & Patient Safety, Stony 
Brook Hospital 
Dennis K. Galanakis, MD (Co-I) 
Professor of Pathology and Medicine 
Director of Blood Bank, Stony Brook Hospital 
Rany Makaryus, MD (Co-I) 
 
 
Clinical Assistant Professor, Department of Anesthesiology 
Jun Lin, MD (Co-I) 
 
 
 
Clinical Associate Professor, Department of Anesthesiology 
Russell Rozensky, MS (Co-I)  
 
Clinical Associate Professor for Respiratory Care Program 
 
 
 
 
 
 
Program Director, Polysomnographic Technology Program 
John Brittelli, MS (Co-I) 
 
 
Clinical Assistant Professor for Respiratory Care Program 
Research Coordinator, Polysomnographic Technology Program 
Ann Cuccia, MPH (Co-I) 
 
 
Clinical Assistant Professor for Respiratory Care Program 
Jamie Romeiser, MPH (Co-I) 
Health Research Scientist, Department of Anesthesiology 
Sabeen Rizwan, BA (Study Coordinator) 
Clinical Research Associate, Department of Anesthesiology 
 
Jean Abbott, MT (Study Coordinator) 
Clinical Research Associate, Department of Anesthesiology 
 
 
 
A. SPECIFIC AIMS 
 
1) To confirm our pilot data in a larger sample size and show that 42-day RBCs do not deliver oxygen as 
effectively as 7-day RBCs, and  
2) To explain the RECESS results, we hypothesize that 28-day RBCs are not inferior to 7-day RBCs in our 
model 
 
B. BACKGROUND AND SIGNIFICANCE 
 
RBCs 
progressively 
develop 
biochemical, 
immunologic, 
structural, 
and 
physiological 
abnormalities during storage, collectively referred 
to as the “storage lesion”.1 For example, 
morphologic differences in fresh vs older stored 
RBCs (photos right) are striking.  
 
These changes may be clinically significant since 
several observational studies have reported that 
transfusion of older RBCs is an independent predictor of morbidity and/or mortality.2-8 Furthermore, in animal 
lethality models, randomization to the oldest RBCs (42 days) results in higher mortality.9,10 Recently completed 
randomized trials, i.e. ABLE and RECESS, essentially comparing “young” (median 7)11 vs “middle-aged (median 
28-day)11 RBCs, show no difference in clinical outcome. These data are important, since most patients are 
exposed to RBCs in this spectrum of age. However, they do not inform us about the safety and effectiveness of 
old RBCs, which some patients receive. Due to pragmatic and ethical reasons a large clinical trial that 
randomizes patients to “old” blood may not be feasible. We have generated compelling preliminary data in a 
healthy volunteer model of strenuous exercise that strongly suggests that old RBCs (42 days) do not deliver 
oxygen as effectively as fresh (7 day) RBCs. The 3 arm (7-, 28-, 42-day RBCs) VO2 max study we propose is 

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designed to address 2 important hypotheses: 1) to confirm our pilot data in a larger sample size and show that 
42-day RBCs do not deliver oxygen as effectively as 7-day RBCs, and 2) to explain the RECESS results, we 
hypothesize that 28-day RBCs are not inferior to 7-day RBCs in our model.  
 
The proposed study is significant for several reasons: 1) We believe that the proposed study will confirm our 
very encouraging preliminary data, showing that the oldest RBCs allowed by FDA regulations (42 days) are 
inferior to 7-day RBCs in a functional assay of oxygen delivery. This is important scientifically since, to our 
knowledge, no studies have definitively shown that storage duration can affect RBC function, i.e. oxygen delivery; 
2) The proposed study is also significant because it is expected to shed light on why the RECESS and ABLE 
outcome trials, comparing young and “middle-aged” RBCs were negative. We hypothesize that 28-day RBCs 
are not inferior to 7 day RBCs, which are exactly the median ages of storage for the 2 study arms in RECESS.11 
3) Finally, we suggest that this model holds promise as a rigorous and quantitative in vivo functional assay of 
RBC function, which can address the quality of the oldest red cells since a clinical trial is likely not feasible and 
also addresses an unmet need for future RBC transfusion research as identified by NHLBI and others. 
 
Potential Importance of RBC Duration of Storage 
The US Food and Drug Administration (FDA) regulations permit additive-containing RBCs (AS-1, AS-3, AS-5) to 
be stored for up to 42 days prior to transfusion. The efficacy of older stored blood, i.e., effective oxygen delivery, 
has not been established in rigorous controlled studies. It is well known that RBCs progressively develop 
biochemical and physiological abnormalities during storage, and our group has been responsible for generating 
some of these data.1 Changes during storage may be of clinical significance since accumulating evidence has 
linked transfusion of RBCs stored for a longer period of time with increased morbidity and mortality compared 
with transfusion of fresher RBCs, although a number of studies have failed to find such an association.12-16 These 
study designs have mostly been observational and retrospective; therefore, residual confounding is a possibility, 
and it may not be possible to establish causality between adverse outcome and transfusion of older blood.17  
 
Recently completed randomized trials, e.g. ABLE and RECESS, essentially comparing “young” (median 7)11 vs 
“middle-aged (median 28-day)11 RBCs, show no difference in clinical outcome. These data are important, since 
most patients are exposed to RBCs in this spectrum of age. However, they do not inform us about the safety 
and effectiveness of older blood. Due to pragmatic and ethical reasons, a large clinical trial that randomizes 
patients to “old” blood may not be feasible.  
 
Therefore, we have developed a model of autologous transfusion in conjunction with strenuous exercise that 
leverages an established quantitative and reproducible test of oxygen delivery (VO2 max) to address important 
gaps in knowledge. Prior to describing our preliminary data, we summarize below previous efforts to use 
physiological tests to assess the impact of storage duration on various endpoints. 
 
Limitations of Previous “Physiologic” Studies of RBC Storage Duration 
Previous studies involving the impact of RBC storage duration on surrogates of tissue perfusion and oxygenation 
showed conflicting results, often due to limitations in the experimental techniques used. In a non-randomized 
study, Kiraly et al showed a significant decrease in peripheral tissue oxygenation (StO2) of anemic, critically-ill, 
trauma patients following transfusion of older RBCs (≥ 21 days) compared to patients who received a transfusion 
of fresher RCBS (≤ 21 days).18 In contrast, in a randomized study comparing transfusion of 2 units of fresh (≤ 5 
days) vs. old (≥ 20 days) RBCs, Walsh et al found that transfusion of older RBCs to euvolemic, anemic, critically-
ill patients had no clinically significant adverse effects on gastric tonometric or global indices of tissue 
oxygenation.19 In their randomized study, Weiskopf et al found that RBCs stored for 3 weeks were as effective 
as RBCs stored for 3.5 hours in reversing a neurocognitive deficit induced by acute anemia (hemoglobin 5 g/dL) 
in healthy volunteers.20 Two studies (Kor et al,21 Weiskopf et al22) focused on the effect of stored blood on 
pulmonary function, and did not assess functional measures of oxygen delivery. Marik and Sibbald failed to 
demonstrate a beneficial effect of transfusing 3 units of RBCs on measured systemic oxygen uptake in patients 
with sepsis.23 Patients in their study who received RBCs stored for more than 15 days developed evidence of 
splanchnic ischemia; however, this study was observational and not randomized. 
 
C. PRELIMINARY STUDIES 
 
Preliminary Data of VO2 Max After 7 vs 42 day Stored AS-3 RBCs 

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Limitations in these previous studies led us to propose strenuous exercise (VO2 max testing) for studying 
potential differences in oxygen delivery attributable to duration of storage. We recently completed a pilot study 
in which 8 healthy subjects had 2 units of leukocyte-reduced RBCs collected (by apheresis) in AS-3 using 
standard methods. This was done at Rex Hospital (a blood collection facility near Durham) since Duke 
Transfusion Service does not collect RBCs. Subjects were randomized to receive both (2) units of their 
autologous RBCs at either 7 or 42 days following blood collection. VO2 max testing was performed 2 days before 
(Monday) and 2 days after (Friday) the transfusion visit (Wednesday). This study design avoids the confounding 
effects on intravascular volume from the 2-unit blood transfusion if VO2 max tests were performed before and 
after transfusion on a single day. In other words, we sought to assess the impact of transfused RBCs on exercise 
performance independent of its impact on intravascular volume. While our research group had experience 
collecting and administering autologous RBCs24 as well as conducting VO2 max tests,25-35 we had never 
combined these interventions and wished to collect preliminary data that would inform the feasibility of 
conducting the study we propose in this grant submission. 
 
Nine subjects were consented but 1 failed screening tests (abnormal pulmonary function testing) and 8 
completed the study. There was strong interest from fit habitual exercise enthusiasts to participate in this type of 
study and enrollment was completed quickly. Subjects in both study arms, encouraged by the same individual 
(M Natoli), exercised to exhaustion with high peak heart rates, e.g. mean of 184 ± 5 vs 184 ± 11. Despite the 
small sample size (4 per study arm) we observed intriguing differences in VO2 max (p=0.2) and exercise duration 
(p=0.02) between study arms (figures below). Indeed in the 42-day arm, in half of the subjects, VO2 max and 
exercise duration actually decreased (was worse) after transfusion with 2 units of 42 day RBCs, whereas all 4 
subjects who received 7 day RBCs exhibited increases in these endpoints. Of note, in order to avoid potential 
confounding from differences in 24-hour viability of 42- vs 7-day RBCs, we only administered 85% of the 7-day 
RBC units, compared with 100% of the 42 day units. Therefore, these preliminary data should reflect differences 
in RBC quality/function and not RBC mass. Indeed, peripheral blood hemoglobin increased similarly (p=0.6) after 
transfusion of 2 units of 7- or 42-day RBCs using this protocol. To our knowledge, no previous study has 
demonstrated functional differences in oxygen delivery/exercise performance due to changes in RBC storage 
duration. We believe these preliminary data demonstrate the feasibility of using rigorous VO2 max testing as an 
in vivo model for RBC function, and are proposing a larger more rigorous study to confirm these observed 
differences. In addition, the proposed study will contain a 3rd study arm (28-day storage). This will add valuable 
information that may help explain the RECESS Trial results.11 Our hypothesis is that there is a difference in VO2 
max between the oldest RBCs (42-day) and fresh blood (7-day), but that, consistent with RECESS results, there 
is no difference in oxygen delivery between 7- and 28-day RBCs, which were the median duration of storage 
from the 2 RECESS study arms.11 
 
Before we describe our experimental approach we will present below general background information on VO2 
max testing and factors that affect it. 
 
Maximal oxygen uptake (VO2 max) testing: Background 
Maximal oxygen uptake (VO2 max) is the maximum rate at which the body can 
consume and use oxygen during incremental to maximum whole-body exercise. 
Oxygen consumption is linearly related to energy expenditure; so, measuring 
oxygen consumption is an indirect way to measure a person’s maximal capacity to 
do work aerobically. In order for oxygen to be used by the body, it must be 
extracted from the atmosphere and delivered to working cells. As shown in the 
Figure, oxygen consumption increases as exercise intensity increases until it 
plateaus, which represents the VO2 max. This occurs approximately 5-20 minutes 
into a standardized exercise protocol, with the duration depending on the subject’s age, level of fitness, and 
genetic factors. 
 

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Although indirect methods of calculating VO2 max 
have been used, the gold standard is graded exercise 
to exhaustion on a treadmill or cycle ergometer with 
direct measurement of O2 consumption using a 
metabolic cart to analyze inspired and expired gases. 
VO2 max can be expressed as L/min but is more 
typically reported as mL/kg/min to adjust for 
differences in body weight. The photo shows a female 
subject on a cycle ergometer. Her nares are occluded 
to force all respiratory gases to pass through the 
mouthpiece that is connected to the metabolic cart. 
She is looking at a monitor that shows her cadence (revolutions per minute), which must be maintained at a 
certain rate (75 rpm in our lab), while the resistance to pedaling increases incrementally. In the photo (right), a 
coach provides encouragement to a subject in our laboratory.  
 
Factors Affecting VO2 max 
The 4 main mechanisms that determine VO2 max are: absorption of O2 from the lungs, O2 transport by the 
circulation, O2 diffusion from the capillary blood to the mitochondria, and mitochondrial capacity.36 As reviewed 
by Pollock,37 VO2 max responds to changes in training. For example, 20 days of bed rest resulted in a decrease 
in VO2 max from 43.0 to 31.8 mL/kg/min, which then increased to 51.1 mL/kg/min after 60 days of training.38 
Elite athletes can have a VO2 max as high as 75-90 mL/kg/min.39 
While VO2 max can change with training and other interventions, it is remarkably constant over a period of days 
in the absence of any significant changes. For example, with a standardized protocol, i.e. absence of any 
interventions such as blood removal or transfusion, and use of the same “coach” to encourage the volunteers to 
exercise to exhaustion, VO2 max test results show very little intra-subject variability over several days. Relevant 
data are below.26,40  
 
 
Therefore, the reproducible and quantitative nature of VO2 max testing makes it ideal for studying interventions 
that may affect oxygen delivery. 
VO2 max: Previous Studies of Anemia/Hemoglobin Reduction and RBC Transfusion 
As previously reviewed,39,40 numerous studies regarding the effects of blood removal and transfusion on VO2 
max and/or exercise performance have been performed, some as early as 1940.41 This research has been driven 
in part by an interest in “blood doping,” a technique used to improve exercise performance.39,40,42,43 Many studies 
report the effects of blood removal and transfusion on both exercise performance and VO2 max; however, some 
report effects on only one of these outcomes.  
 
Blood Removal: Gledhill and others have shown that a reduction in arterial oxygen content (specifically a 
reduction in hemoglobin) results in a reproducible decrease in VO2 max.40 This is attributable to decreased 
arterial oxygen content,29,33,40 and not decreased cardiac output if VO2 max is measured at least 48 hours after 
removal of blood in order to allow the total plasma volume to normalize.40,44,45 Indeed, for hemoglobin levels 
ranging from 11-16 g/dL, each decrease in hemoglobin of 0.3 g/dL results in a 1% decrease in VO2 max.40  
 

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Blood Transfusion: Early studies (1976, 1980) in healthy volunteers failed to 
show an improvement in VO2 max after transfusing liquid RBCs, possibly due 
to the low quality of the stored RBCs.46,47 In contrast, later studies that 
transfused at least 2 units of cryopreserved, deglycerolized autologous RBCs 
to healthy volunteers showed consistent increases in VO2 max, even if pre-
transfusion Hb was as high as 16 g/dL.44,45,48-50 In other words, the increase 
in VO2 max does not require anemia at baseline to show improvements in 
VO2 max. For example, in 8 volunteers, Celsing et al removed 450 mL of 
whole blood 5 times, resulting in lower VO2 max at A (anemia; figure right).48 
One unit of cryopreserved RBCs was transfused on visits 1, 2, 3, and 4 over 
several weeks, resulting in a stepwise increase in both hemoglobin and VO2 
max (L/min). As expected, control subjects (n=8, dotted line) showed no 
change in hemoglobin or VO2 max. 
 
Our preliminary data are consistent with the above studies, since we showed a predictable increase in VO2 max 
after transfusion with 2 units of 7-day RBCs. None of these previous studies, however, determined whether 
storage duration impairs the ability of the RBCs to deliver oxygen, which was the focus of our pilot study and will 
be characterized further in the proposed study. 
 
D. RESEARCH DESIGN AND METHODS 
 
This is a single-center, randomized, partially blinded study.  
1) Overview 
After consent and screening the study interventions last 6 
weeks and consist of 2 phlebotomy visits at Stony Brook 
hospital and transfusion with 2 units of autologous AS-3 
RBCs that are either 7-, 28-, or 42-days old.  Subjects will 
be randomized (1:1:1) to either cycle A (7 day RBCs), 
cycle B (28 day RBCs, or cycle C (42 day RBCs).   
 
 
During week 6 of each cycle, subjects will have 3 study 
visits consistent with the pilot study we just completed.  
 
On Monday subjects will have a VO2 max test.  
 
On Wednesday, they will be transfused with 2 units of 
autologous RBCs (each over 1 hour), followed by a 
VO2 max test 2 hours later to assess the “acute” effects of transfusion (intravascular volume + RBC 
mass), which is a secondary endpoint of the proposed study. 
 
On Friday, they will have a VO2 max test, which is compared with the baseline VO2 max result (Monday) 
in order to calculate a delta VO2 max related to transfusion (primary endpoint), avoiding potential 
confounding from acute intravascular volume effects. 
 
Removal of waste unit(s): During each cycle, subjects will also have a second visit to Stony Brook For 
example, Cycle A has 2 units removed at Week 0, Cycle C has 2 units removed at Week 5. These units are 
removed for 2 reasons: 1) to maintain blinding for the 7- vs 42-day RBC comparison since subjects will not 
know whether they are receiving their 7- or 42- day RBCs; and 2) to induce mild anemia (projected Hb 11-
12 g/dL), which, while still extremely safe, may enhance increases in VO2 max from transfusion in order to 
detect more robust differences between study arms. We have numerous safeguards in place to protect 
human subjects (see Human Subjects section). 
Blinding: We cannot fully blind Cycle B (28-day RBCs). However, we plan to partially blind the subject by 
informing them that they may receive either 2 units of 28-day RBCs or 1 unit of 28-day RBCs and 1 unit of 
7-day RBCs. This “deception” should be allowed since it is inconsequential to the subjects, as they will 
already have agreed to receive 7- and 42-day RBCs in other cycles, and is merely done to improve blinding 
of the study. Notwithstanding the above, we believe this strategy is a reasonable compromise since VO2 max 
is a quantitative measurement and subjects are motivated fit athletes who, in our experience, exercise to 
exhaustion.25-35 Furthermore, subjects are not likely to have preconceived ideas/bias about the efficacy of 

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28-day RBCs.  In cycles A and C, 2 units of blood will be removed 7 days before the transfusion week.  
However, for cycle B, only 1 unit of blood will be removed 7 days before the transfusion week because their 
initial 2 unit phlebotomy will be at day 28 and not at day zero. They will have had 14 fewer days to recover 
hemoglobin compared to cycles A and C. Based on our estimates of RBC mass removal and restoration 
(synthesis), we anticipate that this will result in a pre-transfusion Hb that is similar to cycles A (7-day) and C 
(42-day). 
2) Research Site 
All study procedures will be completed at a single center, Stony Brook University Hospital. 
3) Study Sample 
Consistent with our pilot data, qualified volunteers will be enrolled at Stony Brook University Hospital. 
4) Screening 
Subjects will be preliminarily screened via private phone call made by the study coordinator on a hospital 
phone line, in a private office. In-person screening of subjects will take place in a private office space in the 
Health Sciences Center at Stony Brook University Hospital.  After subjects provide written informed consent, 
eligibility will be confirmed by a questionnaire and screening tests (below). We have used these methods 
before, and they are designed, in large part, to insure the safety of participants. 
Inclusion Criteria:  
i. 
Healthy male or female 
ii. 
Age 18-40 (American College of Sports Medicine Guidelines for Exercise Testing defines this age 
group, with ≤ 1 coronary heart disease risk factor, as low-risk for VO2 max testing.) 
iii. 
Habitual exerciser defined as ≥ 30 minutes of at least moderate or high intensity exercise ≥ 3 times 
per week. After consent, and at the subsequent screening visit, a VO2 max test will be performed, 
and subjects with a low value (< 35 mL/kg/min or unable to achieve at least 95% of maximum HR 
predicted from the standard formula 220 minus age) will be excluded (screen failure). Based on our 
previous experience, we anticipate that <10% of the subjects will fall into this category 
iv. 
Calculated total blood volume (TBV) ≥4,500 mL using an established formula:  
a. Men: (0.006012 x H3) + (14.6 x W) + 604 = TBV 
b. Women: (0.005835 x H3) + (15 x W) + 183 = TBV 
[H=height in inches; W=weight in pounds] 
ii. 
Has access to transportation to visit the blood collection facility and to return to Stony Brook for all 
study visits. 
Exclusion Criteria:  
i. 
Any significant acute or chronic medical illness or problem, including, but not limited to, diabetes, 
hypertension, cardiac disease, asthma, COPD 
ii. 
Current or recent (last 60 days) tobacco or nicotine use  
iii. 
History of sickle cell trait or disease or any other acquired or hereditary hematological abnormality 
iv. 
History of fainting or other significant adverse reaction during phlebotomy or donation of blood 
v. 
Known prolonged QTc (or evidence of such at screening) defined as QTc >470 ms 
vi. 
Known or suspected illicit drug or alcohol abuse 
vii. 
Known or suspected HIV, Hepatitis B, or Hepatitis C infection 
viii. 
History of thrombophilia or anticoagulant therapy 
ix. 
Pregnancy 
x. 
Obesity defined as BMI >30 kg/m2 
xi. 
Recent history of blood donation: a) Single whole blood unit donation within the past 8 weeks; b) 
Double RBC donation by apheresis within the past 16 weeks; or c) Plasma donation by apheresis 
within the past 4 weeks 
xii. 
Inadequate RBC mass based on TBV <4500 ml (above) or screening Hb <14.0 g/dL 
 
Screening tests (performed after consent is obtained): The following will be performed to confirm eligibility 
and optimize the safety of subjects: 1) written questionnaires that provide written confirmation of eligibility for 
blood donation and confirm appropriateness for exercise testing (PAR Q+ questionnaire);51 2) hemoglobin 
must be ≥ 14.0 g/dL; 3) hemoglobin A1c to rule out diabetes; 4) Sickledex to rule out sickle trait or anemia; 
5) ECG to rule out prolonged QTc; 6) height and weight to calculate and confirm required total blood volume 
(above); 7) spirometry to rule out undiagnosed pulmonary disease; and 8) serum hCG test if female 

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Subjects will be informed that during the study they may take a multivitamin but they should not take iron 
supplements. 
 
5) Procedures 
Blood Donation, Processing, and Storage (Visit B on Day 0, Visit C on Day 35) 
We will use the same procedures successfully used in our recently completed pilot study and our TASER 
study.24 Stony Brook’s Transfusion Services is registered and licensed by the FDA and accredited by the 
American Association of Blood Banks. Dr. Galanakis, medical director of Transfusion Services, is a co-
Investigator this study. 
After passing a written screen at Stony Brook University Hospital’s Blood Bank, 2 units of RBCs will be 
collected in CP2D via venous apheresis using a Trima Accel® Automated Blood Collection System: 
(http://www.caridianbct.com/location/emea/products-and-services/Pages/trima-accel-collection-
system.aspx). Blood will be processed by technicians at this center using standard techniques. All RBCs 
will be leukoreduced (pre-storage), tested for all required infectious diseases, stored in AS-3 additive 
solution. AS-3 RBCs will be used since apheresis products collected by Trima Accel® have only been 
validated for AS-3. There are no data to suggest that our results would not be generalizable to AS-1 or AS-
5 RBCs.  Before every blood donation, each subject’s hemoglobin will be measured, and only those with a 
value ≥ 12.5 g/dL will be allowed to donate. 
Each RBC unit will be weighed, and its volume will be calculated according to the Donor Center’s standard 
formula: red cell volume (mL) without AS-3 = product weight/1.08.  
RBC units will be stored in the blood bank under standard conditions (1˚C -6˚C) and under the supervision 
of its medical director (Dr. Galanakis, Co-Investigator). 
MONDAY of Week 6: VO2 max Test (Baseline) 
During week 6 of each cycle, the VO2 max test on Monday will serve as the “baseline” for changes in VO2 
max measured on Friday (primary endpoint) and Wednesday (secondary endpoint) of that same week. 
Although we are targeting mild anemia for this visit (Hb 11-12 g/dL), it is probably not critical that we achieve 
a certain hemoglobin level before transfusion since previous studies have shown increases in VO2 max for 
a very wide range of Hb levels, i.e. up to 16 g/dL.44,45,48-50 
WEDNESDAY of Week 6: Blood Transfusion and VO2 max Testing 
We will use the same procedures for transfusing autologous stored RBCs that we used successfully in our 
recently completed pilot study and TASER study.24 Procedures (details below) have been designed to 
optimize safety of research subjects. 
Before the subject’s visit, a sterile welding device will be used to remove a small aliquot from each RBC unit. 
This aliquot will be sent to Stony Brook Hospital’s Microbiology Laboratory for culture to rule out potential 
bacterial contamination of a stored unit. Based on in-vivo viability at 24 hours after transfusion,52 we anticipate 
that transfusion of 100% of 42-day RBCs, 95% of 28-day RBCs, and 85% of 7-day RBCs, will generally 
compensate for differences in post-transfusion 24-hour viability. In order to confirm that this adjustment is 
appropriate, we will measure the Hb levels for each subject before and after transfusion, and 2 days later 
(Friday visit) to ensure that there are no differences in RBC mass that could confound the results. In the 
unlikely event that this is an issue, our statistician can adjust for this potential interaction. 
Subjects will arrive at Stony Brook Hospital at approximately 8 am for each visit. An intravenous (IV) cannula 
will be placed in an upper extremity vein. Before transfusion, Stony Brook Hospital Transfusion Services will 
confirm compatibility of the autologous units by an extended cross-match, comparing a fresh sample of the 
subject’s blood and blood from the autologous units. Each unit of autologous RBCs will be transfused over 
60 minutes using an Alaris® infusion pump (CareFusion, San Diego, CA). Thus, the 2 units of RBCs (7- or 
28- or 42-day) will be administered over a 2- hour period. The blood will not be diluted or warmed before 
infusion. During the transfusion, vital signs will be monitored by the RN coordinator or physician every 15 
minutes.  

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The subjects will be asked to rest for 2 hours after the end of the transfusion, and will be given a light snack. 
They will then undergo a VO2 max test. Of note, this VO2 max test (2 hours after transfusion) will reflect both 
acute hypervolemia (due to transfusion of the 2 RBC units) and increased hemoglobin mass from the 
transfused RBCs. This will inform our secondary outcome = Δ VO2 max between Wednesday and Monday 
in the 2 study arms. However, since our goal is to focus on the oxygen delivery (not volume) effects of these 
RBCs, our prespecified primary endpoint is the change from Monday’s VO2 max to Friday’s VO2 max, when 
subjects will have equilibrated from the volume effects of the transfusions. 40,44,45 This allows us to rigorously 
assess the in vivo function of RBCs to deliver oxygen independent of intravascular volume effects from 
transfusion. 
FRIDAY of Week 6: VO2 max Test 
This VO2 max test will be used for analysis of the primary endpoint as 
described in Statistical Methods. Briefly, a Δ VO2 max between Friday and 
Monday will be calculated for each subject, and statistical analysis will test 
for a proportional difference from baseline between study intervention 
groups (7- vs 42-day RBCs).  
Detailed Description of VO2 max 
We are experienced in performing VO2 max testing as evidenced by our pilot study. Procedures (details 
below) have been designed to optimize the safety of research subjects while obtaining valid reproducible 
data. 
VO2 max testing will be performed in Level 2 of the Health 
Sciences Center.   Each subject will be instrumented with a 
3-lead ECG to monitor cardiac rhythms and a pulse oximeter 
to record heart rate. Mixed expired oxygen, carbon dioxide, 
breathing frequency, tidal volume, minute ventilation, oxygen 
consumption, and carbon dioxide production will be 
measured with a metabolic cart. Data will be recorded every 
30 seconds as 30-second averages. Subjects will breathe air 
through a two-way non-rebreathing valve connected to the 
metabolic cart.  
Subjects will be seated on a cycle ergometer. The seat 
height will be adjusted so that when sitting squarely on the 
seat with the pedal in the lowest position, a slight bend of 
approximately 10 degrees will be maintained at the knee. 
Digital displays of rpm will be visible to the subjects. 
Three minutes of initial resting baseline measures will be 
collected before the start of exercise. The subject will then 
begin to pedal the cycle ergometer at a cadence of 75 rpm. 
Resistance will be manually set according to the progressive 
protocol shown in the Table (right). A rating of perceived exertion (RPE) will be obtained at the end of each 
exercise stage. The VO2 max test will be terminated when the subject reaches volitional fatigue, or, in the 
very unlikely event, achieves one of the other test termination criteria as defined (see table in Human 
Subjects: Adequacy of Protection Against Risk).  
The screening VO2 max test (Visit A) will allow each subject to become familiar with this testing protocol. 
VO2 max is determined as the highest oxygen consumption averaged over 2 30-second periods, which 
typically occurs in the last stage of the progressive maximal exercise test. Peak heart rate is considered to 
be heart rate at VO2 max. Maximum heart rate (HRmax) is considered to be heart rate at or near VO2 max. 
To minimize variability in VO2 max testing procedures, the same facilitator (Sabeen Rizwan- Study 
Coordinator) will be present at every test.  To further minimize variability, subjects will be required to avoid 
moderate or heavy exercise the weekend before week 6 and during the week of testing (week 6), which will 
eliminate the risk for confounding from recent strenuous exercise, e.g. a 10-mile run the day before testing. 
Additional Measurements During VO2 max Testing 
On 2 of the visits for VO2 max testing (Monday and Friday for our primary endpoint), peripheral blood Hb will 
be measured before each VO2 max test to confirm that our strategy for ensuring similar RBC mass between 

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study arms (7-, 28-. 42-days) has been effective. If necessary our statistician can correct for a possible 
interaction. 
 
Subject Payment 
Subjects will be paid for each study visit that they complete: $50 for completing screening visit, $50 for 
completing each of the two blood donation visits, $50 for completing the day 40 exercise test visit, $150 for 
completing the transfusion and exercise test visit and $350 after completing the final visit of exercise testing 
 
E. STATISTICS 
 
Randomization and Allocation Concealment: The pattern randomization scheme will be generated using a 
computer algorithm. Transfusion Services personnel will assign each subject to an administration 
sequence (above) using a sealed envelope technique. Subjects will be randomized to receive 7-, 28- or 42-
day-old blood at the time of their enrollment.  
 
Statistical Analyses: All analyses will be conducted by the team’s biostatistician (J.R.). Summary statistics, 
including 95% confidence intervals, will be reported on all demographic and outcome data collected, 
including all multiple hemoglobin and VO2 max measures.  
 
The primary outcome of this study is the proportional change from baseline in VO2 max from Monday 
to Friday of week 6, after receiving 7-day or 42-day old blood on the Wednesday of Week 6.  We 
hypothesize that transfusion of RBCs stored for 42 days are less effective at increasing oxygen delivery 
than RBCs stored for 7 days, which would confirm results from our pilot study where we observed an 
82% difference in proportional change between 7- and 42-day VO2 max study arms. We will test the 
change scores for normal distribution, and will compare the 42- to 7-day groups using either a student’s 
t-test or Wilcoxon rank sum test in a one sided superiority hypothesis test at the 0.05 significance level. 
Subjects who withdraw or are excluded before randomization will simply be dropped from analysis. We 
will further examine the treatment effect by adjusting for potential effects of hemoglobin, and BMI in a 
linear regression model. 
 
Secondary outcomes: Our secondary outcomes include mean proportional change between 7- and 
28-day VO2 max study arms, and change in duration of exercise between all three groups. Of note, in 
contrast to the primary endpoint, this secondary analysis is expected to show increases in VO2 max 
attributable to intravascular volume expansion and increased hemoglobin mass. Nevertheless, these 
changes are of interest and it is possible that storage duration may affect VO2 max acutely after 
transfusion.  
 
Sample Size: Sample size and power are estimated for a superiority study design using a t-test with a 
power of 80%, and a one-sided significance level of 0.05. Based on the collected pilot data, we expect the 
mean percent change VO2 max for subjects who receive 7-day blood to be 8.7% (SD = 6%), and the mean 
percent change VO2 max for subjects who receive 42-day blood to be 1.6% (SD = 6%), providing an effect 
size of 1.183. Thus, the sample size needed for each group is 10 participants, for a study total of 30. While 
this calculated sample size may not allow us enough power to test a non-inferiority margin of 20% between 
the 7 day and 28 day groups, it will provide exploratory evidence for a future trial by providing a more 
accurate assessment in the variation of the data. 
 
 
F. FUNDING STATUS, DETAILS 
This study is funded by a grant from the National Institutes of Health/NHLBI. 
 
G. HUMAN SUBJECTS RESEARCH PROTECTION FROM RISK 
 
 
Risks to Subjects 
Numerous aspects of the study procedures are designed to minimize potential risk to subjects. Most of these 
procedures have been used by us in our pilot study and in our TASER study of blood donation/transfusion24 
and maximal exercise testing.25,26 All potential risks will be included in the consent form: 
1. Risks associated with venipuncture and intravenous catheter insertion: Venipuncture and insertion of a 
peripheral intravenous catheter results in momentary discomfort. Infection, excess bleeding, clotting, or 

Version 2c – June 12, 2017 
 
 
fainting is also possible, although unlikely. Very rare complications include hematoma, nerve injury, 
thrombophlebitis, arterial puncture, and compartment syndrome.  
2. Risks associated with blood donation at Stony Brook Hospital Transfusion Services: Approximately 3.5% 
of all donations report an adverse reaction, the majority of which are mild and self-limiting, with less than 
1:3,400 blood donors seeking medical care post-donation. Vasovagal reactions (faintness or loss of 
consciousness) are the most common reactions, with less than 0.01% of reported reactions resulting in 
fainting with injury. There is a 2% risk of adverse reactions to citrate, the anticoagulant used to maintain 
the blood in liquid state (tingling sensation, dizziness, muscle cramping) when undergoing apheresis 
double red cell collections. Nausea, vomiting, and post-donation fatigue are also reported. To minimize 
the chances of this occurring, per standard procedures, all volunteers will be offered some food and liquid 
after donation and will be encouraged to sit for 30 minutes after donating. It should be noted that fewer 
donor adverse events are seen with the double red cell collection we will be using due to the saline blood 
volume replacement they receive. The subject will be informed that they will not be able to donate 
platelets or RBCs for clinical use for 1 year following this study. The subject will also be informed that if 
any of the screening tests for HIV, hepatitis B, or syphilis are positive, these results must be reported to 
the state. This required disclosure could breach the subject’s confidentiality. 
For this research study subjects will donate blood sooner than would be done in routine clinical practice. 
Volunteers, however, should not exhibit deleterious levels of anemia during the study since a hemoglobin 
of at least 12.5 g/dL will be required before donating blood at any time. Based on previous studies we 
expect the lowest hemoglobin levels to be approximately 11-12 g/dL (hematocrit of 33-36%) after 
donation. Volunteers will be told that while anemia of this level is not dangerous, they may feel fatigued, 
be more likely to experience dyspnea on exertion, and have decreased exercise performance until their 
hemoglobin level is increased on the transfusion day. In order to minimize these potential effects they 
will be advised to refrain from moderate or heavy exercise during these times when they may have mild 
anemia.  
Subjects will be informed that during the study they may take a multivitamin but they should not take iron 
supplements. 
3. Risks of blood transfusion (Wednesday visit during week 6 of each cycle):  
a. During blood donation bacteria may be introduced into the donated unit because it is impossible to 
completely sterilize the skin through which venipuncture occurs. Also, donors may have 
asymptomatic infection of bacteria in the blood stream due to recent dental procedures or developing 
illness. The reported rates for allogeneic RBC unit bacterial contamination range from 0.01% to 
0.27%. As the subjects in this study have allogeneic donor criteria applied to them, this would be a 
fair estimation of bacterial contamination risk. If the subject experiences fever within 2 days after 
donating their unit of whole blood, they will be asked to call Dr. Bennett-Guerrero at 919- 812-3975. 
The subject may be excluded from continued participation in the study. If the blood is stored longer 
than 14 days prior to transfusion, a sample of the stored blood will be tested for the presence of 
bacteria 7 days before re-infusion. If tests are positive for the presence of bacteria, the subject will be 
notified and their participation in the study will not continue.  
b. Additional transfusion reactions, such as chills, fever, drop in blood pressure, transfusion associated 
circulatory overload (TACO) or pulmonary edema, and shock may also occur. There may also be 
allergic reactions such as hives, rashes, or (very rarely) anaphylactic reactions that could result in 
death.  
c. The subject may experience pain around the IV site or in the arm with the IV. Investigators will select 
the largest available vein in the forearm or antecubital fossa to minimize pain during blood infusion. 
d. Mistransfusion, arising from errors in unit labeling, component preparation or other clerical errors 
occurs in 1:16,000 - 1:25,000 autologous donations. To minimize the risk of mistransfusion, which in 
the worst possible case may trigger a fatal or serious acute reaction, multiple safeguards to prevent 
this are written into our study protocol and include the following: 
i. 
multiple sample and bag identification checks 
ii. 
2 persons will confirm the subject’s identity with the stored RBCs, using established hospital 
approved standard Stony Brook Hospital transfusion procedures 
iii. 
a standard crossmatch will be performed between the subject’s stored donated unit and a 
fresh sample of the subject’s blood (if an antibody is found, an extended crossmatch will be 
performed) 
iv. 
only one subject will be transfused on a given day to minimize risks 

Version 2c – June 12, 2017 
 
 
v. 
the research subject will be asked to review their signature on the labels for their autologous 
units to confirm they are receiving their own blood 
4. Risks associated with exercise (VO2 max) testing: The most common complaint from those performing 
the exercise test on a bicycle is thigh pain during the test. In healthy volunteers, the likelihood of death 
or a serious complication, e.g. sustained arrhythmia, is low, but not impossible. The following is language 
from a recent publication: 
Risks of Physical Activity, Exercise Training, and Exercise Testing 
Recommendation 1: Maximal exercise stress testing is associated with a very low risk of fatal and non-
fatal cardiac events in either healthy asymptomatic or clinical populations. In healthy, asymptomatic 
individuals, the respective incidences of fatal and nonfatal events are approximately 0.3-0.8/10,000 tests 
and 1.4/10,000 tests (Level 3, Grade B).51 
5. Risks associated with loss of protected health information: As with any participation in a research study 
there is a small chance that a loss of confidentiality could occur, but our use of standard risk management 
procedures will minimize this risk. 
 
Adequacy of Protection Against Risks  
Numerous aspects of the study procedures are designed to minimize potential risk to subjects. Most of these 
procedures have been used by us in previous studies of blood donation/transfusion including our pilot study 
and our TASER study24 and maximal exercise testing.25,26 
1. Safety Monitoring Plan: We will record and analyze both expected and unexpected adverse events in 
order to monitor for and minimize potential risks to human subjects. Adverse events that occur during 
any study procedure will be collected and reported to the IRB, NIH, and FDA as applicable. The Princi-
pal Investigator will review serious and non-serious AEs on a rolling basis as well as in aggregate on a 
monthly basis. Given the long standing safety of VO2 max testing as well as blood collection and trans-
fusion studies (many removing far larger volumes of RBCs) we do not expect to see a significant num-
ber of adverse events. In our recent pilot study and TASER study24 no adverse events were observed. 
2. Protection from Loss of Confidentiality: Study records that identify the subject will be kept confidential 
as required by law. All participants will be given a unique code. The key to the code will be kept in a 
locked file in the Study Coordinator’s or Dr. Bennett-Guerrero’s office. All computer records will be se-
cured with password protection and appropriate firewalls will ensure confidentiality and safety of all pa-
tient materials and data. Study documents will be kept in a locked file cabinet in either the Study Coor-
dinator’s or Dr. Bennett-Guerrero’s office. Source documents sent to the NIH and FDA as applicable 
will have all direct identifiers masked with opaque black ink. Except when required by law, the subject 
will not be identified by name, social security number, address, telephone number, or any other direct 
personal identifier in study records disclosed outside of Stony Brook University. The results of this re-
search study may also be presented at meetings or in publications; however, identity will never be dis-
closed in those presentations. In no way will any subjects’ name be used in any published form. Medi-
cal records that identify the subject and the consent form may be inspected by the FDA, other regula-
tory agencies, and the Institutional Review Board (IRB), in order to meet federal and state regulations. 
If the volunteer’s research record is reviewed by any of these groups, they may need to review the en-
tire medical record. The primary purpose of such review is to ensure the protection of the rights and 
welfare of the human subjects. 
3. Protection from Risks associated with blood donation: Our eligibility criteria are designed in part to 
minimize the likelihood of individuals who may be at higher risk for blood donation. For example, 
individuals with anemia, medical problems, or total blood volume less than 4,500 will not be eligible. In 
addition, we will exclude individuals with a history of fainting or similar adverse reactions during blood 
donation. Subjects will only be allowed to donate blood (2 unit apheresis collection) if their pre-donation 
hemoglobin is at least 12.5 g/dL measured using SBUH Blood Bank’s standard FDA approved device. A 
hemoglobin of 12.5 is consistent with the following guideline published by AABB: “The minimum 
acceptable hemoglobin for both men and women is defined by the FDA as 12.5 g/dL, with the essentially 
equivalent hematocrit requirement of 38%.” Per standard SBUH Blood Bank procedures, all volunteers 
will be offered some food and liquid after donation and will be encouraged to sit for 30 minutes after 
donating. The SBUH Blood Bank has routine emergency resuscitation equipment in the collection area, 
which further minimizes risk. In addition, volunteers will be told that while the mild anemia they are 
expected to have after blood collection is not dangerous for fit healthy volunteers, they may feel fatigued, 
be more likely to experience dyspnea with exertion, and have decreased exercise performance until their 

Version 2c – June 12, 2017 
 
 
hemoglobin level is increased on the transfusion visit in week 6 of each cycle. They will be advised to 
refrain from moderate or heavy exercise during the weekend prior to week 6 and during week 6 of each 
cycle. Since subjects will agree to this exercise restriction at the time they provide informed consent, we 
believe few to none of them will drop out due to anemia symptoms or restricted exercise during this short 
(9 days) period of time each cycle. Subjects are unlikely to have significant symptoms of anemia during 
light exercise, e.g. walking. 
4. Protection from Risks associated with blood transfusion: To minimize risks, the following study proce-
dures will be used: 
a. If the subject experiences fever within 2 days after donating their unit of whole blood, they will be 
asked to call Dr. Bennett-Guerrero at 919-812-3975. The subject may be excluded from continued 
participation in the study.  
b. If the blood is stored longer than 14 days prior to transfusion, a sample of the stored blood will be 
tested for the presence of bacteria 7 days before re-infusion. If tests are positive for the presence of 
bacteria, the subject will be notified and their participation in the study will not continue.  
c. During blood transfusion (which will occur in a Health Sciences Center lab or the SBUH Blood Bank), 
subjects will be monitored by an experienced RN study coordinator and one of the physician 
investigators will be either at the bedside or immediately available. We will monitor temperature, blood 
pressure, ECG, and pulse oximetry in order to detect possible transfusion reactions. In addition, 
consistent with our previous pilot study, each RBC unit will be administered over 60 minutes to 
minimize the likelihood of transfusion associated circulatory overload (TACO) or pulmonary edema, 
which is unlikely in healthy, fit subjects. 
d. Investigators will select the largest available vein in the forearm or antecubital fossa to minimize pain 
during blood infusion. 
e. To minimize the risk of mistransfusion, which in the worst possible case may trigger a fatal or serious 
acute reaction, multiple safeguards to prevent this are written into our study protocol and include the 
following: 
i. 
multiple sample and bag identification checks 
ii. 
2 persons will confirm the subject’s identity with the stored RBCs, using standard Stony Brook 
Hospital transfusion procedures 
iii. 
a standard crossmatch will be performed between the subject’s stored donated unit and a 
fresh sample of the subject’s blood (if an antibody is found, an extended crossmatch will be 
performed) 
iv. 
only one subject will be transfused on a given day to minimize risks 
v. 
the research subject will be asked to review their signature on the labels for their autologous 
units to confirm they are receiving their own blood 
 
5. Protection from Risks associated with exercise (VO2 max) testing: To minimize risks of VO2 max testing, 
we will only enroll healthy, habitually exercising, non-obese subjects between 18-40 years of age. This 
represents a group at very low risk for adverse events. Nevertheless, during testing all subjects will be 
monitored by a physician with appropriate monitors and emergency resuscitation, e.g. defibrillator, will 
be immediately available. All exercise tests will be performed in a co-investigator’s lab in the Health 
Sciences Center of Stony Brook University Hospital.  Additional safeguards include a screening ECG to 
rule out long QT interval, and spirometry to rule out occult pulmonary disease. In addition, we will use 
standard criteria to terminate the VO2 max test. 
 
Termination Criteria for VO2 max Test 
1. Subject fatigue or subject request for any reason 
2. Inability to maintain the prescribed cadence (75-80 rpm) at the current work rate 
3. Onset of chest pain or equivalent angina symptoms, such as unusual or extreme shortness of breath, or signs of 
poor perfusion, as noted by the subject or determined by the monitoring physician 
4. Sequential ventricular beats of two or more (i.e., 2 beats of ventricular tachycardia) 
5. New onset of sustained (>15 seconds) atrial fibrillation, supraventricular tachycardia (SVT), or other 
supraventricular tachyarrhythmias 
6. Development of new left bundle branch block (LBBB) 
7. Systolic blood pressure >240 mm Hg, or diastolic blood pressure >130 mm Hg 
8. Progressive decrease in heart rate or systolic blood pressure during increasing exercise intensity accompanied by 
clinically significant signs or symptoms 
9. Second-degree Type II or third-degree heart block. 
 

Version 2c – June 12, 2017 
 
 
 
Potential Benefits of the Proposed Research to the Subject and Others 
There is no benefit to the subject for participating in this study. There may be a benefit to future patients 
based on results from this study.  
 
Importance of the Knowledge to be Gained  
The proposed study is significant for several reasons: 1) We believe that the proposed study is likely to 
confirm our very encouraging preliminary data, showing that the oldest RBCs allowed by FDA regulations 
(42 days) are inferior to 7 day RBCs in a functional assay of oxygen delivery. This is important scientifically 
since, to our knowledge, no studies have definitively shown that storage duration can affect RBC function, 
i.e. oxygen delivery; 2) The proposed study is also significant because it may shed light on why the RECESS 
and ABLE outcome trials, comparing young and “middle-aged” RBCs were negative. We hypothesize that 
28 day RBCs are not inferior to 7 day RBCs, which are exactly the median ages of storage for the two study 
arms in RECESS.11 3) Finally, we suggest that this model holds promise as a rigorous and quantitative in 
vivo functional assay of RBC function, which is an unmet need for future RBC transfusion research as 
identified by NHLBI and others. 
 
Risk Benefit Assessment 
We believe that the information to be gained from this study is reasonable compared with the small risks of 
transfusion of autologous blood to healthy volunteers and VO2 max testing in healthy volunteers. 

Version 2c – June 12, 2017 
 
 
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