A Pilot Study on the Impact of Rejuvenated Autologous Blood Transfusion on VO2 Max in Healthy Subjects
Phase: PHASE4
Conditions: Erythrocyte Transfusion
Sponsor: Duke University

RESEARCH Summary 
Pro00078468 
NCT03089047 
PI:  Ian Welsby 
 
Page | 1  ver. Date 2/21/18 
 
1.  Protocol Title: A Pilot Study on the Impact of Rejuvenated Autologous Blood 
Transfusion on VO2max in Healthy Subjects 
 
2.  Purpose of the Study: To compare the physiologic benefits of rejuvenated RBCs 
(rejRBCs) to standards RBCs (PRBCs), we will emulate critical conditions by safely 
maximizing stress on the cardiovascular system, in an elective, feasible volunteer 
study. Maximal oxygen uptake (VO2max) will be measured in an anemic, post-
donation, pre-transfusion state, and also after transfusing 14-day-old, autologous 
blood randomized to standard storage or standard storage with rejuvenation. We 
hypothesize that transfusion of standard PRBCs is less effective at increasing oxygen 
delivery (measured by VO2max) compared with transfusion of rejRBCs. 
 
3.  Background & Significance: Transfusion is one of the most commonly performed 
medical procedures, designed to improve oxygen delivery to tissues throughout the 
body, yet there are few data to support the efficacy of this therapy in terms of its 
proposed purpose – oxygen delivery.  
 
Previous studies have shown that a reduction in arterial oxygen content, by reducing 
hemoglobin, results in reproducible decreases in individuals’ maximal oxygen uptake 
or consumption (VO2 max),1 a measure that reflects the body’s capacity to both deliver 
and utilize oxygen. While an increase in VO2 max has been demonstrated after 
transfusing autologous, cryopreserved, RBCs, these data cannot be extrapolated to 
the standard, liquid stored RBCs used clinically2-6 and previous testing of liquid stored 
units did not demonstrate a benefit. The most likely explanation for poor oxygen 
delivery with transfused RBCs is the lowering of p50 (increased affinity for oxygen with 
poor off-loading in the tissues) related to the loss of 2,3 DPG that happens over the 
first 7-10 days of storage.7 
 
This proposal addresses two fundamental questions: does standard RBC transfusion 
even improve oxygen delivery and can rejuvenated RBCs (with a high/normal p50) 
maximize the oxygen delivery capacity of transfused RBCs. Oxygen delivery is 
considered to be dependent upon hemoglobin concentration, cardiac output and 
hemoglobin saturation. Transfusion of blood with a lower than normal p50 (such as 
stored RBCs) is expected to be less effective at delivering oxygen as supported by 
tissue pO2 (tpO2) being dependent on modulating p50 in an animal exchange 
transfusion model.8 Modulation of p50 is possible in animal models and increases 
exercise capacity in mice with heart failure9 and ischemic hearts,10,11 consistent with a 
physiological benefit to increased O2 off-loading in the tissues. One unit of optimized, 
rejuvenated RBCs (rejRBCs), could be as valuable as multiple units of standard RBCs, 
especially for patients unable to further compensate for reduced oxygen delivery with 
increased cardiac output, such as the burgeoning heart failure population. Decreasing 
RBC exposures, costs, and adverse effects in high-risk populations could improve 
patient safety and revolutionize transfusion medicine. Evaluating volunteers stressed 
by the demands of exercise is an essential, proof-of-principle, pilot step. We will test 
the hypothesis that: Repurposing an FDA approved rejuvenation process to increase 
the p50 of stored RBCs and increase their ability to off-load oxygen at the tissue level, 
will translate into better oxygen delivery, as measured by exercise capacity (VO2max). 

RESEARCH Summary 
Pro00078468 
NCT03089047 
PI:  Ian Welsby 
 
Page | 2  ver. Date 2/21/18 
 
Red blood cells are rejuvenated according to the procedure in the labeling of the 
Rejuvesol product ( FDA approved). 
 
4.  Design & Procedures:  
Study type: Prospective randomized “healthy volunteer” study 
 
Site(s):  
1) Duke University where consenting, screening, exercise testing, and transfusion of 
RBCs will take place. 
 
2) The Blood Connection (TBC) is located in Piedmont, SC with a satellite in Raleigh, 
NC and was established in 1962.  The Blood Connection is registered and licensed by 
the US FDA and accredited by the American Association of Blood Banks. TBC has two 
blood mobiles, which travel throughout the Triangle and one fixed site where blood is 
collected and products are produced.  Blood collection is performed by single whole 
blood donation and automated apheresis for the collection of red cell, plasma and 
platelets.  TBC (Raleigh) processes approximately 18,000 donations per year, 
manufactures red blood cell products, frozen plasma products, and platelet products; in 
North Carolina TBC is the primary supplier to Wake Med and Rex Hospitals in Raleigh. 
They also supplyDuke University Medical Center, as inventory and need allows, 
especially for autologous units.  The VP Business Development/Chief Technical Officer 
of TBC (Tracy Bridges) and the medical director ( Robert Rainer,MD) has agreed to 
allow the blood collections and processing for this study to take place at TBC using their 
standard procedures for autologous blood collection by apheresis.  A fee is being 
charged to Duke for these services as if this were a standard autologous donation. 
 
Study size: 
 
 
In this pilot study, we will plan to enroll 20 subjects. 
 
Eligibility Criteria:   
 
Healthy, habitually exercising, non-smoking, non-diabetic males and females (as 
described in the inclusion/exclusion criteria below): 
 
Qualified volunteers will be enrolled at a single center (Duke University Medical Center). 
After subjects provide written informed consent, eligibility will be confirmed with 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 (the American College of Sports Medicine Guidelines for Exercise 
Testing defines this age group with no or no more than one 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 (< 30 mL/kg/min) will be excluded (screen failure). Based on our 

RESEARCH Summary 
Pro00078468 
NCT03089047 
PI:  Ian Welsby 
 
Page | 3  ver. Date 2/21/18 
 
previous experience, we anticipate that up to 25% of the subjects will fall into 
this category; therefore, 20 subjects will be screened to obtain 10 participants 
who go on to donate their blood. 
iv. 
Calculated total blood volume of at least 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] 
v. 
Has access to transportation to visit the blood collection facility and to return 
to Duke for all study visits 
vi. 
Weighs at least 130 pounds 
 
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 
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 
c.  Plasma donation by apheresis within the past 4 weeks 
d. Any  self-reported blood donation  within a year  
xii. 
Inadequate red blood cell mass evidenced by total blood volume <4500 mL 
(above) or screening hemoglobin <13.3 g/dL 
xiii. 
Known hypersensitivity to lithium compounds. And known taking lithium salts 
for treatment of other medical/psychiataric conditions 

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   xiv.     Students and employees who are under direct supervision of the investigators 
listed in this study. 
VISIT A ( – Screening) Written informed consent will be obtained from subjects who 
respond to an IRB-approved advertisement/flyer and fulfill eligibility requirements. The 
following video will help explain the study: vimeo.com/167291799. Individuals who 
respond to an IRB approved advertisement/flyer (uploaded to eIRB) will be given 
information about the study and asked to read an IRB approved consent form.  An 
investigator or designee will obtain written informed consent from each individual 
participating in this study, after adequate explanation of the methods, objectives, and 
potential hazards of the study.  The investigator or designee will also explain to the 
subjects that they are completely free to refuse to enter the study and free to withdraw 
from it at any time. A copy of the signed consent form will be given to the subject and will 
be shared with The Blood Connection to ensure proper labeling of autologous units.  
 
After subjects have provided written informed consent, they will be asked to answer 
several questions in 2 written questionnaires (uploaded to eIRB) in order to confirm that 
they are eligible to donate blood.  These questions are similar to those routinely asked to 
people prior to a blood donation.  These are focused upon keeping the risks of blood 
donation to the lowest possible level.  Subject will undergo screening lab tests for 
baseline hemoglobin, sickle cell trait, serum pregnancy tests only for women of child-
bearing potential, ECG, exercise test. Subject will receive a phone confirmation from the 
investigator or delegated research staff after review of the screening lab results.  Subject 
will bring a copy of the signed consent form to The Blood Connection if qualified.  
  
VISIT B (Day 0 – Blood donation) The Blood Connection (TBC), Raleigh, NC, is 
registered and licensed by the US FDA and accredited by the American Association of 
Blood Banks (AABB) to manufacture RBCs and other blood products. Two units of blood 
will be withdrawn from each subject according to standards for double, AS-3, apheresis 
units, and will be marked for autologous donation, and purchased by Duke Transfusion 
Services, as arranged with the medical director of TBC (Robert Rainer, MD). RBC 
handling and storage will meet FDA and AABB standards; units will be stored with Duke 
Transfusion Services c/o Dr. Poisson. 
 
VISIT C (Day 14 – “Anemic” VO2 max, Transfusion (Tx) and “post-Tx” VO2max))  
Small amount of blood [3 lavender topped tubes] will be collected from subject to test for 
hemoglobin, type & screen and 1 cross match to verify the donated blood received from 
Study structure: study visits (A-E).  
 
A 
Screen  
Screen, consent, H&P, ECG, screening labs, and VO2max  
B 
Day 0  
Donate 2 units of PRBCs by autologous apheresis 
C 
Day 14  
8am     Measure pre-transfusion, “anemic” VO2max  
     
9 am    Transfuse 2 units of standard  or rejuvenated PRBCs 
 
 
 
3 pm  Measure post-transfusion VO2max (end of study) 
 

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PI:  Ian Welsby 
 
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The Blood Connection belongs to the same subject who arrives at the appointed date at 
Duke facility 
Anemic VO2max 
Three minutes of resting baseline measures will be recorded 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 a standardized, progressive protocol. A 
rating of perceived exertion (RPE) will be obtained at the end of each exercise stage. 
Vital signs will be recorded every 3 minutes, and ECG monitoring will be continuous. 
Exercise will be terminated when the subject reaches volitional fatigue. VO2max will be 
reported as the highest oxygen consumption averaged over two 30-second periods, 
which typically occurs in the last stage of the progressive maximal exercise test. 
Maximum heart rate will be the heart rate at or near VO2max. To minimize variability in 
VO2max testing procedures, the same 2 facilitators (an engineer/technician and a 
research staff) will be present at every test.  Radial arterial access will be obtained for 
cardiac output monitoring (LiDCO pulse contour analysis) at this visit. 
Tx and post-Tx VO2max 
Duke Transfusion Services will randomize each subject to receive 2 units of standard 
PRBCs or rejRBCs over 120 minutes using a standard infusion pump. All units will be 
washed to equalize the transfusion volumes; rejuvenation includes a mandatory washing 
step. Rejuvenation of donated blood will be done according to the procedures in the 
labeling of the Rejuvesol product. Vital signs will be monitored and recorded every 15 
minutes. After a total 4-hour rest after completing the first VO2max testing, and a light 
snack, the exercise test will be repeated. 
Laboratory testing: The p50 will be measured in one of the blood units transfused and in 
the subject before the exercise test. Radial arterial access will be obtained for serial 
cardiac output monitoring (LiDCO Pulse Contour Analysis, LiDCO Products, London, UK), 
arterial blood gas analysis and lactate measurement before and after transfusion, and 
after exercise testing. Final blood collected for a hemoglobin level will be done at the 
conclusion of blood transfusion. This is the final visit and participation is completed.  
 
 
 
5.  Study Interventions: 
Detailed Description of VO2 max Testing 
We have significant experience performing VO2 
max testing (e.g. DUHS IRB Pro00011747).  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 the Center 
for Hyperbaric Medicine and Environmental Physiology, 
which is part of Duke University Medical Center. Dr. 
Moon (Co-Investigator) is medical director of this center,   
 
 

RESEARCH Summary 
Pro00078468 
NCT03089047 
PI:  Ian Welsby 
 
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The photo (right) shows a 
VO2 max test in progress in our 
laboratory. 
Each subject will be instrumented 
with a three-lead ECG (Spacelabs, 
Model 511) to monitor cardiac 
rhythms and a pulse oximeter 
(Ohmeda, model 3740) 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 (Consentius 
Technologies, ParvoMedics 
TrueMax 2400). Data will be 
recorded every 30 seconds as 30-
second averages. Subjects will wear 
a nose-clip and breathe air through a 
two-way non-rebreathing valve 
(Hans Rudolf, model 2700) connected to the metabolic cart.  
Subjects will be seated on a cycle ergometer (Monark, model 818E). 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 and recorded on a computer 
(Macintosh Quadra/MacLab data acquisition).  
Three minutes of initial resting baseline measures will be collected prior to 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 (above). A rating of perceived exertion (RPE) is obtained at the end of each 
exercise stage. The VO2 max test is terminated when the subject reaches volitional 
fatigue, or achieves one of the other test termination criteria as defined in the Table 
above.  
The screening VO2 max test (Visit A) will allow each subject to become familiar with this 
testing. 
VO2 max is determined as the highest oxygen consumption averaged over two 
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. 
 
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 (ie, 2 beats of ventricular tachycardia) 

RESEARCH Summary 
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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. 
 
To minimize variability in VO2 max testing procedures, whenever possible, the same 2 
facilitators will be present at every test (Michael Natoli - engineer/technician for VO2 max 
equipment; a nurse Research Coordinator who will collect data and coach each subject). 
 
Additional Measurements During VO2 max Testing  
On the Day 14 visit for VO2 max testing , the following additional study procedures will 
be performed: 
o A radial arterial catheter will be inserted under local anesthetic prior to 
exercise testing, consistent with previous studies on healthy volunteers 
done in our laboratory.  
o Arterial blood samples (6 mL each) will be obtained from the radial arterial 
catheter immediately before exercise, twice during exercise, and after 
exercise completion (total 24 mL). Blood samples will be analyzed for 
arterial lactate, PO2, PCO2, and pH. We know that arterial lactate 
increases during VO2 max testing, and we will conduct exploratory 
analyses to determine whether there are differences in peak lactate 
between study arms (Standard vs Rejuvenated RBCs). 
o Cardiac output, cardiac index, and stroke volume will be determined 
during VO2 max testing using the validated and FDA-approved LiDCO 
monitor, which includes a lithium dilution determination of cardiac output, 
with subsequent measurements from radial arterial pulse contour 
analysis. These data will be used to confirm that cardiac output is similar 
for subjects in the two study arms, ie, no difference in VO2 max is 
attributable to differences in cardiac output between study arms. We do 
not expect to see any differences in cardiac output between groups since 
the study arms will receive an identical volume of fluid (2 RBC units) over 
the 2-hour transfusion period. 
 
Detailed Description of Rejuvenation Process: Rejuvenation refers to the process of 
adding a mix of solutes (Rejuvesol®, Citra Labs, Braintree, MA; consists of sodium 
pyruvate, inosine, adenine, mono- and dibasic sodium phosphate) to older, stored (i.e., 
2-3 DPG-depleted) blood to immediately restore 2,3-DPG and ATP levels in the stored 
red blood cells15. Rejuvenation was originally developed to prolong the storage life of 
rare-phenotype RBC units. It is FDA-approved for use in RBC units stored in CPD, 
CPDA-1, and AS-1. The major contraindication for the use of Rejuvesol® is in RBC units 
stored for fewer than 6 days due to high baseline 2,3-DPG and ATP levels.16 
Rejuvenated blood should not be used in patients with PaO2 < 40 mmHg due to the 
impaired pulmonary oxygen loading at low arterial oxygen tensions seen with high-p50 
hemoglobin; we will appropriately exclude these patients at the first screening visit. 

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Rejuvenated RBC units will be prepared according to manufacturer’s protocols, which 
include a mandatory washing step on an approved cell-washing device.16 The 
manufacturer reports no adverse effects resulting from use of Rejuvesol® per 
manufacturer’s instructions, and further notes that the risk of toxicity due to Rejuvesol® 
solution components is low since all solution components are common endogenous 
chemicals that are normally metabolized in vivo.16 
 
6. Risk/Benefit Assessment: 
1) 
Risks associated with venipuncture:  Venipuncture results in momentary 
discomfort. Infection, excess bleeding, clotting, or fainting is also possible, 
although unlikely.  Phlebotomy performed for blood donation has complications 
including hematomas, nerve injury, thrombophlebitis, arterial puncture, and 
compartment syndrome.  Excluding the blood collected at The Blood Connection 
an additional 110 ml of blood will be withdrawn from the subject over the 
screening period through Study Day 14. 
 
2) 
Risks associated with blood donation5: 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 citrate toxicity (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 TBC procedures, all volunteers will be offered some food 
and liquid after donation and will be encouraged to sit for 30 minutes after 
donating.  Additionally, fewer donor adverse events are seen with double red cell 
collection due to the saline blood volume replacement they receive.  In addition, 
consistent with The Blood Connection normal routine procedures, blood will only 
be collected from subjects with a hemoglobin of at least 12.5 g/dL. 
 
3) 
Decreased hemoglobin level: Volunteers should not exhibit significant anemia 
during the study since a hemoglobin of at least 12.5 g/dL is  required at The 
Blood Connection prior to donation of blood.  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 and have decreased exercise 
performance until their hemoglobin level is increased on Day 14(Study visit C). 
 
4) 
Risks of blood transfusion:   
 
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 

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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. Welsby.  The subject may be excluded from continued 
participation in the study   
b. 
Additional transfusion reactions, such as chills, fever, drop in blood 
pressure, shortness of breath, and (very rarely) 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 this 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 standard Duke Medical Center transfusion procedures 
iii) an extended crossmatch will be performed between the subject’s 
stored donated unit and a fresh sample of the subject’s blood 
iv) only one subject per day will be transfused to minimize risks 
v) the research subject will be asked to review the autologous RBC unit 
label including their own name, and date of birth to confirm they are 
receiving their own blood 
     e.        Additional risk of rejuvenated blood: If lung function is abnormal, use 
of rejuvenated blood may lower the subject’s oxygen levels making 
him/her short of breath.   
5) 
Risks associated with exercise 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).14 
6) 
Risks associated with arterial catheter placement: Arterial catheter insertion in 
the wrist can cause occlusion of the radial artery, although most instances occur 
after many hours or days of a catheter in place. Radial artery catheterization has 
been used extensively in our laboratory, with more than 200 being performed in 
healthy volunteer studies with no significant complications. The arterial line 
insertion (standard 20 g) will be performed by an experienced anesthesiologist 
under sterile technique with local anesthetic. 
7) 
Risks associated with LiDCO cardiac output monitoring: 

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The LiDCO cardiac output monitor is an FDA cleared monitor, which is used 
routinely in critically ill patients (e.g. ICU) and other high risk patients, e.g. during 
surgery. It has been used extensively in patients in the U.S. for over 10 years. 
Cardiac output is measured by injecting a tiny, non-therapeutic dose (0.15-0.3 mM) 
of lithium intravenously (through an IV) and then approximately 5 ml of blood is 
sampled from the arterial catheter over the next few seconds. A dilution curve is 
calculated from which the cardiac output is determined. This lithium calibration will 
be done a total of 2 times. We are not aware of any risks associated with injection 
of this dose of lithium for the LiDCO monitor except individuals with lithium 
hypersensitivity. The device cannot be used in persons taking lithium salts for 
treatment of other medical/psychiatric conditions, persons who weigh less than 
40kg.  According to the manufacturer’s instructions the maximum dose is 20 ml of 
the lithium solution (0.15 mM/ml). and we will be using approximately 2 two-ml 
doses on any given study day (Day 14).  
8) 
We are not aware of any risks associated with the ECG and spirometry done for 
screening. 
9) 
The subject will not be able to donate platelets or RBCs for 1 year following this 
study. 
10) 
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.   
11) 
Risks associated 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 standard risk management procedures will be used to minimize this 
risk. 
 
7. Subject Identification, Recruitment, and Compensation: Healthy volunteers 
who respond either to our advertisement or by word of mouth will be shown the 
eligibility criteria and asked if they believe they are likely to be eligible. Volunteers 
who responded to advertisement and left their phone numbers in voicemail will be 
returned call by investigator/delegated key personnel.  Investigator/ designee will use 
IRB-approved phone script as reference to discuss the study activities over the 
phone and at the same time ask permission to PHI use prior to written consent form. 
Volunteers who ended up enrolling in the study but are screen failures (after lab 
results out) will be compensated $100. Participants will received up to total of $1000 
by completion of all study activities, inclusive of screening visit and blood donation . 
Volunteers who has donated blood and missed subsequent visits   will be 
compensated $100 for that specific visit.  In addition, subjects will be provided with 
supplemental oral fluids (juices) and a light snack on day 14 during the break after 
the autologous blood transfusion and before the second exercise testing .  
 
A member of the study team will conduct the consent process in an office at Duke 
Medical Center.  He/she will describe the study and ask the volunteer to read the 
consent form and ask any questions they may have.  As much time as the subject 
needs will be provided for conducting the consent process, and the potential subject 
will have as much time as they need to consider whether or not to participate. 
 

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8. Costs to the Subject: Subjects will incur no direct costs as a result of this study. 
Subjects will only be responsible for indirect costs such as parking fees. 
 
9. Data Analysis & Statistical Considerations: The primary outcomes of this study 
are the change in VO2max and lactate between the “anemic” VO2max and after 
transfusion of 2 units of either standard PRBCs or rej-PRBCs “post-Tx” VO2max . We 
hypothesize that transfusion of RBCs is less effective at increasing oxygen delivery 
(measured by VO2max and lactate) compared with transfusion of rej-PRBCs. We 
expect the changes to be normally distributed and will compare the treatment groups 
with a 2-group t-test or a rank-sum test if the distribution is non-normal. We will test at 
a 2-sided significance level of 0.05. The primary analysis will be on an intent-to-treat 
basis, with follow-up as-treated analysis if necessary. Subjects who withdraw or are 
excluded before randomization (Day 14) will simply be dropped from analysis. We will 
further examine the treatment effect in a multivariable regression adjusting for potential 
effects of baseline VO2max, lactate, hemoglobin, and BMI. 
 
Secondary outcomes include the change in “post-Tx” VO2max  from baseline and 
differences in these changes will be compared between groups with t-tests or rank-
sum tests. Of note, in contrast to the primary endpoint, this secondary analysis may 
show larger increases in VO2max attributable to hypervolemia after transfusion. 
Duration of exercise and peak lactate  “post-Tx” will also be compared between study 
groups. 
 
Based on preliminary data (Richard Moon, personal communication), we know that 2 
units of autologous, stored PRBCs increase VO2max on the day of transfusion by 2.4 
+/-0.6ml/kg/min. Assuming that rej-PRBCs exceed the oxygen delivery of native fresh 
blood (due to their p50 of 40mmHg compared to 18mmHg for stored PRBCs and 
28mmHg for normal blood), we anticipate that:  
- Pre-transfusion/“anemic” VO2max will be decreased by 4.6 ml/kg/min from 
baseline by day 14.  
- 2 units rej-RBC will increase VO2max by > 6ml/kg/min from “anemic” VO2max.  
- 2 units control PRBC will only increase VO2max by 2.5ml/kg/min from “anemic” 
VO2max.  
- Therefore we will set our expected, measured difference (δ) at 3.5 ml/kg/min. 
 
Preliminary data also show that the variability of VO2max has a narrow range, with 
standard deviation ranging from 0.8-1.9 for a given subject (Richard Moon, personal 
communication). Combining with our preliminary data (which showed and increase 
after transfusion of 2.4 +/-0.6 ml/kg/min – see above), we set our standard deviation 
for the power analysis (σ) at +/-1.5 mL/kg/min. 
 
Using this data, we can expect 80% power to detect a significant (p<0.05) intergroup 
difference in the change in VO2max from anemic to post-transfusion state with 3 
volunteers in each group. More patients will be studied as unexpected variability may 
occur. 
 
10. Data & Safety Monitoring: We will record and analyze both expected and 
unexpected adverse events in order to monitor for and minimize potential risks to 

RESEARCH Summary 
Pro00078468 
NCT03089047 
PI:  Ian Welsby 
 
Page | 12  ver. Date 2/21/18 
 
human subjects. Adverse events that occur during any study procedure will be 
collected and reported to the IRB, and FDA as appropriate. The Principal 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 transfusion studies (many removing far larger volumes of RBCs) we 
do not expect to see a significant number of adverse events. 
 
11. Privacy, Data Storage & Confidentiality: Study records that identify the patient 
will be kept confidential as required by law. They will be kept in a locked file cabinet 
in either the Study Coordinator’s or Dr. Welsby’s office.  Data will also be stored in 
password protected computers.  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 Duke University Health 
System (DUHS). The results of this research study may also be presented at 
meetings or in publications; however, identity will never be disclosed in those 
presentations.  In no way will any subjects’ name be used in any published form. 
 
As part of the study, Dr. Welsby and the study team will report the results of the 
study-related laboratory tests named below.  These test results will be recorded in 
the study record and results of tests and studies done solely for this research study 
will not be included in the volunteer’s medical record. 
 
Medical records that identify the subject and the consent form may be inspected by 
the FDA, DUHS Institutional Review Board (IRB), and Rex Healthcare’s regulatory 
bodies, 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 entire 
medical record.  The primary purpose of such review is to ensure the protection of 
the rights and welfare of the human subjects. 
 
The study results will be retained in the study research for at least six years.  At that 
time, either the research information may be destroyed or information identifying the 
volunteer will be removed from such study results at DUHS.  Any research 
information in the volunteer’s medical record will be kept indefinitely.   
 
12. Bibliography 
 
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erythrocythemia on aerobic work capacity. J Appl Physiol Respir Environ Exerc 
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induced polycythaemia on maximal aerobic power in individuals with high and low 
haemoglobin concentrations. Acta Physiol Scand. Jan 1987;129(1):47-54.  
4. Robertson RJ, Gilcher R, Metz KF, et al. Effect of induced erythrocythemia on 
hypoxia tolerance during physical exercise. J Appl Physiol Respir Environ Exerc 
Physiol. Aug 1982;53(2):490-495.  

RESEARCH Summary 
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PI:  Ian Welsby 
 
Page | 13  ver. Date 2/21/18 
 
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13. Roberson RS, Lockhart E, Shapiro NI, et al. Impact of transfusion of autologous 
7- versus 42-day-old AS-3 red blood cells on tissue oxygenation and the 
microcirculation in healthy volunteers. Transfusion. Nov 2012;52(11):2459-2464. 
14. Warburton DER, Gledhill N, Jamnik VK, et al. INTERNATIONAL LAUNCH OF 
THE PAR-Q+ AND ePARmed-X+ The Physical Activity Readiness Questionnaire 
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