Rejuvenated RBC and VO2 Max in Healthy Subjects

VISIT A (Day 0 - 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.

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.

VISIT B (Day 2 - Blood donation) Rex Blood Services (RBS), 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 Two units of blood will be withdrawn from each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 RBS (Tim Carter, 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 16 - "Anemic" VO2 max) 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 nurse/coach) will be present at every test.

VISIT D (Day 18 after "Anemic" VO2max - Transfusion (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. Vital signs will be
monitored and recorded every 15 minutes. After a 2-hour rest 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.

Subjects will have blood collected for a hemoglobin level. After this visit, subjects'
participation in the study is complete.

Inclusion Criteria:

- Healthy male or female

- 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)

- 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) will be
excluded (screen failure). Based on our previous experience, we anticipate that up to
25% of the subjects will fall into this category; therefore, 10 subjects will be
screened to obtain 6-8 participants who go on to donate their blood.

- Calculated total blood volume of at least 4,500 mL using an established formula:

1. Men: (0.006012 x H3) + (14.6 x W) + 604 = TBV

2. Women: (0.005835 x H3) + (15 x W) + 183 = TBV [H= Height in inches; W=Weight in
pounds]

- Has access to transportation to visit the blood collection facility and to return to
Duke for all study visits

- Weighs at least 130 pounds

Exclusion Criteria:

- Any significant acute or chronic medical illness or problem, including, but not
limited to, diabetes, hypertension, cardiac disease, asthma, COPD

- Current or recent (last 60 days) tobacco or nicotine use

- History of sickle cell trait or disease or any other acquired or hereditary
hematological abnormality

- History of fainting or other significant adverse reaction during phlebotomy or
donation of blood

- Known prolonged QTc (or evidence of such at screening) defined as QTc >470 ms

- Known or suspected illicit drug or alcohol abuse

- Known or suspected HIV, Hepatitis B, or Hepatitis C infection

- History of thrombophilia or anticoagulant therapy

- Pregnancy

- Obesity defined as BMI>30

- Recent history of blood donation:

1. Single whole blood unit donation within the past 8 weeks

2. Double RBC donation by apheresis within the past 16 weeks

3. Plasma donation by apheresis within the past 4 weeks

- Inadequate red blood cell mass evidenced by total blood volume <4500 mL (above) or
screening hemoglobin <13.3 g/dL

- Known hypersensitivity to lithium compounds.