Clinical trial NCT02731157
Conditions: Sickle Cell Disease
Sponsor: Duke University
Start date: 2016-12-01; Completion date: 2017-12-31
Enrollment: 4 participants; Allocation: NON_RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
The objective of this proposal is to test the feasibility of red blood cell (RBC) rejuvenation to chronic transfusion in sickle cell disease (SCD) and the potential benefit of RBC rejuvenation in this population to determine if a larger clinical trial powered to definitively characterize the benefits of rejuvenation is warranted.

This is a small pilot study is to see if restoring important energy molecules (ATP and 2,3,DPG) in stored red blood cells before they are transfused, with a rejuvenating solution (Rejuvesol), offers any advantages to individuals over standard blood transfusion. Subjects will receive either rejuvenated (R) or standard (S) RBCs with each transfusion for 6 transfusions (over approximately a 6-month period) in a pre-defined order to maximize detection of any signal.

Detailed description:
Blood transfusion is part of the standard care for individuals that have sickle cell disease. Often these transfusions become needed quite frequently. The purpose of the red blood cells in the blood is to deliver oxygen to the organs and tissues of the body. People with sickle cell disease have abnormal red blood cells. Stored blood undergoes some changes that may make it less effective in achieving this goal.

The purpose of this study is to see if restoring important energy molecules (ATP and 2,3,DPG) in stored red blood cells before they are transfused, with a rejuvenating solution (Rejuvesol), offers any advantages to individuals over standard blood transfusion. This is a Food and Drug Administration (FDA) approved process that is described by the American Association of Blood Banks for prolonging blood storage but not used for everyday transfusions. The investigators want to use this process to improve blood transfused to individuals who need frequent transfusions. Potential advantages include better delivery of oxygen by the transfused red blood cells and easier release of oxygen to the tissues. In addition the study will assess how using the rejuvenated blood affects the interval between transfusions. This means possibly that transfusions may not be needed as often.

Although Rejuvesol has been previously approved by the FDA, it is not routinely used to prepare standard blood transfusions to individuals who have sickle cell disease. Use of Rejuvesol in this study is considered investigational.

Arms: arm 1: Subjects with sickle cell disease will receive RBCs treated with Rejuvesol®. Scheduled red cell exchanges performed with the last 4 units of the exchange having been incubated with Rejuvesol® solution. Only transfusions necessary for the treatment of the sickle cell disease will be given for the purpose of the study. arm 2: Subjects with sickle cell disease will receive standard RBCs. Only transfusions necessary for the treatment of the sickle cell disease will be given for the purpose of the study.
Interventions: intervention 1: Rejuvesol intervention 2: Blood transfusion
Intervention descriptions: intervention 1: None intervention 2: Only transfusions necessary for the treatment of the sickle cell disease will be given for the purpose of the study.

Protocol documents:
Prot_SAP_000.pdf: 
Rejuvesol® Washed RBC in Sickle Cell Patients Requiring Frequent Transfusions 
Research Summary 
 
PI:  Ian Welsby, MD  
Research Summary Version -2- 26-2017 
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Rejuvesol® Washed RBC in Sickle Cell Patients Requiring Frequent 
Transfusions 
 
NCT02731157 
 
Document Date: 2017-02-26 
 
 
 
 
 


Rejuvesol® Washed RBC in Sickle Cell Patients Requiring Frequent Transfusions 
Research Summary 
 
PI:  Ian Welsby, MD  
Research Summary Version -2- 26-2017 
2
Protocol Title: Rejuvesol® Washed RBC in Sickle Cell Patients Requiring Frequent 
Transfusions 
 
Purpose of the Study – The long-term goal of reducing morbidity and increasing 
treatment intervals for chronically transfused patients, the objective of this study is to test 
the feasibility of the novel application of red blood cell (RBC) rejuvenation to chronic 
transfusion in sickle cell disease (SCD) and the potential benefit in terms of RBC 
longevity, prothrombotic microparticle (MP) formation, and oxygen (O2) delivery 
capacity of transfused RBCs. 
 
The purpose of this study is to:  
1.  To demonstrate that Rejuvenation improves in vivo RBC survival. 
We hypothesize that the loss of HbA over a transfusion interval will be reduced after a 
transfusion treatment with rejuvenated compared to standard RBCs. 
This will allow prolonging transfusion intervals to maintain the same %HbA target. 
 
2.  To determine whether Rejuvenation reduces in vivo, RBC microparticles. 
We hypothesize that in vivo RBC-MP counts and RBC/RBC-MP-mediated thrombin 
generation before transfusion therapy will be lower after a prior treatment with 
rejuvenated compared to standard RBCs (“trough level”). Similarly, RBC-MP counts and 
RBC/RBC-MP-mediated thrombin generation will be lower immediately after transfusion 
therapy with rejuvenated compared to standard RBCs (“peak level”). This will minimize 
the contribution of transfusion therapy to the pre-exisiting, prothrombotic MP burden. 
 
3.  To demonstrate optimal in vivo O2 delivery capacity after Rejuvenation 
We hypothesize that, compared to standard RBCs, transfusion of rejuvenated blood will 
not reduce the p50 of patients’ blood. A high p50 is a compensatory mechanism to 
maintain tissue O2 delivery in SCD, since standard RBCs have a low p50 from 2,3-DPG 
loss. 
 
Background & Significance –SCD is a group of inherited RBC disorders that affects 
approximately 100,000 SCD patients in the United States and millions worldwide, 
making it a major national and international health concern. Patients with SCD that 
require RBC therapy carry the burden of approximately monthly transfusions to maintain 
their health due to clearance of transfused cells. In contrast to other medical treatments 
for SCD complications, repeated RBC therapy via RBC-ST or RBC-XT is considered for 
those with the most severe disease manifestations. These patients have often suffered or 
have an elevated risk of developing SCD complications such as stroke, transient ischemic 
attack, acute chest syndrome, priapism, or persistent chronic pain. While beneficial, 
transfusion is also associated with potential risks and adverse effects, including immune 
hemolysis, RBC alloimmunization, iron overload, transfusion reactions, and infection 
with bacteria, viruses, and parasites are all potential risks of transfusion; therefore 
maximizing the benefit and minimizing the risk of each transfusion is necessary. 
RBC transfusion was approved on the basis of short-term quantitative parameters, 
including <1% hemolysis in the unit at time of infusion and an in vivo recovery >75% of  
 


Rejuvesol® Washed RBC in Sickle Cell Patients Requiring Frequent Transfusions 
Research Summary 
 
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Research Summary Version -2- 26-2017 
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transfused cells 24 hours after infusion. There was no regard for the RBC quality, longer-
term in vivo survival kinetics, burden of RBC byproducts with repeated transfused doses, 
or tissue oxygenation capacity. Subsequent research has demonstrated that RBCs 
exhausted by refrigerated storage are structurally and functionally abnormal. These cells 
are more rigid, less tolerant of stress, form increased pathogenic ex vivo RBC-MPs, and 
abnormally adhere to the vascular endothelium.1,2,6,20 Intracellular ATP levels decrease 
during storage and can impair the ability of RBCs to effect local vasodilation in hypoxic 
microcirculatory environments to promote O2 delivery.24,25 Furthermore, ATP losses 
decrease the function of vital intracellular ATP-dependent enzymes, such as the flippases 
that maintain normal membrane phospholipid asymmetry.26 Stored RBCs are also 
depleted of 2,3-DPG and have lower p50, thus are less effectively able to transport and 
release O2 where it is needed in the tissues.3 Once transfused, these altered RBCs are 
cleared from circulation and produce additional burdens of RBC byproducts and 
RBC-MPs already present in SCD patients.4-6 Rejuvenation of stored RBCs in the Blood 
Bank is a FDA-approved process that improves several cell parameters that are directly 
relevant to SCD pathophysiology, including RBC recovery, RBC-MP burden, in vivo 
RBC-MP formation, ATP and 2,3-DPG levels, p50, microcirculatory oxygenation, 
membrane deformability, and vascular endothelial adhesion.2,3,14-20 Surprisingly, 
benefits of RBC rejuvenation for transfusion in the SCD population specifically have not 
been explored. We postulate that RBC rejuvenation increases the in vivo survival of 
transfused RBCs, decreases pathogenic RBC-M formation in vivo, and can improve in 
vivo tissue oxygenation via normalization of p50. 
 
Our proposed, pilot cross-over, single-blind study seeks to demonstrate the feasibility that 
RBC rejuvenation can improve the longevity and quality of transfused RBCs in 
chronically transfused SCD patients. Power analysis (Dr. Yi-Ju Li, Duke University) was 
performed based on the 2015 %HbA results from the monthly transfusion records of 15 
UNC SCD patients. Persistence of HbA during the transfusion interval represents 
survival of transfused RBCs. From these data, the mean (±SD) decrement of %HbA per 
day (total %HbA decrement per transfusion interval in days) was 0.76% (±0.16). 
Therefore, the average %HbA decrement of 22.5% over 30 days can 
be considered a “standard decrement”. In order to increase the interval by 7 days, while 
maintaining a “standard decrement”, we must reduce the %HbA decrement/day from 
0.76% to 0.62% per day (a 19% reduction). This could decrease transfusions visits from 
12 to 10 per year, with a projected cost saving of $10,000 per year per patient. Our null 
hypothesis is that the equivalence limit of mean differences of %HbA decrement per day 
between two treatments (standard versus rejuvenated) is ±19%.  
This study offers to benefit both SCD patients and potentially other massively transfused 
populations, such as trauma patients. The use of an FDA-approved process permits 
immediate application from bench to bedside upon completion of the research.  
RBC-ST and RBC-XT, as detailed in the study timeline. This has the potential to 
revolutionize RBC therapy and benefit millions of SCD patients worldwide. 
 
We additionally are interested in exploring the effectiveness of rejuvenation at 
reversing the deleterious biochemical changes that occur in stored RBCs, and have 
discussed the possibility of collaboration with Dr. Angelo D’Alessandro at the 


Rejuvesol® Washed RBC in Sickle Cell Patients Requiring Frequent Transfusions 
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University of Colorado-Denver (UC-Denver). Dr. D’Alessandro has extensive 
experience analyzing the metabolic changes that occur during RBC storage34,35, and is 
interested in identifying the biochemical differences between RBCs of sickle-cell 
patients receiving rejuvenated transfusions compared to standard transfusions. As such, 
we propose an external specimen repository, to be stored securely at UC-Denver under 
the supervision of Dr. D’Alessandro, which will be used to conduct future research that 
is unspecified at this time but will generally involve metabolomic analyses of subject 
RBCs. Investigators at Duke will contribute only leftover blood samples to this 
repository, and subjects will be given the option to opt-in or opt-out of this repository. 
Subjects will not receive additional needlesticks for this repository and no additional 
blood will be drawn. All biological samples will be de-identified and no personnel 
outside of DUHS will be provided with information that can be used to identify subject 
samples. All sample transfer will occur within the context of a pending materials 
transfer agreement, and Dr. D’Alessandro will obtain IRB approval or waiver from his 
institution for this repository. 
 
Design & Procedures – This is a pilot study. In this prospective, single blind, cross-over 
clinical trial, patients with sickle cell disease will receive either rejuvenated (R) or 
standard (S) RBCs with each treatment over a 6-treatment period as follows to maximize 
detection of any signal: SRR|RSS. Each patient will receive 1 transfusion per treatment.  
Each transfusion consists of 2 units of RBCs or RBC apheresis exchange consisting of 
approximately 5 units of RBCs.  Treatment 1 after enrollment the patient will receive the 
standard RBC’s (in all units) Treatment 2 and treatment 3 the patient will receive the 
RBCs treated with Rejuvesol (in the last 4 units).  The patient then crosses over to the 
RSS arm. Treatment 4 the patient receives Rejuvesol treated RBCs (in the last 4 units). 
Treatments 5 and 6 the patient receives standard RBCs (in all units). For patients 
receiving RBC-XT, only the final 4 RBC units used in the treatment will be rejuvenated 
as they are the most likely to persist in circulation following treatment completion. 
The Duke site (Dr. Shah) will enroll 4 chronically transfused patients receiving RBC-ST 
(2 units each month) or RBC-XT (approximately 5 units each month). Total patient 
enrollment at Duke is expected to be 4 subjects  and will be treated as a single site pilot 
study. 
Patients at Duke undergo RBC-ST with 2 units or RBC-XT with approximately 5 units 
every 4-6 weeks. This established care pattern and current medications will be recorded 
but not altered. Only the RBC units will be modified. 
RBCs provided by the Blood Bank 
All patients will receive crossmatch-compatible leukoreduced RBCs that have been 
phenotypically matched for at least ABO, D(Rh), C, E, and Kell antigens. For sessions in 
which standard units are used, no additional manipulations will be performed. For 
sessions in which rejuvenated units are needed, rejuvenation will be performed with 
Rejuvesol® RBC Processing Solution (Citra Labs, LLC, Braintree, MA) per 
manufacturer’s protocol.30 Briefly, RBC units are incubated with rejuvenating solution 
for 1 hr at 37 C in the blood bank under the supervision of co-director Dr. Jessica 
Poisson, washed with saline in a C.A.T.S. (Fresenius Kabi AG; Bad Homburg, 
Germany), and suspended in 0.9% saline/0.2% dextrose for transfusion. The C.A.T.S. is 


Rejuvesol® Washed RBC in Sickle Cell Patients Requiring Frequent Transfusions 
Research Summary 
 
PI:  Ian Welsby, MD  
Research Summary Version -2- 26-2017 
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an intra-operative cell-salvage device maintained by the Biomedical Engineering 
Department and routinely used in the ORs. The C.A.T.S. is FDA-approved for the 
preparation of washed RBC units, and thereby meets Rejuvesol® manufacturer’s 
specifications for the required washing step. Due to logistical challenges, the wash 
procedure will be performed  at the bedside by study personnel specifically trained in the 
use of the C.A.T.S. apparatus, and the C.A.T.S. apparatus will be maintained using 
standard blood bank QC procedures. Flowsheets designed in conjunction with the blood 
bank will be used during these wash procedures to maintain the safety and integrity of the 
RBC units and wash process. All flowsheets and QC will be maintained in study binder 
and reviewed monthly by Dr. Poisson of the blood bank. Consistent with AABB 
regulations, all units washed with the C.A.T.S device will be transfused within 4 hours. 
Demographics and standard clinical and laboratory data 
Demographic data include age, sex, race, height, and weight. Pre- and post-transfusion 
session vital signs will be recorded (temperature, heart rate, respiratory rate, blood 
pressure, and pulse oximetry), complete blood count with differential, peripheral blood 
film analysis (Advia 120; Siemens Healthcare, Malvern, PA) and HbA/S analysis 
routinely drawn. ABO type, RBC antibody screen, calculated patient total blood volume, 
calculated patient total RBC volume, and volume and storage age of RBCs transfused for 
each session will also be recorded. 
Study specific laboratory data 
With no additional blood draw time points or venipunctures, an additional 18 ml whole 
blood will be drawn into 4 citrated tubes and 2 EDTA tubes for RBC-MPs and p50 
measurements at the time of routine blood draws pre- and post-transfusion. This means 
the total monthly volume of study blood is 36ml.  
Specific procedures to accomplish each objective 
1.  Hb A/S determinations. Samples will be whole blood in EDTA. Bio-Rad Variant II 
HPLC system with Variant II β-Thalassemia Short Program Reorder Pack (Bio-Rad 
Laboratories, Inc.; Hercules, CA) will be used per manufacturer’s instructions. Briefly, 
samples will be mixed, diluted with specific hemolyzing/wash buffer, and injected into an 
analytic cartridge. Absorbance will be measured at 415 nm. Background noise will be 
reduced with use of a second wavelength at 690 nm. Raw data will be integrated by 
manufacturer’s software, and a chromatogram will be generated. Integrated peaks will be  
 
assigned to manufacturer-defined windows derived from retention time of the peaks. The 
%Hb will be calculated by determining the area of a peak as a fraction of the total area of 
all Hb peaks on the HPLC chromatogram. 
 2: RBC-MP determinations. Samples will be whole blood in citrate. The first blood 
tube will be discarded to avoid contamination during venipuncture. Blood will be 
centrifuged at 1,500g for 10 min at 4 C. The supernatant will be carefully removed into 
microcentrifuge tubes, taking care not to disturb the buffy coat layer. A second 
centrifugation will be performed at 2,000g for 5 min to obtain platelet free plasma (PFP) 
to be frozen at -80 C. PFP aliquots (30 μL) will be stained with the following 
markers/antibodies: 10 μL FITC-labeled AnnexinV mixed 1:1 with Annexin binding 
buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2) and 2 μL PE-labeled anti-
CD235a. All markers/antibodies will be centrifuged at 20,000g x 20 min prior to 
use.RBCMs will be defined as Annexin+/CD235+ events. Calcium-free samples and 


Rejuvesol® Washed RBC in Sickle Cell Patients Requiring Frequent Transfusions 
Research Summary 
 
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Research Summary Version -2- 26-2017 
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single-stained MP-free plasma samples (supernatant from normal, healthy donor PFP 
spun at 100,000g x 60min and stored at -80 C until use) will be used as negative controls. 
BD CompBeads® will be run as compensation controls per manufacturer’s instructions. 
Stained samples will be incubated in the dark for 20 min and then further diluted with 
500 μL of Annexin binding buffer or phosphate buffered saline prior to analysis. All 
buffers will be filtered through a 0.1 μm filter prior to use. CytoCount® beads will be 
added to each sample in equal volume as PFP (30 μL) immediately prior to analysis to 
enable MP enumeration. The concentration of MPs in each sample will be calculated as 
follows: (MPs counted x concentration of beads) / number of beads counted. All samples 
will be analyzed on a Stratedigm S1000Ex (Stratedigm; San Jose, CA, USA) high-
sensitivity flow cytometer equipped with a forward scatter photomultiplier tube (FSC 
PMT) using a side scatter (SSC) trigger and 0.1 μm filtered sheath fluid. MP size gate for 
analysis will be set between 200 and 1000 nm utilizing Megamix Plus FSC/SSC® beads,  
which are a mixture of 900, 500, 300, 240, 200, 160 and 100 nm fluorescent beads. Event 
rates will be <8000 events/sec for all samples to minimize coincident events. 
RBC-mediated thrombin generation determinations. Phosphatidylserine (PS) has 
been found to directly correlate with rate of production of meizothrombin-antithrombin 
(mTAT) complexes and thrombin generation on RBCs. Whole blood samples will be 
collected in EDTA. For positive controls, washed RBCs will be diluted to 20% 
hematocrit in RBC wash buffer and incubated with 10mM N-ethylmaleimide (NEM) at 
37 C for 30 min. NEM-treated cells will then be washed 3 times before incubation with a 
4μM concentration of the calcium ionophore A23187 at 37 C for 30 min. Ionophore-
treated cells will be washed 3 times and diluted to 2x106 cells/mL in RBC wash buffer. 
Similar to controls, samples will be incubated and washed but without NEM or ionophore 
treatment. RBCs will be detected using PE-anti-glycophorin A (CD235a). RBC PS will 
be measured via binding of FITC-labeled Annexin V on a FACS Canto II flow cytometry 
(BD Biosciences Franklin Lakes, NJ). 
RBC-MP-mediated thrombin generation determinations. Whole blood samples will 
be collected in citrate. PFP will be prepared as described above and frozen at -80 C. The 
Zymuphen ELISA (Aniara Inc., West Chester, OH) is a functional assay that measures 
the procoagulant activity of MPs in PFP. A microtiter plate is pre-coated with annexin V-
streptavidin. PS+ MPs bind to the plate and expose their phospholipid surface, allowing  
 
pro-thrombinase complex to cleave prothrombin into thrombin. Phospholipid in the 
sample is the limiting factor, so there is a direct relationship between MP phospholipid 
concentration and thrombin generation. Thrombin generation will be measured by a 
specific chromogenic substrate. Thawed PFP samples and kit controls will be diluted 
1:20 in the kit sample diluent and supplemented with calcium, Factor Xa (FXa), and 
thrombin. A calibrator curve, diluted controls and diluted samples will be added to the 
ELISA plate and incubated for 1 hr at 37 C. The plate will be washed five times, then 100 
μl of Reagent 1 (FXa–FVa) and 50 μl Reagent 2 (prothrombin) will be added and 
incubated for 10 min at 37 C, then 50 μl of Reagent 3 (thrombin substrate) will be added, 
and incubated for 3 min at 37 C. Finally, 50 μl of stop solution will be added and 
absorbance read at 405 nm. Results will be calculated from the calibrator curve. 
3: O2-binding curves and P50 determinations. RBCs will be washed and PBS 


Rejuvesol® Washed RBC in Sickle Cell Patients Requiring Frequent Transfusions 
Research Summary 
 
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Research Summary Version -2- 26-2017 
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containing DTPA. A 10% hematocrit suspension will be prepared in Krebs buffer. The 
RBC suspension will be placed in a specially designed, temperature- and humidity 
controlled rotating tonometer. RBCs will be progressively exposed to varying gas 
mixtures in order to produce fixed solution O2%, typically including 2%, 5%, 8%, and 
20%. After 10 min of gas mixture at each pO2% sample will be removed from the 
tonometer for blood gas measurement. An Abbott i-Stat (Abbott Laboratories; Abbott 
Park, IL) will be used to measure pO2 and directly measures including Hb O2 saturation. 
The Hb O2 saturation versus pO2 curve will be constructed and p50 extrapolated for each 
sample. p50 results will be generated and O2-binding curves displayed graphically. 
4: Preparation of samples for external repository. Any blood remaining after the 
above analyses are complete will be spun to separate RBCs and plasma at 2000g for 10 
minutes at 4 degrees Celsius. Once separated, plasma and RBCs will be stored in separate 
deidentified cryovials at -80 degrees Celsius. These specimens will be batch-shipped to 
Dr. Angelo D’Alessandro at the University of Colorado-Denver according to the terms of 
the pending materials transfer agreement. 
 
Selection of Subjects – Inclusion criteria: Adults 18 years of age and older who are 
undergoing frequent RBC transfusions as part of their routine care for Sickle cell disease 
will be approached for participation. Exclusion criteria: Patients with acute pain crisis 
with 2 weeks prior to enrollment.   Duke expects to enroll 4 subjects for this pilot study. 
 
Subject Recruitment and Compensation – Subjects will be identified by the Co-
investigator Dr. Shah for the Sickle cell patients after review of the sickle cell transfusion 
schedule. All subjects are Dr. Shah’s patients.  The consent form will be presented to the 
prospective subjects/parents by the physician investigator in this study or the study 
coordinators known to the subject/parents. Participation in the research study will not 
significantly alter the routine clinical management as currently practiced at Duke 
University Medical Center. Patients will not be compensated for participation in the 
research study. 
 
Consent Process – See section 14 of the eIRB submission 
 
Subject’s Capacity to Give Legally Effective Consent – All subjects must have the 
capacity to give legal consent. 
 
Study Interventions – Please refer to item 4 
 
 
 
Risk/Benefit Assessment –  It is impossible to estimate without conducting this study 
what the benefit or risk of participation in this study is.  There are no apparent risks for 
subjects participating in this study. Rejuvenation is an FDA approved process and an 
AABB standardized procedure; furthermore pediatric patients may often receive washed 
units of packed red blood cells. Risks are no more than receiving a unit of PRBCs as all 
these products are approved. No subject will receive a transfusion specifically for the 


Rejuvesol® Washed RBC in Sickle Cell Patients Requiring Frequent Transfusions 
Research Summary 
 
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purpose of this study. RBC rejuvenation will only involve units of blood that would have 
been used for the patient regardless of the study. 
No additional blood draws will be done specifically for the study.  An additional 36ml of 
blood (18ml pre-transfusion and 18 ml post transfusion) will be drawn with the standard 
of care labs.. 
 
Costs to the Subject – There is no cost to the subject for participation in this study. 
 
Data Analysis & Statistical Considerations –Routine clinical measurements will be 
compared with repeated measures analysis of variance. As this is a pilot study and there 
is no control group, the only relevant changes to analyze will be pre- and post-procedure. 
Statistical data obtained (noted and described in the Design and Procedures section) will 
be used for future planning of a larger comparison study.  
No interim analysis is planned but adverse events will be reported to the IRB as required.   
1: To demonstrate that Rejuvenation improves in vivo RBC survival. We 
hypothesize that the loss of HbA over a transfusion interval will be reduced after a 
transfusion treatment with rejuvenated compared to standard RBCs. HbA/S 
determination is routinely performed pre- and post- treatments. The %HbA decrement is 
the current pre-treatment HbA – previous post treatment HbA in %. Our primary outcome 
variable will be HbA decrement per day to account for minor variability. A generalized 
linear model will be applied to determine whether the mean difference in HbA exceeds 
+/-19%. Secondary outcomes will evaluate actual HbA decrement (g/dl) with and without 
indexing to calculated circulating blood volume. If %HbA decrement with rejuvenated 
units exceeds -19% we will reject the null hypothesis and conclude that rejuvenation will 
permit prolonging transfusion intervals by a week to maintain the same %HbA target. 
Whether or not we reject the null hypothesis, these data will inform treatment effect size 
and prospective collection of %HbA variability can validate preliminary data 
strengthening future sample size estimate calculations. 
2: To determine whether Rejuvenation reduces in vivo, RBC microparticles. We 
hypothesize that in vivo RBC-MP counts and RBC/RBC-MP-mediated thrombin 
generation before transfusion therapy will be lower after a prior treatment with 
rejuvenated compared to standard RBCs (“trough level”). Similarly, RBC-MP 
counts and RBC/RBC-MP-mediated thrombin generation will be lower immediately 
after transfusion therapy with rejuvenated compared to standard RBCs (“peak 
level”). These secondary outcomes will inform on effect size of rejuvenation on the 
immediate changes in RBC-MP counts after treatment. If reduction in RBC-MPs is 
augmented by rejuvenation, better efficacy of treatment could be postulated and 
subsequently tested, as this will minimize the contribution of transfusion therapy to the  
 
preexisiting, prothrombotic MP burden. Phosphatidylserine (PS) has been found to 
directly correlate with rate of production of meizothrombin-antithrombin (mTAT) 
complexes and thrombin generation on RBCs. The capacity of RBCs and RBC-MPs to 
generate thrombin could inform evaluation of future anticoagulation needs and targets. 
Wilcoxon Rank Sum tests will be used to compare RBC-MP counts and thrombin 
generation. 
3:  To demonstrate optimal in vivo O2 delivery capacity after Rejuvenation. We 


Rejuvesol® Washed RBC in Sickle Cell Patients Requiring Frequent Transfusions 
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hypothesize that, compared to standard RBCs, transfusion of rejuvenated RBCs 
will not reduce the p50 of the patients’ blood. The effect of transfused RBCs on 
overall, in vivo p50should be dose related; this study is uniquely poised to demonstrate 
this phenomenon by comparing low-dose RBC-ST to high-dose RBC-XT. 
Determinations of O2-binding curves and, therefore, accurate p50 measurements will 
inform the potential for rejuvenation to optimize tissue oxygenation. Invasive probes are 
required to measure the increased tissue O2 and VO2 max of muscle as p50 is 
increased.31,32 Identifying a dose-dependent modulation of p50 with rejuvenation will 
support future testing in SCD and other critical populations such as trauma, sepsis, or 
cardiogenic shock. Wilcoxon Rank Sum tests will be used to compare change in p50 pre- 
and post-transfusion within groups. 
In the future, if the study team wishes to share any data with UNC or any other external 
researchers an amendment will be submitted. 
 
Data & Safety Monitoring –  
Rejuvesol is a blood product additive that is washed out of the unit prior to 
administration per AABB and FDA standards, it is not given as an iv drug. The effect of 
Rejuvesol is to increase/normalize the p50 of the RBCs to a level that is seen in normal 
and anemic individuals. Rejuvenation is a AABB standardized and FDA approved 
procedure and as such we believe a DSMB is not indicated. AEs and SAEs will be 
reported to the IRB, per policy,  
 
Specific, product related adverse effect would include be to further decrease SaO2 in 
hypoxic individuals, especially if the p50 was markedly increased. For this reason the 
exclusion criterion (per package insert) include not using on units stored for < 10 days 
(2,3, DPG and p50 would be supranormal) and not enrolling patients with a pO2 
<40mmHg or a room air SaO2 <92%. 
 
Data and safety monitoring will be ongoing as part of the standard of care protocol.  
There are no additional procedures or interventions as part of this study. Transfusion 
SOC monitoring will be done according to guidelines and should there be any type of 
reaction the transfusion will be stopped and the appropriate actions taken. 
Any adverse events will be reported to the IRB as required. 
 
Privacy, Data Storage & Confidentiality – Study records that identify subjects will be 
kept confidential as required by law.  Federal Privacy Regulations provide safeguards for 
privacy, security, and authorized access.  Except when required by law, the subjects 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). For records disclosed outside of DUHS, subjects will be 
assigned a unique code number.  The key to the code will be kept in a locked file in Dr. 
Shah’s office. The study results will be retained in the subject’s research record for a 
minimum of 6 years.  Any research information in the medical record will also be kept 
indefinitely. Some information collected about subjects only for this research study may 


Rejuvesol® Washed RBC in Sickle Cell Patients Requiring Frequent Transfusions 
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be kept in a research study record separate from subject’s medical record, and some 
research information may also be part of the subject’s medical record.  Subjects will not 
have access to this research information until the end of the study.   
 
 


Rejuvesol® Washed RBC in Sickle Cell Patients Requiring Frequent Transfusions 
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References- 
1.   Raval JS, Waters JH, Seltsam A, et al. The use of the mechanical fragility test in 
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Rejuvesol® Washed RBC in Sickle Cell Patients Requiring Frequent Transfusions 
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