Conditions: Sickle Cell Disease Sickle Cell Anemia
Keywords: Red Cell Survival Biotin Sickle Cell Anemia Sickle Cell Disease Sickle Cell Trait
Allocation: NON_RANDOMIZED | Intervention model: SINGLE_GROUP
Enrollment: 22
Healthy volunteers: yes

Brief summary:
Background:

Sickle cell disease (SCD) is an inherited blood disorder. It results from a single genetic change (mutation) in red blood cells (RBCs). RBCs are the cells that carry oxygen to the body. In people with SCD, some RBCs are abnormal and die early. This leaves a shortage of healthy RBCs. Researchers want to learn more about how long RBCs live in the human body.

Objective:

To study how long RBCs live in people with and without SCD.

Eligibility:

People age 18 and older who either have SCD, had SCD but were cured with a bone marrow transplant, have the sickle cell trait (SCT), or are a healthy volunteer without SCD or SCT

Design:

Participants will be screened with a medical history and physical exam. They will give a blood sample.

Participants will have a small amount of blood drawn from a vein. In the laboratory, the blood will be mixed with a vitamin called biotin. Biotin sticks to the outside of RBCs without changing their function, shape, or overall lifetime. This process is known as biotin labeling of RBCs. The biotin labeled RBCs will be returned to the participant via vein injection.

Participants will give frequent blood samples. Their RBCs will be studied to see how many biotin labeled RBCs remain over time. This shows how long the RBCs live. Participants will give blood samples until no biotin labeled RBCs can be detected.

During the study visits, participants will report any major changes to their health.

Participation lasts for up to 6 months.

Detailed description:
Study Description:

This study will use biotin-labeling of red blood cells (RBCs) to determine the mean potential lifespan (MPL) of RBCs in patients with sickle cell disease (SCD) compared to patients who have successfully undergone curative bone marrow transplantation (BMT, allogeneic or autologous), participants with sickle cell trait, and healthy donors without SCD. Previous studies have corroborated the MPL of healthy donor RBCs to be approximately 115 days while RBCs from patients with SCD have a much more variable but consistently shorter MPL of approximately 32 days. Allogeneic BMT is a curative therapy for the treatment of severe SCD with stable, mixed donor recipient chimerism after BMT sufficient to reverse the sickle cell phenotype by virtue of improved donor red cell survival compared to the ineffective erythropoiesis of SCD. We predict that the hematologic variables associated with red cell survival among patients with SCD vs. participants with SCT and healthy donors can be used to determine the necessary amount of corrected hemoglobin required to overcome the red cell pathology of SCD. Data generated will be used to determine the utility of performing a population study of RBC lifespan in gene therapy treated patients to ultimately target the percentage of transferred globin gene needed to reverse SCD. The data generated will refine our understanding of the degree of correction necessary to reverse the phenotype of SCD.

Objectives:

Primary Objective: To determine and compare red blood cell survival in patients with SCD, patients with SCD who have undergone BMT, participants with SCT, and healthy donors, and validate the association of red cell survival with known markers of increased red cell survival.

Secondary Objectives: To evaluate correlation of markers of hemolysis (reticulocyte count), number of alpha globin genes, and fetal hemoglobin with RBC survival.

Endpoints:

Primary Endpoint: Red blood cell survival

Secondary Endpoints: Relationship of red blood cell survival to hematologic parameters. Antibody detection to biotin.

Arms:
arm 1: Autologous cells will be collected in participants with Sickle Cell Disease Pre-Transplantation and biotin-labeled ex vivo and reinfused to measure red cell survival arm 2: Autologous cells will be collected in participants with Sickle Cell Disease Post-Transplantation and biotin-labeled ex vivo and reinfused to measure red cell survival arm 3: Autologous cells will be collected in participants with Sickle Cell Trait (HbAS) and biotin-labeled ex vivo and reinfused to measure red cell survival arm 4: Autologous cells will be collected in participants with HbAA (Healthy volunteers) and biotin-labeled ex vivo and reinfused to measure red cell survival

Interventions: intervention 1: Biotin label
Intervention descriptions: intervention 1: Autologous cells will be collected and biotin-labeled ex vivo and reinfused to measure red cell survival

Protocol document:
Prot_SAP_000.pdf: 
 
 Protocol with Statistical Analysis Plan Cover Page:   
 
Official Title: 
 
Red Cell Half Life Determination in Patients with and without Sickle Cell 
Disease 
NCT number: 
 
NCT04476277 
Document Type:  
Protocol with Statistical Analysis Plan (SAP)  
Document Date: 
September 20, 2023 
 


Protocol Number: 20-H-0080
PI: John Tisdale, MD
September 20, 2023 Amendment I
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Clinical Research Project: P184438
National Heart, Lung & Blood Institute
 
Title:  Red Cell Half Life Determination in Patients with and without Sickle Cell Disease
Abbreviated Title: Red Cell Survival
Protocol #: 20H0080
Version Date:   20 SEP 2023
Title: Red Cell Half Life Determination in Patients with and without Sickle Cell Disease
NIH Principal Investigator:
John F. Tisdale, MD, CMTB, NHLBI (E)
National Heart, Lung & Blood Institute (NHLBI)
National Institutes of Health (NIH)  
Building 10
9000 Rockville Pike
Bethesda, MD 20814
Phone: (301)402-6497
E-mail: johntis@nhlbi.nih.gov
Other identifying words:  Red cell survival, cohort label, biotin, sickle cell anemia, sickle cell disease
Principal Investigator:   John F. Tisdale, MD, CMTB, NHLBI (E)
  
Estimated duration of study:  18 months
Subjects of study: 
Number 
       Sex
Age range 
Patients with Sickle Cell Disease (SCD) and controls
   21*
      either
18 and above
(*The accrual goal is 21 evaluable subjects; however, to cover screenfails, withdrawal, drop-outs and 
loss-to-follow ups, an accrual ceiling of 40 subjects is allowed). 
Cohorts
-
SCD pre-transplantation
-
SCD post-transplantation
-
HbAS
-
HbAA (Healthy volunteers)
Project involves ionizing radiation?  
No
Project uses “Durable Power of Attorney”?  
No
Offsite project?  
Yes
Multi-institutional project?  
No


Protocol Number: 20-H-0080
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September 20, 2023 Amendment I
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Investigational Agent:
Drug Name:
CC TAG BIOTIN ®
IND Number:
IND 19550
Sponsor:
NHLBI OCD
Manufacturer:
ChemConnection BV


Protocol Number: 20-H-0080
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September 20, 2023 Amendment I
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PROTOCOL SUMMARY
Synopsis
Title:
Red Cell Half Life Determination in Patients with and without Sickle 
Cell Disease
Study Description:
This study will use biotin-labeling of red blood cells (RBCs) to 
determine the mean potential lifespan (MPL) of RBCs in patients with 
sickle cell disease (SCD) compared to patients who have successfully 
undergone curative bone marrow transplantation (BMT, allogeneic or 
autologous), participants with sickle cell trait, and healthy donors 
without SCD. Previous studies have corroborated the MPL of healthy 
donor RBCs to be approximately 115 days while RBCs from patients 
with SCD have a much more variable but consistently shorter MPL of 
approximately 32 days. Allogeneic BMT is a curative therapy for the 
treatment of severe SCD with stable, mixed donor–recipient chimerism 
after BMT sufficient to reverse the sickle cell phenotype by virtue of 
improved donor red cell survival compared to the ineffective 
erythropoiesis of SCD. We predict that the hematologic variables 
associated with red cell survival among patients with SCD vs. 
participants with SCT and healthy donors can be used to determine the 
necessary amount of corrected hemoglobin required to overcome the 
red cell pathology of SCD. Data generated will be used to determine the 
utility of performing a population study of RBC lifespan in gene 
therapy treated patients to ultimately target the percentage of 
transferred globin gene needed to reverse SCD. The data generated 
will refine our understanding of the degree of correction necessary to 
reverse the phenotype of SCD.
Objectives:
Primary Objective: To determine and compare red blood cell survival 
in patients with SCD, patients with SCD who have undergone BMT, 
participants with SCT, and healthy donors, and validate the association 
of red cell survival with known markers of increased red cell survival.
Exploratory Objective: To create a mathematical model incorporating 
RBC survival and reticulocyte count to determine the necessary 
amount of normal hemoglobin, and therefore vector copy number or 
amount of transferred globin, required for gene therapy protocols. 
Endpoints:
Primary Endpoint: Red blood cell survival
Secondary Endpoints: Relationship of red blood cell survival to 
hematologic parameters. Antibody detection to biotin.
Study Population:
Healthy volunteers, participants with known SCT or SCD, and patients 
with SCD who have successfully undergone curative hematopoietic 
stem cell transplant. No selection will be based upon gender, race, or 
age (in those 18 years or greater in age). Participants must be available 
for frequent blood draws required by and for the duration of this 
study.
Phase:
Pilot
Description of 
Sites/Facilities Enrolling 
Participants:
This is a pilot single site study. Participants will be enrolled at the 
Clinical Center at the National Institutes of Health. 
Description of Study 
Intervention:
The MPL of RBCs from patients with SCD will be compared to 
patients who have successfully undergone curative BMT, participants 
with SCT, and healthy donors without SCD, the latter two who serve as 
donors for patients with SCD who undergo BMT. RBCs will be isolated 


Protocol Number: 20-H-0080
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September 20, 2023 Amendment I
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from participant whole blood. RBCs will be biotinylated ex vivo and 
reinfused the following day. Biotin can be easily tracked via flow 
cytometry therefore venous sampling will occur at regular intervals 
until the percentage of biotinylated RBCs has decreased to the lower 
limit of detection.
Study Duration:
Estimated 18 months
 


Protocol Number: 20-H-0080
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September 20, 2023 Amendment I
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Table of Contents
PROTOCOL SUMMARY...............................................................................................................3
Synopsis.......................................................................................................................................3
STATEMENT OF COMPLIANCE ................................................................................................5
1.0
Introduction and Background ...............................................................................................7
2.0
Objectives .............................................................................................................................8
3.0
Scientific and Clinical Justification......................................................................................8
4.0
Subject Eligibility .................................................................................................................9
5.0
Study Design and Methods...................................................................................................9
6.0
Procedures, Blood Collection, Laboratory Tests and Processing.......................................10
7.0
Analysis of the Study..........................................................................................................14
8.0
Drug Formulation, Availability, and Preparation...............................................................17
9.0
Data and Safety Monitoring Plan .......................................................................................18
10.0
Adverse Events, Deviations, and Unanticipated Problem Assessments.............................18
11.0
Specimen and Data Storage, Tracking and Disposition .....................................................20
12.0    Confidentiality and Privacy ................................................................................................22
13.0
Human Subjects Protections...............................................................................................23
14.0
Compensation for Participation ..........................................................................................26
15.0
Reimbursement for Travel..................................................................................................26
References......................................................................................................................................28
APPENDIX A: NIH INFORMATION SHEET ON STAFF RESEARCH PARTICIPATION 
(April 2016) ...................................................................................................................................29
APPENDIX B: SOE SCD Pre-transplant Patient……………………………………………….30 
APPENDIX C:  SOE HbAA, HbAS, SCD Post-transplant Volunteers ………………………...35 


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STATEMENT OF COMPLIANCE
The trial will be carried out in accordance with International Conference on Harmonisation Good Clinical 
Practice (ICH GCP) and the following: 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 CFR Part 
46, 21 CFR Part 50, 21 CFR Part 56, and/or 21 CFR Part 312) 
National Institutes of Health (NIH)-funded investigators and clinical trial site staff who are responsible for 
the conduct, management, or oversight of NIH-funded clinical trials have completed Human Subjects 
Protection and ICH GCP Training.
The protocol, informed consent form(s), recruitment materials, and all participant materials will be 
submitted to the Institutional Review Board (IRB) for review and approval.  Approval of both the protocol 
and the consent form must be obtained before any participant is enrolled.  Any amendment to the protocol 
will require review and approval by the IRB before the changes are implemented to the study.  In addition, 
all changes to the consent form will be IRB-approved; a determination will be made regarding whether a 
new consent needs to be obtained from participants who provided consent, using a previously approved 
consent form.


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1.0 Introduction and Background
Sickle cell disease (SCD) is a severe hereditary form of anemia in which a mutated form of the beta globin 
gene distorts red blood cells and decreases red cell survival. Unstable sickle hemoglobin rapidly 
polymerizes into gelled, aggregated hemoglobin S (HbS) tetramers at low oxygen levels, leading to 
increased red cell rigidity, oxidative damage of the red cell membrane, distorted red cell morphology, and 
a shortened red blood cell (RBC) survival. The altered rheology of erythrocytes and chronic hemolysis 
leads to the numerous downstream effects including activation of monocytes, leukocytes, platelets, plasma 
proteins and endothelium. This red cell rigidity and subsequent inflammation is responsible for the 
devastating clinical manifestations of SCD including chronic anemia, viscosity changes, intravascular 
sludging, stroke, end organ damage, and ultimately, decreased overall survival (1). 
Inherent instability of the HbS polymer leads to oxidative damage of the red cell membrane, with 
subsequent activation of ion channels leading to a dehydrated and acidotic RBC with increased risk for 
hemolysis and decreased lifespan. In contrast with the lifespan of RBCs from healthy donors (HD) or even 
individuals with sickle cell trait (SCT) who have been shown to have an RBC lifespan of approximately 
107-115 days (2-4), the mean life span of RBCs in patients with SCD is approximately 10-32 days (2, 5-
6). Certain molecular factors are thought to affect the severity of anemia in patients with SCD, likely 
reflected by different rates of hemolysis and therefore different hemoglobin levels at steady state. Several 
biochemical markers of hemolysis have a correlation to erythrocyte survival in patients with SCD, including 
a strong inverse correlation with absolute reticulocyte count (ARC) (r=-0.84, p<0.0001), percent of 
reticulocytes (r=-0.78, p=0.002), α globin gene number (r=-0.15, p=0.008), and a positive correlation with 
percent fetal hemoglobin (HbF) (r=0.58, p=0.038) (2,4). The range of HbF content in fetal hemoglobin 
containing cells (F cells) appears to be important, with cells on the high end of the range surviving longer 
than those on the low end (7). Furthermore, the survival of non-F cells correlates inversely with the fraction 
of F cells, with shorter non-F cell RBC lifespan in patients with a high percentage of F cells independent 
of hydroxyurea therapy (7).  
There are two methods to determine red cell survival; in vivo cohort methodology or in vitro population 
labeling. Cohort labeling involves labeling hemoglobin with oral glycine containing radioactive or stable 
isotopes (15N-glycine) which becomes metabolically incorporated into hemoglobin-synthesizing 
normoblasts in the bone marrow (BM). The resulting labeled RBCs are similarly aged, and mature and are 
released from the BM at the same time. The quantification of RBC lifespan would therefore require a full 
time-course study of approximately 150 days (based on HD data and time to maximum 15N-glycine 
detection) and analysis by mass spectrometry. While this method allows study of single age cohort and does 
not require ex vivo manipulation, samples are not analyzed until the completion of the study and would 
therefore not be available live throughout the study. 
In contrast to the cohort method, current population labels include biotin and 51Cr, which tag a 
representative population of circulating RBCs irrespective of RBC age. The ideal label has minimal RBC 
manipulation, stability throughout the life of the RBC, lack of RBC shortening due to labeling, and lack of 
toxicity to the individual receiving labeled RBCs. Biotinylated RBCs (BioRBCs) confer advantages over 
radiolabeled methods by upholding these ideals in addition to posing no radiation risks and providing a 
more acceptable method to study vulnerable populations such as women, fetuses, infants, and children. A 
further advantage of the population study method is the label (ie. biotin) can be easily tracked via flow 
cytometry, and the analysis can be complexed with additional characterization parameters such as 
intracellular staining for different hemoglobin moieties. The labeled population of cells is representative of 
what is found in circulation at the time of labeling and therefore the RBCs are of different ages. This type 
of study has allowed for measurement of phosphatidylserine exposure on RBCs as they age in vivo (8) and 
the quantification of lifespan of F cells versus non-F cells in SCD patients as previously described (7). 


Protocol Number: 20-H-0080
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Additionally, a retrospective analysis of a population study data set suggests that a 28-day window may be 
sufficient for accurate quantification of MPL(4).
The only currently available cure for SCD is bone marrow transplantation (BMT), with gene therapy 
research active and underway. Stable, mixed donor–recipient chimerism after BMT is sufficient to reverse 
the sickle cell phenotype by virtue of improved donor red cell survival compared to the ineffective 
erythropoiesis of SCD (9). A previously developed mathematical model to test the hypothesis that the 
percentage of necessary donor myeloid chimerism is determined solely by the differences between donor 
and recipient RBC half-lives has been described (10). As stable mixed chimerism is intrinsically linked to 
the differences in RBC survival in healthy vs. the SCD genotype, understanding the variability and 
predictors of red cell survival among patients with SCD vs. individuals with SCT and healthy donors is 
critical for applied work in gene therapy research which seeks to generate sufficient corrected hemoglobin 
to overcome the red cell pathology of SCD. 
Here we described a method to study the MPL of RBCs from patients with SCD, compared to patients who 
have successfully undergone curative BMT, individuals with SCT, and healthy donors without SCD, the 
latter two who serve as donors for patients with SCD who undergo BMT. Unlike biotinylation data from 
patients with SCD and healthy donors, the MPL of RBCs from participants with SCT has only previously 
been validated using the 51Cr population method (3). As individuals with SCT are valuable donors to 
patients with SCD undergoing HSCT, validation of the biotinylation method in this important population 
is critical. Understanding the differences in MPL of RBCs from these groups using a uniform method may 
ultimately guide gene therapy protocols to determine adequate hemoglobin content, ie. vector copy number 
(number of lentiviral vector copies carrying the therapeutic gene of interest in individual target cells, VCN) 
and/or the amount of corrective fetal or adult globin, required to overcome the SCD phenotype as it relates 
directly to red cell survival. Such application in the gene therapy patients will help target the percentage of 
transferred globin needed to reverse SCD based on a patient’s engraftment, VCN or transferred globin 
editing, and red cell survival.
2.0 Objectives
2.1 
Primary: To determine and compare red blood cell survival in patients with SCD, patients with SCD 
who have undergone hematopoietic stem cell transplantation, participants with SCT, and healthy 
donors, and validate the association of red cell survival with known markers of increased red cell 
survival including ARC, % HbF, and F cell percentage.
 
2.2
Exploratory: To create a mathematical model incorporating RBC survival and reticulocyte count to 
determine the necessary amount of normal hemoglobin, and therefore VCN or amount of 
edited/transferred globin, required for gene therapy protocols. This model can be validated by 
studying lifespan in patients after gene therapy treatment. 
3.0 Scientific and Clinical Justification
The safety of biotinylating RBCs in SCD patients is known as is a shortened RBC survival. Known 
influencers of RBC survival such as ARC, %F, and F cells, and mixed chimerism after BMT is sufficient 
to reverse the sickle phenotype by virtue of improved donor red cell survival compared to the ineffective 
erythropoiesis of SCD. The percentage of transferred globin necessary to cure SCD through a gene therapy 
approach and the relationship among ARC, amount of HbA and/or HbF, and measured red cell survival is 
currently unknown. Furthermore, RBC survival in patients with SCD who have successfully undergone 
HSCT has never been performed, as has the biotin methodology for individuals with SCT who are a 
critically important donor source for patients undergoing BMT. 


Protocol Number: 20-H-0080
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Collection and biotinylation of RBCs in this study will allow the measurement and study of red cell survival 
in patients with and without SCD. The study of RBC survival and its relation to known influencers of red 
cell survival including ARC, % HbF, and F cell percentage will allow for the creation of a mathematical 
model that will help determine necessary amount of normal hemoglobin required to reverse the SCD 
phenotype. Such determination has the potential to influence current and future gene therapy protocols. 
Future questions include the ability to measure RBC lifespan in gene therapy patients, and the relationship 
of transferred globin (VCN) and percent engraftment of gene modified cells to the patient’s RBC survival. 
As has been previously described (10), the reticulocyte fraction is a good proxy for RBC lifetime, and 
therefore gene therapy may be most efficacious in patients in which RBC survival is lowest (with highest 
reticulocyte count) as they would require lower amounts of normal hemoglobin production (lower levels of 
gene correction) to sufficiently reverse the SCD phenotype. 
The population method will be used here to study RBC lifespan due to the safety, potential for a 
substantially shorter study period, and the possibility of being able to complex the labeling of the RBCs 
with other markers of interest.
4.0 Subject Eligibility
4.1
Inclusion criteria:
4.1.1
Age 18 or greater with a confirmed diagnosis of homozygous SCD (HbSS, HbSC, 
HbSB0), sickle cell trait (HbAS), or healthy volunteer (HbA)
4.1.2
Normal renal function: creatinine <1.5 mg/dL
4.1.3
Negative direct antiglobulin test (DAT)
4.1.4
Ability to give informed consent to participate in the protocol
4.2 Exclusion criteria:
4.2.1
Any uncontrolled chronic illness other than sickle cell disease
4.2.2
Active viral, bacterial, fungal, or parasitic infection
4.2.3
Consumption of biotin supplements or raw eggs within 30 days
4.2.4
Blood loss within the previous 8 weeks >540mL
4.2.5
Pregnancy
4.2.6
Pre-existing, naturally occurring antibodies against biotin
5.0 Study Design and Methods
 
5.1 Description of Study Population and Recruitment 
This is a pilot single site study and participants will be enrolled at the NIH Clinical Center. Recruitment 
will occur from a pool of normal volunteers, individuals with known SCT or SCD, and patients with SCD 
who have successfully undergone curative hematopoietic stem cell transplant. Volunteers will be recruited 
through the Clinical Center Volunteer Office.  
The recruitment ceiling is an N=40 to account for screen failures.  Accrual will be stopped once the 
numbers of active participants who have  completed data collection have been reached:

Normal Volunteers: 
 N=3

Individuals with SCT: 
 N=6

Individuals with SCD pre-transplantation:
 N=6

Individuals with SCD post-transplantation:
 N=6-10


Protocol Number: 20-H-0080
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Accrual will be based solely up on protocol entry criteria and no selection will be based upon gender, 
race, or age (in those 18 years or greater in age).  The study will be listed on the clinicaltrials.gov, Clinical 
Center research studies, and the National Heart, Lung and Blood Institute patient recruitment websites.  
5.2 Study Design
Figure 1. Schematic diagram of the study design 
*Participants with SCD pre-HSCT will have samples collected weekly until completion of study.
6.0 Procedures, Blood Collection, Laboratory Tests and Processing
6.1
Screening: Potential and enrolled participants may have had pre-study evaluation procedures 
conducted for clinical and research purposes under the IRB-approved NHLBI screening protocol 
97H0041. Prior, concurrent and subsequent results from pre-study evaluation procedures 
performed under this screening protocol may be utilized to determine eligibility for this protocol.  
If previous pre-study evaluations are not available from 97H0041, then all screening activities 
will be conducted under this protocol. 
First, all volunteers will be evaluated with a medical history and a physical examination.  
Next, the volunteer will undergo a venous assessment in the Department of Transfusion Medicine 
(DTM) on the day of screening. Approximately, 50 mL of venous blood will be required for 
screening. Baseline laboratory tests, including complete blood count (CBC) and differential, 
reticulocyte count, hemoglobin electrophoresis, type and screen,  acute care panel, mineral panel, 
hepatic panel, markers of hemolysis (DAT, LDH, AST, ALT, and bilirubin), DTM viral screen, 
existence of pre-existing antibodies to biotin, urinalysis, and a complete medical history will be 
performed (Table 2). All patients who have received a transplant on a Gene Therapy protocol 


Protocol Number: 20-H-0080
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may require additional confirmatory HIV testing which will also be performed under this 
protocol.
Participants with naturally occurring antibodies against BioRBCs will be excluded. All female 
volunteers of childbearing age will be required to undergo a serum pregnancy test. The screening 
visit will be used to determine initial eligibility for the protocol.  Final subject eligibility will be 
confirmed once all lab results are noted in CRIS and eligibility criteria are met.
Once eligibility is confirmed, the participant will be scheduled for biotinylation of patient RBCs, 
and will have α-Globin genotype determined by Southern blot hybridization of genomic DNA 
extracted from peripheral blood leukocytes if the genotype is not already known.
Screen Failures:  If a subject is found ineligible during the screening process and the subject will 
not meet the requirements within 30 days of the screening visit, the subject will be counted as a 
screen failure and removed from study.  
The subject may re-screen if/when it is believed eligibility criteria may be met in accordance with 
the PI’s discretion.  Subjects will need to repeat the DTM viral screen during re-screening, as 
these lab results are no longer valid after 30 days.  Any other test(s) or procedure(s) whose prior 
result rendered the subject ineligible needs to be repeated to see if more recent results would 
allow the subject to meet eligibility criteria.  A new subject number will be assigned to a subject 
who is re-screened.
6.2
Biotinylation General: Collection of RBCs (as per section 6.3), biotinylation of patient RBCs, 
and re-infusion back to the patient within 48 hours as an outpatient. In the event the collection of 
RBCs occurs late in the day and biotinylation is unable to be performed the same day, RBCs will 
be stored overnight at 2-8°C. Biotinylation will be performed at room temperature under sterile 
conditions by the DTM. The goal total time during which the BioRBCs are at room temperature 
(i.e. during biotinylation and the transfusion procedure) should not exceed 4 hours. If immediate 
transfusion is not possible, the BioRBCs should be kept in a monitored refrigerator set at 4°C 
(range, 1-6°C).
6.3 
Collection: On the day of blood collection, approximately 100 mL of venous blood will be 
collected via venipuncture for biotinylation of RBCs (~80 +/- 5mL ), routine labs and alpha-
Globin genotype (~20 mL).   Alpha-Globin genotype will be determined by Southern blot 
hybridization of genomic DNA extracted from peripheral blood leukocytes if genotype is not 
already known. For participants without central venous access, an 18- or 20-gauge catheter will 
be placed in an adequate patient vein.   Routine labs include approximately 20 mL of venous 
blood to provide a baseline complete blood count, reticulocyte count, DAT, and repeat pregnancy 
test (females only). In addition, whole blood is submitted for the purpose of DTM storage and 
will be held per DTM policies.  DTM isolation procedures as defined in BB-DMF 11054 will be 
followed. The remaining approximately 70mL of whole blood will be collected in ACDA (Baxter 
healthcare Corp, Deerfield, IL) to obtain >15mL of packed RBCs (pRBCs). After verification 
that the DAT is negative, biotinylation of the collected sample may proceed.  
6.4
Pre-Biotinylation Washing: The whole blood product will be visually inspected carefully; if 
clots/clumps are observed, product will be filtered before further processing. Blood will be 
centrifuged at 1000 x g (2142 rpm), deceleration (DEC) 4 at room temperature (RT; 17°C –25°C) 
for 10 minutes in table top centrifuge. The plasma layer will be aspirated and saved for use in 
reconstituting RBCs after biotin labeling. RBCs will be washed three times with 4 vol (4:1 wash: 


Protocol Number: 20-H-0080
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September 20, 2023 Amendment I
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RBCs) of PlasmaLyte A (Baxter healthcare Corp, Deerfield, IL). Washed RBCs will be 
resuspended in sufficient wash buffer to yield a 25% suspension of RBCs.
6.5
Preparation of Biotin: The CC-TAG-Biotin (ChemConnection BV, Netherlands) is stored in a -
20ºC freezer and should be allowed to come to room temperature (~10-15min) before opening of 
the vial to avoid saturation of the biotin reagent. To prevent hydrolysis of the biotin reagent before 
addition to the RBCs, the biotinylation reagent will be prepared in wash buffer before use (within 
20 minutes). A solution containing 1 mg/ml biotin will be prepared by dissolving 1 mg biotin in 
1 mL PlasmaLyte A. This solution will then be sterilized by filtration through a 0.22-µm syringe 
filter (21062-25; Corning Glassware, Corning, NY). 
6.6
Biotinylation Reaction: Biotinylation will be performed under a sterile Class 2 laminar flow hood 
in DTM. The appropriate volume of biotin solution will be added with gentle agitation to the 
25% RBC suspension to give the desired final concentration of biotin of 18 µg/mL, incubated at 
room temperature for 30 minutes (Preclinical data shows efficiency of biotinylation of sickle 
RBCs from 1-80 µg/mL, however RBC morphology becomes deformed at concentrations >54 
µg/mL. Flow cytometry peaks separating biotin labeled RBCs from unstained control is best at 
18 µg/mL compared to lower biotin concentrations, without any evidence of RBC morphology 
changes at 18 µg/mL). The reaction will be terminated by sedimentation of RBCs at 1000 g (2142 
rpm), deceleration (DEC) 4 at RT for 10 minutes followed by three 4:1 (vol/vol) buffer washes 
with PlasmaLyte A, sedimentation, and resuspension; this removes the remaining biotinylating 
reagent as well as any reaction and hydrolysis byproducts. The washed BioRBCs will be passed 
through an 18um filter (Hemo-Nate Syringe Filter, Utah Medical Products, Inc, Midvale, UT) 
and resuspended in autologous plasma at a hematocrit (Hct) of 50% (range 40-60%). 150 µL 
(range 50-500 µL) of pre-biotinylated RBCs and 150 µL of post-biotinylated RBCs will be 
withheld and sent to the Tisdale laboratory for pre-clinical studies (to ensure all cells are labeled 
and that there is a baseline separation of labeled and unlabeled cells on flow cytometric 
histogram) via methodology described below (see section 7.1).
6.7 
Final Product: Final samples are removed for CBC, biotin labelling efficiency determination via 
flow cytometry, and safety assays (sterility and endotoxin). Release of the drug product is 
contingent on meeting the criteria listed in the table below (Table 1), which are also listed in the 
Certificate of Analysis in Module 3.2.P.5.4. For Safety testing, the final drug product must have 
an endotoxin level of ≤ 5 EU/mL and be negative for contamination in the sterility tests. Since 
the product is infused fresh, release for use is based on gram stain for sterility, with post-infusion 
testing based on a 14 day culture. The percent hematocrit and the percent of biotinylated RBC’s 
will serve as a measure of purity and identity of the product.
Table 1: Drug Product Release Criteria
bioRBC Drug Product 
Release 
Testing
Assay type
Method
Acceptable
Limit
Purity
Appearance
Visual check
Red; no clots or 
aggregates
Identity/Purity
% HCT
Advia CBC
50 ± 10%
Identity/Purity
% Biotinylated 
RBC
Flow
≥ 80%
Safety
Endotoxin
Limulus amebocyte 
lysate (LAL)
≤ 5 EU/mL


Protocol Number: 20-H-0080
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Safety
Sterility
Gram stain
No organisms seen
Safety
Sterility*
Bacterial, BacT/ALERT 
bacteria 
culture*
No growth in 14 days*
Safety
Sterility*
Fungal culture *
No growth in 14 days*
*Pending at release
6.8
Infusion of Product: For participants without central venous access, an 18- or 20-gauge catheter 
will be placed in an adequate patient vein for infusion of biotinylated RBCs (max volume 50 mL) 
and for obtaining immediate post-transfusion blood samples. Biotinylated RBCs will be reinfused 
per Nursing SOP for Cellular Therapy Products Administration; the time 20 minutes after the 
end of the infusion will be designated time zero (time for equilibration). The catheter will be 
flushed with 5mL normal saline at completion of the infusion. The time zero research lab can be 
drawn from the same IV in which biotinylated RBCs were infused.
6.9
Sample Collection: Study design, assessment parameters, and sample collection intervals (with 
approximate volume of blood required) are shown in Figure 1, Table 2, and Table 3, respectively.  
Approximately 5 mL of venous blood will be collected at 20 minutes after infusion for all 
participants, then approximately 10 mL will be collected at intervals as specified in Table 3 (+/- 
3 days); non-transplant SCD participants will have samples collected bi-weekly for the first two 
weeks, then weekly until the percentage of biotinylated RBCs has decreased to the lower limit of 
detection (approximately less than 0.06%; estimate 1-8 weeks); all other participants (SCD post-
transplant, sickle cell trait, healthy volunteers) will have samples drawn weekly for the first 4 
weeks (starting week 2 through week 4) then every other week until the percentage of biotinylated 
RBCs has decreased to the lower limit of detection (approximately less than 0.06%; estimate 12-
16 weeks) (Table 3).  All participants will return for anti-biotin antibody detection at 6 months 
post-infusion (end of study). Samples will be analyzed as described below (sections 7.1-7.5) for 
% survival biotinylated RBCs, hemoglobin content, F cell percentage, and delay time analysis, 
with the remainder of the sample sent to the clinical lab, DTM, and the Tisdale lab for 
determination of Hb, Hct, reticulocyte count, LDH, AST, ALT, bilirubin, DAT, and detection of 
antibodies to biotinylated RBCs (see section 6.10)(Table 2). At every encounter, participants will 
be asked about any illness since their last visit.
Table 2 Assessment Parameters
Visit
Parameter
Additional History
Screening
CBC
Reticulocyte count
Hemoglobin electrophoresis
Acute Care Panel
Mineral Panel
Hepatic Panel
DAT
ABO/Rh and antibody screen
LDH
Biotin antibody screen*
Serum pregnancy test (females)
**HIV-1/HCV/HBV (NAT)
Hepatitis B (surface antigen)
Hepatitis B (core antibody)
Hepatitis C (antibody)
Complete history including past 
medical history, transfusion 
history, bleeding history, and 
medications. A complete SCD 
history (where applicable) will 
include hydroxyurea use and 
dose, pain crisis frequency, ER 
and hospitalization utilization, 
and transfusion history. 


Protocol Number: 20-H-0080
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September 20, 2023 Amendment I
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HTLV I and II (antibody)
**HIV 1 and 2 (antibody)
West Nile Virus
T cruzi (antibody) 
Treponema pallidum (antibody) or 
equivalent (such as RPR)
Urinalysis
Week 1/Day -1
                        (Blood 
Collection)
CBC
Reticulocyte count
DAT
Serum pregnancy test (females)
α-Globin genotype
Infectious screening of whole blood 
per DTM (see 6.3)
Interim illness update 
(specifically for SCD patients, 
history of pain crisis, 
transfusion, ER visit, or recent 
hospitalization)
For Tisdale Lab& bluebird bio
Week 1, Day 1
(Infusion Day)
% survival biotinylated RBCs
Hemoglobin content
F cell percentage
Delay time analysis
HbF staining
HbS single cell Western Blot
Assay development
Clinical Lab & 
DTM
Tisdale & Eaton 
Labs, bluebird bio
Study Assessments 
(Time Zero – End of Study*)
CBC
Reticulocyte 
count
Acute Care 
Panel
Mineral Panel
Hepatic Panel
LDH
DAT
ABO/Rh and 
antibody screen
Hemoglobin 
electrophoresis
Biotin 
Antibody 
Detection*
% survival 
biotinylated RBCs
Hemoglobin 
content
F cell percentage
Delay time analysis
HbF staining
HbS single cell 
Western Blot
Assay development
Interim illness update as above
*Biotin antibody detection will be done at initial screen, during the course of the study if bioRBC degradation is faster than predicted, 
and at 3 and 6 months post-infusion for all patients regardless of end of study date.
                  **Gene Therapy patients may have other HIV testing, including HIV ½ Antibody/Ag Combo.


Protocol Number: 20-H-0080
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^All patients will follow the sample interval until the percentage of biotinylated RBCs has decreased to the lower limit of detection 
(approximately <0.06%), at which time the next interval for that patient will be the final 6 month (week 24) interval.
2*= Bi-week 2
6.10 Antibody Detection: To test for antibodies to biotinylated RBCs, plasma samples will be tested 
using gel card antibody detection(MTS anti-IgG Card, MTS084024, Ortho Clinical Diagnostics) 
as previously described (11). The detection of specific antibodies to bioRBC does not determine 
whether these antibodies are physiologically active or are clinically significant. Thus far, all 
identified pre-existing, naturally occurring anti-bioRBC antibodies are neutralized by biotin 
compounds, while induced anti-bioRBC antibodies are expected to be inhibited by bioRBC and 
not by free biotin as previously published (11). It is speculated that this is analogous to penicillin-
type drug-dependent antibodies with biotin covalently attached to RBC surface proteins acting 
as immunogenicity enhancing haptens. In those reported cases of anti-bioRBC antibody 
detection, biotinylation occurred at higher concentrations (54-162 µg/mL), antibodies were 
transient and no longer detected after 6 months, and did not alter the red cell survival (4,11).
7.0 Analysis of the Study
7.1 
Flow Cytometry Analysis: Enumeration of BioRBCs will be performed by flow cytometry. 
Samples will be collected pre and post biotinylation prior to infusion (as per section 6.6), at 20 
minutes post infusion (time zero), and as described in section 6.9 & Figure 1. To isolate the RBC 
fraction, 40 uL of whole blood aliquoted in triplicate will be mixed with 1 mL of wash buffer, 
centrifuged at 10,000 x g until the rpm value is reached, and supernatant will be discarded (repeat 
x1). The final washed RBC pellet will be resuspended in 80 µL of wash buffer, mixed with 20 µl 
of a 1:20 dilution (in PBS) of streptavidin-phycoerythrin (SA-PE; S-866; Molecular Probes, 
Eugene, OR), and incubated for 30 min at room temperature in the dark. The staining reaction is 
stopped by adding 3 mL of wash buffer, centrifuging at 1500 rpm for 5 minutes, and removing 
the aspirate. RBCs will then be suspended in 500 µL of wash buffer, and will be filtered through 
a 0.45 µm filter prior to flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA). 
Percentages of BioRBCs will then be measured by flow cytometry.
7.2 
F-cell and S-cell measurement: Simultaneously with the measurement of BioRBCs by flow 
cytometry (see section 7.1), samples will be analyzed with an additional color (3-color flow 
cytometry) for Biotin, HbF, and HbS. After incubation with SA-PE and two washes, the cells 
will be mixed with a 2µL/mL solution of glutaraldehyde/PBS. Cells will be gently mixed and 
incubated at room temperature for 10 minutes. Cells will be centrifuged at 2500 rpm x 5 min, 
then incubated at room temperature with 1mL 0.1% Triton X solution for 10 minutes. After 
centrifugation at 2500 rpm x 5 minutes, cells will be stained for 30 minutes at room temperature 
with a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody to HbS and 
allophycocyanin (APC)-conjugated monoclonal antibody to HbFprior to analysis by flow 
Table 3. Sample Collection Intervals
Week 1/Day 0, 
Infusion Day
Week^
Screening
Pre-
infusion
Post-
infusion
1
2
2*
3
4
5
6
7
8
9
10
11
12
13
14
15
16
24
SCD Pre-
Transplantation
X
X
X
X
X
X
X
X X X
X
X
X
X
X
X
X
X
X
X
X
SCD Post-
Transplantation
X
X
X
X
X
X
X
X
X
X
X
X
X
HbAS
X
X
X
X
X
X
X
X
X
X
X
X
X
HbAA
X
X
X
X
X
X
X
X
X
X
X
X
X
Approximate Volume 
of Blood (mL)
50
100
5
10 (per sample collected)


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cytometry. The F per F cell can be calculated from the percent F cells, the percent HbF 
(determined by HPLC, see section 7.3), and the MCH.
7.3 
Hb Content: BioRBCs will be isolated from post-infusion blood samples using streptavidin-
coated magnetic beads or flow cytometry separation. The collected BioRBCs and the negative 
fraction will be analyzed by high performance liquid chromatography (HPLC) for hemoglobin 
content. 
7.4 
Measurements of delay time for sickling of individual red cells: Blood from patients will be 
diluted 1500 to 3000-fold, depending on the hematocrit, into PBS. 10 µL of the cell suspension 
is added to the wells of a 384-well plate. The well plate is inserted into a humidified chamber of 
a Biotek “Lionheart FX Automated Microscope System” and, for patients with SS disease, 
deoxygenated with a mixture of 96% nitrogen and 4% oxygen to induce sickling. Everything that 
follows is fully automated. Images taken in 430 nm light (the absorption peak of 
deoxyhemoglobin) of 100-200 cells settled on the bottom of each of the 384 wells are measured 
every 15 minutes for up to 8 hours (see Figure 2). A single scan of all 384 wells takes 15 minutes 
because the autofocusing system of the instrument requires about 2.3 seconds to obtain a focused 
image from each well and 56 seconds to scan a row of 24 wells. Because images are taken of 
40,000 to 80,000 cells, excellent statistics can be obtained on more than 20 different blood 
samples in a single experiment (using less than about 100 nanoliters from each patient). Sickling 
times are determined with our in-house developed, robust image analysis software (12), which 
compares several metrics of the cell image to determine in which 15 minute interval the cell has 
become distorted, i.e. sickled. These include the eccentricity (deviation from a circular 
projection) and ratio of the optical density at the periphery of the cell to that at the cell center 
(unsickled cell is more transparent in the center). From the fraction sickle vs time (Figure 3), the 
final output for each blood sample is a t50 for sickling. The comparison between SS and AS cells 
shows the enormous sensitivity to the intracellular hemoglobin composition (> 90% Hb S in SS 
cells; 60/40 HbA/Hb S mixture in AS cells).
Figure 2.  Images of cells at 135 and 150 minutes after the start of deoxygenation with 
96% nitrogen and 4% oxygen.   Cells that sickle between 135 and 150 minutes are 
circled.  At 150 minutes 37 % of the 84 cells are sickled. 


Protocol Number: 20-H-0080
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Figure 3. Mean fraction sickled as a function of time following deoxygenation of SS and AS cells with 96% nitrogen and 4% 
oxygen. The error bars correspond to one standard deviation from the mean of the fraction sickled in 96 wells. They are about twice 
the expected value due to the finite number (~100) of cells in each well. The higher standard deviations compared to AS is mainly 
caused by small differences in the geometry of each of the 10 microliter droplets, which result in differences in the fraction sickled 
at the same deoxygenation time due to differences in the rate of oxygen diffusion out of the droplet. The fraction sickled vs 
deoxygenation time for the SS cells is very similar to the kinetics for AS cells when deoxygenated with 100% nitrogen.
7.5 
Other Analysis: A portion of collected RBCs will be used for imaging flow cytometry, HbF 
staining, HbS single cell Western Blot, and assay development. Duplicate samples of 
approximately 2 mL (range 1-5 mL) of pre-infusion, time zero, and subsequent patient samples 
will be sent to our collaborators at bluebird bio Inc, for parallel analysis and assay development. 
No samples will be sent to our collaborators until a Material Transfer Agreement (MTA) has 
been established.
Another portion of collected RBCs will be used for measurement of glycolytic metabolites. 
Duplicated coded samples of approximately 450 uL will be sent to our collaborators at Agios 
Pharmaceuticals, Inc. for parallel analysis.  The results of these findings will be reidentified at 
the NIH to answer this new analysis. An MTA for this collaboration has been fully executed. 
7.6   Data and Statistical Analysis: This is an exploratory study designed to test methodology and 
assay development for the purposes of studying red cell survival in patients with SCD. RBC 
survival curves will be generated for each participant. Mean potential RBC life span (MPL) will 
be determined by plotting the percentage of remaining biotinylated RBCs in circulation against 
time after transfusion. Median RBC survival and mean RBC age will be calculated from these 
curves. Correlation of mean RBC survival with known markers of red cell survival will be 
performed using Pearson Correlation. The mean of each participant’s clinical laboratory values 
during the study period will be used for correlation with RBC survival. Average MPL among the 
groups studied will be examined descriptively using means and standard deviations. The 
differences between groups will be used to examine the feasibility and guide the design of larger, 
definitive studies; in this pilot study, statistically significant differences are not generally 
anticipated. Similarly, information regarding hydroxyurea use, α globin genotype, etc are likely 
to not be powered to see an effect. 
7.7 
Mathematical Model: We assume that in erythropoietic homeostasis, RBC lifetime is 
proportional to the inverse of the reticulocyte fraction such that RBC survival is lowest in patients 
with the highest reticulocyte counts. We will subsequently develop a mathematical model to 
predict the percentage of necessary hemoglobin to reverse SCD from reticulocyte count and RBC 
half-life.


Protocol Number: 20-H-0080
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7.8
Off Study Criteria: Patients reserve the right to come off study for any reason during the study 
period. Patients may be taken off-study by the PI if there is a health-related event during the 
course of the study that affects RBCs lifespan or production of RBCs including hemorrhage or 
need for transfusion. In the event of pregnancy after infusion of biotinylated RBCs, the patient 
will remain on study unless directed otherwise by the patient.
8.0 Drug Formulation, Availability, and Preparation

Biotin
CC TAG BIOTIN ® (Biotin 3 sulfo N Hydroxysuccinimide ester sodium salt) is derived from 
the natural form of biotin, which is the D enantiomer (Vitamin B7 or B8 according to 
nomenclature of each country). Because of its small molecular size CC TAG Biotin (small 
spacer arm: 13.5 Å, between the biotin and the sulfo NHS group) can be conjugated to many 
proteins without altering their biological activities.
Sulfo NHS activated biotin reacts efficiently with primary amino groups (-NH2) in pH 7-9 
buffers to form stable and covalent amide bonds. Proteins from plasma membrane cells 
generally have several primary amines in the side chain of lysine (K) residues that are available 
as targets for attachment of biotin. Biotin binds with high affinity to avidin and streptavidin 
proteins. Therefore the tracking of biotin labelled cells with the CC TAG BIOTIN ® can be 
performed easily by flow cytometry with fluorescent streptavidin. Biotin labelled proteins can 
also be easily detected in ELISA, dot blot or Western blot applications using streptavidin or 
avidin conjugated probes.
The new cGMP CC-TAG-BIOTIN® has been demonstrated to be efficient in labeling blood 
cells (RBC, WBC and platelets) ex vivo. It will reduce the safety concerns and contribute to 
overcome the need for non- radioactive methods within the framework of in vivo studies in 
humans.
Supply: ChemConnection. (commercially available)
Preparation: As per section 6.5, CC-TAG-Biotin is stored in a -20ºC freezer and should be allowed 
to come to room temperature (~10-15min) before opening of the vial to avoid saturation of 
the biotin reagent. To prevent hydrolysis of the biotin reagent before addition to the RBCs, 
the biotinylation reagent will be prepared in wash buffer before use (within 20 minutes). A 
solution containing 1 mg/ml biotin will be prepared by dissolving 1 mg biotin in 1 mL 
PlasmaLyte A. This solution will then be sterilized by filtration through a 0.22-µm syringe 
filter (21062-25; Corning Glassware, Corning, NY).
Storage: CC-TAG-Biotin is moisture-sensitive. Store reagent at -20°C in the original container with 
desiccant. To avoid moisture condensation onto the product, vial must be equilibrated to room 
temperature before opening.
Route of Administration: As per section 6.6 and 6.8, biotin will be applied ex vivo to patient RBCs, 
then biotinylated RBCs will be reinfused intravenously to the patient. 
9.0 Data and Safety Monitoring Plan
The side effects of blood donation and transfusion are well described and vast experience with the 
procedure exists in the Clinical Center. 
Principal Investigator: Accrual and safety data will be monitored by the Principal Investigator, Dr. 
John Tisdale, who will provide oversight for the conduct of this study. The protocol will be 


Protocol Number: 20-H-0080
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continuously evaluated for any unusual or unpredicted complications with the aim of detecting and 
preventing unacceptable increase in morbidity and mortality over and above that anticipated from 
sickle cell disease or other hemoglobinopathies.
NIH IRB: Accrual and safety data will be monitored and reviewed annually by the Institutional 
Review Board (IRB). Prior to implementation of this study, the protocol and the proposed patient 
consent and assent forms will be reviewed and approved by the properly constituted Institutional 
Review Board (IRB) operating according to the 45 CFR 46 Code of Federal regulations. This 
committee must approve all amendments to the protocol or informed consent, and conduct 
continuing annual review so long as the protocol is open to accrual or follow up of subjects. Quality 
assurance and control monitoring will be consistent with the NHLBI Division of Intramural 
Research Clinical Research Quality Assurance and Quality Control Policy. 
10.0 Adverse Events, Deviations, and Unanticipated Problem Assessments
10.1 Definitions 
Please refer to definitions provided in Policy 801: Reporting Research Events.
10.2 Adverse Event Assessment 
The following adverse event management guidelines are intended to ensure the safety of each participant 
while on the study. The AEs will be attributed (unrelated, unlikely, possibly, probably or definitely) to study 
procedure and/or disease and graded by severity utilizing CTCAE version 5.0. A copy of the criteria can be 
downloaded from the CTEP home page at http://ctep.cancer.gov/reporting/ctc.html. 
Biotin labeling of RBCs is not expected to result in adverse events. However, all  Grade 2 and above adverse 
events (AE) that are related to diagnostic and interventions directly related to this study whether volunteered 
by the participant, discovered by study personnel during questioning, or detected through physical 
examination, clinically significant laboratory test, or other means will be recorded in the participant’s 
medical record. 
Only Grade 2 and above adverse events (AE) that are related to diagnostic and interventions directly related 
to this study will be captured in the research database.  Adverse events associated with the natural history 
of sickle cell disease will not be captured in the database.  Expected sequelae of sickle cell disease such as 
vaso-occlusive pain crisis, acute chest syndrome or cytopenias associated with hydroxyurea therapy will 
not be captured in the database.  
10. 3
NIH Intramural and IRB Reporting
Expedited Reporting
Events requiring expedited reporting will be submitted to the IRB per Policy 801 “Reporting Research 
Events”.
Reports to the IRB at the time of Continuing Review: 
The PI or designee will refer to HRPP Policy 801 “Reporting Research Events” to determine IRB reporting 
requirements.
Reports to the CD:
The PI or designee will refer to NHLBI DIR guidelines to determine CD reporting requirements and 
timelines.


Protocol Number: 20-H-0080
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September 20, 2023 Amendment I
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10.4
Protocol Deviations
It is the responsibility of the investigator to use continuous vigilance to identify and report deviations to the 
NIH Institutional Review Board as per Policy 801.  
NIH Definition 
A protocol deviation is any changed, divergence, or departure from the IRB-approved research 
protocol. 

Major deviations: Deviations from the IRB approved protocol that have, or may have the potential 
to, negatively impact the rights, welfare or safety of the participant, or to substantially negatively 
impact the scientific integrity or validity of the study.

Minor deviations: Deviations that do not have the potential to negatively impact the rights, safety 
or welfare of participants or others, or the scientific integrity or validity of the study.
10.5
Unanticipated Problems
The investigator will report unanticipated problems (UPs) to the NIH Institutional Review Board (IRB) as 
per Policy 801.
Definition:
Any incident, experience, or outcome that meets all of the following criteria:

Unexpected in terms of nature, severity, or frequency given (a) the research procedures that are 
described in the protocol-related documents, such as the Institutional Review Board (IRB)-
approved research protocol and informed consent document; and (b) the characteristics of the 
participant population being studied; and

Related or possibly related to participation in the research (“possibly related” means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research); and

Suggests that the research places participants or others (which many include research staff, family 
members or other individuals not directly participating in the research) at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously known or 
expected.
Reporting:
The investigator will report unanticipated problems (UPs) to the NIH Institutional Review Board (IRB) as 
per Policy 801 and to the NHLBI Clinical Director per NHLBI guidelines.
11.0 Specimen and Data Storage, Tracking and Disposition 
11.1 Intended Use of the Samples/Specimens/Data 
Biospecimen Management
Specimens and their derivatives (e.g., genomic material, cell lines) will be coded and stored in conformity 
with DIR Policy. Coded biospecimens may be sent to collaborators outside of the NIH with IRB approval 
in accordance with applicable NIH and DIR Policy for sharing research resources, including an executed 
material transfer agreement. Biospecimens with participant personal identifiers may be sent to associate 
investigators and collaborators outside of the NIH only after approvals of both NHLBI and local IRBs, an 
executed reliance agreement with NHLBI’s IRB, or an extension of the NIH’s FWA through an Individual 
Investigator Agreement.


Protocol Number: 20-H-0080
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Any future research use of biospecimens not defined in the protocol in which NHLBI investigators 
are engaged in research (e.g., they are undertaking research activities and hold the key that 
identifies research subjects) requires IRB review and approval.  Coded biospecimens (NHLBI 
investigators hold the key that identifies research subjects) to be shared outside of NIH for future 
research use where results will not be returned to the Principal Investigator does not require IRB 
review or approval. 
11.2 How Samples will be Stored 
Data Management 
The PI will be responsible for overseeing entry of these data into an in-house password protected 
electronic system at the NIH and ensuring data accuracy, consistency, and timeliness. The principal 
investigator and associate investigators, research nurses and/or a contracted data manager will assist 
with the data management efforts. Data will be abstracted from Clinical Center progress notes as well 
as reports in CRIS from tests performed at the Clinical Center. Laboratory data from NIH will be 
reviewed using CRIS or an approved NHLBI database. 
All human subjects’ personally identifiable information (PII), eligibility and consent verification will 
be recorded. Primary data obtained during the conduct of the protocol will be kept in secure network 
drives or in approved alternative sites that comply with NIH security standards. Primary and final 
analyzed data will have identifiers so that research data can be attributed to an individual human subject 
participant, (e.g., a unique code or minimum PII required for subject identification). Genetic 
information will not be shared with anyone besides the protocol analysis team, and these research data 
will be maintained on a secure server at NIH. 
Sample Storage 
Access to research samples will be limited. The samples will be stored in the secure laboratory of the 
principal investigator and placed in locked freezers. Only those authorized can retrieve samples. 
Samples and data will be stored using codes assigned by the investigators or their designee(s). Only 
investigators or their designee(s) will have access to the samples and data. The PI will be responsible 
for overseeing entry of data into an in-house password protected electronic system. Research samples 
will be stored in accordance with NHLBI DIR Biospecimen policy. 
11.3 How Specimens/Data will be Tracked 
Samples collected at the NIH will be ordered and tracked through CRIS Research Screens. Should a 
CRIS screen not be available, the NIH form 2803-1 will be completed and will accompany the 
specimen and be filed in the medical record. 
All samples collected at the NIH under this protocol will be coded with a specific patient number (i.e. 
161-1, 161-2, etc.). They will be logged in on an excel spreadsheet containing the sample code, date 
drawn, and location of storage. 
All medical information collected from study participants at the NIH will be kept in a locked file at the 
Clinical Center at the NIH. Unique patient identifiers will be used to label all data. Strict standards of 
confidentiality will be upheld at all times. 
11.4. Publication, Data Sharing/Future Use of Data 
De-identified human data generated for use in future and ongoing research will be shared through a 
NIH-funded or approved repository (ClinicalTrials.gov) and BTRIS. At the completion of data 


Protocol Number: 20-H-0080
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analysis, data will be submitted to ClinicalTrials.gov either before publication or at the time of 
publication or shortly thereafter. 
Research data may be shared with qualified non-collaborator recipients following publication of the 
primary research results after removal of PII. Future research use of identifiable data not defined in the 
research protocol may occur only after IRB review and approval. Refusal of a research subject 
participant to permit future use of data will be honored. 
Blood samples may be used for other research purposes related to the study of SCD. Volunteer clinical 
data will be protected and tracked using standard operating procedures in the medical record 
department.  No volunteer data will be stored in the research office. 
Following collection and processing, identifiers will be stripped from the samples prior to their 
distribution to other study investigators.  The IRB will be notified if research samples are 
unintentionally destroyed or lost.
Transmission of Data to Outside Investigators: Coded samples and data will be sent to our 
collaborators for analysis. No samples or data collected on this study will be sent outside NIH without 
a fully executed material transfer agreement (MTA).
11.5 End of Study Procedures 
The study will remain open so long as sample or data analysis continue. Samples from consenting 
participants will be stored until they are no longer of scientific value or if a participant withdraws 
consent for their continued use, at which time they will be destroyed. A file note will be made to 
document the withdrawal / destruction, and existing data will not be used in future studies. 
At the completion of the protocol (termination), samples and data will either be destroyed, or after IRB 
approval, transferred to another existing protocol or a repository. 
11.6 Loss or Destruction of Specimens/Data 
Any loss or unanticipated destruction of samples (for example, due to freezer malfunction) or data (for 
example, misplacing a printout of data with identifiers) will be reported to the IRB. Additionally, 
participants may decide at any point not to have their samples stored. In this case, the principal 
investigator will destroy all known remaining samples and report what was done to both the participant 
and to the IRB. This decision may not affect the participant’s participation in this protocol or any other 
protocols at NIH. 
12.0 Confidentiality and Privacy
Participant confidentiality and privacy is strictly held in trust by the participating investigators, their staff, 
and the sponsor(s) and their interventions. This confidentiality is extended to cover testing of biological 
samples and genetic tests in addition to the clinical information relating to participants. Therefore, the study 
protocol, documentation, data, and all other information generated will be held in strict confidence. No 
information concerning the study or the data will be released to any unauthorized third party without prior 
written approval of the sponsor. 
All research activities will be conducted in as private a setting as possible.


Protocol Number: 20-H-0080
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September 20, 2023 Amendment I
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The study monitor, other authorized representatives of the sponsor, representatives of the Institutional 
Review Board (IRB), and/or regulatory agencies may inspect all documents and records required to be 
maintained by the investigator, including but not limited to, medical records (office, clinic, or hospital) and 
pharmacy records for the participants in this study. The clinical study site will permit access to such records.
The study participant’s contact information will be securely stored at each clinical site for internal use 
during the study. At the end of the study, all records will continue to be kept in a secure location for as long 
a period as dictated by the reviewing IRB, Institutional policies, or sponsor requirements.
To further protect the privacy of study participants, a Certificate of Confidentiality has been issued by the 
National Institutes of Health (NIH).  This certificate protects identifiable research information from forced 
disclosure. It allows the investigator and others who have access to research records to refuse to disclose 
identifying information on research participation in any civil, criminal, administrative, legislative, or other 
proceeding, whether at the federal, state, or local level. By protecting researchers and institutions from 
being compelled to disclose information that would identify research participants, Certificates of 
Confidentiality help achieve the research objectives and promote participation in studies by helping assure 
confidentiality and privacy to participants.
13.0 Human Subjects Protections
13.1 Rationale for Exclusion of Children 
This is a non-treatment study with no potential for direct benefit to the subject. 
13.2 Rationale for Exclusion of Subjects Unable to Consent 
This is a non-treatment study with no potential for direct benefit to the subject. 
13.3 Rationale for Exclusion of Pregnant Women
This is a non-treatment study with no potential for direct benefit to the subject. Furthermore, the natural 
increase in circulating blood volume associated with pregnancy may render the small volume of 
biotinylated RBCs described in this study unable to be detected by analysis. 
13.4 Rationale for Exclusion of Subjects with Pre-existing Antibodies to Biotin
This is a non-treatment study with no potential for direct benefit to the subject. Though the existence 
of antibodies to BioRBCs has not demonstrated a shortened RBC lifespan (4, 11), data is limited in 
subjects with sickle cell disease. 
13.5 Inclusion of NIH Staff 
NIH staff (employees, NIH contractors, special volunteers, guest researchers, and trainees) may 
voluntarily participate in this protocol. 
• If the individual requesting to participate in the protocol is a co-worker, the consent from the NIH 
staff member (co-worker) will not be obtained by the staff member’s direct supervisor but by another 
research staff member approved for obtaining informed consent who is not a co-worker. 
• Recruitment, enrollment and compensation of NIH employee subjects will be consistent with the 
Guidelines for the Inclusion of Staff in NIH Intramural Research Studies (NIH HRPP Policy 404) and 
NIH Policy Manual Chapter 2300-630-3 and the Leave Policy for NIH Employees Participating in 
NIH Medical Research Studies. 
• Neither participation nor refusal to participate as a subject in this protocol will have an effect, either 
beneficial or adverse, on the participant’s employment or position at NIH. 


Protocol Number: 20-H-0080
PI: John Tisdale, MD
September 20, 2023 Amendment I
24
Known Potential Risks
13.6 Known Potential Risks 
Blood Draw: A potential risk of participation in the protocol involves the risk associated with drawing 
blood. The potential complications of phlebotomy involve minimal risk and consist of pain, mild 
bruising at the site of venipuncture and potential local infection. 
HIV Testing: Testing for infection with the human immunodeficiency virus (HIV) may be performed 
more than once for this study.  If the subject is infected with HIV, they will not be able to participate 
in this study.  We will inform the subject of the results, what they mean, how to find care, how to avoid 
infecting others, how we report HIV infection, and the importance of informing their partners at 
possible risk because of the subject’s HIV infection.
Risks related to protection of confidentiality: The information from this study will be maintained 
confidential. Samples will be assigned a unique code prior to testing that will serve as a link to the 
individual’s identity and other information collected as part of this research protocol. The actual testing 
will be performed on coded samples. 
Any breach of the secure database or accidental release of a code key for linked samples will be 
reported to the IRB immediately as an adverse event. All efforts will be made to protect confidentiality 
of subjects to the extent possible by law. 
Risks related to Biotin
Background:
Biotin is a water-soluble B-vitamin, also called vitamin B7. Biotinylation is the rapid and specific process 
of covalently attaching biotin to a protein, nucleic acid, or other macromolecule, and is unlikely to perturb 
the natural function of the molecule due to the small size of biotin (MW = 244.31 g/mol) (13). Biotin forms 
a covalent amido bond between the carboxyl group of biotin and the epsilon amino group of lysine residues 
in the RBC membrane proteins (Figure 4). Biotin binds to streptavidin and avidin with an extremely high 
affinity, fast on-rate, and high specificity, and is a useful tool in nonradioactive methods of purification, 
detection, immobilization, labeling, viral vector-targeting, and drug targeting systems. Proteins that are 
biotin labeled are routinely detected or purified with avidin conjugates in many protein research 
applications, including cell surface labeling and flow cytometry/fluorescence-activated cell sorting.
Figure 4 Biotin Structure. The reaction of sulfo-NHS biotin targets the amino groups on RBC surface proteins.
Expected Side Effects:
Previous studies with identical or similar protocols have shown the safety of biotinylated RBCs in human 
subjects for determining red cell survival (4, 14-15). As these groups have surmised, because the biotin 
reaction with the amino group produces the same biotinylation of the epsilon amino group of lysine seen in 
biotin-dependent carboxylases and biotinylated histones and is not known to generate toxic byproducts, we 
speculate that no toxicity will be observed with the use of biotinylated RBC in human subjects. 


Protocol Number: 20-H-0080
PI: John Tisdale, MD
September 20, 2023 Amendment I
25
Furthermore, biotin itself will not be administered to subjects, nor will the biotinylating reagent. Only RBCs 
after they have been biotinylated using the sulfo-NHS biotinylating reagent and washed (thus eliminating 
the excess biotinylating reagent) will be used. 
Common Side Effects:
We do not expect side effects directly from the use of biotin. Adverse reactions related to blood draws may 
occur, include lightheadedness, nausea, and pre-syncope due to vasovagal reactions. Vasovagal reactions 
are managed by postural manipulation and volume administration. Further, pain and bruising can result 
from needle sticks required for blood draws. 
Rare Side Effects: 
Many lab tests use biotin technology due to its ability to bond with specific proteins which can then be 
measured to detect certain health conditions. Patients who are ingesting high levels of biotin in dietary 
supplements (ingested at 650 times the recommended daily intake promoted for hair, skin, and nail benefits, 
or recommended for patients with certain conditions such as multiple sclerosis) can cause clinically 
significant incorrect lab test results (16). Biotin levels higher than the recommended daily allowance (RDA, 
0.03mg) may cause interference with lab tests. The amount of biotin used in this study is well below the 
RDA, therefore is not expected to interfere with any clinical laboratory tests.
A theoretical safety issue is the development of anti‐BioRBCs after BioRBC exposure. Previous studies 
have detected transient BioRBC‐induced anti‐BioRBC, however these antibodies have not caused clinical 
abnormalities (4,11). It is speculated that this is analogous to penicillin‐type drug‐dependent antibodies 
with biotin covalently attached to RBC surface proteins acting as immunogenicity enhancing haptens. 
Clinical features of hemolytic reactions have not been seen—presumably because the volumes of BioRBCs 
transfused for RBC kinetic studies are small (approx. 0.40 mL of BioRBC/kg body weight) and unlikely to 
cause a clinically significant transfusion reaction, even if the entire BioRBC transfusion was acutely 
hemolyzed (11).
13.7 Known Potential Benefits
This is a non-treatment study with no potential for direct benefit to the subject. The collection and 
biotinylation of RBCs in this study will allow the measurement and study of red cell survival in patients 
with and without SCD. The study of RBC survival and its relation to known influencers of red cell survival 
including ARC, % HbF, and F cell percentage will allow for the creation of a mathematical model that will 
help determine necessary amount of normal hemoglobin required to reverse the SCD phenotype. Such 
determination has the potential to influence current and future gene therapy protocols. With the success of 
this pilot study, this methodology can be expanded to include the ability to measure RBC lifespan in gene 
therapy patients, and the relationship of transferred globin (VCN) and percent engraftment of gene modified 
cells to the patient’s RBC survival. As has been previously described (10), the reticulocyte fraction is a 
good proxy for RBC lifetime, and therefore gene therapy may be most efficacious in patients in which RBC 
survival is lowest (with highest reticulocyte count) as they would require lower amounts of normal 
hemoglobin production (lower levels of gene correction) to sufficiently reverse the SCD phenotype. A study 
of red cell survival will aid in this determination of the amount of hemoglobin transfer necessary for any 
particular individual with SCD. 
13.8 Assessment of Potential Risks and Benefits
Risks involved in participation in this study are low and include risks related to blood draws, risks 
related to protection of confidentiality, and risks related to biotin (see section 12.6). We do not expect 
to see side effects related to biotinylation of RBCs, however the theoretical safety issue of development 
of anti‐BioRBCs after BioRBC exposure is addressed by screening before, during, and after completion of 
the study for anti-BioRBCs. Previous studies that have detected BioRBC‐induced anti‐BioRBC describe 


Protocol Number: 20-H-0080
PI: John Tisdale, MD
September 20, 2023 Amendment I
26
these antibodies as transient with no clinical changes presumably because the volumes of BioRBCs 
transfused for RBC kinetic studies are small (approximately 0.40 mL of BioRBC/kg body weight) and 
unlikely to cause a clinically significant transfusion reaction, even if the entire BioRBC transfusion was 
acutely hemolyzed (4,11). The knowledge gained from this study including RBC survival in patients with 
SCD before and after BMT, and RBC survival in subjects with SCT (not previously done with 
biotinylation), will provide essential information that will inform future gene therapy studies while 
exposing participants to minimal risk.
13.8 Informed Consent Processes and Procedures 
Informed consent will be conducted following OHSRP Policy 301- Informed Consent.
An IRB-approved consent form will be provided to the subject electronically or by hard copy for 
review prior to consenting. The investigational nature and objectives of this trial, the procedures, 
and their attendant risks and discomforts and potential benefits will be carefully explained to the 
subject in a private setting. The subject will be given as much time as they need to review the 
document and to consult with their family, friends, and personal health care providers. In addition, 
a study team member will be available to answer any questions.
A signed and dated informed consent document will be obtained by any investigator authorized to 
consent prior to entry onto the study. Consent may be obtained with required signatures on the 
hard copy of the consent  or on the electronic document.  
When a document that is in electronic format is used for obtaining  consent, this study may use the 
iMed platform which is 21 CFR, Part 11 compliant, to obtain the required signatures.
During the consent process, participants and investigators may view the same approved consent 
document simultaneously when participant is being consented in person at the Clinical Center or 
both may view individual copies of the approved consent document on screens in their respective 
locations remotely. Signatures may be obtained either by both directly signing on the device that 
the consenting investigator is using (when in person) or through iMed Mobile Signature Capture  
(remotely) which allows texting or emailing a link to the participant. That link allows the 
participant to review the consent, then proceed to sign on the device they are using.
Whether hard copy or electronic consent, both the investigator and the participant will sign the 
document with a hand signature using a pen, finger, stylus, or mouse.
When done remotely, if the participant prefers to sign a hard copy, they may be instructed to sign 
and date the consent document during the discussion and mail, secure email or fax the signed 
document to the consenting investigator.
Whether in person or remotely, the privacy of the participant will be maintained.
Finally, the fully signed informed consent document will be stored in the electronic medical record, 
and the subject will receive a copy of the signed informed consent document.
13.9 Patient Advocate 
A patient’s rights representative is available to patients on this protocol. The representative is 
located in Building 10 and can be reached by phone at (301) 496-2626. Patients may ask any 
questions about the study and may withdraw their consent at any time. 


Protocol Number: 20-H-0080
PI: John Tisdale, MD
September 20, 2023 Amendment I
27
14.0 Compensation for Participation
Clinical research volunteers evaluated at the Clinical Research Center will receive monetary 
compensation in accordance with NIH guidelines.  NIH employees may take part in this study as 
volunteers and receive compensation, as outlined below, in accordance with the NIH leave policy.  
Patients with a disease may be compensated as volunteers if testing is done solely for use as 
controls to study other disease processes. 
Outpatients (on-site): $20 for first hour or part thereof and $10 each additional hour or part 
thereof (up to 4 hours).
*Dependent on the subject’s red cell half- life, the number of research samples needed to collect until study completion, although rare, may 
exceed the projected 16 week collection timeline. Therefore total compensation may exceed $1,600.00.
**Unless screened under another NIH protocol. 
15.0 Reimbursement for Travel
Reimbursement will be consistent with NHLBI DIR Travel and Lodging Compensation of Clinical 
Research Subjects policy.  Please reference the table above in Section 13.0 Compensation for 
Participation, this study offers a $40 voucher for travel per visit; however, it does not offer 
reimbursement for, or payment for lodging and/or meals.
Procedure
Inconvenience units ($10 for each 
inconvenience unit)
Outpatient Visit (first hour)
$20 (2 units)
Outpatient Visit (additional hours, 
 up to 4 hours)
$10 (1 unit)
Screening History & Physical
$25 (2.5 units)**
Research blood sampling (per blood draw)
$40 (4 units)
IV Placement
$10 (1 unit)
Drug Infusion/Administration by IV
$50 (5 units)
Completion Bonus
$500 (50 units)
Travel Voucher (per visit)
$40 (4 units)
Total Potential Compensation
Up to $1,600.00*


Protocol Number: 20-H-0080
PI: John Tisdale, MD
September 20, 2023 Amendment I
28
     References
1.
Lanzkron S, Carroll CP, Haywood C. Mortality Rates and Age at Death from Sickle Cell Disease: 
U.S., 1979–2005. Public Health Reports 2013; 128(2):110-116. 
2.
Quinn CT, Smith EP, Arbabi S, Khera PK, Lindsell CJ, Niss O, Joiner CH, Franco RS, and 
Cohen RM. Biochemical surrogate markers of hemolysis do not correlate with directly measured 
erythrocyte survival in sickle cell anemia. Am J Hematol 2016; 91, 1195-1201.
3.
Barbedo MM and McCurdy PR. Red cell life span in sickle cell trait. Acta Haematol 1974; 51, 
339-43.
4.
Mock DM, Matthews NI, Zhu S, Strauss RG, Schmidt RL, Nalbant D, Cress GA, Widness JA. 
Red blood cell (RBC) survival determined in humans using RBCs labeled at multiple biotin 
densities. Transfusion 2011; 51, 1047-57
5.
Franco RS, Lohmann J, Silberstein EB, Mayfield-Pratt G, Palascak M, Nemeth TA, Joiner CH, 
Weiner M, Rucknagel DL.Time-dependent changes in the density and hemoglobin F content of 
biotin-labeled sickle cells.  J Clin Invest. 1998;101(12):2730-2740
6.
Ballas SK and Marcolina MJ. Determinants of red cell survival and erythropoietic activity in 
patients with sickle cell anemia in the steady state. Hemoglobin 2000; 24: 277–286. 
7.
Franco RS, Yasin Z, Palascak MB, Ciraolo P, Joiner CH, Rucknagel DL. The effect of fetal 
hemoglobin on the survival characteristics of sickle cells. Blood 2006; 108, 1073-6
8.
Franco RS, Yasin Z, Lohmann JM, Palascak MB, Nemeth TA, Weiner M, Joiner CH, Rucknagel 
DL. The survival characteristics of dense sickle cells. Blood. 2000 Nov 15;96(10):3610-7
9.
Hsieh MM, Kang EM, Fitzhugh CD, Link MB, Bolan CD, Kurlander R, Childs RW, Rodgers GP, 
Powell JD, Tisdale JF. Allogeneic hematopoietic stem-cell transplantation for sickle cell disease. 
N Engl J Med. 2009 Dec 10;361(24):2309-17.
10. Fitzhugh CD, Cordes S, Taylor T, Coles W, Roskom K, Link M, Hsieh MM, Tisdale JF. At least 
20% donor myeloid chimerism is necessary to reverse the sickle phenotype after allogeneic 
HSCT. Blood. 2017 Oct 26;130(17):1946-1948. 
11. Schmidt RL, Mock DM, Franco RS, Cohen RM, North AK, Cancelas JA, Geisen C, Strauss RG, 
Vlaar AP, Nalbant D, Widness JA. Antibodies to biotinylated red blood cells in adults and 
infants: improved detection, partial characterization, and dependence on red blood cell-biotin 
dose. Transfusion. 2017 Jun;57(6):1488-1496.
12. Li Q, Henry ER, Hofrichter J, Smith JF, Cellmer T, Dunkelberger EB, Metaferia BB, Jones-
Straehle S, Boutom S, Christoph GW, Wakefield TH, Link ME, Staton D, Vass ER, Miller JL, 
Hsieh MM, Tisdale JF, Eaton WA. Kinetic assay shows that increasing red cell volume could be 
a treatment for sickle cell disease. Proc Natl Acad Sci U S A. 2017 Jan 31;114(5):E689-E696.
13. Thermo Fisher Scientific. Protein Biology Resource Library. Available at: 
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-
center/protein-biology-resource-library/pierce-protein-methods/biotinylation.html
14. Mock DM, Lankford GL, Matthews NI, Burmeister LF, Kahn D, Widness JA, Strauss RG. 
Accelerated removal of antibody-coated red blood cells from the circulation is accurately tracked 
by a biotin label. Transfusion. 2012 May;52(5):1097-105. 
15. Widness JA, Nalbant D, Matthews NI, Strauss RG, Schmidt RL, Cress GA, Zimmerman MB, 
Mock DM. Tracking donor RBC survival in premature infants: agreement of multiple populations 
of biotin-labeled RBCs with Kidd antigen-mismatched RBCs. Pediatr Res. 2013 Dec;74(6):689-
97. 
16. Holmes EW, Samarasinghe S, Emanuele MA, Meah F. Biotin Interference in Clinical 
Immunoassays: A Cause for Concern. Arch Pathol Lab Med. 2017 Nov;141(11):1459-1460.


Protocol Number: 20-H-0080
PI: John Tisdale, MD
September 20, 2023 Amendment I
29
APPENDIX A: NIH INFORMATION SHEET ON STAFF RESEARCH PARTICIPATION (April 
2016)
As an NIH employee, contractor, Special Volunteer, Guest Researcher, or trainee, you may participate in 
intramural research studies unless it is prohibited by your Institute or Center (IC), or if you are excluded 
by the criteria of the protocol in which you want to enroll. The inclusion of NIH staff in a particular 
protocol must also be approved by the IRB. You may be motivated by altruism, a commitment to research 
in your own or related fields, or want access to clinical trials of potential direct therapeutic benefit. When 
deciding, you should make an informed decision about participation. This information sheet offers some 
points to consider for NIH staff who are considering research participation at NIH.
First, similar to any individual who is considering research participation, you should seek adequate 
information about the study purpose, what is required of you in terms of procedures, interventions and 
time, and the potential risks and benefits of participation. For more information, see the NIH Clinical 
Center’s public website “Are Clinical Studies for You?” at: 
http://www.cc.nih.gov/participate/studies.shtml.
When you are thinking about participation in a research study that is being conducted by your supervisor, 
or others with whom you work closely in your laboratory, branch, or unit, you should consider some 
additional factors:
A.
Possible bias: Are you confident that you can be unbiased about reporting answers, side effects, 
or other information that could influence the study outcome or risk to you? 
B.
Confidentiality: Has the principal investigator (PI) spoken about what information will be 
collected from you as part of the study? Has the PI discussed what information will be available to those 
within, and outside, the study team? If applicable, are you comfortable sharing your medical history 
(including, for example, mental health history or STDs) and your social history (e.g. substance use) with 
study investigators who may be your coworkers, or with the possibility of them discovering something 
about your health during the study (e.g. pregnancy status or a new diagnosis)? Although every effort will 
be made to protect your information and keep it private and confidential, your information may, 
depending on the nature of the protocol, become available in medical records or to authorized users 
outside of the study team. Discuss any concerns with the PI. 
C.
Pressure: Do you perceive any pressure or expectations from your supervisor or colleagues 
regarding participation? Could that pressure influence your decision or make it difficult for you to choose 
whether or not to participate? Remember that it is your choice whether or not to participate and that your 
decision to participate or not should not have an effect, either beneficial or adverse, on your position at 
NIH.
D.
Time and Compensation: Can you take time off from work to complete the study requirements or 
participate solely during non-duty hours? Can you receive compensation for your participation in this 
study? Will your supervisor give you permission to participate during work hours?  See the NIH Policy 
Manual 2300-630-3 “Leave Policy for NIH Employees Participating in NIH Medical Research Studies.”
E. 
Consent Process: Is the person obtaining your consent for the study your supervisor, a 
subordinate, or co-worker? If so, is there an independent person monitoring the consent process? If the 
study PI is a supervisor and intends to obtain consent from you, an independent person (e.g., through 
Bioethics or the NIMH Human Subjects Protections Unit [HSPU], or others as approved by the IRB) 
must monitor the consent process. If the person obtaining consent from you is a co-worker then an 


Protocol Number: 20-H-0080
PI: John Tisdale, MD
September 20, 2023 Amendment I
30
independent person (e.g., through Bioethics or the NIMH HSPU, or others as approved by the IRB) may 
be required to monitor the consent process, as determined by the IRB for the specific study.
If you are thinking of enrolling as a subject at the NIH Clinical Center and you have any questions or 
concerns, please contact the Office of Human Subjects Research Protections (OHSRP) at (301) 402-3444 
and/ or the Patient Representative if you are thinking of enrolling as a subject at the NIH Clinical Center 
on (301) 496-2626. If you are at a NIH site outside the Clinical Center then please contact local site 
leadership.


31
Protocol 20-H-0080: Red Cell Half Life Determination in Patients with and without Sickle 
Cell Disease
Schedule of Events: SCD Pre-transplant Patients
Turquoise: To be done after 20-H-0080 protocol signed.
Procedure: 
Consent Forms
Screening 
Eval
Day 
-1
W 
1, 
Day 
0
W
1.5
W
2
W 
2.5
W
3
W 
4
W 
5
W
6 
W
7
W
8
W 
9
W
10
W
11
W
12 
W
 24
(6 
mos)
20-H-0080 
Eligibility checklist

20-H-0080 
Protocol consent

Procedure: 
History & Physical
Screeni
ng Eval
Day 
-1
W
1, 
Day 
0
W
1.5
W
2
W 
2.5
W
3
W 
4
W 
5
W
6 
W
7
W
8
W 
9
W
10
W
11
W
12 
W
 24
(6 
mos
)
Medical history 

Physical examination 
       
Vital signs, 
height/weight

Interim illness update















Name
MRN
DOB
Genotype


32
Procedures/ Consults:
Screeni
ng Eval
Day 
-1
W
1, 
Day 
0
W
1.5
W
2
W 
2.5
W
3
W 
4
W 
5
W
6 
W
7
W
8
W 
9
W
10
W
11
W
12 
W
 24
(6 
mo
s)
DTM for Consult and
Venous Assessment

DTM for Blood 
Collection
(total vol: ~ 80 mL)

VADD Service 
for 18G or 20G IV
(ONLY IF NEEDED)


Infusion of Biotinylated 
RBCs (max vol 50 mL)

Procedure: Blood for 
Clinical Laboratory 
Testing
Screeni
ng Eval
Day 
-1
W
1, 
Day 
0
W
1.5
W
2
W 
2.5
W
3
W 
4
W 
5
W
6 
W
7
W
8
W 
9
W
10
W
11
W
12 
W
 24
(6 
mo
s)
CBC w/diff















Reticulocyte count















Hemoglobin 
electrophoresis














Type and antibody 
screen 














DAT

















33
Acute care panel (CMP)














Mineral panel (CMP)














Hepatic panel (CMP)














LDH














Β-HCG serum 
pregnancy (females 
only)
  

Urinalysis

HIV-1/HCV/HBV NAT

HBsAg

HCV antibody

HTLV I/II

HIV 1/2

HB core antibody

West Nile Virus

T. cruzi antibody

Treponema palladium 
(antibody) or equivalent 
(such as RPR)



34
Blood for Research 
Samples
Screeni
ng Eval
Day 
-1
W
1, 
Day 
0
W
1.5
W
2
W 
2.5
W
3
W 
4
W 
5
W
6 
W
7
W
8
W 
9
W
10
W
11
W
12 
W
 24
(6 
mos
)
α-Globin Genotype 
Analysis 
(LVV 3mL tube, 3 mL volume)
Alexis pick up for OJ

Biotin Antibody Screen 
(LVV 3mL tube, draw 1 mL vol) 
Alexis Leonard pick up
*will be done during the course 
of the study if bioRBC 
degradation is faster than 
predicted



Bluebird Bio Sample 
(LVV 3mL tube, draw 1-3 mL)
Alexis pick up for OJ 













Eaton Lab
(LVV 3mL tube, draw 1 mL)
Qaun Li pick up













Tisdale Lab 
(LVV 3 mL tube, draw 1-3 mL)
Alexis pick up
















35
Protocol 20-H-0080: Red Cell Half Life Determination in Patients with and without Sickle 
Cell Disease
Schedule of Events: HbAA, HbAS, and SCD Post-transplant Volunteers
Turquoise: To be done after 20-H-0080 protocol signed.
Procedure: 
Consent Forms
Screening 
Evaluation
Day -1
Week 
1, 
Day 0
Week 
2
Week 
3
Week 
4
Week 
6
Week
8 
Week 
10
Week
12 
Wee
k 14
Wee
k 16
Week 
24
(6 mos)
20-H-0080 
Eligibility checklist

20-H-0080 
Protocol consent

Procedure: 
History & Physical
Screening 
Evaluatio
n
Day -
1
Week 
1, 
Day 0
Week 
2
Week 
3
Week 
4
Week 
6
Week
8 
Week 
10
Week
12 
Wee
k 14
Wee
k 16
Week 
24
(6 
mos)
Medical history 

Physical examination 
       
Vital signs, height/weight

Interim illness update











Procedures/ Consults:
Screening 
Evaluatio
n
Day -
1
Week 
1, 
Day 0
Week 
2
Week 
3
Week 
4
Week 
6
Week
8 
Week 
10
Week
12 
Wee
k 14
Wee
k 16
Week 
24
(6 
mos)
DTM for Venous 
Assessment and Consult

DTM for Blood 
Collection
(total vol: ~ 80 mL)

Name
MRN
DOB
Genotype


36
VADD Service 
for 18G or 20G IV
(ONLY IF NEEDED)


Infusion of Biotinylated 
RBCs (max vol 50 mL)

Procedure: Blood for 
Clinical Laboratory 
Testing
Screening 
Evaluatio
n
Day -
1
Week 
1, 
Day 0
Week 
2
Week 
3
Week 
4
Week 
6
Week
8 
Week 
10
Week
12 
Wee
k 14
Wee
k 16
Week 
24
(6 
mos)
CBC w/diff











Reticulocyte count











Hemoglobin 
electrophoresis










Type and antibody screen 










DAT











Acute care panel (CMP)










Mineral panel (CMP)










Hepatic panel (CMP)










LDH










Β-HCG serum pregnancy 
(females only)
  

Urinalysis

HIV-1/HCV/HBV NAT



37
HBsAg

HCV antibody

HTLV I/II

HIV 1/2

HB core antibody

West Nile Virus

T. cruzi antibody

Treponema palladium 
(antibody) or equivalent 
(such as RPR)

Blood for Research 
Samples
Screening 
Evaluatio
n
Day -
1
Week 
1, 
Day 0
Week 
2
Week 
3
Week 
4
Week 
6
Week
8 
Week 
10
Week
12 
Wee
k 14
Wee
k 16
Week 
24
(6 
mos)
α-Globin Genotype 
Analysis 
(LVV 3mL tube, 3 mL volume)
Alexis pick up and deliver to OJ

Biotin Antibody Screen 
(LVV 3mL tube, draw 1 mL 
vol) 
Alexis Leonard Pick-up
*will be done during the course 
of the study if bioRBC 
degradation is faster than 
predicted



Bluebird Bio Sample 
(LVV 3mL tube, draw 1-3mL)
Alexis pick up and deliver to OJ 









Eaton Lab
(LVV 3mL tube, draw 1 mL)
Qaun Li Pick-up











38
Tisdale Lab 
(LVV 3mL tube, draw 1-3mL)
Alexis pick-up











ICF_001.pdf: 
Informed Consent Cover Page for FDAAA consent posting:  
 
Official Title: 
 
Red Cell Half Life Determination in Patients with and without Sickle Cell 
Disease 
 
NCT number: 
 
NCT04476277 
Document Type:  
Informed Consent Form  
Document Date: 
11/30/2021 
 


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: November 23, 2021
Page 1 of 14
CC ICF template v. 09.27.19
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
PRINCIPAL INVESTIGATOR:  John F Tisdale, MD
STUDY TITLE: Red Cell Half Life Determination in Patients with and without Sickle Cell 
Disease
STUDY SITE:  NIH Clinical Center
Cohort: Standard 
Consent Version:  November 23, 2021
WHO DO YOU CONTACT ABOUT THIS STUDY? 
Principal Investigator: John Tisdale, M.D., Ph. (301)402-6497, Email johntis@nhlbi.nih.gov 
Study Coordinator: Christina Luckett, RN, Ph. (301)827-7901, Email christina.luckett@nih.gov
KEY INFORMATION ABOUT THIS RESEARCH 
This consent form describes a research study and is designed to help you decide if you would 
like to be a part of the research study.  
You are being asked to take part in a research study at the National Institutes of Health (NIH). 
This section provides the information we believe is most helpful and important to you in making 
your decision about participating in this study. Additional information that may help you make 
a decision can be found in other sections of the document. Taking part in research at the NIH is 
your choice.
You are being asked to participate in this research study because you either have Sickle Cell 
Disease (SCD), had SCD but were cured with a bone marrow transplant, have Sickle Cell Trait 
(SCT), or are a volunteer without SCD or SCT.
SCD is an inherited blood disorder that results from a single genetic change (mutation) in red 
blood cells (RBCs). RBCs are the cells that carry oxygen to the body. In people with SCD, the 
mutated RBCs can become hard, rigid, and contorted into a sickle shape. These abnormal RBCs 
die early, leaving a shortage of healthy RBCs (sickle cell anemia). RBCs typically survive in 
the human body for approximately 120 days. However, this duration is shortened to an average 
of 32 days in patients with SCD. Currently SCD can be cured with a bone marrow transplant. 
This can result in restoration of the red cell lifetime to greater than 100 days. 
Our aim is to study how long RBCs live in the human body in patients with SCD compared to 
patients with SCD who have been cured by a bone marrow transplant, people with Sickle Cell 
Trait (SCT) or non-SCD/SCT volunteers. To do this, we will collect a small volume of blood 
(approximately 5 tablespoons) from your vein. In the laboratory we will mix your blood with a 
vitamin called Biotin. This vitamin is much smaller than the size of a single red blood cell and 
sticks to the outside of your RBCs without changing their function, shape, or overall lifetime. 
This process is known as “biotin labeling of RBCs”. We will then return your “biotin labeled 
RBCs” to you via vein injection. The collection of your RBCs, the labeling in the laboratory, 
and the reinfusion of your RBCs back to you will occur on the same day; however in rare 
IRB NUMBER: 20H0080
IRB APPROVAL DATE: 11/30/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: November 23, 2021
Page 2 of 14
CC ICF template v. 09.27.19
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
circumstances the reinfusion of your biotin labeled RBCs may occur the following day if we 
collect your RBCs too late in the day on that first day. 
There are three phases of this study: I. Screening, II. Collection and Infusion, and III. Follow-
up phases. 
You will be asked to provide blood samples at the following times:
I.
Screening: 
A single sample of venous blood will be required for laboratory tests and other screening 
procedures. 
II.
Collection and Infusion:
Day of blood collection
A blood sample will be collected for laboratory tests and other screening procedures prior to 
blood collection for biotin labeling. 
Day of infusion of biotin labeled RBCs 
A research blood sample will be taken 20 minutes after the end of the infusion.
III.
Follow-up:
Post-infusion day-  Month 1

SCD participants who have not had a transplant 
o
For the first two weeks (W1 and W2), participants will have samples drawn twice 
weekly
o
For the next two weeks (W3 and W4), participants will have samples drawn once 
weekly 

All other participants
o
For the - three weeks post infusion (W2, W3, and W4), participants  will have 
samples drawn once a week
Post-infusion day -  Month 2 until completion of the study (W24)

SCD Participants who have not had a transplant 
o
Will have a sample drawn weekly until biotinylation can no longer be detected 
in your RBCs, and once again during W24

All other participants
o
No sample will be drawn for W5
o
Starting on W6, participants will have a sample drawn every other week until 
biotinylation can no longer be detected in your RBCs, and once again during 
W24
There are a few known risks associated with Biotin, IV placement, blood draws, learning your 
HIV testing results, and the infusion. Although there is no direct benefit to you, by participating 
IRB NUMBER: 20H0080
IRB APPROVAL DATE: 11/30/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: November 23, 2021
Page 3 of 14
CC ICF template v. 09.27.19
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
in this study we will learn about RBC lifespan in patients with SCD and what factors influence 
this red blood cell survival.
The remaining document will now describe more about the research study.  This information 
should be considered before you make your choice. Members of the study team will talk with you 
about the information described in this document. Some people have personal, religious, or ethical 
beliefs that may limit the kinds of medical or research treatments they would want to receive (such 
as blood transfusions). Take the time needed to ask any questions and discuss this study with NIH 
staff, and with your family, friends, and personal health care providers.
IT IS YOUR CHOICE TO TAKE PART IN THE STUDY
You may choose not to take part in this study for any reason. If you join this study, you may change 
your mind and stop participating in the study at any time and for any reason. In either case, you 
will not lose any benefits to which you are otherwise entitled. However, to be seen at the NIH, you 
must be taking part in a study or are being considered for a study. If you do choose to leave the 
study, please inform your study team to ensure a safe withdrawal from the research.  
WHY IS THIS STUDY BEING DONE?
This is a research study. The purpose of this research study is to investigate how long red blood 
cells (RBCs) live in patients with and without Sickle Cell Disease (SCD).
SCD is an inherited   blood disorder that results from a single genetic change (mutation) in RBCs. 
RBCs are the cells that carry oxygen to the body. In people with SCD, the mutated RBCs can 
become hard, rigid, and contorted into a sickle shape. These abnormal RBCs die early, leaving a 
shortage of healthy RBCs (sickle cell anemia). RBCs typically survive in the human body for 
approximately 120 days. However, this duration is shortened to an average of 32 days in patients 
with SCD. Currently SCD can be cured with a bone marrow transplant. This can result in 
restoration of the red cell lifetime to greater than 100 days. We aim to study how long RBCs live 
in the human body in patients with SCD compared to patients with SCD who have been cured by 
a bone marrow transplant, people with Sickle Cell Trait (SCT) or non-SCD/SCT volunteers. We 
hope the knowledge gained from this study will allow us to better understand and enhance curative 
therapies for patients with SCD. 
To study how long RBCs live, we will collect a small volume of blood (approximately 70 mL; this 
is equivalent to approximately 5 tablespoons) from your vein. In the laboratory, we will mix your 
blood with a vitamin called Biotin which sticks to the outside of your RBCs. This process is known 
as “biotin labeling of RBCs”. We will then return your “biotin labeled RBCs” to you via vein 
injection. We can then measure the amount of biotin labeled RBCs remaining over the lifespan of 
those RBCs by drawing a small amount of blood (approximately 10 mL; this is equivalent to 2 
teaspoons) until we can no longer detect any biotin labeled RBCs.
We are asking you to join this research study because you either have SCD,  had SCD but were 
cured with a bone marrow transplant, have Sickle Cell Trait (SCT), or are a volunteer without SCD 
or SCT. 
IRB NUMBER: 20H0080
IRB APPROVAL DATE: 11/30/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: November 23, 2021
Page 4 of 14
CC ICF template v. 09.27.19
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
Although biotin is a vitamin we consume regularly in our diets, the use of biotin in this manner to 
label RBCs is considered investigational. 
WHAT WILL HAPPEN DURING THE STUDY?
If you decide to take part in this study, you will be asked to donate blood and will receive a 
transfusion of your own blood so we can measure how long your RBCs live. This procedure will 
take no more than 10 total hours for completion. 
here are three phases required in participating on this protocol:
I.
Screening
II.
Collection and Infusion
III.
Follow-up
Screening: After the consent is reviewed and signed, a medical history is collected and a physical 
exam is performed. We will collect approximately 50 mL of blood (approximately 3-4 
tablespoons) for blood tests that will tell us about your blood counts, liver and kidney functions. 
We may also test your blood for certain diseases: hepatitis B and C viruses, HIV, syphilis, and 
others that can be transmitted by blood. You will be asked to provide a urine sample to see if there 
are any abnormal cells, protein, or sugar. Pregnancy testing will be performed for all female 
subjects of childbearing age. You will be seen in the Department of Transfusion Medicine, where 
we will look at your arms to determine whether your veins are suitable for placing needles for the 
collection. All patients who have received a transplant on a Gene Therapy protocol may require 
additional confirmatory HIV testing during screening which will be performed under this protocol.
Collection and Infusion: If eligible, we will collect about 100 mL of blood (about 7 tablespoons) 
from your vein. In the laboratory we will mix your blood with a vitamin called Biotin. This vitamin 
is much smaller than the size of a single red blood cell and sticks to the outside of your RBCs 
without changing their function, shape, or overall lifetime. This process is known as “biotin 
labeling of RBCs”. We will then return your “biotin labeled RBCs” to you via vein injection. The 
collection of your RBCs, the labeling in the laboratory, and the reinfusion of your RBCs back to 
you will occur on the same day; however in rare circumstances the reinfusion of your biotin labeled 
RBCs may occur the following day if we collect your RBCs too late in the day on that first day. 
Some of the blood will be taken for lab tests and screening (approximately 20 mL, about 1.5 
tablespoons), while the rest of the blood will be used for biotin labeling (approximately 80 mL, 
about 5.5 tablespoons). A single blood sample will be taken 20 minutes after infusion 
(approximately 5 mL of whole blood, about 1 teaspoon) to measure the biotin labeling of RBCs.
Follow-up: We will calculate how long your RBCs live by measuring the amount of biotin that 
remains on your RBCs at frequent intervals until we can no longer detect any biotin labeled RBCs. 
Our bodies are naturally producing millions of RBCs every day, therefore over time your body 
naturally removes old blood cells. Just as the body removes old RBCs, it will remove the biotin 
that was attached to it. We will therefore be able to measure less and less biotin on your RBCs 
over time until all of it has been removed. 
To do this, you will provide a small sample of blood (approximately 10 mL; this is equivalent to 
2 teaspoons) according to the following schedule below until the completion of the study. For 
some patients, this may be as short as a week, for others it may be up to 16 weeks. 
IRB NUMBER: 20H0080
IRB APPROVAL DATE: 11/30/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: November 23, 2021
Page 5 of 14
CC ICF template v. 09.27.19
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
Number of blood samples
Post-infusion day-  Month 1
SCD participants who have not had a transplant: 
o
A total of 5 samples will be drawn.
o
Participants will have samples drawn once on W1 and twice on W2, (total of both weeks 
will be approximately 30 mL (about 2 tablespoons).
o
For W3 and W4, participants will have samples drawn once weekly (total of both weeks 
will be  approximately 20 mL (about 1.5 tablespoons).
All other participants:
o
A total of 3 samples will be drawn. 
o
No sample will be drawn on W1.
o
For W2, W3, and W4 participants will have samples drawn once weekly (total of three 
weeks will be  approximately 30 mL (about 2 tablespoons).
Post-infusion day -  Month 2 until completion of the study (W24)
SCD Participants who have not had a transplant: 
o
Will have samples drawn weekly until the percentage of biotinylated RBCs has decreased 
(up until week 8), and once again at W24 (weekly draw will be approximately 10 mL, 
(about 2 teaspoons);  a total of  approximately 50 mL (about 3-4 tablespoons).
All other participants:
o
There will be a sample drawn every other week, starting on W6 until the percentage of 
biotinylated RBCs has decreased (until approximately weeks 14-16), and once again at 
W24 (every other week draw will be approximately 10 mL (about 2 teaspoons); for a total 
up to 70 mL (about 4.5 tablespoons).
When you come to the NIH to give these blood samples, we will ask you if there have been any 
major changes to your health (for examples, a crisis or need for transfusion if you have SCD). We 
need to be aware of any changes as these could influence how long your RBCs live. Otherwise we 
do not expect any unwanted side effects from the biotin itself. 
 HOW LONG WILL THE STUDY TAKE?
If you agree to take part in this study, your involvement is expected to last for up to 6 months. You 
will no longer need to come in for regular blood draws once we can no longer detect your biotin 
labeled RBCs, but will require all participants to come in for a final blood draw at 6 months.
HOW MANY PEOPLE WILL PARTICIPATE IN THIS STUDY?
We plan to have approximately 40 people participate in this study at the NIH. 
WHAT ARE THE RISKS AND DISCOMFORTS OF BEING IN THE STUDY?
From biotin: The use of biotin labeling of RBCs has been studied in adults and babies with and 
without SCD and is very well tolerated with minimal to no side effects. 
IRB NUMBER: 20H0080
IRB APPROVAL DATE: 11/30/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: November 23, 2021
Page 6 of 14
CC ICF template v. 09.27.19
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
Biotin is a water-soluble B-vitamin (also called vitamin B7) that we receive in our diets every day. 
It is an essential vitamin that helps metabolize fats and carbohydrates and is involved in various 
metabolic processes in our bodies. The recommended daily allowance of biotin is 0.03 mg. 
Although no safety events related to biotin have been reported, very high levels of biotin (650 
times the recommended intake) can interfere with certain laboratory test results. The amount of 
biotin used in this study is well below the recommended daily allowance, and there is no need to 
modify your diet based on this study. 
In previous studies that have looked at biotin labeling of RBCs in adult and babies, rarely 
participants have developed an antibody to biotin (<5%). This antibody has not caused any 
problems in these participants including no breakdown of their red blood cells. None had changes 
in their complete blood counts (CBC) or any clinical signs or symptoms. One participant 
experienced accelerated removal of biotin labeled RBCs when this participant participated in a 
second biotin labeled study 5 years later. It is therefore possible that if an antibody to biotin 
develops, you would be excluded from future studies investigating biotin labeling of RBCs.
From IV placement and Blood Draws: Insertion of a needle into a vein for drawing blood and 
for injection of RBCs may cause mild pain, and there is a small risk of bleeding with bruising at 
the site. Side effects commonly seen in a normal blood donation (however the volume of blood 
taken in this study is less than blood donation) includes sweating, light headedness, vomiting, low 
blood pressure, rapid pulse, and fainting. Although all material used to draw blood are sterile, very 
rarely infection may occur at the site of the blood draw. Blood samples will be drawn by 
experienced study personnel using standard measures to minimize each of these risks. 
If you are a healthy volunteer, it is recommended that you do not donate blood during this study 
and for 8 weeks after you complete this study.
From the Infusion: The injection of the treated RBCs is similar to a blood transfusion and could 
cause some bruising, irritation, or infection. Study infusions will be done by experienced study 
personnel using standard measures to minimize each of these risks. Infusion of blood may cause 
fever, chills, nausea, hives, rash, itching, swelling of the face or neck, difficulty breathing, and low 
blood pressure. 
From HIV testing: As part of this study, we will test you for infection with the human 
immunodeficiency virus (HIV), the virus that causes AIDS.  If you are infected with HIV you will 
not be able to participate in this study.  We will tell you what the results mean, how to find care, 
how to avoid infecting others, how we report HIV infection, and the importance of informing your 
partners at possible risk because of your HIV infection.
What are the risks related to pregnancy? 
If you are capable of becoming pregnant, we will ask you to have a pregnancy test before beginning 
this study. You must use effective birth control methods and try not to become pregnant while 
participating in this study. If you become pregnant, there may be unknown risks to the fetus or 
unborn child, or risks that we did not anticipate. There may be long-term effects of the treatment 
being studied that could increase the risk of harm to a fetus. You must tell the study doctor if your 
birth control method fails while you are in the study. If you think or know you have become 
pregnant while participating in this research study, please contact the research team member 
identified at the top of this document as soon as possible. If you plan to become pregnant in the 
IRB NUMBER: 20H0080
IRB APPROVAL DATE: 11/30/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: November 23, 2021
Page 7 of 14
CC ICF template v. 09.27.19
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
future, please discuss with the research team how long you need to wait before becoming pregnant 
after completing the course of this study drug or procedures on this study.
If you are a sexually active person with a partner capable of becoming pregnant, it is important 
that your partner not become pregnant during your participation in this study. There may be 
unknown risks to a fetus or risks we did not anticipate. You and your partner must agree to use 
birth control if you want to take part in this study. If you think your partner has become pregnant 
during your participation in this study, please contact the research team member identified at the 
top of this document as soon as possible.  If you and your partner plan for your partner to become 
pregnant after your participation in this study, please discuss this with the study team.
WHAT ARE THE BENEFITS OF BEING IN THE STUDY?
You will not benefit from being in this study.
Your participation will help us develop a better understanding of the shortened red blood cell 
lifespan in patients with SCD and what factors influence this red blood cell survival. We know 
that patients with SCD can be cured with a bone marrow transplant, in part because the donor red 
blood cells survive longer than sickle RBCs. There is no direct benefit to you to participate in this 
study. 
Are there any potential benefits to others that might result from the study?
In the future, other people might benefit from this study because information from this study will 
allow us to better understand this phenomenon and help us improve our curative therapies 
including bone marrow transplantation and gene therapy methods. 
WHAT OTHER OPTIONS ARE THERE FOR YOU?
Before you decide whether or not to be in this study, we will discuss other options that are 
available to you. Instead of being in this study, you could choose not to take part in this study. If 
you do not take part in this study, you can still receive your regular medical care by your doctor. 
Taking part in this trial is voluntary. You have the right to stop being a part of this study (this is 
called withdrawal) at any time, even if you have already signed this consent form and without 
giving a reason.  You will not lose any benefits to which you are entitled; you can continue 
regular medical care and can go back to your regular doctor and treatment.  If you decide you 
want to stop being a part of this study, you must first tell the study doctor right away.
DISCUSSION OF FINDINGS
New information about the study
If we find out any new information that may affect your choice to participate in this study, we 
will get in touch with you to explain what we have learned. This may be information we have 
learned while doing this study here at the NIH or information we have learned from other 
scientists doing similar research in other places.
Return of research results
The research team will not provide you with any research results unless they are meaningful. 
Additional research may be needed before these results are meaningful.
IRB NUMBER: 20H0080
IRB APPROVAL DATE: 11/30/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: November 23, 2021
Page 8 of 14
CC ICF template v. 09.27.19
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
By agreeing to participate in this study, you do not waive any right that you may have regarding 
access to and disclosure of your medical records.  For further information on those rights, please 
contact Dr. Tisdale
The investigators will give you information about the overall results of this study after completion 
of the study. Published research results will only describe groups of people who took part in the 
study. Information that points out a single person will not appear in research journals or other 
reports. You will never personally be identified in any of the publications.
EARLY WITHDRAWAL FROM THE STUDY 
You can withdraw from this study at any time. This decision will not affect your legal rights or 
the quality of health care that you will/may receive at NIH. At any time, the study doctor may 
tell you to stop taking part in the study. This may happen if you have a health-related event 
during the course of the study that affects how long your RBCs live or how many RBCs are 
produced, you do not follow the instructions given by the study doctor, or if the study doctor 
believes it is in your best interest. 
WILL YOUR SPECIMENS OR DATA BE SAVED FOR USE IN OTHER RESEARCH 
STUDIES?
As part of this study, we are obtaining specimens and data from you. We will remove all the 
identifiers, such as your name, date of birth, address, or medical record number and label your 
specimens and data with a code so that you cannot easily be identified. However, the code will 
be linked through a key to information that can identify you. We plan to store and use these 
specimens and data for studies other than the ones described in this consent form that are going 
on right now, as well as studies that may be conducted in the future. These studies may provide 
additional information that will be helpful in understanding SCD, or other diseases or conditions. 
This could include studies to develop other research tests, treatments, drugs, or devices, that may 
lead to development of a commercial product by the NIH and/or its research or commercial 
partners. There are no plans to provide financial compensation to you if this happens. Also, it is 
unlikely that we will learn anything from these studies that may directly benefit you.
I give permission for my coded specimens and data to be stored and used for future research as 
described above.
_____ Yes
_____ No
Initials
Initials
WILL YOUR SPECIMENS OR DATA BE SHARED FOR USE IN OTHER RESEARCH 
STUDIES?
We may share your deidentified biospecimens and data with other researchers.  If we do, while 
we will maintain the code key, we will not share it, so the other researchers will not be able to 
identify you.  They may be doing research in areas similar to this research or in other unrelated 
areas. These researchers may be at NIH, other research centers and institutions, or commercial 
entities.
IRB NUMBER: 20H0080
IRB APPROVAL DATE: 11/30/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: November 23, 2021
Page 9 of 14
CC ICF template v. 09.27.19
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
I give permission for my coded specimens and data to be shared with other researchers and used 
by these researchers for future research as described above.
_____ Yes 
_____ No
Initials
Initials
If you change your mind and do not want us to store and use your specimens and data for future 
research, you should contact the research team member identified at the top of this document. 
We will do our best to comply with your request but cannot guarantee that we will always be 
able to destroy your specimens and data. For example, if some research with your specimens and 
data has already been completed, the information from that research may still be used. Also, for 
example, if the specimens and data have been shared already with other researchers, it might not 
be possible to withdraw them. 
In addition to the planned use and sharing described above, we might remove all identifiers and 
codes from your specimens and data and use or share them with other researchers for future 
research at the NIH or other places.  When we or the other researchers access your anonymized 
data, there will be no way to link the specimens or data back to you.  We will not contact you to 
ask your permission or otherwise inform you before we do this.  We might do this even if you 
answered "no" to the above questions.  If we do this, we would not be able to remove your 
specimens or data to prevent their use in future research studies, even if you asked, because we 
will not be able to tell which are your specimens or data.
NIH policies require that your clinical and other study data be placed in an internal NIH database 
that is accessible to other NIH researchers for future research. These researchers will not have 
access to any of your identifiers, such as your name, date of birth, address, or medical record 
number; and your data will be labeled with only a code. We cannot offer you a choice of whether 
your data to be placed in this database or not.  If you do not wish to have your data placed in this 
database, you should not enroll in this study.  
How Long Will Your Specimens and Data be Stored by the NIH?
Your specimens and data may be stored by the NIH possibly indefinitely or until they become 
irrelevant and are destroyed with the IRB approval. A small amount of your blood sample may 
be used for research purposes to develop better laboratory techniques for SCD. In addition, we 
will store certain information in your medical record. We may be interested in using your 
samples to pursue other research into SCD or similar disorders. We may send your samples 
elsewhere for analysis. If we do so, we will not reveal your identity, but there will be a code to 
link your samples with your name and other personal information. The code will be stored in a 
password protected database under the control of Dr. Tisdale.
Risks of Storage and Sharing of Specimens and Data
When we store your specimens and data, we take precautions to protect your information from 
others that should not have access to it. When we share your specimens and data, we will do 
everything we can to protect your identity, for example, when appropriate, we remove information 
that can identify you. Even with the safeguards we put in place, we cannot guarantee that your 
identity will never become known or someone may gain unauthorized access to your information. 
IRB NUMBER: 20H0080
IRB APPROVAL DATE: 11/30/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: November 23, 2021
Page 10 of 14
CC ICF template v. 09.27.19
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
New methods may be created in the future that could make it possible to re-identify your specimens 
and data.
COMPENSATION, REIMBURSEMENT, AND PAYMENT
Will you receive compensation for participation in the study?
We will pay you for participating in this research study.  The amount will be dependent on the 
tests performed. We calculate your compensation based on the inconvenience we will cause you 
for the hours you spend participating on this study, for the procedures and other forms of 
participation in the study up to a total of $1,600.00 (for at least 12 visits). 
Procedure
Inconvenience units ($10 for each inconvenience 
unit)
Outpatient Visit (first hour)
$20 (2 units)
Outpatient Visit (additional hours, 
 up to 4 hours)
$10 (1 unit)
Screening History & Physical
$25 (2.5 units)**
Research blood sampling (per blood draw)
$40 (4 unit)
IV Placement
$10 (1 unit)
Drug Infusion/Administration by IV
$50 (5 units)
Completion Bonus
$500 (50 units)
Travel Voucher (per visit)
$40 (4 units)
Total Potential Compensation
up to $1600.00*
*Dependent on the subject’s red cell lifetime, the number of blood draws needed to be collected 
until study completion, although rare, may exceed the projected 16 week collection timeline. 
Therefore total compensation may exceed $1,600.00.
**Unless you were screened under another NIH protocol. 
If you are unable to finish the study, you will receive payment for the parts you completed.  
With few exceptions, study compensation is considered taxable income that is reportable to the 
Internal Review Service (IRS). A “Form 1099-Other Income” will be sent to you if your total 
payments for research participation are $600 or more in a calendar year. If you have unpaid debt 
to the federal government, please be aware that some or all of your compensation may be 
automatically reduced to repay that debt on your behalf.
Will you receive reimbursement or direct payment by NIH as part of your participation?
This study offers vouchers for travel (per visit) but does not offer reimbursement for, or payment 
of, lodging or meals. 
Will taking part in this research study cost you anything? 
NIH does not bill health insurance companies or participants for any research or related clinical 
care that you receive at the NIH Clinical Center.
IRB NUMBER: 20H0080
IRB APPROVAL DATE: 11/30/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: November 23, 2021
Page 11 of 14
CC ICF template v. 09.27.19
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
Participation in this study will not cost you.  
CONFLICT OF INTEREST (COI) 
The National Institutes of Health (NIH) reviews NIH staff researchers at least yearly for conflicts 
of interest.  This process is detailed in a COI Guide.  You may ask your research team for a copy 
of the COI Guide or for more information.  Members of the research team who do not work for 
NIH are expected to follow these guidelines or the guidelines of their home institution, but they 
do not need to report their personal finances to the NIH.
No NIH investigator involved in this study receives payments or other benefits from any 
company whose drug, product or device is being tested.
CLINICAL TRIAL REGISTRATION AND RESULTS REPORTING 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required 
by U.S. Law. This Web site will not include information that can identify you. At most, the Web 
site will include a summary of the results. You can search this Web site at any time.
CONFIDENTIALITY PROTECTIONS PROVIDED IN THIS STUDY  
The people working on the study will do whatever they can to keep your information private and 
not share it with other people unnecessarily.  The National Institute of Health (NIH) and its 
representatives may need to look at and/or make copies of your information and health records.  
If this happens, the people who review your records will be very careful to protect your identity.  
In rare cases, people who look at your records may be required by law to share information about 
you with others.  
Federal privacy regulations provide safeguards for privacy, security, and authorized access.  
Except when required by law, you will not be identified by name, social security number, 
address, telephone number, or any other direct personal identifier in study records. NIH may 
give your health data, without identifiers, to other researchers or use it for other approved 
research projects not listed in this form. The NIH, Dr. Tisdale, and study staff will comply with 
any and all laws regarding the privacy of such information. There are no plans to pay you for the 
use or transfer of this information.
Will your medical information be kept private? 
We will do our best to make sure that the personal information in your medical record will be kept 
private.  However, we cannot guarantee total privacy.  Organizations that may look at and/or copy 
your medical records for research, quality assurance, and data analysis include:

The NIH and other government agencies, like the Food and Drug Administration (FDA), 
which are involved in keeping research safe for people.

National Institutes of Health Intramural Institutional Review Board

The study Sponsor National Heart Lung and Blood Institute or their agent(s) 
When results of an NIH research study are reported in medical journals or at scientific meetings, 
the people who take part are not named and identified.  In most cases, the NIH will not release any 
information about your research involvement without your written permission.  However, if you 
sign a release of information form, for example, for an insurance company, the NIH will give the 
IRB NUMBER: 20H0080
IRB APPROVAL DATE: 11/30/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: November 23, 2021
Page 12 of 14
CC ICF template v. 09.27.19
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
insurance company information from your medical record.  This information might affect (either 
favorably or unfavorably) the willingness of the insurance company to sell you insurance.
If we share your specimens or data with other researchers, in most circumstances we will remove 
your identifiers before sharing your specimens or data. You should be aware that there is a slight 
possibility that someone could figure out the information is about you. 
Further, the information collected for this study is protected by NIH under a Certificate of 
Confidentiality and the Privacy Act. 
Certificate of Confidentiality
To help us protect your privacy, the NIH Intramural Program has received a Certificate of 
Confidentiality (Certificate). With this certificate, researchers may not release or use data or 
information about you except in certain circumstances.  
NIH researchers must not share information that may identify you in any federal, state, or local 
civil, criminal, administrative, legislative, or other proceedings, for example, if requested by a 
court.  
The Certificate does not protect your information when it:  
1. is disclosed to people connected with the research, for example, information may be used 
for auditing or program evaluation internally by the NIH; or 
2. is required to be disclosed by Federal, State, or local laws, for example, when information 
must be disclosed to meet the legal requirements of the federal Food and Drug 
Administration (FDA); 
3. is for other research;
4. is disclosed with your consent. 
The Certificate does not prevent you from voluntarily releasing information about yourself or your 
involvement in this research. 
The Certificate will not be used to prevent disclosure to state or local authorities of harm to self or 
others including, for example, child abuse and neglect, and by signing below you consent to those 
disclosures. Other permissions for release may be made by signing NIH forms, such as the Notice 
and Acknowledgement of Information Practices consent.
Privacy Act
The Federal Privacy Act generally protects the confidentiality of your NIH medical information 
that we collect under the authority of the Public Health Service Act.  In some cases, the Privacy 
Act protections differ from the Certificate of Confidentiality.  For example, sometimes the Privacy 
Act allows release of information from your record without your permission, for example, if it is 
requested by Congress. Information may also be released for certain research purposes with due 
consideration and protection, to those engaged by the agency for research purposes, to certain 
federal and state agencies, for HIV partner notification, for infectious disease or abuse or neglect 
reporting,  to tumor registries, for quality assessment and medical audits, or when the NIH is 
involved in a lawsuit.  However, NIH will only release information from your medical record if it 
is permitted by both the Certificate of Confidentiality and the Privacy Act.
IRB NUMBER: 20H0080
IRB APPROVAL DATE: 11/30/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: November 23, 2021
Page 13 of 14
CC ICF template v. 09.27.19
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
POLICY REGARDING RESEARCH-RELATED INJURIES  
The NIH Clinical Center will provide short-term medical care for any injury resulting from your 
participation in research here. In general, no long-term medical care or financial compensation for 
research-related injuries will be provided by the NIH, the NIH Clinical Center, or the Federal 
Government. However, you have the right to pursue legal remedy if you believe that your injury 
justifies such action.
PROBLEMS OR QUESTIONS
If you have any problems or questions about this study, or about your rights as a research 
participant, or about any research-related injury, contact the Principal Investigator, John Tisdale, 
M.D., Ph.: (301)402-6497, Email: johntis@nhlbi.nih.gov. Another researcher you may call is 
Christina Luckett, RN, Ph. (301)827-7901. You may also call the NIH Clinical Center Patient 
Representative at (301)496-2626, or the NIH Office of IRB Operations at (301)402-3713, if you 
have a research-related complaint or concern.
CONSENT DOCUMENT 
Please keep a copy of this document in case you want to read it again.
IRB NUMBER: 20H0080
IRB APPROVAL DATE: 11/30/2021


PATIENT IDENTIFICATION
Consent to Participate in a Clinical Research Study
NIH-2977 (4-17)
File in Section 4: Protocol Consent (1)
Version Date: November 23, 2021
Page 14 of 14
CC ICF template v. 09.27.19
MEDICAL RECORD
CONSENT TO PARTICIPATE IN AN NIH CLINICAL RESEARCH STUDY
Adult Research Participant: I have read the explanation about this study and have been given the opportunity 
to discuss it and to ask questions. I consent to participate in this study.
Signature of Research Participant
Print Name of Research Participant
Date
Witness to the oral short-form consent process only: This section is only required if you are doing the oral 
short-consent process with a non-English speaking subject and this English consent form has been approved 
by the IRB for use as the basis of translation.
Witness:
Signature of Witness*
Print Name of Witness
Date
Investigator:
Signature of Investigator
Print Name of Investigator
Date
*NIH ADMINISTRATIVE SECTION TO BE COMPLETED REGARDING THE USE OF AN 
INTERPRETER:
An interpreter, or other individual, who speaks English and the participant’s preferred language facilitated
the administration of informed consent and served as a witness. The investigator obtaining consent may not 
also serve as the witness.
An interpreter, or other individual, who speaks English and the participant’s preferred language facilitated
the administration of informed consent but did not serve as a witness. The name or ID code of the person 
providing interpretive support is:
.
IRB NUMBER: 20H0080
IRB APPROVAL DATE: 11/30/2021