Quantification of Peripheral Hematopoietic Progenitor Cell Circulation Following Repeated Acute, Vascular Restriction Resistance Exercise Using the Delfi Tourniquet System
Phase: NA
Conditions: Blood Flow Restriction (BFR) Training Effects
Sponsor: Andrews Research & Education Foundation

 
 
Quantification of Peripheral Hematopoietic Progenitor Cell Circulation Following Repeated 
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Title:   
Quantification of Peripheral Hematopoietic Progenitor Cell 
Circulation Following Repeated Acute, Vascular Restriction 
Resistance Exercise Using the Delfi Tourniquet System  
 
Principal Investigator:  
Tyler Opitz, DPT, SCS, CSCS 
 
Co-Investigators:  
 
James Andrews, MD 
Adam Anz, MD 
Jessica Jewell, MAT, L/ATC, CSCS 
Steve Jordan, MD 
 
 
Research Team: 
 
Chandler Bridges, MS 
Joshua Cook, BS 
Victoria Williams 
 
 
Carter Sheppard 
 
 
Funding Mechanism:   
State of Florida  
 
Institutions:   
 
Andrews Research and Education Foundation (AREF) 
 
 
 
 
1020 Gulf Breeze Parkway 
 
 
 
 
Gulf Breeze, FL 32561 
 
 
 
 
(850) 916-8584 
 
 
 
 
 
Andrews Institute 
 
 
 
 
1040 Gulf Breeze Parkway 
 
 
 
 
Gulf Breeze, FL 32561 
 
 
 
 
(850) 916- 8464 
 
 

 
 
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Table of Contents 
1 
Scientific Rationale / Background ........................................................................................................ 6 
2 
Study Design ........................................................................................................................................ 7 
3 
Objectives ............................................................................................................................................ 8 
3.1 
Primary Objective ........................................................................................................................ 8 
3.2 
Secondary Objectives .................................................................................................................. 8 
3.3 
Primary Effectiveness Endpoint .................................................................................................. 8 
3.4 
Secondary Effectiveness Endpoints ............................................................................................. 9 
4 
Hypotheses .......................................................................................................................................... 9 
4.1 
Null Hypothesis: .......................................................................................................................... 9 
4.2 
Alternative Hypothesis: ............................................................................................................... 9 
5 
Participant Recruitment and Screening ............................................................................................... 9 
5.1 Screening Process .............................................................................................................................. 9 
5.2 Participant Eligibility ....................................................................................................................... 10 
5.2.1 
Inclusion Criteria ................................................................................................................... 10 
5.2.2 
Exclusion Criteria .................................................................................................................. 10 
5.3 
Informed Consent 21 CFR 50 .................................................................................................... 11 
5.4 
Consent Withdrawal: ................................................................................................................. 11 
5.5 
Benefits ...................................................................................................................................... 11 
5.6 
Compensation ............................................................................................................................ 11 
6 
Treatment Plan .................................................................................................................................. 11 
6.1 
Familiarization Session: ............................................................................................................. 12 
6.2 
Experimental Testing Sessions (12 Total): ................................................................................ 14 
6.3 Final Session: ................................................................................................................................... 15 
7 
Review of Safety ................................................................................................................................ 15 
7.1 
Adverse Event (AE) ................................................................................................................... 15 
7.2 
Serious Adverse Event (SAE) .................................................................................................... 16 
7.3 
Unanticipated Problem (UP): ..................................................................................................... 16 
7.4 
AE & SAE Collection and Reporting ........................................................................................ 16 

 
 
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7.5 
Risks and Discomforts ............................................................................................................... 17 
8 
Data Management Procedures .......................................................................................................... 17 
9 
Data Analysis ...................................................................................................................................... 17 
 

 
 
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Abbreviations and Definitions 
AE 
Adverse Event 
ANOVA Analysis of Variance 
AREF Andrews Research and Education Foundation 
BFR 
Blood Flow Restriction 
CBC Complete Blood Count 
Cc 
Cubic Centimeter 
CD 
Cluster Differentiation 
CFR 
Code of Federal Regulations 
EDC Electronic Data Capture 
G-CSF Granulocyte Colony Stimulating Factor 
HIPAA Health Insurance Portability and Accountability Act 
HIT 
High-Intensity Training 
HPC 
Hematopoietic Progenitor Cells 
HSD Tukey’s Honestly Significant Difference 
HSC 
Hematopoietic Stem Cells 
ICF 
Informed Consent Form 
IRB 
Institutional Review Board 
mL 
Milliliters 
MSC Mesenchymal Stem Cells 
NIRS  Near-infrared Spectroscopy 
NSAIDS Non-steroidal anti-inflammatory drugs 
PI  
Primary Investigator 
SAE  Serious Adverse Event 
SAS 
Statistical Analysis System 

 
 
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UP 
Unanticipated Problem 
1-RM One-repetition Maximum 
 
 

 
 
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1 
Scientific Rationale / Background  
Intense interest in the areas of orthopedic surgery and recovery science has been placed 
on progenitor cell research. The ability to transplant these cells in successful patient 
engraftments has been sought after due to properties of hematopoietic stem cells (HSCs) 
that allow for long-term self-renewal and multipotency (1). The obstacle of an 
immunocompetent immune system has been a source of difficulty for some time within 
the scientific community (2). Using stem cells from different regions of the same patient 
prevents the need for long-term immune system suppression (2, 3). Currently, HSCs are 
collected through two primary methods: bone marrow aspiration and peripheral harvest 
following pharmaceutically induced mobilization using granulocyte colony-stimulating 
factor (G-CSF). These methods suffice but less invasive methods are preferred. By 
quantifying peripheral hematopoietic progenitor cell presence within the blood following 
repeated blood flow restriction exercise, we hope to uncover a more convenient method 
for harvesting these hematopoietic progenitor cells. We aim to accomplish this with the 
aid of exercise-induced elevation of progenitor cell concentration in peripheral blood (4, 
5). Prior studies with BFR and stem cell mobilization have shown increased cell 
concentration immediately post a single bout of exercise (6). 
Also present within peripheral blood following exercise are adult mesenchymal stem 
cells (MSCs). MSCs can monitor their local and systemic environments for pertinent 
stimuli. These stimuli such as often determine the fate and commitment of MSC 
differentiation (7). MSCs utilize paracrine effects to conduct this interaction with their 
surrounding (8). MSCs can also respond to changes in environmental conditions and 
hypoxia, as seen in animal studies (9), as well as exercise, seen in human studies (4). 
MSCs have the same primary methods of collection as HSCs. 
Blood Flow Restriction Therapy (BFR) has been successfully implemented during the 
post-operative rehabilitation period, for patients with osteoarthritis or muscle atrophy, 
and in instances of limb salvage following injury (10-13). This type of training has been 
shown to also be effective in producing hypertrophic skeletal muscle growth that can 
parallel growth seen in High-Intensity Training (HIT), but without the mechanical muscle 
damage that occurs with HIT (13-18). Blood flow restriction training is an alternative 
method to traditional exercise for the goal of retaining and increasing muscle mass 
without the musculoskeletal stress placed on the body by traditional training 
methodologies. The application of BFR uses low-intensity resistance training with high 
repetition volume to mimic the exertion required of the body by higher-intensity, lower-
repetition training (19). 
The mechanism by which BFR training acts is not fully understood. Some of the leading 
theories for the mechanism are the metabolite theory, which includes, increased cellular 
swelling through the accumulation of metabolites and blood within the muscle cells, 
increased protein synthesis and growth hormone production, elevated metabolic stress, 

 
 
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and heightened motor unit recruitment (20, 21). The influx of blood carrying metabolites 
during BFR to the area of injury facilitates elevation of growth hormone levels locally 
(21, 22). Local accumulation of metabolites has been indicated to influence the 
production of growth hormone more so than total metabolite production. (23, 24). These 
growth hormones can exhibit an anabolic effect on muscle. Increased metabolic stress 
results in the local accumulation of lactate, hydrogen ions among other metabolic 
byproducts (25, 26). Analysis of BFR from a physiological perspective have been 
consistent in determining that levels of lactate and growth hormone increase following 
BFR training, ultimately peaking in a time window immediately to 40 minutes after BFR 
(23, 27-29). Exercise using BFR has also been linked to extremely low oxygenation 
saturation (StO2) levels within the skeletal muscle (<10%) as measured via near-infrared 
spectroscopy (NIRS). This finding suggests that the working tissue experiences severe 
hypoxia during BFR exercise (30). There have been inconsistent findings on if exercise 
within this hypoxic state is better suited to stimulate greater gains in muscle strength and 
physiologic structure change (31, 32). Further investigation on muscular and physiologic 
response to hypoxic training conditions could provide insight into a new way of 
conditioning the body without needing excessive load, a more ideal approach for the 
therapeutic patient recovery process. The final theory behind the action mechanism of 
BFR is motor unit recruitment. Motor unit recruitment using Henneman’s size principle 
theorizes that motor unit recruitment within a motor pool begins with the smallest motor 
unit(s) and progresses to larger and larger motor units depending on the targeted force. 
Recruiting progressively higher threshold motor units is influential in generating muscle 
hypertrophy as external load determines motor unit recruitment (21). This idea paired 
with accompanying adjustments in repetitions has been at the core of much of the 
traditional approaches to hypertrophy development within musculature. Thus, the aim of 
this study is to quantify the cellular content (in vitro) of mobilized hematopoietic stem 
cells extracted from peripheral blood before exercise and at 0-, and 20-minute intervals 
following exercise. This study will be useful in the continued advancement of localized, 
peripheral hematopoietic progenitor cell research and patient application, development of 
effective non-surgical or post-surgical therapeutic treatments, and incorporation of 
knowledge in the implementation of performance training. 
 
2 
Study Design  
The proposed study is a prospective, quasi-experiment, dual-center (laboratory and 
exercise facility) study involving 10 healthy male and 10 healthy female volunteers. A 
potential subject must clear the screening, consent to the procedures of this study, and 
complete the medical interview before proceeding with the familiarization session. The 
familiarization process will include an introduction to the Delfi BFR Tourniquet System 
(Delfi Medical Innovations Inc., Vancouver, BC) and the exercises (seated leg extension, 

 
 
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semi-reclined leg press, prone hamstring curl) that the subject will complete. The weight 
each subject will use for each exercise will be determined during this time. All 
familiarization procedures must be completed prior to the scheduling and execution of the 
first of twelve experimental testing sessions.  
The experimental testing sessions will occur twice a week for six weeks (12 sessions) and 
will involve the BFR training utilizing the Delfi BFR Tourniquet System, as further 
described in the Experimental Testing Session section of the treatment plan (6.2).  At the 
beginning of the first, the sixth, and the twelfth testing sessions there will be a blood draw 
requiring 6 cc of blood to be taken prior to exercise. Post-exercise, at each testing session, 
additional 6cc blood draws will be conducted immediately post exercise (time point 0), 
and 20 minutes after the conclusion of the workout. Following every testing session 
workout, finger-prick blood samples for lactate testing will be taken. These blood 
samples are to be taken at Time point 0- (T 0), 10-, and 20-minutes following exercise. 
Once the twelfth experimental testing session has been completed, the subjects will have 
one final session that is to take place no more than five days following the twelfth testing 
session. This final session will include one final blood draw of 6 cc. Throughout the 
duration of the study, the blood drawn will be used for obtaining a complete blood count 
(CBC) and for cellular analysis to quantify peripheral hematopoietic progenitor cell 
concentration. The blood samples collected from subject’s fingers throughout the study 
will be used for analysis using a portable lactate analyzer. Lactate analysis will allow 
researchers insight into each subject’s exertion level reflected by the amount of lactate 
found within the samples collected. 
3 
Objectives 
3.1 
Primary Objective 
The primary objective of this study is to quantify the cellular content (in vitro) of 
mobilized hematopoietic stem cells and CBC data extracted from peripheral blood before 
exercise and at 0-, and 20-minute intervals following exercise.  
3.2 
Secondary Objectives 
Post-exercise lactate levels and demographic information will be recorded to provide 
insight into the fitness development and endurance shift of each subject. Each peripheral 
blood sample will also be analyzed for MSC concentration if enough sample is available 
after primary testing. 
3.3 
Primary Effectiveness Endpoint 
The primary effectiveness endpoint will be the quantification of mobilized hematopoietic 
stem cells and CBC data extracted from peripheral blood before exercise and at 0- and 
20-minute intervals following exercise.  

 
 
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3.4 
Secondary Effectiveness Endpoints 
Secondary effectiveness endpoints include post-exercise lactate levels, participant 
demographics, and MSC marker concentrations.   
4 
Hypotheses 
4.1 
Null Hypothesis:  
H0 - Subjects will have equal levels of hematopoietic progenitor cells (HPCs) before and 
after undergoing twelve experimental testing sessions with the Delfi BFR tourniquet 
system. 
4.2 
Alternative Hypothesis: 
H1- Subjects will not have equal levels of hematopoietic progenitor cells (HPCs) before 
and after undergoing twelve experimental testing sessions with the Delfi BFR tourniquet 
system. 
5 
Participant Recruitment and Screening 
The research study team intends to enroll a total of 25 volunteers (with 5 expected screen 
fails or withdraws) 
Subject recruitment will occur through the Andrews Institute physician practices and 
physical therapy departments via word of mouth. Recruitment of subjects will also utilize 
a flyer displaying the opportunity for subjects to enroll in this study. Flyers will be placed 
in easily viewed spaces around the Andrews Institute campus in Gulf Breeze, FL. 
Potential study subjects will undergo a prescreening process covering inclusion and 
exclusion criteria through standard of care medical evaluations. Potential subjects 
meeting all inclusion criteria will have the study described to them by a member of the 
investigating team. This description will include potential risks, discomforts, and 
benefits. Subjects will then be provided an opportunity to read and sign an approved 
informed consent form. The subject will be provided sufficient time to consent and sign 
the informed consent form (ICF). The principal investigator (PI) will be available to 
answer any questions or provide clarifications during the informed consent process. This 
process will be completed before conducting any study related activities.  
5.1 Screening Process 
Once consented, each participant can be scheduled for the initial screening visit. During 
the initial visit, a screening form will be completed and reviewed. If an individual 
answers "yes" to any of the initial screening exclusion questions, they will be informed 
that they do not qualify, and they will be informed that they can keep their screening 
form. If all answers are "no" then the form will be placed in the study documents. A 

 
 
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medical interview will also be conducted. This medical interview will include a complete 
physical examination and subjects will also be asked to fill out the Tegner Activity Level 
Scale to obtain a measure of their physical activity. The physical examination will 
include head/neck, cardiovascular, lung, and abdominal examinations. Standard vitals 
will also be taken. 
5.2 Participant Eligibility 
5.2.1 Inclusion Criteria 
1. Volunteer population of healthy males and females aged 18-45.  
2. Subjects consent to attending a total of 14 visits over a time span of seven to 
nine weeks. The breakdown of the 14 visits is as follows: 1 familiarization 
session, 12 experimental testing sessions, 1 final session.  
5.2.2 Exclusion Criteria 
Volunteers who have medical history involving one or more of the following 
medical conditions:  
1. Uncontrolled diabetes (accepted methods of control include medication, diet, 
exercise, or another form of intervention following guidelines from the 
American Diabetes Association)(33).  
2. Uncontrolled hypertension (accepted methods following guidelines from the 
International Society of Hypertension)(34).  
3. Autoimmune disorders 
4. Blood disorders 
5. Ongoing infectious disease 
6. Cancer 
7. Any disorder requiring immunosuppression treatment 
8. Steroid usage 
9. Significant cardiovascular, pulmonary, hepatic, or renal disease. 
10. Volunteers where 20 minutes of intense exercise is contra-indicated will 
also be excluded from the study. 
11. Positive pregnancy test 

 
 
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5.3 
Informed Consent 21 CFR 50 
In adherence to the 21 CFR 50, Protection of Human Subjects Guidelines, the informed 
consent process will be performed by one of the study investigators or research team 
member, in the physician clinic or research office that has received training on the 
informed consent process. No aspects of the study will be conducted prior to obtaining 
informed consent from each subject.  The purpose and methods of the study along with 
the expected effects will be reviewed with each potential subject. Each subject will be 
provided a copy of the consent and sufficient time will be given for the opportunity to 
read and ask any questions about the study.  After signing of the informed consent 
document, subjects will be given a copy for their records.  
The designee will review with each subject that they are free to refuse to the study or to 
withdraw from it at any time. 
5.4 
Consent Withdrawal: 
During the informed consent procedure, subjects will be informed that if at any point 
during the study, consent may be withdrawn. To withdraw consent, subjects can request 
via phone, email, or in writing to withdraw HIPAA authorization and the research site 
will not use or provide any health information to researchers. At this time, the link 
between the subject’s health information will be severed with the research team. This 
process for consent withdrawal will be reviewed with each subject and identified barriers 
will be addressed at the time of informed consent. Any samples obtained as part of the 
study will be destroyed per the site’s standard operating procedures, unless broad consent 
was obtained. 
5.5 
Benefits 
There are no direct benefits for subjects who participate in the study. Indirect benefits 
include additional medical care and physical therapy care not associated with any medical 
procedures or injury. Results from the study may help clinicians to develop a better 
understanding of BFR and its effects on blood metabolites and the mobilization of 
peripheral hematopoietic progenitor cells (aspirational benefit).  
5.6 
Compensation 
Participants will be provided a stipend for their time spent participating in this study. A 
$20.00 gift card will be provided at each of the 14 sessions described below 
(Introductory, 12 Experimental, Final). 
6 
Treatment Plan 
Each subject will attend a total of 14 sessions. 

 
 
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Subjects will attend a familiarization session three to fourteen days prior to the beginning 
of the experimental sessions during which testing will be conducted.  
Following the familiarization session, subjects will attend two experimental testing 
sessions per week for six weeks.  
During the seventh week following the first experimental testing session, subjects will 
attend the final session for a blood draw.  
Sessions will not occur within 24 hours of another session. Blood samples will be 
collected from the same visit within the week if possible (either the first in the week or 
second in the week depending on scheduling).  
6.1 
Familiarization Session:  
• Subjects will undergo a standard physical examination that includes answering 
questions pertaining to their medical history to rule out any exclusion criterion.  
 
• Subjects assigned female at birth with no history of surgical sterilization, including 
but not limited to hysterectomy, tubal ligation, or oophorectomy, must have a 
negative pregnancy test at screening prior to enrollment. 
 
• Subjects will also be asked to fill out the Tegner Activity Level Scale to obtain a 
measure of their physical activity.  
 
• Subjects will be introduced to each of the exercise machines and will be taught how 
to properly use the equipment by a study team member. Subjects will be instructed on 
a seated leg extension machine, a semi-reclined leg press machine, and a prone 
hamstring curl machine.  
 
• The Delfi BFR tourniquet system will be introduced to the subject. Subjects will 
become familiarized with the pressure that the tourniquet system will produce on their 
legs during the experimental testing session.  
 
•  The study team will find the one repetition maximum (1-RM) for each exercise 
(Figure 1). This process will be performed by the physical therapist trained in the use 
of the exercise equipment. The resistance of each exercise will be modified as needed 
until the physical therapist finds the suitable amount of weight required for the 
subject to be able to perform one and only one repetition. The progression for 
obtaining a 1-RM weight will be repeated for each exercise (seated leg extension, 
semi-reclined leg press, prone hamstring curl.) 
 

 
 
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• The study team will answer any questions the subject may have about any of the 
exercises or the Delfi tourniquet system.  
 
 
Figure 1. Protocol for determining 1-RM.  
 
 
 
Warm Up: 5-min stationary bike
Warm Up: 8-10 repetitions with 50% of 
estimated 1-RM
1 Minute Rest
1 Repetition with 80% of estimated 1-
RM
3 Minute Rest
Increase weight 20% and re-attempt 1-
RM
•Continue to increase weight and attempt 1-RM 
with 3 minute rest periods until failure

 
 
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6.2 
Experimental Testing Sessions (12 Total): 
• Subjects will be asked to refrain from consumption of alcohol and caffeine for 12 
hours before testing and strenuous exercise 24 hours prior to testing.  
 
• 
Subjects will be advised to avoid the consumption of non-steroidal anti-inflammatory 
drugs (NSAIDS) in response to muscle soreness. NSAIDS include but are not limited 
to ibuprofen, high dose aspirin, naproxen, and diclofenac. 
 
• Data will be collected upon presentation for testing and the final experimental testing 
session. Data collected will include height, weight, and seated blood pressure. 
 
• At the first, sixth, and twelfth experimental testing sessions, subjects will then have 6 
cc of blood collected via forearm/arm vein access once 15 minutes of rest in the 
sitting position have elapsed. The first sample will be used to establish a pre-BFR 
complete blood count (CBC) with differential and flow cytometry to analyze the 
concentration of HPCs and MSCs (if available) in the blood.  
 
• During all experimental testing visits, subjects will, under the supervision of an 
individual trained in BFR, undergo a BFR session. The Delfi Personalized Tourniquet 
System (PTS) (Delfi Medical Innovations Inc., Vancouver, BC) is the BFR system 
that will be utilized. Bilateral proximal thigh bands will be applied and inflated to a 
pressure of 80% of occlusive pressure as determined by the automated tourniquet. 
The three exercises (seated leg extension, semi-reclined leg press, prone hamstring 
curl) will be formatted with 4 sets of 30-15-15-15 repetitions per exercise. The 
resistance will be set as 30% of one repetition maximum (1-RM).  
 
• At the first, sixth, and twelfth experimental testing sessions, after the BFR session 
concludes, additional 6 cc blood draws will be obtained immediately post-exercise 
and after 20-minutes have elapsed. Subjects will be offered the insertion of a 
peripheral intravenous blood draw or multiple percutaneous blood draws. The blood 
collected post-BFR will also be analyzed using a CBC with differential and flow 
cytometry.  
 
• Following all experimental testing sessions, blood samples will be taken for lactate 
testing. These samples will be collected prior to the exercise session, immediately 
after exercise, 10 and 20-minute time points post-exercise and analyzed via a portable 
lactate analyzer. To acquire the blood samples, a finger will be cleaned with an 
alcohol swab and a single-use lancet will be used to puncture the finger for blood 
testing. Both sides of the puncture site will be gently pressed to develop a drop of 

 
 
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blood. Care will be taken to avoid a vigorous squeeze. The first drop of blood will be 
wiped off using a sterile cotton swab as it may contain interstitial fluid. When the 
second drop of blood develops, the test strip with the lactate meter will be touched to 
the blood drop until the lactate meter beeps. Different testing sites (fingers) will be 
used for each lactate test. 
All samples will be handled under Universal Precautions. Samples will be stored and 
disposed of according to site standard operating procedures. 
 
6.3 Final Session: 
• The final session will take place no sooner than six weeks after the first experimental 
session and within five days of the twelfth and final experimental session.  
 
• Within the seventh week following completion of the familiarization session, subjects 
will be brought in for a blood draw post-BFR. Subjects will be asked to refrain from 
strenuous exercise 24 hours leading up to the final session. Subjects will also be 
asked to refrain from the consumption of alcohol and caffeine for 12 hours before the 
blood draw.  
 
• Subjects will be advised to avoid the consumption of non-steroidal anti-inflammatory 
drugs (NSAIDS) in response to muscle soreness. NSAIDS include but are not limited 
to ibuprofen, high dose aspirin, naproxen, and diclofenac. 
 
• Data will be collected upon presentation for the blood draw. Data collected will 
include height, weight, and seated blood pressure. 
 
• Subjects will then have 6 cc of blood collected via forearm/arm vein access once 15 
minutes of rest in the sitting position have elapsed. This sample will be utilized to 
obtain a post-BFR CBC with differential and flow cytometry to evaluate the 
concentration of HPCs and MSCs (if available) within the blood.  
 
All samples will be handled under Universal Precautions. Samples will be stored and disposed 
of according to site standard operating procedures.  
7 Review of Safety 
7.1 
 Adverse Event (AE) 
An adverse event is any untoward or unfavorable medical occurrence in the human 
subject, including any abnormal sign, symptom, or disease, temporally associated with 

 
 
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the subject’s participation in the research, whether considered related to the subject’s 
participation in the research. 
Subjects must agree to notify the investigator or study team member of potential or 
confirmed pregnancy, either of which may result in being withdrawn from the study or 
having limited participation in the study.  Subjects who become pregnant while in the 
study will be followed for AEs/SAEs that may occur for up to 30 days following the birth 
of the child. 
7.2 
 Serious Adverse Event (SAE) 
Serious adverse events are any events that: 
• Result in death 
• Is life threatening, or places the subject at immediate risk of death from the event 
as it occurred 
• Requires or prolongs hospitalization 
• Causes persistent or significant disability or incapacity 
• Results in congenital anomalies or birth defects 
• Is another condition which investigators just to represent significant hazards 
7.3 
 Unanticipated Problem (UP): 
Defined by DHHS 45 CFR part 46 as any incident, experience, or outcome that meets 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 study population.  
• related or possibly related to participation in the research (in this guidance 
document, 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). 
• suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized. 
7.4 
 AE & SAE Collection and Reporting 
Throughout the study the research team will monitor the occurrence of AE and SAE. 
Data will be collected if an instance occurs, and the PI will be notified. All AE data, such 

 
 
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as onset date, resolution date, outcome and treatments given will be documented in the 
source documents and will be recorded in the Electronic Data Capture (EDC) and 
analyzed for severity to follow reporting protocol if severity level. 
Follow-up will occur using the provided safety monitoring form if AE occurs. The follow 
up will end either when the symptoms resolve or up to 30 days past the end of the study 
participation. 
7.5 
 Risks and Discomforts 
This study includes the risks of blood draws, mild exercise, and vascular occlusion. The 
risks associated with the taking of blood include pain, bruising at the point where the 
blood is taken, redness and swelling of the vein and infection, and a risk of fainting 
although rare. These risks will be mitigated via utilization of standard universal 
precautions.  During the exercise, the following may occur abnormal blood pressure, 
fainting, dizziness, disorders of heart rhythm, and in very rare cases heart attack, stroke, 
or even death. Bodily injury is also a possible risk including, but not limited to, injuries to 
the muscles, ligaments, tendons, and joints of the body. Exercise could also cause fatigue 
and soreness of the muscles. The risk of vascular occlusion is discomfort. The 
aforementioned risks are minimized by conducting the proper assessments before 
exercise and having properly trained staff. With the implementation of the Delfi BFR 
tourniquet, nerve conduction deficits or pinching of the superficial skin may occur. 
However, these are transient effects and can be mitigated with the usage of a proper 
fitting cuff placed in the correct location and an adequate amount of pressure.  
8 Data Management Procedures 
All personal information is strictly confidential, and no names will be disclosed except as 
required by law. All information and data collected during this research will be recorded 
in spreadsheeting source and within the EDC. Records related to this study will be 
securely retained in a secure location for a period of 3 years after the completion of the 
study or longer as required by law. At that time, all records will be properly destroyed.  
Data will be collected using the EDC system. Reports of data will be used by internal site 
monitors to ensure accuracy of data elements. 
9 Data Analysis 
All participant data will be entered into the Electronic Data Capture (EDC) system. The 
study team will meet at appropriate intervals to evaluate and analyze the data. All 
compiled data will be de-identified.  A combination of Excel and Statistical Analysis 
System (SAS) Studio (Version 3.8 on SAS 9.4, SAS Institute Inc., Cary, NC) will be 

 
 
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used. Outcome measures will be those from pre and post exercise blood samples 
collected according to the study procedures (Section 5). 
Outcome measures will be as follows: 
Complete Blood Counts with differential: Red Blood Cell Count, White Blood Cell 
Count with differential, Total Nucleated Cell Count (number of nucleated cells per 
milliliter (mL)), Mononuclear Cell Count (number of mononuclear cells per mL), and 
Platelet Count (number of platelets per mL). 
Flow Cytometry: Counts including cluster differentiation (CD) markers for HPCs and 
MSCs (if available).  
Lactate Pro 2 Portable Lactate Analyzer (COSMED USA Inc., Concord, CA) Blood 
lactate level.  
10 
Statistical Considerations  
Descriptive statistics will be compiled for all numeral measures. Analysis will be 
performed with a repeated-measures ANOVA and post-hoc Tukey’s Honestly Significant 
Difference (HSD) for each numerical outcome variable. Significance will be set a priori 
at p < 0.05. With an alpha level of 5%, a beta of 10%, and an effect size of 200%, we 
calculated that a sample size of 10 is sufficient. Sufficient power has been confirmed on 
previous mobilization studies. The sample size of this study has been increased to 25 to 
account for potential subject withdrawal.  
11 
Quality Control and Assurance  
All protocols will be monitored and analyzed data will be checked for accuracy by the 
principal investigator and/or a designated AREF study team member. All medical data 
will be kept in compliance with HIPAA guidelines.   
 
 
 

 
 
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