A Single Center, Prospective Study to Compare the Quality and Quantity of the Cellular Content of M-PRP Harvested After Peripheral Mobilization of Progenitor Cells Using Filgrastim Versus Pegfilgrastim
Conditions: Healthy
Sponsor: Andrews Research & Education Foundation

Version: 2.0  
Version Date: 20Jan2022 
Title: A single center, prospective study to compare the quality and quantity of the cellular 
content of M-PRP harvested after peripheral mobilization of progenitor cells using filgrastim 
versus pegfilgrastim. 
 
 
Principal Investigator:  
Adam Anz, M.D.  
 
 
Co-Investigators:  
 
Steve Jordan, M.D., Roger Ostrander, M.D. 
 
 
Funding Mechanism:   
The State of Florida Grant  
 
 
 
 
 
Institution(s):   
 
Andrews Institute 
 
 
 
 
1020 Gulf Breeze Pkwy. 
 
 
 
 
Gulf Breeze, FL 32561 
 
 
 
 
(850) 916- 8590 
 
 
 
 
 
 

Version: 2.0  
Version Date: 20Jan2022 
 
 
Table of Contents 
1 
Background / Scientific Rationale ......................................................................................................... 5 
1.1 Introduction .......................................................................................................................................... 5 
1.1.1. ............................................... Background on Filgrastim and Pegfilgrastim and the Use of Biosimilars
 
5 
1.1.2 Indication, Dosage and Safety of Filgrastim and Pegfilgrastim ............................................................. 6 
1.2 Rationale for the study .......................................................................................................................... 7 
2 
Protocol Design ..................................................................................................................................... 8 
3 
Objectives.............................................................................................................................................. 8 
3.1 Primary Objective ................................................................................................................................ 8 
3.2 Primary Effectiveness Endpoint .......................................................................................................... 8 
4. Hypotheses................................................................................................................................................ 9 
4.1Null Hypothesis: ................................................................................................................................... 9 
4.2 Alternative Hypothesis: ....................................................................................................................... 9 
5  
Treatment Plan ..................................................................................................................................... 9 
5.1 Participant Eligibility ........................................................................................................................... 9 
5.2 Inclusion Criteria: ................................................................................................................................ 9 
5.3 Exclusion criteria: ................................................................................................................................ 9 
6 
Participant Enrollment ........................................................................................................................ 10 
6.1 Screening Process: ............................................................................................................................ 10 
6.2 Informed Consent 21 CFR 50 ............................................................................................................ 11 
6.3 Consent Withdrawal: ........................................................................................................................ 11 
6.4 Benefits: ............................................................................................................................................ 11 
6.5 Compensation: .................................................................................................................................. 11 
7 
Study Procedures ................................................................................................................................ 11 
7.1 Screening Visit: .................................................................................................................................. 11 
7.2 Filgrastim Treatment Plan: ................................................................................................................ 12 
7.3 Pegfilgrastim Treatment Plan ........................................................................................................... 12 
8 
Review of Safety .................................................................................................................................. 12 
8.1 Adverse Event (AE) ............................................................................................................................ 13 

Version: 2.0  
Version Date: 20Jan2022 
8.2 Serious Adverse Event (SAE) ............................................................................................................. 13 
8.3 Unanticipated Problem (UP): ............................................................................................................ 13 
8.4 AE & SAE Collection and Reporting ................................................................................................... 13 
8.5 Risks and Discomforts: ...................................................................................................................... 14 
8.6 Safety Monitoring Plan: .................................................................................................................... 15 
9 
Data Analysis & Management Procedures ......................................................................................... 16 
9.1 Data Collection .................................................................................................................................. 16 
9.2 Statistical Analysis ............................................................................................................................. 16 
9.3 
Statistical Considerations ............................................................................................................ 16 
10  Quality Control and Assurance ........................................................................................................... 16 
11 Regulatory Requirements ................................................................................................................... 16 
11.1 21 CFR 50- Informed Consent: ........................................................................................................ 16 
11.2 Participant Confidentiality: ............................................................................................................. 17 
11.3 Consent Withdrawal: ...................................................................................................................... 17 
12 References .......................................................................................................................................... 17 
 
 
 
 
 
 
 
 
 
 
 
 

Version: 2.0  
Version Date: 20Jan2022 
 
 
 
 
Abbreviations and Definitions 
AE 
Adverse Event 
ACDA Anti-Coagulant Citrate Dextrose A 
BMA Bone Marrow Aspirate 
cBMA Concentrated Bone Marrow Aspirate 
CBC 
Comprehensive Metabolic Panel 
Cc 
Cubic Centimeter 
CRF 
Case report Form 
EDC 
Electronic Data Capture  
GCP 
Good Clinical Practice 
G-CSF Granulocyte Colony‐Stimulating Factor 
HEENT Head, Eyes, Ears, Nose, and Throat 
examination 
HIPAA Health Insurance Portability and 
Accountability Act 
H0 
Null Hypothesis 
H1 
Alternative Hypothesis 
HPCs  Hematopoietic Stem Cells  
INR 
International Normalized Ratio 
IRB 
Institutional Review Board 
Kg 
Kilograms 
M-PRP Mobilized Platelet-Rich Plasma 
MPCs  Mesenchymal Progenitor Cells  
Mg 
Milligrams 
mL 
milliliters 
mm 
Millimeters 
PI 
Principal Investigator 
PB 
Peripheral blood 
PEG 
Polyethylene glycol 
PPP      Platelet-Poor Plasma 
PRP 
Platelet-Rich Plasma 
PT 
Prothrombin Time 
PTT 
Partial Thromboplastin Time 
QA 
Quality Assurance 
SAE 
Severe Adverse Event 
SOC 
Standard of Care 
SOP 
Standard Operating Procedure 
WBC White Blood Count 

A single center, prospective study to compare the quality and quantity of the cellular content of M-PRP 
harvested after peripheral mobilization of progenitor cells using filgrastim versus pegfilgrastim.  
Adam Anz, M.D. 
Page 5 of 20 
 
 
 
 
1 
Background / Scientific Rationale 
1.1 Introduction 
Musculoskeletal injuries are a significant public health concern globally, contributing a large 
burden of disability and suffering.[1] Progenitor/stem cell therapies have shown great 
potential for addressing acute, traumatic and chronic orthopedic injury. [2] Orthopedic 
surgeons have started to augment surgical procedures and treat degenerative conditions such 
as osteoarthritis, with injections of bone marrow aspirate (BMA) and platelet rich plasma 
(PRP). [3-7] Several clinical application studies have concluded that the success of the 
intervention is dependent upon the number of progenitor cells harvested and utilized. While 
bone marrow aspiration is frequently utilized for orthopedic applications, the number of cells 
harvested is variable and dependent on several factors including individual, aspiration 
technique and location of harvest. The most efficient method involves rapid, small-volume, 
and multiple aspirations from multiple locations on the iliac crest.[8, 9] In addition, bone 
marrow aspiration is an invasive procedure requiring more clinical resources and logistics 
demands. [10] As a result, orthopedic studies have begun to investigate different methods and 
locations for progenitor cells for orthopedic purposes including injury effusion fluids and 
arthroscopic by products [11], adipose tissues [12], synovial fluid [13, 14], subacromial 
bursal cells [15], and mobilized peripheral blood cells. [7, 16, 17]  
Bone marrow (BM) contains a variety of stem/progenitor cells, including hematopoietic 
progenitor cells (HPCs), endothelial progenitor cells (EPCs), and mesenchymal progenitor 
cells (MPCs). [18] CD34 is a common membrane marker for HPCs and studies have shown 
very low level of circulating CD34+ cells (10-100 CD34+ cells/ml) in PB under 
physiological steady-state conditions [19-21]. A mobilization regime is needed to effectively 
shift progenitor cells from BM niche to PB. Various interleukins, the chemokines, or the 
hematopoietic growth factors have shown to increase mobilization of progenitor cells with 
various efficiencies and kinetics. [22, 23]  In this study, filgrastim and pegfilgrastim will be 
used as HPCs mobilizing agents.  
1.1.1. Background on Filgrastim and Pegfilgrastim and the Use of Biosimilars 
Filgrastim is a human granulocyte colony-stimulating factor (G-CSF) protein with a molecular 
weight of approximately 19 kilodaltons (kD) and consists of 175 amino acids. Filgrastim is 
obtained from the bacterial fermentation of a strain of E. coli. transformed with a genetically 
engineered plasmid containing the human G-CSF gene. Pegfilgrastim Fulphila® is a long-
acting 
covalent 
conjugate 
of 
recombinant 
methionyl 
human 
filgrastim, 
and 
monomethoxypolyethylene glycol (PEG). To produce pegfilgrastim, a 20 kD PEG molecule is 
covalently bound to the N-terminal methionyl residue of filgrastim. The average molecular 
weight of pegfilgrastim is approximately 39 kD. Attachment of PEG increases the size of 
filgrastim as a result it becomes too large for renal clearance. Consequently, neutrophil-

A single center, prospective study to compare the quality and quantity of the cellular content of M-PRP 
harvested after peripheral mobilization of progenitor cells using filgrastim versus pegfilgrastim.  
Adam Anz, M.D. 
Page 6 of 20 
 
 
 
 
mediated clearance predominates in elimination of the drug. A single injection of pegfilgrastim 
produces increased serum concentrations to 42 hours, compared with between 3.5 and 3.8 hours 
for multiday administration of filgrastim [24, 25].  
Granix® (tbo-filgrastim), an FDA-approved biosimilar of filgrastim, will be used for the 
filgrastim mobilization (see appendix A). Fulphila® (pegfilgrastim-jmdb), an FDA-approved 
biosimilar of pegfilgrastim, will be used for the pegfilgrastim mobilization (see appendix B). 
Biosimilars are biological products that have no clinically meaningful differences from a 
reference product [26]. Biosimilars are approved by the FDA after rigorous evaluation and 
testing by the applicant [26]. The above biosimilars were selected by the study investigator and 
a consulting board-certified oncologist based on the similarity of indication, risk/benefit ratio, 
previous clinical testing, and general availability. 
1.1.2 Indication, Dosage and Safety of Filgrastim and Pegfilgrastim 
Filgrastim and Pegfilgrastim are indicated to increase survival in patients acutely exposed to 
myelosuppressive doses of radiation and to decrease the incidence of infection in patients with 
non-myeloid malignancies receiving myelosuppressive anti-cancer drugs associated with a 
clinically significant incidence of febrile neutropenia.  
Previous work has determined that the optimal dosage of filgrastim for cell mobilization is 10 
mcg/kg/day for four or more consecutive days which is inconvenient for the patients [27].  
According to the FDA label, patients undergoing autologous peripheral blood progenitor cell 
collection and therapy 10 mcg/kg/day subcutaneous injection of filgrastim is administered for 
at least 4 days before first leukapheresis procedure. For pegfilgrastim according to the FDA 
label, 6 mg of pegfilgrastim is administered subcutaneously once per chemotherapy cycle to 
patients with cancer receiving myelosuppressive chemotherapy. The above suggested dosage 
will be used in the study. Pegfilgrastim may be preferred over filgrastim, due to its convenient 
once-per-cycle subcutaneous administration as compared to daily administration of filgrastim 
for at least 4 days. 
Several clinical studies have shown filgrastim and pegfilgrastim to be safe and effective for 
mobilizing peripheral blood progenitor cells in patients and healthy donor. [28-31] Both the 
pharmaceutical agents increase the production of progenitor cells in the bone marrow leading 
to increased release and circulation of progenitor cells in the peripheral blood [24, 32, 33]. The 
long-term safety of filgrastim mobilization has been reported with bone pain as a common 
adverse effect and no association with neoplastic risks identified. [27, 34-37] In one study 
involving 126 cancer patients, 44% reported mild-to-moderate muscle and bone pain and 7% 
reported headaches.[33] The Spanish National Donor Registry reports that out of 736 donors, 
90% reported bone pain and 33% had headaches.[34]  In preclinical and clinical studies, 
pegfilgrastim has been shown to have comparable efficacy and safety profile to filgrastim with 
pegfilgrastim only requiring one injection to achieve the same effect as multiple day injections 
of filgrastim. [24, 38-42] Pegfilgrastim is comparable to filgrastim for mobilization of 

A single center, prospective study to compare the quality and quantity of the cellular content of M-PRP 
harvested after peripheral mobilization of progenitor cells using filgrastim versus pegfilgrastim.  
Adam Anz, M.D. 
Page 7 of 20 
 
 
 
 
peripheral blood progenitor cells as shown by CD34+ (marker for hemopoietic stem cells) cells. 
[43, 44] A single dose of pegfilgrastim was able to mobilize a sufficient number of CD34+ in a 
multiple myeloma patient not responsive to two previous attempts with high or standard dose 
chemotherapy followed by filgrastim.[45]   
1.2 Rationale for the study 
Mainly the previous study of mobilization of progenitor cells to PB is focused on hematologic 
oncologic clinical practice of hematopoietic stem cell transplant. Pharmaceutical mobilization 
by filgrastim, followed by peripheral harvest of cells with apheresis is used more commonly 
than bone marrow aspirate for the hematologic oncologic clinical practice of hematopoietic 
stem cell transplant [46].  These previous studies have evaluated the potential of filgrastim and 
pegfilgrastim to improve apheresis harvest. However, combining pegfilgrastim mobilization 
with peripheral blood harvest and concentration with a PRP device has not been fully evaluated. 
The PI recently completed a study on developing M-PRP product created by peripheral blood 
mobilization of progenitor cells with filgrastim and processing with Arthrex Angel system and 
comparing it with concentrated bone marrow aspirate (cBMA).  The most significant finding 
of this study was that the M-PRP product contained a higher concentration of platelets and 
monocytes when compared to cBMA. As expected, M-PRP had significantly greater WBC, 
monocytes, and HPCs compared to PRP alone. This study found overall a similar cellular 
product with an easier harvest with M-PRP when compared to cBMA [47].  
The multiday administration of filgrastim is inconvenient for the patients; therefore, the PI in 
this study aims to utilize pegfilgrastim as a mobilizing agent for creating M-PRP product in 
one subcutaneous administration. In this study the M-PRP product created by either 
pegfilgrastim or filgrastim will be evaluated in vitro or bench top. The PI hypothesizes that 
both of the pharmaceutical mobilization agents would result in similar M-PRP product. It is 
proposed that the progenitor cells isolated in M-PRP would be of similar property to cultured 
bone marrow derived cells (when cultured in hypoxia), with established multi-potentiality and 
differentiation potential to the mesodermal lineage. Previous studies have shown that 
circulating cells in PB are multipotent and possess differentiation potential. [16, 48, 49] Due to 
convenience, pegfilgrastim is a more practical mobilizing agent; thus, could be preferred in 
creating potent point of care M-PRP product for application in orthopedic medicine.  
In this study, in conjunction with the mobilizing agent, the M-PRP processing requires a 
concentrating PRP system, Arthrex Angel system.  The Arthrex Angel system uses 
centrifugation and optics to more precisely separate cell types using buffy coat method. All 
cells have a density range and non-uniformly after centrifugation. Arthrex Angel system has 
the unique ability to isolate specific cells using inherent properties of cells that absorb differing 
wavelengths of light. The settings on the system can be controlled to adjust the proportion of 
cells versus plasma. Increasing the setting from 7% to 15%, the Angel system isolates more 
cells from a deeper portion of the buffy coat, which results in capturing more HPCs per volume. 

A single center, prospective study to compare the quality and quantity of the cellular content of M-PRP 
harvested after peripheral mobilization of progenitor cells using filgrastim versus pegfilgrastim.  
Adam Anz, M.D. 
Page 8 of 20 
 
 
 
 
Also, the volume of the cPRP can be increased with autologous PPP, which consists of growth 
factors and other beneficial proteins. The Arthrex Angel system combined with peripheral 
mobilization could be used as point of care method for creating effective M-PRP product. 
(Device pamphlet, Appendix H) 
2 Protocol Design 
The proposed study is a prospective, randomized controlled, single-center laboratory study 
involving 10 healthy volunteers. Once the potential participant has cleared the screening, consented 
to the study procedures, completed the medical interview, and laboratory blood testing, the subject 
will undergo two serial mobilization events.  The scheduling of the mobilization events will be 
varied across the 10 participants to counter sequencing effects of the mobilization events. 5 heathy 
donors will be administered standard filgrastim mobilization regimen of 10 mcg/kg per day for 4 
days will be followed by a standard pegfilgrastim mobilization regimen consisting of one 6 mg 
injection separated by 8 weeks for 5 of the participants. The other 5 healthy donors will receive the 
reverse order of the pharmaceutical agent, first pegfilgrastim followed by filgrastim. 
On the first day of the study, a first blood draw of 130 mL will be performed which will be used to 
create standard PRP for laboratory testing and subjects will begin a filgrastim or pegfilgrastim 
dosage series. After the specified time (4 days for filgrastim and 7 days for pegfilgrastim), a second 
130 mL of blood will be harvested and processed with the Arthrex Angel system to create M-PRP 
for laboratory testing.  The standard PRP and M-PRP cellular content will be studied and quantified 
in vitro with cell counting, cell culturing and protein analysis. 8 weeks after the second blood 
harvest, the subjects will return for a third 130 mL of blood draw, followed by administration of a 
second mobilizing agent (pegfilgrastim or filgrastim). After the specified time (4 days for filgrastim 
and 7 days for pegfilgrastim), the patients will return for a fourth blood draw of 130mL. The sample 
will be processed with the Arthrex Angel system to create M-PRP for laboratory testing. The 
cellular content of the M-PRP product will be studied and quantified in vitro with cell counting, 
cell culturing and protein analysis.  Thereafter, the cellular content of M-PRP product will be 
compared between filgrastim and pegfilgrastim mobilization agents.   
3 
Objectives 
3.1 Primary Objective 
The objective of this study is to compare and quantify the cellular content (in vitro) of the mobilized 
M-PRP products harvested after a standard four-day filgrastim mobilization regimen versus one-
day pegfilgrastim regimen when combined with Arthrex Angel System. This study would be useful 
to further develop optimized point of care orthopedic product in future.  
 3.2 Primary Effectiveness Endpoint 
The Automated hematological analysis will be performed on all M-PRP products to quantify total 
red blood cell (RBC), white blood cell (WBC), monocyte, platelet, and hematopoietic progenitor 

A single center, prospective study to compare the quality and quantity of the cellular content of M-PRP 
harvested after peripheral mobilization of progenitor cells using filgrastim versus pegfilgrastim.  
Adam Anz, M.D. 
Page 9 of 20 
 
 
 
 
cell (HPC) concentration. The cell count of the M-PRP products will be performed and cells will 
be cultured and characterized under different differentiation conditions. The protein analysis of 
the M-PRP will be carried out via ELISA and/or flow cytometry. 
 
4. Hypotheses 
 
4.1Null Hypothesis:  
H0-Particpants will have not a similar cellular content (in vitro) of the mobilized M-PRP 
products harvested after a standard when comparing a 4-day filgrastim mobilizing treatment 
to a one-day pegfilgrastim mobilizing treatment. 
 
4.2 Alternative Hypothesis:  
H1- Participants will have a similar cellular content (in vitro) of the mobilized M-PRP products 
harvested after a standard when comparing a 4-day filgrastim mobilizing treatment to a 
one-day pegfilgrastim mobilizing treatment. 
5  
Treatment Plan 
5.1 Participant Eligibility 
The proposed study is a controlled laboratory study involving 10 healthy male volunteers 
between the ages of 19 and 39 years with weight 50-100 kg.  The age was limited because the 
cellular components of PRP have been found to be dependent on these variables. [50, 51]  The 
weight was restricted due to the logistics of pre-filled syringes and the cost of Filgrastim. Female 
volunteers will be excluded to avoid the potential of any fetal risks from administration of 
filgrastim during pregnancy. The general exclusion criteria were based on associated risk factors 
with mobilizing agents. Subjects will be selected using the following inclusion and exclusion 
criteria: 
5.2 Inclusion Criteria:  
1. Healthy Male 19-39 of age and weight 50-100 kg  
2. Subject consents to coming 5 serial days for filgrastim treatment and additional blood 
draw and then 8 weeks later two addition visits for pegfilgrastim treatment and 7 days 
later blood draws. The above order of administration will be provided to 5 participants 
and the additional 5 will receive pegfilgrastim treatment first followed by filgrastim to 
counter sequencing effects.  
5.3 Exclusion criteria: 
1. Female 
2. Weight < 50kg or  > 100kg 

A single center, prospective study to compare the quality and quantity of the cellular content of M-PRP 
harvested after peripheral mobilization of progenitor cells using filgrastim versus pegfilgrastim.  
Adam Anz, M.D. 
Page 10 of 20 
 
 
 
 
3. Previous allergic reaction to filgrastim, PEG, lidocaine, latex, acrylic or any other 
injectable numbing agent  
4. History of Diabetes 
5. Abdominal tenderness to palpation 
6. Unclear lung fields on physical exam 
7. Splenomegaly 
8. Significant cardiovascular, renal, hepatic, or pulmonary disease 
9. WBC over 20,000/mcL upon initial screening CBC 
10. Blood disorders, autoimmune disorders, disorders requiring immunosuppression, 
cancer, an ongoing infectious disease, or sickle cell or other blood disorders. 
6 
Participant Enrollment 
10 healthy male participants will be recruited through the Andrews Institute physician practices via word 
of mouth and utilizing a recruitment flyer (see appendix F) at the Andrews Institute campus. Potential 
participants will be prescreened for inclusion and exclusion criteria through standard of care medical 
evaluations. Participants meeting the inclusion criteria will have the study explained to them by one of the 
members of the investigating team, and they will be given an opportunity to participate if they are 
interested. Once a potential participant has agreed to be involved in the study, they will go through the 
described informed consent process.  
 
6.1 Screening Process: 
Once interested participants are identified and prescreened, a screening visit will be scheduled. 
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. 
Once the screening requirements are met, the informed consent will be provided to ensure the 
volunteer understands the details of the study including the benefits and risk factors. 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.  After the volunteer has consented to participate in the 
study, a medical interview, and a blood test will be administered (CBC, CMP) will be conducted.  
The medical interview will include a complete physical examination and review of the blood test 
to ensure that no exclusion conditions exist.  Physical examination will include height/weight, 
head/neck, cardiovascular, lung, and abdominal examinations. Standard vitals will be taken.  

A single center, prospective study to compare the quality and quantity of the cellular content of M-PRP 
harvested after peripheral mobilization of progenitor cells using filgrastim versus pegfilgrastim.  
Adam Anz, M.D. 
Page 11 of 20 
 
 
 
 
6.2 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 staff, in the 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 participant.  The purpose and methods of the study 
along with the expected effects will be reviewed with each potential participant. Each participant 
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, 
participants will be given a copy for their records.  
The designee will review with each participant that they are free to refuse to the study or to 
withdraw from it at any time. 
6.3 Consent Withdrawal: 
During the informed consent procedure, participants will be informed that if at any point during 
the study, consent may be withdrawn. To withdraw consent, participants can request 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 participant’s health information will 
be severed with the research team. This process for consent withdrawal will be reviewed with 
each participant and identified barriers will be addressed at the time of informed consent.  
6.4 Benefits:  
There is no direct benefit to participants participating in this study. This study may help direct 
clinical practice in alternative method to extract stem cells that can be used to augment surgical 
procedures. 
6.5 Compensation: 
Compensation will be provided to participants on a pre-set schedule. A $50.00 dollar stipend 
will be provided to participants on the first day of receiving the mobilization agents. Each 
subsequent scheduled study treatment visit will award the participant a $75.00 stipend until 
the total of $500.00 is met. 
7 
Study Procedures 
7.1 Screening Visit: 
Upon obtaining consent from the participant, the Research Team will complete the screening 
process along with the screening form. The subject will be provided the filgrastim and 
pegfilgrastim label handout (Appendix A & B) to continue to best practices. The Research Team 
will obtain history of the participant to check inclusion and exclusion criteria. If not excluded, the 
study physician will complete another physical examination including height/weight, head/neck, 
cardiovascular, lung, and abdominal examinations. Standard vitals will be taken. The blood draw 
will be completed (CBC, CMP)  and sent to the lab. Once labs are reviewed and are within the 

A single center, prospective study to compare the quality and quantity of the cellular content of M-PRP 
harvested after peripheral mobilization of progenitor cells using filgrastim versus pegfilgrastim.  
Adam Anz, M.D. 
Page 12 of 20 
 
 
 
 
acceptable limits, WBC <20k and coagulation studies are normal, the subject may be scheduled 
for the initial mobilizing regimen which may take place on the same day as screening.  
7.2 Filgrastim Treatment Plan: 
During the collection/treatment visit, the physician will complete another physical examination 
including height/weight, head/neck, cardiovascular, lung, and abdominal examinations. Standard 
vitals will be taken. A standard venipuncture will be performed on the left or right upper 
extremity.  Before the treatment 130.0 ml of blood will be drawn and processed with the Arthrex 
Angel® System (Arthrex, Inc, Naples, FL USA).  The PRP will be analyzed for cellular 
components total cell count, cell growth and protein (chemokine/cytokine) analysis. .  
After the blood draw, the volunteers will undergo 4 consecutive days of mobilization with 
filgrastim.  The 10 mcg/kg dose of filgrastim will be administered to the volunteers 
subcutaneously into the thigh.  Dosages will be rounded to 300 mcg, 600 mcg, 780 mcg, 840 
mcg. Appendix A, a filgrastim information handout will be given after filgrastim administration. 
After 4 doses of the filgrastim, 130.0 mL of blood will be drawn and will be processed with the 
Arthrex Angel system.  The M-PRP samples will be analyzed for cellular components total cell 
count, cell growth and protein (chemokine/cytokine) analysis.  The safety of the volunteer will be 
followed up via a phone call at 7 day and 28 days after mobilized blood collection.   
 
There will be 8 weeks interval between the two treatments.  
7.3 Pegfilgrastim Treatment Plan 
During the pegfilgrastim visit, the physician will complete another physical examination 
including height/weight, head/neck, cardiovascular, lung, and abdominal examinations. Standard 
vitals will be taken. A standard venipuncture will be performed on the left or right upper 
extremity.  Before the treatment 130.0 ml of blood will be drawn and processed with the Arthrex 
Angel® System (Arthrex, Inc, Naples, FL USA).The PRP samples will be analyzed for cellular 
components total cell count, cell growth and protein (chemokine/cytokine) analysis. After the 
blood draw, the volunteers will undergo 1 injection of pegfilgrastim.  The 6mg dose will be 
administered to the volunteers subcutaneously into the thigh. Appendix B, a pegfilgrastim 
information handout will be given after pegfilgrastim administration.   
One week after the pegfilgrastim administration, another 130 mL blood draw will occur by a 
standard venipuncture on the left or right upper extremity. The blood will be processed with the 
Arthrex Angel system to generate M-PRP.  The M-PRP samples will be analyzed for cellular 
components total cell count, cell growth and protein (chemokine/cytokine) analysis.  The safety 
of the volunteer will be followed up via a phone call at 7 day and 28 days after mobilized blood 
collection.   
8 
Review of Safety  

A single center, prospective study to compare the quality and quantity of the cellular content of M-PRP 
harvested after peripheral mobilization of progenitor cells using filgrastim versus pegfilgrastim.  
Adam Anz, M.D. 
Page 13 of 20 
 
 
 
 
8.1 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 the subject’s 
participation in the research, whether considered related to the subject’s participation in the 
research. 
8.2 Serious Adverse Event (SAE) 
Serious adheres events are any events that: 
• 
Result in death 
• 
Is life threatening, or places the participant 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 
8.3 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 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 participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized. 
8.4 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 
as onset date, resolution date, outcome and treatments given will be documented in the 
source documents and will be recorded in the 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. 

A single center, prospective study to compare the quality and quantity of the cellular content of M-PRP 
harvested after peripheral mobilization of progenitor cells using filgrastim versus pegfilgrastim.  
Adam Anz, M.D. 
Page 14 of 20 
 
 
 
 
8.5 Risks and Discomforts: 
 As with any research involving participants, there is the inherent risk of a breach in 
patient confidentiality though this will be minimized using participant code numbers and 
adherence to all HIPAA guidelines. Standard sterile precautions will be utilized for the 
administration of filgrastim and pegfilgrastim, but with any procedure there is risk of 
infection, bleeding, and swelling.  
Filgrastim is administered subcutaneously via a single prefilled syringe for manual use or 
for use with the On-body Injector for filgrastim which is co-packaged with a single 
prefilled syringe. 
 
Anticipated Discomforts of Filgrastim 
Aching in the bones and muscles 
 
Possible Serious Adverse Events (SAE) of Filgrastim 
Splenic rupture 
Serious lung problem called acute respiratory distress syndrome (ARDS). 
Serious allergic reaction 
Sickle cell crises 
Kidney injury (glomerulonephritis).  
Capillary leak syndrome. 
 
Additional response to these adverse events can be found in Appendix B. 
Anticipated Discomforts of Pegfilgrastim 
Aching in the bones and muscles 
 
Possible Serious Adverse Events (SAE) of Pegfilgrastim 

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Splenic rupture.  
Serious lung problem called acute respiratory distress syndrome (ARDS). 
Serious allergic reaction. 
Glomerulonephritis 
Leukocytosis 
Thrombocytopenia 
Aortitis  
Capillary leak syndrome. 
 
Contraindications to the use of Filgrastim and pegfilgrastim include patients with a history of 
serious allergic reactions to human granulocyte colony-stimulating factors such as Filgrastim or 
Pegfilgrastim. Most common adverse reactions in patients undergoing peripheral blood progenitor 
cell mobilization and collection (≥ 5% incidence) are bone pain, pyrexia (fever) and headache. 
Although extremely rare, cases of splenic rupture, acute respiratory distress syndrome, serious 
allergic reactions, fatal sickle cell crisis, capillary leak syndrome, cutaneous vasculitis, and 
glomerulonephritis have been reported in patients undergoing treatment with Filgrastim and 
pegfilgrastim. For this reason, patients will be monitored with a review of symptoms and a 
physical examination upon each visit, including ascertaining blood pressure and pulse, as well as a 
lung, heart, and abdomen examination.  
The approximate duration of the study will be a 10 week period. During this 10 week period a total 
of four blood draws will occur with the amount of 130 mLs each draw with a total amount of 520 
mL of blood drawn. To review the safety of this practice, a comparison between the acceptable 
blood donation practice was reviewed. For whole blood donation, one pint (~473.16 mL) of blood 
is drawn in one sitting with the acceptable inter-response time of 56 days. During the duration of 
this entire study, a total of 520 mL of blood will be drawn over the 10 week duration, lowering the 
risk of the blood draws significantly less than the risks with blood draw regimes.  
8.6 Safety Monitoring Plan:   
To ensure close monitoring and support of the participants due to the use of filgrastim, a safety and 
data monitoring plan will be implemented.  The participant safety monitoring plan will include 
follow up via a phone call at 7 day and 28 days after mobilized blood collection.  At this time, 
patients will be asked an open-ended question by the research team: “How has your general health 
been and did you find the study uncomfortable?” Responses will be summarized in Appendix C 

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“Monitoring Form.”   Expected side effects include bone pains and muscle aches.  Any unexpected 
severe adverse events will be documented and reported to the PI and the IRB notified.    
9 
Data Analysis & 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.  
Laboratory data will include CBC with differentials, characterization of cell content and results 
from ELISA testing. All data will by organized by an anonymous identifier and will not be linked 
or identifiable to the study participants.  
9.1 Data Collection 
Data will be collected using the EDC system. Reports of data will be used by internal site 
monitors to ensure accuracy of data elements.  
9.2 Statistical Analysis 
All patient data will be entered into EDC. The investigators will meet at appropriate intervals to 
evaluate and analyze the data. All compiled data will be de-identified. The numbers of cells will 
be determined per microliter of total volume collected. Cytokine levels will be determined 
according to volume collected. 
9.3 
Statistical Considerations 
Calculations of standard metrics including means, ranges, and standard deviations will be 
performed.  
10  
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 research team member. All medical data will be kept in 
compliance with HIPAA guidelines. 
11 
Regulatory Requirements 
 
11.1 21 CFR 50- Informed Consent: 
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 staff, in the office. No aspects of 
the study will be conducted prior to obtaining informed consent from each participant.  The 
purpose and methods of the study along with the expected effects will be reviewed with each 
potential participant. Each participant will be provided a copy of the consent and sufficient time 

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will be given for the opportunity to read and ask any questions about the study.  After signing of 
the informed consent document, participants will be given a copy for their records.  
The designee will review with each participant that they are free to refuse to the study or to 
withdraw from it at any time.  
 
11.2 Participant Confidentiality: 
Signed consent forms for each subject will be deidentified by a coding system with the subject’s 
unique study identification system.  Authorization to use each subject’s personal health 
information will be obtained during the informed consent procedure to adhere to the federal 
Health Insurance Portability and Accountability Act (HIPAA). The consent will specifically grant 
permission to use health information obtained as part of the presented study. 
11.3 Consent Withdrawal: 
During the informed consent procedure, participants will be informed that if at any point during 
the study consent may be withdrawn. To withdraw consent participants can request 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 participant’s health information will 
be severed with the research team. This process for consent withdrawal will be reviewed with 
each participant and identified barriers will be addressed.  
12 
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