A Phase I/II Clinical Trial To Determine Safety And Feasibility Of Using An Acellular Amniotic Fluid Application To Expedite Healing In Chronic Wounds
Phase: PHASE1
Conditions: Wounds and Injuries
Sponsor: University of Utah

Study Title 
A PHASE I/II CLINICAL TRIAL TO DETERMINE 
SAFETY AND FEASIBILITY OF USING AN 
ACELLULAR AMNIOTIC FLUID APPLICATION TO 
EXPEDITE HEALING IN CHRONIC WOUNDS 
ClinicalTrials.gov ID (NCT Number) 
NCT04438174 
Principal InvesƟgator (PI) 
Giavonni Lewis, MD 
Document (ICF, Protocol, SAP) 
Protocol 
Update Date (Approval Date) 
February 2, 2022 
 

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TITLE:  A PHASE I/II CLINICAL TRIAL TO DETERMINE SAFETY AND 
FEASIBILITY OF USING AN ACELLULAR AMNIOTIC FLUID 
APPLICATION TO EXPEDITE HEALING IN CHRONIC WOUNDS  
 
 
 
Principal Investigator 
 
Giavonni Lewis, MD 
University of Utah Health 
Burn Center 
50 N Medical Dr 
Salt Lake City, UT 84132 
giavonni.lewis@hsc.utah.edu  
 
Sub-Investigators 
 
Crystal Webb, PA-C 
University of Utah Health 
Burn Center  
50 N Medical Dr. 
crystal.webb@hsc.utah.edu 
 
Molly Baily, PA-C 
University of Utah Health 
Burn Center  
50 N Medical Dr. 
molly.baily@hsc.utah.edu 
 
Kathleen Ewanowski, PA-C 
University of Utah Health 
Burn Center  
50 N Medical Dr. 
kathleen.ewanowski@hsc.utah.edu 
 
Callie Thompson, MD 
University of Utah Health 
Burn Center  
50 N Medical Dr. 
Callie.thompson@hsc.utah.edu  
 
Irma Flemming, MD 
University of Utah Health 
Burn Center  
50 N Medical Dr. 
Irma.flemming@hsc.utah.edu 
 
West Hunsaker, PA/C 

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University of Utah Health 
Burn Center  
50 N Medical Dr. 
West.hunsaker@hsc.utah.edu 
 
David Souza, PA/C 
University of Utah Health 
Burn Center  
50 N Medical Dr. 
David.souza@hsc.utah.edu 
 
Mindy Orr, DMP 
University of Utah Health 
Burn Center  
50 N Medical Dr. 
Mindy.orr@hsc.utah.edu 
 
 
 
 
 
 
 
John Phillips, PhD 
Division of Hematology and 
Hematologic Malignancies 
675 Arapeen Drive, Suite 300 
Salt Lake City, Utah 84108 
 
john.phillips@hsc.utah.edu 
 
Jan Pierce, MBA 
Cell Therapy & Regenerative Medicine 
University of Utah Health  
675 Arapeen Drive, Suite 300 
Salt Lake City, Utah 84108 
jan.pierce@hsc.utah.edu 
 
 
 
 
 
 
 
 
 
 
 
 

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Protocol Version: 5.00, dated 02FEB2022 
 
1- SYNOPSIS 
 
Title 
A phase I/II clinical trial to determine safety and 
feasibility of using an acellular human amniotic fluid 
(pAF) application to expedite healing of chronic 
wounds 
Short Title 
pAF for the Treatment of Chronic Wounds 
IRB Number 
IRB_00128708 
IND 
IND # 19330 
Phase 
Phase I/II 
Design 
This is a prospective randomized controlled study to 
determine the safety and feasibility of using processed 
sterile filtered amniotic fluid (pAF) to expedite 
healing in chronic wounds. 
Study Duration 
4 years 
Study Center(s) 
University of Utah  
Objectives 
Primary Objective: 
• To determine the safety of using processed 
amniotic fluid (pAF) to treat patients with chronic 
wounds.  
Secondary Objective: 
• To determine efficacy by examining a reduction in 
wound size after application of pAF compared to 
Standard of Care (SOC). 
Number of Subjects 
60 patients 
Diagnosis and Main Eligibility Criteria Inclusion: 
1. Patients 18-85 years old. 
2. Patients with chronic upper and/or lower 
extremity wounds (including thermal) that are 
greater than 3 months, but less than 12 months 
old. 
3. Patients with full thickness wounds. 
4. Patients with at least one wound that is ≥5 cm2 
and<75 cm2 in size. 
5. Patient who is able to complete required site study 
visits and procedures in good faith 
 
Exclusion: 
1. Patients admitted to the hospital at the time of 
enrollment. 
2. Patients who are pregnant, nursing or plan to 
become pregnant while participating in the study. 
If of child-bearing potential, unwillingness to use 
effective birth control while participating in the 

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study. 
3. Suspicion of or diagnosis of osteomyelitis 
underlying the wound. 
4. Patients who have received an investigational 
agent or intervention within the prior 30 days or 
plan to use within the study period. 
5. Patient with sinus tracts, enterocutaneous fistulas 
or other epithelialized tracts. 
6. Patients who require skin grafting. 
7. Patients diagnosed with a highly disruptive, non-
controlled mental health disorder (e.g.., bipolar, or 
schizophrenia.  
8. Patients with a history of prior drug abuse. 
Study Product, Dose, Route, Regimen 
pAF will be delivered to the clinic on the date of 
application. 
Dose is 1ml/5cm2; Route: injected directly into 
wound; Limited to two injections. 
Statistical Methodology 
The primary goal of the analysis is to demonstrate 
safety, which does not require statistical significance. 
 
The secondary goal of the analysis is to assess 
efficacy by comparing percent reduction in wound 
area. Additional exploratory analyses will be 
conducted, such as subgroup analyses.  
 
 
2- OBJECTIVES 
 
-Primary Objective: 
• The Burn Outpatient Clinic sees multiple types of wounds from burn to non-burn.  
The care of patients with chronic wounds is complex and difficult.  To date, we 
have not found any treatments, including various dressings or other stem cell 
based therapies, which consistently reduce time of wound closure and improve 
pain. We would like to investigate the safety of using pAF to treat these wounds. 
 
-Secondary Objective: 
• We would like to determine efficacy of using pAF to treat chronic wounds by 
examining a reduction in wound size after application of pAF compared to 
Standard of Care (SOC). 
 
3- ENDPOINTS 
 
-Primary Endpoint: 
• Whether the patient experienced any post-randomization, study-related SAEs 
while on study (up to Visit 5, defined later).  

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-Secondary Endpoint: 
• The percent reduction in wound area at Visit 5 relative to the size at randomization 
visit.  
 
4- BACKGROUND AND RATIONALE 
 
The Burn Outpatient Clinic patient demographic includes 7-10% non-burn wounds per 
month. These wounds range from chronic venous stasis ulcers, pressure ulcers, diabetic foot 
ulcers, to rare genetically predisposed wounds. These wounds are slow to heal, they require 
large amounts of health care resources, and cause disability for the patient. We have yet to 
find a standard treatment modality or wound care regimen that consistently lessens time to 
wound closure, improves pain, and reduces scarring. 
 
 
Human amniotic fluid and amniotic membrane have been used for many years in 
regenerative medicine. Literature review shows that amniotic fluid with the cellular 
components included 1, 2 and an additional study using granulized amniotic membrane with 
amniotic fluid have shown improved wound healing rates in select populations 3.  Amniotic 
fluid contains cytokines, nutrients, and growth factors that are required for development of 
the fetus. Components of the amniotic fluid that make it important to wound healing include 
its anti-inflammatory properties (38%), immune regulation (25%) and anti-microbial 
properties (9%) 4. 
 
While amniotic membranes have been used for over 100 years in burns and other chronic 
wounds, amniotic fluid has not been rigorously studied.  With the continuous rise of 
healthcare costs, price is another factor to consider with amniotic fluid and membranes. It is 
reported that the US spends more than 25 billion dollars annually on chronic wounds 5. This 
is on the rise due to the aging of the population and increases in obesity and diabetes. In an 
article titled " Wound Care Outcomes and Associated Costs Among Patients Treated in US 
Outpatient Wound Centers: Data from the US Wound Registry", it was estimated that the 
mean healing cost per wound ranged from $3,927-$9,358 depending on the type of wound 6. 
In other words, on average 2.6 - 6.3 million people require treatment for chronic wounds 
annually, a heavy toll for national healthcare costs. 
 
The goal of our study is to assess, in an outpatient setting, the safety and healing in chronic 
wounds treated with pAF.  We will include patients with upper or lower extremity full 
thickness wounds (including thermal) that are greater than 3 months old and less than 12 
months old and who are able to complete required site study visits and procedures in good 
faith.  The wound size must be greater than or equal to 5cm2 and less than 75 cm2. We will 
exclude anyone that is admitted to the hospital at the time of enrollment, is less than 18 years 
old or older than 85 years old, is known to be pregnant or planning to become pregnant 
(including women of childbearing potential who are unwilling to use effective birth control 
while participating in the study), is breastfeeding, has suspicion of osteomyelitis, has 
received or plans to receive an investigational agent or intervention,  requires skin grafting, 
has wounds with sinus tracts, has a diagnosed highly disruptive uncontrolled mental health 
disorder, or has a known history of prior drug abuse. Our primary objective is to determine 

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the safety of using pAF to treat patients with chronic wounds and our secondary objective is 
to assess the reduction in wound size after application of pAF compared to standard of care. 
 
Cases #1 & 2 show preliminary results that we have obtained with pAF applications when 
applied to refractory chronic wounds (>12 months without healing).  
 
Case #1 
 
A patient with a Martorell’s ulcer with tendon exposure received 4 injections of pAF (Fig. 
3A).  The first injection consisted of 8 mL of pAF (Fig. 3B).   
 
 
 
Approximately, 1 month later, the patient received a second injection of 9 mL of pAF (Fig. 
3C).   Subsequent injections of 9 mL of pAF were given at approximately 2 and 4 months 
after the initial injection of pAF (Fig. D & E).    Four months after the first pAF injection, 
there was no tendon exposure and re-epithelialization of the wound was apparent.    
 
Case #2 
 
A patient was involved in a bombing terrorist attack and sustained the following injuries: a 
35% TBSA burn to the left leg, right leg, right hand, and posterior scalp; traumatic 
pseudoaneurysm of left popliteal artery; left fibular fracture; and left calcaneal fracture. He 
developed multiple infections of the left lower leg and heel including mucormycosis 
infection.  He eventually healed however, 1 year from his injury and definitive treatment, a 
heel and Achilles non-healing post-traumatic ulcer persisted. He underwent traditional local 

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wound care strategies and peripheral vascular evaluation.  The ulcers were open for greater 
than 1 year without evidence of healing.       
                                           
 
He underwent eight amniotic fluid treatments within 1 year (Fig. 4).   His wound was 
completely healed after seven treatments within 9 months (Fig. 4D).  Unfortunately, he 
underwent another orthopedic procedure with prolonged casting of the left leg and suffered 
breakdown of the heel ulcer (Fig. 4E) requiring subsequent amniotic fluid injection to 
complete healing (Fig. 4F).  
 
Amniotic fluid (AF): Preclinical Data 
 
Early after conception and until the mother’s water breaks for the delivery of their infant, the 
fetus is bathed in amniotic fluid. AF functions as a supportive cushion to the fetus and 
provides a protective environment. AF is a rich source of nutrients, cytokines and growth 
factors that are required for fetal development and maturation 7. AF also contains stem cells 
with the potential to differentiate along multiple cell lineages 8, 9. The protective and 
regenerative properties of AF are achieved via the exchange of water and solutes with 
surrounding tissues. This is accomplished via the utilization of different pathways during the 
course of a pregnancy that likely contribute to changes in the composition of the AF with 
gestational age7.   

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Early reports showing that concentrates of AF inhibit the development of peritonitis were 
among some of the first evidence that AF has protective biological properties 10.  This was 
followed by a publication by Shimberg and co-workers that AF accelerates defense-repair 
mechanisms within damaged joints 10, 11.  Since these early publications, more sophisticated 
evaluations have revealed the presence of antimicrobial, immunomodulatory, and growth-
promoting activities in AF 7, some of which are likely to contribute to its protective 
biological properties.   
 
Reports about the antimicrobial activity of AF differ among investigators 12.  It is reported 
that AF with low antimicrobial activity is associated with a high incidence of an infectious 
syndrome in pregnant women 13.   Some studies show that AF is inhibitory, while others 
show no effect against the same microorganisms.  Components with antimicrobial, antiviral 
and antifungal activity that are present in AF include lysozyme, peroxidase, transferrin,       
β-lysin, immunoglobulins and zinc-peptide complexes 12.  
 
Immunomodulatory properties of AF are evident from studies showing that enteral feeding of 
AF suppresses the pro-inflammatory responses in preterm pigs with necrotizing enterocolitis 
14.   Growth promoting activities of AF are supported by animal studies as well as by in vitro 
studies.  These studies show that AF can enhance neochondrogenesis 15, regenerate 
peripheral nerves 16 and bone 17, accelerate re-epithelialization in corneas 18, and promote 
healing of human skin wounds 19. Some of the factors that are found in AF that may 
contribute to these activities could be inflammatory mediators that include, but are not 
limited to TNF-a, IL-6, IL8, and IL-10 20, trophic factors that include EGF, IGF-1, FGF, 
HGF and TGF-a 21-25, and HA (hyaluronic acid), an important factor in promoting re- 
epithelialization in human skin wounds 19.  
 
Based on the hypothesis that nutrients, cytokines and growth factors contained in the non-
cellular fraction of AF are useful for reparative and regenerative treatments in patients, Pierce 
et al. conducted a study at the University of Utah to address three issues 4. The first was to 
determine the feasibility of consenting and screening volunteer donors for the routine 
collection of AF from full-term pregnant women who were scheduled for Caesarean sections 
(C-sections), and to then process the AF for clinical application. The second aim was to 
develop a processing method that resulted in a cell-free AF preparation suitable for clinical 
application. The third goal was to gain a better understanding of the components of the 
processed AF (pAF).  
 
Goal #1: AF was collected by the staff of the Department of Obstetrics/Gynecology at the 
University of Utah hospital and was processed by technical staff of the Cell Therapy and 
Regenerative Medicine (CTRM) facility at the University of Utah.   Physician-executed 
abdominal incisions were performed through the abdominal and uterine muscles without 
cutting into the amnion membrane. Using a sterile suction catheter connected to a sterile 
MediVac suction container (Cardinal Health, Waukegan, IL), a blunt end insertion with the 
catheter was made through the amniotic membrane and the AF aseptically suctioned into the 
MediVac container. The container was labeled, wrapped in frozen Insul-ice mats (Fisher 
Scientific, Hanover Park, IL) and placed in a temperature-monitored shipper that was 

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validated for transport between 2- 8 °C.  The AF was transported to the CTRM facility at the 
University of Utah. Upon arrival at the CTRM facility, the tissue was immediately placed 
into a refrigerator at 2–8 °C until processing occurred.  
 
Goal #2:  At the time of processing, the MediVac container with AF was aseptically placed 
in a biological safety cabinet and the AF was aseptically transferred into sterile centrifuge 
tubes. The total volume and gross appearance of the AF were recorded and samples removed 
for sterility testing, cell counts and other relevant testing. The AF was centrifuged, the 
supernatant was collected, and the AF underwent a proprietary filtration process to eliminate 
insoluble components and sterilize the AF.   The resulting product, processed AF (pAF), was 
evaluated for total volume, fluid chemistry, total protein, and hyaluronic acid (HA) levels. 
Final pAF products were also assessed for their cellular content and protein profiles using 
quantitative antibody arrays.  
 
To validate the above described approach for collecting (Goal #1) and processing (Goal #2) 
AF, 36 pregnant women consented and passed the donor screening criteria.  AF was 
successfully collected and processed from 17 of these individuals. Median AF volumes were 
70 mL (range 10–815 mL; n = 17) 4. 
 
Goal #3: Fluid chemistries were similar among the 17 pAFs, but some differences were noted 
in HA levels and cytokine profiles. Cytokine arrays revealed that an average of 304 ± 20 
ng/mL (mean ±SD; n=3) of 400 proteins tested were present in pAF, with a majority of 
cytokines associated with host defense. Some of the peptides encountered and classified 
according to their function are found in table 1.  
 
Experience with using pAF in Clinical Settings:  
pAF manufactured at the University of Utah has been clinically used in over 2000 
applications for over 100 different conditions.  A majority of treatments have been for 
wounds and burns.  No unexpected and related adverse events have been directly associated 
with the injection or topical application of pAF in these previous uses (Table 2). 

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ELIGIBILITY CRITERIA 
Inclusion Criteria: 
1. Patients who are 18-85 years old 
2. Patients with chronic upper and/ or lower extremity wounds (including thermal) that 
are greater than 3 months, but less than 12 months old 
3. Patients with full thickness wounds 
4. Patients with at least one wound that is ≥5 cm2 and <75 cm2 in size  
5. Patient who is able to complete required site study visits and procedures in good faith. 
 
Exclusion Criteria: 
1. Patients admitted to the hospital at the time of enrollment  
2. Patients who are pregnant, nursing or plan to become pregnant while participating in 
the study. If of child-bearing potential, unwillingness to use effective birth control 
while participating in the study.  
3. Suspicion of or diagnosis of osteomyelitis underlying the wound. 
4. Patients who have received an investigational agent or intervention within the prior 
30 days or plan to use within the study period. 
5. Patient with sinus tracts, enterocutaneous fistulas or other epithelialized tracts 
6. Patients who require skin grafting 
7. Patients diagnosed with highly disruptive, non-controlled mental health disorder (e.g. 
bipolar, or schizophrenia.  
8. Patients with a history of prior drug abuse 
 
 
5- STUDY DESIGN AND PROCEDURES 
 
A written Informed Consent will be obtained from all subjects before any study-related 
procedures are executed.   Subjects will be randomized into two groups at a 1:1 ratio. The 
randomization will employ random block sizes to limit the ability to predict the next subject’s 

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treatment prior to randomization. Because of the random block sizes, it is not guaranteed that 
exactly 30 subjects will be randomized to each arm. There may be as few as 28 in the 
treatment arm (32 in the control arm) or as many as 32 in the treatment arm (28 in the control 
arm). Because of the modest number of overall patients and the anticipated slow accrual rate, 
stratification will not be used in the randomization procedure. 
 
If a subject has more than one qualifying wound, the largest wound will be randomized to one 
of the two study arms (pAF or SOC) and included in the primary and secondary endpoint 
analyses. All other wounds will be treated with standard of care.  
 
Whether a wound is randomized to pAF or SOC, many of the components of the treatment 
regimen are the same. The overall treatment is described, with differences unique to the pAF 
arm emphasized. 
 
• Study Overview:  
a. Visit 1: 
i. Screening of subject and wounds for eligibility 
ii. Informed consent 
iii.  Pregnancy Test (only applicable to women of childbearing potential) 
iv. Subject/Wound evaluation and assessment including measurements 
(includes local cleansing and debridement if needed, digital image 
capture, manual measurements, and maximal wound depth) 
v. Wound-associated pain assessment 
vi. Laboratory tests: 
1. Parvovirus B19 serology and nucleic acid testing 
2. Complete blood count including platelet count with white 
blood cell (WBC) differential 
3. Chemistry panel to include aminotransferases, serum creatinine  
4. Hemoglobin A1c 
5. HLA Type 1 antibody (Only done on the first 10 patients 
enrolled) 
6. PT/INR 
7. PTT 
8. Wound cultures 
vii. Standard Burn Center wound care treatment 
viii. Schedule subject’s next visit to occur within 6 weeks (+/- 
1 week) of consent 
 
If a subject’s wound reduces in size greater than or equal to 50% between Visit 1 and Visit 2, 
or if the subject received any out-of-study investigational agent/intervention to the wound 
after Visit 1 but before Visit 2, the subject will not be randomized into the study and will be 
excluded. Because such subjects are not to be randomized, they are not considered enrolled 
and do not count towards the target enrollment of 60 subjects. 
 
 
b. Visit 2 (6 weeks +/- 1 week from Visit 1): 

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i. Subject/Wound evaluation and assessment including measurements 
(includes local cleansing and debridement if needed, digital image 
capture and upload to the Burn Unit image storage, manual 
measurements, and maximal wound depth) 
ii. Wound-associated pain assessment 
iii. Wound cultures 
iv. Determination of whether largest qualifying wound is 
eligible for randomization (see note following Visit 5) 
v. If applicable, randomization of largest qualifying wound to 
pAF or SOC arm 
 
If in pAF arm, 
1. The amount of processed amniotic fluid to be injected 
into the randomized wound will be calculated based 
on the area of the wound (1ml/5cm2). 
2. To assist with discomfort, topical or injectable 
anesthetic will be applied to the wound based on 
standard dosing recommendations. Pliaglis (Lidocaine 
7%, tetracaine 7%; emulsion cream) is the topical 
anesthetic applied 30-45 min prior to procedure in the 
clinic setting. If this is insufficient we will inject 1% 
lidocaine directly into the study site. All dosing is 
based on weight based recommendations in adults. 
Some patients may not need any of the above and 
some may require all. 
3. The wound will be cleansed and debrided if 
needed prior to application of pAF and then 
prepped with chloraprep. 
4. pAF is thawed and drawn into a 10ml syringe and, 
using a 25g needle, injected into the wound. 
5. pAF is injected at the skin-wound base interface 
and base of the wound in a grid pattern 
(approximately 0.1 ml of pAF spaced evenly apart 
throughout the wound). 
6. Standard burn center wound treatment regimen is 
applied to the wound per standard dressing care 
procedures (see Standard Burn Center Wound 
Treatment Regimen below). 
vi. Standard Burn Center wound care treatment of all other 
wounds (if any), including the wounds randomized to 
standard of care. 
vii. Schedule subject’s next visit to occur 6 weeks (+/-1 week) 
later. 
viii. Subject will receive a follow-up phone call within 24 
hours AND 5-7 days after visit to identify any adverse 
events that occur in relation to study participation and 

14 
 
record additional medications that were taken. 
 
c. Visit 3 (6 weeks +/- 1 week from Visit 2):  
i. Subject/Wound evaluation and assessment including measurements 
(includes local cleansing and debridement if needed, digital image 
capture and upload to the Burn Unit image storage, manual 
measurements, and  maximal wound depth) 
ii. Wound-associated pain assessment 
iii. Wound cultures 
 
If in pAF Arm 
1. The amount of processed amniotic fluid to be injected 
into the randomized wound will be calculated based 
on the area of the wound (1ml/5cm2). 
2. To assist with discomfort, topical or injectable 
anesthetic will be applied to the wound based on 
standard dosing recommendations. Pliaglis (Lidocaine 
7%, tetracaine 7%; emulsion cream) is the topical 
anesthetic applied 30-45 min prior to procedure in the 
clinic setting. If this is insufficient we will inject 1% 
lidocaine directly into the study site. All dosing is 
based on weight based recommendations in adults. 
Some patients may not need any of the above and 
some may require all. 
3. The wound will be cleansed and debrided if 
needed prior to application of pAF and then 
prepped with chloraprep. 
4. pAF is thawed and drawn into a 10ml syringe and, 
using a 25g needle, injected into the wound. 
5. pAF is injected at the skin-wound base interface 
and base of the wound in a grid pattern 
(approximately 0.1 ml of pAF spaced evenly apart 
throughout the wound). 
6. Standard burn center wound treatment regimen is 
applied to the wound per our standard dressing care 
procedures (see Standard Burn Center Wound 
Treatment Regimen below). 
 
iv. Standard Burn Center wound care treatment of all other 
wounds if any, including the wounds randomized to 
standard of care 
v. Collect information on adverse events related to study 
participation. 
vi. Schedule subject’s next visit to occur 6 weeks (+/-1 
week) later. 
vii. Subject will receive a follow-up phone call within 24 

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hours AND 5-7 days after visit to identify any adverse 
events that occur in relation to study participation and 
record additional medications that were taken.  
 
d. Visit 4 (6 weeks +/-1 week from Visit 3):  
i. Subject/Wound evaluation and assessment including measurements 
(includes local cleansing and debridement if needed, digital image 
capture and upload to the Burn Unit image storage, manual 
measurements, maximal wound depth) 
ii. Wound-associated pain assessment 
iii. If the wound(s) remains open, Standard Burn Center wound care 
treatment 
iv. Laboratory tests: 
1. Parvovirus B19 serology and nucleic acid testing for subjects testing 
negative for Parvovirus at Visit 1.   
2. Complete blood count including platelet count with WBC 
differential 
3. Chemistry panel to include aminotransferases, serum creatinine 
4. Hemoglobin A1C 
5. HLA Type 1 antibody (Only done on the first 10 patients 
enrolled) 
6. PT/INR 
7. PTT 
8. Wound cultures 
v. Collect information on adverse events related to study participation. 
vi. Schedule the final study visit, 3 months (+/- 1 week) later. 
 
e. Visit 5 (3 months +/-1 week from Visit 4): 
i. Subject/Wound evaluation and assessment including measurements  
(includes local cleansing and debridement if needed, digital image 
capture and upload to the Burn Unit image storage, manual 
measurements, and maximal wound depth). This assessment will 
include status of the wound and if it has closed since Visit 2, wound 
recurrence and hypertrophic scar formation (measured with modified 
Vancouver Scar Scale).  
ii. Wound-associated pain assessment 
iii. If the wound(s) remains open, Standard Burn Center wound care 
treatment 
iv. Collect information on adverse events related to study 
participation. 
v. Wound cultures 
Standard Burn Center Wound Treatment Regimen 
 
For all groups, change dressing as directed. 

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 
Primary dressings are variable and based on the moisture content, microorganism load 
and perception of infection.  Wound cultures will be taken at every visit. In general, 
wounds respond differently to various topical treatments.  Through our clinical practice, 
we have found that wounds plateau with the same topical for greater than 4 weeks, hence 
changing antimicrobial topical helps to manage the bacterial overgrowth.  Dressings for 
both the control group and treatment group include ointment-based dressing and non-
ointment-based dressings consisting of silver product. We will start with our application 
of our slurry, a 1:1:1 ratio of Nystatin ointment, Mupirocin Ointment, and Bacitracin 
Ointment.  This slurry will be applied directly to the cleansed wound, followed by silver 
gauze and/ or foam product to all wounds. Types of silver product, site and comfort 
predict use of Restore, Mepilex-AG, or Mepitel-AG.  If allergies to the above slurry 
occurs, we will use medical honey with or without bacitracin.  If ointment related rashes 
present we will transition to silver product only or silver product plus medical honey. 
 
Secondary dressing: gauze and ace wrap are used to protect wound from shear and 
desiccation. 
 
If a patient is allergic to any of the above dressings, alternative dressings will be supplied 
per standard wound care practice in our clinic.  
Table 3.  PATIENT STUDY CALENDAR: 
 
 
Visit 1 (Screening) 
Visit 2     (6 
weeks +/- 
1 week 
from last 
visit) 
Visit 3     (6 
weeks +/- 
1 week 
from last 
visit) 
Visit 4        (6 
weeks +/- 1 
week from 
last visit) 
Visit 5     (3 
months +/- 
1 week 
from last 
visit) 
Informed Consent 
X 
 
 
 
 
Eligibility 
X 
 
 
 
 
Medical History 
X 
 
 
 
 
Vitals (VAS, HR, BP, RR, O2 
Sat, Weight, Height) 
X 
X 
X 
X 
X 
Randomization 
 
X 
 
 
 
Physical Exam 
X 
X 
X 
X 
X 
Pregnancy Test  
X1 
 
 
 
 
Injection of pAF (1ml/5cm2)-
Treatment group 
 
X 
X 
 
 
Burn Center SOC 
X 
X 
X 
X 
X 
Manual Measurement 
X 
X 
X 
X 
X 
Pain Assessment 
X 
X 
X 
X 
X 
Laboratory Tests 
X 
 
 
X 
 
Wound Culture Swabs 
X 
X 
X 
X 
X 
Digital Image 
X 
X 
X 
X 
X 
Adverse Events Noted 
 
X 
X 
X 
X 
Wound assessment 
X 
X 
X 
X 
X 

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1Only applicable to women of childbearing potential 
2Follow-up phone call within 24 hours AND 5-7 days after visit to assess adverse events and 
medications. 
Applicable study procedures may be completed via telehealth or by phone, at the discretion of 
the treating provider. 
 
DRUG DISPENSATION AND RANDOMIZATION: 
Upon verification at Visit 2 that the patient’s wound is still eligible the patient’s largest 
wound will be randomized to the pAF arm or to the standard-of-care arm, and the CTRM 
facility will be notified.  If the wound is randomized to the pAF arm, the CTRM will package 
the pAF on dry ice and transport the pAF to the Burn Unit.  
 
The amniotic fluid or investigational product (IP) comes frozen in a nunc cryovial. The 
frozen IP is thawed at room temperature until the last visible ice crystal is dissolved.  
6- ASSESSMENTS  
ASSESSMENT OF RESPONSE:  
Assessment of response will be performed at every follow up visit.   
Wound assessment: 
Wound assessments will be performed over the course of 5 exams: Visit 1-screening exam, 
Visit 2, Visit 3, Visit 4 and Visit 5.  
Wound surface area will be calculated. Additionally, maximal length, width and depth of 
wound will also be measured. 
Digital images will be captured at each visit using our image capture software.  
The digital photography will be standardized as follows:  A ruler will be present in photographs 
taken at each study visit.  Photography will proceed using an iPad camera.  We will incorporate 
a standard background for all photographs to help standardize the iPad’s auto color balance. 
Photos will be taken in normal anatomical positions at a defined distance from the site of 
interest according to our standard operating procedure.   Typically, for best wound image 
capture flash or direct lights are contraindicated, hence normal clinic room lighting is 
sufficient.  .  
Wound digital images will be assessed and evaluated by a University Staff member or 
contracted professional blinded to both the control and treatment group. Length and width 
may have substantial variability.  Hence, ImageJ software is used to assess area of the wound 
after the perimeter of the wound is manually outlined (to a reasonable accuracy). It provides 
more objective assessment of the size of the wound, lending more validity to the wound 
measurements.   
Follow-up phone call to 
assess adverse events 
 
X2 
X2 
 
 

18 
 
ASSESSMENT OF SAFETY: 
Safety and tolerability will be evaluated by the PI and/ or trained physician from the results 
of reported signs and symptoms and scheduled physical examinations, for every follow-up 
visit. AEs will also be assessed by phone within 24 hours AND 5-7 days after Visit 2 and 
Visit 3 (injection visits). Unresolved AEs will be monitored for 1 year. More frequent safety 
evaluations may be performed if clinically indicated or at the discretion of the investigator. 
SAEs will be reported within 24 hours of discovery to the central Data Coordinating Center.  
 
7- SAFETY MONITORING 
Adverse Event Reporting 
The site investigator is responsible for evaluating all adverse events at their Clinical Center 
and ensuring that they are properly reported.  Adverse events that occur during this study will 
be recorded.  The nature of each experience, date and time (where appropriate) of onset, 
outcome, course, and relationship to treatment will be documented. 
Definition of Adverse Event and Serious Adverse Event 
Adverse Event (AE) means any untoward medical occurrence associated with the use of an 
intervention in humans, whether or not considered intervention-related (21 CFR 312.32 (a)).  
An event constitutes a disease, a set of related signs or symptoms, or a single sign or 
symptom. 
Serious Adverse Event (SAE): A serious adverse event (SAE) for this population is an 
adverse event that: 
• results in death; or 
• is life-threatening (the patient was, in the view of the site investigator, in immediate 
danger of death from the event as it occurred); or 
• requires inpatient hospitalization or prolongs an existing hospitalization; or 
• results in persistent or significant disability or incapacity; or 
• results in congenital anomaly/birth defect; or 
• is any other event that, based upon appropriate medical judgment, may jeopardize the 
subject's health and may require medical or surgical intervention to prevent one of the 
other outcomes listed in this definition. 
Classification of an Adverse Event (Relatedness, Severity, and Expectedness) 
Relatedness: The suspected relationship between study interventions and any adverse event 
will be determined by the site investigator using the criteria below.  Relatedness must be 
assessed by an investigator and may not be assessed by a research coordinator. 
• Not Related: The event is clearly related to other factors, such as the subject's clinical 
state, therapeutic interventions, or concomitant drugs administered to the subject. 

19 
 
• Possibly Related: The event follows compatible temporal sequence from the time of 
beginning the assigned study intervention, but could have been produced by other 
factors such as the subject's clinical state, therapeutic interventions, or concomitant 
drugs administered to the subject. 
• Probably Related: The event follows a reasonable temporal sequence from the time of 
beginning the assigned study intervention, and cannot be reasonably explained by 
other factors such as the subject's clinical state, therapeutic interventions, or 
concomitant drugs administered to the subject. 
Severity: The severity, which is a measure of intensity, of clinical adverse events and 
laboratory abnormalities will be recorded by the site investigator and categorized. The 
following guidelines will be used to describe severity. 
• Mild: The event requires minimal or no treatment and does not interfere with the 
participant’s daily activities. 
• Moderate: The event results in a low level of inconvenience or concern with the 
therapeutic measures.  Moderate events may cause some interference with functioning. 
• Severe: The event interrupts a participant’s usual daily activity and may require 
systemic drug therapy or other treatment.  Severe events are usually potentially life-
threatening or incapacitating.  Of note, the term ``severe'' does not necessarily equate 
to ``serious''. 
Expectedness of the Event: All adverse events, including serious adverse events, will be 
evaluated as to whether their occurrence was expected or unexpected. An AE will be 
considered unexpected if the nature, severity, or frequency of the event is not consistent 
with the risk information described for the study intervention. Expected adverse events for 
this study are:  
• Infection/ Cellulitis 
• Local bleeding 
• Purulent drainage 
• Inflammation 
• Rash  
Treatment or Action Taken: For each adverse event, the site investigator will record whether 
an intervention was required: 
 
• Medical or surgical procedure 
• Concomitant medication: started, changed, or discontinued 
• Other, specify 
• No action taken 
 
Outcome of Event: Finally, the site investigator will record the clinical outcome of each 
adverse event as follows: 
 

20 
 
• Death 
• Recovered and the patient returned to baseline status 
• Recovered with permanent sequelae 
• Symptoms persist 
 
Time Period for Adverse Events 
For purposes of this study, events that occur following participant consent through the last 
study visit will be reported as adverse events. Serious adverse events, unexpected medically 
attended events, and new onset chronic illnesses will be recorded from consent through Visit 
5 (or the last available status before Visit 5 if the subject does not complete Visit 5).   
 
Data Collection Procedures for Adverse Events 
After patient consent, all adverse events (including serious adverse events) will be recorded 
according to relatedness, severity, and expectedness, as well as their duration and any 
treatment prescribed. Any medical condition present at the time of consent, recorded in the 
patient's baseline history at study entry, which remains unchanged or improves (unless the 
clinician feels it is clinically relevant), will not be recorded as an adverse event at subsequent 
evaluations.  However, worsening of a medical condition that was present at the time of 
consent will be considered a new adverse event and reported. 
 
Adverse events will be coded using the MedDRA coding vocabulary. Coding will be done 
centrally at the Data Coordinating Center as this requires specific training. 
 
Unanticipated Problems (UP) 
Unanticipated problems (UP) are defined as incidents, experiences, or outcomes that are 
unexpected, related or possibly related to participation in the study, and suggest that the 
research places subjects at a greater risk of harm than was previously known or recognized. 
The site investigator will report unanticipated problems to the Data Coordinating Center 
within 24 hours of becoming aware of the event.  A detailed completed report will be 
required to be sent to the Data Coordinating Center within 3 working days of becoming 
aware  of the event. After receipt of the complete report, the Data Coordinating Center will 
report these unanticipated problems to the funding Program Official or Project Officer in an 
expedited manner (as close to 24 hours as possible). 
 
In accordance with local IRB requirements, the site investigator may be required to report 
such unanticipated problems to the IRB in addition to notifying the Data Coordinating 
Center. In the event that the medical monitor believes that such an event warrants emergent 
suspension of enrollment in the trial, and funding staff cannot be reached expeditiously, the 
Data Coordinating Center will notify the study investigator and all site investigators to cease 
enrollment in the trial. 
 
Monitoring Serious Adverse Events 
A qualified physician will be designated to fulfill the function of the medical monitor for this 
study. Site investigators and/or research coordinators will report serious adverse events to the 
Data Coordinating Center within 24 hours of becoming aware of the event. A detailed 
completed report will be required to be sent to the Data Coordinating Center within 3 

21 
 
working days of becoming aware of the event, and the medical monitor will assess all serious 
adverse events reported from site investigators. 
 
For each of these serious adverse events, the site investigator will provide sufficient medical 
history and clinical details for a safety assessment to be made with regard to continuation of 
the trial. The medical monitor will sign off on each SAE report after review.  All SAE reports 
will be retained at the Data Coordinating Center, and all SAE reports will be available for 
review by funding staff. The SAE reporting process may be incorporated into the Electronic 
Data Capture (EDC) System in use for the study. 
 
In the unlikely event that the medical monitor believes an unexpected and study-related SAE 
warrants emergent cessation of enrollment in the trial, funding staff will be immediately 
consulted.  If these individuals concur with the judgment of the medical monitor, or if the 
funding staff cannot be reached expeditiously, the Data Coordinating Center will notify the 
study investigator and all site investigators to cease enrollment in the trial.  Resumption of 
enrollment will not occur without consent of the funding staff. 
 
In accordance with local IRB requirements, the site investigator may be required to report 
such events to the IRB in addition to notifying the Data Coordinating Center. 
 
Follow-up of Serious, Unexpected and Related Adverse Events 
All serious, unexpected and related adverse events, that are unresolved at the time of the 
patient's termination from the study, will be followed by the site investigators until the events 
are resolved, subject is lost to follow-up, the adverse event is otherwise explained or has 
stabilized, or 12 months have passed from the time of last study dose. 
 
PATIENT WITHDRAWAL FROM STUDY: 
A subject may withdraw their consent at any time without effect to their care.  
 
DISCONTINUATION OF STUDY DRUG: 
pAF-specific treatment will be discontinued if any of the following occur:  
• Discovery of new information which makes the subject ineligible to continue 
participation in the study.  This could include systemic sepsis, disseminated infections 
requiring inpatient hospitalization, worsening infection of the wound, such as necrosis 
or surgical intervention, increase in wound size by 50% from Visit 2 measurement, 
development of osteomyelitis, etc. 
• Severe allergic reaction possibly related to the investigational product.  
• Patient receives any investigational agent/intervention to the wound outside the study 
visits or is non-compliant with care between Visits 2 and 3. 
• Any other condition that in the opinion of the treating physician are not compatible 
with participation in this study. 
 
The study PI, treating clinicians and research staff will monitor closely for the above events, 
as well as any additional adverse events that occur while the subject is receiving treatment. 
Adverse events will be reported immediately upon discovery of the event to the Data 
Coordinating Center and assessed for seriousness, relatedness, severity, and expectedness. 

22 
 
The DSMB will meet regularly to review adverse events, safety analyses and study data and 
will have access to the actual treatment assignments for each group.  
 
If study drug is discontinued, we will continue to follow the patient and monitor for safety 
and efficacy endpoints.  
 
DISCONTINUATION OF STUDY: 
The study will be discontinued:   
• If any subject has an adverse event leading to respiratory failure requiring 
resuscitation or death from the investigational product, 
• If two or more incidences in the pAF group of wounds require emergent surgical 
intervention or disseminated infections requiring hospitalization,  
• At the discretion of the Data Safety Monitoring Board (DSMB). The DSMB will 
meet regularly to review adverse events, safety analyses, and study data. The DSMB 
will have access to the actual treatment assignments for each group. 
 
8- STATISTICAL CONSIDERATIONS 
 
Statistical Method 
This is a modest feasibility study with subjects randomized to two groups:  (1) treatment 
group with pAF applied to the randomized wound (n=30 ± 2 patients expected), (2) control 
group, with wounds receiving standard of care treatment (n=30 ± 2 patients expected). 
 
Feasibility will be demonstrated if the study’s clinical wound care providers are able 
to successfully apply pAF to the wounds in this clinical study situation. Any issues 
or concerns that occur will be recorded and reported descriptively in a similar 
fashion to safety outcomes (described below). As no issues or concerns are 
expected, it should be clear from this descriptive analysis if feasibility of applying 
pAF is demonstrated. 
 
Primary Endpoint:  Whether the patient experienced any post-randomization, study-related 
SAEs while on study. 
 
For the definition of the primary endpoint, a serious adverse event (SAE) is considered 
study-related if the medical monitor concludes the SAE is either possibly related or probably 
related to study participation. 
 
Safety will be assessed by descriptive reporting of the incidence proportion of study-related 
serious adverse events in each study group, with one-sided 95% binomial exact confidence 
intervals. No statistical hypothesis test is planned to assess safety, which is consistent even 
with large Phase III trials. The table below displays the number of subjects, out of 30, 
experiencing no related SAE and the corresponding lower-bound of the 95% exact 
confidence interval.  
 

23 
 
Number of Subjects with no 
Study-related Serious 
Adverse Events 
Number of Subjects 
Experiencing any Study-
related Serious Adverse 
Events 
Lower-bound of the 95% 
Exact Binomial Confidence 
Interval for Safety 
30 
0 
90% 
29 
1 
85% 
28 
2 
80% 
27 
3 
76% 
 
If there are 30 subjects in the pAF group and if all subjects in the pAF group complete the 
study without experiencing any study-related SAEs, we will be able to state with 95% 
confidence that the safety of pAF is at least 90%. 
 
Exploratory outcomes for safety include, but are not limited to, the following: 
• 
Whether the patient experienced any post-randomization SAEs while on study 
(regardless of relatedness to study participation) 
• 
Whether the patient experienced any post-randomization related AEs while on 
study 
• 
Whether the patient experienced any post-randomization unexpected AEs while on 
study. 
 
Secondary Endpoint:  The percent reduction in wound area at Visit 5 relative to the size at 
randomization (Visit 2), per the imaging software assessments. 
 
The inclusion criteria limit the study to wounds which are difficult to heal, explaining why 
patients are not randomized if the wound area shrinks significantly between Visits 1 and 2. 
Therefore, greater reduction in wound size with pAF, than with SOC will suggest efficacy of 
pAF for wound treatment.  To describe a trend in a statistical hypothesis testing framework, 
the following two-sided hypothesis will be assessed: 
 
 
 
H0: μpAF = μSOC versus H1: μpAF ≠ μSOC 
  
 
 
The secondary outcome variable will be the reduction in wound size at Visit 5 relative to the 
Visit 2 wound size (i.e., 100% * (1- Visit 5 wound area/ Visit 2 wound area)), with all 
measurements expressed as the approximate area determined by the imaging software. 
Wound size should be measured at all study visits.  To provide an intuitive effect size, 
percentage change from Visit 2 will be computed at visits 3, 4, and 5 (approximately 6, 12, 
and 25 weeks after randomization) with the changes at visits 3 and 4 considered as 
exploratory outcomes.  If there are study visits outside of the visit’s +/- 1 week window, all 
percentages will be divided by the number of elapsed days since Visit 2, so the outcome is 
expressed as a percentage change per day.  If the change in wound size at Visit 5 follows a 
symmetric distribution a two-sample t-test will be performed, otherwise a Wilcoxon rank 
sum test will be used. As exploratory analyses, the prescribed analytic approach will be 
applied to the exploratory endpoints of percent reduction in wound area at Visit 3 and percent 
reduction in wound area at Visit 4.  

24 
 
 
If enrolled subjects have multiple wounds that meet size requirements (according to the 
eligibility criteria) the largest wound will be identified as the primary wound, while all others 
will be identified as target wounds. Data may be collected on the target wounds and used 
only for exploratory purposes. The largest wound that is randomized to the treatment or 
standard of care, will be selected as the primary wound for the hypothesis test just described 
because it is the only wound with randomized treatment; other wounds on the subject are to 
receive standard-of-care treatment. Data will be collected on the primary wound for analyses. 
Wounds that are contiguous are treated as one wound. Over the course of the study, if the 
index wound (i.e., the wound randomized to treatment) becomes contiguous with one or 
more extant wounds, the “merged” wounds will be regarded as the wound going forward but 
not retrospectively. It is not anticipated that disconnected wounds will become contiguous 
over the course of the study. 
 
An exploratory endpoint is whether or not a wound is closed at Visit 5 (the last specified 
follow-up visit). Fisher’s exact test will be used to test for an association between treatment 
arm and whether the wound is closed at Visit 5.   
 
Descriptive summaries will also be provided by treatment arm for the following additional 
exploratory endpoints: the proportion of wounds that have recurred at Visit 5; the percent 
reduction (relative to Visit 2) in the maximal depth of the wound at Visits 3, 4, and 5; the 
change since Visit 2 in wound-associated pain at Visits 3, 4, and 5; the presence of 
hypertrophic scar formation at Visit 5 (measured with modified Vancouver Scar Scale); and 
incidence of wound infection as determined by wound culture/assessment from Visit 4. 
 
The primary analysis of the primary outcome implicitly adopts a last-observation- 
carried-forward approach. Multiple imputation will be used for the primary analysis of the 
secondary outcome and may also be used for other outcomes, as described in the Statistical 
Analysis Plan. Because the sample size is modest, it may not be feasible to use complex 
multiple imputation models, but candidate variables for the imputation of missing values for 
the secondary outcome will include variables such as assigned treatment arm and wound area 
measurements from other study visits. As sensitivity analyses, the analyses of the primary 
and secondary outcomes will be repeated twice, first replacing each missing outcome with 
the lowest observed outcome at that timepoint from among subjects in the same assigned 
treatment arm, and then again replacing each missing outcome with the highest observed 
outcome at that timepoint from among subjects in the same assigned arm. 
 
A number of additional prespecified exploratory analyses will be conducted. Although the 
overall sample size is modest, subgroup analyses were requested by the FDA and will be 
conducted for the primary and secondary outcomes using subgroups defined by the following 
variables: sex; race; ethnicity; diagnosis of diabetes mellitus or a screening hemoglobin A1C 
greater than or equal to 6.5; use of immunosuppressive medications; presence/absence of 
known malignancy other than non-metastatic basal or squamous cell carcinoma; nutritional 
status as defined by albumin levels; presence/absence of blood coagulation conditions 
defined by PT/INR and PTT levels; presence/absence of venous vascular disease affecting 
the wound area; presence/absence of arterial vascular disease affecting the wound area; Visit 

25 
 
1 oxygen saturation <90% or ≥ 90%; and concomitant anticoagulation medication or lack 
thereof. The details of the subgroup analysis will be available in the Statistical Analysis Plan 
(SAP). 
 
Finally, although some additional collected data elements may influence the response, the 
primary and secondary analyses will not adjust for these other characteristics because the 
modest sample size makes such adjustments impractical. The randomization procedure will 
be relied upon to avoid favoring either treatment arm in the safety and efficacy comparisons. 
Exploratory analyses may be conducted to attempt to adjust for one or more additional 
variables. 
 
Sample Size Justification 
 
A sample size of n = 30 patients per group (total n = 60) is expected, though this may differ 
slightly because of the randomization procedure.  Thirty participants in the pAF group with 
no related serious adverse events will provide a lower-bound of the 95% one-sided exact 
binomial confidence interval for safety of 90% and provide enough evidence to move on to 
the next phase of study. 
 
9 - DATA MANAGEMENT 
 
Clinical Site Data Management 
The Data Coordinating Center (DCC) will develop an electronic data capture system for 
this trial. Currently the DCC uses multiple applications, such as OpenClinica or REDCap, and 
will elect to use the most appropriate application at the time of implementation of the study.  
 
The DCC will use an electronic discrepancy management system to notify sites of inconsistent 
or erroneous data entry, which will be corrected by the clinical site. The discrepancy 
management system maintains an audit trail of all discrepancy resolution. 
 
Study Monitoring 
The investigators recognize the importance of ensuring data of excellent quality. Study 
monitoring is critical to this process. Monitoring has been a very effective tool for maintaining 
data quality in previous network studies, and we will utilize this process to ensure excellent 
quality data in the proposed study. The DCC utilizes risk-based methodology to identify and 
correct problems that may arise at sites. The risk-based approach to monitoring focuses on 
oversight activities and preventing or mitigating key risks to data quality, as well as to 
processes critical to human subject protection and integrity of the trial or study. 
 
Study monitors must be provided with appropriate access to study materials and the medical 
records for study subjects. If the medical records are in electronic form, the clinical 
investigator or an authorized individual must provide any assistance necessary to facilitate the 
study monitor's review of data in the electronic medical record. 
 
 
Site Monitoring Plan 

26 
 
A supplemental study-specific risk-based monitoring plan, separate from the protocol will be 
completed which outlines specific criteria for monitoring. This plan may include the number 
of planned site visits, criteria for focused visits, additional visits or remote monitoring, a plan 
for chart review and a follow up plan for non-compliant sites. The monitoring plan also 
describes the type of monitoring that will take place (e.g., sample of all subjects within a site; 
key data or all data), the schedule of visits, how they are reported and a time frame to resolve 
any issues found. 
 
Clinical Site Monitoring 
Site monitoring visits will be performed by a trained site monitor during the study period to 
ensure regulatory compliance, patient safety, and to monitor the quality of data collected. 
Essential document binders, regulatory documents and data collection forms may be 
reviewed.  Interim visits will take place depending on grant budget, site enrollment, and 
compliance issues identified.  The site monitor will provide each site with a written report, 
and sites will be required to follow up on any deficiencies. It is anticipated that the study 
monitoring visits for this protocol will consist of a site initiation visit (prior to patient 
enrollment), interim visits, and a close-out visit. The site initiation may take place as group 
training made up of site investigators and research assistants. 
 
Remote Monitoring 
The Data Coordinating Center may supplement on-site monitoring with remote monitoring 
activities. Remote monitoring involves detailed review of the data entered by the site and 
consultations with the site investigator and/or research coordinator to review safety and data 
quality. This may require uploading copies of medical records, patient study files, regulatory 
documentation, or other source documents for the monitor to review. Alternatively, other 
methods, such as remotely viewing source documentation, may be utilized. This helps assure 
protocol compliance and accurate data collection. The Data Coordinating Center may conduct 
more remote monitoring activities early in the trial to assure protocol compliance and identify 
any training issues that may exist. Remote monitoring documents will be retained in 
accordance with federal requirements. 
 
10 - PROTECTION OF HUMAN SUBJECTS 
 
Institutional Review Board Approval 
The Data Coordinating Center and each clinical center must obtain approval from their 
respective IRBs prior to participating in the study. The Data Coordinating Center will track 
IRB approval status at all participating centers and will not permit subject enrollment without 
documentation of initial IRB approval and maintenance of that approval throughout 
subsequent years of the project. 
 
Amendments 
Any amendments or administrative changes to the IRB approved protocol will not be initiated 
without submission of an amendment for IRB review and approval. 
 

27 
 
Any amendment to the protocol that significantly affects the safety of subjects, the scope of 
the investigation, or the scientific quality of the study are required to submit the amendment 
for FDA review. 
 
Annual Reports 
    An annual progress report will be submitted to the FDA within 60 days of the anniversary of    
    the date that the IND went into effect (21 CFR 312.33) 
 
 
Informed Consent 
Informed consent is required because all eligible subjects are at least 18 years old. Subjects 
who are capable of giving consent and who are alert and competent, will be asked, following 
an appropriate discussion of risks and benefits, to give consent to the study. For those with 
diminished mental capacity, a Legal Authorized Representative will be used. 
 
Potential Risks 
Loss of confidentiality of the subject is a potential risk of the study; however, safeguards are 
in place to protect against this.  There is an additional medical risk of rash around the injection 
site if randomized to pAF treatment. 
 
Protection Against Potential Risks 
Regarding loss/breach of privacy and confidentiality, all applicable parties (e.g. clinical sites, 
DCC) will be responsible for ensuring that appropriate data security procedures are in place. 
 
Potential Benefits 
There may be a potential benefit in helping decrease the size of wounds that are treated with 
pAF and the amount of time it takes to decrease the wound size. 
 
 
11 - REGULATORY CONSIDERATIONS 
 
Food and Drug Administration 
This trial is being conducted under an Investigational New Drug application. The clinical 
investigator at each participating site will complete a Form FDA 1572, ``Statement of 
Investigator.'' 
 
Health Insurance Portability and Accountability Act 
Data elements collected include the date of birth and date of visits. Prior to statistical analyses, 
dates will be used to calculate patient age at the time of the study events. The final data sets 
(used for study analyses and made available at the end of the study) will be de--identified, and 
will exclude these specific dates. 
 
Data elements for race, ethnicity, and gender are also being collected. These demographic 
data are required for Federal reporting purposes to delineate subject accrual by race, ethnicity, 
and gender. 
 

28 
 
Inclusion of Women and Minorities 
There will be no exclusion of patients based on gender, race, or ethnicity. 
 
 
12- DATA COORDINATING CENTER 
 
Overview 
The Data Coordinating Center (DCC) in the Department of Pediatrics at the University Of 
Utah School Of Medicine provides data coordination and management services for a variety 
of national research networks. Anchoring these services is a new state-of-the art, energy 
eﬃcient data center. The data center facility supports more than 3500 users around the 
country and provides a secure, reliable, enterprise-wide infrastructure for delivering mission 
critical DCC systems and services.  
 
Facility, Hardware, Storage, Data Backup and System Availability 
The data center was built using high industry standards and energy eﬃcient cooling solutions. 
The data center is cooled by a LCP (Liquid Cooling Package) inline cooling technology; 
providing eﬃciency, redundancy and the modularity required. Cooling is based upon a 
hot/cold aisle design that allows for even air distribution with minimal hot spots. The data 
center electrical power system contains an uninterruptible power system (UPS) with a diesel 
backup generator. The data center is protected with a FM200 ﬁre suppression system to act as 
a secondary system to the smoke detectors. The data center provides enhanced security to 
safeguard the equipment and the data within it.  Entry into the data center is restricted by card 
access and layered security measures and controls. The data center and external building 
access points are monitored with video surveillance. 
 
The data center has a virtualized environment. The virtualized environment consists of more 
than 400 virtual servers and nearly 25 physical servers.  The data center’s virtualized solution 
provides key advantages; 1) high availability – in the event of hardware failure, virtual servers 
automatically go back online in a seamless process. 2) Flexible infrastructure – disk storage, 
memory and processor capacity can be increased or reallocated at any time. 3) Rapid 
deployment – servers can be provisioned on demand.  
 
Production servers running mission critical applications are clustered and conﬁgured for 
failover events. Servers are backed up with encryption through a dedicated backup server. Our 
storage area networking (SAN) applications, clusters, and switch-to-switch links are on a 10 
gigabit network. Incremental backups occur Monday through Friday.  A full system backup 
occurs weekly. Full backups are taken oﬀ site on a weekly basis to an oﬀ-site commercial 
storage facility.  The data center currently manages over 150 terabytes of data. 
 
Our information systems are available 24 hours a day, 7 days a week to all users, unless a 
scheduled maintenance interruption or mitigation of an unexpected event is required. If this 
occurs, we notify all users of the relevant systems, and data entry can be deferred until after 
the interruption is over.  
 
Security, Support Encryption and Conﬁdentiality 

29 
 
The data center coordinates the network infrastructure and security with the University 
Information Technology (UIT) at the University of Utah. This provides us with eﬀective 
ﬁrewall hardware, automatic network intrusion detection, and the expertise of dedicated 
security experts working at the University.  Centralized authentication and communication 
over public networks is encrypted using transport layer security (TLS) or virtual private 
network (VPN) technologies, both of which provide at least 128 bit encryption.  
 
All network traﬃc is monitored for intrusion attempts, security scans are regularly run against 
our servers, and our IT staﬀ are notiﬁed of intrusion alerts. Security is maintained with 
Windows user/group domain-level security. Users are required to change their passwords 
every 90 days. All ﬁles are protected at user/group levels; database security is handled in a 
similar manner with group-level access to databases, tables, and views in Microsoft SQL 
Server. Finally, all laptop computers in use in the DCC are whole-disk encrypted. 
 
The data center uses control center tools to continuously monitor systems and failure alerts. 
Environmental and network systems are also monitored to ensure up time. And highly trained 
system administrators on staﬀ are available to respond in high risk emergency events.  
 
All personnel involved with the DCC have signed conﬁdentiality agreements concerning data 
encountered in the course of their daily work. All personnel (including administrative staﬀ) 
have received Human Subjects Protection and Health Information Portability and 
Accountability Act (HIPAA) education. We require all users to sign speciﬁc agreements 
concerning security, conﬁdentiality, and use of our information systems, before access is 
provided. 
 
Record Access 
The medical record and study ﬁles (including informed consent) must be made available to 
authorized representatives of the Data Coordinating Center, upon request, for source 
veriﬁcation of study documentation. In addition, medical information and data generated by 
this study must be available for inspection upon request by representatives (when applicable) 
of the Food and Drug Administration (FDA), NIH, other Federal funders, and the Institutional 
Review Board (IRB) for each study site. 
 
13 - APPENDICES 
 
APPENDIX A- REFERENCES 
 
1. Yang, J.D. et al. Effect of amniotic fluid stem cells and amniotic fluid cells on the wound healing process in a 
white rat model. Arch Plast Surg 40, 496-504 (2013). 
2. Kratz, G., Haegerstrand A, Palmer B Growth stimulatory effects of amniotic fluid and amniotic cell conditioned 
medium on human cells involved in wound healing. European Journal of Plastic Surgery 16, 130-133 (1993). 
3. Werber, B. & Martin, E. A prospective study of 20 foot and ankle wounds treated with cryopreserved amniotic 
membrane and fluid allograft. J Foot Ankle Surg 52, 615-621 (2013). 
4. Pierce, J. et al. Collection and characterization of amniotic fluid from scheduled C-section deliveries. Cell and 
Tissue Banking 17, 413-425 (2016). 
5. Sen, C.K. et al. Human skin wounds: a major and snowballing threat to public health and the economy. Wound 
Repair Regen 17, 763-771 (2009). 

30 
 
6. Fife, C.E. & Carter, M.J. Wound Care Outcomes and Associated Cost Among Patients Treated in US Outpatient 
Wound Centers: Data From the US Wound Registry. Wounds 24, 10-17 (2012). 
7. Underwood, M.A., Gilbert, W.M. & Sherman, M.P. Amniotic fluid: not just fetal urine anymore. J Perinatol 25, 
341-348 (2005). 
8. Prusa, A.R., Marton, E., Rosner, M., Bernaschek, G. & Hengstschlager, M. Oct-4-expressing cells in human 
amniotic fluid: a new source for stem cell research? Hum Reprod 18, 1489-1493 (2003). 
9. Bottai, D. et al. Third trimester amniotic fluid cells with the capacity to develop neural phenotypes and with 
heterogeneity among sub-populations. Restor Neurol Neurosci 30, 55-68 (2012). 
10. Johnson, H.L. Peritoneal Immunization. The American Journal of Surgery 34, 266-271 (1936). 
11. Shimberg, M. The Use of Amniotic Fluid Concentrate in Orthopedic Conditions. The Journal of Bone and Joint 
Surgery, 167-177 (1938). 
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