Conditions: Wounds and Injuries
Keywords: chronic wounds
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 1
Healthy volunteers: no

Brief summary:
A prospective randomized controlled study to determine the safety and feasibility of using processed amniotic fluid (pAF) to expedite healing in chronic wounds.

Detailed description:
The goal of our study is to assess, in an outpatient setting, the safety and the time to wound closure in chronic wounds treated with processed amniotic fluid (pAF). We will include patients with lower extremity full thickness wounds that are greater than 3 months old and less than 12 months old. The wound size must be greater than 5cm2 and less than 75 cm2. We will exclude anyone that requires skin grafting, or wounds with sinus tracts. Our primary objective is to determine the safety and feasibility of using pAF to treat patients with chronic wounds. The secondary objective is to determine the efficacy of pAF compared to standard of care in reducing wound area.

Arms:
arm 1: Processed Amniotic Fluid. Dose is 1ml/5cm2; Route: injected directly into wound; Limited to two injections. The wound will then be dressed according to standard of care. arm 2: Primary dressings are variable and based on the moisture content and microorganism load. 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. 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/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 rash present with transition to silver product only or silver product plus medical honey.

Interventions: intervention 1: Processed Amniotic Fluid intervention 2: Standard of Care Wound Treatment Regimen
Intervention descriptions: intervention 1: Injection of 1 ml of processed amniotic fluid per 5 cm2 of wound area intervention 2: ointment-based dressing and non-ointment-based dressings

Protocol document:
ICF_003.pdf: 
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) 
ICF 
Update Date (Approval Date) 
December 8, 2022 


PI:  Giavonni Lewis, MD 
 
Page 1 of 8 
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  
Version:  V4 Chronic Wounds Consent 28Mar2022 
 
FOOTER FOR IRB USE ONLY 
Version: K0218 
 
 
University of Utah 
Institutional Review Board 
Approved 12/8/2022 
Expires 12/7/2023 
IRB_00128708 
 
 
Consent and Authorization Document 
 
BACKGROUND 
You are being asked to take part in a research study. This study is “A Phase I/II Clinical Trial to determine 
safety and feasibility of using an acellular amniotic fluid application to expedite healing in chronic 
wounds”. Before you decide, it is important for you to understand why the research is being done and 
what it will involve. Please take time to read the following information carefully and discuss it with 
friends and relatives if you wish. Ask the research doctor or staff if there is anything that is not clear or if 
you would like more information. Take time to decide whether or not to volunteer to take part in this 
research study.   
 
You may be able to participate if: 
• 
You have a chronic wound or wounds on your lower or upper extremities.    
• 
These wounds have not healed with standard treatments (Standard of Care).   
• 
These wound(s) have affected your quality of life and you have experienced pain, and it has 
limited your ability to function normally.  
 
The purpose of the study is to find out if human amniotic fluid can improve your chronic extremity 
wound, decreasing the inflammation and assisting the wound(s) to heal.  Amniotic fluid is the liquid that 
surrounds the unborn, growing baby during pregnancy.  Amniotic fluid is mainly water, but also has 
other components like proteins and antibodies that protect the baby from infection and help growth. 
Some of these components have been shown to have an anti-inflammatory effect. The fluid is obtained 
during the delivery of the baby and processed so that it is sterile and can be injected into wounds. We 
hope that the beneficial components of the amniotic fluid will help your chronic wound in assisting your 
body to heal the wound.  The Amniotic Fluid used in this research study is considered investigational and 
has not been approved by the Food and Drug Administration (FDA). 
 
This study is being conducted by the Cell Therapy and Regenerative Medicine Program and by the Burn 
and Wound Clinic, which are part of the University of Utah. 
 
CONFLICT OF INTEREST 
In studies like this where there is a conflict of interest, an organization (called the Conflict of Interest 
Office) will create and manage a plan to ensure that the data from this study is accurately reported and 
correctly interpreted. This helps to make certain that the study is conducted correctly, and that 
participants in the study are treated in the most fair way possible. 
 
The University of Utah has a significant institutional financial interest in intellectual property being used 
in this study (U-5650 "Amnion/Chorion Membrane as a Wound Cover to Control Adhesions and Heal 
Wounds, Amniotic Fluid (Supernatant) and Amniotic Fluid (Whole) to Reduce Inflammation, Heal 
Wounds and Stimulate Bone Growth") and a significant institutional financial interest in Eliksa (a non-
publicly traded company which is licensing the technology for commercialization). This is a conflict of 
interest as determined by the University of Utah Institutional Conflict of Interest Officer and a 


PI:  Giavonni Lewis, MD 
 
Page 2 of 8 
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  
Version:  V4 Chronic Wounds Consent 28Mar2022 
 
FOOTER FOR IRB USE ONLY 
Version: K0218 
 
 
University of Utah 
Institutional Review Board 
Approved 12/8/2022 
Expires 12/7/2023 
IRB_00128708 
 
 
management plan has been implemented to ensure transparency, promote data integrity, and to 
safeguard human subjects in the research. 
 
Investigators Dr. John Phillips and Jan Pierce have a financial interest in Eliksa, (a non-publicly traded 
company which is licensing the technology for commercialization), which is a conflict of interest as 
determined by the University of Utah Individual Conflict of Interest Committee, and a management plan 
has been implemented to ensure transparency, promote data integrity, and to safeguard human 
subjects in the research. 
 
STUDY PROCEDURES 
Once we have determined you can be part of the study, you can be enrolled in the study if you so desire.  
At the first follow-up, if your wound has not mostly healed you will either be given amniotic fluid 
injections or standard of care treatment without amniotic fluid injections.  The assignment of which 
treatment you receive is done randomly, which means by chance. This means that a computer will 
decide by chance which group a person is in, not the doctors running the trial. You have a 50:50 chance 
of receiving the study treatment of amniotic fluid or standard treatment.  The reason for this is to 
compare whether participants receive benefits from the amniotic fluid compared to the standard of 
care.  The use of random assignment of treatment will help the interpretation of study results to be 
more reliable. 
 
If you are chosen to have the amniotic fluid injection, you will be given the treatment during your 2nd (6 
weeks after your first clinic visit) and 3rd (6 weeks after your 2nd clinic visit) clinic visit.   If you are not 
chosen to have the amniotic fluid injection, you will be given standard of care treatment at the same 
timepoints. Whether or not you are chosen to have the amniotic fluid injection, you will have to see 
your doctor in the Burn/Wound for your 4th clinic visit approximately 6 weeks after your last treatment 
and then again for your 5th visit 3 months later.  We will train you on how to care for your wound 
between clinic visits.   
 
In this study you will be referred and screened by the Principal Investigator (PI) to participate in this 
research study at the Burn/Wound Clinic within the University of Utah Hospital. 
 
The first visit is called a screening visit. This visit may be split into two visits if needed. At this visit the 
research coordinator and your study doctor will perform the following: 
• 
You will be asked to read and sign this consent form before any study procedures can be done. 
You will receive a copy of this to take home.  
• 
Your medical history and medications will be reviewed 
• 
Your vital signs will be measured including height, weight, blood pressure, pulse and 
temperature. 
• 
The study doctor will perform a physical exam and assess your pain level. 
• 
Blood and urine samples may be collected.  
• 
If you are a woman of child-bearing potential you will be asked to take a pregnancy test 


PI:  Giavonni Lewis, MD 
 
Page 3 of 8 
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  
Version:  V4 Chronic Wounds Consent 28Mar2022 
 
FOOTER FOR IRB USE ONLY 
Version: K0218 
 
 
University of Utah 
Institutional Review Board 
Approved 12/8/2022 
Expires 12/7/2023 
IRB_00128708 
 
 
• 
The study team will ask you to complete questionnaires about yourself and the study doctors 
will ask you questions. 
• 
You must be willing to use effective contraception if you are a person of childbearing potential 
during the study, if you cannot agree to this, you should not consent to be on this study. 
 
Follow Up visits include 4 in-person clinic visits and a follow-up phone call within 24 hours and 5-7 
days after visits 2 and 3. 
• 
Your medical history and medications will be reviewed 
• 
Your vital signs will be measured including height, weight, blood pressure, pulse and 
temperature. 
• 
The study doctor will perform a physical exam 
• 
Blood and urine samples may be collected.  
• 
The study team will ask you to complete questionnaires about yourself and the study doctors 
will ask you questions. 
 
RISKS 
The CTRM screens the amniotic fluid coming from donors for infectious diseases through medical and 
social history questionnaire, medical record review, and testing of the donor’s blood for viruses 
including HIV, Hepatitis B & C, and others. The CTRM Medical Director reviews the records and test 
results to determine if the donor meets eligibility requirements.  Although the risk of transmitting 
infectious disease is minimal, there is a potential for transmission. 
 
Possible risks related to your participation in this study include the following:  
• 
Cellulitis (an infection of the skin caused by bacteria) 
• 
Bleeding 
• 
Drainage 
• 
Inflammation 
• 
Rash  
• 
Allergic Reactions 
• 
Parvovirus B19 infection 
The symptoms of Parvovirus B19 infection in immunocompromised, pregnant, and immunocompetent 
individuals are: Flu-like symptoms of malaise, muscle pain, and fever, rash, chronic infection and 
anemia. If you have these symptoms, please notify the study team.  However, some people have no 
symptoms at all.     
 
If you have a cellulitis infection, skin may appear red, swollen, warm to the touch, and/or may be 
painful.  We do not anticipate that this will happen. However, if it does, please tell the investigator 
immediately and seek treatment.   
 


PI:  Giavonni Lewis, MD 
 
Page 4 of 8 
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  
Version:  V4 Chronic Wounds Consent 28Mar2022 
 
FOOTER FOR IRB USE ONLY 
Version: K0218 
 
 
University of Utah 
Institutional Review Board 
Approved 12/8/2022 
Expires 12/7/2023 
IRB_00128708 
 
 
If you are injured during the course of your study participation, you should seek medical help 
immediately, and contact the study doctor or study staff as soon as possible.  
 
Participation in the study may also involve risks that are currently unforeseeable. 
 
Participation in studies of any kind can result in a loss of confidentiality.  This possibility is remote; 
however, we will take measures to protect you.  Please see the Authorization Section below for further 
details regarding how we will protect your information.   
 
BENEFITS 
It is unclear whether the product will provide any benefit.  The potential benefit from this treatment is 
to assist in the healing of your chronic wound(s).  The information gained in this study will aid in the 
understanding of the use of amniotic fluid to heal wounds and help in the development of new 
approaches to its treatment in the future. 
 
ALTERNATIVE PROCEDURES 
You may choose not to be in this study. If you do not want to take part in the study, there are other 
choices to improve your symptoms, including: 
- 
Standard of Care 
- 
Surgical/procedural intervention 
 
You may have received one or more of these treatments, and you may discuss these options with your 
doctor.  
 
PERSON TO CONTACT 
If you have questions, complaints or concerns about this study, you can contact Dr. Giavonni Lewis at 
801-581-3050. If you think you may have been injured from being in this study, please call Dr. Lewis at 
801-581-3050.  
 
Institutional Review Board: Contact the Institutional Review Board (IRB) if you have questions regarding 
your rights as a research participant. Also, contact the IRB if you have questions, complaints or concerns 
that you do not feel you can discuss with the investigator. The University of Utah IRB may be reached by 
phone at (801) 581-3655 or by e-mail at irb@hsc.utah.edu.   
 
Research Participant Advocate:  You may also contact the Research Participant Advocate (RPA) by 
phone at (801) 581-3803 or by email at participant.advocate@hsc.utah.edu.  
 
Person to Contact:  
 
Giavonni Lewis, M.D. 
50 North Medical Drive 
Salt Lake City, UT 84132 
Phone: 801-581-3050 


PI:  Giavonni Lewis, MD 
 
Page 5 of 8 
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  
Version:  V4 Chronic Wounds Consent 28Mar2022 
 
FOOTER FOR IRB USE ONLY 
Version: K0218 
 
 
University of Utah 
Institutional Review Board 
Approved 12/8/2022 
Expires 12/7/2023 
IRB_00128708 
 
 
 
24-Hour Number:   
Please call the Burn ICU at 801-581-2700 
 
 
RESEARCH-RELATED INJURY 
If you are injured from being in this study, medical care is available to you at University of Utah, as it is 
to all sick or injured people. The University of Utah has not set aside any money to pay the costs for such 
care. The University will work with you to address costs from injuries. Costs would be charged to you or 
your insurance company (if you have insurance), to the study sponsor or other third party (if applicable), 
to the extent those parties are responsible for paying for medical care you receive. Since this is a 
research study, some health insurance plans may not pay for the costs. By signing this consent form you 
are not giving up your right to pursue legal action against any parties involved with this research. 
 
The University of Utah is a part of the government. If you are injured in this study, and want to sue the 
University or the doctors, nurses, students, or other people who work for the University, special laws 
may apply. The Governmental Immunity Act of Utah is a law that controls when a person needs to bring 
a claim against the government, and limits the amount of money a person may recover. See sections 
63G -7-101 to -904 of the Utah Code. 
 
VOLUNTARY PARTICIPATION 
Research studies include only people who choose to take part.  You can tell us that you do not want to 
be in this study.  You can start the study and then choose to stop the study later.  We will still give you 
medical care and answer any questions you have.  Your decision will not affect your relationship with 
your doctor or the study team in any way. 
 
If you want to stop being in this study, please let the research doctor know. That way you can find out 
what should be done about your normal medical care outside of the study. 
 
RIGHT OF INVESTIGATOR TO WITHDRAW PARTICIPANTS 
The investigator can withdraw you without your approval. Possible reasons for withdrawal include: 
- 
Any adverse effect that may affect you, and that in the opinion of the treating doctor justifies 
withdrawing you from the study. 
- 
Noncompliance with the treatment. 
- 
Any other unforeseeable circumstance that in the opinion of the treating doctor justifies 
withdrawing you from the study. 
 
There are always reasonably foreseeable risks of participation in a study. If an adverse effect occurred 
during the study and you were withdrawn from it, the doctors may have to follow-up on the problem 
until it is resolved. 
 
 


PI:  Giavonni Lewis, MD 
 
Page 6 of 8 
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  
Version:  V4 Chronic Wounds Consent 28Mar2022 
 
FOOTER FOR IRB USE ONLY 
Version: K0218 
 
 
University of Utah 
Institutional Review Board 
Approved 12/8/2022 
Expires 12/7/2023 
IRB_00128708 
 
 
COSTS AND COMPENSATION TO PARTICIPANTS 
The parts of your care that would normally be done as standard treatment such as regular doctor visits 
and lab work for the follow-up of your chronic wound will be billed to you or your insurance company. 
Anything strictly related to the study, such as the amniotic fluid or exams required by the study outside 
what is considered standard of care, will not be billed to you or your insurance company. The standard 
of care exams required by the study include your doctor visits, including exams, and your routine blood 
tests. 
 
You will not be compensated for participating in this study.  
 
NEW INFORMATION 
Sometimes during the course of a research project, new information becomes available about the 
amniotic fluid that is being studied. If this happens, your research doctor will tell you about it and 
discuss with you whether you want to continue in the study.   
 
If you decide to withdraw at that time, your research doctor will make arrangements for your medical 
care to continue.  Also, on receiving new information your research doctor might consider it to be in 
your best interests to withdraw you from the study. He/she will explain the reasons and arrange for your 
medical care to continue. 
 
NUMBER OF PARTICIPANTS 
We expect to enroll 60 participants at Burn/Wound Clinic, University of Utah. This study is not 
conducted in any other center outside the University of Utah 
 
AUTHORIZATION FOR USE OF YOUR PROTECTED HEALTH INFORMATION  
Signing this document means you allow us, the researchers in this study, and others working with us to 
use some information about your health for this research study. 
 
This is the information we will use and include in our research records: 
- 
Demographic information like age, gender, name, address, telephone number, and email 
address 
- 
Related medical information about you like allergies, current and past medications or therapies, 
and information from physical examinations, such as blood pressure reading, heart rate, 
temperature, and lab results 
- 
All tests and procedures that will be done in the study 
How we will protect and share your information: 
• 
We will do everything we can to keep your information private but we cannot guarantee this. 
Study information will be kept in a secured manner and electronic records will be password 
protected. Study information may be stored with other information in your medical record. 
Other doctors, nurses, and third parties (like insurance companies) may be able to see this 


PI:  Giavonni Lewis, MD 
 
Page 7 of 8 
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  
Version:  V4 Chronic Wounds Consent 28Mar2022 
 
FOOTER FOR IRB USE ONLY 
Version: K0218 
 
 
University of Utah 
Institutional Review Board 
Approved 12/8/2022 
Expires 12/7/2023 
IRB_00128708 
 
 
information as part of the regular treatment, payment, and health care operations of the 
hospital. We may also need to disclose information if required by law. 
 
• 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required 
by U.S. Law.  This website will not include information that can identify you.  At most, the 
website will include a summary of the results.  You can search this website at any time. 
 
• 
In order to conduct this study and make sure it is conducted as described in this form, the 
research records may be used and reviewed by others who are working with us on this research: 
o Members of the research team at the University of Utah Health Sciences Center. 
o The University of Utah Institutional Review Board (IRB), which reviews research 
involving people to make sure the study protects your rights. 
o National Institutes of Health and Food and Drug Administration  
o Independent contracted professionals to review de-identified wound digital images 
 
• 
If we share your identifying information with groups outside the University of Utah Health 
Sciences Center, they may not be required to follow the same federal privacy laws that we 
follow.  They may also share your information again with others not described in this form. 
 
• 
If you do not want us to use information about your health, you should not be part of this 
research.  If you choose not to participate, you can still receive health care services at the 
Burn/Wound. 
 
What if I decide to Not Participate after I sign the Consent and Authorization Form? 
You can tell us anytime that you do not want to be in this study and do not want us to use your health 
information.  You can also tell us in writing.  If you change your mind, we will not be able to collect new 
information about you, and you will be withdrawn from the research study. However, we can continue 
to use information we have already started to use in our research, as needed to maintain the integrity of 
the research.   
 
This authorization does not have an expiration date.   
 
You have a right to information used to make decisions about your health care. However, some 
information from this study will not be available during the study; it will be available after the study is 
finished. 
 
 
 


PI:  Giavonni Lewis, MD 
 
Page 8 of 8 
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  
Version:  V4 Chronic Wounds Consent 28Mar2022 
 
FOOTER FOR IRB USE ONLY 
Version: K0218 
 
 
University of Utah 
Institutional Review Board 
Approved 12/8/2022 
Expires 12/7/2023 
IRB_00128708 
 
 
CONSENT 
I confirm that I have read this consent and authorization document and have had the opportunity to ask 
questions. I will be given a signed copy of the consent and authorization form to keep. 
 
 
I agree to take part in this research study and authorize you to use and disclose health information 
about me for this study, as you have explained in this document. 
 
 
________________________ 
Participant’s Name 
 
________________________ 
 
 
 
 
____________ 
Participant’s Signature 
 
Date 
 
________________________ 
Name of Person Obtaining Authorization and Consent 
 
________________________ 
 
 
 
 
____________ 
Signature of Person Obtaining Authorization and Consent 
 
Date 
 
 
 
INTERPRETER STATEMENT: (For Non-English Speaking Participants Only):  I confirm that I was present 
as an interpreter for the duration of the consent process for this research study.  I confirm that I am 
qualified and have the necessary skills to provide interpretation between the participant’s language and 
English.  By signing this form, I confirm that I provided a full and complete interpretation of the 
exchange between the researcher obtaining consent and the participant, to the best of my ability. 
 
 
______________________________________________ 
Name of Interpreter 
 
______________________________________________                        ____________________ 
Signature of Interpreter                                                                                  Date 
 
 
 

Prot_000.pdf: 
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 
 


1 
 
 
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 


2 
 
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 
 
 
 
 
 
 
 
 
 
 
 
 


3 
 
 
 
 


4 
 
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 


5 
 
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).  


6 
 
 
-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 


7 
 
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 


8 
 
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.   


9 
 
 
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 


10 
 
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). 


11 
 
 
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 


12 
 
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): 


13 
 
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 


15 
 
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. 


16 
 
 
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 


17 
 
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). 
12. Ismail, M.A., Salti, G.I. & Moawad, A.H. Effect of amniotic fluid on bacterial recovery and growth: clinical 
implications. Obstet Gynecol Surv 44, 571-577 (1989). 
13. Ojo, V.A., Okpere, E.E. & Obaseiki-Ebor, E.E. Antimicrobial properties of amniotic fluid from some Nigerian 
women. Int J Gynaecol Obstet 24, 97-101 (1986). 
14. Siggers, J. et al. Postnatal amniotic fluid intake reduces gut inflammatory responses and necrotizing 
enterocolitis in preterm neonates. Am J Physiol Gastrointest Liver Physiol 304, G864-875 (2013). 
15. Ozgenel, G.Y., Filiz, G. & Ozcan, M. Effects of human amniotic fluid on cartilage regeneration from free 
perichondrial grafts in rabbits. Br J Plast Surg 57, 423-428 (2004). 
16. Ozgenel, G.Y. & Filiz, G. Combined application of human amniotic membrane wrapping and hyaluronic acid 
injection in epineurectomized rat sciatic nerve. J Reconstr Microsurg 20, 153-157 (2004). 
17. Karacal, N., Kosucu, P., Cobanglu, U. & Kutlu, N. Effect of human amniotic fluid on bone healing. The Journal 
of Surgical Research 129, 283-287 (2005). 
18. Castro-Combs, J. et al. Corneal wound healing is modulated by topical application of amniotic fluid in an ex 
vivo organ culture model. Exp Eye Res 87, 56-63 (2008). 
19. Nyman, E., Huss, F., Nyman, T., Junker, J. & Kratz, G. Hyaluronic acid, an important factor in the wound 
healing properties of amniotic fluid: in vitro studies of re-epithelialisation in human skin wounds. J Plast Surg 
Hand Surg 47, 89-92 (2013). 
20. Weissenbacher, T. et al. Influence of maternal age, gestational age and fetal gender on expression of immune 
mediators in amniotic fluid. BMC Res Notes 5, 375 (2012). 
21. Merimee, T.J., Grant, M. & Tyson, J.E. Insulin-like growth factors in amniotic fluid. J Clin Endocrinol Metab 
59, 752-755 (1984). 
22. Watanabe, H. Epidermal growth factor in urine of pregnant women and in amniotic fluid throughout pregnancy. 
Gynecol Endocrinol 4, 43-50 (1990). 
23. Lang, A.K. & Searle, R.F. The immunomodulatory activity of human amniotic fluid can be correlated with 
transforming growth factor-beta 1 (TGF-beta 1) and beta 2 activity. Clin Exp Immunol 97, 158-163 (1994). 
24. Kurauchi, O. et al. The concentration of hepatocyte growth factor (HGF) in human amniotic fluid at second 
trimester: relation to fetal birth weight. Horm Metab Res 27, 335-338 (1995). 
25. Hirai, C. et al. Trophic effect of multiple growth factors in amniotic fluid or human milk on cultured human 
fetal small intestinal cells. J Pediatr Gastroenterol Nutr 34, 524-528 (2002). 
 

SAP_001.pdf: 
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) 
SAP 
Update Date (Approval Date) 
March 9, 2021 
 


 
Statistical Analysis Plan 
 
Protocol 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 
 
Protocol Version and Date: 3.00, 14 October 2020 
Past Versions: 2.00, 31 March 2020; 1.00, 18 March 2020 
 
 
SAP Author: Bradley Barney, PhD (with use of DCC template language) 
SAP Version and Date: 1.0; March 9, 2021 
 
CONFIDENTIAL 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
i 


pAF for the Treatment of Chronic Wounds: Statistical Analysis Plan 
Page ii 
March 9, 2021 
SAP Version 1.0 
CONFIDENTIAL 
 
 
 
Approvals: 
 
Approved By: 
 
 
 
Giavonni Lewis MD (Mar 11, 2021 16:51 MST) 
 
Giavonni Lewis MD 
 
 
Mar 11, 2021 
 
Giovanni Lewis, MD 
Principal Investigator 
Date 
 
 
 
 
 
Bradley Barney, PhD 
Trial Statistician 
Date 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


pAF for the Treatment of Chronic Wounds: Statistical Analysis Plan 
Page iii 
March 9, 2021 
SAP Version 1.0 
CONFIDENTIAL 
 
 
 
Approvals: 
 
Approved By: 
 
 
 
 
              
Giovanni Lewis, MD 
Principal Investigator 
Date 
 
Bradley Barney, PhD 
Trial Statistician 
Date 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


pAF for the Treatment of Chronic Wounds: Statistical Analysis Plan 
Page iv 
March 9, 2021 
SAP Version 1.0 
CONFIDENTIAL 
 
 
 
Contents 
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
v 
1 PREFACE 
1 
1.1 Purpose of SAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
1 
1.2 Auxiliary/Other Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . 
1 
2 STUDY OBJECTIVES AND OUTCOMES 
2 
2.1 Study Objectives  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
2 
2.1.1 Primary Objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
2 
2.1.2 Secondary Objective  . . . . . . . . . . . . . . . . . . . . . . . . . . . 
2 
2.2 Study Outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
2 
2.2.1 Primary Outcome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
2 
2.2.2 Secondary Outcome . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
2 
2.2.3 Exploratory Outcome(s) . . . . . . . . . . . . . . . . . . . . . . . . . 
3 
2.2.4 Safety Outcome(s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
3 
3 STUDY DESIGN AND METHODS 
3 
3.1 Overall Study Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
3 
3.2 Method of Treatment Assignment and Randomization . . . . . . . . . . . . . 
4 
3.2.1 Delivery of Randomization and Emergency Backup . . . . . . . . . . 
4 
3.2.2 Handling of Incorrect Randomization in Study Analyses and Reports 
4 
3.3 Treatment Masking (Blinding) . . . . . . . . . . . . . . . . . . . . . . . . . . 
4 
3.4 Study Intervention Compliance . . . . . . . . . . . . . . . . . . . . . . . . . 
5 
4 STUDY SUBJECTS AND ANALYSIS POPULATIONS 
5 
4.1 Eligibility  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
5 
4.2 Populations for Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
5 
4.2.1 Eligible and Approached for Consent Population . . . . . . . . . . . . 
5 
4.2.2 
Enrolled Population . . . . . . . . . . . . . . . . . . . . . . . . . . . 
5 
4.2.3 Intention-to-Treat Population . . . . . . . . . . . . . . . . . . . . . . 
6 
4.2.4 
Per-Protocol Efficacy Population 
. . . . . . . . . . . . . . . . . . . . 
6 
4.2.5 Closed Wound Efficacy Populations . . . . . . . . . . . . . . . . . . . 
6 
4.2.6 Safety Population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
7 
5 GENERAL ISSUES FOR STATISTICAL ANALYSES 
7 
5.1 Analysis Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
7 
5.2 Methods for Withdrawals, Missing Data, and Outliers . . . . . . . . . . . . . 
7 
5.3 Multiple Comparisons and Multiplicity . . . . . . . . . . . . . . . . . . . . . 
8 
5.4 Planned Subgroups, Interactions, and Covariates . . . . . . . . . . . . . . . . 
8 


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5.5 Derived and Computed Variables  . . . . . . . . . . . . . . . . . . . . . . . . 
9 
5.6 Independent Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
9 
6 INTERIM ANALYSES 
10 
6.1 Frequency of and Timepoints for Interim Analysis .................................................... 10 
6.2 Stopping Rules for Interim Efficacy Analysis .............................................................. 10 
6.3 Subgroups in the Interim Analysis ............................................................................... 10 
6.4 Blinding in the Interim Analysis .................................................................................. 10 
7 PLANNED ANALYSES 
11 
7.1 Description of Subject Characteristics .......................................................................... 11 
7.2 Primary Outcome Analysis ........................................................................................... 11 
7.2.1 Additional Analyses of Primary Outcome ..................................................... 11 
7.3 Secondary Outcome Analysis ........................................................................................ 12 
7.3.1 Additional Analyses of Secondary Outcome .................................................. 13 
7.4 Exploratory Outcome(s) Analyses ................................................................................ 13 
7.4.1 Wound Area Reduction at Visits 3, 4 ............................................................. 13 
7.4.2 Wound Area Reduction at Visits 3, 4, 5, per rectangular wound area 
approximation .................................................................................................... 13 
7.4.3 Wound Closure at Visit 5 ................................................................................. 14 
7.4.4 
Wound Closure Prior to Visit 5 ....................................................................... 14 
7.4.5 Wound Recurrence at Visit 5 ........................................................................... 14 
7.4.6 Hypertrophic Scar Formation at Visit 5 ......................................................... 14 
7.4.7 
Wound Depth Reduction ................................................................................... 14 
7.4.8 Change in Wound-associated Pain ................................................................... 15 
7.4.9 Wound Infection at Visit 4 ............................................................................... 15 
7.5 Technical Approaches for Subgroup Analyses ............................................................. 16 
7.6 Safety Analyses .............................................................................................................. 16 
7.6.1 Formal Safety Outcome(s) ............................................................................... 16 
7.6.2 
Adverse Events .................................................................................................. 17 
8 SAMPLE SIZE DETERMINATION 
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Abbreviations 
Abbreviation 
Definition 
CRF 
Case Report Form 
CW3 
 Closed Wound at Visit 3 (population) 
CW4 
 Closed Wound at Visit 4 (population) 
CW5 
 Closed Wound at Visit 5 (population) 
DCC 
Data Coordinating Center 
DSMB 
Data and Safety Monitoring Board 
ITT 
 Intent-To-Treat 
MI 
Multiple Imputation 
mVSS 
modified Vancouver Scar Scale 
pAF 
processed Amniotic Fluid 
PP 
Per-Protocol 
(S)AE 
(Serious) Adverse Event 
SAFETY 
Safety Population 
SAP 
Statistical Analysis Plan 
SOC 
Standard of Care 


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1 PREFACE 
1.1 
Purpose of SAP 
This Statistical Analysis Plan (SAP) describes the planned analysis and reporting for the 
protocol: A Phase I/II Clinical Trial to Determine Safety and Feasibility of Using an Acellular 
Amniotic Fluid Application to Expedite Healing in Chronic Wounds. 
 
The structure and content of this SAP provides sufficient detail to meet the requirements 
and standards set by the Data Coordinating Center (DCC). 
 
1.2 Auxiliary/Other Documents 
The following documents were reviewed in preparation of this SAP: 
• Protocol (including intended revisions): A Phase I/II Clinical Trial to Determine Safety 
and Feasibility of Using an Acellular Amniotic Fluid Application to Expedite Healing 
in Chronic Wounds 
• Case Report Forms (CRFs) for the pAF for the Treatment of Chronic Wounds protocol 
 
 
The reader of this SAP is encouraged to read the protocol for details on the conduct of this 
study, and the operational aspects of clinical assessments. 
 
The purpose of this SAP is to outline the planned analyses to be completed for the pAF for 
the Treatment of Chronic Wounds trial. The planned analyses identified in this SAP will be 
included in future study abstracts and manuscripts. Also, exploratory analyses not necessarily 
identified in this SAP may be performed. Any post hoc, or unplanned, analyses not explicitly 
identified in this SAP will be clearly identified as such in any published reports from this study. 
 
It is possible that, due to updates or identification of errors in specific statistical software 
discussed in this SAP, the exact technical specifications for carrying out a given analysis may 
be modified. This is considered acceptable as long as the original, prespecified statistical 
analytic approach is completely followed in the revised technical specifications. 


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2 STUDY OBJECTIVES AND OUTCOMES 
2.1 Study Objectives 
2.1.1 
Primary Objective 
The primary objective of the pAF for the Treatment of Chronic Wounds trial is: 
 
1. To determine the safety of using processed amniotic fluid (pAF) to treat patients with 
chronic wounds. 
 
2.1.2 Secondary Objective 
The secondary objective of the pAF for the Treatment of Chronic Wounds trial is: 
 
1. To determine efficacy by examining a reduction in wound size after application of pAF 
compared to Standard of Care (SOC). 
 
2.2 Study Outcomes 
2.2.1 Primary Outcome 
There is one primary outcome, which pertains to safety: whether or not the subject has 
any post-randomization serious adverse events (SAEs) related to study treatment up to and 
including the final visit (Visit 5). 
 
Eligibility for Primary Analysis The primary outcome is to be evaluated in subjects 
who are randomized. Randomization does not occur until Visit 2 to make sure a wound 
is not randomized if the subject has become ineligible between Visit 1 and Visit 2. The 
inclusion/exclusion criteria from baseline should be reevaluated as feasible (without needing 
to perform additional lab tests). A wound is also considered ineligible for randomization if it 
has reduced in area by at least 50% with standard of care since Visit 1. To permit an efficient 
eligibility assessment work-flow at Visit 2, clinician judgment may be used to ascertain if the 
wound has reduced in area by at least 50% since Visit 1. 
 
2.2.2 Secondary Outcome 
The secondary outcome pertains to efficacy: the percentage reduction in wound area at Visit 
5 relative to Visit 2. 
 
Eligibility for Secondary Analysis The secondary outcome is to be evaluated in subjects 
who are randomized. 


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2.2.3 Exploratory Outcome(s) 
Exploratory outcomes include: 
• Wound closure at Visit 5 
• Wound recurrence at Visit 5 
• Percent reduction in wound area at Visits 3, 4 (relative to Visit 2) 
• Percent reduction in maximal wound depth at Visits 3, 4, 5 (relative to Visit 2) 
• Change in wound-associated pain at Visits 3, 4, 5 (relative to Visit 2) 
• Hypertrophic scar formation at Visit 5 (graded with modified Vancouver Scar Scale) 
• Wound infection at Visit 4 (per wound culture exceeding the threshold) 
 
Eligibility for Prespecified Exploratory Analyses The prespecified exploratory out- 
comes are to be evaluated in subjects who are randomized. Recurrence of wounds at Visit 
5 will be assessed only in subjects with wound closure prior to Visit 5. It is possible that 
additional exploratory analyses will be conducted, and such analyses may have different 
eligibility criteria. 
 
2.2.4 Safety Outcome(s) 
The primary outcome of this study is related to safety: the occurrence of any post- 
randomization SAEs related to study participation, up to and including the final visit. 
In addition, other safety outcomes evaluated (from randomization up to and including the 
final visit) include the following: 
 
• Occurrence of any post-randomization SAEs (regardless of relatedness to study partici- 
pation) 
• Occurrence of any post-randomization adverse events (AEs) 
• Occurrence of any post-randomization AEs related to study participation 
• Occurrence of any post-randomization unexpected AEs 
 
3 STUDY DESIGN AND METHODS 
3.1 Overall Study Design 
The pAF Wound trial is a two-arm, randomized controlled trial to assess the safety and efficacy 
of using processed amniotic fluid (pAF) to treat chronic wounds. The largest qualifying 
wound per subject will be assessed at the initial visit. At Visit 2 (about 6 weeks later), if the 


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wound has not decreased by at least 50% in area, the patient’s wound is to be randomized to 
either standard of care or the injection of pAF. The randomized treatment is given to the 
randomized wound at Visit 2 and at Visit 3 (about six weeks after Visit 2). Thereafter, two 
additional visits (Visit 4 about six weeks after Visit 3, Visit 5 about 3 months after Visit 4) 
are used to collect further information. At all study visits, non-randomized wounds are to 
receive standard of care treatment. 
 
3.2 Method of Treatment Assignment and Randomization 
Subjects will be randomized into two groups at a 1:1 ratio. The randomization will employ 
random block sizes of two, four, or six subjects to limit the ability to predict the next 
subject’s treatment prior to randomization. It is not guaranteed that exactly 30 subjects will 
be randomized to each arm due to random block sizes and the unlikely event of administering 
backup randomization for a subject. There may be as few as 28 in the pAF arm (32 in the 
standard of care–SOC–arm) or as many as 32 in the pAF arm (28 in the SOC arm). Because 
of the small number of overall patients and the anticipated slow accrual rate, stratification 
by subject characteristics will not be used in the randomization procedure. 
 
3.2.1 Delivery of Randomization and Emergency Backup 
The randomization treatment assignment will be generated by a DCC faculty statistician and 
uploaded to a REDCap instance. At Visit 2, if a subject is still eligible to have the largest 
qualifying wound randomized to treatment, the treatment assignment will be delivered using 
the randomization module in REDCap. Backup randomization envelopes will be available 
to the research coordinator(s) and/or other authorized study staff, to be used only when 
technical difficulties preclude use of the web-based randomization. 
 
3.2.2 Handling of Incorrect Randomization in Study Analyses and Reports 
The Intention-to-Treat (ITT) principle will be used, in that subjects will be analyzed in 
the arm to which they were randomized. Assuming that misrandomization is infrequent, no 
further adjustments are planned for misrandomizations beyond applying the ITT principle. 
 
3.3 Treatment Masking (Blinding) 
This is an unblinded trial, but the person interacting with the imaging software is to be 
blinded to treatment arm to avoid bias between arms for purposes of measuring the secondary 
outcome. This is because wound area calculations used by the software rely on an individual 
manually outlining the wound within the imaging software. 


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3.4 Study Intervention Compliance 
As part of the work-flow for assessments at Visits 2–5, the subject will be asked if any 
investigational wound care has been received since the last study visit that is external to that 
described in the protocol. Subjects who received investigational wound care between Visits 1 
and 2 are not to be randomized as the protocol does not allow such individuals to receive any 
pAF injections during the study. Subjects randomized to the pAF arm who receive external 
wound care between Visits 2 and 3 are not eligible to receive pAF at Visit 3. Follow-up data 
at Visits 2–5 should still be collected on all individuals who were randomized to a treatment 
arm at Visit 2, regardless of study intervention compliance. The only exceptions to the desire 
to follow-up are for individuals who die or formally withdraw during the study. 
 
4 STUDY SUBJECTS AND ANALYSIS POPULATIONS 
4.1 Eligibility 
The inclusion and exclusion criteria for the pAF for the Treatment of Chronic Wounds Trial 
are described in the protocol. Enrolled subjects may be randomized to a treatment arm at 
Visit 2 if still eligible and if the wound area has not decreased by ≥ 50% since Visit 1. The 
determination of whether the wound has decreased by ≥ 50% since Visit 1 may be per the 
rectangular approximation to the area at Visit 1 and at Visit 2 or per clinician assessment, 
even if subsequent analysis (i.e., wound size per the imaging software) would yield a different 
conclusion. 
 
4.2 Populations for Analyses 
4.2.1 Eligible and Approached for Consent Population 
The CONSORT-like diagram will begin with its most general population being those who 
met all eligibility criteria and who were approached for consent by study staff. 
 
4.2.2 Enrolled Population 
All subjects who meet all inclusion criteria, none of the exclusion criteria, have the ap- 
propriate consent, and are enrolled into the trial at Visit 1 will be considered part of 
the enrolled population. That is, the enrolled population consists of the subset of the eli- 
gible and approached population who provide consent and are enrolled into the trial at Visit 1. 


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4.2.3 Intention-to-Treat Population 
The Intention-to-Treat (ITT) population consists of all enrolled subjects who at Visit 2 are 
randomized to have the study wound be treated with pAF or SOC. 
 
In order to be randomized, the wound area must not have decreased by at least 50% at Visit 
2 relative to Visit 1. Because of the infeasibility of having adjudication of imaging-software- 
derived wound areas in real time at Visit 2, the rectangular approximation to the wound 
area and/or clinician judgment will be used for the Visit 1 and Visit 2 areas to determine if 
the Visit 2 wound area is more than half of the Visit 1 wound area. Furthermore, the study 
inclusion/exclusion criteria are to be reassessed at Visit 2 to ensure the subject is still eligible 
for the study. Receiving investigational treatment to the wound outside of the study between 
Visits 1 and 2 render the subject ineligible to be randomized. 
Even if a ≥ 50% reduction in wound size by Visit 2 causes the subject to be ineligible for 
randomization of one wound to pAF/SOC, the subject will still be considered part of the 
enrolled population. Likewise, even if a subject is not randomized because, e.g., out-of-study 
investigational wound care was received after Visit 1 and before Visit 2, the subject is still 
considered part of the enrolled population. 
 
There is no requirement that the subject receive the randomized treatment (e.g., that pAF 
be injected into the wound of somebody randomized to the pAF treatment arm) to be part of 
this population. Primary and secondary analyses (and exploratory analyses, unless explicitly 
noted otherwise) comparing the arms are to be performed according to the arm to which the 
subject was randomized, even if this differs from the actual treatment received. 
 
4.2.4 Per-Protocol Efficacy Population 
The Per-protocol (PP) population consists of all subjects from the ITT population who meet 
both of the following conditions: 
• The subject did not receive out-of-study investigational wound care between enrollment 
and the earlier of Visit 5 and the time when Visit 5 should have occurred; and 
• The subject received the randomized treatment at Visit 2 and, if applicable, at Visit 
3. Treatment at Visit 3 is applicable unless the wound was healed at the time when 
Visit 3 was to have occurred or the subject had a study-related SAE by the time of 
Visit 3 that precluded treatment administration. Study-related is defined as having a 
classification of “probably related” or “possibly related.” 
 
4.2.5 Closed Wound Efficacy Populations 
Several analyses are only applicable among those whose wound closes between Visits 2 and 
5. The closed wound at Visit 3 (CW3) population consists of all subjects from the ITT 


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population whose wound is closed at Visit 3. The closed wound at Visit 4 (CW4) and closed 
wound at Visit 5 (CW5) populations are analogously defined. 
 
4.2.6 Safety Population 
The safety population (SAFETY) coincides with the ITT population. The SAFETY popula- 
tion admits comparisons between arms. 
 
An additional population for assessing safety will be limited to ITT participants randomized 
to the pAF treatment arm who received at least one pAF injection during the course of the 
study. 
 
5 GENERAL ISSUES FOR STATISTICAL ANALY- 
SES 
 
5.1 Analysis Software 
Analysis will be performed using SASQ
R 
 
 
Software version 9.4 or later whenever possible. 
Other software packages, including R and StatXactQ
R , may be used for particular specialized 
procedures. IVEware is the preferred software for creating the study’s multiply imputed data 
sets. 
 
5.2 Methods for Withdrawals, Missing Data, and Outliers 
The applicability of some measures will depend on whether or not the wound is (or has 
previously) closed. Values that are not observed because they are not applicable will be 
excluded from the pertinent analyses, rather than regarded as missing. Values that are not 
directly obtained but are implied by other measurements will use the implied value. 
 
Multiple imputation (MI) will be applied for analyses of the secondary outcome. Using 
MI leverages partial information and attempts to reduce bias in final inferences that could 
result from a naive complete-case analysis. A minimum of ten data sets will be generated. 
Chained equations will be used along with variable selection; for imputing missing values 
for a given variable, the input variables will be limited to those that increase the R2 value 
by at least 0.01. If feasible, MI will be conducted separately within each treatment arm. 
The pool of variables for the imputation process includes: wound area per the imaging 
software at Visits 1, 2, 3, 4, and 5; treatment arm if infeasible to impute by treatment arm; 
and indicators for comorbidities. The following comorbidities will be considered: diabetes, 
peripheral vascular disease, and immunosuppression. Imputation will exclude individuals 


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who were not randomized. 
 
Visit measurements obtained outside the permissible window may or may not be used in the 
imputation model. We will determine the closest visit number to assign the out-of-window 
visit per the protocol’s timing for follow-up visits. The out-of-window measurement will be 
used for the determined visit number in the case that the visit number does not already have 
a valid measurement (and if there are multiple out-of-window candidate measurements, the 
one closest to the protocol timing will be used). Otherwise, the out-of-window measurement 
will be ignored. 
 
Multiple imputation may also be used for exploratory outcomes, as appropriate. 
 
We do not plan to use different methods for missing data due to withdrawal compared with 
data missing for other reasons. 
 
 
5.3 Multiple Comparisons and Multiplicity 
There is one primary endpoint (occurrence of post-randomization study-related SAEs on 
or before Visit 5), which is not intended to be tested between arms. Instead, descriptive 
summaries and confidence intervals will be constructed by study arm. There is one secondary 
endpoint (percent reduction in wound area at Visit 5 compared to Visit 2), which will be 
tested at α = 0.05. The additional analyses are regarded as exploratory and therefore no 
adjustments are indicated for the multiplicity of testing; each will use α = 0.05. 
 
5.4 Planned Subgroups, Interactions, and Covariates 
The primary and secondary outcomes will be analyzed within the following subgroups; these 
analyses will be considered exploratory even though they are prespecified. Determination of 
subgroups is per Visit 1 assessment, unless stated otherwise. 
• Sex (male, female) 
• Race (white, non-white) 
• Ethnicity (Latinx, non-Latinx) 
• Diabetes Mellitus or high A1C (diagnosis or A1C ≥ 6.5, no diagnosis and A1C < 6.5) 
• Use of immunosuppressive medications (yes, no) 
• Known malignancy other than non-metastatic basal or squamous cell carcinoma (yes, 
no) 
• Nutritional status (albumin level < 3.0, ≥ 3.0) 


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• Blood coagulation conditions (PT/INR > 1.0 or PTT > 15, PT/INR ≤ 1.0 and PTT 
≤ 15) 
• Venous vascular disease affecting the wound area (yes, no) 
• Arterial vascular disease affecting the wound area (yes, no) 
• Visit 1 oxygen saturation (< 90%, ≥ 90%) 
• Concomitant anticoagulation medication (yes, no) 
• Injury type/disease etiology: (ulcer, trauma, post-surgical non-healing wound, other) 
 
For the subgroup analyses, if a subject’s subgroup cannot be ascertained or is not compatible 
with any of the stated options, the individual will be excluded from the analysis of that 
subgroup. 
 
5.5 Derived and Computed Variables 
The definitions of key derived variables will be provided in a separate dataset specifications 
document. For example, age will be calculated as the integer portion of 
Date of informed consent − Date of birth 
. 
365.25 
Primary and secondary outcome definitions are described in Section 7.1. 
 
All derived variables used in analyses will be dual-coded by a second statistician to promote 
quality and minimize error; see also Section 5.6. 
 
5.6 Independent Review 
All statistical analyses for primary reporting of trial results will be independently verified 
through dual programming. Two statisticians will each program all datasets and analyses for 
the DSMB reports and the primary manuscript(s) and the results will be compared. This 
process will begin at the analysis design stage and will continue through writing of abstracts 
and manuscripts for primary analyses. 
 
Additional analyses for secondary abstracts or manuscripts will use a second statistician 
conducting at least code review, if not dual programming. 


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6 INTERIM ANALYSES 
6.1 Frequency of and Timepoints for Interim Analysis 
The only planned interim analyses are for reports to be provided to the Data and Safety 
Monitoring Board (DSMB). The frequency of the interim analyses for the DSMB will be per 
the DSMB charter. 
 
The DSMB will be provided with summaries of adverse events (AEs) and serious adverse 
events (SAEs); some of the AE and SAE summaries coincide with safety outcome analyses 
described in Section 7.6.1. Adverse events will be summarized as to their seriousness, expect- 
edness, relatedness to study, and severity. Overall summaries of the ITT population may 
be provided for the open session of DSMB meetings, but summaries by treatment arm for 
the ITT population will be limited to the closed session for interim analyses. Even overall 
summaries might be limited to the closed session of interim DSMB reports to promote the 
blindedness of study results for the principal investigator until all patients have had time for 
all study-prescribed follow-up. 
 
The closed session will also present adverse event summaries for the subset of ITT subjects who 
were randomized to the pAF treatment arm AND received at least one study-administered 
pAF injection. 
 
6.2 Stopping Rules for Interim Efficacy Analysis 
There is no stopping rule for interim analysis of the secondary efficacy outcome, either for 
demonstrated efficacy or for futility. The study is not adequately powered to spend alpha 
early. 
 
6.3 Subgroups in the Interim Analysis 
No subgroup analyses are planned for the interim analysis. Nonetheless, the DSMB has the 
prerogative to request such subgroup analyses as they deem appropriate. 
 
6.4 Blinding in the Interim Analysis 
Any interim analyses for the DSMB reports will be limited by only including summaries by 
treatment arm in the closed session. Attendance at the closed session will be limited to the 
DSMB members and the DCC biostatisticians. Actual treatment designations will be used in 
the closed session report. 


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7 PLANNED ANALYSES 
7.1 Description of Subject Characteristics 
The primary results will include a summary of the following baseline measures, by randomized 
treatment arm and overall: 
• Sex (male, female) 
• Race (white, non-white) 
• Ethnicity (Latinx, non-Latinx) 
• Age 
• Diabetes mellitus diagnosis or A1C ≥ 6.5 (yes, no) 
Additional measures may also be included. 
 
7.2 Primary Outcome Analysis 
The primary outcome is whether or not the subject has any post-randomization study-related 
SAEs while on study, up to and including the Visit 5 checkpoint (or the last permitted time 
for Visit 5, if Visit 5 was not conducted). The primary analysis of the primary outcome 
implicitly adopts a last-observation-carried-forward approach, in that if such an SAE is not 
identified it will be assumed there was no such SAE. 
 
Safety will be assessed by descriptive reporting of the incidence proportion of study-related 
SAEs in each study group, with upper one-sided 95% binomial exact confidence intervals 
(equivalently, lower one-sided 95% binomial exact confidence intervals for the proportion 
without any study-related SAEs). No statistical hypothesis test is planned to assess safety, 
which is consistent even with large Phase III trials. 
 
 
7.2.1 Additional Analyses of Primary Outcome 
The primary analysis of the primary outcome implicitly adopts a last-observation-carried- 
forward approach. As a sensitivity analysis, it will be assumed all randomized subjects 
without follow-up through Visit 5 had a study-related post-randomization SAE. 
 
The primary endpoint of presence/absence of study-related SAEs will be summarized in the 
ITT population for the primary analysis, but it will also be separately summarized among 
the subset of individuals randomized to the pAF arm who received at least one dose of pAF 
during the study. An exact one-sided 95% binomial confidence interval will be calculated for 
the proportion in this subpopulation who had at least one study-related SAE. 


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1 
  
 
 
7.3 Secondary Outcome Analysis 
The secondary outcome is the percentage reduction in wound area at Visit 5 relative to wound 
area at Visit 2. Because reduction in wound area at Visits 3 and 4 are exploratory outcomes, 
the general definition for deriving percent reduction in wound area at Visit k relative to Visit 
2 is 
1 − Visit k wound area × 100%, 
Visit 2 wound area 
with all measurements expressed as the area determined through the imaging software. 
Wound area will be reported in (cm2), although for purposes of the percent reduction the 
units are immaterial. The percent reductions will be summarized overall and by assigned 
treatment arm for the ITT population, using means and standard deviations or medians and 
interquartile ranges, as appropriate given the observed skewness and presence of extreme 
outliers. 
 
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, any 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. 
 
Note that µpAF (µSOC) represents the average percent reduction in wound area at Visit 5 
compared to Visit 2 for the pAF (SOC) treatment. 
 
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). 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 reasonably symmetric distribution within each 
treatment arm, then a two-sided, two-sample, unpooled t-test will be performed to compare 
the mean percentage reductions at Visit 5 between arms for the ITT population. If the 
distribution is very asymmetrical in one or both arms, a Wilcoxon rank sum test will be used. 
In either case, Rubin’s rules will be used to combine inferences across the multiply imputed 
data sets and the test will be declared statistically significant if the two-sided p-value is ≤ 0.05. 
 
Only the wound randomized to treatment is considered for the secondary outcome. Other 
wounds are to receive standard-of-care and measurements on other wounds are only applicable, 
if at all, to exploratory analyses. See the protocol for further details. 


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7.3.1 
Additional Analyses of Secondary Outcome 
The primary analysis of the secondary outcome explicitly adopts a multiple imputation 
approach. Two sensitivity analyses will be conducted in which missing values are replaced 
with the lowest (highest) observed value in the assigned treatment arm. 
 
 
7.4 Exploratory Outcome(s) Analyses 
7.4.1 Wound Area Reduction at Visits 3, 4 
The percent reduction in wound area at Visit 3 (4) relative to Visit 2 will be summarized 
overall and by assigned treatment arm for the ITT population. 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. The summary 
will include means and standard deviations or medians and interquartile ranges, as appropriate. 
 
If the change in wound size at Visit 3 (4) follows a reasonably symmetric distribution within 
each treatment arm, then a two-sided, two-sample, unpooled t-test will be performed to 
compare the mean reductions at Visit 3 (4) between arms for the ITT population. If the 
distribution is very asymmetrical in one or both arms, a Wilcoxon rank sum test will be used. 
 
7.4.2 Wound Area Reduction at Visits 3, 4, 5, per rectangular wound area 
approximation 
The secondary outcome relies on wound images that are compatible for use with the imaging 
software. However, there is also interest in assessing reduction in wound area for wound areas 
calculated with the rectangular approximation. The area per rectangular approximation at 
visit k, RectangularApproxk, is defined as: 
RectangularApproxk ≡ MaxWidthk × MaxLengthk, 
 
where MaxWidthk and MaxLengthk are the maximal width and length (in cm), at Visit k, 
respectively. 
 
As an exploratory outcome, the percent reductions per rectangular approximation at Visits 
3, 4, and 5 relative to Visit 2 will be analyzed in the same manner as for the percent 
reductions per imaging software. Each analysis of the rectangular wound area approximations 
is considered an exploratory analysis to gauge the sensitivity of the secondary outcome 
analysis. 


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7.4.3 Wound Closure at Visit 5 
An exploratory endpoint is whether or not the randomized wound is closed at Visit 5. Fisher’s 
exact test will be used to test for an association between treatment arm and whether the 
wound is closed at Visit 5. This will be assessed for the ITT population. In the principal 
analysis of this outcome, only participants with a nonmissing Visit 5 would closure status 
will be included. If feasible, multiple imputation will be used to fill in missing wound closure 
statuses, with candidate variables in the imputation model possibly including variables such 
as treatment arm, wound closure status at visits 3 and 4, wound area at Visit 1 per the 
imaging software, and percent reduction in wound area from Visit 1 to Visit 2. 
 
7.4.4 Wound Closure Prior to Visit 5 
Another exploratory endpoint is whether or not the randomized wound was closed at Visit 3 
and/or Visit 4, regardless of whether it later recurred. This will be determined by having the 
wound area per the imaging software assessment be equal to 0. The number and percentage 
of wounds closed prior to Visit 5 will be summarized, overall and by treatment arm, for the 
ITT population. 
 
7.4.5 Wound Recurrence at Visit 5 
This exploratory endpoint is limited to those from the ITT population whose randomized 
wound closed prior to Visit 5. Among this subset, the number and percent whose wound was 
open at the time of Visit 5 will be reported overall and by treatment arm. If the denominator 
is not zero, the corresponding percentage will also be displayed, overall and by treatment 
arm. 
 
7.4.6 Hypertrophic Scar Formation at Visit 5 
This exploratory endpoint is measured by the modified Vancouver Scar Scale (mVSS) total 
score and is applicable to those from the ITT population whose randomized wound is closed 
at Visit 5. Among this subset, the mean and standard deviation (or median and interquartile 
range) will be summarized overall and by treatment arm for the applicable subjects with 
nonmissing mVSS total scores. 
 
7.4.7 Wound Depth Reduction 
The maximal depth of the wound is scheduled to be measured at each study visit. The percent 
reduction in the maximal depth at Visits 3, 4, and 5 relative to Visit 2 will be calculated 
as (1-(maximal depth at Visit k)/(maximal depth at Visit 2))× 100% for k = 3, 4, 5. The 
percent reduction for these visits will be summarized overall and by treatment arm in the 
ITT population. The summaries for each visit number (3, 4, and 5) will use means and 


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standard deviations or medians and interquartile ranges, as appropriate, and will be limited 
at each timepoint to observations with nonmissing values. As for the wound area reduction, 
if any measurements are obtained outside of the permissible follow-up windows the wound 
depth reductions will be standardized by dividing by the number of days since Visit 2. As a 
sensitivity analysis, multiple imputation may be attempted to fill in missing wound depths, 
with summaries of the completed data sets provided, overall and by treatment arm. 
 
7.4.8 Change in Wound-associated Pain 
The wound-associated pain level is scheduled to be measured at each study visit using a 
numerical rating scale: 0–10, with higher numbers indicating more pain. The change in 
wound-associated pain at Visits 3, 4, and 5 relative to Visit 2 will be calculated as (pain 
at Visit k − pain at Visit 2) for k = 3, 4, 5. The change will be summarized overall and by 
treatment arm in the ITT population at timepoints 3, 4, and 5. The summaries will use 
means and standard deviations or medians and interquartile ranges, as appropriate, and will 
be limited at each timepoint to observations with nonmissing values. As a sensitivity analysis, 
multiple imputation may be attempted to fill in missing wound depths, with summaries of 
the completed data sets provided, overall and by treatment arm. 
 
7.4.9 Wound Infection at Visit 4 
An exploratory endpoint is whether or not the randomized wound is infected at Visit 4. 
Version 3.00 of the protocol stated that “incidence of wound infection as determined by 
wound culture assessment from Visit 4” will be descriptively summarized by treatment arm, 
but is open to interpretation as to how wound infection is defined. Wounds that are closed 
at a particular visit will be assumed to not have an infected status at that visit. For open 
wounds, the principal determination will be based on having a culture level of at least 2+ 
per the laboratory’s metric AND having the appearance of an infection, as indicated by a 
database element asking whether the wound appears to be infected; if there is not a valid 
wound culture result for the visit then the database element will be used to determine the 
outcome. As sensitivity analyses, two other endpoint definitions will be considered: one 
ignoring the database element about the wound appearing infected, and the other ignoring 
the laboratory metric in favor of the database element. 
 
The number and percentage of wounds with wound infection at Visit 4 will be summarized, 
overall and by treatment arm. The summaries will be limited to the ITT subjects with 
observable wound infection status at Visit 4. The presence of wound infection at Visit 4 will 
be modeled with ordinary or exact logistic regression, as appropriate, with adjustment for 
infection status at Visit 1 and treatment arm. The two-sided p-value of the treatment effect 
will be compared with α = 0.05 for the principal analysis of this exploratory endpoint, as 
well as for the sensitivity analyses mentioned in the preceding paragraph. 


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7.5 Technical Approaches for Subgroup Analyses 
For each subgroup, the primary outcome and secondary outcome will be analyzed in separate 
models with a main effect for subgroup level, a main effect for treatment arm, and the 
subgroup × treatment interaction effect. The interaction effects are the targets for the 
subgroup analyses. For the primary outcome, exact logistic regression or (ordinary) logistic 
regression will be used, as appropriate. For the secondary outcome, quantile regression for 
the median or linear regression for the mean, as appropriate, will be used. 
 
Because of the modest sample size and large number of subgroups, the exploratory nature of 
these analyses is especially important to emphasize. If any subgroup analyses are not feasible 
to conduct (e.g., no participants have the primary outcome), the infeasible subgroup analyses 
may be omitted. 
 
7.6 Safety Analyses 
7.6.1 Formal Safety Outcome(s) 
In addition to the primary outcome (post-randomization, study-related SAEs by the time of 
Visit 5), there are several exploratory safety outcomes. 
• Whether the subject experienced any post-randomization SAEs while on study (regard- 
less of relatedness to study participation) 
• Whether the subject experienced any post-randomization, study-related AEs while on 
study. 
• Whether the subject experienced any post-randomization unexpected AEs while on 
study. 
The primary outcome is whether or not the subject has any post-randomization study-related 
SAEs while on study, up through and including the Visit 5 checkpoint (or the last permitted 
time for Visit 5, if Visit 5 was not conducted). Safety will be assessed by descriptive reporting 
of the incidence proportion of study-related SAEs in each study group and the confidence 
intervals as described in Section 7.2. 
 
Any Post-randomization SAEs The number and percentage of subjects with at least 
one post-randomization SAE (up through the Visit 5 checkpoint, or through the last permitted 
time for Visit 5, if Visit 5 was not conducted), will be summarized overall and by treatment 
arm for the ITT population. Because this is an ITT analysis, a note will be provided along 
with the summary if there are any subjects randomized to the SOC group who receive one or 
more pAF injections in the wound during the course of the study. If applicable, the note 
would state how many individuals in the SOC arm had pAF administered while on study as 


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well as how many such individuals had any post-randomization SAEs while on study. 
 
As a supplementary analysis, the number and percentage of subjects with at least one 
post-randomization SAE will be calculated for the pAF arm among the subset of the ITT 
population that received at least one study administration of pAF. 
 
Any Post-randomization Study-related AEs The number and percentage of subjects 
with at least one post-randomization study-related AE (up through the Visit 5 checkpoint, 
or through the last permitted time for Visit 5, if Visit 5 was not conducted), will be 
summarized overall and by treatment arm for the ITT population. Note that AEs also 
include SAEs. Because this is an ITT analysis, a note will be provided along with the 
summary if there are any subjects randomized to the SOC group who receive one or more 
pAF injections in the wound during the course of the study. If applicable, the note would 
state how many individuals in the SOC arm had pAF administered while on study as well 
as how many such individuals had any post-randomization study-related SAEs while on study. 
 
In addition, the number and percentage of subjects with at least one post-randomization 
study-related AE will be calculated for the pAF arm among the subset of the ITT population 
that received at least one study administration of pAF. 
 
Any Post-randomization Unexpected AEs The number and percentage of subjects 
with at least one post-randomization unexpected AE (up through the Visit 5 checkpoint, or 
through the last permitted time for Visit 5, if Visit 5 was not conducted), will be summarized 
overall and by treatment arm for the ITT population. Note that AEs also include SAEs, and 
that expectedness is to be noted for each recorded AE. Because this is an ITT analysis, a note 
will be provided along with the summary if there are any subjects randomized to the SOC 
group who receive one or more pAF injections in the wound during the course of the study. If 
applicable, the note would state how many individuals in the SOC arm had pAF administered 
while on study as well as how many such individuals had any post-randomization unexpected 
AEs while on study. 
 
In addition, the number and percentage of subjects with at least one post-randomization 
unexpected AE will be calculated for the pAF arm among the subset of the ITT population 
that received at least one study administration of pAF. 
 
7.6.2 Adverse Events 
The adverse event summaries described for the interim DSMB reports will also be created 
after time has lapsed for all study-related data to have been acquired within the permitted 


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time windows. The final AE summaries may be made available to the principal investigator 
and study sponsor. 
 
8 SAMPLE SIZE DETERMINATION 
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 study-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. 
 
The power calculation depends on several assumptions: 
• That 30 subjects are assigned to each treatment arm and that the secondary outcome 
is observed for all 60 subjects; 
• That each subject has wound closure at Visit 5 (i.e., 100% wound area reduction relative 
to Visit 2) with probability pSOC = 0.2 or ppAF = 0.5 in the SOC and pAF arms, 
respectively; AND 
• That for the remaining subjects in each arm, the wound area reduction percentage is 
uniformly distributed between 0% and 100%. 
Using the Wilcoxon rank sum test (in this scenario, the response is very skewed in the pAF 
treatment arm), the Monte Carlo estimate of power is approximately 54%. 
 
The trial has only modest power to detect statistically significant efficacy of pAF, but this is 
not unexpected for a phase I/II trial. More importantly, the sample size is sufficiently large 
to support the safety of moving on to the next phase of study if no study-related SAEs occur 
in the pAF arm, and the next phase of study would be appropriately powered for assessing 
efficacy. 


 
Created: 
2021-03-11 
By: 
Status: 
Transaction ID: 
Elizabeth Brown (u0843406@utah.edu) 
Signed 
CBJCHBCAABAAvUMP3dZCaNupqQJtavUadHtUrEKt9kc0 
pAF_in_Wounds_SAP_v_1_0 
Final Audit Report 
2021-03-11 
 
 
 
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