Safety and Efficacy of Concentrated Autologous Concentrated Bone Marrow Aspirate (cBMA) in Preventing Wound Complications in Below Knee Amputation (BKA) (The MarrowCHAMP Study)
Phase: PHASE1
Conditions: Peripheral Artery Disease, Vascular Disease, Critical Limb Ischemia
Sponsor: Michael Murphy

Safety and Efficacy of Concentrated Autologous Concentrated Bone Marrow 
Aspirate (BMAC) in Preventing Wound Complications in Amputations 
(The MarrowCHAMP Study)
NCT 02863926
Document Date: 05 November 2021

CHAMP PRO v.05NOV2021 
IRB# 1511774456 
CLINICAL STUDY PROTOCOL 
 Safety and Efficacy of Concentrated Autologous Concentrated Bone Marrow Aspirate (BMAC) in 
Preventing Wound Complications in Amputations 
(The MarrowCHAMP Study) 
Protocol IRB# 1511774456 
Indiana University School of Medicine 
1801 N. Senate Blvd. 
MPC2, #3500 
Indianapolis, IN 46202 
05 Nov 2021 
ETHICS AND REGULATORY COMPLIANCE STATEMENT 
The procedures set forth in this protocol are designed to ensure that the sponsor(s) and principal 
investigator(s) abide by the International Conference on Harmonization (ICH) current Good Clinical 
Practice (cGCP) guidelines, current Good Laboratory Practice (cGLP) guidelines, the Declaration of 
Helsinki, and applicable local regulatory requirements and laws in the conduct, evaluation, and 
documentation of this study. 

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IRB# 1511774456 
Protocol Name 
Safety and efficacy of concentrated autologous concentrated 
bone marrow aspirate (BMAC) in preventing wound 
complications in amputations  
Protocol Number 
1511774456 
Investigational Product Name 
BioCue™ Platelet Concentration System (autologous 
concentrated bone marrow aspirate) 
Author 
Michael P. Murphy, MD 
PROTOCOL HISTORY 
Version Date (dd-mmm-
yyyy) 
Description 
24 Sep 2015 
Initial Release 
12 Apr 2016 
Initial IRB Approval 
25 May 2016 
Amendment 1 
15 Jul 2016 
Amendment 2 
10 Jan 2017 
Annual Renewal + Amendment 3 
15 Mar 2017 
Amendment 4 
24 May 2017 
Amendment 5 
26 Jul 2017 
Amendment 6 
05 Dec 2018 
Amendment 7 – add 6 subjects 
05 Nov 2021 
Amendment 8 – change to BioCue™ device 
CONTACT INFORMATION 
Sponsor 
Indiana University School of Medicine 
Primary Study Contact, Principal 
Investigator 
Michael P. Murphy, MD 
Protocol Author: Michael P. Murphy, MD 
Coordinating Investigator and Principal Investigators   
An updated list of Principal Investigators (PI), investigation sites, and institutions will be 
maintained separately. The definitive list will be provided in the clinical study report. 

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IRB# 1511774456 
PROTOCOL SIGNATURE PAGE 
Protocol #: 151177456 
Safety and efficacy of concentrated autologous concentrated bone marrow aspirate (BMAC) in 
preventing wound complications in amputations  
As an Investigator for this Study, I have read the Clinical Trial Protocol. I agree to make available to the 
Sponsor, Indiana University School of Medicine (or its designee), original source documents and all 
regulatory documents pertaining to this Study.  
By my signature below, I agree to conduct this Study in accordance with the Clinical Trial Protocol, 
current Good Clinical Practice (cGCP) and Good Laboratory Practice (cGLP) guidelines, obligations as 
set forth in Title 21 CFR Parts 812, 54, 56 and 11 (as applicable), and any applicable regulatory laws. I 
will make no changes to protocol-defined procedures without written permission from the Sponsor.   
In compliance with Title 21 CFR Part 812, I will disclose any investigation or research in which I was 
involved that was terminated, with and explanation of the circumstances that led to termination.  
IND 11429 was terminated on June 30, 2011 due to noncompliance with regulatory reporting 
requirements [21 CFR 312.44(b)(viii)]. 
I understand that Investigational Use Products may be used only for the purposes explicitly described in 
this protocol. The BioCue™ Platelet Concentration Kit is investigational. 
CAUTION – Investigational device. Limited by Federal (or United States) law to investigational use. 
I further agree to treat the results of this Study as confidential information and will not submit the 
results of the Study for publication without proper authorization. 
Michael P. Murphy 
11/5/2021 
PRINTED NAME 
SIGNATURE 
DATE 

 
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SYNOPSIS 
Title of Study 
Safety and efficacy of concentrated autologous concentrated bone 
marrow aspirate (BMAC) in preventing wound complications in 
amputations  
Objectives 
 
• 
Determine the safety and explore efficacy of BMAC in preventing 
wound complications in amputations. 
• 
Characterize the host tissue response to, paracrine activity of, and 
fate of transplanted BMAC.  
• 
Determine the effects of chronic limb ischemia and diabetes on 
nerve function and to assess the effects of mesenchymal stem cell 
administration on their function.   
 
Planned Number of 
Subjects and Duration of 
Involvement 
12 patients, 12 month follow up period 
Patient Population 
 
Critical limb ischemia (CLI) patients scheduled for amputation 
Investigational Product 
Name 
Concentrated autologous bone marrow aspirate (BMAC) 
Methodology Overview 
 
This is an open label pilot trial.  
 
Up to twelve (12) patients scheduled for amputation will receive bone 
marrow cells concentrated (BMAC) via the BioCue™ Platelet 
Concentration Kit device injected IM at 25 sites in the leg proximal to 
the amputation in the index limb to prevent ischemic wound 
complications after surgery. BMAC will be injected into the anterior 
tibialis muscle (ATM) below the point of amputation for analytical 
purposes. Patients will be scheduled for amputation at Day 7 post 
injection. Safety will be evaluated by review of treatment related 
adverse events (AE) during the 12-month follow-up period. In subjects 
undergoing BKA, we will compare rates of wound complications and 
conversion from BKA to AKA to historical controls at our institution to 
assess trends in therapeutic efficacy.   
 
Patients will undergo amputation and injection sites will be harvested 
from the ATM at that time. Immunohistochemical staining (IHC) will 
determine capillary density and local host immune responses. 
Angiogenic and inflammatory cytokines will be quantified using a 
multiplex array system and quantitative polymerase chain reaction 
(PCR).    
 
 
 
 
 

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Abbreviations 
ABMNC  
Autologous Bone Marrow Mononuclear Cells  
ACS 
Acute coronary syndrome  
AE 
Adverse event 
AKA  
Above Knee Amputation  
ATM  
Anterior tibialis muscle  
BKA  
Below Knee Amputation  
BMAC 
Concentrated bone marrow aspirate  
cGCP 
Good Clinical Practice 
cGLP 
Good Laboratory Practice 
CHAMP 
Clinical and Histological Analysis of concentrated autologous concentrated bone 
Marrow aspirate (BMAC) in amPutations (MarrowCHAMP) 
CLI 
Critical Limb Ischemia  
CTSI 
Clinical and Translational Sciences Institute 
DMSB 
Data Monitoring and Safety Board  
HIF-1α 
Hypoxia Inducible Factor-1α  
ICA 
Indocyanine angiography  
ICF 
Informed consent form  
ICH 
International Conference on Harmonization  
IHC 
Immunohistochemical staining  
IM 
Intramuscular  
LEADs 
LC-MS 
Lower Extremity Arterial Dopplers 
Liquid Chromatography- Mass Spectrometry  
MHC 
Major Histocompatibility Complex  
MSC 
Mesenchymal Stromal Cell 
PAD 
Peripheral Arterial Disease 
PCR 
polymerase chain reaction  
PHC 
Proangiogenic Hematopoietic Cell 
PI 
Principal Investigator 
SAE 
Serious adverse event 
SDF-1 
Stromal Cell Derived Factor-1  
TcPO2 
Transcutaneous oxygen pressure  

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TABLE OF CONTENTS 
1 
INTRODUCTION ......................................................................................................................................... 8 
2 
DESCRIPTION OF THE INVESTIGATIONAL PRODUCT................................................................................... 9 
2.1 
OVERVIEW 
2.2 
PROPOSED INTENDED USE STATEMENT 
3 
STUDY OBJECTIVES .................................................................................................................................. 11 
4 
STUDY OVERVIEW ................................................................................................................................... 12 
4.1 
STUDY APPROACH 
4.2 
STUDY DURATION 
5 
STUDY POPULATION ............................................................................................................................... 12 
5.1 
SAMPLE SIZE AND TARGET STUDY POPULATION 
5.1.1 
Sample size 
5.1.2 
Study population 
5.1.3 
Alignment with intended study population 
5.2 
RECRUITMENT METHODS 
5.2.1 
Recruitment for Study 
5.2.2 
Duration of study activities 
5.3 
PATIENT SELECTION 
6 
STUDY MATERIALS .................................................................................................................................. 14 
6.1 
INVESTIGATIONAL PRODUCT 
6.1.1 
Identity of the investigational products 
6.1.2 
Safety issues 
6.1.3 
Handling, storage, accountability 
6.1.4 
Required training 
6.1.5 
External studies 
6.2 
OTHER STUDY MATERIALS 
6.2.1 
Materials to be provided by study site 
6.2.2 
Materials to be provided by device manufacturers 
6.2.3 
Materials to be provided by external testing facilities 
7 
STUDY PROCEDURES ............................................................................................................................... 15 
7.1 
WORKFLOW 
7.2 
STUDY DATA 
7.2.1 
Collection of data 
7.3 
PROCEDURES FOR STUDY CLOSURE 
7.3.1 
Routine study close-out 
7.3.2 
Suspension or premature termination of the study 
8 
DATA QUALITY ASSURANCE .................................................................................................................... 20 
9 
STATISTICAL METHODS ........................................................................................................................... 20 
9.1 
DETERMINATION OF SAMPLE SIZE 
9.2 
BIAS MINIMIZATION 
9.3 
PLANNED ANALYSES 

 
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10 
ADVERSE EVENT REPORTING ................................................................................................................... 22 
10.1 
ADVERSE EVENTS 
10.2 
SPONSOR CONTACT FOR SAE REPORTING 
11 
RISK ANALYSIS ......................................................................................................................................... 22 
11.1 
POTENTIAL RISKS OF THE INVESTIGATIONAL PRODUCT AND CLINICAL INVESTIGATION 
11.2 
POTENTIAL BENEFITS OF THE INVESTIGATIONAL PRODUCT AND CLINICAL INVESTIGATION 
11.3 
MINIMIZATION OF RISKS 
12 
INVESTIGATOR RESPONSIBILITIES ........................................................................................................... 23 
12.1 
SITE QUALIFICATION AND STUDY OVERSIGHT 
12.2 
CASE REPORT FORMS/ELECTRONIC DATA RECORDS 
12.3 
ACCESS TO SOURCE DOCUMENTS 
12.4 
FINANCIAL DISCLOSURE 
12.5 
DEVIATIONS FROM THE STUDY PROTOCOL 
12.6 
RECORD RETENTION 
12.7 
PUBLICATION POLICY 
13 
ETHICS AND COMPLIANCE ....................................................................................................................... 25 
13.1 
INFORMED CONSENT AND DE-IDENTIFICATION 
13.1.1 
Prospectively collected data 
13.2 
IRB REVIEW 
13.3 
CONFIDENTIALITY OF DATA AND PATIENT RECORDS 
13.3.1    Provisions to Protect the Privacy Interests of Participants 
14 
REFERENCES ............................................................................................................................................ 27 
 
 
LIST OF TABLES 
Table 1. 
Aspiration Devices and Delivery Accessories Compatible with BioCue™ Device .................... 9 
Table 2. 
Operating Specifications for Biomet Biologics Centrifuge .................................................... 11     
Table 3.  
Inclusion/Exclusion Criteria for Enrollment of Subjects ........................................................ 13 
Table 4. 
Schedule of Events ................................................................................................................ 19 
 
LIST OF FIGURES 
Figure 1. 
BioCue™ Separator Device .................................................................................................... 10 
Figure 2.  
Separator (left); Centrifuge (center); BMAC (right)  .............................................................. 10 
Figure 3.        Scheduling Scheme for Amputations Date and MSC Injections ............................................ 16 
Figure 4.  
Diagram of Injection Sites of BMAC in Below Knee Amputations (BKA)  .............................. 17 
 
 
 

 
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1 
INTRODUCTION 
Critical limb ischemia (CLI) is the endstage of atherosclerotic peripheral arterial disease (PAD) and occurs 
when there is insufficient blood flow to meet the resting metabolic demands of the leg. The clinical 
features of CLI are rest pain and gangrene, both heralding signs of limb loss.1 Despite advances in 
surgical and endovascular revascularization techniques, approximately 40% of patients with CLI will 
require amputation of the affected extremity within one year.2,3 Most surgeons approach amputation in 
neurologically intact patients with the goal to save as much limb length as possible to promote 
independent ambulation with a limb prosthesis. Consequently, below knee amputation (BKA) is the 
most common amputation procedure performed in the U.S.4,5 However, contemporary studies and 
review of our own series demonstrate conversion rates from a BKA to AKA as high as 19% with the 
primary cause being ischemic wound complications of the myocutaneous flaps below the knee.6,7 The 
physiological consequences of loss of the knee joint with AKA, as compared to BKA, is a 40-60% increase 
in energy expenditure in ambulation with a prosthesis.8 This excessive effort results in a decreased rate 
of independent ambulation after AKA as compared to BKA (< 33% vs. > 65%, respectively) and translates 
into decreased quality of life, increased institutionalization, and increased health care costs for AKA 
patients.9,10 Thus, a procedure that may improve perfusion and promote wound healing of a lower 
extremity amputation stump may provide significant benefits to this patient population.  
 
Bone marrow derived progenitor cells have been shown to respond to crosstalk with injured cells to 
limit tissue destruction or enhance repair by a variety of mechanisms that include (a) secretion of 
bioactive proteins that act in a paracrine or autocrine fashion; (b) upregulation of genes that modulate 
excessive inflammatory and immune reactions; and (c) transfer of vesicular components that contain 
mitochondria and microRNAs.11 For example, evidence supports the hypothesis that the predominant 
mechanisms driving tissue repair in a heart post-infarction is angiogenesis orchestrated via the release 
of stem-cell derived paracrine factors.12,13 Cumulative data regarding the bioactivity of bone marrow 
cells has been derived entirely from animal models of disease despite the fact that more than 120 
clinical trials listed in clinicaltrials.gov use bone marrow derived cells for therapeutic endpoints. 
Furthermore, the translation of these data to human conditions is limited by the genomic discordance 
between mouse and human responses in pathological conditions. Seoka and colleagues found that 
among genes that changed significantly in humans in response to inflammatory stresses, the murine 
orthologs were close to random in matching their human counterparts.16 To date, no clinical trials have 
been designed with the ability to harvest human tissue post-cell implantation at time points relevant to 
their tissue reparative activity and survival. Thus the in vivo biological activity and survival of 
transplanted bone marrow derived cells in diseased human tissue remains undefined.14,15 
 
The BioCue™ Blood and Bone Marrow Aspirate (BMAC) Concentration System is used to process blood 
and bone marrow aspirate to produce an autologous PRP output for use in autograft and/or allograft 
therapies. The BioCue™ device is the exact same device as the MarrowStim™ PAD Kit that was used to 
treat subjects in prior versions of this protocol.  Treatment includes removal of approximately 300 mL of 
bone marrow from the patient’s posterior or anterior iliac crest, which is then mixed with an 
anticoagulant solution and processed in the BioCue™ device to concentrate the autologous bone 
marrow cell suspension for delivery.  The subsequent treatment utilizes this BMAC at the point of care 
by injection to multiple sites in the affected leg.  The BioCue™ BMAC product is hypothesized to function 
via angiogenic mechanisms.  To date there are no comprehensive descriptive analyses of the fate of 
BioCue™ BMAC cells after transplantation in human tissue. Thus we propose a pilot trial, Safety and 
Efficacy of Concentrated Autologous Concentrated Bone Marrow Aspirate (BMAC) in Preventing 
Wound Complications in Amputations (The MarrowCHAMP Study), that will help discern the 
mechanisms of therapeutic action in man. MarrowCHAMP will address two critical areas simultaneously: 

 
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assessing the efficacy of BMAC in preventing wound complications in lower extremity amputations and 
providing tissue at BioCue™ injections sites from a controlled time-point for first in man biochemical and 
histological analyses.   
  
 
2 
DESCRIPTION OF THE INVESTIGATIONAL PRODUCT  
2.1 
Overview  
The BioCue™ Kit contains six (6) disposable BioCue™ Separator for the preparation of autologous 
concentrated bone marrow aspirate (BMAC) at the patient’s point of care.  The BioCue™ Kit can be used 
with any legally marketed bone marrow aspiration (BMA) device and associated delivery accessories, 
provided separately (Table 1).  The BioCue™ Kit is used in conjunction with the Biomet Biologics 
Centrifuge, a table-top swinging bucket centrifuge.  
 
Table 1. 
Aspiration Devices and Delivery Accessories Compatible with BioCue™ Kit  
 
Description 
FDA Clearance 
H.S. Hospital Service Bone Marrow Biopsy Needle, or equivalent 
K013070 
Ranfac Bone Marrow Aspiration Needle, or equivalent 
501(k) Exempt Product Code GAA 
StemCor Systems MarrowMiner Biopsy Instrument, or equivalent K071732 
BD 60 ml Luer Lock Syringe, or equivalent 
K980987 
BD 30 ml Luer Lock Syringe, or equivalent 
K980987 
BD 10 ml Luer Lock Syringe, or equivalent 
K980987 
BD 22 G x 1 1/2 in. hypodermic needle, or equivalent 
K021475 
Terumo 23 G x 1 1/2 in. hypodermic needle, or equivalent 
K771203 
Merit Medical 20 ml Luer Lock Syringe, or equivalent 
K981417 
 
The BioCue™ Platelet Concentration Kit is indicated for use in the clinical laboratory or intraoperatively at 
the point of care for the safe and rapid preparation of platelet poor plasma and platelet concentrate 
(platelet rich plasma, or PRP) from a small sample of blood and bone marrow mixture.  The design, 
materials, and processing methods are identical to the previously cleared GPS® II Platelet Concentration 
System in BK090008 and to the MarrowStim™ Concentration Kit and MarrowStim™ Mini Concentration 
Kit in K071934. The BioCue™ Separator is also identical in design to the MarrowStim™ Separators 
previously utilized in this investigation. 
 
The BioCue™ Separator (Figure 1) has a 60 ml capacity and includes a density-tuned dual buoy 
separation system designed for the isolation and separation of nucleated cells. 

 
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Figure 1.  BioCue™ Separator 
 
 
 
 
 
 
 
The use of the BioCue™ device and Zimmer Biomet Biologics centrifuge to concentrate autologous bone 
marrow aspirate is illustrated in Figure 2.  Anticoagulated bone marrow aspirate (60 ml) is loaded into 
the separator through the center port and covered with the white cap (left picture).  The separator is 
placed into the centrifuge and spun at 3,200 RPM for 15 minutes (center picture).  During centrifugation, 
the marrow’s nucleated cells collect between the two buoys within a fixed volume of fluid (6 ml).  The 
red blood cells pack below the bottom buoy and the remaining plasma collects above the top buoy.  
Following centrifugation, the concentrated bone marrow aspirate (BMAC - right picture) containing the 
nucleated cells is re-suspended within the fixed volume of fluid.  The BMAC is then recovered from the 
separator by connecting a syringe to the red capped port and extracting the contents.  
 
Figure 2.  Separator (left); Centrifuge (center); BMAC (right) 
 
 
The operating specifications for the Zimmer Biomet Biologics centrifuge are provided in Table 2.  
 

 
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Table 2. 
Operating Specifications for Zimmer Biomet Biologics Centrifuge 
Principle of Operation  
Separation based on density of liquids  
Table Top  
Yes  
Refrigerated  
No  
Swinging Bucket  
Yes  
Automatic Decanting  
No  
Micro-Processor Controlled 
Yes 
User Programmable 
Yes, preset program automatically displayed when switched on  
Speed Control  
Preset, adjustable  
Acceleration and Braking  
Current-controlled  
Maximum RPM  
3,500 RPM  
Maximum RCF  
2,200 g  
Tube Capacity  
Four 60ml disposable tubes  
Imbalance Detector  
Yes  
Construction  
Anti-torsion construction, metal housing and rotor  
 
 
2.2 
Proposed Intended Use Statement 
Human bone marrow preparations induce angiogenesis, decrease muscle fiber apoptosis, and stimulate 
re-epithelialization of wounds in ischemic hindlimb models in mice. Prior to amputation, bone marrow 
aspirate will be isolated from patients and concentrated using a BioCue™ device (BMAC), thus 
presenting a more clinically feasible and potent cell population that may improve tissue perfusion and 
mitigate ischemic wound complications after amputation.  
 
Concentrated, enriched, autologous, bone marrow aspirate is offered as an option to prevent wound 
complications after amputation. The BioCue™ device is intended for investigational use only to 
concentrate BMA by selected investigators familiar with their use and experienced in conducting clinical 
studies. Human BMAC may only be administered IM to human subjects participating in clinical studies 
sponsored/approved by Indiana University School of Medicine, and who meet all inclusion criteria and 
have provided formal written consent.  
 
3 
STUDY OBJECTIVES 
The principal objectives of this study are as follows:  
 
Determine the safety and explore efficacy of BMAC in preventing wound complications in 
amputations. In a Phase I trial, BMAC (up to 5 mL) will be injected into the ATM below the point of 
amputation in the index limb in an area of approximately 3 x 10 x 3 cm. At the same time, BMAC (1 mL) 
will be injected at approximately 25 sites in the proximal leg to promote tissue perfusion before 

 
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amputation (Figure 4). Patients will be scheduled for amputation at approximately one week post 
injection. Safety will be evaluated by review of treatment related Adverse Events during the 12-month 
follow-up period. Efficacy will be assessed by rates of wound complications and revision of amputation 
to historical controls at our institution to assess trends in therapeutic efficacy.  
 
Characterize the host tissue response to, paracrine activity of, and fate of transplanted BMAC. 
Patients will undergo amputation and injection sites at the anterior tibial muscle (ATM) will be 
harvested at that time. Collection of up to 10 cm of the soleus muscle will serve as control.  IHC staining 
will determine capillary density and local host immune responses. Angiogenic and inflammatory 
cytokines will be quantified using a multiplex array system and quantitative PCR. 
 
The expected outcomes will provide the framework for the design of a Phase II randomized clinical trial 
and critical insights that may direct a future trial to test multi-dosing regimens. This investigation will 
establish a unique clinical model that can expedite safety and mechanistic analyses of projects in PAD 
that include bioscaffolds to enhance cell survival. 16  
 
Determine the effects of chronic limb ischemia and diabetes on nerve function and to assess the 
effects of mesenchymal stem cell administration on their function.  Peripheral nerve tissue (sciatic, 
femoral, tibial, sural and/or plantar nerve segments) will be collected at the time of amputation from 
the amputated portion of the lower limb.  Patients with critical limb threatening ischemia, particularly 
with diabetes, have polyneuropathies that alter motor unit engagement and peripheral sensation 
leading to ambulatory dysfunction and pressure ulceration of the foot.   
 
 
4 
STUDY OVERVIEW 
4.1 
Study Approach 
This protocol is designed to describe a Phase I single center open label trial study that will enroll up to 12 
patients requiring semi-elective amputation within a 30-day period for complications related to CLI. 
 
4.2 
Study Duration 
It is expected that the study will be completed within 5 years.  Each patient will be followed for 52 
weeks. 
 
5 
STUDY POPULATION 
5.1 
Sample Size and Target Study Population 
5.1.1 
Sample size 
The total number of individual subjects is expected to reach approximately twelve (12).   
 
5.1.2 
Study population  
The study population will be representative of adults at least 40 years of age who require major 
amputation, as determined by an independent vascular specialist. It is anticipated that Indiana School of 
Medicine will enroll up to 12 qualifying adults for this study.  
 
5.1.3 
Alignment with intended study population  
The study population includes patients likely to benefit BMAC transplantation. 
 

 
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5.2 
Recruitment Methods 
5.2.1 
Recruitment for Study 
Eligible patients will be invited to participate in the study on a first-come basis, subject to Indiana School 
of Medicine weekly recruitment goals. They will be informed of the possible risks of the procedure and 
will be required to give informed consent before study-specific procedures can proceed. A minimal 
financial inducement will not be offered, and no subject recruitment materials will be used. Each subject 
will be informed that no personally relevant clinical information will be derived from the collected data. 
Medications will be documented, and certain medications may be held. Each subject’s involvement in 
the study will be limited to the period between signing of the informed consent form (ICF) and the 
completion of study specific procedures.   
 
5.2.2 
Duration of study activities 
Enrollment is anticipated to continue for approximately 3 years. In the event that additional studies are 
going to be conducted, new protocols will be developed specifically for those studies. 
 
5.3 
Patient Selection 
The eligibility criteria for prospective enrollment of subjects are shown in Table 1.  
 
Table 3. 
Inclusion/Exclusion Criteria for Enrollment of Subjects  
Inclusion criteria 
1. Be ≥ 40 and ≤90 years of age.  
2. Patients requiring lower extremity major amputation, as determined by 
an independent vascular specialist. 
3. If ulceration or gangrene is present, it is distal to malleoli (to allow 
adequate length of ATM area of approximately 3 cm x 10 cm x 3 cm)  
4. Amputation can safely be performed up to 30 days after screening, as 
determined by an independent vascular or orthopedic surgeon.  
5. Females of childbearing potential must be willing to use one form of 
birth control for the duration of the study. Female participants must 
undergo a blood or urine pregnancy test at screening.  
Exclusion criteria 
1. Patients who are pregnant, planning to become pregnant in the next 12 
months, or lactating. 
2. CHF hospitalization within the last 1 month prior to enrollment.*  
3. Acute coronary syndrome (ACS) in the last 1 month prior to enrollment.*  
4. HIV positive or active, untreated HCV as determined by review of 
medical records.  
5. History of cancer within the last 5 years, except basal cell skin carcinoma.  
6. Inability to provide written informed consent due to cognitive or 
language barriers (interpreter permitted).  
7. Concurrent enrollment in another clinical investigative trial.  
8. Any condition requiring immunosuppressant medications (e.g., for 
treatment of organ transplants, psoriasis, Crohn’s disease, alopecia 
areata).  
9. Presence of any clinical condition that in the opinion of the PI or the 
sponsor makes the patient not suitable to participate in the trial.  
*As defined by the standard definitions of CHF and ACS by the American 
Heart Association. 

 
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Participant 
Withdrawal Criteria 
At the discretion of the investigator or at the request of the participant.  
 
6 
STUDY MATERIALS 
6.1 
Investigational Product 
6.1.1 
Identity of the investigational products  
The BioCue™ Platelet Concentration Device   
6.1.2 
Safety issues 
Safety of the BioCue™ device to concentrate BMA for the prevention of wound complications in 
amputation will be determined by review of treatment related adverse events (AE) and serious 
adverse events (SAE) during the study period of 12 months by the Data Safety Monitoring Board 
(DMSB) appointed by the Clinical and Translational Sciences Institute (CTSI) at the Indiana School of 
Medicine or Data Safety Committee (DMC) designated by the Principal Investigator.  
 
The BMAC dose proposed in this trial was selected based on prior clinical trials.17 We may not see 
any evidence of efficacy in our composite endpoint, which may reflect an inadequate dose or 
sample size. In this case we would propose a future study exploring escalating doses of BMAC. In the 
instance of evolving infection, we would re-assess our inclusion/exclusion criteria and evaluate the 
contribution of pre-amputation procedures on the lower leg. Patients may request earlier 
amputation due to worsening rest pain or anxiety. Consideration for amputation sooner than one 
week post injections will be determined by the PI based on the severity of the subject’s condition, so 
as to offer this research as an option to all eligible subjects.   
 
6.1.3 
Handling, storage, accountability 
Autologous heparin-anticoagulated bone marrow aspirate (360 ml) is loaded into 6 Biocue™ 
separators through their center ports. The separators are placed into the centrifuge and spun at 
3,200 RPM for 15 minutes. Following centrifugation, the BMAC containing the nucleated cells is re-
suspended within the fixed volume of fluid. The BMAC is then recovered from the separator by 
connecting a syringe to the red capped port and extracting the contents. All steps (marrow harvest, 
BioCue™ processing, BMAC administration) are performed at the point-of-care within a single 
procedure. 
Output characterization will be performed on a sample from a single BioCue™ separator chosen 
randomly. A sample will be pulled from one tube, assuming it is representative of all tubes.  The 
number of cells will be enumerated from one separator and multiplied by six (6). In the case that a 
positive culture should result from the BioCue™ product, the PI will notify the patient and schedule 
an examination within 48 hours. The patient will be assessed for evidence of infection including 
evidence of erythema at the site of the injection, fever (temperature > 100.9 F), and/or leukocytosis 
(white blood count >11K). In the event of these clinical and laboratory findings, the appropriate 
antibiotic or antifungal agent will be instituted based on the culture results and recommendations of 
an Infectious Disease specialist. Follow up examinations and laboratory work will be scheduled 
based on the severity of symptoms and laboratory findings at the discretion of the PI and per 
Indiana University School of Medicine procedures. 
Sterility testing of the BioCue™ output will be performed in accordance with 21 CFR 610.12.  This 
information will be recorded on a source document and kept in the subject’s research chart.  Bone 

 
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marrow samples remaining after cell characterization may be subject to additional assessments by 
outside laboratories. Any samples sent outside the site will be identified by subject number only and 
will not contain any identifying information. 
All sterility failures will be reported to the IRB and FDA as adverse events within 30 calendar days. 
The report will include results of the investigation into the cause of the event and any and all 
corrective actions taken to mitigate risks to future study subjects. 
Following the BioCue™ procedure, laboratory analyses will be performed on unconcentrated and 
concentrated bone marrow aspirate samples.  Total white blood cell (WBC) count, mononuclear cell 
(MNC) count, mononuclear cell recovery (concentrated MNC/unconcentrated MNC), granulocyte 
count, platelet count, cell viability, and cell purity (MNC/WBC) will be recorded.  In addition, total 
aspiration volume, total BioCue™ input volume, total BioCue™ output volume, and total delivery 
volume will be recorded.  Additional information, such as CD marker characterization, may also be 
performed.  
6.1.4 
Required training 
Operating room personnel will be instructed on the use of the BioCue™ Kit and the collection and 
transfer of bone marrow preparations. The study coordinator will be trained in the operation and 
management of the centrifugation system. 
6.1.5 
       External studies 
No additional studies are planned at this time.  
 
6.2 
Other Study Materials 
6.2.1 
       Materials to be provided by study site 
• 
Study notebooks to maintain study documents, including signed ICFs, all applicable 
information, and additional forms to be collected and retained by the study institution 
during the course of the study.  
• 
Example spreadsheets for tracking patient information. 
• 
Hank’s Balanced Salt Solution 
• 
Sodium Heparin 
 
6.2.2 
Materials to be provided by device manufacturers 
1) The BioCue™ Platelet Concentration Device 
 
6.2.3 
Materials to be provided by external testing facilities  
There are no external testing facilities associated with this study.  The cellular characterization work 
will be performed at the Indiana University School of Medicine core flow cytometry laboratory.  
 
7 
STUDY PROCEDURES 
7.1 
Workflow  
The necessity for amputation will be determined by a vascular or orthopedic surgeon not participating in 
the study. After that decision has been rendered, the patient will be screened for enrollment. After 
enrollment, 12 patients requiring major amputation for CLI will be scheduled for amputation 
approximately one week post-BMAC injection in the ATM. Autologous BMA will be retrieved per 
standard institution procedures. The BioCue™ device will be used per manufacturer instructions. 
Patients will receive IM injections of BMAC into the ATM below the point of amputation and up to 25 mL 

 
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•
Up to 1 mL BMAC MSC injected IM (each site) above 
amputation at up to 25 sites 
•
Up to 3 mL BMAC MSC injected IM in index leg ATM (3 x 10 x 
3 cm area) 
of BMAC in the proximal leg above the point of amputation. Delivery of up to 25 sites of up to 1 mL each 
of BMAC will be performed in 2 cm increments along the lateral and medial aspects of the upper leg and 
gastrocnemius to ensure an even distribution.  Patients will then undergo amputation approximately 
one week after injections. Patients will be examined at Baseline, BMAC Injection Day, Day of 
Amputation, Day 3 post-op amputation, and Weeks 2, 6, 12, 18, and 24 after amputation.  At any time 
during a subject’s participation, photographs of current wounds (as applicable at baseline visit) and 
photographs of the subject’s amputation site (at follow-up visits) may be taken to assess wound healing.  
There will be a week 52 phone call to assess the patient for primary endpoints. Incidence of wound 
infection, minor (debridement of skin), and major revision (debridement of muscle and bone) of the 
amputation stump, and revision of amputation will be recorded. Rates of revision of amputation at 12 
months will be compared to historical controls. The initiation of treatment will be staggered by a 
minimum of one week for the first 3 subjects to ensure that potential study treatment-related risks to 
subjects are minimized. 
 
Determine the safety and explore efficacy of BMAC in preventing wound complications in 
amputations. In a Phase I trial, BMAC (up to 3 mL) will be injected into the ATM below the point of 
amputation in the index limb. At the same time BMAC (1 mL) will be injected at up to 25 sites in the 
proximal leg to promote tissue perfusion before amputation (Figure3, Figure 4). Patients will be 
scheduled for amputation approximately one week post-injections. Safety will be evaluated by review of 
treatment related AEs during the 12-month follow-up period. Efficacy will be assessed by rates of wound 
complications and revision of amputation to historical controls at our institution to assess trends in 
therapeutic efficacy. 
 
Figure 3. Scheduling Scheme for Amputation Date and MSC Injections 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
                                  
Patients assessed for eligibility 
12 patients enrolled; Scheduled to 
time of amputation approximately one 
week post-MSC injections 

 
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Figure 4. Diagram of Injection Sites of BMAC in Below Knee Amputations (BKA).  
 
 
 
 
                                          
             
 
 
BMAC Administration: Anterior Tibialis Muscle. 
On the day of BMAC injection in the operating room of the Richard L. Roudebush VA Medical Center, the 
index limb will be marked for amputation.  The injection site area is marked on the anterior 
compartment of the lower leg below the point of amputation. The BioCue™ product is injected along a 3 
cm tract into the anterior tibialis muscle (ATM).  The injection site(s) will be marked with permanent ink 
on the ATM. The BMAC suspension will be injected over 3 minutes with the needle retracted 5 mm 
simultaneously to permit infiltration into the muscle.  
 
Ability to Recover BioCue™ Injection Sites in Anterior Tibialis Muscle.     
Retrieval of the BioCue™ injection sites is accomplished at the time of amputation. The area demarcated 
by the surgical marker is excised for histological analysis.   
 
Needle biopsies may be performed below the knee at the time of cell injections or above the knee at the 
time of amputation to obtain skeletal muscle samples for histological and biochemical analysis.  There will 
be no additional MCS injections for this purpose.   
Baseline Screening and Follow-Up Schedule. The following evaluations will be carried out at baseline to 
determine if the patient is eligible for study: (1) Baseline blood tests (complete blood count, serum 
chemistries, eGFR, hsCRP); (2) medical history and physical exam; (3) concomitant medications; (4) 
pregnancy test for women of childbearing age; and (5) 12 lead ECG; and (6) LEADs and/or TcPO2 of 
index leg, as indicated.   In the interest of time to major amputation, we may collect data retroactively to 
determine eligibility.  If eligibility can be determined based on recent prior clinical notes and laboratory 
values, we will use those values for our study purposes and omit corresponding tests from the baseline 
Figure 4. 
Upper Leg and Gastrocnemius Muscle Injections: BMAC will be injected 2-3 cm. deep into the muscle at 
approximately 15 sites 3 cm apart medially and approximately 10 sites laterally, delivering 1 mL of BMAC at each site.   
ATM Injections: BMAC (up to 3 mL) will be injected into the ATM below the anticipated point of amputation in up to 5 
sites within the area of 3 x 10 x 3 cm. 

 
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evaluation.  It will not be considered a protocol deviation if labs are not finalized prior to cell injection 
and amputation; subjects will still be able to participate in the study. 
 
Determination of survival of BioCue™ cells and Quantify angiogenic activity of transplanted BMAC cells. 
Harvested ATM from each time point fixed in 4% paraformaldehyde will be embedded in paraffin and 
sectioned (5µm thickness). Using IHC, sections of ATM will be stained with fluorescent monoclonal 
antibodies against CD34 and CD133 to quantify Proangiogenic Hematopoeitic Cells (PHC) at each injection 
site and each time point. We will further enumerate PHCs in ATM using multiparametric flow cytometry 
(MFC) for CD133+CD34+CD45+CD16-CD14-CD235- cells, as previously described.16,17 Hypoxia-inducible 
factor-1α (HIF-1α), will be quantified and localized in ATM sections with immunostaining, as previously 
performed in ischemic human muscle.16 mRNA for HIF-1α, VEGFF, PDGF, and FGF will be quantified using 
qPCR.16 Chemokine protein expression from muscle homogenates and plasma from blood samples will be 
further quantified with multiplex chemokine analyte panels and liquid chromatography-mass 
spectrometry (LC-MS), focusing on Angiogenin, Angiopoietin-1, Angiopoietin-2, Endostatin, FGF (acidic 
and basic), PDGF-AA, PDGF-BB, PIGF, Thrombospondin-2, VEGF, VEGF-D, Activin, Follistatin, GCSF, TGF-B, 
SDF-1, and HGF, in addition to other proteins associated with the BioCue secretome, as previously 
reported.15  
 
Capillary density will be quantified using IHC for CD31.  Angiogenic and inflammatory cytokines will be 
quantified using multiplex protein arrays from muscle homogenates and peripheral blood samples 
(collected at baseline through week 24 follow up visit). Comparison between BioCue™ injection sites and 
controls will be made. Levels of circulating cytokines from peripheral blood will be compared to baseline.  
Changes in skeletal muscle morphology will be described as compared to healthy controls.  It will not be 
considered a protocol deviation if peripheral blood specimens are missed or outside the window ranges 
of study visits. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Table 4. Schedule of Events 
 
 
Procedures 
Baseline 
Visit 1 
BMAC 
Injection 
Visit 2 
Amp 
Day 
Visit 3 
Day 3 
F/U 
+ 5 
days 
Visit 4 
Week 
2 F/U 
+/- 2 
days 
Visit 5 
Week 
6 F/U 
+/- 4 
days 
Visit 6 
Week 
12 F/U 
+/- 7 
days 
Visit 7 
Week 
18 F/U  
+/- 2 
weeks 
Visit 8 
Week 
24 F/U 
+/-2 
weeks 
Visit 9 
Week 52 
Phone 
Call 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
 
 
Physical Exam 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Vital Signs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Con. Medications 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
AE/SAE Evaluations 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
Laboratory 
evaluations  
X1 
 
 
 
X2 
X2 
X2 
X2 
X2 
 
12 Lead ECG  
X 
 
 
 
X 
 
X 
 
X 
 
LEADs / TcPO2 
X3 
 
 
 
X4 
X4 
X4 
X4 
X4 
 
Photograph of index 
leg / amputation 
site 
X5 
 
 
 
X6 
X6 
X6 
X6 
X6 
 
Schedule 
Amputation 
X 
 
 
 
 
 
 
 
 
 
Research Blood 
Specimen7 
 
X 
X 
 
X 
X 
X 
X 
X 
 
Evaluate for 
infection, gangrene  
 
 
 
X 
X 
X 
X 
X 
X 
X 
Evaluate necessity 
for amputation 
revision 
 
 
 
X 
X 
X 
X 
X 
X 
X 
Screening Laboratory Tests1:  CBC w/diff/plt;  CMP (Sodium, Potassium, Chloride, CO2, Glucose, Calcium,  BUN, 
Creatinine); eGFR, hsCRP 
Baseline only: Serum pregnancy testing (childbearing females)  
Follow-up labs2:  hsCRP  
TcPO2: Transcutaneous Oxygen Pressure; LEADs or Foot TcPO23 will be collected as a baseline measurement, 
TcPO2 of medial and lateral stump site4 will be collected at follow up visits  
Photographs5,6:  Index leg at baseline5, BKA stump site at follow up visits6. It is not considered a protocol deviation 
if photographs were not performed. 
Research blood specimens7: Collected prior to induction of surgery (BMAC Injection Day, and Amputation Day) 
from peripheral vein of up to 30 mL, and at follow-up visits at weeks 2 through week 24. It is not considered a 
protocol deviation if research blood collection is not performed.   

 
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7.2 
Study Data 
Eligible subjects will be invited to participate in the study on a first-come basis. Potential donors must 
provide informed consent to participate. To be eligible for participation, subjects must be at least 40 
years of age.  
 
7.2.1 
Collection of data  
At the discretion of the Investigator and subject to Indiana University School of Medicine 
requirements, data will be collected from each eligible subject providing signed informed consent. 
7.3 
Procedures for Study Closure 
7.3.1 
Routine study close-out 
The study will end when Indiana University School of Medicine has obtained all data necessary to 
complete its studies of the test product. Study close-out will follow Indiana University School of 
Medicine standard procedures and may include, but is not limited to, review of regulatory 
documents, collection of completed case report forms, reconciliation of study records, removal or 
destruction of ancillary study supplies, and informing the Investigator of remaining obligations (e.g., 
record retention, final report submission to the IRB, financial disclosure updates, etc.). 
 
7.3.2 
 Suspension or premature termination of the study 
 
This study may prematurely terminate at any time because of a regulatory authority decision, a 
change in opinion of the IRB, or at the discretion of the Investigator or Sponsor. The study will be 
terminated in the event of a Grade 4-5 unexpected and research related event based on the 
Common Terminology Criteria for Adverse Events (CTCAE), version 4.0.  Enrollment will be 
temporarily suspended for any related or possibly related post-BMAC injection and post-amputation 
infection, bleeding or tissue reaction at the index site that are ≥ Grade 3.  If this trial is temporarily 
suspended or prematurely discontinued, Indiana University School of Medicine will promptly notify 
the Investigator(s) and provide instructions. If the study is temporarily suspended, Indiana University 
School of Medicine will provide guidance on timing and procedures for resuming the study. If the 
study is prematurely discontinued, all study materials must be collected and all study forms 
completed to the extent possible. All such materials must be returned to Indiana University School 
of Medicine upon request with an explanation of the circumstances that led to termination.    
 
8 
DATA QUALITY ASSURANCE  
The study site will be responsible for the accuracy of data.  Indiana University School of Medicine or its 
agent may periodically conduct monitoring visits to ensure the quality of data collection. 
 
9 
STATISTICAL METHODS 
9.1 
Determination of Sample Size  
The study is not powered for efficacy in preventing wound complications of revision of amputation. The 
maximum number of tissue specimens we hope to collect will be twelve (12).  Quantification of 
BioCue™, capillary density and cytokines will be compared between injection sites (between escalating 
doses and control) using a paired Student’s t-test. Serum cytokine levels post BioCue™ injection will be 
compared to baseline levels using a paired Student’s t-test. 
 
9.2 
Bias Minimization 
All subjects meeting the specified eligibility criteria will be enrolled on a first-come basis.  
 

 
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9.3 
Planned Analyses 
The clinical study report will contain only summary data analyses reflecting safety profiles, and summary 
data analyses of efficacy.  All patient data will remain coded during analyses to maintain patient 
confidentiality.  
 
The data management center for this trial will be at the CTSI, or a Data Monitoring Committee (DMC) 
will be appointed by the Principal Investigator. We will use a web-based HIPAA compliant online system 
for data entry and data management, as we are currently doing in our current clinical trials. Data will be 
collected from each enrolled patient using a comprehensive case report form and uploaded into a 
specifically designed database in the OnCore Enterprise Clinical Trial Management System and VA 
REDCap system. All the data will be analyzed and generated using SAS.   
 
Safety Assessment. Primary attention will focus on the safety of the administration of BMAC as 
measured by treatment-related AEs. Treatment-related AEs will be categorized overlapping systems and 
severities. Three categories of systems are cardiovascular, respiratory, or infectious. Two categories of 
severity will be serious adverse (SAE) and major adverse cardiac events (MACE). For completeness, 
instances of AEs may appear in more than one category. Within each of these categories AEs will be 
listed in descending order of frequency for the treatment group. In addition, for each category, the sum 
and difference between the two routes of delivery of the proportions will be reported as percent 
incidence. Binomial confidence Intervals at the 95% confidence level and p-values for these 3 groups will 
be calculated. Since previous trials have not reported AEs with BMAC treatment, confidence intervals 
will be generated by the method of the Wilson Score Interval because they are robust, with good 
coverage probability even for small number of trials and are not degenerate near zero. These intervals 
will be used to summarize the data rather than as any formal inferential statement and to assist the 
Data Monitoring Board in their deliberations. No adjustment for multiplicity will be made.  
 
Exploratory Efficacy. Continuous confidence intervals at the 95% level will be constructed to explore the 
effect of administration of BMAC on the composite endpoint at 6-months of death, revision of 
amputation and gangrene, and will be compared to historical cohorts at our institution. The null 
hypothesis of no change will be rejected if the pairwise confidence intervals does not include zero. The 
critical levels for the multiplicity adjustment will be determined by simple Monte Carlo simulation.  
 
Quantities over time of BMAC will be fit to an exponential decay curve using a residual pseudo-
likelihood procedure and cell half-life (λ) will be estimated.18 Binomial confidence Intervals at the 95% 
confidence level and p-values for these four groups after BMAC transplantation will be calculated for the 
presence or absence of MHC expression and SDF-1 activation. Continuous confidence intervals at the 
95% level will be constructed to explore the differences among the time-tiered administration of BMAC 
for (1) the CD34+CD133+ pro-angiogenic hematopoietic cells recruitment of HIF-1α/SDF-1/CXCR4 to 
ischemic muscle, (2) the quantify of capillary density in muscle fibers using hematoxylin phloxin saffron 
and CD31 counts, (3) VEGF-A,C,D, hepatocyte growth factor, angiopoietin-1 to characterize angiogenic 
cytokine expression, (4) percent coverage, fiber diameter and cross-sectional area to examine changes 
in morphology. The correlation between capillary density (CD31 counts) with tissue perfusion (ICA) for 
each time point will be estimated by Spearman’s rank coefficient.19 
 
10 ADVERSE EVENT REPORTING 
10.1 Adverse Events 
Reporting of adverse events will comply with federal regulations for IDE safety reporting.  AEs occurring 
during the enrollment period should be documented by the Investigator in progress notes but will not 

 
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be collected or analyzed by Indiana University School of Medicine unless considered serious and 
research related by the Investigator. The MedDRA scale will be used to describe adverse events. 
 
Serious adverse events (SAEs) related to the research treatment that are encountered during study 
enrollment will be documented by the Investigator and reported to Indiana University School of 
Medicine within 72 hours upon discovery. SAEs are defined under current cGCP guidelines as events that 
result in one or more of the following: 
• 
life-threatening illness or injury;  
• 
permanent impairment of a body structure or a body function;  
• 
medically necessary in-patient hospitalization;  
• 
medical or surgical intervention necessary to prevent permanent impairment to body 
structure or function; or  
• 
fetal distress, fetal death, or congenital abnormality. 
 
Serious events that affect the rights, safety, or welfare of subjects must be documented on a form and 
be reported within 72 hours to Indiana University School of Medicine and to the Investigator’s IRB 
according to that IRB’s policies. 
 
Any potential deficiencies with the BioCue™ devices (including, but not limited to deficiencies in 
packaging, labelling, and/or device processing) will be reported to Zimmer Biomet for evaluation. 
 
10.2 Sponsor Contact for SAE Reporting 
 
 
 
11 
RISK ANALYSIS 
11.1 
Potential Risks of the Investigational Product and Clinical Investigation 
 
The types of risk associated with IM injection are notably rash, mild fever, and myalgia. These risks are 
mild and rare. These risks are all stated in the consent form.  
 
Further risks include pain, bleeding, and infection following the surgical procedure. Additionally, as with 
any other surgical procedure, the investigational device may be dropped in the surgical theater. Flash 
sterilization will not be allowed, and additional sterile devices will be available to prevent procedure 
termination with minimal delay. 
11.2 
Potential Benefits of the Investigational Product and Clinical Investigation 
Subjects are not expected to benefit in any way from their participation in the study. 
 
The studies made possible by the data collected in this study are expected to lead to the prevention of 
wound complications after major amputation. 
Michael P. Murphy, MD 
1801 N. Senate Blvd. 
MPC2, #3500 
Indianapolis, IN 46202 
 

 
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11.3 
Minimization of Risks 
Although the risk to subjects participating in the study is anticipated to be minimal, the clinician, at 
his/her discretion, will not collect data from those individuals for whom collection is judged to pose an 
unusually high risk of physical or mental harm or discomfort. 
 
An additional risk is the event that a syringe containing the bone marrow product or the centrifugation 
tube is dropped. In this case the syringe or tube would be recovered, swabbed externally with alcohol, 
the needle/cap removed and replaced with a sterile needle, and then the sterile contents containing the 
bone marrow product will be emitted into a sterile container on the sterile surgical field and reloaded 
into a new sterile 10 cc syringe and needle. The dropped syringe will be discarded and not contact the 
sterile field(s). In the rare event that the investigator feels that the bone marrow product comes into 
contact with the non-sterile environment then the bone marrow product will be discarded, which is a 
total volume of 6 mL of the planned 30 mL to be injected. Additional bone marrow will not be aspirated 
and the patient will receive 25 mL rather than 30 mL of bone marrow product. It must be emphasized 
that the sterile field encompasses the area of the patient’s limb continuous by sterile drapes to the 
operating room table where the bone marrow product is loaded into syringes, thus the likelihood of the 
syringe contacting a non-sterile surface is minimal. Likewise, the centrifugation tube is a closed sterile 
system with fixed caps and should it fall to the floor the contents would remain in the closed sterile 
system and recovered as described. 
 
Participation in this study poses no risk to study personnel other than that normally encountered during 
standard practice. These risks will be minimized by adherence to the following guidelines: 
 
• 
Personnel should wear appropriate personal protective equipment to avoid contact of 
the eyes or skin with hazardous materials or products derived from biological sources.  
 
12 INVESTIGATOR RESPONSIBILITIES 
12.1 
Site Qualification and Study Oversight 
The PI is responsible for general administration of the study.  
Before the study, the PI must: 
• 
Obtain approval to conduct the study from the study site’s IRB; 
• 
Sign the Protocol Signature Page themselves and have all sub-investigators sign the 
Protocol Signature Page and return it to Indiana University School of Medicine; 
• 
Provide financial disclosures to Indiana University School of Medicine for themselves 
and all sub-investigators participating in study conduct, per Title 21CFR 54 (see Section 
12.4 below).  
 
During the study, the PI must ensure that:  
• 
The study is conducted ethically;  
• 
Case report forms (CRFs), including Subject ICFs, are provided with each transfer of data 
requiring informed consent; and 
• 
All other study forms are completed as instructed by Indiana University School of 
Medicine. 
• 
Investigators, primary care physicians, and all enrolled diabetic subjects follow the 
American Diabetes Association guidelines for diabetic care throughout the trial. 
 

 
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In the case of completion or termination of the study or an Investigator's role in the study, or at Indiana 
University School of Medicine request, all study materials must be returned to Indiana University School 
of Medicine. 
 
12.2 Case Report Forms/Electronic Data Records 
As used in this protocol, the term CRF should be understood to refer to either a paper form or an 
electronic data record or both, depending on the data collection method(s) used. 
 
Original CRFs are the sole property of Indiana University School of Medicine and should not be made 
available in any form to third parties, except for authorized representatives of Indiana University School 
of Medicine or appropriate regulatory authorities, without written permission from Indiana University 
School of Medicine.  
 
It is the PI's responsibility to ensure completion, review, and approval of all CRFs. CRFs must be signed 
by the PI or by an authorized staff member. These signatures serve to attest that the information 
contained on the CRFs is true. At all times, the PI has final personal responsibility for the accuracy and 
authenticity of all clinical and laboratory data entered on the CRFs.   
 
12.3 Access to Source Documents 
Indiana University School of Medicine or its agents and appropriate regulatory authorities shall be 
granted direct access to all study-related documents to perform verification that the protocol and all 
applicable current cGLPs, GCPs, and regulations are being followed and to confirm that study documents 
are complete and accurate. It is important that Investigator(s) and their relevant personnel be made 
available during monitoring visits and any audits or inspections, and that sufficient time is allotted for 
the process.  
 
12.4 Financial Disclosure 
Investigators must provide Indiana University School of Medicine with sufficient, accurate financial 
information in accordance with local regulations to allow Indiana University School of Medicine to 
submit complete and accurate financial certification or disclosure statements to the appropriate health 
authorities. Investigators are responsible for providing information to Indiana University School of 
Medicine concerning their relevant financial interests during the course of the study and for 1 year after 
completion of the study. Conflicts of interest should be disclosed as required by law.  
 
12.5 Deviations from the Study Protocol 
An Investigator may not deviate from the study protocol without prior approval by Indiana University 
School of Medicine unless the deviations are necessary under emergency circumstances to protect 
the rights, safety, or well-being of human subjects or the scientific integrity of the clinical 
investigation. These deviations must be documented and promptly reported to Indiana University 
School of Medicine and, if applicable, to the IRB providing oversight of the study. Protocol deviations 
may result in corrective and preventive actions and/or disqualification of the Investigator.  If 
research blood draws (Research Blood Specimen†) are not obtained at time points specified in the 
schedule of events, they will not be considered protocol deviations. 
     
12.6 Record Retention 
To enable evaluations and/or audits from regulatory authorities or Indiana University School of 
Medicine, the PI and all sub-investigators agrees to retain all study records, including copies of all CRFs, 
UADE forms, and source documents, for 3 years following completion of the project dependent upon 

 
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the study data. The Investigator must obtain the Indiana University School of Medicine written 
permission before disposing of any records, even if retention requirements have been met. 
 
If an Investigator relocates, retires, or for any other reason withdraws from the trial, Indiana University 
School of Medicine must be notified in advance, and study records must be transferred to a designee 
acceptable to Indiana University School of Medicine. This designee might be another Investigator, 
another institution, or Indiana University School of Medicine itself. 
 
12.7 Publication Policy 
The results of this study will be submitted for publication to a medical journal.  The PI agrees that any 
publication of data from this study will comply with Indiana University School of Medicine publication 
policy, the instructions to authors outlined by the editor of the journal or conference proceedings where 
the data is to be published, and the spirit of recommendations made in the good publication practice 
guidelines (GPP2) of the International Society of Medical Publication Professionals. Indiana University 
School of Medicine has the right to review any manuscripts, presentations, or abstracts that originate 
from this study or that utilize these data before they are submitted for publication or other means of 
communication. 
 
13 ETHICS AND COMPLIANCE 
13.1 
Informed Consent and De-Identification  
13.1.1 Prospectively collected data 
All subjects will be given a copy of the IRB-approved ICF to review before their study participation 
begins. The Investigator or authorized study personnel will explain all aspects of the study in lay 
language and answer all of the potential participant’s questions regarding the study. If the 
participant decides to participate in the study, they will be asked to sign and date the ICF. Subjects 
who refuse to participate or who withdraw from the study will be treated without prejudice. 
 
13.2 IRB Review 
The PI is required to obtain IRB oversight of the research study. The IRB must be provided with the 
Indiana University School of Medicine-approved study protocol. Performance of the study may not begin 
until written evidence of IRB approval has been provided to Indiana University School of Medicine.  
 
The conduct and performance of this study will be in accordance with applicable Sponsor and 
Investigator responsibilities as described in Title 21 CFR 812 and other GCP guidance.  
 
IRB/Ethics Committee oversight will be required as human subjects or data from humans are being used. 
This protocol and the associated informed consent document(s) (if applicable) must be submitted to the 
IRB for review and approval. Performance of the study at a given site may not begin until written 
evidence of IRB oversight has been provided to an Indiana University School of Medicine study manager. 
IRB Review and approval must comply with Title 21 CFR 812 Subpart D. 
 
13.3 Confidentiality of Data and Patient Records 
The study institution shall keep all records associated with this study for at least 3 years, as specified in 
Section 12.6. Investigators will keep all records associated with this study for at least 3 years. 
 

 
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13.3.1    Provisions to Protect the Privacy Interests of Participants 
The PI and/or study institution shall provide sufficient information to allow the IRB to evaluate the 
researcher’s provisions to maintain the confidentiality of data. 
 
Privacy data will be maintained in accordance with HIPAA and other applicable policies and local law.  
 
 
14 REFERENCES 
 
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