ICG Fluorescence Imaging in Trauma Patients
Conditions: Trauma Injury
Sponsor: Dartmouth-Hitchcock Medical Center

Cover Page for ClinicalTrials.gov 
Document: Protocol 
Official Study Title: FLUORESCENCE IMAGING TO GUIDE DEBRIDEMENT OF FRACTURE AND INFECTION 
Document Date: April 23, 2021 
NCT04245111 
 

CONFIDENTIAL
CLINICAL RESEARCH PROTOCOL
FLUORESCENCE IMAGING
TO
GUIDE
DEBRIDEMENT
OF 
FRACTURE AND INFECTION
A Phase I open label, single-center clinical trial of fluorescence imaging for predicting complications in 
orthopaedic surgical patients
Principal Investigators:
Leah Gitajn, MD
Department of Orthopaedic Surgery 
Dartmouth-Hitchcock Medical Center 
One Medical Center Drive
Lebanon, NH 03756
603-650-5133
Shudong Jiang, PhD
Thayer School of Engineering 
Dartmouth College
Hanover, NH 03755
603-646-2884
Regulatory Sponsor:
Shudong Jiang, PhD
Funding Sponsors:
Orthopaedic Department
Dartmouth Hitchcock Medical Center 
Lebanon, NH 03756
(603) 653-6187
Study Product:
ICG fluorescence imaging in trauma patients
Protocol Number:
eIND Number:
Not applicable
Initial version:
Jan. 14 2019

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Table of Contents
LIST OF ABBREVIATIONS ...............................................................................................................................IV
STUDY SUMMARY......................................................................................................................................1
1 INTRODUCTION................................................................................................................................4
1.1
BACKGROUND INFORMATION.................................................................................................................4
1.2
INVESTIGATIONAL IMAGING AGENT......................................................................................................5
1.3
CLINICAL DATA TO DATE...................................................................................................................6
1.4
DOSE RATIONALE..................................................................................................................................6
1.5
POTENTIAL RISKS AND BENEFITS...........................................................................................................7
1.5.1
Potential Risks...........................................................................................................................7
1.5.2
Potential Benefits ...........................................................................................................................8
2 STUDY OBJECTIVES .......................................................................................................................9
3 STUDY DESIGN...................................................................................................................................9
3.1
GENERAL DESIGN ............................................................................................................................9
3.2
PRIMARY STUDY ENDPOINTS.................................................................................................................9
3.3
SECONDARY STUDY ENDPOINTS ...........................................................................................................9
4 SUBJECT SELECTION AND WITHDRAWAL ..................................................................................9
4.1
PATIENT SELECTION..............................................................................................................................9
4.2
INCLUSION CRITERIA............................................................................................................................10
4.3
EXCLUSION CRITERIA..........................................................................................................................10
4.4
SUBJECT RECRUITMENT AND RETENTION ............................................................................................11
4.5
EARLY WITHDRAWAL OF SUBJECTS ................................................................................................11
4.5.1
When and How to Withdraw Subjects....................................................................................11
4.5.2
Data Collection and Follow-up for Withdrawn Subjects..............................................................13
5 STUDY DRUG AND DEVICE...............................................................................................................13
5.1
DESCRIPTION ......................................................................................................................................13
5.2
PREPARATION AND ADMINISTRATION OF STUDY DRUG ......................................................................13
5.3
SUBJECT COMPLIANCE MONITORING ...............................................................................................14
5.4
PRIOR AND CONCOMITANT THERAPY ...................................................................................................14
5.5
PACKAGING.................................................................................................................................14
5.6
BLINDING OF STUDY DRUG .............................................................................................................14
5.7
RECEIVING, STORAGE, DISPENSING AND RETURN.............................................................................14
5.7.1
Receipt of Drug Supplies..............................................................................................................14
5.7.2
Storage..................................................................................................................................14
5.7.3
Dispensing of Study Drug ..........................................................................................................14
5.7.4
Drug Accountability.......................................................................................................................14
5.7.5
Return or Destruction of Study Drug ........................................................................................15
6 STUDY PROCEDURES...................................................................................................................15
7 STATISTICAL PLAN........................................................................................................................17
7.1
SAMPLE SIZE DETERMINATION ............................................................................................................17
7.2
DATA AND STATISTICAL ANALYSIS........................................................................................................17
7.3
SUBJECT POPULATION(S) FOR ANALYSIS ...........................................................................................19
8 SAFETY AND ADVERSE EVENTS .................................................................................................19
8.1
IRB REPORTING REQUIREMENTS FOR ADVERSE EVENTS & UNANTICIPATED PROBLEMS.......................19
8.2
INSTITUTIONAL SAFETY AND DATA MONITORING.................................................................................20

CONFIDENTIAL
9
DATA HANDLING AND RECORD KEEPING .......................................................................................20
9.1
CONFIDENTIALITY ................................................................................................................................20
9.2
CASE REPORT FORMS......................................................................................................................20
9.3
RECORD RETENTION...........................................................................................................................20
10
CLINICAL SITE MONITORING..........................................................................................................20
10.1
ON-SITE MONITORING.........................................................................................................................20
10.2
AUDITING AND INSPECTING ..................................................................................................................20
11
ETHICAL CONSIDERATIONS.................................................................................................................20
12
STUDY FINANCES............................................................................................................................20
12.1
FUNDING SOURCE........................................................................................................................20
12.2
CONFLICT OF INTEREST.......................................................................................................................20
13
REFERENCES...........................................................................................................................................21

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List of Abbreviations
Page IV
AE：
Adverse Event/Adverse Experience
AUC:
Area under the curve
CIS:
Center of Innovation Surgery
CT:
Computed tomography
CTSA:
Clinical and Translational Science Institute 
CCRC:
Clinical Cancer Review Committee CFR： 
Code of Federal Regulations
CMS:
Centers for Medicare and Medicaid Services 
CRF:
Case Report Forms
CPHS:
Committee for the Protection of Human Subjects 
DCC：
Data Coordinating Center
DCE：
Dynamic contrast-enhanced
DCE-MRI:
Dynamic contrast-enhanced magnetic resonance imaging 
DHMC:
Dartmouth Hitchcock Medical Center
DSMC:
Data and safety monitoring committee 
FDA:
Food and Drug Administration
FI
Fluorescence imaging
GCP:
Good Clinical Practice
Gd-DTPA:
gadolinium-diethylenetriamine penta-acetic acid 
GFR:
Glomerular filtration rate
HVHC:
High Value Health Care 
ICG:
Indocyanine green
HIPAA:
Health Insurance Portability and Accountability Act 
IACUC:
Animal Care and Use Committee
ICF：
Informed Consent Form
iFI:
Intraoperative Fluorescence Imaging
IRB:
Institutional Review Board
Imax :
Maximum ICG fluorescence intensity
Imax-post:
ICG fluorescence intensity at post-debridement 
ISM:
Independent Safety Monitor
LAR:
A legally authorized representative 
MRI:
Magnetic resonance imaging
NIASM:
National Institute of Arthritis and Musculoskeletal and Skin Diseases 
NIH:
National Institutes of Health
NIST:
National Institute of Standards and Technology 
NCCC:
Norris Cotton Cancer Center
NSF:
nephrogenic sclerosing fibrosis
OCTOM:
Office of Clinical Trials Operations and Management, NIDCR, NIH 
OHRP:
Office for Human Research Protections
OHSR:
Office of Human Subjects Research 
OTA:
Orthopaedic Trauma Association 
PI:
Principle investigator
PROs:
Patient reported outcomes 
QA:
Quality assurance
QC:
Quality Control
RCTs:
Randomized controlled trials 
REB:
Research Ethics Board
ROC:
Receiver operating characteristic
SAE:
Serious Adverse Event/Serious Adverse Experience 
SOP:
Standard Operating Procedure
SSI :
Surgical site infection

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Study Summary
Title
Fluorescence imaging to Guide Debridement of Fracture and Infection
Short Title
ICG fluorescence imaging in trauma patients
Protocol Number
N/A
Phase
Phase I
Methodology
This will be a prospective observational trial to better understand the range and 
variation associated with bone/soft tissue perfusion in fracture patients and 
examine the relationship between perfusion, measured using quantitative 
Indocyanine green (ICG) fluorescence and complications such as surgical site 
infection (SSI), persistent SSI, and fracture nonunion. Primary outcome 
measure is complication (either infection, recurrent infection or nonunion).
Eligible consenting patients will receive standard of care treatment for 
their fracture or infection including irrigation and debridement of their operative 
site and/or fracture fixation. Participation in this study has no specific benefit to 
the subject. This treatment is not expected to alleviate the need for surgical 
intervention. After exposure, 0.1 mg/Kg ICG will be injected intravenously and 
video rate ICG fluorescence images will be acquired 20 seconds before and 4 
minutes after the injection, each before and after debridement. A subset of 38 
post-fracture complication patients will undergo surgical treatment for their 
infection in Center of Innovation Surgery (CIS) and have either an 
intraoperative DCE-MRI after exposure but before the ICG injection or a 
preoperative DCE-MRI. Patients receiving surgery in the CIS will be required 
to sign an additional CIS-specific consent document, Informed consent with 
MRI.
The need for repeat surgical procedure will be left up to the treating surgeon. 
If repeat procedure is needed, pre- and post-debridement quantitative ICG 
fluorescence images will be obtained at each procedure.
Study participants will be followed at 2 weeks, 6 weeks, 3 months and 6 
months from their index study surgery. Complication including index infection, 
recurrent infection or delaying union/nonunion will be identified at the time of 
diagnosis and/or during each participant’s assessment that occurs during 
routine outpatient clinic visit. Detailed information on the infection including 
date of diagnosis, participant signs and symptoms, culture test results, method 
of treatment(s), and date of resolution will be documented.
Study Duration
Five years
Study Center(s)
Single-center, Dartmouth-Hitchcock Medical Center
Objectives
The primary study objectives are to (1)  establish the range and variation 
associated with bone/soft tissue perfusion in fracture patients and (2) examine 
the relationship between perfusion, measured using quantitative ICG 
fluorescence and  complications such as  SSI, persistent  SSI, and fracture 
nonunion.
Number of Subjects
(1) 176 patients with open fracture patients; (2) 82 patients with a complications 
(infection or nonunion) following surgical treatment of fracture; (3) 100 patients 
with closed fracture.

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Diagnosis and Main 
Inclusion Criteria
Open Fracture Cohort (Cohort 1)
a.
Patients 18 years of age or older.
b.
Open extremity fracture.
c.
Planned definitive  fracture management with  external fixation, internal 
fixation, or joint fusion.
d.
Will have all planned fracture care surgeries performed by a participating 
surgeon or delegate.
e.
Provision of informed consent.
Complication Cohort (Cohort 2)
f.
Patients 18 years of age or older.
g.
Extremityfracture.
h.
Prior definitive fracture management with external fixation, internal fixation, 
or joint fusion.
i.
Superficial, deep, or organ space SSI (as per CDC criteria) at the fracture site 
that requires operative management.
j.
Will have all fracture care surgeries performed by a participating surgeon or 
delegate.
k.
Provision of informed consent.
Closed Fracture Cohort (Cohort 3)
l.
Patients 18 years of age or older
m.
Closed extremity fracture
n.
Planned definitive  fracture management with  external fixation, internal 
fixation, or joint fusion.
p.  Provision of informed consent.
Study Product, Dose, 
Route, Regimen
This protocol is an imaging study which does not specify any change in 
treatment protocol or and will not alter the surgical course for the patient 
enrolled, therefore should not be considered an experimental treatment.. ICG 
is an FDA approved fluorescence probe. After skin exposure, 0.1 mg/Kg ICG 
will be injected intravenously, each before and after debridement. Video rate 
ICG fluorescence images will be acquired 20 seconds before and 4 minutes 
after ICG injection, by an FDA approved fluorescence imaging system, (Spy 
Elite (Novodaq/Stryker US). The imaging is non-contact and the imaging head 
is at least 50 cm away from the bone.
Duration of 
administration
Patients are enrolled in the study from time of consent prior to surgery to the 
end of six months of the follow up period.
Reference methods
N/A
Statistical 
Considerations
Descriptive statistics will be present (mean and standard deviation) with 95% 
confidence intervals. We are planning to collect ICG data before and after the 
debridement. Pearson correlation between ICG parameters (pre-debridement, 
or/and post-debridement, or/and changes between pre- and post-debridement) 
and index infection/recurrent will be calculated for each of three patient 
cohorts). We will also include the corresponding coefficient of determination 
indicating how much variation in index infection/recurrent can be explained in 
ICG parameters. Finally, we will evaluate the significance of the correlation 
coefficient using two sided t-test. P-value less than 0.05 will indicate that the 
correlation is significant. Then, we will form receiver operating characteristic 
(ROC) curves to illustrate graphically the performance and to obtain the 
threshold for differentiating the recurrent infection from non-infection group, of 
each chosen model. Area under the ROC curve will be used to determine the 
optimal property for differentiating the recurrent infection patients from the non- 
infection patients.

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1 Introduction
This document is a clinical research protocol and describes a human research study. This study will 
be conducted according to US and international standards of Good Clinical Practice (FDA Title 21 part 
312 and International Conference on Harmonization guidelines), applicable government regulations and 
Institutional research policies and procedures. All personnel involved in the conduct of this study have 
completed human subject protection training and primary oncologic surgeons performing study 
procedures will be trained on this protocol
1.1 Background Information
The focus of this Phase 1 clinical study is to (1) establish the range and variation associated with 
bone/soft tissue perfusion in fracture patients, using ICG fluorescence imaging; (2) examine the 
relationship between perfusion and complications such as surgical site infection (SSI), persistent SSI, 
and fracture nonunion; (3) to determine whether the quantitative ICG fluorescence can be used to guide 
bony debridement in the setting of open fracture or infected fracture to minimize complications. This 
research has been funded by the SYNERGY Pilot Translational Research Grant and by the Department 
of Orthopaedics.
Infection following trauma is one of the most prevalent and challenging complications faced by 
orthopedic surgeons in both military and civilian populations, occurring after up to 60% of open bone 
fractures1-7. Several factors specific to this trauma place patients at high risk for infectious complications, 
including: traumatized tissues, open contaminated fracture, soft tissue coverage issues, catabolic state 
due to polytrauma, prolonged hospitalization with exposure to nosocomial bacteria, and presence of 
metallic implants8. Infection requires one or more unplanned surgical procedures and leads to prolonged 
morbidity, loss of function, and potential loss of limb1, 9-11. Failed treatment for bone infection results in 
recurrent infection, requiring repeat surgical procedures in approximately 30% of patients.12-15
Deficient perfusion prevents delivery of antibiotics and endogenous immune cells to traumatized 
tissues. In the setting of open fracture and established infection, poorly perfused bone is a nidus for 
biofilm formation creating resistance to antibiotics. Because of this, initial management of open fractures 
and the management of SSIs at the fracture site are based on aggressive, thorough debridement in an 
effort to remove all poorly perfused bone. However, there is a critical lack of data to objectively inform 
bone perfusion and the corresponding amount of tissue to debride. This extensive gap in our 
understanding leads to substantial variation in extent of debridement and places patients at unnecessarily 
high risk for initial SSI and recurrent SSI.
While, to date, there is no gold standard technique established to measure bone perfusion in human 
patients, dynamic contrast-enhanced MRI (DCE-MRI) is the imaging modality that has been used most 
frequently and effectively to measure bone perfusion in patients without metal implants implants16-21.
Because of this, developing a better understanding of the relationship between DCE-MRI, which 
assesses the bone and surrounding soft tissues in a three-dimensional format and ICG-based DCE-FI, 
which is surface-weighted, is an important next step. Data acquired from intraoperative DCE-MRI and 
DCE-FI will be used to create a fluorescence-imaging specific model which will correct for the surface- 
weighted nature of fluorescence imaging and more accurately reflect the fluorescence intensity and 
kinetics throughout the entire depth of the bone.
There are currently no accepted intraoperative tools that can be used to make objective decisions 
about bone perfusion. Methods currently used to guide debridement are quite rudimentary. Clinical 
judgement is based on the gross appearance of soft tissue and bone, including color, turgor, and extent 
of soft tissue stripping. A burr may be used to look for bleeding bone.  More extensive debridement is 
thought to minimize risk of index infection or reduce the rate of persistent infection; however, this comes 
at the cost of increasingly complex reconstructive procedures to fill bony defects22 23, 24. Clearly what is 
needed is a functional imaging system which can assess vascular perfusion of surgical field tissue to  
guide surgeons in the amount of tissue to debride. In turn, this will lead to fewer infections and a more 
effective treatment of SSIs at the fracture site. Both scenarios will allow patients to return to duty or work 
sooner.

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.3 Preclinical Data
1.2 Investigational imaging method
ICG fluorescence imaging has been used to assess tissue perfusion in vivo in real-time intra-operative 
arterial and lymphatic perfusion imaging, 25, 26 osseous flap perfusion imaging27. Compared to the 
conventional medical imaging modalities, such as computed tomography (CT) or magnetic resonance 
imaging (MRI), ICG fluorescence imaging is nonionizing, low-cost, and portable. ICG is a dye that 
absorbs near-infrared light in the wavelength range between 790 to 805 nm and has an emission at the 
peak of 835 nm. ICG is almost 98% plasma protein bound so that once it takes up into the 
microcirculation, it remains within the lymphatic and circulatory vasculature. ICG has been approved for 
clinical use by FDA since 1959.28   Since then, ICG has been used for human in angiography for 
ophthalmology and cardiology applications and is given routinely to patients in these clinical settings. In 
addition, as the clinical trials, ICG fluorescence imaging has been used to guide laparoscopic surgery, 29 
robotic adrenalectomy,30 assess tissue perfusion in vivo in real-time intra-operative arterial and lymphatic 
perfusion imaging,25, 31, 32 and tissue flap perfusion imaging.33, 34 ICG is ideal to measure perfusion/blood 
flow because it binds efficiently to blood lipoproteins and thus does not leak from circulation. In addition, 
the ICG half-life is short making repeated measures possible, and it is indirectly activated, so that the
dynamic fluorescence due to bone and tissue 
perfusion can be captured by a video rate 
imaging system.
The dynamic fluorescence imaging 
systems used in this study will be SPY Elite 
(Novodaq/Stryker). Both of them are the 
publicly available imaging systems that can be 
used to assess tissue perfusion in real time in 
the operating room. The systems have a multi- 
directional imaging arm which contains a near- 
infrared light source that illuminates the 
fluorescent agent within the tissues, an HD 
video camera that captures the intensity of 
fluorescent marker in real-time, and software 
that allows the user to capture relative and 
absolute perfusion values within the surgical
field.  This product has been FDA approved for use to monitor blood flow, plastic surgery, microsurgery, 
reconstructive surgery, gastrointestinal imaging and coronary bypass surgery.
We have conducted work utilizing a 
porcine model to demonstrate measurable, 
reproducible and predictable differences in 
bone perfusion as measured by quantitative 
ICG fluorescence in five conditions: (a) 
baseline, (b) two diaphyseal osteotomies, 
separating 1cm of tibia with circumferential soft 
tissue stripping (disrupting both periosteal and 
endosteal 
blood 
supply), 
(c) 
~2 
cm 
circumferential soft tissue stripping (disrupting 
periosteal blood supply) from the proximal 
bone close to osteotomy, (d) circumferential 
soft tissue stripping of the entire proximal 
bone  segment  (disrupting  periosteal  blood
supply), and (e) circumferential soft tissue stripping of the entire bone (extensively disrupting periosteal 
blood supply). This study protocol has been approved by Dartmouth College’s Institutional Animal Care and 
Use Committee (IACUC). Before imaging each condition, 0.1 mg/kg ICG was injected intravenously and 
the dynamic fluorescence change was captured by a fluorescence surgical microscope (Pentero, Zeiss) for 
4 minutes. Between imaging each condition 20 minutes was allowed for elimination of residual ICG. Figure
1

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1 shows the ICG fluorescence map overlay on the white light bone images as well as dynamic curves at 
the proximal bones (green ROIs in middle bone models). As shown in Figure 1 the ICG intensity and 
dynamic changes are substantially different in different conditions and ROIs. Baseline conditions, without 
disruption of either periosteal or endosteal blood supplies demonstrated the highest ICG intensity. Similarly, 
the next highest intensity was noted in ROIs proximal and distal to the osteotomy, prior to any removal of 
periosteum (again, leaving endosteal and periosteal blood supply intact, however, bone disrupted by an 
osteotomy). As sequential soft tissue stripping was performed, sequentially removing periosteal blood 
supply, the ICG intensity decreased. The osteotomized fragment of bone, with both periosteal and endosteal 
blood supplies disrupted, demonstrated the lowest ICG intensity and a flat dynamic curve. This experiment 
demonstrated that ICG intensity decreased in relationship to progressive disruption in bony blood supply. 
To date, we have collected fluorescence data on five limbs (three pigs) for this preliminary study. Figure 
2 shows the summary data of the maximum intensity of each ROIs at each condition. The data of the first 
leg has been excluded from this summary due to the ICG concentration and experimental condition have 
been changed/optimized after the first pig experiment. From this summary graph, it is clear that ICG 
fluorescence is highest in bone with endosteal and periosteal blood supply intact compared both with bone 
with periosteal blood supply disrupted/endosteal blood supply intact and bone with both periosteal and 
endosteal blood supply disrupted. These studies validate the effectiveness of quantitative ICG fluorescence 
as a method of measuring bone perfusion in a quantitative manner.1.3 Clinical Data to Date
ICG is FDA approved for human use in angiography for ophthalmology and cardiology applications and is 
given routinely to patients in these clinical settings. In addition, as the clinical trials, ICG has been used to 
guide laparoscopic surgery, 29 robotic adrenalectomy,30 assess tissue perfusion in vivo in real-time intra- 
operative arterial and lymphatic perfusion imaging,25, 31, 32 and tissue flap perfusion imaging33, 34. The 
examples of these trials are summarized below.
(1) Guide laparoscopic surgery29 ((0.1–0.5 mg/ml/kg): This study has been carried out in Minimally 
Invasive Surgery Research Center, at University of Insubria, Varese, Italy. 108 ICG-enhanced 
fluorescence guided laparoscopic procedures were performed. ICG imaging provided high- 
resolution real-time images of blood flow in vessels and organs as well as highlighted biliary 
excretion. As the result, “no intra-operative or injection-related adverse effects were reported, and 
the biliary/vascular anatomy was always clearly identified.” The real-time imaging provided 
sufficient information to conduct a safe cholecystectomy and ensure adequate vascular supply for 
colectomy, nephrectomy, or find lymph nodes.
(2) Guide robotic adrenalectomy30: (3.8–20 mg/subject) This study investigates the use of ICG 
florescence to guide an accurate dissection during robotic adrenalectomy (RA). A total of 40 
patients had been involved and the borders of adrenal tumors delineated by ICG imaging was 
superior, equivalent, or inferior to conventional robotic view in 46.5%, 25.6%, and 27.9% of the 
procedures, respectively.
(3) Real-time intra-operative arterial and lymphatic perfusion imaging: 32 (2-6.6 mg/subject) 301 patients 
were involved in the trial, and 589 nodes were removed. 583 nodes (99%) were identified with ICG 
and the identify rate is higher than that the conventional 99mTc (452 nodes). No any significant 
acute side effects were found related to ICG in this study.
(4) Tissue flap perfusion imaging33, 34: (5 mg/subject) 77 single-pedicle breast reconstruction patients 
were involved. Perfusion was measured intraoperatively using ICG angiography following flap 
harvest and before transfer. Significant perfusion difference has been detected in various flaps and 
between zones. The results shown that the lower abdominal free flaps based on the inferior 
epigastric system have better perfusion when compared with pedicled transverse rectus abdominis 
muscle flaps.
1.4 Dose Rationale
We plan a sample size of 300 patients in this open label, single center,  clinical trial of ICG 
fluorescence imaging for predicting recurrent infection in musculoskeletal trauma patients. For each 
subject, during the surgery, prior and after to the debridement, each of 0.1 mg/Kg ICG will be injected 
intravenously and video rate ICG fluorescence images will be acquired for each of 4 minutes. The ICG will

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be supplied directly by Novodaq/Stryker, which is FDA approved. As it shown in Section 1.4, total does of
0.2 mg/kg is within the range of previous studies. Indeed, this is 10 times less than the maximum 
recommended dose by FDA of 2mg/kg.
The subset patients who will undergo surgical treatment in CIS may undergo gadolinium (Gd-DTPA) 
contrast-enhanced MRI either intraoperatively or preoperatively. Gd-enhanced MRI is FDA-approved 
clinical imaging. The Gd-DTPA injection procedures will follow FDA-approved guidelines which are accepted 
as part of standard care in this setting.
1.5 Potential Risks and Benefits
The risks to subjects are minimal in relation to anticipated benefits and/or knowledge that might reasonably 
be expected from the results of this clinical trial.
1.5.1 Potential Risks
Risk of ICG injection: ICG (Indocyanine Green) will be administered intravenously twice: once before and 
once after the debridement. ICG is FDA approved for human use in angiography for ophthalmology and 
cardiology applications and is given routinely to patients in these clinical settings. The risks are considered 
minimal and consist of nausea, vomiting, hives, increased heart rate in subjects with particular sensitivity 
to the dye. ICG does contain sodium iodide and patients with a history of allergy to iodides will be excluded. 
Anaphylactic or urticardial reactions are rare but have been reported in patients both with and without a 
history of allergy to iodides. Anaphylactic deaths have been reported following IGG administration during 
cardiac catheterization. Reported rates of mild, moderate and severe adverse reactions to ICG are 0.15%, 
0.2% and 0.05%. Every effort will be made to minimize this risk as much as possible. It is the standard of 
care at this institution to obtain information related to allergies, sensitivities and past medical histories upon 
patient arrival. Patients will also be monitored throughout their hospitalization, as is the standard of care, 
by everyone involved in their medical care for evidence of new or previously unknown reactions or 
sensitivities. Additionally, at all times ICG fluorescence imaging assessments are taking place, research 
staff and both surgical and anesthesia staff will monitor patients closely for any adverse reaction to ICG. 
Research staff and surgical and anesthesia staff will also monitor patients for at least 30 minutes after all 
ICG fluorescence assessment procedures have been completed. In the event of an unexpected allergic 
reaction, Dartmouth-Hitchcock Medical Center and all affiliated groups within this institution have 
procedures in place for managing patients with unknown or unexpected allergic reactions. If such a reaction 
were to occur, all standard of care treatments will be provided including but not limited to treatment with 
appropriate agents such  as epinephrine, antihistamines and corticosteroids. Risk of infection from IV 
injection is also extremely rare but can occur. Pregnant women are excluded from the study. A pregnancy 
test is administered to women of child-bearing age as part of standard of care pre-operative testing.
Risk of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI): The risks associated 
with Gd contrast-enhanced MR are minimal. Gd-DTPA is an FDA-approved contrast agent and is widely 
used with few complications. The contrast injection procedures follow FDA-approved guidelines which are 
accepted as part of standard care in this setting. Consenting participants will have an intravenous catheter 
placed in an arm vein prior to their MRI examination by a trained MRI technologist conforming to the 
standard clinical methods used at DHMC.
Serious reactions to the gadolinium (Gd) are extremely rare and are primarily limited to nausea and 
vomiting. In response to the FDA notification regarding the potential association of nephrogenic sclerosing 
fibrosis (NSF) and intravenous gadolinium administration (primarily gadodiamide (Omniscan)), the 
Department of Radiology has developed a protocol which we will follow:
1. Identify the patients at higher risk of developing NSF (at the time of scheduling or before). Patients at 
risk include:

Patients > 50y

Patients already on dialysis

Patients with known renal insufficiency but not currently on dialysis

Diabetic patients

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
Patients with known cardiovascular disease

Patients who will be receiving high dose gadolinium (e.g. MR angiograms)

Hypertensive patients
2. Obtain estimated Glomerular filtration rate (GFR) on this higher risk group of patients (unless already on 
dialysis).

If a GFR value has been checked and is available in the patient's medical record within 1 
month of contrast-enhanced MR, that value will be acceptable.

If the participant is not a patient at DHMC, a "stat" GFR will be performed at DHMC one (1) 
hour prior to contrast-enhanced MR scan.
Subjects with a GFR <30 ml/min. will be considered ineligible for the contrast enhanced MRI portion of the 
study.
GFR values will not be sought at the time of scheduling on low risk patients, but the scheduler will check 
medical records at the time of the scan to see if an incidental GFR value is available.
Risk of infection from IV injection is also extremely rare but can occur. MR examination is used daily for 
many conditions and is considered safe. There are no known risks with MR imaging. Some individuals 
may feel claustrophobic inside the MR unit. Metal implants, such as surgical clips, and electronic devices, 
such as pacemakers, can be contra-indications for MR exams. Participants are questioned about these 
conditions and not enrolled accordingly.
Prolonged operating room time: Because the ICG fluorescence assessments will take place in the 
operating room, participation in this study may increase the amount of time spent under surgical anesthesia 
in the operating room. For the average case, an additional 10 minutes at most will be incurred. However, 
the research staff will make every effort to minimize this risk by performing the assessment test while other 
required operative procedures are being performed. For the subset patients who will have DCE-MR, the 
imaging sequence and time for DCE-MRI (~10-15 minutes) is not expected to change significantly from 
standard care of surgical treatment.
Risk of confidentiality breach: Subjects enrolled in research are always exposed to additional risks of a 
breach in confidentiality, for example, of some elements of their personal health information that is made 
available to study investigators as part of their participation. The risk is 100%, but the occurrence rate of an 
actual breach is < 1% (to date, we are not aware of any subject participating in our studies who has 
experienced a detrimental breach of confidentiality or confidential information).
Information about study subjects will be kept confidential and managed according to the 
requirements of the Health Insurance Portability and Accountability Act of 1996 (HIPAA). Risk of breach of 
confidentiality of the medical records and status of participants will be minimized. Databases which are 
used to store subject-sensitive information are password-protected and encrypted during file/data transfers 
from viewing terminals. Access will be limited to research team members who have undergone CPHS 
training at Dartmouth. Whenever possible and practical standard-of-care clinical data used in the research 
will be de-identified when under analysis.
1.5.2 Potential Benefits
Patients enrolling in this study will not benefit directly because no diagnostic or therapeutic decisions will 
occur based on the study, and thus, administration of the study is not intended to alter the surgical 
procedure. However, future patients may benefit from the knowledge gained from the study since this study 
may provide an objective, intraoperative, and real-time methodology to assess bone perfusion and 
thoroughness of debridement. This method may save the patients from unnecessarily high risk for index 
infection or nonunion—in the setting of open fracture—or recurrent infection in the setting of established 
infection, due to the substantial practice variation.

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2Study Objectives
The primary study objective is to establish the range and variation associated with bone/soft tissue 
perfusion in fracture patients, using ICG fluorescence imaging and examine the relationship between 
perfusion and complications such as SSI, persistent SSI, and fracture nonunion.
The secondary study objective is to develop intraoperative hardware and software tools that will be used to 
guide debridement to optimize the quantity of bone/tissue resected to minimize complications.
3
3Study Design
3.1 General Design
This study is a Phase I open label, single-center clinical trial of fluorescence imaging for predicting recurrent 
infection in musculoskeletal trauma patients. Patients who is undergo fracture fixation or joint fusion or 
requiring open surgical debridement/saucerization for osteomyelitis, may recruited into the study. Although 
the SPY Elite imaging system and the use of ICG have not been used in this type of study previously both 
have been established in other clinical settings to assess tissue perfusion. The expected duration for 
enrolling study participants is five years, and up to 300 subjects may be enrolled. Eligible consenting 
patients will receive standard of care treatment for their fracture or complication (infection or nonunion) 
including irrigation and debridement of their operative site. Detail study procedures will be described in the 
following Section 6.
3.2Primary Study Endpoints
The primary study endpoint for the open fracture cohort (cohort 1) is SSI using the CDC National Healthcare 
Safety Network reporting criteria (https://www.cdc.gov/nhsn/pdfs/pscmanual/9pscssicurrent.pdf) within 12 
months of fracture.
The primary study endpoint for the cohort of patients with complications, (cohort 2) is treatment failure 
(either persistent SSI using the CDC National Healthcare Safety Network reporting criteria or persistent 
nonunion requiring repeat surgical procedure).
The primary study endpoint for the closed fracture cohort (cohort 3) is SSI using the CDC National 
Healthcare Safety Network reporting criteria.
3.3 Secondary Study Endpoints
The secondary endpoint for all cohorts is unplanned fracture-related reoperation within 12 months of the 
initial open fracture. This endpoint has been used in previous fracture trials and is defined as any unplanned 
surgery that occurred from the time of index surgery to 12 months post-fracture that is associated with a 
wound healing problem, an infection at the operative site or contiguous to it, or fracture delayed 
union/nonunion.
4
4 Subject Selection and Withdrawal
4.1Patient Selection
Consecutive patients 18 years of age or older who present to DHMC with an open fracture will be 
considered for the open fracture cohort (Cohort 1). Fracture patients who present with a complication 
(either SSI or nonunion) following fracture fixation or joint fusion will be screened for the complication cohort 
(Cohort 2). Patients who present to DHMC with a closed fracture will be screened for the closed fracture 
cohort (Cohort 3).

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4.2Inclusion Criteria
The following criteria must be met by study subjects to be eligible for study enrollment:
Open Fracture Cohort (Cohort 1)
(1) Patients 18 years of age or older.
(2) Open extremity fracture.
(3) Planned definitive fracture management with external fixation, internal fixation, or joint fusion.
(4) Will have all planned fracture care surgeries performed by a participating surgeon or delegate.
(5) Provision of informed consent.
Established SSI Fracture Cohort (Cohort 2)
a.
Patients 18 years of age or older.
b.
Extremity fracture.
c.
Prior definitive fracture management with external fixation, internal fixation, or joint fusion.
d.
Superficial, deep, or organ space SSI (as per CDC criteria) at the fracture site that requires 
operative management.
e.
Will have all fracture care surgeries performed by a participating surgeon or delegate.
f.
Provision of informed consent.
Closed Fracture Cohort (Cohort 3)
a.
Patients 18 years of age or older.
b.
Closed extremity fracture.
c.
Planned definitive fracture management with external fixation, internal fixation, or joint fusion.
d.
Will have all planned fracture care surgeries performed by a participating surgeon or delegate.
e.
Provision of informed consent.
4.3 Exclusion Criteria
The following criteria would exclude a subject from study enrollment:
Open Fracture Cohort (Cohort 1)
(7) Fracture of the hand.
(8) Iodine allergy.
(9) Received previous surgical debridement or management of their fracture at a non-participating 
hospital or clinic.
(10)Open fracture managed outside of the participating orthopaedic service.
(11)Chronic or acute infection at or near the fracture site at the time of initial fracture surgery. 
(12)Burns at the fracture site.
(13)Incarceration.
(14)Expected survival of less than 90 days.
(15)Problems, in the judgment of study personnel, with maintaining follow-up with the patient.
Established SSI Fracture Cohort (Cohort 2)
a.
Fracture of the hand.
b.
Iodine allergy.
c.
Received previous surgical debridement to manage the SSI.
d.
Incarceration.
e.
Problems, in the judgment of study personnel, with maintaining follow-up with the patient.
  Subset: DCE-MRI (Cohort 2-1)
f.
the presence of an electronic implant, such as a pacemaker
g.
the presence of a metal implant, such as an aneurysm clip
h.
the presence of other contraindication(s), as determined by the MRI technologists and radiologists.
i.
A history of allergy to iodides

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j.
A GFR < 30 ml/min as determined by blood test on the day of NIR/MR imaging, or from lab results 
within 3 months of DCE-MRI for this study
Closed Fracture Cohort (Cohort 3)
a.
Fracture of the hand.
b.
Iodine allergy.
c.
Chronic or acute infection at or near the fracture site at the time of initial fracture surgery.
d.
Burns at the fracture site.
e.
Incarceration.
f.
Expected survival of less than 90 days.
g.
Problems, in the judgment of study personnel, with maintaining follow-up with the patient.
4.4 Subject Recruitment and Retention
Participation in this research requires informed consent according to Institutional Review Board (IRB) 
guidelines and a signed IRB-approved Consent Form as the means of documenting this understanding. 
Patients receiving surgery in the CIS will be required to sign an additional CIS-specific surgical consent 
document.
Potential recruits are instructed that their participation is completely voluntary and that their medical care 
will not be altered in any way should they elect not to participate at any time prior to surgery. Subjects are 
recruited from patients presenting or referred to Orthopedic Department at Dartmouth-Hitchcock Medical 
Center for treatment and meeting protocol inclusion criteria. This study requests a partial waiver of HIPAA for 
recruitment purposes. The partial waiver would allow for the surgeon to share with the research coordinator the 
patient name, sex, MRN number, age, underlying conditions as well as type and location of injury. There is an 
adequate plan to protect identifiers from improper use and disclosure as information will only be used by study team 
members to determine whether a patient may be eligible. Information will only be communicated and stored using D-
HH approved secure platforms. Information is only being used to determine if a patient is eligible for the study, for 
recruitment purposes. Once approached, if a patient is not interested, information will be destroyed. Protected health 
information will not be re-used or disclosed for another purpose. It will only be used for recruitment. This research 
could not practically be done without a partial waiver of HIPAA. This information is needed for recruitment. Patients 
will sign a full HIPAA Authorization when consenting to the study. 
Potential subjects may be contacted by Dr. Gitajn, Dr. Henderson or another surgeon in the Orthopaedic 
department to learn about the study and participation. Subjects will be invited to participate in this study by 
a member of the Orthopaedic Department, which will occur either at the time of consultation with the 
surgeon about the candidate’s standard-of-care procedures or at another time agreed to by the 
potential participant, the candidate’s surgeon and/or Dr. Gitajn and/or Dr. Henderson or their designee. No 
advertisements or other promotional material will be used. No finder fees or recruitment incentives will be 
offered. Women of child bearing potential are eligible for enrollment into this study because ICG 
administration is not considered to present any additional risk for these women. The study will exclude 
women who are pregnant or breast-feeding as indicated in the exclusion criteria. Women of child-bearing 
potential, if asked to participate, will be given a pregnancy test as part of their standard of care pre-operative 
testing to confirm pregnancy status before administration of ICG.
Subjects enrolled into this study prior to 01Nov2020 will be re-consented in order to allow for the use of their 
data in the following applicable studies:
Fluorescence Based Measurement of Bone Perfusion in Infection (STUDY 02000374) 
ICG Fluorescence Imaging in Open Trauma (STUDY 02000365)
All follow-up visits are within standard practice for these injuries/diagnoses.
Several additional strategies may be used to maximize follow-up including: 1) at the time of enrollment each 
participant will provide their own telephone number, as well as the name and address of a primary care 
physician, and the names and phone numbers of three people at different addresses with home the 
participant does not live and who are likely to be aware of the patient’s whereabouts; 2) participants will 
receive a reminder card upon discharge for their next follow-up visit by the clinical site study personnel; 3) 

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ICG
White 
ligh
Lens
participants will receive text message reminders; 4) follow-up will coincide with normal surgical fracture 
clinic visits; and 5) if a participant refuses or is unable to return for the follow-up assessment, study 
personnel will determine his/her status with regards to major study outcomes by telephone, text, mail or 
email contact with the participant or the provided alternate contacts.
4.5Early Withdrawal of Subjects
4.5.1 When and How to Withdraw Subjects
Participants will be removed from the protocol if: Study 
imaging is not completed for any reason.
1)
The subject withdraws consent.
The subject has an occurrence of a significant clinical event that precludes imaging.
2)
The subject becomes pregnant.
If a participant is withdrawn from the protocol, the PI will mark the data of this subject as "withdrawn" and 
will add a detailed explanation about the cause of withdrawal in the database.
4.5.2 Data Collection and Follow-up for Withdrawn Subjects
For subjects who withdraw after the intraoperative imaging, we will follow-up to see if they still complete 
their clinical follow up in six months. For subjects who eventually finish their all clinical follow up visits, we 
will acquire their follow up results to include these subjects in the statistical analysis portion that only 
involves the intraoperative image data and infection status.
  5 Study Drug and device
White
&NIR lights
Filter
ICG
(a)
Figure (a): Schematic sketch of ICG fluorescence imaging.
5.1 Description
This  study is  neither  a  drug  nor  a  device  trial.  Patients  will  be 
administered  FDA  approved  ICG  through  intravenous  injection  and 
imaged by a FDA approved surgical microscope (Spy Elite) which is 0.5 
meter away  from  the subject. Both  ICG fluorescence and  the  two 
imaging systems have been used for routine clinical practice for many 
years. Figure (a) shows the Schematic sketch of the imaging systems. 
ICG fluorescence imaging utilizes intravenously injected ICG, which is a 
fluorescent dye that is FDA-approved for clinical use, illuminated with 
near-infrared light. The ICG dye is indirectly activated and the dynamic 
fluorescence due to bone perfusion can be captured by a video rate 
imaging system.

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5.2 Preparation and Administration of Study Drug
ICG used in this study is purchased from Novodaq/Stryker.
ICG Preparation and Administration Procedures:
1)
ICG is prepared by the Surgical and/or Anesthesia teams in the operating room on the day of 
surgery.
2)
From the  vial  containing  ICG, the  required dose is  withdrawn into a  syringe, labeled, and 
administered through a peripheral intravenous line followed by a saline flush.
Prior to ICG administration, patients will undergo standard preoperative monitoring by the Same Day 
nursing staff that includes continuous pulse oximetry, blood pressure monitoring, and heart rate, and 
respiratory rate. These results will be available prior to surgery and prior to administration of ICG. ICG will 
be administered by an anesthetist in the operating room. Patients are under the care and observation of 
the anesthesia and surgical teams during this time. Postoperatively, patients are transferred to the Post- 
Anesthesia Care Unit (PACU) where they are monitored continuously by nursing staff supervised by 
anesthesiologists. During this entire time vital signs (including temperature, pulse, respiratory rate and 
blood pressure) are collected as standard of care.  All clinical data will be reviewed for adverse events for a 
period of 30 minutes post-injection,
Day of surgery Procedures
Pre- 
Infusion
0-30
minutes 
post- 
infusion
Pulse
X
X
Blood Pressure
X
X
Temperature
X
X
Respiratory Rate
X
X
Adverse Events
X
A Pulse dye densometer (Pulsion Medical Systems), similar to a pulse oximetry probe, will be placed on 
the patient’s finger to acquire an arterial blood input function during ICG injection.  After exposure but prior 
to debridement, 0.1 mg/kg ICG will be injected intravenously. Video rate ICG fluorescence images will be 
acquired 20 seconds before and 4 minutes after ICG injection. Debridement will then proceed per standard 
of care. The wound will be irrigated with a minimum of 3 liters irrigation with normal saline. After irrigation 
and debridement is completed, 0.1 mg/kg ICG will be injected intravenously again and video rate ICG 
fluorescence images will be acquired 20 seconds before and 4 minutes after ICG injection. There will be a 
minimum of 5 minutes between ICG infusions to allow ICG to wash out of tissues.
At investigator discretion, only one ICG injection may be performed and only one series of ICG 
fluorescence imaging will be obtained. There will be no increased risk to the subject if the 
investigator opts to only obtain one series of images.
5.3 Subject Compliance Monitoring
Subjects participate in a “one-time” event (i.e., surgery) as part of this study and are monitored through the 
in-patient surgical service during the time of their participation.

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5.4 Prior and Concomitant Therapy
N/A.
5.5
Packaging
N/A
5.6
Blinding of Study Drug
N/A.
5.7 Receiving, Storage, Dispensing and Return
5.7.1
Receipt of Drug Supplies
ICG will be shipped to the Pharmacy at Dartmouth-Hitchcock Medical Center by standard UAB. Pharmacy 
staff will count and verify that the shipment contains all the items noted in the shipment inventory, providing 
temperature monitoring data to UAB if necessary. Pharmacy staff will document receipt into their electronic 
investigational drug management database that keeps a perpetual inventory. Any damaged or unusable 
study drug in a given shipment will be documented in the study files, and destroyed on site following 
standard procedures.
5.7.2 Storage
ICG will be stored as the original packages that shipped from the maker (Stryker).
5.7.3 Dispensing of Study Drug
Once agent assignment is performed, research staff will transport ICG in its original package to the 
operating room. The surgeon or anesthesiologist will prepare the solution for injection per instructions from 
the Stryker. 0.1mg/kg ICG will then be administered per study protocol.
5.7.4 Drug Accountability
A member of the study team will document the amount of ICG administered and any amount remaining in 
the institution’s electronic medical record. 
5.7.5 Return or Destruction of Study Drug
ICG is FDA approved for routine clinical practice. No return or destruction of study drug is needed for this 
study.
6 Study Procedures
Consent Process
Eligible patients regardless of ethnicity or health status, will be identified and recruited subject to inclusion 
and exclusion criteria above. Patients who meet eligibility criteria will be asked to participate in the trial. If 
they agree written informed consent will be obtained from the patient or their healthcare proxy. Patients 
receiving surgery in the CIS will be required to sign an additional CIS-specific surgical consent document. 
To obtain informed consent, study personnel (surgeon or research coordinator) will adhere to the following 
procedures: (1) present study information in a manner that is understandable to the patient; (2) discuss the 
study with the patient and answer any questions; (3) allow the patient an opportunity to discuss participation 
with their family; (4) confirm that the patient understands the risks and benefits of participating in the study 
nd that their participation is voluntary; (5) complete the consent process and obtain signatures from the 
patient and research team.  The process of obtaining and documenting informed consent forms will be 
completed in accordance with local Good Clinical Practice (GCP) recommendations. If the research team 
member obtaining consent is at all unsure about the patient's ability to consent, s/he will consult with the 
study PI.
Recognizing that consent is an ongoing process, the study team will encourage the participants to ask 

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additional questions that may arise during the course of their participation in the study.
This study will not involve the subjects who is under age 18.
The study team will comply with consent procedures outlined in SOP HRP-090.
This study does not involve the assessment of an experimental treatment to the subjects enrolled. The 
subjects will undergo a method for collecting imaging data that has been fully vetted and utilized in other 
clinical settings. The purpose of this research it to evaluate the utility of this imaging technique in 
orthopaedic trauma patients who have a closed orthopaedic trauma.
Cognitively Impaired Adults
Patients with impaired decision-making capacity will be included in this study. If the patient is unable to 
provide informed consent (e.g. due to their injury) at the time they are identified, informed consent may be 
obtained from their legally authorized representative (LAR). Allowing informed consent from a patient’s 
LAR will reduce the risk of recruitment bias against the most severely infected patients.
For patient with impaired decision-making capacity, a LAR with knowledge of the potential participant will 
be approached to consent on the patient’s behalf. If the patient cannot adequately answer at least two 
questions and it is determined that the patient’s level of cognition is not likely to change before surgery, 
their LAR will be approached. The choice of LAR will follow standard procedures. The following will be 
approached in this order of priority:

legal guardian

proxy (health care agent) named in an advanced directive or durable power of attorney for health- 
care

family member or other surrogate identified by state law on health care decisions.
The LAR will be advised to base the decision on the participant’s expressed wishes, or, if these are not 
known, what they believe the participant would have desired under the circumstances of the injury, their 
beliefs and values. Recognizing that consent is an ongoing process, the study team will encourage the 
participants to ask additional questions that may arise during the course of their participation in the study.
Subjects participation in this study completely voluntary. If at any time a patient finds participation to be 
unduly stressful the patient or their legal authorized representative may withdraw the patient from the 
study with no repercussions.
Assent will not be requested from subjects unable to consent for themselves.
The study team will comply with consent procedures for cognitively impaired adults outlined in 
SOP HRP-013.
Surgical Procedures
In the surgery day, the patient will be prepared and transported to surgery as per routine at Dartmouth- 
Hitchcock. In the OR, patient positioning, preparation of the surgical field, and draping will follow standard 
practice, as fracture fixation or joint fusion or requiring open surgical debridement/saucerization for 
osteomyelitis.
A Pulse dye densometer (Pulsion Medical Systems), similar to a pulse oximetry probe, will be placed on 
the patient’s finger to acquire an arterial blood input function during ICG injection.  After exposure but prior 
to debridement, 0.1 mg/kg ICG will be injected intravenously. Video rate ICG fluorescence images will be 
acquired 20 seconds before and 4 minutes after ICG injection. Debridement will then proceed per standard 
of care. The wound will be irrigated with a minimum of 3 liters irrigation with normal saline. After irrigation 
and debridement is completed, 0.1 mg/kg ICG will be injected intravenously again and video rate ICG 
fluorescence images will be acquired 20 seconds before and 4 minutes after ICG injection. There will be a 
minimum of 5 minutes between ICG infusions to allow ICG to wash out of tissues.

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A subset of 30 eligible consenting patients with post-fracture complications will undergo surgical treatment 
for their infection in CIS at DHMC. Patients receiving surgery in the CIS will be required to sign an additional 
CIS-specific consent document. For the patients who didn’t receive contrast MRI in 48 hours prior to the 
operation, after exposure and removal of hardware but prior to pre-debridement ICG injection, four MRI 
fiducials will be placed on the four corners of the damaged bone area and the MRI sequences will be 
acquired. Sequences will include a localizer, coronal T1 fast spin echo (FSE), axial short-tau inversion 
recovery (STIR), a T1 mapping sequence consisting of a T1-weighted 3D FLASH sequence with at least 3 
different flip angles,  and DCE acquisition,  consisting of 3D FLASH sequences  before and after the 
intravenous administration of 0.2 mL/kg Gadoterate meglumine (Dotarem®). The T1 mapping and DCE 
sequences will be acquired in the same plane (either the coronal or axial) for all patients. The protocol for 
the DCE acquisition has already been optimized for current clinical applications in the imaging of both bone 
and soft tissue tumors. For the patients who received MRI within 48 hours prior to the operation, we may 
use their pre-operation MRI, instead of intra-operative MRI.
The need for repeat debridement or tissue cultures will be left up to the treating surgeon. If repeat 
debridement is needed, pre- and post-debridement quantitative ICG fluorescence images will be obtained 
at each procedure. If the skin wound is unable to be closed, it will be left open with a sterile dressing until 
delayed coverage in collaboration with plastic surgery.
Upon admission and throughout the hospital course, patients will be treated with antibiotics per standard of 
care by the orthopaedic service or in collaboration with infectious disease recommendations. Management 
of study patients including clinical and radiographic assessments will not differ from standard of care.
Study participants will be followed at 6 weeks, 3 months, 6 months, 9 months and 12 months from their 
fracture (open and closed fracture cohorts) and from the date of consent (complication cohort), 
respectively. Final follow-up will be at  6 months to capture all infectious complications (Figure c.2)35. If 
a participant does not return to clinic, follow-up may be conducted by telephone. SSI and unplanned 
fracture-related reoperations will be identified at  the time of diagnosis/occurrence and/or during each 
participant clinical assessment and medical record review that will occur during their routine outpatient 
clinic visits.
7 Statistical Plan
7.1 Sample Size Determination
The participating Statisticians on the study are members of the Norris Cotton Cancer Center and/or 
Department of Orthopaedics at Dartmouth-Hitchcock Medical Center. Sample size determination is based 
on the primary objective of the study as a pilot assessment of ICG fluorescence intensity of bone perfusion.
For patient cohort 1, we anticipate that approximately 10% of patients with open fracture will have an SSI 
within 12 months of their fracture1, 2, 32. A total sample size of 160 patients will result in an estimated 16 
patients with an SSI and 144 patients without an SSI. We will have 86% statistical power to detect a mean 
maximum fluorescence intensity (Imax) difference of 24 (a.u.) between the SSI group and the non-SSI group 
with an estimated standard deviation of 30 (a.u.) and 15 (a.u.), respectively. All parameters were obtained 
from our previous porcine study. To be conservative, we increased the standard deviation by 50% for this 
power calculation. The type I error is set to be 5%. If we assume a drop-out rate of 10%, then a total of 
176 patients will be enrolled.
For patient cohort 2, we anticipate approximately 30% of patients with an SSI will go on to develop a 
persistent SSI/treatment failure within 12 months of diagnosis of their SSI12. Based on this estimate, a 
sample size of 22 patients with SSIs and 52 patients without, will have 93% statistical power to detect a 
mean Imax difference of 24 (a. u.) between the persistent SSI/treatment failure group and the non-persistent 
SSI/non-treatment failure group with Imax estimated standard deviation of 30 (a. u.) and 15(a. u.). All 
parameters are obtained from our previous porcine study, which are consistent with a pilot study conducted 
at STC in human participants using ICG fluorescence for a different indication. To be conservative, we 
increased the standard deviation by 50% for this study. The type I error is set to be 5%. If we assume the 
dropout rate is10%, then a total of 82 patients will be recruited.
For patient cohort 2-1, the strength of the relationship between DCE-MR and DCE-FI measurements will 

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be assessed by determining the zero-order correlation and resulting R2 metric. A sample of 21 was 
estimated utilizing formula presented by Bonett & Wright 36 with a Type I error rate of 0.05 and Type II error 
rate of 0.20 (1-Power) to estimate the magnitude of zero-order correlation and interval estimation. Formula
inputs included expected zero-order relationship of r = 0.85 (95% CI 0.55 to 1.00). To optimize the imaging 
parameters of DCE-MRI, extra seven patients has been added into this cohort. Thus, total of 30 patients 
will be involved in this cohort.
Patient cohort 3 will serve as the control group for cohorts 1 and 2.
7.2 Data and Statistical Analysis
Patient cohort 1:
For the primary outcome, SSI is the dependent variable and bone and tissue perfusion, as measured by 
ICG fluorescence, is the independent variable. Unplanned fracture-related reoperation is the secondary 
dependent variable and bone and tissue perfusion as measured by ICG fluorescence, is the independent 
variable. Descriptive statistics, including means and standard deviations, will be presented with 95% 
confidence intervals. To select the most important ICG fluorescence kinetic curve-related variables, we will 
perform a bivariate analysis evaluating whether each variable predicts infection and we will select the 
variable with the highest individual area under the receiver operating curve (AUC ROC).
Our primary null hypothesis is that the post-debridement maximum ICG intensity (Imax-post) among patients 
with SSI is equal to or greater than that in the non-SSI patient group. The primary alternative hypothesis is 
that Imax-post in the SSI group is smaller than that in the non-SSI group. We will then perform Students t- 
tests and chi-square analyses (depending on continuous or categorical variable status) to determine 
unadjusted associations between the  most  important  ICG  fluorescence  variable  and  SSI.  A p-value of 
0.05 or less will be considered significant for differences between SSI and non-SSI groups. We will then 
apply multiple logistic model including ICG fluorescence and previously identified predictors for SSI37-39. 
Wald test will be used to determine if ICG fluorescence or any other predictors are significant in the model. 
We set the type I error at 5%.      Table 7.1 shows a summary of the study outcomes, corresponding
hypotheses, and method of analysis to be performed. We anticipate that we will demonstrate a statistically 
significant difference between the maximum post-debridement ICG fluorescence in patients with and 
without SSI
Table 7.1 Summary of Outcome Analysis Plan for patient cohort 1
Outcome
Objective
Name
Type
Hypothesis
Method of analysis
SSI
Binary
Mean perfusion will be 
lower among patients 
who develop SSI
Students t-test 
Multivariable logistic 
regression
Determine
the
association 
between post-debridement  
bone perfusion as measured by 
quantitative  ICG  fluorescence 
and surgical site infection (SSI) 
in patients with open fracture
Unplanned 
fracture- 
related 
reoperation
Binary
Mean perfusion will be 
lower  among  patients 
who require unplanned 
fracture-related 
reoperation
Students t-test 
Multivariable logistic 
regression
Patient cohort 2:
For the primary outcome, treatment failure will be the dependent variable and bone and tissue perfusion, 
as measured by ICG fluorescence will be the independent variable. Descriptive statistics, including means 
and standard deviations, will be presented with 95% confidence intervals. To select the most important 
ICG fluorescence kinetic curve-related variables, we will perform a bivariate analysis evaluating whether 
each variable predicts infection and we will select the variable with the highest area under the curve (AUC).
Our primary null hypothesis is that the post-debridement maximum ICG intensity (Imax-post) among patients 
with persistent SSI/treatment failure is equal to or greater than that in the non-persistent SSI/non-treatment 
failure patient group. The primary alternative hypothesis is that Imax-post in the persistent SSI/treatment failure 

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group is smaller than that in the non-persistent SSI/non-treatment failure group. We will then perform 
a Students t-test to determine the association between the most important ICG fluorescence variable 
and persistent SSI/treatment failure. A p-value of 0.05 or less will be considered significant for 
differences between persistent SSI/treatment failure and non-persistent SSI/non-treatment failure groups. 
We will then apply multiple logistic model including ICG fluorescence and previously identified predictors 
for persistent SSI/treatment failure37-39. Wald test will be used to determine if ICG fluorescence or any 
other predictors are significant in the model. We set the type I error at 5%. Table 7.2 shows a 
summary of the study outcome, corresponding hypotheses and method of analysis to be performed. 
We anticipate that we will demonstrate a statistically significant difference between the maximum post-
debridement ICG fluorescence 
in patients who fail treatment for their SSI resulting in persistent 
SSI/treatment failure and those who do not.
For subset cohort 2-1, for each DCE-MR and DCE-FI parameter, statistics of median and percentiles 
will be calculated for each of ROIs. Due to the small cohort size, the correlations between DCE-MRI 
and DCE-FI will be utilized to analyze the data of each corresponding ROI, assuming that all ROIs in 
each subject were independent 40.
Table 7.2: Summary of Outcome Analysis Plan of patient cohort 2.
Outcome
Objective
Name
Type
Hypothesis
Method of analysis
Determine  the  association 
between post-debridement 
bone perfusion as measured 
by
quantitative
ICG 
fluorescence and persistent 
SSI/treatment
failure
in 
patientswith
established 
SSI
Persistent 
SSI/Treatment 
failure
Binary
Mean perfusion will be 
lower among fracture 
patients with persistent 
SSI/treatment failure
Students t-test 
Logistic regression
Patient cohort 3: Classification into cohort categories (closed fracture, open fracture, complication) 
is the independent variable and ICG fluorescence magnitude is the dependent variable. 
Descriptive statistics, including means and standard deviations, will be presented with 95% 
confidence intervals.
7.3Subject Population(s) for Analysis
This study will involve consecutive patients 18 years of age or older who present to DHMC with an open 
fracture will be considered for the open fracture cohort (Cohort 1). Similarly fracture patients who present 
with an SSI following fracture fixation or joint fusion will be screened for the fracture SSI cohort (Cohort 2). 
Patients who present to DHMC with a closed fracture will be considered for the closed fracture cohort 
(Cohort 3). The image and clinical outcome data of all patients involved in this study will be analyzed.
8
Safety and Adverse Events
8.1 IRB Reporting Requirements for Adverse Events & Unanticipated Problems
Adverse events that are serious, unexpected, and possibly, probably or definitely related to participation in 
the research study will be reported to the Dartmouth Committee for the Protection of Human Subjects 
(CPHS) and to the sponsor. These events will be reported to the CPHS within 1 week. The sponsor will 
report these events to the FDA within 7 calendar days. All lethal events will be reported to the CPHS, 
Sponsor, and FDA.
Adverse events to be reported to the Sponsor are: any adverse experience, defined as any untoward or 
unfavorable medical occurrence in a human subject, including any abnormal sign (for example, abnormal 
physical exam or laboratory finding), symptom, or disease, temporally associated with the subject’s 

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participation in research, whether or not considered related to the subject’s participation in the research), 
that is considered:

Serious: Death; a life-threatening adverse drug experience; inpatient hospitalization or prolongation 
of existing hospitalization; a persistent or significant disability or incapacity; or a congenital anomaly 
or birth defect; and

Unexpected: Any adverse experience, the specificity or severity of which is not consistent with the 
current investigator brochure or consent form; and

Possibly related: There is a reasonable possibility that the incident, experience, or outcome may 
have been associated with the procedures involved in the research; and

Is experienced by a participant in a trial open at a site subject to review by the CPHS.
An unanticipated problem involving risks to subjects or others is defined as any incident, experience, or 
outcome that meets each of the following criteria:

Unanticipated in terms of nature, severity, or frequency given: (a) the research procedures that are 
described in the protocol-related documents, such as the IRB-approved research protocol and 
consent document; and (b) the characteristics of the subject population being studied; and

Possibly related to participation in the research means there is a reasonable possibility that the 
incident, experience, or outcome may have been associated with research participation; and

The problem suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, emotional, economic, legal, or social harms) than was previously 
known or recognized.
Copies of each report and documentation of IRB notification and receipt will be kept in the Clinical 
Investigator’s study file.
Any unexpected or serious adverse events, or drug reaction beyond those expected from open sarcoma 
surgery that are determined likely to be related to the study procedures, will be reported to the FDA as 
required.
8.2 Institutional Safety and Data Monitoring
This study will be monitored by the Data Safety Monitoring and Accrual  Committee (DSMAC).  The 
Committee meets quarterly to review accrual rates and information for studies that have accrued 
participants. The Clinical Cancer Review Committee (CCRC) determines the frequency of DSMAC review. 
The DSMAC has the authority to suspend or to recommend termination to the CCRC of all research 
activities that fall within its jurisdiction. In the event that a study is suspended or terminated, that information 
will be forwarded to the CPHS (Dartmouth IRB) office.
9
 Data Handling and Record Keeping
9.1 Confidentiality
Information about study subjects will be kept confidential and managed according to the requirements of 
the Health Insurance Portability and Accountability Act of 1996 (HIPAA). Risk of breach of confidentiality of 
the medical records and status of participants will be minimized. Databases which are used to store subject- 
sensitive information are password-protected and encrypted during file/data transfers from viewing 
terminals. Access will be limited to research team members who have undergone CPHS training at 
Dartmouth. Whenever possible and practical standard-of-care clinical data used in the research will be de- 
identified when under analysis.
9.2 Case Report Forms
Study case report forms (CRFs) will be the primary data collection instruments for the study. All data 
requested on CRFs will be recorded. Any missing data will be explained. If a space on the CRF is left 
blank because the procedure was not performed or the question was not asked, a written notation will be 
made. If an item is not applicable to an individual case, written notation will be made. Changes to the 
CRFs will be initialed and dated.
9.3 Record Retention

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Following closure of the study, the investigator will maintain all site study records in a safe and secure 
location. The records are maintained to allow easy and timely retrieval when needed (e.g., audit or 
inspection) and, whenever feasible, to allow any subsequent review of data in conjunction with assessment 
of the facility, supporting systems, and staff. Upon completion of study analysis, research information is 
stored in Dartmouth College Records Management off-site storage located at 6218 Etna Road, Hanover, 
NH 03755. Documents are shredded on site after 50 years of storage.
10 Clinical Site Monitoring
10.1 On-Site Monitoring
Clinical research monitoring for regulatory compliance and data integrity will be conducted. Internal 
monitoring is conducted by appropriately trained staff of the Office of Clinical Research and Dartmouth- 
Hitchcock Medical Center Clinical Trials Office who are not involved in the study. This monitoring will include 
periodic assessment of the regulatory compliance, data quality, and study integrity. Study records will be 
reviewed and directly compared to source documents and the conduct of the study will be discussed with 
the investigator. Monitors may request access to all regulatory documents, source documents, CRFs, and 
other study documentation for on-site inspection. Direct access to these documents is guaranteed by the 
investigator, who must provide support at all times for these activities.
10.2 Auditing and Inspecting
The study investigators will permit study-related monitoring, audits, and inspections by the IRB, the sponsor, 
government regulatory bodies, and university compliance and quality assurance groups of all study related 
documents (e.g. source documents, regulatory documents, data collection instruments, study data, etc.). 
The study investigators will ensure the capability for inspections of applicable study-related facilities (e.g. 
pharmacy, diagnostic laboratory, etc.).
Participation as an investigator in this study implies acceptance of potential inspection by government 
regulatory authorities and applicable university compliance and quality assurance offices.
11 Ethical Considerations
This study is to be conducted according to US and international standards of Good Clinical Practice (FDA 
Title 21 part 312 and International Conference on Harmonization guidelines), applicable government 
regulations and Institutional research policies and procedures.
All subjects for this study will be provided a consent form describing this study and providing sufficient 
information for subjects to make an informed decision about their participation in this study. See Attachment 
for a copy of the Subject Informed Consent Form. This consent form will be submitted with the protocol for 
review and approval by the IRB. The formal consent of a subject, using the IRB-approved consent form, 
will be obtained before that subject undergoes any study procedure. The consent form will be signed by the 
subject or legally acceptable surrogate, and the investigator-designated research professional obtaining 
the consent.
12 Study Finances
12.1 Funding Source
This study is being funded by the Orthopaedics Department at Dartmouth Hitchcock Medical Center.
12.2 Conflict of Interest
All Dartmouth investigators will follow the Dartmouth conflict of interest policy, and will have disclosed 
potential conflicts-of-interest related to the study

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