Clinical trial NCT02006108
Conditions: Renal Transplant Rejection
Keywords: kidney transplant rejection Feraheme ferumoxytol
Sponsor: Stanford University
Start date: 2012-11-27; Completion date: 2017-04-11
Enrollment: 21 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The goal of this study is to develop a non-invasive imaging test for in vivo detection of kidney transplant rejection. The hypotheses are that 1) Ferumoxytol-MRI can generate accurate estimates of tissue iron concentrations and tissue macrophages. 2) The signal given by a renal allograft on Ferumoxytol-MRI demonstrates significant differences between rejected and non-rejected transplants.

Detailed description:
In children with kidney transplants, immunologically mediated rejection is the major cause of allograft failure. Thus, the therapeutic success of kidney transplants is highly dependent on the ability to avoid rejection during both the acute and chronic phase after transplantation. Children with kidney transplants currently undergo at least three routine (protocol) biopsies during the first two years after the transplantation in addition to biopsies required to investigate deterioration of kidney function. These biopsies are invasive and nearly always require general anesthesia, causing anxiety and distress of the patients and their parents, as well as significant costs to our health care system. There is currently no non-invasive diagnostic tool capable of detecting rejection in vivo. Thus, the goal of this study is to develop a non-invasive imaging test for in vivo detection of kidney transplant rejection. The investigators propose to accomplish this goal by detecting macrophage infiltration in kidney transplants with iron oxide nanoparticle-enhanced MR imaging. Macrophages play a major role in transplant rejection. CD68-positive macrophages comprise approximately 50% of the infiltrating leukocyte population in renal allograft rejection, they co-localize with areas of tissue-damage and fibrosis, and are preponderant in more severe forms of rejection. The investigators hypothesize that iron oxide nanoparticle-enhanced MR imaging can detect differences in macrophage infiltrations in renal allografts undergoing rejection as opposed to allografts without significant rejection. This hypothesis is based on the bio-physical properties of intravenously injected superparamagnetic iron oxide nanoparticles, which are phagocytosed by tissue macrophages and cause strong signal effects on MR images.

The specific aims of the study are the following:

Aim #1. Technical Development of a Quantitative Susceptibility Mapping (QSM)-Sequence for in vivo MRI detection and quantification of iron oxide nanoparticle-labeled macrophages.This aim will focus on the technical development of Quantitative Susceptibility Mapping (QSM), a novel MR imaging pulse sequence that will be used to accurately quantify the tissue concentration of free ferumoxytol and ferumoxytol in macrophages in renal allografts. Based on pulse sequence optimizations of phantoms with known concentrations of free and cell-bound iron, we expect to generate accurate estimates of tissue iron concentrations and macrophages with the QSM-MRI method.

Aim #2. Detect rejection in kidney allografts with ferumoxytol-enhanced MRI. The investigators hypothesize that ferumoxytol can detect and quantify macrophages in kidney allografts, based upon the observation that iron oxide nanoparticles can be taken up by macrophages in malignant tumors. The investigators will evaluate the ability of ferumoxytol to map macrophage quantities in renal allografts, with histopathological correlation. We expect significantly higher ferumoxytol-MRI enhancement and macrophage quantities in rejected allografts compared to non-rejected allografts.

Arms: arm 1: Intravenous injection of Feraheme, 5 mg Fe/kg

Interventions:

Drug: Feraheme Procedure: MR Scan
Interventions: intervention 1: Feraheme intervention 2: MRI-GE Healthcare 3 Tesla magnet
Intervention descriptions: intervention 1: Therapeutic classification: iron preparations. Use: Off-label use of ultrasmall paramagnetic iron nanoparticle as contrast agent for magnetic resonance imaging intervention 2: All patients will undergo

Protocol documents:
Prot_000.pdf: 
Non-invasive MR Imaging Diagnosis of Transplant 
Rejection 
NCT02006108 
November 30, 2016 
Heike Daldrup-Link, Principal Investigator 
Stanford University 
Stanford, California 94305 
1. 
PURPOSE OF THE STUDY 
a. 
Brief Summary 
The purpose of this study is to develop a non-invasive imaging test for in vivo detection 
of kidney transplant rejection. We propose to use an FDA-approved iron supplement "off 
label" as a marker for macrophages. After intravenous injection, the iron compound is 
taken up by macrophages and causes a detectable signal on magnetic resonance 
MRimages. Since kidney transplants that undergo a rejection contain macrophages, but 
not unimpaired transplants, this approach should enable us to detect kidney transplant 
rejection with a simple imaging test.  
b. 
Objectives 
If successful, this ferumoxytol-based MR imaging test could reduce the number of renal 
allograft biopsies and associated anesthesia. If at least some of these biopsies of could be 
replaced by a non-invasive imaging test, this would have immense impact on patient 
management and patient quality of life, and reduce health care costs. Exclusion of renal 
allograft rejection based on ferumoxytol-enhanced MRI would help avoid unnecessary 
biopsies, accelerate diagnostic workup of other reasons for an impaired renal function, 
and ultimately, improve patient outcomes. The proposed non-invasive imaging test could 
be in principle also applied to evaluation of other solid organ or stem cell transplants. 
Data derived from this project will be used as preliminary data for a subsequent NIH 
grant application.  
c. 
Rationale for Research in Humans 
Initial studies have been performed in animal models. We now have to verify the animal 
model results can be reproduced in patients.  
2. 
STUDY PROCEDURES 
a. 
Procedures 


November 30, 2016 
 
Page 2 of 12 
The patients and their parents will be informed about the nature of the study and a written 
informed consent will be obtained. 12-24 h before a planned MRI study, Ferumoxytol 
will be administered intravenously at a dose of 5 mg Fe/kg over approximately 1-2 
minutes. The blood pressure and respiratory rate will be monitored before and directly 
after the injection, and the patients will be observed for any potential adverse events for 
at least 30 minutes after the iron oxide administration. The patients will undergo MR 
imaging at 1-7 days after iron oxide injection. For some patients, we have the option to 
get a MR- exam before the injection of the contrast agent for comparison of pre and post 
images. This long interval between injection and imaging is needed in order to allow for 
sufficient time for macrophage phagocytosis to occur. Patients will be placed supine in a 
3T MR scanner, the area of the transplant kidney will be covered by a dedicated surface 
coil, and T1- and T2-weighted MR images will be obtained of the transplant, using axial 
and/or coronal T1-LAVA and T2-FSE sequences as well as multi-TE SE, multi-TI IR 
sequences and QSM sequences for iron quantifications. All MR images will be 
transferred to a postprocessing workstation and T1- and T2-relaxation times will be 
calculated of the kidney transplant as well as adjacent muscle and bone marrow in a 
pelvic bone as an internal standard. The patients will undergo a routine biopsy of their 
transplant within 3 weeks before or preferablyafter the MRI study. Based on renal 
biopsies and histopathology results, patients will be divided into groups of "no rejection" 
and "positive rejection". Various MRI and histopathological parameters will be compared 
between these two groups using student's t-tests, an analysis of variance e.g. for 
comparison of data from multiple time points, multiple pulse sequences, different 
histopathologic stainsand regression analyses. Significant differences will be assigned 
for a p-value of less than 0.05. 
Ferumoxytol is an FDA approved iron supplement, and it is slowly metabolized and 
release iron in vivo. In order to determine the effect and clearance of ferumoxytol, we 
propose to monitor the participant's blood iron levels immediately before and up to 6 
months 3-4 times in totalafter ferumoxytol injection. Furthermore, we want to 
investigate the blood for interaction between blood plasma proteins and iron as well as 
related immune cell responses and potential mechanisms for hypersensitivity reactions. 
We will collect small blood samples <5ml eachfrom the participant when we 
administer ferumoxytol intravenously and when the participant visits Stanford for their 
routine blood tests to avoid additional visits or needle sticks. The tests will be ordered 
through Stanford Clinical Laboratories.  
b. 
Procedure Risks 
Once MRI contraindications have been excluded e.g. MRI incompatible metal devices, 
the imaging test itself does not involve any risks for the patient. Contrast agent injection 
will occur outside of the magnet, in order to allow for close observation of the patient for 
any side effects. Of note, our other clinical imaging studies with ferumoxytol did not 
show any subjective or objective side effects in our patient population so far.  


November 30, 2016 
 
Page 3 of 12 
c. 
Use of Deception in the Study 
NA 
d. 
Use of Audio and Video Recordings 
NA 
e. 
Alternative Procedures or Courses of Treatment 
The alternative procedure to verify a suspected transplant rejection is a transplant biopsy, 
which is performed in children under general anesthesia. A biopsy is associated with risk 
of bleeding, vascular fistulas, other organ injuries and anesthesia complications.  
f. 
Will it be possible to continue the more (most) appropriate therapy for the 
participant(s) after the conclusion of the study? 
yes, all patients undergo a biopsy  
g. 
Study Endpoint(s) 
Endpoint: To generate a non-invasive, easily applicable and widely available MR 
imaging test for evaluation of kidney transplant rejection. Primary endpoint is to develop 
a sensitive and reliable, non-invasive and quantitative imaging test for detection of 
macrophages in kidney transplants as a biomarker for rejection. As a secondary endpoint, 
since different oxygenation states of iron have scaled but measurable changes on the 
inherent tissue magnetic susceptibility, we plan to also evaluate the utility of 
simultaneously generated 3D maps of arterial and venous supplies of kidney transplants, 
provided by our imaging method, in comparison with ultrasound as the current diagnostic 
standard. The ultimate goal is to provide a comprehensive evaluation of the morphology, 
vascular supply and host immune responses in kidney transplants with one single, non-
invasive and comprehensive diagnostic test.  
3. 
BACKGROUND  
a. 
Past Experimental and/or Clinical Findings 
In patients with end stage renal failure, renal transplantation is the treatment modality of 
choice, leading to markedly improved quality of life and survival when compared to 
chronic dialysis. To date, more than 1.4 million adult patients and 70.000 children have 
received renal allografts. However, a major complication of renal allograft transplantation 
in children and adolescents is an acute or chronic rejection, which causes almost half of 
the kidney transplant losses. Currently the diagnosis of rejection relies on allograft 
biopsies, which are invasive, nearly always require general anesthesia in children and are 
prone to sampling errors. A non-invasive diagnostic test, which could visualize and 
monitor allograft rejection directly and longitudinally in vivo would save invasive 
biopsies and anesthesia, reduce potentially associated complications and reduce health 
care costs. 


November 30, 2016 
 
Page 4 of 12 
b. 
Findings from Past Animal Experiments 
We chose macrophages as our target for imaging transplant rejection, because 
macrophages play a critical role in transplant rejection and because macrophages can be 
imaged with immediately clinically applicable MR imaging approaches. Macrophages are 
key inflammatory mediators of the innate immune response that contribute to both acute 
and chronic allograft rejection through a variety of mechanisms. Macrophages are 
attracted to sites of immune complex formation by complement fragments (e.g. C5a) and 
specific cytokines/chemokines. In the transplant, the macrophages are activated by IFN 
gamma (produced by T cells or NK cells) and TNF alpha (produced by APCs) which 
leads to a pro inflammatory cascade with production of reactive oxygen species, 
progressive transplant injury, and ultimately, graft rejection. In renal allografts that 
undergo rejection, CD68 positive macrophages comprise approximately 50% of the 
infiltrating leukocyte population, they co-localize with areas of tissue-damage and 
fibrosis, are preponderant in more severe forms of rejection and represent an independent 
predictor of worse outcomes. As new immune-modulating therapeutic agents enter the 
clinic whose mechanism of action involves diminishing macrophage infiltration or 
presence in allografts, it becomes increasingly important to identify those transplants 
heavily infiltrated by macrophages, as well as monitoring response to these new 
therapies. 
4. 
RADIOISOTOPES OR RADIATION MACHINES 
a. 
Standard of Care (SOC) Procedures 
Identify Week/Month of Study 
Name of Exam 
Identify if SOC or Research 
NA 
NA 
NA 
b. 
Radioisotopes 
i. 
Radionuclide(s) and chemical form(s) 
NA 
ii. 
Total number of times the radioisotope and activity will be administered (mCi) and 
the route of administration for a typical study participant 
NA 
iii. 
If not FDA approved: dosimetry information and source documents (package insert, 
Medical Internal Radiation Dose [MIRD] calculation, and peer reviewed literature) 
NA 
c. 
Radiation Machines – Diagnostic Procedures 
i. 
Examination description (well-established procedures) 
NA 
ii. 
Total number of times each procedure will be performed (typical study participant) 


November 30, 2016 
 
Page 5 of 12 
NA 
iii. 
Setup and techniques to support dose modeling 
NA 
iv. 
FDA status of the machine and information on dose modeling (if procedure is not 
well-established) 
NA 
d. 
Radiation Machines – Therapeutic Procedures 
i. 
Area treated, dose per fraction/number of fractions, performed as part of normal 
clinical management or due to research participation (well-established procedures) 
NA 
ii. 
FDA status of the machine, basis for dosimetry, area treated, dose per fraction and 
number of fractions (if procedure is not well-established) 
NA 
5. 
DEVICES USED IN THE STUDY 
a. 
Investigational Devices (Including Commercial Devices Used Off-Label) 
Investigational Device 1 
Name: 
NA 
 
Description: 
NA 
 
Significant Risk? (Y/N) 
NA 
 
Rationale for Non-Significant Risk 
NA 
 
Investigational Device 2 
Name: 
N/A 
Description: 
N/A 
Significant Risk? (Y/N) 
N/A 
Rationale for Non-Significant Risk 
N/A 
Investigational Device 3 
Name: 
N/A 
Description: 
N/A 
Significant Risk? (Y/N) 
N/A 
Rationale for Non-Significant Risk 
N/A 
 
b. 
IDE-Exempt Devices 
IND-Exempt Device 1 
Name: 
MRI 
 
Description: 
GE Healthcare, is a designated NSR 
device per the published IRB guidance 
GUI-7m 


November 30, 2016 
 
Page 6 of 12 
 
IND-Exempt Device 2 
Name: 
N/A 
Description: 
N/A 
IND-Exempt Device 3 
Name: 
N/A 
Description: 
N/A 
 
6. 
DRUGS, BIOLOGICS, REAGENTS, OR CHEMICALS USED IN THE STUDY 
a. 
Investigational Drugs, Biologics, Reagents, or Chemicals 
Investigational Product 1 
Name: 
Ferumoxytol (Feraheme, AMAG Pharmaceuticals), IND 111 154 
Dosage: 
5mg Fe/kg bodyweight 
Administration Route: 
intravenous 
Investigational Product 2 
Name: 
N/A 
Dosage: 
N/A 
Administration Route: 
N/A 
Investigational Product 3 
Name: 
N/A 
Dosage: 
N/A 
Administration Route: 
N/A 
b. 
Commercial Drugs, Biologics, Reagents, or Chemicals 
Commercial Product 1 
Name: 
N/A 
Dosage: 
N/A 
Administration Route 
N/A 
New and different use? (Y/N) 
N/A 
Commercial Product 2 
Name: 
N/A 
Dosage: 
N/A 
Administration Route 
N/A 
New and different use? (Y/N) 
N/A 
Commercial Product 3 
Name: 
N/A 
Dosage: 
N/A 
Administration Route 
N/A 
New and different use? (Y/N) 
N/A 
7. 
DISINFECTION PROCEDURES FOR MEDICAL EQUIPMENT USED ON BOTH HUMANS AND 
ANIMALS 
We will use MR scanners at Lucas Center or Lucile Packard Childrens Hospital at 
Stanford University. The bed/table and accessories that are used for the animals is 
different than the table humans use. Physiologic monitoring equipment is cleaned with a 
commercial disinfectant such as Roccal, Conflick, Sani-Wipes, or a 10Bleach solution. 
All RF coils and positioning accessories are wrapped in plastic wrap or plastic bags for 
use with animals. Everything, even if it is animal use only, is cleaned with the above 
disinfectants after every use even if they are wrapped in plastic. The Lucas Center is 
checked yearly by several groups at Stanford who approve animal research in human 


November 30, 2016 
 
Page 7 of 12 
systems: Stanford Health & Safety. The facility is reviewed by: Stanford APLAC panel; 
USDA; NIH; and Aaalac. 
8. 
PARTICIPANT POPULATION 
a. 
Planned Enrollment 
30 solid organ transplant recipients, to monitor for transplant rejection and evaluate the 
utility of 3D maps of arterial and venous supplies to the transplant.  
 
b. 
Age, Gender, and Ethnic Background 
We expect all 30 participants to be pediatric kidney transplant patients, any gender, any 
ethnic background. Referrals only accepted from Drs. Grimm and Concepcion.  
 
c. 
Vulnerable Populations 
We expect all 30 participants to be pediatric kidney transplant patients. The study adds on 
a non-invasive imaging study to the standard of care for these patients. Informed Consent 
and Assent practices will be followed.  
 
d. 
Rationale for Exclusion of Certain Populations 
NA 
e. 
Stanford Populations 
NA 
f. 
Healthy Volunteers 
NA 
g. 
Recruitment Details 
Participants will be referred from their treating physician's office, either Dr. Grimm or 
Dr. Concepcion invitation only.  
 
h. 
Eligibility Criteria 
i. 
Inclusion Criteria 
recipient of a solid organ transplant, scheduled for a standard of care rejection survey  


November 30, 2016 
 
Page 8 of 12 
ii. 
Exclusion Criteria 
contraindication for MRI  hemosiderosis/hemochromatosis, diagnosed based on routine 
lab values obtained within 30 days prior to MRI scanand/or precontrast MRI  
i. 
Screening Procedures 
Referring treating physician discusses study with potential participant. Potential 
participant is referred to study coordinator to schedule a screening visit. The protocol 
director or other IRB approved trained personnel complete consent/assent, MRI screen, 
and chart review lab values.  
j. 
Participation in Multiple Protocols 
We will ask the participant and their family if they are enrolled in any other studies. If 
they are enrolled in a drug study, we will consult with the other PI before proceeding to 
avoid any conflicts.  
k. 
Payments to Participants 
Reimbursement for gas/mileage to a max of $50 per visit to the family/driver. A $20 
Amazon gift card to the participant.  
l. 
Costs to Participants 
No research costs will be charged to the participants.  
m. 
Planned Duration of the Study 
The study is expected to enroll patients for two years, with data analysis continuing for 
possibly another year total: three years. iscreening: 20-45 minutes; ii90-120 minutes; 
iii3-5 hours per participant, including 3D reconstructions, plus another 40-100 hours of 
collective data analysis.  
9. 
RISKS 
a. 
Potential Risks 
i. 
Investigational devices 
NA 
ii. 
Investigational drugs 
Dr. Daldrup-Link has applied USPIO ultra small superparamagnetic iron oxidesas MR 
contrast agents in phase II and III clinical trials in adult patients. In addition, Dr. Daldrup-
Link has currently two active clinical trials on the use of ferumoxytol in children for MR 
imaging of bone lesions and for whole body tumor staging. The USPIO contrast agents 


November 30, 2016 
 
Page 9 of 12 
are very well tolerated and show excellent safety profiles. The delivered iron dose via a 
typical ferumoxytol administration is in the order of 150-500 mg iron oxides note that 
these are coated iron particles, not free iron, which is equivalent to or lower than the iron 
dose administered with one blood transfusion. USPIO are slowly metabolized in the liver 
and not excreted via the kidneys. Thus, they are safe to use in patients with renal 
insufficiencies and are not associated with any risk of nephrogenic sclerosis a potential 
adverse event after injections of certain gadolinium chelates. Expected risks include 
dizziness and nausea, possible localized twitching sensation, etc. Anaphylaxis or 
anaphylactoid reactions were reported in 0.2of subjects, which is in the order of other 
MR contrast agents.  
iii. 
Commercially available drugs, biologics, reagents or chemicals 
NA 
iv. 
Procedures 
MRI w/ contrast  Magnetic fields do not cause harmful effects at the levels used in the 
MRI machine. However, the MRI scanner uses a very strong magnet that will attract 
some metals and affect some electronic devices.  In some cases, having those devices 
means the participant should be excluded.  Additionally, when contrast is injected, as 
with any intravenous injection, there are risks of bruising, bleeding, or infection from the 
venipuncture; and allergic reaction to the injected contrast.  
v. 
Radioisotopes/radiation-producing machines 
NA 
vi. 
Physical well-being 
NA 
vii. 
Psychological well-being 
Some small risk of a patient experiencing claustrophobia. This is an infrequent but 
regular occurrence in any MRI facility. Every effort is made to minimize this risk and the 
research and clinical staff are well trained to act appropriately.  
viii. 
Economic well-being 
NA 
ix. 
Social well-being 
NA 
x. 
Overall evaluation of risk 
Low - innocuous procedures such as phlebotomy, urine or stool collection, no therapeutic 


November 30, 2016 
 
Page 10 of 12 
agent, or safe therapeutic agent such as the use of an FDA approved drug or device.  
b. 
International Research Risk Procedures 
NA 
c. 
Procedures to Minimize Risk 
An MRI screening form will be completed prior to participation. Any potential 
contraindication to MRI revealed by the comprehensive screening form will result in 
participant exclusion. The research team will use the clinical systems and the RedCap 
database for data management. RedCap is maintained by Clinical Informatics at Stanford 
University as a HIPAA compliant, secure, encrypted database for research purposes. Data 
exported by the researchers from these secure systems will only be exported in a 
deidentified form to minimize risks to confidentiality.  
d. 
Study Conclusion 
Endpoint: To generate a non-invasive, easily applicable and widely available MR 
imaging test for evaluation of kidney transplant rejection. Primary endpoint is to develop 
a sensitive and reliable, non-invasive and quantitative imaging test for detection of 
macrophages in kidney transplants as a biomarker for rejection. As a secondary endpoint, 
since different oxygenation states of iron have scaled but measurable changes on the 
inherent tissue magnetic susceptibility, we plan to also evaluate the utility of 
simultaneously generated 3D maps of arterial and venous supplies of kidney transplants, 
provided by our imaging method, in comparison with ultrasound as the current diagnostic 
standard. The ultimate goal is to provide a comprehensive evaluation of the morphology, 
vascular supply and host immune responses in kidney transplants with one single, non-
invasive and comprehensive diagnostic test.  
In terms of individual participation, each patient's experimental participation is over 
when their individual MRI is collected, analyzed, and reported. If a patient experiences 
an anaphylactoid reaction to the study agent during initial administration, participation 
would be terminated - administration of the agent would be discontinued, counteracting 
medication would be given, and the patient would not undergo MRI. Such reactions are 
very rare reported in 0.2of subjectsbut anticipated, and as such, the contrast agent is 
only administered in a clinical setting under the direct supervision of an experienced MD, 
with access to a crash cart and appropriate anti-anaphylactoid medication. 
e. 
Data Safety Monitoring Plan (DSMC) 
i. 
Data and/or events subject to review 
Of note, Dr. Daldrup-Link has applied iron oxide nanoparticles as MR contrast agents in 
phase II and III clinical trials in adult patients. These contrast agents are very well 
tolerated and show excellent safety profiles. The delivered iron dose via a typical 


November 30, 2016 
 
Page 11 of 12 
ferumoxytol administration is in the order of 150-500 mg iron oxides note that these are 
coated iron particles, not free iron, which is equivalent to or lower than the iron dose 
administered with one blood transfusion. Iron oxide nanoparticles are slowly metabolized 
in the liver and not excreted via the kidneys. Thus, they are safe to use in patients with 
renal insufficiencies and are not associated with any risk of nephrogenic sclerosis a 
potential adverse event after injections of certain gadolinium chelates. Anaphylaxis or 
anaphylactoid reactions were reported in 0.2of subjects, which is in the order of or lower 
compared to other MR contrast agents see FDA report: Lu M, Cohen MH, Rieves D, 
Pazdur R. FDA report: Ferumoxytol for intravenous iron therapy in adult patients with 
chronic kidney disease. Am J Hematol. 2010;855:315-9. In the unlikely event of an 
anaphylactoid reaction, appropriate actions will be taken as with any other contrast agent 
reaction and the event will be reported to the IRB.  
ii. 
Person(s) responsible for Data and Safety Monitoring 
Dr. Daldrup-Link will serve as the ME. In an unanticipated and unlikely event of any 
adverse event or anaphylactoid reaction to this contrast agent, appropriate actions will be 
taken to treat the reaction and the event will be reported to the IRB.  
iii. 
Frequency of DSMB meetings 
NA 
iv. 
Specific triggers or stopping rules 
Any signs of an anaphylactoid reaction, such as rash, urticaria, nausea, cough, breathing 
difficulty will lead to discontinuation of contrast administration and symptomatic 
treatment.  
v. 
DSMB Reporting 
The ME will forward written reports to the appropriate entities via email  
vi. 
Will the Protocol Director be the only monitoring entity? (Y/N) 
y 
vii. 
Will a board, committee, or safety monitor be responsible for study monitoring? 
(Y/N) 
y 
f. 
Risks to Special Populations 
The study involves an non-invasive imaging study and the off-label use of an FDA 
approved drug, with an FDA IND to do so. Side effects for this drug have been observed 
in less than 0.2of the population. Informed Consent and Assent are acquired 
appropriately and all data and safety precautions are observed. Risks are thus assessed as 
minimal.  


November 30, 2016 
 
Page 12 of 12 
10. 
BENEFITS 
Participants may immediately benefit from more accurate assessment of arterial and 
venous supply to their allograft. In the long term, validating a method for accurately 
assessing rejection status/health of an allograft without general anesthesia or invasive 
biopsy reduces risk and cost to the patient.  
11. 
PRIVACY AND CONFIDENTIALITY 
All participant information and specimens are handled in compliance with the Health Insurance 
Portability and Accountability Act (HIPAA) and privacy policies of Stanford University, Stanford 
Health Care, and Stanford Children’s Health. 

SAP_001.pdf: 
Non-invasive MR Imaging Diagnosis of Transplant 
Rejection 
NCT02006108 
April 6, 2018 
Statistical Analysis Plan 
 
Heike Daldrup-Link, MD, PhD, Principal Investigator 
Stanford University 
Stanford, California 94305 
 
 


April 6, 2018 
 
Page 2 of 2 
Statistical Analysis Plan 
Histopathology  
Patients with allografts underwent routine biopsies either on the same day as the MRI, before the 
MRI, or after the MRI. The biopsy specimens were dissected parasagittally, kept in formalin 
overnight, dehydrated through graded alcohol washes (70, 95, and 100 %), embedded in paraffin 
and sliced on a microtome. Five-micrometer thick tissue slices on glass slides were stained with 
hematoxylin and eosin (HE) and Periodic acid-Schiff (PAS) stains. In addition, the samples were 
stained with antisera against CD163 (Novocastra, Clone 10D6, 1:50 dilution, performed on the 
Ventana XT using HIER solution CC1, pH 8.0). The histological and immunohistochem- ical 
analysis was performed under a light microscope (Olympus BX51). The biopsy samples were 
evaluated for cellular and antibody-mediated rejection as per the Banff 2013 criteria. Evidence 
for other disease processes affecting the allograft was also sought. The CD163 stained slides 
were scored blinded as follows: no stained cells = 0, G10 positive cells/hpf present in G25 % of 
biopsy = 1, 910 positive cells/hpf in 26–50 % of biopsy = 2, and 910 positive cells in 950 % of 
biopsy = 3.  
Data Analysis  
GFR and MRI data were compared between patients with non- rejecting and rejecting allografts 
using the two-sided t test. In addition, mean T2* values of whole kidneys as well as kidney 
cortex and medulla were compared between the three patient groups of native, non-rejecting and 
rejecting allografts using analysis of variance (ANOVA). In addition, T2* relaxation times were 
correlated with the CD163 score, using a linear regression analysis. All statistical computations 
were per- formed with Microsoft Excel software. An alpha level of 0.05 was chosen to indicate 
significant differences.