Novel Cardiac Magnetic Resonance Imaging to Define a Unique Restrictive Cardiomyopathy in Sickle Cell Disease
Conditions: Sickle Cell Disease
Sponsor: Children's Hospital Medical Center, Cincinnati

Title: 
 
 
Novel Cardiac Magnetic Resonance Imaging to Define a Unique Restrictive 
Cardiomyopathy in Sickle Cell Disease 
Registration Number: 
NCT02410811 
Document Date: 
March 16, 2016 

 
 
CLINICAL RESEARCH PROTOCOL 
 
 
 
 
Protocol Title: 
 
 
 
 
Novel Cardiac Magnetic Resonance Imaging to Define a Unique 
Restrictive Cardiomyopathy in Sickle Cell Disease 
 
 
Protocol Version: 
 
 
 
 3.1 
 
Protocol Date: 
 
 
 
 
March 16, 2016 
 
Principal Investigators:  
 
Charles T. Quinn, M.D., M.S. 
 
 
 
 
 
 
 
 
 
Pediatric Hematology, Cincinnati Children’s Hospital Medical Center 
 
 
 
 
 
 
 
 
 
Michael D. Taylor, M.D. 
 
 
 
 
 
 
 
 
 
Pediatric Cardiology, Cincinnati Children’s Hospital Medical Center 
Robert J. Fleck, Jr., M.D. 
 
 
 
 
 
 
 
 
 
Pediatric Radiology, Cincinnati Children’s Hospital Medical Center 
 
Co-Investigators: 
 
 
 
Lin Fei, Ph.D. 
 
 
 
 
 
 
 
 
 
Biostatistics, Cincinnati Children’s Hospital Medical Center 
 
 
 
 
 
 
 
 
 
Punam Malik, M.D., Ph.D. 
 
 
 
 
 
 
 
 
 
Pediatric Hematology, Cincinnati Children’s Hospital Medical Center 
 
 
 
 
 
 
 
 
 
Jeffrey A. Towbin, M.D. 
 
 
 
 
 
 
 
 
 
Pediatric Cardiology, Cincinnati Children’s Hospital Medical Center 
George Atweh M.D. 
 
 
 
 
 
 
 
 
 
Internal Medicine – Hematology, U. of Cincinnati College of Medicine 
 
 
 
 
 
 
 
 
 
Omar Niss, M.D. 
 
 
 
 
 
 
 
 
 
Pediatric Hematology, Cincinnati Children’s Hospital Medical Center 
 
Clinical Study Sites:  
 
 
Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 
 
 
 
 
 
 
 
 
 
University of Cincinnati College of Medicine, Cincinnati, OH 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Protocol Signature Page 
 
 
I, the Site Principal Investigator, agree to conduct this study in full accordance with the provisions of this 
protocol. 
 
I have read and understand the information in this protocol and will ensure that all associates, colleagues, and 
employees assisting in the conduct of the study are informed about the obligations incurred by their 
contribution to the study. 
 
 
 
 
 
_Charles T. Quinn, M.D., M.S.____ 
Name of Site Principal Investigator 
 
 
_______________________________  
_______________________________  
Signature of Site Principal Investigator  
Date of Signature

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Table of Contents 
1. 
Abbreviations ................................................................................................................................................ 4 
2. 
Synopsis ........................................................................................................................................................ 5 
3. 
Background and Rationale ............................................................................................................................ 5 
4. 
Specific Aims ............................................................................................................................................... 10 
4.1. Aim 1: Frequency of RCM phenotype ................................................................................................ 10 
4.2. Aim 2: Stability of RCM phenotype .................................................................................................... 10 
4.3. Aim 3: Association of RCM with TRJV ............................................................................................... 10 
4.4. Aim 4: Biomarkers .............................................................................................................................. 10 
5. 
Primary Endpoint ......................................................................................................................................... 10 
6. 
Study Population ......................................................................................................................................... 10 
6.1. Overview and Age Strata ................................................................................................................... 10 
6.2. Inclusion Criteria ................................................................................................................................ 11 
6.3. Exclusion Criteria ............................................................................................................................... 11 
6.4. Withdrawal and Replacement of Subjects ......................................................................................... 12 
7. 
Study Design and Monitoring ...................................................................................................................... 12 
7.1. Study Procedures ............................................................................................................................... 12 
7.2. Laboratory Studies and Biomarkers ................................................................................................... 13 
7.3. MRI Protocol ...................................................................................................................................... 13 
7.4. Echocardiography (TDI) Protocol ....................................................................................................... 14 
8. 
Sample Size and Analysis Plan .................................................................................................................. 15 
8.1. Aim 1: Frequency of RCM phenotype ................................................................................................ 15 
8.2. Aim 2: Stability of RCM phenotype .................................................................................................... 16 
8.3. Aim 3: Association of RCM with TRJV ............................................................................................... 16 
8.4. Aim 4: Biomarkers .............................................................................................................................. 16 
9. 
Study Sites and Personnel .......................................................................................................................... 16 
10. 
Recruitment .............................................................................................................................................. 16 
11. 
Potential Risk ............................................................................................................................................ 17 
12. 
Procedures to Maintain Confidentiality ..................................................................................................... 17 
13. 
Potential Benefits ...................................................................................................................................... 18 
14. 
Risk/Benefit Analysis ................................................................................................................................ 18 
15. 
Protection of Human Subjects .................................................................................................................. 18 
16. 
Data and Safety Monitoring Plan .............................................................................................................. 18 
17. 
Quality Assurance Monitoring Plan ........................................................................................................... 21 
18. 
Plan for Informed Consent ........................................................................................................................ 21 
19. 
Cost of Participation .................................................................................................................................. 22 
20. 
Compensation ........................................................................................................................................... 22 
21. 
References ............................................................................................................................................... 23 
 
 

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1. 
Abbreviations 
AE ...................... Adverse event 
AT1R .................. Angiotensin-1 receptor 
ALT .................... Alanine transaminase 
AST .................... Aspartate transaminase 
CBC ................... Complete blood count 
CMR ................... Cardiac magnetic resonance imaging 
ECV .................... Extracellular volume fraction 
EF ...................... Ejection fraction 
GCP ................... Good clinical practices 
Hb ...................... Hemoglobin 
HbF .................... Fetal hemoglobin 
HbS .................... Sickle hemoglobin 
HbSb0 ................. Sickle-b0-thalassemia 
HbSS .................. Sickle cell anemia (homozygous) 
HCT .................... Hematocrit 
ICH ..................... International conference on harmonisation 
IRB ..................... Institutional review board 
IVRT ................... Isovolumic relaxation time 
LDH .................... Lactate dehydrogenase 
LGE .................... Late gadolinium enhancement 
LV ....................... Left ventricle (or left ventricular) 
MDA ................... Malonaldehyde 
mPAP ................. Mean pulmonary artery pressure 
MR ..................... Magnetic resonance 
MRI .................... Magnetic resonance imaging 
MR-pro-ANP ...... Mid-regional pro-atrial natriuretic peptide 
NOX ................... NADPH oxidase 
NT-pro-BNP ....... N-terminal pro-hormone of brain natriuretic peptide 
PAH .................... Pulmonary arterial hypertension 
PAI-1 .................. Plasminogen activator inhibitor-1 
PVR .................... Pulmonary vascular resistance 
Rac ..................... Ras-related C3 botulinum toxin substrate (Rho-family GTPase) 
RBC ................... Red blood cell 
RCM ................... Restrictive cardiomyopathy 
RAS .................... Renin-angiotensin system 
ROS ................... Reactive oxygen species 
RV ...................... Right ventricle (or right ventricular) 
RVEDV ............... Right ventricular end diastolic volume 
RVESV ............... Right ventricular end systolic volume 
SAE .................... Severe adverse event 
SCD ................... Sickle cell disease 
TDI ..................... Tissue Doppler imaging 
TGFb1 ................ Transforming growth factor b-1 
TRJV .................. Tricuspid regurgitant jet velocity 
VCG ................... Vectorcardiographic algorithm 
VSD .................... Ventricular septal defect 
WB ..................... Whole blood 
3-d ...................... Three-dimensional 
 
 

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2. 
Synopsis 
Sickle cell disease (SCD) causes progressive cardiopulmonary morbidity, beginning in childhood, which 
can ultimately be fatal. As a group, cardiopulmonary complications, such as acute chest syndrome and 
sudden death, are now the most common causes of death in SCD, especially in adolescents and adults. 
Patients with SCD have features of both an anemia-related, high cardiac output state and a restrictive 
cardiomyopathy (RCM). We propose that this unique RCM is an overlooked and understudied 
complication of SCD. RCM could explain the modest increases in pulmonary artery pressure in patients 
with SCD, as measured by cardiac catheterization or estimated by tricuspid regurgitant jet velocity (TRJV), 
which has often been attributed to a primary pulmonary arterial hypertension (PAH). RCM could also be 
the cause of unexplained sudden cardiac death in SCD, which is a feature of other forms of RCM. 
Our overarching hypothesis is that increased ROS-mediated AT1R-TGFβ1 signaling is pro-fibrotic 
and, in combination with vaso-occlusive ischemia-reperfusion injury, results in an age-dependent, 
progressive RCM that can be detected by non-invasive cardiac imaging. 
This pilot, longitudinal, observational study uses a novel, comprehensive, multimodal cardiac imaging 
strategy, combining cutting-edge cardiac magnetic resonance imaging (CMR) and echocardiographic 
tissue Doppler imaging (TDI), to demonstrate the unique RCM of SCD, characterizing its frequency and 
the temporal evolution over a 2-year period. We will also correlate the RCM phenotype with biomarkers of 
ROS and RAS-TGFβ1 signaling. 
This research could change our understanding of how SCD affects the heart and lungs. We 
propose studies that will change the current concept of primary pulmonary vasculopathy to a 
cardiomyopathy-centered model with secondary pulmonary vascular changes leading to sudden death. 
This translational pilot study will deliver a novel, clear, quantifiable CMR phenotype with 
established diagnostic performance that will be used in phase II/III clinical trials to test anti-fibrotic 
therapy to prevent or reverse SCD-related RCM. 
3. 
Background and Rationale 
SCD is the name for a group of recessive genetic disorders caused by mutant sickle hemoglobin (HbS). 
The most common and severe form of SCD is sickle cell anemia (HbSS), the homozygous state for the 
HbS mutation (b6 gluèval). Other forms of SCD result from compound heterozygous states with HbS and 
other b-hemoglobinopathies, such as b0-thalassemia (sickle-b0-thalassemia). HbS is insoluble in the 
deoxygenated state. So, as HbS-containing red blood cells traverse the circulation undergoing cycles of 
oxygenation and deoxygenation, HbS repeatedly forms rigid polymers that damage the red blood cell 
(RBC) membrane and drastically shorten RBC lifespan. These HbS-containing RBCs can also obstruct 
the microcirculation before their premature demise. Consequently, individuals with SCD have chronic 
hemolytic anemia and episodic vaso-occlusive crises. Approximately 1 in 700 African-Americans has SCD 
at birth, and as many as 100,000 individuals are affected in the United States. [1,2]. 
Elevated Pulmonary Artery Pressure in SCD 
Sickle cell disease (SCD) causes progressive cardiopulmonary morbidity, beginning in childhood, which 
can ultimately be fatal. [3] As a group, cardiopulmonary complications, such as acute chest syndrome and 
sudden death, are now the most common causes of death in SCD, especially in young adults. [4,5] 
Pulmonary arterial hypertension (PAH) has been the focus of much recent research and debate. [6,7] 
Echocardiographic measurements of tricuspid regurgitant jet velocity (TRJV) are commonly used to 
estimate the prevalence of PAH (TRJV > 2.5 m/s), which has consistently been approximately 30% in both 
children and adults. Indeed, an elevated TRJV is associated with early mortality in adults, [8] although not 
children. [9] 
Multiple epidemiologic studies associate elevated right ventricular (RV) pressures with adverse events in 
patients with SCD. [8,10,11] These data have been interpreted to support the idea that hemolysis-related 
endothelial dysfunction causes PAH, which is responsible for the observed increase in RV pressure.  

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However, in a series of recent studies, patients were selected to undergo right heart catheterization based 
on echocardiographic screening. [12-14] These studies showed the prevalence of PAH (defined simply as 
mPAP ≥ 25 mmHg) is low, ranging from 6-10%, and that the prevalence of moderate to severe PAH is 
significantly less. The pulmonary vascular resistance (PVR) of these patients is markedly lower than that 
reported in other pulmonary hypertension populations. [12] In one study, the PVR in all the patients with 
SCD was within the normal range. [15] Consequently, the number of adverse events in these patients with 
very mild elevation in pulmonary pressures exceeds that expected in patients with idiopathic pulmonary 
hypertension. [16] These observations have led us to propose that the primary mechanism for cardiac 
morbidity and mortality including sudden death is not primary PAH, but rather a unique restrictive 
cardiomyopathy (RCM). 
Restrictive Cardiomyopathy 
Several lines of data suggest that increased right ventricular (RV) and pulmonary arterial (PA) 
pressure in SCD is secondary to the restrictive left ventricular (LV) physiology found in restrictive 
cardiomyopathy (RCM) and not a primary form of pulmonary arterial hypertension (PAH). These lines of 
evidence include: (1) It is well known that impaired diastolic filling is common in all SCD patients, 
including young patients, before they develop increased RV pressures.[17] In a study by Hankins et al., all 
children between the ages of 6-18y had an abnormal E/e’ ratio on echocardiography, a marker of 
increased left atrial (LA) pressure.[18] This finding was also seen in a group SCD patients undergoing 
sleep studies.[19] Although it has been postulated that impaired relaxation and restrictive filling is 
secondary to iron deposition, Hankins et al. showed no significant iron deposition by MRI T2* 
measurements. It has also been postulated that the diastolic dysfunction seen in these patients is 
secondary to hypertrophy; however, Hankins et al. also showed that no patients had abnormal age-
corrected indexed LV mass. (2) The diastolic dysfunction seen in SCD appears to precede the PA 
pressure elevation. A study by Eddine et al. showed abnormalities in nearly all diastolic filling parameters 
in a group of 54 children with SCD, but none had significant elevations of tricuspid regurgitant jet velocity 
(TRJV).[20] Dham et al. reported a group of SCD children with minimal TRJV increase but with a high 
prevalence diastolic dysfunction.[21] (3) There is a high rate of sudden death, in excess of what is seen 
in the idiopathic PAH population, in the SCD population, with relatively low PA pressures. While not 
consistent with what is seen in the pediatric and adult PAH population, this high rate of sudden death is 
consistent with that  seen in RCM, in which there is an excessive rate of sudden death (28% in a pediatric 
cohort; 2-year survival 50% and 5-year survival 25% in some series).[22,23] As in SCD, the risk of sudden 
death is particularly high in young patients with idiopathic RCM associated with PAH.  
The standard definition of RCM includes (1) dilated atria, (2) normal or nearly normal left ventricular 
systolic function without significant ventricular dilatation; (3) no echocardiographic evidence of 
hypertrophic cardiomyopathy; and (4) no evidence of 
constrictive pericarditis. Supportive evidence for RCM 
includes a restrictive mitral inflow pattern by pulsed-
wave Doppler. The cardiomyopathy in sickle cell 
disease is characterized by dilated atria, normal or 
supra-normal systolic function, impaired relaxation, 
and restrictive filling patterns. However, the ventricles 
are typically mildly dilated, likely due to the anemia-
related high output state. We propose that the 
cardiomyopathy of SCD is a unique RCM 
phenotype with normal to mildly dilated left ventricle 
with abnormal diastolic relaxation, increasingly 
elevated left atrial pressures, and normal systolic 
function as a result of myocardial fibrosis (Figure 1). 
It is this cardiomyopathy that results in elevated post-
capillary PVR, secondary PAH, and elevated TRJV.  
 
Figure 1. The restrictive cardiomyopathy of SCD. Four 
chamber (A) and two chamber (B) SSFP images from a 20 
y.o. HbSS patient showing moderate bi-atrial dilatation, left 
ventricular dilatation, and normal systolic function. The left 
atrial volume was 79 ml/m2 (normal ~ 45 ± 9 ml/m2).  The 
left ventricular end-diastolic volume was 121 ml/m2 (normal 
~ 92 ±10 ml/m2). The ejection fraction was 66%.   
 
LV 
LA 
RV 
RA 
LV 
LA 

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Cardiac Magnetic Resonance Imaging (CMR) 
CMR will be used to characterize cardiac structure and function in this study of children and adults with 
SCD. CMR provides functional data (systolic and diastolic) and myocardial tissue characterization. CMR is 
ideal for measuring ventricular 
volumes and masses, because it is 
not restricted by geometric 
assumptions, provides high contrast 
images, and is inherently three-
dimensional (3-d). CMR has been 
validated against ex vivo samples for 
measurements in adult and pediatric 
ventricles. [24-27] The intra- and 
inter-observer variability for standard cardiac MRI measures is 3-8%. [28,29] Compared to 
echocardiography, CMR is also less variable and more reproducible, allowing smaller sample sizes for 
clinical studies (Figure 2).  
Diastolic assessment with CMR is validated for assessment of myocardial relaxation and filling dynamics. 
[30,31] Using velocity sensitive phase contrast imaging of the mitral valve inflow and the left ventricular 
myocardium, diastolic parameters including isovolumic relaxation time (IVRT), early to late inflow ratio 
(E/A ratio), deceleration time (DT), 
and mitral valve inflow to septal 
velocity ratio (E/e’) are measured with 
both accuracy and precision 
compared with echocardiography 
(Figure 3). [32,33] Using standard 
planimetry, the left atrial volume 
provides an indirect measure of the 
filling pressure. There are also novel 
deformational diastolic parameters 
that can be measured including 
diastolic strain rate, untwisting, and 
torsion (Figures 4, 5). [34] 
 
Figure 3. Restrictive LV filling in SCD. Myocardial velocity (green) and mitral 
valve (red) inflow phase contrast curves from a SCD patient at CCHMC. The 
mitral valve inflow shows a markedly increased E/A ratio and the E/e’ is 
elevated consistent with restrictive filling. 
Figure 2. CMR is less variable and more 
reproducible than echocardiography in 
patients undergoing repeated LV measurements 
using both modalities. Standard deviations of 
CMR measurements are significantly lower than 
echocardiography. Decreased variability and 
greater reproducibility affords 80-90% smaller 
sample sizes to detect differences in cardiac 
structure and function. 
Figure 4. CMR and regional myocardial strain.  The short axis of the mid-ventricle is obtained from the four-chamber view (a) at 
the level of the papillary muscles with a tag sequence (b).  Mesh overlaying of the tag image using HARP (c). Regional Myocardial 
Strain (d): In this idealized LV diagram, normal strains are represented: wall thickening (radial strain, εrr), circumferential shortening 
(circumferential strain, εcc), and longitudinal compression (longitudinal strain, εll). Peak composite (shown) or regional εcc can be 
shown graphically or exported to a spreadsheet for data analysis. 

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Myocardial tissue characterization by CMR broadly involves determining 
signal characteristics before and after administration of IV contrast and 
comparing quantitative parameters with data from normal myocardium. [35] 
Myocardium can be differentiated from fat, fluid, inflammation, and fibrosis. 
[36] Regions of myocardial inflammation can be identified using T2-
weighted imaging, secondary to higher fluid content in these areas of 
myocardial tissue. Similarly, regions of edema can be seen in active 
myocarditis and infiltrative cardiomyopathies. Gadolinium–diethylene-
triamine pentaacetic acid (Gd-DTPA) is a partially extracted extracellular 
contrast agent that resides in the interstitial space of territories that receive 
myocardial blood flow. Territories with scar have replacement fibrosis, 
more interstitial space, and as a result, they retain gadolinium. Late 
gadolinium enhancement (LGE) is the most widely used and investigated 
CMR technique for myocardial characterization (Figure 6). [37,38] LGE 
sequences obtained approximately 10 minutes after gadolinium injection 
result in distinct image contrast. Gadolinium is paramagnetic resulting in 
significant signal differentiation. In T1-weighted pulse sequences normal 
myocardium is set to appear black with regions of gadolinium accumulation 
appearing bright. Enhanced (white) regions indicate areas of gadolinium accumulation and suggest the 
presence of inflammation, fibrosis, or scar.  
The specific pattern of tissue characteristics can identify a given cardiomyopathy. For example, cardiac 
amyloidosis frequently demonstrates diffuse global subendocardial LGE. In contrast, cardiac sarcoidosis 
demonstrates patchy myocardial LGE of either the LV or right ventricle. Unlike other common non-
ischemic cardiomyopathies, hypertrophic 
cardiomyopathy (HCM) is known to have a unique 
structural phenotype that classically involves 
asymmetric LV wall thickening of the basal 
interventricular septum. In hypertrophic cardiomyopathy, 
LGE is often present in a patchy midwall distribution, 
particularly at the insertion point of the left and right 
ventricles and regions of greatest hypertrophy. Non-
ischemic dilated cardiomyopathies are generally 
recognized to have a typical midwall linear LGE pattern 
that is commonly located in the ventricular septum. In 
contrast to chronic cardiomyopathies, active myocarditis 
typically demonstrates focal increases in midwall and 
subepicardial signal on T2-weighted images, signaling 
the presence of edema in these areas. Farther out from 
the initial onset of myocarditis, epicardial LGE may be 
seen in areas of residual fibrosis. Using these tissue 
characteristics, the etiology of cardiomyopathy can be 
derived in many cases and may obviate invasive 
myocardial biopsy. 
 
Figure 5. CMR-based deform-
ational diastolic parameters. 
Diastolic slope derived from 
longitudinal deformation curve 
allows quantitative measurement 
of the relaxation rate. 
Figure 6. Late gadolinium enhancement (LGE). 
Panel A: CMR of fibrotic myocardium highlights 
differences hydrogen nuclei (spins) present throughout 
tissues. After applying radiofrequency energy in a 
magnetic field, spins in fibrotic tissue relax faster (i.e. 
have a shorter T1) to equilibrium compared to those in 
normal muscle, a difference that can be detected using 
the LGE imaging technique. Panel B: Sample LGE 
image in the 4 chamber from patient with myocardial 
infarction (white area) as indicated by the arrow. 

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The majority of studies of SCD patients showed no 
significant areas of LGE. However, pathology 
(autopsy) data show significant areas of fibrosis. 
Small microscopic areas of fibrosis are not detected 
using standard post-Gd LGE techniques. 
Consequently, new CMR techniques have recently 
been developed to quantify the extent of diffuse 
myocardial fibrosis present at a given time point.  
Microscopic fibrosis has been shown to be 
quantified by the partition coefficient of gadolinium, 
which can be measured with T1 mapping. With this 
technique, decreases in the T1 time of the 
myocardium reflect increased interstitial space (microfibrosis) and increased gadolinium concentration 
throughout the myocardium. Thus, the relative distribution of gadolinium between the blood pool and 
myocardium can be used to estimate the amount of fibrosis within the myocardium. When quantified 
as the extracellular volume fraction (ECV) metric (Figure 7), [39] this relative distribution of gadolinium in 
the myocardium has been shown to correlate with the presence of fibrosis on surgical biopsies in patients 
with HCM. [40]  
Preliminary Data 
In our preliminary study of 7 SCD patients (Table 1), all 7 had dilated atria and LVs, 6/7 had abnormal 
relaxation, and 5/7 had restrictive filling patterns with evidence of increased filling pressures. 
Consequently, 5/7 fit the criteria for the RCM phenotype. None had evidence of LGE by standard 
techniques. However, upon 
implementing the fibrosis imaging 
scheme using ECV parametric 
maps (Figure 7), our data show 
similar findings to those from 
SCD mice, where relaxation is 
impaired and ECV is increased. 
The image data were confirmed 
by histopathology showing 
increased perivascular fibrosis 
and myocardial fibrosis. 
Overview of Research Plan and Deliverables 
We will now apply this novel and comprehensive imaging strategy, CMR with LGE and quantification of 
the extent of diffuse myocardial fibrosis using the ECV metric (with supplementation by echocardiographic 
TDI), for the first time to a representative group of children and adults with SCD to demonstrate the 
unique RCM of SCD. 
This research could change our understanding of how SCD affects the heart and lungs. We 
propose studies that will change the current concept of primary pulmonary vasculopathy to a 
cardiomyopathy-centered model with secondary pulmonary vascular changes leading to sudden death. 
This translational pilot study will generate the prerequisite for phase II/III clinical trials to test anti-
fibrotic therapy to prevent SCD-related RCM; specifically, this study will provide a novel, clear, 
quantifiable CMR phenotype with established diagnostic performance characteristics. 
 
 
Table 1. Selected Cardiac MRI parameters in 7 SCD patients 
referenced to normal values as mean z-scores.   
Parameter 
Value ± SD 
Mean z-score 
LV end-diastolic volume index(ml/m2) 
124 ± 14 
2.96 
LV ejection fraction (%) 
67 ± 6 
0.92 
LV mass (g/m2) 
82 ± 7 
2.56 
LV Diastolic Function 
 
 
LA volume index (ml/m2) 
65 ± 14 
3.85 
Early diastolic strain rate [Inf/Lat] (s-1) 
59 ± 11 
4.17 
Mitral Inflow E /A 
2.8 ± 0.4 
1.26 
Figure 7. CMR evidence of 
microscopic fibrosis. ECV 
map of the LV at 3T in an 8 
y.o. Duchenne Muscular 
Dystrophy patient at CCHMC 
without evidence of significant 
LGE. The mid-ventricular ECV 
was 35±4 % suggesting 
diffuse microscopic fibrosis. 
Normal value from the 
literature is 26±3%. [37] 

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4. 
Specific Aims 
This pilot study will provide the critical data needed to design phase II/III clinical trials to test anti-fibrotic 
therapy, such as losartan, as a means to prevent or reverse SCD-related RCM. 
4.1. Aim 1: Frequency of RCM phenotype 
Estimate the proportion of the RCM phenotype in SCD in a representative sample of children and 
adults with SCD and how the proportion varies by age groups in a cross-sectional sample. We will 
also quantify the extent of diffuse myocardial fibrosis using the extracellular volume fraction (ECV) 
metric. 
Hypothesis: The proportion of the RCM phenotype will be 30% in the overall study population, and 
the proportion increases in older age strata. 
4.2. Aim 2: Stability of RCM phenotype 
Estimate the stability (resolution, non-progression, or progression) of the RCM phenotype over a 2-
year period to determine its suitability as an outcome measure for a future clinical trial in children and 
adults with SCD who are imaged yearly. 
Hypothesis: The RCM phenotype, once detected by imaging, will not spontaneously resolve. 
4.3. Aim 3: Association of RCM with TRJV 
Explore the association between tricuspid regurgitant jet velocity (TRJV) and RCM phenotype. 
Hypothesis: Individuals with a high TRJV (>2.5 m/s) will be more likely to have RCM features and 
more extensive fibrosis than those with a normal TRJV (≤2.5 m/s). 
4.4. Aim 4: Biomarkers 
Estimate the within-patient and between-patient variability of a comprehensive panel of biomarkers 
for heart failure, ROS production, and RAS-TGFb1 signaling, as well as a panel of clinical laboratory 
tests. Explore the correlations between the RCM phenotype and the panel of biomarkers. 
Hypothesis: These biomarkers will have performance characteristics to serve as discriminative 
surrogate markers in future clinical trials. 
5. 
Primary Endpoint 
The primary endpoint is the RCM phenotype, which is defined for this study as the following: 
• 
Normal LV ejection fraction (%); 
• 
Impaired diastolic relaxation:  (dεcc/dt) 2 z-scores below normal; 
• 
Restrictive left ventricular filling:  deceleration time 2 z-scores below normal for age; 
• 
LA indexed volume 2 z-scores above normal; and 
• 
No evidence of infiltrative disease, hypertrophic cardiomyopathy, or constrictive pericarditis. 
All normal values will be age-appropriate when available. 
6. 
Study Population 
6.1. Overview and Age Strata 
We will enroll a maximum of 30 participants in the entire study across three age strata: A, 6 – 13.9 
years; B, 14 – 20.9 years; and C, 21 years and older (Figure 8). 25 participants are required to fill all 
strata, but up to 5 additional participants will be enrolled, if needed, to replace any drop-outs to 
maintain complete, active enrollment of 25 in the study.  Enrollment in all strata should proceed 
simultaneously (until each is filled). Stratum A will have 5 participants, while strata B and C will each 
have 10 participants. Participants in stratum A can have any TRJV. Participants will be selected for 

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strata B and C such that half will have a normal TRJV (≤2.5 m/s) and the other half an elevated 
TRJV (>2.5 m/s). Once filled, each stratum will be closed to enrollment. 
 
Figure 8. Definitions of Strata by Age, Group Size, and TRJV 
 
6.2. Inclusion Criteria 
The following inclusion criteria apply to all study participants: 
1. 
Sickle cell anemia (HbSS) or sickle-b0-thalassemia (HbSb0) confirmed by hemoglobin 
separation and identification techniques. 
2. 
Ability to cooperate with and undergo CMR without sedation or anesthesia. 
3. 
Ability to cooperate with and undergo echocardiogram. 
4. 
Written informed consent in accordance with the institutional policies and federal guidelines 
must be provided by the participant (if ≥18 years of age) or parent or legally authorized guardian 
(if the participant is <18 years of age).  Minor participants ≥11 years of age will be requested to 
provide assent. 
The following additional inclusion criterion applies to Age Stratum A: 
1. 
Age 6 to 13.99 years. 
The following additional inclusion criteria apply to Age Stratum B: 
1. 
Age 14 to 20.99 years. 
2. 
Detectible and quantifiable TRJV with reported value. 
The following additional inclusion criteria apply to Age Stratum C: 
1. 
Age ≥21 years. 
2. 
Detectible and quantifiable TRJV with reported value. 
 
Note: If the tricuspid valve insufficiency peak gradient (TV gradient) is provided on an 
echocardiogram report rather than the TRJV directly, the TRJV can be back-calculated as follows: 
 
𝑇𝑅𝐽𝑉= &(𝑇𝑉 𝑔𝑟𝑎𝑑𝑖𝑒𝑛𝑡)/4  
6.3. Exclusion Criteria 
1. 
Current chronic transfusion therapy (defined as regular, approximately monthly, transfusions of 
packed red blood cells given for at least 6 consecutive months for the treatment of prevention of 
SCD-related complications with the plan to continue this therapy at the time of potential 
enrollment). 
2. 
Any contraindication to MRI or physical or behavioral factor that could degrade the quality of 
MRI data or interfere with a participant’s tolerance of the MRI, such as permanent or semi-
permanent metallic implants, including pacemakers and defibrillators, or severe claustrophobia. 
3. 
Known ventricular septal defect (VSD) documented in medical record. 
4.    Estimated GFR <60 mL/min/1.73 m2 (estimated by serum creatinine or cystatin-C). 
5. 
Pregnancy (documented by serum or urine pregnancy test). 
Stratum A
6-13.9 years
Total N=5
Any TRJV
Stratum B
14-20.9 years
Total N=10
TRJV ≤2.5 m/s, N=5
TRJV >2.5 m/s, N=5
Stratum C
≥21years
Total N=10
TRJV ≤2.5 m/s, N=5
TRJV >2.5 m/s, N=5

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Note: Participants are allowed to participate in the SCD Stable Isotope Protocol (2011-2977) 
concurrently with this study. 
6.4. Withdrawal and Replacement of Subjects 
Participants may withdraw from the study at any time. Study personnel may withdraw participants 
who are deemed to be non-compliant with study procedures. Another eligible participant may replace 
the withdrawn participant. The study data of such withdrawn participants will not be discarded and 
may be analyzed in post hoc analyses. 
 
Although chronic transfusion therapy is an exclusion criterion, it is not a reason for withdrawal of 
subjects if begun after enrollment. 
7. 
Study Design and Monitoring 
7.1. Study Procedures 
Participants will have minimum of 4 study visits. Participants will undergo CMR and 
echocardiography at study entry (visit 1) and then yearly for two years (visits 2 and 3). Study 
procedures are detailed in Table 2. 
 
Visit windows for each of the 4 visits will be ± 3 days. Some of the procedures for a visit can be 
performed on different days, if necessary, within this window. Visits 0 and 1 may also be combined 
for participants’ convenience, if desired. If done separately, the maximum time between visits 0 and 
1 is 3 months. 
 
Table 2. Study Procedures by Study Visit 
Study Procedure 
Visit 0 
(consent/pre-
baseline) 
Visit 1 
(baseline) 
Visit 2 
(12 months) 
Visit 3 
(24 months) 
Confirm eligibility 
X 
 
 
 
Informed consent 
X 
 
 
 
Review of medical records 
X 
X 
X 
X 
History and physical1 
 X1 
X 
X 
X 
Clinical laboratories1 
 X1 
X 
X 
X 
Cardiac biomarkers1 
 X1 
X 
X 
X 
ROS biomarkers1 
 X1 
X 
X 
X 
RAS-TGFβ-1 biomarkers (blood and urine) 1 
 X1 
X 
X 
X 
Genetic testing 
 
X 
 
 
Pre-CMR hydration status2 
 
X 
X 
X 
Pre-CMR pregnancy status2 
 
X 
X 
X 
CMR 
 
X 
X 
X 
Echocardiography 
 
X 
X 
X 
Electrocardiogram 
 
X 
X 
X 
AE monitoring 
 
X 
X 
X 
Interim history 
 
X 
X 
X 
Reimbursement 
 
X 
X 
X 
Notes: 
1Blood and urine tests may be obtained on Visit 0 or Visit 1 after consent obtained. 
2 Prior to CMR there must be assessment and documentation of (1) hydration status—must not be dehydrated; 
(2) renal function—must have eGFR ≥60 mL/min/1.73 m2; and (3) pregnancy status—must have negative 
serum or urine pregnancy test.  These results must be known before the CMR and Echo. 

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7.2. Laboratory Studies and Biomarkers 
1. Clinical laboratories: 
Complete blood count, reticulocyte count, HbF quantitation, LDH, AST, bilirubin fractions, plasma 
free Hb, serum creatinine, ektacytometry, cystatin-C, urinalysis, and first-morning and 24-hour 
urine collection for protein, creatinine, microalbumin, and osmolality. Rationale: these tests are 
markers of SCD-related severity, renal function and/or indirect markers of hemolysis that will be 
correlated with clinical and imaging outcomes. In addition, a pregnancy test will determine if 
females of childbearing potential may proceed with the study. 
2. Cardiac biomarkers: 
Plasma concentrations of the natriuretic peptides, NT-pro-BNP and MR-pro-ANP, and cardiac 
troponins. Rationale: these tests are markers of heart failure (including RCM) that will be 
correlated with clinical and imaging outcomes. 
3. Biomarkers of ROS: 
RBC ROS measured by flow cytometry; plasma MDA by HPLC; RBC glutathione level; relative 
NOX1, NOX2, NOX4 and NOX5 expression in RBCs (WB); activity of Rac GTPases by pull-
down. Rationale: these tests are indicators of oxidative stress that we hypothesize lead to the 
RCM phenotype that will be correlated with clinical and imaging outcomes. 
4. Biomarkers of RAS-TGFb1: 
Plasma for renin activity, aldosterone level, active TGFb1, and PAI-1 activity; urine and serum for 
angiotensin-II and serum forangiotensinogen (oxidized and reduced forms for both); and AT1R 
expression on platelets and RBCs. Rationale: these assays are for markers of signaling through 
the RAS-TGFb1 pathways, that we hypothesize lead to the RCM phenotype that will be 
correlated with clinical and imaging outcomes. 
5. Genetic testing: 
Screen for single nucleotide polymorphisms in ~100 genes that encode components of the RAS-
TGFb1 pathways. Rationale: these are polymorphisms of components in the RAS-TGFb1 
pathways that could modulate the development of the RCM phenotype that we hypothesize 
results from excessive TGFb1 signaling. 
7.3. MRI Protocol 
CMR will be performed on a Philips Achieva 1.5T 70 cm bore scanner with a 32 channel dedicated 
cardiac coil. Parallel imaging with SENSE reconstruction (SENSE factor=2) will be used for 
acceleration. Acquisitions will be gated using a standard vectorcardiographic algorithm (VCG).  
Subjects will be asked to breath hold as tolerated for the scanning sequences; subjects unable to 
breath hold will be scanned using “breathe-through” techniques that have become standard at most 
pediatric CMR centers, and has been shown to be accurate. Standard sequences will be performed 
including localizers and steady-state free precession cine CMR in the ventricular long and short axis 
planes for chamber sizes, EF, and ventricular mass. Once “standard” imaging is completed, cine 
myocardial tag imaging will be performed in short axis and 2 long axis planes of the mid-papillary 
slice of the left ventricle via the SPAMM (spatial modulation of magnetization) technique.  Certified 
CMR technologists will perform all cardiac CMR scans with continuous guidance from pediatric CMR 
physicians.  The following sequences and procedures will be performed on all subjects: 
1. 
Three plane SSFP localizer. 
2. 
Axial Black Blood stack using a single shot technique 
3. 
2 Chamber, 4 Chamber, 3 chamber, and short axis cine SSFP  
4. 
Single short axis T2* image 

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5. 
Phase Contrast imaging of the aortic valve, pulmonary valve, branch pulmonary arteries, 
atrioventricular valves. 
6. 
Tissue PC imaging of the myocardium in the short axis, 4 chamber, and 2 chamber 
7. 
T1 mapping using a MOLLI sequence in the short axis and long axis planes    
8. 
Intravenous injection of 0.2 mmol/kg Gd - DTPA  
9. 
Seven minutes post Gd injection, Look-Locker based TI scout 
10. Eight minutes post Gd injection LGE Short Axis and long axis images 
11. Ten minutes post Gd injection T1 mapping using a MOLLI sequence in the short axis and long 
axis planes       
CMR Image Processing: 
The CMR images will be stored in standard DICOM format in a password protect computer in the 
PI’s office for analysis using cardiac software analysis packages [Mass – Medis, Leiden, Netherlands 
and HARP/SENC – Diagnasoft, Cary, NC].   
CMR Image Analysis:  
Global Ventricular Function: Ventricular and atrial chamber sizes, myocardial mass, and ejection 
fraction will be measured using standard planimetric software. Endocardial and epicardial contours 
will be drawn manually including segmenting large trabeculations (MEDIS, Leiden, The 
Netherlands). The location of the atrioventricular valve planes will be confirmed by referencing the 
images to the 2- and 4-chamber images.  An experienced analyst and CMR physician will draw all 
contours. The ventricular mass will be calculated as the difference between the ventricular epicardial 
and endocardial volumes multiplied by the estimated myocardial density (1.06 g/ml). The volume and 
mass will be indexed to the body surface area (BSA), calculated by the Mosteller algorithm.  As an 
internal data verification, the ventricular stroke volume (RVEDV – RVESV) will be compared with the 
phase contrast derived cardiac output after excluding shunting. 
The left ventricular mid-ventricular systolic circumferential, longitudinal, and radial strains and the 
diastolic relaxation slope will be calculated using HARP algorithm with the 4 chamber and short axis 
CSPAMM images.  (HARP/SENC, Diagnasoft, Cary, NC).  A dedicated research expert will perform 
all strain analyses.  
The ECV will be measured using a combination of MRmap 1.3 (Danial Messroghli, Berlin) and 
MATLAB.  MRmap allows creation of the T1 maps from MOLLI image sets.  Using Matlab 
(Mathworks, Natck, MA), a parametric image is created according to the following equations where R 
is the relaxation rate.  ECV and HCT are expressed as percentages. 
 
7.4. Echocardiography (TDI) Protocol 
All patients will undergo a limited echocardiogram just prior to the MRI.  The echocardiogram will be 
focused to look at the Doppler tissue velocities of the myocardium, the atrioventricular inflows, and 
the TRJV.  All echocardiograms will be performed and all measurements will be made centrally in 
the core echocardiography laboratory at CCHMC.  As noted in our previous losartan in SCD grant, 
agitated saline will be used to increase the contrast of the TRJV.   
Pre-procedure: 
• 
Assess for contraindications to agitated saline: pregnancy and known VSD 
ΔR1myo =1/T1myo−postGd −1/T1myo−preGd
ΔR1blood =1/T1blood−postGd −1/T1blood−preGd
ECV = ΔR1myo / ΔR1blood ∗(100 −HCT)

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• 
All patients will have peripheral IV (20 gauge or larger) in upper extremity for both the 
echocardiogram and the MRI. A topical numbing spray or anesthetic ointment may be used to 
minimize the discomfort of IV placement. 
 
Left Ventricular Diastolic Function 
• 
Mitral valve inflow 
• 
Mitral valve Doppler tissue imaging (DTI) (both lateral wall annulus and septal annulus) 
• 
Pulmonary venous Doppler velocity profile (AP4C, sample volume in pulmonary vein upstream 
from ostium, ensure A wave is clearly seen) 
• 
Describe atrial septal bowing if any, use PFO Doppler velocity for interatrial gradient 
Right Atrial Pressure estimation 
• 
IVC collapse (sniff test) from longitudinal abdominal view -Hepatic vein flow reversal – color 
Doppler from longitudinal abdominal view 
Atrial septum 
• 
2d and color Doppler of the atrial septum interrogating for ASD/PFO 
RV/PA Pressure 
• 
TR Doppler peak velocity -PR peak and end-diastolic velocity -Repeat TR Doppler peak velocity 
during the administration of 10 ml of agitated saline IV 
Pulmonary artery imaging 
• 
MPA Pulmonary Doppler flow profile 
8. 
Sample Size and Analysis Plan 
This pilot study will provide the critical data needed to design phase II/III clinical trials to test anti-
fibrotic therapy, such as losartan, as a means to prevent or reverse SCD-related RCM. Total sample 
size is based upon the degree of precision of the estimate of the true proportion of the RCM phenotype 
(see Aim 1) in combination with pragmatic issues. 
8.1. Aim 1: Frequency of RCM phenotype 
The primary endpoint of this aim is diagnosis of RCM as defined by CMR. Sample proportion and 
confidence interval will be used to make inference on the true proportion of the RCM phenotype in a 
population of children and adults with SCD. We will enroll a total of n=25 subjects across three 
different age strata (n=5 for age 6-13, n=10 for age 14-20 and n=10 for age ≥21). With a 95% level 
of confidence, the proposed sample of 25 would estimate the true proportion with an error 
margin≤0.2. If the RCM prevalence in the SCD population is ≥21% and the prevalence in a normal 
population does not exceed 5%, the proposed sample would also provide a ≥80% power to detect a 
higher RCM frequency in the SCD population. The expected (hypothesized) prevalence is 30%. 
Given that the expected proportion of RCM in a normal population of children and adults would be 
close to zero, this is an excellent power for this pilot study. We will also explore whether the 
proportion of RCM changes across the three age strata using the Freeman-Halton extension of the 
Fisher exact probability test for a 2x3 contingency table. We will also quantify the extent of diffuse 
myocardial fibrosis using the partition coefficient of gadolinium for each participant with RCM, and 
perform secondary exploratory correlations with clinical, laboratory, and imaging variables. 
 
25 participants are required to fill all strata, but up to 5 additional participants will be enrolled, if 
needed, to replace any drop-outs to maintain complete, active enrollment of 25 in the study.   

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8.2. Aim 2: Stability of RCM phenotype 
In this aim, we will estimate the temporal stability (e.g., resolution, non-progression, or progression) 
of the RCM phenotype over a 2-year period to determine its suitability as an outcome measure for a 
future clinical trial.  The primary endpoint is RCM classification repeatedly diagnosed at enrollment 
and every year post enrollment. The proportion of changes in classification from baseline (RCM or 
no RCM) at the time of the 2 yearly follow-up CMR studies will be used to make inference on the 
temporal stability. We hypothesize that the RCM phenotype, once detected by imaging, will not 
spontaneously resolve. Therefore, poor CMR test performance would be the change from RCM to no 
RCM phenotype compared to any preceding study. Acceptable CMR test performance would be the 
persistence of the RCM phenotype from one CMR study to the next (baseline to year 1; year 1 to 
year 2). Whether the degree of the RCM phenotype changes with time (e.g., progresses, or remains 
stable) is not a factor in this analysis, as both outcomes are biologically expected. We will conclude 
that the RCM phenotype by CMR is acceptable for use in clinical trials as an imaging outcome if no 
more than 1 subject (<5% of the total sample size) with the CMR-defined RCM phenotype has 
resolution of the RCM phenotype on any follow-up CMR study. 
8.3. Aim 3: Association of RCM with TRJV 
In this exploratory aim, we will test for association between the RCM phenotype, as the binary 
dependent variable, and the TRJV both as a continuous regressor and as a dichotomous (≤2.5 vs 
>2.5 m/s) independent variable. This dichotomy has already been established as clinically and 
prognostically meaningful in adults with SCD. For this secondary analysis, we will combine all the 
age strata into a single study population, and we will not test for this association across age strata 
because of small sample size in each. We also hypothesize that RCM will be associated with a 
higher TRJV independent of age. Analysis will be by GLM and binary logistic regression. 
8.4. Aim 4: Biomarkers 
In this exploratory aim, we will estimate the within-patient and between-patient variability of a 
comprehensive panel of biomarkers for heart failure, ROS production, and RAS-TGFb1 signaling, as 
well as a panel of clinical laboratory tests. The goal of this aim is to describe the performance 
characteristics of the biomarkers to determine their suitability for future clinical trials. 
9. 
Study Sites and Personnel 
Cincinnati Children’s Hospital Medical Center (CCHMC).  Drs. Charles Quinn, Michael Taylor, and Robert 
Fleck will be responsible for the overall conduct of the study. Dr. Charles Quinn will oversee the 
identification and enrollment of participants at CCHMC and coordinate all multi-center activities. CMR will 
be overseen and interpreted by Drs. Michael Taylor, Robert Fleck, and Jeffrey Towbin. Echocardiography 
with be overseen and interpreted by Drs. Michael Taylor and Jeffrey Towbin. Dr. Lin Fei will be 
responsible for statistical design and analysis. All study investigators (see page 1) will responsible for 
design, analysis, and publication of the study. Children and young adult participants (age ≤21 years) will 
be recruited at this site. 
University of Cincinnati College of Medicine. Adult participants (age ≥18 years) will be recruited at this site, 
overseen by Dr. George Atweh. All other study procedures will take place at CCHMC. 
10. Recruitment 
Study personnel may approach potential participants during clinically indicated inpatient or outpatient 
encounters. IRB-approved brochures will be mailed to potentially available participants and provided to 
them in person during medical and/or research encounters. We will post IRB-approved flyers in approved 
locations in the hospital and clinics of study sites to solicit direct contact by interested participants. The 
study may also be advertised to our patient population by phone calls, the research website, and at 
outreach activities. Recruitment and participation in this study will not interfere with or delay standard 
medical care. 

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11. Potential Risk 
This study involves few risks to subjects, including: (1) discomfort from venipuncture and IV catheter 
placement, (2) anxiety related to echocardiography, (3) anxiety or claustrophobia during the MRI, and (4) 
adverse effects of the MR contrast agent, gadolinium. Echocardiography and cardiac MRI are safe, 
noninvasive procedures that are often performed as standard of care for children and adults with SCD to 
screen for or diagnose cardiopulmonary complications of SCD. The side effects of gadolinium include 
headache, nausea and local discomfort (at the IV site). Rarely (less than 1%) low blood pressure and 
light-headedness occurs. This can be treated immediately with intravenous fluids. Very rarely (less than 
one in one thousand), patients may be allergic to gadolinium. Allergic reactions are most commonly hives 
and itchy eyes, but more severe reactions have been reported. 
Gadolinium contrast is safe for patients with SCD who are not dehydrated. There is some evidence to 
suggest that gadolinium-based contrast agents may be associated with the development of nephrogenic 
systemic fibrosis (NSF) in patients with severe renal insufficiency, i.e. in patients with acute or chronic 
severe renal impairment [estimated GFR (eGFR) <30 mL/min/1.73 m2 for children >2 years of age, 
adolescents, and adults], or acute renal failure of any cause. Adequate hydration status will be ensured 
before gadolinium is administered (by patient questionnaire and examination). Subjects with renal 
impairment, i.e. an eGFR <60 mL/min/1.73 m2, will be excluded. The eGFR result must be available 
before gadolinium-based contrast injection. 
Recent research studies have found that in people who have had 4 or more MRI scans with gadolinium-
based contrast agents, small amounts of gadolinium can be found in the brain after the MRI scan has 
been completed. The Food and Drug Administration has stated that no adverse health effects have been 
identified from this residual gadolinium. The FDA has recommended that gadolinium-based contrast 
agents should be used when it is necessary to provide the required information. We have made the 
determination that the use of a gadolinium-based contrast agent is necessary. Information about this 
potential risk will be included in the informed consent document and process. 
There is also the possible risk of the loss of confidentiality by inadvertent release of protected health 
information (Section 10 details the procedures to minimize this possibility). 
12. Procedures to Maintain Confidentiality  
Subject confidentiality will be maintained by the investigators, the investigators’ associates and co-
workers, and by all administrators who are part of the project. Confidentiality will be maintained according 
to ICH E6; 4.8.10, part O: “Records identifying the subject will be kept confidential and, to the extent 
permitted by the applicable laws and/or regulations, will not be made publicly available. If the results of the 
trial are published, the subject’s identity will remain confidential.” 
 
The investigators, their staff and associates, and the appropriate regulatory agencies may use the 
information included in this protocol as necessary for the conduct of the trial and the safety of subjects.  
The subject/legal guardian may obtain the results of clinical labs, tests and procedures obtained for this 
research study.  
 
Subjects will be identified by a unique identifier. Any publications will reflect only unique identifiers. All 
information related to this study will be kept in secure and locked offices. 
 
Any clinical data on paper forms that needs to be stored separately from a participant’s official medical 
record will be kept in individual study binders that are secured in locked cabinets. Any data on computer 
will be accessible only by password access. Only members of the research team will have access to these 
physical and electronic files.  
 
Study data will be shared between Study Site investigators in accordance with the HIPAA waivers signed 
by each participant/parent/guardian. 

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13. Potential Benefits 
This research will establish improved screening and diagnostic techniques for patients with SCD. The 
potential benefits of this study will mainly accrue to the class (individuals with SCD) rather than the 
participants. It is possible that this research will uncover a medically important issue that would benefit the 
participant if treated as a result of the discovery, but this is not the focus of the study. All potentially 
medically significant findings will be reported to the patient and his or her primary hematologist for 
counseling and decisions about further testing, treatment, or both. 
14. Risk/Benefit Analysis 
The potential benefits (section 11) of participation in the study outweigh the few, generally minor risks 
(section 10). The formal IRB risk assessment is: slightly more than minimal risk but with potential for direct 
benefit to participants. 
15. Protection of Human Subjects 
The following will be done to ensure safety of human subjects: 
• 
IRB approval will be obtained at each institution implementing this study. 
• 
All Informed Consent Forms (ICFs), including parental permission forms and assent forms where 
applicable must also be approved by the sponsor or designee. 
• 
Patients and parents will be provided with the risks and benefits and give informed consent for the 
study. 
• 
Assent will be sought for patients based on local/institutional IRB requirements 
• 
All Institutional (IRB and HIPPA) and NIH requirements for human studies will be met. 
• 
Exclusion criteria are in place that ensure safety of the drug and exclude vulnerable groups prone to 
adverse events (e.g., pregnant or lactating women are not enrolled and pregnancy tests will be 
performed at each visit). 
• 
Adults with diminished capacity will not be enrolled 
• 
Children will be enrolled only with proper parental permission (age <18 years) and assent (age 11 
years and older) 
Compliance with Good Clinical Practice (GCP) guidelines for the conduct and monitoring of this clinical 
trial will occur through observation of the ethical and regulatory requirements presented in ICH E6, Good 
Clinical Practice: Consolidated Guideline.  The study (protocol, informed consent, advertisements, subject 
information sheets, and Investigator CV and credentials) should be reviewed and approved by the 
Institutional Review Board (IRB) or ethics committee. Changes to the protocol will be approved by the IRB 
prior to being implemented.  Subjects must sign written informed consent prior to undergoing any study 
procedures.  
 
The investigators and institutions affiliated with this study will permit trial-related monitoring, audits, 
IRB/IEC review, and regulatory inspection(s) by providing direct access to source documents. 
16. Data and Safety Monitoring Plan 
There is no Data Safety and Monitoring Board for this study because this in an non-interventional study 
with only yearly visits for cardiac imaging with few risks to participants. Adverse event (AE) reporting will 
be limited to the 30 days following each yearly study visit to capture any possible complications, 
whether SCD-related or not, associated with MRI, gadolinium, echocardiography, and phlebotomy/IV 
placement. 
An adverse event (AE) is defined is defined as any untoward medical occurrence in a study subject that 
does not necessarily have a causal relationship with the study. An AE can be any unfavorable and 
unintended sign (including abnormal laboratory findings), symptom, or disease temporally associated with 
the study. 

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The Common Toxicity Criteria (CTC v4.0) criteria will be used, with SCD-specific modifications noted 
below (Table 3). Rationale for the modifications: SCD causes multisystem chronic, progressive organ 
injury and intermittent and unpredictable “crises” or vaso-occlusive complications and infections. Baseline 
and expected laboratory values are also different in this patient population as compared to normal 
individuals. If the event is not found in the CTC v4.0 criteria, the event will be captured and rated mild, 
moderate, severe, life-threatening, or death per the investigator (Table 4).  
 
 
 
Table 3. CTC v4.0 modifications for SCD  
Parameter 
Grade 2 
Grade 3 
Grade 4  
Hemoglobin (g/dL) 
5.5-6.9 
4.0-5.4 
<4.0 
Total WBC (x109/L) 
1.0-1.000 
0.5-0.999 
<0.5 
ANC (x109/L) 
0.5-0.999 
0.2-0.499 
<0.2 
Platelets (x109/L)  
50-79 
20-49 
<20 
Total bilirubin (mg/dL)  
5.0-10.0 
10.1-20.0 
20.0 
AST (IU/L) 
150-300 
301-1000 
>1000 
ALT (IU/L) 
150-300 
301-1000 
>1000 
Creatinine 
Doubling of the baseline serum 
creatinine level OR a value of 
>1.0 mg/dL 
1.6-2.0 
>2.0 
 
Table 4. Severity descriptors for events not found in CTC v4.0 
Severity  
Numerical 
Value  
Description  
Mild 
1 
Aware of sign, symptom, or event, but easily tolerated; does not 
interfere with daily routine 
Moderate 
2 
Discomfort enough to interfere with daily routine and may require 
some therapeutic intervention  
Severe 
3 
Incapacitating, significantly affects clinical status; requires 
therapeutic intervention  
Life 
Threatening 
4 
Life-Threatening; immediate intervention required  
Death  
5 
Adverse event causes death.  
 
The investigator is also responsible for determination adverse event attribution to determine if the event is 
related the protocol (Table 5). The following table outlines these definitions:  
 
Table 5. AE attribution definitions 
Unrelated 
The event is unrelated to the protocol.   
Possibly Related 
The event or severity of event is not usually associated by the 
clinical condition or standard of care for the condition, but there is 
no strong evidence to link the event to the protocol.  
Probably Related 
The event or severity of event is such that it can likely be correlated 
to a protocol activity.  
Definitely Related 
There is a strong correlation between the event and a protocol 
related activity.  
 
Expected AEs are a known sign, symptom, finding, or condition related to SCD, MRI, gadolinium, 
echocardiography, and phlebotomy/IV placement (Tables 6 and 7). Expected events (Grade 2-5) will be 

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recorded and will include the following information only: (a) nature of event; (b) start date/stop date; and 
(c) highest grade. 
Reporting of Expected AEs: grades 2-5 are to be reported to the IRB according to local IRB 
requirements.  
 
 
 
Table 6. Expected events, conditions or findings associated with SCD 
Acute chest syndrome 
Hematuria 
Pyelonephritis 
Albuminuria 
Hemiplegia 
Renal failure  
Amenorrhea 
Hemolysis 
Renal insufficiency 
Anemia 
Hepatic sequestration 
Renal papillary necrosis 
Aplastic crisis 
Hepatomegaly 
Reticulocytosis 
Arthralgia 
Hospitalization  
Retinopathy 
Avascular necrosis of bone 
Hyperbilirubinemia 
Retinal hemorrhage 
Bacteremia 
Hypersplenism 
Rhabdomyolysis 
Bone infarction 
Hypertension 
Seizure 
Cardiac arrhythmia 
Hypocalcemia 
Septicemia/sepsis 
Cardiomegaly 
Hyposthenuria 
Silent infarct (stroke) 
Cerebrovascular accident 
Hypertension 
Skin ulcer 
Cholecystitis 
Hypoxemia  
Splenic sequestration  
Cholelithiasis 
Ileus 
Splenomegaly 
Cognitive dysfunction 
Infection: bacterial or viral 
TIA 
Constipation 
Jaundice 
Transfusion  
Cranial nerve palsy 
Leukocytosis 
Vaso-occlusive crisis  
Decreased renal function 
Meningitis 
 
Decreased lung function 
Nephropathy 
 
Delayed growth/puberty 
Osteomyelitis 
 
Depression 
Pain: any body site 
 
Dizziness 
Priapism 
 
Electrolyte imbalance 
Proteinuria 
 
Elevated urobilinogen 
Pneumonia 
 
Elevated serum transaminases 
Pulmonary embolism  
 
Fever 
Pulmonary hypertension 
 
Hand-foot syndrome (dactylitis) 
Pulmonary radiographic infiltrate 
 
 
Table 7. Expected events, conditions or findings associated with MRI, IV placement/phlebotomy, 
gadolinium, and/or echocardiography 
Allergic reactions 
Erythema 
Nausea 
Anaphylaxis 
Headache 
Pain/discomfort 
Anxiety 
Hives 
Rash 
Claustrophobia 
Hypotension 
Syncope 
Dizziness 
Itching 
Thrombophlebitis 
Edema 
Light-headedness 
Vomiting 
 
Unexpected AEs are those that have not been previously associated with SCD, MRI, gadolinium, 
echocardiography, and phlebotomy/IV placement. Information on unexpected AEs will include a 
description of the event, specific tests and/or treatments and the results thereof, and outcome parameters. 

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Unexpected AEs will be followed until resolution (or study completion) from initial start date at which time 
the event will be defined as: (a) ongoing; (b) resolved or stabilized without sequelae; or (c) resolved or 
stabilized with sequelae. 
Reporting of Unexpected AEs: grades 2-5 are to be reported to the IRB according to local IRB 
requirements unless meeting the definition of a serious adverse event (SAE), described below.  
Serious adverse Events: For the purpose of this study, an SAE is considered one that: 
• Results in death; 
• Is life-threatening (an event in which the subject was at risk of death at the time of the event; it does 
not refer to an event which hypothetically might have caused death if it were more severe); 
• Requires inpatient hospitalization or a prolongation of an existing hospitalization which, in the opinion 
of the investigator, is not attributable to the subject’s SCD (hospitalization is not uncommon for the 
subjects in this study based on the nature of their illness) but is at least possibly related to any study-
related activity; 
• Results in a persistent or significant disability/incapacity greater than that which existed at baseline; 
• Results in a congenital anomaly/birth defect; or 
• Is, in the opinion of the investigator, an important medical event. 
Reporting of SAEs: SAEs will be reported to the IRB according to IRB policy. SAEs that require 
expedited reporting (within 7 calendar days of the investigator’s initial receipt of the information) are: 
• 
Unexpected (in terms of nature, severity, or frequency) given the research procedures that are 
described in the protocol-related documents and given the characteristics of the participant 
population being studied; 
• 
Related or possibly related to a participant’s participation in the research; and 
• 
Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or recognized. 
17. Quality Assurance Monitoring Plan 
Monitoring will be performed to ensure the study is conducted, documented, and reported in accordance 
with the IRB approved protocol, the International Conference on Harmonization (ICH) Good Clinical 
Practice (GCP) Guidelines, and applicable regulatory requirements.  
 
For this observational study (no intervention or therapy involved), the Cancer and Blood Diseases Institute 
at CCHMC will monitor eligibility, consent documentation, and CRF to source data verification for the first 
patient enrolled and for at least 5% of the patients enrolled since the last monitor visit on an annual 
basis. This schedule will be subject to change based on enrollment, the degree of risk or severity of 
monitoring findings, and other study management issues.  At the end of each monitoring visit, findings and 
follow-up actions will be summarized in a monitoring report and will be provided to the study PIs. 
18. Plan for Informed Consent 
Informed consent is an ongoing process that includes the signing of an informed consent document. 
Subjects are required to sign an informed consent prior to undergoing any study procedures or 
assessments, in accordance with International Conference on Harmonisation (ICH) E6; 4.8, “Informed 
Consent of Trial Subjects.”  When substantial modifications are made to the informed consent, the IRB 
may require that all subjects currently enrolled in the study will be re-consented; ICH E6; 4.8 guidelines 
would still apply.   
 
Subjects will be provided with a copy of the informed consent that explains the purpose of the study, the 
study procedures and assessments, risks and benefits and alternative choices in clear, understandable 
language.  Subjects will also be provided with the telephone numbers of the investigator and qualified 
personnel who can assist with their questions and concerns. 

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The following is the consent process that will be used for this project. 
 
• 
Subjects are recruited from patient populations or from eligibility criteria obtained preparatory to 
research. 
• 
A copy of the informed consent is given to the patient or parent before signing the consent.  The 
content of these documents, and the nature of the study, is discussed with the subject, patient and 
parents or guardians before obtaining their signature.  If there are no questions regarding the 
studies or the content of the consent forms, the subject, patient and/or guardian are requested to 
sign the consent in the presence of a member of the investigation team. 
• 
An investigator/designee involved in the study conducts the consent process.    
• 
The consent is obtained by a study investigator/designee in person and the investigator signs the 
consent.   
• 
Subjects are given copies of the consent with signatures to keep. 
• 
Assent to participate in the study will be obtained from children as per each site IRB policy 
• 
Prior to use, advertisements will be reviewed and approved by the IRB for participating institutions.  
• 
Subjects who entered the study as minors with the permission of the parents/legal guardians will 
be asked to provide informed consent upon reaching the age of majority.  
 
To safeguard the participation of children in this study, we will do the following (hereafter “parent” means 
“parent or legal guardian”): 
 
• 
Obtain parent’s written consent 
• 
Assent will be obtained from children of appropriate age (11 years of age and older). 
• 
Inform parents of alternative options, including non-participation and standard of care 
• 
Inform parents that participation is voluntary and that they are free to withdraw from the study at 
any time. 
• 
Inform parents that choosing not to participate in the study will not result in any penalty or loss of 
benefits to which the patient is otherwise entitled 
• 
Whenever possible and appropriate, purposefully solicit that the parent and child are still willing to 
participate (e.g., when new information regarding the risks/benefits is available) 
19. Cost of Participation 
The patient and/or his insurance will be billed for all standard of care treatments. Participants will not be 
charged for any procedures associated with the research. 
20. Compensation 
Participants will be reimbursed for participation in this study to compensate them for their time, 
inconvenience, and meals related to the study procedures and visits.  
 
If additional study visits are required for Visit 1, 2 or 3, (e.g., if a study procedure has to be repeated for 
quality), an additional $180 will be provided per extra visit.  
 
Visit 
Visit 0 
Visit 1 
Visit 2 
Visit 3 
Compensation 
$60 
$180 
$180 
$180 
 
If needed, families can be provided with a travel voucher for transportation to and from the hospital for 
study visits. 
 
 

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