Assessment of valvular calcification and inflammation by positron emission tomography in patients with aortic stenosis.

Abstract

The pathophysiology of aortic stenosis is incompletely understood, and the relative contributions of valvular calcification and inflammation to disease progression are unknown.
Patients with aortic sclerosis and mild, moderate, and severe stenosis were compared prospectively with age- and sex-matched control subjects. Aortic valve severity was determined by echocardiography. Calcification and inflammation in the aortic valve were assessed by 18F-sodium fluoride (18F-NaF) and 18F-fluorodeoxyglucose (18F-FDG) uptake with the use of positron emission tomography. One hundred twenty-one subjects (20 controls; 20 aortic sclerosis; 25 mild, 33 moderate, and 23 severe aortic stenosis) were administered both 18F-NaF and 18F-FDG. Quantification of tracer uptake within the valve demonstrated excellent interobserver repeatability with no fixed or proportional biases and limits of agreement of ±0.21 (18F-NaF) and ±0.13 (18F-FDG) for maximum tissue-to-background ratios. Activity of both tracers was higher in patients with aortic stenosis than in control subjects (18F-NaF: 2.87±0.82 versus 1.55±0.17; 18F-FDG: 1.58±0.21 versus 1.30±0.13; both P<0.001). 18F-NaF uptake displayed a progressive rise with valve severity (r(2)=0.540, P<0.001), with a more modest increase observed for 18F-FDG (r(2)=0.218, P<0.001). Among patients with aortic stenosis, 91% had increased 18F-NaF uptake (>1.97), and 35% had increased 18F-FDG uptake (>1.63). A weak correlation between the activities of these tracers was observed (r(2)=0.174, P<0.001).
Positron emission tomography is a novel, feasible, and repeatable approach to the evaluation of valvular calcification and inflammation in patients with aortic stenosis. The frequency and magnitude of increased tracer activity correlate with disease severity and are strongest for 18F-NaF.
http://www.clinicaltrials.gov. Unique identifier: NCT01358513.

Full text

Page 1

Boon, James H.F. Rudd and David E. Newby
A. Wallace, Donald M. Salter, Graham McKillop, Edwin J.R. van Beek, Nicholas A.
Richardson, Audrey White, Mark Marsden, Renzo Pessotto, John C. Clark, William
Marc Richard Dweck, Charlotte Jones, Nikhil V. Joshi, Alison M. Fletcher, Hamish
Tomography in Patients With Aortic Stenosis
Assessment of Valvular Calcification and Inflammation by Positron Emission
ISSN: 1524-4539
Copyright © 2011 American Heart Association. All rights reserved. Print ISSN: 0009-7322. Online
72514
Circulation is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TX
doi: 10.1161/CIRCULATIONAHA.111.051052
2012, 125:76-86: originally published online November 16, 2011
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Imaging
Assessment of Valvular Calcification and Inflammation by
Positron Emission Tomography in Patients With
Aortic Stenosis
Marc Richard Dweck, MD; Charlotte Jones, BSc; Nikhil V. Joshi, MD; Alison M. Fletcher, PhD;
Hamish Richardson, BSc; Audrey White; Mark Marsden, BSc; Renzo Pessotto, MD;
John C. Clark, DSc; William A. Wallace, PhD; Donald M. Salter, MD; Graham McKillop, MD;
Edwin J.R. van Beek, PhD; Nicholas A. Boon, MD; James H.F. Rudd, PhD; David E. Newby, DSc
Background—The pathophysiology of aortic stenosis is incompletely understood, and the relative contributions of valvular
calcification and inflammation to disease progression are unknown.
Methods and Results—Patients with aortic sclerosis and mild, moderate, and severe stenosis were compared prospectively
with age- and sex-matched control subjects. Aortic valve severity was determined by echocardiography. Calcification
and inflammation in the aortic valve were assessed by 18F-sodium fluoride (18F-NaF) and 18F-fluorodeoxyglucose
(18F-FDG) uptake with the use of positron emission tomography. One hundred twenty-one subjects (20 controls; 20
aortic sclerosis; 25 mild, 33 moderate, and 23 severe aortic stenosis) were administered both 18F-NaF and 18F-FDG.
Quantification of tracer uptake within the valve demonstrated excellent interobserver repeatability with no fixed or
proportional biases and limits of agreement of ?0.21 (18F-NaF) and ?0.13 (18F-FDG) for maximum tissue-to-
background ratios. Activity of both tracers was higher in patients with aortic stenosis than in control subjects (18F-NaF:
2.87?0.82 versus 1.55?0.17; 18F-FDG: 1.58?0.21 versus 1.30?0.13; both P?0.001). 18F-NaF uptake displayed a
progressive rise with valve severity (r2?0.540, P?0.001), with a more modest increase observed for 18F-FDG
(r2?0.218, P?0.001). Among patients with aortic stenosis, 91% had increased 18F-NaF uptake (?1.97), and 35% had
increased 18F-FDG uptake (?1.63). A weak correlation between the activities of these tracers was observed (r2?0.174,
P?0.001).
Conclusions—Positron emission tomography is a novel, feasible, and repeatable approach to the evaluation of valvular
calcification and inflammation in patients with aortic stenosis. The frequency and magnitude of increased tracer activity
correlate with disease severity and are strongest for 18F-NaF.
Clinical Trial Registration—http://www.clinicaltrials.gov. Unique identifier: NCT01358513.
(Circulation. 2012;125:76-86.)
Key Words: aortic stenosis ? calcification ? inflammation ? positron emission tomography
C
sents a major healthcare burden that is projected to increase
with an aging population.1However, the underlying patho-
physiology remains incompletely defined, and there are
currently no effective medical treatments capable of altering
its course.2Unfortunately, histological studies are limited by
the availability of valve tissue from patients with advanced
disease and do not lend themselves to the longitudinal study
of disease progression. Alternative techniques are therefore
alcific aortic stenosis is the most common form of
valvular heart disease in the Western world and repre-
required to investigate the pathogenesis and progression of
this condition.
Editorial see p 9
Clinical Perspective on p 86
Positron emission tomography (PET) combined with com-
puted tomography (CT) is a noninvasive imaging technique
that allows the identification and quantification of specific
biochemical processes within small anatomic structures, such
as the aortic valve. Furthermore, 2 common PET tracers
Received June 29, 2011; accepted October 11, 2011.
From the Centre for Cardiovascular Science (M.R.D., C.J., N.V.J., A.W., M.M., R.P., W.A.W., D.M.S., N.A.B., D.E.N.), Clinical Research Imaging
Centre (M.R.D., C.J., N.V.J., A.M.F., H.R., J.C.C., G.M., E.J.R.v.B., D.E.N.), and Division of Pathology (W.A.W., D.M.S.), University of Edinburgh,
Edinburgh; and Division of Cardiovascular Medicine, University of Cambridge, Cambridge (J.H.F.R.), United Kingdom.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.
111.051052/-/DC1.
Correspondence to Dr Marc Dweck, Centre for Cardiovascular Science, University of Edinburgh, Little France Crescent, Edinburgh, United Kingdom.
E-mail MDweck@staffmail.ed.ac.uk
© 2011 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.111.051052
76
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target calcification and inflammation, which are believed to
play a key role in the development of the disease. PET/CT
therefore holds considerable promise as a means of investi-
gating the pathophysiology of aortic stenosis.
18F-Flurodeoxyglucose (18F-FDG) is a glucose analogue
that is taken up into cells by glucose transport proteins and
enters the glycolytic metabolic pathway. After the initial
hexokinase step, 18F-FDG-6-phosphate cannot be metabo-
lized further and becomes trapped within cells that have high
metabolic requirements, such as macrophages. PET imaging
with the use of 18F-FDG has become an established means of
quantifying vascular inflammation in both the aorta and
carotid arteries,3,4correlating with plaque macrophage bur-
den5and symptomatic status.418F-Sodium fluoride (18F-
NaF) is an alternative PET tracer that is directly incorporated
into exposed bone crystal (hydroxyapatite) via an exchange
mechanism with hydroxyl groups.6It is therefore thought to
detect areas of novel calcification and regions of calcium
remodeling and is used clinically for the detection of primary
osteoblastic tumors and bone metastases.7More recently,
studies have described 18F-NaF uptake as a marker of
calcification within atherosclerotic plaque8,9; however, to
date this tracer has not been used to study patients with aortic
stenosis.
In this PET study, we investigated 18F-NaF and 18F-FDG
uptake in the valves of patients with aortic stenosis with 3
major aims: to examine the feasibility of this approach, to
establish its repeatability, and to assess the relative impor-
tance of inflammation and calcification at different stages of
the disease.
Methods
Consecutive patients aged ?50 years with aortic sclerosis and mild,
moderate, and severe aortic stenosis attending the outpatient depart-
ment of the Royal Infirmary of Edinburgh were considered for
participation in this study. Exclusion criteria included insulin-
dependent diabetes mellitus, blood glucose ?200 mg/dL, and inabil-
ity to undergo PET/CT scanning. Patients were not approached if
they fulfilled any of the exclusion criteria or if their clinician
believed that participation was not appropriate. Of the patients
approached, 52% agreed to take part in the trial. These patients were
then compared with age- and sex-matched control subjects with a
normal aortic valve and a similar range of comorbidity. The study
was conducted with local research ethics committee approval and the
written informed consent of all patients.
Echocardiography
Valve disease severity was assessed by a single dedicated research
ultrasonographer on a dedicated machine (Phillips Medical Systems,
Best, Netherlands) under standardized conditions and according to a
formal protocol.10,11Patients were studied with the use of an S51
pure wave transducer (Phillips Medical Systems) for 2-dimensional,
M-mode, and pulsed and continuous wave Doppler studies. Contin-
uous wave Doppler velocities were confirmed with the use of a D2
CWC transducer (Phillips Medical Systems) from the apex, right
sternal edge, and suprasternal notch. Measurements were determined
online and averaged from 3 cardiac cycles or 5 if the patient was in
atrial fibrillation.
Aortic sclerosis was defined as a thickened aortic valve on
echocardiography in the absence of accelerated flow though the
valve (peak jet velocity ?2 m/s). The severity of aortic stenosis was
graded according to American Heart Association and American
College of Cardiology criteria with the use of peak transvalvular
aortic valve velocity and mean and maximum aortic valve pressure
gradients.12In our clinical laboratory, we previously demonstrated a
coefficient of reproducibility of 0.32 m/s for the Doppler measure-
ment of peak aortic valve velocity in patients with aortic stenosis.10
Aortic stenosis severity was also assessed with the use of the time
velocity integral, the dimensionless index, and the aortic valve area,
calculated with the continuity equation.
PET and CT
Combined PET/CT scans of the aortic valve were performed on 2
occasions in close succession with the use of a hybrid scanner
(Biograph mCT, Siemens Medical Systems, Erlangen, Germany).
On the first occasion, a target dose of 125 MBq 18F-NaF was
injected intravenously, and patients rested in a quiet environment for
the 60-minute uptake period. An attenuation-correction CT scan
(nonenhanced, low dose 120 kV, 50 mAs) was then performed,
followed by PET imaging covering 2 bed positions centered over the
valve in 3-dimensional mode for 10 minutes. On the second
occasion, a target dose of 200 MBq 18F-FDG was injected, and
patients rested in a quiet environment for 90 minutes. Combined
PET/CT imaging was then performed as described for the 18F-NaF
scan but with the use of a 15-minute bed time. Tracer circulation
times were based on previous studies with the use of 18F-FDG and
18F-NaF in atherosclerosis4,8,9and aimed to allow for optimal
contrast between the aortic wall, aortic valve, and blood pool. An
ECG-gated breath-hold CT scan (nonenhanced, 40 mAs per rotation
[CareDose], 100 kV) was performed of the aortic valve immediately
after the 18F-NaF PET/CT scan for calculation of the aortic valve
calcium score.
The PET data were reconstructed with the use of the Siemens
Ultra-HD (time of flight?True X) reconstruction algorithm. Correc-
tions were applied for attenuation, dead time, scatter, and random
coincidences. All image analysis was performed on fused PET/CT
data sets.
Dietary Restrictions
Intense uptake of 18F-FDG by the left ventricle leads to difficulties
in discriminating between activity in the aortic valve and the
myocardium. All patients in our cohort were asked to observe a
carbohydrate-free diet for 24 hours before their 18F-FDG scan
because this suppresses myocardial uptake as the heart switches from
glucose to free fatty acid metabolism.13–15Patients were provided
with a list of food and drink to avoid and reminded of these
restrictions the day before their scan. Dietary diaries were recorded,
and patients were categorized into dietary compliance or noncom-
pliance. Myocardial tracer uptake was assessed by recording the
maximum standardized uptake value (SUV) in the left ventricular
septal myocardium. The SUV is the decay-corrected tissue uptake
divided by the injected dose per body weight and is a semiquanti-
tative dimensionless unit that is a widely used and validated measure
of tissue 18F-FDG and 18F-NaF uptake.4,8,16High myocardial
18F-FDG uptake was prespecified as an SUV value ?5.0, whereas
low uptake, indicating successful myocardial suppression, was de-
fined by measurements ?5.0.13
Quantification of Tracer Uptake in the
Aortic Valve
PET image quantification is usually performed in the axial, coronal,
or sagittal planes. However, the aortic valve is a complex
3-dimensional structure that does not align perfectly with any of
these orthogonal planes, making accurate identification of the bound-
aries of the valve difficult with the use of standard techniques. To try
to overcome this, the PET and CT images were fused and analyzed
with the use of a workstation (OsiriX version 3.5.1 64-bit; OsiriX
Imaging Software, Geneva, Switzerland) that allows for rotation of
the plane of view into the true axis of the valve. This facilitated the
more accurate delineation of regions of interest around the valve, as
described below.
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18F-NaF: Short-Axis Method
The fused PET/CT image was rotated in a 3-dimensional multiplanar
reconstruction mode to provide a coaxial short-axis view of the aortic
valve (Figure 1). Starting superiorly, we drew a circular region of
interest around the aortic valve on 3-mm slices guided by anatomic
information provided by CT and any obvious valvular calcification
(Figure 1C). Further regions of interest were drawn on adjacent
slices until the whole valve had been examined. Mean and maximum
SUVs were calculated for each slice and then for the valve as a whole
after these values were averaged. However, SUV measurements in
vascular structures are influenced by variation in 18F-FDG and
18F-NaF activity in the blood pool. Therefore, SUV measurements
were divided by an averaged mean SUV value derived from 5
circular regions of interest drawn in the central blood pool of the
superior vena cava. This provided mean and maximum tissue-to-
background ratios (TBRs).5,17
18F-FDG Analysis
The coaxial short-axis method was performed for 18F-FDG as
described above. Whereas the prescan dietary restrictions sought to
minimize the difficulties cause by myocardial 18F-FDG uptake, we
also explored 2 additional image analysis approaches to define better
and to assess more specifically the valvular uptake (Figure 1). In the
long-axis technique, images were reoriented into a modified coronal
view, from which it was hoped the boundaries of myocardial uptake
would be more clearly observed and therefore avoided. In the center
valve technique, regions of interest were again drawn on the coaxial
short-axis view but in the center of the valve, thereby excluding the
base of the valve leaflets and any potential incorporation of myo-
cardial or aortic wall uptake.18For all techniques, measurements
were taken on adjacent 3-mm slices, and mean and maximum SUV
and TBR values were calculated for the valve.
Repeatability Studies
Twenty-five patients with a range of aortic valve disease were
selected at random from the cohort. After establishment of the aortic
valve image analysis methodology, all scans from these patients
were analyzed independently by 2 trained observers (M.R.D., C.J.).
For each technique, this provided measures of interobserver repeat-
Figure 1. Method for the quantification
of 18F-sodium fluoride (18F-NaF) and
18F-fluorodeoxyglucose (18F-FDG)
uptake in the aortic valve. 18F-NaF: A,
Coronal view (blue axis) of the thorax.
Note the intense 18F-NaF uptake (white,
red, and yellow areas) in the calcified
aortic valve as well as in the ribs, clavi-
cles, and arch of the aorta. The purple
axis has been rotated so that it lies per-
pendicular to the aorta and parallel to
the aortic valve. B, Modified sagittal view
of the valve (yellow axis). The purple axis
has again been rotated so that it lies
perpendicular to the aorta and parallel to
the aortic valve. C, A coaxial short-axis
view of the aortic valve is now obtained
along the purple axis. White areas
denote areas of existing calcium, and
yellow/red regions denote areas of
increased 18F-NaF uptake. A region of
interest has been drawn around the
valve (white line). 18F-FDG: D, Short-
axis technique. The imaging plane has
been reoriented as described for 18F-
NaF to provide the coaxial short-axis
view shown. This patient has intense
myocardial 18F-FDG uptake, which
appears to spill into the aortic valve
(white arrows) and makes appreciation
of the less intense activity in the valve
difficult. In the short-axis technique, the
green region of interest has been drawn
to include as much of the valve as pos-
sible while avoiding the myocardial activ-
ity. E, Long-axis technique. A region of
interest has been drawn on the modified
coronal images, attempting to avoid the
myocardial uptake. F, Center valve tech-
nique. In the same patient, the region of
interest has been drawn in the center of
the valve well away from myocardial
uptake around the periphery. Purple bor-
ders indicate images taken in the coaxial
short-axis view of the valve. Blue bor-
ders indicate images taken in the modi-
fied coronal view. Yellow borders indi-
cate images taken in the modified
sagittal view of the valve.
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ability for mean and maximum SUV and TBR values. To assess
intraobserver variation, both observers repeated the analyses at least
2 weeks later to minimize recall bias.
Statistical Methods
Continuous variables were expressed as mean?SD and compared
with the unpaired Student t test or 1-way ANOVA when appropriate.
All continuous variables were tested for normal distribution with the
Shapiro-Wilk test. In cases in which data were not normally
distributed, they were presented as the median?interquartile range.
Categorical variables were expressed as percentages and analyzed
with the ?2test. Correlations between normally distributed data were
performed with Pearson correlation and presented as r2values.
Spearman correlation was used for nonparametric data. The 95%
normal range for differences between sets of SUV and TBR
measurements (the limits of agreement) were estimated by multiply-
ing the SD of the mean of the differences by 1.96.19Intraclass
correlation coefficients with 95% confidence intervals were calcu-
lated for intraobserver and interobserver variation. Statistical analy-
sis was performed with the use of SPSS version 18 (SPSS Inc,
Chicago, IL). A 2-sided P?0.05 was regarded as statistically
significant.
Results
Patient Population
A total of 121 patients were recruited (aged 72?8 years; 69%
male; peak aortic valve velocity 2.8?1.2 m/s) and had both
18F-NaF (66?7 minutes after 124?10 MBq) and 18F-FDG
(94?7 minutes after 197?14 MBq) scans of their aortic
valve ?1 month apart. This cohort comprised 20 control
subjects, 20 patients with aortic sclerosis, and 25 patients
with mild, 33 with moderate, and 23 with severe aortic
stenosis. Patients were well matched for age, sex, and
comorbidity (Table 1).
Dietary Restrictions and Blood Pool Uptake
Average myocardial SUV across the entire cohort was
4.6?3.6, and dietary restrictions effectively suppressed 18F-
FDG myocardial uptake (SUV ?5) in 67% of patients,
similar to that observed in previous studies.20On the basis of
dietary diaries, 61% of patients complied with the dietary
Table 1.Baseline Subject Characteristics
ControlAortic Sclerosis
Aortic Stenosis
PMildModerateSevere
No.
Age, y
Male sex, %
Body mass index, kg/m2
Ischemic heart disease, %
Cardiovascular disease, %
Smoking, %
Diabetes mellitus, %
Hypertension, %
Chronic kidney disease stage ?3, %
Creatinine, mg/dL
Urea (BUN), mg/dL
Calcium, mg/dL
Phosphate, mg/dL
Alkaline phosphatase, U/L
Total cholesterol, mg/dL
LDL cholesterol, mg/dL
HDL cholesterol, mg/dL
Triglycerides, mg/dL
Statin therapy, %
ACE inhibitor therapy, %
Peak aortic jet velocity, m/s
Peak aortic valve gradient, mm Hg
Mean aortic valve gradient, mm Hg
Time-velocity integral, m
Aortic valve area, cm2
Dimensionless index
Agatston aortic valve calcium score, AU
20 202533 23. . .
0.726
0.687
0.051
0.445
0.373
0.566
0.886
0.203
0.927
0.314
0.651
0.228
0.973
0.314
0.040*
0.036*
0.165
0.613
0.262
0.604
?0.001
?0.001
?0.001
?0.001
?0.001
?0.001
?0.001
70?8
65
26?3
35
35
50
10
40
20
0.91?0.20
20.2?5.1
9.2?0.2
3.7?0.5
75?19
191?42
98?44
58?15
63?35
35
35
1.3?0.2
7.1?2.2
3.7?1.0
0.29?0.05
2.70?0.67
0.73?0.11
1.6?3.8
71?9
75
29?6
40
45
35
15
55
20
0.99?0.26
19.0?6.8
9.2?0.7
3.6?0.6
85?30
194?53
101?41
60?35
72?33
50
40
1.7?0.2
11.1?2.6
5.9?1.4
0.38?0.05
2.37?0.54
0.62?0.11
343?377
73?8
60
27?3
48
48
48
20
64
20
0.97?0.32
20.5?10.4
9.2?0.5
3.6?0.5
79?21
210?59
121?47
57?19
69?41
52
52
2.5?0.2
25.5?4.6
13.2?2.7
0.58?0.07
1.49?0.37
0.44?0.99
702?485
72?7
76
29?5
36
39
52
13
73
24
1.05?0.26
21.0?4.6
9.3?0.4
3.6?1.5
82?22
171?41
89?37
49?11
77?52
67
36
3.4?0.3
46.2?7.7
24.9?4.4
0.81?0.10
1.19?0.31
0.31?0.06
2084?1324
73?11
65
28?4
22
22
61
17
61
30
1.09?0.41
22.3?7.8
9.5?0.9
3.6?0.4
102?89
203?52
119?48
49?13
82?45
57
30
4.6?0.6
84.3?24.0
48.8?15.4
1.13?0.19
0.81?0.28
0.24?0.07
3956?2300
Values are mean?SD unless indicated otherwise. BUN indicates blood urea nitrogen; LDL, low-density lipoprotein; HDL,
high-density lipoprotein; ACE, angiotensin-converting enzyme; and AU, Agatston unit.
*There was no correlation between peak aortic valve velocity and either serum total cholesterol (Pearson correlation, r2?0.000,
P?0.976) or LDL cholesterol (r2?0.007, P?0.36) concentrations.
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restrictions and had lower myocardial 18F-FDG uptake than
noncompliers (SUV 3.2?2.3 versus 6.7?4.2; P?0.001).
Across the cohort, blood pool uptake in the SVC was
0.99?0.18 for 18F-NaF and 1.26?0.20 for 18F-FDG.
Repeatability Studies
18F-Sodium Fluoride
Among the 25 patients selected (aged 74?10 years; 64%
male; aortic valve peak velocity 3.8?1.1 m/s), aortic valve
18F-NaF uptake showed excellent interobserver repeatability
for the mean and maximum SUV and TBR values. There
were no fixed or proportional biases, and the majority of data
fell within narrow limits of agreement: ?0.20 for mean and
?0.21 for maximum TBR measurements (Table 2 and Figure
2). Limits of agreement for intraobserver measurements were
similarly good, and intraclass coefficients for interobserver
and intraobserver repeatability were all ?0.95, indicating
excellent agreement (Table 3).
18F-Fluorodeoxyglucose
Avoiding myocardial uptake was difficult with the use of the
short- and long-axis techniques, particularly for the latter.
Reproducibility statistics reflected this and demonstrated that
the variability was much greater than for 18F-NaF. The
interobserver limits of agreement for the short-axis technique
were ?0.28 and ?0.72 for the mean and maximum TBR
values, respectively, and were ?0.78 and ?1.18 with the
long-axis approach (Table 2 and Figure 2). The intraclass
coefficients for the short-axis technique were 0.76 and
0.59 for the mean and maximum TBR values, respectively,
and were 0.39 and 0.52 with the long-axis approach (Table
3). Intraobserver repeatability measures were similarly
poor.
The center valve analysis was more reproducible. There
were no fixed or proportional biases in the differences
between interobserver measurements, and the data fell
within narrow limits of agreement: ?0.11 for mean and
?0.13 for maximum TBR values (Table 2 and Figure 2).
Intraobserver repeatability was similarly good, and intra-
class coefficients were all ?0.90, indicating excellent
agreement (Tables 2 and 3).
There were concerns that the center valve technique might
underestimate 18F-FDG activity in the valve by excluding the
valve ring. However, there was no difference between mean
uptake values calculated by the center valve technique and
the short-axis method (center valve TBR: 1.43?0.17; short
Table 2.
Quantification in the Aortic Valve
Interobserver and Intraobserver Repeatability Statistics for 18F-NaF and 18F-FDG
Mean Difference Between Aortic
Valve SUV and TBR Measurements
Interobserver
Intraobserver
M.R.D.C.J.
Mean SUV
18F-NaF short axis
18F-FDG short axis
18F-FDG long axis
18F-FDG center valve
Maximum SUV
18F-NaF short axis
18F-FDG short axis
18F-FDG long axis
18F-FDG center valve
Mean TBR
18F-NaF short axis
18F-FDG short axis
18F-FDG long axis
18F-FDG center valve
Maximum TBR
18F-NaF short axis
18F-FDG short axis
18F-FDG long axis
18F-FDG center valve
0.04 (?0.12 to 0.20)
0.02 (?0.18 to 0.20)
0.05 (?0.57 to 0.67)
0.01 (?0.05 to 0.07)
0.02 (?0.10 to 0.14)
?0.01 (?0.10 to 0.12)
0.05 (?0.37 to 0.47)
0.01 (?0.05 to 0.07)
0.05 (?0.14 to 0.24)
?0.02 (?0.19 to 0.15)
0.00 (?0.14 to 0.14)
?0.02 (?0.16 to 0.12)
0.02 (?0.16 to 0.20)
0.02 (?0.80 to 0.84)
?0.03 (?1.06 to 1.00)
0.03 (?0.07 to 0.13)
0.04 (?0.11 to 0.19)
?0.11 (?0.64 to 0.42)
0.12 (?0.44 to 0.68)
0.02 (?0.13 to 0.17)
0.00 (?0.19 to 0.19)
?0.09 (?0.89 to 0.72)
0.10 (?0.41 to 0.61)
?0.06 (?0.26 to 0.14)
0.07 (?0.13 to 0.27)
0.06 (?0.22 to 0.34)
0.06 (?0.72 to 0.84)
?0.01 (?0.12 to 0.10)
?0.01 (?0.15 to 0.17)
0.00 (?0.15 to 0.15)
0.07 (?0.50 to 0.64)
0.02 (?0.06 to 0.10)
0.05 (?0.17 to 0.27)
0.01 (?0.12 to 0.14)
0.02 (?0.22 to 0.26)
?0.01 (?0.07 to 0.05)
0.02 (?0.19 to 0.23)
0.06 (?0.66 to 0.78)
0.01 (?1.17 to 1.19)
?0.02 (?0.15 to 0.11)
?0.04 (?0.16 to 0.24)
?0.09 (?0.39 to 0.57)
0.12 (?0.50 to 0.74)
0.02 (?0.11 to 0.15)
0.00 (?0.21 to 0.21)
0.07 (?0.06 to 0.20)
0.06 (?0.33 to 0.39)
?0.05 (?0.14 to 0.04)
Mean difference between standard uptake value (SUV) and tissue-to-background ratio (TBR) measurements for
18F-sodium fluoride (18F-NaF) valve uptake with the use of the short-axis technique and 18F-fluorodeoxyglucose
(18F-FDG) uptake with the use of the short-axis, center valve, and long-axis techniques is shown (95% confidence
intervals in parentheses). The short-axis technique for 18F-NaF and the center valve technique for 18F-FDG display
no fixed proportional bias with narrow limits of agreement.
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axis: 1.47?0.45; P?0.473). Maximum TBR values were
lower with the use of the former approach (center valve:
1.60?0.20; short axis: 1.80?0.45; P?0.041). However, this
difference was no longer apparent when patients with high
myocardial uptake (n?11) were excluded from the analysis
(center valve: 1.56?0.18; short axis: 1.64?0.20; P?0.245),
reflecting the wide limits of agreement (?1.05) for the
short-axis technique when myocardial suppression was poor.
By contrast, limits of agreement for the center valve tech-
nique were equally good in patients with low and high
myocardial uptake (?0.13 versus ?0.14, respectively;
P?0.919). Given this and other advantages, subsequent
analysis of the entire cohort was performed with the center
valve method for 18F-FDG.
Aortic Valve Uptake
18F-Sodium Fluoride
Focal 18F-NaF uptake was observed in the valves of patients
with calcific aortic valve disease in areas overlying, adjacent
to, and remote from existing calcification. Areas of estab-
lished calcium were also observed frequently in the absence
of increased 18F-NaF activity (Figure 3). Compared with
control subjects, valvular 18F-NaF uptake was higher in
patients with both aortic sclerosis (maximum TBR:
1.55?0.17 versus 1.92?0.31; P?0.001) and aortic stenosis
(maximum TBR: 1.55?0.17 versus 2.87?0.82; P?0.001).
The highest maximum TBR value in the control group was
1.97, which was used to divide patients with aortic valve
disease into those with increased 18F-NaF uptake (?1.97)
Figure 2. Bland-Altman plots of interobserver variability. A, Maximum 18F-sodium fluoride (18F-NaF) tissue-to-background ratio (TBR)
values with the use of the short-axis technique. The dark central line is the mean difference between the 2 analyses, which does not
show any fixed or proportional biases. The light outer lines show the limits of agreement (mean of the differences ?1.96?SD), which
are narrow. Maximum 18F-fluorodeoxyglucose (18F-FDG) values with the use of the short-axis (B), long-axis (C), and center valve tech-
niques (D) are shown. Note the wide limits of agreement for the short-axis and long-axis 18F-FDG techniques but the excellent mea-
sures of repeatability for the center valve technique. Also note that there is a single outlier not plotted on the long-axis graph (C); the
mean difference for this patient is given in parentheses.
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and those without (?1.97). Overall, 45% of patients with
aortic sclerosis and 91% of those with aortic stenosis had
increased uptake. The proportion of patients with increased
activity rose sharply with increasing disease severity such
that 100% of patients with severe disease had increased
uptake (Table 4).
All measures of 18F-NaF uptake displayed a progressive
rise with increasing aortic jet velocity (maximum TBR:
r2?0.540, P?0.001; Table 4 and Figure 4), the aortic valve
calcium score (r2?0.641, P?0.001), and other echocardio-
graphic measures of aortic stenosis severity (time-velocity
integral: r2?0.546, P?0.001; aortic valve area: r2?0.387,
P?0.001; dimensionless index: r2?0.527, P?0.001).
18F-Fluorodeoxyglucose
18F-FDG showed a more diffuse pattern of activity within the
valve (Figure 3), and compared with control subjects, uptake
was increased in patients with aortic sclerosis (maximum
TBR: 1.30?0.13 versus 1.47?0.15; P?0.001) and aortic
stenosis (maximum TBR: 1.30?0.13 versus 1.58?0.21;
P?0.001). The highest maximum TBR value in the control
group was 1.63, which was used to divide patients with aortic
valve disease into those with increased 18F-FDG uptake
(?1.63) and those without (?1.63). Overall, 20% of patients
with aortic sclerosis and 35% of patients with aortic stenosis
had increased uptake. The proportion of patients with in-
creased activity in the valve again rose with increasing aortic
valve disease; however, this rise was more gradual than for
18F-NaF, with only 52% of patients with severe disease
demonstrating increased activity (Table 4).
All measures of 18F-FDG activity displayed a progressive
rise with increasing aortic jet velocity (maximum TBR:
r2?0.218, P?0.001; Table 4 and Figure 4), the aortic valve
Agatston score (r2?0.138, P?0.001), and other echocardio-
graphic measures of aortic stenosis (time-velocity integral:
r2?0.246, P?0.001; aortic valve area: r2?0.184, P?0.001;
dimensionless index: r2?0.229, P?0.001). These correla-
tions were weaker and more modest than for 18F-NaF. A
modest correlation was also observed between valvular 18F-
NaF and 18F-FDG activities (maximum TBR: r2?0.174,
P?0.001).
Discussion
In this PET study, we have established the feasibility of
evaluating 18F-NaF and 18F-FDG activity in patients with
aortic stenosis. Moreover, we have demonstrated excellent
repeatability for the quantification of these tracers in the
valve as measures of calcification and inflammation, respec-
tively. 18F-NaF and 18F-FDG activity was increased in
patients with both aortic sclerosis and stenosis, displaying a
Table 3.
in the Aortic Valve
Intraclass Coefficient Values for 18F-NaF and 18F-FDG Quantification
ICC Values Comparing Aortic
Valve SUV and TBR Measurements
Intraobserver
Interobserver M.R.D.C.J.
Mean SUV
18F-NaF short axis
18F-FDG short axis
18F-FDG long axis
18F-FDG center valve
Maximum SUV
18F-NaF short axis
18F-FDG short axis
18F-FDG long axis
18F-FDG center valve
Mean TBR
18F-NaF short axis
18F-FDG short axis
18F-FDG long axis
18F-FDG center valve
Maximum TBR
18F-NaF short axis
18F-FDG short axis
18F-FDG long axis
18F-FDG center valve
0.98 (0.96–0.99)
0.95 (0.88–0.98)
0.58 (0.24–0.79)
0.99 (0.97–0.99)
0.99 (0.98–1.00)
0.98 (0.96–0.99)
0.68 (0.39–0.84)
1.00 (0.99–1.00)
0.98 (0.94–0.99)
0.96 (0.91–0.98)
0.96 (0.91–0.98)
0.99 (0.98–1.00)
0.99 (0.99–1.00)
0.62 (0.30–0.81)
0.53 (0.18–0.76)
0.98 (0.94–0.99)
1.00 (0.99–1.00)
0.88 (0.75–0.95)
0.77 (0.54–0.89)
0.98 (0.95–0.99)
0.99 (0.98–1.00)
0.58 (0.24–0.79)
0.80 (0.60–0.91)
0.96 (0.90–0.98)
0.98 (0.96–0.99)
0.76 (0.52–0.89)
0.39 (0.01–0.68)
0.96 (0.90–0.98)
0.99 (0.97–0.99)
0.94 (0.86–0.97)
0.56 (0.22–0.78)
0.97 (0.93–0.99)
0.97 (0.93–0.99)
0.94 (0.87–0.97)
0.93 (0.85–0.97)
0.84 (0.66–0.92)
0.99 (0.99–1.00)
0.59 (0.26–0.80)
0.52 (0.17–0.76)
0.92 (0.83–0.97)
0.99 (0.99–1.00)
0.89 (0.76–0.95)
0.85 (0.69–0.93)
0.94 (0.87–0.97)
0.99 (0.99–1.00)
0.71 (0.44–0.86)
0.92 (0.83–0.97)
0.91 (0.80–0.96)
Intraclass correlation coefficient (ICC) values (95% confidence intervals in parentheses) for the
short-axis 18F-sodium fluoride (18F-NaF) technique and the center valve 18F-fluorodeoxyglucose
(18F-FDG) method are shown. SUV indicates standard uptake value; TBR, tissue-to-background ratio.
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progressive rise in uptake with increasing disease severity.
However, calcification rather than inflammation appears to be
the predominant process affecting the valve, particularly in
the latter stages of the disease, in which a more marked
progression in 18F-NaF activity was observed that was
disproportionate to 18F-FDG.
Valve calcification plays a key role in the development of
aortic stenosis. Hydroxyapatite becomes deposited on a
bonelike matrix containing collagen, osteopontin, and other
bone matrix proteins21–23to form nodules that progress until,
by the end stage of the disease, lamellar bone, microfractures,
and hemopoietic tissue can all be identified.23This appears to
occur as part of a highly regulated process, coordinated by
increased osteoblast activity24,25and the local production of
osteopontin, osteocalcin, bone sialoproteins, and bone mor-
phogenic protein-2, all of which are more commonly associ-
ated with skeletal bone formation.21,23,25,26Established aortic
valve calcium can be measured accurately with the use of
CT,10but measurement of 18F-NaF uptake offers, for the first
time, the possibility of detecting areas of developing calcifi-
cation within the valve. In this study, 18F-NaF uptake was
observed in regions adjacent to and remote from existing
calcium, suggesting expansion of the calcific process to new
areas of the valve. In addition, uptake was observed in regions
Figure 3. 18F-Sodium fluoride (18F-NaF) and 18F-fluorodeoxyglucose (18F-FDG) uptake in patients with aortic stenosis. 18F-NaF:
Fused positron emission tomography/computed tomography scans demonstrating uptake of 18F-NaF on coaxial short-axis views of
the aortic valve in patients with a normal aortic valve (A), aortic sclerosis (B), and mild (C), moderate (D), and severe aortic stenosis (E
and F). White areas show regions of existing calcium, and yellow and red areas show areas of 18F-NaF uptake. Focal areas of uptake
are observed in regions overlying existing calcium as well as in areas remote from it. Furthermore, areas of existing calcification are
observed in the absence of overlying 18F-NaF uptake. Note the increased activity with increasing severity of valve disease. Regions of
interest have been drawn around the periphery of the valve (white lines) with the use of the short-axis technique. 18F-FDG: Fused posi-
tron emission tomography/computed tomography scans demonstrating uptake of 18F-FDG on coaxial short-axis views of the aortic
valve in patients with a normal aortic valve (G), mild aortic stenosis (H), and severe aortic stenosis (I). Patients all have excellent myo-
cardial suppression, allowing uptake to be visualized in the patients with aortic valve disease. Regions of interest have been drawn with
the use of both the short-axis and center valve techniques (green lines).
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overlapping with that of established calcium, and, in these
areas, activity is likely to represent calcium remodeling and
maturation of the calcific process.
18F-NaF activity was increased in the valves of patients
with aortic sclerosis and stenosis compared with control
subjects and demonstrated a marked progressive rise with
increasing disease severity accounting for ?50% of the
variance associated with valve stenosis. Moreover, increased
valvular 18F-NaF activity was observed in 45% of patients
with aortic sclerosis, 91% of patients with aortic stenosis, and
all patients with severe stenosis. Calcium accumulation is the
predominant mechanism by which valve cusp rigidity in-
creases and aortic stenosis advances. As such, this technique
offers considerable promise as a biomarker of disease activity
and as a means of predicting disease progression. Longitudi-
nal studies are now required to determine whether calcifica-
tion activity quantified by 18F-NaF uptake is an accurate
predictor of disease progression and superior to baseline
measures of valve severity and calcium scores. If confirmed,
these studies would pave the way for mechanistic studies of
medical interventions to interrupt progressive calcific disease
with the use of 18F-NaF activity as a surrogate biomarker and
end point.
In the early stages of aortic stenosis, endothelial damage
secondary to mechanical stress and lipid deposition triggers
an inflammatory response within the valve. This is charac-
terized by increased macrophage27and T-cell activity within
the valve leaflets and the expression of a range of proinflam-
matory cytokines including transforming growth factor-?1,28
tumor necrosis factor-?, and interleukin-1?.29The inflamma-
tory response is thought to trigger the fibrotic and calcific
processes that subsequently drive valve orifice narrowing.
Thus, identifying and quantifying valvular inflammation with
18F-FDG have the potential to be critical in the evaluation of
aortic stenosis. In the present study, 18F-FDG uptake was
higher in patients with aortic sclerosis and stenosis compared
with control subjects, and activity again rose with increasing
valve severity. However, this association was weaker and the
increase in activity was more modest than for 18F-NaF.
Indeed, increased valvular 18F-FDG activity was only ob-
served in 20% of patients with aortic sclerosis, 35% of
patients with aortic stenosis, and 52% of patients with severe
stenosis. Although this may reflect the high cutoff used for
increased activity or an insensitivity of 18F-FDG in detecting
inflammation, these data suggest that calcification is the
predominant pathogenic process in aortic stenosis and a better
target for novel therapeutic strategies. It might also explain
the disappointing results of statin therapy in this condition,
which has consistently failed to modify vascular calcification
even in the coronary circulation despite reducing systemic
markers of inflammation.11,30–32
Table 4. Correlation Between Aortic Stenosis Severity and Radiotracer Uptake
ControlAortic Sclerosis
Aortic Stenosis
Correlation With
Peak Aortic
Jet Velocity
MildModerateSeverer2
P
18F-NaF
Mean SUV
Mean TBR
Maximum SUV
Maximum TBR
Patients with increased uptake %
18F-FDG
Mean SUV
Mean TBR
Maximum SUV
Maximum TBR
Patients with increased uptake, %
1.20 (1.10–1.55)
1.23 (1.20–1.36)
1.54 (1.33–1.86)
1.56 (1.41–1.64)
0
1.35 (1.24–1.59)
1.53 (1.34–1.59)
1.77 (1.58–2.09)
1.96 (1.63–2.11)
45
1.59 (1.38–1.73)
1.73 (1.45–1.92)
2.21 (1.84–2.45)
2.45 (1.94–2.71)
76
1.82 (1.67–2.05)
2.03 (1.71–2.28)
2.57 (2.27–2.99)
2.89 (2.31–3.24)
95
2.10 (1.78–2.51)
2.17 (1.82–2.36)
3.25 (2.47–4.42)
3.25 (2.62–3.63)
100
0.461
0.534
0.551
0.540
. . .
?0.001
?0.001
?0.001
?0.001
. . .
1.49 (1.33–1.56)
1.18 (1.09–1.26)
1.62 (1.47–1.68)
1.27 (1.21–1.40)
0
1.73 (1.46–1.88)
1.35 (1.19–1.44)
1.91 (1.64–2.07)
1.47 (1.31–1.61)
20
1.66 (1.53–1.88)
1.29 (1.21–1.45)
1.85 (1.72–2.07)
1.44 (1.37–1.63)
24
1.71 (1.61–1.91)
1.33 (1.26–1.47)
1.95 (1.81–2.18)
1.58 (1.41–1.65)
30
1.76 (1.61–2.18)
1.42 (1.36–1.62)
2.07 (1.88–2.25)
1.65 (1.55–1.85)
52
0.142
0.203
0.213
0.218
. . .
?0.001
?0.001
?0.001
?0.001
. . .
Values are median?interquartile range, with Pearson correlation values. 18F-NaF indicates 18F-sodium fluoride; 18F-FDG, 18F-fluorodeoxyglucose; SUV, standard
uptake value; and TBR, tissue-to-background ratio.
Figure 4. Uptake of 18F-fluorodeoxyglucose (18F-FDG) and
18F-sodium fluoride (18F-NaF) according to the severity of aor-
tic stenosis. Box plots show the median and interquartile ranges
of the tissue-to-background ratios (TBR) for 18F-NaF (white
boxes) and 18F-FDG (gray boxes) with whiskers to 1.5?inter-
quartile range.
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A recent retrospective study described increased 18F-FDG
uptake in the valves of 42 patients with cancer who were
coincidentally found to have aortic stenosis.18However, in
contrast to our study, a reduction in activity was observed in
patients with severe compared with moderate disease. This
difference is likely to reflect the small number of subjects in
the severe subgroup (n?8), the retrospective nature of the
study analysis, and the confounding effects of coexistent
malignancy. In contrast, we have prospectively recruited a
larger, well-defined cohort of patients with aortic stenosis
who are more likely to be representative of those seen in
cardiology practice. Moreover, we studied patients following
dietary restrictions to minimize the effects of myocardial
uptake and spillover into the valve. Our data are in agreement
with a modest yet sustained and progressive increase in
inflammation even in those with advanced disease.
Study Limitations
With exclusion of part of the valve, it is possible that the
center valve technique employed for 18F-FDG analysis may
underestimate valvular inflammation. However, there were
no differences in mean uptake values compared with the
short-axis technique. This reflects the diffuse nature of
18F-FDG activity in stenotic valves and the equal distribution
of lesions between the base of the valve leaflets (54%) and the
mid portion and tips (46%).33
This study has not validated 18F-FDG and 18F-NaF
activity against histological samples. Although the mecha-
nism of uptake for both tracers has been investigated in other
tissues, further work is required in the valve to address this
issue (see the online-only Data Supplement).
Conclusion
The evaluation of aortic stenosis with the use of PET is
feasible and highly reproducible, with 18F-FDG and partic-
ularly 18F-NaF holding considerable promise as novel bio-
markers of disease activity. Both calcification and inflamma-
tion are increased in patients with aortic valve disease
compared with control subjects, and the activity of both rises
steadily with increasing disease severity. However, calcifica-
tion appears to be the predominant pathological process,
particularly in the latter stages of the disease, and would
therefore appear to be a better target for future potential
medical therapies.
Acknowledgments
We are grateful to the Wellcome Trust Clinical Research Facility and
the Clinical Research Imaging Centre for their help with this study.
Sources of Funding
This work and Dr Dweck are supported by a British Heart Founda-
tion Clinical PhD Training Fellowship (FS/10/026) and the British
Heart Foundation Centre of Research Excellence Award. The work
of Dr Rudd is supported by the Higher Education Funding Council
for England, the British Heart Foundation, and the Cambridge
National Institute for Health Research Biomedical Research Centre.
Dr van Beek is supported by the Scottish Imaging Network, a
Platform of Scientific Excellence. Dr Newby is supported by the
British Heart Foundation. The Wellcome Trust Clinical Research
Facility and the Clinical Research Imaging Centre are supported by
National Health Service Research Scotland through National Health
Service Lothian.
Disclosures
None.
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studies.
Circulation.
CLINICAL PERSPECTIVE
Aortic stenosis is the most common form of valvular heart disease in the Western world and represents a major healthcare
burden that is projected to increase with an aging population. However, there are currently no effective medical treatments
or biomarkers of disease activity. The pathogenesis of aortic stenosis is incompletely understood, and defining the various
stages of this process will be highly important to develop the therapies and biomarkers that are required. Positron emission
tomography combined with computed tomography is a noninvasive imaging technique that allows the identification and
quantification of specific pathological processes within small anatomic structures, such as the aortic valve. In this study,
we sought to test the feasibility, repeatability, and validity of this technique in the evaluation of aortic valve stenosis.
Positron emission tomography/computed tomography imaging of the aortic valve was performed to assess inflammation
(18F-fluorodeoxyglucose) and active calcification (18F-sodium fluoride) of the valve leaflets. The positron emission
tomography/computed tomography findings were compared in 121 patients with a full spectrum of disease severity. Our
data have clearly established that this technique is both feasible and repeatable, indicating that these tracers may prove to
be useful biomarkers of disease activity. Furthermore, we have demonstrated that 18F-fluorodeoxyglucose and 18F-sodium
fluoride activity increase with progressive disease severity. However, uptake of 18F-sodium fluoride appears to
predominate in both the early and latter stages of the disease. This may explain the disappointing effects of statin therapy
in this condition and indicates that calcification might represent a better target for novel therapeutic interventions.
86 Circulation
January 3/10, 2012
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Supplemental??  Material??  
Supplemental??  Methods??  
One??  patient??  underwent??  an??  aortic??  valve??  replacement??  3??  months??  after??  their??  PET??  scans.??  The??  valve??  
was??  harvested??  and??  incubated??  with??  18F-­‐FDG,??  Dulbecco’s??  modified??  eagle??  medium??  (Invitrogen,??  
Paisley,??  UK)??  and??  10%??  fetal??  calf??  serum??  for??  90??  min??  before??  PET/CT??  imaging??  using??  the??  same??  protocol??  
as??  the??  in??  vivo??  clinical??  scans.??  The??  next??  day,??  PET/CT??  imaging??  was??  repeated??  after??  incubation??  with??  
18F-­‐NaF??  for??  60??  min.??  Finally??  the??  valve??  was??  fixed??  in??  formalin??  and??  decalcified??  using??  ethylene??  
diamine??  tetracetic??  acid??  before??  immunohistochemistry??  was??  performed??  to??  examine??  for??  
macrophage??  accumulation??  (CD68)??  and??  active??  calcification??  (osteocalcin).??  ??  
??  
Supplemental??  Results??  
18F-­‐NaF??  uptake??  was??  consistent??  on??  both??  the??  in??  vivo??  and??  ex??  vivo??  PET/CT??  scans.??  Furthermore??  
uptake??  co-­‐localized??  with??  the??  distribution??  of??  osteocalcin??  staining??  on??  histology,??  extending??  beyond??  
the??  boundaries??  of??  existing??  macroscopic??  calcification??  (Supplemental??  Figure??  1).??  The??  pattern??  of??  
valvular??  18F-­‐FDG??  uptake??  was??  again??  consistent??  between??  in-­‐vivo??  and??  ex-­‐vivo??  PET/CT??  scans.??  
Furthermore??  activity??  mapped??  closely??  to??  areas??  of??  increased??  macrophage??  density??  on??  
immunohistochemistry??  (Supplementary??  Figure??  2).??  

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Supplemental??  Figure??  Legends??  
Supplemental??  Figure??  1.??  18F-­‐NaF??  studies??  on??  excised??  aortic??  valve.??  ??  
??  
A??  Excised??  portion??  of??  a??  stenotic??  bicuspid??  aortic??  valve??  removed??  at??  the??  time??  of??  an??  aortic??  valve??  and??  
root??  replacement.??  The??  aortic??  side??  of??  one??  of??  the??  valve??  leaflets??  is??  shown.??  ??  
B??  Immunohistochemistry??  of??  the??  valve??  for??  osteocalcin??  in??  a??  region??  of??  the??  valve??  adjacent??  to??  a??  
calcific??  nodule.??  Osteocalcin??  is??  incorporated??  in??  to??  the??  bone??  matrix??  where??  it??  binds??  to??  
hydroxyapatite??  during??  active??  mineralization.??  Osteocalcin??  immunoreactivity??  can??  be??  seen??  at??  the??  
periphery??  of??  the??  existing??  calcified??  nodule.??  Inset??  (black??  border)??  shows??  cytoplasmic??  staining??  of??  
cells??  adjacent??  to??  this.??  C.??  Immunohistochemistry??  in??  a??  region??  remote??  from??  existing??  calcification??  
with??  no??  staining??  present.??  This??  focal??  distribution??  of??  staining??  matches??  the??  pattern??  of??  18F-­‐NaF??  
activity??  in??  the??  valve??  on??  both??  the??  in??  vivo??  and??  ex??  vivo??  scans??  described??  below.??  ??  
D.??  In??  vivo??  18F-­‐NaF??  PET/CT??  scan??  performed??  3??  months??  prior??  to??  the??  operation.??  ??  Note??  the??  focal??  
areas??  of??  uptake??  overlying??  the??  area??  of??  calcification??  at??  the??  bottom??  left??  of??  the??  valve??  and??  adjacent??  
to??  the??  smaller??  area??  of??  calcification??  in??  the??  top??  right.??  This??  closely??  matches??  the??  pattern??  of??  uptake??  
observed??  on??  the??  ex??  vivo??  PET/CT??  scan??  performed??  on??  the??  excised??  valve??  (E).??  ??  
??  
??   ??  
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Supplemental??  Figure??  2.??  18F-­‐NaF??  studies??  on??  excised??  aortic??  valve.??  ??  
A.??  Excised??  portion??  of??  the??  same??  aortic??  valve??  as??  in??  Figure??  1.??  ??  
B.??  Immunohistochemistry??  for??  CD??  68??  in??  a??  region??  of??  the??  valve??  adjacent??  to??  a??  calcific??  nodule??  
demonstrating??  the??  presence??  of??  macrophages??  around??  the??  lesion??  in??  the??  fibrosa??  of??  the??  valve??  
leaflet.??  C.??  Immunohistochemistry??  in??  a??  region??  remote??  from??  existing??  calcification,??  which??  again??  
displays??  staining??  for??  macrophages.??  ??  D.??  In??  vivo??  18F-­‐FDG??  PET/CT??  of??  the??  valve??  shows??  a??  more??  diffuse??  
pattern??  of??  uptake??  than??  for??  18F-­‐NaF??  matching??  the??  distribution??  of??  macrophages??  as??  well??  as??  the??  
pattern??  of??  uptake??  observed??  in??  the??  ex??  vivo??  PET/CT??  scan??  of??  the??  excised??  valve??  tissue??  (E).??  
??  
??  ??  
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