Author + information
- Received March 25, 2011
- Accepted March 28, 2011
- Published online June 1, 2011.
- Claudio Rapezzi, MD⁎,⁎ (, )
- Candida C. Quarta, MD⁎,
- Pier Luigi Guidalotti, MD†,
- Cinzia Pettinato, MD†,
- Stefano Fanti, MD†,
- Ornella Leone, MD‡,
- Alessandra Ferlini, MD§,
- Simone Longhi, MD⁎,
- Massimiliano Lorenzini, MD⁎,
- Letizia Bacchi Reggiani, MSc, MStat⁎,
- Christian Gagliardi, MD⁎,
- Pamela Gallo, MD⁎,
- Caterina Villani, MD⁎ and
- Fabrizio Salvi, MD∥
- ↵⁎Reprint requests and correspondence:
Prof. Claudio Rapezzi, Istituto di Cardiologia, Policlinico S. Orsola-Malpighi, via Massarenti 9, 40138 Bologna, Italy
Objectives In a cohort of patients with hereditary transthyretin-related amyloidosis (ATTR), we aimed to assess the role of 99mTc-3,3-diphosphono-1,2-propanodicarboxylic acid (99mTc-DPD) in detecting myocardial amyloid infiltration across a wide spectrum of cardiac involvement and in predicting major adverse cardiac events (MACE).
Background Hereditary transthyretin-related amyloidosis is a challenging and underdiagnosed condition where both early diagnosis and prognosis remain problematic.
Methods We evaluated 63 patients with ATTR: 40 with and 23 without echocardiographically diagnosed amyloidotic cardiomyopathy (AC). Myocardial uptake of 99mTc-DPD scintigraphy was semiquantitatively and visually assessed at 5 min and 3 h.
Results All patients with AC showed moderate-to-severe myocardial tracer uptake (i.e., visual score ≥2). Within the subgroup without AC, only 4 patients (with Ala36Pro, Gly47Ala, Thr49Ala, and Glu89Gln transthyretin mutations) showed myocardial tracer uptake and abnormal heart/whole body retention (H/WB) values: in all these cases endomyocardial biopsies showed amyloidotic infiltration. The H/WB was positively correlated with left ventricular (LV) mean wall thickness (Pearson's r = 0.695, p < 0.001) and negatively with LV ejection fraction (r = −0.368, p = 0.004). The H/WB was an unfavorable predictor of MACE-free survival at Cox univariate analysis and contributed to the multivariate model. Notably, LV wall thickness >12 mm in combination with H/WB >7.5 was associated with the highest event rate.
Conclusions In ATTR, 99mTc-DPD scintigraphy can identify myocardial infiltration across a wide spectrum of morphologic/functional cardiac involvement, allowing an early diagnosis of the disease (even before the appearance of echocardiographic abnormalities). The 99mTc-DPD myocardial uptake is a prognostic determinant of “cardiac” outcome in ATTR, either alone or in combination with LV wall thickness.
- 99mTc-DPD scintigraphy
- cardiac amyloidosis
- hereditary transthyretin-related amyloidosis
Hereditary transthyretin-related amyloidosis (ATTR) is the most frequent form of familial systemic amyloidoses, a group of severe diseases with neurological and polyvisceral involvement (1–3). Hereditary transthyretin-related amyloidosis is challenging and widely underdiagnosed, because of an extreme phenotypic variability that ranges from almost exclusive neurologic involvement to cases with a strictly cardiologic presentation (1–3). Such heterogeneity is mainly linked to specific transthyretin (TTR) mutations, geographic distribution, endemic/nonendemic type of aggregation (1–5). Echocardiography and electrocardiography (ECG) have an established role in the diagnosis of overt forms of amyloidotic cardiomyopathy (AC) (4–7). Unfortunately, none of them gives information with regard to the etiologic type of amyloidosis; furthermore, their diagnostic role in the early phases or in the mild forms of the disease is limited by a low specificity (4,5,7,8).
99mTc-3,3-diphosphono-1,2-propanodicarboxylic acid (99mTc-DPD) scintigraphy has proved to be useful in differential diagnosis between TTR (both mutant and wild-type) and immunoglobulin light-chain (AL) etiologies in patients with echocardiographically defined AC (9–11): 99mTc-DPD uptake is absent in unaffected control subjects, strong in TTR-related AC, and absent or weak in AL-derived AC (10,11). However, apart from the contribution in tissue diagnosis, the diagnostic possibilities of 99mTc-DPD in ATTR have not yet been fully assessed. It also remains to be determined whether 99mTc-DPD myocardial uptake has major prognostic implications on cardiac events.
The present study was designed to evaluate the diagnostic and prognostic contribution of 99mTc-DPD in ATTR across a wide spectrum of morphologic and functional cardiac involvement, including early stages of the disease.
Setting and study design
We enrolled all patients diagnosed with ATTR who presented to our center from March 2004 to March 2010. In our referral center for the diagnosis and treatment of cardiomyopathies, clinical examination, echocardiogram, ECG, and 99mTc-DPD scintigraphy are part of the usual diagnostic workup of patients with suspected or confirmed cardiac amyloidosis. Each patient was required to provide standard informed consent for the execution of 99mTc-DPD scintigraphy. Cardiac catheterization and endomyocardial biopsy (EMB) were performed in selected cases and in all potential candidates for liver or combined liver/heart transplantation.
All patients provided informed consent for anonymous publication of scientific data. Formal ethical approval was not applicable for this retrospective study involving only routinely performed procedures.
Our study included a cross-sectional analysis of baseline clinical and instrumental profiles and a longitudinal analysis of ATTR patients to assess the role of myocardial 99mTc-DPD uptake in predicting major adverse cardiovascular events (MACE).
Diagnosis of systemic amyloidosis was based on histological documentation of Congo-red staining and apple-green birefringence under cross-polarized light in at least 1 involved organ (12,13). The AC was echocardiographically defined as end-diastolic thickness of the interventricular septum >1.2 cm (in the absence of any other plausible cause of ventricular hypertrophy) (4,14). Other echocardiographic signs suggesting cardiac amyloidosis were: granular sparkling appearance of ventricular myocardium, increased thickness of right ventricular free wall, atrioventricular valves or interatrial septum, and pericardial effusion (4–6). Diagnosis of ATTR was defined by a documented TTR mutation at deoxyribonucleic acid analysis (15). Renal insufficiency was defined as glomerular filtration rate <60 ml/min (14). The assessment of peripheral nervous system involvement was based on characteristic neurologic signs and symptoms (typical symmetric ascending sensorimotor peripheral neuropathy) (14). Autonomic impairment was defined by presence of orthostatic hypotension, gastric-emptying disorder, pseudo-obstruction, and voiding dysfunction not related to direct organ infiltration (14).
The ECG and echocardiographic measures were based on standard definitions (16,17). Left ventricular (LV) mass was calculated according to Devereux and was classified as increased when >130 g/m2 in men and >110 g/m2 in women (18). The LV restrictive filling pattern was defined in terms of E-wave deceleration time <150 ms accompanied by E/A-wave ratio >2.5 at pulsed Doppler echocardiography (19).
Histology and immunohistochemistry
Histological documentation of amyloid deposition was obtained either in subcutaneous adipose tissue from abdominal fat or from EMB. All myocardial samples (5 per patient) were microwave fixed/processed, and multiple 2-μm sections were tested for presence of amyloid by Congo-red staining and apple-green birefringence under cross-polarized light microscopy. The amount of amyloid infiltration at EMB was semiquantitatively classified as: mild (<30% of the analyzed tissue specimen), moderate (30% to 60% of the analyzed tissue specimen), or severe (more than 60% of the analyzed tissue specimen). Immunohistochemical analysis was performed by the labeled Streptavidin-Biotin LSAB method with an antibody against TTR (R.P. Linke, Max Planck Institute of Biochemistry, Martinsried, Germany), or by immunoelectron microscopy with specific antibodies proteins (DAKO, Ely, United Kingdom).
Scintigraphy protocol and image analysis
Patients received 740 MBq of 99mTc-DPD intravenously; a dual-head camera (ECAM, Siemens Medical Systems, Hoffman Estates, Illinois) equipped with low-energy, high-resolution collimators was used. Whole body scans were obtained 5 min (early) and 3 h (late) after injection. In patients showing cardiac uptake, a myocardial single-photon emission computed tomography study was acquired after the late whole-body scan. Image analysis was independently performed by experienced analysts unaware of all patient data. Visual scoring of cardiac retention was routine: score 0, absent cardiac uptake and normal bone uptake; score 1, mild cardiac uptake, inferior to bone uptake; score 2, moderate cardiac uptake associated with attenuated bone uptake; or score 3, strong cardiac uptake with mild/absent bone uptake. Semiquantitative analysis of heart retention, whole-body retention, and heart/whole body ratio (H/WB) were evaluated from region-of-interest (ROI) drawings in the standard manner. In brief, rectangular ROIs were drawn over the heart, and irregular ROIs were drawn over the kidneys and bladder on anterior images. These ROIs were copied and mirrored on posterior images, and geometric means of the 2 projections were calculated for each ROI. All ROIs were corrected for background counts. Total counts in the images were considered as whole-body counts. Early whole-body counts were used to represent the injected activity. Whole-body retention was evaluated by comparing counts in the late images (corrected for decay and scan speed, and subtracting the activity in the urinary tract and bladder) with the counts in the early whole-body images. Heart retention was evaluated by comparing decay-corrected counts of the heart in late images with counts in early whole-body images. The H/WB ratios were calculated by dividing counts in the heart by counts in late whole-body images.
Planning of follow-up visits was based on clinical needs (or 6 monthly). Data collection was closed in September 2010; for patients who had not attended a visit in the previous 6 months, vital status was ascertained by telephone and/or by contacting referring physicians.
We defined MACE as the occurrence of death from cardiovascular causes (due to either fatal arrhythmias or severe worsening heart failure), hospital stay due to congestive heart failure, complete atrioventricular block, atrial fibrillation/flutter, myocardial infarction, or stroke. Each prognostic outcome was assessed by contacting the patients, the family members, or the referring physician.
Summary statistics were expressed as median [interquartile range] or frequencies (percentages). Independence of categorical variables was tested using Fisher exact test or Pearson chi-square test. Continuous variables were analyzed using Mann-Whitney U test. Pearson correlation coefficient (r) was used to study the possible correlation of H/WB with mean LV wall thickness and LV ejection fraction. MACE-free survival was analyzed with Kaplan-Meier curves. Patients were censored at time of solid organ transplantation. To explore possible associations with MACE-free survival, univariate Cox regression analysis was initially performed with clinical, echocardiographic, ECG, and scintigraphic variables. Multivariate analysis was then performed by entering into the model the variables that were significant (p < 0.05) on univariate analysis but unrelated to each other. A formal search for colinearity (with the variance inflation factor test) and interactions (with the test of proportional hazard assumption) was performed to guide the construction of the final statistical model. Analyses were conducted with STATA (version 11.1 SE, Stata Corporation, College Station, Texas). Values of p < 0.05 were considered significant.
A total of 63 patients with ATTR fulfilled the study protocol and entered the analysis. Twenty-eight of these patients were included in a previous study (11) analyzing the role of 99mTc-DPD scintigraphy in the differential diagnosis between AL and TTR-related AC. Fourteen patients with ATTR evaluated during the same period were excluded from this study due to lack of informed consent or to a severe global clinical impairment (that was judged incompatible with the execution of scintigraphy).
Distribution of TTR mutations was as follows: Ile68Leu (n = 18), Val30Met (n = 11), Glu89Gln (n = 10), Thr49Ala (n = 9), Phe64Leu (n = 3), Ser23Asn (n = 2), Ala36Pro (n = 2), Arg34Thr (n = 2), Phe33Val (n = 1), His88Arg (n = 1), Thr59Lys (n = 1), Ser50Arg (n = 1), Gly47Ala (n = 1), and Val14Leu (n = 1), a novel TTR mutation not previously described.
At baseline, 40 patients (63%) fulfilled the echocardiographic diagnostic criteria of AC (14), whereas 23 (37%) patients did not. In particular, among patients without echocardiographically defined cardiomyopathy, echocardiogram was normal in 16 (69%) patients, whereas in 7 cases (31%) some echocardiographic abnormalities were detectable: interventricular septal thickness = 12 mm in 3 cases (13%), and increased interatrial septum thickness (>5 mm) in 2 cases (9%). Tables 1 and 2⇓ summarize the main clinical and instrumental (ECG/echocardiographic) profiles of the patients. Patients with AC were older than those without, with a higher prevalence of male sex and a higher prevalence of TTR Ile68Leu mutation. The most represented mutation in patients without AC was the Val30Met. Only 43% of patients received the (final) diagnosis of ATTR due to neurologic manifestations; the diagnostic route was cardiologic in 33% and related to family screening in 24%. Of note, approximately 40% of cases without AC showed ECG abnormalities (vs. 100% in the other group, p < 0.001), most frequently consisting in nonspecific repolarization abnormalities, first degree atrioventricular block or QRS axis deviation.
Scintigraphic findings and scintigraphic-echocardiographic correlations
Figure 1 shows examples illustrating the spectrum of 99mTc-DPD uptake among the groups under investigation, with the corresponding echocardiographic and ECG images. Semiquantitative and visual scintigraphic findings are summarized in Table 3 and Figure 2, in the overall population and according to the presence of cardiomyopathy. At semiquantitative analysis, heart retention and H/WB were both higher in patients with cardiomyopathy. A visual myocardial uptake of the scintigraphic tracer (i.e., visual score ≥1) was present in all patients with echocardiographically defined cardiomyopathy. Within the subgroup without cardiomyopathy, 19 patients (81%) did not show any visible myocardial tracer uptake, whereas 4 patients (18%) showed myocardial tracer uptake (mild in 1 case, moderate in 2 cases, strong in 1 case) and higher H/WB values (Fig. 2, Table 3). In all these 4 cases, LV wall thickness, left ventricular ejection fraction (LVEF), and trans-mitral Doppler profile were normal, whereas E/E′ was abnormal in 1 case. The ECG showed nonspecific T-wave abnormalities in 1 patient. All 4 cases carried non-Val30Met TTR mutations (Thr49Ala, n = 1; Ala36Pro, n = 1; Glu89Gln, n = 1; Gly47Ala, n = 1). As can be seen in Figure 3, in the overall population, H/WB was positively correlated with LV mean wall thickness (Pearson r = 0.695, p < 0.001) and inversely correlated with LVEF (Pearson r = −0.368, p = 0.004).
An EMB was performed in 28 (70%) patients with echocardiographically defines AC. Among patients without AC, EMB was performed only in the 4 cases with myocardial 99mTc-DPD tracer uptake (all candidates for orthotopic liver transplantation) and showed a mild-to-moderate amyloid infiltration in all cases. We did not find any correlation between the extent of the histological infiltration at EMB and the degree of myocardial tracer uptake (p = 0.86 at Kruskal-Wallis test) (Fig. 4).
Outcome and prognosis
The median duration of follow-up was 14 (6.2 to 32) months. No patients were lost to follow-up. Sixteen patients (25%) were submitted to solid organ transplantation (liver transplantation, n = 11; heart-liver transplantation, n = 4; heart-liver-kidney transplantation, n = 1). With regard to MACE, we recorded 15 total events: deaths from cardiovascular causes were recorded in 2 (3%) patients; hospital stay due to heart failure was recorded in 9 (14%) patients; complete atrioventricular block was recorded in 2 (3%) patients; new-onset atrial fibrillation/flutter was recorded in 1 (2%) patient; and sustained ventricular tachyarrhythmia was recorded in 1 (2%) case. Annualized rate of MACE was 0.121/year.
Figure 5 shows MACE-free survival in ATTR patients according to (Fig. 5A) mean LV wall thickness (≤ or >12 mm), (Fig. 5B) H/WB (≤ or >7.5, which represented the median value), and (Fig. 5C) the combination of the previous variables. Notably, the combination of LV wall thickness >12 and H/WB >7.5 was associated with the highest event rate (Fig. 5C).
Table 4 reports univariate and multivariate Cox models on MACE-free survival. At univariate analysis, increasing age, left atrial diameter, mean LV wall thickness, and LV mass/volume ratio as well as increasing New York Heart Association functional class at first evaluation, presence of low QRS voltage, presence of restrictive filling pattern, and increasing scintigraphic heart retention and H/WB were unfavorable predictors of MACE-free survival, whereas increasing LVEF was a favorable predictor (a trend was also recorded for increasing glomerular filtration rate). Therefore, we constructed 2 multivariate analysis models: the first one included only significant and unrelated variables on univariate analysis (i.e., age and filling pattern). The second was obtained by forcing H/WB into the previous one. Likelihood ratio chi-square was 24.13 in the first model and 20.10 in the second (likelihood ratio test, p = 0.044) (Table 4). No interactions emerged between the variables included in the models.
Our study highlights the clinical relevance of 99mTc-DPD scintigraphy in the evaluation of ATTR patients with assessed or suspected AC. Indeed, our data provide 2 main findings: 1) in patients with ATTR, 99mTc-DPD scintigraphy can identify myocardial infiltration across a wide spectrum of morphologic and functional cardiac involvement, increasing the accuracy of echocardiographic diagnosis of AC and allowing early diagnosis of the disease; and 2) 99mTc-DPD myocardial uptake is a prognostic determinant of “cardiac” outcome in ATTR.
99mTc-DPD detection of myocardial involvement in ATTR
In clinical practice, scintigraphy has provided conflicting results in the diagnosis of cardiac amyloidosis. 123I-labeled serum amyloid P (the so-called “SAP”) protein binds in a calcium-dependent way to all amyloid deposits, irrespective of the protein of origin (20–22). Unfortunately, the myocardium does not apparently uptake the tracer, probably due to high background (blood) activity and decreased permeability of the cardiac tissue to the tracer (9,20). 99mTc-aprotinin has been mainly studied in AL patients, in whom it accumulates in various organs, including the myocardium (23,24). However, available experience is limited, and quantitative assessment is not feasible (20,23–25). 123I-metaiodobenzylguanidine does not bind directly to amyloid deposits and has proved useful only in the functional evaluation of amyloid cardiomyopathy, because it indirectly reveals the impairment of cardiac sympathetic nerve endings due to amyloid deposition (20,26,27). Studies on bone tracers in cardiac amyloidosis have produced conflicting results, with significant variations in diagnostic performance (20,28–33). Our previous studies showed that this variability was mainly related to the etiology of amyloid deposits and that this characteristic could be used for noninvasive differential diagnosis between AL and TTR-related AC at least in cases with intense or null tracer uptake (10,11).
In the present study, 99mTc-DPD was able to disclose cardiac amyloidosis across a wide spectrum of morphologic involvement, ranging from patients with overt cardiomyopathy to cases with normal echocardiograms and normal or near-normal ECGs. Our data suggest that the myocardial tracer uptake is an expression of the extent of amyloidotic infiltration: amyloidosis was detected at myocardial histology in all cases with “positive” myocardial scintigraphy (visual score >0), and mean LV wall thickness was linearly and positively correlated to the cardiac tracer retention (Fig. 3). The absence of a significant correlation between the extent of amyloid infiltration at EMB and the degree of myocardial tracer uptake (Fig. 4) does not necessarily contrast with this interpretation, considering the intrinsic “spotty” nature of the EMB and the lack of a quantitative morphometric evaluation of amyloidotic infiltration in our protocol.
Interestingly, 99mTc-DPD scintigraphy was positive in 4 cases with normal LV wall thickness and circumferential function (diastolic longitudinal function was slightly depressed in only 1 case). All these patients were affected by TTR mutations other than Val30Met. This finding is consistent with the current knowledge that some non-Val30Met TTR mutations are more aggressive in the development and progression of myocardial involvement, even in the absence of overt neurologic manifestations or clear familial transmission.
The possibility of identifying myocardial involvement before overt morphological abnormalities are echocardiographically visible is potentially relevant from a clinical perspective, particularly when considering that drugs that stabilize the tetramer and possibly slow disease progression are starting to be available (4,5,8). Diagnostic performance of 99mTc-DPD scintigraphy favorably compares with the other noninvasive techniques. With regard to 2-dimensional echocardiography, data and diagnostic criterion reported so far all refer to cases with overt cardiomyopathy (14,34). Similar considerations can be made with regard to standard ECG (4,35) and magnetic resonance (36). Tissue-Doppler and deformation imaging techniques have proved to identify early impairment of LV mechanics (37). However, these functional (all nonspecific) abnormalities have been described mainly in patients with AL-amyloidosis (38) and are shared by other pathological conditions (such as hypertensive heart disease). The possibilities offered by scintigraphy could usefully integrate with those of Doppler echocardiography, increasing its specificity and therefore its clinical usefulness. Furthermore, the integration between echocardiography and scintigraphy could also be decisive in cases with echocardiographically overt cardiomyopathy. Indeed, it must be considered that in many cases (one-third in our series) patients present with an exclusively cardiologic phenotype, typically with an LV hypertrophy compatible with different interpretations ranging from hypertensive heart disease to hypertrophic cardiomyopathy. This is the case of some TTR mutations including Val122Ile (probably the most frequent TTR mutation, because its prevalence is more than 4% in African Americans), Leu111Met (the so called “FAC,” familial AC, of Danish origin), Thr60Ala, and Ile68Leu. The documentation of myocardial 99mTc-DPD tracer uptake would be decisive in the diagnostic workup of such cases.
Prognostic implication of 99mTc-DPD myocardial uptake
This is the first study that investigates the prognostic role of 99mTc-DPD in ATTR. The natural history of ATTR is very difficult to assess due to the high genotypic heterogeneity and the rarity of the disease. These 2 factors explain the paucity of large systematic studies of ATTR as a whole. The vast majority of data with regard to outcome and prognostic stratification of cardiac amyloidosis derive from studies of patients with AL amyloidosis (4,39,40). In this setting, the main known (negative) prognostic factors are advanced congestive heart failure, increased N-terminal pro– B-type natriuretic peptide, increased parietal thickness, and trans-mitral restrictive filling pattern at Doppler evaluation (41). In general, groups of patients with ATTR AC caused by non-Val30Met mutations tend to have a worse prognosis (compared with Val30Met), even after liver transplantation (3,5,42). Because amyloidotic infiltration is responsible for the myocardial tracer uptake, it is reasonable to hypothesize that the amount of tracer uptake could be a prognostic determinant of cardiac events. Our data seem to confirm this hypothesis: H/WB is not only a significant predictor of MACE-free survival at Cox univariate analysis, but it also interacts with the mean LV wall thickness in predicting cardiovascular events: when mean LV wall thickness is >12 mm, a high myocardial tracer uptake is associated with a particularly poor MACE-free survival.
However, it should be noted that, in this relatively small case series with a relatively low number of events, only increasing age and the presence of LV restrictive filling pattern were independently associated with MACE-free survival. To correctly interpret these prognostic data, it is necessary to consider that our series was mostly formed by patients with TTR mutations other than Val30Met, with a high prevalence of cardiologic abnormalities even at first evaluation. The prognostic contribution could be quite different in patients with Val30Met TTR mutation where myocardial involvement is infrequent (and age-dependent) and neurologic progression is generally the main prognostic determinant.
Although this study represents the largest report of patients with ATTR ever studied with myocardial scintigraphy, the relatively small population (typical of a rare disease) might represent an unavoidable limitation. In particular, only 4 cases in our study showed myocardial tracer uptake, despite normal echocardiography. So, the statement that 99mTc-DPD scintigraphy can be useful in the early diagnosis of ATTR-related myocardial disease needs to be confirmed by further studies. In this retrospective study, where all the examinations were performed on the basis of a clinical indication, EMB was not performed when echocardiogram and 99mTc-DPD scintigraphy were both normal. Therefore, the study does not provide information on the sensitivity of 99mTc-DPD scintigraphy in this context.
B-type natriuretic peptide or N-terminal pro–B-type natriuretic peptide collection (which was not systematically performed in this study) could have been usefully integrated with DPD uptake data. Finally, it should be emphasized that the clinical relevance of 99mTc-DPD might only apply to ATTR patients and not to cases with AL amyloidosis.
In patients with ATTR, 99mTc-DPD scintigraphy can identify myocardial infiltration across a wide spectrum of morphologic and functional cardiac involvement, increasing the accuracy of the echocardiographic diagnosis of AC and allowing an early diagnosis of the disease (even before the appearance of echocardiographic abnormalities), at least in cases with non-Val30Met TTR mutations. 99mTc-DPD myocardial uptake is a prognostic determinant of “cardiac” outcome in ATTR, either alone or in combination with LV wall thickness.
All authors have reported that they have no relationships to disclose.
- Abbreviations and Acronyms
- 99mTc-3,3-diphosphono-1,2-propanodicarboxylic acid
- amyloidotic cardiomyopathy
- immunoglobulin light-chain
- hereditary transthyretin-related amyloidosis
- endomyocardial biopsy
- heart/whole body retention
- left ventricle/ventricular
- major adverse cardiac events
- region of interest
- Received March 25, 2011.
- Accepted March 28, 2011.
- American College of Cardiology Foundation
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