# Test-retest reproducibility of myocardial 99mTc-HMDP uptake quantification by SPECT-CT in suspected transthyretin cardiac amyloidosis

Damien Legallois, Lucas Turcan, Denis Agostini, Alain Manrique

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Damien Legallois, Lucas Turcan, Denis Agostini, Alain Manrique. Test-retest reproducibility of myocardial 99mTc-HMDP uptake quantification by SPECT-CT in suspected transthyretin cardiac amyloidosis. Journal of Nuclear Cardiology, 2025, 51, pp.102273. 10.1016/j.nuclcard.2025.102273. hal-05409798

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ORIGINAL RESEARCH

# Test-retest reproducibility of myocardial ⁹⁹mTc-HMDP uptake quantification by SPECT-CT in suspected transthyretin cardiac amyloidosis

Damien Legallois, MD, PhD <fnref n="1" />*, Lucas Turcan, MD <fnref n="2" />, Denis Agostini, MD, PhD <fnref n="3" />,
Alain Manrique, MD, PhD <fnref n="3" />

## Abstract

**Background:** Diagnosing transthyretin-related cardiac amyloidosis (ATTR-CA) relies on excluding plasma cell dyscrasia and performing bone-avid tracer cardiac scintigraphy. However, visual assessment of cardiac uptake has limitations in evaluating amyloid burden. Whole-body single-photon emission computed tomography-computed tomography (SPECT-CT) may overcome these limitations by enabling quantitative assessment, but data on test-retest reproducibility are lacking.

**Objectives:** This study aimed to evaluate the test-retest, intraobserver and interobserver reproducibility of cardiac amyloid quantification in suspected ATTR-CA using whole-body cadmium zinc telluride SPECT-CT.

**Methods:** Twenty patients with suspected ATTR-CA underwent repeated planar bone-avid tracer cardiac scintigraphy and thoracic SPECT-CT one week apart (October 2020–December 2022; Clinical Trial: NCT04535349). The Perugini score was assessed using planar acquisition. Cardiac 99mTc-HMDP uptake was quantified on SPECT images with CT-based cardiac contouring and attenuation correction. The evaluated metrics included maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean), percentage of injected dose (%ID), and cardiac amyloid activity (CAA). Concordance was evaluated by Lin's concordance correlation coefficient (CCC). Test-retest reproducibility was assessed using the mean absolute difference, coefficient of variation, and intraclass correlation coefficient (ICC).

**Results:** Intraobserver and interobserver reproducibility was excellent for all metrics (CCC ≥.99). Test-retest reproducibility demonstrated substantial agreement for SUVmean (CCC: .79) and almost perfect agreement for SUVmax (CCC: .81), %ID (CCC: .88), and CAA (CCC: .93). All parameters correlated with the Perugini score. Test-retest reliability was high across metrics, with ICC values of .80-.97.

**Conclusions:** Bone-avid tracer cardiac SPECT-CT shows excellent reproducibility for quantifying cardiac amyloid burden in suspected ATTR-CA, with %ID and CAA being especially reliable for future longitudinal studies.

**Clinical trial registration:** NCT04535349.

<footnote n="1">¹Normandie Univ, UNICAEN, INSERM U1086 ANTICIPE, Centre Hospitalier Universitaire de Caen Normandie, Department of Cardiology, 14000 CAEN, France</footnote>

<footnote n="2">²Centre Hospitalier Universitaire de Caen Normandie, Department of Nuclear Medicine, 14000 CAEN, France</footnote>

<footnote n="3">³Normandie Univ, UNICAEN, UR 4650 PSIR, Centre Hospitalier Universitaire de Caen Normandie, Department of Nuclear Medicine, Avenue de la Côte de Nacre, 14000 CAEN, France</footnote>

<footnote>*Corresponding author. Caen-Normandy University Hospital, Department of Cardiology, Avenue de la Côte de Nacre, 14000 Caen, France. E-mail address: damien.legallois@unicaen.fr (Damien Legallois).</footnote>
**Keywords:** Quantification, Reproducibility, Bone-avid tracer cardiac scintigraphy, SPECT, Transthyretin cardiac amyloidosis

ABBREVIATIONS

<table><tr><td>ATTR-CA</td><td>transthyretin-related cardiac amyloidosis</td></tr><tr><td>CAA</td><td>cardiac amyloid activity</td></tr><tr><td>CCC</td><td>concordance correlation coefficient</td></tr><tr><td>CZT</td><td>cadmium zinc telluride</td></tr><tr><td>%ID</td><td>percentage of injected dose</td></tr><tr><td>ICC</td><td>intraclass correlation coefficient</td></tr><tr><td>SPECT-CT</td><td>single-photon emission computed tomography -computed tomography</td></tr><tr><td>SUV</td><td>standardized uptake value</td></tr><tr><td><sup>99m</sup>Tc-HMDP</td><td>technetium 99 metastable-Hydroxymethylene diphosphonate</td></tr><tr><td>VOI</td><td>volume of interest</td></tr></table>

INTRODUCTION

Transthyretin-related cardiac amyloidosis (ATTR-CA) is an increasingly recognized cause of left ventricular hypertrophy resulting from the deposition of transthyretin amyloid fibrils and can lead to heart failure. The diagnosis of ATTR-CA is typically based on histological findings or, more commonly, with the presence of typical cardiac imaging features suggestive of amyloidosis (on echocardiography and/or cardiac magnetic resonance), combined with grade 2 or 3 myocardial uptake of bone-avid tracers on cardiac scintigraphy, and in the absence of a plasma cell dyscrasia [1]. In clinical routine, cardiac uptake is evaluated using a visual 4-point grading system, ranging from 0 (no uptake) to 3 (intense myocardial uptake with reduced apparent bone uptake) as proposed by Perugini et al. [2] However, this visual assessment has several limitations. Previous studies have reported significant overlap between grades [3], and follow-up studies have demonstrated that a substantial proportion of grade 1 patients eventually progress to higher grades over time [4].

Semi-quantitative approaches have been proposed, typically based on the ratio of cardiac uptake to whole-body or contralateral lung activity, and are inherently derived from planar imaging [5]. However, this technique is limited by low spatial resolution and may be further confounded by systemic amyloid deposition, which can elevate

background signal. As a result, its contribution to diagnostic accuracy is limited and definitive interpretation should rely primarily on single-photon emission computed tomography (SPECT) imaging.

With the advent of solid-state single-photon emission computed tomography with computed tomography (SPECT-CT) cameras, a quantitative approach for cardiac uptake of bone radiopharmaceuticals has become feasible [6,7]. This approach offers the potential to assess disease progression and therapeutic response in patients with ATTR-CA [8,9]. However, the reproducibility of quantitative parameters, which is essential for longitudinal studies, remains unknown due to the lack of test-retest studies.

Thus, the aim of this study was to evaluate the test-retest reproducibility of different quantitative metrics in patients with suspected ATTR-CA referred for bone-avid tracer cardiac scintigraphy.

METHODS

Study design

The prospective REMOD-TTR study (Quantitative Analysis of Myocardial Uptake of Bone Radiopharmaceuticals in Patients With Cardiac ATTR Amyloidosis) was conducted in the Nuclear Medicine department of the Caen University Hospital (Caen, France). The study was approved by an ethics committee (CPP, Ile de France 1, number: 19.12.20.64457) and conducted in accordance with the Declaration of Helsinki. All procedures followed were in accordance with institutional guidelines. All patients provided written informed consent prior to enrollment and the study protocol was registered on [ClinicalTrials.gov (NCT04535349)].

Patients were screened for eligibility between October 2020 and December 2022. The inclusion criteria were as follows: patients with a suspected diagnosis of ATTR-CA and with at least one prior hospitalization for heart failure or clinical evidence of heart failure indicated by the following factors: (i) signs or symptoms of volume overload or elevated intracardiac pressures requiring diuretic treatment, (ii) an increase in brain natriuretic peptide >200 pg/mL and/or N-terminal prohormone of brain natriuretic peptide >500 pg/mL, and (iii) evidence of cardiac involvement demonstrated by an echocardiographic septal wall thickness >12 mm. Exclusion criteria were as follows: the New York Heart Association functional class IV despite
diuretic treatment, life expectancy <6 months due to severe cardiac amyloidosis and/or comorbidities, recent aortic valve surgical replacement or percutaneous replacement within 30 days or planned replacement within 3 months.

### Whole-body planar scintigraphy and chest cadmium zinc telluride SPECT-CT

Patients received an intravenous injection of 10 MBq/kg of ⁹⁹mTc-hydroxymethylene diphosphonate (⁹⁹mTc-HMDP), with a maximum dose of 740 MBq, followed by a saline flush. The syringe activity was measured before and after injection using a well counter and the injected dose was calculated after decay correction. A whole-body planar acquisition was performed using an Anger camera (Symbia, Siemens, Erlangen, Germany) 3 hours postinjection. SPECT-CT imaging was then conducted, beginning with a low-dose computed tomography scan for attenuation correction, followed by a 10-min tomographic list mode acquisition using a 360° dedicated volumetric cadmium zinc telluride (CZT) camera (Veriton; Spectrum Dynamics, Biosensors, Caesarea, Israel) and a 10% asymmetrical (–7

to +9 keV) energy windows centered on 140.5 keV. The same procedure was repeated within 7 to 10 days (Fig 1). All SPECT data were corrected for attenuation and reconstructed using a dedicated workstation using the standard parameters recommended by the manufacturer. As part of quality control, the automated co-registration of SPECT and CT images was evaluated, and additional reconstruction was performed using a manually corrected co-registration matrix in case of misregistration.

### Postprocessing and analysis

Planar images were analyzed using the Perugini scale. Reconstructed SPECT images were processed using MIM software (version 7.2.7, MIM Software Inc, USA). A single volume of interest (VOI) encompassing both ventricles was manually delineated on axial computed tomography slices, using the segmentation tools provided in the MIM software, which allow contouring across multiple slices simultaneously. The VOI was adjusted by the operator when necessary to avoid overlap with the sternum or rib cage and then transferred to the SPECT images. The average

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Study Protocol. Panel A: Protocol for assessing the reproducibility of cardiac amyloid burden quantification in patients with suspected transthyretin-related cardiac amyloidosis using planar bone-avid tracer cardiac scintigraphy and chest-centered SPECT-CT. Each patient underwent two imaging sessions, with a 7-10 day interval between scans. Panel B: Absolute quantification of cardiac amyloid burden was performed independently by two observers, each conducting two rounds of analysis for every acquisition. This process involved the reconstruction and contouring of volumes of interest, including the ventricles, on computed tomography images. Intraobserver reproducibility, interobserver reproducibility, and test-retest reproducibility were analyzed for all quantification metrics (see main document). SPECT-CT, single-photon emission computed tomography–computed tomography.</figcaption></figure>
time required to complete the VOI definition for one study was approximately 10 minutes. Cardiac amyloid activity (CAA) was calculated as the product of SUVmean and VOI volume, and the percentage of injected dose (%ID) was calculated as the ratio of the activity measured in the VOI to the injected dose as previously described [6].

## Reproducibility assessment

The Perugini score was estimated by a consensus of two observers (DL and LT). Intraobserver and interobserver reproducibility of quantitative SPECT measurements was assessed through two independent analyses performed by each observer for all acquisitions (including both test and retest sessions). To ensure blinded conditions, the second analysis was performed at least one week after the first and all image datasets were anonymized. Observers were not informed of whether the scans corresponded to test or retest sessions, had no access to their previous measurements, and were also blinded to the results of the other observer. Test–retest reproducibility was evaluated using two imaging sessions conducted 7–10 days apart, with the same protocol for radiopharmaceutical administration and image acquisition.

## Statistical analysis

At the time of the writing of this protocol, no published data were available on the quantitative assessment of myocardial uptake of technetium-labeled bone tracers using 3D CZT-based whole-body SPECT cameras. Consequently, the sample size was defined as 20 patients, based on previous test–retest assessments in oncology positron emission tomography studies [10–13].

Quantitative variables are presented as median [interquartile range], except for group characteristics based on the Perugini grades presented as the median [min-max]. Alternatively, variables are displayed as mean ± standard deviation when appropriate and compared using Student's paired t-test or Wilcoxon rank-sum test. Comparisons between multiple quantitative parameters were performed using nonparametric Kruskal–Wallis test.

Repeatability was assessed using Bland–Altman plots analysis [14], Lin's concordance correlation coefficient (CCC) test [15], and intraclass correlation coefficient (ICC). Lin's CCC is similar to the kappa coefficient but is applicable to continuous data. It evaluates both accuracy and precision, indicating how far measurement pairs deviate from the line of identity. Lin's CCC scale ranges from 0 (no agreement) to +1 (perfect agreement), where .21-.40 indicates fair

concordance, .41-.60 indicates moderate concordance, .61-.80 indicates substantial concordance, and .81-1.00 indicates almost perfect concordance. The ICC and its 95 % confidence interval (95 % CI) were calculated using a 2-way model. The coefficient of variation was calculated as the mean relative difference between the two imaging sessions and indicated reproducibility when it was below 20 %. A P value < .05 was considered significant. All analyses were performed using R (version 4.3.1, R Foundation for Statistical Computing, Vienna, Austria).

## RESULTS

### Patient characteristics and SPECT-CT data

Patient characteristics are summarized in Table 1. The median septal thickness was 16 mm [13-16.5]. Additional echocardiographic parameters are provided in the Supplementary Data (Supplementary Table 1). No significant changes were observed between the two examinations for patient weight (69.5 kg [63-90.8] vs 69.5 kg [63-82.5], P = .333), the delay between tracer injection and SPECT-CT acquisition (205 minutes [184-223] vs 199 minutes [176-230], P = .706), and the injected dose (662 MBq [609-704] vs 710 MBq [660-758], P = .077). Cardiac

<table><caption>Table 1. Patient characteristics. Data are expressed as median [interquartile range] or n (%). NYHA: New York Heart Association.</caption><thead><tr><th>Baseline characteristics</th><th>n = 20</th></tr></thead><tbody><tr><td>Age, years</td><td>84 [80-86]</td></tr><tr><td>Sex, male</td><td>9 (45 %)</td></tr><tr><td>Hypertension</td><td>15 (75 %)</td></tr><tr><td>Carpal tunnel syndrome</td><td>5 (25 %)</td></tr><tr><td>Dyspnea</td><td></td></tr><tr><td>NYHA I</td><td>3 (15 %)</td></tr><tr><td>NYHA II</td><td>13 (65 %)</td></tr><tr><td>NYHA III</td><td>4 (20 %)</td></tr><tr><td>NYHA IV</td><td>0</td></tr><tr><td>Prior hospitalization for heart failure</td><td>6 (30 %)</td></tr><tr><td>Atrial fibrillation</td><td>11 (55 %)</td></tr><tr><td>Pace maker</td><td>4 (20 %)</td></tr><tr><td>Biological parameters</td><td></td></tr><tr><td>- Brain natriuretic peptide, ng/L</td><td>473 [294-789]</td></tr><tr><td>- Troponin I, ng/L (normal value &lt; 17.5; n = 19)</td><td>38.2 [24.1-87.3]</td></tr><tr><td>- Estimated glomerular filtration rate (eGFR), mL/min (n = 19)</td><td>52 [40-80]</td></tr><tr><td>Left ventricular ejection fraction, %</td><td>56.5 [48.5-65.5]</td></tr><tr><td>Interventricular septal thickness, mm</td><td>16 [14.5-19]</td></tr><tr><td>Perugini grade</td><td></td></tr><tr><td>0</td><td>6 (30 %)</td></tr><tr><td>1</td><td>3 (15 %)</td></tr><tr><td>2</td><td>6 (30 %)</td></tr><tr><td>3</td><td>5 (25 %)</td></tr><tr><td>Dose of furosemide, mg/day</td><td>40 [35-80]</td></tr></tbody></table>
SUVmax, SUVmean, %ID, and CAA ranged from 1.16 g/mL to 6.70 g/mL, .44 g/mL to 3.14 g/mL, .19 % to 2.04 %, and 158 to 1734 g, respectively, depending on the Perugini grade (Fig 2 and Table 2). All quantitative parameters showed a significant correlation with the Perugini grade (P < .001, Fig 2). Notably, among the three patients with a Perugini grade 1, one exhibited quantitative parameters similar to those of Perugini 0 patients, another had parameters resembling those of Perugini grade 2, and the third displayed values at the threshold between grades 1 and 2 (Fig 3), depending on the specific metric.

## Reproducibility and test-retest

With regard to postprocessing, the intraobserver reproducibility was excellent for all quantitative parameters, with Lin's CCC = 1.00 for all metrics. The interobserver reproducibility was also excellent, with Lin's CCC values of .99 [95%CI: .98-.99] for SUVmax, .99 [95%CI: .99-1.00] for SUVmean, 1.00 [95%CI: .99-1.00] for %ID and 1.00 [95%CI: .99-1.00] for CAA.

Regarding test-retest reproducibility, the absolute difference between the two sessions was minimal for SUVmax, SUVmean, %ID, and CAA. However, a significant increase in cardiac CT

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Association between quantitative parameters of cardiac amyloid burden and the Perugini grades. Each dot represents a patient, with the plotted value corresponding to the mean of all measurements (ie, two evaluations per observer for each imaging session, totaling eight measurements per patient).</figcaption></figure>
<table><caption>**Table 2.** Quantitative assessment of the different metrics representative of cardiac ⁹⁹mTc-HMDP uptake, categorized by Perugini grades. maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean), percentage of injected dose (%ID), and cardiac amyloid activity (CAA, defined as SUVmean multiplied by the volume of interest [VOI]). Data are presented as median [min-max].</caption><thead><tr><th></th><th>Perugini 0 (n = 6)</th><th>Perugini 1 (n = 3)</th><th>Perugini 2 (n = 6)</th><th>Perugini 3 (n = 5)</th><th>P value</th></tr></thead><tbody><tr><td>SUVmax, g/mL</td><td>1.80 [1.16-2.48]</td><td>3.62 [1.90-5.05]</td><td>4.72 [2.90-6.70]</td><td>5.50 [4.21-6.20]</td><td>.004</td></tr><tr><td>SUVmean, g/mL</td><td>.60 [.44-1.15]</td><td>1.46 [.60-2.33]</td><td>2.03 [1.21-2.75]</td><td>2.53 [2.09-3.14]</td><td>.004</td></tr><tr><td>%ID</td><td>.30 [.19-.46]</td><td>1.05 [.40-1.70]</td><td>1.08 [.74-1.96]</td><td>1.64 [1.36-2.04]</td><td>.004</td></tr><tr><td>CAA, g</td><td>204 [158-323]</td><td>1010 [427-1592]</td><td>785 [477-1265]</td><td>1277 [883-1734]</td><td>.003</td></tr></tbody></table>

volume was observed at retest compared with that of the initial test (Table 3). This increase in cardiac volume was dependent on the Perugini grade (P = .004), with grade 1 patients exhibiting the largest volumes and reduced reproducibility for volume measurement (Fig 4). Despite the observed differences in cardiac CT volume, the coefficient of variation remained within the expected limits for all metrics, and high test–retest reproducibility was observed for all parameters. Lin’s CCC demonstrated a nonsignificant trend toward improved reproducibility for %ID (Lin’s CCC: .88 [95%CI: .74-.95]) and CAA (Lin’s CCC: .93 [95%CI: .85-.97]) compared with SUVmax (Lin’s CCC: .81 [95%CI: .58-.92] and SUVmean (Lin’s CCC: .79 [95%CI: .55-.91] (Fig 5 and graphical abstract). The ICC values for test–retest reproducibility were good for SUVmean (.80), SUVmax (.82), and %ID (.89) and excellent for CAA (.94) and cardiac volumes (.97). The ICC values for test–retest reproducibility were similar between the two observers, with ICC values of .81 and .77 for SUVmean, .83 and .80 for SUVmax, .89 and .89 for %ID, .94 and .93 for CAA, and .97 and .97 for cardiac volumes (Table 4).

DISCUSSION

This is the first study analyzing the test–retest reproducibility of the absolute quantification of cardiac 99mTc-HMDP uptake in patients with suspected ATTR-CA. Among the various metrics evaluated, CAA and %ID yielded better results compared with those of cardiac SUVmax and SUVmean, with a high reproducibility and a narrow 95 % CI for the absolute difference between the two SPECT acquisitions.

Several factors may influence the quantitative assessment of cardiac 99mTc-HMDP uptake. SUV (in g/mL), initially proposed as an estimate of metabolic activity in 18F-fluorodeoxyglucose positron emission tomography, represents a relative tissue uptake (in KBq/mL) normalized to the decay-corrected injected dose and patient weight. SUVmax reflects the hottest voxel within a given organ or VOI, while SUVmean accounts

for the size of the VOI. The %ID may also be influenced by the size of the VOI, while CAA is calculated as the product of SUVmean and VOI volume. Consequently, heart segmentation may bias the uptake measurement and alter its reproducibility.

Several methods have been proposed for the segmentation of the heart, including segmentation of the whole heart [16], segmentation of the left ventricle and its cavity only using non-contrast CT [9], and a hybrid method combining automatic isocontour of left ventricular tracer uptake with manual left ventricle contouring using CT in patients with no cardiac uptake [3]. We observed a moderate but significant difference in cardiac volumes between test and retest, particularly in patients with low Perugini grades. Previous phantom studies demonstrated the accuracy of left ventricular volume measurements based on an automated threshold set at 39 % of maximal activity [17], a method proposed for monitoring therapy in patients with a high Perugini grade [9]. Although most candidates for longitudinal follow-up would fall under grades 2 or 3, applying segmentation based on the SPECT tracer uptake may be difficult when tracer uptake decreases significantly over time. Using segmentation based on noncontrast CT, Dorbala et al. demonstrated excellent values for SUVmax, SUVmean, %ID, and CAA for diagnosing ATTR-CA [6]. These metrics were correlated with left ventricular mass and extracellular volume as measured by cardiac magnetic resonance, suggesting the robustness of the technique. Our results are encouraging, demonstrating that despite variability in cardiac volume measurements, the SPECT indices of ⁹⁹mTc-HMDP uptake were highly reproducible. Moreover, as recently suggested, the accuracy of cardiac segmentation using non-contrast CT may be further improved by employing automated deep learning-based algorithms [18,19].

To overcome possible limitations, several authors have used a SUV retention index based on peak SUV, normalized to muscle and/or bone SUVs to avoid radiotracer competition from soft
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Example of a patient stated as Perugini 1 with SPECT-CT quantification parameters. Note: Segmentation of the left ventricle may be challenging due to the absence of contrast enhancement and the low level of amyloid burden. Of note, cardiac MRI was not contributive due to artifacts caused by a pacemaker. Using transthoracic echocardiography, left ventricular ejection fraction was 68 %. Septal wall thickness measured 15 mm. Labial salivary gland biopsy revealed only grade IV focal lymphocytic sialadenitis. Troponin was elevated (5 × the upper limit of normal) and brain natriuretic peptide (BNP) was 2430 pg/mL. Work up for monoclonal gammopathy was negative and no mutation in the transthyretin gene was identified. In cases of grade 1 uptake and absence of monoclonal gammopathy, current guidelines recommend performing biopsy to confirm transthyretin cardiac amyloidosis. In the present case, tafamidis therapy was initiated following multidisciplinary discussion, based on quantitative SPECT/CT findings and in the context of negative peripheral biopsies and significant comorbidities (chronic respiratory failure, dialysis-dependent renal failure, and long-term anticoagulation) precluding endomyocardial biopsy. MRI, magnetic resonance imaging; SPECT-CT, single-photon emission computed tomography–computed tomography.</figcaption></figure>
<table><caption>**Table 3.** Reliability of the different metrics for the test–retest analysis. Bland Altman results, coefficient of variation and intraclass correlation coefficient (ICC).</caption><thead><tr><th></th><th>Test mean ± SD</th><th>Retest mean ± SD</th><th>Absolute<br>difference<br>mean [95%CI]</th><th>Test–retest<br>P value</th><th>Coefficient<br>of variation (%)<br>median</th><th>ICC [95%CI]</th></tr></thead><tbody><tr><td>Cardiac CT volume, mL</td><td>440 ± 137</td><td>459 ± 142</td><td>−19 [−78; 39]</td><td>.009</td><td>3.32</td><td>.97 [.89; .99]</td></tr><tr><td>SUVmax, g/mL</td><td>3.93 ± 1.88</td><td>3.79 ± 1.83</td><td>.14 [−2.09; 2.38]</td><td>.582</td><td>−5.31</td><td>.82 [.60–.92]</td></tr><tr><td>SUVmean, g/mL</td><td>1.67 ± .99</td><td>1.57 ± .85</td><td>.1 [−1.06; 1.26]</td><td>.459</td><td>.00</td><td>.80 [.56–.91]</td></tr><tr><td>%ID</td><td>1.03 ± .65</td><td>1.07 ± .71</td><td>−.04 [−.68; .60]</td><td>.566</td><td>6.16</td><td>.89 [.75–.96]</td></tr><tr><td>CAA, g</td><td>737 ± 481</td><td>762 ± 532</td><td>−24 [−383; 334]</td><td>.557</td><td>5.16</td><td>.94 [.85–.97]</td></tr></tbody></table>

%ID, percentage of injected dose; CI, confidence interval; CT, computed tomography; SD, standard deviation; SUVmax, maximum standardized uptake value; SUVmean, mean standardized uptake value; 99mTc-HMDP, Technetium 99 metastable-hydroxymethylene diphosphonate.

<figure><img src="image_5.png" /><figcaption>**Figure 4.** Test–retest absolute difference in ventricular volume depending on the Perugini grade. Each dot represents a test and retest evaluation of ventricular volume for each patient and observer. The horizontal line within each box represents the median absolute difference in ventricular volumes, and the box itself displays the interquartile range.</figcaption></figure>

tissues and to provide a global means of monitoring therapy [9,20,21]. However, peak SUV represents the average SUV within a 1 cm³ volume centered on the hottest voxel. To ensure accurate quantification, particular attention must be paid during VOI delineation as inclusion of voxels adjacent to bone structures (e.g. sternum or ribs) may result in measurement artifacts due to spillover from bone uptake [22].

The intraobserver and interobserver reproducibility of SPECT postprocessing of the same acquisition was excellent [6,23,24]. To our best knowledge, this is the first study evaluating the test–retest reproducibility of whole-heart

amyloid burden measurement. The four metrics tested (SUVmax, SUVmean, %ID, and CAA) were highly reproducible across test and retest, with no variation between observers. However, SUV-derived measures (including CAA) depend on patient weight, which is an important cause of variability in SUV measurements as observed in ¹⁸F-fluorodeoxyglucose positron emission tomography [25]. By contrast, %ID is independent of patient weight, which is particularly relevant for patients with heart failure and amyloid cardiomyopathy, where significant weight variations may result from edema or diuretic treatment. In addition, the test–retest reproducibility of %ID was not impacted by the variability of cardiac volume measurement. The 95%CI of the test–retest difference in %ID [−.68; .60] suggests that a threshold of .7 could be proposed as a cut-off value for significant changes in whole-heart amyloid burden. While defining a threshold is necessary to determine what constitutes a significant change in amyloid burden quantification using SPECT/CT in recent [26] and future longitudinal studies, it is important to keep in mind that the mechanisms underlying the affinity of bone-avid tracers for TTR amyloid fibrils remain incompletely understood [27]. Recently, Vijayakumar et al. demonstrated a decrease in quantitative amyloid burden parameters in a cohort of 23 patients receiving transthyretin-stabilizing therapy [24]. However, no significant changes were observed in echocardiographic or biomarker parameters, highlighting the lack of a close correlation between tracer uptake on SPECT/CT and actual cardiac function.

## Study limitations

We investigated the test–retest reproducibility of amyloid burden measurement using a 360° CZT camera with increased detection sensitivity compared with conventional cameras [28]. Beside attenuation and scatter corrections, this increased
<figure><img src="image_6.png" /><figcaption>**Figure 5.** Test-retest repeatability of the quantitative parameters of cardiac amyloid burden. The upper row displays maximum cardiac standardized uptake values (SUVs), then mean SUV, the percentage of injected dose within the ventricular volume of interest (%ID) and the lower row shows cardiac amyloid activity (CAA, defined as SUVmean multiplied by the volume of interest). The left panel presents Lin's CCC concordance plots and the right panel shows Bland-Altman plots. CCC, concordance correlation coefficient.</figcaption></figure>

sensitivity may influence image quality and quantification. We acknowledge that the sample size (20 patients from one single center) is limited. However, similar cohort sizes are commonly used in studies assessing amyloid burden quantification [24,26]. As in such studies, generalizability should be approached with caution, particularly regarding gender differences. In addition, the use of 99mTc-HMDP—a radiopharmaceutical less commonly used in the United States compared with 99mTc-

pyrophosphate—may also limit the broader applicability of our findings. Furthermore, our results may not be directly transferable to the conventional SPECT/CT systems, widely used in the previous studies [3,9,16,20]. Replicating this work using these systems would be valuable to support external validity. Nevertheless, despite these limitations, our results demonstrated minimal variation and high correlation across parameters, consistent with prior test-retest studies using
<figure><img src="image_7.png" /><figcaption>**Figure 5.** (continued).</figcaption></figure>

<table><caption>**Table 4.** Test–retest repeatability of 99mTc-HMDP cardiac uptake measurement, depending on observer. Data are expressed as intraclass correlation coefficient (ICC) and 95 % CI.</caption><thead><tr><th>ICC [95 % CI]</th><th>Cardiac CT volume</th><th>SUVmax</th><th>SUVmean</th><th>%ID</th><th>CAA</th></tr></thead><tbody><tr><td>Observer 1</td><td>.97 [.86-.99]</td><td>.83 [.61-.93]</td><td>.81 [.58-.92]</td><td>.89 [.74-.96]</td><td>.94 [.86-.98]</td></tr><tr><td>Observer 2</td><td>.97 [.91-.99]</td><td>.80 [.57-.92]</td><td>.77 [.52-.90]</td><td>.89 [.74-.95]</td><td>.93 [.84-.97]</td></tr></tbody></table>

CI, confidence interval; CT, computed tomography; SUVmax, maximum standardized uptake value; SUVmean, mean standardized uptake value; ⁹⁹mTc-HMDP, Technetium 99 metastable-hydroxymethylene diphosphonate.
positron emission tomography (PET) imaging, even
those involving fewer than 20 patients [10–13].

The aim of this study was to assess the repro-
ducibility of quantitative amyloid burden mea-
surements; it does not inform treatment
decisions. The patient illustrated in Figure 3 rep-
resents an exceptional case and should not be
interpreted as justification for avoiding biopsy in
cases of grade 1 uptake without monoclonal
gammopathy. The role of quantitative imaging in
the initial diagnostic pathway requires further
dedicated investigation.

At last, although the acquisition delay was set
at 3 hours after tracer injection, the SPECT ac-
quisitions were performed at an average of
200 minutes after injection. Using 99mTc-DPD,
Wollenweber et al. demonstrated that cardiac
tracer activity and myocardial SUVpeak are time-
dependent with a significant decrease in SUV-
peak between 1 and 3 hours after injection [29].

CONCLUSIONS

This study demonstrated the high test-retest reproducibility of quantitative assessment of cardiac amyloid burden using a 360° CZT SPECT-CT camera. Our results suggest that %ID and CAA are the most reproducible quantitative parameters and may serve as reliable metrics to define thresholds for significant changes in amyloid burden in longitudinal SPECT/CT studies.

NEW KNOWLEDGE GAINED

* We demonstrate that quantitative bone-avid tracer cardiac single-photon emission computed tomography/computed tomography yields reproducible measurements of cardiac amyloid burden in patients with transthyretin cardiac amyloidosis using a test-retest protocol.

* In particular, the percentage of injected dose and cardiac amyloid activity showed the highest reproducibility among the evaluated quantitative parameters.

CLINICAL PERSPECTIVE

* Quantitative SPECT/CT using bone-avid tracers—particularly percentage of injected dose and cardiac amyloid activity—may offer a potential framework for monitoring patients with transthyretin cardiac amyloidosis.

* However, further studies are needed to clarify the clinical significance of uptake changes and to integrate quantitative imaging with clinical, biological, and multimodality imaging data into treatment algorithms.

FUNDING AND SUPPORT

This study was granted by Pfizer as part of the
2019 ATTR-CM Global ASPIRE program and is also
supported by the Groupement de coopération
sanitaire (GCS) G4 as part of the Fédération
Hospitalo-Universitaire (FHU) CARNAVAL,
labeled by AVIESAN.

AUTHOR DISCLOSURES

The authors declare that they have no personal
financial interests or professional relationships
related to the subject matter.

DISCLOSURES

The authors declare the following financial in-
terests/personal relationships which may be
considered as potential competing interests:
Damien Legallois reports that financial support
was provided by Pfizer. The other authors declare
that they have no competing financial interests or
personal relationships that could have appeared
to influence the work reported in this paper.

ACKNOWLEDGMENTS

The authors gratefully thank Mrs. Delphine Rots
for her technical assistance.

APPENDIX A. SUPPLEMENTARY DATA

Supplementary data to this article can be found
online at https://doi.org/10.1016/j.nuclcard.2025.
102273.

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