Method Article
This novel protocol entails the quantification of cardiovascular calcification progression from serial micro positron emission tomography (PET)/micro computed tomography (CT) images in small research animals.
Micro positron emission tomography (PET) and micro computed tomography (CT) imaging are powerful, ideal research tools for following the progression of cardiovascular calcification. Due to their non-invasive nature, small research animals can be imaged at multiple time points. The challenge lies in the accurate quantification of cardiovascular calcification. Here, we provide a protocol, using images from the later disease stages as a template, to accurately quantify the progression of cardiovascular calcification in longitudinal studies. The protocol involves 1) the alignment of the chest area in multiple images from the same animal during a longitudinal study as the first step, 2) the definition of a region of interest (ROI) situated within the heart and the aorta at the site of larger calcium deposits that become apparent in later images, and 3) simultaneous segmentation and quantification of calcium deposits across all images acquired during the longitudinal study. This streamlined method enhances the accuracy of image analysis in following the progression of cardiovascular calcification by improving the precision of ROI definition and reducing the variability associated with earlier techniques that analyze individual scans independently.
Cardiovascular disease is a leading global cause of morbidity and mortality, demanding rigorous exploration to uncover its mechanisms and to devise effective preventive and therapeutic strategies. Coronary artery calcification (CAC) is widely acknowledged by the experts in the field as a predictive factor for cardiovascular disease, significantly elevating the risk of cardiovascular mortality1,2,3,4,5. Microscopic calcifications are considered the earliest stages of calcific atherosclerosis, and the term "microcalcifications" has been used to refer to calcium deposits between 0.5 to 50 µm6,7,8,9 in diameter. These small calcifications are believed to coalesce to form larger calcium deposits, fueling the progression of calcific plaque6,7.
Positron emission tomography (PET) and computed tomography (CT) serve as valuable research tools, frequently employed for the non-invasive assessment of cardiovascular calcification in vivo5,10,11,12,13,14,15,16,17,18,19. These imaging modalities prove particularly advantageous for tracking vascular calcification progression in longitudinal studies involving small research animals11,12,13,19. MicroCT imaging has demonstrated effectiveness in providing anatomical images of relatively large calcium deposits11,12,13,19,20. However, its utility for imaging small calcium deposits in living animals is constrained by its spatial resolution of ~100 µm8,14, making it challenging to investigate calcification during its initial phases.
A notable advancement is the adoption of combined microPET/microCT imaging with the PET tracer, fluoride-18-labeled sodium fluoride (18F-NaF), as the standard method for the detection of calcification based on its binding to the mineral surface area. This approach uses radiolabeled 18F-NaF, shown to identify the calcium mineral surface10,13 as the fluoride ions bind covalently to calcium hydroxyapatite, replacing hydroxyl groups to form fluoroapatite21. Consistent with the slower exchange rate relative to the radioactive decay of the 18F (half-life ~110 min) and to the clearance of the tracer through kidneys22, Irkle and colleagues13 found that 18F-NaF binding to calcified carotid specimens was limited to the surface at the time of detection. Thus, tracer uptake should relate directly with mineral surface area, which is greater when a given amount of mineral occurs in multiple small foci or in porous form than when present in few, large, solid deposits. By accentuating the earliest stages of mineralization with high sensitivity, 18F-NaF PET imaging may provide valuable insights into the early stages of disease, making it particularly useful in studying preventive as well as therapeutic strategies13,14,15.
Even with recent advancements in the combined microPET/microCT imaging of vascular calcification, there are opportunities for improving the accuracy of image analysis in longitudinal, cardiovascular calcification studies. Conventional approaches use labor-intensive, manual delineation of regions of interest (ROI) around each visible calcified region in every mouse at each individual time point throughout the longitudinal study. This method reduces precision, particularly in the early disease stages when the sizes of calcium deposits approach the scanners' detection limits, potentially making invisible some areas with minuscule deposits arranged in low density.
In the realm of imaging, alignment generally refers to the spatial alignment of a series of images. Introducing alignment as a novel solution to the existing challenges, our proposed method allows investigators to use a consistent location for following the progression of calcification in serial images from individual subjects throughout a longitudinal study. Given that tissue calcification is known to arise from nanosized matrix vesicles (50-150 nm), which coalesce to form microscopic, then macroscopic hydroxyapatite mineral23, one may retrospectively identify where microcalcifications would have been located in early images before they are discernable.
Following that same location over time, made possible by image alignment, is the basis for this method. It obviates the need to identify the earliest calcification stages directly, as the ROI is assigned based on the latest stages when mineralization is readily identified. In this protocol, we present an improved, streamlined data analysis method that incorporates the alignment of a time series of images as a vital step, enhancing accurate quantitation of calcium deposition in longitudinal, combined, microPET/microCT imaging studies (Figure 1). While we use PET/CT data analysis as an example, this method can be applied to the analyses of other longitudinal imaging data, including single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), and optical imaging24.
Figure 1: Protocol overview flowchart. Flowchart summarizing the novel protocol for quantifying cardiovascular calcification. General steps include longitudinal imaging, alignment of images acquired at different time points, and segmentation and quantification of the calcified region. Please click here to view a larger version of this figure.
The representative images present a female apolipoprotein-E-deficient (Apoe-/-) mouse. Experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of California, Los Angeles.
1. Animal scanning
2. Import DICOM files in DICOM viewer software
NOTE: While this representative protocol uses ORS Dragonfly software under a non-commercial license, its flexibility extends to other DICOM viewer software options.
3. Adjust DICOM viewer settings to optimize image visualization
Figure 2: Adjustment of DICOM viewer settings to optimize image visualization. (A,B) Coronal view of CT images (A) before and (B) after contrast/brightness adjustment. (C,D) Coronal view of PET/CT images (C) before and (D) after Lookup Table adjustment. Abbreviations: CT = computed tomography; PET = positron emission tomography. Please click here to view a larger version of this figure.
4. Align the chest area in CT images
NOTE: For simplicity, one CT image will serve as the "base" image and will not be translated or rotated. The second CT image (and eventually subsequent serial images) will be translated and/or rotated to align with the base image; this will be called the "overlay" image for the purpose of the demonstration. Throughout alignment, it is important to distinguish between the base image and all other overlay images.
Figure 3: Alignment of all CT images. (A,B) Coronal view of base (gray) and overlay (red) CT images (A) before and (B) after alignment. (C,D) Sagittal view of CT images (C) before and (D) after alignment. Blue arrows indicate the mouse (1) thoracic cage, (2) spine, and (3) sternum. Abbreviation: CT = computed tomography. Please click here to view a larger version of this figure.
5. Co-register PET images with corresponding CT images
NOTE: Alignment of the overlay CT image and base CT image will initially result in misalignment of the overlay CT image with its respective PET image seen in Figure 4. The PET image needs to be co-registered with its corresponding CT image once again.
Figure 4: Alignment of PET images with corresponding CT image. A representative PET image and its corresponding CT image (A, C) before and (B, D) after co-registration. Following completion of co-registration, the PET and CT images should be aligned as demonstrated in Panel B. Abbreviations: CT = computed tomography; PET = positron emission tomography. Please click here to view a larger version of this figure.
6. Identify cardiovascular calcification
Figure 5: Identifying calcified regions in the heart. Calcification regions depicted in representative (A) sagittal, (B) transverse, and (C) coronal CT images, as well as (D) a coronal PET/CT image. Yellow arrows indicate calcium deposits. Blue arrows identify reference structures used for determining the location of calcification. Abbreviations: CT = computed tomography; PET = positron emission tomography. Please click here to view a larger version of this figure.
7. Draw ROIs around the calcified regions
Figure 6: Using segmentation to restrict the ROI to calcified regions. (A) The arrows guide through the steps required to eliminate undesired density data from the ROI. The blue arrow points to the Segment tab; the yellow arrow highlights the Define Range feature; green arrows indicate the selected range inputs; and the red arrow points to the Remove button. (B) Following completion of protocol steps 7.4-7.4.3, the ROI should specifically delineate the calcified regions, as depicted in this panel. Abbreviation: ROI = region of interest. Please click here to view a larger version of this figure.
8. Quantify the ROI for each image
Figure 7: ROI quantification. The Statistical Properties tab for a selected ROI. The red arrows illustrate steps required to obtain statistical information for a dataset based on the ROI. The red boxes identify the volume and the mean Hounsfield unit value, essential for further analysis calculations. Abbreviation: ROI = region of interest. Please click here to view a larger version of this figure.
Analysis methods
This section illustrates successful utilization through representative results. Here, we showcase the combined microPET/microCT image of a single mouse scanned at 15 and 18 months of age, after subjected to a Western diet (21% fat, 0.2% cholesterol) from 12 through 14 months of age. Following protocol sections 2-8 for calcification quantification, four independent researchers separately measured volumetric calcium content and 18F-NaF PET activity using the same representative microPET/microCT images obtained at the 15 and 18 month time points. Statistical analyses, including mean, standard deviation, intraclass correlation coefficient (ICC)28,29, were calculated to determine the inter-rater reliability and reproducibility of the protocol. ICC estimates and their 95% confidence intervals were calculated using IBM SPSS statistical package based on a mean-rating (k = 4), consistency, 2-way random-effects model (ICC(C,1)) (Supplemental File 1-Supplemental Table S1).
Results
The volumetric calcium content, which is derived from the volume and density of the calcified region, increased in one mouse between the 15 month (mean = 876.08 vHU, standard deviation = 27.18 vHU) and 18 month (mean = 1253.13 vHU, standard deviation = 7.61 vHU) image time points (Figure 8). In contrast, the 18F-NaF PET activity, which measures the surface area of the calcified region, decreased in the same mouse between the 15 month (mean = 24173.90 Bq/mm3, standard deviation = 1426.60 Bq/mm3) and 18 month (mean = 13849.94 Bq/mm3, standard deviation = 1524.67 Bq/mm3) image time points (Figure 8).
These results are consistent with previous findings that 18F-NaF PET and CT imaging provide what appears to be divergent information about atherosclerotic plaques12,13,14. A longitudinal study on calcification showed a similar trend, suggesting that the reduction in 18F-NaF signal may be due to coalescence of small calcium deposits, ultimately decreasing the surface area despite an increase in content12.
Figure 8: Representative microPET/CT images. Transverse (left), coronal (middle), and sagittal (right) views illustrating fluoride-18 (F-18) uptake in 15 month versus 18 month representative microPET images. White arrowheads indicate regions of calcification. Abbreviations: CT = computed tomography; PET = positron emission tomography. Please click here to view a larger version of this figure.
The small standard deviations observed in our results suggest a high degree of agreement and consistency between the measurements obtained by different researchers. Further, the ICC(C,1) value of 0.997 with 95% confidence interval = 0.983-1.000 obtained in our study (Supplemental File 1-Supplemental Table S1) indicates excellent agreement between the measurements made by different researchers. This level of agreement suggests that the measurements are reliable and consistent across researchers, indicating the high reproducibility of this novel quantification protocol.
This novel protocol is an improved approach to the quantification of cardiovascular calcification. Due to the non-invasive nature of imaging, longitudinal microCT images may be acquired to follow the progression of cardiovascular calcification in small animals. Although microCT images alone can show the progression of calcium content, microPET images, when available, can provide additional levels of information, especially its enhanced detection of small calcium deposits due to tracer binding to surface area. For further improved accuracy and reproducibility in this method, incorporating contrast-enhanced cardiac-gated CT imaging is recommended. Contrast-enhanced CT imaging streamlines the application of this protocol in larger animal and clinical studies, where lower-resolution contrast-enhanced CT with 18F-NaF PET imaging is the standard for quantification of cardiovascular calcification30,31. Furthermore, cardiac-gated PET and cardiac-gated CT reduce motion artifacts using electrocardiogram (ECG) data, resulting in clearer, higher-quality images of the heart32,33. Our image analysis method can be applied to cardiac-gated PET and cardiac-gated CT data in longitudinal studies.
DICOM viewers universally offer the functions essential to this protocol. Basic functions, including 2D and 3D image viewing, histograms, windowing controls, look-up tables, translocation, rotation, overlay, and navigation tools, are standard among most DICOM viewing software34,35 and allow for precise alignment of images and pinpointing of calcifications. More advanced functions such as segmentation are also common to most DICOM viewing software34,35,36 , enabling precise ROI delineation and accurate quantification of selected ROIs. Overall, this adaptable protocol ensures compatibility across DICOM viewing platforms, allowing researchers to use their preferred software without compromising methodology.
While this protocol is generally straightforward, successful utilization and reliable results hinge on several critical considerations. Prior to image reconstruction and analysis, it is imperative to include the decay-corrected injected dose and its unit of 18F-NaF PET tracer in the DICOM file to ensure the accuracy of quantification. Additionally, maintaining a consistent and stable positioning of the mice within the scanner bore greatly facilitates digital alignment of images — a process fundamental for maintaining analytical consistency across serial images in a longitudinal study.
Other critical steps are embedded within the protocol. The ROI should be selected carefully to exclude skeletal bone and other calcified structures in the upper chest (such as thyroid cartilage). Before initiating the drawing of an ROI, a pivotal step is careful selection of the time point featuring the largest visible calcification as the "reference" image (refer to protocol step 6.1). To further increase accuracy of the ROI, it is best to extend the sphere dimension beyond the visible calcification boundaries while excluding any adjacent bone, as exemplified in Supplemental Figure S7 (Supplemental File 1). These strategic choices ensure that the boundaries of the ROI comprehensively encompass the calcification regions in all subsequent scans. An equally crucial step is to select a threshold value of voxel density that will identify calcium mineral (refer to protocol step 7.4); this may vary due to the specifications of a particular study, such as imaging equipment and DICOM viewer software used. In the illustrative example, the minimum microCT density threshold for the ROI was defined as 300 HU based on our microPET/microCT experience.
Troubleshooting for this protocol mainly involves verifying that the correct images, objects, and ROIs are selected or appropriately toggled on/off at each step. Common errors in ROI quantification involve users mistakenly selecting an image instead of an ROI, or choosing a dataset under the statistical properties dropdown menu that does not align with the chosen ROI. Image visualization can present another challenge. For instance, without using a suitable color scale, the images may be overly dark or bright, obscuring calcium deposits. Fine-tuning visualization settings is crucial, as the optimal configuration may vary from those employed in this protocol. An additional protocol modification that may prove beneficial involves exploring various shapes during the ROI drawing step to best surround the calcified region. Depending on the study's requirements, alternative shapes, such as the cylinder or capsule, may prove more effective than the standard sphere used in the example protocol. For more detailed assistance in troubleshooting errors specific to DICOM viewing software, it is recommended to consult the software's technical support.
While this protocol simplifies the analysis of cardiovascular calcification in rodents, there are certain limitations. Despite being an excellent method for detecting cardiovascular calcifications, 18F-NaF PET imaging may face challenges in capturing the smallest calcium deposits due to resolution limitations and the partial volume effect, potentially leading to an underestimation of PET signal8,27. In CT images, the presence of noise may impact detection37. Although noise may be mitigated by setting a higher HU threshold, the tradeoff is the exclusion of the smallest low-density deposits. When calcium mineral deposits of the usual density are smaller than voxel dimensions, the partial volume effect causes them to appear to be of lower density and greater size than actual38,39. Consequently, there is a delicate balance in setting the minimum density threshold for a study, with a tradeoff between potential overestimation due to noise and potential underestimation due to exclusion of small deposits.
Many established methods have quantified calcification through CT imaging11,12,20,40,41,42. This typically includes identifying the CAC score in research subjects and patients, following the Agatston score protocol43. However, serious limitations of the Agatston score make it highly unreliable, due to post-acquisition truncation, reassignment, and thresholding of the data39. In addition, the Agatston score protocol can only identify regions above the scanner's detection limit of 100-200 µm8. Recognizing the growing importance of calcification as a risk factor for cardiovascular disease, it is imperative to develop methods that accurately quantify calcification. Advancements in 18F-NaF PET/CT imaging have improved the visualization of cardiovascular calcification; however, challenges persist in developing accurate data analysis methods for longitudinal studies. Current methods for quantification of calcification involve manually drawing ROIs around each visible calcified area in every subject at every individual time point in the longitudinal study 11,12,15. These current methods may hinder accuracy, particularly when deposits are near the scanner's detection limit. Without alignment to the time point showing the largest calcification, defining ROI in images with smaller calcifications becomes challenging. In our novel protocol, we present a refined data analysis approach integrating image alignment and ROI consistency to increase the consistency of data analysis in sequential images, thereby improving the validity of calcification assessment in longitudinal PET/CT studies. This method not only addresses challenges in data analysis, but it may also provide insights into the process of cardiovascular calcification progression.
The authors have no conflicts of interest to declare.
We thank all members of the UCLA Crump Preclinical Imaging Technology Center for their help with data acquisition and procession as well as equipment and infrastructure maintenance. We thank Jeffrey Collins for his help with cyclotron operation and 18F-NaF synthesis. We thank the UCLA Statistical Consulting Group for their help with statistical analysis. This work is supported by the NIH Cancer Center Support Grant (2 P30 CA016042-44 to MT) and National Institutes of Health, Heart, Lung, and Blood Institute (HL137647 and HL151391 to YT and LLD). The GNEXT PET/CT scanner was funded by NIH S10 Shared Instrumentation for Animal Research Grant (1S10OD026917-01A1 to Arion Chatziioannou).
Name | Company | Catalog Number | Comments |
0.5 cc Sterile Insulin Syringes | Exel International | #26028 | Used for IV injection of 18F-NaF PET Tracer |
18F-NaF PET Tracer | CNSI Cyclotron | ||
Biorender | Biorender | Used for figure 1 | |
Female Apoe-/- mouse | Jackson Laboratories | #002052 | B6.129P2-Apoetm1Unc/J |
GNEXT PET/CT | Sofie Biosciences, Dulles, Virginia | ||
Isoflurane | Piramal Critical Care | Used as anesthesia for mouse imaging | |
ORS Dragonfly | Comet Technologies Canada Inc. | ||
SPSS Statistics | IBM | ||
Western diet for mice | Envigo | #TD88139 | 21% fat, 0.2% cholesterol |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved