Surgical removal of atherosclerotic plaques from the carotid artery highly reduces the risk of future stroke in symptomatic patients with ≥ 70% stenosis.1 However, most of these patients will not have a new event when treated with best medical therapy.2 Furthermore, the role of surgery in moderate symptomatic stenosis (50% to 69%) and asymptomatic stenosis is under debate.3‐5 Therefore, taking into account the potential risk for surgical complications, the selection of patients who will benefit most from surgery is challenging.

In order to improve risk stratification, research has been focused on the identification of plaques at risk for rupture, so-called vulnerable plaques.6‐8 Currently, plaque thickness and intraplaque processes, such as inflammation and microcalcification, are seen as important contributors to vulnerability. These processes have become targets of various molecular imaging techniques, as they potentially allow non-invasive risk stratification of individual patients with carotid artery stenosis.9,10

Recently, several studies have shown the feasibility of ¹⁸F-sodium fluoride (¹⁸F-NaF) positron emission tomography (PET) for imaging of atherosclerotic plaques.11‐13¹⁸F-NaF predominantly binds to areas of microcalcification within the plaque.14 Appearance of microcalcifications indicates the active formation of calcification and is associated with plaque vulnerability.15,16 In contrast, established calcifications are seen as atherosclerotic end stage products and are associated with plaque stability.17‐20

It has been suggested that ¹⁸F-NaF may additionally be a useful marker for plaque vulnerability.21 Indeed, a clinical study by Joshi et al. showed that ruptured and high-risk coronary plaques have a significantly higher ¹⁸F-NaF uptake than non-culprit and low-risk coronary plaques.22 However, data on ¹⁸F-NaF uptake in carotid plaques are limited and their usefulness for the prediction of future stroke is unclear.23‐25 Additionally, limited data have been published on the relation between active microcalcifications and established calcifications in human carotid plaques.26,27

The primary objective of this study is to compare in vitro microPET assessed ¹⁸F-NaF uptake between culprit and non-culprit carotid plaques from stroke patients, using non-macrocalcified renal arteries from healthy kidney donors as negative controls. The secondary objective is to compare the distribution of ¹⁸F-NaF uptake on microPET with calcification visualized on a high-resolution microcomputed tomography (microCT) in carotid plaques.

The average ¹⁸F-NaF uptake was similar in culprit and non-culprit carotid plaques (median 2.32 %Inc/g [IQR 1.98 to 2.81] vs. median 2.35 %Inc/g [IQR 1.77 to 3.00], P = 0.916), while the uptake in carotid plaques was significantly higher than in renal arteries (median 2.32 %Inc/g [IQR 1.86 to 2.80] vs. median 0.44 %Inc/g [IQR 0.18 to 0.68], P < 0.001) (Figure 3).

The present study investigated in vitro microPET assessed ¹⁸F-NaF uptake, in culprit and non-culprit human carotid plaques. We hypothesized that ¹⁸F-NaF uptake in culprit plaques was higher than in non-culprit carotid plaques, based on previous results in coronary plaques, and the concept of microcalcification and plaque rupture.15,22 Our results, however, demonstrate comparable ¹⁸F-NaF uptake in culprit and non-culprit carotid plaques. Interestingly, we found that ¹⁸F-NaF uptake was present in regions without evidence of calcification on CT scan. Furthermore, most of the CT calcification VOIs had low ¹⁸F-NaF uptake, confirming that both techniques represent a different stage of calcification.14,23,32

Recently, Vesey et al. showed that in vivo ¹⁸F-NaF uptake was higher in culprit carotid artery stenosis than in the contralateral non-culprit stenosis in 18 patients with recent CVA (log₁₀ standardized uptake value, mean 0.29 ± 0.10 vs 0.23 ± 0.11 respectively, P < 0.001).23 These findings are consistent with the results of a clinical study of Quirce et al., in nine patients, where ¹⁸F-NaF uptake reported as mean target-to-background ratio was higher in culprit plaques (2.12 ± 0.44) than in contralateral non-culprit plaques (1.85 ± 0.46, P = 0.220).24 Age and sex distribution were comparable between these two studies and our study. Only Vesey et al. provided information about the cardiovascular history and other cardiovascular risk factors of the included patients. The prevalence of smoking and diabetes mellitus was similar. Furthermore, more than 50% of the patients had other manifestations of atherosclerosis, as in our study. Since the included patients in both studies were comparable, the remaining question is how these contradictive results can be explained.

First, although Vesey et al. did find a significant difference between ¹⁸F-NaF uptake in culprit and contralateral non-culprit plaques, the differences were small and a substantial overlap between the uptake values in both groups was present, as was in the study of Quirce et al. Furthermore, the ¹⁸F-NaF uptake between patients scheduled for CEA and control patients differed to a larger extent (delta 0.17 SUV_mean) than the difference between culprit and non-culprit uptake (delta 0.07 SUV_mean).23 Therefore, we believe that no absolute cut off value for the diagnosis of culprit plaques based on ¹⁸F-NaF uptake can be determined, only when compared with control patients there is a relevant difference. This is in line with the results of our study. Although the sample size is small, our data show that ¹⁸F-NaF uptake between culprit and non-culprit carotid plaques was comparable while the uptake in plaques was significantly higher than in control renal arteries.

Second, there may be differences in the degree of stenosis of the carotid arteries between patients in the aforementioned studies and our patients. Vesey et al. found that ¹⁸F-NaF uptake was related to the degree of stenosis on CT, but they did not report the stenosis degree in the separate groups, neither did Quirce et al. In our study, the stenosis degree in both, non-culprit and the culprit plaques, was high and all plaques showed high-risk features based on histology. This could explain the similar ¹⁸F-NaF uptake.

In contrast, Joshi et al. did find a difference between culprit and non-culprit coronary plaques of patients with myocardial infarction.22 This might be the consequence of local differences in the mechanical forces exerted to the artery wall and the endothelium of the coronary vascular bed, causing local differences in plaque initiation and progression.33 In contrast to the coronary arteries, both carotid arteries are exposed to similar blood flow patterns and mechanical stress. This might result in the same pattern of plaque development and progression.

¹⁸F-NaF activity was increased in areas without calcification on CT and most of the CT calcification VOI showed minimal ¹⁸F-NaF uptake. This supports the idea that microcalcification, as visualized with ¹⁸F-NaF PET, and established calcification visualized on CT may reflect different stages of the calcification processes in atherosclerotic plaques.23,27,34

Established calcification is a well-known marker of total plaque burden and is strongly associated with the risk for cardiovascular events.35 However, the amount of established calcification, as detected by CT, only provides information about the processes in the past and not about the actual biological activity of the plaque.36 Moreover, larger and denser areas of calcification may even stabilize the plaque.37 This has, for example, been suggested by Shalaan et al., who found a higher CT assessed calcification volume in non-culprit than in culprit carotid plaques.38 The average percentage of plaque volume that was calcified was comparable with our study. Unfortunately, in our study CT assessed calcification volume could not be compared between culprit and non-culprit plaques, due to limited availability of CT images in the non-culprit group (n = 1) because of image reconstruction failures.

Another important example can be derived from the work of Puri et al., who performed a post-hoc patient-level analysis of 8 prospective trials in which coronary atheroma was measured with intravascular ultrasound. They showed that although statins had a clear plaque-regressive effect, they also promoted coronary atheroma calcification, indicating stabilization.39

In this study increased ¹⁸F-NaF uptake was related to the calcium volume on CT in the majority of the carotid plaques. This is probably caused by binding of ¹⁸F-NaF at only the surface of the calcifications.14 The binding of fluoride to hydroxyapatite is based on ion exchange, rather than incorporation by active transport. This probably also explains why ¹⁸F-NaF uptake can still be found in vitro.

However, in our study a few plaques with low calcium volume had a high ¹⁸F-NaF uptake and vice versa. This suggests ¹⁸F-NaF accumulation in areas without any evidence of calcification, or at least no calcification with a size above the detection limit of the microCT scan.14 The presence of calcifications smaller than the CT detection limit, i.e. microcalcifications was indeed confirmed by histological staining of ¹⁸F-NaF positive and CT negative segements. ¹⁸F-NaF probably binds to the relatively large surface of microcalcifications , causing an intense signal on PET images.14

These observations indicate that ¹⁸F-NaF imaging can detect biologically active plaques, before they can be visualized on CT. This implies that ¹⁸F-NaF imaging may be useful in evaluating disease progression, as was further shown in patients with aortic stenosis, where baseline ¹⁸F-NaF uptake correlated well with the calcium progression after 1 year.40 Especially, ¹⁸F-NaF uptake in areas without established calcification on CT was the best predictor of calcium progression.

Furthermore, Derlin et al. found a positive correlation between ¹⁸F-NaF uptake in the carotid arteries and, age and various cardiovascular risk factors in 269 patients with no history of stroke.32 Derlin et al. included a heterogeneous population, consisting of patients with low or minimal cardiovascular risk as well. In contrast, we included only patients with already a history of cardiovascular disease and, therefore, at a high-risk for a cardiovascular event. These findings further highlight the possibility of ¹⁸F-NaF imaging to identify patients at high-risk for cardiovascular disease in a low-risk population. In addition, the finding that ¹⁸F-NaF uptake in carotid plaques exceeded that of controls (renal arteries) and no calcification was visible in renal arteries on microCT or with histological staining adds evidence to the hypothesis that the presence of microcalcification identified by ¹⁸F-NaF is a feature of atherosclerosis. The relation between the extent of ¹⁸F-NaF uptake in bilateral carotid plaques and features of vulnerability needs to be further investigated.

Strengths of our study are the inclusion of a control group, and the scanning of calcium phantoms in order to accurately determine the calcium threshold. Furthermore, by comparing calcification identified by ¹⁸F-NaF PET imaging with calcification visualized on microCT, this study adds knowledge to the relatively new field of ¹⁸F-NaF imaging in atherosclerosis. Our study has some limitations. First, it should be considered that the number of plaques, especially non-culprit plaques, is small and a type II statistical error might be introduced. However, given the similar distribution of ¹⁸F-NaF uptake in both groups, a high number of plaques would need to be recruited to find a statistical significant difference if present. We believe that small differences in uptake will not have any clinical implication.

Second, the tracer uptake could be underestimated due to partial volume effects, as with every imaging study. Third, CT images of 16 plaques (one non-culprit) out of 23 were available for analysis due to practical and technical issues. Fourth, we did not assess the plaque morphology of the right and left side in the same patient. Culprit and non-culprit plaques were derived from different patients.

We have demonstrated that the calcification patterns on ¹⁸F-NaF PET images and CT images are different. Clearly, ¹⁸F-NaF PET visualizes a different stage of the calcification process than CT. ¹⁸F-NaF uptake in carotid plaques exceeded the uptake in non-calcified renal arteries, but was comparable between culprit and non-culprit carotid plaques, probably due to the advanced nature of atherosclerotic disease in our patients.

We conclude that ¹⁸F-NaF has the potential to identify carotid plaques with active calcification. Further prospective studies on ¹⁸F-NaF uptake and symptomatology are required to assess the predictive and diagnostic value of ¹⁸F-NaF imaging in patients with early stage atherosclerosis.