We would like to thank Vrachatis et al . for their detailed comments on our original article, “Detection of Carotid Atherosclerotic Plaque Neovascularization Using Contrast Enhanced Ultrasound: A Systematic Review and Meta-Analysis of Diagnostic Accuracy Studies,” focusing on the detection of carotid atherosclerotic intraplaque neovascularization (IPN). The aim of our review was to evaluate the diagnostic accuracy (pooled sensitivity and specificity) of both visual and quantitative analysis of IPN compared with histologic and/or clinical diagnosis of symptomatic plaque, as this had not been evaluated in a meta-analysis of published studies. We highlighted that the ultrasonic detection of microbubbles within carotid plaques, and surrounding tissues, is feasible, with pooled sensitivity and specificity for visual analysis of 80% (95% CI, 72%–87%) and 83% (95% CI, 76%–89%), respectively, while for quantitative analysis, sensitivity and specificity were 77% (95% CI, 71%–83%) and 68 (95% CI, 62%–73%), respectively. Our meta-analysis further supports the role of contrast-enhanced ultrasound (CEUS )in correctly identifying IPN, which is a surrogate for the underlying inflammatory process that could potentially aid in the risk stratification of patients with carotid stenosis.

Vrachatis et al . accurately point out that our report also outlined the challenges of CEUS in evaluating the dynamicity of IPN, some of which include the multiple heterogeneous factors that affect the visualization of microbubbles within carotid plaques, homeostatic variability of vascular regulation, plaque topography, the technical limitations of delivering a uniform ultrasonic field, and the need for development and standardization of technology to optimize acquisition and interpretive analysis of IPN. More specifically, it might be challenging for CEUS to differentiate between contrast microbubbles located in the neovessels (as in IPN) and those freely moving within the plaque after acute neovessel rupture (intraplaque hemorrhage). Intraplaque hemorrhage contributes to plaque growth and instability and may be suspected on an image when there is an intense prolonged enhancement of the plaque.

The precise pathophysiology of IPN and plaque instability is complex; however, it has been shown in prior studies that the prevalence of unstable plaques has been shown to be higher in symptomatic (i.e., those with cerebral ischemic events) than asymptomatic patients. Hence, in the design of our meta-analysis, we opted to use “symptom” status as a surrogate for plaque instability, including IPN (which is a prerequisite for maintaining inflammatory cell trafficking within the plaque). In our meta-analysis, we were further limited by the information that was available within the included reports.

Vrachatis et al . refer to a study performed by their own group assessing excised plaques histologically and immunohistochemically (measuring the vascular marker CD34 as a surrogate for microvessel density within the plaque) and correlating those findings with plaque enhancement on CEUS. They reported a significant correlation only for stable plaques. When comparing the degree of IPN enhancement on CEUS with the degree of neovascularization seen on histologic characterization, we concur with the authors that confounding factors (as detailed above) may contribute to interpretive variability. However, we also want to draw attention to the specificity of the vascular markers (CD31, CD34, and CD105) used to identify the presence and degree of IPN. A study by Feinstein’s group demonstrated a good correlation of plaque enhancement on CEUS with the vascular marker CD31, while a weaker correlation with CD34 was noted. This highlights the issue of histologic sampling variability and bias (i.e., specific volume and spatial orientation) when comparisons are made among CEUS imaging studies, which can influence the degree of statistical correlation between the neovascularization observed on the ultrasound images and correlative extent of neovascularization within the tissue specimens as detected by vascular markers. Hence, we agree with the authors that there is much work to do in this field. Certainly, detailed understanding of the precise pathophysiology that creates a vulnerable carotid plaque, as well as the optimal means for its detection and characterization, continue to evolve through multimodality imaging research; advanced imaging techniques using three-dimensional vascular ultrasound imaging may potentially help reduce such sampling bias. Additionally, the sensitivity of conventional vascular endothelial markers used for assessing neovascularization in atherosclerotic plaques may be improved upon by novel biomarkers; CD105 (endoglin), which is universally expressed in microvessels within the atheroma, may be a more appropriate biomarker than CD31.

We further acknowledge the importance of improving the meta-analysis search methodology criteria using different terms, such as “neoangiogenesis,” “plaque neovessels,” and “vasa vasorum,” as emphasized in the authors’ comments. In the interest of focusing our study on the most relevant terminology, we chose the search terms as stated, but we agree that this may have limited our catchment of additional articles. The relevant literature in this field is quite nascent, and the results of our meta-analysis and review can be considered a fair benchmark of the contemporary status of CEUS assessment of carotid plaques. We would agree that accurate and precise identification and quantification of IPN by CEUS remains at present “an unsolved mystery,” which is an active area of ongoing research with the aim of establishing uniform methodology and standardized evaluation of carotid atherosclerotic plaques using the relatively inexpensive, widely available, nontoxic, nonionizing, and readily applied technique of CEUS.