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Abstract

Molecular imaging is a form of nanotechnology that enables the noninvasive examination of biological processes in vivo. Radiopharmaceutical agents are used to target biochemical markers, permitting their detection and evaluation. Early visualization of molecular variations indicative of pathophysiological processes can aid in patient diagnoses and management decisions. Molecular imaging is performed by introducing into the body molecular probes, which are often contrast agents that have been nanoengineered to target and tether to molecules, thus enabling their radiologic identification. Through a nanoengineering process, ultrasound contrast agents can be targeted to specific molecules, extending ultrasound’s capabilities from the tissue to molecular level. Molecular ultrasound, or targeted contrast-enhanced ultrasound (TCEUS), has recently emerged as a popular molecular imaging technique due to its ability to provide real-time anatomic and functional information without ionizing radiation. However, molecular ultrasound represents a novel form of molecular imaging and consequently remains largely preclinical. This review explores the commonalities of TCEUS across several molecular targets and points to the need for standardization of kinetic behavior analysis. The literature underscores evidence gaps and the need for additional research. The application of TCEUS is unlimited but needs further standardization to ensure that future research studies are comparable.

Keywords

molecular imaging inflammation targeted contrast-enhanced ultrasound selectins

Introduction

As the clinical translation of contrast-enhanced ultrasound (CEUS) is applied to the liver and other structural anatomic locations, it is important to look at the next experimental application of contrast microbubbles. To this end, a review of the literature was needed not only to understand the history of CEUS but also to capture the growing body of literature surrounding the ability to capture imaging of cellular-level processes. Diagnostic pressure to visualize disease at its earliest stage of development has pushed researchers to descend from the structural anatomy to the level of cellular pathways of disease.

The first application of CEUS imaging was reported in 1968 by Gramiak and Shah,¹ in which a saline mixture was discovered to improve visualization of the aortic root. Today, the use of ultrasound contrast agents is common practice during echocardiograms for left ventricular opacification and left ventricular endocardial border definition.² In addition, the American Society of Echocardiography practice guidelines recommend its use in a variety of other clinical scenarios to enhance echocardiographic image quality.^3,4 More recently in the United States, CEUS has been approved for its second indication, which is to further aid in the characterization of liver lesions.

However, ultrasound contrast agents are used extensively in Europe, Canada, and Asia as an enhanced method of detecting blood perfusion at the tissue level.^5–9 This is being done in a wide variety of tissue types and pathologies, as the 2011 European Federation of Societies for Ultrasound in Medicine and Biology Guidelines recommend its use in 18 different applications, spanning more than 75 indications.¹⁰ The most notable noncardiac applications for CEUS involve the evaluation of abdominal lesions, specifically those of the liver and kidney.^2,11 It has even been hypothesized that further research in this area will allow ultrasound to play a competitive role in abdominal imaging, relative to computed tomography (CT) and magnetic resonance imaging (MRI).¹² Supporting this idea, Ding et al.¹³ reported a sensitivity and specificity of 96% and 97%, respectively, in the detection of liver lesions. These findings were corroborated in a 2013 review of the literature, which reported the overall effectiveness of CEUS for the detection of liver lesions to maintain a sensitivity of 86% to 100% and a specificity of 80% to 100%.¹⁴

Its widespread use in these countries is supported by multiple years of in vitro and in vivo research, which have established the properties of ultrasound contrast agents.^15,16 Therefore, current research in this area focuses on developing innovative applications for CEUS imaging.² One area in which such innovative applications are being investigated is imaging of the musculoskeletal system. This line of inquiry is based on ultrasound contrast’s ability to enhance visualization of the macro- and microvasculature, which affords the potential of detecting hyperemia present in the early stages of many musculoskeletal pathologies.^17–19 Studies investigating musculoskeletal CEUS applications have demonstrated significant differences in microvasculature blood flow detection within a variety of joints in the digits, wrists, and knees of individuals with rheumatoid arthritis compared with normal controls.^20–22 This research has facilitated its clinical use in the evaluation of a variety of pathologies, beyond rheumatoid arthritis, such as bone erosions, synovitis, tendinopathies, enthesopathies, carpal tunnel syndrome, and osteoarthritis.^23–25

Another research focus in the area of ultrasound contrast explores its ability to provide biochemical and physiological information at the molecular level. Through a nanoengineering process, ultrasound contrast agents are able to be targeted to specific biological markers in vivo.^26,27 This is known as targeted contrast-enhanced ultrasound (TCEUS), or molecular ultrasound imaging, and its applications are currently being investigated in a variety of tissue types and pathologies.

Targeted Contrast-Enhanced Ultrasound Imaging

The first use of molecular ultrasound imaging, or TCEUS, was reported by Unger et al.²⁸ for the detection of thrombus formation. Since then, numerous studies have investigated TCEUS applications for the targeting and detection of a variety of biological markers that are thought to be present in the early stages of various disease processes.^29,30 Ultrasound contrast agents are restricted to the vascular compartment; therefore, they are ideal for investigating disease processes taking place on the endothelial surface.^9,31,32 Subsequently, disease processes such as angiogenesis, inflammation, and thrombus formation are the primary focus of TCEUS research.^33–35

One such application being investigated is molecular imaging of the angiogenic process, in an attempt to establish an early diagnostic tool for pathological processes such as tumor formation. Multiple preclinical animal studies have reported successful findings in this area by targeting various integrins, as these are present in the initial stages of angiogenesis.^36–44 Other preclinical animal studies investigating angiogenesis have also reported successful findings while targeting different vascular endothelial growth factor receptors for the application of early tumor detection.^40,42–53 The most commonly targeted angiogenic markers are alpha-v beta-3 integrin (α_vβ₃) and vascular endothelial growth factor receptor 2 (VEGFR-2), and they could have major implications in the future of cancer diagnosis and treatment.⁵⁴ Although most research has been directed at targeting angiogenic markers, the targeting of inflammatory markers is another application for which molecular ultrasound imaging is being investigated. This stems from the hypothesis that, due to the vast number of diseases in which inflammation presents, the future clinical advantages of this application could outweigh those of angiogenesis.⁵⁵

Proof of principle

The inflammatory process is mediated by the initial capturing and adhesion of rolling leukocytes by cell adhesion molecules (CAMs) on the endothelial surface.^29,56–60 Such CAMs include P-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). Each of these CAMs has been found to correlate with various stages of inflammation severity and are therefore felt to be early inflammatory biomarkers.^9,34,61–65 To this end, the determination of their presence could aid in an early diagnosis of inflammation.⁶⁶ For molecular ultrasound imaging to play a role in the early detection of inflammation, its ability to detect inflammatory biochemical markers must be investigated. This has led to numerous preclinical proof of principle studies that have explored microbubbles targeted to various inflammatory biochemical markers, in an assortment of tissues (Table 1).

Table 1.

In Vitro and In Vivo Molecular Ultrasound Imaging Proof of Principle Studies Investigating Inflammation While Targeting P-Selectin, VCAM-1, and ICAM-1.

Study and Year	In Vitro/In Vivo	Model	Tissue Type	Molecular Target	Contrast Agent	Conjugation Technique	Burst (Y/N)	Reference No.
Villanueva et al., 1998	In vitro	NA	Human coronary artery endothelium	ICAM-1	Lipid MB	Carboxyl group	NA	69
Weller et al., 2002	In vitro	NA	Human coronary artery endothelium	ICAM-1	Lipid MB	Avidin-biotin	NA	67
Weller et al., 2003	In vitro/in vivo	Rat	Cardiac tissue rejection	ICAM-1	Lipid MB	Avidin-biotin	Yes	68
Demos et al., 1997	In vitro	NA	Miniswine atherosclerosis	ICAM-1, fibrinogen	Liposome	NA	NA	70
Barreiro et al., 2009	Ex vivo	NA	Human umbilical vein	ICAM-1	Lipid MB	Avidin-biotin	NA	71
Hamilton et al., 2004	In vivo	Swine	Femoral and carotid artery atherosclerosis	ICAM-1, VCAM-1, fibrin, fibrinogen	Liposome	Avidin-biotin	Yes	72
Davis et al., 2003	In vitro	NA	Atherosclerosis	VCAM-1	Lipid MB	NA	NA	73
Tlaxca et al., 2013	In vivo	Murine	Mesentery	VCAM-1, MAdCAM-1	Lipid MB	Avidin-biotin	Yes	74
Kaufmann et al., 2007	In vivo	Murine	Thoracic aortic ischemia	VCAM-1	Lipid MB	Avidin-biotin	Yes	75
Hernot et al., 2012	In vitro/in vivo	Murine	Tumor	VCAM-1, eGFP	Lipid MB	Avidin-biotin	Yes	39
Khanicheh et al., 2013	In vivo	Murine	Aortic atherosclerosis	VCAM-1	Lipid MB	Avidin-biotin	Yes	76
Ferrante et al., 2009	In vitro	NA	Inflammation	VCAM-1, P-selectin	Lipid MB	Avidin-biotin	NA	77
Chadderdon et al., 2014	In vivo	Macaque	Carotid artery atherosclerosis	VCAM-1, P-selectin	Lipid MB	Avidin-biotin	Yes	78
Hoyt et al., 2015	In vivo	Rat	Renal	VCAM-1, P-selectin	Lipid MB	Avidin-biotin	Yes	79
Lindner et al., 2001	In vivo	Murine	Renal	P-selectin	Lipid MB	Avidin-biotin	Yes	80
Takalkar et al., 2004	In vitro	NA	Rat vasculature	P-selectin	Lipid MB	Avidin-biotin	NA	81
Rychak et al., 2006	In vitro/in vivo	Murine	Cremaster muscle	P-selectin	Lipid MB	Avidin-biotin	NA	82
Klibanov et al., 2006	In vitro/in vivo	Murine	Femoral vein and artery	P-selectin	Lipid MB	Avidin-biotin	NA	83
Guenther et al., 2010	In vitro	NA	Mouse arterial vasculature	P-selectin	Lipid MB	Avidin-biotin	NA	84
Rychak et al., 2007	In vivo	Murine	Femoral vein and artery	P-selectin	Lipid MB	Avidin-biotin	Yes	85
Kaufmann et al., 2007	In vivo	Murine	Myocardial ischemia	P-selectin	Lipid MB	Avidin-biotin	Yes	87
Villanueva et al., 2007	In vivo	Rat	Coronary artery ischemia	P-selectin	Lipid MB	Avidin-biotin	NA	88
Warram et al., 2011	In vitro/in vivo	Murine	Mouse breast tumor	P-selectin, VEGFR-2, α_vβ₃	Lipid MB	Avidin-biotin	No	44
Davidson et al., 2012	In vivo	Murine	Myocardial ischemia	P-, E-selectin	Lipid MB	Avidin-biotin	Yes	89
Bettinger et al., 2012	In vivo	Rat	Hind limb inflammation	P-, E-selectin	Lipid MB	Avidin-biotin	Yes	86
Hyvelin et al., 2014	In vivo	Rat	Myocardial ischemia	P-, E-selectin	Lipid MB	Lipid-thiolate	Yes	91
Davidson et al., 2014	In vivo	Macaque	Myocardial ischemia	P-, E-selectin	Lipid MB	Thioether	Yes	92
Wang et al., 2015	In vivo	Swine	Bowel (acute terminal ileitis)	P-, E-selectin	Lipid MB	Avidin-biotin	Yes	93

Abbreviations: α_vβ₃, alpha v beta 3 integrin; eGFP, enhanced green fluorescent protein; ICAM-1, intercellular adhesion molecule 1; MAdCAM-1, mucosal vascular addressin cell adhesion molecule 1; MB, microbubble; NA, not applicable; VCAM-1, vascular cell adhesion molecule 1; VEGFR-2, vascular endothelial growth factor receptor 2.

Weller et al.^67,68 investigated TCEUS targeted to ICAM-1 in cardiac tissue, for the detection of early stage cardiac transplant rejection, and reported a statistically significant increase in microbubble binding both in vitro and in vivo. In a similar study, signal intensities obtained in TCEUS imaging targeted to ICAM-1 were found to be significantly elevated in an inflamed coronary artery.⁶⁹ Successful detection of ICAM-1 targeted microbubbles has also been reported while investigating markers of atherosclerosis.⁷⁰ VCAM-1 targeted TCEUS imaging has also been investigated to determine its role in the early detection of inflammation. Promising results have been reported while investigating inflammation of the bowel, kidneys, vasculature, and soft tissue, as well as in tumor detection.^39,71–79 Last, particular success has been attained while targeting P-selectin with TCEUS during preclinical in vivo studies. In a study examining renal tissue inflammation, microvascular adhesion of ultrasound contrast microbubbles to P-selectin was found within an inflamed kidney, resulting in an enhanced sonographic signal.⁸⁰ Another application has been established by the successful targeting of P-selectin proteins in inflamed muscle tissue and vasculature.^81–86 Furthermore, Kaufmann et al.⁸⁷ demonstrated 10 times more binding of microbubbles targeted to P-selectin than controls while examining myocardial ischemia and reperfusion. Similar findings were reported by Villanueva et al.,⁸⁸ also examining P-selectin targeted microbubbles in myocardial ischemia and reperfusion. This has been further supported by other comparable studies.^89
–
92 Last, the ability of TCEUS to detect molecular alterations consistent with inflammation has been reported in the bowel.⁹³

These studies have provided evidence of the ability of targeted ultrasound contrast to bind to P-selectin, ICAM-1, and VCAM-1 for the early evaluation of inflammation. Successful visualization of these inflammatory biochemical markers has been accomplished in a variety of pathophysiological processes and tissues. However, the absence of the exploration of TCEUS imaging for the application of evaluating neural tissue inflammation following a traumatic spinal cord injury was notable, thus providing rationale for its investigation.

Longitudinal

In addition to serving as a diagnostic tool, ultrasound imaging has advantages that create an opportunity for targeted microbubbles to be used as a method of disease monitoring.^35,66,93,94 Due to its acoustic properties, molecular ultrasound poses no threat of adverse effects caused by overexposure to ionizing radiation, making its use as a surveillance tool ideal.^9,27,94,95 Repeated molecular ultrasound imaging studies provide information regarding disease progression, which can be used to gauge disease advancement and monitor therapy effectiveness.^66,94 This will provide clinicians with the unique opportunity of modifying patient management at an early stage, likely before clinical symptoms have manifested.^31,35,46

Proof of principle studies have provided evidence that molecular ultrasound imaging is capable of detecting biochemical markers in a variety of tissues, for the advancement of its use as a diagnostic tool. Disease monitoring via molecular ultrasound imaging would require a high level of sensitivity for the detection of biochemical markers at all levels of expression. Therefore, it is unfortunate that most proof of principle studies have been cross-sectional in design and provide evidence of molecular detection effectiveness at only a single stage of disease and severity. Furthermore, this is often performed during the acute phase of disease, in which biochemical markers are expressed at high levels. This provides no evidence of molecular ultrasound imaging’s capabilities for detecting late stages of disease and makes this information unsuitable for its translational use in disease surveillance. To appropriately provide evidence for this potential application, it is necessary that preclinical longitudinal studies be performed to evaluate TCEUS’s ability to detect targeted molecules at varying levels of expression. Longitudinal designed studies represent the next step in molecular ultrasound research, as information gained will advance the knowledge of its effectiveness and aid in proper bench-to-bedside translation.^43,45,96 It is unfortunate that the number of published longitudinal studies is minimal compared to that of proof of principle studies (Table 2).

Table 2.

In Vitro and In Vivo Studies Investigating the Utility of Molecular Ultrasound Imaging for Detecting Molecular Markers of Disease at Varying Levels of Expression.

Study and Year	In Vitro/In Vivo	Model	Pathophysiologic Process	Tissue Type	Molecular Target	Time Points/Levels of Severity	Reference No.
Longitudinal studies
Ellegala et al., 2003	In vivo	Murine	Angiogenesis	Malignant human gliomas xenograft	α_vβ₃ integrin	14, 28 days	41
Behm et al., 2008	In vivo	Murine	Vasculogenesis/arteriogenesis	Proximal adductor muscle tumor/iliac artery ischemia	VCAM-1, α_vβ₃ integrin	3, 6, 10 days/2, 4, 7 days	98
Pysz et al., 2010	In vivo	Murine	Angiogenesis	Human colon carcinoma tumor xenograft	VEGFR-2	0, 1, 2, 3, 6 days	45
Leguerney et al., 2015	In vivo	Murine	Angiogenesis	Melanoma tumor	Endoglin, integrin, VEGFR-2	0, 3 days	42
Deshpande et al., 2012	In vivo	Murine	Inflammation	Bowel tissue (IBD)	P-selectin	0, 1, 2, 3, 4, 5 days	99
Wang et al., 2013	In vivo	Murine	Inflammation	Bowel tissue (acute colitis)	P-selectin, E-selectin	0, 1, 5 days	100
Varying severity cross-sectional studies
Weller et al., 2005	In vitro	NA	Inflammation	Human coronary artery endothelial cells	ICAM-1, selectins	4 levels of inflammation (normal, low, medium, high)	101
Deshpande et al., 2011	In vivo	Murine	Angiogenesis	Breast, ovarian, pancreatic tumor xenografts	Endoglin, integrin, VEGFR-2	3 tumor sizes (small, medium, large)	43
Kaufmann et al., 2010	In vivo	Murine	Atherosclerosis	Aortic arch	P-selectin, VCAM-1	0, 10, 20, 40 weeks	102
Masseau et al., 2012	In vivo	Swine	Atherosclerosis	Carotid artery	VCAM-1	0, 16-20 weeks	103

Abbreviations: α_vβ₃, alpha v beta 3 integrin; IBD, irritable bowel disease; ICAM-1, intercellular adhesion molecule 1; NA, not applicable; VCAM-1, vascular cell adhesion molecule 1; VEGFR-2, vascular endothelial growth factor receptor 2.

The majority of these studies were performed in vivo and investigated the longitudinal monitoring of tumor growth by examining angiogenesis. This has been accomplished most frequently by targeting microbubbles to various integrins and/or VEGFR-2.^41,42,45,97 A few longitudinal TCEUS studies have been performed using antibodies targeted against P- and E-selectin for the evaluation of inflammation; however, these investigations are limited to tissues of the bowel.^98,99,100

In addition to these longitudinal studies, other cross-sectional studies do exist that incorporate subjects with different levels of disease. Although not longitudinal, these studies were conducted with the same goal of providing evidence of molecular ultrasound’s use in disease surveillance. Early research using this model was performed by Weller et al.¹⁰¹ in 2005, who demonstrated in vitro that the adhesion strength of targeted microbubbles to ICAM-1 had a strong linear relationship with inflammation severity (level of ICAM-1 expression). In vivo studies have investigated carcinoma by using tumors of different sizes to represent severity.⁴³ This was accomplished by targeting multiple angiogenic markers in various tumor types.⁴³ Other in vivo studies have explored molecular ultrasound’s use in the evaluation of atherosclerosis, often targeting CAMs such as P-selectin and VCAM-1.^102,103

These studies represent innovation in molecular ultrasound imaging research. They were conducted with the objective of extending knowledge beyond proof of principle information, by providing evidence regarding molecular ultrasound’s ability to detect biochemical markers at varying levels of expression. By doing so, they have taken the next step toward the clinical translation of TCEUS imaging.^43,45,96

The variety of tissue types in which targeted ultrasound contrast agents have been successfully detected during inflammation is indeed impressive; further research is needed to provide evidence on the appropriate dosing of contrast agents. Contrast dosing information must be obtained for ultrasound contrast imaging to be appropriately, effectively, and safely translated to humans.^92,96 The volume of contrast agent administered is directly related to obtained signal intensities and, in turn, the sensitivity of the TCEUS.^104,105 This further entails the need for established dosing parameters, so as to ensure accurate interpretation of the scan.^104,105 Dosing parameters also need to be considered for pediatric applications, so that toxicity risks are minimized, while still achieving a clinically appropriate scan.

Analytics of Kinetic Behavior

In addition to investigations of TCEUS applications, techniques of image analysis and interpretation should also be examined. One popular method of ascertaining information from contrast imaging is through the analysis of the contrast agent’s temporal behavior.¹⁰⁶ Temporal behavior refers to the activity of the contrast agent over time. This idea was first introduced in the 1990s and is primarily used and investigated in MRI.¹⁰⁶ Enhancement kinetics are represented semiquantitatively in the form of time intensity curves (TICs).¹⁰⁷ Time intensity curves are generated by placing a region of interest over the area being examined. The signal intensity of the image, which has been enhanced via the contrast agent, is calculated and plotted over time to provide a visual depiction of the contrast agent’s temporal behavior.¹⁰⁷ Known by a variety of terms, TIC shape analysis can then be conducted, in which the curve representing signal intensity is classified based on its shape.¹⁰⁸

As mentioned previously, the use of TICs to obtain information regarding the perfusion of an area has largely been in the field of MRI.¹⁰⁶ Time intensity curves are primarily used in situations in which malignancy of a tumor is suspected, and knowledge of its perfusion characteristics is necessary.¹⁰⁷ Researchers who investigate TIC analysis applications attempt to define distinctive TIC patterns and assign them to physiologic or pathologic findings.^106,109 Classification of TICs focuses more on the shape of the curve and relies less on their quantitative absolute values.¹⁰⁷ This idea has been referred to as TIC shape analysis and will be referenced as such for the remainder of this article.¹⁰⁷ Yankeelov and Gore¹¹⁰ have coined this idea “curve-ology,” whereas Kuhl et al.¹⁰⁷ referred to this method of TIC analysis as “enhancement kinetics.” Kuhl et al.¹¹¹ continued to describe three classifications of TIC shape analysis, based on the shape of the initial signal intensity wash-in phase and the wash-out phase components of the TIC. A type 1 curve shape depicts a continuous enhancement of signal intensity that increases over time.¹¹¹ A type 2 curve is characterized by a wash-in phase that reaches a peak and then maintains a plateau during the wash-out phase after approximately 2 to 3 minutes of imaging.¹¹¹ And finally, Kuhl et al.¹¹¹ defined a type 3 curve as a wash-out curve, where a decrease in signal intensity is seen approximately 2 to 3 minutes after peak signal intensity is achieved in the wash-out phase. These TIC shape analysis classifications were determined through evidence obtained by the same researchers, which showed that 57% of malignant breast lesions produced a type 2 curve, whereas 83% of benign lesions produced either a type 1 or type 3 curve.¹¹¹

In a more recent study, van Rijswijk et al.¹⁰⁹ investigated the use of TIC shape analysis in the evaluation of soft tissue sarcomas. In this study, researchers used contrast-enhanced MRI to obtain TICs representing soft tissue sarcoma perfusion. These TICs were then subjectively placed into five different categories based on their shape.¹⁰⁹ A type 1 curve was categorized by the absence of a slope in the wash-in phase and therefore the absence of enhancement.¹⁰⁹ A type 2 curve depicts a gradual and continuous increase in signal intensity for approximately 5 minutes, with no steep slope in the wash-in phase.¹⁰⁹ Type 3 was defined as a curve containing a rapid slope in the wash-in phase, followed by a plateau in the wash-out phase.¹⁰⁹ Similar to type 3, type 4 was characterized by a rapid slope in the wash-in phase; however, a decrease in signal intensity is seen in the wash-out phase.¹⁰⁹ Finally, van Rijswijk et al.¹⁰⁹ defined a type 5 TIC as a curve with a rapid signal intensity slope in the wash-in phase, followed by a sustained signal intensity increase in the wash-out phase. Throughout this study, curve types 3 through 5 were seen, as the investigators concluded that the only curve feature that was consistently associated with malignancy was early enhancement.¹⁰⁹

Recently, the utility of TIC shape analysis has been investigated in CEUS imaging. As briefly described previously, ultrasound contrast agents comprise microbubbles consisting of a gaseous core, stabilized by an outer shell made of lipid, protein, or polymer.^{9,15,104,112–115} Once introduced into circulation, ultrasound contrast microbubbles interact with the incident ultrasonic beam, which causes them to oscillate and produce an enhanced sound wave that is returned to the transducer. This is due primarily to the large difference in acoustic impedance between the gas inside the contrast microbubble and the surrounding tissue.^{9,15,17,66,94,116–119} The returned enhanced sound wave produces an enhanced signal intensity, which can then be displayed in the form of a TIC, just as in MRI. Using this analysis technique in CEUS, multiple clinical conditions have been evaluated, including the monitoring of liver lesions.¹²⁰ In a review conducted by Cosgrove and Lassau,¹⁹ TICs were obtained from CEUS imaging studies that had been performed longitudinally on a metastatic breast tumor while the patient underwent antivascular therapy. It is interesting that TIC shape analysis demonstrated a decrease in size and a difference in shape of the curve as treatment continued.¹⁹ In this case, CEUS TIC shape analysis was found to be a more sensitive measure than CT.¹⁹ Furthermore, Lassau et al.¹²¹ reported very similar findings, in which changes in CEUS TIC shape analysis representing liver perfusion were observed longitudinally in an individual receiving treatment for hepatocellular carcinoma. Last, it is important to note that, just as demonstrated in the MRI literature, a wide spectrum of nomenclature has been used to describe CEUS TIC shape analysis. Terms such as perfusion kinetics, contrast uptake curves, and time variance imaging were all reported in studies that examined CEUS TICs.^121–123

Conclusion

Molecular imaging has the potential to detect protein activity at the cellular level. This pathophysiologic information can be leveraged to better recognize the formation of disease processes while still in the acute stages. Targeted contrast-enhanced ultrasound makes use of nanoengineering to create microbubbles that can be targeted and tethered to molecules at the site of diagnostic inquiry. Once tethered, the microbubbles are insonated, which provides a resonance frequency that increases the signal-to-noise ratio through the amplification of reflections toward the transducer. Raw signal data have the potential to predict the behavior of cellular processes below structural anatomy.

Analysis of the temporal behavior of the TCEUS signal allows for both quantitative and qualitative measures. This technology is still in its infancy and therefore is in need of standardization to ensure that the microbubbles’ kinetic behavior can be reproducibly analyzed.

Since a lack of standardization exists for analyzing microbubble kinetic behavior, MRI TICs were investigated to determine if these techniques translate. An actual comparison of the analytic techniques would be required to make an informed determination on which are the most sensitive for describing TCEUS kinetics.

Footnotes

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

References

Gramiak

Shah

: Echocardiography of the aortic root. Invest Radiol 1968;3(5):356–366.

Appis

Tracy

Feinstein

: Update on the safety and efficacy of commercial ultrasound contrast agents in cardiac applications. Echo Res Pract 2015;2(2):R55–R62.

Mulvagh

Rakowski

Vannan

: American Society of Echocardiography consensus statement on the clinical applications of ultrasonic contrast agents in echocardiography. J Am Soc Echocardiogr 2008;21(11):1179–1201.

Porter

Abdelmoneim

Belcik

: Guidelines for the cardiac sonographer in the performance of contrast echocardiography: a focused update from the American Society of Echocardiography. J Am Soc Echocardiogr 2014;27(8):797–810.

Greenbaum

Burns

Copel

: American Institute of Ultrasound in Medicine recommendations for contrast-enhanced liver ultrasound imaging clinical trials. J Ultrasound Med 2007;26(6):705–716.

Klibanov

: Molecular imaging with targeted ultrasound contrast microbubbles, in Bogdanov

Licha

(eds): Molecular Imaging: An Essential Tool in Preclinical Research, Diagnostic Imaging, and Therapy. Vol. 49. New York, NY, Springer Science + Business Media, 2004, pp 171–191.

Kono

Holscher

Mattrey

: Use of ultrasound microbubbles for vascular imaging, in Bulte

JWM

Modo

MMJ

(eds): Nanoparticles in Biomedical Imaging. New York, NY, Springer, 2008, pp 311–326.

Wilson

Burns

: Microbubble-enhanced US in body imaging: what role? Radiology 2010;257(1):24–39.

Deshpande

Willman

: Microparticle- and nanoparticle-based contrast-enhanced ultrasound imaging, in Chen

(ed): Nanoplatform-Based Molecular Imaging. Hoboken, NJ, Wiley, 2011, pp 233–262.

10.

Piscaglia

Nolsoe

Dietrich

: The EFSUMB guidelines and recommendations on the clinical practice of contrast enhanced ultrasound (CEUS): update 2011 on non-hepatic applications. Ultraschall Med 2012;33(1):33–59.

11.

Unnikrishnan

Klibanov

: Microbubbles as ultrasound contrast agents for molecular imaging: preparation and application. AJR Am J Roentgenol 2012;199(2):292–299.

12.

Albrecht

Blomley

Burns

: Improved detection of hepatic metastases with pulse-inversion US during the liver-specific phase of SHU 508A: multicenter study 1. Radiology 2003;227(2):361–370.

13.

Ding

Wang

Huang

: Imaging of focal liver lesions: low-mechanical-index real-time ultrasonography with SonoVue. J Ultrasound Med 2005;24(3):285–297.

14.

Alzaraa

Gravante

Chung

: Contrast-enhanced ultrasound in the preoperative, intraoperative and postoperative assessment of liver lesions. Hepatology 2013;43(8):809–819.

15.

Voigt

: Ultrasound molecular imaging. Methods 2009;48(2):92–97.

16.

van Rooij

Daeichin

Skachkov

de Jong

Kooiman

: Targeted ultrasound contrast agents for ultrasound molecular imaging and therapy. Int J Hyperthermia 2015;31(2):90–106.

17.

Bloch

Dayton

Ferrara

: Targeted imaging using ultrasound contrast agents. IEEE Eng Med Biol Mag 2004;23(5):18–29.

18.

De Zordo

Mlekusch

Feuchtner

Mur

Schirmer

Klauser

: Value of contrast-enhanced ultrasound in rheumatoid arthritis. Eur J Radiol 2007;64(2):222–230.

19.

Cosgrove

Lassau

: Imaging of perfusion using ultrasound. Eur J Nucl Med Mol Imaging 2010;37(1):65–85.

20.

Doria

Kiss

MHB

Lotito

APN

: Juvenile rheumatoid arthritis of the knee: evaluation with contrast-enhanced color Doppler ultrasound. Pediatr Radiol 2001;31(7):524–531.

21.

Klauser

Frauscher

Schirmer

: The value of contrast-enhanced color Doppler ultrasound in the detection of vascularization of finger joints in patients with rheumatoid arthritis. Arthritis Rheum 2002;46(3):647–653.

22.

Carotti

Salaffi

Morbiducci

: Colour Doppler ultrasonography evaluation of vascularization in the wrist and finger joints in rheumatoid arthritis patients and healthy subjects. Eur J Radiol 2012;81(8):1834–1838.

23.

Backhaus

Burmester

Gerber

: Guidelines for musculoskeletal ultrasound in rheumatology. Ann Rheum Dis 2001;60(7):641–649.

24.

Ishizaka

Nishida

Motomiya

: Reliability of peripheral intraneural microhemodynamics evaluation by using contrast-enhanced ultrasonography. J Med Ultrason 2014;41(4):481–486.

25.

Song

Burmester

Backhaus

: Knee osteoarthritis. Efficacy of a new method of contrast-enhanced musculoskeletal ultrasonography in detection of synovitis in patients with knee osteoarthritis in comparison with magnetic resonance imaging. Ann Rheum Dis 2008;67(1):19–25.

26.

Cai

Chen

: Nanoplatforms for targeted molecular imaging in living subjects. Small 2007;3(11):1840–1854.

27.

Klibanov

: Targeted microbubbles: ultrasound contrast agents for molecular imaging, in Bulte

JWM

Modo

MMJ

(eds): Nanoparticles in Biomedical Imaging. New York, NY, Springer, 2008, pp 327–341.

28.

Unger

McCreery

Sweitzer

Shen

: In vitro studies of a new thrombus-specific ultrasound contrast agent. Am J Cardiol 1998;81(12):58G–61G.

29.

Lindner

: Assessment of inflammation with contrast ultrasound. Prog Cardiovasc Dis 2001;44(2):111–120.

30.

Weissleder

Mahmood

: Molecular imaging 1. Radiology 2001;219(2):316–333.

31.

Klibanov

: Microbubble contrast agents: targeted ultrasound imaging and ultrasound-assisted drug-delivery applications. Invest Radiol 2006;41(3):354–362.

32.

Kee

McPherson

: Use of acoustically active contrast agents in imaging of inflammation and atherosclerosis, in Bulte

JWM

Modo

MMJ

(eds): Nanoparticles in Biomedical Imaging: Emerging Technologies and Applications. New York, NY, Springer, 2008, pp 343–370.

33.

Kircher

Willmann

: Molecular body imaging: MR imaging, CT, and US. Part II. Applications. Radiology 2012;264(2):349–368.

34.

Kaufmann

Lindner

: Molecular imaging with targeted contrast ultrasound. Curr Opin Biotechnol 2007;18(1):11–16.

35.

Deshpande

Needles

Willmann

: Molecular ultrasound imaging: current status and future directions. Clin Radiol 2010;65(7):567–581.

36.

Leong-Poi

Christiansen

Klibanov

: Noninvasive assessment of angiogenesis by ultrasound and microbubbles targeted to αv-integrins. Circulation 2003;107(3):455–460.

37.

Willmann

Kimura

Deshpande

Lutz

Cochran

Gambhir

: Targeted contrast-enhanced ultrasound imaging of tumor angiogenesis with contrast microbubbles conjugated to integrin-binding knottin peptides. J Nucl Med 2010;51(3):433–440.

38.

Anderson

Tlaxca

: Ultrasound molecular imaging of tumor angiogenesis with an integrin targeted microbubble contrast agent. Invest Radiol 2011;46(4):215–224.

39.

Hernot

Unnikrishnan

: Nanobody-coupled microbubbles as novel molecular tracer. J Control Release 2012;158(2):346–353.

40.

Palmowski

Huppert

Ladewig

: Molecular profiling of angiogenesis with targeted ultrasound imaging: early assessment of antiangiogenic therapy effects. Mol Cancer Ther 2008;7(1):101–109.

41.

Ellegala

Leong-Poi

Carpenter

: Imaging tumor angiogenesis with contrast ultrasound and microbubbles targeted to αvβ3. Circulation 2003;108(3):336–341.

42.

Leguerney

Scoazec

Gadot

: Molecular ultrasound imaging using contrast agents targeting endoglin, vascular endothelial growth factor receptor 2 and integrin. Ultrasound Med Biol 2015;41(1):197–207.

43.

Deshpande

Ren

Foygel

Rosenberg

Willmann

: Tumor angiogenic marker expression levels during tumor growth: longitudinal assessment with molecularly targeted microbubbles and US imaging. Radiology 2011;258(3):804–811.

44.

Warram

Sorace

Saini

Umphrey

Zinn

Hoyt

: A triple-targeted ultrasound contrast agent provides improved localization to tumor vasculature. J Ultrasound Med 2011;30(7):921–931.

45.

Pysz

Foygel

Rosenberg

Gambhir

Schneider

Willmann

: Antiangiogenic cancer therapy: monitoring with molecular US and a clinically translatable contrast agent (BR55) 1. Radiology 2010;256(2):519–527.

46.

Rychak

Graba

Cheung

: Microultrasound molecular imaging of vascular endothelial growth factor receptor 2 in a mouse model of tumor angiogenesis. Mol Imaging 2006;6(5):289–296.

47.

Anderson

Rychak

Backer

Klaus

Klibanov

: scVEGF microbubble ultrasound contrast agents: a novel probe for ultrasound molecular imaging of tumor angiogenesis. Invest Radiol 2010;45(10):579–585.

48.

Fischer

Thomas

Tardy

: Vascular endothelial growth factor receptor 2-specific microbubbles for molecular ultrasound detection of prostate cancer in a rat model. Invest Radiol 2010;45(10):675–684.

49.

Pochon

Tardy

Bussat

: BR55: a lipopeptide-based VEGFR2-targeted ultrasound contrast agent for molecular imaging of angiogenesis. Invest Radiol 2010;45(2):89–95.

50.

Tardy

Pochon

Theraulaz

: Ultrasound molecular imaging of VEGFR2 in a rat prostate tumor model using BR55. Invest Radiol 2010;45(10):573–578.

51.

Bzyl

Lederle

Rix

: Molecular and functional ultrasound imaging in differently aggressive breast cancer xenografts using two novel ultrasound contrast agents (BR55 and BR38). Eur Radiol 2011;21(9):1988–1995.

52.

Knowles

Heath

Saini

: Molecular targeting of ultrasonographic contrast agent for detection of head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg 2012;138(7):662–668.

53.

Wei

Sun

: Targeted contrast-enhanced ultrasound imaging of angiogenesis in an orthotopic mouse tumor model of renal carcinoma. Ultrasound Med Biol 2014;40(6):1250–1259.

54.

Kiessling

Fokong

Koczera

Lederle

Lammers

: Ultrasound microbubbles for molecular diagnosis, therapy, and theranostics. J Nucl Med 2012;53(3):345–348.

55.

Kiessling

: Science to practice: molecularly targeted US of inflammation—important steps toward clinical translation. Radiology 2015;276(3):621–623.

56.

Butcher

: Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 1991;67(6):1033–1036.

57.

Ley

: The role of selectins in inflammation and disease. Trends Mol Med 2003;9(6):263–268.

58.

Ley

Laudanna

Cybulsky

Nourshargh

: Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol 2007;7(9):678–689.

59.

Man

Ubogu

Ransohoff

: Inflammatory cell migration into the central nervous system: a few new twists on an old tale. Brain Pathol 2007;17(2):243–250.

60.

Kolaczkowska

Kubes

: Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol 2013;13(3):159–175.

61.

Kiessling

Huppert

Palmowski

: Functional and molecular ultrasound imaging: concepts and contrast agents. Curr Med Chem 2009;16(5):627–642.

62.

Kiessling

: Science to practice: exploring new indications for molecular US imaging. Radiology 2013;267(3):661–662.

63.

Nakashima

Raines

Plump

Breslow

Ross

: Upregulation of VCAM-1 and ICAM-1 at atherosclerosis-prone sites on the endothelium in the ApoE-deficient mouse. Arterioscler Thromb Vasc Biol 1998;18(5):842–851.

64.

Gutiérrez-López

Ovalle

Yanez-Mo

: A functionally relevant conformational epitope on the CD9 tetraspanin depends on the association with activated β1 integrin. J Biol Chem 2003;278(1):208–218.

65.

King

: Adhesion receptors as therapeutic targets for circulating tumor cells. Front Oncol 2012;2:79.

66.

Christiansen

Lindner

: Molecular and cellular imaging with targeted contrast ultrasound. Proceedings of the IEEE 2005;93(4):809–818.

67.

Weller

GER

Villanueva

Klibanov

Wagner

: Modulating targeted adhesion of an ultrasound contrast agent to dysfunctional endothelium. Ann Biomed Eng 2002;30(8):1012–1019.

68.

Weller

Csikari

: Ultrasound imaging of acute cardiac transplant rejection with microbubbles targeted to intercellular adhesion molecule-1. Circulation 2003;108(2):218–224.

69.

Villanueva

Jankowski

Klibanov

: Microbubbles targeted to intercellular adhesion molecule-1 bind to activated coronary artery endothelial cells. Circulation 1998;98(1):1–5.

70.

Demos

Onyusel

Gilbert

: In vitro targeting of antibody-conjugated echogenic liposomes for site-specific ultrasonic image enhancement. J Pharm Sci 1997;86(2):167–171.

71.

Barreiro

Aguilar

Tejera

: Specific targeting of human inflamed endothelium and in situ vascular tissue transfection by the use of ultrasound contrast agents. JACC Cardiovasc Imaging 2009;2(8):997–1005.

72.

Hamilton

Huang

Warnick

: Intravascular ultrasound molecular imaging of atheroma components in vivo. J Am Coll Cardiol 2004;43(3):453–460.

73.

Davis

Christiansen

Klibanov

: Microbubbles targeted to endothelial VCAM-1 adhere to atherosclerotic plaques at physiologic shear rates. Circulation 2003;108(17):624.

74.

Tlaxca

Rychak

Ernst

: Ultrasound-based molecular imaging and specific gene delivery to mesenteric vasculature by endothelial adhesion molecule targeted microbubbles in a mouse model of Crohn’s disease. J Control Release 2013;165(3):216–225.

75.

Kaufmann

Sanders

Davis

: Molecular imaging of inflammation in atherosclerosis with targeted ultrasound detection of vascular cell adhesion molecule-1. Circulation 2007;116(3):276–284.

76.

Khanicheh

Xie

: Molecular imaging reveals rapid reduction of endothelial activation in early atherosclerosis with apocynin independent of antioxidative properties. Arterioscler Thromb Vasc Biol 2013;33(9):2187–2192.

77.

Ferrante

Pickard

Rychak

Klibanov

Ley

: Dual targeting improves microbubble contrast agent adhesion to VCAM-1 and P-selectin under flow. J Control Release 2009;140(2):100–107.

78.

Chadderdon

Belcik

Bader

: Pro-inflammatory endothelial activation detected by molecular imaging in obese non-human primates coincides with the onset of insulin resistance and progressively increases with duration of insulin resistance. Circulation 2014;129(4):471–478.

79.

Hoyt

Warram

Wang

Ratnayaka

Traylor

Agarwal

: Molecular ultrasound imaging of tissue inflammation using an animal model of acute kidney injury. Mol Imaging Biol 2015;17(6):786–792.

80.

Lindner

Song

Christiansen

Klibanov

Ley

: Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin. Circulation 2001;104(17):2107–2112.

81.

Takalkar

Klibanov

Rychak

Lindner

Ley

: Binding and detachment dynamics of microbubbles targeted to P-selectin under controlled shear flow. J Control Release 2004;96(3):473–482.

82.

Rychak

Lindner

Ley

: Deformable gas-filled microbubbles targeted to P-selectin. J Control Release 2006;114(3):288–299.

83.

Klibanov

Rychak

Yang

: Targeted ultrasound contrast agent for molecular imaging of inflammation in high-shear flow. Contrast Media Mol Imaging 2006;1(6):259–266.

84.

Guenther

von zur Muhlen

Ferrante

Grundmann

Bode

Klibanov

: An ultrasound contrast agent targeted to P-selectin detects activated platelets at supra-arterial shear flow conditions. Invest Radiol 2010;45(10):586–591.

85.

Rychak

Klibanov

Ley

Hossack

: Enhanced targeting of ultrasound contrast agents using acoustic radiation force. Ultrasound Med Biol 2007;33(7):1132–1139.

86.

Bettinger

Bussat

Tardy

: Ultrasound molecular imaging contrast agent binding to both E- and P-selectin in different species. Invest Radiol 2012;47(9):516–523.

87.

Kaufmann

Lewis

Xie

Mirza-Mohd

Lindner

: Detection of recent myocardial ischaemia by molecular imaging of P-selectin with targeted contrast echocardiography. Eur Heart J 2007;28(16):2011–2017.

88.

Villanueva

Bowry

: Myocardial ischemic memory imaging with molecular echocardiography. Circulation 2007;115(3):345–352.

89.

Davidson

Kaufmann

Belcik

Xie

Lindner

: Detection of antecedent myocardial ischemia with multiselectin molecular imaging. J Am Coll Cardiol 2012;60(17):1690–1697.

90.

Bettinger

Bussat

Tardy

: Ultrasound molecular imaging contrast agent binding to both E- and P-selectin in different species. Invest Radiol 2012;47(9):516–523.

91.

Hyvelin

Tardy

Bettinger

: Ultrasound molecular imaging of transient acute myocardial ischemia with a clinically translatable P- and E-selectin targeted contrast agent: correlation with the expression of selectins. Invest Radiol 2014;49(4):224–235.

92.

Davidson

Chadderdon

Belcik

Gupta

Lindner

: Ischemic memory imaging in nonhuman primates with echocardiographic molecular imaging of selectin expression. J Am Soc Echocardiogr 2014;27(7):786–793.

93.

Wang

Machtaler

Lutz

: Quantitative assessment of inflammation in a porcine acute terminal ileitis model: US with a molecularly targeted contrast agent. Radiology 2015;276(3):809–817.

94.

Frangioni

Hajjar

: In vivo tracking of stem cells for clinical trials in cardiovascular disease. Circulation 2004;110(21):3378–3383.

95.

Hausner

: Basic principles of molecular imaging. Nanoplatform-based molecular imaging, in Chen

(ed): Nanoplatform-Based Molecular Imaging. Hoboken, NJ, Wiley, 2011, pp 3–24.

96.

Kircher

Willmann

: Molecular body imaging: MR imaging, CT, and US. Part I. Principles. Radiology 2012;263(3):633–643.

97.

Borden

Qin

Ferrara

: Ultrasound contrast agents, in Weissleder

(ed): Molecular Imaging: Principles and Practice. Shelton, CT, People’s Medical Publishing House–USA, 2010, pp 425–444.

98.

Behm

Kaufmann

Carr

: Molecular imaging of endothelial vascular cell adhesion molecule-1 expression and inflammatory cell recruitment during vasculogenesis and ischemia-mediated arteriogenesis. Circulation 2008;117(22):2902–2911.

99.

Deshpande

Lutz

Ren

: Quantification and monitoring of inflammation in murine inflammatory bowel disease with targeted contrast-enhanced US. Radiology 2012;262(1):172–180.

100.

Wang

Machtaler

Bettinger

: Molecular imaging of inflammation in inflammatory bowel disease with a clinically translatable dual-selectin–targeted US contrast agent: comparison with FDG PET/CT in a mouse model. Radiology 2013;267(3):818–829.

101.

Weller

GER

Villanueva

Tom

Wagner

: Targeted ultrasound contrast agents: in vitro assessment of endothelial dysfunction and multi-targeting to ICAM-1 and sialyl Lewisx. Biotechnol Bioeng 2005;92(6):780–788.

102.

Kaufmann

Carr

Belcik

: Molecular imaging of the initial inflammatory response in atherosclerosis implications for early detection of disease. Arterioscler Thromb Vasc Biol 2010;30(1):54–59.

103.

Masseau

Davis

Bowles

: Carotid inflammation is unaltered by exercise in hypercholesterolemic swine. Med Sci Sports Exerc 2012;44(12):2277–2289.

104.

Hammoud

Hoffman

Pomper

: Molecular neuroimaging: from conventional to emerging techniques. Radiology 2007;245(1):21–42.

105.

James

Gambhir

: A molecular imaging primer: modalities, imaging agents, and applications. Physiol Rev 2012;92(2):897–965.

106.

Gordon

Partovi

Muller-Eschner

: Dynamic contrast-enhanced magnetic resonance imaging: fundamentals and application to the evaluation of the peripheral perfusion. Cardiovasc Diagn Ther 2014;4(2):147–164.

107.

Kuhl

: Concepts for differential diagnosis in breast MR imaging. Magn Reson Imaging Clin N Am 2006;14(3):305–328.

108.

Lavini

Buiter

Maas

: Use of dynamic contrast enhanced time intensity curve shape analysis in MRI: theory and practice. Reports Med Imaging 2013;6:71–82.

109.

van Rijswijk

CSP

Hogendoorn

PCW

Taminiau

AHM

Bloem

: Synovial sarcoma: dynamic contrast-enhanced MR imaging features. Skeletal Radiol 2001;30(1):25–30.

110.

Yankeelov

Gore

: Dynamic contrast enhanced magnetic resonance imaging in oncology: theory, data acquisition, analysis, and examples. Curr Med Imaging Rev 2009;3(2):91–107.

111.

Kuhl

Mielcareck

Klaschik

: Dynamic breast MR imaging: are signal intensity time course data useful for differential diagnosis of enhancing lesions? Radiology 1999;211(1):101–110.

112.

Kolishetti

Alexis

Pridgen

Farokhzad

: Biodistribution and pharmacokinetics of nanoprobes, in Chen

(ed): Nanoplatform-Based Molecular Imaging. Hoboken, NJ, Wiley, 2011, pp 75–106.

113.

Osborn

Jaffer

: Advances in molecular imaging of atherosclerotic vascular disease. Curr Opin Cardiol 2008;23(6):620–628.

114.

Massoud

Gambhir

: Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev 2003;17(5):545–580.

115.

Albrecht

Blomley

Bolondi

: Guidelines for the use of contrast agents in ultrasound. Ultraschall Med 2004;25(4):249–256.

116.

Klibanov

: Targeted microbubbles: ultrasound contrast agents for molecular imaging, in Bulte

JWM

Modo

MMJ

(eds): Nanoparticles in Biomedical Imaging: Emerging Technologies and Applications. New York, NY, Springer, 2008, pp 327–341.

117.

Frinking

Bouakaz

Kirkhorn

Ten Cate

de Jong

: Ultrasound contrast imaging: current and new potential methods. Ultrasound Med Biol 2000;26(6):965–975.

118.

Wilson

Greenbaum

Goldberg

: Contrast-enhanced ultrasound: what is the evidence and what are the obstacles? Am J Roentgenol 2009;193(1):55–60.

119.

Ziegler

O’Brien

: Harmonic ultrasound: a review. Vet Radiol Ultrasound 2002;43(6):501–509.

120.

Tang

Mulvana

Gauthier

: Quantitative contrast-enhanced ultrasound imaging: a review of sources of variability. Interface Focus 2011;1(4):520–539.

121.

Lassau

Koscieiny

Chami

: Advanced hepatocellular carcinoma: early evaluation of response to bevacizumab therapy at dynamic contrast-enhanced US with quantification—preliminary results. Radiology 2011;258(1):291–300.

122.

Lavisse

Lejeune

Rouffiac

: Early quantitative evaluation of a tumor vasculature disruptive agent AVE8062 using dynamic contrast-enhanced ultrasonography. Invest Radiol 2008;43(2):100–111.

123.

Eyding

Wilkening

Reckhardt

: Contrast burst depletion imaging (CODIM): a new imaging procedure and analysis method for semiquantitative ultrasonic perfusion imaging. Stroke 2003;34(1):77–83.

Targeted Contrast-Enhanced Ultrasound for Inflammation Detection

Abstract

Keywords

Introduction

Targeted Contrast-Enhanced Ultrasound Imaging

Proof of principle

Longitudinal

Analytics of Kinetic Behavior

Conclusion

Footnotes

Declaration of Conflicting Interests

Funding

References