1. Targeted Contrast-Enhanced Ultrasound: An Emerging Technology in Abdominal and Pelvic Imaging 
Marybeth A. Pysz, Jürgen K. Willmann 
Gastroenterology 
Volume 140, Issue 3, Pages e6 (March 2011)
DOI: /j.gastro
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2. Figure 1
Principles of nontargeted and molecularly targeted contrast-enhanced ultrasound imaging with contrast microbubbles. (A) The gas core of lipid- or protein-shelled microbubbles (left) makes them highly echogenic compared to surrounding tissue and blood. Right, The pulse-inversion technique is commonly used for detection of microbubbles (see Supplementary Material). Two inverse-phase (red) pulses are transmitted through the tissue, and different echoes are reflected back from either microbubbles (MBs; white) or tissue (black). The resulting echo (summation from each pulse) from microbubbles (nonlinear behavior) is a distinct signature (white wave), whereas the waves reflected from the tissue (linear) signal cancel out (white flat line).49 The resulting ultrasound contrast-mode image shows a pixel-by-pixel distribution of the microbubble signal in a subcutaneous human colon cancer xenograft in a mouse. (B) Example of imaging sequence for quantification of molecular ultrasound imaging signal intensities within a region of interest. Please refer to text for more details.
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3. Figure 2
Possible approaches for molecular target discovery (step 1; A) and validation (step 2; B), testing of targeted contrast microbubbles (step 3; C), and clinical-grade contrast microbubble design (step 4; D). See text for more details. Example micrographs for target validation are immunohistochemically stained normal and diseased colon (C, crypt; SM, submucosa) tissues with hematoxylin-stained cell nuclei (blue) and target-stained (brown) vascular endothelial cells (black arrows); note that in this example, imaging targets are only expressed on vascular endothelial cells in diseased but not in normal tissue. Targeted microbubble can then be tested preclinically for binding specificity both in cell culture with flow chamber (schematic; brightfield micrograph [original magnification, ×100] shows white microbubbles attaching to cells2) and in animal models in vivo in a subcutaneous human colon cancer xenograft (green outline) in a mouse. Target expression is also verified ex vivo by immunostaining; a 100-× micrograph shows brightly stained green blood vessels. Example of clinical-grade targeted microbubble design shows direct incorporation of binding ligand (eg, peptides identified by phage display; see also Supplementary Figure 1) into the microbubble shell.
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4. Figure 3
Nontargeted and molecularly targeted contrast-enhanced ultrasound imaging techniques can be used for several applications: Primary diagnostic imaging (detection and characterization of disease foci), monitoring disease activity and therapeutic treatment, and highly focused therapeutic delivery. (A) Early detection of cancer in a subcutaneous mouse xenograft by visualizing KDR, a marker of tumor angiogenesis expressed at early tumor stages (few mm of size; yellow bar, 3 mm), using KDR-targeted BR55 microbubbles.2 (B) Transverse ultrasound images show inflamed mouse colon (green region of interest around colon wall) visualized with contrast microbubbles (red and white colormetric map overlaid on B-mode image) targeted at inflammatory marker P-selectin, which is over-expressed in inflammatory bowel disease.46 (C) Nontargeted and/or disease-targeted (more focused delivery) microbubbles carrying therapeutics combined with ultrasound can be used to enhance therapeutic delivery to highly localized anatomical regions. Delivery process is described in text (also see Tinkov et al48).
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5. Supplementary Figure 1
Ligands (eg, antibody, protein, or peptide; blue oval) can be conjugated either directly to the phospholipid/protein shell (green arc) of microbubbles or to the polyethylene glycol (PEG; red line) arm using several types of chemistries: (1) Streptavidin/avidin–biotin bridge; (2) amide/NH2 linkage; (3) thioether/maleimide linkage; or (4) PDP/disulfide linkage (adapted from Klibanov11,12). A recently introduced clinical-grade KDR-targeted microbubble (BR55; not drawn to scale) was constructed as follows: (1) Two peptides were isolated by phage display and found to bind to human KDR with high affinity (KDs of 0.22 and 4.8 nmol/L)59,61; (2) the peptides were linked together via disuccinimidylglutarate (DSG) to form a heteropeptide (5.5 kDa molecular weight; KD of 0.5 nmol/L; prepared as an acetate salt)59–61; (3) the heteropeptide was combined to a 2000-unit polymer of PEG by mixing the 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[amino PEG2000] ammonium salt, [DSPE-PEG2000-NH2]; (4) DSPE-PEG2000-NH-glutaryl-heterodimer peptide acetate salt was then linked to a phospholipid to form a heterolipopeptide that could be directly incorporated into microbubbles.59 KDR-MBs (BR55; 1–3 μm diameter range) were constructed to contain 34,200 ± 1300 heteropeptides per square micrometer of MB.2
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6. Supplementary Figure 2
Three different methods can be used for assessment of tissue perfusion or vascularity using ultrasound. (A) The time–intensity curve analysis method involves recording the real-time ultrasound signal intensity measurements immediately after intravenous microbubble injection. Within seconds, a rapid enhancement and wash-out pattern is observed, and parameters used for measurement include the peak enhancement intensity (green bar), time needed to reach peak intensity (orange bar), wash-in rate (purple bar, representing the slope of influx rate), and wash-in (blue lines, corresponding to the area under the curve). Example ultrasound images of a subcutaneous colon xenograft in a mouse include B-mode, contrast-mode acquired before microbubble (MB) injection, and contrast mode acquired after MB injection at peak enhancement intensity. (B) Maximum intensity persistence (MIP) imaging analysis also uses MB influx, but, instead records the cumulative “history” of ultrasound intensity in the imaging plane; therefore, the displayed image represents a sum of intensities over all frames acquired during a fixed time interval. The MIP imaging method effectively records MB tracks and creates a vascular map. After a few minutes, the MB tracks reach a saturation point, and the plateau or MIP intensity is recorded. Example ultrasound MIP images of a subcutaneous colon xenograft (same as in A) include B-mode, contrast-mode acquired before MB injection, and contrast mode acquired after MB injection where MIP imaging plateau has been reached. (C) Reperfusion analysis involves continuously injecting MBs (eg, via an injection pump) to obtain a steady-state concentration of MBs in the blood circulation; then, a high-powered pulse (as in Figure 1B) is delivered to destroy all the MBs within the beam elevation. The influx (reperfusion) of MBs into the field of view is then analyzed. The curve shape is given by the equation y (signal intensity) = A (1 – exp(−βτ)), where A is the video intensity at steady-state, and β is the inverse of the time, τ, it takes to reach the steady-state intensity (with linear slope; black dashed line). Blood flow can then be measured as A × β.
Adapted from Wei et al.54
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7. Supplementary Figure 2
Three different methods can be used for assessment of tissue perfusion or vascularity using ultrasound. (A) The time–intensity curve analysis method involves recording the real-time ultrasound signal intensity measurements immediately after intravenous microbubble injection. Within seconds, a rapid enhancement and wash-out pattern is observed, and parameters used for measurement include the peak enhancement intensity (green bar), time needed to reach peak intensity (orange bar), wash-in rate (purple bar, representing the slope of influx rate), and wash-in (blue lines, corresponding to the area under the curve). Example ultrasound images of a subcutaneous colon xenograft in a mouse include B-mode, contrast-mode acquired before microbubble (MB) injection, and contrast mode acquired after MB injection at peak enhancement intensity. (B) Maximum intensity persistence (MIP) imaging analysis also uses MB influx, but, instead records the cumulative “history” of ultrasound intensity in the imaging plane; therefore, the displayed image represents a sum of intensities over all frames acquired during a fixed time interval. The MIP imaging method effectively records MB tracks and creates a vascular map. After a few minutes, the MB tracks reach a saturation point, and the plateau or MIP intensity is recorded. Example ultrasound MIP images of a subcutaneous colon xenograft (same as in A) include B-mode, contrast-mode acquired before MB injection, and contrast mode acquired after MB injection where MIP imaging plateau has been reached. (C) Reperfusion analysis involves continuously injecting MBs (eg, via an injection pump) to obtain a steady-state concentration of MBs in the blood circulation; then, a high-powered pulse (as in Figure 1B) is delivered to destroy all the MBs within the beam elevation. The influx (reperfusion) of MBs into the field of view is then analyzed. The curve shape is given by the equation y (signal intensity) = A (1 – exp(−βτ)), where A is the video intensity at steady-state, and β is the inverse of the time, τ, it takes to reach the steady-state intensity (with linear slope; black dashed line). Blood flow can then be measured as A × β.
Adapted from Wei et al.54
Gastroenterology  , e6DOI: ( /j.gastro )
Copyright © 2011 AGA Institute Terms and Conditions