Name: contrast-enhanced subharmonic aided pressure estimation (shape) using ultrasound imaging with a focus on identifying portal hypertension
Uploaded: 2020-12-05T15:00:00
Description: Watch this Scientific Journal Video about Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension at JoVE.com

This work is supported in part by the U.S. Army Medical Research Material Command under W81XWH-08-1-0503, and W81XWH-12-1-0066, by AHA grants no 0655441U and 15SDG25740015 as well as by NIH R21 HL081892, R21 HL130899, R21 HL089175, RC1 DK087365, R01 DK098526, R01 DK118964, R01 CA140338, R01 CA234428, by Lantheus Medical Imaging and by GE Healthcare, Oslo, Norway.

The institutional review boards of both Thomas Jefferson University and the Hospital of the University of Pennsylvania approved this protocol. The protocol is compliant with the Health Insurance Portability and Accountability Act. The United States Food and Drug Administration (FDA) issued an Investigational New Drug approval (IND # 124,465 to F. Forsberg) for this protocol. GE Healthcare (Oslo, Norway) provided the UCA used in this research (Sonazoid; Table 1). Sonazoid is not approved by the FDA for an...

<ol>
	<li>Lyshchik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamentals of CEUS. , (2019).</li><li>Leighton, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Acoustic Bubble. , (1994).</li><li>Forsberg, F., Shi, W. T., Goldberg, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subharmonic+imaging+of+contrast+agents.">Subharmonic imaging of contrast agents.</a> Ultrasonics. 38 (1-8), 93-98 (2000).</li><li>Forsberg, F., Piccoli, C. W., Merton, D. A., Palazzo, J. P., Hall, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+lesions:+imaging+with+contrast-enhanced+subharmonic+US+-+initial+experience.">Breast lesions: imaging with contrast-enhanced subharmonic US - initial experience.</a> Radiology. 244 (3), 718-726 (2007).</li><li>Sridharan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+breast+lesions+using+quantitative+parametric+3D+subharmonic+imaging:+a+multi-center+study.">Characterizing breast lesions using quantitative parametric 3D subharmonic imaging: a multi-center study.</a> Academic Radiology. 27 (8), 1065-1074 (2020).</li><li>Forsberg, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subharmonic+and+endoscopic+contrast+imaging+of+pancreatic+masses:+a+pilot+study.">Subharmonic and endoscopic contrast imaging of pancreatic masses: a pilot study.</a> Journal of Ultrasound in Medicine. 37 (1), 123-129 (2018).</li><li>Delaney, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+adnexal+masses+using+contrast-enhanced+subharmonic+imaging:+a+pilot+study.">Characterization of adnexal masses using contrast-enhanced subharmonic imaging: a pilot study.</a> Journal of Ultrasound in Medicine. 39 (5), 977-985 (2020).</li><li>Eisenbrey, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast-enhanced+subharmonic+and+harmonic+ultrasound+of+renal+masses+undergoing+percutaneous+cryoablation.">Contrast-enhanced subharmonic and harmonic ultrasound of renal masses undergoing percutaneous cryoablation.</a> Academic Radiology. 22 (7), 820-826 (2015).</li><li>Gupta, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transrectal+subharmonic+ultrasound+imaging+for+prostate+cancer+detection.">Transrectal subharmonic ultrasound imaging for prostate cancer detection.</a> Urology. 138 (4), 106-112 (2020).</li><li>Shi, W. T., Forsberg, F., Raichlen, J. S., Needleman, L., Goldberg, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure+dependence+of+subharmonic+signals+from+contrast+microbubbles.">Pressure dependence of subharmonic signals from contrast microbubbles.</a> Ultrasound in Medicine and Biology. 25 (2), 275-283 (1999).</li><li>Halldorsdottir, V. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subharmonic+contrast+microbubble+signals+for+noninvasive+pressure+estimation+under+static+and+dynamic+flow+conditions.">Subharmonic contrast microbubble signals for noninvasive pressure estimation under static and dynamic flow conditions.</a> Ultrasonic Imaging. 33 (3), 153-164 (2011).</li><li>Nio, A. Q. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+control+of+SonoVue+microbubbles+to+estimate+hydrostatic+pressure.">Optimal control of SonoVue microbubbles to estimate hydrostatic pressure.</a> IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control. 67 (3), 557-567 (2020).</li><li>Eisenbrey, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+liver+disease:+noninvasive+subharmonic+aided+pressure+estimation+of+hepatic+venous+pressure+gradient.">Chronic liver disease: noninvasive subharmonic aided pressure estimation of hepatic venous pressure gradient.</a> Radiology. 268 (2), 581-588 (2013).</li><li>Gupta, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosing+portal+hypertension+with+noninvasive+subharmonic+pressure+estimates+from+an+ultrasound+contrast+agent.">Diagnosing portal hypertension with noninvasive subharmonic pressure estimates from an ultrasound contrast agent.</a> Radiology. , (2020).</li><li>Nam, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+neoadjuvant+chemotherapy+for+breast+cancer+by+using+three-dimensional+subharmonic+aided+pressure+estimation+and+imaging+with+US+contrast+agents:+preliminary+experience.">Monitoring neoadjuvant chemotherapy for breast cancer by using three-dimensional subharmonic aided pressure estimation and imaging with US contrast agents: preliminary experience.</a> Radiology. 285 (1), 53-62 (2017).</li><li>Dave, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+intra-cardiac+pressure+measurements+using+subharmonic-aided+pressure+estimation:+proof+of+concept+in+humans.">Non-invasive intra-cardiac pressure measurements using subharmonic-aided pressure estimation: proof of concept in humans.</a> Ultrasound in Medicine and Biology. 43 (11), 2718-2724 (2017).</li><li>Esposito, C., Dickie, K., Forsberg, F., Dave, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+an+interface+and+investigating+optimal+parameters+for+real-time+intra-cardiac+subharmonic+aided+pressure+estimation.">Developing an interface and investigating optimal parameters for real-time intra-cardiac subharmonic aided pressure estimation.</a> IEEE Transactions on Ultrasonics, Ferroelectrics and Frequency Control. , (2020).</li><li>Bosch, J., Groszmann, R. J., Shah, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+in+the+understanding+of+the+pathophysiological+basis+of+portal+hypertension:+How+changes+in+paradigm+are+leading+to+successful+new+treatments.">Evolution in the understanding of the pathophysiological basis of portal hypertension: How changes in paradigm are leading to successful new treatments.</a> Journal of Hepatology. 62, 121-130 (2015).</li><li>Procopet, B., Berzigotti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+cirrhosis+and+portal+hypertension:+imaging,+non-invasive+markers+of+fibrosis+and+liver+biopsy.">Diagnosis of cirrhosis and portal hypertension: imaging, non-invasive markers of fibrosis and liver biopsy.</a> Gastroenterology Report. 5 (2), 79-89 (2017).</li><li>Dietrich, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+and+good+clinical+practice+recommendations+for+contrast-enhanced+ultrasound+(CEUS)+in+the+liver-update+2020+WFUMB+in+cooperation+with+EFSUMB,+AFSUMB,+AIUM,+and+FLAUS.">Guidelines and good clinical practice recommendations for contrast-enhanced ultrasound (CEUS) in the liver-update 2020 WFUMB in cooperation with EFSUMB, AFSUMB, AIUM, and FLAUS.</a> Ultrasound in Medicine and Biology. , (2020).</li><li>Gupta, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pulse+shaping+on+subharmonic+aided+pressure+estimation+in+vitro+and+in+vivo.">Effect of pulse shaping on subharmonic aided pressure estimation in vitro and in vivo.</a> Journal of Ultrasound in Medicine. 36 (1), 3-11 (2017).</li><li>Dave, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+implementation+of+an+automated+acoustic+output+optimization+algorithm+for+subharmonic+aided+pressure+estimation.">On the implementation of an automated acoustic output optimization algorithm for subharmonic aided pressure estimation.</a> Ultrasonics. 53 (4), 880-888 (2013).</li><li>Gupta, I., Eisenbrey, J. R., Machado, P., Stanczak, M., Wallace, K., Forsberg, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+factors+impacting+subharmonic-+aided+pressure+estimation+(SHAPE).">On factors impacting subharmonic- aided pressure estimation (SHAPE).</a> Ultrasonic Imaging. 41 (1), 35-48 (2019).</li><li>Eisenbrey, J. R., Daecher, A., Kramer, M. R., Forsberg, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+needle+and+catheter+size+on+commercially+available+ultrasound+contrast+agents.">Effects of needle and catheter size on commercially available ultrasound contrast agents.</a> Journal of Ultrasound in Medicine. 34 (11), 1961-1968 (2015).</li></ol>

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. The protocol for SHAPE measurements presented here achieves this goal. The most critical component of the SHAPE procedure is the optimization algorithm, since subharmonic data not acquired at the optimal acoustic power output will correlate poorly with hydrostatic pressures.17,22,23&#160;The initial version ...

A number of different ultrasound contrast agents (UCAs) are approved for clinical use in cardiology (in particular left ventricular opacification) and radiology (in particular adult and pediatric liver lesion characterization) across the world.1&#160;The sensitivity and specificity of ultrasound imaging can be improved by intravenous (IV) injection of gas-filled microbubbles (diameter &#60; 10 &#956;m) encapsulated by a lipid or protein shell as UCAs that traverse the entire vasculature and enhance signals by up to 30 dB.1&#160;These UCAs not only enhance the backscattered ultrasound signals, but at sufficient acoustic pressures (&#62; 200 kPa) they also act as nonlinear oscillators. Hence, significant energy components will be produced in the received echoes ranging from subharmonic and harmonic to ultraharmonic frequencies.1,2&#160;These nonlinear signal components can be extracted from tissue and linear bubble echoes (e.g., using pulse inversion) and used to create contrast-specific imaging modalities such as subharmonic imaging (SHI), which receives at half the transmit frequency (i.e., at f0/2).3&#160;Our group has demonstrated in human clinical trials that SHI can detect the blood flow in neovessels and arterioles associated with a variety of&#160;tumors and tissues.4,5,6,7,8,9
We have advocated the use of UCAs not as vascular tracers, but as sensors for noninvasive pressure estimation in the circulatory system by monitoring subharmonic contrast bubble amplitude variations.10&#160;This innovative technique, called subharmonic-aided pressure estimation (SHAPE), relies on the inverse linear correlation between the amplitude of the subharmonic signals and hydrostatic pressure (up to 186 mmHg) measured for most commercial UCAs in vitro (r2 &#62; 0.90) as summarized in Table 1.10,11&#160;However, it should be noted that not all UCAs exhibit this behavior. Most notably, it has been shown that subharmonic signals from the UCA SonoVue (known as Lumason in the USA) initially rise with hydrostatic pressure increases, followed by a plateau and a decreasing phase.12&#160;Nonetheless, SHAPE offers the possibility of allowing pressure gradients in the heart and throughout the cardiovascular system as well as interstitial fluid pressure in tumors to be obtained noninvasively.13,14,15,16,17&#160;Recently, we implemented a real-time version of the SHAPE algorithm on a commercial ultrasound scanner and provided proof-of-concept that SHAPE can provide in vivo pressure estimates with errors of less than 3 mmHg in the left and right ventricles of patients.16,17
The most experience with SHAPE to date has been for&#160;diagnosing portal hypertension with more than 220 subjects enrolled &#160;and initial findings confirmed in a multi-center trial.13,14&#160;Portal hypertension is defined as an increase in the pressure gradient between the portal vein and hepatic veins or the inferior vena cava exceeding 5 mmHg, while clinically significant portal hypertension (CSPH) requires a gradient or its equivalent, a hepatic venous pressure gradient (HVPG) &#8805; 10 mmHg.18&#160;CSPH is associated with an increased risk of gastroesophageal varices, ascites, hepatic decompensation, post-operative decompensation, and hepatocellular carcinoma.18,19&#160;Patients who develop ascites have a 50% three-year mortality and those who develop spontaneous infection of the ascites fluid carry a 70% one-year mortality. Patients with cirrhosis have a 5-10% yearly incidence of gastroesophageal variceal formation, and a 4-15% yearly incidence of bleeding; each bleeding episode carries up to a 20% risk of death.18,19
This manuscript describes how to conduct a SHAPE study using commercially available equipment and UCAs with an emphasis on identifying portal hypertension in the liver of patients. The critical calibration procedure required to achieve the highest sensitivity to estimating pressure changes is explained in detail.

<table>
	<tbody>
		<tr>
			<td>2 mL syringe</td>
			<td>Becton Dickinson</td>
			<td>309637</td>
			<td>Used for reconstituting Sonazoid</td>
		</tr>
		<tr>
			<td>10 mL saline-filled syringe</td>
			<td>Becton Dickinson</td>
			<td>306545</td>
			<td>Used for flushing line to verify IV access</td>
		</tr>
		<tr>
			<td>500 mL saline bag</td>
			<td>Baxter Healthcare Corp</td>
			<td>2131323</td>
			<td>Used for co-infusion with Sonazoid</td>
		</tr>
		<tr>
			<td>C1-6-D curvi-linear proble</td>
			<td>GE Healthcare</td>
			<td>H40472LT</td>
			<td>Used for liver imaging</td>
		</tr>
		<tr>
			<td>Chemoprotect Spike</td>
			<td>Codan USA</td>
			<td>C355</td>
			<td>Chemospike used for reconstituting Sonazoid</td>
		</tr>
		<tr>
			<td>Discofix C Blue</td>
			<td>B. Braun Medical Inc</td>
			<td>16494C</td>
			<td>3-way stopcock</td>
		</tr>
		<tr>
			<td>Intrafix Safeset 180 cm</td>
			<td>B. Braun Medical Inc</td>
			<td>4063000</td>
			<td>Infusion tubing</td>
		</tr>
		<tr>
			<td>Logiq E10 ultrasound scanner</td>
			<td>GE Healthcare</td>
			<td>H4928US</td>
			<td>Used for conventional ultrasound imaging as well as for SHI and SHAPE</td>
		</tr>
		<tr>
			<td>Luer lock 10 mL syringe</td>
			<td>Becton Dickinson</td>
			<td>300912</td>
			<td>For infusion of Sonazoid</td>
		</tr>
		<tr>
			<td>Medfusion 3500 syringe pump</td>
			<td>Smiths Medical</td>
			<td>3500-500</td>
			<td>Used for infusing Sonazoid at 0.18 mL/kg/hour</td>
		</tr>
		<tr>
			<td>Perfusor-leitung tubing 150 mm</td>
			<td>B. Braun Medical Inc</td>
			<td>8722960</td>
			<td>Extension line enabling syringe connection to patient&#39;s IV access</td>
		</tr>
		<tr>
			<td>SHI/SHAPE software</td>
			<td>GE Healthcare</td>
			<td>H4920CI</td>
			<td>Contrast-specific imaging software</td>
		</tr>
		<tr>
			<td>Sigma Spectrum infusion system</td>
			<td>Baxter Healthcare Corp</td>
			<td>35700BAX</td>
			<td>Pump used for co-infusing saline at 120 mL/hour</td>
		</tr>
		<tr>
			<td>Sonazoid</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>Gas-filled microbubble based ultrasound contrast agent</td>
		</tr>
		<tr>
			<td>sterile water, 2 mL</td>
			<td>B. Braun Medical Inc</td>
			<td></td>
			<td>Used for reconstituting Sonazoid</td>
		</tr>
		<tr>
			<td>ultrasound gel</td>
			<td>Cardinal Health</td>
			<td>USG-250BT</td>
			<td>Used for contact between probe and patient</td>
		</tr>
		<tr>
			<td>Venflon IV cannula 22GA</td>
			<td>Becton Dickinson</td>
			<td>393202</td>
			<td>Cannula needle for obtaining IV access</td>
		</tr>
	</tbody>
</table>

contrast-enhanced subharmonic aided pressure estimation (shape) using ultrasound imaging with a focus on identifying portal hypertension

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound imaging are gas-filled, encapsulated microbubbles (diameter &#60; 10 &#956;m) that traverse the entire vasculature and enhance signals by up to 30 dB. These microbubbles also produce nonlinear oscillations at frequencies ranging from the subharmonic (half of the transmit frequency) to higher harmonics. The subharmonic amplitude has an inverse linear relationship with the ambient hydrostatic pressure. Here an ultrasound system capable of performing real-time, subharmonic aided pressure estimation (SHAPE) is presented. During ultrasound contrast agent infusion, an algorithm for optimizing acoustic outputs is activated. Following this calibration, subharmonic microbubble signals (i.e., SHAPE) have the highest sensitivity to pressure changes and can be used to noninvasively quantify pressure. The utility of the SHAPE procedure for identifying portal hypertension in the liver is the emphasis here, but the technique has applicability across many clinical scenarios.

As with all ultrasound imaging examinations, the first consideration for liver SHAPE is to obtain the best possible baseline grayscale images of the target region and to ensure (using Doppler imaging) that there are no intrahepatic portal venous shunts or other vascular abnormalities present. In the case of liver imaging for diagnosing portal hypertension the key is to visualize both the portal vein and a hepatic vein at the same depth to minimize the impact of attenuation (Figure 1).
<p...

Watch this Scientific Journal Video about Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension at JoVE.com

Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

A protocol for noninvasively estimating ambient pressures utilizing subharmonic ultrasound imaging of infused contrast microbubbles (following appropriate calibration) is described with examples from human patients with chronic liver disease.

Contrast-Enhanced Subharmonic Aided Pressure Estimation (SHAPE) Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound ...

This technique is designed for measuring pressures non-invasively within the body. It's particularly useful for monitoring pressures in the heart, for monitoring treatment of cancers, and in this particular project for measuring pressures in the liver. To prepare the ultrasound contrast agent, remove the protective cap from the syringe port of the chemo spike and use a chemo spike to perforate the stopper of the first of three vials of the agent.Add two milliliters of sterile water to the vial and immediately shake the vial for one minute. When a homogenous product has been obtained, withdraw the product into the syringe. The reconstituted ultrasound contrast agent contains microbubbles at a concentration of eight microliters per milliliter.After repeating the reconstitution for the other two vials in the same manner, use saline to fill up connecting tubes and connect the tubes to a three-way stopcock. Connect the stopcock to the extension tubing leading to the cannula and connect the syringe directly to the stopcock. Then mount the syringe onto a syringe pump at the same level or below the patient.To acquire an initial set of ultrasound images, power up an ultrasound scanner and select the C1-6-D curvilinear probe. Select an abdominal preset on the scanner. Use a subcostal approach and a curvilinear array to acquire grayscale images of both the portal and hepatic veins in the same imaging plane and at similar depths.Optimize the images based on good clinical practice, taking care to select the hepatic vein region away from the inferior vena cava to avoid the influence of retrograde flow. After the initial ultrasound imaging, open the stopcock and infuse the saline solution at a rate of 120 milliliters per hour while co-infusing the contrast at a rate of 0.024 microliters per kilogram body rate per minute. For activating subharmonic imaging in dual display mode, use the subharmonic contrast touch panel button to activate contrast mode.Select subharmonic imaging amplitude modulation on the rotary control. Perform subharmonic imaging at a transmit frequency of 2.5 megahertz and obtain the received signals at 1.25 megahertz. Confirm the patency of the portal and the hepatic veins as well as the presence of microbubbles, which can take up to one to two minutes from the start of the infusion.Select time intensity curve analysis on the touch panel followed by F6 and K to compensate for varying depths and attenuations to activate the SHAPE automated optimization code to optimize the SHAPE imaging. The SHAPE optimization algorithm will acquire subharmonic data for every acoustic output level. Once the data acquisition is complete, position a region of interest on the portal vein in the contrast sample window.Plot the average subharmonic data within the region as a function of acoustic output and fit a logistic curve to the data. Then select the inflection point of this curve as the optimized power, as this has been shown to be the point of greatest SHAPE sensitivity. Adjust the acoustic output power to the inflection point value to maximize the sensitivity of the SHAPE and acquire subharmonic data from the microbubbles in five to 15 second segments during the infusion of the UCA suspension.When imaging for diagnosing portal hypertension in the liver, the key is to visualize both the portal and hepatic veins at the same depth to minimize the impact of attenuation. After a UCA induced equilibrium enhancement, the optimization algorithm should be activated and a region of interest in the portal vein should be selected to produce subharmonic amplitude and output power selection curves. In these SHAPE images from patients with and without clinical significant portal hypertension, the main visual difference is the marked subharmonic signal present in the hepatic vein in the patient with hypertension that is absent in the patient without hypertension.Note that some patients present with clinical and laboratorial signs of portal hypertension but have HVPG values that are normal or zero. This can be attributed to a number of anatomical and/or vascular variations, resulting in an incorrect SHAPE diagnosis. This technique is non-invasive, safe, and accurate.It allows the acquisition of relative as well as absolute pressure estimates, and it's something that no other imaging technique can currently do.

contrast-enhanced-subharmonic-aided-pressure-estimation-shape-using

Research

JoVE Journal

Bioengineering

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

Fabrication of a Bioactive, PCL-based &quot;Self-fitting&quot; Shape Memory Polymer Scaffold

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create anatomically accurate head phantoms with realistic brain imaging properties because standard fabrication methods are not optimized to replicate any patient-specific anatomical detail and 3D printing materials are not optimized for imaging properties. In order to test and validate a novel navigation system for use during brain tumor surgery, an anatomically accurate phantom with realistic imaging and mechanical properties was required. Therefore, a phantom was developed using real patient data as input and 3D printing of molds to fabricate a patient-specific head phantom comprising the skull, brain and tumor with both ultrasound and X-ray contrast. The phantom also had mechanical properties that allowed the phantom tissue to be manipulated in a similar manner to how human brain tissue is handled during surgery. The phantom was successfully tested during a surgical simulation in a virtual operating room.
The phantom fabrication method uses commercially available materials and is easy to reproduce. The 3D printing files can be readily shared, and the technique can be adapted to encompass many different types of tumor.

This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material.

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.