Advanced Techniques for Liver Fibrosis Detection: Spectral Photoacoustic Imaging and Superpixel Photoacoustic Unmixing Analysis for Collagen Tracking.

Liver fibrosis, a major global health issue, is marked by excessive collagen deposition that impairs liver function. Noninvasive methods for the direct visualization of collagen content are crucial for the early detection and monitoring of fibrosis progression. This study investigates the potential of spectral photoacoustic imaging (sPAI) to monitor collagen development in liver fibrosis. Utilizing a novel data-driven superpixel photoacoustic unmixing (SPAX) framework, we aimed to distinguish collagen presence and evaluate its correlation with fibrosis progression. We employed an established diethylnitrosamine (DEN) model in rats to study liver fibrosis over various time points. Our results revealed a significant correlation between increased collagen photoacoustic signal intensity and advanced fibrosis stages. Collagen abundance maps displayed dynamic changes throughout fibrosis progression. These findings underscore the potential of sPAI for the noninvasive monitoring of collagen dynamics and fibrosis severity assessment. This research advances the development of noninvasive diagnostic tools and personalized management strategies for liver fibrosis.

Dynamic contrast-enhanced MRI and Doppler sonography in patients with squamous cell carcinoma of head and neck treated with induction chemotherapy.

In view of the inherent limitations associated with performing dynamic contrast enhanced-magnetic resonance imaging (DCE-MRI) in clinical settings, current study was designed to provide a proof of principle that Doppler sonography and DCE-MRI derived perfusion parameters yield similar hemodynamic information from metastatic lymph nodes in squamous cell carcinomas of head and neck (HNSCCs). Strong positive correlations between volume fraction of plasma space in tissues (Vp ) and blood volume (r = 0.72, p = 0.02) and between Vp and %area perfused (r = 0.65, p = 0.04) were observed. Additionally, a moderate positive correlation trending towards significance was obtained between volume transfer constant (Ktrans ) and %area perfused (r = 0.49, p = 0.09).

Diagnosing Portal Hypertension with Noninvasive Subharmonic Pressure Estimates from a US Contrast Agent.

Background The current standard for assessing the severity of portal hypertension is the invasive acquisition of hepatic venous pressure gradient (HVPG). A noninvasive US-based technique called subharmonic-aided pressure estimation (SHAPE) could reduce risk and enable routine acquisition of these pressure estimates. Purpose To compare quantitative SHAPE to HVPG measurements to diagnose portal hypertension in participants undergoing a transjugular liver biopsy. Materials and Methods This was a prospective cross-sectional trial conducted at two hospitals between April 2015 and March 2019 (ClinicalTrials.gov identifier, NCT02489045). This trial enrolled participants who were scheduled for transjugular liver biopsy. After standard-of-care transjugular liver biopsy and HVPG pressure measurements, participants received an infusion of a US contrast agent and saline. During infusion, SHAPE data were collected from a portal vein and a hepatic vein, and the difference was compared with HVPG measurements. Correlations between data sets were determined by using the Pearson correlation coefficient, and statistical significance between groups was determined by using the Student t test. Receiver operating characteristic analysis was performed to determine the sensitivity and specificity of SHAPE. Results A total of 125 participants (mean age ± standard deviation, 59 years ± 12; 80 men) with complete data were included. Participants at increased risk for variceal hemorrhage (HVPG ≥12 mm Hg) had a higher mean SHAPE gradient compared with participants with lower HVPGs (0.79 dB ± 2.53 vs -4.95 dB ± 3.44; P < .001), which is equivalent to a sensitivity of 90% (13 of 14; 95% CI: 88, 94) and a specificity of 80% (79 of 99; 95% CI: 76, 84). The SHAPE gradient between the portal and hepatic veins was in good overall agreement with the HVPG measurements (r = 0.68). Conclusion Subharmonic-aided pressure estimation is an accurate noninvasive technique for detecting clinically significant portal hypertension. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Kiessling in this issue.

Hepatic Vein Contrast-Enhanced Ultrasound Subharmonic Imaging Signal as a Screening Test for Portal Hypertension.

BACKGROUND: Portal hypertension is the underlying cause of most complications associated with cirrhosis, with the hepatic venous pressure gradient (HVPG) used for diagnosis and disease progression. Subharmonic imaging (SHI) is a contrast-specific imaging technique receiving at half the transmit frequency resulting in better tissue suppression. AIMS: To determine whether the presence of optimized SHI signals inside the hepatic vein can be used as a screening test for portal hypertension. METHODS: This prospective trial had 131 patients undergoing SHI examination of portal and hepatic veins using a modified Logiq 9 scanner (GE, Waukesha, WI). Images acquired after infusion of the ultrasound contrast agent Sonazoid (GE Healthcare, Oslo, Norway) were assessed for the presence of optimized SHI signals in the hepatic vein and compared to the HVPG values obtained as standard of care. RESULTS: Of 131 cases, 64 had increased HVPG values corresponding to subclinical (n = 31) and clinical (n = 33) portal hypertension (> 5 and > 10 mmHg, respectively), and 67 had normal HVPG values (< 5 mmHg). Two readers performed independent, binary qualitative assessments of the acquired digital clips. Reader one (experienced radiologist) achieved for the subclinical subgroup sensitivity of 98%, specificity of 88%, and ROC area of 0.93 and for the clinical subgroup sensitivity of 100% and specificity of 61%, with an ROC area of 0.74. Reader two (less experienced radiologist) achieved for the subclinical subgroup sensitivity of 77%, specificity of 76%, and ROC area of 0.76 and for the clinical subgroup sensitivity of 88% and specificity of 63%, with an ROC area of 0.70. Readers agreement was of 83% with kappa value of 0.66. CONCLUSION: The presence of optimized SHI signals inside the hepatic vein can be a qualitative screening test for portal hypertension, which could reduce the need for invasive diagnostic procedures.

Reliability of acoustic radiation force impulse shear wave elastography in the evaluation of liver stiffness in morbidly obese patients.

PURPOSE: To evaluate technical and patient-related factors that can affect the reliability of acoustic radiation force impulse shear wave elastography (ARFI-SWE) in morbidly obese patients. METHODS: A prospective single-center study was performed on 41 patients (32 females, 78%) presenting for preoperative evaluation for bariatric surgery. ARFI-SWE was performed using a 6 to 1.5 MHz curved (6C1) transducer. Hepatic steatosis was mild, moderate, severe, and absent in 24.4%, 12.2%, 43.9%, and 19.5% of patients, respectively. Interquartile range/median (IQR/M) ranged from 0.05 to 2.07 (0.78 ± 0.56 m/s). Twenty patients (48.7%) had reliable measurements (IQR/M < 0.3). Shear wave velocity (SWV) values were >1.34 m/s (clinically significant fibrosis) in 25 of 41 patients (61%) and >2.2 m/s (advanced fibrosis) in 19 patients (46%). RESULTS: Median SWV was correlated with body mass index (BMI; correlation coefficient [CC] = .37; 95% CI, 0.07-0.61; P-value = .03) and skin-to-liver capsule distance (SLD) (CC = .38; 95% CI, 0.09-0.62; P-value = .01). IQR/M was higher in patients with BMI > 40 (0.24 ± 0.11 vs 0.39 ± 0.25, P-value = .031) and SLD > 3 cm (0.46 ± 0.27 vs 0.23 ± 0.08, P-value = .001), and there was higher number of unreliable examinations among patient with SLD > 3 cm (16/23 vs 5/18, P-value = .01). CONCLUSION: ARFI-SWE is technically more challenging among patients with higher BMI and SLD, resulting in a higher number of unreliable studies, which highlights the need for further advancement of ARFI technology.

Machine learning for diagnostic ultrasound of triple-negative breast cancer.

PURPOSE: Early diagnosis of triple-negative (TN) breast cancer is important due to its aggressive biological characteristics, poor clinical outcomes, and limited options for therapy. The goal of this study is to evaluate the potential of machine learning with quantitative ultrasound image features for the diagnosis of TN breast cancer. METHODS: Ultrasonic and clinical data of 140 surgically confirmed breast cancer cases were analyzed retrospectively for the diagnosis of TN and non-TN (NTN) subtypes. The subtypes were classified based on the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Ultrasound image features were measured from the grayscale and color Doppler images and used with logistic regression for classification by machine learning. Leave-one-out cross validation was used to train and test the differentiation. Diagnostic performance was measured by the area under receiver operating characteristic (ROC) curve, and sensitivity and specificity determined at the Youdons index. RESULTS: Of the twelve grayscale and Doppler features measured, eight were found to be statistically different for the TN and NTN subtypes (p < 0.05). The area under the ROC curve (AUC) of the statistically significant grayscale (GS) and color Doppler (CD) features was 0.85 and 0.65, respectively. The AUC increased to 0.88 when the GS and CD features were used together, with sensitivity of 86.96% and specificity of 82.91%. Consideration of patient age in the analysis did not improve discrimination of TN and NTN. CONCLUSIONS: The analysis of breast ultrasound images by machine learning achieves high level of differentiation between the TN and NTN subtypes, exceeding the diagnostic performance by standard visual assessments of the images.

Fluid flow rate dictates the efficacy of low-intensity anti-vascular ultrasound therapy in a microfluidic model.

OBJECTIVE: Low-intensity anti-vascular ultrasound therapy is an effective means of disrupting the blood supply in the tumor microenvironment. Its diminished effect on the surrounding vasculature is thought to be due to higher blood flow rates outside the tumor that decreases the interaction time between the endothelial lining and the microbubbles, which transduce acoustic energy to thermal heat. However, investigating the effect of circulation rate on the response to low-intensity ultrasound is complicated by the heterogeneity of the in vivo vascular microenvironment. Here, a 3D microfluidic model is used to directly interrogate the dynamics of ultrasound stimulation. METHODS: A 3D in vitro vessel consisting of LifeACT transfected endothelial cells facilitate real-time analysis of actin dynamics during ultrasound treatment. Using an integrated testing platform, both the flow rate of microbubbles within the vessel and the magnitude of insonation can be varied. RESULTS: Morphological measurements and dextran transport assays indicate that lower flow rates exacerbate the effect of low-intensity ultrasound on vessel integrity. Additionally, immunostaining for VE-cadherin and transmission electron microscopy provide further insight into structural changes in cell-cell junctions following insonation. CONCLUSIONS: Overall, these results reveal that blood flow rate is an important parameter to consider during the refinement of anti-vascular low-intensity ultrasound therapies.

Engineering the Echogenic Properties of Microfluidic Microbubbles Using Mixtures of Recombinant Protein and Amphiphilic Copolymers.

Microbubbles are used as ultrasound contrast agents in medical diagnosis and also have shown great promise in ultrasound-mediated therapy. However, short lifetime and broad size distribution of microbubbles limit their applications in therapy and imaging. Moreover, it is challenging to tailor the echogenic response of microbubbles to make them suitable for specific applications. To overcome these challenges, we use microfluidic flow-focusing to prepare monodisperse microbubbles with a mixture of a recombinant amphiphilic protein, oleosin, and a synthetic amphiphilic copolymer, Pluronic. We show that these microbubbles have superior uniformity and stability under ultrasonic stimulation compared to commercial agents. We also demonstrate that by using different Pluronics, the echogenic response of the microbubbles can be tailored. Our work shows the versatility of using the combination of microfluidics and protein/copolymer mixtures as a method of engineering microbubbles. This tunability could potentially be important and powerful in producing microbubble agents for theranostic applications.

Multispectral photoacoustic imaging for the detection of subclinical melanoma.

BACKGROUND: Photoacoustic imaging (PAI) is a noninvasive technique for melanin detection within melanoma metastases. While ex vivo and early studies suggest promising clinical application, there are no standardized parameters for defining presence of melanoma metastases. METHODS: Following flank/hindlimb melanoma induction in BRaf-PTEN transgenic mice, bilateral inguinal lymph nodes (LN) were imaged in vivo at 4 to 8 weeks using PAI. Fourteen diagnostic parameters for in vivo detection of LN metastases were compared using the receiver operating characteristic and area-under-the-curve (AUC). Limits of detectability were assessed in ex vivo and in vitro phantom studies. RESULTS: Forty-nine LNs were imaged in 25 mice. Among metastatic LNs, tumor size ranged from scattered cells to 2.8 mm. The strongest predictor of LN metastasis was the ratio of peak 10% PA melanin signal in the LN compared with adjacent soft tissue (median 4.22 for positive LNs vs 1.07 for negative LNs, P < 0.0001). The AUC was 0.95 (95% CI, 0.90-1.00). In phantom studies, B16 tumor cells were detectable at a concentration of 10 to 25 cells/µL and at a tissue depth of 2.5-3 cm. CONCLUSIONS: We identified a simple, objective diagnostic parameter for identifying melanoma LN metastases in vivo. These findings may help inform the design of future clinical trials.

Placental Pulsatility: Quantitative Assessment of Placental Bed Vasculature by 2-Dimensional Doppler Cine Imaging.

OBJECTIVES: Vascular resistance is known to be one of the determinants of pulsatile flow. This study aimed to investigate whether quantitative 2-dimensional Doppler ultrasound can capture and evaluate the pulsatility within the placental bed vasculature. METHODS: The placental bed vasculature was imaged by directional power Doppler ultrasound. Ten-second cine clips were recorded by using standardized machine settings. A region of interest with a prominent Doppler signal in the uteroplacental interface was analyzed for the percentage of vascularity to generate a time-vascularity waveform. A vascular pulsatility index representing variation over the cardiac cycle was calculated by the ratio of the systolic-diastolic difference in vascularity to the mean vascularity. The acquisitions were repeated with 6 different pulse repetition frequencies (PRFs) and 3 wall motion filter (WMF) settings to evaluate their impact on the Doppler measurements. RESULTS: Ten sets of cine clips were analyzed for this study. The pulsatile nature of the vascularity was readily apparent in each cine clip. The measured time-vascularity waveforms showed uniform cyclic variation in vascularity over the cardiac cycle, with systolic vascularity significantly higher than diastolic vascularity at each combination of PRF and WMF (P < .05). A gradual increase in the vascular pulsatility index was observed with an increasing PRF or WMF. Normalization of systolic-to-diastolic measurement provided a stable vascular assessment across the range of PRFs. CONCLUSIONS: Doppler cine clips provide a dynamic representation of the placental bed vasculature and a novel analytic approach to quantitatively evaluating the pulsatility of this critical vascular network. Further work is warranted to explore the reproducibility and clinical potential of this approach.

Assessment of Age-related Oxygenation Changes in Calf Skeletal Muscle by Photoacoustic Imaging: A Potential Tool for Peripheral Arterial Disease.

Peripheral artery disease is often asymptomatic, and various imaging and nonimaging techniques have been used for assessment and monitoring treatments. This study is designed to demonstrate the ability of photoacoustic imaging to noninvasively determine changes in tissue oxygenation that occur in mice's hind limb skeletal muscle as they age. Mice from two age cohorts were scanned bilaterally with a pulsed laser. The photoacoustic signal was unmixed to generate a parametric map of estimated oxygen saturation and then overlaid on grayscale ultrasound images. Tissue oxygenation measured in young and old mice was compared. Photoacoustic imaging visually and quantitatively showed the decrease in skeletal muscle oxygenation that occurs with age. Percent tissue oxygenation decreased from 30.2% to 3.5% (p < 0.05). This reduction corresponded to reduced fractional area of oxygenation, which decreased from 60.6% to 6.0% (p < 0.05). The change in oxygenation capacity of the still active vascular regions was insignificant (p > 0.05). Intrasubject, intra-, and interobserver comparisons showed low variability in measurements, exhibited by high regression and intraclass correlations exceeding 0.81 for all ages. The decrease in oxygenation detected by photoacoustic imaging paralleled the known oxygenation decrease observed in aging tissues, demonstrating that photoacoustic imaging can assess age-related changes in a mouse calf muscle. These intramuscular changes could potentially act as a strong diagnostic marker for peripheral artery disease. This study thus opens the doors for a novel, affordable, noninvasive method of evaluation free of radiation or exogenous material.

Feed-forward active contour analysis for improved brachial artery reactivity testing.

The object of this study was to utilize a novel feed-forward active contour (FFAC) algorithm to find a reproducible technique for analysis of brachial artery reactivity. Flow-mediated dilation (FMD) is an important marker of vascular endothelial function but has not been adopted for widespread clinical use given its technical limitations, including inter-observer variability and differences in technique across clinical sites. We developed a novel FFAC algorithm with the goal of validating a more reliable standard. Forty-six healthy volunteers underwent FMD measurement according to the standard technique. Ultrasound videos lasting 5-10 seconds each were obtained pre-cuff inflation and at minutes 1 through 5 post-cuff deflation in longitudinal and transverse views. Automated segmentation using the FFAC algorithm with initial boundary definition from three different observers was used to analyze the images to measure diameter/cross-sectional area over the cardiac cycle. The %FMD was calculated for average, minimum, and maximum diameters/areas. Using the FFAC algorithm, the population-specific coefficient of variation (CV) at end-diastole was 3.24% for transverse compared to 9.96% for longitudinal measurements; the subject-specific CV was 15.03% compared to 57.41%, respectively. For longitudinal measurements made via the conventional method, the population-specific CV was 4.77% and subject-specific CV was 117.79%. The intraclass correlation coefficient (ICC) for transverse measurements was 0.97 (95% CI: 0.95-0.98) compared to 0.90 (95% CI: 0.84-0.94) for longitudinal measurements with FFAC and 0.72 (95% CI: 0.51-0.84) for conventional measurements. In conclusion, transverse views using the novel FFAC method provide less inter-observer variability than traditional longitudinal views. Improved reproducibility may allow adoption of FMD testing in a clinical setting. The FFAC algorithm is a robust technique that should be evaluated further for its ability to replace the more limited conventional technique for measurement of FMD.

Diagnostic accuracy of hepatorenal index in the detection and grading of hepatic steatosis.

PURPOSE: The objectives of our study were to assess the accuracy of hepatorenal index (HRI) in detection and grading of hepatic steatosis and to evaluate various factors that can affect the HRI measurement. METHODS: Forty-five patients, who had undergone an abdominal sonographic examination within 30 days of liver biopsy, were enrolled. The HRI was calculated as the ratio of the mean brightness levels of the liver and renal parenchymas. The effect of the measurement technique on the HRI was evaluated by using various sizes, depths, and locations of the regions of interest (ROIs) in the liver. The measurements were obtained by two observers. The HRI was compared with the subjective grading of steatosis. RESULTS: The optimal HRI cutoff to detect steatosis was 2.01, yielding a sensitivity of 62.5% and specificity of 95.2%. Subjective grading had a sensitivity of 87.5% and specificity of 62.5%. HRIs of the hepatic steatosis group were statistically different from the no-steatosis group (p < 0.05). However, there was no statistically significant difference between mild steatosis and no-steatosis groups (p value = 0.72). There was a strong correlation between different HRIs based on variable placements of ROIs, except when the ROIs were positioned randomly. Interclass correlation coefficient for measurements performed by two observers was 0.74 (confidence interval: 0.58-0.86). CONCLUSIONS: The HRI is an effective tool for detecting hepatic steatosis. It provides similar accuracy for different methods of ROI placement (except for random placement) and has good interobserver agreement. It, however, is unable to effectively differentiate between absent and mild steatosis. © 2016 Wiley Periodicals, Inc. J Clin Ultrasound 44:580-586, 2016.

Validation of semiautomated and locally resolved aortic wall thickness measurements from computed tomography.

OBJECTIVE: Aortic wall thickness (AWT) is important for anatomic description and biomechanical modeling of aneurysmal disease. However, no validated, noninvasive method for measuring AWT exists. We hypothesized that semiautomated image segmentation algorithms applied to computed tomography angiography (CTA) can accurately measure AWT. METHODS: Aortic samples from 10 patients undergoing open thoracoabdominal aneurysm repair were taken from sites of the proximal or distal anastomosis, or both, yielding 13 samples. Aortic specimens were fixed in formalin, embedded in paraffin, and sectioned. After staining with hematoxylin and eosin and Masson's trichrome, sections were digitally scanned and measured. Patients' preoperative CTA Digital Imaging and Communications in Medicine (DICOM; National Electrical Manufacturers Association, Rosslyn, Va) images were segmented into luminal, inner arterial, and outer arterial surfaces with custom algorithms using active contours, isoline contour detection, and texture analysis. AWT values derived from image data were compared with measurements of corresponding pathologic specimens. RESULTS: AWT determined by CTA averaged 2.33 ± 0.66 mm (range, 1.52-3.55 mm), and the AWT of pathologic specimens averaged 2.36 ± 0.75 mm (range, 1.51-4.16 mm). The percentage difference between pathologic specimens and CTA-determined AWT was 9.5% ± 4.1% (range, 1.8%-16.7%). The correlation between image-based measurements and pathologic measurements was high (R = 0.935). The 95% limits of agreement computed by Bland-Altman analysis fell within the range of -0.42 and 0.42 mm. CONCLUSIONS: Semiautomated analysis of CTA images can be used to accurately measure regional and patient-specific AWT, as validated using pathologic ex vivo human aortic specimens. Descriptions and reconstructions of aortic aneurysms that incorporate locally resolved wall thickness are feasible and may improve future attempts at biomechanical analyses.

Effects of age and smoking on endothelial function assessed by quantitative cardiovascular magnetic resonance in the peripheral and central vasculature.

BACKGROUND: Both age and smoking promote endothelial dysfunction and impair vascular reactivity. Here, we tested this hypothesis by quantifying new cardiovascular magnetic resonance (CMR)-based biomarkers in smokers and nonsmokers. METHODS: Study population: young non-smokers (YNS: N = 45, mean age = 30.2 ± 0.7 years), young smokers (YS: N = 39 mean age 32.1 ± 0.7 years), older non-smokers (ONS: N = 45, mean age = 57.8 ± 0.6 years), and older smokers (OS: N = 40, mean age = 56.3 ± 0.6 years), all without overt cardiovascular disease. Vascular reactivity was evaluated following cuff-induced hyperemia via time-resolved blood flow velocity and oxygenation (SvO2) in the femoral artery and vein, respectively. SvO2 dynamics yielded washout time (time to minimum SvO2), resaturation rate (upslope) and maximum change from baseline (overshoot). Arterial parameters included pulse ratio (PR), hyperemic index (HI) and duration of hyperemia (TFF). Pulse-wave velocity (PWV) was assessed in aortic arch, thoracoabdominal aorta and iliofemoral arteries. Ultrasound-based carotid intimal-medial thickness (IMT) and brachial flow-mediated dilation were measured for comparison. RESULTS: Age and smoking status were independent for all parameters. Smokers had reduced upslope (-28.4%, P < 0.001), increased washout time (+15.3%, P < 0.01), and reduced HI (-19.5%, P < 0.01). Among non-smokers, older subjects had lower upslope (-22.7%, P < 0.01) and overshoot (-29.4%, P < 0.01), elevated baseline pulse ratio (+14.9%, P < 0.01), central and peripheral PWV (all P < 0.05). Relative to YNS, YS had lower upslope (-23.6%, P < 0.01) and longer washout time (13.5%, P < 0.05). Relative to ONS, OS had lower upslope (-33.0%, P < 0.01). IMT was greater in ONS than in YNS (+45.6%, P < 0.001), and also in YS compared to YNS (+14.7%, P < 0.05). CONCLUSIONS: Results suggest CMR biomarkers of endothelial function to be sensitive to age and smoking independent of each other.

Antivascular ultrasound therapy: magnetic resonance imaging validation and activation of the immune response in murine melanoma.

OBJECTIVES: The purpose of this study was to investigate the treatment effects of antivascular ultrasound (US) with dynamic contrast-enhanced magnetic resonance imaging (MRI), contrast-enhanced sonography, and histopathologic analysis in a murine melanoma model. METHODS: Subcutaneous K1735 murine melanoma tumors were grown in syngeneic C3H/HeN mice. Quantitative tumor perfusion characteristics were measured before antivascular US treatment with both dynamic contrast-enhanced MRI and high-resolution contrast-enhanced sonography. Tumors were subsequently treated with 1 or 3 minutes of continuous low-intensity US after intravenous administration of a US contrast agent. Treatment effects were assessed by quantitative dynamic contrast-enhanced MRI, contrast-enhanced sonography, histopathologic analysis, and immunohistochemistry. RESULTS: Low-intensity antivascular US treatment resulted in approximately a doubling and tripling of the time to peak enhancement on dynamic contrast-enhanced MRI in the 1- and 3-minute treatment groups, respectively, along with a significant decrease in contrast wash-out (P < .01). There was a potent reduction in tumor perfusion on contrast-enhanced sonography, with approximately 40% and 70% reductions in the tumor area perfused as assessed by contrast-enhanced sonography after 1 (P < .05) and 3 (P < .01) minutes of antivascular US. The pathologic and histologic changes spatially correlated with the regions of diminished perfusion seen on contrast-enhanced sonography and dynamic contrast-enhanced MRI. Antivascular US therapy resulted in a significant increase in the number of hypoxia-inducible factor 1A(+) cells, indicating tumor hypoxia (P < .01), and of CD45(+)/CD3(+) cells in tumors after treatment, in keeping with increased T-cell infiltration (P < .01). CONCLUSIONS: Antivascular US treatment effects extend beyond direct cytotoxicity from hemorrhagic necrosis to include ischemia-mediated cytotoxicity, enhanced small molecule retention, and intratumoral immune activation.

Recombinant protein-stabilized monodisperse microbubbles with tunable size using a valve-based microfluidic device.

Microbubbles are used as contrast enhancing agents in ultrasound sonography and more recently have shown great potential as theranostic agents that enable both diagnostics and therapy. Conventional production methods lead to highly polydisperse microbubbles, which compromise the effectiveness of ultrasound imaging and therapy. Stabilizing microbubbles with surfactant molecules that can impart functionality and properties that are desirable for specific applications would enhance the utility of microbubbles. Here we generate monodisperse microbubbles with a large potential for functionalization by combining a microfluidic method and recombinant protein technology. Our microfluidic device uses an air-actuated membrane valve that enables production of monodisperse microbubbles with narrow size distribution. The size of microbubbles can be precisely tuned by dynamically changing the dimension of the channel using the valve. The microbubbles are stabilized by an amphiphilic protein, oleosin, which provides versatility in controlling the functionalization of microbubbles through recombinant biotechnology. We show that it is critical to control the composition of the stabilizing agents to enable formation of highly stable and monodisperse microbubbles that are echogenic under ultrasound insonation. Our protein-shelled microbubbles based on the combination of microfluidic generation and recombinant protein technology provide a promising platform for ultrasound-related applications.

Bayesian probability of malignancy with BI-RADS sonographic features.

OBJECTIVES: The purpose of this study was to develop a quantitative approach for combining individual American College of Radiology Breast Imaging Reporting and Data System (BI-RADS) sonographic features of breast masses for assessing the overall probability of malignancy. METHODS: Sonograms of solid breast masses were analyzed by 2 observers blinded to patient age, mammographic features, and lesion pathologic findings. BI-RADS sonographic features were determined by using American College of Radiology criteria. A naïve Bayes model was used to determine the probability of malignancy of all the sonographic features together and with age and BI-RADS mammographic features. The diagnostic performance for various combinations was evaluated by using the area under the receiver operating curve (Az). RESULTS: Sonographic features had high positive and negative predictive values. The Az values for BI-RADS sonographic features for the 2 observers ranged from 0.772 to 0.884, which increased to 0.866 to 0.924 when used with patient age and BI-RADS mammographic features. The benefit of adding age and mammographic information was more marked for the observer with lower initial diagnostic performance. Age-specific analysis showed that diagnostic performance varied with age, with higher performance for patients aged 45 years and younger and patients older than 60 years compared to those aged 46 to 60 years. In 85% of cases, the diagnosis of the observers matched. When the consensus between the observers was used for diagnostic decisions, a high level of diagnostic performance (Az, 0.954) was achieved. CONCLUSIONS: A naïve Bayes model provides a systematic approach for combining sonographic features and other patient characteristics for assessing the probability of malignancy to differentiate malignant and benign breast masses.

In Vivo Photoacoustic Ultrasound (PAUS) Assay for Monitoring Tendon Collagen Compositional Changes during Injury and Healing.

Tendon injury and healing involve significant changes to tissue biology and composition. Current techniques often require animal sacrifice or tissue destruction, limiting assessment of dynamic changes in tendons, including treatment response, disease development, rupture risk, and healing progression. Changes in tendon composition, such as altered collagen content, can significantly impact tendon mechanics and function. Analyses of compositional changes typically require ex vivo techniques with animal sacrifice or destruction of the tissue. In vivo evaluation of tendons is critical for longitudinal assessment. We hypothesize that photoacoustic ultrasound detects differences in collagen concentration throughout healing. We utilized photoacoustic ultrasound, a hybrid imaging modality that combines ultrasound and laser-induced photoacoustic signals to create detailed and high-resolution images of tendons, to identify its endogenous collagen composition. We correlated the photoacoustic signal to picrosirius red staining. The results show that the photoacoustic ultrasound-estimated collagen content in tendons correlates well with picrosirius red staining. This study demonstrates that photoacoustic ultrasound can assess injury-induced compositional changes within tendons and is the first study to image these targets in rat Achilles tendon in vivo.