Quantitative Analysis of Contrast-Enhanced Ultrasound Imaging in Invasive Breast Cancer: A Novel Technique to Obtain Histopathologic Information of Microvessel Density

We examined whether enhancement area ratios obtained by the new bubble detection method correlate with histologic microvessel density in invasive breast cancer. Forty consecutive patients with invasive breast cancer lesions underwent contrast-enhanced ultrasound. The ratio of enhanced area to manually segmented tumor area (enhancement area ratio) was obtained with the new method at peak and delayed phases (50–54, 55–59, 60–64 and 65–69 s). We also analyzed time–intensity curves to obtain peak intensity and area under curve. Enhancement area ratios in both peak and delayed phases (50–54, 55–59, 60–64 and 65–69 s) were significantly correlated with microvessel density (r = 0.57, 0.62, 0.68, 0.61 and 0.58; p = 0.0001, <0.0001, <.0001, <.0001 and 0.0001, respectively). In time–intensity curve analysis, peak intensity was significantly correlated (r = 0.43, p = 0.0073), whereas area under the curve was not (r = 0.29, p = 0.0769). Enhancement area ratios obtained by the new method were correlated with microvessel density in invasive breast cancer.     

Contrast-Enhanced Ultrasound Imaging of Carotid Plaque Neo-vascularization: Accuracy of Visual Analysis

The aim of our study was to evaluate whether neo-vascularization of the carotid plaque can be accurately assessed by visual analysis of contrast-enhanced ultrasound images and whether these findings correlate with intensity-over-time curve analysis (ITC) and histopathology. Patients with ≥50% symptomatic or ≥60% asymptomatic stenosis according to European Carotid Surgery Trial criteria were included. Four investigators evaluated contrast enhancement visually (three grades), with positive agreement when three or more investigators were unanimous. ITC analysis of contrast enhancement was performed in the plaque and in the lumen. Histopathology (microvessel density with CD34 + staining) was completed when endarterectomy was performed. Visual grading (33 patients, inter-observer agreement = 94%) correlated significantly with ITC analysis (p = 0.03). Histopathology (n = 19) revealed a larger CD34 + area in patients with grade 1/2 versus grade 0 (p = 0.03). Visual analysis of neo-vascularization by means of contrast-enhanced ultrasound imaging is accurate and reproducible, with significant correlations with ITC and histopathology.     

超声造影与乳腺癌肿瘤微血管密度测定的相关性

目的 研究乳腺癌超声造影与肿瘤微血管密度(MVD)的相关性.方法 乳腺癌49例术前接受超声造影检查,应用时间-强度曲线分析软件测定定量参数,所得参数与术后病理组织免疫组化染色测得的MVD和血管内皮生长因子(VEGF)表达对照,进行相关性分析.结果 峰值强度、增强强度指数与MVD、VEGF表达显著正相关(P<0.01),始增时间、峰值时间与VEGF表达负相关(P<0.05),但与MVD无相关性(P>0.05),曲线上升支斜率、平台持续时间与MVD、VEGF表达均无相关性(P>0.05).结论 超声造影峰值强度、增强强度指数与MVD、VEGF表达显著相关,可以作为定量评价肿瘤血管生成的依据.     

Quantification of Carotid Plaque Neovascularization Using Contrast-Enhanced Ultrasound With Histopathologic Validation

We sought to evaluate contrast-enhanced ultrasound (CEUS) imaging for the quantification of carotid plaque neovascularization. Seventeen patients underwent carotid endarterectomy after standard ultrasound and CEUS. Semiquantitative and quantitative analyses of contrast enhancement within the plaque were performed using a visual interpretation scale and quantitative analysis software, respectively. Enhancement intensity (dB) was measured at the plaque (EI_plaque). Each specimen was stained with CD34 and CD68 to assess for microvessels and macrophages, respectively. Semiquantitative CEUS analyses were correlated with neovascularization at histology (r = 0.70, p = 0.002). Quantitative analysis was also correlated with neovascularization at histology (EI_plaquer = 0.81, p < 0.001). EI_plaque (r = 0.64, p = 0.01) was correlated with the degree of enhancement as assessed visually. Semiquantitative and quantitative analyses were not correlated with macrophage infiltration at the plaque. Contrast enhancement in the carotid plaque was correlated with neovascularity at the histopathologic exam. Furthermore, semiquantitative and quantitative measurements were highly correlated with each other, suggesting that either can be used to detect intraplaque neovascularization.     

Longitudinal Changes in Knee Joint Synovial Vascularity in a Rabbit Model of Rheumatoid Arthritis: Quantification Using Power Doppler Ultrasound and Contrast-Enhanced Ultrasound

We studied a rabbit model of rheumatoid arthritis (RA) to examine the time course of changes in synovial neovascularization based on quantitative power Doppler ultrasound and contrast-enhanced ultrasound (CEUS). Twenty-five male New Zealand rabbits were in the ovalbumin-induced arthritis (OIA) group, and 5 were in the control group. Both rear knee joints of all rabbits were examined using conventional US and CEUS over 16 weeks. The knee synoviums of OIA rabbits were sampled by US-guided biopsy, and expression of CD31 and vascular endothelial growth factor (VEGF) was determined by immunohistochemistry. The correlation of joint damage based on multimodal US with microvessel density (CD31 positivity) and VEGF expression at different times was analyzed. OIA rabbits had increased synovial expression of CD31 and VEGF from weeks 6 to 12 (p < 0.01). During the early stage of CEUS enhancement, “dot enhancement” was more common at weeks 6 and 8, and “stripe enhancement” was more common at weeks 12 and 16 (p < 0.05). There were significant positive correlations of synovial CD31 and VEGF expression with power Doppler image grade, CEUS grade and peak intensity (p < 0.05 for all). Thus, OIA rabbits mimicked early-stage RA at 6 to 8 weeks, middle-stage RA at 8 to 12 weeks and late-stage RA at 12 to 16 weeks. Power Doppler image grade, CEUS grade and peak intensity, especially when combined with CD31 expression data, accurately characterized the extent of synovial vascularization in a rabbit model of RA. Increased vascularity based on CEUS may have value for the early diagnosis of RA.     

An Animal Model Allowing Controlled Receptor Expression for Molecular Ultrasound Imaging

Reported in this study is an animal model system for evaluating targeted ultrasound (US) contrast agents binding using adenoviral (Ad) vectors to modulate cellular receptor expression. An Ad vector encoding an extracellular hemagglutinin (HA) epitope tag and a green fluorescent protein (GFP) reporter was used to regulate receptor expression. A low and high receptor density (in breast cancer tumor bearing mice) was achieved by varying the Ad dose with a low plaque forming unit (PFU) on day 1 and high PFU on day 2 of experimentation. Targeted US contrast agents, or microbubbles (MB), were created by conjugating either biotinylated anti-HA or IgG isotype control antibodies to the MB surface with biotin-streptavidin linkage. Targeted and control MBs were administered on both days of experimentation and contrast-enhanced US (CEUS) was performed on each mouse using MB flash destruction technique. Signal intensities from MBs retained within tumor vasculature were analyzed through a custom Matlab program. Results showed intratumoral enhancement attributable to targeted MB accumulation was significantly increased from the low Ad vector dosing and the high Ad vector dosing (p = 0.001). Control MBs showed no significant differences between day 1 and day 2 imaging (p = 0.96). Additionally, targeted MBs showed a 10.5-fold increase in intratumoral image intensity on day 1 and an 18.8-fold increase in image intensity on day 2 compared with their control MB counterparts.     

Contrast-Enhanced Ultrasound Characteristics of Breast Cancer: Correlation with Prognostic Factors

The purpose of the work described here was to investigate the correlation of contrast-enhanced ultrasound (CEUS) characteristics with prognostic factors in breast cancer. Forty-five consecutive breast cancer patients were studied with CEUS. All patients were diagnosed with invasive ductal carcinoma on the basis of biopsy or surgery results. Lack of blood perfusion of the tumor was identified in 2 cases; blood perfusion was observed in 43 cases. Enhancement was heterogeneous in 25 cases, and centripetal in 25 cases. A blood perfusion defect was present in 27 cases. Enhancement shape was irregular in 37 cases, margins were poorly defined in 34 cases, and penetrating vessels were present in 32 cases. Compared with the surrounding normal tissue, tumor tissue had faster rising times and times to peak and higher peak intensities and wash-in slopes; the differences between the two groups were statistically significant (p < 0.05). Compared with the interior of the tumor, the periphery had faster times to peak and higher peak intensities and wash-in slopes (p < 0.05). Heterogeneous enhancement, perfusion defect, centripetal enhancement and penetrating vessels were correlated with prognosis (p < 0.05). Overall, some CEUS characteristics of breast cancer were associated with prognostic factors that can predict breast cancer prognosis in vivo.     

Clinical and Imaging Characteristics of Papillary Neoplasms of the Breast Associated with Malignancy: A Retrospective Cohort Study

Papillary neoplasms of the breast comprise a broad range of pathologies ranging from papillomas to papillary carcinomas and have been associated with breast cancers. In this study, we evaluated the clinical, mammographic and sonographic features of papillary breast neoplasms from benign papillary breast lesions to malignancy-associated papillary lesions. A total of 194 lesions in 179 patients were analyzed, including 117 benign papillomas, 24 atypical papillomas, 41 benign papillomas with malignancies and 12 papillary carcinomas found between January 2003 and August 2011 in our institution. Statistically significant clinical factors included patient age (p = 0.001), lesion multiplicity (p = 0.009) and peripheral location (p = 0.003). Among these factors, the odds ratio for malignancy was 8.9 for bilateral multiple lesions. Visibility (p = 0.001) and density (p = 0.039) were significant factors for malignancy in mammograms, and echo patterns (p = 0.006), boundary (p = 0.001) and vascularity (p = 0.005) were significant features on ultrasound that differentiated malignancies from benign lesions. Overall, when papillary breast lesions are located bilaterally and peripherally in older patients, they are correlated with breast cancers. Additionally, for papillary breast lesions that appear highly dense on mammograms and/or exhibit positive vascularity on ultrasound, the probability of malignancy is relatively high.     

Quantitative Contrast-Enhanced Ultrasonic Imaging Reflects Microvascularization in Hepatocellular Carcinoma and Prognosis after Resection

Our aim was to evaluate the correlation between tumor vasculature detected by pre-surgical contrast-enhanced ultrasonography and the post-surgical prognosis of patients with hepatocellular carcinoma. One hundred ninety-five patients with hepatocellular carcinoma who had undergone curative resection and pre-operative contrast-enhanced ultrasonography were enrolled. Intra-tumoral microvessels were evaluated by immunohistochemical staining for anti-CD31 and anti-CD34. On the basis of the immunohistochemical staining and morphology patterns, tumors were divided into capillary-like and sinusoid-like microvessel subtypes. The rise time of tumors was shorter in the capillary-like microvessel subtype than in the sinusoid-like microvasculature subtype (p = 0.026). Intra-tumor microvascular density (p < 0.001, hazard ratio = 0.137) and rise time (p = 0.006, hazard ratio = 2.475) were independent factors corresponding to different microvasculature types. Microvascular density, vascular invasion and wash-in perfusion index were determined to be independent factors in recurrence-free survival and overall survival. In conclusion, contrast-enhanced ultrasonography may serve as a means of non-invasive assessment of tumor angiogenesis and may be associated with the survival of patients with hepatocellular carcinoma after resection.     

Potential Application Value of Contrast-Enhanced Ultrasound In Neoadjuvant Chemotherapy of Breast Cancer

The purpose of this study was to investigate the value of contrast-enhanced ultrasound (CEUS) in evaluating the response of breast cancer to neoadjuvant chemotherapy (NAC). The study included 31 breast cancer patients who were treated with NAC between August 2010 and October 2011. All patients were evaluated by both conventional ultrasound (US) and CEUS. The tumor sizes measured by CEUS were larger and more accurately imaged than those evaluated by US. Necrosis at the tumor center could be detected by CEUS, which showed a local blood perfusion defect in 26 cases (83.9%) before NAC and 27 cases (87.1%) after NAC, whereas US did not show liquefaction in any patient. The CEUS time-intensity curve displayed quantitatively the tumors' blood-perfusion changes; after NAC, blood perfusion reduced, enhancement intensity decreased, time to peak increased, peak intensity reduced, and the wash-in slope reduced (p < 0.05). Overall, the CEUS is a promising tool for evaluating the response of breast cancer to NAC.     

动态增强MRI评价肺癌肿瘤血管生成

目的评价动态增强磁共振成像(DCE-MRI)的各种强化参数与肺癌组织标本中肿瘤血管生成的相关性。方法对经手术病理证实的周围型肺癌患者14例行DCE-MRI检查,描绘时间-信号强度曲线,测量各强化参数,包括早期强化峰值(SIep)、强化峰值(SImax)、最大强化比(MER)和最大下降率(washout)。对肿瘤组织取材进行CD34和VEGF的免疫组织化学染色,分析各强化参数与肺癌组织标本的MVD和VEGF的相关性。结果 SIep、SImax与肺癌组织标本的MVD呈正相关(r=0.811、0.841,P均<0.01);而SIep、SImax、MER和最大下降率与VEGF均无相关性(P均>0.05)。结论应用SIep和SImax可预测肺癌组织标本的MVD水平,但根据DCE-MRI各强化参数均无法预测VEGF表达水平。     

11C-methionine uptake correlates with tumor cell density rather than with microvessel density in glioma: A stereotactic image-histology comparison

¹¹C-methionine positron emission tomography (¹¹C-methionine PET) provides accurate detection of brain tumors. Several reports have analyzed the correlation between uptake of ¹¹C-methionine and Ki-67 index or microvessel density non-stereotactically and suggested that ¹¹C-methionine uptake reflects both proliferation potential and angiogenic capability in gliomas. As gliomas possess heterogeneous histological architecture, non-stereotactic comparison of the histology and ¹¹C-methionine PET image may not be accurate. In the present study, the correlation between ¹¹C-methionine uptake and cell or microvessel density was analyzed using histological specimens obtained by stereotactic biopsy, and an exact local comparison of ¹¹C-methionine PET image and histological specimens was conducted. The tumor/normal tissue (T/N) ratio of ¹¹C-methionine positron emission tomography was found to correlate better with cell density (R = 0.747, p = 0.000042) and Ki-67 index (R = 0.675, p = 0.00041) than with microvessel density (R = 0.467, p = 0.025) in a histological comparison using a stereotactic image. Furthermore, multiple linear regression analysis revealed that cell density was the key determinant for predicting ¹¹C-methionine level while microvessel density was not. These results suggest that cell density contributes more to ¹¹C-methionine uptake than microvessel density in glioma tissues and that the previously reported correlation of ¹¹C-methionine uptake and microvessel density in glioma patients requires reevaluation.     

Sentinel Lymph Node Identification in Patients With Breast Cancer Using Lymphosonography

The objective of the work described here was to evaluate the efficacy of lymphosonography in identifying sentinel lymph nodes (SLNs) in patients with breast cancer undergoing surgical excision. Of the 86 individuals enrolled, 79 completed this institutional review board–approved study. Participants received subcutaneous 1.0-mL injections of ultrasound contrast agent (UCA) around the tumor. An ultrasound scanner with contrast-enhanced ultrasound (CEUS) capabilities was used to identify SLNs. Participants were administered with blue dye and radioactive tracer to guide SLN excision as standard-of-care. Excised SLNs were classified as positive or negative for the presence of blue dye, radioactive tracer and UCA, and sent for pathology. Two hundred fifty-two SLNs were excised; 158 were positive for blue dye, 222 were positive for radioactive tracer and 223 were positive for UCA. Comparison with blue dye revealed accuracies of 96.2% for radioactive tracer and 99.4% for lymphosonography (p > 0.15). Relative to radioactive tracer, blue dye had an accuracy of 68.5%, and lymphosonography achieved 86.5% (p < 0.0001). Of 252 SLNs excised, 34 were determined to be malignant by pathology; 18 were positive for blue dye (detection rate = 53%), 23 for radioactive tracer (detection rate = 68%) and 34 for UCA (detection rate = 100%) (p < 0.0001). Lymphosonography was similar in accuracy to radioactive tracer and higher in accuracy than blue dye in identifying SLNs. All 34 malignant SLNs were identified by lymphosonography.     

乳腺癌超声造影边缘带增强特征及定量参数与其免疫组化指标的相关性分析

目的分析乳腺癌超声造影边缘带增强特征及定量参数与其免疫组化指标的相关性。方法选取经术后病理及免疫组化证实的乳腺癌女性患者76例(76个病灶),均行超声造影检查,观察乳腺癌病灶边缘带增强特征,获取时间-强度曲线(TIC)的定量参数,包括峰值强度、基础强度、梯度、上升支斜率、达峰时间、曲线下面积,分析乳腺癌超声造影特征及定量参数与免疫组化指标雌激素受体(ER)、孕激素受体(PR)、人类表皮生长因子受体-2(HER-2)、增殖细胞核抗原(Ki-67)的相关性。结果乳腺癌病灶超声造影边缘带放射状增强与ER、Ki-67阳性表达相关,穿支增强与HER-2阳性表达相关,造影后范围扩大与ER、HER-2、Ki-67阳性表达相关,边缘增强特征与Ki-67阳性表达相关,差异均有统计学意义(均P<0.05)。乳腺癌超声造影边缘带定量参数峰值强度、TIC曲线下面积及梯度与Ki-67阳性表达均呈正相关,达峰时间与ER、PR阳性表达均呈负相关,差异均有统计学意义(均P<0.05)。结论乳腺癌超声造影边缘带增强特征及定量参数与ER、PR、HER-2、Ki-67具有一定的相关性,可为乳腺癌患者术前选择精准的治疗方式提供一定的参考依据。     

超声造影在乳腺叶状肿瘤与纤维腺瘤鉴别诊断中的价值

目的探讨超声造影在乳腺叶状肿瘤与纤维腺瘤鉴别诊断中的应用价值。 方法选取我院病理诊断明确且术前进行过超声造影检查的患者（叶状肿瘤23例、纤维腺瘤57例）,回顾性分析其超声造影表现,采用χ²检验或t检验分析乳腺叶状肿瘤与纤维腺瘤在超声造影表现和病理中微血管密度间的关系。 结果乳腺叶状肿瘤与纤维腺瘤比较,边界是否清晰,差异无统计学意义（P>0.05）,而增强顺序、增强强度、均匀性三者比较,差异均有统计学意义（χ²=22.960、24.419、4.901,P均<0.05）。叶状肿瘤的超声造影表现为增强后边界清晰、散在的离心性不均匀低增强。纤维腺瘤超声造影表现为增强后边界清楚、向心性均匀高增强。 结论超声造影对乳腺叶状肿瘤与纤维腺瘤有一定的鉴别诊断价值,可为临床手术方法的选择提供参考。     

Characterization and diagnostic confidence of contrast-enhanced ultrasound for solid renal tumors

The objective was to determine whether contrast-enhanced ultrasound (CEUS) could improve the diagnostic confidence of solid renal masses. CEUS examinations were performed on 51 patients with renal tumors. Histologic findings from surgical specimens (n = 24) or magnetic resonance imaging follow-up (n = 27) were used as reference procedures for definitive diagnosis. Diffuse heterogeneous/homogeneous enhancement and quick peripheralnodularenhancement were found to be characteristic patterns in renal cell carcinoma (RCC). Dotlike or diffuse heterogeneous/homogeneous enhancement and slow peripheral nodular enhancement were observed as typical enhancement patterns in angiomyolipoma. The results show that CEUS combined with conventional ultrasound significantly improves diagnostic confidence. The sensitivity for RCC diagnosis with this imaging approach was 86% and the specificity was 93%. Both positive and negative predictive values of detection were 90% and the overall accuracy was 90%.     

Increased expression of vascular endothelial growth factor and hypoxia inducible factor-1α in lung tissue of patients with chronic bronchitis

ObjectivesVascular endothelial growth factor (VEGF) seems to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD), but its site-specific expression in lung tissue and the relationship with hypoxia inducible factor-1 alpha (HIF-1α) expression in chronic bronchitis (CB) type COPD have not been studied.Design and methodsWe evaluated the expression of VEGF and its receptors in various compartments of lung tissue in three groups: non-smokers with normal lung function (non-smokers, n = 10), smokers without COPD (healthy smokers, n = 10) and smokers with CB (CB, n = 10), using immunohistochemical staining and Western blotting. The expression of HIF-1α was assessed by enzyme-linked immunosorbent assay.ResultsCompared with healthy smokers, VEGF expression in CB was significantly increased in bronchiolar epithelium, vascular endothelium and vascular smooth muscle (p < 0.05). VEGF receptor (VEGFR)-2 expression in CB was also increased in bronchiolar smooth muscle, vascular endothelium and vascular smooth muscle compared with healthy smokers (p < 0.05). The level of HIF-1α was increased in CB compared with healthy smokers and positively correlated with those of VEGF (r = 0.64, p < 0.05).ConclusionVEGF and VEGFR-2 expressions were up-regulated in CB and increased expression of VEGF was related with HIF-1α. HIF-1α-regulated VEGF overexpression may be a characteristic of chronic bronchitis.     

Expression level of VLDL receptor and VLDL-c levels in the malignant and benign breast tumors: The correlation with miRNA-4465 and miRNA-1297

Breast cancer as one of the most prevalent cancers has high morbidity and mortality. Very low-density lipoprotein receptor (VLDLR) is a multifunctional receptor which plays a principal role in the tumor development through affecting cell metastasis and proliferation. The VLDLR as a target for miRNA-4465 and miRNA-1297 was predicted using bioinformatics analysis. Tissue specimens of malignant (n = 50), benign (n = 35) and corresponding normal breast (n = 20) were considered to evaluate the expression of VLDLR using RT-qPCR and western blotting. The VLDL cholesterol (VLDL-C) levels were quantified using a colorimetric assay. The relative VLDLR expression was found in the malignant tumors, which was significantly lower than that in the normal tissues (P<0.05). The expression levels of VLDLR had no significant difference between malignant and benign tissues (P>0.05). Correlation analysis revealed that the VLDLR expression level had a direct correlation with miRNA-1297 (R=0.566, P<0.05), but a reverse one with miRNA-4465 (R = −0.663, P<0.0001). The VLDL-C level in the malignant and normal tissues was lower than that in the benign tumors, which was not significant (P>0.05). The expression levels of VLDLR in E⁺P–H^- (ER⁺,PR⁻,HER2^-) tumors were higher than those in other subtypes (P<0.05). Furthermore, a negative correlation was found between the VLDLR expression level and the Ki 67% score. These data revealed that the lower expression of VLDLR mediated by the high expression levels of miRNA-4465 may be involved in the development of breast cancer. These results might provide some evidence for the effect of VLDLR on the breast cancer.     

Value of Contrast-Enhanced Ultrasound Using Perflubutane Microbubbles for Diagnosing Liver Fibrosis and Cirrhosis in Rats

We determined the ability of contrast-enhanced ultrasound (CEUS) using perflubutane microbubbles to diagnose liver fibrosis and cirrhosis in rats using histology as the reference standard. Fibrosis was induced by oral administration of carbon tetrachloride to 32 Wistar rats. Features with baseline ultrasound (US) and enhancement level of liver and spleen with CEUS were obtained. In the post-vascular phase of CEUS, images of normal livers (n = 5) were significantly brighter than images of fibrotic (n = 6) and cirrhotic livers (n = 13) by quantitative analysis (all p < 0.05). The contrast between livers and spleens in rats with cirrhosis was quantitatively greater than that in normal rats and rats with fibrosis (all p < 0.05). Compared with US, CEUS improved sensitivity from 63% to 84% and accuracy from 71% to 88%. Specificity was 100% for both. The increased value of CEUS in diagnosing liver fibrosis and cirrhosis in rats supports its evaluation in clinical trials.     

Flow Quantification with Nakagami Parametric Imaging for Suppressing Contrast Microbubbles Attenuation

Flow quantification with contrast-enhanced ultrasound is still limited by the effects of contrast microbubble attenuation. Nakagami parametric imaging (NPI) based on the m parameter, which is related to the statistical property of echo envelope, is implemented to suppress contrast attenuation. Flow velocity (FV) and volumetric flow rate (VFR) are estimated through the least square fitting of burst depletion kinetic model to time m parameter curves (TMCs). A non-recirculating flow phantom is imaged as contrast microbubbles are infused at 10, 15, 20, 25, and 30 mL/min. Contrast microbubbles with two different concentrations are used to generate variations of contrast microbubble attenuation. The results suggest that 4 × 4 mm² is the optimal size of a sliding window of NPI for flow quantification under current experiment condition. At a lower microbubble concentration, the FV calculated from TMCs correlates strongly with actual FV in both unattenuated (R² = 0.97; p < 0.01) and attenuated regions (R² = 0.92; p < 0.01) within phantom. And there is a strong correlation (R² = 0.98; p < 0.01; slope = 0.96; intercept = 0.68) between VFR calculated from TMCs and actual VFR within the whole phantom. Similar results are obtained at higher microbubble concentrations. Compared with conventional ultrasound imaging that is intensity dependent, NPI achieves better performance on flow quantification in the presence of contrast microbubble attenuation.