Abstract
Introduction
It is challenging to diagnose liver masses in the context of a rough liver background using computed tomography and magnetic resonance imaging. Perfluorobutane CEUS microbubble, an ultrasound (US) contrast agent, has a unique phase called the Kupffer phase. This study aimed to explore whether the diagnostic ability of Kupffer-CEUS is affected by the rough liver background.
Methods
A prospective analysis was conducted on patients with access to histological pathology of FLLs and liver parenchyma from 23 centers between August 2020 and March 2021. Kupffer-CEUS was performed on 552 patients, who were divided into two groups (normal and abnormal) based on the pathological results of liver parenchyma, and the abnormal group was further divided into four groups (sight liver fibrosis, serious liver fibrosis, fatty liver, mixed). Kupffer-CEUS was divided into vascular phase and vascular and Kupffer phase.
Results
In Kupffer-CEUS, differences in diagnostic efficiency of FLLs between normal and abnormal liver backgrounds were not statistically significant. However, in patients with abnormal liver backgrounds, the specificity (57.45% vs. 74.47%, p = 0.008) and accuracy (89.81% vs. 96.92%, p < 0.001) of the vascular phase in distinguishing malignant from benign lesions were statistically lower than those of the vascular and Kupffer phase (V&KP). Subgroup analysis revealed that the sensitivity, specificity, and accuracy of V&KP in screening FLLs were all higher than those of vascular phase, except for the accuracy in patients with serious liver fibrosis (98.51% vs. 94.06, p < 0.001).
Conclusion
This study indicated that although there is no obvious restriction on the choice of Kupffer-CEUS methods when diagnosing liver masses in patients with normal or abnormal liver backgrounds, Kupffer phase CEUS can effectively reduce the misdiagnosis of FLLs. Particularly for patients with serious liver fibrosis, contrast-enhanced US combined with the Kupffer phase can provide a more accurate diagnosis than conventional contrast-enhanced ultrasonography.
Keywords: Kupffer phase, Vascular phase, Liver tumor, Coarse liver parenchyma, Diagnosis, CEUS
Introduction
Coarse liver parenchyma, commonly found in chronic liver disease patients, is characterized by diffuse or focal fibrosis, hepatocyte proliferation, and hepatocyte keratinization in liver tissue [1], leading to fibrosis and loss of normal liver structure and function [2, 3]. Coarse liver parenchyma is usually a manifestation of chronic liver diseases such as hepatitis and cirrhosis [4, 5], severely affecting liver function and health [6, 7], potentially leading to various lesions, such as cirrhosis, regenerative nodule, tumors, liver cysts, etc. Timely diagnosis, controlling disease progression, and implementing comprehensive treatment measures are essential for managing these lesions, with imaging techniques including computed tomography (CT), magnetic resonance imaging (MRI), ultrasound (US), and contrast-enhanced ultrasound (CEUS) playing critical roles in diagnosis. Gadolinium-based extracellular contrast-enhanced MRI and iodine-based contrast-enhanced CT (CECT) have long been the imaging gold standards for preoperative assessment of focal liver lesions (FLLs) [8].
FLLs exhibit numerous physiological variations in CT or MRI images, such as pathological blood vessels and bile duct hyperplasia, which can easily interfere with the diagnosis of lesions [9]. Some lesions may not be easily detected in the early stages and require a combination of clinical symptoms and laboratory tests for accurate diagnosis. Overlapping blood supply patterns in cirrhotic nodules further challenge definitive diagnosis [10, 11]. CEUS is recognized as a second-line option for diagnosis hepatocellular carcinoma (HCC) when CT or MRI results are inconclusive by the Japanese, Canadian, Italian, and Asia-Pacific hepatology or liver cancer societies, after CT or MRI when these are inconclusive or contraindicated [12–14].
The World Federation for Ultrasound in Medicine and Biology (WFUMB) CEUS guidelines do not address the diagnostic effectiveness of liver nodules in coarse liver parenchyma caused by various etiologies [14, 15]. The CEUS LI-RADS guidelines demonstrate diagnostic accuracy in differentiating malignant from benign FLLs in at-risk patients, with pooled sensitivity of 0.92 and pooled specificity of 0.87 and are vital in assessing cirrhotic individuals for HCC [16, 17]. Additionally, the AFSUMB Consensus Statements highlight the diagnostic difficulties in differentiating FLLs in a cirrhotic background [12].
Commonly used contrast agents for US include sulfur hexafluoride and perfluorobutane (PFB). PFB microbubbles (Sonazoid, GE Healthcare, Oslo, Norway), a second-generation ultrasound contrast agent, allow similar depictions of the macrovasculature and microvasculature as seen with sulfur hexafluoride and act as blood pool agents in the early vascular phase [16]. PFB is engulfed by Kupffer cells in the liver sinusoidal endothelial system and some benign solid lesions, providing high-contrast functional imaging in the post vascular phase, known as “Kupffer phase.” It has a unique phase compared to traditional contrast agents, with imaging time exceeding 1 h, and it can withstand an intermediate mechanical index (MI) which implies greater penetration power, thus providing additional diagnostic time and information. This may help improve the diagnostic performance of CEUS in cases with FLLs and rough liver backgrounds [13, 18–20].
Therefore, we aimed to explore whether the ultrasound enhancement technology with the unique Kupffer phase can optimize the diagnosis process and improve the diagnostic efficiency of benign and malignant lesions in different liver backgrounds. Due to the diversity of different liver backgrounds, in order to avoid patient population selection bias and make the research design more scientific and accurate, we conducted a multicenter large sample study.
Materials and Methods
This was a prospective, multicenter, noncontrolled clinical study; a summary of the protocol is available online (Identifier: NCT04563897, https://clinicaltrials.gov/). The study protocol involved 23 centers (online suppl. Table E1; for all online suppl. material, see https://doi.org/10.1159/000543501). The detailed research protocol, including eligibility criteria and principles of accessing standardized data, was used at every institution.
Patients
All patients scheduled to undergo CEUS for FLLs between August 2020 and March 2021 were considered for enrollment. Patients were excluded from the study if CEUS or histological examinations of target lesions and liver background could not be performed. The flow diagram of the study is shown in Figure 1.
Fig. 1.
Flowchart indicating patient selection according to the diagnosis procedures based on different liver backgrounds. CEUS, contrast-enhanced ultrasound.
Based on Kupffer Phase Contrast-Enhanced US Technology
Real-time contrast-enhanced ultrasound examinations were performed with a contrast-specific ultrasound platform (including GE, Mindray, Siemens, Philips) with a C5-1 MHz convex or 4V-1 MHz convex probe. PFB (Sonazoid; GE Healthcare, Oslo, Norway) was used as the contrast agent. The CEUS parameters followed the recommendations of the contrast agent and ultrasound machine manufacturers. The mechanical index (MI) was ranged from 0.16 to 0.21, and the dynamic range were set at 40 dB. The acoustic focus was placed below the target lesions, and a dual-screen format was used for all CEUS examinations, displaying a gray-scale image. The examined sections included the largest diameter of the target lesion and the surrounding liver parenchyma. The regions were imaged continuously for the first 120 s and then intermittently imaged for 5 s per minute until 12 min had elapsed or the liver parenchymal enhancements had disappeared. All CEUS procedures were performed by radiologists with over 2 years of experience performing CEUS examinations.
The radiologists at each center, with more than 2 years of experience in CEUS, provided initial diagnoses immediately after imaging was completed. Senior radiologists, all with more than 8 years of experience in CEUS, then review the vascular phase images to obtain the diagnosis results, and then review the Vascular and Kupffer phase to obtain the diagnosis results, and the final results (vascular phase and Vascular and Kupffer phases) will be summarized to the main center. The radiologists were blinded to the MRI/CT and pathologic results at the time of operation. The clinical, imaging, and pathologic data uploaded by each center were summarized by another physician. If there was any disagreement between the CEUS and pathologic diagnosis, arbitration was conducted by another radiologist (X.L., Yu.) with 30 years of experience in abdominal ultrasound and 15 years in CEUS, who was blinded to the previous CEUS diagnostic results, other imaging and histopathology results, and independently reviewed the stored images. The operators and reviewers followed the World Federation for Ultrasound in Medicine and Biology (WFUMB) CEUS guidelines outlined in online supplementary Table E2 [14].
The Reference Standard
Diagnosis of target lesions and liver background is based on histopathologic findings (biopsy or surgical excision). Pathology reports on FLLs and liver background are typically described by two experienced pathologists from each hospital. For questionable pathologic diagnoses, another pathologist with 10 years of experience in hepatic pathology was asked to verify the diagnosis. Liver background definitions: (1) normal liver background: percentage of hepatocytes containing large or medium-sized lipid droplets is less than 5%, and degree of liver fibrosis is equal to 0; (2) slight liver fibrosis background: percentage of hepatocytes containing large or medium-sized lipid droplets is less than 5%, and degree of liver fibrosis is ≥1; (3) serious liver fibrosis background: percentage of hepatocytes containing large or medium-sized lipid droplets is less than 5%, and degree of liver fibrosis is ≥3; (4) fatty liver background: percentage of hepatocytes containing large or medium-sized lipid droplets is ≥5%, and degree of liver fibrosis is ≤1; (5) mixed liver background: percentage of hepatocytes containing large or medium-sized lipid droplets is ≥5%, and degree of liver fibrosis is ≥2. The histopathologic diagnoses were routinely described in the pathologic reports at each hospital by one of two experienced pathologists. For questionable pathologic diagnoses, another pathologist with 10 years of experience in hepatic pathology was asked to verify the diagnosis. For the flow diagram, see Figure 2.
Fig. 2.
Flowchart indicating classification of different liver backgrounds according to the CEUS imaging examinations.
Statistical Analysis
Descriptive analyses were used to describe the data, with frequency and percentage for categorical variables, and mean ± SD for continuous variables. The diagnostic efficacy of FLLs across normal and abnormal liver backgrounds, as well as vascular and Kupffer phases, was evaluated by calculating the sensitivity, specificity, accuracy, positive predictive value (PPV), negative predictive value (NPV), and area under the curve (AUC). Chi-square tests and Fisher’s exact tests were used to compare the difference of sensitivity, specificity, and accuracy between different liver backgrounds and diagnosis approaches (vascular phase vs. Kupffer phase). Subgroup analyses with stratified diagnostic approaches and abnormal liver backgrounds were conducted to further explore the diagnostic performance of CEUS. For comparing diagnostic performance between two groups, the following statistical methods were used: Pearson’s chi-square test when n ≥ 40 and T ≥ 5; Yates’ continuity correction test when n ≥ 40 and 1 ≤ T ≤ 5; and Fisher’s exact test when n < 40 or any cell has T < 1. For comparisons involving more than two groups, Pearson’s chi-square test was applied when n ≥ 40 and T ≥ 5, and Fisher’s exact test was used in all other cases. Here, “n” refers to the total sample size, and “T” denotes the expected frequency in each cell of the contingency table. A p value of <0.05 was considered statistically significant for two-group comparisons. For multiple-group comparisons, pairwise comparisons were performed with Bonferroni correction (in this study, with five groups, statistical significance was set at p < 0.005). All statistical analyses were performed using SPSS 25.0.
Results
Of these, 408 patients were excluded due to poor image quality (n = 4), undefined CEUS diagnosis (n = 11), unavailability of target lesion pathological results (n = 164), or lack of defined liver background pathological results (n = 199). The remaining 552 patients were included in the study. The patient characteristics are summarized in Table 1.
Table 1.
Demographics classified by existence of newly identified nodules
| Characteristic | Malignant (n = 473) | Benign (n = 79) |
|---|---|---|
| Age, years, median (range) | 57 (25–92) | 50 (20–79) |
| Sex, men/women | 380/93 | 45/34 |
| Preoperative serum AFP level, ng/mL | ||
| <20, n (%) | 289 (61.1) | 75 (94.9) |
| 20–400, n (%) | 100 (21.1) | 1 (1.3) |
| >400, n (%) | 84 (17.8) | 3 (3.8) |
| Tumor size, mm, mean (range) | 44.32±27.9 (6–167) | 45.03±28.9 (12–129) |
| Liver background | ||
| Normal, n (%) | 98 (20.7) | 32 (40.5) |
| Slight liver fibrosis, n (%) | 103 (21.8) | 11 (13.9) |
| Serious liver fibrosis, n (%) | 191 (40.4) | 11 (13.9) |
| Fatty liver, n (%) | 29 (6.1) | 15 (19.0) |
| Mixed, n (%) | 52 (11.0) | 10 (12.7) |
| Pathological classification | ||
| HCC, n (%) | 333 (70.4) | |
| Metastatic, n (%) | 99 (20.9) | |
| ICC, n (%) | 31 (6.6) | |
| Other malignant, n (%) | 10 (2.1) | |
| FNH, n (%) | 18 (22.8) | |
| Hemangioma, n (%) | 16 (20.2) | |
| DN/RN, n (%) | 14 (17.7) | |
| Abscess, n (%) | 10 (12.7) | |
| Other benign, n (%) | 21 (26.6) | |
Unless otherwise noted, data are numbers of patients or observations, with percentages in parentheses.
Comparative Analysis of Diagnostic Efficacy in FLLs across Normal-Abnormal Liver Backgrounds and Vascular-V&K Phases
During the vascular phase, CEUS diagnostic performance was similar across different liver backgrounds. Differences in sensitivity (90.82% vs. 93.87%, p = 0.284), specificity (75.00% vs. 57.45%, p = 0.109), and accuracy (86.92% vs. 89.81%, p = 0.335) between the combines with normal and abnormal liver backgrounds were not statistically significant. However, in the V&KP, sensitivity in the normal liver background combine was statistically lower than in the abnormal liver background combine (96.94% vs. 99.73%, p = 0.029). Differences in specificity (90.63% vs. 74.47%, p = 0.072) and accuracy (95.38% vs. 96.92%, p = 0.573) between the two combines were not statistically significant.
In patients with a normal liver background, the sensitivity (90.82% vs. 96.94%, p > 0.999), specificity (75.00% vs. 90.63%, p = 0.147), and accuracy (86.92% vs. 95.38%, p = 0.176) of the vascular phase were less than in the V&KP, though the differences were not statistically significant. For patients with abnormal liver backgrounds, specificity (57.45% vs. 74.47%, p = 0.008) and accuracy (89.81% vs. 96.92%, p < 0.001) of the vascular phase was statistically lower than in the V&KP, while the difference in sensitivity (93.87% vs. 99.73%, p > 0.999) between the two combines was not statistically significant. Therefore, for patients with rough liver background, CEUS diagnostic performance can be enhanced by incorporating the Kupffer phase. The detailed results are summarized in Table 2, and a representative case is provided in Figure 3.
Table 2.
Comparison of diagnostic efficacy between different combines
| Sensitivity | Specificity | Accuracy | PPV | NPV | AUC | |
|---|---|---|---|---|---|---|
| Combine I (N = 130) | 90.82% (89/98) | 75.00% (24/32) | 86.92% (113/130) | 91.75% (89/97) | 72.73% (24/33) | 0.829 |
| Combine II (N = 422) | 93.87% (352/357) | 57.45% (27/47) | 89.81% (379/422) | 94.62% (352/372) | 54.00% (27/50) | 0.757 |
| Combine III (N = 130) | 96.94% (95/98) | 90.63% (29/32) | 95.38% (124/130) | 96.94% (95/98) | 90.63% (29/32) | 0.938 |
| Combine IV (N = 422) | 99.73% (374/375) | 74.47% (35/47) | 96.92% (409/422) | 96.89% (374/386) | 97.22% (35/36) | 0.871 |
| I vs. II (p value) | 0.284 | 0.109 | 0.335 | – | – | – |
| III vs. IV (p value) | 0.029 | 0.072 | 0.573 | – | – | – |
| I vs. III (p value) | >0.999 | 0.147 | 0.176 | – | – | – |
| II vs. IV (p value) | >0.999 | 0.008 | <0.001 | – | – | – |
Combine I: vascular phase and normal liver background; combine II: vascular phase and abnormal liver background; combine III: V&K phase and normal liver background; combine IV: V&K phase and abnormal liver background; PPV, positive predictive value; NPV, negative predictive value; AUC, area under curve.
Fig. 3.
A contrast-enhanced ultrasound of a case was pathologically confirmed dysplastic nodule (DN) in serious liver fibrosis liver background. In vascular phase examination, this lesion was diagnosed as HCC. In V&K phase examination, it was diagnosed as DN. Arterial phase hyperenhancement (APHE) observation in the liver (a, arrows). In portal venous, the lesion shows isoenhancing (b). And hypoenhancement was noted in the late phase (the late phase is initiated after 120 s and ends within 4-8 min) (c). Because DN contains Kupffer cells, contrast agents are taken up during the Kupffer phase. So, in Kupffer phase, the lesion shows mild hypoenhancement (d).
Comparative Analysis of Diagnostic Efficacy in HCC, Metastasis, Hemangioma, and FNH across Normal-Abnormal Liver Backgrounds and V&KP
Patients with HCC, metastasis, hemangioma, and FNH were stratified, and the results are presented in online supplementary Tables E3–E5. During the vascular phase, differences in sensitivity and specificity between normal and abnormal liver backgrounds for malignant lesions were not statistically significant, except for accuracy in metastatic nodules, which was significant (p = 0.015). In the V&KP, sensitivity for diagnosing HCC was statistically lower in patients with normal liver backgrounds compared to those with abnormal backgrounds. Conversely, specificity was statistically higher in patients with normal liver backgrounds. And, sensitivity for diagnosing metastatic was statistically higher in patients with normal liver backgrounds compared to those with abnormal backgrounds. See online supplementary Tables E3–E4 for details. For benign lesions, differences in sensitivity, specificity, and accuracy between normal and abnormal liver backgrounds were not statistically significant in either the vascular or V&KP.
For malignant lesions in patients with normal liver backgrounds, differences in sensitivity and specificity between the vascular and V&KP were not statistically significant. However, for benign lesions, accuracy in the vascular phase was statistically lower than in the V&KP. In the abnormal liver background, in patients with both malignant and benign lesions, specificity and accuracy in the vascular phase were statistically lower than in the V&KP, while differences in sensitivity between the phases were not statistically significant.
Subgroup Analysis Based on Vascular-V&K Phases
In the vascular phase examination, the differences in sensitivity and specificity for FLLs screening among patients with normal liver background, slight liver fibrosis, serious liver fibrosis, fatty liver, and mixed liver background were not statistically significant. However, differences in accuracy among these groups were statistically significant (p = 0.021). A multi-comparison analysis revealed that patients with serious liver fibrosis showed higher accuracy than those with fatty liver (94.06% vs. 79.55%, Bonferroni corrected p = 0.0047). See Table 3 for details.
Table 3.
Comparison of diagnostic efficacy of vascular phase between different liver background
| Liver background | Sensitivity | Specificity | Accuracy | PPV | NPV | AUC |
|---|---|---|---|---|---|---|
| Normal (N = 130) | 90.82% | 75.00% | 86.92% | 91.75% | 72.73% | 0.829 |
| Serious liver fibrosis (N = 202) | 96.86% | 45.45% | 94.06% a | 96.86% | 45.45% | 0.712 |
| Slight liver fibrosis (N = 114) | 91.26% | 63.63% | 88.60% | 95.92% | 43.75% | 0.774 |
| Fatty liver (N = 44) | 89.66% | 60.00% | 79.55% a | 81.25% | 75.00% | 0.748 |
| Mixed (N = 62) | 90.38% | 60.00% | 85.48% | 92.16% | 54.55% | 0.752 |
| p | 0.073 | 0.495 | 0.021 | – | – | – |
PPV, positive predictive value; NPV, negative predictive value; AUC, area under curve.
aBonferroni corrected p < 0.005.
In V&KP examination, differences in sensitivity, specificity, and accuracy for FLLs screening among patients with normal liver background, slight liver fibrosis, serious liver fibrosis, fatty liver, and mixed liver background were not statistically significant. See Table 4.
Table 4.
Comparison of diagnostic efficacy of V&K phase between different liver background
| Liver background | Sensitivity | Specificity | Accuracy | PPV | NPV | AUC |
|---|---|---|---|---|---|---|
| Normal (N = 130) | 96.94% | 90.63% | 95.38% | 96.94% | 90.63% | 0.938 |
| Serious liver fibrosis (N = 202) | 100% | 72.72% | 98.51% | 98.45% | 100.00% | 0.864 |
| Slight liver fibrosis (N = 114) | 99.03% | 72.73% | 96.49% | 97.14% | 88.89% | 0.859 |
| Fatty liver (N = 44) | 100% | 80.00% | 93.18% | 90.60% | 100% | 0.900 |
| Mixed (N = 62) | 100% | 70.00% | 95.16% | 94.55% | 100% | 0.850 |
| p | 0.098 | 0.339 | 0.185 | – | – | – |
PPV, positive predictive value; NPV, negative predictive value; AUC, area under curve.
Subgroup Analysis Based on Abnormal Liver Background
The sensitivity, specificity, and accuracy of the V&KP in screening FLLs were all above than those of vascular phase, regardless of the cause of the abnormal liver background. However, the differences were not statistically significant, except for the accuracy in patients with serious liver fibrosis (98.51% vs. 94.06, p < 0.001). See Table 5 for details.
Table 5.
Comparison of diagnostic efficacy between vascular phase and V&K phase in different abnormal liver background subgroup
| Liver background | Sensitivity | Specificity | Accuracy | PPV | NPV | AUC | |
|---|---|---|---|---|---|---|---|
| Serious liver fibrosis (N = 202) | VP | 96.86% | 45.45% | 94.06% | 96.86% | 45.45% | 0.712 |
| V&KP | 100% | 72.72% | 98.51% | 98.45% | 100.00% | 0.864 | |
| p value | NA | 0.182 | <0.001 | – | – | – | |
| Slight liver fibrosis (N = 114) | VP | 91.26% | 63.63% | 88.60% | 95.92% | 43.75% | 0.774 |
| V&KP | 99.03% | 72.73% | 96.49% | 97.14% | 88.89% | 0.859 | |
| p value | >0.999 | 0.491 | 0.063 | – | – | – | |
| Fatty liver (N = 44) | VP | 89.66% | 60.00% | 79.55% | 81.25% | 75.00% | 0.748 |
| V&KP | 100% | 80.00% | 93.18% | 90.60% | 100.00% | 0.9 | |
| p value | NA | 0.523 | 0.101 | ||||
| Mixed (N = 62) | VP | 90.38% | 60.00% | 85.48% | 92.16% | 54.55% | 0.752 |
| V&KP | 100% | 70.00% | 95.16% | 94.55% | 100% | 0.85 | |
| p value | NA | 0.5 | 0.053 | – | – | – | |
VP, vascular phase; V&KP, vascular and Kupffer phase; PPV, positive predictive value; NPV, negative predictive value; AUC, area under curve.
Discussion
This large, prospective, multicenter study used pathology gold standards for validation and optimized and improved the diagnostic process and performance or assessing rough liver parenchyma through the application of novel technologies. This ensures that CEUS provides more accurate diagnostic information for patients with FLLs. The large sample size of 552 patients and the use of histological examination as the reference standard also make the results reliable. All the liver nodules and liver parenchyma were ultimately diagnosed by histological examination, considered the most robust reference method [8, 21]. To the best of our knowledge, this is the first prospective study to compare the diagnostic performance of contrast-enhanced ultrasound for FLLs in different liver backgrounds [22–24]. This study indicated that although there is no significant restriction on the choice of Kupffer-CEUS methods when diagnosing liver masses in patients with normal or abnormal liver background (p > 0.05), the presence of the Kupffer phase may facilitate a more accurate diagnosis in patients with diffuse liver disease.
In our study, although the diagnostic specificity (75.00% vs. 57.45%, p = 0.109; 90.63% vs. 74.47%, p = 0.072) of both types of CEUS decreased in the abnormal group, there was no significant difference in diagnostic efficiency between the normal group and abnormal group in either the vascular phase or V&KP examination. Therefore, we can conclude that these two methods of contrast-enhanced ultrasonography are equally effective in diagnosing FLLs in patients with normal or abnormal liver backgrounds. However, in patients with diffuse liver lesions, the combination of the Kupffer phase improved the diagnostic efficiency, especially in terms of specificity (57.45% vs. 74.47%, p = 0.008) and accuracy (89.81% vs. 96.92%, p < 0.001). Further stratification analysis of specific lesion pathology types yielded similar results. The presence of diffuse liver disease is more likely to increase the false-positive rate of HCC lesions and the false-negative rate of metastatic lesions, although its impact on the diagnosis of benign lesions was not significant. Similarly, we also found that when combined with the Kupffer phase, especially when patients suffer from diffuse liver disease, the diagnostic performance for various diseases is improved to varying degrees. Therefore, we believe that V&K phase CEUS can effectively reduce the misdiagnosis of hepatic space-occupying lesions.
The superiority of CEUS combined with the Kupffer phase may be attributed to the unique characteristics of the PFB contrast agent, such as gas solubility, bubble size distribution, and bubble stability. PFB microbubbles have high toughness: they can pass through a filter screen with a smaller diameter than their own microbubbles without deformation, and their flexible but stable nature also means that the microbubbles have good elasticity, can reach deep tissues and tumor microvessels, and can be evenly distributed [25]. Therefore, when using PFB contrast agent for liver imaging, it can withstand an intermediate MI [12]. An intermediate MI means higher ultrasound energy, which implies greater penetration power. Fixed scanning for long periods and repeated scanning of the whole liver can be accomplished, reducing the false-positive rate and the number of missed lesions [26, 27]. This can overcome the influence of a rough liver background on diagnosis FLLs. Therefore, there is no difference in diagnostic performance between the normal group and abnormal group using the vascular phase of PFB-CEUS. In addition, PFB microbubbles can be phagocytosed by the reticuloendothelial system, particularly the liver sinusoid Kupffer cells, and microbubbles containing PFB may provide long-term (up to 2 h post-injection) parenchyma-specific enhancement [28, 29]. The normal liver parenchyma contains a normal number of Kupffer cells, but malignant lesions usually contain fewer or no Kupffer cells, resulting in little or no engulfment of microbubbles [30–32]. This long-lasting Kupffer phase, markedly different from the vascular phase imaging mechanism, can provide additional diagnostic information. Therefore, the unique features of PFB can improve its diagnostic accuracy, especially in rough liver background.
Subgroup analysis revealed that relying solely on vascular phase diagnosis is more likely to be misled by the background of fatty liver and severe liver fibrosis, resulting in a deviation in diagnostic accuracy (79.55% vs. 94.06%, Bonferroni corrected p = 0.0047). This finding is consistent with actual clinical knowledge. Further research demonstrated that the contrast-enhanced ultrasound of V&KP can effectively reduce diagnostic errors. The diagnostic performance among patients with normal liver background, slight liver fibrosis, serious liver fibrosis, fatty liver, and mixed liver background were not statistically significant (p > 0.05). Specifically, for patients with serious liver fibrosis, contrast-enhanced ultrasound combined with Kupffer phase can provide more accurate diagnosis than conventional contrast-enhanced ultrasonography (98.51% vs. 94.06, p < 0.001). Therefore, for patients with severe liver fibrosis, contrast-enhanced ultrasound with the Kupffer phase should be the first choice to obtain more accurate diagnostic results.
Our study has several limitations. First, this study included 552 FLL patients from 23 research centers, and in order to obtain clear pathological results, the number of benign lesions in each group was relatively small. However, actual clinical practice is still assessed through the above clinical setting. Another limitation is the exclusive use of perflubutane microbubbles as an ultrasonic contrast agent. Sulfur hexafluoride microbubbles were often used in comparative studies in some previous reports [27, 33–35]. However, some studies have suggested that the application of perflubutane microbubbles can obtain vascular phase diagnostic results similar to those of sulfur hexafluoride microbubbles, and the Kupffer phase can improve the diagnosis [27, 33, 35]. Therefore, this study focuses on whether the new Kupffer phase can provide more diagnostic information, exploring whether V&K CEUS can be used as a supplementary method to CT/MRI and other imaging technologies to achieve better diagnostic efficiency, optimize the diagnostic process of rough liver backgrounds, and better assist the clinic practice. Similarly, this study also lacks research data on CECT or CEMRI. We will further investigate the diagnostic performance differences of CECT or CEMRI sequential or contrast-enhanced ultrasound in patients with specific liver focal lesions on different rough liver backgrounds.
Acknowledgments
We would like to thank for the support of clinical cases from Prof. Yuanyuan Yan (Zhengzhou Central Hospital), Prof. Kai Zhang (The First Affiliated Hospital of Xinxiang Medical University), Prof. Shaochun Wang (Affiliated Hospital of Jining Medical University), Prof. Zubang Zhou (Gansu Provincial Hospital), Prof. Bing Hu & Lei chen (The Sixth Affiliated Hospital of Shanghai Jiao Tong University), Prof. Gaiqin Xue (Shanxi Provincial Cancer Hospital), Prof. Ruiqing Du (Shijiazhuang Fifth Hospital), Prof. Mingxing Xie (Wuhan Union Hospital), the effort of clinical data collection from Xu Wang (Tianjin Third Central Hospital), Jinshu Pang (The First Affiliated Hospital of Guangxi Medical University) and Pingping Zhou (Tongji Hospital, Tongji Medical College).
Statement of Ethics
This study protocol was reviewed and approved by [Ethics Committee of Chinese PLA General Hospital], Approval No. (S2020-300-01). Written informed consent to participate in the study has been obtained from all adult participants.
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
Funding Sources
This study has received funding by Grants 92159305, 82227804, 82172027 from the National Scientific Foundation Committee of China, Grants ZZ16-XRZ-022 from the CACMS Fundamental Research Business Funds.
Author Contributions
Concept and design: P.L, X.L.Y, and Y.L.L; experiments and procedures: Y.L.L, X.J., H.Y., K.Y.L., W.W, S.C.L., H.D., G.J.L., W.C., G.Z.H., K.L., Y.L., L.P.L., T.A.J., G.M.Z., X.Y.X., Z.G.S., F.Y. L., J.Y., Z.Y.H., Z.G.C., S.L.T., J.Q.X., X.J.D., E.P.Q., J.D.Y., and X.C.D.; writing of article: Y.L.L. and X.L.Y.; statistical analysis: Y.L.L.; supervision and graduate mentor: P.L. and X.L.Y.
Funding Statement
This study has received funding by Grants 92159305, 82227804, 82172027 from the National Scientific Foundation Committee of China, Grants ZZ16-XRZ-022 from the CACMS Fundamental Research Business Funds.
Data Availability Statement
The data that support the findings of this study are not publicly available due to their containing information that could compromise the privacy of research participants but are available from the corresponding author upon reasonable request.
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Associated Data
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Data Availability Statement
The data that support the findings of this study are not publicly available due to their containing information that could compromise the privacy of research participants but are available from the corresponding author upon reasonable request.