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doi:10.1111/jgh.12789
H E PAT O L O G Y
Assessment of liver fibrosis: The relationship between point shear wave elastography and quantitative histological analysis Hong Ding,* Jiao-jiao Ma,* Wen-ping Wang,* Wen-jiao Zeng,† Tao Jiang,† Bei-jian Huang* and Shi-yao Chen‡ Departments of *Ultrasound and ‡Gastroenterology, Zhongshan Hospital, Fudan University, and †Department of Pathology, Shanghai Medical College, Fudan University, Shanghai, China
Key words collagen proportionate area, liver fibrosis, liver stiffness measurement, point shear wave elastography. Accepted for publication 21 August 2014. Correspondence Professor Wen-ping Wang, Department of Ultrasound, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, China. Email: [email protected] Potential conflicts of interest: No conflict of interest is declared.
Abstract Background and Aim: Traditional pathological scoring systems for liver fibrosis progression are predominantly based on the description of architectural changes with no consideration of the amount of collagen fiber deposition. Our purpose was to explore a true histological standard in accordance with the liver stiffness measured by point shear wave elastography (PSWE) in patients with chronic hepatitis B. Methods: A total of 78 patients with liver neoplasms underwent liver stiffness measurements with PSWE as well as biochemical investigations within 3 days before partial hepatectomy. One tissue section of the liver specimens was stained with HE trichrome and evaluated traditionally with the Scheuer scoring system. The other tissue section was stained with picroSirius red and was evaluated according to the semiquantitative Chevallier et al. scoring system. In addition, this second tissue section was evaluated for the collagen proportionate area (CPA) with computer-assisted digital image analysis. The reproducibility of PSWE technology was explored through the intra-class correlation coefficient of a reliability analysis. Results: The PSWE technology revealed good reproducibility in liver stiffness measurements, and the PSWE values increased with the pathological severity of liver fibrosis on both the Scheuer scoring system and the semiquantitative Chevallier et al. scoring system. PSWE values exhibited more reasonable relationships with CPA (r = 0.628, P = 0.00 < 0.05) than with the Scheuer scoring system (r = 0.473, P = 0.00 < 0.05) or the Chevallier et al. semiquantitative scoring system (r = 0.487, P = 0.00 < 0.05). Conclusion: CPA is a better pathological parameter than traditional semiquantitative scoring systems in accordance with liver stiffness measured by PSWE technology.
Introduction The continuous development of liver fibrosis is a common histopathological characteristic of all chronic liver diseases. Significant advancements in the understanding of the cellular and molecular mechanisms of liver fibrogenesis have revealed the progressive substitution of the functioning liver parenchyma with fibrotic tissue; currently, major efforts are directed at translating these acquisitions with diagnostic and therapeutic applications.1–5 The standard pathological examination of liver tissue is invasive; furthermore, the common histological scoring system describes the morphological features based on architectural changes of liver lobules, rather than the amount of collagen, which represents the direct cause of liver fibrosis.6–10 Therefore, the amount of collagen plays a key role in personalized treatment, follow-up and the evaluation of liver fibrosis to accurately and non-invasively assess the progression of fibrosis and non-invasively in the clinical setting.
Several investigators had reported non-invasive approaches for the quantitative diagnosis of liver fibrosis, such as routine laboratory tests, serum markers of fibrosis, radiological imaging, and elastography. Elastography is an exciting new technology that estimates liver fibrosis through the measurement of liver stiffness. Elastography is accurate, safe, cheap, quick and widely applicable, and could reduce the need for liver biopsy in chronic hepatitis patients. Point shear wave elastography (PSWE), which is integrated in a conventional ultrasound machine and can be performed with conventional ultrasound probes during an abdominal ultrasound scan, is a novel shear wave-based elastography technology. Previous studies had reported that PSWE (or ElastPQ technique, Philips Healthcare, Seattle, WA, USA) is a valid and reproducible non-invasive technology to measure liver stiffness among chronic hepatitis patients and liver fibrosis is the dominating factor affecting liver stiffness measured by PSWE.11–14 However, PSWE is limited in differentiating the stage of fibrosis (i.e. differentiating S0 from S1
Journal of Gastroenterology and Hepatology 30 (2015) 553–558 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
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Ultrasound elastography of liver fibrosis
H Ding et al.
and S2 from S3) according to the Scheuer scoring system. Researchers have reported that other elastography technologies are also unable to accurately differentiate the stages of fibrosis according to traditional histological scores system, which have been widely used as the “golden standard” to validate non-invasive markers of liver fibrosis and to evaluate the therapeutic effects of antifibrotic treatment. Thus, traditional histological scores systems may be inappropriate for the assessment of liver stiffness. The traditional histological scores systems use numerical category labels to evaluate the stage of liver fibrosis instead of measuring the amount of liver fibrosis quantitatively. The assigned numbers or scores of categories are not quantitatively related and are not continuous variables. Applying these fibrosis stage scores as numerical data in statistics is incorrect and misleading. As scoring systems do not quantify fibrosis, fibrosis progression or regression has historically been evaluated in terms of a one- or two-stage change. Furthermore, cirrhosis is only classified as a single stage histologically, representing the most severe categorical assignment, with no histological subclassification.15 An alternative to numerical fibrosis scoring systems is the direct measurement of the amount of fibrosis in the biopsy specimen by computer-assisted digital image analysis. Although the methodology is not standardized, a number of publications have described methods for quantifying liver fibrosis by imaging analysis, and all publications have yielded similar results. The methods, however, are time consuming compared with simple histological scoring, and the necessary equipment and expertise are not widely available. However, in a study to evaluate the progression or regression of fibrosis, measuring the amount of fibrosis as precisely as possible seems appropriate.16–18 The traditional histological scores systems largely assess the predominant architectural changes and sites of fibrosis irrespective of collagen fiber thickness or density, which was constrained by trichrome or reticulin stains. Routine histological evaluation of fibrosis is often performed with trichrome or reticulin stains, which are generally used for connective tissue and are not selective for collagen; the amount of trichrome or reticulin staining did not necessarily correspond to the amount of liver collagen. Computerassisted digital image analysis of histological sections, histochemically stained by the picroSirius red technique, is a method to measure fibrosis morphologically. PicroSirius red staining primarily identifies tissue collagen. The quantity of bound stain correlates well with chemically determined collagen content and morphometrically determined liver fibrosis.19–21 Shear wave elastography imaging including ElastPQ technology is a method for detecting liver stiffness, which largely corresponds to the amount of liver collagen. The main purpose of our study was to discuss the relationship between liver stiffness measured with the PSWE technology and the amount of liver collagen based on the picroSirius red stain, and to explore a true histological standard in accordance with the liver stiffness measurement to validate non-invasive markers of liver fibrosis in a series of patients with chronic hepatitis B.
Methods Patients. Between October 2012 and February 2013, we prospectively enrolled patients with hepatic neoplasms with clinical 554
Table 1
Characteristics of the included patients (n = 78)
Characteristics
Measured values/normal range
Age (years) Gender (male) ALT (IU/L) AST (IU/L) γ-GT (IU/L) Globulins (g/L) γ-Globulins (g/L) Platelet count ( × 109/L)
49 ± 18 (24∼78) 52 (66.7%) (36 ± 29)/(9∼40) (39 ± 30)/(15∼40) (86 ± 132)/(10∼60) (28 ± 5)/(20∼40) (5 ± 2)/(3∼5) (162 ± 51)/(125∼350)
Results are reported as means ± standard deviation or n (%). ALT, alanine aminotransferase; AST, aspartate aminotransferase; γ-GT, Gamma-glutamyltransferase.
indications for hepatic surgery in our hospital. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki, and all patients gave informed consent for the procedures and the histological evaluation. All patients underwent conventional ultrasound examination and elastography, as well as hematological, biochemical, and virological investigations within 3 days before surgery. We excluded patients who failed to meet the inclusion criteria of partial hepatectomy because of hypohepatia and obese patients (BMI ≥ 35 kg/m2). The characteristics of the enrolled patients are summarized in Table 1. Ultrasound measurements. Measurements of liver stiffness were obtained during the conventional ultrasound scan by two sonographers (with 24 and 2 years of experience on ultrasound examination). They sonographers received training in liver stiffness measurements for 2 days before the study began. An ultrasound system (iU elite; Philips Healthcare, Bothell, WA, USA) with a convex transducer (C5-1) was used for the conventional ultrasound scan and liver stiffness measurement. The subjects were requested to lie supine with their right arm abducted. The operator positioned the probe on the right lobe the liver through the seventh to the 10th intercostal space using real-time B-mode imaging to locate a fixed region of interest (0.5 cm × 1.5 cm) in the liver free of visible ducts or vessels. The measurement location was selected with a cursor moved by a trackball. The subject was instructed to hold his or her breath while the operator hit the “update” button that launched the ultrasound data acquisition sequence. Approximately 5 s were required for each data acquisition, and the parameter of liver stiffness was expressed in kilopascals (kPa) of Young’s modulus. Ten measurements of liver stiffness were obtained in the same location of the right liver lobe of every patient. The median value of the successful measurements was taken as representative of the liver stiffness in that individual. The entire examination of each participant took less than 10 min. Histopathological analysis. Liver specimens obtained from surgical operations were fixed in formalin, embedded in paraffin and sectioned at 3 μm in thickness. One tissue section was stained with HE trichrome and liver fibrosis staging was evaluated semiquantitatively according to the Scheuer scoring system.22,23 Fibrosis was staged on a scale from 0
Journal of Gastroenterology and Hepatology 30 (2015) 553–558 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
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Ultrasound elastography of liver fibrosis
to 4 according to the Scheuer scoring system (Table 2) as follows: stage 0 (S0), the absence of fibrosis; stage 1 (S1), fibrous portal expansion; stage 2 (S2), periportal or rare portal-portal septa; stage 3 (S3), fibrous septa with architectural distortion; and stage 4 (S4), cirrhosis. The other tissue section was stained with picroSirius red for collagen semiquantification and quantification. The semiquantitative Chevallier et al.24 method is designed to reflect morphometric measurements of fibrosis, including centrilobular veins, perisinusoidal space, portal tract, and the number and width of septa, separately taking into account fibrosis (Table 3). The Chevallier et al. semiquantitative score is the sum of the score in the centrilobular veins, perisinusoidal space, portal tract, and the numbers and width of septa. The quantitative assessment of collagen was evaluated as the collagen proportionate area (CPA) using computer-assisted digital image analysis. The image acquisition system was composed of a Nikon E clipse 80i upright microscope, a Nikon Digital Sight DS-U2 camera controller, and an NIS Elements BR 2.30 microscope camera image processing software (Nikon Corp., Tokyo, Japan). Image-Pro plus 6.0 software (Media Cybernetics Co., Ltd, Bethesda, MD, USA) was used
Table 2 The fibrosis grade distribution in the Scheuer scoring system of histological findings (n = 78) The scoring systems
n (%)
S0, S1, S2, S3, S4,
20 8 17 14 19
the absence of fibrosis fibrous portal expansion periportal or rare portal-portal septa fibrous septa with architectural distortion cirrhosis
(25.6) (10.3) (21.8) (17.9) (24.4)
Table 3 The fibrosis grades distribution in the Chevallier et al. semiquantitative score system of histological findings (n = 78) The scoring systems CLV (centrilobular veins)
PS (perisinusoidal space)
PT (portal tract)
NS (numbers of septa)
WS (width of septa)
n (%) 0, normal vein or the absence of vein 1, moderately thickened 2, markedly thickened 0, normal 1, localized fibrosis 2, diffuse fibrosis 0, normal 1, enlarged without septa 2, enlarged with septa 3, cirrhosis 0, absence 1, < 6 septa/10 mm 2, > 6 septa/10 mm 3, nodular organization 0, absence 1, thin and/or incomplete 2, thick and loose 3, very thick and dense connective matrix 4, > 2/3 biopsy area
21 (26.9) 34 (43.6) 23 (29.5) 18 (23.1) 32 (41.0) 28 (35.9) 5 (6.4) 25 (32.1) 37 (47.4) 11 (14.1) 22 (28.3) 31 (39.7) 8 (10.2) 17 (21.8) 22 (28.3) 7 (8.9) 21 (26.8) 13 (16.7) 15 (19.3)
in the image analysis system. The results were obtained by the same professional personnel under the same defined conditions: the objective magnification was 10 × , the light intensity was adjusted to the same level, and three non-overlapping visual fields were chosen randomly. Subsequently, areas of red collagen fibers (type I collagen) and green collagen fibers (type III collagen) were measured and recorded in five hot spots. CPAn = (type I collagen + type III collagen)/total area (area of each visual field). CPA = (CPA1 + CPA2 + . . . + CPA5)/5; CPA1–5 were obtained from the same sample.16–18 All specimens were analyzed by an experienced liver pathologist unaware of the results of ultrasound and laboratory tests. Statistical analysis. All data were analyzed using the statistical package SPSS (version 17.0; SPSS Inc, Chicago, IL, USA). Continuous variables were expressed as the mean ± standard deviation or median ± interquartile range (M ± IQR) as appropriate. The reproducibility of PSWE technology was explored by the intra-class correlation coefficient (ICC) of reliability analysis. The differences between fibrosis stages according to the Scheuer scoring system or Chevallier et al. semiquantitative score were analyzed by nonparametric Kruskal–Wallis test. Correlation between the variables was evaluated by Spearman correlation. Linear regression analysis was used to determine the effect of parameters in predicting liver fibrosis. Significance testing was two sided, and type 1 error rates were set to 0.05.
Results A total of 78 (26 women and 52 men; age range, 24–78 years) patients met the inclusion criteria in this study. The patient characteristics are summarized in Table 1. Among the 78 hepatic neoplasms that were surgically removed, 68 were malignant (41 hepatocellular carcinomas, eight intrahepatic cholangiocarcinomas, two neuroendocrine carcinomas, one hepatic epithelioid hemangioendothelioma and 16 metastatic carcinomas), and 10 were benign (four hepatic hemangiomas, two hepatic angiomyolipomas, two focal nodule hyperplasias and two hepatic adenomas). Laboratory examination revealed 48 patients with positive serology tests for serum hepatitis B surface antigen. The fibrosis grade distribution of all patients according to the semiquantitative scoring systems is presented in Table 2 and Table 3. The reproducibility of PSWE technology. Liver stiffness measurements in all 78 patients were successfully examined using the PSWE technique. The ICC of 10 measurements of PSWE was 0.724 (95% confidence interval: 0.692, 0.761), which indicated the good reproducibility of PSWE technology. There was no significant differences in PSWE measurements between the two sonographers (P = 0.34 > 0.05). The relationship between PSWE and histological parameters. The distribution of PSWE values in different fibrosis stages according to the Scheuer scoring system is presented Figure 1a. The PSWE values (M ± IQR) were S0, 4.43 ± 0.85 kPa; S1, 5.22 ± 1.99 kPa; S2, 6.63 ± 3.47 kPa; S3,
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(a)
(b)
(c)
Figure 1 Correlation between point shear wave elastography (PSWE) and different histological systems: (a) PSWE for each fibrosis stage in the Scheuer scoring system. (b) PSWE for each fibrosis stage in the Chevallier et al. semiquantitative scoring system; the box plots present the interquartile range (box), median (thick line), and range (thin lines) of the median velocity measured using PSWE, the asterisk presents the outlier. (c) PSWE for different collagen proportionate areas (CPAs) pathologically.
7.45 ± 2.63 kPa; and S4, 9.32 ± 8.07 kPa; respectively. The PSWE values increased with the severity of liver fibrosis, and there were significant differences among fibrosis stages according to the Scheuer scoring system (χ2 = 23.11, P = 0.00 < 0.05). The distribution of PSWE values in different fibrosis stages according to the Chevallier et al. semiquantitative scoring system (the sum of the score in centrilobular veins, perisinusoidal space, portal tract, and the numbers and width of septa) is presented in Figure 1b. There were significant difference among fibrosis stages according to the Chevallier et al. semiquantitative scoring system (χ2 = 40.979, P = 0.00 < 0.05). The relationship between PSWE values and CPA is presented in Figure 1c, and the PSWE was positively correlated with CPA 556
(r = 0.628, P = 0.00 < 0.05). According to the correlation coefficients presented in Table 4, PSWE exhibited a more reasonable relationship with CPA than with the stages evaluated by Scheuer scoring system (r = 0.473, P = 0.00 < 0.05) or the Chevallier et al. semiquantitative scoring system (r = 0.487, P = 0.00 < 0.05).
Discussion In this study, we evaluated 78 patients who underwent liver segmental resection and described the assessment of the histological progression of liver fibrosis using morphometric collagen quantification relative to the Scheuer scoring system and the Chevallier et al. semiquantitative scoring system. Furthermore, we evaluated
Journal of Gastroenterology and Hepatology 30 (2015) 553–558 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
H Ding et al.
Ultrasound elastography of liver fibrosis
Table 4 The comparison of correlation coefficients among pathological parameters and PSWE values Coefficient
SS
CS
CPA
PSWE
SS CS CPA
— 0.543* 0.438*
0.543* — 0.396*
0.438* 0.396* —
0.473* 0.487* 0.628*
*Correlation is significant at the 0.05 level (two-tailed). CPA, collagen proportionate area; CS, fibrosis stage according to the Chevallier et al.; PSWE, point shear wave elastography semiquantitative scoring system; SS, fibrosis stage according to the Scheuer scoring system.
the association of these three different histological parameters with liver stiffness measured by the shear wave elastography ElastPQ technology as the relevant end point, to assess the potential clinical applicability of the methodology. The Scheuer scoring system, a widely used histopathological method in the evaluation of liver fibrosis, was chosen to semiquantitatively evaluate the tissue section stained with HE trichrome. The Chevallier et al. semiquantitative scoring system and CPA were used to evaluate the amount of specific collagen type I and III in liver fibrosis with picroSirius red staining. CPA differed from the traditional histological scoring systems for fibrosis including the Scheuer scoring system and the Chevallier et al. semiquantitative scoring system, which assigned numerical symbols to descriptive categories of architectural changes in liver fibrosis. The numerical symbols were not quantitatively related, nor were they continuous variables. We assessed the correlation among the results of the Scheuer scoring system, the Chevallier et al. semiquantitative scoring system, and CPA, and compared each with liver stiffness measured by the PSWE technology in patients, the majority of whom were diagnosed with chronic hepatitis B. We observed that CPA correlated better with liver stiffness measurements than the Scheuer scoring system or the Chevallier et al. semiquantitative scoring system, and there was poor consistency between liver stiffness and the Scheuer scoring system and the Chevallier et al. semiquantitative scoring system, which were all semiquantitative methods. Our data suggest that liver stiffness, which was detected by PSWE technology, largely correlated with the amount of liver collagen, which is distinct from liver fibrosis. Traditional histological scores systems might not be proper for the assessment of liver stiffness, and CPA may represent a true standard in accordance with the liver stiffness measurement to validate non-invasive markers of liver fibrosis in a series of patients with chronic hepatitis B. Furthermore, the Chevallier et al. semiquantitative scoring system, which evaluated the tissue sections stained with picroSirius red, performed slightly better than the Scheuer scoring system, which evaluated the tissue section stained with HE trichrome. Simultaneously, the opinion that morphometry is a more sensitive tool than histological staging to demonstrate the progression of fibrosis16,21 was confirmed in this study. Furthermore, CPA has been reported25 to be a better predictor of clinical outcomes than progression by Ishak stage in patients with recurrent chronic hepatitis after transplantation. CPA is a unique, independent predictor of hepatic venous pressure gradient ≥ 10 mmHg and may represent a practical tool for staging fibrosis in patients after liver transplantation.26,27
However, numerous other complex factors in addition to liver collagen influence liver stiffness. Previous research has demonstrated recently that an alanine aminotransferase (ALT) flare can influence liver stiffness measurements because of hepatic inflammatory activity.28,29 To minimize the interference of necroinflammatory activity, we enrolled patients who were clinically indicated for hepatic surgery. Table 1 demonstrates that their laboratory test results including ALT and aspartate aminotransferase were close to normal levels. The limitation of this study was the small number of cases. Only 78 patients were included in this study so it is difficult to make strong conclusions. Further research with an increased number of cases is ongoing to verify the results. In conclusion, in the current study, we demonstrate for the first time that CPA is a good histological parameter in accordance with liver stiffness measurements and is a better discriminator than semiquantitative scoring systems.
Acknowledgment The research was supported by a grant from the Shanghai Natural Science Foundation of China (No. 14ZR1406800).
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chronic hepatitis B virus patients: a comparison with liver pathological results. J. Gastroenterol. Hepatol. 2014; 29: 814–19. Germani G, Burroughs AK, Dhillon AP. The relationship between liver disease stage and liver fibrosis: a tangled web. Histopathology 2010; 57: 773–84. Goodman ZD, Becker RL, Pockros PJ, Afdhal NH. Progression of fibrosis in advanced chronic hepatitis C: evaluation by morphometric image analysis. Hepatology 2007; 45: 886–94. Fontana RJ, Goodman ZD, Dienstag JL et al. Relationship of serum fibrosis markers with liver fibrosis stage and collagen content in patients with advanced chronic hepatitis C. Hepatology 2008; 47: 789–98. Calvaruso V, Burroughs AK, Standish R et al. Computer-assisted image analysis of liver collagen: relationship to Ishak scoring and hepatic venous pressure gradient. Hepatology 2009; 49: 1236–44. Bedossa P, Dargère D, Paradis V. Sampling variability of liver fibrosis in chronic hepatitis C. Hepatology 2003; 38: 1449–57. Jimenez W, Parés A, Caballería J et al. Measurement of fibrosis in needle liver biopsies: evaluation of a colorimetric method. Hepatology 1985; 5: 815–8. Isgro G, Calvaruso V, Andreana L et al. The relationship between transient elastography and histological collagen proportionate area for assessing fibrosis in chronic viral hepatitis. J. Gastroenterol. 2013; 48: 921–9.
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doi:10.1111/jgh.12789
H E PAT O L O G Y
Assessment of liver fibrosis: The relationship between point shear wave elastography and quantitative histological analysis Hong Ding,* Jiao-jiao Ma,* Wen-ping Wang,* Wen-jiao Zeng,† Tao Jiang,† Bei-jian Huang* and Shi-yao Chen‡ Departments of *Ultrasound and ‡Gastroenterology, Zhongshan Hospital, Fudan University, and †Department of Pathology, Shanghai Medical College, Fudan University, Shanghai, China
Key words collagen proportionate area, liver fibrosis, liver stiffness measurement, point shear wave elastography. Accepted for publication 21 August 2014. Correspondence Professor Wen-ping Wang, Department of Ultrasound, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, China. Email: [email protected] Potential conflicts of interest: No conflict of interest is declared.
Abstract Background and Aim: Traditional pathological scoring systems for liver fibrosis progression are predominantly based on the description of architectural changes with no consideration of the amount of collagen fiber deposition. Our purpose was to explore a true histological standard in accordance with the liver stiffness measured by point shear wave elastography (PSWE) in patients with chronic hepatitis B. Methods: A total of 78 patients with liver neoplasms underwent liver stiffness measurements with PSWE as well as biochemical investigations within 3 days before partial hepatectomy. One tissue section of the liver specimens was stained with HE trichrome and evaluated traditionally with the Scheuer scoring system. The other tissue section was stained with picroSirius red and was evaluated according to the semiquantitative Chevallier et al. scoring system. In addition, this second tissue section was evaluated for the collagen proportionate area (CPA) with computer-assisted digital image analysis. The reproducibility of PSWE technology was explored through the intra-class correlation coefficient of a reliability analysis. Results: The PSWE technology revealed good reproducibility in liver stiffness measurements, and the PSWE values increased with the pathological severity of liver fibrosis on both the Scheuer scoring system and the semiquantitative Chevallier et al. scoring system. PSWE values exhibited more reasonable relationships with CPA (r = 0.628, P = 0.00 < 0.05) than with the Scheuer scoring system (r = 0.473, P = 0.00 < 0.05) or the Chevallier et al. semiquantitative scoring system (r = 0.487, P = 0.00 < 0.05). Conclusion: CPA is a better pathological parameter than traditional semiquantitative scoring systems in accordance with liver stiffness measured by PSWE technology.
Introduction The continuous development of liver fibrosis is a common histopathological characteristic of all chronic liver diseases. Significant advancements in the understanding of the cellular and molecular mechanisms of liver fibrogenesis have revealed the progressive substitution of the functioning liver parenchyma with fibrotic tissue; currently, major efforts are directed at translating these acquisitions with diagnostic and therapeutic applications.1–5 The standard pathological examination of liver tissue is invasive; furthermore, the common histological scoring system describes the morphological features based on architectural changes of liver lobules, rather than the amount of collagen, which represents the direct cause of liver fibrosis.6–10 Therefore, the amount of collagen plays a key role in personalized treatment, follow-up and the evaluation of liver fibrosis to accurately and non-invasively assess the progression of fibrosis and non-invasively in the clinical setting.
Several investigators had reported non-invasive approaches for the quantitative diagnosis of liver fibrosis, such as routine laboratory tests, serum markers of fibrosis, radiological imaging, and elastography. Elastography is an exciting new technology that estimates liver fibrosis through the measurement of liver stiffness. Elastography is accurate, safe, cheap, quick and widely applicable, and could reduce the need for liver biopsy in chronic hepatitis patients. Point shear wave elastography (PSWE), which is integrated in a conventional ultrasound machine and can be performed with conventional ultrasound probes during an abdominal ultrasound scan, is a novel shear wave-based elastography technology. Previous studies had reported that PSWE (or ElastPQ technique, Philips Healthcare, Seattle, WA, USA) is a valid and reproducible non-invasive technology to measure liver stiffness among chronic hepatitis patients and liver fibrosis is the dominating factor affecting liver stiffness measured by PSWE.11–14 However, PSWE is limited in differentiating the stage of fibrosis (i.e. differentiating S0 from S1
Journal of Gastroenterology and Hepatology 30 (2015) 553–558 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
553
Ultrasound elastography of liver fibrosis
H Ding et al.
and S2 from S3) according to the Scheuer scoring system. Researchers have reported that other elastography technologies are also unable to accurately differentiate the stages of fibrosis according to traditional histological scores system, which have been widely used as the “golden standard” to validate non-invasive markers of liver fibrosis and to evaluate the therapeutic effects of antifibrotic treatment. Thus, traditional histological scores systems may be inappropriate for the assessment of liver stiffness. The traditional histological scores systems use numerical category labels to evaluate the stage of liver fibrosis instead of measuring the amount of liver fibrosis quantitatively. The assigned numbers or scores of categories are not quantitatively related and are not continuous variables. Applying these fibrosis stage scores as numerical data in statistics is incorrect and misleading. As scoring systems do not quantify fibrosis, fibrosis progression or regression has historically been evaluated in terms of a one- or two-stage change. Furthermore, cirrhosis is only classified as a single stage histologically, representing the most severe categorical assignment, with no histological subclassification.15 An alternative to numerical fibrosis scoring systems is the direct measurement of the amount of fibrosis in the biopsy specimen by computer-assisted digital image analysis. Although the methodology is not standardized, a number of publications have described methods for quantifying liver fibrosis by imaging analysis, and all publications have yielded similar results. The methods, however, are time consuming compared with simple histological scoring, and the necessary equipment and expertise are not widely available. However, in a study to evaluate the progression or regression of fibrosis, measuring the amount of fibrosis as precisely as possible seems appropriate.16–18 The traditional histological scores systems largely assess the predominant architectural changes and sites of fibrosis irrespective of collagen fiber thickness or density, which was constrained by trichrome or reticulin stains. Routine histological evaluation of fibrosis is often performed with trichrome or reticulin stains, which are generally used for connective tissue and are not selective for collagen; the amount of trichrome or reticulin staining did not necessarily correspond to the amount of liver collagen. Computerassisted digital image analysis of histological sections, histochemically stained by the picroSirius red technique, is a method to measure fibrosis morphologically. PicroSirius red staining primarily identifies tissue collagen. The quantity of bound stain correlates well with chemically determined collagen content and morphometrically determined liver fibrosis.19–21 Shear wave elastography imaging including ElastPQ technology is a method for detecting liver stiffness, which largely corresponds to the amount of liver collagen. The main purpose of our study was to discuss the relationship between liver stiffness measured with the PSWE technology and the amount of liver collagen based on the picroSirius red stain, and to explore a true histological standard in accordance with the liver stiffness measurement to validate non-invasive markers of liver fibrosis in a series of patients with chronic hepatitis B.
Methods Patients. Between October 2012 and February 2013, we prospectively enrolled patients with hepatic neoplasms with clinical 554
Table 1
Characteristics of the included patients (n = 78)
Characteristics
Measured values/normal range
Age (years) Gender (male) ALT (IU/L) AST (IU/L) γ-GT (IU/L) Globulins (g/L) γ-Globulins (g/L) Platelet count ( × 109/L)
49 ± 18 (24∼78) 52 (66.7%) (36 ± 29)/(9∼40) (39 ± 30)/(15∼40) (86 ± 132)/(10∼60) (28 ± 5)/(20∼40) (5 ± 2)/(3∼5) (162 ± 51)/(125∼350)
Results are reported as means ± standard deviation or n (%). ALT, alanine aminotransferase; AST, aspartate aminotransferase; γ-GT, Gamma-glutamyltransferase.
indications for hepatic surgery in our hospital. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki, and all patients gave informed consent for the procedures and the histological evaluation. All patients underwent conventional ultrasound examination and elastography, as well as hematological, biochemical, and virological investigations within 3 days before surgery. We excluded patients who failed to meet the inclusion criteria of partial hepatectomy because of hypohepatia and obese patients (BMI ≥ 35 kg/m2). The characteristics of the enrolled patients are summarized in Table 1. Ultrasound measurements. Measurements of liver stiffness were obtained during the conventional ultrasound scan by two sonographers (with 24 and 2 years of experience on ultrasound examination). They sonographers received training in liver stiffness measurements for 2 days before the study began. An ultrasound system (iU elite; Philips Healthcare, Bothell, WA, USA) with a convex transducer (C5-1) was used for the conventional ultrasound scan and liver stiffness measurement. The subjects were requested to lie supine with their right arm abducted. The operator positioned the probe on the right lobe the liver through the seventh to the 10th intercostal space using real-time B-mode imaging to locate a fixed region of interest (0.5 cm × 1.5 cm) in the liver free of visible ducts or vessels. The measurement location was selected with a cursor moved by a trackball. The subject was instructed to hold his or her breath while the operator hit the “update” button that launched the ultrasound data acquisition sequence. Approximately 5 s were required for each data acquisition, and the parameter of liver stiffness was expressed in kilopascals (kPa) of Young’s modulus. Ten measurements of liver stiffness were obtained in the same location of the right liver lobe of every patient. The median value of the successful measurements was taken as representative of the liver stiffness in that individual. The entire examination of each participant took less than 10 min. Histopathological analysis. Liver specimens obtained from surgical operations were fixed in formalin, embedded in paraffin and sectioned at 3 μm in thickness. One tissue section was stained with HE trichrome and liver fibrosis staging was evaluated semiquantitatively according to the Scheuer scoring system.22,23 Fibrosis was staged on a scale from 0
Journal of Gastroenterology and Hepatology 30 (2015) 553–558 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
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to 4 according to the Scheuer scoring system (Table 2) as follows: stage 0 (S0), the absence of fibrosis; stage 1 (S1), fibrous portal expansion; stage 2 (S2), periportal or rare portal-portal septa; stage 3 (S3), fibrous septa with architectural distortion; and stage 4 (S4), cirrhosis. The other tissue section was stained with picroSirius red for collagen semiquantification and quantification. The semiquantitative Chevallier et al.24 method is designed to reflect morphometric measurements of fibrosis, including centrilobular veins, perisinusoidal space, portal tract, and the number and width of septa, separately taking into account fibrosis (Table 3). The Chevallier et al. semiquantitative score is the sum of the score in the centrilobular veins, perisinusoidal space, portal tract, and the numbers and width of septa. The quantitative assessment of collagen was evaluated as the collagen proportionate area (CPA) using computer-assisted digital image analysis. The image acquisition system was composed of a Nikon E clipse 80i upright microscope, a Nikon Digital Sight DS-U2 camera controller, and an NIS Elements BR 2.30 microscope camera image processing software (Nikon Corp., Tokyo, Japan). Image-Pro plus 6.0 software (Media Cybernetics Co., Ltd, Bethesda, MD, USA) was used
Table 2 The fibrosis grade distribution in the Scheuer scoring system of histological findings (n = 78) The scoring systems
n (%)
S0, S1, S2, S3, S4,
20 8 17 14 19
the absence of fibrosis fibrous portal expansion periportal or rare portal-portal septa fibrous septa with architectural distortion cirrhosis
(25.6) (10.3) (21.8) (17.9) (24.4)
Table 3 The fibrosis grades distribution in the Chevallier et al. semiquantitative score system of histological findings (n = 78) The scoring systems CLV (centrilobular veins)
PS (perisinusoidal space)
PT (portal tract)
NS (numbers of septa)
WS (width of septa)
n (%) 0, normal vein or the absence of vein 1, moderately thickened 2, markedly thickened 0, normal 1, localized fibrosis 2, diffuse fibrosis 0, normal 1, enlarged without septa 2, enlarged with septa 3, cirrhosis 0, absence 1, < 6 septa/10 mm 2, > 6 septa/10 mm 3, nodular organization 0, absence 1, thin and/or incomplete 2, thick and loose 3, very thick and dense connective matrix 4, > 2/3 biopsy area
21 (26.9) 34 (43.6) 23 (29.5) 18 (23.1) 32 (41.0) 28 (35.9) 5 (6.4) 25 (32.1) 37 (47.4) 11 (14.1) 22 (28.3) 31 (39.7) 8 (10.2) 17 (21.8) 22 (28.3) 7 (8.9) 21 (26.8) 13 (16.7) 15 (19.3)
in the image analysis system. The results were obtained by the same professional personnel under the same defined conditions: the objective magnification was 10 × , the light intensity was adjusted to the same level, and three non-overlapping visual fields were chosen randomly. Subsequently, areas of red collagen fibers (type I collagen) and green collagen fibers (type III collagen) were measured and recorded in five hot spots. CPAn = (type I collagen + type III collagen)/total area (area of each visual field). CPA = (CPA1 + CPA2 + . . . + CPA5)/5; CPA1–5 were obtained from the same sample.16–18 All specimens were analyzed by an experienced liver pathologist unaware of the results of ultrasound and laboratory tests. Statistical analysis. All data were analyzed using the statistical package SPSS (version 17.0; SPSS Inc, Chicago, IL, USA). Continuous variables were expressed as the mean ± standard deviation or median ± interquartile range (M ± IQR) as appropriate. The reproducibility of PSWE technology was explored by the intra-class correlation coefficient (ICC) of reliability analysis. The differences between fibrosis stages according to the Scheuer scoring system or Chevallier et al. semiquantitative score were analyzed by nonparametric Kruskal–Wallis test. Correlation between the variables was evaluated by Spearman correlation. Linear regression analysis was used to determine the effect of parameters in predicting liver fibrosis. Significance testing was two sided, and type 1 error rates were set to 0.05.
Results A total of 78 (26 women and 52 men; age range, 24–78 years) patients met the inclusion criteria in this study. The patient characteristics are summarized in Table 1. Among the 78 hepatic neoplasms that were surgically removed, 68 were malignant (41 hepatocellular carcinomas, eight intrahepatic cholangiocarcinomas, two neuroendocrine carcinomas, one hepatic epithelioid hemangioendothelioma and 16 metastatic carcinomas), and 10 were benign (four hepatic hemangiomas, two hepatic angiomyolipomas, two focal nodule hyperplasias and two hepatic adenomas). Laboratory examination revealed 48 patients with positive serology tests for serum hepatitis B surface antigen. The fibrosis grade distribution of all patients according to the semiquantitative scoring systems is presented in Table 2 and Table 3. The reproducibility of PSWE technology. Liver stiffness measurements in all 78 patients were successfully examined using the PSWE technique. The ICC of 10 measurements of PSWE was 0.724 (95% confidence interval: 0.692, 0.761), which indicated the good reproducibility of PSWE technology. There was no significant differences in PSWE measurements between the two sonographers (P = 0.34 > 0.05). The relationship between PSWE and histological parameters. The distribution of PSWE values in different fibrosis stages according to the Scheuer scoring system is presented Figure 1a. The PSWE values (M ± IQR) were S0, 4.43 ± 0.85 kPa; S1, 5.22 ± 1.99 kPa; S2, 6.63 ± 3.47 kPa; S3,
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(a)
(b)
(c)
Figure 1 Correlation between point shear wave elastography (PSWE) and different histological systems: (a) PSWE for each fibrosis stage in the Scheuer scoring system. (b) PSWE for each fibrosis stage in the Chevallier et al. semiquantitative scoring system; the box plots present the interquartile range (box), median (thick line), and range (thin lines) of the median velocity measured using PSWE, the asterisk presents the outlier. (c) PSWE for different collagen proportionate areas (CPAs) pathologically.
7.45 ± 2.63 kPa; and S4, 9.32 ± 8.07 kPa; respectively. The PSWE values increased with the severity of liver fibrosis, and there were significant differences among fibrosis stages according to the Scheuer scoring system (χ2 = 23.11, P = 0.00 < 0.05). The distribution of PSWE values in different fibrosis stages according to the Chevallier et al. semiquantitative scoring system (the sum of the score in centrilobular veins, perisinusoidal space, portal tract, and the numbers and width of septa) is presented in Figure 1b. There were significant difference among fibrosis stages according to the Chevallier et al. semiquantitative scoring system (χ2 = 40.979, P = 0.00 < 0.05). The relationship between PSWE values and CPA is presented in Figure 1c, and the PSWE was positively correlated with CPA 556
(r = 0.628, P = 0.00 < 0.05). According to the correlation coefficients presented in Table 4, PSWE exhibited a more reasonable relationship with CPA than with the stages evaluated by Scheuer scoring system (r = 0.473, P = 0.00 < 0.05) or the Chevallier et al. semiquantitative scoring system (r = 0.487, P = 0.00 < 0.05).
Discussion In this study, we evaluated 78 patients who underwent liver segmental resection and described the assessment of the histological progression of liver fibrosis using morphometric collagen quantification relative to the Scheuer scoring system and the Chevallier et al. semiquantitative scoring system. Furthermore, we evaluated
Journal of Gastroenterology and Hepatology 30 (2015) 553–558 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
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Table 4 The comparison of correlation coefficients among pathological parameters and PSWE values Coefficient
SS
CS
CPA
PSWE
SS CS CPA
— 0.543* 0.438*
0.543* — 0.396*
0.438* 0.396* —
0.473* 0.487* 0.628*
*Correlation is significant at the 0.05 level (two-tailed). CPA, collagen proportionate area; CS, fibrosis stage according to the Chevallier et al.; PSWE, point shear wave elastography semiquantitative scoring system; SS, fibrosis stage according to the Scheuer scoring system.
the association of these three different histological parameters with liver stiffness measured by the shear wave elastography ElastPQ technology as the relevant end point, to assess the potential clinical applicability of the methodology. The Scheuer scoring system, a widely used histopathological method in the evaluation of liver fibrosis, was chosen to semiquantitatively evaluate the tissue section stained with HE trichrome. The Chevallier et al. semiquantitative scoring system and CPA were used to evaluate the amount of specific collagen type I and III in liver fibrosis with picroSirius red staining. CPA differed from the traditional histological scoring systems for fibrosis including the Scheuer scoring system and the Chevallier et al. semiquantitative scoring system, which assigned numerical symbols to descriptive categories of architectural changes in liver fibrosis. The numerical symbols were not quantitatively related, nor were they continuous variables. We assessed the correlation among the results of the Scheuer scoring system, the Chevallier et al. semiquantitative scoring system, and CPA, and compared each with liver stiffness measured by the PSWE technology in patients, the majority of whom were diagnosed with chronic hepatitis B. We observed that CPA correlated better with liver stiffness measurements than the Scheuer scoring system or the Chevallier et al. semiquantitative scoring system, and there was poor consistency between liver stiffness and the Scheuer scoring system and the Chevallier et al. semiquantitative scoring system, which were all semiquantitative methods. Our data suggest that liver stiffness, which was detected by PSWE technology, largely correlated with the amount of liver collagen, which is distinct from liver fibrosis. Traditional histological scores systems might not be proper for the assessment of liver stiffness, and CPA may represent a true standard in accordance with the liver stiffness measurement to validate non-invasive markers of liver fibrosis in a series of patients with chronic hepatitis B. Furthermore, the Chevallier et al. semiquantitative scoring system, which evaluated the tissue sections stained with picroSirius red, performed slightly better than the Scheuer scoring system, which evaluated the tissue section stained with HE trichrome. Simultaneously, the opinion that morphometry is a more sensitive tool than histological staging to demonstrate the progression of fibrosis16,21 was confirmed in this study. Furthermore, CPA has been reported25 to be a better predictor of clinical outcomes than progression by Ishak stage in patients with recurrent chronic hepatitis after transplantation. CPA is a unique, independent predictor of hepatic venous pressure gradient ≥ 10 mmHg and may represent a practical tool for staging fibrosis in patients after liver transplantation.26,27
However, numerous other complex factors in addition to liver collagen influence liver stiffness. Previous research has demonstrated recently that an alanine aminotransferase (ALT) flare can influence liver stiffness measurements because of hepatic inflammatory activity.28,29 To minimize the interference of necroinflammatory activity, we enrolled patients who were clinically indicated for hepatic surgery. Table 1 demonstrates that their laboratory test results including ALT and aspartate aminotransferase were close to normal levels. The limitation of this study was the small number of cases. Only 78 patients were included in this study so it is difficult to make strong conclusions. Further research with an increased number of cases is ongoing to verify the results. In conclusion, in the current study, we demonstrate for the first time that CPA is a good histological parameter in accordance with liver stiffness measurements and is a better discriminator than semiquantitative scoring systems.
Acknowledgment The research was supported by a grant from the Shanghai Natural Science Foundation of China (No. 14ZR1406800).
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