PATIENTS WITH BETA-THALASSEMIA frequently

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1 bs_bs_banner Hepatology Research 2013 doi: /hepr Original Article Transient elastography in hepatitis C virus-infected patients with beta-thalassemia for assessment of fibrosis Hossein Poustchi, 1 Masoomeh Eslami, 2 Mohammad Reza Ostovaneh, 1 Amirhossein Modabbernia, 1 Fatemeh Sima Saeedian, 2 Shervin Taslimi, 1 Jacob George, 3 Reza Malekzadeh 1 and Farhad Zamani 2 1 Digestive Diseases Research Institute, Shariati Hospital, 2 Gastrointestinal and Liver Disease Research Center, Firoozgar Hospital, Tehran University Of Medical Sciences, Tehran, Iran; and 3 Storr Liver Unit, Westmead Hospital, University of Sydney, Sydney, New South Wales, Australia Aim: We sought to evaluate the performance of transient elastography (TE) for the assessment of liver fibrosis in chronic hepatitis C (CHC) patients with beta-thalassemia. Methods: Seventy-six CHC patients with beta-thalassemia underwent TE, liver biopsy, T 2-weighted magnetic resonance imaging (MRI) for the assessment of liver iron content (LIC) and laboratory evaluation. The accuracy of TE and its correlation with the other variables was assessed. Results: TE values increased proportional to fibrosis stage (r = 0.404, P < 0.001), but was independent of T 2-weighted MRI-LIC (r = 0.064, P = 0.581). In multivariate analysis, fibrosis stage was still associated with the log-transformed TE score- (standardized b=0.42 for F4 stage of METAVIR, P = 0.001). No correlation was noted between LIC and TE score (standardized b=0.064, P = 0.512). The area under the receiver operating characteristic curve for prediction of cirrhosis was 80% (95% confidence interval, %). A cut-off TE score of 11 had a sensitivity of 78% and specificity of 88.1% for diagnosing cirrhosis. The best cut-off values for TE-FIB-4 cirrhosis score comprising TE and FIB-4 and TE-APRI cirrhosis score combining TE with aspartate aminotransferase-to-platelet ratio index (APRI) both had 87.5% sensitivity and 91.04% specificity for the diagnosis of cirrhosis. Conclusion: Regardless of LIC, TE alone or when combined with FIB-4 or APRI, is a diagnostic tool with moderate to high accuracy to evaluate liver fibrosis in CHC patients with betathalassemia. However, because splenectomy in a proportion of our subjects might have affected the platelet count, the scores utilizing APRI and FIB-4 should be interpreted cautiously. Key words: hepatitis C virus, liver cirrhosis, liver iron content, thalassemia, transient elastography Correspondence: Dr Farhad Zamani, Gastroenterology and Liver Disease Research Center, Firouzgar Hospital, Valadi Street, Valiasr Square, Tehran 14117, Iran. farhadzamani79@yahoo.com Conflict of interest: No conflict of interest exists in relation to the submitted manuscript and there was no source of extrainstitutional commercial funding. An abstract of this paper was presented at Digestive Disease Week (DDW) Received 17 December 2012; revision 31 January 2013; accepted 4 February INTRODUCTION PATIENTS WITH BETA-THALASSEMIA frequently develop progressive liver fibrosis due to hepatic iron overload and coexistent chronic hepatitis C (CHC) infection because most have received transfusions prior to the advent of effective screening for the hepatitis viruses. 1 FibroScan, or transient elastography (TE), noninvasively assesses liver fibrosis 2 11 and presents comparable performance to liver biopsy to predict liver-related outcomes in patients with CHC. 12 However, the role of TE technology in patients with hepatic iron overload and CHC, to our knowledge, has not been extensively investigated. Liver biopsy evaluates hepatic architecture, steatosis, inflammation and iron deposition, and thus is likely to play a role in the management of patients with liver disease. However, if the aim of biopsy is to evaluate the extent of fibrosis, then non-invasive techniques such as TE are likely to be important, which evaluates liver stiffness over a larger portion of the liver and therefore may be even more representative of changes in hepatic fibrosis than liver biopsy, as stated by Castera et al. 7 In this regard, a first step is the evaluation of the performance of TE in specific clinical settings as most published data concerns its role in CHC. There is limited data on the 1

2 2 H. Poustchi et al. Hepatology Research 2013 performance of TE in patients with beta-thalassemia and there is debate whether iron overload per se affects the TE score. 1,13,14 Previous studies have not provided adequate data, principally due to limitations in the number of patients with liver biopsy. Mirault et al. 13 found a modest but non-significant correlation between iron load and fibrosis in a study of 13 patients with post-transfusional iron overload. In contrast, Di Marco et al. reported no association between TE score and iron load, while a study by Fraquelli et al., 14 in which only 14 had liver biopsy, was inconclusive on the utility of TE in the context of iron overload to assess fibrosis stage. In this study, we sought to: (i) evaluate the performance of TE for the assessment of liver fibrosis in hepatitis C virus (HCV)-infected patients with betathalassemia; (ii) assess the influence of liver iron on TE performance; and (iii) define TE thresholds to discriminate between fibrosis stages in these patients. METHODS Participants WE UNDERTOOK A prospective cohort study on CHC patients with beta-thalassemia referred to a tertiary referral center over a 2-year period ( ). HCV RNA was assessed by polymerase chain reaction (Amplicor; Roche Molecular Systems, Branchburg, NJ, USA) and beta-thalassemia was already confirmed on hemoglobin electrophoresis. All patients underwent TE, liver biopsy and T 2-weighted magnetic resonance imaging (T2*MRI). T2*MRI and liver biopsy were performed at a maximum of 3 months after the TE assessment. All patients received regular transfusions every 2 4 weeks, with pre-transfusion hemoglobin levels of 9 10 g/dl and post-transfusion levels of g/dl. Chelation therapy was additionally undertaken using subcutaneous deferoxamine. Exclusion criteria were acute hepatitis or the presence of ascites which would preclude accurate performance of TE. All patients had biochemical laboratory assessments at the time of TE, including a blood count, liver enzymes, liver tests and serum ferritin. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the ethics committee of Tehran University of Medical Sciences. All patients provided written informed consent. TE Transient elastography was performed using FibroScan (Echosens, Paris, France) by a single operator on the right lobe of the liver within 3 months of liver biopsy. As per the manufacturer s instructions, TE was performed with the patient in the dorsal decubitus position with the right arm in maximal abduction. Using the FibroScan ultrasound guide, the operator located a liver portion with a thickness of at least 6 cm and free of large vessels. Success rate was calculated as the ratio of the number of valid measurements divided by the total number of measurements. The median value of at least 10 validated measurements with a success rate of at least 60% and interquartile range (IQR) of less than 30% of the median TE value was considered as reliable representative of the elasticity of the liver. The results were expressed in kilopascals (kpa). T2*MRI Magnetic resonance imaging were performed using a 1.5 Tesla Magnetom Siemens Symphony scanner (Siemens Medical Solution, Erlangen, Germany). A standard radiofrequency body coil was used in all measurements for both excitation and signal detection. Respiratory triggering was used to monitor respiration. Spatial presaturation slabs were used to suppress motion-related artifacts. The T2*MRI value of the liver was determined using a single 10-mm slice through the center of the liver scanned at 12 different echo times (TE, msec). Each image was acquired during a s breath-hold using a gradient-echo sequence with a repetition time of 200 msec, flip angle of 20, base resolution matrix of 128 pixels, a field of view of 39.7 cm 19.7 cm and a sampling bandwidth of 125 khz per pixel. The liver iron content (LIC) was calculated using Cardiovascular Magnetic Resonance Tools software (CMR Tools; Imperial College, London, UK) using the formula 1 / (T2* / 1000) The T2*MRI method to evaluate LIC has been previously validated and correlated with biochemically measured LIC. 17,18 Liver biopsy A 16-G Tru-cut needle (TSK Laboratory, Tochigi-Ken, Japan) was used for liver biopsy under ultrasound guidance. Biopsy specimens were examined by pathologists expert in liver histopathology. Specimens were considered adequate if they were at least 20 mm in length and contained 10 or more portal tracts. Liver fibrosis stage (scores, F0 F4) and necroinflammatory grade (scores, A0 A3) were scored semiquantitatively according to METAVIR scoring system. 4,19,20

3 Hepatology Research 2013 Transient elastography in beta-thalassemia 3 Statistical analysis STATA ver. 11 (StataCorp, College Station, TX, USA) was used for statistical analysis. Continuous data were presented as median (IQR) or mean (1standard deviation) when appropriate. The difference of categorical variables was assessed by Pearson s c 2 -test. Due to non-normality of the continuous variables, non-parametric tests (Mann Whitney U-test, Kruskal Wallis H-test and Spearman s rank correlation coefficient) were used as appropriate. Linear regression modeling was performed to assess the independent effect of clinical factors on the log-transformed TE score. Variables with P < 0.2 in univariate regression were included in the multivariate model. The assumptions of the linear regression were assessed through testing normality and homogeneity of variance of residuals and the linear relation between outcome variable and predictors. Variance inflation factor and correlation coefficients were calculated to assess multi-collinearity. For variables which violated the normality of residuals in regression analysis, appropriate transformations were used. Robust regression estimates were reported when the conditions of homogeneity of variance for residuals did not hold. Receiver operating characteristic curve (ROC) analysis was undertaken to evaluate the predictive accuracy of TE at the different stages of liver fibrosis, and to compare its accuracy with the aspartate aminotransferase (AST)-toplatelet ratio index (APRI) 21 and FIB-4, 22,23 two other non-invasive tools for the assessment of liver fibrosis. The best cut-off scores were chosen according to Youden s J index. To improve the predictive power of TE in combination with APRI or FIB-4 for the diagnosis of cirrhosis, we developed weighted linear combinations based on the coefficients obtained from logistic regression analysis, with cirrhosis (METAVIR stage 4) as the outcome variable and TE score and APRI or FIB-4 as predictors. Independence of TE and APRI or FIB-4 was assessed by Spearman s rank correlation coefficient test. P < 0.05 was considered statistically significant. RESULTS SEVENTY-SIX (37 male and 39 female) chronic HCV RNA positive patients with beta-thalassemia were included in the study. Serological anti-hcv antibody and HCV RNA in polymerase chain reaction were detected in all subjects. Only seven out of 76 subjects had body mass index (BMI) of more than 25. Baseline clinical and laboratory findings are presented in Table 1. Median T2*MRI-LIC was 3.96 mg iron/g dry liver (IQR, ) and this significantly correlated with Table 1 Characteristics of patients Variable Value Age (years) Female sex, n (%) 37 (48.7) Thalassemia 59 major, 17 intermedia HCV genotype, n (%) 1, 1a/1b, 1b 57 (75) 3 19 (25) HCV RNA load ( 10 5, IU/mL) 3.15 ( ) Positive HCV antibody, n (%) 76 (100) BMI (kg/m 2 ) Ferritin (mg/l) 1580 ( ) ALT (IU/L) 28 (21 52) AST (IU/L) 28 (21 42) Bilirubin (mg/dl) 2.5 ( ) ALP (IU/L) 241 ( ) Hemoglobin (g/dl) Platelet ( 10 3,mm 3 ) Previous splenectomy, n (%) 35 (46) Prothrombin time (s) Albumin (g/dl) 5 ( ) TE score (kpa) 6.7 ( ) Stage, n (%) F0 9 (12) F1 9 (12) F2 27 (35) F3 22 (29) F4 9 (12) Grade, n (%) A0 7 (9.21) A1 39 (51.3) A2 22 (28.9) A3 8 (10.5) T2*MRI (msec) 3.34 ( ) Iron load (mg/g based on MRI) 3.96 ( ) All continuous values are presented as median (interquartile range), other than age, BMI, hemoglobin, platelet and PT which are presented as mean 1 SD. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; HCV, hepatitis C virus; T2*MRI, T 2-weighted magnetic resonance imaging; PT, prothrombin time; SD, standard deviation; TE, transient elastography. serum ferritin (r = 0.725, P < 0.001). The median value of T2*MRI-LIC did not differ with respect to fibrosis stage of necroinflammatory grade. (P = 0.954, = 0.515, respectively, Fig. 1, upper panel). There were no cases of failure in the performance of TE and we reached at least 60% success rate and IQR of less than 30 of the median TE value for all patients. The median TE score of 6.7 (IQR, ) did not differ

4 4 H. Poustchi et al. Hepatology Research 2013 MRI-based liver iron concentration mg iron/g dry liver TE score (kpa) METAVIR stage of fibrosis As shown in Table 3, in linear regression modeling, sex, stage of fibrosis, necroinflammatory grade, T2*MRI-LIC, log-transformed alanine aminotransferase (ALT), log-transformed AST and BMI were correlated to log-transformed TE score with P < 0.2 in univariate analysis. Hence, they were included in the multivariate model. Serum ferritin was excluded from the multivariate model due to collinearity with T2*MRI-LIC (r = 0.725, P < 0.001). As shown in Table 3, in multivariate analysis, TE score associated with fibrosis stage, independently. The TE score was more efficient to identify cirrhosis or higher stages of fibrosis than lower stages (Tables 3,4). Furthermore, sex, log-transformed ALT and BMI were significantly and independently associated with log-transformed TE score (Table 3), however, there was no association of TE score with T2*MRI-LIC. Receiver operating characteristics curve analysis showed that TE score was accurate for the diagnosis of cirrhosis (stage F4, area under ROC [AUROC], 80%), but performed less well at the lower stages of fibrosis. The TE score was also superior to FIB-4 (AUROC, 69%) and APRI (AUROC, 66.6%) for the assessment of cirrhosis (Table 4). A TE cut-off score of 11 had a sensitivity of METAVIR stage of fibrosis Figure 1 Correlation of T 2-weighted magnetic resonance imaging (MRI) and transient elastography (TE) values with fibrosis. Upper panel, correlation of MRI-based liver iron content and METAVIR stage of fibrosis; lower panel, correlation of TE values and METAVIR stage of fibrosis. between patients with thalassemia major (TM) and intermedia (TI) (6.5 [IQR, ] and 8 [IQR, ], respectively, P = 0.24). The TE value was 6.75 (IQR, 5.9 8) for patients with ferritin level of less than 500 ng/ml and 6.85 (IQR, 5 9.7) and 6.7 (IQR, ) for those with ferritin levels of and more than 1000 ng/ml, respectively (P = 0.96). With regard to the association of TE with liver histology parameters, TE increased proportionally to METAVIR fibrosis stages (r = 0.418, P < 0.001), with significant difference between stages (P < 0.001, Fig. 1, lower panel), but was independent of T2*MRI-LIC (r = 0.064, P = 0.581) (Table 2). TE score also correlated with necroinflammatory grade (r = 0.4, P < 0.001). The correlation coefficients of the other variables are listed in Table 2. Table 2 Spearman s rank correlation coefficient between TE values and other variables Variable Correlation coefficient P-value Age Stage <0.001 Grade 0.4 <0.001 Hepatic iron load (T2*MRI) BMI AST <0.001 ALT <0.001 ALP Albumin Prothrombin time Bilirubin APRI FIB Platelet AST to platelet ratio index = 100*AST (U/L) / (upper limit of normal for AST * platelets [10 9 /L]). FIB-4 = age (years) * AST (U/L) / (platelets [10 9 /L] * ALT [U/L] 1/2 ). ALP, alkaline phosphatase; ALT, alanine aminotransferase; APRI, aspartate aminotransferase-to-platelet ratio index; AST, aspartate aminotransferase; BMI, body mass index; TE, transient elastography.

5 Hepatology Research 2013 Transient elastography in beta-thalassemia 5 Table 3 Variables associated with TE values in regression analysis Variable Univariate analysis Multivariate analysis Un-standardized coefficients (standardized beta) P-value Un-standardized coefficients (standardized beta) P-value Age (0.08) 0.48 Male sex 0.28 (0.31) (0.33) Thalassemia (M/I) (0.001) 0.99 Stage (F0 as reference) F (-0.02) (0.001) F (0.04) (0.018) F (0.31) (0.22) F (0.53) (0.42) Grade (A0 as reference) A (0.2) (0.04) A (0.47) (0.15) A (0.38) (0.15) 0.1 Iron load (MRI) 0.02 (0.16) (0.06) ALT* 0.23 (0.40) (0.26) 0.03 AST* 0.23 (0.41) < (-0.04) 0.75 ALP (0.14) BMI (0.18) (0.31) PT (0.10) Bilirubin (-0.002) Ferritin (0.22) *Log-transformed values were used. Variance inflation factor for all variables is <4, adjusted R 2 = ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; F, Female. M, male; MRI, magnetic resonance imaging; PT, prothrombin time; TE, transient elastography. 78% and specificity of 88.1%, while for FIB-4, a cut-off of 0.29 had 87.5% sensitivity and 48% specificity for diagnosing cirrhosis. The best APRI cut-off score of 0.14 had 87.5% sensitivity and 37.3% specificity for diagnosing cirrhosis. TE score was independent of APRI (r = 0.195, P = 0.094) or FIB-4 (r = 0.12, P = 0.299). Accordingly, using the related coefficients of logistic regression analysis, the linear combinations of TE-APRI cirrhosis score calculated as TE score + (1.02 APRI) or TE-FIB-4 cirrhosis score calculated as TE score + (3.3 FIB-4) were able to diagnose cirrhosis with AUROC of 84.1% and 84.5%, respectively. The cut-off TE-APRI cirrhosis score of and TE-FIB-4 cirrhosis score of 13, both had 87.5% sensitivity and 91.04% specificity for diagnosing cirrhosis. DISCUSSION IN THE PRESENT study, we have shown that: (i) TE is an alternative diagnostic tool with moderate to high accuracy for the assessment of liver fibrosis and cirrhosis in HCV-infected patients with beta-thalassemia; (ii) TE score is not influenced by liver iron content; and (iii) the combination of TE with FIB-4 or APRI in these subjects allows for the non-invasive identification of patients with cirrhosis with higher accuracy. In this group of patients with CHC and thalassemia, HCV virus and LIC are the most important drivers of hepatic injury and consequent fibrosis. 14,24 Although liver biopsy remains the gold standard in assessing hepatic injury, biopsy-related complications in thalassemic patients, especially in those with concomitant HCV infection or cirrhosis-related coagulopathies is problematic. In these individuals, the assessment of fibrosis stage and LIC (which can now be assessed noninvasively by MRI techniques) is most critical for the long-term prediction of liver-related adverse outcomes. Such prediction is also critical for the institution of best practice and liver cancer screening. While TE has shown promising results for the non-invasive evaluation of liver fibrosis of different etiologies, 2 8,10,28,29 adequate data is lacking in patients with thalassemia. 1,13,14

6 6 H. Poustchi et al. Hepatology Research 2013 Table 4 ROC analysis to find cut-off scores of TE scores, FIB-4 and APRI to diagnose a certain fibrosis stage TE score APRI FIB-4 Fibrosis stage Sensitivity Specificity Best cut-off score Sensitivity Specificity AUROC (95% CI) Best cut-off score Sensitivity Specificity AUROC (95% CI) Best cut-off score AUROC (95% CI) (0.59 1) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) AST to platelet ratio index = 100 * AST (U/L) / (upper limit of normal for AST * platelets [10 9 /L]). FIB-4 = age (years) * AST (U/L) / (platelets [10 9 /L] * ALT [U/L] 1/2 ). APRI, aspartate aminotransferase-to-platelet ratio index; AST, aspartate aminotransferase; AUROC, area under the receiver operating characteristics curve; CI, confidence interval; ROC, receiver operating characteristics curve; TE, transient elastography. The present study comprises a relatively large cohort (n = 76), all of whom had liver biopsy, and hepatic iron quantification. It was of particular interest for us to clarify the relationship between liver iron content and liver stiffness. We demonstrated that TE score increased proportionally to fibrosis stage and necroinflammatory grade, but did not find any evidence for an effect of T2*MRI-LIC or serum ferritin on TE score. The correlation of TE and biopsy parameters (stage and grade) remained significant even after adjusting for the effect of other variables. Although most of the previous studies did not show a significant effect of LIC on TE score, some of these reports are limited by small numbers of patients with liver biopsy. In the study by Fraquelli et al., 14 the cohort size of 14 patients with liver biopsy limited their ability to determine the independent effect of iron on TE values and to assess the accuracy of TE in predicting fibrosis. In a study of 56 patients with thalassemia, Di Marco et al. 1 found no correlation between hepatic iron measured by atomic absorption spectrometry and TE score. The present study supports these results. However, in contrast to Di Marco et al., we used T2*MRI to assess LIC. In cirrhosis, iron deposition is heterogeneous and biochemically assessed LIC (atomic absorption spectrometry) can vary greatly from one nodule to another. 17,18,30 32 Accordingly, a recent study stated that in patients with cirrhosis, T2*MRI-LIC may be more representative than biopsy quantification of liver iron 18 which does not have the risk of biopsy-related complications. Moreover, unlike the study by Di Marco et al., 1 which comprised a relatively heterogeneous group (45 adults and 11 children) of whom only 23 (41%) were HCV positive, 1 all of the subjects in the current study were HCV positive. In another study, Adhoute et al. assessed the performance of TE in hemochromatosis and showed that these patients have higher TE scores than controls, 33 however, they did not address the effect of LIC on TE score and did not evaluate for a correlation between TE score and fibrosis stage. We did not have any cases of failure in performance of TE in the present study which might have been partly due to increased liver size and relatively low BMI in thalassemic patients (only seven patients with BMI > 25). A cut-off TE score of 11 showed moderate sensitivity (78%) and specificity (88.1%) for the diagnosis of cirrhosis. Although less accurate, these results are comparable to those reported in a meta-analysis by Talwalkar et al. (pooled sensitivity 91%, specificity 87%) for cirrhosis detection from different etiologies. 11 The AUROC

7 Hepatology Research 2013 Transient elastography in beta-thalassemia 7 of TE for detection of cirrhosis in our study was also lower than that reported in previous studies. 1 However, the observed difference may partly be explained by specific characteristics of the subjects in our study and limitation in generalizability of similar studies including ours due to small sample size. In the present study, cut-off values for the diagnosis of cirrhosis were significantly lower than those in other liver diseases, 5,34 possibly due to the younger age of our cohort, as suggested previously. 13 We showed that TE was superior to FIB-4 or APRI in predicting liver fibrosis. However, two separate cirrhosis scores combining TE with these two measures demonstrated improved performance for the prediction of cirrhosis (sensitivity 87.5% and sensitivity 91.04% for both scores) providing a relatively high degree of confidence, especially to exclude cirrhosis. However, 46% of our subjects had already undergone splenectomy which might have resulted in increase in platelet count and influenced the FIB-4 and APRI calculation according to the formulas. Accordingly, the scores utilizing FIB-4 and APRI should be interpreted cautiously. Despite the difficulty in performing similar studies on large numbers of patients with CHC and betathalassemia, a small cohort size is still a limitation of our study. Nevertheless, to our knowledge, it constitutes the largest cohort reported to date (although with a moderate increase in sample size compared to previous studies). LIC quantification by atomic absorption spectrometry as gold standard, would have been also beneficial for the assessment of the accuracy of T2*MRI-LIC, but was not undertaken. In conclusion, we have shown that TE is a an alternative diagnostic tool with moderate to high accuracy for the assessment of liver fibrosis in HCV-infected patients with beta-thalassemia. The performance characteristics of TE in this setting, is not influenced by liver iron content. Combining TE scores with the APRI or FIB-4 allows for the exclusion of cirrhosis in HCV-infected patients with beta-thalassemia with a relatively high degree of confidence and is useful for their clinical management. ACKNOWLEDGMENTS WE THANK MS Mansoureh Maadi and Ms Farzaneh Zamani for secretarial assistance and Nikan Teb Sasan Company for providing access to the FibroScan machine. REFERENCES 1 Di Marco V, Bronte F, Cabibi D et al. Noninvasive assessment of liver fibrosis in thalassaemia major patients by transient elastography (TE) lack of interference by iron deposition. Br J Haematol 2010; 148 (3): Ziol M, Handra-Luca A, Kettaneh A et al. Noninvasive assessment of liver fibrosis by measurement of stiffness in patients with chronic hepatitis C. Hepatology 2005; 41 (1): Coco B, Oliveri F, Maina AM et al. Transient elastography: a new surrogate marker of liver fibrosis influenced by major changes of transaminases. J Viral Hepat 2007; 14 (5): Fraquelli M, Rigamonti C, Casazza G et al. Reproducibility of transient elastography in the evaluation of liver fibrosis in patients with chronic liver disease. Gut 2007; 56 (7): Rigamonti C, Donato MF, Fraquelli M et al. Transient elastography predicts fibrosis progression in patients with recurrent hepatitis C after liver transplantation. Gut 2008; 57 (6): Arena U, Vizzutti F, Corti G et al. Acute viral hepatitis increases liver stiffness values measured by transient elastography. Hepatology 2008; 47 (2): Castera L, Forns X, Alberti A. Non-invasive evaluation of liver fibrosis using transient elastography. J Hepatol 2008; 48 (5): Castera L, Vergniol J, Foucher J et al. Prospective comparison of transient elastography, Fibrotest, APRI, and liver biopsy for the assessment of fibrosis in chronic hepatitis C. Gastroenterology 2005; 128 (2): Foucher J, Chanteloup E, Vergniol J et al. Diagnosis of cirrhosis by transient elastography (FibroScan): a prospective study. Gut 2006; 55 (3): Ganne-Carrie N, Ziol M, de Ledinghen V et al. Accuracy of liver stiffness measurement for the diagnosis of cirrhosis in patients with chronic liver diseases. Hepatology 2006; 44 (6): Talwalkar JA, Kurtz DM, Schoenleber SJ, West CP, Montori VM. Ultrasound-based transient elastography for the detection of hepatic fibrosis: systematic review and meta-analysis. Clin Gastroenterol Hepatol 2007; 5 (10): Vergniol J, Foucher J, Terrebonne E et al. Noninvasive tests for fibrosis and liver stiffness predict 5-year outcomes of patients with chronic hepatitis C. Gastroenterology 2011; 140 (7): Mirault T, Lucidarme D, Turlin B et al. Non-invasive assessment of liver fibrosis by transient elastography in post transfusional iron overload. Eur J Haematol 2008; 80 (4): Fraquelli M, Cassinerio E, Roghi A et al. Transient elastography in the assessment of liver fibrosis in adult thalassemia patients. Am J Hematol 2010; 85 (8):

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