Assistant Professor of Gastroenterology Liver stiffness measurement (Fibroscan®) Principles - indications - results - limitations Samir Haffar M.D. Assistant Professor of Gastroenterology

Imaging (US, MRI, endoscopy) Clinical Examination Biological work-up Blood markers Fibrose Hepatic biopsy Imaging (US, MRI, endoscopy) FibroScan®

Ideal non-invasive test for diagnosis of liver fibrosis Simple Reproducible Readily available Less expensive than biopsy Predicts full spectrum of fibrosis Reflects changes occurring with therapy

Evaluation of chronic liver injury according to health care level Physical examination Liver function tests Serum Hyaluronate APRI or other simple tests Primary health care Ultrasound Fibroscan® Serum markers & algorithms Secondary health care Fibroscan® ARFI* MR elastography* Tertiary health care Liver biopsy HVPG * Promising but currently under investigation ARFI: Acoustic Radiation Force Impulse Imaging HVPG: Hepatic Venous Pressure Gradient Castéra L et al. Gut 2010 ; 59 : 861 – 866.

Bedossa P. Liver Int 2009 ; 29 (s1): 19 – 22. Liver stiffness Assessed by US (FibroScan®) & more recently by MRI Evaluates velocity of propagation of a shock wave within liver tissue (examines a physical parameter of liver tissue which is related to its elasticity) Rationale Normal liver is viscous Not favorable to wave propagation Fibrosis increases hardness of tissue Favors more rapid propagation Bedossa P. Liver Int 2009 ; 29 (s1): 19 – 22.

Fibroscan® (Echosens, Paris, France) Fibroscan® device Electronic platform Ultrasonic signals acquisition Numerical signal processing Integrated computer Stiffness measurement Examinations database Dedicated probes with unique technology Vibrator (50 Hz) US Transducer (3,5 MHz) Fibroscan® (Echosens, Paris, France)

Position of probe & explored volume Cylinder of 1 cm wide & 4 cm long From 25 mm to 65 mm below skin surface This volume is at least 100 times bigger than a biopsy sample

Interval around median Contains 50% of valid shots Results Stiffness (kPa) Median value of 10 shots 3.9 Kilo Pascals  IQR * (kPa) Interval around median Contains 50% of valid shots ≤ 25% of median value  At least 10 shots  Success Rate: ≥ 60%

Manufacturer’s criteria for LSM interpretation First step Number of shots ≥ 10 Second step Success rate ≥ 60 % Third step Interquantile range (IQR) ≤ 25% Failure Zero valid shot Unreliable results < 10 valid shots Success rate ≤ 60% IQR ≥ 25%

Liver stiffness values in healthy subjects 429 subjects 5.2 ± 1.5 kPa 5.8 ± 1.5 kPa p = 0.0002 Roulot D et al. J Hepatol 2008 ; 48 : 606 – 613.

Roulot D et al. J Hepatol 2008; 48 : 606 – 613. Liver stiffness values in healthy subjects with & without metabolic syndrome 5.3 ± 1.5 kPa 6.5 ± 1.6 kPa p < 0.0001 Roulot D et al. J Hepatol 2008; 48 : 606 – 613.

Liver stiffness cut-offs in chronic liver diseases Matavir F0-F1 Mild Fibrosis F2 Sign F3 Severe F4 Cirrhosis Liver stiffness values are expressed in kilopascals Range from 2.5 – 75 kPa Values around 5.5 kPa were recently shown to reflect normality LSM 2.5 – 7 kPa → Mild or absent fibrosis is likely LSM > 12.5 kPa → Cirrhosis is likely Castéra L et al. J Hepatol 2008 ; 48 : 835 – 847.

Faria SC et al. RadioGraphics 2009 ; 29 : 1615 – 1635. Progression of fibrosis in viral hepatitis Photomicrographs (magnification ×40; trichrome stains) F 0 F 1 F 2 F 3 F 4 Normal portal triads with no signs of fibrosis (stage F0) Portal fibrous expansion (stage F1) Thin fibrous septa emanating from portal triads (stage F2) Fibrous septa bridging portal triads and central veins (stage F3) Cirrhosis (stage F4), which appears as nodules of liver parenchyma separated by thick fibrous bands. Faria SC et al. RadioGraphics 2009 ; 29 : 1615 – 1635.

No significant fibrosis Perform LSM ≤ 6 kPa No significant fibrosis No biopsy F0 F1 F Intermediate values Grey area Biopsy if results influence management F2 Implementation of other NI tests ≥ 12 kPa Advanced fibrosis No biopsy F4 Treatment or Follow-up F3 The experience accumulated so far suggests that the performance of LSM is optimal for advanced stages of fibrosis (ie, >F3 according to the METAVIR scoring system). This is likely due to the proportionally reduced presence of necrosis and inflammation, which are known to affect LSM. On the contrary, within intermediate stages of fibrosis the occurrence of inflammation, steatosis, cholestasis and passive/active congestion of the liver, may affect LSM. Therefore, patients with advanced fibrosis or compensated cirrhosis represent an optimal setting for the use of LSM. The ideal candidate for TE is a lean patient with severe fibrosis. Vizzutti et al. Gut 2009;58:156-60.

Sandrin L. Ultrasound Med Biol 2003 ; 29 : 1705 – 1713. Shear wave propagation velocity according to severity of hepatic fibrosis (Metavir score) E = 3.0 kPa F 0 E = 7.7 kPa F 2 E = 27 kPa F 4 Sandrin L. Ultrasound Med Biol 2003 ; 29 : 1705 – 1713.

Liver stiffness for each Metavir stage in CHC Box-and-whiskers plot Gastroenterology 2005; 28:343 – 350. Hepatology 2005;41:48 – 54. Vertical axis is in logarithmic scale (wide range of F4 values)

Correlation between LSM & fibrosis stage * Gastroentérol Clin Biol 2008;32,58-67. ** J Hepatol 2009;49:1062-68, Aliment Pharmacol Ther 2008;28:1188-98. *** Hepatology 2010;51:454-62. Gastroentérol Clin Biol 2008;32:58-67.

Accuracy of a diagnostic test Dichotomous test (only 2 results) Sensibility (Sn) Specificity (Sp) Positive Predictive Value (PPV) Negative Predictive Value (NPV) Likelihood Ratios + & – (LRs) Diagnostic Odds Ratio (OR) Multilevel test (> 2 results) Receiver Operating Characteristic (ROC) CIs Newman TB & Kohn MA. Evidence-based diagnosis. Cambridge University Press, Cambridge, UK, 1st edition, 2009.

Hypothetical ROC curve Pines JM & Everett WW. Evidence-Based emergency care: diagnostic testing & clinical decision rules. Blackwell’s publishing, West Sussex, UK, 2008.

Accuracy of diagnostic test using AUC of ROC Value Accuracy 0.90 - 1.00 Excellent 0.80 - 0.90 Good 0.70 - 0.80 Fair 0.60 - 0.70 Poor AUROC of a ‘‘good” test should be ≥ 0.80 Pines JM & Everett WW. Evidence-Based emergency care: diagnostic testing & clinical decision rules. Blackwell’s publishing – West Sussex – UK – 2008.

Meta-analysis of TE for staging liver fibrosis 50 studies – random effect – all type of CLD Cirrhosis (F4): 0.94 (95% CI: 0.93 – 0.95) Cut-off value: 13.0 kPa Severe fibrosis (F3): 0.89 (95% CI: 0.88 – 0.91) Significant fibrosis (F2): 0.84 (95% CI: 0.82 – 0.86) Cut-off value: 7.7 kPa Friedrich R et al. Gastroenterology 2008 ; 134 : 960 – 974.

Significance of wide range of LSM in cirrhosis 13 - 75 kPa 2.5 13 26 36 49 53 62 75 EV stage 2 or 3 Child-Pugh B or C Additional advantage of Fibroscan is that it provides a wide range of stiffness values within the group of cirrhotic livers that would overcome one major limitation of the biopsy (i.e. one histological stage for all types of cirrhosis), and could thus provide an additional prognostic value within this group (i.e. one histological stage for all types of cirrhosis), and could thus provide an additional prognostic value within this group. Background: Transient elastography (FibroScan) is a new, non-invasive, rapid, and reproducible method allowing evaluation of liver fibrosis by measurement of liver stiffness. In cirrhotic patients, liver stiffness measurements range from 12.5 to 75.5 kPa. However, the clinical relevance of these values is unknown. The aim of this prospective study was to evaluate the accuracy of liver stiffness measurement for the detection of cirrhosis in patients with chronic liver disease. Methods: A total of 711 patients with chronic liver disease were studied. Aetiologies of chronic liver diseases were hepatitis C virus or hepatitis B virus infection, alcohol, non-alcoholic steatohepatitis, other, or a combination of the above aetiologies. Liver fibrosis was evaluated according to the METAVIR score. Results: Stiffness was significantly correlated with fibrosis stage (r = 0.73, p,0.0001). Areas under the receiver operating characteristic curve (95% confidence interval) were 0.80 (0.75–0.84) for patients with significant fibrosis (F.2), 0.90 (0.86–0.93) for patients with severe fibrosis (F3), and 0.96 (0.94–0.98) for patients with cirrhosis. Using a cut off value of 17.6 kPa, patients with cirrhosis were detected with a positive predictive value and a negative predictive value (NPV) of 90%. Liver stiffness was significantly correlated with clinical, biological, and morphological parameters of liver disease. With an NPV > 90%, the cut off values for the presence of oesophageal varices stage 2/3, cirrhosis Child-Pugh B or C, past history of ascites, hepatocellular carcinoma, and oesophageal bleeding were 27.5, 37.5, 49.1, 53.7, and 62.7 kPa, respectively. Conclusion: Transient elastography is a promising non-invasive method for detection of cirrhosis in patients with chronic liver disease. Its use for the follow up and management of these patients could be of great interest and should be evaluated further. Retrospective study of 711 patients with chronic liver diseases (95 with histologically proven cirrhosis). These preliminary findings need to be confirmed in long-term prospective follow-up studies to see whether liver stiffness values can predict the occurrence of clinical events in patients with compensated cirrhosis. Ascites HCC ? Variceal bleeding Foucher J et al. Gut 2006 ; 55 : 403 – 408.

Cumulative incidence of HCC based on LSM 866 CHC – Mean follow-up 3 years LSM > 25 kPa HR 45.5 (p< 0.001) 20 < LSM ≤ 25 HR 25.6 (p< 0.001) 15 < LSM ≤ 20 kPa HR 20.9 (p< 0.001) 10 < LSM ≤ 15 kPa HR 16.7 (p< 0.001) LSM ≤ 10 kPa HR 0 Hazard ratio as compared to LSM<10 kPa ≤ LSM 10.1-15 kPa 16.7 (95% CI, 3.71-75.2; P<0.001 LSM 15.1-20 kPa 20.9 (95% CI, 4.43-98.8; P<0.001 LSM 20.1-25 kPa 25.6 (95%CI, 5.21-126.1; P < 0.001 LSM >25 kPa 45.5 (95% CI, 9.75-212.3; P < 0.001 Liver stiffness, noninvasively measured by transient elastography, correlates well with liver fibrosis stage. The aim of this prospective study was to evaluate the liver stiffness measurement (LSM) as a predictor of hepatocellular carcinoma (HCC) development among patients with chronic hepatitis C. Between December 2004 and June 2005, a total of 984 HCV-RNA positive patients, without HCC or a past history of it, visited the University of Tokyo Hospital. LSM was performed successfully in 866 patients, who gave informed consent. During the follow-up period (mean, 3.0 years), HCC developed in 77 patients (2.9% per 1 person-year). The cumulative incidence rates of HCC at 1, 2, and 3 years were 2.4%, 6.0%, and 8.9%, respectively. Adjusting for other significant factors for HCC development, patients with higher LSM were revealed to be at a significantly higher risk, with a hazard ratio, as compared to LSM<10 kPa, of 16.7 (95% confidence interval [CI], 3.71-75.2; P<0.001) when LSM 10.1-15 kPa, 20.9 (95% CI, 4.43-98.8; P<0.001) when LSM 15.1-20 kPa, 25.6 (95%CI, 5.21-126.1; P < 0.001) when LSM 20.1-25 kPa, and 45.5 (95% CI, 9.75-212.3; P < 0.001) when LSM >25 kPa. Conclusions: This prospective study has shown the association between LSM and the risk of HCC development in patients with hepatitis C. The utility of LSM is not limited to a surrogate for liver biopsy but can be applied as an indicator of the wide range of the risk of HCC development. LSM: Liver Stiffness Measurement – HR: Hazard Ratio Masuzaki R et al. Hepatology 2009 ; 49 : 1954 – 1961.

Reproducibility of TE in assessing hepatic fibrosis. Bland Altman Plot Upper limit Lower limit Mean 95% limit of agreement 200 patients with chronic liver disease 2 different operators within 3 days (800 exams) 8 patients scored outside limits of agreement Fraquelli M et al. Gut 2007 ; 56 : 968 – 973.

Cost of FibroScan® versus liver biopsy Cost of liver biopsy 703 – 1 566 € in a French hospital with a one day observation period Fibroscan® ** FibroScan equipment 70 000 € Low running cost except probe calibration twice/year Cost per FibroScan exam 100 € with 150 exams annually * Blanc J et al. Hepatol Res 2005 ; 32 : 1 – 8. ** Canadian Agency for Drugs and Technologies in Health (CADTH). Transient Elastography (FibroScan) for Non-invasive Assessment of Liver Fibrosis; 2006.

AASLD guidelines. Hepatology, 2009 ; 49 : 1017 – 1044. Liver biopsy size Because grading, & staging of nonneoplastic diffuse parenchymal liver disease is dependent on adequate sized biopsy, a biopsy of at least 2-3 cm in length & 16-gauge in caliber is recommended Presence of fewer than 11 complete portal tracts in pathology report may be incorrect in recognition of grading, & staging due to insufficient sample size In a study of 10 000 liver biopsies, Bedossa demonstrated that the stage of fibrosis was correctly diagnosed in 65% of biopsies with length of 15 mm 75% of biopsies with length of 25 mm Bedossa P et al. Hepatol 2003 ; 38 : 1449 – 57. AASLD guidelines. Hepatology, 2009 ; 49 : 1017 – 1044.

Limitations of liver biopsy Sampling errors Extremely small portion of liver (1/50 000) Intraobserver & interobserver variation Even when widely validated systems used for score Invasive procedure Morbidity: pain in 20% of patients Major complications: bleeding or hemobilia in 0.5% Mortality:

Grading & staging systems for chronic hepatitis IASL1 Batts–Ludwig2 Metavir3 Grading system Minimal activity Mild activity Moderate activity Marked activity Marked activity & bridging Grade 1 Grade 2 Grade 3 Grade 4 A1 A2 A3 (kappa 0.2 – 0.6) Staging system No fibrosis Fibrous portal expansion Few bridges or septa Numerous bridges Cirrhosis Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 F0 F1 F2 F3 F4 (kappa 0.5 – 0.9) 1 Desmet VJ et all. Hepatology 1994;19:1513-1520. 2 Batts KP et all. Am J Surg Pathol 1995;19:1409-1417. 3 Bedossa P et all. Hepatology 1996;24:289-293.

Interpretation of different values of kappa Kappa from Greek letter κ Value of kappa Strength of agreement 0 – 0.20 Poor 0.21– 0.40 Fair 0.41– 0.60 Moderate 0.61– 0.80 Good 0.81–1.00 Very good kappa score ≥ 0.6 indicates good agreement Perera R, Heneghan C & Badenoch D. Statistics toolkit. Blackwell Publishing & BMJ Books, Oxford, 1st edition, 2008.

Liver biopsy is not the “gold standard” but is the “best available gold standard”

Contraindications of liver biopsy Uncooperated patients Disorders in coagulation profile Severe ascites Cystic lesions Vascular tumors (hemangiomas) Amiloidosis Congestive liver disease

R0C curves for FibroScan, FibroTest, & APRI for cirrhosis (F0 – F3 vs F4) Castera L et al. Gastroenterology 2005 ;128 : 343 – 50. Castera L et al. Lancet 2010 ; 375 : 1419 – 20.

Pitfalls of liver stiffness measurement  Obesity  Operator experience  Acute liver injury  Extrahepatic cholestasis  Increased CVP  Ascites  Narrow intercostal spaces

Obesity & operator experience

Unreliable results (16%) Limitations of liver stiffness measurement 13 369 examinations – 5 year prospective study – 5 operators BMI > 30 kg/m2 (OR 7.5) Operator experience (OR 2.5) Age > 52 years (OR 2.3) Type 2 diabetes (OR 1.6) Failure (3%) BMI > 30 kg/m2 (OR 3.3) Operator experience (OR 3.1) Age > 52 years (OR 1.8) Female sex (OR 1.4) Hypertension (OR 1.3) Type 2 diabetes (OR 1.1) Unreliable results (16%) Waist circumference 80 cm in women or 94 cm in men. Specific probe is developed for obese patients. LSM uninterpretable in one of five cases Main raisons: obesity ( WC) – operator experience Castéra L et al. Hepatology 2010 ; 51 : 828 – 835.

Castéra L et al. Hepatology 2010 ; 51 : 828 – 835. Failure rates according to BMI 7261 patients at the time of first examination Castéra L et al. Hepatology 2010 ; 51 : 828 – 835.

Castéra L et al. Hepatology 2010 ; 51 : 828 – 835. Unreliable results according to BMI 6968 patients at the time of first examination Castéra L et al. Hepatology 2010 ; 51 : 828 – 835.

Feasibility of LSM with FibroScan® using XL probe New probe for obese patients Feasibility of liver transient elastography with FibroScans using a new probe for obese patients de Lédinghen V et al. Liver International 2010 ; : 1043 – 1048. Background & Aims: Liver stiffness measurement (LSM) failure when using transient elastography occurs in 2–10% of patients, and is generally related to obesity. The aim of this prospective study was to assess the feasibility of LSM when using a new XL probe on patients with a body mass index(BMI)Z30 kg/m2. Methods: For each patient, LSM was performed using both M probe (currently available and dedicated to patients with standard morphology) and XL probe (dedicated to overweighed patients). A blood sample was taken to assess usual biological variables and simple readily available fibrosis blood tests. Results: Ninety-nine patients were included (27 men, mean age 52 years, mean BMI 40.5 kg/m2). LSM was successful (10 valid measurements) in 45% of the cases with the M probe, vs 76% of the cases with the XL probe (P<0.001). Fifty-nine percent of those who could not be measured (< 10 valid measurements) using the M probe could successfully be measured using the XL probe. In the 44 patients successfully measured with both probes, LSM was correlated with the platelet count, prothrombin time, g-glutamyltransferase, aspartate aminotransferase, fasting glucose, AST platelet ratio index, Forns score and FIB-4. Conclusion: The new XL probe allows providing a higher rate of LSM than the M probe in patients with an increased BMI and shows promising results for the evaluation of liver fibrosis. 60% not measured by M probe successfully measured by XL probe de Lédinghen V et al. Liver International 2010 ; : 1043 – 1048.

 Acute liver injury

Arena U et al. Hepatology 2008 ; 47 : 380 – 384. Acute viral hepatitis increases liver stiffness 18 patients with acute viral hepatitis When interpreting LSMs, the clinical question should be defined and LSM should be interpreted in the context of a defined diagnosis, biochemical data and appropriate cutoff points. Conclusion: LSM does not represent a reliable instrument to detect the presence of advanced fibrosis and cirrhosis in patients presenting with a clinical picture of acute hepatitis. I Peak increase in aminotransferase II Aminotransferase ≤ 50% of the peak III aminotransferase levels ≤ 2 ULN Arena U et al. Hepatology 2008 ; 47 : 380 – 384.

Arena U et al. Hepatology 2008 ; 47 : 380 – 384. Acute viral hepatitis increases liver stiffness 18 patients with acute viral hepatitis When interpreting LSMs, the clinical question should be defined and LSM should be interpreted in the context of a defined diagnosis, biochemical data and appropriate cutoff points. Conclusion: LSM does not represent a reliable instrument to detect the presence of advanced fibrosis and cirrhosis in patients presenting with a clinical picture of acute hepatitis. I Peak increase in aminotransferase II Aminotransferase ≤ 50% of the peak III aminotransferase levels ≤ 2 ULN Arena U et al. Hepatology 2008 ; 47 : 380 – 384.

 Extrahepatic cholestasis

Obstructive jaundice due to GIST occluding CBD Bilirubin 3.5 mg/dL Stiffness 5.7 kPa Stent placement Stent occlusion Bilirubin 8 mg/dL Stiffness 10 kPa Bilirubin 2 mg/dL Stiffness 5 kPa Transient elastography (FibroScan [FS]) is a novel non-invasive tool to assess liver fibrosis/ cirrhosis. However, it remains to be determined if other liver diseases such as extrahepatic cholestasis interfere with fibrosis assessment because liver stiffness is indirectly measured by the propagation velocity of an ultrasound wave within the liver. In this study, we measured liver stiffness immediately before endoscopic retrograde cholangiopancreatography and 3 to 12 days after successful biliary drainage in patients with extrahepatic cholestasis mostly due to neoplastic invasion of the biliary tree. Initially elevated liver stiffness decreased in 13 of 15 patients after intervention, in 10 of them markedly. In three patients, liver stiffness was elevated to a degree that suggested advanced liver cirrhosis (mean, 15.2 kPa). Successful drainage led to a drop of bilirubin by 2.8 to 9.8 mg/dL whereas liver stiffness almost normalized (mean, 7.1 kPa). In all patients with successful biliary drainage, the decrease of liver stiffness highly correlated with decreasing bilirubin (Spearman’s 0.67, P < 0.05) with a mean decrease of liver stiffness of 1.2± 0.56 kPa per 1 g/dL bilirubin. Two patients, in whom liver stiffness did not decrease despite successful biliary drainage, had advanced liver cirrhosis and multiple liver metastases, respectively. The relationship between extrahepatic cholestasis and liver stiffness was reproduced in an animal model of bile duct ligation in landrace pigs where liver stiffness increased from 4.6 kPa to 8.8 kPa during 120 minutes of bile duct ligation and decreased to 6.1 kPa within 30 minutes after decompression. Conclusion: Extrahepatic cholestasis increases liver stiffness irrespective of fibrosis. Once extrahepatic cholestasis is excluded (e.g., by liver imaging and laboratory parameters) transient elastography is a valuable tool to assess liver fibrosis in chronic liver diseases. Millonig G et al. Hepatology 2008 ; 48 : 1718 – 1723.

Millonig G et al. Hepatology 2008 ;48 : 1718 – 1723. Liver stiffness as a function of bile duct ligation 10 German landrace pigs: 5 controls – 5 BD ligation 120 min after Bile duct ligation 8.8 kPa 30 min after decompression 6.1 kPa Control 4.6 kPa Conclusion: Extrahepatic cholestasis increases liver stiffness irrespective of fibrosis. Once extrahepatic cholestasis is excluded (e.g., by liver imaging and laboratory parameters) transient elastography is a valuable tool to assess liver fibrosis in chronic liver diseases. Millonig G et al. Hepatology 2008 ;48 : 1718 – 1723.

 Increased CVP

Representation of clamping site of the IVC 5 German landrace pigs Experiment approved by local committee for Animal Welfare University of Heidelberg – Germany Millonig G et al. J Hepatol 2010 ; 52 : 206 – 210.

LSM after clamping & reopening of IVC 5 anesthesized landrace pigs 27.8 kPa 5 min after clamping 5 min after reopening 5.1 kPa Before clamping 3.1 kPa Millonig G et al. J Hepatol 2010 ; 52 : 206 – 210.

Millonig G et al. J Hepatol 2010 ; 52 : 206 – 210. Liver stiffness directly influenced by CVP 10 patients with CHF before & after recompensation Median 40.7 Median 17.8 p = 0.004 Median weight loss of 3.0 kg LS is a direct function of central venous pressure which should be considered when assessing the degree of fibrosis. The presence of enlarged liver and/or hepato-jugular reflux on physical examination together with dilated hepatic veins and inferior vena cava, suggestive of chronic heart failure, would have helped to anticipate the findings of liver biopsy. Millonig G et al. J Hepatol 2010 ; 52 : 206 – 210.

 Ascites

Ascites in liver cirrhosis Diagnosis of cirrhosis is obvious Ascites grade 1: detectable only by ultrasound

 Narrow intercostal spaces

Position of FibreScan® probe Dorsal decubitus position Right arm in maximal abduction

Interpretation of the results of LSM should always be done by expert clinicians according to clinical context

Transient elastography in clinical practice Examination quality 10 shots at least Success rate ≥ 60% IQR ≤ 25% of median value Liver disease Not used in acute hepatitis Not used in acute exacerbation Not used in ascites & EH cholestasis Choice of cutoff point Cutoffs different for each CLD Range of value rather than cutoff De Lédinghen V et al. Gastroentérol Clin Biol 2008 ; 32 : 58 – 67.

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