Chapter 5

NON INVASIVE

EVALUATION OF LIVER FIBROSIS

Contents

Introduction Serum Markers Indirect Markers AST/ALT Ratio PGA Index Fibrotest Acti Test Steato Test Forns index APRI (AST to Platelet Ratio Index) Gteborg University Cirrhosis Index (GUCI) Direct Markers of Liver Fibrogenesis Hyaluronic Acid YKL-40 (Chondrex) Collagen Matrix metalloproteinases and inhibitors Cytokines FibroSpect FibroSpect II European Liver Fibrosis Group assay Fibrometer Hepascore SHASTA Index

282 283 285 285 286 287 290 291 291 292 294 294 297 299 300 301 302 303 303 304 304 306 307

APRICOT Clinical Investigators Assay

13

307 308
280

C- Caffeine Breath Test

Fibrosis Markers to Assess Effect of Treatment Fibrosis Markers to Predict Disease Progression Gene Markers and Liver Fibrosis Radiology in the Noninvasive Assessment of Liver Fibrosis Measurement of Hepatic Stiffness (Elastography) Platelets in Chronic Liver Disease The role of Spleen Platelet Auto antibodies and Thrombocytopenia Thrombopoietin and Chronic Liver disease Platelet Parameters and Chronic Liver Disease

309 312 313 316 317 322 322 323 326

5.1 Introduction
Chronic liver diseases, CLD, are a major cause of morbidity and mortality in the present day world
1-6

. Chronic infections with hepatitis B and C viruses, alcoholic and 281

non-alcoholic fatty liver diseases are important causes of CLD

7-13

. The major

determinant in their prognosis is the progressive accumulation of fibrosis, with distortion of the hepatic architecture, and ultimate progression to cirrhosis and its allied complications
3,8,9-21

. In the recent years, with the great advancement in therapeutic

modalities to treat chronic liver diseases, accurate assessment of fibrosis has become of paramount importance; to guide management decisions, predict outcome (prognosis), and monitor disease activity in individual patients 21-26. Chronic hepatitis from HBV and HCV are the most common causes of cirrhosis and hepatocellular carcinoma in the world today. Of approximately 2 billion people who have been infected with HBV worldwide, more than 350 million, or about 5% of the worlds population are chronic carriers, and with an annual incidence of more than 50 million 1,27. HBV accounts for 500,000 to 1.2 million deaths per year 4. Similarly more than 3 % of the world population or about 170-5 million people may be infected with HCV 2,3,28,29. The prevalence of HCV is increasing and estimates of the future burden of chronic hepatitis C predict at least a 3 fold rise in chronic liver disease and cirrhosis by the year 2020 3,30-32. Nonalcoholic fatty liver disease (NAFLD) affects 10-30% of the general population in various countries. 10% of these people, or ~ 2-3% of the general population, satisfy the criteria for nonalcoholic steatohepatitis, (NASH). The prevalence of NAFLD is also expected to rise in developed countries given the epidemic of its major determinant, obesity 6,11,12,20,25,33-36. Studies indicate that advanced fibrosis and cirrhosis will develop in 20-40% of patients with chronic hepatitis B or C and in a similar proportion of patients with nonalcoholic fatty liver disease 3,4,5,7, 9,10,11,12,37,38. Therefore it is imperative for both clinicians and patients to acquire accurate information about the degree of fibrosis to guide management decisions predict outcome and monitor disease activity 23. Hepatic fibrosis is the final common pathway for a multitude of liver injuries. Viral, immune, toxin mediated liver injuries all result in expansion of the extracellular matrix, with the deposition of fibrous tissue, distortion of hepatic architecture and ultimately the development of cirrhosis 39.

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Percutaneous liver biopsy is considered the gold standard for grading and staging the liver disease. But it has its limitations, which include: small but significant morbidity and mortality rates, sampling error, inter- and intra-observer variability, limits of the histopathological scoring systems, and the provision of a static picture of liver architecture in a dynamic disease process 19,23. With the expanding knowledge of fibrosis, more accurate, reproducible, and noninvasive methods of determining liver fibrosis are required. Understanding the pathogenesis of hepatic fibrosis at molecular level has led to the identification of several potential serum markers of hepatic fibrosis. Either individually or in combination, these serum markers appear capable of determining early and advanced fibrosis 39. Serum markers of hepatic fibrosis offer an attractive alternative to liver biopsy; as they are less invasive than biopsy, may allow dynamic calibration of fibrosis and may be more cost effective 23. Additionally, radiological means can also noninvasively assess liver fibrosis. Ultrasonography and cross-sectional imaging with CT, MRI though enables detailed images of the liver and surrounding structures, but resolution of hepatic parenchyma is insufficient to determine any of the earlier stages of fibrosis before the establishment of cirrhosis and portal hypertension 40. Transient hepatic elastography is a novel technology demonstrating promise as a noninvasive means of fibrosis determination 41.

5.2 Serum Markers

According to Afdhal N 39, serum markers of hepatic fibrosis refer to the measurement of one or more molecules within blood or serum sample as markers of fibrosis in the liver
42

. There are several proposed biomarkers or combinations of

biomarkers. Ideal features of serum markers are 39:

Liver specific Independent of metabolic alterations Easy to perform Minimally influenced by urinary and biliary excretion

283

Reflective of fibrosis irrespective of cause Sensitive enough to discriminate between stages of fibrosis Correlate with dynamic changes in fibrogenesis or fibrosis resolution

According to Afdhal N 19, none of the available markers fulfill, these ideal criteria. None are liver specific, so there may be significant contribution from non-hepatic sources, including bone, joints, skin, and lungs. Levels of these markers are altered by changes in their clearance, metabolism, and excretion. For instance, both the liver (80%) and the kidneys (20%) clear hyaluronan and the removal from circulation is also dependent upon binding to specific receptors by hepatic endothelial cells for these receptors 44. Their relationship to the total matrix content of the liver and to the activity of fibrogenesis or degradation is usually mixed. Indeed, in the absence of a golden standard of matrix turnover, it is not possible to assess the relationship of the levels of these markers to ongoing matrix remodeling and new matrix deposition or removal. Thus from a practical and research standpoint, it would be ideal to have marker(s) that can (a) noninvasively stage the degree of liver fibrosis, (b) reflect the rate of matrix deposition or removal, both to monitor the impact of therapies and to give prognostic information. It is unlikely that any single marker can meet the acid test, and can fulfill both of these ideal criteria 19. Proposed serum markers of hepatic fibrosis can be categorized broadly as either direct or indirect. Indirect markers reflect alterations in hepatic function but do not directly reflect extracellular matrix (ECM) metabolism, for instance platelet count, coagulation studies, and assessment of liver transaminases. Direct markers of fibrosis reflect ECM turnover. Their discovery has been linked directly to advances in understanding of the molecular mechanisms of hepatic fibrosis. Serum assays for products of matrix synthesis or degradation and the enzymes involved in these processes have been investigated as markers of liver fibrosis in several studies routine liver biopsy for many patients with liver disease 54-57.
45-53 43

. Increased

hyaluronan levels occur in the post prandial state, presumably as a result of competition

. Combinations

of these markers, both direct and indirect, are emerging as a promising alternative to

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5.3 Indirect Markers

According to Afdhal N 39, various indirect markers of liver fibrosis have been used in clinical practice for many years, such as on physical examination, hepatomegaly and splenomegaly. Patients who have elevated AST, ALT, coagulopathy, hypersplenism with thrombocytopenia invariably have cirrhosis and portal hypertension. Initial efforts attempted to correlate these routine investigations with stage of hepatic fibrosis
39

. For

example the AST/ALT ratio 58, platelet count 59, and the prothrombin index 60. It should be reiterated that the indirect markers reflect the disturbance of hepatic function or structure, rather than the deposition or the removal of ECM 19. One of the main limitations to the clinical use of direct markers of liver fibrosis is that they are not routinely available in all hospital settings. While direct markers of liver fibrosis have shown promise in detecting liver fibrosis, the indirect markers satisfy the request for a simple and easy to perform marker with diagnostic accuracy for detecting liver fibrosis that is equal or better than direct markers.

5.4 AST/ALT Ratio

According to Afdhal N 39, Park and colleagues compared the AST/ALT ratio between 30 known cirrhotics and 123 controls without cirrhosis and reported a ratio greater than or equal to 1 had a 95.9% specificity and 73.7% positive predictive value in distinguishing patients who had cirrhosis from those who did not have cirrhosis, with 46.7% sensitivity and 88.1% negative predictive value. It appeared that though relatively insensitive, an AST/ALT greater than or equal to 1 is highly specific but not diagnostic for the presence of cirrhosis in patients who have chronic HCV infection 61. In a cohort of patients who had hepatitis C, Assy reported a moderate correlation between AST levels and the presence of advanced fibrosis
63 62

. A study of 252 patients

who had hepatitis C evaluated the diagnostic and prognostic value of the AST/ALT ratio . Diagnostically, the ratio performed well; an AST/ALT ratio of greater than 1 was

strongly suggestive of cirrhosis with sensitivity of 78% and specificity 97%. Combined use of the AST/ALT ratio with a platelet count of less than 130 x 109/l increased the

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diagnostic accuracy of the test with positive and negative predictive values of 97% and 86% respectively 39. Furthermore, a progressive increase in AST/ALT ratio was observed in patients with more advanced liver disease as determined by Child Pugh and MELD scores as well as by lidocaine metabolism (MEGX test) 19. The relative increase in AST is probably related to both reduced clearance of AST by hepatic sinusoidal cells as well as to mitochondrial dysfunction 64-66. The value of this ratio is greatest for the noninvasive diagnosis of cirrhosis, where a ratio of >1.0 is suggestive of this diagnosis disease
67 61

. This has been shown for both viral

and nonalcoholic fatty liver disease

68

. The efficacy of this ratio, is however,

not relevant to all forms of the liver disease, in particular alcoholic liver disease (associated with elevated AST) or conditions associated with predominantly with hepatic inflammation (autoimmne hepatitis) 39. The prognostic value of AST/ALT ratio was assessed in subset of 63 cirrhotic patients who were followed for at least one year. In this subgroup, an AST/ALT ratio greater than 1.16 was found to predict mortality and performed similarly to the Child Pugh and MELD scores. It should be noted that this study included a significant proportion of patients with clinically diagnosed cirrhosis and hence it would be expected that the diagnostic accuracy of this ratio for the detection of asymptomatic cirrhosis and earlier stages of fibrosis would be lower 19.

5.5 PGA Index

According to Afdhal N 39, PGA index was the original index of hepatic fibrosis described in 1991. It was devised as a simple biological index for detecting alcoholic liver disease in drinkers
69

. It combines the measurement of the prothrombin index,

gamma glutamyl transferase (GT) and apolipoprotein A1 (PGA). It has been validated in patients with various chronic liver diseases, in particular alcohol (hence the use of GT)
70,71

. The diagnostic accuracy of the PGA score for detecting cirrhosis is reported between
69, 70

66% and 72%

. This was subsequently modified to the PGAA index by the addition

of 2- macroglobulin, which resulted in some improvement in its performance 72. Oberti, in a study of 243 patients with chronic viral or alcoholic liver disease, assessed the utility of a large number of clinical and ultrasonographic parameters as well

286

as indirect and direct markers of liver fibrosis to diagnose cirrhosis

70

. Of the clinical

features of cirrhosis, a firm liver with a thin lower edge had the highest diagnostic accuracy of 83%. Of the indirect markers of fibrosis, the prothrombin index had the highest diagnostic accuracy 86%, while the performance of the PGA and PGAA scores were somewhat less good at 78.5% and 80%, respectively. In this study
70

, of all the specific markers of fibrosis being assessed, including

laminin, procollagen III N-peptide (PIIINP), hyaluronan, and TGF- 1, only hylauronan performed as well as the prothrombin index with a diagnostic accuracy of 86%. Of importance, it was noted that the aetiology of the liver disease affected the performance of some of these tests. For example analysis of the AST/ALT ratio had a diagnostic accuracy of 79% in viral liver disease, but as expected performed poorly in alcoholic liver disease, 65%. The PGA and PGAA scores performed better in alcoholic than viral liver disease, whereas, the prothrombin index and hyaluronan levels performed similarly in both disease groups 19.

5.6 Fibrotest
Fibrotest 54, is the most widely known, and the most validated of the tests of the noninvasive evaluation of liver fibrosis, with over 20 studies reported in the literature 24. Developed by Poynard and colleagues, several biochemical markers of liver fibrosis were assessed in 339 patients with hepatitis C. The aim of this study was to find out whether a panel of biochemical markers could reliably identify patients with clinically significant fibrosis (METAVIR F2 or greater) and thus be used to limit the need for liver biopsy in the selection of patients for treatment of hepatitis C. Of the assessed markers, 2 macroglobulin, 2 globulin (or haptoglobin), globulin, apolipoprotein A 1, glutamyl transferase, and total bilirubin were the most informative, and were used to derive a calculated index; now known as the Fibrotest. In the study, a total of 11 markers were assessed: 2 macroglobulin, AST, ALT, glutamyl transferase, total bilirubin, albumin, 1 globulin, 2 globulin, globulin, globulin, and apolipoprotein A1. The 2 globulins mainly consisted of 2 macroglobulin and haptoglobin. In the second period, Interleukin 10 (IL-10), tumor (transforming) growth

287

factor 1(TGF 1), hepatocyte growth factor, apolipoprotein A2, and apolipoprotein B were also assessed. However, the most informative markers were, in the decreasing rank: 2 macroglobulin, haptoglobin, GGT, globulin, total bilirubin, and apolipoprotein A1 54. The areas under the receiver operating characteristic curve, ROC, for the first year (0.836), and second year (0.870) did not differ (p=0.44). By selecting the upper and lower cut off values, the authors were able to categorize their patients into three groups: (1) those in which there was a high certainty of mild liver disease (METAVIR F0-1), (2) those with high certainty of significant fibrosis (METAVIR F2-4), (3) and a group of patients who could not be adequately characterized and in whom liver biopsy would be necessary, the indeterminate group 39. With the best index, a high negative predictive value (100% certainty of absence of F2, F3, or F4 fibrosis) was obtained in scores ranging from zero to 0.10 ( 12% of all patients), and high positive predictive value ( >90% certainty of presence of F2, F3, or F4 fibrosis) for scores ranging from 0.60 to 1.00 (34% of all patients). The detection of significant fibrosis F2 or greater had a 75% sensitivity and 85% specificity. The assay performed somewhat better for the assessment of more advanced liver disease (METAVIR stages 3 and 4). Thus the correct identification of the disease as either mild or severe was made in 46% of the patients overall, hence obviating the need for liver biopsy in these patients. This included 100% negative predictive value for the exclusion of METAVIR stages F2-4 fibrosis and a greater than 90% positive predictive value 54. The same authors have validated this score in other hepatitis C positive cohorts including those with HIVinfection same results . Rossi et al
76 76 73-75

, but an independent study did not achieve the

, studied 125 hepatitis C positive patients who had undergone liver

biopsy. Using local assays and the original authors computer program for the calculation of Fibrotest score, the performance of the assay was somewhat less impressive. The area under the receiver operating characteristic curve, ROC for significant fibrosis staging (> F2 on Metavir Index) was 0.739; which was smaller than the area under the ROC in the principal study. The negative and positive predictive values were 85% and 78%, respectively. Using these cut off values only 33 of the 125 patients would

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have been saved liver biopsy. Six of these patients would have been misclassified as having mild fibrosis, while the results of the liver biopsy demonstrated more significant disease requiring treatment 76. The reason for this discrepancy in results is unclear, since the original authors have shown that the assay and score appear to be reproducible when performed in different laboratories 77,78. Commenting on the Rossi et al study, in another study the Fibropaca study,
79

the

authors had the opinion that the smaller area under the ROC may be partially explained by non respecting the analytical recommendations for performing the Fibrotest, the low number of patients, and the lack of information concerning biopsy sample. More importantly, Rossi et al, did not discuss the causes of failures for the Fibrotest and biopsy. Various studies have highlighted the importance of the pre-analytical and analytical steps in the validation of the values of biochemical markers for carrying out the Fibrotest 77,80. The Fibropaca Study 79, an independent prospective multicenter study confirmed the diagnostic value of Fibrotest and Actitest found in the principal study and suggested that both the tests could be an alternative to liver biopsy in most patients with chronic HCV. In this study 504 patients were followed, 46% were classified as F2-F4 fibrosis and 39% as A2-A3 activity. The area under the ROC for the diagnosis of activity (A2-A3) was 0.73 (0.69-0.77), for significant fibrosis (F2-F4) was 0.79 (0.75-0.82), and for severe fibrosis (F3-F4) was 0.80 (0.76-0.83). Among the 92 patients (18%) with 2 fibrosis stages of discordance between Fibrotest and biopsy, the discordance was attributable to Fibrotest in 5.4% of patients, to biopsy in 3.8%, and undetermined in 9.1%. In analyzing the discordance between liver biopsy and biomarkers, the main difficulty is the absence of a true gold standard of liver injury. The most frequent failures attributable to the markers were false positives due to Gilberts disease and inflammation and false negatives due to inflammation. The most frequent failures due to biopsy were false negatives due to small biopsy size. Among the eight cirrhotic patients well defined by radiological, endoscopic, or biochemical criteria with discordant Fibrotest and liver biopsy, five were not diagnosed by liver biopsy and three were not diagnosed by Fibrotest, underlining the absence of a gold standard in determination of liver fibrosis.

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Nevertheless, both the Fibrotest and Acitest bring new insights in the diagnosis of fibrosis in patients with chronic hepatitis C. However, the identification of risk factors of Fibrotest and Actitest failures such as Gilberts syndrome, inflammation, and haemolysis must be considered before and after the Fibrotest-Actitest interpretation. The combined use of Fibrotest and Fibroscan (an instrument used to detect transient hepatic elastography) among 183 patients with chronic hepatitis C demonstrated an area under the ROC curve of 0.88 for F > 2, 0.95 for F > 3, and 0.95 for F > 4. When the Fibrotest and Fibroscan results agreed, liver biopsy examination confirmed them in 84% cases for F > 2, in 95% of cases for F > 3, and in 94% of cases for F > 4 complement each other and enhance accuracy of fibrosis detection 39.
81

Therefore it is likely that a combination of serum markers and Fibroscan will

5.7 Acti Test

Acti Test is a modification of the Fibrotest that incorporates ALT and reflects both necroinflammatory activity and liver fibrosis. According to the original study 54, the best logistic regression for diagnosis of clinically significant fibrosis (F2, F3, or F4) or substantial activity (A2 or A3) combined the same six markers as for fibrosis alone plus ALT ( R2 =0.297, p<0.0001). Acti Test appears to demonstrate improved identification of more advanced fibrosis associated with greater histologic inflammation
77

. Both Acti Test and Fibrotest

have shown reductions among those with sustained virological response to interferon and ribavirin therapies, supporting a role in following response to therapy 82. A meta-analysis of 16 publications and 1570 patients, by the same group
83

concluded that: at a cut off of 0.31, the Fibrotest negative predictive value for excluding significant fibrosis was 91%. At a cut off of 0.36, the Acti Test negative predictive value for excluding significant necrosis was 85%. Additionally, there was no difference between the area under the ROCs of Fibrotest/Acti Test according to the genotype or viral load. Thus the use of biochemical markers of liver fibrosis (Fibrotest), and necrosis (Acti Test) can be recommended as an alternative to liver biopsy for the assessment of liver injury in patients with chronic hepatitis C 83.

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5.8 Steato Test

Developed by Poynard and colleagues 84, Steato Test incorporates the five components of Fibrotest (2 macroglobulin, haptoglobin, apolipoprotein A1, glutamyl transferase, total bilirubin) and Acti Test (ALT) plus body mass index, serum cholesterol, triglycerides, and glucose adjusted for age and gender. A cut off of 0.30 had a 90% sensitivity and a cut off of 0.72 had a 90% specificity permitting to achieve a useful predictive value, 93% NPV and 63% PPV for a steatosis prevalence of 30%. Although the PPV of the Steato test is not that high, but still is significantly higher than those of previous markers to evaluate steatosis, such as, GGT, ALT, and ultrasonography 84.

5.9 Forns index

Forns et al
55

, reported an index based on four readily available variables; age,

platelet count, glutamyl transferase, and cholesterol levels. The study was confined to patients with hepatitis C and included both test and validation cohorts. The authors constructed a simple score system applying a constant to the obtained formula: 7.811- 3.131.In (platelet count) + 0.781.In (GGT) + 3.467.In (age) 0.014. (cholesterol) Using the test cohort of 125 patients, in a manner similar to the Imbert-Bismut study (Fibrotest) 54, the authors set thresholds for defining individuals with a high or low probability of significant fibrosis (METAVIR F2-4). The area under the ROC curve was 0.86 for the estimation group and 0.81 for the validation group
55

.When these criteria

were applied to the validation cohort, 51% of the population could be classified using these upper and lower cut off values. The lower cut of value had a 96% negative predictive value, whereas the upper cut off value had a positive predictive value of 66%. Hence this test was useful at excluding patients with minimal fibrosis, but was of limited value for the identification of patients with more advanced liver disease
19

. In summary

half of the patients with chronic hepatitis C without significant liver fibrosis can be identified with high accuracy with this panel; and liver biopsy could have been avoided in more than one third of the patients 55. One important aspect of the index is the use of very basic clinical parameters, and the fact that the accuracy of this model compares with models based on more

291

sophisticated variables (such as Fibrotest). In comparing with the study by Imbert-Bismut (Fibrotest)
54

some of the variables used do provide a high level of certainty of the

presence or absence of significant fibrosis, but the usefulness may be curtailed by the fact that some of the predictive markers (2 macroglobulin, haptoglobin, and apolipoprotein A1) are research tools not readily available in routine clinical practice in most centers. In addition, the patient population analyzed in the Imbert-Bismut study included a high proportion of patients with advanced hepatitis C infection, as indicated by the presence of significant fibrosis in up to 40% of the subjects. In the authors series, significant fibrosis was present in only 25% of the subjects a figure possibly closer to the spectrum of the disease in the community at large 55. Additionally, the performance of this index was compared to the Fibrotest by the authors of the later test
57

. They found that the results of the Forns index were

reproducible but performed slightly less well than Fibrotest. Major caveats of the Forns index include concerns about the impact of lipid abnormalities in patients with hepatitis C reproducibility of platelet estimations 19.
57

, cholesterol altering medicines, and the

5.10

APRI (AST to Platelet Ratio Index)

Reported by Wai and colleagues 56, APRI refers to the AST to platelet ratio index.

In this retrospective study, a total of 270 patients were evaluated, which included training (n=192), and validation cohorts (n=78). The authors looked at a number of simple laboratory studies and their relationship to two end points, significant liver fibrosis and cirrhosis. Based on their analysis of simple laboratory measurements, they found that the APRI was the simplest and the most accurate test for the evaluation of two end points 19. It is calculated as follows:

APRI = AST level / ULN x 100 Platelet count (109/l)

292

Platelet counts and AST levels are the most important predictors of significant fibrosis and cirrhosis. This novel index was developed to amplify the opposing effects of fibrosis on AST and platelet count. The area under the ROC curve of APRI for predicting significant fibrosis and cirrhosis in the training set were 0.80 and 0.89, respectively. In the validation cohort the same values were 0.88 and 0.94, respectively. While the upper and lower cut off levels were selected with the aim of correctly characterizing this cohort into those with significant fibrosis and cirrhosis, the index performed reasonably well. Thus, using the optimized cut off values, significant fibrosis could be predicted accurately in 51% and cirrhosis in 81% of patients 56. APRI test successfully and simply can exclude those with and without significant fibrosis and cirrhosis with negative predictive values of 86% and 98%, respectively; and corresponding positive predictive values of 88% and 57% 56. Its most important aspect is the use objective and readily available laboratory values. Both platelet count and AST levels are routine tests performed in chronic hepatitis C patients in clinical practice, so no additional tests are required. The finding of decreased platelet count and increased AST level with progression of liver fibrosis has been reported in many studies. With increasing fibrosis and worsening portal hypertension, there is increased sequestration and destruction of platelets in the enlarging spleen 85. In addition, studies in liver transplant patients showed that the progression of liver fibrosis is associated with decreased production of thrombopoietin by hepatocytes, and hence reduced platelet production 86,87. Progression of liver fibrosis may reduce the clearance of AST, 65 leading to increased serum AST levels. In addition, advanced liver disease may be associated with mitochondrial injury, resulting in more marked release of AST, which is present in mitochondria and cytoplasm, relative to ALT 64,66. One caveat of the study is that: it included a sufficient proportion of patients with significant fibrosis (47%) and cirrhosis (15%) from a tertiary care center; so the results may not be totally generalizeable to community based practice.

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Though slightly inferior, nevertheless, its simplicity (APRI can be determined by the bedside with the help of a calculator), matched with performance to the more sophisticated Fibrotest and Forns index is a great advantage 39.

5.11

Gteborg University Cirrhosis Index (GUCI)

In this study 88, samples from 179 patients with chronic hepatitis C were analyzed

using routinely available biochemical markers of liver disease, and compared with liver biopsy using the Ishak protocol. By multivariate logistic regression analysis the authors found strong association between AST, platelet count and prothrombin-INR. They developed the Gteborg University Cirrhosis Index (GUCI) according to the formula:

Normalized AST x prothrombin-INR x 100 Platelet count (x 109/l)

Using a cut-off value of 1.0, the sensitivity was 80%, and the specificity 78% for the diagnosis of cirrhosis, and the negative predictive value and the positive predictive values were 97% and 31% respectively. The authors concluded that the GUCI score proved slightly superior for sensitivity, specificity, NPV, PPV, and the area under the receiver operating characteristic curve (ROC) for the prediction of cirrhosis and bridging fibrosis compared with AST to platelet ratio index (APRI) 88.

5.12

Direct Markers of Liver Fibrogenesis

According to Afdhal N
39

, the extracellular matrix refers to a group of

macromolecules (both soluble and insoluble) that comprise the supporting framework of the normal and fibrotic liver. The liver fibrosis results in qualitative and quantitative changes in ECM markers. Some ECM markers reflect fibrogenesis, and others reflect fibrosis regression, a dynamic evaluation of liver fibrosis reflecting ECM activity is possible. The direct markers of liver fibrosis include a number of serum or urinary markers which have been shown to be, or are thought to be directly involved in the deposition or

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removal of ECM (see table 17) 19. The potential markers of fibrosis include products of collagen synthesis or degradation, enzymes involved in matrix biosynthesis or degradation, extracellular matrix glycoproteins, proteoglycans and glycosaminoglycans
39

. None of the currently available direct markers completely fulfill the ideal criteria.

In particular, none is liver specific, and all are affected by changes in their clearance, metabolism, and excretion. The serum half lives of these molecules is short lived, so levels probably reflect the activity of ECM metabolism. Since the ECM turnover is related to both new ECM deposition and removal, as well as remodeling of established ECM, serum levels probably reflect both the activity of the process as well as the total mass of ECM undergoing metabolism 19. This is supported by at least three features. First levels of these markers are most often elevated in conditions with rapidly progressing fibrosis (e.g., severe alcoholic hepatitis or more active hepatitis) and may be high prior to the deposition of ECM
91,92 89,90

Second, levels tend to fall in response to treatment of the underlying disease process, often before to any discernible reduction in the stage of fibrosis . Third in chronic

liver diseases, elevations of several, but not all of these markers correlate independently with the stage of fibrosis, rather than with either biochemical or histological features of inflammation 53, 93-95. However, in some studies serum levels of these markers correlated more strongly with the degree of histological inflammation or transaminases96,97. The observation that markers of ECM metabolism are increased in parallel with markers of liver inflammation and necrosis may reflect the importance of these processes in upregulating fibrogenesis
19

. According to Afdhal N 19, the direct markers of liver fibrosis can be conveniently

subdivided into three groups: Those reflecting (a) matrix deposition, (b) matrix removal, or (c) where the relationship to the matrix deposition or removal is unclear. One approach to the evaluation of matrix deposition is to simultaneously measure markers of both matrix deposition and removal, with a view to define the net effect upon the fibrotic process. However, using combination of these markers has added

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little diagnostic accuracy, while increasing both the expense and the complexity of interpreting results19.

Table 17. Direct markers of liver fibrosis19

Markers of matrix removal

Procollagen IV C peptide Procollagen IV N peptide (7-S collagen) Collagen IV Metalloproteinase MMP Undulin Urinary desmosine and hydroxylysylpyridinoline
Markers of matrix deposition

Procollagen I carboxy terminal peptide (PICP) Procollagen III amino terminal peptide (PIIINP) Tissue inhibitor of metalloproteinase (TIMP) Transforming growth factor beta (TGF-) Tenascin
Uncertain

Hyaluronic acid YKL-40 (Chondrex) Laminin

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Fibrosis markers can also be classified according to their molecular structure

19

These include: (a) the collagens; procollagen I and III, propeptides released into the serum during matrix deposition and remodeling. Type IV collagen, which is released during interstitial filament degradation, reflects matrix degradation and remodeling. Several assays for type IV collagen are available but their performance tends to be similar; (b) The glycoproteins and polysaccharides including hyaluronan, laminin, tenascin, and YKL-40, which are found in association with the basement membranes and in regions of matrix deposition. The relationship between these markers and matrix metabolism has not been clearly defined; (c) Collagenases and their inhibitors, include the metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs); (d) Cytokines involved in liver fibrosis, the best studied of these is TGF-. Others including platelet derived growth factor and the antifibrotic cytokine IL-10, have been less well evaluated 19.

5.13

Hyaluronic Acid
According to Afdhal N
39

, in evaluating single marker assays that reflect ECM

concentration, the best individual test appears to be hyaluronic acid (HA), which has been validated in many clinical trials. Studies have demonstrated that serum hyaluronic acid levels correlate with the degree of hepatic fibrosis in patients who have chronic hepatitis C 53,98. HA is a high molecular weight glycosaminoglycan, which is an essential component of extracellular matrix in virtually every tissue in the body 99. In the liver, HA is mostly synthesized by the hepatic stellate cells and degraded by the sinusoidal endothelial cells
94

. HA levels are increased in chronic liver diseases 94. In patients with

chronic hepatitis C virus, HA levels increase with the development of liver fibrosis. Moreover, in patients with cirrhosis, HA levels correlate with clinical severity 100,101,102. Serum hyaluronic acid can identify those without cirrhosis accurately. A level of 60 g/l had a 99% negative predictive value for the absence of cirrhosis. It had low accuracy for diagnosing cirrhosis (30% positive predictive value) 53.

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A study by Halfon et al,

103

focused on the diagnostic accuracy of HA alone,

(instead of combination of markers) in predicting fibrosis and cirrhosis in HCV infected patients. In all 405 patients were studied. Absence of significant fibrosis, severe fibrosis, and cirrhosis can be predicted by HA levels of 16, 25, and 50 g/l respectively (with NPV of 82%, 89%, and 100% in the same order). Presence of significant fibrosis, severe fibrosis, and cirrhosis can be predicted by HA levels of 121, 160, and 237 g/l respectively (with PPV of 94%, 100%, and 57% in the same order). Thus serum HA is a clinically useful as a noninvasive marker of liver fibrosis and cirrhosis. It suffers from the need to limit, as much as possible, potential confounding variables such as the effects of exercise and eating. According to Afdhal N
70

, Oberti evaluated four specific markers of fibrosis

(PIINP, HA, Laminin, TGF-). These specific markers of fibrosis were studied in 243 patients with viral and alcoholic liver disease 70. In this prospective study HA performed best with a diagnostic accuracy of 86%, whereas the performance of other specific markers of fibrosis was less impressive; PIIINP 74%, laminin 81%, and TGF- 67%. Overall, the results of this study did not demonstrate any diagnostic advantage of the specific over the nonspecific markers of fibrosis19. Other comparative studies have also supported the superiority of HA over PIIINP for the diagnosis of cirrhosis 98,104,105. For example in a comparative study of 326 patients with hepatitis C, areas under the receiver operating curve (ROC) for the diagnosis of fibrosis and cirrhosis were 0.86 and 0.92 for HA, while they were 0.69 and 0.73 for PIIINP respectively 98. One explanation for the difference in performance may be related to the observation that PIIINP levels correlate more closely with histological and serum markers of hepatic inflammation than HA 104. The greatest clinical utility of HA may be its ability to exclude patients with significant fibrosis and cirrhosis
53, 103

. In a study of 486 patients who entered the

hepatitis C Consensus Interferon Study, an HA level of <60 mg/l was found to exclude patients with cirrhosis and significant hepatic fibrosis with predictive values of 99% and 93% respectively. Hence a low HA level may have a special role in identifying patients with early fibrosis and hence reduce the need for biopsy in this subgroup of patients 19,103.

298

5.14

YKL-40 (Chondrex)
YKL-40 is a novel marker of liver fibrosis. YKL-40 (chondrex, human cartilage
106

glycoprotein-39), according to Saitou et al belongs to the chitinase family

110 107

, is a recently described glycoprotein that

109

. YKL-40 m RNA was strongly expressed in human ,

106

liver and arthritic articular cartilage107,108 and was elevated in the synovial fluid alcoholic liver disease , recurrent breast cancer, and colorectal cancer .

Although its physiologic function is not known in detail, YKL-40 is thought to contribute to tissue remodeling or degradation of the extracellular matrix in liver fibrosis
111

. YKL-40 is produced by a wide variety of cells, including: chondrocytes, synovial

109

cells

, activated macrophages, neutrophils, and in particular from cells located in

111 112

tissues with increased remodeling/degradation or inflammation of the extracellular matrix, such as hepatic stellate cells . It is growth factor for fibroblasts , ,
113

chondrocytes and synovial cells. It is a chemo-attractant for endothelial cells tubules, indicating that YKL-40 may have a role in angiogenesis 106. The authors
106

modulates vascular endothelial cell morphology by promoting the formation of branching

studied Type IV collagen, amino-terminal peptide of type III

procollagen (PIIIP), hyaluronic acid (HA), YKL-40 and biochemical parameters. The authors concluded that HA and YKL-40 were more useful than other markers for assessing the fibrosis stage. In particular, YKL-40 was the most useful for monitoring the fibrosis of liver disease and for distinguishing extensive liver fibrosis from mild stage of liver fibrosis, enabling to predict severe stage of fibrosis at 80% positive predictive value. It appears that serum levels of YKL-40 represented ongoing fibrosis like HA, in addition to fibrogenesis similar to type IV collagen and PIIIP of the liver disease. Serum YKL-40 levels is valuable for diagnosing mild stage of fibrosis (value < 186.4), severe stage of fibrosis (186.4 < value < 284.8), and F4 (284.8 < value). HA appeared to be slightly better for prediction of cirrhosis (F4) from chronic hepatitis (F0-3) than YKL-40. Additionally, after Interferon therapy, only YKL-40 values significantly decreased not only in the responder group, but also in the non-responder group 106. Further studies have also shown that YKL-40 appears to be reduced in patients with chronic hepatitis C undergoing treatment, in conjunction with several other direct markers of hepatic fibrosis, including PIIINP, MMP-2, and TIMP-1
114

. In a study of

299

YKL-40 in alcoholic liver disease suggested that it could function as an independent marker of clinical outcomes 115.

5.15

Collagen
According to Afdhal N
39

, collagen, the major component of hepatic fibrosis, is

synthesized in a precursor form known as procollagen. Enzymatic cleavage by two distinct enzymes liberates carboxy and amino terminal procollagen peptides that have proposed correlation with fibrogenesis. Procollagen type III amino terminal peptide (PIIINP) is known to be elevated in acute hepatitis, and levels reflect stage of fibrosis in chronic liver disease
71,97,116

. PIIINP does not perform as well as HA for predicting

fibrosis but does reflect inflammatory score better 98. Type IV collagen has been immunolocalized to the periportal interstitium and large fibrotic bands in alcoholic liver disease chronic liver diseases relative to controls inconsistent48,105,119. According to Afdhal N 19, Murawaki et al, assessed the diagnostic performance of two forms of type IV collagen (7s domain and central triple helix domain) in 151 patients with viral hepatitis 119. While the two assays performed similarly, the 7s domain achieved slightly greater diagnostic accuracy for identification of patients with cirrhosis, with positive and negative predictive values of 75% and 92% respectively. In another study from the same group, the correlation between two assays for type IV collagen and fibrosis scores were similar to HA but better than PIIINP. However, the correlation to histological inflammatory scores and ALT values were greater, suggesting that type IV collagen may be a marker of both inflammation and fibrosis 104. At this time there appears to be no clear advantage to the use of assays of type IV collagen over HA for fibrosis staging 19.
118 117

. Serum type IV collagen is increased in

. Some studies have demonstrated equivalent

efficacy in fibrosis determination to that seen with HA, but results have been

300

5.16

Matrix metalloproteinases and inhibitors

According to Afdhal N
39

, the matrix metalloproteinases (MMPs) and their

inhibitors are a group of proteins involved in controlling matrix degradation. MMPs are enzymes that are produced intracellularly and secreted in a proenzyme form that requires cleavage by cell surface mechanisms for functional activity. The action of MMPs is counteracted by tissue inhibitors of metalloproteinases (TIMPs) 39. As such these proteins act to both degrade ECM and to permit new matrix deposition. However, the relationship is complex and in addition to the actions cited above; these molecules have multiple other activities including: activation of growth factors, effects on cell proliferation, and inhibition of apoptosis. Thus the association between peripheral levels and liver fibrosis is not clear19. The hypothesis that hepatic fibrogenesis is associated with disrupted matrix degradation stems from hepatic stellate cell research. MMP-13 is decreased in activated stellate cell culture, while TIMP activity increases, promoting accumulation of extracellular matrix. This corresponds to findings in human cirrhotic livers explants that demonstrate increased expression of TIMPs
120,121

. Therefore, it seems that imbalance

between MMPs and TIMPs affects rate of fibrosis progression, and their estimation correlates with stage of fibrosis. Results from studies of these markers, however, have been variable and dependent upon the MMP/TIMP being assessed 51,115,122,123. According to NA
19 122

, Boeker et al

, reported on the diagnostic accuracy of

TIMP-1 and pro-MMP-2, the free precursor molecule of MMP-2, and their relationship to histological inflammatory scores. In this study of 78 patients with hepatitis C infection, both of these assays performed as well or better than HA for the diagnosis of cirrhosis, but only TIMP-1 showed diagnostic value for the identification of patients with earlier stages of fibrosis. MMP-2 levels become elevated only once cirrhosis has developed. Disturbing characteristics of these assays include the observation that TIMP-1 levels correlate strongly with histological inflammatory scores and that in the noncirrhotic liver MMP-2 levels demonstrated no relationship to the stage of fibrosis. These results are similar to other studies and significantly limit their value for staging liver disease
123,124

. Serum MMP-1 and MMP-3 levels have not been shown to be of

significant diagnostic value51,125.

301

5.17

Cytokines
Cytokines are thought to mediate hepatic fibrogenesis have also been evaluated in

determining hepatic fibrogenesis in a limited number of studies with mixed results19. TGF- is the dominant stimulus for producing extracellular matrix by hepatic stellate cells. In a study of 88 patients who had chronic hepatitis C, there was a correlation between total TGF- 1 and the degree of hepatic fibrosis 126. In another study of 39 patients with two liver biopsies, a close correlation was noted between TGF- serum levels and the rate of fibrosis progression, which may suggest the use of TGF- to determine those with progressive disease suitable for antiviral therapy
127

. But the performance of TGF- appears to be less than the other

already discussed markers 54,70,126. According to Afdhal N 19, a few studies have evaluated the relationship between the levels of soluble adhesion molecules and liver inflammation and fibrosis
128,129,130

Increased expression of intracellular adhesion molecule-1 (ICAM-1) occurs in virus infected hepatocytes; whereas expression of vascular adhesion molecule-1 (VCAM-1) may be seen in association with progressive hepatic fibrosis
128-130

. It is thought that

increased VCAM-1 expression occurs in association with capillarization of the sinusoidal spaces and fibrous septa. In a study of 52 patients with hepatitis C virus, soluble VCAM-1 was found to have excellent discriminant power for the detection of advanced fibrosis, sensitivity 100%, and specificity 85%. In this small study, measurement of soluble VCAM was of greater diagnostic value than PIIINP128. Further studies are needed to confirm these results, in larger patient populations and in other disease cohorts 19.

Panels with Indirect & Direct markers of liver fibrosis

Combinations of direct markers have been tried with indirect markers to enhance the accuracy of liver fibrosis detection. Some of the assays are:

302

5.18

FibroSpect
McHutchison and colleagues in a retrospective cohort study evaluated the
131

FibroSpect assay

. The aim of the study was to evaluate the diagnostic accuracy of a

panel of markers in chronic hepatitis C patients, develop a predictive algorithm that differentiates no/mild (METAVIR F0-F1) from moderate/severe (F2-F4) fibrosis and validate the model in external cohorts. The assay involves three parameters: hyaluronic acid, TIMP-1, and 2 macroglobulin. These three markers were selected as having the best predictive accuracy for F2-F4 fibrosis (combined AUROC = 0.831) 131. At an index cut off of >0.36 and prevalence for F2-F4 of 52%, results in all 696 patients indicated positive and negative predictive values of 74.3% and 75.8% with an accuracy of 75%. All patients were evaluable by the FibroSpect with no indeterminate values, and this is an advantage of the assay 39. Maximum sensitivity and specificity were seen at the two extreme spectrums of disease, stage 0 and stage 4
39

. The authors

concluded that the three marker panel may reliably differentiate chronic hepatitis C patients with moderate/ severe fibrosis from those with no/mild fibrosis; although accurate delineation between stages was not possible the score 39.
131

. In clinical use, the test gives a

likelihood of prediction as to whether the patient has mild or advanced disease based on

5.19

FibroSpect II
In a recently reported retrospective study 132, hyaluronic acid, YKL 40, and

FibroSpect II (comprising hyaluronic acid, TIMP-1 (tissue inhibitor of metalloproteinase 1), and alpha 2 macroglobulin) were assessed with Ishak stages and digital quantification of fibrosis (DQF). Among the serum markers, hyaluronic acid was effective in discriminating Ishak stages 0-1 and Ishak stages 2-3 compared with FS-II, with area under the ROC curve of 0.76 versus 0.66 respectively. All three serum markers predicted advanced fibrosis and cirrhosis. YKL-40 had the highest false positive rates in all categories of fibrosis.

303

5.20

European Liver Fibrosis Group assay

The ELF group
133

reported a novel assay in an international multi-center cohort

of 1021 patients with hepatitis C, nonalcoholic fatty liver disease, and alcoholic liver disease. The authors aimed to develop a panel of sensitive automated immunoassays to detect matrix constituents and mediators of matrix remodeling in serum to evaluate their performance in the detection of liver fibrosis. Serum levels of 9 surrogate markers of liver fibrosis were compared with fibrosis stage in liver biopsy specimens obtained from all 1021 subjects with chronic liver disease. Discriminant analysis of a test set of samples was used to identify an algorithm combining age, hyaluronic acid, amino-terminal propeptide of type III collagen (PIIINP), and tissue inhibitor of matrix metalloproteinase 1. The sensitivity for the detection of Scheuer stage 3 or 4 fibrosis was 90% at a threshold score of 0.102, and accurately detected the absence of fibrosis (negative predictive value for significant fibrosis, 92%; area under the curve of a receiver operating characteristic plot, 0.804; standard error, 0.02; p < 0.0001; 95% confidence interval, 0.758-0.851). The algorithm performed equally well in comparison with each of the pathologists. In contrast, the pathologists agreement over histological scores ranged from very good to moderate (kappa = 0.97-0.46). Thus the algorithm achieved a similar level of sensitivity and specificity when compared with the scoring of three different pathologists, providing evidence that it could be used with similar accuracy in different settings. The authors concluding that assessment of liver fibrosis with multiple serum markers used in combination is sensitive, specific, and reproducible, suggesting they may be used in conjunction with liver biopsy to assess a range of chronic liver diseases 133.

5.21

Fibrometer
In this landmark study 134, the authors studied 383 patients with viral hepatitis, 95

patients with alcoholic liver disease in the exploratory step, and the validating population consisted of 120 patients with chronic liver disease due to HCV. The objective was to develop tests to characterize different fibrosis parameters (Fibrometer) in viral and

304

alcoholic liver diseases. Measurements included 51 blood markers, and Fibrotest, Fibrospect, ELFG, APRI, and Forns scores. The clinically significant fibrosis was evaluated via Metavir staging (F2-F4), and image analysis was used to determine the area of fibrosis (AOF). In patients with chronic viral hepatitis, the area under the receiving operator characteristic (AUROC) curve for stages F2-F4 in a test termed the Fibrometer test, combining platelets, prothrombin index, AST, 2 macroglobulin (A2M), hyaluronate, urea, and age was 0.883 compared with 0.808 for the Fibrotest (p = 0.01), 0.820 for the Forns test (p = 0.005), and 0.794 for the APRI test (p < 10-4). The Fibrometer AUROC curve was 0.892 in the validating step in 120 patients. In patients with alcoholic liver disease the AUROC curve for stages F2-F4 in a test combining prothrombin index, A2M, hyaluronate, and age was 0.962. The area of fibrosis was estimated in viral hepatitis by testing for hyaluronate, glutamyl transferase, bilirubin, platelets, and apolipoprotein A1, the adjusted R hyaluronate, prothrombin index, A2M, and platelets (aR2 = 0.836). The 95 patients with alcoholic CLD were significantly older and had more marked fibrosis than patients with viral CLD, something that could have influenced the statistical results. The authors claimed that their study was original because it measures the area of fibrosis, AOF, in addition to histological staging, takes into account the cause of CLD, and includes a large number of blood variables (n = 51). The results of blood tests for clinically significant fibrosis were better in the largest specimens, thus test results could be even better with new criteria for specimen length, > 20 mm
135,136 2

coefficient in linear regression (aR2 = 0.645), and in alcoholic liver diseases by testing for

. It has been suggested that the variability is greater for AOF

135

evaluation than it is for histological staging

. AOF is the only quantitative

morphological method of determining the amount of liver fibrosis, and it has been suggested that it is superior to histological staging 95. For instance, there is restriction of cirrhosis to one stage (F4), whereas the amount of fibrosis in cirrhosis is four times that of the other four stages 95.

305

Although there was less observer variability for histological staging when a consensual reading was performed by two experts
137

, blood test results were not perfect

(e.g., the diagnostic accuracy, DA, for the Fibrometer test was 83.3% in the validation population). This might be due to preanalytical or analytical variability in blood variables or variability in the pathological reference. Studies with the Fibrotest have suggested that most errors are due to histological staging itself 80,138. The authors commented that the plots of the blood test results and of interobserver agreement in relation to F stages
137

, all had the same V-shape with a nadir at the F2

stage, suggesting that the difficulty in distinguishing F2 from F1 or F3 stages via histological staging was the main cause of misclassification by the blood tests 134. The use of blood tests to estimate the area of fibrosis is new. Measurement of area of fibrosis via image analysis is limited to clinical research. Unlike histological staging, the AOF provides precise quantification of the extensive variations in fibrosis during cirrhosis. AOF estimation via blood test is the only statistically validated quantitative test for the noninvasive diagnosis of fibrosis. However, the aim of AOF evaluation is not to distinguish mild stages due to overlap of AOF values in these stages 134. The authors concluded that: the pathological staging and the area of liver fibrosis can be estimated using different combinations of blood markers in viral and alcoholic liver diseases. The Fibrometer has a high diagnostic accuracy for clinically significant fibrosis; blood tests for the area of liver fibrosis provide a quantitative estimation of the amount of fibrosis, which is especially useful in cirrhosis 134.

5.22

Hepascore
In this study
139

, the authors evaluated 10 biochemical biomarkers at the time of

liver biopsy in 117 untreated hepatitis C patients, and further validated in 104 patients. Multivariate logistic regression and ROC curve analyses were used to create a predictive model for significant fibrosis (METAVIR F2, F3, and F4), advanced fibrosis (F3 and F4), and cirrhosis (F4). A model, Hepascore, consisting of bilirubin, glutamyl transferase, hyaluronic acid, 2 macroglobulin, age and sex produced areas under the ROC curves of 0.85, 0.96 and 0.94 for significant fibrosis, advanced fibrosis, and cirrhosis, respectively. In the 306

training set, the model was 92% specific and 67% sensitive for significant fibrosis, 81% specific and 95% sensitive for advanced fibrosis, and 84% specific and 71% sensitive for cirrhosis. Thus a model consisting of four serum markers plus age and sex provides clinically useful information regarding different fibrosis stages among hepatitis C patients.

5.23

SHASTA Index
Developed by Afdhal N and colleagues 140, the SHASTA index consists of serum

hyaluronic acid, AST, and albumin. It was evaluated in a cohort of 95 patients with HIV/HCV coinfection. As with other biomarker assays, optimal results were noted in the extreme categories. Using a cut off of 0.8 resulted in a specificity of 100% and a positive predictive value of 100%, but this applied to less than 5% of patients. At the other end of the spectrum, a cutoff of less than 0.30 was associated with a sensitivity of more than 88% and a negative predictive value of more than 94%. Overall 42% of patients could be correctly classified at either extreme, but 58% would not be classifiable with scores between 0.3 and 0.8. The SHASTA index in HIV/HCV has similar accuracy to the Fibrotest and in this study performed significantly better than the APRI test.

5.24

APRICOT Clinical Investigators Assay

A retrospective analysis of liver biopsies was performed in 832 patients with

HIV/HCV co-infection, who were randomly assigned to training (n = 555), and validation (n = 277) sets. The authors derived a simple index (FIB-4) 141: Age ([yr] x AST [U/L]) / ((Plt [109/L]) x (ALT [U/L]) (1/2))

The AUROC of the index was 0.765 for differentiation between Ishak stage 0-3 and 4-6. At a cut off of < 1.45 in the validation set, the negative predictive value to exclude advanced fibrosis (stage 4-6) was 90% with a sensitivity of 70%. A cut off of

307

>3.25 had a positive predictive value of 65% and a specificity of 97%. Using these cutoffs, 87% of the 198 patients with FIB-4 values outside 1.45-3.25 would be correctly classified, and liver biopsy could be avoided in 71% of the validation group.

5.25

13

C- Caffeine Breath Test

142

According to Park et al

, the properties of caffeine render it an ideal substrate

for a quantitative test of liver function. It has high oral bioavailability, and undergoes almost exclusive hepatic metabolism, principally via demethylation by cytochrome P450 1A2 (CYP1A2) to CO2143. It has a low extraction ratio and low plasma binding 144, and at the test doses used may be considered innocuous. These characteristics render caffeine an ideal substrate for a breath test of hepatic function 142. The authors studied that whether the caffeine breath test (CBT) using orally administered
13

C-caffeine correlates reliably with plasma caffeine clearance and reflects

varying degrees of liver dysfunction. The CBT was performed in 25 healthy controls; 20 subjects with non-cirrhotic, chronic hepatitis B or C; and 20 subjects with cirrhosis. Cirrhotic patients were characterized by significantly reduced CBT values (1.15 + 0.75 o/oo mg-1) compared with controls (2.23 + 0.76; p = 0.01), and hepatic patients (1.83 + 1.05; p = 0.04). There was a significant inverse relationship between the CBT and Child-Pugh score (r = -0.74, p = 0.002). The intraclass correlation between repeated CBTs in 20 subjects with normal and cirrhotic livers was 0.89. Multivariate analysis revealed that only smoking (p< 0.001) and disease state (p = 0.001) were significant predictors of CBT. The authors concluded that the results from their study support the application of CBT as a test of quantitative liver assessment. 1) The elimination of caffeine is impaired in cirrhosis, characterized by a reduction in plasma clearance and a prolongation in caffeine half-life; 2) the oral
13

C-CBT using a single 1-hour measure correlates very

closely with plasma caffeine clearance; 3) cirrhotic subjects show significantly reduced CBT results compared with control and hepatitis subjects; 4) the CBT values are increased in smokers but still distinguish smokers of varying hepatic functional impairment; 5) the CBT is administered easily and safely in subjects with liver disease; 6) the CBT appears to be a reproducible assay142. 308

The CBT exhibited significant correlations with serum albumin and platelet count and correlated modestly with INR, bilirubin, glutamyl transferase, and AST/ALT ratio, markers traditionally associated with hepatic dysfunction. The CBT was associated inversely with the Child-Pugh score and differentiated the grades of cirrhosis. In addition, it was able to predict the cirrhotic state in two patients with biopsy proven cirrhosis who had completely normal physical and laboratory findings but significantly reduced CBT values. It thus may be viewed as a complementary test in the overall assessment of liver status, to be taken in context with laboratory, radiologic, and histologic data where available 142.

5.26

Fibrosis Markers to Assess Effect of Treatment

According to Afdhal N 19, an attractive use of fibrosis markers would be to assess

the effects of treatment, and if they can be validated, they could be valuable tools in the search of new antifibrotic treatments. However, none of the currently available markers have yet been validated for use in this way, but several studies have shown that levels of these markers are altered by treatment and they have prognostic value. Poynard and colleagues reported on two studies describing the relationship between response to interferon therapy and the results of Fibrotest performed before and after therapy 78, 82. In the larger of these studies (n = 352)
82

, the authors studied the effect of

interferon plus ribavirin on both the Fibrotest and Actitest scores. Actitest is a modification of Fibrotest that incorporates ALT and reflects both liver fibrosis and necroinflammatory activity. All patients had two interpretable liver biopsies and stored serum sample before and after treatment. Of the patients, 208 patients received peginterferon alfa-2b, 1.5 mcg per kg and ribavirin. The remaining 144 patients received interferon alfa-2b, 3 MU and ribavirin. All patients received treatment for 48 weeks 82. At baseline, Fibrotest performed fairly well for the diagnosis of significant hepatic fibrosis, defined as METAVIR score of F2-4. The area under the receiver operating curve was 0.733 + 0.03, but was lower than that which had been reported in the original study by this group (0.827) 54. It increased to 0.766 + 0.03 at the end of follow up. For bridging

309

fibrosis and/or moderate necroinflammatory activity, the area under the ROC was 0.76 + 0.03 at baseline and 0.82 + 0.02 at the end of follow up. The index had 90% sensitivity and 88% for the diagnosis of bridging fibrosis and moderate necroinflammatory activity
82

. Actitest showed slightly greater diagnostic value for the identification of

individuals with more advanced fibrosis and with greater histological activity as determined by Knodell score, and may thus be of value in guiding treatment decisions. Of interest, the performance of Actitest improved with longer liver biopsies, those greater than 15 mm or which contained six or more portal tracts, suggesting that these assays truly reflect the more global liver histology 82. On follow up, patients who had a sustained virological response to interferon had a substantial reduction in Fibrotest and Acitest scores, as compared to those who were either primary nonresponders or relapsed following therapy. There was a significant decrease of Fibrotest among the 184 sustained virological responders, from (0.39 + 0.02 to 0.28 + 0.02) at 72 weeks; in comparison with 126 nonresponders from (0.59 + 0.03 to 0.55 + 0.02) at 72 weeks, p<0.001; and with 42 relapsers, from (0.49 + 0.04 to 0.45 + 0.02) at 72 weeks, p<0.001 82. There was also a significant decrease of Actitest among the 184 sustained virological responders, from (0.55 + 0.02 to 0.08 + 0.020) at 72 weeks, in comparison with 126 nonresponders from 0.58 + 0.02 to 0.50 + 0.03 at 72 weeks, and in comparison with 42 relapsers, from (0.58 + 0.03 to 0.43 + 0.03) at 72 weeks 82. Furthermore, there was a significant concordance between Fibrotest and fibrosis stage variations. At baseline 32 patients had cirrhosis. Fibrotest scores fell substantially in 17 patients with cirrhosis at baseline and a post treatment reduction of 1 or more stages; from 0.68 to 0.44 for 3 stages improvement, from 0.60 to 0.47 for 2 stages improvement and from 0.61 to 0.56 for 1 stage improvement. For 15 patients, the fibrosis stage remained F4 and Fibrotest did not significantly change (0.67 + 0.05 vs 0.64 + 0.05). These data support the concept that these assays may be useful not only in the initial staging of the liver disease, but may also be of value in following the histological response to therapy 82.

310

According to Afdhal N
93,105,114,145-153

19

, many other studies have evaluated the effect of

interferon therapy on levels of fibrosis markers and have compared them to histological findings . Most of these studies have shown that serum levels of several of

these markers including HA, PIIINP, YKL-40, and TIMP-1 fall in patients who achieve a sustained virological and biochemical response. In these patients, levels continue to fall following treatment and often return to normal levels. In patients who relapse following treatment, the levels frequently fall during therapy but most often return to pretreatment levels with virological and biochemical relapse
145

. Where follow up biopsies have been

performed, levels of HA and PIIINP have often been found to correlate better with improvement in fibrosis stage, than with biochemical or histological markers of inflammation
94,105,151,152

. Furthermore, levels of these markers often become normal

despite evidence of fibrosis on liver biopsy, suggesting that these markers do reflect the underlying fibrogenic activity within the liver. According to Afdhal N 19, treatment with interferon has been associated with a fall in serum markers of fibrosis, independent of a biochemical or virological response
145,148,149,153

, while other studies have not confirmed these findings 93. And this reduction

in the levels of these markers tends to be less than which is observed in patients who achieve a biochemical and virological response. This has been used as evidence that interferon has a beneficial direct antifibrotic effect, presumably through direct inhibition of TGF- expression 154,155. Serum TGF- levels and tissue expression has been shown to decline in response to successful therapy of hepatitis C and following treatment of autoimmune hepatitis
156,157,158

. Based on the available data it is not possible to discern whether interferon has a

clinically important antifibrotic effect and hence ongoing long-term low dose interferon studies and studies of interferon and interferon with more potent antifibrotic activity and less antiviral effect are awaited with interest 19.

311

5.27

Fibrosis

Markers
19

to

Predict

Disease

Progression
According to Afdhal N , if noninvasive markers of fibrosis truly reflect fibrogenic activity and the rate of underlying disease progression then they should have prognostic value, both for predicting progression of fibrosis, and clinical outcomes. In this regard, the available data are more limited. However, several studies looked at the prognostic value of these fibrosis markers in patients with more advanced liver disease
101,102,159

Guechot evaluated the predictive value of HA in a cohort of 91 patients with hepatitis C associated cirrhosis followed for a median of 38 months 159. During this time, severe complications including death, liver transplantation, and severe complications of cirrhosis occurred in 24 patients. Of all the laboratory tests evaluated, HA was found to have the greatest predictive value and was equivalent to Child-Pugh score. Similarly, in a cohort of 97 patients with primary biliary cirrhosis, Poupon demonstrated that by multivariate analysis HA, PIIINP, bilirubin, and prothrombin time were independently predictive of disease progression
101

. Interestingly, in the subgroup of 49 patients who

were treated with ursodeoxycholic acid therapy, only HA was predictive of a poor outcome. Nojgaard reported on the ability of PIIINP and YKL-40 to predict survival in a cohort of 370 patients with alcoholic liver disease
115

. In this study, elevated levels of

PIIINP and YKL-40 were predictive of shorter survival and carried an increased relative risk of dying of 3.32 (95% CI 1.05-10.5), and 4.24 (95% CI 2.18-8.26), respectively. As prognostic assays, it is not possible to discern whether these markers simply reflect more advanced liver disease or whether they identify individuals whose fibrotic disease is progressing more rapidly and hence have a worse prognosis 19,115. According to Afdhal N disease progression
160,161 19

, serum TGF- levels have also been used to assess

. In a 12 month study of 39 patients with hepatitis C infection,

who underwent paired liver biopsies, Kanzler showed that those patients who had progressive liver fibrosis had higher levels of serum and tissue expression of TGF- than

312

those who did not progress transaminases, or viral load.

160

. No such correlation could be found for PIIINP, serum

161

Neuman and colleagues

, using a slightly different study design, obtained

similar results. They assessed two patient cohorts, a group of 56 patients with minimal or no fibrosis and no inflammation and a second group of 103 patients with no fibrosis but mild histological disease activity. When followed over 3 years, those patients who showed progressive hepatic fibrosis had a parallel increase in TGF- levels. Hence, TGF levels were found to identify the subgroup of patients with mild liver disease that subsequently progressed 19,161. Similarly, prognostic value of the Fibrotest was compared with biopsy staging for predicting cirrhosis decompensation and survival in patients with chronic HCV infection. The investigators concluded that the Fibrotest measurement of HCV biomarkers has a 5year prognostic value similar to that of liver biopsy 26. According to the authors, Fibrotest was a better predictor than biopsy staging for HCV complications, with the area under the ROC, 0.96 vs 0.91, respectively; it was also a better predictor for HCV related deaths, AUROC 0.96 vs 0.87 respectively. The prognostic value of Fibrotest was also significant in multivariate analyses after taking into account histology, treatment, alcohol consumption, and HIV coinfection 26.

5.28

Gene Markers and Liver Fibrosis

The hepatic fibrogenesis is a complex process requiring an intricate balance

between extracellular matrix (ECM) deposition and removal. Indeed, the ECM metabolism is a very dynamic process, influenced by factors/cytokines/molecules that contribute to its deposition and by others that mediate its degradation 24. The complexity of the fibrogenetic process and the high number of factors/cytokines/molecules involved imply that several genetic polymorphisms could influence progression of liver fibrosis. The genetic polymorphisms linked to hepatic fibrogenesis have been investigated mainly in chronic hepatitis C and in alcoholic fatty

313

liver disease. There are many studies that report gene polymorphisms to either favor or reduce fibrogenesis in patients with different forms of chronic liver disease 24. While these studies clearly indicate that many genetic factors have a definitive influence on the risk of developing a more or less active and progressive fibrogenesis, very few of them have found an application as a diagnostic/prognostic marker in clinical practice, due to their complexity, difficulty to test and variable behavior in different patient populations 24. One such study, consists of a set of seven marker genes, the cirrhosis risk score (CRS), was found to be a better predictor of high risk versus low risk for cirrhosis in Caucasian patients than clinical factors
162

. The clinical factors such as age, gender,

163-165

alcohol use, and age at infection, obesity and hepatic steatosis, patients with chronic hepatitis C 16.

influence the

progression to cirrhosis, but cannot accurately predict the risk of developing cirrhosis in

The authors hypothesized that host genetic factors, such as single nucleotide polymorphisms (SNPs) could play a primary role in determining fibrosis risk. The preliminary results from their previous study suggested that replicated SNPs identified by large association studies had several advantages over clinical risk factors, such as higher odds ratios, consistent percentages of risk population across different study cohorts, and objective genotyping calls that are available for all patients
166

. The key

question remained as to how these markers could be utilized in clinical settings. The aim of the study was to develop and validate a predictive signature (of genes) alone or in combination with standard clinical factors to assess the risk of cirrhosis in Caucasian patients with chronic hepatitis C virus infection 162. In the training set (n = 420), all patients had well characterized liver histology and clinical factors. DNA was extracted from the whole blood for genotyping. The authors validated all significant markers from a genome scan in the training cohort, and selected 361 markers for the signature building. Subsequently, a signature consisting of 7 markers most predictive for cirrhosis risk in Caucasian patients was developed. The Cirrhosis Risk Score (CRS) was calculated to estimate the risk of developing cirrhosis for each patient. The CRS performance was then tested in an independently enrolled validation cohort of 154 Caucasian patients.

314

The area under the receiver operating characteristic (ROC) curve was 0.75 in the training cohort. In the validation cohort, the ROC was only 0.53 for clinical factors, increased to 0.73 for CRS, and 0.76 when CRS and clinical factors were combined. A low CRS cutoff of < 0.50 to identify low risk patients would misclassify only 10.3% of high risk patients, while a high cut off of > 0.70 to identify high risk patients would misclassify 22.3% of low risk patients. Thus more importantly fewer of the high risk patients would be misclassified. In conclusion, CRS is a better predictor than clinical factors in differentiating high risk versus low risk for cirrhosis in Caucasian patients 162. According to the authors, for the first time, one can estimate the risk of cirrhosis rather than using findings of liver biopsy to project the future course of disease. Liver biopsy represents only one time point in the long natural history of hepatitis C, whereas the genetic markers are intrinsic and life long. Potentially the CRS could be used to stratify patients cirrhosis risk prior to liver biopsy 162. Unlike rare Mendelian disorders, and similar to other complex human diseases, liver cirrhosis is caused by the interactions among multiple genetic and environmental factors. Yang et al, estimated that for genes with very common genotype frequencies (>30%) and moderate ORs (1.2-1.5), 10-15 markers are needed to achieve appreciable population attributable fraction (PAF) for disease occurrence
167

. Consistent with this

finding, the CRS signature is comprised of seven markers with high frequencies (18.5%87.3%) and significant ORs (1.86-3.23). In contrast, only two clinical factors, sex and age, had significant associations with cirrhosis risk. Moreover, the CRS measurements are objective 162. In the CRS, each of the 7 most predictive markers only provide moderate predictability; whereas the combination of these 7 was more robust and predictive. This finding is consistent with multiple biological pathways known to be involved in hepatic fibrogenesis 168. Of the 7 genes, antizyme-Inhibitor-1 (AZIN1) and Toll-like receptor 4 (TLR4) have an identified role in hepatic fibrosis. AZIN1 binds to ornithine decarboxylase (ODC) antizyme and stabilizes ODC, thus inhibiting antizyme-mediated ODC degradation 169. Regarding TLR4, it is expressed in all hepatic cell types in response to its ligand lipopolysaccharide (LPS)
170

. In patients with chronic hepatitis C, NS5A induces

315

the elevated expression of TLR4 in B cells 3- to 7- fold pathway plays an important role in the hepatic fibrogenesis the applicability of the CRS in other liver diseases162.

171

. Moreover, LPS-TLR4

172

. The authors were in the

process of determining the functional mechanisms of the other 5 genes in fibrognesis, and

Nevertheless, improved understanding of the genetic influence on liver fibrogenesis is of paramount importance as it may lead in the near future to identification of patients for high risk for fibrosis/cirrhosis, diagnosis of liver fibrosis, and new therapeutic targets and strategies for development of effective antifibrotic treatments 24.

5.29

Radiology in the Noninvasive Assessment of Liver Fibrosis

According to Afdhal N
19

, radiological evaluation of liver to assess fibrosis has

been limited to the identification of individuals with cirrhosis and its complications. The advent of cross sectional imaging with CT, MRI, and ultrasound enables detailed images of the liver and surrounding structures to be made. Resolution of the hepatic parenchyma with any of the available modalities, however, is insufficient to determine any of the earlier stages of fibrosis before the establishment of cirrhosis and portal hypertension. Established cirrhosis with portal hypertension can be determined with a high specificity by identifying splenomegaly, an enlarged caudate lobe, or the presence of large varices
39

. Studies using CT, ultrasound, and MRI, have identified reduction in the

size of the right lobe of the liver with relative enlargement of the left and caudate lobes as reliable markers of cirrhosis 40,173,174. Harbins original description of this technique measured the ratio of transverse width of the caudate lobe, to the transverse width of the right lobe of the liver. Using this technique, cirrhosis could be correctly diagnosed with sensitivity 84%, a specificity of 100%, and diagnostic accuracy of 94% advanced disease 19. Further studies using up to 11 sonographic and Doppler parameters, including: measurements of liver morphology, assessment of portal venous blood flow, spleen size,
40

. These studies report a high specificity but

limited sensitivity because these morphological changes are only present in more

316

and liver surface nodularity; reported accuracy of cirrhosis detection between 82% and 88%
175,176

, however anatomical limitations and interobserver variability remained

limiting factors 39. In addition, it should also be noted that a large number of patients had decompensated liver disease, and the diagnosis of cirrhosis could have been made using simple clinical criteria, including palpation of a nodular liver, or the presence of a hard and enlarged left lobe 175. When Oberti compared the ability of ultrasound to diagnose cirrhosis to clinical examination and biochemical studies, he found ultrasonography to be of less diagnostic value 70. It should be noted that ultrasound also identified cirrhosis in a substantial number of patients in which a definite diagnosis of cirrhosis was not made on liver biopsy, in the study cited above
175

. It can therefore, be argued that ultrasound examination has

substantial complementary value, i.e., a properly performed examination can identify patients with cirrhosis where the biopsy findings are equivocal, or at variance with clinical impression complications177.
19

. Furthermore, progression in the ultrasonographic changes,

including an enlarging spleen has been associated with an increased risk of

5.30

Measurement (Elastography)

of

Hepatic

Stiffness

Accepting the limitations of standard radiological techniques to accurately determine fibrosis, transient elastography is an emerging technology that is more sensitive for staging hepatic fibrosis. This technique rapidly and noninvasively measures mean hepatic tissue stiffness 178,179. Hepatic stiffness is related to the degree of fibrosis; palpation of a firm liver edge has been used for centuries as a marker of hepatic injury. Using a probe (Fibroscan, Echosens, Paris) that includes an ultrasonic transducer, a vibration of low frequency (50 MHz) and amplitude is transmitted into the liver. The vibration wave induces an elastic shear wave that propagates through the organ. The velocity of this wave as it passes through the liver correlates directly with tissue stiffness and by simultaneously using a

317

pulse-echo ultrasound by means of the same probe, the velocity of the wave is measured. The harder or stiffer the tissue, the faster the shear wave propagates. Results are expressed in kilopascals (kPa). Fibroscan measures liver stiffness of a volume that is approximately a cylinder of 1 cm diameter, and 2 cm long, 100 times greater in size than a standard liver biopsy. Thus it is more representative of the entire hepatic parenchyma
39,41

. According to Afdhal N
19

, initial studies of hepatic elastography in 19

180

hepatectomy specimens correlated with histologic analysis

. In vivo study of a cohort

of 15 patients with hepatitis C virus (HCV) showed excellent intra- and inter-operator reproducibility. Furthermore, in 91 patients who had HCV reported by the same authors, the receiver operating characteristic (ROC) curve, which estimates the diagnostic performance of elastography, were 0.90, 0.88, 0.91, and 0.99, respectively, for the hepatic fibrosis grade superior and equal to F1, F2, F3, and F4, respectively. Of the studied patients with a score of less than 5.1 kPa, 93% were METAVIR F0 or F1, whereas for those with a score of 7.6 kPa or higher, 94% were F2 or more 178. In a large multicenter study 41, the authors enrolled 327 patients. The aim of the study was to investigate the use of liver stiffness measurement in the evaluation of liver fibrosis in patients with chronic hepatitis C. Patients underwent both liver biopsy and liver stiffness measurement. METAVIR liver fibrosis stages were assessed on biopsy specimens. The areas under the receiver operating characteristic curve (ROC); for F > 2 was 0.79 (95% CI, 0.73-0.84), for F > 3 was 0.91 (0.87-0.96), and for F > 4 was 0.97 (0.931.0). For larger biopsy specimens (longer than the median value in each category), these values were, 0.81, 0.95, and 0.99 respectively. Optimal cut of values of 8.7 and 14.5 kPa showed F > 2, and F = 4, respectively 41. The authors concluded that they found significant positive correlation between liver stiffness measurement and fibrosis stages in patients with chronic hepatitis C. Although there was overlap in scores for those with early stages of fibrosis; yet there was significant areas under the ROC curves for F > 3, and F = 4 fibrosis, and with high total sensitivity, specificity, and high likelihood ratios suggest that liver elastometry is a

318

reliable method for the diagnosis of extensive fibrosis (F > 3), and cirrhosis (F = 4). In the study 30% of patients were F > 3, or F = 4. However, in the community practice, where the proportion of patients with F4 may be lower than in referral centers, liver stiffness measurement accuracy in predicting patients with F2 or more METAVIR fibrosis stage might be lower 41. The study also confirmed that in chronic hepatitis C patients, the correlation between liver stiffness and fibrosis stage is not affected by steatosis or activity grade. Indeed, activity was not expected to modify the liver stiffness, whereas steatosis could have been expected to soften the liver because it consists of fat deposits in the liver parenchyma. But in the study the multivariate analysis showed that the potential effect of steatosis on liver stiffness was hidden by the strong effect of fibrosis. These findings support a study on elastic modulus measurements of ex vivo human liver samples that reported a correlation between liver stiffness and fibrosis but did not show any obvious correlation between steatosis and elastic modulus 180. Results show that the diagnostic performances of liver stiffness measurement were better in the larger specimens than in the smaller specimens. This suggests that the real diagnostic performance of liver elastometry may be underestimated because of the sampling error of the biopsy 41. According to the authors41, the diagnostic performance of liver elastometry appears to be equivalent to that of the best biochemical scores for patients with significant fibrosis (F > 2), and is better than these tests for the diagnosis of extensive fibrosis (F > 3) and cirrhosis (F = 4), (there are studies which report better performance of serum markers in earlier stages). The main advantage of liver elastometry over the fibrosis markers is that it measures a quantitative physical parameter directly on the liver and there is no interference from extrahepatic disorders. It, therefore, is complementary to the fibrosis markers to better assess liver fibrosis without resorting to liver biopsy 41. There are certain limitations to the procedure. Elastometry cannot be applied to patients with ascites, even if clinically undetected. Ascites is a physical limitation to the technique because elastic waves do not propagate through the liquids. In addition, elastometry is unsuccessful in patients with narrow intercostals spaces and in patients with morbid obesity. In obese patients the fatty thoracic belt attenuates both elastic waves

319

and ultrasound, rendering liver stiffness measurement more difficult or even impossible. Probes with smaller size and elongated shaped transducer tips are currently available for these patients 41. In another study, a total of 711 patients with chronic liver disease were evaluated. 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 the prospective study was to evaluate the accuracy of liver stiffness measurement for the detection of cirrhosis in patients with chronic liver disease
181

Etiologies of chronic liver diseases were hepatitis C virus or hepatitis B virus infection, alcohol, non-alcoholic steatohepatitis, other, or a combination of the above etiologies. Liver fibrosis was evaluated according to the METAVIR score. Areas under the receiver operating characteristic curve were 0.80 for patients with significant fibrosis (F>2), 0.90 for patients with severe fibrosis (F3), and 0.96 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 complications of liver disease. With an NPV >90%, the cut off values for the presence of esophageal varices stage 2/3, cirrhosis ChildPugh B or C, past history of ascites, hepatocellular carcinoma, and esophageal bleeding were 27.5, 37.5, 49.1, 53.7, and 62.7 kPa, respectively181. The authors concluding that 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. The limitation is that it requires a costly device to measure liver stiffness
24

Similarly, some of the direct markers of noninvasive evaluation of fibrosis are only available in highly specialized research laboratories and are thus not routinely available. Thus in conclusion, this simple noninvasive technique has proved beneficial in detecting patients with advanced fibrosis or cirrhosis and more generally in assessing fibrosis in patients with chronic hepatitis C. Studies underway will evaluate the applicability of hepatic elastography across a range of liver diseases. Further research also will determine its use for assessing hepatic

320

fibrosis over time and its responsiveness to detecting changes in fibrosis associated with specific therapies, in particular the response to antiviral therapies for chronic viral hepatitis. For now, the preliminary results and its ease of use predict a promising future in the reliable staging of fibrosis 39.

321

5.31

Platelets in Chronic Liver Disease The role of Spleen

Thrombocytopenia is a commonly encountered hematological condition in

chronic liver disease and cirrhosis

182

. It is a risk factor for gastrointestinal bleeding and

other life threatening hemorrhagic events 182. Historically it has been attributed to the sequestration and destruction of platelets in the enlarged spleen with an impaired ability of bone marrow to compensate by increasing platelet production
85,183,184

. Hypersplenism occurs in a varying percentage of

patients with advanced liver disease and is a common complication of portal hypertension. Radiolabeled platelet studies have shown that human spleen contains a sizeable fraction (about one third) of the total body platelet mass in the form of exchangeable pool
85

. The spleen can increase its pool by 30-90% when pathologically

enlarged. This redistribution of cells from the peripheral circulation to the spleen appears sufficient to produce thrombocytopenia despite the normal platelet life span, normal total body mass and unimpaired platelet production, as might be expected form the number of megakaryocytes in the marrow 85,87. However attempts to correct the low platelet levels by splenectomy and portal decompression procedures have failed to consistently improve the platelet count in the long term 85,185.

5.32

Platelet Autoantibodies and Thrombocytopenia

It has been postulated that auto-antibody mediated platelet destruction, as seen in

patients with idiopathic thrombocytopenia purpura (ITP) may also contribute to cirrhotic thrombocytopenia186. In this situation, the thrombocytopenia is principally mediated by enhanced platelet clearance in the periphery, resulting in accelerated platelet turnover 182. Autoimmune mechanism mediated by platelet associated Ig may play an important role in thrombocytopenia associated with viral hepatitis187.

322

In another study, antiplatelet autoantibodies were determined in blood serum with the use of ELISA method in 15 patients with cirrhosis and thrombocytopenia (mean platelet count 67.9 +/- 24.9 x 103/l). Three patients (20%) presented with anti-GPIIb/IIIa antibodies and 2 patients with anti-GPIa/IIa. These patients had liver failure (stage C according to Child-Pugh classification) and splenomegaly. Platelet morphological parameters were also evaluated. The significant decrease of plateletcrit as well as the decrease of mean platelet volume (MPV) was observed in liver cirrhosis with thrombocytopenia. The increase of megathrombocyte population (MPV > 20fl) up to 5.5% of all platelets was also observed. Megathrombocytes in healthy individuals were 2.25% of platelet population. Examinations confirmed that autoimmunological factors play an important role in the development of thrombocytopenia in liver cirrhosis188. The authors postulating that immunological disorders in patients with liver cirrhosis, loss of tolerance to own antigens, and the change of platelet antigenicity enable antiplatelet antibody formation under the influence of continuous activation188. Another study has shown that serum thrombopoietin levels may not be directly associated with thrombocytopenia in patients with chronic hepatitis and liver cirrhosis. In contrast spleen volume and platelet associated immunoglobulin (PAIgG) are associated with thrombocytopenia in such patients, suggesting that hypersplenism and immune mediated processes are predominant thrombocytopenic mechanisms189.

5.33

Thrombopoietin and Chronic Liver disease

Impaired platelet production due to thrombopoiten (TPO) deficiency has also

been proposed as another cause of thrombocytopenia in cirrhosis patients. Thrombopoietin, a principal regulator of megakaryopoiesis is predominantly produced by the liver
190,191 182

. Its levels are insufficient in advanced liver failure

. This is further

supported by the observation that reduced circulating level of TPO in cirrhosis patients is restored in conjunction with an increase in platelet count after orthotopic liver transplantation 192.

323

Adinolfi et al, has shown that in patients without splenomegaly, the thrombocytopenia was associated with the stage of fibrosis; platelets counts were the highest in patients with fibrosis stage 0-2 (Knodell HAI), lower in those with stage 3 (p< 0.008) and lowest in those with stage 4 (p< 0.05). These findings were independent of demographic, biochemical, hepatic necroinflammatory activity, portal hypertension and splenomegaly. Patients with normal platelet counts showed higher thrombopoietin levels than those with low platelet counts (p< 0.0001). An inverse correlation between thrombopoietin levels and fibrosis grade was observed (r= -0.50; p, 0.0001). The authors postulated that advanced hepatic fibrosis causing an altered production of thrombopoietin and portal hypertension, plays a central role in the pathogenesis of thrombocytopenia in chronic viral hepatitis 87. Evaluating the association between the degree of fibrosis and the platelet count, another study included seven hundred eighty-four patients (265 chronic viral hepatitis C and 519 chronic viral hepatitis B). In an effort to avoid the effects of hypersplenism, patients with splenomegaly and/or bi- or pancytopenia were excluded. In multivariate analysis, the peripheral platelet count had a negative correlation with the fibrosis score and age, but not with necroinflammatory activity, in both groups. The authors concluding that, a decrease in peripheral platelet count may be a sign of an increase in the degree of fibrosis during the course of chronic viral hepatitis B and C and factors other than hypersplenism may play a role in this decrease in the peripheral platelet count 193. However, several other studies have found that the circulating TPO level is maintained or even increased in patients with cirrhosis Kajihara et al
182 86,194

including the study by

In their study when compared with healthy controls, cirrhosis patients presented with, (i) normal or slightly increased plasma TPO, (ii) accelerated platelet turnover based on elevated %RP (reticulated platelets) and GCI (glycocalicin index), and (iii) reduced platelet production based on decreased absolute RP count and plasma GC 182. Reticulated platelets (RP) are young platelets that contain higher levels of nucleic acid components than mature platelets. The absolute RP count is a reliable indicator of the thrombopoiesis rate, analogous to the reticulocyte count to evaluate erythropoiesis. Glycocalicin is a proteolytic fragment of the -chain of glycoprotein (GP) Ib, which is

324

cleaved from the surface of megakaryocytes and platelets. The plasma glycocalicin concentration is decreased in patients with aplastic anemia and greatly increased in patients with essential thrombocythemia, indicating that it is a marker of platelet production195. In contrast, the proportion of reticulated platelets in total platelets (%RP) and the plasma glycocalicin level normalized to the individual platelet count (GC Index; GCI) have been shown to reflect platelet turnover196. These markers in ITP and cirrhosis patients were comparable, but significantly different from those in aplastic anemia patients. The bone marrow megakaryocyte density in cirrhosis and ITP patients was similar, and significantly higher than in aplastic anemia patients182. The authors concluded that cirrhotic thrombocytopenia is a multifactorial condition involving accelerated platelet turnover and moderately impaired

thrombopoiesis. Thrombopoietin deficiency is unlikely to be the primary contributor to cirrhotic thrombocytopenia182. It has also been shown in one of the studies that in acute liver failure (ALF) the inverse relationship between platelet count and TPO levels was not observed. Despite severe hepatic dysfunction, serum TPO levels were initially normal and increased during hospitalization in acetaminophen-induced ALF, but did not prevent the development of thrombocytopenia197. TPO is synthesized primarily in the liver as a single 353-amino acid precursor protein. Following removal of the 21 amino acid signal peptide, the remaining 332 amino acids undergo glycosylation to produce a 60-70 kDa protein. Thrombopoietin is produced at a constant rate by the liver and enters the circulation where most of it is removed by avid thrombopoietin (c-mpl) receptors on normal platelets. The residual amount of thrombopoietin (50-150 pg/ml) provides basal stimulation of megakaryocytes and a basal rate of platelet production198. It is the circulating platelet mass, not the platelet count, which is regulated by the body. A practical demonstration of this principle is the effect of changes in the size of the spleen, (which normally sequesters one third of the platelet mass), on the platelet count. With increasing splenomegaly, thrombocytopenia becomes progressively more severe,

325

but the total body mass of platelets (circulating + splenic pools) remains constant. How the body maintains this constant circulating platelet mass has been the subject of considerable investigation198. Thrombopoietin is released into the circulation at a constant rate. In the absence of platelets, there is little clearance of thrombopoietin by platelets, levels rise, bone marrow megakaryocytes are stimulated and platelet production increases. In contrast, in the presence of platelets, thrombopoietin clearance increases, levels are low, megakaryocytes are not stimulated and basal platelet production ensues. Unlike the mechanism for red blood cells, there is no "sensor" of the platelet mass; instead, as occurs in the regulation of neutrophils and monocytes where the regulated cells bind and clear their regulatory cytokine, the circulating platelet mass directly determines the circulating level of thrombopoietin198,199. Since it is the total number of circulating platelet c-mpl receptors that determines the clearance of thrombopoietin, the constancy of normal circulating platelet mass, and not the platelet count can now be explained. Which is the body defends the total mass of platelets, and not the platelet count. The platelet count decreases proportionally to the increase in the size of spleen, but the total mass of platelets remains normal and unchanged. This could be one explanation for low platelets and not having increased thrombopoietin levels.

5.34

Platelet Parameters and Chronic Liver Disease

The quantitation of platelets in peripheral blood is a well-recognized tool.

Recently, new indices related to platelet counts have been provided by hematologic analyzers. Concerning the platelet parameters, the important parameters are mean platelet volume (MPV), platelet distribution width (PDW)
200

. The evaluation of these two

parameters does constitute part of the routine evaluation of complete blood count (CBC) in modern automated analyzers, but clinical inferences are usually not drawn. However, there are interesting observations.

326

One observer

200

, found a significant correlation between PDW and RDW. And

the anisocytosis of red blood cells and platelets might co-occur. However, these data are basic observations; further in-depth evaluation of the platelet parameters is recommended. The mean platelet volume is the geometric mean of the transformed lognormal platelet volume data in impedence technology systems. In some, optical systems, (e.g., Bayer), MPV is the mode of the measured platelet volume 201. Under normal circumstances, there is an inverse relationship between platelet size and number. Therefore, the total platelet mass, the product of MPV and the platelet count (plateletcrit) is closely regulated. When platelets decrease in number, bone marrow megakaryocytes are stimulated by thrombopoietin and produce larger platelets. Thus platelets with a larger volume are expected to be seen in destructive thrombocytopenia when megakaryocytic stimulation is present. Conversely, platelets with low MPV are expected to be seen in thrombocytopenic states seen in marrow hypoplasia or aplasia 200. A very important exception occurs in splenic sequestration, in which a low MPV is seen, because spleen sequesters large platelets. A decrease of mean platelet volume (MPV) was observed in liver cirrhosis with thrombocytopenia 188. This fact has been known from before, for instance in a study from 1984 202, MPV in patients with liver disease (9.25 + 1.14 fl) was significantly lower than that of controls (10.52 + 0.74 fl, p< 0.001). In control subjects, the MPV and platelet counts were inversely correlated 202. It has also been shown that, platelet distribution width is a better indicator of altered platelet homeostasis than MPV in liver cirrhosis
203

. In the study, platelet count,

mean platelet volume, giant platelet percentage, expressed as megathrombocytic index (MTI), and platelet distribution width (PDW) were evaluated in 32 controls, and in 27 patients with cirrhosis and thrombocytopenia. MTI and PDW were linearly and inversely correlated to platelet count both in controls and patients. MTI and PDW were markedly increased in cirrhosis as compared to controls, while MPV was not significantly different. The authors concluded that: MTI and PDW were good indicators of thrombopoietic stimulus both in controls and in cirrhosis and are better indicators of altered platelet homeostasis than MPV in cirrhosis203.

327

These platelet parameters are altered in similar direction in immune thrombocytopenia, as in cirrhotic thrombocytopenia. Likewise, it has been postulated that auto-antibody mediated platelet destruction, as seen in patients with idiopathic thrombocytopenia purpura (ITP) may also contribute to cirrhotic thrombocytopenia 186, as already discussed. In a study the authors, investigated the significance of the platelet indices, mean platelet volume (MPV), platelet size deviation width (PDW), and platelet-large cell ratio (P-LCR), in the diagnosis of thrombocytopenia by comparing these levels in 40 patients with hypo-productive thrombocytopenia (aplastic anaemia; AA) and 39 patients with hyper-destructive thrombocytopenia (immune thrombocytopenia; ITP). The sensitivity and specificity of platelet indices to make a diagnosis of ITP were also compared. All platelet indices were significantly higher in ITP than in AA, and platelet indices showed sufficient sensitivity and specificity. The area under the curve (AUC) of the receiver operating characteristics curve of platelet indices was large enough to enable the diagnosis of ITP. P-LCR and PDW had the largest AUCs, which indicated that these values were very reliable for immune thrombocytopenia. The authors concluded that, these indices provide clinical information about the underlying conditions of thrombocytopenia. More attention should be paid to these indices in the diagnosis of thrombocytopenia 204.

328

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