Introduction

The poor survival of patients with decompensated cirrhosis has driven physicians to a constant search for good prognostic markers.

The Child-Pugh Score (CPS) is the cornerstone prognostic marker in patients with decompensated cirrhosis, due to its simplicity in daily clinical practice⁽¹⁾. The determination of the CPS, which ranges from 5 to 15 scoring points, is based on the presence and severity of ascites and hepatic encephalopathy, the prolongation of the prothrombin time (PT), and the levels of serum bilirubin and albumin. Patients' classes are three (A, B, and C with scores of 5-6, 7-9 and 10-15 respectively ⁽²⁾).

Recently, a modified CPS taking into account serum creatinine level as a sixth categorical variable was evaluated ⁽³⁾, since renal function in patients with decompensated cirrhosis has been shown to affect survival and no renal parameter was included in the original CPS ⁽⁴⁾.

Later, another modified CPS added four parameters to the original CPS [serum sodium, serum creatinine, white blood count (wbc) and arterial/alveolar oxygen tension ratio (Pa/AO₂)]. The score ranged between 9 and 27 points, and patients were classified into three classes (A, B, and C, with scores of 9-10, 11-14, and 15-27 respectively). This score proved to be more accurate and specific in a prospective study including 210 surgical cirrhostic patients. ⁽⁵⁾

However, the inclusion of two subjective parameters in these scores (ascites, and encephalopathy), together with a ceiling effect in class C patients (10-15 scoring points in CPS, and 15-27 scoring points in the modified CPS) prompted the search for purely objective scores.

The model for end-stage liver disease (MELD) was developed for predicting survival and to provide more accurate prognosis than the CPS in patients with decompensated cirrhosis ⁽⁶⁾. It is based on the etiology of cirrhosis and three objective laboratory variables, serum bilirubin, serum creatinine, and PT expressed as the international normalized ratio (INR), but it includes logarithmic transformations and multiplication by several factors, which make it more sophisticated than CPS. The etiology of the disease as a parameter was later omitted due to its insignificant prognostic value ⁽⁶⁾.

Lastly, a new model for end-stage liver disease (NMELD), has been suggested, based on seven laboratory variables; serum bilirubin, serum albumin, serum creatinine, INR, serum sodium, wbc, and P(a/A)O₂, with no logarithmic transformations ⁽⁷⁾. It has been validated, and proved to have excellent diagnostic accuracy, compared to the original MELD score (AUROC: 0.85 versus 0.78).

Despite the use of scoring systems as prognostic markers for decompensated cirrhosis, liver biopsy is still the gold standard for diagnosing liver disease as well as for assessing the stage of fibrosis and the grade of inflammation and necrosis ⁽⁸⁾.

Pathogenesis of fibrosis:

Hepatic fibrosis refers to the accumulation of tough fibrous scar tissue in the liver. It is the principal feature of liver cell injury, and determines the major clinical events that lead to liver related-deaths. Heptocyte injury may be due to viral or parasitic infection, heavy alcohol consumption, toxins, trauma, autoimmune disease or other factors. Liver fibrosis represents a nonspecific wound-healing response to an inflammatory process.

Although multiple liver cell types including periportal and pericentral fibroblasts and myofibroblasts are involved in the healing process, the stellate perisinosoidal cells which are widely distributed in the hepatic lobule, play the main role. This is because the immune system is activated and the healing process swings its gear. The inflammatory immune cells including kupfer cells together with platelets stimulate the release of cytokines, growth factors, and other mediators that mainly transfer the stellate cells from a quiescent to an active state.

Activated stellate cells produce collagen, proteoglycans, and glycoproteins such as fibronectin leading to a striking increase in the building up of the non-functioning connective tissue known as the extracellular matrix (ECM) ⁽⁹⁾.

At the same time, a number of cytokines appears to have anti-activation properties towards the stellate cells. So, the normal process of breaking down of collagen is impaired. An imbalance between fibrogenesis and fibrinolysis leads to a faster building-up of fibrous tissue than it can be broken and removed by the liver. A prominent feature of chronic liver disease is that fibrosis is a disturbance of ECM turnover including its synthesis and degradation ⁽¹⁰⁾.

Stellate cell contraction is important in the injured liver because it may contribute in the shrunken state of the cirrhotic liver, and appears to play a role in portal hypertension and its sequalae.

The fibrogenesis process is accompanied by an inflammatory necrosis process denoting activity.

The liver disease, as is clear in HCV infection, can run a variable course, from decades of viremia with little fibrosis to a rapid onset of cirrhosis within 10-15 years. It seems that the host factors rather than the viral factors correlate with fibrosis progression in HCV disease.

Regression of fibrosis is associated with increased apoptosis of activated stellate cells. This can be addressed by elimination of the underlying cause as treatment of HCV ⁽¹¹⁾.

Progression of fibrosis can be noted in neonatal liver disease, HCV-infected patients after liver transplantation, or some drug induced hepatitis. Such “fulminant” fibrosis reflects a dysregulation in the pathway due to defective immunity, massive necrosis and inflammation and/or altered ECM resorption leading to a highly dynamic nature of scar accumulation and degradation. Accelerating factors include also older age, concurrent  liver disease, and increased body mass index.

Liver fibrosis can be viewed as a clinical problem whose diagnosis and treatment will soon have rationale evidence-based approaches. In the early stages of fibrosis the liver functions relatively well, and few people experience symptoms. But, as the liver injury and inflammation continue, scar builds-up and connects with existing scar tissue, which can eventually disrupt the metabolic functions of the liver. If the disease progresses, it can lead to cirrhosis, a condition in which the liver is severely scarred, its blood flow is restricted, and its ability to function is impaired.

Once, cirrhosis and its complications develop, its prognosis is predicted by scoring systems such as the CPS, ⁽²⁾, the MELD ⁽⁶⁾, and the NMELD ⁽⁷⁾ irrespective of the etiology of the liver disease. However, it is important to regularly monitor the major histological features in the form of fibrosis and activity which characterize the healing process through invasive or non-invasive methods.

Methods to measure fibrosis:

The level of liver fibrosis plays an important role in the clinical management and determines patients’ prognosis. With growing evidence that fibrosis is reversible, methods will need to assess its progression and regression accurately.

Lever biopsy, an invasive method, has been considered the gold standard test to assess liver fibrosis. However, a variety of non-invasive tests have been advanced as potential alternatives to liver biopsy.

(I) Liver biopsy

The current gold standard for determining the extent of liver disease is the percutaneous ultrasound guided liver biopsy. A small sample of tissue is removed with a needle, stained, and examined under the microscope. In order to monitor the disease progress in a timely manner, most experts recommend to repeat biopsies every 5-7 years.

In the fibrotic liver, there are quantitative and qualitative ECM changes, that can provide information for the diagnosis of the histological lesions during the natural history of the disease ⁽¹²⁾. They also help the therapeutic decision and the evolution of the disease under treatment ⁽¹¹⁾.

Staging and grading

Liver biopsy aims at staging and grading of the disease. Staging represents the amount of fibrosis or scarring, while grading is an indication of the activity or the amount of inflammation.

Staging is a measure of how far the disease has progressed in its natural history, with the end-stage resulting in death of the patient or failure of the liver. Grading is meant to reflect how quickly the disease is progressing to the end-stage. The end-stage in chronic liver disease is cirrhosis with clinical decompensation. The grade relates the severity of the disease with features that vary with the type and pattern of injury. Ideally, both staging and grading should predict prognosis. So, pathologists are asked to assign the stage and the grade of the disease with the intention that this will predict patient outcome ⁽¹³⁾.

Interpretation

 The natural history of the liver disease is characterized by fibrosis progression and necro-inflammatory activity. Fibrosis alone is the best marker of ongoing fibrogenesis, while the activity grade predicts the level of necrosis. It is the combination of the two factors, as well as the age, the gender, and certain risk factors that determine the appropriate diagnosis and progression of the liver disease. It is important that the length of the liver specimen and the knowledge of the pathologist reading the biopsy, may influence the interpretation of the report.

There are a variety of methods to interpret a liver biopsy. The most common examples are the METAVIR⁽¹⁴⁾, the histologic activity index (HAI) also known as the Knodell scoring system ⁽¹⁵⁾, and the Ishak scoring system ⁽¹⁶⁾.

The METAVIR scoring system ⁽¹⁴⁾ consists of staging the fibrosis and grading the activity. For staging fibrosis, F0 = no fibrosis, F1 = portal fibrosis without septa, F2 = fibrosis with few septa, F3= fibrosis with numerous septa,  and  F4 = cirrhosis.

This means that in F0 there is no scarring, in F1 there is minimal scarring, in F2 scarring extends outside the area in the liver that contains blood vessels, in F3 bridging fibrosis is spreading and connecting to other areas that contain fibrosis, and in F4 there is cirrhosis or advanced scarring of the liver ⁽¹⁴⁾.

The grading of the METAVIR score is also assigned a number from 0-4, based on the degree of activity or inflammation, zero being no activity, and 3 or 4 representing severe activity ⁽¹⁴⁾.

The Knodell or HAI scoring system ⁽¹⁵⁾, also denotes staging and grading of the liver disease. For staging fibrosis, the amount of scarring in the liver is scored from zero being no scarring to 4 representing extensive scarring or cirrhosis.

For grading activity or the amount of inflammation, zero = no inflammation, 1-4 = minimal inflammation, 5-8 = mild  inflammation, 9-12 = moderate inflammation, and 13-18 = marked inflammation.

The Ishak fibrosis scoring system ⁽¹⁶⁾ has 7 stages (0-6), and provides more information than other fibrosis scores which include only 5 stages (0-4) ^{(14, 15)}

Limitations and risks:

Liver biopsy for determining staging and grading of liver disease has limitations ⁽¹⁷⁾. Limitations are due to its complications, sampling errors, intra and inter-observer variability, expense, and patient reluctance to undergo serial monitoring ⁽¹⁷⁾. Sampling errors result from heterogenous distribution of pathological changes in the liver. False negative results occur in patients with known sepsis or inflammations. The risks of liver biopsy include pain, severe adverse events as hemoperitoneum, pneumothorax, and death ^{(8, 18)}

Liver biopsy is 80% accurate in staging fibrosis, and may miss advanced fibrosis in 30% of patients ⁽¹⁷⁾.

Non-invasive markers:

Due to the limitations and risks of liver biopsy, interest continues to grow in new non-invasive methods for assessing live fibrosis, including serum markers assay and imaging techniques.

  (1) Serum markers assay:

Serum markers for hepatic fibrosis includes two categories:

1-     Direct markers: which reflect ECM turnover, as well as cytokine levels. Assay of ECM include collages I, IV and VI; glycoproteins as fibronectin; and collagenases and their inhibitors. An example of cytokine assay is the transforming growth factor beta-1 (TGF-B1). This platelet-derived cytokine is the most potent chemotactic agent known to stimulate migration of inflammatory cells and fibroblasts. It plays a key-role in the synthesis of ECM during wound healing ⁽¹⁹⁾.

2-     Indirect markers: which reflect alterations in hepatic function ⁽²⁰⁾. Examples are aspartate aminotransferase (AST),alanine aminotransferase ( ALT), and platelet count.

Other authors prefer to divide serum markers into other two categories:

1-     Biomarkers: which directly reflect the biologic process of fibrosis and can be measured, as the FibroTest ⁽²¹⁾.

2-     Surrogate markers: which may correlate with fibrosis, but do not directly reflect pathophysiologic events leading to fibrosis as the AST and the ALT levels ⁽²²⁾

Estimation of single markers or combinations of markers specially focusing or ECM turnover (fibrogenesis and fibrinolysis) have been designed to evaluate the whole fibrosis process. Examples are the aspartate aminotransferase/platelet ratio index (APRI) ⁽²³⁾ and the FibroTest-ActiTest (FT-AT) ⁽²⁴⁾.

Serum markers assay:

Generally speaking a number of routine laboratory tests have been used to determine advanced liver disease with portal hypertension and/ or oesophageal varices ⁽²⁵⁾. These include PT, serum albumin level, platelet count, and aspartate aminotransferase/alanine aminotransferase ratio (AST/ALT) ⁽²⁶⁾. Forn's et al ⁽²⁷⁾ developed a model using age, gamma glutamyl transferase (GGT), cholesterol, and platelet count. The APRI is a simple test that was designed to diagnose or to exclude cirrhosis. The PGA index combined PT, GGT, and apolipoprotein A₁, and has been examined in patients with alcoholic cirrhosis ⁽²⁸⁾. It’s diagnostic accuracy was later improved by the addition of α₂ – macroglobulin, and hence termed PGAA index ⁽²⁹⁾. Analysis of serum markers of ECM includes many proteins which are important in fibrogenesis such as fibronectin, collagen I, Collagen IV, Collagen VI, and hyaluronic acid (HA) ⁽²¹⁾.

    Showing the value of the serum marker HA as an example, an Iranian study including a small  number of patients  with chronic hepatitis B (n = 65) reported that serum hyaluronate was the best predictor of extensive liver fibrosis and inflammation. It concluded that it can be used as a non-invasive test to monitor patients more closely with developing antiviral agents in clinical trials⁽³⁰⁾. In an Egyptian study including a relatively larger number of patients with chronic hepatitis C (n = 220), young patients with low levels of HA proved to be at low risk of fibrosis. It was concluded that this can limit the number of liver biopsies to those whose clinical findings conflict with the biomarker results ⁽³¹⁾.Again, a Japanese study concluded that HA could discriminate non-alcoholic steato-hepatitis (NASH) from the benign non-alcoholic fatty liver disease (NAFLD), and could be used as a predictor test for the degree of hepatic fibrosis in patients with NAFLD ⁽³²⁾.

The biomarkers or biopredictive tests are innovative diagnostic and staging tools for the most common liver diseases. They include the FibroTest (FT), the ActiTest (AT), the SteatoTest, the alcoholic steatohepatitis test (AshTest),  the non-alcoholic steatohepatitis  test (Nash Test).

The FibroTest (FT) was reported by the French MULTIVIRC group ^{(21, 33)} to diagnose hepatic fibrosis. It includes five markers to predict liver fibrosis; α₂macroglobulin, haptoglobin, GGT, apolipoprotein A₁, and total bilirubin. The FT provides a numerical quantitative estimate of liver fibrois ranging from 0.001 to 1.000, corresponding to the METAVIR scoring system, which grades fibrosis from F0 (no fibrosis) to F4(cirrhosis). An algorithm classifies patients into three groups. FT 0-0.31 = no or minimal fibrosis, FT 0.31-0.58 = moderate fibrosis, and FT 0.58-1.00 = severe fibrosis ⁽³⁴⁾.

  The FT could correctly identify clinically advanced or minimal liver fibrosis in many studies, and could be considered a good alternative to liver biopsy in patients with HBV, HCV, alcoholic liver disease (ALD) and NAFLD ^{(21, 35-37)}. However, a study showed that the FT could not accurately predict the presence or absence of significant liver fibrosis in patients with HCV ⁽³⁸⁾. The FT was shown to be superior to HA, APRI, Forn’s test and Child-Pugh score in diagnosing liver fibrosis^{(39, 40)}. The practical combination of FT with APRI and Forn’s test could predict the stage with accuracy, potentially avoiding liver biopsy in the majority of haemophilic patients ⁽⁴⁰⁾. An interesting Italian study ( n = 110) has recently suggested that the FT and liver biopsy should be considered as agonists and not antagonists towards the common goal of estimating liver fibrosis. The FT combined with APRI in a stepwise manner could be the most useful to decrease the need for liver biopsy. ⁽⁴¹⁾

Different studies reported that the FT-AT provides a more accurate picture of fibrogenic and necrotic events occurring within the liver than liver biopsy ^{(24, 42, 43)}. The FT-AT was derived by the addition of ALT to the five FT markers for the prediction of METAVIR necro-inflammatory activity^{(24, 34)}. A meta-analysis of 16 randomized controlled studies on patients with HCV, proved that the FT-AT can be used as an alternative to liver biopsy, which should be recommended only as a second line test as in the presence of high risk error of the biochemical tests⁽³³⁾. Limitations of the FT-AT included false positive results due to increases in bilirubin or decreases in haptoglobin with ribavirin-induced hemolysis during therapy, or Gilbert's syndrome or cholestasis. Acute inflammation also affects these tests due to changes inα₂ macroglobulin or haptoglobin ^{(23, 34)}.

The SteatoTest assesses fatty liver disease through a marker of 0.001-1.000 for hepatic steatosis grades of  S0 to S3 ^{(44).     .}                                      .

The AshTest diagnoses severe alcoholic steato-hepatitis ⁽⁴⁵⁾. It also diagnoses steatosis in patients who are overweight, insulin-dependent diabetics or hyper-lipidemics ⁽⁴⁵⁾. It is a marker of 0.001 to 1.000 for steatosis grades of A1 to A 3. The AshTest includes age, gender, weight, and height added to serum levels ofα₂ macroglobulin, haptogobin, apolipoprotein A₁, total bilirubin, GGT, ALT, AST, total cholesterol, and triglycerides ⁽⁴⁵⁾.

The Nash Test diagnoses non-alcoholic steato-hepatitis ⁽⁴⁶⁾. It can discriminate patients with the benign NAFLD from those with fibrosis. So, it can motivate them for dieting and life-style modification. The Nash Test includes fasting blood sugar added to the Ash Test markers. It includes a marker of 0.001 to 1.000 for three categories; N  0 = no Nash, N 1 = borderline Nash, and 2 = Nash.

Combinations for biomarker tests for broad spectrum diagnosis of liver disease include the FT-AT⁽²⁴⁾ and the FibroMax (FT, AT, SteatoTest, Ash Test, and Nash Test) ⁽²¹⁾. In the USA, a Nash-FibroSURE test includes FT, SteatoTest and NashTest, while an Ash-FibroSURE test includes FT, SteatoTest and AshTest. These combinations provide algorithms for diagnosis and management of chronic hepatic patients with different etiologies.

The biopredictive tests are implemented through four steps. In step1, the physician prescribes the FT-AT, the Fibro-Max or other tests. In step 2, a blood sample is taken from the patient at a local biochemical laboratory. In step 3, the result of the biochemical test is directly entered on line at: www.biopredictive.comwebsite, by the biologist and the result is immediately generated. In step 4, the biologist provides the report back to the physician.

Ideally, non-invasive serum biomarkers must be reliable, accurate, reproducible, and easy to perform. In addition, they must reflect the total mass of liver collagen and ECM, and be able to reflect both fibrogenesis and necrosis turnover. They should be able to accurately stage and grade the liver disease, and also be sensitive to changes in fibrosis induced by therapy or the natural history of the liver disease progression. So, further research is needed to attain the goal of ideal serum biomarkers.

(2) Imaging Tests:

A wide variety of radiographic tests have been used to image patients with fibrosis/ cirrhosis. Included in this group are ultrasound, CT, and MRI. In general, these tests are capable of detecting evidence of portal hypertension, and have the ability to improve the diagnosis and differential diagnosis of advanced liver disease ^(47-49).

Transient elastography (FibroScan), which uses pulse-echo ultrasound acquisions to measure liver stiffness and predict fibrosis stage, has gained interest as a method to quantify fibrosis as it appears that liver “stiffness” may accompany the fibrogenic response ⁽⁵¹⁾. The FibroScan offers good reproducibility with low inter-and intra-observer variability. The procedure is performed by obtaining multiple validated measurements in each patient, further reducing the potential for sampling errors. The FibroScan reports a value that is measured in kilopascals, and this value can be extrapolated to a fibrosis score. The FibroScan yielded results that showed perfect agreement with liver biopsy⁽⁵¹⁾. A study on chronic HCV patients, showed that the FibroScan compared favourably with the FT and APRI.⁽⁵²⁾ When biomarkers and FibroScan were combined, the predictive value for fibrosis was improved, and such combination could be considered as first-line non-invasive assessment for liver fibrosis, being quite sensitive and specific ^{(52, 53)}.

A novel approach using sonography-based-real-time-elastography, which can be performed with conventional ultrasound probes during a routine sonographic examination is promising is assessing fibrosis ⁽⁵⁴⁾. It is the first application for this goal after its successful application for the characterization and the detection of focal lesions in the breast, prostatic gland and thyroid gland ^(55-57). Results were compared with fibrosis stage obtained by assessing biopsy samples and with APRI. The areas under the receiver operator curves showed high diagnostic accuracy of real-time elastography in diagnosis of liver fibrosis.

An imaging study revealed that ultrasound had more value than CT and/ or MRI in the diagnosis of liver fibrosis ⁽⁵⁸⁾. It recommended more research so as to increase the sensitivity, specificity, and accuracy of these diagnostic tests. However, the study reported that the combination of serum markers, and imaging tests are quite sensitive and specific in diagnosing hepatic fibrosis⁽⁵⁸⁾.However, a recent meta-analysis of 30 studies with pooled 6378 subjects, stresses that neither non-invasive markers nor biopsy are sufficient alone for decision-making  in a given patient ⁽⁵⁹⁾.

Finally, an experimental approach to utilize advances in the molecular understanding of liver fibrosis, tried to identify activated stellate cells by tagging them with cell-specific markers ⁽⁶⁰⁾.

Conclusions:

The major pitfall of all the currently used non-invasive methods measuring liver fibrosis (serum markers and imaging tests), is their lack of ability to differentiate small changes in the state of the ECM. So, more research is required to improve biomarkers assay and imaging techniques before we retire liver biopsy from our diagnostic armamentarium in the evolution of liver disease. This is because neither non-invasive markers nor biopsy are sufficient alone to take definitive decision in a given patient,  and  all  the clinical  and biological  data  must  be taken  into account .

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