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Markers of liver fibrosis - ELF test (enhanced liver fibrosis)

The risk of errors in the interpretation of liver biopsy has necessitated the search for alternative methods to assess the severity of liver fibrosis. Markers present in serum are of particular interest in this regard due to the possibility of standardisation and automation of their determination. The ELF™ test is a routine biological marker blood test that accurately diagnoses serious liver disease.

Liver fibrosis

Liver fibrosis is the process of connective tissue formation in the liver due to liver damage. Similar to the healing process of skin damage, the liver repairs itself by depositing collagen and other matrix components and remodelling the tissue. Over time, this process can lead to cirrhosis, with impaired liver function, portal hypertension, liver failure and liver cancer. With the development of cirrhosis, which should be considered a precancerous condition, the risk of liver cancer increases dramatically. Currently, cirrhosis and liver cancer are among the top ten causes of death worldwide, and in many developed countries, liver pathology is one of the five leading causes of death in middle-aged patients.

Pathogenesis of liver fibrosis

Fibrotic neomatrix in the liver is formed in stellate cells (Ito cells). In the normal liver, the resident population of these cells exists in a quiescent state and is the main depot of vitamin A in the body. Upon activation, stellate cells transform into myofibroblasts capable of secreting collagen. This fibrous tissue is then modified by matrix cleavage by matrix metalloproteinases (MMP). Matrix cleavage, in turn, is regulated by MMP inhibition by tissue inhibitors of MMPs (TIMPs), among which TIMP-1 plays a special role. Liver fibrosis, previously thought to be solely an accumulation of scar tissue, is now recognised as a dynamic process that can progress or regress in a matter of months.

Causes of liver fibrosis

Liver fibrosis can be caused by any chronic liver disease (CLD). For many years, chronic viral hepatitis B (CHB) and alcoholic liver disease (ALD) were the main causes of CLD. While alcoholism and ALD are declining in most countries, there is an alarming increase in ALD due to alcohol abuse among young people in several Nordic countries. In the last few decades, two other diseases have emerged that significantly increase the burden of CLD. Chronic hepatitis (CHC) C and steatohepatitis (NASH) have been found to strongly influence the prevalence of CLD.

Hepatitis C virus (HCV) is transmitted through blood and blood products through unsafe injections and therapy with infected blood products. It is estimated that approximately 200 million people worldwide are infected with HCV.

Due to the dramatic increase in obesity rates in the developed world, NASH is recognised as a major cause of advanced fibrosis. It is likely that only a small percentage of patients with NASH (perhaps 20%) develop significant fibrosis, but because of the large number of overweight individuals, the disease may be epidemic in liver fibrosis.

Liver biopsy

Traditionally, abdominal puncture liver biopsy is considered the standard technique for detecting and assessing liver fibrosis. During this procedure, a hollow needle is inserted into the liver to take 1/50,000th of the organ for subsequent histological test. Due to the small sample size and the heterogeneous distribution of pathological liver tissue, this method is characterised by a significant margin of error. The procedure can be painful (in almost 30% of cases) and dangerous (bleeding in one in 1,000 cases and even death in one in 10,000 cases). Histological test of a biopsy specimen takes time and may be interpreted differently by different researchers.

disadvantages of the liver biopsy technique:
painfulness;
sampling error (25-35%);
single study to assess an evolving biological process;
expensive and time-consuming procedure, requiring a hospital visit and the involvement of a highly qualified specialist;
variability of results.

In addition, repeated sampling of liver tissue for research purposes is considered inappropriate and therefore cannot be used to determine the course of the disease or the effect of therapy directed at the fibrotic process itself or at the underlying cause of CLD, such as antiviral treatment. So many problems in obtaining and interpreting liver biopsy specimens have necessitated the search for alternative methods of assessing the severity of liver fibrosis. Markers present in serum are of particular interest in this regard due to the possibility of standardisation and automation of their determination.

Markers of liver fibrosis

Fibrosis markers detected in serum can be divided into direct and indirect markers. Direct markers are fragments of hepatic matrix components produced by hepatic stellate cells during fibrogenesis and molecules involved in the regulation of fibrosis progression and regression. These include hyaluronic acid (HA), collagens IV and VI, N-terminal fragments of procollagen III (P3NP), as well as MMP and TIMP-1.

Indirect markers of fibrosis include molecules released into the blood during liver inflammation (e.g., the aminotransferases AlAT and AsAT), molecules synthesised, regulated or secreted by the liver (e.g., clotting factors, cholesterol and bilirubin), and processes that are impaired when liver function deteriorates, such as insulin resistance.

ELF markers

In 1997, a group of European researchers assembled by Professor Michael Arthur began work on a project (led by Professor William Rosenberg and funded by Bayer Healthcare) to identify serum markers of liver fibrosis. This research programme, which has been ongoing for almost a decade, has resulted in the identification of a panel of direct markers that have been validated, CE marked and marketed in Europe by iQur Limited (UK) (the Enhanced Liver Fibrosis test, or ELF™).

The original European Liver Fibrosis Study Project involved over 1,000 patients in 13 centres across Europe who underwent liver biopsy. Participants were found to have a wide range of CLD, with more than 40% of participants having CHC or CHB. All patients had fasting serum samples taken at the same time as the biopsy. These samples were sent to the laboratory for determination of direct and indirect markers. A sandwich immunoassay test method was developed for each of the direct markers. It was found that a combination of HA, P3NP and TIMP-1 combined in an algorithm that initially took age into account could be used to determine the severity of liver fibrosis with high accuracy. The group subsequently established the feasibility of removing age from the algorithm to develop the ELF™ test.

After the initial study, validation studies in independent populations were conducted in many countries to further evaluate the efficacy of the markers. All studies in patients with CHC, NASH and primary biliary cirrhosis (PBC) confirmed that the markers accurately reflect the severity of fibrosis as determined by liver biopsy.

Marker studies have also been conducted to predict long-term clinical outcomes of CLD, including the development of portal hypertension, decompensation of CLD, development of hepatic cell cancer, liver transplantation and death from liver disease, and mortality from any cause. Even an incomplete interim test of a seven-year follow-up of 500 patients showed that ELF markers were at least as good as liver histological findings in predicting clinical outcome. Similar work has been done with a cohort of patients with PBC.

Advantages of comprehensive marker detection

Analysis of the literature consistently shows that individual fibrosis markers can detect cirrhosis with some accuracy. The great potential of ELF markers is particularly evident in the screening and management of patients with CLD.

The ability to identify patients with mild to moderate fibrosis (usually asymptomatic) is important. This allows changes in lifestyle or treatment to be made before liver damage becomes irreversible. Despite the good performance of many indirect markers, most give unreliable results when examining patients undergoing treatment for viral hepatitis, where transaminase activity is altered, or where there may be an increase in bilirubin levels (e.g. on ribavirin) or cholestasis.

Application in clinical practice

The ELF test has already been introduced into clinical practice in a number of countries and its impact on the quality of patient diagnosis is appreciated. The ability to use routine blood tests to accurately identify patients with serious liver disease is of great help in making a diagnosis and prescribing appropriate therapeutic measures.

The ELF test is not a substitute for liver biopsy in the detailed assessment of inflammatory and structural pathological changes. However, it can be used to prioritise patient evaluation, to determine the severity of fibrosis in patients who are unwilling or unable to undergo biopsy, and to supplement biopsy by minimising histological bias.

Once a diagnosis of CLD has been made, most patients receive some course of treatment with long-term follow-up. For the vast majority of patients, further repeat biopsies are unacceptable, but the additional knowledge of fibrosis severity, progression or regression is of particular value to both patient and clinician. The ELF test will provide this information through a simple blood test that can be repeated at short intervals.

Development prospects

New lines of research, such as metabonomics, proteomics and transcriptomics, which link individual molecules to pathological processes, may discover new markers that complement or improve existing panels. Diagnostic imaging of the liver using a variety of modalities, including ultrasound, elastography and magnetic resonance imaging, is evolving in parallel with non-invasive marker test. Combining these complementary techniques will further expand the diagnostic options available to clinicians, but determining the optimal combination of techniques and their suitability for the diagnosis of specific CLD will not be an easy task.

The discovery of ELF markers marks the beginning of a new era of early detection of liver disease and effective monitoring of CLD to assess the impact of therapeutic intervention and disease course.