Liver disease is increasing globally. In addition to being an insidious condition, there are few tools to evaluate the severity of the disease besides liver biopsy (which is a highly invasive test), neither is it easy to monitor the impact of current treatments.
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What is a biomarker?
According to the National Institute of Health (NIH), a biomarker is “a characteristic that can be objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.”
Biomarkers help to screen for, evaluate, monitor and forecast the prognosis of specific conditions.
Specific liver biomarkers are difficult to find due to the common pathways of liver injury.
Non-alcoholic fatty liver disease (NAFLD) and its consequences
NAFLD is a term that covers a spectrum of diseases, ranging from pure steatosis, through steatosis with mild liver inflammation, to early non-alcoholic steatohepatitis (NASH), fibrotic NASH, NASH-cirrhosis and, finally, hepatocellular carcinoma (HCC). At present, a liver biopsy is a gold standard to diagnose NASH.
NAFLD biomarkers may be classified as:
- Imaging biomarkers
- Molecular and biochemical biomarkers
- Novel biomarkers in the form of extracellular vesicles (EVs) and microRNAs (miRNAs)
Imaging biomarkers include
- Ultrasound scan – a simple, available, inexpensive method to screen for the condition, but operator-dependent and less reliable in obesity;
- Computed tomography scan – widely available, can assess the whole liver but involves radiation, is insensitive for mild steatosis, and is expensive;
- Magnetic resonance spectroscopy (MRS) – a highly reproducible technique with excellent accuracy, but is costly and time-consuming, requires operator expertise and has restricted availability;
- Magnetic resonance imaging proton density fat fraction (MRI-PDFF) – superior to MRS, very accurate, sensitive and offers complete liver assessment, but not freely available and very expensive;
- Proton-MRS – allows quantitative estimation of liver fat, but is expensive and used for research only.
Molecular and biochemical biomarkers
Serum biomarkers and disease scores are used to diagnose the presence of hepatic steatosis where imaging is unavailable or not feasible, such as in large epidemiological studies. The characteristics of a useful biomarker include:
- Specific for the disease and the outcome
- Safe, convenient and inexpensive
- Must correlate with disease activity scores
The liver aminotransferases
Serum aminotransferases include aspartate and alanine aminotransferase (AST and ALT respectively), two liver enzymes that escape into the circulation upon liver damage. AST is also made in the heart and skeletal muscles and increases with strenuous exercise. It is induced by many drugs as well.
High ALT levels are therefore more specific for liver injury. However, ALT is normal in up to 80% of NAFLD patients. An ALT threshold value of 23 IU/L could pick up as many as 94% of NAFLD cases.
In the NAFLD spectrum, the AST/ALT ratio is below 1 in all diseases but advanced fibrosis.
The AST/ALT ratio also helps diagnose obstruction of the biliary tract, along with high levels of ALP (alkaline phosphatase), GGT (gamma-glutamyl transpeptidase) and bilirubin. A ratio of less than 1.5 suggests extrahepatic obstruction, while if AST/ALT is more than 1.5 the obstruction might be intrahepatic.
Again, the AST/ALT is above 1 in the 92% of alcoholic liver disease due to severe inflammation and underlying liver disease, and above 2 in the 70%.
The liver function test (LFT) consists of blood tests that measure a range of substances made by the liver, often in a single blood sample. Besides aminotransferases, this may include:
- Albumin (the major protein in the blood) made by the liver
- Total protein - the total amount of protein in the blood
- ALP–a widespread enzyme family, released from the cells lining the bile ducts and canaliculi, it is raised in cholestasis, but also bone disease and kidney disease. In drug-induced cholestasis, ALP is higher than GGT, with an ALT/ALP ratio above 2. Normal aminotransferases with high ALP may indicate primary biliary cirrhosis
- GGT is another enzyme produced by the liver microsomes, but also the kidney, pancreas, and intestine. High ALP and GGT levels indicate cholestasis. GGT also rises with alcohol and medication intake due to enzyme induction.
- Bilirubin is a waste product formed in the liver from hemoglobin
- Lactate dehydrogenase (LDH) which is found in multiple tissues
- Prothrombin time (PT), a liver protein that takes part in blood clotting
These include extracellular vesicles (EVs) and miRNAs. EV production and clearance are imbalanced in liver disease for various reasons, like overproduction from inflamed or dying cells, and reduced macrophage phagocytosis. High plasma EVs indicate progressive NAFLD, while EV content helps differentiate various etiologies.
MicroRNAs (miRNAs) are small, non-protein coding molecules that bind to mRNA and modulate many different biological functions like apoptosis, cell proliferation, and differentiation. They play an important role in chronic liver disease. NAFLD markers include miR-122, miR-34a, and its target SIRT1, and miR-29.
Liver panels in NAFLD
As of now, no non-invasive tests are approved for NASH diagnosis. Researchers have developed various ratios and indices to move towards a non-invasive diagnosis of NAFLD and fibrosis:
- AST/platelet ratio (APRI) for advanced fibrosis and cirrhosis
- FIB-4 score, with age, AST, ALT and platelet count for fibrosis in HIV/HCV coinfection
- FibroIndex using platelet count, AST and gamma globulin
- FibroTest using 6 parameters (total bilirubin, GGT, alpha-2 macroglobulin, haptoglobin, ALT, and apolipoprotein-A1) can accurately detect NASH
- Forms index using age, platelet count, serum cholesterol and GGT for distinguishing mild and advanced fibrosis
- PGA Index using PT, GGT, apolipoprotein A, and alpha-2 macroglobulin
- SteatoTest that tests 12 parameters
- NASH diagnostic panels such as the Shimada index, cytokeratin-18, and the NASH diagnostic panel, that reflect liver inflammation
Several diagnostic algorithms have been developed for the diagnosis of hepatic fibrosis in chronic hepatitis C:
- SAFE (sequential algorithm for fibrosis evaluation) uses APRI initially, followed by FibroTest as a second-line test, and finally, a liver biopsy in up to 30%–50% of cases. This is not only inconvenient, requiring successive testing, but often invasive for the diagnosis of significant fibrosis and cirrhosis
- The Bordeaux algorithm (BA), based on a blood test and transient elastography for liver stiffness, for significant fibrosis or cirrhosis
- Another recent algorithm uses transient elastography with a 5-parameter (platelets, PT, AST, alpha-2-macroglobulin, GGT) blood test, with 87% sensitivity for the non-invasive diagnosis of liver fibrosis while offering 6 diagnostic classes.
Other proposed biomarkers include aPA/1 for NASH, adiponectin, IGFII, and tPA/1 for mild to moderate steatosis and MCP-1 for lobular inflammation.
New non-invasive diagnostic panels are awaited to replace liver biopsy in this field.
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- Cales, P., Boursier, J., Oberti, F., Moal, V., et al. A single blood test adjusted for different liver fibrosis targets improves fibrosis staging and especially cirrhosis diagnosis. Hepatology Communications 2018, Volume 2, Issue 4. https://doi.org/10.1002/hep4.1161. https://aasldpubs.onlinelibrary.wiley.com/doi/full/10.1002/hep4.1161
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