Most Indirect biomarkers of fibrosis are integrated with one or more fibrosis predicting biomarker panels.
AST/ALT ratio
Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) are hepatic enzymes that are released into the bloodstream from damaged hepatocytes. The predictive value of the AST/ALT ratio has been validated in non-alcoholic liver disease, chronic viral hepatitis, primary sclerosing cholangitis, and primary biliary cirrhosis [
74]. In many forms of acute and chronic liver injury or steatosis (fatty infiltration of the liver), this ratio is less than or equal to 1, while in alcoholic hepatitis, an AST/ALT ratio is often greater than 2. While these ratios are suggestive of certain etiology of liver conditions, there is too much overlap between groups to rely on AST/ALT exclusively when making a diagnosis - for example, in patients with both hepatitis C and history of alcohol abuse [
75].
The
PGA index combines the measurement of the Prothrombin Index, γ glutamyl transferase levels and apolipoprotein A1. It was subsequently modified to the PGAA index by the addition of α2- macroglobulin, which resulted in marginal if any improvement in its performance. In chronic liver diseases, the PGA index has a relationship to both the inflammation and the fibrosis (
P < 0.01,
P < 0.05 respectively). However, overall accuracy of this index is relatively low [
76,
77].
The
AST-to-Platelet Ratio Index (APRI) is calculated as (AST/upper limit of normal range)/platelet count (10
9/L) × 100. This index has previously been validated as a surrogate marker of significant hepatic fibrosis in HIV/HCV-coinfected patients, and has recently been used to determine advanced fibrosis in HIV-monoinfected patients [
78]. However, recent large meta-analysis suggested that APRI can identify hepatitis C-related fibrosis with only a moderate degree of accuracy [
79].
The
Forns index is based on 4 routine clinical variables: age, platelet count, cholesterol levels, and γ glutamyl transferase. This method can be used to differentiate patients with mild (F0-F1) fibrosis from those with severe (F2-F4) fibrosis, but it is less accurate in distinguishing patients with grades F2 versus F4. The Forns index has been validated in other cohorts as a predictive tool for response to anti-HCV therapy [
80].
The
HepaScore combines age, gender, bilirubin, γ glutamyl transferase, hyaluronic acid, and γ2-macroglobulin into a score from 0.00 to 1.00 [
81]. In 512 chronic HCV patients, automated HepaScores showed good predictive performances for significant fibrosis (AUROC = 0.81), severe fibrosis (AUROC = 0.82), and cirrhosis (AUROC = 0.88). Importantly, HepaScore test can be automated using a single analyzer [
81].
The
FIB-4 score which combines platelet count, ALT, AST and age, was originally developed for use in HIV-HCV co-infection. Use of this index correctly classified 87% of patients with FIB-4 values outside 1.45-3.25 and avoided biopsy in 71% of the validation set with an AUROC of 0.765, sensitivity of 70% and a specificity of 97% for differentiating Ishak 0-3 from 4-6 [
82]. This model was subsequently validated by Vallet-Pichard in a large cohort of HCV mono-infected patients, with the finding that using these ranges, 78% of 847 biopsies were correctly classified [AUROC 0.85 for severe fibrosis and 0.91 for cirrhosis] [
83].
The
SHASTA Index which consists of serum hyaluronic acid (HA), AST, and albumin was evaluated in a cohort of 95 patients with HIV/HCV co-infection [
84]. 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; however 58% were classifiable with scores between 0.3 and 0.8. However, the SHASTA index in HIV/HCV in this cohort has performed significantly better than the APRI test [
85].
The
13
C-methacetin breath test (MBT) is amongst several
13C breath tests used for the quantitative non-invasive assessment of cytochrome P450-dependent hepatocellular function [
86]. MBT is rapidly metabolized by healthy liver cells into acetaminophen and
13CO
2 in a single dealkylation event, so the increase of
13CO
2 in breath samples can be quantified by isotope ratio mass spectrometry or non dispersive isotope-selective infrared spectroscopy [
86]. MBT has been shown to have high sensitivity (92.6%) and specificity (84.1%) in predicting liver cirrhosis. The areas under the curve were found to be 0.958 for predicting cirrhosis and 0.827 for identifying patients with advanced fibrosis [
87]. MBT does not involve a blood test and can provide an immediate result at the point-of-care.
The
FIBROSpect II test uses a combination of components in the fibrogenic cascade, such as hyaluronic acid, TIMP-1 (tissue inhibitor of metalloproteinase), and α-2-macroglobulin to calculate a composite score. The test is intended to differentiate mild fibrosis (Metavir stages F0 to F1) from more severe disease (Metavir stages F2 to F4), and had been shown to do well in chronic hepatitis C cohorts [
88,
89].
The
FibroTest and
FibroSure are identical tests marketed under different names in Europe and America for the assessment of fibrosis and necroinflammatory activity. The FibroTest score is computed by accessing a proprietary website and entering the patient's age, sex, and results for serum haptoglobin, α
2-macroglobulin, apolipoprotein A1, γ-glutamyltransferase, and bilirubin analyses [
90]. It generates a score that is correlated with the degree of liver damage in people with a variety of liver diseases. Due to the variability of component of assays and analyzers, FibroTest assays can only be performed in validated laboratories [
91]. A recent study showed an AUROC of 0.69 and 0.91 for the diagnosis of significant fibrosis (F≥2) and liver cirrhosis in 74 patients comprising of 36 with HCV, 10 with HBV, and 28 with primary biliary cirrhosis [
92]. The sensitivity and specificity values for FibroTest based detection of primary severe fibrosis were found to be 75% and 85%, respectively [
77].
The
FibroIndex was developed by Koda and co-authors [
92] for liver fibrosis in chronic hepatitis C. This test relies on platelet count, AST and serum IgG. FibroIndex showed high predictive values for significant fibrosis, including in a subgroup of HCV cases with normal alanine aminotrasferase (NALT) [
93]. The sensitivity and specificity of FibroIndex for detecting fibrosis in patients with HCV were 78% and 74% [
94]. In a comparative study, the validated AUROC of the FibroIndex for predicting significant fibrosis was found to be 0.83 and 0.82, which is better than those of the Forns index and APRI in patients with chronic hepatitis C [
94].
The
FibroMeter is a combination of the platelet count, prothrombin index, AST, γ2 macroglobulin, hyaluronate, blood urea nitrogen and age. The good performance and applicability of FibroMeter was validated in a number of chronic liver diseases, including chronic viral hepatitis B or C, alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD). An important feature of the FibroMeter is that it presents the amount of liver fibrosis as a percentage of fibrous tissue within the liver. Another significant feature of FibroMeter is that it validates the results through an expert system that detects erroneous results. FibroMeter has two main diagnostic targets - fibrosis stage corresponding to the histological staging system Metavir and the amount of fibrosis which corresponds to morphometric determinations of the fibrotic area [
95].
Two additional panels have been developed to assess hepatic fibrosis specifically in NAFLD. First, the so called "Simple Test" for fibrosis in NAFLD is a relatively easy to use panel that includes age, hyperglycemia, body mass index, platelet count, albumin, and AST/ALT [
96]. When the purpose of performing a liver biopsy in NAFLD is to determine the extent of hepatic fibrosis, using the Simple Test can correctly stage 90% of patients, obviating the need for liver biopsy in approximately 75% of patients. In addition to the "Simple Test", another panel for hepatic fibrosis is the Original European Liver Fibrosis (OELF) panel [
97], which includes age, hyaluronic acid, amino-terminal propeptide of type III collagen, and tissue inhibitor of matrix metalloproteinase 1. A simplified version OELF is ELF, which does not include age, and seems to perform well in patients with NAFLD [
98]. The performances of ELF and OELF were found to be almost identical. In a recent study, when the ELF panel was used for "ruling in" severe fibrosis, only 14% of NAFLD patients in this cohort required a liver biopsy. The combination of Simple/ELF panel reached an AUROC of 0.98 for distinguishing severe fibrosis from initial stages of the fibrotic disease in patients with NAFLD [
98].
The
Proteomics based tests assess patterns of protein or glycoprotein by mass spectroscopy using serum samples. Importantly, while a series of 'peaks' generated, the precise identities of these peaks remain unknown. For example, Callewaert N et al., 2004 developed tests based on the altered N-glycosylation of total serum protein (GlycoCirrhoTest and GlycoFibroTest) [
99], which could be both cost-effective and could rapidly determine a signature profile for n-glycans. At first, it was reported that the combination of GlycoCirrhoTest with the FibroTest produced a sensitivity of 79% and specificity of 86% in distinguishing cirrhosis from non cirrhotic disease. However, later tests showed limited applicability of the test to discern the etiology of liver diseases. Specifically, under galactosylation did not show a significantly different quantitative alteration in the cirrhotic and noncirrhotic population of all etiologies [
100]. Moreover, the same modifications seem to continuously reappear in all liver diseases: hyperfucosylation, increased branching and a bisecting N-acetylglucosamine [
100]. Larger prospective studies are necessary to determine the clinical application of these new technologies.
The recently developed
Phosphoproteomics tests serve the goal of improving and understanding the pathogenesis of liver fibrosis to more than actually contributing to the practicality of clinical diagnostics. For example, phosphoproteomics based tests predict the fibrosis of the liver have been used to profile the phosphorylated (i.e. activated) forms of the major signaling proteins in visceral adipose samples of patients with NAFLD [
101].
Recently, a number of attempts were made to improve sensitivity and specificity of non-invasive biomarker-based tests through combining them using sequential algorithms. One example of such studies is a work of Sebastiani et al, who combined APRI, Forns' index and Fibrotest to reduce need for liver biopsy in hepatitis C patients by 50-70% [
102]. Later, the same group of authors combined APRI with FibroTest-FibroSure and validated this approach known as SAFE (sequential algorithm for fibrosis evaluation) biopsy in a very large cohort of hepatitis C patients (N = 2035) [
103]. For the detection of fibrosis, SAFE biopsy had remarkable accuracy of 92.5% (95% confidence interval, 0.89-0.94), obviating the need for 81.5% of liver biopsies [
103].