Non-alcoholic fatty liver disease (NAFLD) represents a spectrum of conditions characterized histologically by excessive accumulation of hepatic fat in the absence of alcohol consumption. Two main histological patterns of NAFLD are described: fatty liver alone and steatohepatitis (NASH). NAFLD is an increasingly recognized cause of liver-related morbidity and mortality 123. Although the majority of patients do not develop complications, 28% may develop serious liver sequelae, including end-stage liver disease and hepatocellular carcinoma 12345. Those at highest risk include patients with significant hepatic necro-inflammation and fibrosis 126. Therefore liver biopsy had been recommended for confirming diagnosis and for providing prognostic information 78.

There are several drawbacks in using liver biopsy for this purpose. This procedure is invasive, costly, and prone to complications, some minor, such as pain, others more severe with a recorded risk of death of 0.01% 91011. Notably, just as is the case in other chronic liver diseases, there is considerable sampling variability (40% for fibrosis staging), and a high intra and inter-pathologist variability1213. Most importantly, the number of patients at risk for NAFLD is high enough that liver biopsy is not a practical and efficient tool for identifying those at risk of advanced fibrosis. Indeed an estimated 15 to 20% of the Western European population has steatosis 14 while more than half of Americans are overweight or obese.

Because liver biopsy is impossible to perform in such large cohorts of individuals, some investigators have tried to identify simple non-invasive markers of liver injury, in particular fibrosis, in patients with NASH. Different studies have shown that an age of 45 years or more, the extent of obesity, type 2 diabetes, high levels of alanine aminotransferase and triglycerides, high HOMA indices of insulin resistance, systemic hypertension, and high level of C-peptide 61516 are associated with advanced fibrosis in patients with NASH. However, these findings are not consistent between studies and have been generated through retrospective studies, all amenable to biases known and unknown. Imaging techniques have moderate predictive values for advanced steatosis but not for bridging fibrosis 1718.

In the last 3 years, we developed 2 panels of simple biochemical markers, named FibroTest (FT) and Actitest, (Biopredictive Paris, France, FibroSURE in the US patented artificial intelligence algorithm USPTO 6,631,330 http://www.uspto.gov/patft/index.html). The fibrosis index includes α₂-macroglobulin (A2M), apolipoprotein A1, haptoglobin, total bilirubin, and γ-glutamyl-transpeptidase (GGT), and the necrotico-inflammatory activity index combines the same 5 markers, plus ALT. These panels demonstrated high predictive values for significant lesions in patients with chronic hepatitis C 19202122232425262728, chronic hepatitis B 2529 and alcoholic liver disease 30. These diagnostic values were also confirmed by independent groups and versus glycomics and elastometry 31323334. Because fibrosis features are similar between patients with alcoholic liver disease and NAFLD, it was logical to hypothesize that the diagnostic value of FT would be the same.

The objective of the current study was to validate the diagnostic utility of FT for the detection of advanced fibrosis in patients with NAFLD in two prospective validation groups, one in a single center (Group 1) and one in multicenter study (Group 2). The specificity has been also assessed in a large prospective cohort of blood donors.

Mass screening for significant liver injury in patients with NAFLD will be an important medical challenge in the years to come because of the epidemics of obesity and diabetes. The inability of liver biopsy to meet this challenge makes the development of non-invasive, readily available, and easy to perform serum markers, a high priority. This study highlights the potential utility of FT for the prediction of fibrosis in patients with NAFLD, as previously observed for patients infected with HCV, HBV and for patients with alcoholic liver disease.

The first validation group included patients of a secondary care center, which makes it liable to referral selection bias but the second validation group was most representative of less specialized centers. The demographic characteristics of our patients, including age and gender distribution, prevalence of cirrhosis, components of the metabolic syndrome, are similar to those reported by other studies from France638. We have taken less limited inclusion criteria concerning the alcohol consumption with inclusion of patients up to 40 g of alcohol per day for male and up to 25 g for female. There is no consensual limit. However when males with 30–45 g or women with 20–25 g or more per day were excluded following Guideline for diagnosis of NAFLD 39, the diagnostic value of FT was even better although not significantly (Table 4).

An important limitation of liver biopsy is its sampling variability 12. The ideal gold standard should be not a 15 mm fragment but rather a 25 mm or sample 12. In chronic hepatitis C 18 % of the discordant results between liver biopsy results and FT values have been attributed to liver biopsy failures (mostly because of small sample size) and 2% only to FT 27. Histological lesions of NAFLD including perisinusoidal fibrosis were unevenly distributed throughout the liver parenchyma 12. Discordant results of one stage or more between biopsy and FT were at high as 41%. Being a serum marker, FT has the advantage of representing a more global estimate of liver fibrosis throughout the whole liver. One case was emblematic of the weakness of even a 20 mm-long biopsy: a patient had severe fibrosis (F3) on the first biopsy, no fibrosis on the second biopsy (F0) and intermediate FT values (F1-F2). Although not significant, the AUROC for FT was higher when the mean fibrosis stage between the 2 biopsies was taken into account (Table 4). Contrary to histological staging systems which are all semi-quantitative, a serum biochemical marker provides a continuous quantitative assessment of liver fibrosis in 100% of patients without indeterminate cases 28.

Another drawback of liver biopsy is that for most practitioners it seems almost unethical for it to be performed in patients with normal serum transaminases values. Unfortunately, many patients with NAFLD or NASH have normal ALT and some of them have advanced liver fibrosis 4041. In the present study ALT was lower than 50 IU/L in 43% of patients with advanced fibrosis. As in chronic hepatitis C, FT AUROCs for the diagnosis of advanced fibrosis in NAFLD were unchanged in patients with ALT values lower than 50 IU/L (Table 4); Therefore FT could allow the diagnosis of fibrosis even in patients that are not eligible for liver biopsy.

A few other markers have been evaluated in NAFLD for the diagnosis of fibrosis. Sakugawa et al, demonstrated in 112 patients with NAFLD, good diagnostic values for hyaluronic acid and type IV collagen 7S for stage F3 and F4 with AUROCs of 0.80 and 0.83 respectively 42. Lainé et al combined hyaluronic acid and the carbohydrate-deficient transferrin/transferrin ratio in 173 patients with increased serum aminotransferases and features of metabolic syndrome 38. Hyaluronic acid AUROC for F2F3F4 fibrosis was 0.92. In our experience in patients with chronic hepatitis C and alcoholic liver disease FT had a higher sensitivity than hylauronic acid, especially for the diagnosis of moderate fibrosis 2030. Rosenberg et al. studied a panel combining age, hyaluronic acid, amino terminal propeptide of type III collagen and tissue inhibitor of matrix metaloprotein-1 in 81 patients with NAFLD 43. They observed an AUROC of 0.87 for advanced fibrosis, similar to FT AUROC in the present study 43.

When comparing the performance of different serum markers for liver fibrosis the diagnostic yield is far from being the only aspect that needs to be considered 44. Equally important are the description of analytical conditions for serum measurements including intra patient and intra sample variability, the description of precautions of use and the identification of cases with discordant results between serum markers and liver biopsy as well as risk factors for these discordances 22232745. In the present study we observed 5% of discordances due to FT failure versus 4% due to biopsy failure. We recognize that in the analysis of discordance, we assert that in patients with no known cause of false negative FT, and a small length biopsy, we consider because of biopsy sampling error that the discordance was due to failure of biopsy. We acknowledge that in these cases there is no direct prove of failure of biopsy when no second biopsy and no other independent marker have been performed. As previously described Gilbert's syndrome and acute inflammation were the most frequent causes of FT failures. We observed a possible cause of false negative FT failure, not previously described: an unusual high serum apoA1 concentration due to high serum HDL-cholesterol (correlation between ApoA1 and HDL cholesterol R = 0.77). This condition was rare (3 cases out of 170, 1.8%) but is probably more frequent than in other chronic liver diseases without lipids abnormalities.

None of the FT components is a direct marker of hepatic extracellular matrix nevertheless the overall score is correlated with liver fibrosis. A2M is a protease inhibitor, but also has multiple functions as a binding, carrier and targeting protein 46. A2M is associated with several growth factors: fibroblast, vascular endothelial, epidermal, transforming and platelet derived growth factors 19. Interestingly, in patients with NAFLD, the present study demonstrated a very significant association between A2M and insulin levels, a hallmark of insulin resistance. Relationships between A2M and insulin have been described for more than 40 years 47. Some studies have observed an increase of A2M in diabetic patients 48. Insulin is covalently bound to A2M in plasma 49 and A2M is a binding protein of Insulin-like Growth Factor Binding Protein-1 (IGFBP-1) which modifies the IGFBP-1/IGF interaction 50. Therefore A2M can be directly involved both in the hepatic mechanisms of insulin resistance and fibrogenesis 5051.

When compared to alcoholic liver disease the decrease of apolipoprotein A1 was not significant in patients with advanced fibrosis and NAFLD. The interpretation of this negative observation must be prudent because of the small number of patients with cirrhosis included in our NAFLD population. We observed a dramatic decrease in ApoA1 serum levels in patients with alcoholic liver disease which was associated with necrosis, polymorphonuclear infiltrate and Mallory bodies 30. In the present NAFLD population the prevalence of patients with severe necrosis was small, and only 2 patients had a polymorphonuclear infiltrate (1.2%). In the first case there was a dramatic decrease in ApoA1 (0.05 g/L), as observed in alcoholic steato-hepatitis. In the second case the absolute value of ApoA1 was not decreased (1.72 g/L) but was relatively low in comparison with HDL cholesterol (1.64 mmol/L).

Although in NAFLD there is no specific treatment approved to treat liver injury, the diagnosis of advanced fibrosis could be very important to motivate the patient for diet or lifestyle modifications, for intensive treatment of complications of the metabolic syndrome or for weighing in favour of anti-obesity surgery. The early detection of advanced fibrosis is the first step to reduce future cirrhosis-related deaths. Diagnosing silent cirrhosis has important consequences in terms of screening for portal hypertension and hepatocellular carcinoma, of preventing complications and of timely indication for liver transplantation.

Based on our data, a preliminary algorithm for the use of FT as a screening tool in patients at risk for NAFLD can be suggested. Below 0.30 the probability of cirrhosis is very low and there is no need for ultrasonography or endoscopy. Between 0.30 and 0.70 it is mandatory to help the patient in reducing all metabolic factors (overweight, diabetes, dyslipidemia and maybe complete alcohol abstinence). Follow-up of these patients with FT can be advised. When the FT value is 0.70 or higher, the patient should be managed as a patient with cirrhosis, and surveillance by ultrasonography and endoscopy should be implemented in order to prevent potentially severe complications of cirrhosis.

In patients with NAFLD, FibroTest-FibroSURE, a simple and non-invasive quantitative estimate of liver fibrosis reliably predicts advanced fibrosis

A2M alpha2macroglobulin; GGT, γ-glutamyl-transpeptidase; ALT, alanine aminotransferase; ROC, receiver operating characteristic; AST, aspartate aminotransferase; NPV, negative predictive value; PPV, positive predictive value; ULN, upper limit of normal.

TP is a consultant and has a capital interest in Biopredictive, the company marketing FibroTest-Actitest.

VR and TP conceived and wrote the manuscript. VR, TP, LB, MM, DT, JFC, and VDL were responsible for the patient drafting, and participated in the coordination of the study. DM and FIB drafted the paper. FC and BLB were responsible of histological analysis. TP performed the statistical analysis. All authors read and approved the final manuscript