Diagnostic value of biochemical markers (FibroTest-FibroSURE) for the prediction of liver fibrosis in patients with non-alcoholic fatty liver disease

A total of 954 blood donors prospectively included were used as controls.

All patients and controls gave informed consent for use of data and serum for research purposes.

Liver biopsies were fixed, paraffin-embedded, and stained with at least hematoxylin-eosin-safran, iron staining, and Masson's trichrome or picrosirius red for collagen. A single pathologist unaware of patient characteristics analyzed the histological features (FC) in Group 1 and in Group 2 (BLB). A scoring system recently published by Kleiner et al [35] who studied inter observer variability was used. Fibrosis was staged as follows: stage 0: no fibrosis; stage 1: perisinusoidal or periportal fibrosis with 3 different patterns: 1A: mild, zone 3, perisinusoidal; 1B: moderate, zone 3, perisinusoidal fibrosis, and 1C portal/periportal fibrosis; stage 2: perisinusoidal and portal/periportal fibrosis; stage 3: bridging fibrosis; stage 4: cirrhosis. Steatosis was scored from 0 to 3 with a four grades scoring system from S0 to S3: S0_no steatosis or less than 5% (low to medium -power evaluation of parenchymal involvement by steatosis), S1_5%–33%, S2_>33%–66%, S3_>66%. Steatohepatitis was defined as a NASH score (NAS) greater than 5. The histological NAS score is defined as the unweighted sum of the scores for steatosis (0–3), lobular inflammation (0–3), and ballooning (0–2); thus ranging from 0 to 8. Cases with NAS of 0 to 2 were largely considered not diagnostic of NASH; on the other hand, most cases with scores of 5 or greater were diagnosed as NASH. Cases with activity scores of 3 and 4 were considered as borderline (probable) NASH [35].

The two panel markers for the prediction of activity and fibrosis were the same as those validated in patients with chronic hepatitis C [28], B [25, 29] and alcoholic liver disease [30]: 1) FibroTest (FT) includes total bilirubin, GGT, α₂-macroglobulin, apolipoprotein A1, and haptoglobin, corrected for age and gender and is designed for a quantitative assessment of fibrosis; and 2) Actitest which includes ALT in addition to the above specified markers and is designed for a quantitative assessment of histological activity in chronic viral hepatitis. Values of FibroTest and Actitest range from zero to 1.00 with higher values indicating a greater probability of significant lesions.

AST, ALT, GGT, cholesterol, triglycerides, uric acid and total bilirubin were measured by autoanalyzer Hitachi 917 Automate (Mannheim, Germany) and Roche Diagnostics reagents (Mannheim, Germany). α₂-macroglobulin, apolipoprotein A1, and haptoglobin were measured using an automatic nephelometer (BNII, Dade Behring; Marburg, Germany). Insulin was measured by autoanalyzer Axsym (Abbott, Irwin Texas, USA) and C-peptide by autoanalyzer IMMULIT (DPC, Los Ageles California, USA). HDL and LDL cholesterol were measured by autoanalyzer Kone (Thermo, Vantaa, Finland). The laboratory followed the recommended and validated procedures to insure reproducibility between FT components [22, 23]. All the biochemical components have been prospectively assessed and assays were performed on fresh serum.

The primary outcome was advanced fibrosis (F2, F3 and F4). In a secondary analysis, patients were classified according to the presence of severe fibrosis or cirrhosis (F3F4). Sensitivity analysis compared patients without or with moderate alcohol consumption, patients with elevated or not baseline ALT, patients without high risk of FibroTest failures, patients with baseline biopsy length smaller than 25 mm or greater, with or without fragmented biopsy and patients with paired biopsies.

The cause of discordance between presence of advanced fibrosis predicted by biochemical markers and biopsy (first biopsy if two) was attributed according to respective risk factors of failure as previously detailed [27]. Risk factors of FT failure were hemolysis, Gilbert's disease, acute inflammation and extrahepatic cholestasis. Risk factors of biopsy failure were biopsy size (less than 25 mm) and fragmentation (more than one fragment). Failure attributable to biopsy (false negative) was suspected when the biopsy was smaller than 15 mm and fragmented and without risk failure of FT [27].

Statistical analysis used Fisher's exact test, the chi-square test, Student's t test, the Mann-Whitney test and variance analysis using the Bonferroni all-pair wise and Tukey-Kramer multiple-comparison tests to take into account the multiple comparisons and multiple logistic regression for multivariate analysis. The diagnostic values of the markers were assessed using sensitivities, specificities, positive (PPV) and negative predictive values (NPV), and the areas under the receiver operating characteristic curves (AUROC). AUROC curves were calculated including FT quantitative values using empirical non-parametric method according to Delong et al [36] and compared using the method of Zhou et al [37]. For all analyses, two-sided statistical tests were used; a P-value of 0.05 or less was considered significant. Number Cruncher Statistical Systems 2003 software (NCSS, Kaysville, Utah, USA) was used for all analyses.

A total of 170 patients were included in Group 1 and 97 in Group 2. Characteristics of patients included in Group 1 and Group 2 as well as those of the non-included groups were similar (Table 1). The only significant differences observed between Group 1 and Group 2 was for Group 2 younger mean age, less metabolic factors, more fibrosis stage 1, more severe steatosis and more NASH than in Group 1. The biopsy size in Group 1 was longer with more portal tracts than in Group 2. One case in Group 1 had a severe adverse event of biopsy with gallbladder perforation. The histological diagnosis was made during the surgical operation.

Among 267 included patients, 24 (9%) patients declared "at risk" daily alcohol consumption, between 30 g and 40 g for 17 males and between 20 g and 25 g for 7 females.

When compared to patients with no or mild fibrosis (F0-F1), those with advanced fibrosis (F2-F4) were older, without difference for gender (Table 2). Mean levels of α₂-macroglobulin, total bilirubin and FT were higher and apoA1 lower. By multivariate analysis, age (P < 0.0001), α₂-macroglobulin (P < 0.0001), GGT (P = 0.01), bilirubin (P = 0.02) and apoA1 (P = 0.048) were independently predictive of advanced fibrosis (model r², 0.38; P < 0.0001).

ROC curves of FT for predicting fibrosis are illustrated in Figure 1. For the primary outcome, the detection of F2 to F4 fibrosis, the area under the ROC curve was 0.86 ± 0.04. For the detection of F3-F4, the area under the ROC curve was 0.92 ± 0.04.

The distribution of FT according to the stage of fibrosis is illustrated in Figure 2. In 954 blood donors the median FT value was 0.085 (95% CI: 0.082–0.090). The median in 77 patients without fibrosis (F0) was 0.18 (0.14–0.21); in 53 patients with mild fibrosis (F1), 0.23 (0.16–0.26); in 20 patients with moderate fibrosis (F2), 0.36 (0.28–0.60); in 11 patients with bridging fibrosis (F3), 0.58 (0.45–0.80); and in 9 patients with cirrhosis (F4), 0.63 (0.52–0.91). All differences were significant (P < 0.05) except between F0 and F1 and between F3 and F4. Among blood donors the security algorithms permitted to exclude 29 (3%) patients with high risk of false positive or negative. Among the remaining 925 controls 24 (2.6%) patients had FT between 0.30 and 0.48 and none above.

Diagnostic values of FT for predicting Fibrosis in the two groups are given in Table 3 and for the overall population in Table 4. An FT score of 0.30 had 77% sensitivity and 90% negative predictive value for advanced fibrosis. An FT score of 0.70 had 98% specificity and 76% positive predictive value. FT was highly sensitive for the detection of bridging fibrosis or cirrhosis (F3F4): an FT equal to or higher than 0.30, had 92% sensitivity and 98% negative predictive value for F3F4.

In group 1, There was a clinically significant (2 stages or more) discordance in 17 patients (10%): 5% due to FT failure (9/170), 4% due to biopsy failure (6/170) and indeterminate in 1% (2/170). For 7 out of these 17 patients, the fibrosis stage estimated by biopsy was higher than the one estimated by FT. In one case the cause of failure was certainly attributable to FT since a well known cause of false negative was present: a case with an acute inflammation (urinary sepsis) and high haptoglobin. There was a possible cause of FT false negative in three cases with highly elevated ApoA1. In three cases there was a possible cause of failure of biopsy (poor quality: 7 mm with 3 fragments, 13 mm with 2 fragments and 21 mm with 3 fragments, respectively) with no known cause of false negative of FT. For the remaining 10 patients the fibrosis stage was two stages lower than estimated by FT. In four cases there was a certain cause of failure of FT namely Gilbert syndrome. In four cases there was a possible false negative for liver biopsy in the absence of a cause of false positive FT values: a case (25 mm, 2 fragments) with a low platelet count of 130,000/mm³ and gastric varices at endoscopy; a case with a biopsy of 20 mm (1 fragment) and a low platelet count of 143,000/mm³; a case with a biopsy of 15 mm (5 fragments) and a low platelet count of 146,000/mm³; another with a biopsy of 25 mm and 2 fragments. Two cases had no attributable cause of failure: a good quality biopsy (20 mm, one fragment for one and 30 mm, one fragment for the other) with no risk factors for false positive FT values.

In group 2 there was a clinically significant (2 stages or more) discordance in 10 patients (10%): 2% due to FT failure (2/97), 2% due to biopsy failure (2/97) and indeterminate in 6% (6/97). For 9 out of these 10 patients, the fibrosis stage estimated by biopsy was higher than the one estimated by FT. In two cases the cause of failure was attributable to FT with inflammation (CRP elevated) and high haptoglobin. In one case there was a possible cause of failure of biopsy, a possible false positive with poor quality biopsy 10 mm with 3 fragments and no known cause of false negative of FT. In one case the fibrosis stage estimated by FT was higher than the one estimated by biopsy. The biopsy was small 10 mm and platelet count was low (120.000), without cause of false positive FT and therefore a false negative of the biopsy is possible.

FT had no significant diagnostic value for NASH (AUROC = 0.59 se = 0.06; p = 0.15). ActiTest had a statistically significant (AUROC = 0.62 se = 0.06; p = 0.04) but very weak diagnostic value for NASH.

All patients included with histological diagnosis of NASH (borderline or NASH) had steatosis. A total of 75 patients (44%) had steatosis without NASH (borderline or NASH) in group 1 and 29 (30%) in group 2. Advanced fibrosis (F2F3F4) was more frequent in patients with NASH than in those with steatosis alone: 30/95 (32%) and 30/68 (44%) in patients with NASH, versus 10/75 (13%; P = 0.005) and 1/29 (3%;P < 0.0001) in patients with steatosis alone, in group 1 and 2 respectively.

Sensitivity analyses revealed that the FT AUROCs for the diagnosis of advanced fibrosis were not affected by ALT values (Table 5). ALT was not significantly higher in patients with advanced fibrosis than in those with early fibrosis (Table 2). Seventeen patients out of 40 (43%) with advanced fibrosis had ALT lower than 50 IU/L. AUROCS were higher but not significantly in patients with a biopsy size greater than 25 mm, and after exclusion of patients with Gilbert syndrome and acute inflammation (Table 5). When the 24 patients who drank at least 20 g alcohol/day in women, and 30 g/day in men have been excluded, the FT diagnostic value of the FibroTest was even higher (not significantly) with an AUROC for F2F3F4 = 0.82 (95%CI 0.74–0.87) in the remaining 243 patients (Table 5).

A total of 47 patients had 2 liver biopsy samples collected on the same day. Fibrosis staging was concordant in 27 (57%) and discordant in 20 (43%) out of the 47 patients. The discordance was 3 stages in one case, with bridging fibrosis in one sample (F3) and no fibrosis (F0) in the other; FT was 0.40 that is F1-F2 in the conversion system. In 2 cases the discordance was of 2 stages: in one case F4 versus F2 with a FT = 0.60 (F3); one case with F3 versus F1 with a FT = 0.61 (F3). In the remaining 17 cases the discordances were of one stage. The AUROC of FT was slightly higher but not significantly when the mean between the two biopsies stages performed in the same patient was taken as the endpoint (Table 5).

As expected ApoA1 was highly correlated with HDL cholesterol and total cholesterol, and negatively correlated with triglycerides. A2M was highly correlated with insulinemia and C-peptide. GGT was associated with total cholesterol and LDL-cholesterol but not with triglycerides. FT was associated with insulinemia and C-peptide. There was no correlation between FT and its components with glucose and uricemia (data not shown).