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FibroMAX™: towards a new
universal biomarker of
liver disease?
Rachel Morra, Mona Munteanu, Françoise Imbert-Bismut,
Djamila Messous, Vlad Ratziu and Thierry Poynard†

Among the noninvasive alternatives to liver biopsy, several studies have demonstrated the
predictive value and a better benefit-to-risk ratio than biopsy of five combinations of

simple serum biochemical markers (the super combination being FibroMAX™
[BioPredictive, Paris, France) in patients at risk of chronic liver diseases: FibroTest™
What are the unmet needs
of currently available tests?

(BioPredictive) for the quantitative assessment of fibrosis; SteatoTest™ (BioPredictive) for
the quantitative assessment of steatosis; ActiTest™ (BioPredictive) for the quantitative

Which tests are currently
under study?

assessment of necroinflammatory activity in chronic viral hepatitis C and B; NashTest™
(BioPredictive) for the categorical diagnosis of nonalcoholic steatohepatitis; and AshTest™

How the test works
for the quantitative assessment of alcoholic steatohepatitis (also known in the USA as
HCV-FibroSURE™, HBV-FibroSURE™, ASH-FibroSURE™ and NASH-FibroSURE™; LabCorp, NC,
Sensitivity & specificity
USA). The possible causes of false-negative and false-positive results are also better
Clinical profile
identified. These tests, which are now available in 50 countries, can facilitate the screening
Alternative tests
and management of the most frequent liver diseases.
Expert Rev. Mol. Diagn. 7(5), 481–490 (2007) Expert commentary
The consensus conference statements recom- and with a subsequent increase in the number Five-year view
of patients treated [11]. A recent overview by FInancial disclosure
almost all patients with chronic liver diseases French health authorities officially approved Key issues
related to hepatitis C, hepatitis B, alcoholic noninvasive biomarkers FT and elastography fatty liver diease (AFLD) and nonalcoholic (FibroScan®) as first-line estimates of fibrosis References
fatty liver disease (NAFLD), but also underline in patients with chronic hepatitis C, recom- Affiliations
the necessity of developing reliable noninvasive mended reimbursement by social security and tests [1]. Due to the limitations [2–7] and risks of approved liver biopsy only as a second-line biopsy [8], as well as the improvement of the estimate in cases of discordance or noninter- pretability of noninvasive markers [12]. An Salpêtrière, 47-83 Boulevard de biomarkers, numerous studies strongly sug- updated overview is pending for other chronic l’Hôpital, 75651 Paris Cedex 13, gest that liver biopsy should no longer be con- sidered mandatory as a first-line estimate of injury in these most frequent chronic liver USA) was initially used in chronic hepatitis C, and thereafter validated in chronic hepatitis B, Practices are evolving rapidly and a nation- hepatitis-delta, hepatitis/HIV co-infection, and KEYWORDS:
ActiTest™, activity biomarker,
wide survey in France recently found that alcoholic liver disease (ALD) and nonalcoholic liver disease with associated steatosis. Thus, FT fatty liver, FibroMAX™, fibrosis biomarker, FibroSURE™, invasive biomarker (FibroTest™ [FT]–Acti- is a universal marker of liver fibrosis, at least for Test™ [AT], BioPredictive, Paris, France) and the four most frequent chronic liver diseases.
biopsy limit, NashTest™, nonalcoholic fatty liver disease, AT is the only marketed noninvasive biomarker decrease in the use of liver biopsy for more of necroinflammatory activity. It is used only in steatohepatitis biomarker, steatosis biomarker, SteatoTest™ than 50% of patients with chronic hepatitis C, chronic viral hepatitis (B and C) patients. Morra, Munteanu, Imbert-Bismut, Messous, Ratziu & Poynard
Table 1. FibroTest diagnostic values according to the cause of liver disease and the independency of authors.
n Observed area under the ROC curve
Standardized area under the ROC curve
In order to improve this diagnostic tool, worsening factors of fibrosis: liver steatosis (SteatoTest™), nonalcoholic steatohepati- of concomitant calculation of all these fibro- sis-related tests in a single procedure. This and, according to the risk factors, also pro- alcoholic steatosis and AshTest for alcoholic steatosis (ASH-FibroSURE™, LabCorp).
ALD. NashTest is intended only for patients NAFLD Ave
patients with excessive alcohol consumption and ALD and is used to detect the presence of severe alcoholic steatohepatitis. The early AUROC minus 0.50
detection of hepatic fibrosis and its worsen- ing associated factors is essential, becausethere are now effective treatments that can Figure 1. Diagnostic value (AUROC) of FibroTest™ in the most frequent liver disease.
prevent the occurrence of cirrhosis and thus ALD: Alcoholic liver disease; AUROC: Area under the ROC curve; HBV: Hepatitis B virus; HCV: Hepatitis C its complications (liver cancer, digestive virus; NAFLD: Nonalcoholic fatty liver disease.
hemorrhage and hepatic insufficiency).
Expert Rev. Mol. Diagn. 7(5), (2007) FibroMAX™
For the diagnosis of fibrosis, FT has been extensively studied short length) and 2% to FT-AT failure [30], and that the and validated both alone and combined with elastography or 5-year prognostic value of FT was at least equal to that of liver other indexes in various algorithms. Initially, most studies have been performed in hepatitis C (>5000 patients) [9,12], but alsoin hepatitis B (>2000 patients) [13–19], in patients with NAFLD What are the unmet needs of currently available tests?
(>1000 patients) [15,20–23] and 360 patients with ALD [15,24–27].
No available tests have been extensively validated for the diagno- Between 2001 and 2006, FT has been assessed in sis of fibrosis, steatosis and specific activity grades. Different 51 publications. A total of 29 studies have been conducted: combinations of liver tests have been suggested, mostly for fibro- 15 independent from the inventor group, nine by the inventor sis staging and in patients with hepatitis C. No available test has group and five mixed with independent and nonindependent demonstrated a continuous and linear correlation with fibrosis authors (TABLE 1 & FIGURE 1). There was no significant difference stage and steatosis and activity grades in the most frequent in the FT accuracy according to the cause of liver disease or the independency of authors. We demonstrated that most of the Isolated biomarkers have smaller diagnostic value than panels variability of the observed area under the ROC curves of biomarkers [9,32]. Serum alanine aminotransferase (ALT) was (AUROCs: 0.65–0.89) is related to the variability of fibrosis the most commonly investigated marker, with low sensitivity of stage prevalences defining advanced and nonadvanced fibrosis 61–71%. The diagnostic value was lower than the combination [29]. When standardized, the AUROCs were even more similar of markers in all direct comparisons. Among the extracellular matrix tests, hyaluronic acid correlated best with fibrosis stage Very few patients (n = 19) with hepatitis D have been studied overall, but has been demonstrated only for extensive fibrosis.
among HBV studies. In HIV co-infected cases, only two full The AUROCs for extensive fibrosis range from 0.65 to 0.86. Pro- papers have been published and one of these is independent. In collagen type III peptide and tissue inhibitor of metalloproteinase ALD patients, only two of the studies are full papers, none are (TIMP)-1–4 were less predictive than hyaluronic acid [9,32]. independent. In NAFLD patients, only two studies are full Numerous other panels have been elaborated for fibrosis staging. Five panels have been covered in three publications, To date, other diagnostic tests have not been so extensively and not validated by health authorities. studied and validated, particularly in ALD and NAFLD The aspartate aminotransferase (AST)-to-platelet ratio index [9,12,29]. A prospective study in HCV observed that 18% of (APRI) has many publications, but the accuracy is lower than discordances were attributable to biopsy failure (mostly due to Fibrotest™
Figure 2. Notched box plots showing the relationship between FibroTest™ and the stage of fibrosis (A) and between ActiTest™ and the grade of
activity. (A)
FibroTest values according to status, from blood donors to patients with cirrhosis. (B) ActiTest values according to status, from blood donors to
patients with severe necrosis. (B). The horizontal line inside each box represents the median and the width of each box the median ± 1.57 interquartile range/
to assess the 95% level of significance between group medians. Failure of the shaded boxes to overlap signies statistical signicance (p < 0.05). The horizontal lines above and below each box encompass the interquartile range (from 25th to 75th percentile), and the vertical lines from the ends of the box encompass the adjacent values (upper: 75th percentile plus 1.5-times interquartile range; lower: 25th percentile minus 1.5-times interquartile range). Consensus conferences recommend treatment in patients with either F stage or A grade.
A : No activity; A : Minimal activity; A : Moderate; A : Severe; F : No fibrosis; F : Portal fibrosis; F2: Some septa; F : Many septa; F : Cirrhosis. Morra, Munteanu, Imbert-Bismut, Messous, Ratziu & Poynard
Table 2. Conversion between FibroTest™ and fibrosis stages using METAVIR, Knodell and Ishak fibrosis scoring systems.
METAVIR fibrosis stage estimate
Knodell fibrosis stage estimate
Ishak fibrosis stage estimate
One panel of biomarkers combining α2-macroglobulin, in several proteomic studies [43–46]. A combination of proteomic hyaluronic acid and TIMP-1 is also on the US market peaks has been identified with a 7% increase in the AUROC in (FIBRO-spect I and II™, Prometheus, USA) [33–35]. The other tests are FibroMeter [15,36,37], Hepascore [36,37,38], and Forns index [39–41]. Few separate studies have been pub- How the test works
lished in different chronic liver diseases. These panels are A physician prescribes the tests according to the possible liver designed only for fibrosis diagnosis without diagnostic value for disease, the agreed laboratories measures the components, necroinflammatory activity, steatosis and steatohepatitis. Con- enters the results into the BioPredictive network, the algo- trary to FT [9,31], no study has been presented in community- rithms are computed, and the results are provided to the labora- based population, there are no prognostic studies and the risks tory. FT-AT is a noninvasive blood test that combines the of false negatives and positives has not been identified. quantitative results of six serum biochemical markers(α2-macroglobulin, haptoglobin, γ-glutamyl-transpeptidase Which tests are currently under study?
[GGT], total bilirubin, ApoA1 and ALT) with the patient’s age A study using profiles of serum protein N-glycans found that a and gender in a patented artificial intelligence algorithm profile has a similar AUROC to FT for the diagnosis of com- (USPTO 6631330) to generate a measure of fibrosis stage and pensated cirrhosis. When combined with FT, this marker had necroinflammatory grade in the liver.
100% specificity and 75% sensitivity for the diagnosis of com- FT-AT is a continuous linear biochemical assessment of pensated cirrhosis, which was not significantly different from fibrosis stage and necroinflammatory activity grade. It pro- the 92% specificity and 67% sensitivity of FT alone [42]. vides a numerical quantitative estimate of liver fibrosis ranging Proteomic studies are ongoing, but even if the diagnostic value from 0.00 to 1.00, corresponding to the well-established can be significantly greater than reference panels, cost–utility METAVIR scoring system of stages F0–4 and of grades A0–3 studies are mandatory. Components of FT have been identified Table 3. Conversion between ActiTest™ and activity grades using METAVIR, Knodell and Ishak necroinflammatory
activity scoring systems.

METAVIR activity grade estimate
Knodell necrosis estimate
Ishak necrosis estimate
Expert Rev. Mol. Diagn. 7(5), (2007) FibroMAX™
SteatoTest, NashTest and AshTest each combine six to 12 biomarkers in several patented expert algorithms (USPTO Table 4. Conversion between SteatoTest™ and steatosis
6,631,330, 0060172286 and 0060173629). The biomarkers grades in hepatocytes with steatosis.
include the six components of FT-AT (α2-macroglobulin, hap- SteatoTest™
Estimate of steatosis percentage
toglobin, ApoA1, GGT, total bilirubin and ALT), AST, fastingglucose, total cholesterol, triglycerides and the weight, height, The analyses should preferably be made on fresh serum, but can be performed with plasma if necessary (blood sample onlithium heparinate). The measurements of the six parameters are made preferably on fresh serum (or plasma) or serum thathas been stored between 2 and 8°C for a maximum of 4 days inan unlit area (for the protection of bilirubin). For deferred Table 5. Conversion between NashTest™ and nonalcoholic
measurements, the serum should be quickly frozen to -80°C.
steatohepatitis categories in patients with nonalcoholic
After thawing, it should be centrifuged for 10 min at 15,000 g fatty liver disease.
[47,48]. A standardization of the parameters is important becauseof the worldwide distribution of these tests. Laboratories must NashTest™
Estimate of nonalcoholic steatohepatitis
follow a quality chart in order to be allowed to use the Bio- Predictive algorithms [101]. Several works have validated these preanalytical and analytical recommendations in order toreduce inter- and intralaboratory variability [47–48], fasting con- ditions [49], transferability between analyzers [50–52] and enzy-matic calibration [53]. It has been prospectively demonstrated that FT-AT can be Table 6. Conversion between AshTest™ and alcoholic
performed on fasting or nonfasting serum samples [49]. Steato- hepatitis categories in patients with alcoholic liver disease.
Test, NashTest and AshTest must be performed on fastingserum samples. The results are given in a few days.
Estimate of alcoholic steatohepatitis grades
No real cost–effectiveness analysis of FibroMAX has been per- formed. However, because of the efficacy of FT-AT, the compli- cations of liver biopsy and the difference in direct costs, the results of such cost-effectiveness analysie appears trivial. The costof FibroMAX varies from €150 to 500 in different countries, patients 4.89%). Among these patients there were 272 cases versus €1000–2000 for an uncomplicated liver biopsy (TABLE 7).
with high-risk profile of false positives (0.84%) for which theother components were not concordant in favor of significant Sensitivity & specificity
fibrosis. Patients with extremely low haptoglobin, especially A summary of the FT-AT diagnostic values in chronic when other exams were hardly modified, could have had hemo- hepatitis C according to cut-offs is given in TABLE 8 [54]. In all lysis. A high-risk profile of false positives due to possible Gil- chronic liver diseases, the liver biopsy is far from a true gold bert syndrome was observed in 409 (1.26%) cases. The most standard and a high percentage of false negatives and positives frequent cause of abnormally elevated values of bilirubin was could be due to errors of biopsy. A prospective study observed Gilbert disease, and acute sepsis for α2-macroglobulin and that 18% of discordances were attributable to biopsy failure haptoglobin. In the presence of acute inflammation (i.e., sepsis) (mostly due to short length) and 2% to FT-AT failure [30,31].
or of acute hemolysis, FT-AT analysis must be postponed. Therefore, the classical expression of accuracy stating that dis-cordances are always due to failure of the biomarkers is obso- Clinical profile
lete. Classical overviews are interesting [55], but are obsolete if FT-AT has been extensively studied in patients with chronic they do not include the discordances data [30,31] as well as stand- hepatitis C [9,12,54], in more than 2000 patients with ardization according to the prevalence of stages defining hepatitis B [13–19] and less studied in NAFLD [15,20–23] and advanced and nonadvanced fibrosis [28].
ALD [15,24–27]. In these four frequent chronic liver diseases Between September 1, 2002, and May 31, 2004, a total of (HCV, HBV, ALD and NAFLD), similar diagnostic value has 32,527 tests have been carried out on the secured internet site been observed (TABLE 1 & FIGURE 2). Similar diagnostic values [56]. The tests included 55% male and 16% older than 65 years.
have also been observed in preliminary studies of patients The most frequent abnormal value observed during postmar- with portal hypertension [57] and hemochromatosis [58].
keting follow-up was haptoglobin under 0.12 g/l in 1589 Screening strategies using FibroMAX are ongoing. In patients Morra, Munteanu, Imbert-Bismut, Messous, Ratziu & Poynard
Table 7. Summary of advantages and limits of liver biopsy and biochemical markers.
Liver biopsy
Biochemical markers
Fibrosis, activity, steatosis, steatohepatitis, iron, suspected or Fibrosis, activity, steatosis, steatohepatitis unexpected comorbidities, special staining for iron and copper Hemolysis, Gilbert disease, acute hepatitis, extrahepatic cholestasis, acute inflammation Three deaths out of 10,000, three severe adverse events out of 33% discordance in fibrosis staging, 24% discordance in Fibrosis stage discordance (20%), activity grade discordance (40%) Co-efficient variation <10% Coagulation disorder, risk of respiratory insufficiency €1032 for uncomplicated biopsy, €2745 for complicated biopsy €90–300 for Fibrotest™–ActiTest™–FibroSURE™, €150–500 for FibroMAX™ with diabetes and no history of liver disease, a prospective and two patients with portal hypertension [22]. No studies have screening using FibroMAX has identified five patients with cir- been published to date in patients with cholestatic liver diseases rhosis, including four patients with hepatocellular carcinoma Table 8. Integrated database, with predictive values for significant fibrosis according to METAVIR conversion cut-offs
in patients with chronic hepatitis C.

Cut-off used for METAVIR Sensitivity
mean (SE)
stages conversion
predictive value
predictive value
FibroTest™ with blood donors (n = 1570) FibroTest™ without blood donors (1270) Expert Rev. Mol. Diagn. 7(5), (2007) FibroMAX™
Alternative tests
elastography [37]. Elastography is complementary because of Two biomarkers are on the market for the diagnosis of fibrosis; its specificity for severe fibrosis in case of FT high-risk profile hyaluronic acid (worldwide) and a panel combining α2-mac- of false positives or negatives [59].
roglobulin, hyaluronic acid and TIMP-1 (USA only). Fordiagnosis of necroinflammatory activity, only transaminases Conclusion
Several published studies and overviews have demonstrated Studies directly compared FT with hyaluronic acid, Forns the predictive value and better benefit-to-risk ratio than index and APRI in the same patients [9,12,32,54]. FT had biopsy of these combinations of simple serum biochemical higher diagnostic values (the AUROC was significantly markers in patients with the four most frequent liver disease.
higher). In particular, FT was more sensitive for discriminat- They allow a quantitative assessment of both fibrosis and stea- ing between F1 and F2, and more linearly correlated to stages tosis and, according to the cause of liver disease, an assessmentwhen compared with those three other markers. An advan- of the necroinflammatory histological activity. The possible tage of APRI is its availability and low cost. However, a weak- causes of false negatives and positives are also better identified.
ness of APRI is the absence of standardized methods and These tests, which are now available worldwide, can facilitate assay calibration, and thus expression of aminotransferase or the screening and management of fibrotic liver disease, GGT in multiples of the upper limit of reference values including hepatitis C, hepatitis B, AFLD and NAFLD. should not be employed [18]. An additional weakness of theForns index (combining age, platelets, GGT and cholesterol) Expert commentary
is the inclusion of cholesterol, which varies greatly in patients According to its poor benefit-to-risk ratio, liver biopsy should be abandoned as a first-line assessment of liver injury in The FibroScan is another way to perform a histological chronic hepatitis C. For chronic hepatitis B, AFLD and assessment, using elastography. Elastography has been NAFLD, similar results are increasingly observed, but few approved together with FT as a first-line estimate of liver independent validations are still pending. Two noninvasive fibrosis in patients with chronic hepatitis C [9,12]. In patients methods, FT and elastography, have been validated as nonin- with HCV infection, the sensitivity of FT appears higher for vasive methods for the assessment of liver fibrosis, but markers early fibrosis stages, and the combination of FibroScan and of other liver injuries, steatosis and necroinflammatory activ- FT improves the overall diagnostic value [59]. Preliminary ity, were lacking. The new available markers will better help data have been published in other chronic liver diseases and the clinician. These tests must be interpreted in the overall had similar accuracy in chronic hepatitis C [60]. The combi- clinical and biological context. They also have limitations, but nation of different biological tests [9] or the complementary one advantage for the FibroMAX combinations is that the pro- use of FT and elastography [59] could also add value for more files of patients at risk of false positives or negatives are well accurate fibrosis evaluation. The sensitivity of FT appears to identified. The analytical recommendations are also important be an advantage in comparison with other biomarkers [9] and to limit the interlaboratory variability of these combinations.
Key issues
• Numerous studies strongly suggest that, due to the limitations and risks of biopsy, as well as the improvement of the diagnostic accuracy of biochemical markers, liver biopsy should no longer be considered mandatory. • In some countries, noninvasive methods are already approved as first-line assessment of liver fibrosis and are reimbursed.
• Among the noninvasive alternatives to liver biopsy, several studies have demonstrated the predictive value and the better benefit-to-risk ratio than biopsy of combinations of simple serum biochemical markers in patients infected with hepatitis C virus and hepatitis B virus: FibroTest™ for the quantitative assessment of fibrosis; and ActiTest™ for the quantitative assessment of necroinflammatory activity.
• New studies have observed the predictive value of combinations of simple serum markers for the quantitative assessment of steatosis (SteatoTest™), alcoholic (AshTest™) and categorical diagnosis of nonalcoholic (NashTest™) steatohepatitis. • A prospective study observed that 18% of discordances were attributable to biopsy failure (mostly due to small length) and 2% to FibroTest–ActiTest failure (hemolysis, Gilbert syndrome and acute inflammation). The 5-year prognostic value of FibroTest was better than that of liver biopsy.
• These tests, which are now available worldwide, can facilitate the screening and management of the four most frequent chronic liver diseases: chronic hepatitis C and B, and alcoholic and nonalcoholic fatty liver disease.
Morra, Munteanu, Imbert-Bismut, Messous, Ratziu & Poynard
New studies suggest that these tests can facilitate the screen- decrease the risk of false positives and false negatives. Markers ing of the most frequent liver diseases. The development of identified by proteomics or glycomics must be validated in biomarkers derived from proteomics or glycomics is likely to be These biomarkers will replace liver biopsy in patients with chronic liver diseases, not only for routine assessment of liver Five-year view
injury but also as endpoints in clinical trials. Biopsy will be still Our speculative viewpoint in the next 5 years is that panels of useful but as a second line assessment, in very complicated cases.
simple biochemical markers, such as as FT-AT, SteatoTest,NashTest and AshTest, will replace the classical liver tests.
Financial disclosure
Large-scale validation in chronic liver diseases other than Thierry Poynard is the inventor and has a capital interest in hepatitis C are ongoing, including prospective screening in BioPredictive, the company marketing FibroTest, ActiTest, general populations. The combination of these markers with SteatoTest, NashTest and AshTest. Mona Munteanu is a Bio- elastography and new security algorithms will probably References
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Fachausschuss Varizellen der Deutschen Vereinigung zur Bekämpfung der Viruskrankheiten e.V. Druckerei Kempkes, Offset- und Buchdruck GmbH, 35075 GladenbachVarizellen sind eine akute, hochkontagiö-se Viruserkrankung, die weltweit auftritt. Subklinische Infektionen sind selten. Eine tion latent in den spinalen und zentralen Ganglien persistierenden Varicella-Zoster-rone und Satellitenzelle

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