1. FT in diagnostic of HBV Analytical & pre-analytical variability FibroTest and FibroMax

2. FT in diagnostic of HBV In this presentation Pre-analytical variability 1. Fasting versus non-fasting 2. Transport, pre treatment and storage 3. USA specifications for FibroSure (USA trade mark for FibroTest) Analytical variability 4. Inter-laboratory variability 5. Intra-laboratory variability 6. Transferability between analyzers

3. FT in diagnostic of HBV Pre-analytical variability Fasting, Storage, US specifications

4. FT in diagnostic of HBV Fasting versus non fasting Munteanu et al, Comp Hep 2004 Intra-individual fasting versus postprandial variation of biochemical markers of liver fibrosis (FibroTest, FT) and (ActiTest, AT) Patients and methods  64 subjects  Measurement of variation +/- 1 hour from meal Results  No significant impact on FT/AT components  ANOVA >0.09 and coefficient of variation (CV) from 0,09 for FT to 0,13 for AT  Spearman correlation coefficient from 0,87 to 0,995  Significant impact on triglycerides and glucose  ANOVA P=0.01, CV= 0.65 and ANOVA P=0.04, CV=0.31  Concordance between fasting and predicted histological stages and grades  FT: kappa=0.91, AT: kappa=0.80  almost perfect Conclusion For FibroTest: Fasting is not a requirement for to perform FibroTest, which make the screening for patients at risk of liver diseases more convenient For FibroMax: Due metabolic components of the test, fasting is required to perform a valid FibroMax (especially NashTest and SteatoTest

5. FT in diagnostic of HBV Transport, pre treatment and storage - Messous et al, Clinica Chimica 2005 Time impact of serum storage temperature on stability of important liver enzymes and proteins and on FibroTest-ActiTest Conclusions FibroTest  ActiTest results diminish more than fifty percent with -20°C and -35°C storage.  Sera storage at –80°C  Sera storage at –80°C is recommended for retrospective analyses.  To avoid the risk of false negative or false positive results for non-invasive liver markers like FibroTest-ActiTest, we recommend validation studies on fresh sera. ActiTest

6. FT in diagnostic of HBV USA specifications for FibroSure (USA trade mark for FibroTest) FIBROSURE™ 550123 CPT  Pending Synonyms  HCV FIBROSURE™; FIBROSURE™, FibroTest, ActiTest Test Includes  FibroTest (alpha2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, gamma glutamyl transpeptidase [GGT]);  ActiTest (alpha2-macroglobulin, haptoglobin, apolipoprotein A1, bilirubin, gamma glutamyl transpeptidase [GGT], alanin aminotransferases [ALT]) Specimen Serum  Volume 4 mL  Minimum Volume 2 mL  (Note: This volume does not allow for repeat testing.) Container  Red-stopper tube or serum-separator tube Collection  Separate serum and refrigerate at 2°C to 8°C. Specimen is stable for as long as three days. Freeze if  Storage longer than 72 hours is needed. Storage Instructions  Refrigerate –80°C

7. FT in diagnostic of HBV USA specifications for FibroSure (USA trade mark for FibroTest) Reference Interval METAVIR scale Fibrosis stage  F0: no fibrosis  F1: portal fibrosis  F2: bridging fibrosis with few septa  F3: bridging fibrosis with many septa  F4: cirrhosis Necroinflammatory activity grade  A0: no activity  A1: minimal activity  A2: moderate activity  A3: severe activity Use noninvasive measure  of fibrosis and necroinflammatory activity in the liver of individuals with chronic hepatitis C virus Limitations  Because this procedure is new, Medicare and other carriers may not yet recognize it as a covered benefit for patients. Methodology  Patented Artificial Intelligence algorithm combines patient’s age, gender, and the results of six biomarkers to generate a measure of fibrosis and necroinflammatory activity in the liver

8. FT in diagnostic of HBV Analytical variability Fasting, Storage, US specifications

9. FT in diagnostic of HBV Inter-laboratory Variablity Halfon et al, Comp Hep 2002 A prospective assessment of the inter-laboratory variability of biochemical markers of fibrosis (FibroTest) and activity (ActiTest) in patients with chronic liver disease Concordance (kappa coefficient) between laboratories and the reference center. FibroTest calculated with GGT expressed in International UnitActiTest calculated with GGT-ALT, expressed in International Units Conclusions  Standardized methods against a reference method, as well as calibrated analyzers should be used to increase inter-laboratory coherence, particularly for GGT and ALT measurements.  ALT and GGT expressed as multiples of the upper limit of normal should be avoided.

10. FT in diagnostic of HBV Intra-laboratory variabilty - Imbert-Bismut et al, Clin Chem Lab Med 2004 Intra-laboratory analytical variability of biochemical markers of fibrosis (FibroTest) and activity (ActiTest) and reference ranges in healthy blood donors. Total imprecision for FT-AT and its components on pooled sera (NCCLS-EP5-T)  The total imprecision is correct for the different parameters.  A VC of 8.8% for total bilirubin was acceptable considering that this variation occurred for the low mean values (mean 7.5 micromoles/L).

11. FT in diagnostic of HBV Intra-laboratory variabilty - Imbert-Bismut et al, Clin Chem Lab Med 2004 Intra-patient reproducibility of components and of FibroTest-ActiTest  Concordance between two consecutive measurements was strong (kappa=0.88) for cirrhosis (F4) versus non-cirrhosis.  Stages F0/F1, F2 and F3F4 had kappa coefficients of 0.83 and the concordance was moderate for the repeated ActiTests (kappa=0.44)

12. FT in diagnostic of HBV Intra-laboratory variabilty - Imbert-Bismut et al, Clin Chem Lab Med 2004 Conclusions  The reproducibility of two FibroTests in the same patient evaluated four days apart is good despite some isolated variations of the components. This however, has no impact due to the linearization of the results.  The homogeneity of the results could be attributed to the homogeneity of adapted techniques on the different analyzers (standardized techniques compared to reference techniques), as well as the same conditions of calibration of the enzymatic activity measurements.  The study demonstrated the analytical reliability of FibroTest and ActiTest and the importance of the continued development of homogeneity and good transferability of the results between analyzers and laboratories.

13. FT in diagnostic of HBV Transferability between analysers - Imbert-Bismut et al, Ann Biol Clin 2005 Results transferability on RXL, ARX, X-Pand, BN2 (Dade Behring) and modular DP (Roche Diagnostics) analyzers: application to component assays of FibroTest and ActiTest (on 150 serum samples) Methods of measurement adapted on different automates and evaluation methods

14. FT in diagnostic of HBV Transferability between analysers - Imbert-Bismut et al, Ann Biol Clin 2005 Results of inter-platform comparisons of Dade Behring: RXL, ARX, X-Pand and results of BN2-Modular / RXL: regression models according to Passing- Bablok. Conclusions  This study showed harmonious results for each parameter between the Dimension analysers and between RXL and BN2- Modular DP.  Disregarding alpha-2 macroglobulin, which cannot be assayed on RXL, the results of FibroTest and ActiTest were similar to those performed on BN2- Modular DP and RXL.

15. FT in diagnostic of HBV Transferability between analyzers All validated analyzers and reagents are listed in the technical recommendations available on www.oneliver.com in the “For laboratories” section