the " gold standard " for assessing fibrosis , liver biopsy , is recommended prior to the initiation of antiviral therapy ; in addition , it is vital for monitoring fibrosis progression . unfortunately , this procedure is invasive , prone to complications , including hemorrhage and death , and has a high risk of sampling error .
biochemical markers for liver fibrosis ( fibrotest ) and necroinflammatory features ( actitest ) are an alternative to liver biopsy , in patients with chronic hepatitis c . since the first publication , which included a validation period , those tests have been validated in different populations by the same reference laboratory   and by an independent group .
the tests combine five components ( 2-macroglobulin , haptoglobin , apolipoprotein a1 , -glutamyl transpeptidase ( ggt ) , and total bilirubin ) for fibrotest and same plus alanine aminotransferase ( alt ) for actitest .
the aim of this study was to assess the inter - laboratory variability of fibrotest and actitest , including their six serum liver components , in patients with chronic liver disease , and to identify factors associated with that variability .
our concern was to assess whether the analytical methods adapted on the different analyzers were associated with significant variability in fibrotest and/or actitest values .
moreover , we aimed to compare the variability of fibrotest and actitest in relation to the method of expressing enzymatic activity ; in particular , in terms of absolute values or as multiples of the upper limit of normal . since we and others have demonstrated that current definitions of normal values may be inappropriate [ 8 - 10 ]
in routine practice , the definition of the upper limit of normal ( uln ) of alt and ggt varies between laboratories , but is rarely detailed . because numerous medical guidelines make reference to alt and ggt expressed as multiples of the uln ( uln units ) , variations in the definition of normal may have important practical consequences .
the main characteristics of the included patients are outlined in table 1 . according to each patient and laboratory , details of the fibrotest and actitest assays
there was no significant difference between centers for fibrotest using ggt expressed in international units [ mean ( sd ) = 0.57 ( 0.26 ) , range = 0.480.65 , f - ratio = 0.27 , p = 0.27 ] .
for fibrotest using ggt expressed in uln units [ mean ( sd ) = 0.55 ( 0.27 ) , range = 0.450.68 , f - ratio = 1.26 , p = 0.27 ] , there was a significant difference between three centers ( center 5 had higher means values than center 2 and 4 ; p = 0.02 for both comparisons ) .
fibrotest and actitest variability according to laboratories ( centers ) and units of enzymatic expression : international units ( iu ) and upper limit of normal ( unl ) . characteristics of included patients hcv  hepatitis c virus ; hiv  human immunodeficiency virus ; sd  standard deviation .
there was no significant difference between centers for actitest using alt and ggt expressed in international units [ mean ( sd ) = 0.32 ( 0.26 ) , range = 0.380.53 , f - ratio = 1.21 , p = 0.30 ] and for actitest using alt and ggt expressed in uln units [ mean ( sd ) = 0.44 ( 0.27 ) , range = 0.270.43 , f - ratio = 0.81 , p = 0.59 ) .
the details of the liver proteins and total bilirubin assays according to each patient and laboratory are outlined in figure 2 .
there were no significant differences according to testing center for any of these assays ( between centers or versus the reference center ) : ( 2-macroglobulin [ mean ( sd ) = 2.89 ( 1.16 )
g / l , range = 2.693.33 , f - ratio = 0.72 , p = 0.67 ] , haptoglobin [ mean ( sd ) = 0.98 ( 0.58 )
g / l , range = 0.921.03 , f - ratio = 0.07 , p = 0.99 ) , apolipoprotein a1 [ mean ( sd ) = 1.30 ( 0.51 ) g / l , range = 1.161.42 , f - ratio = 1.21 , p = 0.30 ] and bilirubin [ mean ( sd ) = 28.8 ( 66 ) micromol / l , range = 15.851.1 , f - ratio = 0.51 , p = 0.85 ] .
one analyzer ( advia ) gave lower mean apoliprotein a1 levels [ 1.06 ( 0.43 )
g / l ) than the other analyzers [ 1.33 ( 0.52 ) g / l ; p = 0.02 ] .
the details of the alt and ggt assays , according to each patient and laboratory and expressed in international or uln units , are given in figure 3 .
there was no significant difference between centers for alt expressed in international units [ mean ( sd ) = 70 ( 47 ) iu / ml , range = 5786 , f - ratio = 1.30 , p = 0.25 ] .
however , when the assays used pyridoxal phosphate as in the reference center , the mean alt was higher [ 78 ( 50 ) iu / ml ] than assays not using pyridoxal phosphate [ 60 ( 42 ) iu / ml ; p = 0.003 ] .
for alt expressed in uln units [ mean ( sd ) = 48 ( 37 ) , range = 3771 , f - ratio = 1.65 , p = 0.12 ] , there was a significant difference between center 1 and all centers ( p = 0.009 vs center 2 , p = 0.008 vs center 3 , p = 0.04 vs center 4 , p = 0.04 vs center 5 , p = 0.02 vs center 6 , p = 0.03 vs center 7 , p = 0.01 vs center 10 and p = 0.001 vs center 11 ) .
there were no significant differences between centers for ggt expressed in international units [ mean ( sd ) = 130 ( 158 ) iu / ml , range = 5786 , f - ratio = 1.30 , p = 0.25 ] or in uln units [ mean ( sd ) = 109 ( 121 ) iu / ml , range = 78154 , f - ratio = 1.46 , p = 0.17 ] .
however , and despite the use of the same szasz method , one automate ( dade behring rxl ) gave higher ggt mean values [ 165 ( 200 ) iu / ml ] than the others [ 120 ( 143 ) iu / ml ; p = 0.06 ] .
alanine aminotransferase ( alt ) and -glutamyl transpeptidase ( ggt ) variability according to laboratory and units of enzymatic expression : international units ( iu ) and upper limit of normal ( unl ) .
passing - bablok linear regression analyses   of all samples between laboratories and the reference center are summarized in table 2 .
the intercept and slope between the reference center and other laboratories were excellent for the three proteins with only one decrease for apolipoprotein a1 in a single center using the advia analyzer .
for ggt , centers using the rxl analyzer had a higher slope ( greater than 1 ) . passing - bablok analysis between laboratories and reference center ( lab 1 ) for each component concordance rates ( kappa statistics ) among laboratories
are given in table 3 ; all were statistically significant . when ggt and alt were expressed in international units , fibrotest and actitest kappa statistics were all greater than 0.50 with only 0.8% cases ( 3 out of the 384 comparisons ) with a discordance of more than one fibrosis stage .
in contrast , when ggt and alt were expressed in uln units , fibrotest and actitest kappa statistics were lower than 0.50 in 11 comparisons ( out of the 16 comparisons versus the reference laboratory ) with 5% of cases ( 21 out of the 384 comparisons ) with a discordance of more than one fibrosis stage or greater than one activity grade .
concordance rates ( kappa statistics ) of laboratories with reference center ( lab 1 ) , according to the expression of ggt and alt activities
this study showed that the variability of fibrotest and actitest values , among nine different laboratories , was acceptable and without clinical consequences for the prediction of the stage of liver fibrosis or grade of activity .
this finding is important since it confirms that those tests can be routinely computed from the results of the six individual components obtained by non - centralized measurements .
online assessment is available using the website . to guarantee the quality of this assessment , it was necessary to identify the factors associated with the variability of the six components .
this study confirms that the expression of alt and ggt in multiples of the upper limit of reference values should be avoided . despite efforts to standardize enzymatic assay methods , homogeneity of alt results
has not been achieved as attested by external quality controls , and identical limits of reference values can not be defined .
many clinicians believe that expression of the results as multiples of the upper limits of reference ranges can reduce inter - laboratory variability .
our study confirms previously observed results , that this method of expression is , in fact , worse than that using international units both for alt and ggt . in our reference center ,
the reference limit recorded was similar to the described mean value from a recent study   and lower than in the other laboratories .
if actitest was expressed in a standardized way , using the upper limit of each laboratory for ggt and alt , this induced lower concordance rate than actitest using international units . to increase inter - laboratory coherence in the results of enzymatic activities , standardized assays against a reference method should be employed , with calibration of the assay using a commutable enzymatic material .
the values of this calibrator must be assigned by a reference method . for proteins and bilirubin assays
this was anticipated for 2-macroglobulin since the same analyzer was used in all laboratories . although the use of three different analyzers , haptoglobin has the best homogeneity .
in fact , the assays of these two proteins are standardized against the crm 470 reference material .
this reference product is now used in different measurement procedures to attain results numerically the same , whatever the clinical conditions . for apolipoprotein a1 ,
this is due to the use of a different reference material to standardize the assay .
overall , the data from the laboratories were linearly related with the reference center with a slope close to 1 and a non - significant analytical imprecision ; there were few pairs of assays outside the confidence limits and the samples were adequately distributed over the investigated range .
as previously observed , when ordinary linear regression ( in combination with correlation analysis in the passing bablock method ) gave poor estimates , in particular for ggt and alt assays , we found several analytical reasons for the poor performance .
enzymatic measurement with the szasz method ( standardized against the original for ggt ) , and with the standardized method according to the international federation of clinical chemistry ( using pyridoxal phosphate for alt ) , would probably reduce the variability .
because of their predictive values and their reproducibility in different populations , biochemical markers could be used as surrogate markers for liver biopsy both for the initial decision of liver biopsy and for the follow - up of chronic hepatitis c patients . to date ,
liver biopsy has been considered mandatory for the management of patients infected by hepatitis c virus ( hcv ) .
reviews of morbidity and mortality of intercostal liver biopsy observed a mean occurrence of pain in 30 % of patients , 3 out of 1,000 endured severe adverse events , and 3 out of 10,000 died .
even liver biopsy is dependant on the inter- and intra - observer ( pathologist ) differences .
there are also potential problems with liver biopsy sampling variation . in a study with three consecutive samples through a single entry site ,
only 50 % of patients with cirrhosis were scored as cirrhosis on the three samples .
it is therefore possible that biochemical markers such as those described may provide a more accurate ( quantitative and reproducible ) picture of fibrogenic events occurring within the liver . furthermore , and because treatment is now so effective in patients with genotype 2 or 3 infection , the utility of biopsy in this setting could be challenged .
when ggt and alt are expressed in international units , fibrotest and actitest can be computed from different laboratories with acceptable variability . to increase inter - laboratory coherency , standardized methods and enzymatic calibration
expression of alt and ggt in multiples of the upper limit of reference values should be avoided .
the serum of 24 informed patients ( 21 with chronic hepatitis c and 3 with decompensated alcoholic cirrhosis ) were prospectively collected in the department of hepato - gastroenterology of the piti - salptrire hospital , in paris , france .
sera were separated in the above reference laboratory , conserved at + 4c and distributed to ten different laboratories , in france , within 24 hours . for two laboratories ,
serum was missing for at least one patient ; therefore , these laboratories have been excluded from the core analysis .
sensitivity analyses including these two excluded laboratories did not change the results or conclusions ( data not shown ) .
characteristics of the analyzer , reagents and analytical methods employed used in the nine included laboratories are detailed in table 4 .
eleven different analyzers were used . for the measurement of alt activity , five laboratories used a standardized method according to the ifcc , with pyridoxal phosphate , and four without pyridoxal phosphate . for the measurement of ggt activity ,
the nine laboratories used the szasz method ; including in four a recommended method of standardization .
laboratory analyzers and biochemical methods bn2 , rxl , vitros 250 : dade behring , marburg , germany .
immage , cx5 , cx7 : beckman coulter , brea , california , usa . advia 1650 : bayer diagnostics , tarrytown , new jersey , usa .
analytical measurements of 2-macroglobulin and haptoglobin were standardized against the certified international reference material 470 ( crm 470 ) .
apolipoprotein a1 assays adapted on the different analyzers were standardized against the reference material of world health organization - international federation of clinical chemistry sp1 - 01 ( who - ifcc sp1 - 01 ) , except on the advia - bayer - analyzer ( advia ) .
total bilirubin was assayed by diazoreactions methods . statistical analysis used multiple measure variance analyses and passing - bablok linear regression analyses for the comparison of inter - laboratory results , and kappa statistics for the predicted histological features .
multiple comparisons used bonferroni ( versus control ) and tukey - kramer multiple - comparison tests .
number cruncher statistical systems software was used . the linear relationship between laboratories and reference center
were assessed with confidence limits for the slope and the intercept and the number of pairs out of bounds ; they were used to determine whether there was only a chance difference between the slope and 1 and between the intercept and 0 .
ph , fib , dm , ga , db , pcl , gd , dd , tk , ms , dt and et performed the assays .
 backgroundbiochemical markers for liver fibrosis ( fibrotest ) and necroinflammatory features ( actitest ) are an alternative to liver biopsy in patients with chronic hepatitis c. our aim was to assess the inter - laboratory variability of these tests , and their 6 components ( -glutamyl transpeptidase , alanine aminotransferase , 2-macroglobulin , haptoglobin , apolipoprotein a1 , and total bilirubin ) and to identify factors associated with this variability.resultsserum of 24 patients with chronic hepatitis c or severe alcoholic liver disease were prospectively recorded and analyzed in one reference center and in 8 additional laboratories . 
 when -glutamyl transpeptidase and alanine aminotransferase were expressed in international units , there was no significant difference between laboratories in the results of fibrotest or actitest ; kappa statistics were greater than 0.50 with only 0.8% of cases ( 3/384 ) with a discordance of more than one stage . 
 the main factor significantly associated with variability was the expression of -glutamyl transpeptidase and alanine aminotransferase , as multiples of upper limit of reference values . 
 the use of standardized method with pyridoxal phosphate reduced the variability of alanine aminotransferase expression , and standardized original szasz method reduced the variability of -glutamyl transpeptidase expression.conclusionsthe variability of fibrotest and actitest was acceptable without clinical consequences for the prediction of the stage of liver fibrosis and grade of activity . 
 standardized methods and assay calibration should be used and expression of alanine aminotransferase and -glutamyl transpeptidase in multiples of the upper limit of reference values should not be employed . 