This is an author version of the contribution published on:
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[J Clin Virol, 60(4), 2014, 10.1016/j.jcv.2014.05.013.]
ovvero [Burdino E, Ruggiero T, Proietti A, Milia MG, Olivero A, Caviglia GP, Marietti M,
Rizzetto M, Smedile A, Ghisetti V., 60, Elsevier, 2014, pagg.341-346]
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Quantification of hepatitis B surface antigen with the novel DiaSorinLIAISON XL Murex HBsAg Quant: Correlation with the ARCHITECTquantitative assays
Elisa Burdinoa, Tina Ruggieroa, Alex Proiettia, Maria Grazia Miliaa,Antonella Oliverob, Gian Paolo
Cavigliab, Milena Mariettib, Mario Rizzettob,Antonina Smedileb, Valeria Ghisettia,∗ aLaboratory of Microbiology and Virology, Amedeo di Savoia Hospital, Torino, Italy
bDivision of Gastroenterology and Hepatology, Department of Medical Sciences, University of Torino,
Torino, Italy
Abstract
Background: Recent technologic innovations allow for quantitative assessment of hepatitis B surface anti-gen (HBsAg) levels in serum; this has been used to monitor the course of chronic HBV hepatitis (CHB) andpredict treatment response. LIAISON-XL Murex HBsAg Quant assay (DiaSorin, Saluggia, I) is the newest immunoassay CE approved to quantify HBsAg. Objectives: To compare LIAISON-XL performances with ARCHITECT-QT HBsAg (Abbott Diagnostics, IL,USA), as reference test. Study design: Sequential serum samples (n = 152) from 14 HBe-negative patients with CHB, the majority of them infected by HBV genotype D undergoing antiviral treatment, were retrospectively tested with both assays. The 2nd WHO Standard 00/588 for HBsAg was used as reference. Results: LIAISON-XL and ARCHITECT-QT correlated by r = 0.95, p < 0.0001; by Bland–Altman analysis agreement of mean difference was 0.21 ± 0.15 log10IU/mL, 95% CI: −0.07 to 0.5). Performance of LIAISON-XL against the 2nd WHO Standard was r = 0.998, p < 0.0001 (95% CI: 0.993–0.999) with results nearer to the expected WHO values compared to ARCHITECT-QT. Median baseline HBsAg level was similar with the two methods before antiviral treatment, throughout fluctuations of HBsAg level in treatment non-responders and during the decrease of HBsAg titer in treatment responders. Correlation between HBsAg levels and HBV DNA was statistically significant for both the two immunoassays (LIAISON-XL: r = 0.4988, 95% CI:0.3452–0.6264, p < 0.0001; ARCHITECT-QT: r = 0.480, 95% CI: 0.3233– 0.6111, p < 0.0001).Conclusions: Correlation between HBsAg measurement with LIAISON-XL and ARCHITECT-QT was high. LIAISON-XL accurately quantified HBsAg in clinical samples at baseline or during antiviral therapy; it can be applied for HBsAg quantification in clinical practice and decision making in CHB.
1. Background
There is renewed interest in measuring serum levels of the hep-atitis B virus (HBV) surface antigen (HBsAg) as a surrogate markerto monitor the natural history of chronic HBV infection and predict the efficacy of therapy [1–5].The level of HBsAg correlates with the amount of covalently closed
intrahepatic circular (ccc)DNA of HBV, this representing the template of HBV gene transcription [6,7]. The production of HBV proteins runs through a molecular pathway separate from HBVDNA replication. HBsAg synthesis derives from the translation of two sub genomic specific RNAs that are distinct from the pregenomic RNA; this mechanism is consistent with the hypothesis that serum HBsAg is a marker for the transcriptional activity of the cccDNA [8]. In the course of chronic HBV infection, HBsAg serum levels decline progressively from the immune-tolerant to the low replicative phase; in clinical practice,
spontaneous or treatment induced clearance of HBsAg is associated with an improved clinical outcome, longer survival and reduced incidence of cirrhosis and hepatocellular carcinoma. It has been shown that the combination of HBsAg quantification with HBV DNA load is useful to distinguish specific virologic conditions such as the inactive HBV carrier (HBeAg-negative, HBV DNA <2000 IU/mL, normal ALT) fromthe carrier of active disease [3].In HBeAg-positive patients, HBsAg levels correlate well withthe amount of intrahepatic cccDNA, thus reflecting the burden of HBV infection; however the correlation is poor in HBeAg-negative subjects [9,10], who represent a distinctive virological and immunological categories [9–12]. EASL guidelines for HBeAg-positive HBV infection have focus on the decline of HBsAg titer<1,500 IU/mL at week 12 of PEG-IFN treatment as a strong predictor of anti-HBe
seroconversion, while HBsAg levels >20,000 IU/mL or no decline at week 12 or 24 are associated with treatment failure and stopping PEG-IFN therapy could be considered in these patients [13]. In HBeAg-negative patients, no decline of HBsAg levels by week 12 is a strong predictor of no response to PEG-IFN and alternative treatment should be considered [10,12,14]. Based on these findings, there is an increasing interest in the role of quantitative HBsAg measurement in the management of CHB and the need for accurate, simple, standardized and widely available quantitative HBsAg assays to ensure optimal clinical decision making is becoming very important. A new pipeline of immunoassay for the quantification of HBsAg has been recently developed [15]. At present, three diagnostics assays are available for HBsAg quantification: the ARCHITECT HBsAg QT (ARCHITECT-QT, Abbott Diagnostics, Abbott Park, IL, USA)[16], the Elecsys HBsAg II Quant (Roche Diagnostics, Indianapolis, IN, USA) [11,17,18] and the LIAISON-XL Murex HBsAg Quant(LIAISON-XL, DIASORIN, Saluggia, Italy), the latter approved for CE marketing in 2011 (Table 1). The ARCHITECT-QT assay has been the first one to appear on the market for HBsAg quantification and is the most frequently cited in published studies. Consensus stud-ies have showed a good correlation between HBsAg measurements with ARCHITECT-QT and Elecsys. The LIAISON-XL immunoassay is a new CE approved, fully integrated chemiluminescence system for the detection of HBsAg in the setting of blood transfusion; it is also useful to quantify HBsAg level for clinical purposes using on board dilution. The assay is standardized against the 2nd World Health
Organization (WHO) International Standard (analytical sensitivity:0.03 IU/mL, dynamic range from 0.03 to 150 IU/mL). The design of LIAISON-XL is based on a panel of murine monoclonal antibodies(Mabs) capable of binding the unfolded antigen in the presence of a high concentration of detergents. These
Mabs are directed toward the classical “a” loop region as well as to highly conserved domains derived from both the internal hydrophilic loop and the transmembrane regions. At present, only few data are available on the performance of the LIAISON-XL [19].
2. Objectives
Aim of the present study was to evaluate LIAISON-XL and com-pare the performance of the new system with the first licensed quantitative ARCHITECT-QT immunoassay, as reference. We deter-mined the correlation between the two assays for the accurate measure of the concentration of the HBsAg in sequential serum specimens from patients with CHB undergoing antiviral therapy. 3. Study design
3.1. Study population and specimens
Sequential serum samples (n = 152) were collected from 14patients with chronic HBe-negative hepatitis B, 12 infected by HBV genotype D (129 serum samples, 84.8%), one with HBV genotype A (13 samples, 8.5%) and one with HBV genotype B (10 samples,6.7%), who were followed at the Department of Gastroenterology and Hepatology, University of Turin. Twelve patients underwent antiviral treatment (3 with Lamivudine, 5 with Entecavir and 4with PEG-INF). Serum samples were extracted from the laboratory serum bank, thawed, and subsequently analyzed in singlicate for HBsAg levels using both LIAISON-XL and ARCHITECT-QT. The 2nd HBsAg WHO Standard subtype adw2, genotype A 00/588 was used to assess assay performances. Serial dilutions of this standard within a range of 0.041–10 IU/mL were analyzed in triplicates.
3.2. Immunoassay for HBsAg serum quantification
The LIAISON-XL for the quantitative determination of HBsAg is a direct two-step
chemiluminescence-based immunoassay (CLIA).The assay employs a set of monoclonal antibodies directed against highly conserved epitopes of the superficial antigen’s internal region. A mixture of mouse Mabs is used for coating magnetic particles and a different mixture of mouse Mabs directed to different epitopes is linked to an isoluminol derivative. During the first incubation, the HBsAg present in samples, calibrators or controls binds to the solid phase. During the second incubation, the conjugate reacts with the HBsAg bound to the solid phase. Subsequently, a flash chemiluminescence reaction is induced. The light signal and hence the amount of isoluminol-antibody conjugate, is measured as relative light units (RLU) and is directly proportional to HBsAg concentration. The LIAISON-XL allows the user to select on board dilution for specimens containing HBsAg concentrations above the assay range. These samples can be automatically diluted using the specific sample diluent loaded in the ancillary reagent area of the instrument. The recommended dilution factor is 1:400. The assay
quantitation range is set from 0.03 to 150 IU/mL and is standardized against the 2nd WHO HBsAg International Standard (subtypeadw2, genotype A; code number 00/588).The ARCHITECT-QT is a two-step fully automated chemiluminescent microparticle immunoassay for the quantitative
determination of human serum and plasma HBsAg concentrations using acridinium-labeled anti-HBs conjugate. The ARCHITECT-QT has a dynamic range of 0.05–250.0 IU/mL with onboard dilution for
results outside of the range. Standardized calibration of ARCHITECT-QT has been obtained with the 1st WHO International Standard for HBsAg (subtype adw2, genotype A; code number80/549).
3.3. Molecular assays for HBV DNA testing
A fully automated real-time polymerase chain reaction system was used to measure HBV DNA levels during patient follow-up: the COBAS AmpliPrep/COBAS TaqMan HBV test, version 2.0
(RocheMolecular Diagnostics, Branchburg, NJ, USA) whose detection limit is 20 IU/mL. 3.4. Statistical analysis
Correlation study and Bland–Altman analyses were evaluated with the GraphPad Prism software version 4.0. Bland–Altman analyses was conducted after transforming the results of the two assays to log10IU/mL. A p value of <0.05 was considered statistically significant.
4. Results
Correlation between LIAISON-XL and ARCHITECT-QT was excellent (r = 0.9508, 95% CI: 0.932– 0.964, p < 0.0001) (Fig. 1a). Bland–Altman analysis revealed a close agreement between the two assays and mean quantitation difference was0.21 ± 0.15 log10IU/mL (95% CI: −0.07 to 0.5 log10IU/mL) (Fig. 1b).In 59% of paired samples the difference between the two assays was ≤0.25 log10IU/mL, while in 74% ≤0.3 log10IU/mL, in 86.8% ≤0.4 log10IU/mL and in 98% ≤0.5
log10IU/mL. Four sample sshowed a quantitation difference above ±2 SD (N = 4) and were re-tested on both platform; observed results in retested samples were highly correlated with the original results. Three samples whose HBsAg diverted ≥0.5 log10IU/mL underwent retesting, but differences between first and second measurements were within ±0.02 log10IU/mL with both assays. High divergence was not related to HBV genotype.
Overall mean level of HBsAg as detected by LIAISON-XL and ARCHITECT-QT in the study-series was similar: 3.54 log10IU/mL(3,498 IU/mL) and 3.74 log10IU/mL (5,512 IU/mL), respectively(range 0.04–54,916 IU/mL). Distribution of HBsAg concentrations with ARCHITEC-QT and LIAISON-XL is shown in Fig. 2: 12.5% of samples measured ≤1 log10IU/mL with both the two immunoassays, 4% and 6.5% tested ≤2 logs, 13.8% and 18.5% ≤3 logs, 49.3%and 59.3% ≤4 logs and 20.4% and 3.2% ≤5 logs with the two assays, respectively, constantly giving ARCHITEC-QT higher values than LIAISON-XL across the entire series of samples. Dilution experiments with nine different concentrations ofthe 2nd WHO Standard from 10 to 0.041 IU/mL showed excel-lent concordance to the expected values with both the systems(LIAISON-XL: r = 0.998, p < 0.0001, 95% CI: 0.993–0.999; ARCHITEC-QT: r = 0.996, p < 0.0001, 95% CI: 0.981–0.999). LIAISON-XL results were nearer to the expected WHO Standards when compared to the results of ARCHITECT-QT for all the standard concentrations (Fig. 3),even if not statistically significant (Mann–Whitney test p = 0.077).With LIAISON-XL, in 83.5% of samples the final result was given with a single analysis because they had a final level which was measurable within the initial onboard dilution (1:400). In 16.5% of samples, the first measurement was below the detection range, and re-analysis of undiluted serum was required.
In patients undergoing antiviral treatment median base-line HBsAg levels before antiviral treatment was very similar with the two methods: 3.65 and 3.9 log10IU/mL (4,550 and8,003 IU/mL) with LIAISON-XL and ARCHITECT-QT, respectively. At1 year of antiviral treatment, HBsAg decrease from baseline was−0.3 log10IU/mL with ARCHITECT-QT and -0.2 with LIAISON-XL. Fig. 4 shows some representative courses of quantitative HBsAg of different patients to demonstrate the variety of the courses with thet wo assays. Both assays detected a fluctuating course of HBsAg with pronounced decreases over a short time (<1 year) only in patients undergoing PEG-IFN treatment. The correlation between HBsAg levels, as obtained with both the two immunoassays, and HBV DNA in patients undergoing antiviral treatment with different combination of therapy was studied. In HBV DNA positive patients, median HBV DNA and HBsAg levels were, respectively, 3.49 log10copies/mL (IQR2.79–5.35), 3.34 log10IU/mL (IQR 3.21–3.66) with LIAISON-XL and3.65 log10IU/mL (IQR 3.45–3.92) with ARCHITECT-QT. The correlation between HBsAg levels and HBV DNA was statistically significant for both the two immunoassays (LIAISON-XL: r = 0.4988,95% CI: 0.3452– 0.6264, p < 0.0001; ARCHITECT-QT: r = 0.480, 95%CI: 0.3233–0.6111, p < 0.0001).
5. Discussion
The quantitative measurement of HBsAg is becoming a tool in the clinical assessment and management of CHB. The clinical relevance of HBsAg level stems from the apparent correlation with intrahepatic cccDNA levels and influences treatment response. [12].The HBsAg decline may portend an
immunological response independently from HBV DNA suppression; monitoring HBsAg levels provides additional information over the measure of HBV DNA alone and may be used to establish stopping rules for the antiviral treatment of CHB. In particular, in PEG-IFN treated patients,
differences in HBsAg decline are now used to predict the success of therapy and stopping rules based on the level of HBsAg have been proposed to optimize patient management [9,10,12,16–18].A new pipeline of immunoassays for the quantification ofHBsAg has been recently developed; the LIAISON-XL is the most recently CE approved either for screening test in blood transfusion or for HBsAg quantitative measurement in clinical settings. The ARCHITECT-QT assay was the first commercial assay available for HBsAg quantification and represents the historical reference. We compared the newly developed LIAISON-XL against the ARCHITECT-QT, as reference. The LIAISON-XL format differs from the other assays, in particular from ARCHITECT, because it consists of a panel of seven murine Mabs against HBsAg. The new design makes LIAISON-XL highly sensitive and tolerant to HBsAg variants as recently proved [19].We have shown that the two assays provide comparable results and that there is a close agreement between the two platforms, with a mean quantitation difference of 0.21 log10IU/mL and 98% of samples within ±0.5 log IU/mL. The LIAISON-XL values were slightly lower than ARCHITECT-QT, and closer to the WHO 2nd International Standard than those of
ARCHITECT-QT at all the reference standard concentrations. In our study, the quantitation of HBsAg levels in routine clinical samples by LIASON-XL appeared overall to be accurate, with low variability and little discrepancy com-pared with ARCHITEC-QT. Therefore, threshold or prediction rules established for the ARCHITEC-QT platform may be transferred on the LIAISON-XL, without losing predictive accuracy. The kinetics of HBsAg with LIAISON-XL was also similar to ARCHITECT-QT
in patients undergoing antiviral treatment, confirming that LIAISON-XL can be used for HBsAg quantification in clinical practice for the management of patients with CHB.
However, the samples analyzed in our study were obtained from patients mainly infected by HBV genotype D, which is largely predominant in Southern Europe; further studies are required to establish if there is agreement also for other genotypes. A major advantage of LIAISON-XL is its validation also for HBsAg screening in the setting of blood transfusion. ARCHITECT-QT is meant only for the quantitative measurement of HBsAg level in clinical settings, due to a higher sensitivity of its
qualitative HBsAgtest counterpart, the ARCHITECT HBsAg Qualitative II whose analytical sensitivity ranges from 0.017 to 0.022 IU/mL against the WHO2nd International Standard. In conclusion, our study shows a high correlation and agreement between quantitative HBsAg measurement with
ARCHITECT-QT and LIAISON-XL. LIAISON-XL is a reliable test for HBsAg quantitation providing results that are standardized on the WHO 2ndInternational Standard. Onboard dilution has the
advantage of lowering labor costs, turn-around-time and laboratory errors, improving accuracy and precision. LIAISON-XL is therefore suitable for routine clinical use and can be applied for HBsAg quantification in the clinical practice and decision making for patients with chronic hepatitis B.
Funding
Reagents for HBsAg quantitative testing with LIAISON-XL andARCHITECT-QT were kindly provided by DiaSorin (Saluggia, I).
Competing interests None declared.
Ethical approval Not required.
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