Overrange dilution for improvement of hepatitis B core related antigen as a biomarker: protocol validation and examples for application

Abstract

Background: Hepatitis B core related antigen (HBcrAg) measurement predicts treatment outcomes and reflects intrahepatic HBV replication. The commercially available automated assay for HBcrAg has a linear range of 3.0 - 7.0 logU/mL, with higher levels requiring dilution. However, using different diluents across studies may impact comparability and cross-reactivity, which has not been thoroughly investigated. This study aims to validate a dilution method for specimens above the upper limit of quantification (7.0 logU/mL) to improve comparability.

Methods: The dilution procedure was two-site tested with three matrices for practicability, accuracy and repeatability using samples from HBV-infected patients with high HBcrAg levels. Samples were tested undiluted or diluted when overrange using Fujirebio's specific dilution reagent (SD1) with reflex testing of pre-treated samples, or manually diluted with fetal calf serum (FCS) or human serum (HS) of samples before restarting pre-treatment. Overrange dilution was further validated in three patient cohorts: untreated HBV-infected patients (n = 157) and patients treated with nucleos(t)ide analogues (NA, n = 19), or pegylated interferon-2alpha (PEG-IFN, n = 80) RESULTS: On-board dilution with SD1 showed higher background signals compared to HS or FCS. The dilution process was reproducible across sites, but SD1 underestimated HBcrAg levels. Dilution with FCS showed an early decrease in HBcrAg levels in patients with HBeAg SC during NA treatment (after 3 months, p = 0.022) and PEG-IFN treatment, whereas no change in HBcrAg levels was found without overrange dilution.

Conclusion: Validation showed high background and underestimating levels of HBcrAg with SD1, while FCS-based overrange dilution resulted in significant early HBcrAg decreases and better correlation with treatment response.

Keywords: Biomarkers; HBV; HBcrAg.