Pfefferkorn Maria, Brehm Jessica, Brehm Martin, Honshoven Fanny, Deichsel Danilo, Vernoux Laura, Pavlovic Vedran, Wat Cynthia, Berg Thomas, van Bömmel Florian
Division of Hepatology, Department of Medicine II, Leipzig University Medical Center, Leipzig, Germany.
MVZ Laboratory Dessau, Dessau, Germany.
Virology. 2025 Aug;609:110576. doi: 10.1016/j.virol.2025.110576. Epub 2025 May 14.
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.
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.
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.
乙肝核心相关抗原（HBcrAg）检测可预测治疗结果并反映肝内乙肝病毒复制情况。市售的HBcrAg自动化检测方法线性范围为3.0 - 7.0 logU/mL，更高水平则需稀释。然而，不同研究使用不同稀释剂可能会影响可比性和交叉反应性，对此尚未进行充分研究。本研究旨在验证针对高于定量上限（7.0 logU/mL）的标本的稀释方法，以提高可比性。
使用来自高HBcrAg水平的乙肝感染患者的样本，对稀释程序进行双中心测试，针对实用性、准确性和重复性测试三种基质。样本不稀释或超出范围时使用富士瑞必欧的特定稀释试剂（SD1）进行稀释，并对预处理样本进行复检，或在重新开始预处理前用胎牛血清（FCS）或人血清（HS）手动稀释样本。在三个患者队列中进一步验证超出范围的稀释：未治疗的乙肝感染患者（n = 157）以及接受核苷（酸）类似物（NA，n = 19）或聚乙二醇化干扰素-2α（PEG-IFN，n = 80）治疗的患者。结果：与HS或FCS相比，使用SD1进行板上稀释显示出更高的背景信号。稀释过程在各中心之间具有可重复性，但SD1会低估HBcrAg水平。在NA治疗（3个月后，p = 0.022）和PEG-IFN治疗期间，用FCS稀释显示HBeAg血清学转换患者的HBcrAg水平早期下降，而未进行超出范围稀释时未发现HBcrAg水平变化。
验证表明，使用SD1时背景高且HBcrAg水平被低估，而基于FCS的超出范围稀释导致HBcrAg早期显著下降，且与治疗反应的相关性更好。
