Abstract: Objective To investigate the true positive rate of hepatitis B surface antigen (HBsAg) enzyme-linked immunosorbent assay (ELISA) samples within the gray zone, and to propose a standardized strategy for processing such results in blood banks and transfusion services nationwide. Methods A retrospective analysis of HBsAg ELISA gray zone samples from the past 5 years was conducted. For samples from the past 6 months, supplementary testing was done using two additional HBsAg ELISA reagents, anti-HBc ELISA, anti-HBs ELISA, and quantitative nucleic acid testing. In cases with limited plasma volume, the extracted plasma volume for nucleic acid testing was increased. Results The gray zone rates of four HBsAg ELISA reagents were compared. Reagent 3 exhibited a significantly higher gray zone rate (1.56‰) than the others. Intermittent significant differences were noted in performance between different laboratory equipment and reagent batch numbers. Additionally, donor-specific factors and specimen characteristics were correlated with the gray area results. The positivity rate of nucleic acid in HBsAg gray zone samples was significantly higher than the total positivity rate of nucleic acid in routine ELISA-negative samples, with 10% of gray zone samples showing positive anti-HBc nucleic acid results. Quantitative viral nucleic acid testing revealed viral loads exceeding the minimum detection limit for hepatitis B virus in blood screening reagents. For 7 plasma bag samples with positive anti-HBc but negative results in routine nucleic acid screening, additional nucleic acid testing by increasing plasma volume showed all results to be negative. Conclusion Blood banks and transfusion services can eliminate the HBsAg ELISA gray zone on the premise of using nucleic acid detection systems with high sensitivity. Alternatively, incorporating anti-HBc testing for HBsAg gray zone samples may serve as an effective strategy to rule out false reactivity, thereby supporting the conservation of valuable blood resources.

Key words: Blood screening, HBsAg testing, ELISA gray zone, Anti-HBc testing, Nucleic acid testing, Blood donor reserve