Motshosi, PatiencePhinius, Bonolo B.Jongman, MosimanegapeBaruti, KaboBhebhe, LynnetteMulenga, GracefulChoga, Wonderful T.Moyo, SikhulileGaseitsiwe, SimaniAnderson, Motswedi2025-12-032025-12-032025-05Motshosi, P., Phinius, B.B., Jongman, M., Baruti, K., Bhebhe, L., Mulenga, G., et al. (2025) Optimization of dried blood spot for hepatitis B virus surface antibody quantification. PLoS One 20(5): e0304931. https://doi.org/10.1371/journal.pone.0304931.1932-6203 (online)10.1371/journal.pone.0304931http://hdl.handle.net/2263/107080DATA AVAILABILITY STATEMENT : All data are fully available without restriction. The datasets used to replicate the study results are available at https://figshare.com/articles/dataset/Motshosi_et_al_2024_DBS_Optimization_Study_Data/27219687 or https://doi.org/10.6084/m9.figshare.27219687.Dried blood spot (DBS) cards can be used as an alternative sample collection method to plasma, however, there is no optimized elution protocol for DBS cards specifically for hepatitis B surface antibody (anti-HBs) testing. The study aimed to develop a DBS elution protocol for anti-HBs quantification. Our study sought to determine the ideal phosphate-buffered saline (PBS) volume to use by comparing three PBS volumes (300 µL, 450 µL, and 500 µL), and the optimal time to agitate DBS discs on a plate shaker (1, 2, 3, and 4 hours) to yield DBS anti-HBs concentrations that are comparable to corresponding plasma anti-HBs concentrations. The optimal DBS storage temperature (25°C, -20°C, and -80°C) was investigated to determine the ideal long-term storage temperature of the cards. Residual samples were used for optimization (2019-2021). A total of 50 DBS-plasma pairs were used throughout the study, with plasma anti-HBs concentrations being used as the golden standard to compare. A two-way analysis of variance (ANOVA) was also performed to determine the impact of PBS elution volumes, elution time, and storage temperature on the anti-HBs concentration of DBS samples. No statistically significant difference between the DBS-plasma anti-HBs pairs was observed when using 450 or 500 µL of PBS and when samples were agitated for 3 hours (p = 0.59, p = 0.50) respectively. The optimal storage temperature for DBS cards was 25°C because the results showed no statistically significant difference between DBS-plasma anti-HBs titers (p = 0.59). The two-way ANOVA analysis showed that elution volumes and time had no statistically significant impact on the DBS anti-HBs concentrations, p = 0.95 and p = 0.38 respectively. Storage temperature had a statistically significant impact on the DBS anti-HBs concentrations, p = 0.002. The optimized DBS elution protocol for anti-HBs quantification will help monitor vaccine efficacy in infants due to the low sample volumes required compared to plasma and also can be used for anti-HBs testing in resource-limited areas around the country.en© 2025 Motshosi et al. This is an open access article distributed under the terms of the Creative Commons Attribution LicenseDried blood spot (DBS)PlasmaHepatitis BANOVAPhosphate-buffered saline (PBS)Article