Optimization of dried blood spot for hepatitis B virus surface antibody quantification

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.