Motshosi Patience, Phinius Bonolo B, Jongman Mosimanegape, Baruti Kabo, Bhebhe Lynnette, Mulenga Graceful, Choga Wonderful T, Moyo Sikhulile, Gaseitsiwe Simani, Anderson Motswedi
Botswana Harvard Health Partnership, Gaborone, Botswana.
School of Allied Health Professions, Faculty of Health Sciences, University of Botswana Gaborone, Botswana.
PLoS One. 2025 May 27;20(5):e0304931. doi: 10.1371/journal.pone.0304931. eCollection 2025.
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.
干血斑（DBS）卡片可作为血浆样本采集的替代方法，然而，目前尚无专门针对乙肝表面抗体（抗-HBs）检测的优化DBS卡片洗脱方案。本研究旨在开发一种用于抗-HBs定量的DBS洗脱方案。我们的研究试图通过比较三种磷酸盐缓冲盐水（PBS）体积（300µL、450µL和500µL）以及在平板振荡器上搅拌DBS圆盘的最佳时间（1、2、3和4小时），以获得与相应血浆抗-HBs浓度相当的DBS抗-HBs浓度。研究了DBS的最佳储存温度（25°C、-20°C和-80°C），以确定卡片的理想长期储存温度。使用剩余样本进行优化（2019 - 2021年）。整个研究共使用了50对DBS-血浆样本，以血浆抗-HBs浓度作为比较的金标准。还进行了双向方差分析（ANOVA），以确定PBS洗脱体积、洗脱时间和储存温度对DBS样本抗-HBs浓度的影响。当使用450µL或500µL PBS且样本搅拌3小时时，DBS-血浆抗-HBs对之间未观察到统计学显著差异（p = 0.59，p = 0.50）。DBS卡片的最佳储存温度为25°C，因为结果显示DBS-血浆抗-HBs滴度之间无统计学显著差异（p = 0.59）。双向ANOVA分析表明，洗脱体积和时间对DBS抗-HBs浓度无统计学显著影响，p分别为0.95和0.38。储存温度对DBS抗-HBs浓度有统计学显著影响，p = 0.002。用于抗-HBs定量的优化DBS洗脱方案将有助于监测婴儿的疫苗效力，因为与血浆相比所需样本量较少，并且还可用于全国资源有限地区的抗-HBs检测。
