Development and validation of a Hematocrit-insensitive volumetric absorptive paper disc for dried blood microsampling and genomic DNA recovery

Dried blood spots (DBSs) are widely used in biomedical analyses; however, accurate volume collection remains challenging because of hematocrit (Hct)-dependent blood spreading. To overcome this limitation, we developed an Hct-insensitive volumetric absorptive paper disc (VAPDm6) designed to collect a defined blood volume by saturating a fixed-area paper disc while allowing the removal of excess blood using a surrounding sheet. The analytical performance of VAPDm6 was evaluated using caffeine and its metabolites as UV-active model analytes, which were quantified using HPLC–UV. Analyte recovery from samples with 30–60% Hct remained within ±15%. The calibration curves were linear between 250 and 5000 ng/mL (R 2 > 0.99). The intra- and inter-assay precision ranged from 2% to 14%, and the accuracy met the ±15% criterion. The agreement between VAPDm6 and venous blood concentrations in human samples was confirmed using the Bland–Altman analysis. In addition, VAPDm6 enables genomic DNA recovery with long-term stability, indicating its applicability beyond small molecule analysis. Compared to existing volumetric devices, VAPDm6 provides simpler fabrication, lower cost, and accurate volumetric uptake independent of hematocrit, offering a robust alternative to conventional DBS methodology.