Comparison of Dried Blood Spot Sampling Methods for RNA-Based Biomarker Measurement in Anti-Doping

Dried blood spot (DBS) sampling offers logistical advantages, but it remains unexplored whether analytical results are comparable across DBS sampling methods. This study investigated whether venous or capillary blood spotted on cellulose cards or collected with the Tasso-M20 device provides comparable results for the anti-doping RNA-based biomarkers of erythropoiesis, 5-aminolevulinic acid synthase (ALAS2), and carbonic anhydrase 1 (CA1). In the population of Swiss athletes (n = 12), DBS samples (venous-cellulose and Tasso-M20) were collected in routine anti-doping procedures. In healthy volunteers (n = 12), DBS samples (venous-cellulose, capillary-cellulose and Tasso-M20) were collected at baseline, after 1 week and 10 days post a 3-week recombinant human erythropoietin (rEPO) administration study. ALAS2 and CA1 mRNA were quantified using RT-qPCR, and agreement between matrices was assessed via Passing-Bablok regression. ALAS2 and CA1 expression showed strong linear agreement (r ≥ 0.96) across matrices. Passing-Bablok regression indicated no constant or proportional bias for ALAS2 across all comparisons. For CA1, no bias existed between venous-cellulose and Tasso-M20 in the athlete population, whereas a proportional bias of ~9%-22% was observed when comparing Tasso-M20 DBS with venous- or capillary-cellulose DBS in the rEPO study. No significant differences in relative RNA expression were observed across matrices at any timepoint in the administration study. ALAS2 and CA1 are reliably quantified across venous- and capillary-cellulose-based DBS and Tasso-M20 DBS samples. Strong agreement and minimal bias, with the modest CA1 bias being small relative to expected biological or treatment-induced variability, support their use for longitudinal monitoring, enabling less invasive, flexible, and decentralized sampling in research and anti-doping.