Analytical Validation of Dried Blood Spot Cards for Assessing COVID-19 Seropositivity.

BACKGROUND

Serological testing frequently utilizes high-sensitivity assays, which are very amenable to microsampling. Herein, we describe the conversion of a COVID-19 immunobead assay from a plasma matrix to the dried blood spot (DBS) card matrix with key analytical parameters reported.

METHODS

Plasma samples were obtained for 96 seronegative patients and 97 seropositive patients and spotted onto Advanced Diagnostics 100 (ADx100) lateral-flow DBS cards. All cards were stored in opaque pouches with desiccant at room temperature, with extractions performed on days 1, 30, and 120 for analyte stability assessments. Luminex assays were used to measure antibody titers against the SARS CoV-2 Spike and Nucleocapsid glycoproteins in both plasma and matched DBS extracts. Optimal thresholds were established for seropositivity assessments. Agreement was assessed between timepoint analyte measurements. Robust linear models (RLM) were created to predict DBS levels from measured plasma and vice versa, with classification accuracy predictions determined.

RESULTS

Assay accuracy for the DBS matrix was calculated at 94.9%, which was <1% different from plasma and with limited observed proportional bias. Total IgG normalization improved extract level accuracy to 97.3%. Storage experiments revealed excellent agreement (>0.9) for both the Spike and Nucleocapsid titers when evaluated over a 120-day period. Finally, utilizing the RLM-predicted plasma and DBS levels, equivalent assay performance was observed in a test set for predicted vs measured value classification.

CONCLUSIONS

DBS cards represent a convenient and cost-effective means of specimen delivery for serological testing with analyte stability documented for up to 4 months without loss of diagnostic performance.