Performance of Dried Blood Spot Cards for Serologic Detection of HPV16 Antibodies in Oropharyngeal Squamous Cell Carcinoma Patients

Antibodies against the human papillomavirus type 16 (HPV16) E6 oncoprotein are promising biomarkers for the early detection of HPV-driven oropharyngeal squamous cell carcinoma (HPV+OPSCC). Standard serologic testing is challenging in underserved regions with high HPV+OPSCC incidence. Dried blood spot (DBS) cards offer a low-resource alternative but remain unevaluated for HPV antibody detection. A total of 25 OPSCC patients who provided paired serum (venipuncture) and DBS (finger-prick) samples were recruited from the University of Kentucky. HPV16 antibodies (L1, E1, E2, E4, E6, E7) were measured using multiplex serology, quantified as median fluorescence intensity (MFI) and dichotomized using established cutoffs. Correlation between serum and DBS MFI values was evaluated using linear regression and Bland-Altman plots, whereas sensitivity, specificity, and Cohen's kappa assessed agreement. Mean MFI levels were lower in DBS than serum but were strongly correlated (R = 0.73 to 0.96; HPV16 E6 = 0.83). For HPV16 E6, DBS sensitivity was 90% (95% CI: 68-99) and specificity 100% (95% CI: 48-100), with kappa = 0.787. Specificity was 100% across all markers, while sensitivity varied from 0% (L1) to 100% (E2). DBS cards are accurate, inexpensive, and a scalable alternative for HPV16 E6 antibody detection, particularly in medically underserved regions, though further validation is needed.