Epigenetic CD4⁺ T-Cell Quantification from Dried Blood Spots Using a Real-Time Quantitative PCR-Based Assay

Despite the clinical importance of CD4 testing for identifying advanced HIV disease, access to conventional flow cytometry remains limited in many settings. Epigenetic real-time quantitative PCR (qPCR)-based immune cell quantification represents a molecular alternative that may be compatible with simplified sample types such as dried blood spot (DBS) specimens. This study evaluated the analytical performance of an epigenetic CD4⁺ T-cell qPCR assay using DBS samples. Residual EDTA whole blood specimens (N = 150) from patients with HIV were applied to Whatman 903 DBS cards and tested following manual DNA extraction and qPCR using the i.Mune CD4 assay. CD4 counts derived from DBS specimens were compared with reference flow cytometry using the AQUIOS PanLeucogating platform. Agreement was assessed by using concordance correlation, Bland-Altman analysis, and percentage similarity. Assay repeatability, batch-related variability, and classification performance at clinically relevant CD4 thresholds were also evaluated. DBS specimen-based epigenetic CD4 quantification showed good agreement with flow cytometry (concordance correlation coefficient, 0.91; 95% confidence interval, 0.88-0.93) with a mean bias of -28 cells/μL (-6.9%). Repeatability was acceptable across the measurement range (%CV, 4.0%-11.2%). At a threshold of 200 cells/μL, sensitivity and specificity were 93.8% and 88.9%, respectively. Increased variability was observed in larger manual extraction batches. These findings show the technical feasibility of epigenetic qPCR-based CD4 quantification from DBS samples and support further optimization and validation.