Epigenetic subtyping of white blood cells using a thermoplastic elastomer-based microfluidic emulsification device for multiplexed, methylation-specific digital droplet PCR

Epigenetic markers attract increasing attention for the study of phenotypic variations, which has led to the investigation of cell-lineage DNA methylation patterns that correlate with human leukocyte populations for obtaining counts of white blood cell (WBC) subsets. Current methods of DNA methylation analysis involve genome sequencing or loci-specific quantitative PCR (qPCR). Herein, a multiplexed digital droplet PCR (ddPCR) workflow for determining epigenetic-based WBC differential count is described for the first time. A microfluidic emulsification device fabricated from a commercially available thermoplastic elastomer (e.g., Mediprene) promotes customizability and cost-effectiveness of the methodology, which are prerequisites for translation into clinical and point-of-care diagnostics. Bisulfite-treated DNA from peripheral blood mononuclear cells and whole blood is encapsulated in droplets with ddPCR reagents containing primers and fluorescent hydrolysis probes specific for CpG loci correlated with WBC sub-population types. The method enables multiplexed detection of various methylation sites within a single droplet. Both qPCR and immunofluorescence staining (IF) were conducted to validate the capacity of the ddPCR methodology to accurately determine WBC sub-populations using epigenetic analysis of methylation sites. ddPCR results correlated closely to cell proportions obtained using IF, whereas qPCR significantly underestimated these values for both high and low copy number gene targets.