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A plug-based microfluidic approach was used to perform multiple agglutination assays in parallel without cross-contamination and using only microliter volumes of blood. To perform agglutination assays on-chip, a microfluidic device was designed to combine aqueous streams of antibody, buffer, and red blood cells (RBCs) to form droplets 30-40 nL in volume surrounded by a fluorinated carrier fluid. Using this approach, proof-of-concept ABO and D (Rh) blood typing and group A subtyping were successfully performed by screening against multiple antigens without cross-contamination. On-chip subtyping distinguished common A1 and A2 RBCs by using a lectin-based dilution assay. This flexible platform was extended to differentiate rare, weakly agglutinating RBCs of A subtypes by analyzing agglutination avidity as a function of shear rate. Quantitative analysis of changes in contrast within plugs revealed subtleties in agglutination kinetics and enabled characterization of agglutination of rare blood subtypes. Finally, this platform was used to detect bacteria, demonstrating the potential usefulness of this assay in detecting sepsis and the potential for applications in agglutination-based viral detection. The speed, control, and minimal sample consumption provided by this technology present an advance for point of care applications, blood typing of newborns, and general blood assays in small model organisms.

ABO, D Blood Typing and Subtyping Using Plug-Based Microfluidics