Paper-based fluorescent assay for blood typing and antibody titer determination using long-term ambient-stored bioengineered RBCs

Methods for accurate, rapid blood typing and antibody titration at the point of care are constrained by cold-chain dependent red blood cell (RBC) reagents, time-consuming serial dilutions, and subjective visual interpretation. To overcome these limitations, here we develop a method based on bioengineered red-blood cells. We engineer hemoglobin-depleted RBC membranes that preserve ABO surface antigens, label them with fluorescent nanoparticles, and lyophilize them, demonstrating at least 2 years of stability at room-temperature. We capture hemagglutination on a dual-layer paper pad and quantify it by fluorescence in approximately 8 minutes, eliminating the need for serial dilution. We conduct a registered clinical trial (ChiCTR2300078864) and benchmark performance against the gel microcolumn assay. In a clinical cohort (n = 641), ABO typing achieves 100% concordance. In antibody-titration samples, quantitative titers agree with the gel microcolumn assay (coincidence 80.75%, κ = 0.8623). In summary, we develop a cold-chain-independent, handheld paper-based assay that enables simultaneous ABO grouping and quantitative antibody titration with clinical-grade performance, supporting scalable manufacturing, cross-site standardization, and transfusion decision-making in emergency and resource-limited settings.