Abstract: Objective To establish a novel flow cytometry-based method for quantifying residual RhD antigen on red blood cells (RBCs) in apheresis platelets, aims to assess the immunogenic risk of anti-D immunization in RhD-negative patients who receive RhD-positive platelet transfusion. Methods The method quantified residual RBCs based on anti-D antibody consumption. Simulated samples were prepared by serial dilution of RhD-positive RBCs, then incubated with a low-titer anti-D antibody. After anti-D absorption, the remaining free anti-D was quantitative detected by flow cytometry to generate a standard curve, which correlating antibody test value with RBC count. This method was applied to 30 donor apheresis platelet samples. Results The measurable range of the novel method is 2×10⁸ to 1.25×10¹⁰ RBCs/U. In 12 of the 30 tested samples, the RBC levels were below the limit of detection. In the remaining 18 quantifiable samples, 4 samples contained residual RBC counts>1×10⁹ RBCs/U, 3 samples contained 5×10⁸ to 1×10⁹ RBCs/U, and 11 samples contained 2×10⁸ to 5×10⁸ RBCs/U.All samples met the national standard requirements (<8×10⁹ RBCs/U), while the residual RBC levels were significantly higher than prior reports. Conclusion The novel flow cytometric method offers enhanced sensitivity and accuracy for detecting residual RhD-positive RBCs in platelet products. There is a risk of anti-D alloimmunization in non-DEL RhD-negative patients receiving repeated transfusions of RhD-positive platelet products in a short term.

Key words: Flow cytometry, Apheresis platelet, RhD negative, Erythrocyte antigen