Rapid Electrochemical Impedance Detection of the Mia Antigen on Red Blood Cells.

The Mia antigen on red blood cells (RBCs) of the MNS blood group system is clinically significant in Southeast Asian populations and is associated with a risk of alloimmunization and hemolytic transfusion reactions. Conventional serological typing for Mia requires large reagent and sample volumes and subjective interpretation, highlighting the need for alternative analytical approaches. In this study, a novel simple electrochemical impedance spectroscopy-based immunoassay was developed for Mia antigen detection using a "mix-and-drop" strategy involving packed RBCs and specific anti-Mia antibodies on a screen-printed carbon electrode (SPCE). The analytical signal was defined as the change in charge transfer resistance (ΔR ct) measured on an SPCE in the presence of a ferri/ferrocyanide redox probe. Key assay parameters, including the RBC:antibody volume ratio, antibody dilution, and incubation time, were systematically optimized to maximize ΔR ct net in the agglutinating and non-agglutinating samples. Under optimized conditions, Mia -positive agglutination samples produced a distinct and reproducible increase in impedance, attributed to the formation of hemagglutination, which impeded electron transfer at the electrode interface. The assay was evaluated using 232 clinical blood samples (122 Mia-positive and 110 Mia-negative), containing internal (70%) and external (30%) validation sets. The ΔR ct values for Mia-positive and Mia-negative samples demonstrated significant discrimination between groups (Mann-Whitney U test, p < 0.0001). Receiver operating characteristic analysis revealed excellent diagnostic performance (p < 0.0001, AUC = 1.0), with an optimal ΔR ct cutoff of 71.10 kΩ, yielding 100% sensitivity and specificity within the internal and external validation sets. This label-free electrochemical assay requires low volumes of reagent and sample and is a rapid and practical approach for screening for the Mia antigen without electrode surface modification, supporting its potential use in pre-transfusion blood testing.