| Summary: | The MNS7 (Mi<sup>a</sup>) blood group antigen is found at a different prevalence among different ethnic groups. Anti-Mi<sup>a</sup> can cause hemolytic disease of the fetus and newborn (HDFN) and both acute- and delayed-type hemolytic transfusion reactions (HTR). Mi<sup>a</sup> typing should be performed in donors to prevent life-threatening hemolytic transfusion reactions. The gel card and standard tube methods still need specialized equipment, centrifugation, and expertise for result interpretation. We used a novel paper-based analytical device (PAD) pre-coated with monoclonal IgM anti-Mi<sup>a</sup> for Mi<sup>a</sup> phenotyping. We measured grey pixel intensity in blood typing results for interpretation processing using OpenCV at the sample (SP) and elution parts (EP); furthermore, we used the SP: EP ratio and F-score as analysis criteria. We typed 214 blood EDTA samples with PAD–Mi<sup>a</sup> and then compared with gel card results for setting an analysis criterion. We observed 100% sensitivity, specificity, and accuracy when we applied the SP: EP ratio and F-score with the optimal criterion (1.07 and 0.17 for SP: EP ratio and F-score, respectively). The validation of PAD–Mi<sup>a</sup> typing for blood donor samples (<i>n</i> = 150) via F-score gave 100% sensitivity and specificity when compared with the gel card method; therefore, we argue that PAD–Mi<sup>a</sup> typing can be used for Mi<sup>a</sup> phenotyping without sero-centrifugation. Moreover, to study the correlation between genotype and phenotype, PCR-SSP was performed to identify <i>GYP(B-A-B)</i> hybrids. The results revealed that all Mi<sup>a</sup>+ blood samples gave a positive with GP. Hut, GP. HF, GP. Mur, GP. Hop, and GP. Bun. Results of the gel card method and PCR-SSP were concordant. Hence, using PAD–Mi<sup>a</sup> typing in blood donors would be helpful for creating a phenotype database of blood donors for reducing alloimmunization risks.
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