Mixed‐field reactivity in the ABO blood group system: A quantitative manifestation of weak antigen expression

BACKGROUND: subgroup, but whether MF reflects a subgroup-specific finding or a quantitative serologic phenomenon, and what mechanisms underlie it across contexts, remains uncertain. STUDY DESIGN AND METHODS: A prospective analysis was performed on 30 individuals referred for ABO discrepancies with MF reactivity from Brazilian institutions between 2019 and 2025. The cohort included donors, newborns, and patients with hematologic disorders. Serologic evaluation included routine column agglutination testing, MF separation techniques, and systematic assessment with monoclonal anti-A, anti-B, and anti-A,B reagents from different clones at predefined titers (1:512-1:2048). Complete ABO gene sequencing was performed in all cases, and serologic and molecular findings were integrated to determine the mechanism of each discrepancy. RESULTS: All samples showed weak ABO antigen expression with MF patterns, predominantly graded as 1+ to 2+ in gel testing. Reactivity varied by antibody clone and titer, and weak reactions frequently shifted to MF or stronger agglutination as reagent concentration increased. Discrepancies were attributed to ABO genetic variants in 50% of cases, neonatal immaturity in 23%, disease-related conditions in 13%, biological mechanisms in 7%, and analytical interference in 7%. Higher-titer monoclonal reagents improved weak A/B detection across all categories, achieving complete phenotype-genotype concordance. CONCLUSION: Mixed-field reactivity is a quantitative manifestation of reduced ABO antigen expression rather than a discrete subgroup-defining characteristic. A selected panel of higher-titer monoclonal antibodies may improve diagnostic accuracy and guide supplementary serologic and molecular investigation in unresolved or clinically relevant ABO discrepancies.