HPA-3 allele-specific MKs can detect sialic acid (SA) dependency of anti-HPA-3a alloantibody binding. (A) Schematic of SA dependency of different anti-HPA-3a alloantibody binding. Neuraminidase treatment, which removes terminal SA from nearby O-glycans on GPIIb, can reduce SA-dependent anti-HPA-3a alloantibody binding but has no effect on SA-independent alloantibody binding. PNA is a lectin that specifically binds to unsialylated Galβ1,3GalNAc structure. (B) Flow cytometry analysis of PNA binding to iPSC-derived MKs confirmed successful removal of terminal SA after neuraminidase treatment. (C) Reactions of anti-HPA-3a patient sera with HPA-3 allele-specific iPSC-derived MKs in the presence or absence of neuraminidase treatment in flow cytometry analysis. Binding to HPA-3b cells serves as background binding for individual patient sample. Color-coded numbers in (B) and (C) indicate median fluorescence intensity of corresponding peaks. NeuAc, N-acetylneuraminic acid; Gal, galactose; GalNAc, N-acetyl-D-galactosamine; IgG, immunoglobulin G; Neu, neuraminidase; PE, phycoerythrin.