Rapid phenotyping of HPA‐1a using either diabody‐based hemagglutination or recombinant IgG1‐based assays

BACKGROUND: The HPA‐1 system is carried on the β3 integrin. HPA‐1a (Zw a , Pl A1 ) is immunogenic in an HPA‐1b homozygote ( HPA‐1b1b ) . In pregnancy, 1 of 365 women forms anti‐HPA‐1a, which causes severe thrombocytopenia in 1 in 1100 neonates. Identification of women at risk of forming anti‐HPA‐1a and the screening of donors to obtain HPA‐1a‐negative platelets for therapy need reliable, low‐cost, automated assays. STUDY DESIGN AND METHODS: A diabody with dual specificity for HPA‐1a × D and an IgG1 anti‐HPA‐1a have been constructed by the use of the genes encoding the first anti‐HPA‐1a fragment. With these reagents, two complementary HPA‐1a phenotyping assays have been developed. RESULTS: This diabody was used in a simple hemagglutination technique to perform HPA‐1a phenotyping on soluble glycoprotein IIb/IIIa from EDTA plasma samples. Over 1000 unselected donors have been correctly HPA‐1a‐phenotyped by use of the diabody. The human recombinant IgG1 anti‐HPA‐1a was produced in a rat myeloma cell line and was fluorescein labeled for use in a whole‐blood flow cytometric HPA‐1a phenotyping assay. This IgG1 anti‐HPA‐1a shows a clear differential between HPA‐1a‐positive and HPA‐1a‐negative platelets at n M antibody concentrations. CONCLUSIONS: The two recombinant reagents described are highly suitable for screening and confirmatory HPA‐1a phenotyping. They permit rapid determination of the HPA‐1a phenotype and are amenable to automation.