Rapid phenotyping of HPA-1a using either diabody-based hemagglutination or recombinant IgG1-based assays.
BACKGROUND: The HPA-1 system is carried on the beta3 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 x 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 nM 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.