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

• Published in: Transfusion (Philadelphia, Pa.) Vol. 39; no. 7; pp. 781 - 789
• Main Authors: Watkins, N.A., Armour, K.L., Smethurst, P.A., Metcalfe, P., Scott, M.L., Hughes, D.L., Smith, G.A., Williamson, L.M., Clark, M.R., Ouwehand, W.H.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Inc 01-07-1999
• Subjects: Antigens, CD - genetics; Blood Platelets - chemistry; BSA = bovine serum albumin; Female; FITC = fluorescein isothiocyanate; GP = glycoprotein; Hemagglutination; Humans; Immunoglobulin G - blood; Immunophenotyping; Integrin beta3; Integrins - genetics; MAIPA = monoclonal antibody immobilization of platelet antigens; MoAb(s) = monoclonal antibody(ies); PBS = phosphate-buffered saline; PCR = polymerase chain reaction; Phenotype; Platelet Glycoprotein GPIIb-IIIa Complex - analysis; Platelet Membrane Glycoproteins - genetics; Pregnancy; RBC(s) = red cell(s); Recombinant Proteins - blood; scFv(s) = single-chain V domain fragment(s); Solubility; TBS = Tris-buffered saline; Thrombocytopenia - blood; Thrombocytopenia - immunology
• ISSN: 0041-1132; 1537-2995
• DOI: 10.1046/j.1537-2995.1999.39070781.x

BACKGROUND: The HPA‐1 system is carried on the β3 integrin. HPA‐1a (Zwa, PlA1) 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 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.