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.


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.
Department of Haematology, University of Cambridge, UK.
N A
NA
Watkins
K L
KL
Armour
P A
PA
Smethurst
P
P
Metcalfe
M L
ML
Scott
D L
DL
Hughes
G A
GA
Smith
L M
LM
Williamson
M R
MR
Clark
W H
WH
Ouwehand
D015703:Antigens, CD / Q000235:genetics*; D001792:Blood Platelets / Q000737:chemistry; D005260:Female; D006384:Hemagglutination; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood*; D016130:Immunophenotyping; D039661:Integrin beta3; D016023:Integrins / Q000235:genetics; D010641:Phenotype; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000032:analysis; D010980:Platelet Membrane Glycoproteins / Q000235:genetics*; D011247:Pregnancy; D011994:Recombinant Proteins / Q000097:blood; D012995:Solubility; D013921:Thrombocytopenia / Q000097:blood / Q000276:immunology
D015703:Antigens, CD; D007074:Immunoglobulin G; D039661:Integrin beta3; D016023:Integrins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins