A simple and rapid competitive enzyme-linked immunosorbent assay to identify HPA-1a (PlA1)-negative donor platelet units.
BACKGROUND
Alloantibodies to HPA-1a (PlA1) are the major cause of neonatal alloimmune thrombocytopenia and posttransfusion purpura and have been implicated in refractoriness to random-donor platelet transfusions. However, most assays used to phenotype platelets are cumbersome or time-consuming for large numbers of samples.


METHODS
A simple, competitive (inhibition) enzyme-linked immunosorbent assay for HPA-1a phenotyping of donor platelets was developed. A segment from the donor platelet unit transfer line was sealed to obtain a small aliquot of platelets. These platelets were washed once and added to a predetermined dilution of serum containing alloantibodies to HPA-1a. Residual anti-HPA-1a binding to the glycoprotein IIb/IIIa purified by lectin and high-performance liquid chromatography and coated on microtiter wells was detected with a conjugated antihuman IgG. A lack of inhibition equivalent to control (no platelets) was used to determine that the platelets were HPA-1b/b.


RESULTS
Of the 557 platelet units tested, 14 (2.5%) were found to be HPA-1a negative, and they were confirmed to be HPA-1b/b by DNA genotyping. Two of the 14 HPA-1b/b units were also HPA-3b/b (approx. 0.35% of the random population). Use of the microtiter format allows 100 to 200 samples to be processed per day.


CONCLUSIONS
This simple and inexpensive assay is useful for identifying HPA-1b/b units for platelet-compatible transfusions or for platelet antibody investigations.
Denomme
G
G
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
Horsewood
P
P
Xu
W
W
Smith
J
J
Kelton
J
J
D016824:Antigens, Human Platelet / Q000032:analysis*; D001667:Binding, Competitive; D001792:Blood Platelets / Q000276:immunology*; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D039661:Integrin beta3; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000276:immunology; D017713:Platelet Transfusion / Q000379:methods
D016824:Antigens, Human Platelet; C061248:ITGB3 protein, human; D039661:Integrin beta3; D019039:Platelet Glycoprotein GPIIb-IIIa Complex