Rapid detection of HPA-1 alloantibodies by platelet antigens immobilized onto microbeads

Neonatal alloimmune thrombocytopenia (NAIT) is one of the most common bleeding disorders in neonates. It occurs when alloantibodies from an immunized mother react with paternally inherited alloantigens, mostly human platelet antigen 1a (HPA-1a), on the fetal platelets (PLTs). Currently, monoclonal antibody-immobilized PLT antigen (MAIPA) assay represents the standard technique for the serologic diagnosis of NAIT. MAIPA is time-consuming, however, and limited by the availability of monoclonal antibodies (MoAbs). Here, a gel antigen-specific assay (GASA) was developed, which allows rapid detection of HPA-1 alloantibodies without the use of MoAbs.

Glycoprotein (GP) IIb/IIIa was purified by affinity chromatography from outdated PLT concentrates derived from HPA-1aa or HPA-1bb donors. Purified GPs were biotinylated, immobilized onto streptavidin beads, and used for the analysis of HPA-1a alloantibodies by a microtyping system. HPA-1a serum samples derived from mothers with NAIT (n = 36) and from posttransfusion purpura patients (n = 2) as well as HPA-1b (n = 4), HPA-5b (n = 2), HPA-3a (n = 4), and HLA Class I (n = 2) alloantiserum samples from multitransfused patients were investigated in GASA and MAIPA assays.

GASA was able to detect all HPA-1a and -1b alloantibodies recognized by MAIPA. Cross-reactivity with other PLT-reactive alloantibodies was not observed. Interestingly, 3 of 36 serum samples, which showed only moderate reactivity in MAIPA, reacted strongly in GASA.

GASA has proved to be a rapid method for the detection of HPA-1a alloantibodies and maybe useful for PLT antibody screening, especially in initial assessment of suspected NAIT cases.