IMMUNOHEMATOLOGY Rapid detection of HPA-1 alloantibodies by platelet antigens immobilized onto microbeads Tamam Bakchoul, Oliver Meyer, Ashraf Agaylan, Stephane Bombard, Gregor Bein, Ulrich J.H. Sachs, Abdulgabar Salama, and Sentot Santoso BACKGROUND: Neonatal alloimmune thrombocytope- nia (NAIT) is one of the most common bleeding disor- ders in neonates. It occurs when alloantibodies from an immunized mother react with paternally inherited alloan- tigens, mostly human platelet antigen 1a (HPA-1a), on the fetal platelets (PLTs). Currently, monoclonal antibody–immobilized PLT antigen (MAIPA) assay rep- resents the standard technique for the serologic diagno- sis of NAIT. MAIPAis 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. STUDY DESIGN AND METHODS: Glycoprotein (GP) IIb/IIIa was purified by affinity chromatography from out- dated 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. RESULTS: 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. CONCLUSION: 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. N eonatal alloimmune thrombocytopenia (NAIT) is characterized by thrombocytopenia induced by the destruction of fetal and neo- natal platelets (PLTs) through transplacental passage of maternal PLT-specific alloantibodies. 1 Clini- cally, NAIT manifestations range from asymptomatic to severe bleeding events including intracranial hemorrhage, which is the most common cause of mortality and mor- bidity in 15 to 20 percent of affected cases. 2 In Caucasian persons, NAIT is caused by alloantibody against human PLT antigen 1a (HPA-1a) in approximately 75 percent of all cases. 3 In addition, alloantibodies against HPA-1a are the most common cause of PLT destruction in patients with posttransfusion purpura (PTP). 4 The epitopes of HPA-1a are localized on the glycoprotein IIIa (GPIIIa) subunit of the PLT fibrinogen receptor, GPIIb/IIIa complex. 5 Recent evidence from the crystal structures of GPIIb/IIIa showed that the N-terminal PSI (plexins, semaphorins, integrins) ABBREVIATIONS: GASA = gel antigen-specific assay; GP = glycoprotein; MAIPA assay = monoclonal antibody–immobilized platelet antigen assay; NAIT = neonatal alloimmune thrombocytopenia; PTP = posttransfusion purpura. From the Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University Giessen, Germany; the Insti- tute for Transfusion Medicine, Charite-Medical University of Berlin, Berlin, Germany; and DiaMed AG, Cressier-sur-Morat, Switzerland. Address reprint requests to: Sentot Santoso, PhD, Institute for Clinical Immunology and Transfusion Medicine, Justus- Liebig University Giessen, Langhansstrasse 7, 35385 Giessen, Germany; e-mail: sentot.santoso@immunologie.med.uni- giessen.de. This work was supported by a grant of the German Research Foundation (SFB 547) and is part of the PhD thesis of TB. Received for publication October 26, 2006; revision received January 24, 2007, and accepted January 29, 2007. doi: 10.1111/j.1537-2995.2007.01274.x TRANSFUSION 2007;47:1363-1368. Volume 47, August 2007 TRANSFUSION 1363