YS04 Recombinant Mini‐Β3 Integrin Fragments for the Detection of HPA Antibodies

Abstract

Background The β3 integrin [Glycoprotein (GP) IIIa] gene is highly polymorphic and encodes eight of the Human Platelet Antigen (HPA) systems. HPA immunisation is of clinical relevance in neonatal alloimmune thrombocytopenia (NAIT), post‐transfusion purpura (PTP) and platelet refractoriness. However, the current methods of antibody detection are cumbersome and rely upon the use of platelets from genotyped donors. In the case of the rare HPA antigens, obtaining donor platelets is particularly problematic. We therefore set out to develop an antibody screening assay that uses recombinant, soluble β3‐integrin fragments expressing the HPA‐1, –4, –6, –7, –10, –11 and –16 epitopes.Methods A total of four integrin fragments of different lengths (ΔSDL, ΔβA, PSI‐Hybrid and PSI) were designed using structural data and expressed in Drosophila S2 cells. HPA‐1a (33) and HPA‐1b (P33) forms of all four proteins were expressed as were the fragments ΔSDL‐4b and ΔSDL‐SuperRare, the latter two for the detection of HPA‐4b and the rare HPA antigens (HPA‐6, 7, 10, 11 and 16) respectively. The recombinant proteins were then screened for reactivity with monoclonal antibodies (mAb) and polyclonal antisera containing anti‐HPA in an ELISA based assay.Results The reactivity pattern seen with murine mAbs Y2/51, SZ21 and CRC54 was as expected. Two of three human monoclonal HPA‐1a antibodies (19–7, 23–15, both from a PTP patient) reacted with the four L33 fragments but the third (CamTran007, derived from a NAIT patient) only reacted with the ΔSDL‐L33 and ΔβA‐L33 fragments, defining an epitope split. The reactivity of both potency (NIBSC‐03/152) and sensitivity (NIBSC‐93/710) standards for anti‐HPA‐1a detection were comparable or better than those seen by MAIPA. In addition, samples from 132 NAIT patients with anti‐HPA‐1a [15 with cerebral haemorrhage (CH), 49 with platelet counts <20 × 109 L−1 but no CH, 35 with counts ≥ 20 × 109 L−1 and 33 with antenatal treatment] were tested. Anti‐HPA‐1a was detected in 126 samples including all CH cases. The reactivity patterns against the four different β3‐integrin fragments did not correlate with clinical outcome. Finally, mini‐β3 integrins Δ SDL‐4b and ΔSDL‐SuperRare allowed the detection of 14 of the 15 anti‐HPA samples tested (5 HPA‐4a, 5 HPA‐4b, 1 HPA‐6b, 1 HPA‐7b and 2 of 3 HPA‐11b).Conclusions Recombinant fragments of the β3‐integrin expressing HPA antigens can be used to successfully detect platelet alloantibodies. These fragments with be of use in the screening and diagnosis of suspected cases of NAIT.