Sensitivity of assays for the detection of HPA-1a antibodies: results of an international workshop demonstrating the impact of cation chelation from integrin αIIbβ3 on three widely used assays.

Background and Objectives HPA-1a antibodies account for 70-80% of cases of fetal-neonatal alloimmune thrombocytopenia (FNAIT) in Caucasians. However, numerous workshops have demonstrated variability in their detection. We recently showed that exposure of alpha IIb beta 3 to ethylene diamine tetraacetic acid (EDTA) affected binding of many anti-alpha IIb beta 3 monoclonal, and HPA-1a allo-, antibodies; this adversely affected sensitivity of the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay and indirect platelet immunofluorescence test (PIFT). This study presents results from an international workshop studying the impact of cation chelation on HPA-1a antibody detection in routine diagnostic laboratories. Materials and Methods Serum and EDTA-anticoagulated plasma samples containing anti-HPA-1a were distributed to 39 laboratories. Participants were asked to detect and identify any HPA antibodies present. Results 2/39 (5.1%) participants were able to detect and identify anti-HPA-1a in the serum, but not in the plasma sample. EDTA plasma reduced MAIPA assay sensitivity by >= 20% in 17/24 (70.8%) laboratories and by >= 50% in 9/24 (37.5%) when using HPA-1a1a platelets (mean: 27.7%, range 0-85.1%); when using HPA-1a1b platelets 3/4 (75%), participants reported >= 50% loss of sensitivity (mean 65.6%, range 0-96.6%). A small but significant increase in optical densities was observed in antigen capture ELISA assays when using plasma (mean difference: 0.081, P < 0.01). Insufficient PIFT data were returned to draw firm conclusions. Conclusion Use of EDTA plasma significantly affects the sensitivity of the MAIPA assay and can affect detection of even potent, FNAIT-causing examples of anti-HPA-1a. These data highlight the importance of use of alpha IIb beta 3 in an appropriate conformation for the sensitive detection of anti-HPA-1a.