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

Authors


Correspondence: David L. Allen, Blood Research Group, NHS Blood and Transplant, John Radcliffe Hospital, Headington, Oxford OX3 9BQ, UK.

E-mail: dave.allen@ndcls.ox.ac.uk

Abstract

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 αIIbβ3 to ethylene diamine tetraacetic acid (EDTA) affected binding of many anti-αIIbβ3 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, < 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 αIIbβ3 in an appropriate conformation for the sensitive detection of anti-HPA-1a.

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