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Sensitivity of assays for the detection of HPA ‐1a antibodies: results of an international workshop demonstrating the impact of cation chelation from integrin α II bβ3 on three widely used assays
Author(s) -
Allen D. L.,
Metcalfe P.,
Kaplan C.,
Kekomaki R.,
de Haas M.,
Yusuf R.,
Ouwehand W. H.
Publication year - 2013
Publication title -
vox sanguinis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.68
H-Index - 83
eISSN - 1423-0410
pISSN - 0042-9007
DOI - 10.1111/vox.12043
Subject(s) - antibody , platelet , monoclonal antibody , neonatal alloimmune thrombocytopenia , ethylene diamine , antigen , medicine , chelation , immunology , immunofluorescence , chemistry , microbiology and biotechnology , fetus , pregnancy , biology , nuclear chemistry , organic chemistry , genetics
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 α II bβ3 to ethylene diamine tetraacetic acid ( EDTA ) affected binding of many anti‐α II bβ3 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 α II bβ3 in an appropriate conformation for the sensitive detection of anti‐ HPA ‐1a.