title: 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.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford, UK. dave.allen@ndcls.ox.ac.uk
authors.forename: D L
authors.initials: DL
authors.lastname: Allen
authors.forename: P
authors.initials: P
authors.lastname: Metcalfe
authors.forename: C
authors.initials: C
authors.lastname: Kaplan
authors.forename: R
authors.initials: R
authors.lastname: Kekomaki
authors.forename: M
authors.initials: M
authors.lastname: de Haas
authors.forename: R
authors.initials: R
authors.lastname: Yusuf
authors.forename: W H
authors.initials: WH
authors.lastname: Ouwehand
mesh_terms: D016824:Antigens, Human Platelet; D002614:Chelating Agents / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology*; D004493:Education; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D039421:Integrin alpha2 / Q000097:blood*; D039661:Integrin beta3 / Q000097:blood*; D007518:Isoantibodies / Q000097:blood*; D008297:Male; D012680:Sensitivity and Specificity; D054098:Thrombocytopenia, Neonatal Alloimmune / Q000097:blood*; D044465:White People
chemical_list: D016824:Antigens, Human Platelet; D002614:Chelating Agents; C061248:ITGB3 protein, human; D039421:Integrin alpha2; D039661:Integrin beta3; D007518:Isoantibodies; D004492:Edetic Acid
keywords: fetal-neonatal alloimmune thrombocytopenia; platelet antigens; platelet immunobiology; proficiency testing