title: Implication of transfected cell lines for the detection of alloantibodies against human neutrophil antigen-3.
abstract: BACKGROUND
  Alloantibodies against human neutrophil antigen-3 (HNA-3) are responsible for the fatalities reported in transfusion-related acute lung injury. Consequently, reliable detection of these alloantibodies is mandatory to improve blood transfusion safety. In this study, we developed stable cell lines for the detection of HNA-3 antibodies.
  
  
  METHODS
  HEK293T were transfected with HNA-3a or HNA-3b constructs and sorted by flow cytometry according to high surface expression. Transfected cells were tested with sera containing HNA-3 antibodies in flow cytometry and antibody capture assay (ACA). The results were compared with granulocyte agglutination test and granulocyte immunofluorescence test.
  
  
  RESULTS
  In flow cytometry, 12 of 14 HNA-3a sera reacted specifically with HNA-3aa cells. One serum sample showed positive reaction with HNA-3bb cells. All HNA-3b sera recognized HNA-3bb cells. No reaction was observed with broad reactive antibodies against HLA Class I. In ACA, all HNA-3a sera (12/12) showed positive reactivity with HNA-3aa cells with no cross-reactivity with HNA-3bb cells. Again, all HNA-3b sera reacted with HNA-3bb cells only. Furthermore, genotyping of 249 individuals detected a new HNA-3 allele caused by a nucleotide substitution C>T at Position 457 leading to L(153)F mutation in choline transporter-like protein-2. This mutation impairs polymerase chain reaction with sequence-specific primers based HNA-3a typing. However, analysis with cells expressing F(153) isoform showed that this mutation did not alter the binding of HNA-3 antibodies.
  
  
  CONCLUSIONS
  This study demonstrated that HEK293T cells expressing stable recombinant HNA-3 are suitable for the detection of HNA-3 alloantibodies allowing reliable screening of blood products.
authors: Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University Giessen, Giessen, Germany.
authors: Behnaz
authors: B
authors: Bayat
authors: Yudy
authors: Y
authors: Tjahjono
authors: Silke
authors: S
authors: Werth
authors: Heike
authors: H
authors: Berghöfer
authors: Angelika
authors: A
authors: Reil
authors: Hartmut
authors: H
authors: Kroll
authors: Ulrich J
authors: UJ
authors: Sachs
authors: Sentot
authors: S
authors: Santoso
mesh_terms: D055371:Acute Lung Injury / Q000209:etiology / Q000276:immunology / Q000517:prevention & control; D000372:Agglutination Tests / Q000379:methods; D016913:Blood Component Transfusion / Q000009:adverse effects / Q000592:standards*; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D005455:Fluorescent Antibody Technique / Q000379:methods; D005838:Genotype; D057809:HEK293 Cells; D006801:Humans; D007518:Isoantibodies / Q000276:immunology* / Q000302:isolation & purification*; D007519:Isoantigens / Q000235:genetics* / Q000276:immunology*; D008403:Mass Screening / Q000379:methods / Q000592:standards; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology; D026901:Membrane Transport Proteins / Q000235:genetics / Q000276:immunology; D017354:Point Mutation / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D014162:Transfection
chemical_list: C518134:HNA-3a antigen, human; D007518:Isoantibodies; D007519:Isoantigens; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C484524:SLC44A2 protein, human