Implication of transfected cell lines for the detection of alloantibodies against human neutrophil antigen-3

Transfusion. 2012 Mar;52(3):613-21. doi: 10.1111/j.1537-2995.2011.03303.x. Epub 2011 Aug 24.


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.

Study design and 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.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acute Lung Injury / etiology
  • Acute Lung Injury / immunology
  • Acute Lung Injury / prevention & control
  • Agglutination Tests / methods
  • Blood Component Transfusion / adverse effects
  • Blood Component Transfusion / standards*
  • Flow Cytometry / methods*
  • Flow Cytometry / standards
  • Fluorescent Antibody Technique / methods
  • Genotype
  • HEK293 Cells
  • Humans
  • Isoantibodies / immunology*
  • Isoantibodies / isolation & purification*
  • Isoantigens / genetics*
  • Isoantigens / immunology*
  • Mass Screening / methods
  • Mass Screening / standards
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / immunology
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / immunology
  • Point Mutation / immunology
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Transfection


  • HNA-3a antigen, human
  • Isoantibodies
  • Isoantigens
  • Membrane Glycoproteins
  • Membrane Transport Proteins
  • Recombinant Proteins
  • SLC44A2 protein, human