Flow cytometry method for platelet-specific antibody detection by acid treatment through human leukocyte antigen-1 removal

Abstract

Background: Platelet (PLT) transfusion has been a crucial for clinical blood transfusion and bleeding prophylaxis. Identification of antibody against PLT-specific antigen is important for accurately diagnosing posttransfusion purpura, neonatal alloimmune thrombocytopenia, and platelet refractoriness due to PLT-specific antibodies. However, the presence of anti-human leukocyte antigen (HLA) antibody makes the detection of PLT-specific antibody more complex. Materials and Methods: The influence of treatment of PLTs with phosphate-buffered solution (PBS) or citric acid at pH 3.0 on the expression of human-specific antigen and HLA Class I was tested by flow cytometry. PLTs were treated with citric acid at room temperature for 5 min, and all solutions were precooled at 4°C. Results: Acid treatment reduced more than 90% of HLA Class I complexes when compared with PBS-treated ones on the surface of PLT membrane without any significant cell damage. The antigenicity of HLA Class I was lowered with time, but the antigenicity residue was little with PLT fragments after more than 5 min. However, human-specific antigen involving CD61 was not decreased after appropriate acid treatment, which was confirmed by corresponding monoclonal antibody. The use of standard serum from National Institute of Biological Standards and Control (NIBSC) containing only anti-human PLT antigen-1a antibody or anti-HLA antibody-positive control was applied to confirm the effect of acid treatment on PLT antigen. Conclusion: These findings suggested that acid treatment method could be useful for detecting PLT-specific antibodies, guiding clinical transfusion.