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. 2020 Feb;115(2):202-206.
doi: 10.1111/vox.12843. Epub 2019 Nov 27.

A novel simple assay system for the detection of human platelet antigen 15 (HPA-15) alloantibodies based on three techniques: an HPA-15 expressing cell line, a monoclonal antibody-specific antigen-capture method and mixed-passive haemagglutination

Yangsook Koh  1 , Hiroko Nishimiya  1 , Hiroko Inoue  1 , Ayaka Kishigami  1 , Yukari Tsuji  1 , Rumi Sakamoto  1 , Fumiya Hirayama  1 , Ayumu Kuroishi  1
Affiliations

A novel simple assay system for the detection of human platelet antigen 15 (HPA-15) alloantibodies based on three techniques: an HPA-15 expressing cell line, a monoclonal antibody-specific antigen-capture method and mixed-passive haemagglutination

Yangsook Koh et al. Vox Sang. 2020 Feb.

Abstract

Background and objectives: To detect HPA-15 alloantibodies, we previously developed a human platelet antigen 15 (HPA-15)-expressing cell line-based modified rapid monoclonal antibody immobilization of platelet antigen (CL-MR-MAIPA) assay. In this study, the protocol was modified for easier performance by introducing the mixed-passive haemagglutination (MPHA) principle.

Material and methods: In total, 20 samples that tested negative for HPA alloantibodies and eight that tested positive for HPA-15 alloantibodies (two and six positive for HPA-15a and HPA-15b antibodies, respectively) by CL-MR-MAIPA assay were used in this study. HPA-15 cell lines were incubated with serum/plasma and then solubilized. The lysate was transferred to a round-bottom well, which was coated with anti-human CD109 monoclonal antibodies. After incubation and repeated washings, sheep red blood cells, coated with anti-human IgG, were added to the wells. Haemagglutination was assessed the next day.

Results: The proposed cell line-based immune complex capture-dependent mixed-passive haemagglutination (CL-IC-MPHA) assay consisted of four steps, but required only 2 h to perform, except for overnight incubation for haemagglutination. Two HPA-15a alloantibody samples were reactive only for HPA-15a cells, and six HPA-15b alloantibody samples were reactive only for HPA-15b cells with the CL-IC-MPHA assay. The 20 samples that tested negative for HPA alloantibodies did not react with HPA-15a or HPA-15b cells. These data indicated that the CL-IC-MPHA assay was highly specific and sensitive. Unfortunately, the CL-IC-MPHA assay's analytic sensitivity was twofold to eightfold lower than that of the CL-MR-MAIPA assay.

Conclusion: A novel, easy-to-perform protocol was successfully developed to detect HPA-15 alloantibodies with high specificity and sensitivity.

Keywords: neonatal alloimmune thrombocytopenia; platelet antigens; refractoriness (platelets); serological testing.

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