Establishment of Cell Lines Stably Expressing HNA-1A, -1B, -2A, -4A, -4B, -5A and -5B Antigen with Low Background Reactivity in Flow Cytometric Analysis.

Antibodies to neutrophil antigens have been implicated in neonatal alloimmune neutropenia, autoimmune neutropenia, and transfusion-related acute lung injury. Although FCM analysis with a typed panel of neutrophils has been used to detect such antibodies, this procedure has two major limitations. First, only freshly isolated neutrophils can be used for the assay. Second, the assay cannot identify an HNA antibody in the presence of anti-HLA class I antibodies that react to panel neutrophils. To overcome these limitations, we established a panel of cell lines in which the HNA-1a (−1b; polymorphisms of HNA-1), HNA-2a, HNA-4a (−4b), or HNA-5a (−5b) gene was transduced using a retrovirus vector to confer stable transgene expression in K562 cells that dose not express HLA or HNA and not exhibit background reactivity to normal human serum samples. We confirmed the specificity of the one of each HNA expression on the K562 panel cells by using mAbs. We further showed that several well-characterized human sera containing antibodies against several HNAs were unambiguously identified by our panel cell lines. We also observed a lower background reactivity of the K562 panel cell lines compared with isolated neutrophils, which have been used for the panel to identify antibodies against HNA in human sera. These results indicate that the K562 panel cell lines provide a good panel for detecting HNA-reactive neutrophil antibodies in human sera.