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Differentiation of ABO mosaics from chimeras is performed using flow cytometry (FCM) analysis. Although mosaics and chimeras have been distinguished by presence or absence of clear resolution using FCM analysis, the lack of quantitative metrics and definitive criteria for this differentiation has made some cases difficult to differentiate. In this study, therefore, we attempted to establish a definitive and quantitative criterion for this differentiation. When FCM histogram gates for group "A" or "B" antigen-negative and -positive red blood cells (RBCs) were set such that group O RBCs were classified as 99 percent negative and group A or B RBCs as 99 percent positive, the percentages of RBCs in the middle region of six chimeras and 23 mosaics (12 A mosaics and 11 B mosaics) were 0.1-0.6 percent and 7.0-19.0 percent, respectively. This results suggested that ABO mosaics and chimeras can be unambiguously differentiated when the cutoff point of the intermediate region is set to 1 percent.

Background: Antibodies to neutrophil antigens have been implicated in neonatal alloimmune neutropenia, autoimmune neutropenia, and transfusion-related acute lung injury. Most often, neutrophil-specific antibodies are directed toward human neutrophil antigen (HNA)-1 (Fcgamma receptor 3b) and HNA-2a (CD177) in these disorders., Study Design and Methods: To detect the alloantibodies in the serum samples, a panel of cell lines was established in which the HNA-1a, HNA-1b (polymorphisms of HNA-1), or HNA-2a gene was transduced with a retrovirus vector to confer stable transgene expression in K562 cells that exhibited low background reactivity to human serum samples obtained from healthy donors in flow cytometric analysis., Results: It was shown that several well-characterized human serum samples containing antibodies against HNA-1a, -1b, and -2a were unambiguously identified by the established panel cell lines and observed a lower background reactivity and longer shelf life of the K562 panel cell lines compared with isolated neutrophils, which have been used for the cell panel to identify antibodies against HNA in human serum samples., Conclusion: These results indicate that the K562 panel cell lines provide a good panel for detecting HNA-reactive neutrophil antibodies in human serum samples.

Although flow cytometric (FCM) analysis is one of the most widely used approaches to screen the presence of leucocyte antibodies, it has several drawbacks. First, neutrophils and, especially, monocytes exhibit high background reactivity. Second, to determine antibody specificity, it is often necessary to examine not only neutrophils and monocytes but also other lineage cells including T cells, B cells and platelets. Therefore, we attempted to establish an FCM analysis system in which four lineages of leucocytes and platelets are simultaneously tested with low background. FCM analysis was performed using ethylene diamine tetraacetic acid-anticoagulated whole blood as cell sample without any cell preparation. Discrimination of five cell lineages was carried out based on the differences in forward vs. side scatter distribution and in the expression of CD4, CD20 and CD14. When anti-HNA (human neutrophil antigen) 1b antiserum was applied to HNA 1b-positive blood samples, only neutrophils were unambiguously positive. When anti-Naka (anti-CD36) antiserum was applied, only platelets and monocytes were positive. The background reactivity of neutrophils and monocytes was low enough. When anti-human leucocyte antigen (HLA) class II antiserum was tested, only B-lymphocytes and monocytes were positive. When anti-HLA class I antiserum was tested, all the five-lineage cells were positive.