Simultaneous five cell‐lineage flow cytometric analysis system for detection of leucocyte antibodies

Abstract

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.

Evaluation of HNA-expressing cell line-based antigen capture systems and a solid-phase system for detecting HNA-1a antibodies

“…The KY-MAIGA system for HNA-1a Abs detection based on KY-HNA-1a panel whole blood of HNA-1a/a was used as a neutrophil source. After staining with the sera, followed by incubation with anti-human IgG-FITC, the neutrophil population was analysed [6]. Thin lines and bold lines represent autologous negative control sera derived from panel blood and test sera, respectively.…”

“…Although the International Granulocyte Immunology Workshop recommends using a combination of the granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) [5], it can still be difficult to determine antibody specificity in some cases. Therefore, we previously developed a GIFT-like flow cytometric analysis system, the 5 cell-lineage immunofluorescence test (5 cell-lineage IFT), in which four lineages of leukocytes and platelets could be simultaneously tested with low background reactivity [6]. However, conventional neutrophil-based tests have two further drawbacks: firstly, a typed panel of neutrophils is required; secondly, the presence of other leukocyte antibodies, such as human leukocyte antigen (HLA) Abs, may affect the detection of HNA Abs.…”

Adult-onset autoimmune neutropenia with antineutrophil antibodies to an unknown neutrophil-specific antigen analyzed by using five cell-lineage immunofluorescence test and reactivity against cell lines expressing human neutrophil antigens

“…Herein, we report the case of a patient with adult-onset AIN harboring antineutrophil antibodies to an unknown neutrophil-specific antigen (or antigens). These antibodies were analyzed by using two newly developed assay methods: five cell-lineage immunofluorescence test (IFT) [6] and reactivity against a panel of cell lines expressing particular human neutrophil antigens (HNAs), named KY-cell lines [7,8].…”

“…We analyzed the patient's serum for the presence of antineutrophil antibodies by using the five cell-lineage IFT [6], a modified granulocyte IFT (G-IFT) in which antibodies against neutrophils, monocytes, T cells, B cells, and platelets from healthy volunteers can be detected simultaneously with low background interference (Fig. 1).…”

“…For measuring antineutrophil antibodies, the five celllineage IFT offers some advantages over G-IFT, which is considered to be the standard assay [6,11]. The representative reactivity patterns of anti-HNA antisera or anti-HNA monoclonal antibodies (anti-HNA-1b, anti-HNA-2a, and anti-HNA-3a antisera, and anti-HNA-4 and anti-HNA-5 monoclonal antibodies) against a blood sample from a healthy volunteer in the five cell-lineage IFT are presented in Fig.…”