Matsuyama N, Kojima Y, Hirayama F, Yasui K, Taniue A, Fukumori Y, Yoshimura K, Tabata N, Sakata N, Tani Y, Shibata H
Japanese Red Cross Osaka Blood Center, Kinki University School of Medicine, Osaka, Japan.
Transfus Med. 2006 Apr;16(2):111-8. doi: 10.1111/j.1365-3148.2006.00642.x.
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.
尽管流式细胞术（FCM）分析是筛查白细胞抗体存在的最广泛使用的方法之一，但它有几个缺点。首先，中性粒细胞，尤其是单核细胞表现出高背景反应性。其次，为了确定抗体特异性，通常不仅需要检测中性粒细胞和单核细胞，还需要检测包括T细胞、B细胞和血小板在内的其他谱系细胞。因此，我们试图建立一种FCM分析系统，其中同时对白细胞和血小板的四个谱系进行低背景检测。使用乙二胺四乙酸抗凝全血作为细胞样本进行FCM分析，无需任何细胞制备。基于前向散射与侧向散射分布以及CD4、CD20和CD14表达的差异对五个细胞谱系进行区分。当将抗人中性粒细胞抗原（HNA）1b抗血清应用于HNA 1b阳性血样时，只有中性粒细胞明确呈阳性。当应用抗中田（抗CD36）抗血清时，只有血小板和单核细胞呈阳性。中性粒细胞和单核细胞的背景反应性足够低。当检测抗人白细胞抗原（HLA）II类抗血清时，只有B淋巴细胞和单核细胞呈阳性。当检测抗HLA I类抗血清时，所有五个谱系的细胞均呈阳性。
