Enumeration of micronucleated CD71-positive human reticulocytes with a single-laser flow cytometer
Enumeration of micronucleated CD71-positive human reticulocytes with a single-laser flow cytometer
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DOI：
10.1016/s1383-5718(02)00009-8
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发表时间：
2002-03-25
影响因子：
1.9
通讯作者：
Tometsko, CR
中科院分区：
文献类型：
--
作者：
Dertinger, SD;Torous, DK;Tometsko, CR
未切除脾脏的人外周血中微核网织红细胞（RET）极其罕见，因此无法对这些细胞进行简单计数，也无法将该终点作为细胞遗传学损伤的指标进行评价。在这份报告中，我们描述了一个高通量，单激光流式细胞仪系统评分微核（MN）在新形成的人类RET的发病率。该程序基于一种免疫化学试剂，该试剂根据转铁蛋白受体（也称为CD 71）的表达水平差异标记来自成熟红细胞的RET的最不成熟部分。对于用藻蓝蛋白缀合的抗CD 71、RNA酶和SYTOX绿色或SYBR绿色I核酸染料处理的人血细胞，实现了四种红细胞群体（年轻RET和成熟红细胞，有和没有MN）的分辨率。红系细胞的抗血型糖蛋白A标记（CyChrome缀合物）也被纳入染色程序中，以确保碎片或其他潜在伪影不会对分析产生不利影响。仪器校准程序，利用疟疾感染的啮齿动物红细胞也被开发，并进行了描述。使用该分析系统，采用单激光流式细胞仪分析了10名健康非脾切除志愿者的血液样本的微核频率。在成熟和最不成熟的部分RET的平均微核率分别为0.016和0.19%。还评价了来自三名健康脾切除志愿者的血液样品。正如预期，这些样本在成熟红细胞亚群中显示出较高的微核频率（范围为0.03-0.18%）。由此产生的数据表明，MN可以在人红细胞群体中用单激光流式细胞仪进行定量，并且最年轻的网织红细胞群体中MN细胞的频率接近在不存在针对MN-红细胞的脾选择的情况下预期的值。该高通量系统对于评价微核网织红细胞终点作为人群中染色体断裂和/或染色体分离异常指标的价值具有潜在重要性。(C)2002 Elsevier Science B. V.保留所有权利。
The extreme rarity of micronucleated reticulocytes (RETs) in the peripheral blood of non-splenectomized humans has precluded facile enumeration of these cells, as well as evaluation of this endpoint as an index of cytogenetic damage. In this report, we describe a high-throughput, single-laser flow cytometric system for scoring the incidence of micronuclei (MN) in newly formed human RETs. The procedure is based on an immunochemical reagent that differentially labels the most immature fraction of RETs from mature erythrocytes based on the expression level of the transferrin receptor (also known as CD71). The resolution of four erythrocyte populations (young RETs and mature erythrocytes, with and without MN) was achieved for human blood cells treated with phycoerythrin-conjugated anti-CD71, RNase, and either SYTOX Green or SYBR Green I nucleic acid dyes. Anti-glycophorin A labeling of erythroid cells (CyChrome conjugate) was also incorporated into the staining procedure to ensure that debris or other potential artifacts did not adversely impact the analyses. Instrument calibration procedures utilizing malaria-infected rodent erythrocytes were also developed, and are described. Using this analytical system, blood samples from 10 healthy non-splenectomized human volunteers were analyzed for micronucleus frequencies with a single-laser flow cytometer. Average micronucleus frequencies in the mature and most immature fraction of RETs were 0.016 and 0.19%, respectively. Blood samples from three healthy splenectomized volunteers were also evaluated. As expected, these samples exhibited higher micronucleus frequencies in the mature subset of erythrocytes (range 0.03-0.18%). The resulting data suggest that MN can be quantified in human erythrocyte populations with a single-laser flow cytometer, and that the frequency of MN cells in the youngest reticulocyte population approaches values expected in the absence of splenic selection against MN-erythrocytes. This high throughput system is potentially important for evaluating the value of the micronucleated reticulocyte endpoint as an index of chromosome breakage and/or chromosome segregational abnormalities in human populations. (C) 2002 Elsevier Science B.V. All rights reserved.