ANALYSIS OF MICRONUCLEATED CELLS BY FLOW-CYTOMETRY .1. ACHIEVING HIGH-RESOLUTION WITH A MALARIA MODEL
ANALYSIS OF MICRONUCLEATED CELLS BY FLOW-CYTOMETRY .1. ACHIEVING HIGH-RESOLUTION WITH A MALARIA MODEL
复制标题
DOI：
10.1016/0165-1161(93)90140-u
复制
发表时间：
1993-10-01
期刊：
影响因子：
--
通讯作者：
DERTINGER, SD
中科院分区：
文献类型：
--
作者：
TOMETSKO, AM;TOROUS, DK;DERTINGER, SD
微核细胞（MN 细胞）作为罕见事件存在于血液中（即每 1000 个总数中约 2 个 MN 细胞）。手动对 MN 细胞进行评分既耗时又乏味，这也是为什么每个样本通常只对 1000-2000 个总细胞 (PCE) 进行评分的主要原因。人们普遍认为，对大量细胞进行评分将改善测定统计数据，并且是可取的，但对于手工评分来说是不切实际的。相比之下，自动评分方法可以处理大量细胞，从而改进统计分析。为了准确、快速地评估断裂活性，我们开发了一种基于流式细胞术的微核细胞评分方法。开发自动化检测的第一步是证明该方法解析感兴趣细胞的能力。在这种情况下，必须从缺乏 DNA 的红细胞 (RBC) 中分离出微核细胞。由于微核是异质稀有事件，其大小和 DNA 含量各不相同，因此我们选择使用更丰富和均质的生物模型来优化该测定的实验变量，从而实现稀有细胞的高分辨率。实验描述了鼠疟原虫伯氏疟原虫作为微核模型，并促进了基于流式细胞术的精确评分方法的开发。这种寄生虫存在于红细胞群中，并赋予细胞微核大小范围内的同质（基因决定的）DNA 成分。疟疾寄生虫产生的条件很容易应用于血液样本中微核事件的分析。通过疟原虫获得的双变量概况可用于定义高速微核评分的分析区域。
Micronucleated cells (MN cells) are present in the blood as rare events (i.e. about 2 MN cells/1000 total). Scoring MN cells by hand is both time-consuming and tedious, which is the primary reason why only 1000-2000 total cells (PCEs) are routinely scored for each sample. It is generally recognized that scoring larger numbers of cells would improve assay statistics and is desirable, but impractical with hand-scoring. In contrast, automated scoring methods can process large numbers of cells, thus improving statistical analysis. In order to accurately and quickly evaluate clastogenic activity, we have developed a flow cytometry based method of scoring micronucleated cells. One of the first steps in developing an automated assay is to demonstrate the ability of the method to resolve the cells of interest. In this case, micronucleated cells must be resolved from DNA-deficient red blood cells (RBCs). Since micronuclei are heterogeneous rare events which vary in both size and DNA content, we chose to use a more enriched and homogeneous biological model for optimizing the experimental variables of this assay, leading to high resolution of the rare cells. Experiments are described in which the murine malaria parasite, P. berghei, served as a micronucleus model and facilitated the development of an accurate flow cytometry based scoring method. This parasite resides in the red blood cell population and endows the cells with a homogeneous (genetically determined) DNA component in the micronucleus size range. The conditions developed with the malaria parasite are readily applied to the analysis of micronucleus events in blood samples. Bivariate profiles that are obtained with the malaria parasite can be used to define the analysis area for high-speed micronucleus scoring.