Decordier Ilse, Papine Alexander, Plas Gina, Roesems Sam, Vande Loock Kim, Moreno-Palomo Jennifer, Cemeli Eduardo, Anderson Diana, Fucic Aleksandra, Marcos Ricardo, Soussaline Françoise, Kirsch-Volders Micheline
Laboratorium voor Cellulaire Genetica, Vrije Universiteit Brussel, Brussels, Belgium.
Mutagenesis. 2009 Jan;24(1):85-93. doi: 10.1093/mutage/gen057. Epub 2008 Oct 14.
Micronuclei (MN) frequencies in peripheral blood lymphocytes have been used worldwide as a biomarker of chromosomal damage for genotoxicity testing and biomonitoring studies. Automation of MN analysis would provide faster and more reliable results with minimizing subjective MN identification. We developed an automated facility for the scoring of the in vitro MN cytokinesis-block assay for biomonitoring on Giemsa-stained slides, fulfilling the following criteria: applicable to the cytokinesis-block micronucleus methodology, discriminating between mono-, bi- and polynucleated cells, MN scoring according to HUMN scoring criteria, false-negative MN rate <10% and false-positive (FP) MN rate <1%. We first adapted the slide preparation protocol to obtain an optimal cell density and dispersion, which is important for image analysis. We developed specific algorithms starting from the cell as a detection unit. The whole detection and scoring process was separated into two distinct steps: in the first step, the cells and nuclei are detected; then, in the second step, the MN are searched for in the detected cells. Since the rate of FP MN obtained by the automatic analysis was in the range of 0.5-1.5%, an interactive visual validation step was introduced, which is not time consuming and allows quality control. Validation of the automated scoring procedure was undertaken by comparing the results of visual and automated scoring of micronucleated mono- and binucleated cells in human lymphocytes induced by two clastogens (ionizing radiation and methyl methane-sulphonate), two aneugens (nocodazole and carbendazim) and one apoptogen (staurosporine). Although the absolute MN frequencies obtained with automated scoring were lower as compared to those detected by visual scoring, a clear dose response for MNBN frequencies was observed with the automated scoring system, indicating that it is able to produce biologically relevant and reliable results. These observations, together with its ability to detect cells, nuclei and MN in accordance with the HUMN scoring criteria, confirm the usability of the automated MN analysis system for biomonitoring.
外周血淋巴细胞中的微核（MN）频率已在全球范围内用作遗传毒性测试和生物监测研究中染色体损伤的生物标志物。MN分析的自动化将提供更快、更可靠的结果，同时最大限度地减少主观的MN识别。我们开发了一种用于对吉姆萨染色玻片上的体外MN胞质分裂阻滞试验进行评分的自动化设备，满足以下标准：适用于胞质分裂阻滞微核方法，区分单核、双核和多核细胞，根据HUMN评分标准进行MN评分，假阴性MN率<10%，假阳性（FP）MN率<1%。我们首先调整了玻片制备方案，以获得最佳的细胞密度和分散度，这对图像分析很重要。我们从细胞作为检测单元开始开发特定算法。整个检测和评分过程分为两个不同的步骤：第一步，检测细胞和细胞核；然后，在第二步中，在检测到的细胞中搜索MN。由于自动分析获得的FP MN率在0.5-1.5%范围内，因此引入了一个交互式视觉验证步骤，该步骤不耗时且允许进行质量控制。通过比较两种断裂剂（电离辐射和甲基磺酸甲酯）、两种非整倍体剂（诺考达唑和多菌灵）和一种凋亡剂（星形孢菌素）诱导的人淋巴细胞中微核化单核和双核细胞的视觉评分和自动评分结果，对自动评分程序进行了验证。尽管与视觉评分相比，自动评分获得的绝对MN频率较低，但自动评分系统观察到MNBN频率有明显的剂量反应，表明它能够产生生物学相关且可靠的结果。这些观察结果，连同其根据HUMN评分标准检测细胞、细胞核和MN的能力，证实了自动MN分析系统用于生物监测的可用性。
