Netten H, Young I T, van Vliet L J, Tanke H J, Vroljik H, Sloos W C
Faculty of Applied Physics, Delft University of Technology, The Netherlands.
Cytometry. 1997 May 1;28(1):1-10.
Fluorescence in situ hybridization allows the enumeration of chromosomal abnormalities in interphase cell nuclei. This process is called dot counting. To estimate the distribution of chromosomes per cell, a large number of cells have to be analyzed, especially when the frequency of aberrant cells is low. Automation of dot counting is required because manual counting is tedious, fatiguing, and time-consuming. We developed a completely automated fluorescence microscope system that can examine 500 cells in approximately 15 min to determine the number of labeled chromosomes (seen as dots) in each cell nucleus. This system works with two fluorescent dyes, one for the DNA hybridization dots and one for the cell nucleus. After the stage has moved to a new field, the image is automatically focused, acquired by a Photometrics KAF 1400 camera (Photometrics Ltd., Tuscon, AZ, USA), and then analyzed on a Macintosh Quadra 840AV (Apple Computer, Inc., Cupertino, CA, USA) computer. After the required number of cells has been analyzed, the user may interact to correct the computer by working with a gallery of the cell images. The automated dot counter has been tested on a number of normal specimens where 4,'6-diamidino-2-phenylindole (DAPI) was used for the nucleus counterstain and a centromeric 8 probe was used to mark the desired chromosome. The slides contained lymphocytes from cultured blood. We compared the results of the dot counter with manual counting. Manually obtained results, published in the literature, were used as the "ground truth." For a normal specimen, 97.5% of cells will have two dots. Fully automated scanning of 13 slides showed that an average of 89% of all nuclei were counted correctly. In other words, an average of 11% has to be interactively corrected, using a monitor display. The machine accuracies, after interactive correction, are comparable to panels of human experts (manual). The fully automatically obtained results are biased with respect to manual counting. An error analysis is given, and different causes are discussed.
荧光原位杂交技术可对间期细胞核中的染色体异常进行计数。这个过程称为点计数。为了估计每个细胞中染色体的分布情况，必须分析大量细胞，尤其是当异常细胞的频率较低时。由于手动计数繁琐、易疲劳且耗时，因此需要实现点计数的自动化。我们开发了一种完全自动化的荧光显微镜系统，该系统可以在大约15分钟内检查500个细胞，以确定每个细胞核中标记染色体（视为点）的数量。该系统使用两种荧光染料，一种用于DNA杂交点，另一种用于细胞核。载物台移动到新视野后，图像会自动聚焦，由Photometrics KAF 1400相机（美国亚利桑那州图森市Photometrics有限公司）采集，然后在美国加利福尼亚州库比蒂诺市苹果电脑公司的Macintosh Quadra 840AV计算机上进行分析。在分析完所需数量的细胞后，用户可以通过处理细胞图像库与计算机交互以进行校正。该自动点计数器已在多个正常标本上进行了测试，其中使用4,6-二脒基-2-苯基吲哚（DAPI）对细胞核进行复染，并使用着丝粒8探针标记所需的染色体。载玻片上含有来自培养血液的淋巴细胞。我们将点计数器的结果与手动计数结果进行了比较。手动获得的结果（已发表在文献中）用作“真实值”。对于正常标本，97.5%的细胞将有两个点。对13张载玻片进行全自动扫描显示，平均89%的细胞核被正确计数。换句话说，平均有11%需要使用显示器进行交互式校正。经过交互式校正后，机器的准确性与专家小组（手动）相当。全自动获得的结果相对于手动计数存在偏差。给出了误差分析并讨论了不同的原因。
