FISH and chips: automation of fluorescent dot counting in interphase cell nuclei

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.