Fluorescent dot counting in interphase cell nuclei

Abstract

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 analysed, particularly when the frequency of aberrant cells is low. Automation of dot counting is desirable because manual counting is tedious, fatiguing, and time consuming. We have developed a completely automated fluorescence microscope system that counts fluorescent hybridization dots for one probe in interphase cell nuclei. This system works with two fluorescent dyes—one for the DNA hybridization dots and one for the cell nucleus. A fully automated scanning procedure has been used for the image acquisition. After an image is acquired it has to be analysed in order to find the nuclei and to detect the dots. This article focuses upon the dot detection procedure. Three different algorithms are presented. The problems of ‘overlapping’ dots and split dots are discussed. The automated dot counter has been tested on a number of normal specimens where DAPI was used for the nucleus counter stain and a centromeric probe was used to mark the chromosome 12. The slides contained lymphocytes from cultured blood. The performance of the different algorithms has been evaluated and compared with manually obtained results. The automated counting results approximate the results of manual counting.