Automatic classification of cells in cell cycle phases based on Ki‐67 antigen quantification by fluorescence microscopy

. Ki‐67 antigen is thought to be a marker of cell proliferation, as it can be detected in cycling cells, i.e. cells in G 1 , S, G 2 and M phases, but not in resting cells. The immunocytochemical staining pattern obtained by the Ki‐67 monoclonal antibody varies, depending on the cell cycle phases. Analysis of double staining of Ki‐67 antigen and DNA in the MCF‐7 cell line by videomicrofluorometry allows the description of both the level and the pattern of Ki‐67 staining in the form of a set of parameters defining each cell. These parameters were measured in MCF‐7 cell populations characterized according to their position in the cell cycle. They were submitted to a statistical analysis (principal component and discriminant analysis) which allowed the determination of the optimal parameters to characterize a given cellular group and permitted the use of these parameters for an automatic classification of cells in the different cell cycle phases. In G 1 , S, G 2 , prophase + metaphase and anaphase + telophase cells, these parameters allowed a classification of cells with a good‐classification rate of 94·37%. A comparison of this method with methods based on the DNA histogram and bromodeoxyuridine uptake was performed. The classification coefficients stemming from the discriminant analysis were introduced into a program to obtain, automatically, the Ki‐67 labelling index and the percentages of cells in each phase. This method, which allows a quick evaluation of the proliferation and the phase indices, may be more widely applicable.