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A cell counting method for MIB-1 immunohistochemistry, using an image analysis program, NIH Image, on a personal computer, has advantages over manual cell counting by microscopy. MIB-1 slides were photographed at a magnification of x 50 on 24 x 36 mm color films and the photographs were then enlarged to adequate size for observation. The MIB-1-positive cells, on the enlargements, were marked with a white pen, and negative cells with a black pen. Subsequently, the image of an enlargement was converted into data that can be processed by the program using a flatbed image scanner. Threshold levels were adjusted according to the respective white and black markings, and the program automatically counted the number of MIB-1-positive and negative tumor cells. Our method has the following significant advantages: Once cells had been marked and the image converted into the NIH Image data, the cell count process was completed within a few minutes; in contrast to manual microscopic cell counting, count overlapping or cells missed by the observer are eliminated; overlapping nuclei could be separated by the marking procedure; our method required no elaborate and costly image analysis system. The present method may be recommended to researchers undertaking cell kinetic studies utilizing immunohistochemical methods.

Tumor Cell Counting Using an Image Analysis Program for MIB-1 Immunohistochemistry