Tumor cell counting using an image analysis program for MIB-1 immunohistochemistry.

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.