A modified harlequin stain for cell kinetics

Lymphocytes cultured with 4 X 10(-4) M bromodeoxyuridine (BrdU) and stained by the harlequin procedure show wide variations in the colour and texture of their interphase nuclei. We demonstrate that we are able, by inspection, to identify four sets of nuclei: small dark nuclei (D) in cells that have not transformed in response to mitogenic stimulation; red nuclei (R) in transformed cells that have not synthesized DNA in culture; speckled nuclei (S)--red speckles on a bluish background--in cells that are synthesizing DNA for the first time in culture; blue nuclei (B) in cells that have completed one or more cycles of DNA synthesis. By using this modified harlequin stain to score the fraction of proliferating cells, and using the modified or conventional harlequin stain to determine the relative frequencies of mitotic cells in the first, second, or later divisions, we are able to estimate the fraction of lymphocytes in the original blood sample that can divide in culture. In addition, we show that the technique lends itself to automation. For this we have digitized black and white video images of the cells and extracted features based on the distribution of optical densities in the nuclei. Discriminant analysis carried out on these descriptors resulted in the correct classification of the three major groups R, S and B with an accuracy of 80.3%, 58.5% and 85.1%, respectively.