We have calculated the distribution of DNA contents in micronuclei (MN) induced by ionizing radiation in human lymphocytes on two assumptions: the MN arise from acentric chromosome fragments (ACF), and the ACF result from the random breakage and rejoining of chromosomes. Measurements show that about 80 per cent of MN have a DNA content in the range of 0.5-6 per cent of the G1 nucleus. This group is consistent with the model and shows little dependence on radiation dose over the dose range of 0.5-4 Gy, or on lymphocyte culture time, varying from 48 to 76 hours. The MN with DNA content from 6 to 20 per cent of the G1 nucleus are probably the result both of spindle defects and of DNA synthesis in MN.
In order to quantitatively study cellular and nuclear properties, we have assembled a low cost video /microscope densitometric system. The system consists of a conventional light microscope coupled to a low light level TV camera whose output is sequentially processed by two microcomputers, one for image acquisition and one for data analysis.The system allows us to a) acquire and store microscope images, b) make spatial measurements such as size and shape, and c) make grayness measurements needed to compute optical density and texture descriptors. Despite the inherent limitations of a video micrometry system (e.g. poor signal -to -noise ratio, automatic gain compensations, etc.) we have been able to collect reproducible data with satisfactory accuracies.
Lymphocytes cultured with 4 × 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.
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