The use of harlequin staining to measure delay in the human lymphocyte cell cycle induced by in vitro X-irradiation

Samples of human peripheral blood were given X-ray doses of 1, 2, 3 or 4 Gy at 37°C with a further sample remaining unirradiated. Lymphocytes were then stimulated to divide in cultures containing BrdU for 40-72 h. After harlequin staining the metaphases were recorded as being in their 1st, 2nd or 3rd post-irradiation division. It was confirmed that irradiation delays the proliferation of lymphocytes in culture. A linear relationship between dose and mitotic delay of approximately 1 h per Gray was obtained. This finding of a small effect on cell proliferation is particularly important for biological dosimetry. All in vivo exposures are more or less non-uniform and the lymphocytes in a blood sample therefore possess a spectrum of induced delay characteristics. However, in the great majority of overdose investigations it should not be necessary to increase the normal culture time for the most highly irradiated cells to reach metaphase. The trend towards using harlequin preparations to ensure that only first-division cells are analysed is briefly discussed and it is noted that in this experiment 2nd-cycle metaphases accounted for a maximum of 14% of the cells scored after 48 h in culture.