Molecular dosimetry by flow cytometric detection of thymine dimers in mononuclear cells from extracorporally UV-irradiated blood

Abstract

UV-induced DNA damage in mononuclear leucocytes can be quantified by flow cytometry of fluorescence from a labelled monoclonal antibody that specifically binds to thymine dimers (T ↔ T): specific fluorescence is already detectable after exposures of 1–2 J m −2 of 254 nm radiation and shows a linear relationship with dose. The distribution of UV fluences over an irradiated volume can thus be ascertained by measuring the UV-induced T ↔ T loads of the individual cells from that volume. After irradiation of mononuclear cells in a phosphate buffer solution in a Petri dish, most cells showed a similar intensity of specific T ↔ T fluorescence, forming a single sharp peak in the fluorescence histogram. This signifies an even distribution of fluences over the cells. It was noticed, however, that a variable minor fraction of mononuclear cells (usually less than 10%) could be resistant to immunostaining; this fraction was rejected from the calculation of the specific fluorescence. The flow cytometric technique was also applied to blood cells exposed in an ISOLDA device, which is in use in Russian clinics for UV irradiation of whole blood for therapeutical purposes. Only a small fraction of mononuclear cells in a sample of whole blood treated in ISOLDA acquired a detectable T ↔ T load after exposure to lamps which emit predominantly either UVC or UVB light ((3.6 ± 1.0)% and (1.8 ± 0.4)% of all analysed cells respectively). This small fraction had received a large variation in fluences, resulting in differences in nuclear T ↔ T loads by a factor of 200. There had apparently not been enough mixing of cells during the UV irradiation of the blood to even out differences in UV fluences. Hence the trigger of the reported curative effects of reinfused, UV-irradiated blood would appear to originate from the initial photomodification of a minor fraction of the extracted blood volume (1–2 ml per kilogram of body mass).