RAPID ASSAY OF INTRINSIC RADIOSENSITIVITY BASED ON APOPTOSIS IN HUMANCD4 AND CD8 T-LYMPHOCYTES

Purpose: An assay for radiosensitivity has numerous applications in th e clinic. Avoidance of acute responses, prediction of normal tissue to xicity, and individualization of patient radiotherapy are included amo ng these. We have developed a rapid assay (about 24 h) able to predict intrinsic radiosensitivity of CD4 and CD8 T-lymphocytes based on radi ation-induced apoptosis. Methods and Materials: Fresh blood samples (1 -2 ml in heparinized tubes) were irradiated with 0-, 2-, and 8- Gy X r ays at a dose rate of approximately 3 Gy/min. Following irradiation, t he cells were collected and prepared for flow-cytometric analysis and cell sorting. In conjunction with the CellQuest software available wit h the FACSVantage cell sorter (Becton-Dickinson), two T-lymphocyte typ es were analyzed on the basis of their cell-specific antigens (CD4 and CD8), and DNA was stained with DAPI. Following the separation of thes e cell types, radiation-induced cell death was assessed. Cytotoxicity was characterized by gradual degradation of internucleosomal DNA which results in a sub-G1 peak on the DNA histogram, and by the associated loss of surface antigens causing an intermediate positive peak in the antibody histogram. Using the assay, we investigated the interdonor va riation in a cohort of 45 healthy adult blood donors and 5 children [o ne had immunodeficiency, centromeric instability, and facial anomalies syndrome (ICF), and one had ataxia telangiectasia (AT)]. Intradonor v ariation was assessed with 10 different experiments from a single dono r. Results: CD4 and CD8 T-lymphocyte radiosensitivities were correlate d (r = 0.63 and 0.65 for 2 and 8 Gy, respectively) in 45 adult donors. Both for CD4 and CD8 cells, 2 and 8 Gy irradiation responses showed a good correlation (r = 0.77 for both). Interdonor variation was signif icantly higher than intradonor variation (p < 0.0005) for all CD4 and CD8 data. We observed a decrease in the antigen fluorescence of dying cells, a phenomenon referred to as antigen-ebb. Antigen-ebb was clearl y observed in both cell types, and correlated significantly with cytot oxicity. A trend was observed between radiosensitivity and donor age, but there was no correlation for gender. Blood from a cl-year-old girl presenting with ICF demonstrated compromised radiation-induced cytoto xicity in her CD4 T-lymphocytes, and an Ii-year-old boy presenting wit h AT demonstrated compromised radiation-induced cytotoxicity in both h is CD4 and CD8 T-lymphocytes.Conclusion: We conclude that the assay pr ovides a rapid means of determining radiosensitivity, can discriminate differences in radiation-induced cytotoxicity between individuals, an d can be used as a rapid screen for genetically hypersensitive patient s. Antigen-ebb offers interesting possibilities far molecular biologic al investigations, permitting characterization and isolation of abnorm al but vital cells in the absence of clastogenic agents. (C) 1997 Else vier Science Inc.