Gamma-H2AX biodosimetry for use in large scale radiation incidents: comparison of a rapid lyse/fix protocol with a routine method

Following a radiation incident, preliminary dose estimates made by γ-H2AX foci analysis can supplement the early triage of casualties based on clinical symptoms. Sample processing time is important when many individuals need to be rapidly assessed. A protocol was therefore developed for high sample throughput that requires less than 0.1 ml blood, thus enabling finger prick sampling. The technique combines red blood cell lysis and leukocyte fixation in one step on a 96 well plate, in contrast to the routine protocol, where lymphocytes are separated by Ficoll density gradient centrifugation with subsequent washing and fixation steps. The rapid lyse/fix method reduced the estimated sample processing time for 96 samples to about 4 h compared to 15 h using the routine protocol. However, scoring 20 cells in 96 samples prepared by the rapid protocol took longer than for the routine method (3.1 versus 1.5 h at zero dose; 7.0 versus 6.1 h for irradiated samples). Similar foci yields were scored for both protocols and reliable dose estimates were obtained for coded samples, with mean absolute differences from the actual doses of 0.26 and 0.27 Gy for the routine and lyse/fix method, respectively. The lyse/fix protocol can therefore facilitate high throughput processing for γ-H2AX biodosimetry for use in large scale radiation incidents, at the cost of somewhat longer foci scoring times.