Purpose: To characterize the intra- and inter-measurement variability of a magnetic bead-based DNA dosimeter for quantifying DNA double strand break (DSB) damage from ionizing radiation.
Methods: Biotin molecules were attached to one end of the 5’ terminal of linear DNA by Integrated DNA Technology (IDTDNA). This allowed us to attach magnetic streptavidin particles (Promega, Inc.) to the biotinylated end of the DNA. After incubation, we washed the dosimeters and resuspended them at a concentration of 250 ng/µL in 20 mM potassium phosphate. We performed measurements in triplicate and kept controls (0 Gy) for comparison. We irradiated the dosimeter solutions at 100 kVp and 5.0 mA in polycarbonate phantoms at 27, 56, and 113 Gy using a CellRad irradiator (Precision X-ray, Inc.). The phantoms (ρ=1.19 g/cm³) held 10 µL of dosimeter solutions in wells 1 mm in depth and 5 mm in diameter. After irradiation, we extracted the dosimeter solutions from the phantoms and magnetized them with a magnetic stand in 1.5 mL microcentrifuge tubes. This separated bead-attached DNA from cut-off fragments. We withdrew the remaining solutions and used an agarose gel electrophoresis process to visualize damage and observe intra- and inter-variability between phantoms.
Results: Under both conditions, we observe a linear trend of increasing DSB damage with increasing dose. The intra-detector variability quantified by the slope uncertainties in detectors 1, 2, and 3 varied by ~55%. Inter-observer variability quantified by the standard deviation of slopes was ~3 times greater than the average intra-detector variability, possibly due to variances in DNA amounts recovered from the phantoms after irradiation.
Conclusion: Intra-detector measurement variability was ~3 to 4 times less than inter-detector measurement variability. Future work will investigate methods of reducing inter-detector measurement variability by ensuring the same mass of DNA for each data point in each gel well.