Establishment and Validation of Dose Response Curve for Micronucleus

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Introduction

Chromosome aberrations in lymphocytes are used to estimate absorbed dose to overexposed persons. The estimation of absorbed dose is done by reference to a dose response calibration curve established by in-vitro irradiation study of human blood samples from volunteer donors. Micronucleus is one of the chromosome aberrations that can be used as a biomarker to measure absorbed radiation dose in accidentally exposed persons. It is formed from lagging chromosomal fragments or whole chromosomes at anaphase which are not included in the nuclei of daughter cells. They are therefore seen as distinctly separate small spherical objects that have the same morphology and staining properties of nuclei, within the cytoplasm of the daughter cells (IAEA, 2011).

Materials and Methods

Three (3) volunteer donors who were non-smoker males, aged 23 to 35 years old, with no history of radiation exposure and have signed informed consent forms were the source of blood samples. The Blood samples and Optical Stimulated Luminescence (OSL) dosimeters were irradiated at 0.0, 0.1, 0.25, 0.5, 0.75, 1, 2 and 4Gy using the Gamma cell irradiator of St. Luke's Medical Center. Irradiation time was computed based on the dose rate of the source at the time of irradiation. Blood samples were incubated and harvested following the procedure of IAEA EPR 2011- Biodosimetry manual. Five to six drops of mixed cell suspension were dropped on the slide. Four slides were prepared per sample. The slides were coded for identification and stained in 2% Giemsa staining solution for 8 -10 minutes and were rinsed in distilled water and air-dried. The slides were analyzed by microscopy and the micronucleus images were captured using the built-in microscope camera, saved and counting of binucleated cells and micronucleus scoring were done in the computer.

Results and Discussion

Analysis of the pooled data from the three donors resulted to a dose response curve that fits well into the data but the outlier at dose 2 was not captured at 95% confidence interval. Donors 2 and 3 computed individually and combined have chi-square p-value which indicated that fitted data points were statistically not different from the observed ones, indicating good fit. The best dose response curve was the result from the combination of donors 2 and 3, shown in Figure 1. The purple color is the dose-response curve. The dark green lines are the bounds of the 95% confidence interval estimated using Equation (7) of Section 9.7 from (IAEA, 2011).

Conclusion

The model fits well into the data however, the 95% confidence interval did not capture the outlier at dose 2. The number of cells (micronucleus in binucleated cells) has to be increased in order to increase statistical confidence of dose response curve.