G0-PCC-FISH derived multi-parametric biodosimetry methodology for accidental high dose and partial body exposures

Abstract

High dose radiation exposures are rare. However, medical management of such incidents is crucial due to mortality and tissue injury risks. Rapid radiation biodosimetry of high dose accidental exposures is highly challenging, considering that they usually involve non uniform fields leading to partial body exposures. The gold standard, dicentric assay and other conventional methods have limited application in such scenarios. As an alternative, we propose Premature Chromosome Condensation combined with Fluorescent In-situ Hybridization (G₀-PCC-FISH) as a promising tool for partial body exposure biodosimetry. In the present study, partial body exposures were simulated ex-vivo by mixing of uniformly exposed blood with unexposed blood in varying proportions. After G₀-PCC-FISH, Dolphin's approach with background correction was used to provide partial body exposure dose estimates and these were compared with those obtained from conventional dicentric assay and G₀-PCC-Fragment assay (conventional G₀-PCC). Dispersion analysis of aberrations from partial body exposures was carried out and compared with that of whole-body exposures. The latter was inferred from a multi-donor, wide dose range calibration curve, a-priori established for whole-body exposures. With the dispersion analysis, novel multi-parametric methodology for discerning the partial body exposure from whole body exposure and accurate dose estimation has been formulated and elucidated with the help of an example. Dose and proportion dependent reduction in sensitivity and dose estimation accuracy was observed for Dicentric assay, but not in the two PCC methods. G₀-PCC-FISH was found to be most accurate for the dose estimation. G₀-PCC-FISH has potential to overcome the shortcomings of current available methods and can provide rapid, accurate dose estimation of partial body and high dose accidental exposures. Biological dose estimation can be useful to predict progression of disease manifestation and can help in pre-planning of appropriate & timely medical intervention.

Keywords: G0-PCC-FISH; High dose biodosimetry; Partial body exposure; Premature chromosome condensation; Whole chromosome painting.

Figure 1

Methodology for partial body simulation study.

Figure 2

Ex-vivo radiation response of chromosome 1, 2 & 4 associated breaks & interchanges in human PBMCs assessed using G₀-PCC-FISH. (a) Microscopic images of PCC spreads showing increasing number of aberrations with increasing dose (left to right). (b) Calibration curve from pooled data of aberrations of three donors. (c) Individual response curves of three donors and d) Individual response curves of the three chromosomes, pooled from three donors. The error bar represents mean ± Poisson Error.

Figure 3

Sensitivity of different methods for detection of aberrant cells from the exposed fraction in PBE relative to WBE of the same dose in in-vitro simulated exposures using human blood lymphocytes. The proportion 1:1 refers to samples with 50% exposed proportion. (a) dicentric assay (b) G₀-PCC-Fragments (c) G₀-PCC-FISH. (d) Typical 10 X fields of metaphase preparation in dicentric assay with increasing uniform exposure. Metaphases are encircled in red boundaries. For both the PCC methods, data was corrected for background level of aberrated cells in case of PBE. Data bars represent mean ± SD.

Figure 4

Dose estimates among simulated PBE samples (a) Dose estimates of three different donors for all the three methods. 1:0, 1:1, 1:3, 1:5 refers to ratio of exposed vs. unexposed fraction of the blood. Small and bigger orange ellipticals refer to 20% and 30% error vertically. Doses were heavily underestimated in case of dicentric assay at 12 Gy, 1:1 in all the three donors and at 8 Gy, 1:5 in two of the three donors. (b) Impact of background correction in dose estimation using G₀-PCC- methods. (i) G₀-PCC-FISH (ii) G₀-PCC-Fragments. While there is no significant impact of background correction in G₀-PCC-FISH, dose estimation error is significantly reduced in G₀-PCC-Fragments, specially at higher unexposed proportions.

Figure 5

Distribution of aberrations for a given dose with WBE and after 1:1 mixing with unexposed cells (50% PBE). (a) 4 Gy, (b) 8 Gy, (c) 12 Gy andd) Quantification of dispersion with U-test among three donors. U-value within ± 1.96 refers to Poisson Distribution (dark green cells) greater value refers to greater deviation from Poisson (red cells).

Figure 6

Radiation response of the key parameters under WBE condition: (i) P1, Median of aberrations among aberrated cells shows a linear quadratic response (ii) P2, proportion of multi-aberrant cells (X ≥ 3) among aberrated cells shows a sigmoidal response and (iii) P3, proportion of un-aberrated cells among total cell population depicts an exponential decrease.

Figure 7

Distribution of aberration among irradiated lymphocytes after WBE and PBE with similar dose estimate in G₀-PCC-FISH. (a). Samples with ~ 2 Gy D_WBE; arising from 2 Gy WBE, or from 4 Gy, 1:1 and 8 Gy, 1:5 PBE. (b) and in samples with ~ 6 Gy D_WBE arising from WBE 6 Gy exposure or 12 Gy 1:1 PBE.