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

Time Stamp: July 11, 2024 8:00 PM
Source Node: 635580

Uniform exposure studies

Calibration curve

Radiation response under uniform exposure was a pre-requisite for studying PBE. To obtain the same, calibration curve was generated for Co-60 gamma radiation in the dose range of 0–15 Gy, from three donors. Dose dependent increase in chromosomal aberrations was distinctly visible. Representative images of G₀-PCC-FISH spreads with increasing frequency of aberrations are presented in Fig. 2a. The calibration curve from pooled data of the three donors is plotted in Fig. 2b while the individual data of the three donors is plotted in Fig. 2c. Further, radiation response for each of the chromosomes 1, 2 & 4 for each of the donors is plotted in Fig. 2d. The calibration curve follows a linear quadratic response, with the Yield, Y, being given as:

$$Y=alpha D+beta {D}^{2}+c$$

(3)

where α = 0.28 ± 0.037 Gy^-1, β = 0.039 ± 0.004 Gy^-2 and c = 0.0568 respectively (R² = 0.985).

Response of aberrations in each donor and each of the chromosomes is also found to be linear quadratic, with negligible inter-individual variations. Overall aberrations combined for all doses, were highest for chromosome 1, followed by that for chromosomes 2 & 4 respectively. The frequencies of aberrations in chromosomes 1 & 2 were not statistically significant, although, the response of chromosome 4 was significantly lower than the other two chromosomes at multiple dose points, particularly beyond 2 Gy. Chromosome dependent responses are largely attributed to the genome size of the chromosomes. Similar results were reported earlier for metaphase based translocation assay³⁵. Linear quadratic response for breaks and interchanges has been reported for chromosomes 3 and 4 using G₀-PCC-FISH²⁹, wherein the response was generated 8 h after irradiation to doses 1, 3, 5 & 7 Gy of gamma radiation. Another study has reported linear quadratic response in human chromosome no. 8 using G₀-PCC-FISH in chromosome³². A study limited to doses 2, 4 & 6 Gy for Chromosomes 1, 2 & 4 reported similar radiation responses³³. In constrast to our data, one study has reported linear response for chromosomes 1 & 2 in the dose range 0–5 Gy using chemically induced G₀-PCC-FISH³⁶. In this study, relatively higher aberration yield for chromosome 2 than for chromosome 1 was also reported. The differential sensitivity between two chromosomes was not consistent in our data. The disagreement in the observation could be attributed to (1) difference in the degree of chromosome condensation between chemically vs. cell fusion induced G₀-PCC, (2) scoring of limited number of cells from single individual and (3) limited dose range.

In G₀-PCC-FISH, any number and combinations of chromosomes can be painted with chromosome specific color probes. The most popular ones are; one color, two color, three-color and Multi-plex FISH, which paint one, two, three chromosomes and entire genome (24 colors with 22 autosomes, X and Y) respectively. In this study, three color FISH has been carried out. Two, major considerations for the choice are; processing duration of FISH and sensitivity of the assay. Three color FISH requires same duration as one- or two-color FISH (< 24 h), and has more sensitivity (aberration/spread) due to higher genome proportion coverage (~ 22% compared to ≤ 8% and ≤ 16% in one- and two-color FISH respectively). With its higher sensitivity, it requires lesser number of spreads to be scored for dose estimation. While Multiplex FISH can be more sensitive needing only small number of spreads to be scored (20–25 for a uniform exposure), it demands longer duration (~ 48 h) for FISH processing, thus defeating the purpose of rapid biodosimetry. It is also likely that, for a localized exposure, counting < 50–100 cells may result in false negative dose estimation or incorrect dose, thereby not serving the purpose of scoring lesser number of cells either. Hence, three-color FISH appeared as a balanced option.

It is worth emphasizing here that this is the first study on G₀-PCC-FISH with multi-donor, broad range exhaustive calibration curve, enabling a robust statistical distribution of aberrations and accurate estimates of curve coefficients. Further, extensive dispersion analysis of aberrations from this data were performed which helped in identifying parameters to discern PBE from WBE. These results are discussed in a later section.

Validation of calibration curve

For validation of the calibration curve, dose estimations of five double blinded samples were carried out and the estimated doses were compared with those obtained from conventional dicentric assay (Table 1). Yield of aberrations (Y) was calculated from both the methods and dose was estimated for G₀-PCC-FISH using Eq. 3, whereas for dose estimation with dicentrics, published curve equation (Eq. 4)³⁷ was used:

Table 1 Comparison of true dose and the doses estimated using conventional dicentric assay and G₀-PCC-FISH assay.

Full size table

$$Y=0.027D+0.065 {D}^{2}+0.0005$$

(4)

The G₀-PCC-FISH method was found to be as accurate as dicentric assay in the tested range of 1- 8 Gy, with all the estimates lying within 20% of the true value. Error in the dose estimates reduced with increasing dose. At lower doses, aberrations are relatively lower and contribution by background and individual response variations can result in higher errors compared to that at higher doses.

Simulated partial body exposure studies

Sensitivity of G₀-PCC-FISH for partial body exposure

Sensitivity of a method for PBE refers to efficiency to detect cells from the exposed fraction from the mixed pool. While un-aberrated cells may come from both exposed as well as un-exposed fraction, aberrated cells majorly represent the exposed fraction. Hence to evaluate the sensitivity of the three methods viz., dicentric assay, G₀-PCC-Fragment and G₀-PCC-FISH assay, we estimated proportion of aberrated cells in case of partial exposure (50%, 4 Gy, 8 Gy and 12 Gy) and plotted with that of corresponding uniform WBE of the same dose (Fig. 3).

It was observed that in case of dicentrics, sensitivity to detect aberrated cells from the exposed fraction was lower relative to the two G₀-PCC methods. For instance, 4 Gy (WBE) sample has shown 60% ± 5.2 cells having dicentrics whereas in 50% exposed sample, only ~ 15% cells are found to have dicentrics instead of the expected ~ 30% (Fig. 3a). Efficiency has further declined with increasing doses; in case of 8 Gy and 12 Gy uniformly exposed samples, 100% cells are found to be aberrated whereas 50% exposed samples showed only < 10% and < 5% spreads having dicentrics. Further, in case of both G₀-PCC methods, the proportion of cells with aberrations were in accordance with the proportion of exposed cells within ± 10% error (Fig. 3b, c). The lower sensitivity of dicentric assay can be explained on the basis of dose dependent decrease in cell division rate of exposed fraction and apoptosis in interphase, leading to a reduction in metaphase index (Fig. 3d). Hence, in such cases, while using conventional methods specialized equipment with automated high throughput imaging and scoring may be required to analyze huge number of cells^6,38,39,40. However, owing to higher sensitivity, G₀-PCC has the potential to overcome this shortcoming of conventional assay with scoring of 50–100 spreads, which, can be easily scored in a simple, manual fluorescent microscope as well.

Dose estimations

T_O evaluate accuracy of this method in partial body dosimetry, dose estimations from dicentric, G₀-PCC-FISH and G₀-PCC-Fragment assays were carried out and error in dose estimates were also calculated. For dose estimation, yields of aberration in the exposed fraction were calculated as described in the methodology section using Eqs. (1) and (2) and were subsequently used for calculation of corresponding dose estimates using appropriate curve equations. For G₀-PCC-FISH and dicentric assay Eqs. (3) and (4) were used, whereas, for G₀-PCC-Fragments the following equation, from a previously reported calibration curve²⁷ was used:

$$Y=1.09 D+0.19$$

(5)

Obtained estimates for each donor, with each method are plotted in Fig. 4a.

In case of dicentric assay, severe underestimation (> 30% error) was observed at 12 Gy and 8 Gy, 1:5 proportion. Nevertheless, for 0, 1:1 & 1:3 proportion, dose estimates were close to true dose with scoring of ≤ 500 cells. Hence, it can be concluded that with increasing dose and decreasing exposed fraction, sensitivity of dicentric assay decreases and accuracy of dose estimate gets compromised.

Dose estimates in both G₀-PCC-Fragments and G₀-PCC-FISH did not suffer dose or proportion dependent underestimation. G₀-PCC-FISH is found to be more accurate than G₀-PCC-Fragment based dose estimation and background correction was needed to reduce the error at 1:5 proportion. In case of G₀-PCC-FISH, all the dose estimates, except one, were within ± 20% error, with or without background correction. Further, with G₀-PCC methods, due to high sensitivity, substantially low cell counts are sufficient. Hence, it can be inferred that G₀-PCC methods are more suitable for high dose, PBE dosimetry, with the added advantage of being rapid. Specifically, G₀-PCC-FISH estimated the doses with higher accuracy compared to G₀-PCC-Fragment based technique. It is worth mentioning that while the calibration curves for G₀-PCC-Fragments and G₀-PCC-FISH are established up to 15 Gy, Dicentric assay is only up to 6 Gy. This raises a doubt whether the calibration curve could have contributed in underestimation of the doses in dicentric assay. The experimental results indicate that calibration curve holds good till 8 Gy although metaphase index may be lower; for 8 Gy dose point, as the proportion of unexposed fraction increased, accuracy of dose estimation was compromised, nevertheless, accurate estimates were obtained for uniform and 1:1 proportion. As anticipated, at 12 Gy, for uniform exposures, ~ 20% underestimation was observed while for PBE cases it was > 30%. In such a case, underestimation can be partly attributed to the calibration curve and partly to lower metaphase index from the exposed fraction.

Furthermore, in the methodology, we used background correction for both the PCC methods (Eq. 2) with the assumption that aberrated cell proportion also contains background level of aberrated cells from the unexposed fraction. When, unexposed fraction is large, the contribution of these cells resulted in considerable underestimation of doses at higher unexposed fractions, especially in G₀-PCC-Fragment assay.

For G₀-PCC fragment background level of aberration is ~ 20 per 100 cells (Eq. 5), corresponding to 1 aberration for every 4 un-aberrated cells (n₀). Similarly, for G₀-PCC-FISH considering the background level of aberration of 0.057 cells (c in Eq. 3), 6 aberrations per 100 n₀ were estimated as background.

Accordingly, for every background count, one single aberrated cell (n₁) was considered as background (n_bkg) and added to the cells with n₀. The associated aberrations were also subtracted from total aberrations. Cells with more than 1 aberrations were counted as background when n₁ were inadequate. It is noteworthy that, for low dose exposures the un-aberrated cells are contributed from exposed fraction as well. Hence, background correction should be done only after ascertaining PBE of doses ≥ 4 Gy.

To demonstrate utility of the background correction, % errors in dose estimates were plotted with or without background correction as shown in Fig. 4b. Background correction was more impactful in keeping the errors within 30% in G₀-PCC-Fragment method, especially where un-exposed fractions were higher (8 Gy, 1:5) as observed in Fig. 4b(i). This can be attributed to higher background aberration frequency in unexposed fraction in G₀-PCC-fragment compared to that in G₀-PCC-FISH. G₀-PCC-FISH practically did not benefit from background correction in our study, where the probability of contribution of background is very low at all proportions (Fig. 4b(ii)). Error remained within 20% for all the dose estimates except one in both corrected as well as un-corrected estimates. However, the equation has been maintained with background correction so it remains independent of the exposed fraction and can be used for both the PCC methods.

It is noteworthy that all G0-PCC assays have been conducted 24 h after exposure with scientific and practical reasons. Previously it was reported that optimal duration between exposure and processing or collection of samples is about 8–24 h based on chromosomal break repair kinetics²⁸. In addition, for a PBE, lymphocyte pool recirculation time also needs to be considered. One circulation of peripheral blood takes only < 1 min to travel from and back to the heart. Hence, for a localized exposure, lasting for more than couple of minutes, immediately collected blood will not represent true PBE. However, only about 2% of lymphocytes are present in the peripheral blood while the remaining are present in lymphoid pool, which is relatively stagnant. Equilibration of lymphocytes between these two pools requires a duration of about 8–12 h, and the blood can be collected following this period. Furthermore, considering the delays attributed to detection of incident, reporting, logistic responses and transporting the exposed individual or reaching the site for collection of samples, it is also a most practical time window wherein the sample can be collected by biodosimetry team or medical professionals.

Dose estimation for PBE based on the discerning key parameters

Differentiation of PBE from WBE is very crucial for dose estimation. For this, we compared the dispersion of aberrations among WBE and PBE of the same dose and their deviations from Poisson were quantified by Papworth’s U-test (Fig. 5a, b, c). On applying Papworth’s U-test, it was observed that, in case of WBE, though overdispersion was observed, the distribution was closer to Poisson whereas, in all the PBE samples, considerable deviation from Poisson was observed (U-value range: 5.4–32.9). Deviations in WBE were moderate and hence the Dolphin’s method was able to provide dose estimates within reasonable uncertainty. U-test has been considered to be a good indicator of PBE in case of dicentrics. However, as observed in Fig. 5, signals counted in G₀-PCC may not strictly follow Poisson distribution and large overdispersions may be observed at lower doses. As a result, U-value can only be indicative of PBE. Therefore, instead of exclusive reliance on U-test, a multi-parametric approach should be considered when using G₀-PCC for discerning PBE.

After comparing the distribution of aberrations among WBE and PBE, we could identify three key parameters that can be used in combination to differentiate the two types of exposures for a given yield of aberrations; (i) median of aberrations among aberrated cells (P1), (ii) proportion of multi-aberrant cells (with ≥ 3 aberrations) among aberrated cells (P2) and (iii) proportion of un-aberrated cells among total scored cells (P3). The first two are dependent more on the dose to the exposed fraction and remain largely un-affected by the amount of exposed fraction. The three parameters show a specific response under uniform exposure which is plotted in Fig. 6. For a given whole body dose estimate (D_WBE), substantial deviation from the expected response of key parameters can confirm non-uniformity. Figure 6 shows that P1 was 1 for doses ≤ 2 Gy, and further increased in a linear quadratic manner. On the other hand, P2 follows a sigmoidal pattern wherein, up to 2 Gy, majority of the aberrations are contributed by single aberrated cells; while beyond 2 Gy, multi-aberrant cells increase exponentially up to 8 Gy before saturating to ~ 100%. In contrast, P3 is > 90% for doses below 1 Gy, and decreases exponentially with increasing dose.

In Fig. 7, we have demonstrated that different WBE and PBE samples, with different exposure scenarios, can result in similar yield leading to similar D_WBE; nevertheless they can still be differentiated based on the key parameteres. For example, D_WBE of 2 ± 0.1 Gy is possible under any one of the following three scenarios, (i) 2 Gy WBE, (ii) 4 Gy 1:1 PBE and (iii) 8 Gy 1:5 PBE. For this example, the total cell population and aberrant cell population, along with the key parameters are plotted in Fig. 7a. As may be seen from the aberrant cell population, the P1is 1, 2 and 4 for 2 Gy and 4 Gy 1:1 and 8 Gy 1:5 respectively while the corresponding P2 values are 10%, 33% and 63%. The dose estimates corresponding to these two paramters (see Fig. 6) are ≤ 2 Gy, 3.3–4.0 Gy and 6.8–7.0 Gy respectively. On the other hand, the data from total cell population shows unexpectedly high value of P3 which is also indicative of PBE. Similarly, in Fig. 7b, 6 Gy WBE and 12 Gy 1:1 PBE yields give rise to similar dose estimates but difference in the three key parameters again confirms the non-uniformity of exposure in the latter case. Hence, with the above example, it can be concluded that key parameters have potential to distinguish a partial exposure from a uniform exposure.

It is noteworthy that besides discerning the nature of exposure, median and % multi-aberrant cells can also be used for dose estimation. Once, PBE is confirmed, dataset can be corrected for background for further dose estimation with Dolphin’s approach. Also, P1 and P2 from the corrected dataset can provide more accurate dose estimation.

A step by step methology for discerning the type of exposure and dose estimation of an unknown sample is disscused in the next section.

Stepwise protocol for partial body dosimetry of an unknown sample post G₀-PCC-FISH: an example

Sample preparation: Blood sample with unknown dose is collected. PBMCs are isolated and fusion with mitotic cells is performed. 2 h post fusion, cells are harvested, treated with hypotonic, and fixative. Slides are prepared and FISH is performed. Excess fluorescence signals are counted. For details of G₀-PCC-FISH refer to methodology section and²⁸.

Example dataset:

Assume that the scoring of aberration results in the following dataset:

n₀	n₁	n₂	n₃	n₄	n₅	n₆	n₇	n₈	n₉	n₁₀	X	Ν
182	11	6	6	2	5	5	4	4	1	2	193	228

Where n₀, n₁… refer to cells with 0, 1 … aberrations, X refers to total number of aberrations and N is total number of cells.

Initial dose estimation: Yield, Y is calculated as X/N. Assuming WBE and the dose estimate (D_WBE) of is obtained by solving Eq. (3).

D_WBE = 2 Gy.

Decision on WBE/PBE: All the key parameters expected from 2 Gy WBE were deduced from Fig. 6. Observed values of key parameters were extracted from the scored data. The doses corresponding to the observed values were estimated from standard curves in Fig. 6. Disagreement between observed and expected values was an indication of PBE as presented below:

Key parameters	Expected	Observed (D_WBE : 2 Gy)	Dose corresponding to observed values	WBE/PBE
1. P1	1	4	6.8 Gy	PBE
2. P2	10%	63%	6 Gy	PBE
3. P3	62%	79%	NA	PBE

PBE dose estimation: Once PBE is confirmed, dose estimation is performed with background correction. Background aberrations were calculated as 6% of n₀ hence, 11 single aberrated cells from n₁ population were considered as background (n_bkg). These cells are added with n₀ cells and their aberrations subtracted from total aberrations (corrected X = 182, N − (n₀ + n_bkg) = 35). Further, corrected P1 and P2 were calculated to be 5 and 78.3% respectively. Yield of the exposed fraction was calculated from Dolphin’s approach by solving Eq. 2 & dose estimates were obtained using Eq. 3, P1 and P2 as shown below:

(1)

Dolphin’s approach: D = 8.4 Gy
(2)

Using P1: D = 8.2 Gy
(3)

Using P2: D = 7 Gy

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Source: https://www.nature.com/articles/s41598-024-65330-8

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

Uniform exposure studies

Calibration curve

Validation of calibration curve

Simulated partial body exposure studies

Sensitivity of G₀-PCC-FISH for partial body exposure

Dose estimations

Dose estimation for PBE based on the discerning key parameters

Stepwise protocol for partial body dosimetry of an unknown sample post G₀-PCC-FISH: an example

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

Uniform exposure studies

Calibration curve

Validation of calibration curve

Simulated partial body exposure studies

Sensitivity of G0-PCC-FISH for partial body exposure

Dose estimations

Dose estimation for PBE based on the discerning key parameters

Stepwise protocol for partial body dosimetry of an unknown sample post G0-PCC-FISH: an example

Latest Intelligence

Sensitivity of G₀-PCC-FISH for partial body exposure

Stepwise protocol for partial body dosimetry of an unknown sample post G₀-PCC-FISH: an example