Development and validation of an ex vivo electron paramagnetic resonance fingernail biodosimetric method

Abstract

There is an imperative need to develop methods that can rapidly and accurately determine individual exposure to radiation for screening (triage) populations and guiding medical treatment in an emergency response to a large-scale radiological/nuclear event. To this end, a number of methods that rely on dose-dependent chemical and/or physical alterations in biomaterials or biological responses are in various stages of development. One such method, ex vivo electron paramagnetic resonance (EPR) nail dosimetry using human nail clippings, is a physical biodosimetry technique that takes advantage of a stable radiation-induced signal (RIS) in the keratin matrix of fingernails and toenails. This dosimetry method has the advantages of ubiquitous availability of the dosimetric material, easy and non-invasive sampling, and the potential for immediate and rapid dose assessment. The major challenge for ex vivo EPR nail dosimetry is the overlap of mechanically induced signals and the RIS. The difficulties of analysing the mixed EPR spectra of a clipped irradiated nail were addressed in the work described here. The following key factors lead to successful spectral analysis and dose assessment in ex vivo EPR nail dosimetry: (1) obtaining a thorough understanding of the chemical nature, the decay behaviour, and the microwave power dependence of the EPR signals, as well as the influence of variation in temperature, humidity, water content, and O₂ level; (2) control of the variability among individual samples to achieve consistent shape and kinetics of the EPR spectra; (3) use of correlations between the multiple spectral components; and (4) use of optimised modelling and fitting of the EPR spectra to improve the accuracy and precision of the dose estimates derived from the nail spectra. In the work described here, two large clipped nail datasets were used to test the procedures and the spectral fitting model of the results obtained with it. A 15-donor nail set with 90 nail samples from 15 donors was used to validate the sample handling and spectral analysis methods that have been developed but without the interference of a native background signal. Good consistency has been obtained between the actual RIS and the estimated RIS computed from spectral analysis. In addition to the success in RIS estimation, a linear dose response has also been achieved for all individuals in this study, where the radiation dose ranges from 0 to 6 Gy. A second 16-donor nail set with 96 nail samples was used to test the spectral fitting model where the background signal was included during the fitting of the clipped nail spectra data. Although the dose response for the estimated and actual RIS calculated in both donor nail sets was similar, there was an increased variability in the RIS values that was likely due to the variability in the background signal between donors. Although the current methods of sample handling and spectral analysis show good potential for estimating the RIS in the EPR spectra of nail clippings, there is a remaining degree of variability in the RIS estimate that needs to be addressed; this should be achieved by identifying and accounting for demographic sources of variability in the background nail signal and the composition of the nail matrix.

Figure 1.

The three spectral components of the MIS: MIS-doublet (dotted), MIS-broad (hashed) and MIS-singlet (solid).

Figure 2.

Decay of the MIS in fingernail clippings exposed to ambient air (A) or carbon dioxide (B) over 60 min following cutting. Under air, the MIS-doublet undergoes rapid fading as seen by the decrease the intensity of the peak at 3520 G, with some minor fading of the MIS-broad as seen in the peak centred at 3460 G. However, under carbon dioxide the fading is dramatically slowed for all the MIS spectral components. The peak at 3545 G is the Bruker reference standard.

Figure 3.

Signal amplitudes of the MIS-doublet (triangle), MIS-broad (square) and MIS-singlet (circle) as a function of time after harvesting nail clippings that are then placed under an atmosphere of dry nitrogen gas. The signal amplitudes of the MIS-broad and the MIS-singlet remain constant for at least 7 d when the nail clipping is kept under nitrogen gas within the mylar storage bags (data not shown). The MIS-doublet achieves a constant positive value after 25–30 min under nitrogen and remains stable for at least 7 d.

Figure 4.

Correlation of the signal amplitudes of the MIS spectral components after 30 min under dry nitrogen in mylar bags. Good correlation is shown between the MIS-singlet and MIS-broad (A) and the MIS-doublet and MIS-broad (B) amplitudes in 60 individual nail samples.

Figure 5.

Background signal intensity in nail clippings that have been soaked in water for 20 min and then air-dried under nitrogen (A) or oxygen (B) for 4 h. Exposure of nail clippings to nitrogen after the water treatment prevents the ‘rebound’ in the background signal amplitude. The peak at 3545 G is the Bruker reference standard.

Figure 6.

The basis spectra used in the two-component linear regression spectral fitting model. The MIS (solid) is the composite of the MIS-doublet, MIS-broad and MIS-singlet signals and is the average of 30 MIS spectra obtained from the post-cut spectra of the unirradiated nail samples, after subtraction of the pre-IR (background) spectrum. The RIS (hashed) is a simple Lorentzian function.

Figure 7.

Two-component fit of the MIS and RIS basis functions (red) to the post-cut spectrum of a nail clipping that received a 6 Gy dose (blue) after subtraction of the background.

Figure 8.

Residual spectrum after the linear regression fit of the two-component model to the post-cut spectra acquired from the six nail samples (irradiated to doses of 0, 1, 2, 4 or 6 Gy) in five donor nail sets.

Figure 9.

Comparison of the dose response of the estimated RIS (square), determined from spectral fitting and the dose response of the actual RIS (circle) in all 15-donor nail sets. The actual RIS was obtained by subtracting the pre-IR spectrum from the post-IR spectrum.

Figure 10.

Plots of the mean estimated RIS (circle) and actual RIS (diamond) amplitudes for all 15-donor nail sets calculated without the background included during the spectral fitting. The error bars represent the standard error of the mean. The lines represent the linear regression analysis of the dose response of the estimated and actual RIS amplitudes calculated for all 90 nail clipping samples.

Figure 11.

Plots of the mean estimated RIS (solid lines) and actual RIS (hashed lines) amplitudes for all 16-donor nail sets with the background included during the fitting analysis but no post-fitting correction for background (circles) and with a post-fitting correction for background (diamonds). The error bars represent the standard error of the mean. The lines represent the linear regression analysis of the dose response of the estimated and actual RIS amplitudes calculated for all 96 nail clipping samples.