Whole fingernail parings are typically 1–2 mm wide, 1–2 cm long and slightly curved. This shape allows them to be inserted into a standard quartz EPR tube for spectral analysis. However, as indicated in , this places only a portion of the sample at the center of the EPR resonator where optimal sensitivity is obtained. Further, we have observed that the intensity and shape of the EPR spectrum from whole parings varies with their orientation in the EPR cavity, indicating an anisotropy of the EPR-detectable species when fingernails are in this configuration.
Cut-away representation of the configuration of a whole fingernail sample in an EPR tube positioned in the EPR resonator cavity adjacent to an EPR standard.
Reproducible EPR spectra can be obtained when fingernail parings are cut into ~five smaller pieces that pack in a random orientation at the bottom of an EPR tube. This allows the entire sample to be positioned in the region of maximum instrument sensitivity. Further, as indicated in , the entire sample is also now located immediately adjacent to an EPR standard that is used to normalize the spectrometer response from sample to sample and has the potential to serve as a spin standard for quantitative measurements. Two EPR standards have been used in this research, the Mn+2 that is a dilute impurity in samples of CaO, which has two well-characterized hyperfine lines (MI = −½ and MI = ½) that bracket the g = 2.00 region of interest, and a proprietary standard provided by Bruker, which has a single feature at g = 1.97 that does not overlap the EPR signals from fingernails.
Since all fingernail parings have MIS from harvesting, it is important to understand this signal to account for its contribution to the EPR spectrum of irradiated fingernails. shows the time evolution of the EPR spectrum of two representative fingernail samples, one that had not been irradiated (top) and one that had received a 10 Gy dose of radiation (bottom). In each case the spectra were collected starting immediately after the fingernail paring was cut into 5 small pieces, which allows the entire sample to be positioned in the EPR cavity at the position of optimal sensitivity and adjacent to the EPR standard (Mn+2 in CaO, in this case). Both the intensity and shape of the pure MIS spectrum (, top) change dramatically within the first hour, evolving into a feature at g = 2.003 and broad weaker features at higher g value (lower field). Of particular interest in the early spectra is the high field minimum that is part of a doublet (vide infra) that decreases with time. The irradiated sample (, bottom) has the same MIS features as the non-irradiated sample but also has a dominant symmetric feature at g = 2.003 that does not change with time and is attributed to the RIS. Unfortunately, the RIS is very similar to the final MIS feature at g = 2.003, which poses a challenge to accurately quantifying the RIS. Comparing these spectra, however, there are two regions where the MIS is unique in spectra obtained promptly after cutting. One is the high field minimum feature and the other is the low field side of the maximum, but these MIS features decay within the first ~60 min.
Figure 2 EPR spectra (1 mW) of a fingernail sample that had not been irradiated (top) and a fingernail sample that had received 10 Gy radiation (bottom); in each case the spectra were taken at the indicated time after the sample was cut into five pieces; features (more ...)
The transient MIS features can be isolated by subtraction of the final persistent MIS spectrum from spectra collected within the first 60 min, and results of this spectral decomposition are shown in . The transient MIS spectrum, which we designate MIS3, is a symmetric doublet with g = 2.007 and A = 17 G. Its shape does not change as it decays, suggesting a quenching and/or migration of this radical created by mechanical cleavage of the alpha-keratin of fingernails.
EPR spectra (1 mW) of the initial transient MIS of a fingernail sample at the indicated time after it was cut into five pieces; EPR spectra of the final (persistent) MIS and the EPR standard have been subtracted from each spectrum.
The MIS spectrum that remains after decay of MIS3 has a major feature at g = 2.003 and additional features at lower field (higher g value). To determine if this EPR spectrum consists of multiple components, it has been measured over a range of microwave power, as shown in . While the derivative at g = 2.003 is the major feature at power < 4 mW, the spectrum shifts to a broader derivative at lower field, and it and other low field features increase with increasing power. This indicates that the persistent MIS spectrum consists of at least two components with different power saturation behavior.
EPR spectra at the indicated microwave power of the final (persistent) MIS of a fingernail sample ≥2 hr after it was cut into five pieces; features at 3455 and 3545 G are two of the six hyperfine lines of the Mn+2 in CaO EPR standard.
Since different EPR spectra dominate at lower and higher power, normalized (scaled) low and high power spectra can be subtracted from the EPR spectra to isolate the two components and their power dependence, as shown in . The derivative at g = 2.003 that is the major feature at 1 mW saturates at higher power, as does the RIS (data not shown). The broad low-field features, however, exhibit negligible saturation over a wide range of microwave power, as does the Mn+2 standard. The g = 2.003 feature that dominates the persistent MIS spectrum at lower microwave power and that closely matches the RIS is designated MIS1, while the broad features that are a minor contribution to the persistent MIS at low power but dominate at higher power are designated MIS2.
Figure 5 EPR spectra at the indicated microwave power of the g = 2.003 feature (top) and the broad features (bottom) of the final (persistent) MIS of a fingernail sample ≥2 hr after it was cut into five pieces; the g = 2.003 spectra were obtained by subtracting (more ...)
As indicated in , it is now possible to resolve an experimental MIS spectrum into three components, transient MIS3, which dominates shortly after cutting, MIS1, which is the major residual MIS feature at 3 mW and closely matches the RIS, and MIS2, which is broad and has a unique power saturation behavior. With this new understanding of the EPR spectral contributions from different mechanically generated radicals, it is possible to evaluate the EPR spectrum of irradiated fingernail parings for the RIS.
Figure 6 EPR spectrum (3 mW) of a fingernail sample 2 min after it was cut into five pieces (black), and EPR spectra of its three MIS components; MIS2 (red) was obtained by subtraction of a 3 mW spectrum from a 12 mW spectrum, and MIS1 (green) and MIS3 (lavender) (more ...)
The first goal is to identify a spectral signature of the RIS that can be used to rapidly screen individuals for radiation exposure above a threshold dose. Because of the need to cut fingernails for optimum reproducible positioning of the sample in the EPR spectrometer, this requires a feature or features that allow the RIS to be distinguished from the three MIS signals. shows representative EPR spectra of irradiated (10 Gy) and non-irradiated fingernail parings taken 2, 15 and 120 min after cutting into five pieces. While the 120 min spectra are nearly indistinguishable, except for the intensity of the signal at g = 2.003, and the 15 min spectra are generally similar, the 2 min spectra are both qualitatively and quantitatively different. This MIS spectrum, which consists primarily of MIS3, has two unique and promising features for this spectral analysis. First is the higher field minimum of the MIS3 doublet. Since the RIS contributes only to the lower field minimum of the doublet, the relative intensities of the two minima can indicate whether RIS is present or not. Second is the position of the maximum, which occurs at lower field for MIS3 than for RIS, causing a broadening to higher field in this spectral region and a shift in the crossover when RIS is present. Thus, perturbation of the 2 min MIS spectrum by the RIS provides features that have the potential to screen fingernail samples for the presence of radiation-induced radicals above a concentration (dose) threshold.
Figure 7 EPR spectra (1 mW) of a fingernail sample that had not been irradiated (top) and a fingernail sample that had received 10 Gy radiation (bottom) taken 2, 15 and 120 min after the sample was cut into five pieces; constant derivative features on both ends (more ...)
The second goal is to accurately determine the dose of radiation from the magnitude of the RIS. Because of the necessary presence of MIS, this requires the decomposition of the fingernail EPR spectrum into the MIS and RIS contributions, so the latter can be isolated and quantified. In contrast to the final (persistent) MIS spectrum, which consists primarily of the g = 2.003 MIS1 feature that is indistinguishable from the RIS, the MIS observed shortly after cutting is significantly different than the RIS, and data collected at an early time (e.g., 2 min) are more promising for this analysis. The early (transient) MIS spectrum consists predominantly of MIS3, although there are minor contributions from MIS1 and MIS2. However, MIS2 has a unique power dependence and features that are well separated to lower field, both of which can be used to subtract its minor contributions from the whole MIS spectrum at ~1 mW. (Note, the Mn+2 standard and the g = 1.97 proprietary Bruker standard also do not saturate at higher power and are subtracted at the same time.) The remaining MIS spectrum, which consists primarily of MIS3 with a small contribution from MIS1, which may be an intrinsic background signal, is remarkably consistent in shape from sample to sample at 2.0 min after cutting, as shown in (top) for fingernail samples from 11 different volunteers. Therefore, as shown in (bottom), an average 2 min MIS spectrum, which has the unique higher field minimum, can be scaled and used to subtract the remaining MIS contribution to the EPR spectrum of irradiated fingernails, leaving only the RIS. Comparison of the magnitude of this isolated RIS signal to that obtained from dose-response data with irradiated fingernail parings obtained under similar conditions can then be used to determine the radiation dose to which the fingernails were exposed.
Figure 8 Top: normalized EPR spectra (3 mW) of the initial transient MIS in fingernail samples from 11 volunteers obtained 2 min after the fingernails were cut into five pieces; Bottom: EPR spectrum of a 10 Gy irradiated fingernail sample obtained 2 min after (more ...)