Using Stable Free Radicals to Obtain Unique and Clinically Useful Data In Vivo in Human Subjects

Abstract

This paper attempts to: (1) provide a critical overview of the challenges and opportunities to extend electron paramagnetic resonance (EPR) into practical applications in human subjects, based on EPR measurements made in vivo; (2) summarize the clinical applications of EPR for improving treatments in cancer, wound healing and diabetic care, emphasizing EPR's unique capability to measure tissue oxygen repeatedly and with particular sensitivity to hypoxia and (3) summarize the capabilities of in vivo EPR to measure radiation dose for triage and medical guidance after a large-scale radiation exposure. The conclusion is that while still at a relatively early stage of its development and availability, clinical applications of EPR already have demonstrated significant value and the field is likely to grow in both the extent of its applications and its impact on significant problems.

INTRODUCTION AND OVERVIEW

Since the invention of electron paramagnetic resonance (EPR, also called electron spin resonance, ESR) spectroscopy by Zavoisky in 1944, EPR has been utilized in many fields to study systems containing unpaired electrons, especially free radicals. However, because most free radicals are very reactive and do not occur in high concentrations in tissues and also because of the problems with non-resonant absorption of microwaves by tissues, in vivo clinical applications of EPR have not been widely implemented to date. Nonetheless, there is a growing recognition of the rich potential for applying in vivo EPR in human subjects both in biodosimetry and for improving clinical treatment. This burgeoning field is well illustrated by the articles in this special issue stemming from the international gathering of scientists held at Dartmouth in 2015.

Both because of the author's involvement in the organization of this meeting and because of the author's long-term personal involvement in promulgating and contributing to the clinical applications of EPR (as Gallez’ article in this special issue⁽¹⁾ has exceedingly generously noted), the author feels well-positioned to provide an overview of the current status and potential future developments of clinical applications of EPR.

To that end, this paper undertakes to critically overview the challenges and opportunities to extend EPR's capabilities into practical applications in human subjects, based on measurements made in vivo and to summarize the clinical applications of in vivo EPR. The first type of clinical application reviewed has the potential to improve treatments in cancer, wound healing and diabetic care (which highlights the value added by EPR's unique capability to measure tissue oxygen repeatedly and with particular sensitivity for hypoxia). The second application is to measure radiation dose of individuals after a large-scale radiation exposure (which emphasizes its particular strengths for estimating dose in the circumstances needed for triage and medical guidance).

This review will of necessity draw heavily from the body of work with which the author has been involved, which, it is gratefully noted, is the product of the research accomplishments and intellectual input from many, many people. While too numerous to acknowledge all, an attempt to name many of the sources of the key contributions and collaborations is provided in the ‘Acknowledgements’ section.

Strengths of EPR for clinical applications

If EPR is to significantly impact clinical care, it is essential to identify not just what types of measurements could be made, but to focus on those that EPR can do better than other techniques. ‘Better’ in this context needs to be judged by factors that are critical to their clinical usefulness including: (1) the quality of the measurements, (2) their feasibility to be applied within the constraints of clinical medicine and (3) their comparative effectiveness to achieve the desired clinical goals.

Fortunately, EPR has a number of capabilities that fit these criteria. Table 1 summarizes several features of EPR spectroscopy that are of particular importance for studying biological systems in general and of potential practical importance for clinical applications. The two most promising applications are for measuring tissue oxygen (oximetry) and assessing unknown and clinically significant exposure to radiation (dosimetry). These features and capabilities, including the development of the specialized instruments and techniques needed for their effective clinical applications, are discussed below in subsequent sections and detailed further in the references cited in the section for oximetry^(2–21) and dosimetry^(22–31).

Table 1.

Unique or exceptional capabilities of EPR for studies in live biological systems.

General capabilities of what EPR can measure in biological systems	Capabilities specific to clinical applications of oximetry (in clinic or hospital settings)	Capabilities related to clinical applications of dosimetry by EPR (for unplanned exposures)
Free radicals important in biological systems Nitric oxide (e.g. important in vasodilation, neurotransmission, immune response) Reactive radicals, especially oxygen centered (e.g. important in therapeutic response) Free radical intermediates of drugs, metabolites, etc. Radiation-induced free radicals (useful especially for dosimetry)	Measures oxygen in tissue and responsiveness of tissue to inspired oxygen	Repeated in vivo measurements as needed, valid indefinitely Measurements can be accomplished in 5 minutes
Sensitivity to the physical environment of cells and tissuesa(21) Oxygen Motion Viscosity pH Electrical charge and potential Polarity (hydrophilicity) Temperature	Particularly sensitive to hypoxic levels in tissues (which are critical for causing pathophysiology and impact efficacy of treatment)	Sensitive to clinically meaningful levels of exposure Unaffected by co-occurring trauma such as burns, physical trauma, disease
Sensitivity to redox state (i.e. changes in oxidative state in biological systems) Redox sensitive metabolism(17, 20, 21) Redox sensitive contrast agents for MRI(32)	Measurements are non-invasive (after injecting an initial oxygen-sensitive paramagnetic material)	Non-invasive measurements based on teeth or nails
Paramagnetic states of metal ions in biological systems(16, 18)	Instrument is classified as a non-significant risk device Instrument can be made transportable, for use at the bedside, intraoperatively or during therapy	Instrument is transportable, easily operated by untrained operators, capable of operating off one power outlet or generator and usable at the point-of-care (i.e. movable to temporary facilities at the site of disaster) Instrument is a non-significant risk device for use in human subjects

^aUsually using nitroxides.

While all of the capabilities in Table 1 have been shown to be feasible (and usually valuable) in preclinical studies, two features have been especially productive for direct applications in human subjects: repeated measurements of oxygen in tissues⁽³³⁾ and after-the fact measurements of radiation-induced free radicals⁽²⁸⁾. The remainder of this review, therefore, focuses on these two capabilities with initial evidence of their successful use. It seems quite plausible that, once clinical use of EPR spectroscopy is more widely established in these initial uses, other uses also may be successfully introduced into clinical care, including imaging based on EPR.

OVERVIEW OF CLINICAL APPLICATIONS BASED ON EPR'S UNIQUE CAPABILITY TO MEASURE TISSUE OXYGEN DIRECTLY AND REPEATEDLY

The clinical value of the capability of EPR oximetry to make repeated measurements of oxygen in tissues is based on the fact that oxygen levels are critical for a large range of physiological and pathophysiological processes^(7–9). Despite its centrality, it is seldom measured directly in vivo in patients’ tissues. If oxygen is measured at all, measurements usually assess the level of oxygen in the vascular system, while the greater need for many clinical applications is to know the level in tissues. For example, in the treatment of cancer by radiation, the level of oxygen in the tumor is the single most important variable that affects the outcomes, and it has been shown to be an important variable in outcomes of chemotherapy and immunotherapy^(33–39).

Oxygen also can be a critical variable in surgical outcomes for cancer patients, affecting the success of the initial surgery, especially following neoadjuvant treatment by radiation or previous surgery in the operative area. It is also important for healing of all surgical wounds and can be crucially important for cancer surgery where skin or transplanted tissue flaps may not be well oxygenated. Ischemia per se and ischemia-reperfusion injury are also both intrinsically connected to the availability of oxygen; this is especially important in treating peripheral vascular disease, including that due to complications of diabetes⁽⁵⁾.

Measurement of oxygen by EPR is based on the effects of molecular oxygen on the EPR spectra of paramagnetic materials⁽¹⁰⁾. The two unpaired electrons of molecular oxygen generate a fluctuating magnetic field that perturbs the EPR spectra in a concentration-dependent manner, enabling the changes in the EPR spectra to be quantitatively related to the local concentration of oxygen.

Soluble oxygen-sensitive materials can be imaged with the use of appropriate magnetic field gradients and image reconstruction methods similar to those used for magnetic resonance imaging (MRI)^(40–42). Some very promising results using this approach have already been obtained in animals, but there are challenges in the translation of these capabilities to direct clinical applications because of the need to obtain regulatory approval for the imaging agent and the relatively low sensitivity at the lower frequencies that must be used to obtain sufficient depth penetration for imaging with external resonators.

It has instead been focused on the use of particulate oxygen-sensitive EPR materials and measurements made via spectroscopy instead of imaging⁽⁵⁾. There are two practical reasons for this approach. The first is because of this focus on the importance of being able to apply measurements to patients as soon as feasible. The second is due to practical problems (e.g. the financial cost and time required to complete the clinical studies needed to gain FDA approval). The latter is well illustrated by the processes that are needed to gain regulatory approval for soluble imaging agents, which necessarily need to follow a New Drug Application (NDA) process to obtain FDA approval similar to that for other new drugs used in diagnostic studies including new MRI imaging agents.

One such particulate, India ink, made of paramagnetic carbon particulates, is already in clinical use for other purposes (such as permanently marking the edges of radiation fields for guiding placement during fractionated therapy and pre-surgical marking for identification of thyroid or breast or colon cancer tumors). The FDA does not require regulatory approval of India ink for injections in humans, recognizing its long history of safety in cosmetic tattooing and a few decades of use as a ‘medical device’ for injecting into patients⁽⁴³⁾. Therefore, it has been able to initiate clinical studies using oximetry with a paramagnetic India ink as the sensor, without needing to undergo the FDA's laborious premarket approval process as noted above for new drugs.

At Dartmouth (as of mid-2016), 28 subjects (16 patients and 12 healthy volunteers) have been successfully measured repeatedly on a total of 37 different sites using India ink. Emory University in Atlanta, GA, in collaboration with Dartmouth, has measured an additional six tumor patients using India ink.

Dartmouth is also conducting an EPR oximetry study using Lithium octa-n-butoxy-naphthalocyanine (LiNc-BuO) (referred to as ‘the OxyChip’). Because this sensor has not been approved for use in humans, it has required obtaining an Investigational Device Exemption (IDE) from the FDA. An early Phase 1 clinical study to establish its safety is underway, with an additional six patients, whose tumor (and therefore OxyChip) is planned to be resected, have been enrolled as of mid-2016. An additional six patients will be enrolled in order to examine the OxyChip's safety and use during the period when they receive neoadjuvant radiation therapy or chemotherapy prior to surgery.

Data on these patients have provided very encouraging information regarding the ability to measure oxygen levels in tumors and their response to patient's breathing a gas mixture that is enriched in oxygen. Moreover, these results in patients directly demonstrate the feasibility of making such measurements under clinically practicable conditions and, importantly, confirm the potential value of knowing such information during the course of treatment. (See the overview of clinical problems potentially benefitting from oximetry in Table 2..

Table 2.

Current clinical applications of EPR oximetry.

Clinical problem	Parameter to be measured	Status of measurements in human subjects	Rationale for using in vivo EPR
Peripheral vascular disease	Oxygen at sites of likely pathophysiology (e.g. plantar surface of the foot in diabetics)	Measurements underway in normal volunteers Patients to be studied in near future	Oxygen in the tissues is the most significant pathophysiological variable. No other method is available to make such direct measurements. Oxygen measurements will facilitate evaluating success of therapeutic intervention.
Cancer	Oxygen content in tumors Responsiveness of tumor oxygen to inspiring enriched oxygen	Measurements underway in patients with tumors within 25 mm of the surface Intensive developments underway for measurements at deeper sites	The response of tumors to cytotoxic therapy, especially ionizing radiation, is critically dependent on oxygen. Anti-tumor therapies are given repeatedly and often change oxygen, so repeated measurements are desirable. Knowledge of the changes in individual patients could significantly optimize the type and timing of the therapy.
Wound healing	Oxygen content at various sites in wounds Later on, may also measure reactive oxygen species	Existing clinical protocols use an apparatus compatible with EPR measurements No EPR study has been initiated to date	The oxygen content is a critical variable for successful healing of wounds. Direct measurements would identify patients likely to have poor healing and allow responses to therapy aimed at improving oxygen in the healing wound.
Changes in tumor beds caused by cancer therapy, e.g. fibrosis or neuropathy	Oxygen content in irradiated tumor beds and peripheral normal tissue	Initial measurements underway for effects during radiation and following both radiation and surgery	Radiation-induced hypoxia may play a critical role in the signaling of pro-inflammatory, pro-fibrotic, and pro-angiogenic growth factors and cytokines that lead to tissue fibrosis or debilitating nerve pain.

Furthermore, based on these clinical studies using India ink and on additional preclinical studies in tumor and muscle, the National Cancer Institute (NCI) provides further evidence of the external recognition of the potential clinical importance of making direct, repeated measurements in tissue by way of a study section's very favorable reviews of the proposed work. Subsequently, the NCI awarded funding to Dartmouth to continue these developments via a 5 y Program Project Grant (PPG), in collaboration with colleagues at Emory and the Catholic University of Louvain (CUL) in Brussels, Belgium.

In addition to these important practical considerations associated with India ink (i.e. the immediate feasibility of making measurements in human subjects using India ink without the delays associated with seeking IDEs or NDAs from the FDA and fulfilling other regulatory requirements), EPR oximetry based on carbon particulates has two inter-related features that make it potentially very valuable for clinical applications, i.e. the capability to provide both repeated and direct quantitative measurements of oxygen in the tissue. Both features are very unusual, and the combination is unique to EPR oximetry with particulates (irrespective of the type of sensor).

Because EPR oximetry gives direct measurements in the tissue of interest, there is no ambiguity about the relevance of the measured value to the site of interest. This is in contrast to techniques such as near infrared which give information on oxygen saturation in the vasculature or MRI techniques which provide parameters that often affect tissue oxygen but do not directly assess the oxygen levels in the tissue.

The ability to repeat the measurements at the same site and as often as desired without requiring readministration of the paramagnetic sensor also provides uniquely reliable and accurate measurements of oxygen that track the effects of treatment and disease progression. Because the India ink is permanent (it has measured for >10 y in some subjects) and the OxyChip is left intact until surgically removed (for up to 1 month to date, probably can be indefinite) these repeated in vivo EPR oximetry measurements are also completely non-invasive.

Summary of EPR oximetry's unique clinical capabilities

EPR oximetry measurements have several features that are quite useful for routine clinical applications (summarized in Table 1). As noted above, the measurements themselves are non-invasive (although the initial introduction of the sensor-probe is minimally invasive). Measurements are made in the tissue at the site of interest with sub-mm resolution, where the spatial resolution is determined principally by the size of the paramagnetic material that is injected/implanted. Under some circumstances, the spatial resolution can be further enhanced by using multiple magnetic field gradients^{(11, 12)}.

EPR oximetry measurements are especially sensitive and accurate (i.e. with sensitivity of <1 mm Hg) at the very low (hypoxic) levels of tissue oxygen that are most important for assessing the pathophysiology and they have a strong effect on the response to treatment⁽¹⁰⁾. Acquisition of these data and outputting of the results can be fully automated, making it very feasible for the measurements to be obtained routinely by technicians and with results made immediately available to the clinical team. The output consists of the levels of oxygen at one or more sites whose location is well characterized because the oxygen-sensitive material will have been placed in the desired sites before or at the start of the clinical study.

Ongoing efforts in EPR oximetry

The current emphasis on clinical oximetry at Dartmouth is centered on applications for cancer that are being carried out in the aforementioned PPG funded by NCI and illustrates the capabilities and challenges of clinical EPR oximetry. The aim of the PPG is to provide the first systematic data on the clinical use of EPR oximetry to obtain the oxygen measurements in tumors, tumor beds and relevant (usually adjacent) normal tissues. During each measurement session, oximetry can be used to assess whether the tumor oxygen responds to inspiring enriched oxygen.

Importantly, oxygen measurements can be obtained prior to and during treatment (e.g. radio- or chemotherapy) that is typically carried out over several weeks or months. These data can then be used clinically to optimize a treatment plan by selection of appropriate patients for the therapy and by delivering the most effective therapy at the most effective time (i.e. especially if the tumor responds to enriched oxygen therapy or has been shown to vary over time).

There are several key considerations when designing such an initial study to ensure a successful translation to clinical practice, including demonstrating the capability to obtain clinically pertinent data, showing that the data can be obtained within the constraints of ongoing clinical care and meeting regulatory requirements. The approach in Dartmouth's PPG is to use three complementary approaches (India ink, the OxyChip and the implantable resonator) for the oxygen-sensitive paramagnetic materials that offer different advantages to achieving these considerations. Namely, India ink is already approved for use in human subjects without requiring exemption from the FDA because of its past and current widespread use in human subjects⁽⁴³⁾. It already has been shown to provide very useful data in both tumors and normal tissues within 5–10 mm of the surface, and the material was used for the pivotal studies that were an important part of the data for the funding of the PPG⁽¹⁹⁾.

The second approach (OxyChip) uses one of the paramagnetic phthalocyanines that have been developed to provide very narrow and intense EPR signals, whose linewidth is significantly affected by oxygen⁽⁴⁾. It has the potential to be implanted into tumors at greater depth, up to 2.5 cm. Although it requires IDE exemption from the FDA and first-in-human Phase 1 studies prior to more general investigational use, it is anticipated that this will become a widely used approach.

The third approach uses an implantable resonator with coated phthalocyanines attached to a wire that, after placement in the site(s) of interest, can provide highly sensitive measurements of tissue oxygen from virtually any site and depth^{(2, 6)}.

The path forward for clinical oximetry

The initial clinical oximetry studies are being carried out with a principal focus on enhancing the treatment of cancer by using information gained by repeated measurements of tissue oxygen to select patients who would benefit from interventions to increase oxygenation and to optimize the timing and type of treatment to be used. It is very likely that the applications of EPR oximetry to the management of peripheral vascular disease in diabetics will be another main focus of EPR clinical oximetry in the immediate future, building on oximetry measurements in 16 volunteers in 24 injection sites in the feet of normal volunteers, with repeated measurements extending over 10 y to date.

Pilot clinical studies applied to other clinical problems, such as the role of hypoxia in severe peripheral neuropathy associated with high-dose cancer chemotherapy and measuring the effect of hyperbaric oxygen on tissue oxygen, are also being pursued at Dartmouth.

While the use of India ink as the paramagnetic probe for oxygen will continue to be very important, ongoing studies are using lithium phthalocyanine (and related derivatives) as alternative implantable sensor-probes. The probes, in microcrystalline form, are first coated by an FDA-approved biocompatible material that is oxygen permeable, i.e. coated by polydimethylsiloxane prior to implantation⁽⁴⁾.

If these probes succeed clinically and are approved by the FDA for continued evaluation, they are likely to become very widely used. Such materials would extend the ability to make measurements from the surface, to as deep as 25 mm. This depth would very significantly extend the applicability of EPR oximetry for tumors.

The depth achievable with India ink (5–10 mm) already is sufficient for uses for peripheral vascular disease. Its clinical parallels to tattoos also make its use on the foot especially non-perturbing. Thus, this application using India ink will probably continue to be pursued because of its ease of administration and other practical advantages.

For more deeply located tumors, plans are underway to use implantable resonators^{(2, 6)}. In preliminary discussions with the FDA, it appears that there will be a straightforward and feasible regulatory approval path to obtain prompt IDE approval, using a strategy for these resonators that is based on using the same paramagnetic material as the OxyChip and using a biocompatible coating that is similar to that already approved by the FDA for an initial first in humans IDE trial of the OxyChip.

The challenges for clinical EPR oximetry

Regulatory challenges

As noted above, because of the time, effort and cost of introducing new materials for use in human subjects, it was focused initially on a sensor that does not require IDE approval (India ink) and then used these clinical studies along with preclinical studies using the OxyChip to obtain IDE approval for a Phase 1 study. The OxyChip thereby can expand the tissues measured to a greater depth but currently require their surgical removal.

The regulatory strategy for overcoming the challenge of making measurements at deeper sites is being addressed by developing implantable resonators, using a similar paramagnetic material as in the OxyChip, but with a resonant circuit that can be placed at any depth in the human body. The regulatory process will be facilitated by using the ‘OxyChip’ as the paramagnetic material, building on the acceptance of the FDA for its use as a direct implant. It has two additional potential advantages over the OxyChip used as a direct implant. First, having a small loop placed immediately below the body surface with a hard wire attachment to the rest of the structure will facilitate the ease of removing it if needed. And having several sensor sites on the same resonator will permit independent assessments of oxygen levels at several sites within the tissue.

Clinical challenges

The widespread clinical adoption of a new device or drug requires that sufficient evidence be obtained to demonstrate to the potential users that this will be a significant advancement over that which is currently available and that it will be practical to use. Clinicians treating the diseases listed in Table 2 generally recognize the potential for improving treatment that comes from having repeated measurements of tissue oxygen.

Yet it remains challenging to show that the techniques can be implemented within the usual constraints of safe and effective clinical practice and will be acceptable to both patients and clinical teams, i.e. that it can fit well into the clinical flow of care. The latter requires that the measurements can be made quickly, comfortably, and with procedures and apparatus that does not hinder the flow of clinical care.

Conclusions regarding the future of clinical oximetry

The opportunities and challenges have shaped which clinical applications that are pursuing initially and how these are being proceeding, including being cognizant of the human factors involved in engineering designs for successful operation of the instrument. Clinical interest in the PPG is driven by the widespread understanding among clinical oncologists that the amount of oxygen in a tumor is a very major factor in determining its response to therapy.

This method of measurements includes assessing whether a subject's tissue oxygen responds to a simple therapy: using a non-rebreather facemask to deliver normobaric oxygen-enriched breathing mixtures. This approach was chosen because it can readily be built into oximetry assessments without significantly perturbing the flow of clinical care. Later, assuming that the value of this approach has been demonstrated and adopted widely, it will likely be feasible to add in other methods to improve oxygen in tissues and perhaps include more complex procedures. These initial key challenges, therefore, are to show that the measurements can be made under clinically applicable conditions, and then show that the data obtained can be used to enhance therapeutic outcomes. The future looks very bright.

OVERVIEW OF CAPABILITIES OF IN VIVO EPR TO MEASURE RADIATION DOSE FOR TRIAGE AND FOR MEDICAL GUIDANCE AFTER A LARGE-SCALE RADIATION EXPOSURE

The key issue for medical management of victims in a large-scale radiation event centers on the need to rapidly sort out those who are significantly exposed from those who are not, in a group that can exceed 1 million people^(44–46). The accuracy (and credibility for victims) of this sorting requires knowing the dose at the level of the individual. The need is to determine who may have received a dose of radiation that puts them at a significant risk for experiencing a potentially fatal acute radiation syndrome response if not treated. The cutoff dose usually used for this sorting decision (referred to as a triage decision) is two gray, although plausibly a somewhat higher dose could be used as the cutoff⁽⁴⁴⁾.

In recognition that the existing guidelines for triage, which are based on methods found to be useful for very small radiation accidents involving only a few individuals, federal agencies especially in the USA, have undertaken programmes to develop improved methods based on ‘biodosimetry’^(44–46); also see comments from NIAID in the introduction to this special issue⁽⁴⁷⁾. In biodosimetry, the radiation-induced changes in the tissues of potentially exposed individual are used to estimate the dose of radiation actually received⁽²⁹⁾. Biodosimetry has the advantage of providing dose estimates at the level of the individual, without requiring everyone to have had prior access to an external monitor such a radiation badge to measure the individual's dose.

This approach for biodosimetry is based on the fact that ionizing radiation generates large numbers of unpaired electron species⁽²⁸⁾. While most of these react immediately and disappear, in some materials in which diffusion is limited, the unpaired electrons can persist for long periods. EPR specifically and sensitively responds to the presence of unpaired electrons. This phenomenon has been recognized for more than 50 y to occur in bone and teeth and has been shown to be a feasible method for retrospective dosimetry in teeth and nails⁽⁴⁸⁾. The basic advantages of EPR for addressing these dosimetry needs are summarized in Table 1.

These recent contributions have focused on making these measurements in vivo and under conditions that are suitable for use for triage of large-scale radiation events, using radiation-induced free radicals in teeth or nails for the measurements^{(30, 31)}. The overall goal of these developments at Dartmouth is to produce an effective prototype for one or more dosimetry devices that meet fully the need for effective triage after a large-scale radiation event, with operation by minimally trained personnel. The level of development should be sufficient to be the basis for rapid production of FDA approvable instruments by an appropriate medical device company.

Biodosimetry based on EPR signals in teeth in vivo

The most developed use of EPR for in vivo biodosimetry is based on the radiation-induced stable free radicals generated by ionizing radiation in the hydroxyapatite matrix of teeth. (See Flood et al.⁽³⁰⁾ for a description of the current instrument and clinical studies using it; see Schreiber et al.⁽⁴⁹⁾ for advanced developments underway for a new resonator approach.)

The resultant EPR signal in teeth is extremely stable, with small changes in the first few hours and then a persistent signal lasting millions of years. (See Kinoshita et al.⁽⁵⁰⁾ in this issue for uses for dating teeth.) For these measurements in teeth in vivo, the two central upper incisors are used. These teeth have a relatively smooth surface, which facilitates placing the detection loop on them reproducibly, and they are less likely to have had extensive dental restorations. Using these teeth also allows easy access by operators with no previous training, using a highly automated transportable instrument. Using such an apparatus, it has been demonstrated that, with <5 minutes of data acquisition, it can be satisfactorily resolved whether the dose received is likely to be above the threshold set for initial triage.

In regard to testing these devices in clinical studies, the number of subjects available who have received homogenous irradiation of the upper incisors is necessarily limited, but useful data can be obtained from a few such patients who receive total body irradiation (TBI) as part of their treatment prior to receiving bone marrow transplants. Figure 1 demonstrates that, in human subjects measured in vivo, a linear relationship between dose and the magnitude of the EPR signal was observed; this observed relationship is as expected based on numerous studies done with human teeth irradiated in vitro⁽⁵¹⁾.

Figure 1.

Dose response of EPR measurements made in vivo in teeth. For each measurement, three to five independent sets of data were collected; data collection for each set required 60 seconds, and results from the sets were averaged to provide a single dose estimate. In total, 59 measurements were made in 46 people; 18 measurements were made in 11 TBI patients (2 at 0 Gy, 1 at 1.5 Gy, 12 at 2 Gy and 3 at 12 Gy). For boxes at 0 and 2 Gy, the central mark is the median, the edges of the box are the 25th and 75th percentiles, and the whiskers extend to the most extreme data points. At 1.5 and 12 Gy crosses denote individual measurements. These data establish an in vivo calibration curve (EPR amplitude in V versus dose in Gy) with a 1.25 Gy standard error of inverse dose prediction (reproduced from Figure 7⁽²⁵⁾).

Fortunately, precise measurements of the ability to resolve dose can be estimated by observing the variability of the EPR signal in in vivo measurements of the teeth of normal subjects, i.e. people who have not been exposed to significant therapeutic irradiation. Some of these results from normal subjects are included in Figure 1; another clinical study that was focused on normal subjects is detailed in Figure 2 in Flood et al.⁽³⁰⁾ in this same issue.

Because a large-scale radiation incident will be accompanied by many unusual clinical circumstances, the instrument has been designed to be transportable to and operable in a makeshift and temporary facility, perhaps without electrical power. Figure 2 illustrates the field-ready use of the EPR tooth dosemeter; a measurement is being made at a local fundraising event outdoors and inside a tent located on an actual field, using electrical power provided by a generator.

Figure 2.

Seated volunteer being measured by in vivo tooth dosemeter in 2014 inside a tent and using generator power for the instrument.

Figure 3 shows two variations planned for the final prototype: a standalone version that requires no additional furniture and a tabletop version that can be used with any table/desk surface and chair. These designs take into account the ergonomic needs and variability in size of the operator as well as the subjects. Because it will be necessary to utilize previously untrained personnel to carry out required functions in an actual radiation event, the process is also almost fully automated so that non-experts can readily operate it.

Figure 3.

Prototypes of standalone (on left) and tabletop (on right) versions of in vivo tooth dosimetry instrument. The standalone is a completely self-contained unit where the subject sits on the stool facing the dosemeter and the operator stands in front of the instrument. The tabletop version is designed to be secured onto any table and the subject can sit in any simple chair. The instruments are otherwise identical and be adjusted up and down to accommodate subject size for comfort. The magnet poles (the semicircular arms) are shown rotated out of the way in the standalone version; this allows for easy access and placement of the subject's teeth for measurement. The tabletop version illustrates the magnet poles rotated up (to surround the subject's head) for measurement.

Summary of current status of tooth dosimetry

Using the upper incisor teeth for the measurements, it has a fully functional and field deployable instrument able to determine dose with accuracy sufficient for the needs of initial triage, with <5 minutes of data acquisition. A partially functional mock-up of the final prototypes of the initial design of a fully automated, field deployable system that is suitable for use by non-expert operators to carry out the triage has been built. While there are some remaining steps including full implementation and testing of the final design and obtaining regulatory approval for manufacturing the device for initial triage, this could be ready for response to a large-scale radiation incident within 1–2 y.

Biodosimetry based on EPR signals in nails in vivo

EPR biodosimetry based on nails builds on a fairly extensive literature and this own experience in using radiation-induced EPR signals in nail clippings to estimate radiation dose for triage^{(26, 27, 31, 52)}. While the use of nail clippings is very promising, the current capabilities of using it for response to a large radiation event have some significant challenges. The method currently requires fairly complex measurements that are difficult to carry out in the field, because of relatively low sensitivity of nails compared to teeth and especially because of the presence of a potentially confounding EPR signal from the process of clipping the nails. The latter both induces a potentially interfering set of EPR signals and leads to complex changes over time. Therefore, the methods have been developed to make the measurements in vivo, eliminating the need to carry out complex analyses in a laboratory⁽²³⁾. Instead, a transportable EPR device has been designed that could be deployed in the field and make the measurements directly in the nails in situ on the hands and feet without clipping.

Because of the lower intensity of EPR detectable signals in irradiated nails compared to teeth, nail dosimetry usually is carried out at a higher frequency than tooth dosimetry, i.e. at 9.5 GHz versus 1.2 GHz. The higher frequency is rapidly absorbed in lossy materials such as the normal tissues in the fingers and toes. When making measurements in clippings of nails, this is not a problem because the nail clippings have very low content of water and are detached from the body. When making measurements of nails in situ, however, there is a significant technical challenge to constrain the electric field of the 9.5 GHz microwaves so that they do not interact strongly with the soft tissue beneath the nails.

A systematic set of studies has, therefore, been carried out to develop a resonator that can have sufficient intensity of the magnetic field component of the microwaves to make measurements in the nails while limiting the electrical field component in the underlying tissue of the nail bed^{(22, 23)}. The basic process and representative spectra are shown in Figure 4.

Figure 4.

Illustration of EPR spectra obtained in vivo with a resonator developed for measuring nails in vivo. The inset shows the resonator with the thumb in place. The spectrum is the EPR signal seen when a piece of char, with the intensity of an EPR signal equivalent to about 30 Gy, had been placed on a fingernail.

The nature of how measurements can be made on finger and toenails readily lend themselves to designing a field deployable dosemeter in which it would be feasible to measure one or more nails on multiple limbs simultaneously, as depicted in Figure 5.

Figure 5.

Schematic of a potentially field deployable dosemeter based on making simultaneous in vivo EPR measurements in nails. Small local magnets are used for measurements that could be made on all four limbs to provide spatially resolved estimates of dose.

Multiple measurements per se have potentially significant added-value for triage and acute medical care for two reasons. First, since EPR measurements provide direct evidence about exposure to a specific tissue, having measurements on each limb can provide independent, spatially separated data about the homogeneity/heterogeneity of the exposure to the person. If the exposure was heterogeneous, it implies that some bone marrow may have been spared, thereby reducing the likelihood of having acute radiation syndrome and needing treatment for blood cell death. Second, if there is evidence of homogeneity, the measurements can be averaged, enhancing the sensitivity of the dose estimate.

Summary of current status of in vivo nail dosimetry

The availability of in vivo nail biodosimetry would be very valuable independently of as well as in complement to tooth dosimetry, especially because it can provide information on the homogeneity of the exposure and potentially provide even greater resolution of total dose. The options of using intact nails and teeth also increase the likelihood that an individual can be successfully measured, regardless of injury or prior disease.

The level of technology development of in vivo nail dosimetry, however, is far from complete. While its feasibility has been demonstrated, full achievement of dose resolution needs to be verified. There also is a need to fully implement the very promising field deployable versions. Rapid progress leading to a deployable instrument in 2–3 y could be achieved with a dedicated and adequately funded effort.

OVERALL CONCLUSIONS

In vivo measurements using EPR in human subjects, for both oximetry and dosimetry, have now been established as feasible and valuable. The current applications of oximetry involve repeated measurements of oxygen in pathophysiologically compromised tissues, especially in clinical treatment of tumors and peripheral vascular disease, and in radiation dosimetry for initial triage for large-scale radiation events.

As these developments and other articles in this special issue illustrate, the needs for these developments remain strong^{(44, 46, 53)} and the future appears bright for the increasing evolution and diffusion of these techniques. In both of these very different clinical applications, i.e. standard settings for providing clinical care for oximetry and disaster medical response for dosimetry, the specific applications have demonstrated their potential for making significant improvements to outcomes and their value and practical capacity to be integrated into the clinical context.

DISCLOSURE

H.M.S. is an owner of Clin-EPR, LLC, which manufactures EPR investigational devices for clinical applications.