Method for validating radiobiological samples using a linear accelerator
© Brengues et al.; licensee Springer on behalf of EPJ. 2014
Received: 14 August 2013
Accepted: 20 December 2013
Published: 29 April 2014
There is an immediate need for rapid triage of the population in case of a large scale exposure to ionizing radiation. Knowing the dose absorbed by the body will allow clinicians to administer medical treatment for the best chance of recovery for the victim. In addition, today’s radiotherapy treatment could benefit from additional information regarding the patient’s sensitivity to radiation before starting the treatment. As of today, there is no system in place to respond to this demand. This paper will describe specific procedures to mimic the effects of human exposure to ionizing radiation creating the tools for optimization of administered radiation dosimetry for radiotherapy and/or to estimate the doses of radiation received accidentally during a radiation event that could pose a danger to the public. In order to obtain irradiated biological samples to study ionizing radiation absorbed by the body, we performed ex-vivo irradiation of human blood samples using the linear accelerator (LINAC). The LINAC was implemented and calibrated for irradiating human whole blood samples. To test the calibration, a 2 Gy test run was successfully performed on a tube filled with water with an accuracy of 3% in dose distribution. To validate our technique the blood samples were ex-vivo irradiated and the results were analyzed using a gene expression assay to follow the effect of the ionizing irradiation by characterizing dose responsive biomarkers from radiobiological assays. The response of 5 genes was monitored resulting in expression increase with the dose of radiation received. The blood samples treated with the LINAC can provide effective irradiated blood samples suitable for molecular profiling to validate radiobiological measurements via the gene-expression based biodosimetry tools.
Since September 11, 2001, the possibilities of a radiological or nuclear terrorist attack have been a central focus for both governmental agencies and communities throughout the world. There is an urgent need to have the adequate infrastructure to rapidly assess radiation injury in such a mass casualty scenario. Biodosimetry measurements after a radiation incident will be an immensely helpful tool in order to perform screenings, triages and management of clinical facilities in a mass casualty incident . In case of an accidental radiation exposure, the effects of ionizing radiation can be wide-ranging and involve either the entire body or specific organs. Depending of the dose received, different medical treatments can be adapted and in most cases, the deadly effects of high radiation exposure (>2 Gy) can be mitigated by early triage and treatment decision. Unfortunately, as of today, no single time point measurement that is diagnostic of radiation exposure can reliably or rapidly discriminate the different levels of radiation received. Different platforms are currently under development along with the discovery and evaluation of radiation induced biomarkers to achieve that same goal of being able to measure the dose of radiation absorbed by the body [2–5]. The dicentric chromosome assay (DCA) is currently considered as the goal standard method because of its high sensitivity and accuracy . The dose estimation of this method is based on the frequency of radiation specific aberrant chromosomes with two centromeres (dicentrics) in an irradiated individual’s peripheral blood lymphocytes. But this assay is not applicable in mass casualty incident because it is very labor intensive and time consuming (several days) . The γ-H2AX assay also used in radiation biodosimetry is a direct measure of the number of DNA double strand breaks (DSB) induced by ionizing radiation [8, 9]. The yield of γ-H2AX foci has been shown to be linearly related to dose over a very wide dose range. The number of foci per cell in macaque lymphocytes after total body irradiation with doses of 1, 3.5, 6.5 and 8,5 Gy increase linearly with the irradiation dose (especially at doses greater than 1 Gy) . This assay gives a same day result, but requires that the blood samples are available within about 36 hours of irradiation. Gene expression based assay have been extensively used by many laboratories for biodosimetry measurement of radio-responsive genes in human peripheral blood lymphocytes and is a potential method that fits the criteria to provide high-throughput data with an accurate measurement and in a timely manner [10–18].
The monitoring of the dose administrated and received in radiotherapy is also needed as the possibility of radiation induced cancer exists for patients exposed intentionally to radiation. Being also able to measure the radio-sensitivity of each individual before starting radiotherapy treatment would be of great benefit not only to help control the late toxicity effect of treatment but also to integrate a personalized approach to tumor treatment based upon the chances of recovery and recidivism. One common tool needed for all of these studies is appropriate clinical samples in order to test the platforms under development. When studying the effects of ionizing radiation, it is not always possible to use in vivo samples. Several governmental research programs including NASA, Armed Forces, and BARDA agencies are pursuing work with non human primates. In addition to costs ranging in the millions of dollar and ethical issues, there is not really a standardized animal model in place to compare the results between studies . While there is a need for animal models in radiation research, it might not always be necessary and not for every stage of the research. Very often cancer patients themselves are volunteering to participate in these studies to provide irradiated blood samples while on radiotherapy treatment.
The present study focused on the technique to provide irradiated samples to study biodosimetry. The main sources to irradiated samples are either a Gammacell-40 with a Cesium-137 source or a Cobalt-60 source, as well as X-rays or electron beams for a linear accelerator which does not contain radionuclide sources. The use of cesium-137 has been discontinued for practical reason and safety concerns in radiotherapy. Cobalt-60 is currently used in external beam radiotherapy devices found mostly in developing countries. Based on a report in 2006, there were several thousand radiotherapy devices in the United States in over 2,400 institutions and clinics . Fewer than 250 cobalt-60 teletherapy devices are licensed in the United States and most of those are thought to be in storage for decay, in use for other purposes (such as fixed radiography), or in use for teaching. This is because the linear accelerator (LINAC) is considered a better, more accurate and versatile radiotherapy tool, and has largely supplanted cobalt-60 teletherapy devices in the United States and other developed countries . Another advantage of the LINAC is that it does not require replacement of the radiation source such as the cobalt or cesium sources that have an issue with radioactive decay contributing to dose inhomogeneities and errors in dose calculation. The LINAC is the device most commonly used for external beam radiation treatments for patients with cancer. It is used to treat all parts/organs of the body by delivering high-energy X-rays or electron beams to the region of the patient’s tumor. These treatments can be designed in such a way that they destroy the cancer cells while sparing the surrounding normal tissue. More recently, the LINAC has been utilized to irradiate blood components . Blood component irradiation is the only method of preventing a risk of transfusion associated graft versus host disease . It has been demonstrated that by utilizing the LINAC, the internal irradiation procedures has been proven to be safe and feasible, and along with the significant cost/time reduction suggested that it is more advantageous than external procedures in hospitals without dedicated devices . Our goal being to provide samples that will mimic real life situation as radiotherapy treatment, we irradiated our samples using the linear accelerator (LINAC). Using the LINAC for ex-vivo irradiation to study biodosimetry will allow a direct comparison with future data obtained from in-vivo irradiation. Comparing samples irradiated with the same source will eliminate false interpretations that could rise from using different irradiation methodologies.
This paper describes the process to perform irradiation on human blood samples using the LINAC in order to provide the appropriate irradiated samples as a tool for radiation biology study. To validate our method, the ex-vivo irradiated samples were analyzed to monitor the changes due to irradiation. We chose gene expression assay to analyze the data as it has been extensively used by many laboratories for biodosimetry measurement of radio-responsive genes in human peripheral blood lymphocytes, it has also been the method of choice in our laboratory for many years and it provides a result in less than 3 hours, however any assay could be used to analyze the data once the samples are irradiated with the LINAC. Validation of the use of LINAC can expand the opportunity to use this instrument for other biodosimetry studies and help to standardize the methodology for radiation trials in the future.
Results and discussion
Dose variations due to depth doses and profiles for 21EX radiation beams
Depth of Max Dose (dmax)
Depth dose variation (dmax + 1.5 cm)
Profile variation (±4 cm from center axis )
Dose measurement on blank sample
Gene expression analysis
The authors described in detail how a LINAC at a standard Radiation Oncology Department from a hospital has been implemented in order to provide samples for validating the performance of a radiogenomic test that could be used for guiding medical countermeasures against acute exposure to ionizing radiations. A customized tube holder phantom was made to position the blood sample in a way that will mimic a body material heterogeneity and have an irradiation dose distributions and delivery process as accurate and similar as possible to an in-vivo irradiation. The LINAC was also calibrated in order to distribute the most homogenous dose to the sample tubes and the result showed a 3% variation in dose distribution. The phantom is actually designed to irradiate five tubes at a time but can be adapted to irradiate a larger number of tubes if needed.
By utilizing the LINAC installed in most radiotherapy departments, it is possible to provide irradiated samples that can be used to optimize and validate the radiosensitive biomarkers panel and assay chemistry platform for gene expression measurement. Not only does the use of the LINAC for that purpose allow for unlimited quantity of irradiated blood samples, it also avoids collecting extra blood from cancer patients and the use of animal models at multiple validation phases of the study. Our methodological approach using ex-vivo irradiation of blood samples could also be an advantage to evaluate relative biological effectiveness (RBE). The general procedure for evaluating RBE of most beam equipments is standardized with survival curves on cell culture. Using ex-vivo irradiation directly on blood samples instead of cell culture would allow accessing additional biomarkers representative of the biological interactions at the tissue or organ level. The use of the LINAC is a simple, fast and reliable way to provide ex-vivo irradiated samples with a similar dose accuracy as the one delivered during radiotherapy treatment on patients. In the future, we will assess samples from total body irradiation patients to validate various gene expression signatures and assay chemistries. Since the LINAC is the instrument used for radiotherapy treatment, these in vivo samples will have the same irradiation source as our preliminary study, which is important in order to compare data. The combined data will allow correlating the expression patterns of genes that are involved in biochemical and cellular responses to the irradiation dose by using the ex-vivo LINAC approach reported in this paper.
Design of experiment
Whole blood samples were collected from volunteers and ex-vivo irradiated at 0, 2, 4, and 6 Gy using the Varian Millennium Linear Accelerator (LINAC) with a customized phantom. The irradiated blood samples were placed in culture for allowing gene expression in response to radiation after 6, 24 and 48 hours. Following exposure, the samples were harvested and the samples were processed with the CLPA and the gene expression was analyzed on the CE instrument.
Customized phantom for blood sample irradiation
Blood collection, irradiation and culture
These experiments were approved by the institutional review board (IRB#2008-036) and were conducted according to the principles expressed in the Declaration of Helsinki. Written consent for participation in the study was obtained from all the subjects voluntarily. A total of 3 healthy donors, 1 male and 2 female between the age of 21 and 55 years old participated in this study. On the day of blood irradiation, informed consent was obtained from the volunteers. Prior to phlebotomy, a single certified radiation physicist programmed the Varian 21 EX (3100 Hansen Way, Palo Alto, CA 94306) at the following settings.: 20 MeV, 20 cone, and the following Monitor Unit (MU): 212, 414 and 636 MU for 2, 4 and 6 Gy. The blood irradiation phantom was put in the center of the table and raised to 100 cm source to surface distance (SSD). Samples containing 4 ml of peripheral blood samples were collected from healthy volunteers in glass vacutainer tubes (12.35 mg Sodium Citrate, 2.21 mg Citric Acid) (VWR International, Pittsburg, PA). The vacutainers were transported by batches of 5 for irradiation. The blood was exposed to 0, 2, 4 and 6 Gy X-rays using the Varian 21EX LINAC. After irradiation, blood samples were diluted 1:1 with RPMI 1640 medium (Invitrogen) supplemented with 10% heat inactivated fetal bovine serum (Invitrogen) and incubated for 6, 24 and 48 hours at 37°C in a humidified incubator with 5% CO2. After the indicated exposure times, the sample tubes were removed from the incubator and set up in a sterile field. The blood samples were aliquoted in 2 ml Eppendorf tubes and an equal volume of CLPA reaction buffer was added (1:1). The samples were stored at 4°C.
Gene expression assay – chemical ligation dependent probe amplification (CLPA)
MB: Research Associate Scientist – Biologist – University of Arizona.
DL: Medical Physicist responsible for patient radiotherapy treatment delivery at Scottsdale Healthcare.
RK: Radio oncologist and Chief Medical Officer and Director of Virginia Piper Cancer Center at Scottsdale Healthcare.
FZ: Professor of Basic Medical Sciences - University of Arizona College of Medicine and Director of the Center for Applied Nanobioscience and Medicine (ANBM) at the University of Arizona.
We thank the members of Scottsdale Medical Imaging Ltd (SMIL) Research Institute and Scottsdale Healthcare Research Institute especially Brenda Culver for the samples irradiation, Deandra O’Connor and Joanne Saczynski for collecting the blood samples and all the volunteers who enrolled in the study. We also thank Carla Brooks for help in the laboratory and Dxterity Diagnostics for providing the assay reagents. This work was supported by the Center for High-Throughput Minimally-Invasive Radiation Biodosimetry (National Institute of Allergy and Infectious Diseases Grant U19 AI067773).
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