This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.
The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.
The aim of radiobiology is to establish links between the delivered dose and the biological effects; dosimetry is a crucial aspect in the design of radiobiological experiments. For over 30 years, the importance of dosimetry standards and the harmonization of practices have been highlighted1,2,3,4,5. To establish a dose rate reference, several protocols exist6,7,8,9,10; however, as shown by Peixoto and Andreo11 , there can be differences of up to 7% depending on the dosimetric quantity used for the dose rate determination. Moreover, even if protocols exist, it is sometimes difficult to know which protocol is the most suitable for a particular application, if any, because the dose rate for the cells depends on parameters such as the cell container, quantity of cell culture media or beam quality, for example. The scattering and the backscattering for this type of irradiation is also a very important parameter to take into account. Indeed, for low and medium energy X-rays, in the AAPM TG-61 reference protocol10, the absorbed dose in water is measured at the surface of a water phantom. Taking into account the very specific cell irradiation conditions, the small volume of cell culture media surrounded by air is closer to kerma conditions than those defined for an absorbed dose with a large water equivalent phantom as in the TG-61 protocol. Therefore, we have chosen to use the kerma in water as a dosimetric quantity for reference rather than the absorbed dose in water. Thus, we are proposing a new approach to provide a better determination of the actual dose delivered to cells.
Moreover, another crucial aspect for radiobiological studies is the complete reporting of the methods and protocols used for irradiation in order to be able to reproduce, interpret and compare experimental results. In 2016, Pedersen et al.12 highlighted the inadequate reporting of dosimetry in preclinical radiobiological studies. A larger recent study from Draeger et al.13 highlighted that even though some dosimetry parameters such as the dose, energy, or source type are reported, a large part of the physics and dosimetry parameters that are essential to properly replicate the irradiation conditions are missing. This large scale review, of more than 1,000 publications covering the past 20 years, shows a significant lack of the reporting of the physics and dosimetry conditions in radiobiological studies. Thus, a complete description of the protocol and the method utilized in radiobiological studies is mandatory in order to have robust and reproducible experiments.
Taking into account these different aspects, for the radiobiological experiments carried out at IRSN (Institute of Radiation Protection and Nuclear Safety), a stringent protocol was implemented for cell irradiations in an orthovoltage facility. This dosimetry protocol was designed in order to simulate the real cell irradiation conditions as much as possible and thus, to determine the actual dose delivered to cells. To this end, all the irradiation parameters are listed, and the beam quality index was evaluated by measuring the half value layer (HVL) for which some adaptations have been made as the standard recommendations from the AAPM protocol10 cannot be followed. The absolute dose rate measurement was then performed with the ionization chamber inside the cell container used for cell irradiations, and the attenuation and the scattering of the cell culture media was also quantified with EBT3 radiochromic films. As the modification of only one single parameter of the protocol can significantly impact the dose estimation, a dedicated dosimetry is performed for each cell irradiation configuration. Moreover, the HVL value must be calculated for each voltage-filter combination. In this present work, a voltage of 220 kV, an intensity of 3 mA, and an inherent and an additional filtration of 0.8 mm and 0.15 mm of beryllium and copper, respectively, are used. The cell irradiation configuration chosen is on a T25 flask, where cells were irradiated with 5 mL of cell culture media.
1. Irradiation platform and determination of irradiation parameters
2. Beam quality index: determination of the half value layer
NOTE: The HVL is defined as the thickness of an attenuator (usually copper or aluminum) to reduce the intensity of the beam by a factor of two compared with the original value.
3. Evaluation of the irradiation field (no dose estimation)
4. Dose rate measurement with ionization chamber
5. Measurement of cell culture media attenuation and scattering
NOTE: Handle EBT3 films with gloves throughout the procedure.
6. Reading of EBT3 radiochromic films
7. Determination of the dose rate at the level of the cell monolayer
In this work, we used a platform dedicated to small animal irradiation19; however, this platform can be used to irradiate other types of samples such as cells. The irradiation source is a Varian X-ray tube (NDI-225-22) having an inherent filtration of 0.8 mm of beryllium, a large focal sport size of 3 mm, a high voltage range of about 30 to 225 kV and a maximal intensity of 30 mA.
The parameters used for this study are reported in Table 1. We have chosen to show an example of the use of this protocol for cell irradiation in a T25 flask with 5 mL of cell culture media.
Half value layer
Table 2 reports the measurements performed to estimate the attenuator thickness needed to decrease the intensity of the beam by a factor of two. For this, 10 reference measurements were taken to estimate the average Mraw reading on the electrometer (in Coulombs), corrected by the temperature and pressure correction factor (KT,P).
Different thickness of attenuators were then tested to find the thickness that decreased the beam intensity by a factor of two. When this thickness was found, five measurements were taken to evaluate the average Mraw value corrected by KT,P.
For this configuration, a half value layer of 0.667 mm of copper was found. From the HVL measurement, we can calculate the effective energy of the beam, which is about 69 keV in our case.
Dose rate measurement
Before to these measurements, an EBT3 film was irradiated in order to determine the surface on which the irradiation field is homogeneous, allowing us to correctly place the cell container. This area is about 10 x 10 cm² excluding penumbra regions shown by dotted lines in Figure 2. Then, dose rate measurement was performed using a 31002 (equivalent to 31010) cylindrical ionization chamber calibrated in air kerma. For this configuration, with an open field irradiation field at 35 cm to the source in a T25 cell container placed on a carbon plate, the dose rate was about 0.626 Gy.min-1 in Kair.
To determine the exact dose on cells, the measured Kair was converted in water kerma. Figure 5 shows the X-rays energy spectrum obtained with dedicated software17. From this energy spectrum and the NIST table, we can convert the dose rate in Kair to Kwater, which was 0.659 Gy.min-1.
The overall uncertainty of the absolute dose rate measurement was about 3% at a 95% confidence level.
Cell culture media attenuation and scattering
For the quantification of cell culture media attenuation and scattering, dosimetry measurements with EBT3 radiochromic films were performed at room temperature. From the measurement with the ionization chamber, the dose rate was determined. Calibration films were then irradiated at the same position. EBT3 radiochromic films were calibrated between 0 and 3 Gy with 0.25 Gy steps between 0 and 1 Gy and 0.5 Gy steps between 1 and 3 Gy (nine dose points to construct the calibration curve) as shown in Figure 6. The dose points were fitted with a 4th-degree polynomial curve. The EBT3 films were then irradiated with and without the exact quantity of cell culture media inside the cell container to evaluate the attenuation and the scattering due to the cell culture media. For this configuration, the attenuation of the cell culture media was about 1.5%.
The overall uncertainty of the EBT3 film measurements was about 4% at a 95% confidence level.
Routine measurements
Before performing the cell irradiations, the dose rate was measured each time in the same container used for irradiation. Thus, we used the daily dose rate to estimate the irradiation time. If we closely follow the protocol and do not change any parameters, the HVL measurement and the attenuation due to the cell culture media do not need to be repeated. As an example, the table used for the daily measurement is given in Table 3.
Figure 1: Scheme of the configuration take in place on the SARRP enclosure for HVL measurements. Please click here to view a larger version of this figure.
Figure 2: Evaluation of the irradiation field size. Dose profile obtained at 35 cm to the source without collimator. Dotted lines show the area considered for the irradiation. Please click here to view a larger version of this figure.
Figure 3: Photographs of cell containers with the ionization chamber for dose rate measurement. Upper part: example for measurement with a 31002 cylindrical ionization chamber. Lower part: example for measurement with a TM23342 ionization chamber. Please click here to view a larger version of this figure.
Figure 4: Photographs of the T25 used for the measurement of the cell culture media attenuation. The upper part of the T25 was cut out to be able to place the film inside the flask. Please click here to view a larger version of this figure.
Figure 5: Simulated energy spectra for a 220 kV high voltage with 0.8 mm of Be and 0.15 mm of Cu filtrations17 . Please click here to view a larger version of this figure.
Figure 6: EBT3 films irradiated to construct the calibration curve and the corresponding calibration curve. Please click here to view a larger version of this figure.
High voltage (kV) | 220 |
Intensity (mA) | 3 |
Filtrations (inherent and additional) | 0.8 mm of Be + 0.15 mm Cu |
Half value layer (mm Cu) | Determined below |
Effective energy (keV) | Determined below |
Detector used | Cylindrical ionization chamber + EBT3 radiochromic films |
Source sample distance | 35 cm |
Irradiation field (shape, size, geometry) | Open field (no collimator), square, 20 x 20 cm |
Dosimetry quantity | Kair and Kwater |
Dosimetry method | As described on the protocol section |
Cell container | T25 |
Quantity of cell culture media | 5 ml |
Dose rate (Gy/min) | Determined below |
Table 1: A List of the configuration parameters.
Attenuator (mm Cu) | IC measure (nC) | Temperature (°C) | Pressure (hPa) | kT.P | IC measure corrected by kT.P (nC) | Corrected Mean value (nC) | ST deviation | Attenuation estimation (M / Mref) | |
reference measurements (Mref) | 0 | 10.480 | 21.6 | 993.2 | 1.026 | 10.752 | 10.761 | 0.005 | – |
10.480 | 21.6 | 993.1 | 1.026 | 10.752 | |||||
10.490 | 21.6 | 993.1 | 1.026 | 10.763 | |||||
10.490 | 21.6 | 993.1 | 1.026 | 10.763 | |||||
10.490 | 21.6 | 993.2 | 1.026 | 10.763 | |||||
10.490 | 21.6 | 993.2 | 1.026 | 10.763 | |||||
10.490 | 21.6 | 993.1 | 1.026 | 10.763 | |||||
10.490 | 21.6 | 993.2 | 1.026 | 10.763 | |||||
10.490 | 21.6 | 993.2 | 1.026 | 10.763 | |||||
10.490 | 21.6 | 993.1 | 1.026 | 10.763 | |||||
Finding of attenuatior thickness (M) | 0.514 | 5.840 | 21.7 | 993.2 | 1.026 | 5.992 | – | – | 0.557 |
0.564 | 5.651 | 21.7 | 993.2 | 1.026 | 5.798 | – | – | 0.539 | |
0.584 | 5.569 | 21.7 | 993.2 | 1.026 | 5.714 | – | – | 0.531 | |
0.604 | 5.491 | 21.7 | 993.2 | 1.026 | 5.634 | – | – | 0.524 | |
0.615 | 5.441 | 21.7 | 993.2 | 1.026 | 5.582 | – | – | 0.519 | |
0.627 | 5.380 | 21.7 | 993.2 | 1.026 | 5.520 | – | – | 0.513 | |
0.647 | 5.307 | 21.7 | 993.2 | 1.026 | 5.445 | – | – | 0.506 | |
0.667 | 5.240 | 21.8 | 993.2 | 1.026 | 5.376 | – | – | 0.500 | |
Measurments with the right attenuator (M) | 0.667 | 5.231 | 21.8 | 993.4 | 1.026 | 5.368 | 5.373 | 0.003 | 0.499 |
0.667 | 5.236 | 21.8 | 993.1 | 1.026 | 5.375 | ||||
0.667 | 5.235 | 21.8 | 993.2 | 1.026 | 5.373 | ||||
0.667 | 5.236 | 21.8 | 993.2 | 1.026 | 5.374 | ||||
0.667 | 5.235 | 21.8 | 993.3 | 1.026 | 5.373 |
Table 2: Measurement for the Half Value Layer determination.
IC measure (nC) | Temperature (°C) | Pressure (hPa) | kT.P | IC measure corrected by kT.P (nC) | Corrected Mean value by kT.P (nC) | ST deviation | Corrected mean value by all correction factors | Dose rate in air kerm (Gy/min) | Dose rate at cell level in Kwater (Gy/min) |
2.495 | 22.3 | 1001 | 1.020 | 2.545 | 2.546 | 0.001 | 2.536 | 0.626 | 0.659 |
2.496 | 22.3 | 1001 | 1.020 | 2.546 | |||||
2.497 | 22.3 | 1001 | 1.020 | 2.547 | |||||
2.498 | 22.3 | 1001 | 1.020 | 2.548 | |||||
2.496 | 22.3 | 1001 | 1.020 | 2.546 | |||||
2.495 | 22.3 | 1000.9 | 1.020 | 2.545 | |||||
2.494 | 22.3 | 1000.9 | 1.020 | 2.544 | |||||
2.495 | 22.3 | 1000.9 | 1.020 | 2.545 | |||||
2.496 | 22.3 | 1000.9 | 1.020 | 2.546 | |||||
2.496 | 22.3 | 1000.9 | 1.020 | 2.546 |
Table 3: Daily dose rate measurements for cell irradiation.
This work presents the protocol used and implemented for cell irradiations using low energy X-ray facility. Nowadays, many radiobiology experiments are performed with this type of irradiator as they are easy to use, cost effective and with very few radioprotection constraints, compared to cobalt source for example. Although these setups have many advantages, as they use a low X-ray energy source, a modification of only one irradiation parameter can significantly impact the dosimetry. Several studies have already highlighted the importance of dosimetry standards and protocols for radiobiology studies2,5,20,21. Even though several protocols have already been well defined in the literature1,5, we decided to develop a new protocol to perform dosimetry measurements to simulate real cell irradiation conditions as much as possible and take in account all the parameters that can influence the physical dose, especially for low energy X-rays21,22. Thus, we have chosen to implement a stringent protocol to minimize uncertainties. To this end, irradiation parameters were set (Table 1). The following three steps are then necessary: i) determination of the beam quality index, ii) measurement of the absolute dose rate with an ionization chamber and iii) measurement of the attenuation and scattering due to the cell culture medium with EBT3 radiochromic films.
The beam quality index corresponded to the voltage-half value layer (HVL) couple used to characterize low energy X-ray beams. The HVL is a practical indicator to describe poly energetic radiation and is defined as the thickness of an attenuator (usually copper or aluminum) to reduce the air kerma dose rate by a factor of two from the original value. HVL measurements were performed using the following recommendations of the AAPM protocol for a 40–300 kV X-ray beam10. However, some adaptations had to be made because in the irradiator enclosure it is not possible to achieve a distance of 1 meter between the source and the ionization chamber. Therefore, in the present work, we used a distance of 58 cm between the source and the detector for HVL measurements, as illustrated in Figure 1. We decided to let 25 cm after the ionization chamber because a lot of electronical material, support, and metallic elements are present at the bottom of the enclosure to limit the backscatter effect of these elements. Measurement of the HVL is one of the critical aspects of this protocol. Indeed, for many X-ray irradiators, the inside of enclosures is very restricted and these are not the optimal conditions to perform the measurements or it becomes impossible. Although experimental measurements are the best way to evaluate the HVL, when these measurements are too difficult or even impossible to perform, dedicated software17 can be used to provide a good estimation for the HVL, or a Monte Carlo simulation can be used23. In the present work, we used a dedicated software to obtain the X-ray energy spectrum (Figure 5). We were also able to compare the measured and calculated HVL, which was the same, and to also compare the effective energy.
For dosimetry measurements, we then chose to simulate real cell irradiation conditions as much as possible. For this, we directly performed the absolute dose rate measurements with the ionization chamber inside the cell container used for cells irradiation (Figure 3). However, as we used a cylindrical ionization chamber calibrated for beams over 100 kV, we were not exactly at the same position as the cells because of the thickness of the ionization chamber. For lower beams (15–70 kV), where plane parallel chamber can be used, we can be even closer to the real cell irradiation conditions. Then, relative dosimetry measurements were performed to evaluate the attenuation and the scattering due to the cell culture medium. The results presented on this work do not highlight a significant variation in the dose deposited with or without the exact quantity of cell culture media as we used a voltage of 220 kV, an additional filtration of 0.15 mm of Cu and we only had 5 mL of cell culture medium. However, in a previous study21 carried out at 80 kV, we pointed out that a variation of the cell culture media and filtration significantly impacts the physical dose, up to 40% compared to the reference configuration when we used a 1 mm aluminum filtration. This impact was also demonstrated in terms of biological effects by measuring the surviving cell fraction using a clonogenic assay21,23. Thus, depending on the voltage, additional filtration, the container and quantity of cell culture media, the dose deposited on the cells can be different if the protocol is not closely followed for all irradiations.
Consequently, a dedicated dosimetry should be set up for all cell irradiation configurations. Although this is restrictive and the modification of only a single parameter requires the implementation of a new configuration, we have decided to make this choice to be as close as possible to the real cell irradiation conditions. This requires a close collaboration between the physicists and the radiobiologist in order to set up the best design for the configuration. At our institute, a dozen protocols were established on our platform for a voltage range of 40 to 220 kV for which T25, T75, 6- to 96-plate wells or Petri dishes can be irradiated.
Although this protocol seems quite long to implement, once the configuration is established, the only measurement to be taken on the day of irradiation is the measurement of the dose rate with the ionization chamber inside the cell container. This measurement is also a quality control that enables us to ensure that the dose rate is as expected.
To ensure the reproducibility of radiobiological studies, and to be able to compare and interpret experiments, it is important to rigorously follow established protocols and report all the dosimetry and configuration aspects, particularly for facilities using low or medium energy X-rays. The new protocol proposed here is for cell irradiations, applicable to many X-ray facilities, and takes into account all the parameters influencing the dosimetry and provides a better estimation of the actual dose delivered to the cells.
The authors have nothing to disclose.
None
31010 ionization chamber | PTW | ionization Radiation, Detectors including code of practice, catalog 2019/2020, page 14 | https://www.ptwdosimetry.com/fileadmin/user_upload/DETECTORS_Cat_en_16522900_12/blaetterkatalog/index.html?startpage=1#page_14 |
EBT3 radiochromic films | Meditest | quote request | https://www.meditest.fr/produit/ebt3-8×10/ |
electrometer UNIDOSEwebline | PTW | online catalog, quote request | https://www.ptwdosimetry.com/en/products/unidos-webline/?type=3451&downloadfile=1593& cHash= 6096ddc2949f8bafe5d556e931e6c865 |
HVL material (filter, diaphragm) | PTW | online catalog, page 70, quote request | thickness foils: 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 and 10 mm of copper, https://www.ptwdosimetry.com/fileadmin/user_upload/Online_Catalog/Radiation_Medicine_Cat_en_ 58721100_11/blaetterkatalog/index.html#page_70 |
scanner for radiochromic films | Epson | quote request | Epson V700, seiko Epson corporation, Suwa, Japan |
temperature and pressure measurements, Lufft OPUS20 | lufft | quote request | https://www.lufft.com/products/in-room-measurements-291/opus-20-thip-1983/ |