1. Irradiation platform and determination of irradiation parameters
<ol>
	<li>Use an irradiation platform delivering low to medium energy X-rays. Determine the parameters of the experiment to ensure the robustness and the reproducibility of the radiobiological experiment: High voltage, Intensity, Filtration (inherent and additional), Half Value Layer (HVL), Effective energy, Detector used for dosimetry measurements, Source Sample Distance (SSD), Irradiation field (shape, size, geometry), Dosimetry quantity, Dosimetry m...

<ol>
	<li>Zoetelief, J., Broerse, J. J., Davies, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+X-ray+dosimetry+EULEP.+Report+No.+Report+EUR+9507.">Protocol for X-ray dosimetry EULEP. Report No. Report EUR 9507.</a> Commission of the European Communities. , (1985).</li><li>Zoetelief, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+X-ray+dosimetry+in+radiobiology.">Protocol for X-ray dosimetry in radiobiology.</a> International Journal of Radiation Biology. 77 (7), 817-835 (2001).</li><li>Zoetelief, J., Jansen, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calculated+energy+response+correction+factors+for+LiF+thermoluminescent+dosemeters+employed+in+the+seventh+EULEP+dosimetry+intercomparison.">Calculated energy response correction factors for LiF thermoluminescent dosemeters employed in the seventh EULEP dosimetry intercomparison.</a> Physics in Medicine and Biology. 42 (8), 1491-1504 (1997).</li><li>Coleman, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Education+and+training+for+radiation+scientists:+radiation+research+program+and+American+Society+of+Therapeutic+Radiology+and+Oncology+Workshop,+Bethesda,+Maryland.">Education and training for radiation scientists: radiation research program and American Society of Therapeutic Radiology and Oncology Workshop, Bethesda, Maryland.</a> Radiation Research. 160 (6), 729-737 (2003).</li><li>Desrosiers, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+dosimetry+standardization+in+radiobiology.">The importance of dosimetry standardization in radiobiology.</a> Journal of Research of National Institute of Standards and Technology. 118, 403-418 (2013).</li><li>DIN. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Klinische+Dosimetrie:+Teil+4.">Klinische Dosimetrie: Teil 4.</a> Anwendung von R&#246;ntgenstrahlen mit R&#246;hrenspannungen von 10 bis 100 kV in der Strahlentherapie und in der Weichteildianostik. , (1988).</li><li>DIN. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Klinische+Dosimetrie:+Teil+5.">Klinische Dosimetrie: Teil 5.</a> Anwendung von R&#246;ntgenstrahlen mit R&#246;hrenspannungen von 100 bis 400 kV in der Strahlentherapie. , (1996).</li><li>NCS. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dosimetry+of+low+and+medium+energy+x-rays:+A+code+of+practice+for+use+in+radiotherapy+and+radiobiology.">Dosimetry of low and medium energy x-rays: A code of practice for use in radiotherapy and radiobiology.</a> NCS. , (1997).</li><li>International Atomic Energy Agency. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absorbed+Dose+Determination+in+External+Beam+Radiotherapy.">Absorbed Dose Determination in External Beam Radiotherapy.</a> International Atomic Energy Agency. , (2000).</li><li>Ma, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAPM+protocol+for+40-300+kV+x-ray+beam+dosimetry+in+radiotherapy+and+radiobiology.">AAPM protocol for 40-300 kV x-ray beam dosimetry in radiotherapy and radiobiology.</a> Medical Physics. 28 (6), 868-893 (2001).</li><li>Peixoto, J. G., Andreo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+absorbed+dose+to+water+in+reference+conditions+for+radiotherapy+kilovoltage+x-rays+between+10+and+300+kV:+a+comparison+of+the+data+in+the+IAEA,+IPEMB,+DIN+and+NCS+dosimetry+protocols.">Determination of absorbed dose to water in reference conditions for radiotherapy kilovoltage x-rays between 10 and 300 kV: a comparison of the data in the IAEA, IPEMB, DIN and NCS dosimetry protocols.</a> Physics in Medicine and Biology. 45 (3), 563-575 (2000).</li><li>Pedersen, K. H., Kunugi, K. A., Hammer, C. G., Culberson, W. S., DeWerd, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+biology+irradiator+dose+verification+survey.">Radiation biology irradiator dose verification survey.</a> Radiation Research. 185 (2), 163-168 (2016).</li><li>Draeger, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dose+of+reality:+how+20+years+of+incomplete+physics+and+dosimetry+reporting+in+radiobiology+studies+may+have+contributed+to+the+reproducibility+crisis.">A dose of reality: how 20 years of incomplete physics and dosimetry reporting in radiobiology studies may have contributed to the reproducibility crisis.</a> International Journal of Radiation Oncology, Biology, Physics. 106 (2), 243-252 (2020).</li><li>Devic, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+radiochromic+film+dosimetry+using+a+flat-bed+document+scanner.">Precise radiochromic film dosimetry using a flat-bed document scanner.</a> Medical Physics. 32 (7), 2245-2253 (2005).</li><li>Micke, A., Lewis, D. F., Yu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multichannel+film+dosimetry+with+nonuniformity+correction.">Multichannel film dosimetry with nonuniformity correction.</a> Medical Physics. 38 (5), 2523-2534 (2011).</li><li>Poludniowski, G., Landry, G., DeBlois, F., Evans, P. M., Verhaegen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SpekCalc:+a+program+to+calculate+photon+spectra+from+tungsten+anode+x-ray+tubes.">SpekCalc: a program to calculate photon spectra from tungsten anode x-ray tubes.</a> Physics in Medicine and Biology. 54 (19), 433-438 (2009).</li><li>Hubbell, J. H., Seltzer, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-Ray+mass+attenuation+coefficients+-+Tables+of+X-ray+mass+attenuation+coefficients+and+mass+energy-absorption+coefficients+1+keV+to+20+MeV+for+elements+Z+=+1+to+92+and+48+additional+substances+of+dosimetric+interest+(version+1.4).">X-Ray mass attenuation coefficients - Tables of X-ray mass attenuation coefficients and mass energy-absorption coefficients 1 keV to 20 MeV for elements Z = 1 to 92 and 48 additional substances of dosimetric interest (version 1.4).</a> NIST Standard Reference Database. , 126 (1995).</li><li>Wong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution,+small+animal+radiation+research+platform+with+x-ray+tomographic+guidance+capabilities.">High-resolution, small animal radiation research platform with x-ray tomographic guidance capabilities.</a> International Journal of Radiation Oncology, Biology, Physics. 71 (5), 1591-1599 (2008).</li><li>Trompier, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+the+influence+of+calibration+practices+on+cytogenetic+laboratory+performance+for+dose+estimation.">Investigation of the influence of calibration practices on cytogenetic laboratory performance for dose estimation.</a> International Journal of Radiation Biology. , 1-9 (2016).</li><li>Dos Santos, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+dosimetry+protocol+for+cell+irradiation+on+a+low+X-rays+facility+and+consequences+for+the+biological+response.">Importance of dosimetry protocol for cell irradiation on a low X-rays facility and consequences for the biological response.</a> International Journal of Radiation Biology. , 1-29 (2018).</li><li>Noblet, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Underestimation+of+dose+delivery+in+preclinical+irradiation+due+to+scattering+conditions.">Underestimation of dose delivery in preclinical irradiation due to scattering conditions.</a> Physica Medica. 30 (1), 63-68 (2014).</li><li>Paixao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monte+Carlo+derivation+of+filtered+tungsten+anode+X-ray+spectra+for+dose+computation+in+digital+mammography.">Monte Carlo derivation of filtered tungsten anode X-ray spectra for dose computation in digital mammography.</a> Radiologia Brasileira. 48 (6), 363-367 (2015).</li></ol>

The authors have nothing to disclose.

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 highligh...

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.

<table>
	<tbody>
		<tr>
			<td>31010 ionization chamber</td>
			<td>PTW</td>
			<td>ionization Radiation, Detectors including code of practice, catalog 2019/2020, page 14</td>
			<td>https://www.ptwdosimetry.com/fileadmin/user_upload/DETECTORS_Cat_en_16522900_12/blaetterkatalog/index.html?startpage=1#page_14</td>
		</tr>
		<tr>
			<td>EBT3 radiochromic films</td>
			<td>Meditest</td>
			<td>quote request</td>
			<td>https://www.meditest.fr/produit/ebt3-8x10/</td>
		</tr>
		<tr>
			<td>electrometer UNIDOSEwebline</td>
			<td>PTW</td>
			<td>online catalog, quote request</td>
			<td>https://www.ptwdosimetry.com/en/products/unidos-webline/?type=3451&#38;downloadfile=1593&#38; cHash= 6096ddc2949f8bafe5d556e931e6c865</td>
		</tr>
		<tr>
			<td>HVL material (filter, diaphragm)</td>
			<td>PTW</td>
			<td>online catalog, page 70, quote request</td>
			<td>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</td>
		</tr>
		<tr>
			<td>scanner for radiochromic films</td>
			<td>Epson</td>
			<td>quote request</td>
			<td>Epson V700, seiko Epson corporation, Suwa, Japan</td>
		</tr>
		<tr>
			<td>temperature and pressure measurements, Lufft OPUS20</td>
			<td>lufft</td>
			<td>quote request</td>
			<td>https://www.lufft.com/products/in-room-measurements-291/opus-20-thip-1983/</td>
		</tr>
	</tbody>
</table>

dosimetry for cell irradiation using orthovoltage (40-300 kv) x-ray facilities

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.

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 chose...

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Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

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.

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose ...

This protocol was developed to allow dosimetry measurements to be performed as close as possible to real cell irradiation conditions for radiobiological studies. With this protocol, we can determine the exact dose received by cells making it applicable to many x-ray facilities and can take into account all of the parameters and translate to dosimetry. All irradiation parameters must be set upstream to allow an optimal acquisition of the dosimetry measurement requiring a close collaboration between physicists and radiobiologist.To perform an irradiation field evaluation, place a self-developing dosimetry film onto the support used for irradiation and irradiate the film with at least two grays to obtain a well-marked irradiation field. Scan the self-developing dosimetry film using a dedicated scanner and use analyze and plot profile to plot the dose profile in ImageJ. Then, mark the irradiation support surface to ensure that the cell container will be placed in the correct position.To perform a dose rate measurement, place a modified cell container into the irradiation support enclosure and place the ionization chamber into the container in the correct position according to the marks made on the support surface. Confirm that all of the irradiation parameters have been appropriately set and pre-irradiate the ionization chamber for five minutes. Zero the electrometer and obtain 10 one-minute measurements.Then, use the formula to determine the average dose rate in air kerma. Number all of the films for their downstream identification. At least 24 hours before the irradiation, cut pieces of self-developing dosimetry film according to the size of the cell container.To construct a calibration curve, set aside three pieces of film for the non-irradiated controls and place the first film inside the cell container. Then, irradiate the film to obtain the first dose point. When all of the films have been irradiated in triplicate at the appropriate selected doses, a calibration curve can be generated.To measure the cell culture medium attenuation and scattering, select the same irradiation time for all of the irradiations and irradiate three pieces of self-developing dosimetry films in the container without water. Next, place a single piece of film into the container and fill the container with the same volume of water as the volume of medium that will be irradiated. Using small pieces of tape as necessary, position the container within the enclosure.When the irradiation is complete, dry the films with absorbent paper and store the films protected from light. 24 hours after their irradiation, on the scanner, set the TIFF format to 48-bit red green blue and the transmission mode to 150 dots per inch and select no image correction. To warm up the scanner, place a non-irradiated film on the scanner and launch a preview of the scan.At the end of the preview, start a timer and wait 30 seconds before starting the scan. At the end of the scan, start a 90-second timer. At the same time, register the scan and open the image in ImageJ.Trace a square region of interest within the scan and measure the average red pixel level of the area. At the end of the 90 seconds, repeat the scan preview at least 30 times to warm up and stabilize the scanner. Once the scanner has been stabilized, place a film into the center of the scanner bed and launch a preview of the scan.Wait 30 seconds before starting the scan. At the end of the scan, wait 90 seconds before starting the next scan. Measurements to estimate the attenuator thickness were then performed and the different attenuator thicknesses were tested to find the thickness that decreased the beam intensity by a factor of two.When this thickness was determined, five measurements were taken to evaluate the average mRAW value corrected by the temperature and pressure correction factor. For this configuration, a half-value layer of 0.667 millimeter of copper was found. In this representative analysis, a self-developing dosimetry film was irradiated to determine the surface on which the irradiation field is homogeneous, allowing correct placement of the cell container.To determine the exact dose for the cells, the measured air kerma dose rate was converted to water karma using the ratio of the mean mass energy absorption coefficient for water to air evaluated over the photon fluence spectrum, which for this analysis was determined to be 0.659 grays per minute. In this cell culture medium attenuation and scattering analysis, the self-developing dosimetry radiochromic films were first calibrated between zero and three grays with 0.25 gray steps between zero and one gray and 0.5 gray steps between one and three grays. The dose points were then fitted with a fourth-degree polynomial curve.Here, a representative table used for the daily measurement can be observed. Make sure that all of the configuration parameters are respected, that the does rate is measured inside the right cell container, and that the influence of cell culture medium is evaluated. Several protocols exist to establish a dose reference measurement.The key point is to select the appropriate protocol for a specific application, especially when using low x-ray facilities.

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4.8K Views.  Institute for Radiological Protection and Nuclear Safety (IRSN). This protocol was developed to allow dosimetry measurements to be performed as close as possible to real cell irradiation conditions for radiobiological studies. With this protocol, we can determine the exact dose received by cells making it applicable to many x-ray facilities and can take into account all of the parameters and translate to dosimetry. All irradiation parameters must be set upstream to allow an optimal acquisition of the dosimetry measurement requiring a close collaboration be...