This protocol is a novel combination of several radio biological and radiation dosimeteric methodologies to ensure accurate, reproducible, preclinical radiation therapy experiments. These methods link experimental conditions to national dose standards and allow for the accurate measurements of doses that match radiation treatment doses. Focusing on the design of reproducible radiotherapy experiments, this protocol provides researchers with the tools and methods necessary to perform studies with a high translational relevance.
Dosimetric calibration protocols can be challenging for novices, especially for those without the background in medical physics. We suggest consulting a radiation therapy physicist when first attempting these experiments. The techniques in this protocol are common to both radiation physicists and biomedical researchers.
However, some of these are not typically utilized in combination. To determine the appropriate dose output, set the irradiator to deliver radiation at a 220 peak kilovoltage and 13 milliamps with a 17 by 17 centimeter open field positioned 35 centimeters from the source. Filter the beam with a 0.15 millimeter copper filter with a broad focus and align the one centimeter slab, the two centimeter slab, the two centimeter slab with the ionization chamber and the one centimeter slab.
After setting up the phantom stack, insert the ionization chamber into the phantom and adjust the stacks such that the source to surface distance is 33 centimeters when appropriately leveled. Then confirm that these measurements are correctly placed within the ionization chamber at 35 centimeters from the isocenter. To create a radio chromic film calibration curve, prepare several additional pieces of film of the same size and orientation and place the film at a two centimeter depth in the solid water phantom stack.
To begin the irradiation, place one piece of film on top of four centimeters of solid water and place the remaining two centimeters of solid water above the films. After requiring post-exposure film scans, import the image files into ImageJ in a tiff file format and select Image, Color and select Split Channels. In the red image channel only, use the rectangle tool to draw a region of interest and press Ctrl+M to allow the mean values to be transcribed from the results window.
Once all of the pixel values have been obtained for the unexposed and exposed films, calculate the net optical density using the equation as indicated, then plot the net optical density against the dose at which the film was exposed and fit the plot with a quadratic curve. To determine the alpha/beta value for specific cancer cell lines via a clonogenic assay, count the resultant number of colonies in each treatment group to allow calculation of the survival fraction of each plate, then plot the natural log of the survival fraction against the corresponding dose delivered and fit the curve with a quadratic function. To determine the specific dose output for variable experimental designs, select a desired field size and distance from the source and use solid water phantoms to provide buildup and backscatter as needed, positioning the piece of film in the orientation that best portrays the experimental design.
The dose can then be determined from the net optical density of the film using the film calibration curve for the dose. To determine the appropriate beam position for a tumor bearing mouse treatment, after confirming a lack of response to pedal reflex, use an onboard portal camera and an aluminum filter to obtain a radiogram of the experimental mouse without collimation, then obtain a radiogram with collimation in place and overlay the radiograms in ImageJ to determine the beam positioning. Utilizing the calibration curve as demonstrated, two film samples can be generated that can be used to estimate the required experimental irradiation times.
Overlaying these images will reveal the exact positioning of the collimated radiation beam relative to the small animal being treated. Successful dose deposition can then be confirmed as illustrated by the positive gamma-H2AX staining observed within the treated hemisphere only in this representative analysis. Remaining consistent when moving from determining irradiator output to generating a film calibration curve and when determining dose for your ideal experimental design is critical.
Following these protocols enables researchers to study a wide variety of clinically applicable radiobiological questions in a preclinical setting. These questions may lead to a better understanding of patient outcomes. This protocol isn't a standalone new technique.
However, combining these methodologies will lead to direct clinical relevance and give insights into the outcomes for doses seen in the clinic.
