**3.3. Irradiation setup**

Due to the requirement for a mixed H/D beam, the irradiation facility was set up using an undeflected (0° angle) beam line in the RARAF accelerator facility (**Figure 8a**). The target assembly, shown in Figure 8b consists of a 2-mm-thick Cu disk onto which a 500-μm-thick beryllium foil was diffusion bonded. The dimensions of the copper disk were chosen so that it can be used in lieu of a standard 2.75" CF gasket. The target is clamped between two Conflat flanges and doubles as a vacuum window. The target is cooled by water impinging on the copper backing plate [9].

**Figure 8.** (a) Photo of the irradiation facility. Beam arrives from the right and impinges on the beryllium target generat‐ ing neutrons. Samples to be irradiated (blood or mice) are placed in sample holders (shown here empty) and mounted on a Ferris wheel rotating around the target cooling chamber. At the left is the TE gas ionization chamber used as a beam monitor and the right angle prism used for aligning the beam line. (b) Cross-section of target and cooling cham‐ ber. Direction to the center of the sample holders (190 mm away) and the monitor chamber (610 mm away) are shown.

As the target cooling water lines, supporting structures, shielding, and other surrounding materials were seen to give about a 20% azimuthal variation in dose rate, a vertical Ferris wheel-like fixture (**Figure 8a**) is used to rotate the sample holder tubes (for either blood or mice) around the target. Customized tubes mounting from the rods on the wheel are used to hold the samples with a constant horizontal orientation at a distance of 190 mm from the target center. The fixture rotates up to 18 samples around the beam axis. The sample holders for both mice and ex-vivo irradiated blood are based on standard 50-ml conical centrifuge tubes (BD, Franklin Lakes, NJ) which were modified to allow hanging horizontally from rods on the wheel, so that the tubes maintain a constant orientation as the wheel rotates. This provides an isotropic irradiation, while maintaining the mice in an upright orientation, reducing stress. During irradiations, the wheel is rotated at a speed of about 2 min per revolution and the dose rate adjusted so that the minimal dose is delivered in 10 rotations (20 min) with the sample tubes flipped end-to-end half-way through.

For pure photon exposure, a 250 kVp Westinghouse Coronado X-ray machine, placed within 15 m of the neutron irradiation facility, is used. This proximity allows for future mixed-field studies, where each sample may be immediately transported to the X-ray machine and irradiated after neutron exposure, with a time gap between the two irradiations of less than 5 min.

#### **3.4. Neutron dosimetry**

**3.3. Irradiation setup**

122 Radiation Effects in Materials

copper backing plate [9].

Due to the requirement for a mixed H/D beam, the irradiation facility was set up using an undeflected (0° angle) beam line in the RARAF accelerator facility (**Figure 8a**). The target assembly, shown in Figure 8b consists of a 2-mm-thick Cu disk onto which a 500-μm-thick beryllium foil was diffusion bonded. The dimensions of the copper disk were chosen so that it can be used in lieu of a standard 2.75" CF gasket. The target is clamped between two Conflat flanges and doubles as a vacuum window. The target is cooled by water impinging on the

**Figure 8.** (a) Photo of the irradiation facility. Beam arrives from the right and impinges on the beryllium target generat‐ ing neutrons. Samples to be irradiated (blood or mice) are placed in sample holders (shown here empty) and mounted on a Ferris wheel rotating around the target cooling chamber. At the left is the TE gas ionization chamber used as a beam monitor and the right angle prism used for aligning the beam line. (b) Cross-section of target and cooling cham‐ ber. Direction to the center of the sample holders (190 mm away) and the monitor chamber (610 mm away) are shown.

As the target cooling water lines, supporting structures, shielding, and other surrounding materials were seen to give about a 20% azimuthal variation in dose rate, a vertical Ferris wheel-like fixture (**Figure 8a**) is used to rotate the sample holder tubes (for either blood or mice) around the target. Customized tubes mounting from the rods on the wheel are used to hold the samples with a constant horizontal orientation at a distance of 190 mm from the target center. The fixture rotates up to 18 samples around the beam axis. The sample holders for both mice and ex-vivo irradiated blood are based on standard 50-ml conical centrifuge tubes (BD, Franklin Lakes, NJ) which were modified to allow hanging horizontally from rods on the wheel, so that the tubes maintain a constant orientation as the wheel rotates. This provides an isotropic irradiation, while maintaining the mice in an upright orientation, reducing stress.

The total dose measurement for the IND-like neutron/gamma mixed-field irradiations was performed using a custom A-150 muscle TE gas ionization chamber (**Figure 9**), as described by Rossi et al. [26]. This chamber is intended for use in a mixed neutron and γ field measure‐ ment and features an interchangeable internal TE plastic sleeve. In the dosimetry measure‐ ments reported here, we used a 3.5-mm-thick sleeve to model the dose deposited at the center of the blood samples used. The chamber was filled with methane TE gas at ~700 mm Hg before the dosimetry measurement and sealed. The detector was then attached directly to an elec‐ trometer system and calibrated using a 50 mg 226Ra γ-ray source, which had been previously calibrated by the National Bureau of Standards. The dose rate was ~36 μGy/h at 1 m from the source. After calibration, the dosimeter was mounted on the sample wheel for the IND-like neutron irradiator at 60° with respect to the ion beam axis and 190 mm away from the target.

**Figure 9.** Custom TE gas ionization chamber for neutron dosimetry shown here with 3.5 mm TE plastic sleeve. This figure was originally published by Rossi et al. [26] and has been modified and re-published with the permission of *Radiation Research*.

To extract the dose due to neutrons, γ-ray dosimetry was performed separately with a compensated Geiger–Mueller dosimeter, which is 20 times more sensitive to photons than to monoenergetic neutrons in the range of 0.68–4.2 MeV [22]. The γ-ray dosimetry was conducted in the same manner as the total dose measurement and then subtracted from the latter. Since the γ-ray dose from the target rate is essentially isotropic, only inverse-square law corrections were performed.

The neutron dose rate at the sample position was ~8.6 × 10−2 Gy/h/μA, representing ~79% of the total dose rate, with the remaining 21% due to γ-rays. During the irradiation, the beam current was tuned to and kept at about 17.5 μA, which is equal to a neutron dose rate of ~1.5 Gy/h. Because of the possible variation of the dose rate relative to the beam current, a second TE gas ionization chamber was added as a monitor at a fixed location downstream of the neutron target at an angle of ~12° relative to the ion beam direction. The monitor ionization chamber was filled with flowing TE gas, which was regulated with a constant-density control system. The incident primary particle beam current was recorded with an electrometer coupled to the end of the beam line, which is a Faraday cup-like isolated beam pipe with the target at the end.

### **3.5. Micronucleus assay analysis**

Micronucleus formation in peripheral blood lymphocytes is a well-established marker of ionizing-radiation-induced DNA damage. We have used a recently established cytokinesisblock micronucleus (CBMN) assay protocol by Fenech [23] for accelerated sample processing by performing a miniaturized version of the assay in a multi-tube plate system [27].

Peripheral blood samples were collected from three healthy donors after informed consent (IRB protocol #AAAF2671) and exposed (3 ml aliquots) to nominal neutron doses of approxi‐ mately 0.25, 0.5, 1, and 1.5 Gy (plus the concomitant 0.06, 0.1, 0.2, and 0.3 Gy of γ-rays). Blood sample aliquots were also exposed to 1, 2, and 4 Gy of 250 kVp X-rays.

Two hours post-irradiation, triplicate blood sample aliquots (50 μl) from each dose point were placed into culture in 1.0 ml 2D-barcoded matrix storage tubes (Thermo Scientific, Waltham, MA) with 500 μl of PB-MAX Karyotyping medium (Life Technologies, Grand Island, NY). Following 44 h of incubation, cytochalasin-B (Sigma Aldrich, St Louis, MO) was added at a final concentration of 6 μg/ml to inhibit cell cytokinesis and the tubes returned to the incubator. After a total incubation period of 72 h, the cells were harvested. Following hypotonic treatment, the cells were fixed using ice cold 4:1 fixative (methanol–acetic acid). The fixed samples were stored at 4°C (at least overnight), dropped on slides and stained with Vectashield mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). The slides were imaged using a Zeiss fluorescent microscope (Axioplan 2; Carl Zeiss MicroImaging Inc., Thornwood, NY) with a motorized stage and a 10× air objective. Quantification of lymphocyte micronuclei yields were determined by automatic scanning and analysis by the MetaferMN Score software (MetaSystems, Althaussen, Germany). Between 1800 and 6000, binucleate cells were analyzed for each data point.

**Figure 10a** shows the comparison of the micronucleus yields for lymphocytes exposed to different neutron or X-ray doses. Overall, a dose-dependent increase in micronuclei yields was observed with increasing dose with both radiation fields with the yields induced by the X-rays following a linear quadratic behavior with dose and the neutron data following a linear trend.

The neutron data are actually induced by a mixed neutron + photon field. The dashed line shows that what we would expect from a pure neutron irradiation. This estimate was obtained by calculating excess micronucleus yields over controls from the photon component (21% of the total dose) based on the linear quadratic fit to the X-ray data. This value was then subtracted from the mixed-field yields at the same total dose. The resultant difference corresponds to the micronuclei yields that would be seen in a pure neutron irradiation.
