**3.6. Relative biological effectiveness (RBE) calculations**

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

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

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

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

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

by performing a miniaturized version of the assay in a multi-tube plate system [27].

sample aliquots were also exposed to 1, 2, and 4 Gy of 250 kVp X-rays.

were performed.

124 Radiation Effects in Materials

the end.

**3.5. Micronucleus assay analysis**

for each data point.

The potential biological effects and damage caused by radiation depend not only on the radiation dose received but also on the type of radiation. RBE was introduced to normalize the radiobiological effects caused by different types of radiation. RBE is defined as the ratio of photon dose (in our case 250 kVp X-rays) to the dose of the radiation field of interest (in our case neutrons), providing the same biological effect [28]. The biological effects caused by neutrons vary with energy and produce greater damage than X- or γ-rays. In general, for the same dose, neutrons are much more effective in damaging cells because neutron-induced secondary particles, for example, low-energy protons, have high LET (linear energy transfer) and photon-induced particles are electrons having low-LET. In this paper, we describe experiments aimed at measuring the neutron RBE for micronucleus formation in peripheral blood lymphocytes as part of the irradiator testing. Ongoing experiments using a variety of cytogenetic, transcriptomic, and metabolomic endpoints, in human blood and in mice, will be published separately.

The RBE for the mixed-field irradiation (neutrons and γ-rays; **Figure 10b**) was calculated from the linear and linear-quadratic regression curves, fitted to the neutron and X-ray data,

**Figure 10.** (a) Micronucleus frequency in human peripheral blood lymphocytes exposed *ex vivo* to neutrons or X-rays. The data show mean micronuclei per binucleate cell yields from three healthy volunteers. Error bars show ± SEM. The mixed-field yields plotted vs total dose (neutrons + γ-rays). The solid lines indicate a linear (neutrons) or linear-quad‐ ratic (X-rays) fit. The dashed line indicates the estimated pure neutron component. (b) RBE values for the mixed field and for the pure neutrons, calculated from panel a (see text for details).

respectively, by solving for the X-ray dose that would give the same micronucleus yield as a given dose of the mixed neutron/γ-ray field. The pure neutron RBE will be slightly higher than the RBE value of the mixed field. This was evaluated in the same way from the dashed line in **Figure 10a**.

The limiting RBE value of 4 is higher than the value of 2.5 reported for 6 MeV monoenergetic neutrons [24] but lower than the value of 6 calculated for a reactor fission spectrum [25], in accordance with what we would expect, based on the neutron energies.
