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A NOVEL BIOLOGICAL DOSIMETRY ASSAY AS A POTENTIAL TOOL FOR TRIAGE DOSE ASSESSMENT IN CASE OF LARGE-SCALE RADIOLOGICAL EMERGENCY

A NOVEL BIOLOGICAL DOSIMETRY ASSAY AS A POTENTIAL TOOL FOR TRIAGE DOSE ASSESSMENT IN CASE OF... Abstract In case of mass radiological emergencies, new strategies involving biological and clinical endpoints are requested for an efficient triage classification of casualties. For this purpose, we developed a novel protocol combining the two most established cytogenetic methods used in biological dosimetry (dicentric and micronucleus assays) into a single one, in order to have a time-saving, inexpensive and potentially automatable instrument to be used for triage purposes in case of large-scale radiological events. This method could be considered as a ‘three in one’ assay allowing the simultaneous scoring of chromosome aberrations and micronuclei on a single slide, and also enabling to discriminate between metaphases in first and second cell division without the Fluorescence plus Giemsa staining. This method needs further validation through inter-comparisons involving biological dosimetry laboratories, to verify its reproducibility. Moreover, the possibility to apply the already existing software for automation for dicentric and micronucleus assays could be also verified. INTRODUCTION In large-scale radiological emergencies, a considerable number of individuals can receive a range of radiation doses spanning from background levels to those large enough to cause medical consequences. These individuals need to be rapidly assessed for exposure levels to determine whether medical intervention is required(1, 2). At this time, efficient and reliable triage classification of casualties, using biological and clinical endpoints that initially and rapidly can identify individuals suspected of exposure to life threatening doses, as well as to provide a triage dose, is needed and new strategies to increase its throughput are requested. In this regard, we performed a new combined biological dosimetry assay, specifically designed for triage dose assessment in case of large-scale nuclear accident. The method here proposed results from a modification of a protocol previously established in our laboratory for in situ radiobiology studies on mammalian cells, originally developed for the analysis of chromosome damage induced by charged-particle microbeam irradiation or other radiobiological applications characterized by critical irradiation conditions(3). Thanks to its versatility, this method has been properly modified according to biological dosimetry purposes. This protocol combines in itself three separate techniques: (1) the dicentric assay (DCA), (2) the cytokinesis-block micronucleus (CBMN) assay and (3) a method for identifying the metaphases in the first cell cycle without the need to use the Fluorescence plus Giemsa (FPG) staining but, simply, by means of the difference of the chromosome number due to the cytokinesis inhibition of the Cytochalasin B(4). In such preparations, the metaphases in the first cell cycle (M1) consist of 46 chromosomes while those in the second cell cycle (M2) consist of 92 chromosomes (Figure 1). In summary, by using this combined protocol here described, we can reach on a single slide a satisfactory number of M1 and binucleated cells (BN) to perform a triage mode dose assessment simultaneously for DCA and MN (Figure 2). Figure 1. Open in new tabDownload slide Simultaneous visualization of a metaphase in the first cell cycle (M1), consisting of 46 chromosomes, and a metaphase in the second cell cycle (M2), consisting of 92 chromosomes. Figure 1. Open in new tabDownload slide Simultaneous visualization of a metaphase in the first cell cycle (M1), consisting of 46 chromosomes, and a metaphase in the second cell cycle (M2), consisting of 92 chromosomes. Figure 2. Open in new tabDownload slide Simultaneous visualization of M1 and BN at 20x (a) and at 60x magnification (b). Figure 2. Open in new tabDownload slide Simultaneous visualization of M1 and BN at 20x (a) and at 60x magnification (b). In order to start a validation of the method for dose assessment, a comparison between the combined protocol and the two standard methods for DCA and MN(5) has been performed on blood samples irradiated ex vivo with different X-ray doses. MATERIALS AND METHODS Whole blood samples from two healthy donors (mean age 40 y), collected after receiving informed consent, were put in heparinized tube and irradiated by using a Gilardoni CHF 320 G X-ray generator operated at 250 kVp, 15 mA, at the following doses: 0, 0.25, 0.5, 0.75, 1, 2 Gy (dose rate 0.84 Gy/min). After irradiation, for each dose, whole blood samples were divided in three different aliquots and after 2 h of recovery time, cell culture procedures started according to the standard protocols for DCA and MN(5) and to the combined protocol described in detail below. Combined protocol: for each cell culture, 0.5 ml of whole blood were dispensed in 15 ml round-bottomed sterile disposable tubes containing 4.5 ml RPMI-1640 Dutch Modification medium (Sigma-Aldrich) supplemented with 20% fetal bovine serum (Sigma-Aldrich), 2 mM L-glutamine (Euroclone), 100 units/ml of penicillin and 100 μg/ml of streptomycin solution (Euroclone). T-lymphocytes were stimulated adding 2% phytohaemagglutinin, M form (Gibco) and cultured in an incubator at 37°C keeping the tubes at about a 45° angle with closed caps. Cytochalasin B (Sigma-Aldrich) was added to the culture at 24 h (final concentration: 6 μg/ml). Colcemid solution (Sigma-Aldrich) was added 3 h before terminating the cultures (final concentration: 0.1 μg/ml). The fixation procedure conditions were optimized in order to obtain good yields of both M1 and BN cells. Fixation was performed at 58 hours: cell cultures were centrifuged at 1000 rpm for 10 min and the supernatant removed, then 4 ml of pre-warmed hypotonic solution (0.075 M potassium chloride) were added dropwise to each culture, the pellet was resuspended with a glass Pasteur pipette and the tubes were incubated at 37°C for 20 min. Then, 4 ml of cold fixative (3:1 methanol/acetic acid) were added dropwise to each culture, the suspension was resuspended with a glass Pasteur pipette and the tubes were incubated at 4°C for 30 min. Then the tubes were centrifuged at 1000 rpm for 20 min and the supernatant was removed. About 4 ml of cold fixative were added to each culture, the pellet was resuspended with a glass Pasteur pipette and the tubes were centrifuged at 1000 rpm for 10 min. Further washes with fixative can be performed until the solution becomes clear. The fixed cells may be stored in fixative at −20°C. For slide preparation, the cell suspension was centrifuged at 1000 rpm for 10 min and the supernatant was removed, leaving a suitable quantity of fixative. The pellet was resuspended with a glass Pasteur pipette and a few drops of suspension were dispensed on a cold and wet slide, previously cleaned and degreased. The slides were allowed to air dry overnight. Slides were stained with 5% Giemsa stain (Carlo Erba) prepared in Gurr phosphate buffer, pH 6.8 (VWR): slides were immersed in Giemsa solution for 8–9 min and then washed in water and allowed to air dry overnight. Slides were mounted with a cover glass using Eukitt (Sigma-Aldrich). RESULTS AND DISCUSSION By using the combined protocol, at least 100 M1 and 1000 BN could be simultaneously scored on a single slide. This amount of cells is considered adequate to perform a triage mode dose assessment suggesting that this method could represent a time-saving and inexpensive biological dosimetry assay to be used in case of massive radiological accidents. The application of this combined protocol could have several advantages in comparison to the single methods: a single blood culture is enough for both DCA and CBMN assays leading to a reduction by half of the number of cell cultures to be processed for each individual. This results in a reduced manual work which is generally a critical point in large-scale emergencies. Moreover, there is a considerable reduction in costs related to chemical reagents and less quantity of blood per subject required. It is worth noting that the use of a simple Giemsa staining instead of the FPG method, determines a further reduction in processing times and expenses. The overall PBL culture time (58 h) is shorter in comparison to the standard CBMN assay requiring 72 h, but longer compared with the standard DCA assay (48–52 h). Preliminary results, as shown in Table 1, showed a very good reproducibility between the proposed combined protocol and the standard methods in terms of dicentrics and micronuclei induction, in relation to the doses of X-ray delivered (range 0–2 Gy). Table 1. Dicentrics and micronuclei induction in X-ray irradiated samples: comparison between the combined and standard protocols. . Combined protocol . Standard protocol . . Combined protocol . Standard protocol . Dose (Gy) . M1 . DIC + CR . DIC + CR/cell (±SE) . DIC + CR . DIC + CR/cell (±SE) . BN . MN . MN/cell (±SE) . MN . MN/cell (±SE) . 0 500 0 0.00 (±0.000) 0 0.00 (±0.000) 2000 7 0.003 (±0.000) 8 0.004 (±0.001) 0.25 500 32 0.06 (±0.007) 29 0.06 (±0.007) 2000 30 0.015 (±0.002) 32 0.016 (±0.002) 0.50 500 60 0.12 (±0.024) 50 0.10 (±0.014) 2000 40 0.020 (±0.003) 44 0.022 (±0.003) 0.75 500 75 0.15 (±0.041) 65 0.13 (±0.029) 2000 84 0.042 (±0.006) 72 0.036 (±0.005) 1 200 33 0.16 (±0.005) 34 0.17 (±0.011) 2000 171 0.085 (±0.018) 149 0.074 (±0.014) 2 200 62 0.31 (±0.033) 66 0.33 (±0.007) 2000 350 0.175 (±0.025) 420 0.210 (±0.030) . Combined protocol . Standard protocol . . Combined protocol . Standard protocol . Dose (Gy) . M1 . DIC + CR . DIC + CR/cell (±SE) . DIC + CR . DIC + CR/cell (±SE) . BN . MN . MN/cell (±SE) . MN . MN/cell (±SE) . 0 500 0 0.00 (±0.000) 0 0.00 (±0.000) 2000 7 0.003 (±0.000) 8 0.004 (±0.001) 0.25 500 32 0.06 (±0.007) 29 0.06 (±0.007) 2000 30 0.015 (±0.002) 32 0.016 (±0.002) 0.50 500 60 0.12 (±0.024) 50 0.10 (±0.014) 2000 40 0.020 (±0.003) 44 0.022 (±0.003) 0.75 500 75 0.15 (±0.041) 65 0.13 (±0.029) 2000 84 0.042 (±0.006) 72 0.036 (±0.005) 1 200 33 0.16 (±0.005) 34 0.17 (±0.011) 2000 171 0.085 (±0.018) 149 0.074 (±0.014) 2 200 62 0.31 (±0.033) 66 0.33 (±0.007) 2000 350 0.175 (±0.025) 420 0.210 (±0.030) M1, metaphase in first cell division; DIC, dicentric; CR, centric ring; BN, binucleated cell; MN, micronucleus; SE, standard error. Open in new tab Table 1. Dicentrics and micronuclei induction in X-ray irradiated samples: comparison between the combined and standard protocols. . Combined protocol . Standard protocol . . Combined protocol . Standard protocol . Dose (Gy) . M1 . DIC + CR . DIC + CR/cell (±SE) . DIC + CR . DIC + CR/cell (±SE) . BN . MN . MN/cell (±SE) . MN . MN/cell (±SE) . 0 500 0 0.00 (±0.000) 0 0.00 (±0.000) 2000 7 0.003 (±0.000) 8 0.004 (±0.001) 0.25 500 32 0.06 (±0.007) 29 0.06 (±0.007) 2000 30 0.015 (±0.002) 32 0.016 (±0.002) 0.50 500 60 0.12 (±0.024) 50 0.10 (±0.014) 2000 40 0.020 (±0.003) 44 0.022 (±0.003) 0.75 500 75 0.15 (±0.041) 65 0.13 (±0.029) 2000 84 0.042 (±0.006) 72 0.036 (±0.005) 1 200 33 0.16 (±0.005) 34 0.17 (±0.011) 2000 171 0.085 (±0.018) 149 0.074 (±0.014) 2 200 62 0.31 (±0.033) 66 0.33 (±0.007) 2000 350 0.175 (±0.025) 420 0.210 (±0.030) . Combined protocol . Standard protocol . . Combined protocol . Standard protocol . Dose (Gy) . M1 . DIC + CR . DIC + CR/cell (±SE) . DIC + CR . DIC + CR/cell (±SE) . BN . MN . MN/cell (±SE) . MN . MN/cell (±SE) . 0 500 0 0.00 (±0.000) 0 0.00 (±0.000) 2000 7 0.003 (±0.000) 8 0.004 (±0.001) 0.25 500 32 0.06 (±0.007) 29 0.06 (±0.007) 2000 30 0.015 (±0.002) 32 0.016 (±0.002) 0.50 500 60 0.12 (±0.024) 50 0.10 (±0.014) 2000 40 0.020 (±0.003) 44 0.022 (±0.003) 0.75 500 75 0.15 (±0.041) 65 0.13 (±0.029) 2000 84 0.042 (±0.006) 72 0.036 (±0.005) 1 200 33 0.16 (±0.005) 34 0.17 (±0.011) 2000 171 0.085 (±0.018) 149 0.074 (±0.014) 2 200 62 0.31 (±0.033) 66 0.33 (±0.007) 2000 350 0.175 (±0.025) 420 0.210 (±0.030) M1, metaphase in first cell division; DIC, dicentric; CR, centric ring; BN, binucleated cell; MN, micronucleus; SE, standard error. Open in new tab Main disadvantage of the described protocol concerns the need for further validation through robust calibration curves, performed on a wide panel of doses (spanning from very low to extremely high), different qualities of radiation and higher number of donors. Moreover, inter-comparison exercises involving different biological dosimetry laboratories should be planned in order to verify the reproducibility of the method and to validate it for triage purposes, assuming radiation emergency scenario. The possibility to combine the already existing software for automation designed for the standard dicentric and micronucleus assays into a single one to be applied to this novel protocol should be verified. If this is not possible, it should be possible to scan the slide twice, performing the two analyses separately. Depending on the needs determined by the size of the accident, it would be possible to carry out a first categorization of overexposed subjects by MN scoring and, subsequently, a more precise dose assessment through the analysis of the dicentrics, by scanning again the same slide, could be performed. In conclusion, this protocol, once further validated, could represent a useful method for the categorization of subjects overexposed to ionizing radiation in case of mass radiological emergencies. Acknowledgements We are grateful to the personnel of the Medicine Service of ENEA Casaccia for their collaboration in collecting blood samples from healthy donors. References 1 Jaworska , A , et al. . Operational guidance for radiation emergency response organisations in Europe for using biodosimetric tools developed in EU MULTIBIODOSE project . Radiat. Prot. Dosim. 164 ( 1–2 ), 165 – 169 ( 2015 ). Google Scholar Crossref Search ADS WorldCat 2 Roy , L. , Roch-Lefevre , S. , Vaurijoux , A. , Voisin , P. , Martin , C. , Grégoire , E. and Voisin , P. Optimization of cytogenetic procedures for population triage in case of radiological emergency . Radiat. Meas. 42 ( 6–7 ), 1143 – 1146 ( 2007 ). Google Scholar Crossref Search ADS WorldCat 3 Patrono , C. , Monteiro Gil , O. , Giesen , U. , Langner , F. , Pinto , M. , Rabus , H. and Testa , A. ‘BioQuaRT’ project: design of a novel in situ protocol for the simultaneous visualisation of chromosomal aberrations and micronuclei after irradiation at microbeam facilities . Radiat. Prot. Dosim. 166 ( 1–4 ), 197 – 199 ( 2015 ). Google Scholar Crossref Search ADS WorldCat 4 Hayata , I. , Kajima , J. and Okabe , N. Distinction of metaphases in the first cell cycle for automated system in radiation dosimetry . Radiat. Phys. Chem. 39 ( 6 ), 517 – 520 ( 1992 ). OpenURL Placeholder Text WorldCat 5 International Atomic Energy Agency . Cytogenetic dosimetry: applications in preparedness for and response to radiation emergencies ( Vienna : IAEA ) ( 2011 ). Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC © The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Radiation Protection Dosimetry Oxford University Press

A NOVEL BIOLOGICAL DOSIMETRY ASSAY AS A POTENTIAL TOOL FOR TRIAGE DOSE ASSESSMENT IN CASE OF LARGE-SCALE RADIOLOGICAL EMERGENCY

Testa,, A; Palma,, V; Patrono,, C
Radiation Protection Dosimetry , Volume 186 (1) – Dec 31, 2019
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Abstract

Abstract In case of mass radiological emergencies, new strategies involving biological and clinical endpoints are requested for an efficient triage classification of casualties. For this purpose, we developed a novel protocol combining the two most established cytogenetic methods used in biological dosimetry (dicentric and micronucleus assays) into a single one, in order to have a time-saving, inexpensive and potentially automatable instrument to be used for triage purposes in case of large-scale radiological events. This method could be considered as a ‘three in one’ assay allowing the simultaneous scoring of chromosome aberrations and micronuclei on a single slide, and also enabling to discriminate between metaphases in first and second cell division without the Fluorescence plus Giemsa staining. This method needs further validation through inter-comparisons involving biological dosimetry laboratories, to verify its reproducibility. Moreover, the possibility to apply the already existing software for automation for dicentric and micronucleus assays could be also verified. INTRODUCTION In large-scale radiological emergencies, a considerable number of individuals can receive a range of radiation doses spanning from background levels to those large enough to cause medical consequences. These individuals need to be rapidly assessed for exposure levels to determine whether medical intervention is required(1, 2). At this time, efficient and reliable triage classification of casualties, using biological and clinical endpoints that initially and rapidly can identify individuals suspected of exposure to life threatening doses, as well as to provide a triage dose, is needed and new strategies to increase its throughput are requested. In this regard, we performed a new combined biological dosimetry assay, specifically designed for triage dose assessment in case of large-scale nuclear accident. The method here proposed results from a modification of a protocol previously established in our laboratory for in situ radiobiology studies on mammalian cells, originally developed for the analysis of chromosome damage induced by charged-particle microbeam irradiation or other radiobiological applications characterized by critical irradiation conditions(3). Thanks to its versatility, this method has been properly modified according to biological dosimetry purposes. This protocol combines in itself three separate techniques: (1) the dicentric assay (DCA), (2) the cytokinesis-block micronucleus (CBMN) assay and (3) a method for identifying the metaphases in the first cell cycle without the need to use the Fluorescence plus Giemsa (FPG) staining but, simply, by means of the difference of the chromosome number due to the cytokinesis inhibition of the Cytochalasin B(4). In such preparations, the metaphases in the first cell cycle (M1) consist of 46 chromosomes while those in the second cell cycle (M2) consist of 92 chromosomes (Figure 1). In summary, by using this combined protocol here described, we can reach on a single slide a satisfactory number of M1 and binucleated cells (BN) to perform a triage mode dose assessment simultaneously for DCA and MN (Figure 2). Figure 1. Open in new tabDownload slide Simultaneous visualization of a metaphase in the first cell cycle (M1), consisting of 46 chromosomes, and a metaphase in the second cell cycle (M2), consisting of 92 chromosomes. Figure 1. Open in new tabDownload slide Simultaneous visualization of a metaphase in the first cell cycle (M1), consisting of 46 chromosomes, and a metaphase in the second cell cycle (M2), consisting of 92 chromosomes. Figure 2. Open in new tabDownload slide Simultaneous visualization of M1 and BN at 20x (a) and at 60x magnification (b). Figure 2. Open in new tabDownload slide Simultaneous visualization of M1 and BN at 20x (a) and at 60x magnification (b). In order to start a validation of the method for dose assessment, a comparison between the combined protocol and the two standard methods for DCA and MN(5) has been performed on blood samples irradiated ex vivo with different X-ray doses. MATERIALS AND METHODS Whole blood samples from two healthy donors (mean age 40 y), collected after receiving informed consent, were put in heparinized tube and irradiated by using a Gilardoni CHF 320 G X-ray generator operated at 250 kVp, 15 mA, at the following doses: 0, 0.25, 0.5, 0.75, 1, 2 Gy (dose rate 0.84 Gy/min). After irradiation, for each dose, whole blood samples were divided in three different aliquots and after 2 h of recovery time, cell culture procedures started according to the standard protocols for DCA and MN(5) and to the combined protocol described in detail below. Combined protocol: for each cell culture, 0.5 ml of whole blood were dispensed in 15 ml round-bottomed sterile disposable tubes containing 4.5 ml RPMI-1640 Dutch Modification medium (Sigma-Aldrich) supplemented with 20% fetal bovine serum (Sigma-Aldrich), 2 mM L-glutamine (Euroclone), 100 units/ml of penicillin and 100 μg/ml of streptomycin solution (Euroclone). T-lymphocytes were stimulated adding 2% phytohaemagglutinin, M form (Gibco) and cultured in an incubator at 37°C keeping the tubes at about a 45° angle with closed caps. Cytochalasin B (Sigma-Aldrich) was added to the culture at 24 h (final concentration: 6 μg/ml). Colcemid solution (Sigma-Aldrich) was added 3 h before terminating the cultures (final concentration: 0.1 μg/ml). The fixation procedure conditions were optimized in order to obtain good yields of both M1 and BN cells. Fixation was performed at 58 hours: cell cultures were centrifuged at 1000 rpm for 10 min and the supernatant removed, then 4 ml of pre-warmed hypotonic solution (0.075 M potassium chloride) were added dropwise to each culture, the pellet was resuspended with a glass Pasteur pipette and the tubes were incubated at 37°C for 20 min. Then, 4 ml of cold fixative (3:1 methanol/acetic acid) were added dropwise to each culture, the suspension was resuspended with a glass Pasteur pipette and the tubes were incubated at 4°C for 30 min. Then the tubes were centrifuged at 1000 rpm for 20 min and the supernatant was removed. About 4 ml of cold fixative were added to each culture, the pellet was resuspended with a glass Pasteur pipette and the tubes were centrifuged at 1000 rpm for 10 min. Further washes with fixative can be performed until the solution becomes clear. The fixed cells may be stored in fixative at −20°C. For slide preparation, the cell suspension was centrifuged at 1000 rpm for 10 min and the supernatant was removed, leaving a suitable quantity of fixative. The pellet was resuspended with a glass Pasteur pipette and a few drops of suspension were dispensed on a cold and wet slide, previously cleaned and degreased. The slides were allowed to air dry overnight. Slides were stained with 5% Giemsa stain (Carlo Erba) prepared in Gurr phosphate buffer, pH 6.8 (VWR): slides were immersed in Giemsa solution for 8–9 min and then washed in water and allowed to air dry overnight. Slides were mounted with a cover glass using Eukitt (Sigma-Aldrich). RESULTS AND DISCUSSION By using the combined protocol, at least 100 M1 and 1000 BN could be simultaneously scored on a single slide. This amount of cells is considered adequate to perform a triage mode dose assessment suggesting that this method could represent a time-saving and inexpensive biological dosimetry assay to be used in case of massive radiological accidents. The application of this combined protocol could have several advantages in comparison to the single methods: a single blood culture is enough for both DCA and CBMN assays leading to a reduction by half of the number of cell cultures to be processed for each individual. This results in a reduced manual work which is generally a critical point in large-scale emergencies. Moreover, there is a considerable reduction in costs related to chemical reagents and less quantity of blood per subject required. It is worth noting that the use of a simple Giemsa staining instead of the FPG method, determines a further reduction in processing times and expenses. The overall PBL culture time (58 h) is shorter in comparison to the standard CBMN assay requiring 72 h, but longer compared with the standard DCA assay (48–52 h). Preliminary results, as shown in Table 1, showed a very good reproducibility between the proposed combined protocol and the standard methods in terms of dicentrics and micronuclei induction, in relation to the doses of X-ray delivered (range 0–2 Gy). Table 1. Dicentrics and micronuclei induction in X-ray irradiated samples: comparison between the combined and standard protocols. . Combined protocol . Standard protocol . . Combined protocol . Standard protocol . Dose (Gy) . M1 . DIC + CR . DIC + CR/cell (±SE) . DIC + CR . DIC + CR/cell (±SE) . BN . MN . MN/cell (±SE) . MN . MN/cell (±SE) . 0 500 0 0.00 (±0.000) 0 0.00 (±0.000) 2000 7 0.003 (±0.000) 8 0.004 (±0.001) 0.25 500 32 0.06 (±0.007) 29 0.06 (±0.007) 2000 30 0.015 (±0.002) 32 0.016 (±0.002) 0.50 500 60 0.12 (±0.024) 50 0.10 (±0.014) 2000 40 0.020 (±0.003) 44 0.022 (±0.003) 0.75 500 75 0.15 (±0.041) 65 0.13 (±0.029) 2000 84 0.042 (±0.006) 72 0.036 (±0.005) 1 200 33 0.16 (±0.005) 34 0.17 (±0.011) 2000 171 0.085 (±0.018) 149 0.074 (±0.014) 2 200 62 0.31 (±0.033) 66 0.33 (±0.007) 2000 350 0.175 (±0.025) 420 0.210 (±0.030) . Combined protocol . Standard protocol . . Combined protocol . Standard protocol . Dose (Gy) . M1 . DIC + CR . DIC + CR/cell (±SE) . DIC + CR . DIC + CR/cell (±SE) . BN . MN . MN/cell (±SE) . MN . MN/cell (±SE) . 0 500 0 0.00 (±0.000) 0 0.00 (±0.000) 2000 7 0.003 (±0.000) 8 0.004 (±0.001) 0.25 500 32 0.06 (±0.007) 29 0.06 (±0.007) 2000 30 0.015 (±0.002) 32 0.016 (±0.002) 0.50 500 60 0.12 (±0.024) 50 0.10 (±0.014) 2000 40 0.020 (±0.003) 44 0.022 (±0.003) 0.75 500 75 0.15 (±0.041) 65 0.13 (±0.029) 2000 84 0.042 (±0.006) 72 0.036 (±0.005) 1 200 33 0.16 (±0.005) 34 0.17 (±0.011) 2000 171 0.085 (±0.018) 149 0.074 (±0.014) 2 200 62 0.31 (±0.033) 66 0.33 (±0.007) 2000 350 0.175 (±0.025) 420 0.210 (±0.030) M1, metaphase in first cell division; DIC, dicentric; CR, centric ring; BN, binucleated cell; MN, micronucleus; SE, standard error. Open in new tab Table 1. Dicentrics and micronuclei induction in X-ray irradiated samples: comparison between the combined and standard protocols. . Combined protocol . Standard protocol . . Combined protocol . Standard protocol . Dose (Gy) . M1 . DIC + CR . DIC + CR/cell (±SE) . DIC + CR . DIC + CR/cell (±SE) . BN . MN . MN/cell (±SE) . MN . MN/cell (±SE) . 0 500 0 0.00 (±0.000) 0 0.00 (±0.000) 2000 7 0.003 (±0.000) 8 0.004 (±0.001) 0.25 500 32 0.06 (±0.007) 29 0.06 (±0.007) 2000 30 0.015 (±0.002) 32 0.016 (±0.002) 0.50 500 60 0.12 (±0.024) 50 0.10 (±0.014) 2000 40 0.020 (±0.003) 44 0.022 (±0.003) 0.75 500 75 0.15 (±0.041) 65 0.13 (±0.029) 2000 84 0.042 (±0.006) 72 0.036 (±0.005) 1 200 33 0.16 (±0.005) 34 0.17 (±0.011) 2000 171 0.085 (±0.018) 149 0.074 (±0.014) 2 200 62 0.31 (±0.033) 66 0.33 (±0.007) 2000 350 0.175 (±0.025) 420 0.210 (±0.030) . Combined protocol . Standard protocol . . Combined protocol . Standard protocol . Dose (Gy) . M1 . DIC + CR . DIC + CR/cell (±SE) . DIC + CR . DIC + CR/cell (±SE) . BN . MN . MN/cell (±SE) . MN . MN/cell (±SE) . 0 500 0 0.00 (±0.000) 0 0.00 (±0.000) 2000 7 0.003 (±0.000) 8 0.004 (±0.001) 0.25 500 32 0.06 (±0.007) 29 0.06 (±0.007) 2000 30 0.015 (±0.002) 32 0.016 (±0.002) 0.50 500 60 0.12 (±0.024) 50 0.10 (±0.014) 2000 40 0.020 (±0.003) 44 0.022 (±0.003) 0.75 500 75 0.15 (±0.041) 65 0.13 (±0.029) 2000 84 0.042 (±0.006) 72 0.036 (±0.005) 1 200 33 0.16 (±0.005) 34 0.17 (±0.011) 2000 171 0.085 (±0.018) 149 0.074 (±0.014) 2 200 62 0.31 (±0.033) 66 0.33 (±0.007) 2000 350 0.175 (±0.025) 420 0.210 (±0.030) M1, metaphase in first cell division; DIC, dicentric; CR, centric ring; BN, binucleated cell; MN, micronucleus; SE, standard error. Open in new tab Main disadvantage of the described protocol concerns the need for further validation through robust calibration curves, performed on a wide panel of doses (spanning from very low to extremely high), different qualities of radiation and higher number of donors. Moreover, inter-comparison exercises involving different biological dosimetry laboratories should be planned in order to verify the reproducibility of the method and to validate it for triage purposes, assuming radiation emergency scenario. The possibility to combine the already existing software for automation designed for the standard dicentric and micronucleus assays into a single one to be applied to this novel protocol should be verified. If this is not possible, it should be possible to scan the slide twice, performing the two analyses separately. Depending on the needs determined by the size of the accident, it would be possible to carry out a first categorization of overexposed subjects by MN scoring and, subsequently, a more precise dose assessment through the analysis of the dicentrics, by scanning again the same slide, could be performed. In conclusion, this protocol, once further validated, could represent a useful method for the categorization of subjects overexposed to ionizing radiation in case of mass radiological emergencies. Acknowledgements We are grateful to the personnel of the Medicine Service of ENEA Casaccia for their collaboration in collecting blood samples from healthy donors. References 1 Jaworska , A , et al. . Operational guidance for radiation emergency response organisations in Europe for using biodosimetric tools developed in EU MULTIBIODOSE project . Radiat. Prot. Dosim. 164 ( 1–2 ), 165 – 169 ( 2015 ). Google Scholar Crossref Search ADS WorldCat 2 Roy , L. , Roch-Lefevre , S. , Vaurijoux , A. , Voisin , P. , Martin , C. , Grégoire , E. and Voisin , P. Optimization of cytogenetic procedures for population triage in case of radiological emergency . Radiat. Meas. 42 ( 6–7 ), 1143 – 1146 ( 2007 ). Google Scholar Crossref Search ADS WorldCat 3 Patrono , C. , Monteiro Gil , O. , Giesen , U. , Langner , F. , Pinto , M. , Rabus , H. and Testa , A. ‘BioQuaRT’ project: design of a novel in situ protocol for the simultaneous visualisation of chromosomal aberrations and micronuclei after irradiation at microbeam facilities . Radiat. Prot. Dosim. 166 ( 1–4 ), 197 – 199 ( 2015 ). Google Scholar Crossref Search ADS WorldCat 4 Hayata , I. , Kajima , J. and Okabe , N. Distinction of metaphases in the first cell cycle for automated system in radiation dosimetry . Radiat. Phys. Chem. 39 ( 6 ), 517 – 520 ( 1992 ). OpenURL Placeholder Text WorldCat 5 International Atomic Energy Agency . Cytogenetic dosimetry: applications in preparedness for and response to radiation emergencies ( Vienna : IAEA ) ( 2011 ). Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC © The Author(s) 2019. Published by Oxford University Press. All rights reserved. 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Radiation Protection Dosimetry – Oxford University Press

Published: Dec 31, 2019

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