telomeric PNA probes and Giemsa staining to score dicentrics or excess fragments in non-stimulated lymphocyte prematurely condensed chromosomes

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Triage biodosimetry using centromeric/telomeric PNA probes and Giemsa staining to score dicentrics or excess fragments in non-stimulated lymphocyte prematurely condensed chromosomes Ioanna Karachristou a , Maria Karakosta a , Antonio Pantelias a , Vasiliki I. Hatzi a , Pantelis Karaiskos b , Panagiotis Dimitriou b , Gabriel Pantelias a , Georgia I. Terzoudi a,∗ a Laboratory of Health Physics, Radiobiology & Cytogenetics, Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety, National Centre for Scientific Research “Demokritos”, Athens, Greece b Medical Physics Laboratory, Medical School, University of Athens, Greece

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Article history: Received 15 June 2015 Accepted 17 June 2015 Available online xxx Keywords: Triage biodosimetry Premature chromosome condensation Centromere/telomere staining PNA-FISH Cell fusion Excess PCC fragments

a b s t r a c t The frequency of dicentric chromosomes in human peripheral blood lymphocytes at metaphase is considered as the “gold-standard” method for biological dosimetry and, presently, it is the most widely used for dose assessment. Yet, it needs lymphocyte stimulation and a 2-day culture, failing the requirement of rapid dose estimation, which is a high priority in radiation emergency medicine and triage biodosimetry. In the present work, we assess the applicability of cell fusion mediated premature chromosome condensation (PCC) methodology, which enables the analysis of radiation-induced chromosomal aberrations directly in non-stimulated G0 -lymphocytes, without the 2-day culture delay. Despite its advantages, quantification of an exposure by means of the PCC-method is not currently widely used, mainly because Giemsa-staining of interphase G0 -lymphocyte chromosomes facilitates the analysis of fragments and rings, but not of dicentrics. To overcome this shortcoming, the PCC-method is combined with fluorescence in situ hybridization (FISH), using simultaneously centromeric/telomeric peptide nucleic acid (PNA)-probes. This new approach enables an accurate analysis of dicentric and centric ring chromosomes, which are formed within 8 h post irradiation and will, therefore, be present in the blood sample by the time it arrives for dose estimation. For triage biodosimetry, a dose response curve for up to 10 Gy was constructed and compared to that obtained using conventional metaphase analysis with Giemsa or centromeric/telomeric PNA-probes in metaphase. Since FISH is labor intensive, a simple PCC-method scoring Giemsa-stained fragments in excess of 46 was also assessed as an even more rapid approach for triage biodosimetry. First, we studied the rejoining kinetics of fragments and constructed a dose-response curve for 24 h repair time. Then, its applicability was assessed for four different doses and compared with the PCC-method using centromeric/telomeric PNA-probes, through the evaluation of speed of analysis and minimum number of cells required for dose estimation and categorization of exposed individuals. © 2015 Published by Elsevier B.V.

1. Introduction In radiation accidents, and particularly in large scale exposures to ionizing radiation, a considerable number of individuals may be exposed to a wide distribution of doses. There is, therefore, an immediate need for population triage biodosimetry, which must be both fast and reliable. Predominantly, it is important to assess as quickly and precisely as possible the doses received by potentially overexposed people. Subsequently, if possible, there is

∗ Corresponding author. Fax: +30 210 6534710. E-mail address: [email protected] (G.I. Terzoudi).

a need to predict or reflect the clinically relevant response, i.e., the biological consequences of the dose, in order to adopt the best medical arrangement and obtain risk estimates in a prospective way [1–4]. The current “gold standard” method available for triage biodosimetry in case of a mass-casualty event is based on the interpolation of the frequency of dicentrics scored in blood lymphocytes at metaphase to a pre-established dose-effect calibration curve [4–7]. Nevertheless, the analysis of dicentrics at metaphase, which is the most validated technique, presupposes lymphocyte stimulation and a two-day culture, failing thus the requirement in triage biodosimetry for rapid estimate of the dose, which is a high priority in radiation emergency medicine. Furthermore, another limitation of the analysis of dicentrics at metaphase is its use after

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exposures to very high doses. The activation of different cell-cycle checkpoints to ensure genome integrity, results in reduced cell proliferation and, as a result, only a low number of metaphases will be available for the analysis, following high dose exposure. In fact, at doses above 5 Gy, cellular damage may hamper or even prevent, particularly the highly damaged lymphocytes, to reach mitosis [8–10]. To overcome the shortcomings of the conventional method, in our early work we induced premature chromosome condensation (PCC) in blood lymphocytes by means of their fusion with Chinese Hamster Ovary (CHO) mitotic cells using polyethylene glycol, enabling thus the analysis of radiation-induced chromosomal aberrations directly in non-stimulated G0 -lymphocytes [11–13]. At present, the PCC-method is considered as an important alternative assay in radiation emergency medicine, particularly for high-dose exposures [5,7,14,15]. Specifically, following exposure to ionizing radiation, the PCC-method enables the measurement of chromosomal aberrations directly in G0 -lymphocytes shortly after blood sampling, without the need for their mitogen stimulation and the 2-day culture delay [12]. Indeed, Giemsa staining of lymphocyte prematurely condensed chromosomes (PCCs) allows a rapid quantification of an exposure by means of PCC fragments and rings, using appropriate calibration curves [13,16,17]. However, even though solid Giemsa-staining of G0 - lymphocyte PCCs facilitates the analysis of fragments and rings, it does not allow detection of centric ring and dicentric chromosomes, which are also very important biomarkers of exposure. Combining the PCC-method with C-banding [18] or with fluorescence in situ hybridization (FISH) techniques using specific DNA libraries for centromeric regions [19], this shortcoming is overcome and the analysis of dicentrics and centric ring chromosomes directly in non-stimulated lymphocytes becomes possible. Nonetheless, these early attempts, and particularly the use of C-banding, gave poor accuracy and reproducibility and despite its advantages, quantification of an exposure by means of the PCC-method is not at present widely used. Recently, the development of centromeric/telomeric (C/T) peptide nucleic acid (PNA) probes and their simultaneous use in lymphocyte PCCs in combination with FISH, has offered a new dynamic to the PCC-method, enabling the analysis of dicentrics and centric ring chromosomes directly in non-stimulated lymphocytes, with a level of accuracy and ease not possible previously [20–22]. In the present work, we make use of the PCC-method in combination with PNA probes and the FISH technique in order to construct a dose response curve for up to 10 Gy. The usefulness, reliability and applicability of this approach in triage biodosimetry is compared with the conventional method through the scoring of dicentrics at metaphase, by means of Giemsa stain or the use of C/T PNA probes [23]. Furthermore, given that the application of the C/T PNA probes in combination with the FISH technique is inherently labor intensive and time consuming, a simple PCC-method, based on the scoring of Giemsa stained fragments in excess of 46, is also assessed and proposed here as an even more rapid alternative approach for triage biodosimetry. The great advantage of this approach is that dose estimates can be obtained within only 2 h after collection of blood sample for analysis. The rejoining kinetics of excess fragments is studied for up to 24 h and a dose–response curve for 24 h repair time is constructed and applied to obtain dose estimates. In addition, the reliability of this method for its triage application and potential is assessed at four different doses of exposure. The results are compared to those obtained using the PCC-method with C/T PNA probes, through the evaluation of speed of analysis and minimum number of cells required for dose estimation and categorization of exposed individuals in case of a radiation accident.

2. Material and methods 2.1. Cell cultures and irradiation conditions Peripheral blood from healthy individuals was drawn in heparinized tubes. Informed consent was obtained for each donor. Cultures were set up by adding 0.5 ml of whole blood to 5 ml of RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% phytohemagglutinin (PHA), 1% glutamine and antibiotics [penicillin: 10,000 U/ml; streptomycin: 10,000 ␮g/ml (Biochrom)]. Chinese hamster Ovary (CHO) cells were grown in McCoy’s 5A (Biochrom), culture medium supplemented with 10% FBS, 1% l-gLutamine and antibiotics, incubated at 37 ◦ C in a humidified atmosphere with 5% CO2 . CHO cultures were maintained as exponentially growing monolayer cultures in 75 cm2 plastic flasks at an initial density of 4 × 105 cells/flask. Colcemid (Gibco) at a final concentration of 0.1 ␮g/ml was added to CHO cultures for 4 h and the accumulated mitotic cells were harvested by selective detachment. Once a sufficient number of mitotic cells had been obtained, they were used as mitotic promoting factors (MPF) supplier and PCC inducer in human lymphocytes. Irradiation was carried out in a Gamma Cell 220 irradiator (Atomic Energy of Canada Ltd., Ottawa, Canada) at room temperature and at a dose rate of 40 cGy/min. Different irradiation times were used in order to administer to the blood samples doses ranging from 1 to 10 Gy.

2.2. Cell fusion mediated premature chromosome condensation Human lymphocytes were separated from heparinized blood samples according to a slightly modified Ficoll–Paque method using the biocoll separating solution, following the procedures suggested by the manufacturer (Biochrom). The whole blood was carefully layered on top of an equal amount of biocoll separating solution in a test tube before centrifugation. The isolated lymphocytes were kept in culture medium (RPMI-1640) supplemented with 10% FBS, 1% glutamine and antibiotics. Thereafter, irradiation was carried out and for the repair of DNA damage lymphocytes were incubated at 37 ◦ C. Generally, lymphocytes isolated from 1 ml of blood were used for 1–2 experimental points. Cell fusion and PCC induction was performed using polyethylene glycol (PEG) as previously described [11,24]. Briefly, mitotic CHO cells harvested from a 75 cm2 flask were used for 2–3 fusions. Lymphocytes and mitotic CHO cells were mixed in serum-free RPMI-1640 medium in a 15 ml round-bottom culture tube in the presence of colcemid. After centrifugation at 1000 rpm for 6 min, the supernatant was discarded without disturbing the cell pellet, keeping the tubes always inverted in a test tube rack on a paper towel to drain the pellet from excess liquid. While holding the tubes in an inverted position, 0.15 ml of 50% (w/v) polyethylene glycol (PEG 1500, Boehringer Mannheim) or 45% of PEG (p5402, Sigma–Aldrich) was injected forcefully against the cell pellet using a micropipette, and immediately after the tube was turned in an upright position and held for about 1 min. Subsequently, 1.5–2 ml of PBS was slowly added, the tube was shaken gently and the cell suspension was centrifuged at 1000 rpm for 6 min. The supernatant was discarded and the cell pellet was resuspended gently in 0.7 ml RPMI-1640 complete growth medium with colcemid. To optimize cell fusion when a low number of lymphocytes is available, complete lymphocyte growth medium, containing PHA, was used [11,12]. After 60–75 min at 37 ◦ C, cell fusion and PCC induction was completed, cells were treated with hypotonic KCl (0.075 M), and fixed with methanol: glacial acetic acid (3:1, v/v). The chromosome spreads were prepared by standard cytogenetic procedures and air-dried slides were stained in 3% Giemsa solution. For the PCC analysis, the excess PCC fragments per cell (exceeding 46 pieces/cell) were scored for each

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experimental point, using light microscopy coupled with an image analysis system (Ikaros MetaSystems, Germany). 2.3. Centromeric and telomeric staining with PNA probes Staining of centromeres and telomeres in lymphocyte PCCs or at metaphase, was performed using the Q-FISH technique with a Cy3-labeled PNA probe specific for telomere sequences and a FAM-labeled PNA probe specific for centromere sequences (both from Panagene, Daejon, South Korea). Briefly, the slides were kept in an oven at 60 ◦ Cfor at least 1 h, washed in phosphate buffered saline (PBS) solution for 15 min, fixed in formaldehyde 4% solution for 2 min, washed again in PBS twice for 5 min and digested in a pre-warm pepsin solution (1 mg/ml) for 3 min at 37 ◦ C. After 3 PBS washes, slides were washed and refixed, dehydrated with 70%, 90%, 100% ethanol and air dried. PNA probes for centromere and telomere staining were applied, co-denaturated for 3 min at 80 ◦ C and incubated for 2 h in a humidified chamber at room temperature in the dark. After hybridization, slides were washed with 70% Formamide, 1% Tris 1 M pH7.2, 1% BSA 10%, H2 O, twice for 15 min, then in TBS/Tween 0.08% three times for 5 min each, dehydrated with 70%, 90%, 100% ethanol series, and finally counterstained with DAPI (1 ␮g/ml) and mounting medium. 2.4. Analysis and scoring criteria Lymphocyte PCC spreads and metaphases were located manually and their analysis was greatly facilitated by the use of a semi-automated image analysis system (MetaSystems). Following uniform Giemsa staining of lymphocytes at metaphase demonstrating 46 centromeres, the yields of dicentrics plus centric ring chromosomes were obtained based on the morphology of chromosomes. When C/T staining with PNA probes was applied, dicentrics plus centric ring chromosomes were quantified based on the detection of centromeric regions and telomeric sequences. Only metaphases or PCC spreads with 46 centromeres were analyzed. We detected dicentrics with 4 telomeres at metaphase or with 2 telomeres in the PCC spreads. The analysis of excess PCC fragments in lymphocyte PCC spreads stained with Giemsa, is greatly facilitated by the appearance of the PCCs, which are lighter stained than the CHO mitotic cells and, therefore, easily distinguished from the mitotic chromosomes of the CHO cells. In unirradiated lymphocytes, 45–46 elements were scored in PCCs spreads and for calculating the frequency of excess PCC fragments, this number was subtracted from the one obtained in the irradiated lymphocyte PCCs. Generally, a number of 30 PCC-spreads was considered adequate for dose estimation following a single exposure. 2.5. Calibration curves Appropriate calibration curves were constructed for dose estimation for each of the different endpoints of chromosomal aberrations analyzed (Fig. 5), i.e., dicentric and ring analysis after conventional Giemsa staining or C/T-metaphase staining; dicentric and centric ring chromosomes using C/T-PCC staining with PNA probes;and for comparison, excess PCC fragments using Giemsa staining of PCC-spreads (Fig. 7). The dose-response curves were generated by in vitro irradiation of unstimulated lymphocytes under conditions as close as possible to in vivo situations. Using C/T staining in lymphocyte PCCs, the dose–response curves for PCC-dicentric and centric ring chromosomes fitted linear-quadratic relationships, similar to that obtained for dicentric and centric ring chromosomes at metaphase. However, for excess PCC fragments using Giemsa staining, a linear dose–response curve was obtained.

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3. Results Cell fusion mediated premature chromosome condensation enables the visualization and quantification of interphase chromosomes in peripheral blood lymphocytes,without the need for their mitogen stimulation and the 2-day culture delay. In lymphocytes obtained from non-irradiated individuals, 46 distinct single chromatid prematurely condensed chromosomes (PCCs) can be scored (Fig. 1A). Following an in vitro exposure of blood lymphocytes e.g., 4 Gy, 5 excess (over 46) fragments on the average can be scored at 8 to 24 h after irradiation (Fig. 1B). Even though solid Giemsastaining of G0 -lymphocyte PCCs facilitates the analysis of fragments and rings, it does not allow detection of dicentrics and centric ring chromosomes (Fig. 1C). The arrow in Fig. 1C points to a probable dicentric chromosome but detection of the centromeric regions is necessary in order to confirm it. The simultaneous use of (C/T) peptide nucleic acid (PNA) probes in lymphocyte PCCs in combination with FISH enabled the analysis of dicentrics and centric ring chromosomes directly in non-stimulated lymphocytes. Using this C/T-PCC-FISH method in lymphocytes obtained from non-irradiated individuals, not only the 46 distinct single chromatid PCCs can be visualized but also all their centromeres and telomeres (Fig. 2A). Following in vitro exposure of blood lymphocytes e.g., to 8 Gy, at 8 to 24 h post-irradiation, excess fragments can be scored in non-stimulated lymphocytes, and also the dicentrics and centric ring chromosomes, with a level of accuracy and ease not previously achievable (Fig. 2B). Using the C/T-PCC method, a dose response curve was constructed for the frequency of dicentrics plus centric ring chromosomes for 8 h and 24 h post irradiation repair and for doses up to 10 Gy (Fig. 3). The frequency of dicentrics plus centric rings increased with dose according to a linear quadratic relationship. There was no significant difference between the 8 h and 24 h doseresponse curves. The reliability and applicability of this cytogenetic approach in triage biodosimetry was evaluated through the comparison of the dose-response constructed for the C/T-PCC method with those obtained when scoring dicentrics and centric rings at metaphase using Giemsa stain (Fig. 4A) or C/T PNA probes in lymphocytes at metaphase (Fig. 4B). Fig. 5 shows the dose-response curves obtained for the three different biodosimetry methodologies used. The application of C/T PNA probes in lymphocyte PCCs in combination with the FISH technique shows a better sensitivity, particularly at high doses. However, since this methodology is inherently labor intensive, a simple PCC-method, based on the scoring of Giemsa stained PCC fragments (Fig. 1) in excess of 46, was also assessed and tested as an even more rapid alternative approach for triage biodosimetry. The rejoining kinetics of excess PCC fragments after a 4 Gy in vitro irradiation of blood lymphocytes and for up to 24 h repair time, is shown in Fig. 6. At 0 h the yield of excess fragments was high (14 excess PCC fragments/ cell), due to the unrejoined chromosome breaks, after 6 h it dropped (5 excess PCC fragments/ cell) and reached a plateau after 8 h (4 excess PCC fragments/ cell). The dose-response curve for excess PCC fragments at 24 h post irradiation repair period and for doses up to 9 Gy is shown in Fig. 7. The excess PCCs frequencies observed were adjusted to a linear relationship (Y = Y0 + aX), with linear coefficient a = 1.22. In order to determine which approach is more applicable in triage biodosimetry, a simulation of a real accident was performed. Whole blood was irradiated with 4 different doses i.e. 1.0, 2.0, 3.5 and 7.0 Gy of gamma irradiation and the samples were then coded blindly. The PCC assay was performed and the PCC spreads were stained either with Giemsa or with C/T peptide nucleic acid (PNA) probes. The yields of excess PCC fragments Giemsa stained or dicentrics plus centric rings in lymphocyte PCCs visualized by the FISH technique were obtained scoring 10 cells, 20 cells or 30 cells.

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Fig. 1. Giemsa stained PCCs demonstrating 46 single chromatid chromosomes in a non-irradiated lymphocyte (A). Five excess (over 46) PCC fragments can be visualized in an irradiated lymphocyte with 4 Gy (B). Using Giemsa staining, dicentrics and centric ring chromosomes cannot be identified. The arrow points to a probable dicentric chromosome but detection of the centromeric regions is necessary in order to confirm it(C). Table 1 Simulated whole body exposure to 1, 2, 3.5 and 7 Gy and dose estimation for triage biodosimetry purposes using the C/T – PCC- FISH methodology or the analysis of excess PCC fragments Giemsa stained and scoring only 10, 20 or 30 non stimulated lymphocyte PCC spreads. Mean doses are shown with upper and low confidence levels (UCL/LCL) for dicentric plus centric ring (Dic + CR) analysis, and upper and low dose levels (UDL/LDL) for the excess fragment analysis. 10CELLS

20CELLS

30CELLS

Estimated dose (Gy)

Estimated dose (Gy)

Estimated dose (Gy)

Given Dose (Gy)

Dic + CR

Dose

UCL

LCL

Dic + CR

Dose

UCL

LCL

Dic + CR

Dose

UCL

LCL

1.0 2.0 3.5 7.0

0 3 12 33

0.00 2.06 4.27 7.19

2.30 3.63 5.70 8.55

0.00 0.86 3.03 5.94

1 5 23 77

0.76 1.87 4.18 7.77

1.98 2.93 5.15 8.71

0.04 1.00 3.30 6.89

2 11 31 116

0.89 2.29 3.95 7.79

0.23 3.12 4.74 7.10

1.83 1.58 3.23 8.55

Estimated dose (Gy)

Estimated dose (Gy)

Estimated dose (Gy)

Given Dose (Gy)

Excess fragms

Dose

UDL

LDL

Excess fragms

Dose

UDL

LDL

Excess fragms

Dose

UDL

LDL

1.0 2.0 3.5 7.0

9 22 47 86

0.81 1.87 3.92 7.12

1.10 2.31 4.42 7.74

0.52 1.44 3.43 6.51

17 37 82 157

0.76 1.58 3.43 6.51

1.05 2.02 3.92 7.12

0.48 1.15 2.93 5.90

27 52 122 250

0.81 1.49 3.40 6.91

1.09 1.92 3.90 7.52

0.52 1.05 2.91 6.29

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Fig. 3. Using the C/T-PCC-FISH methodology,dose response curves for doses up to 10 Gy were constructed for 8 h and 24 h post irradiation repair periods. The frequency of dicentrics plus centric rings increased with dose according to a linear quadratic relationship.

Fig. 2. Lymphocyte PCCs visualized by means of C/T staining with PNA probes. In a non-irradiated lymphocyte, not only the 46 distinct single chromatid PCCs can be now visualized, but their centromeres and telomeres as well (A). Dicentrics and centric ring chromosomes can be detected in non-stimulated lymphocytes with a level of accuracy and ease not previously achievable. Dicentrics and a centric ring chromosome are shown in a lymphocyte exposed to 8 Gy, following a 24 h post irradiation repair period (B).

The yields from this analysis were then assigned to the calibration curve for each method and the estimated doses are presented in Table 1. In order to examine whether there is a statistically significant difference between the two methods used, the statistical method of t–test was applied. 4. Discussion The rationale for triaging large populations based on dose is to set a reasonable cutoff of dose absorbed, below which treatment is not expected to impact survival rates and above which treatment is necessary to improve survival rates. This cutoff is generally set at 2 Gy, but this threshold could plausibly be set higher, e.g., 3 Gy, if the numbers of affected individuals were beyond the capabilities of the medical system [1,25–27]. Essentially, the aim in triage

biodosimetry is to classify the exposed individuals into three categories: those who have suffered radiation injury, for whom immediate medical intervention could mean the difference between life and death; those with intermediate doses, for whom medical intervention will be necessary to mitigate the short, medium and long term effects of exposure, and the “worried well” with probable low doses, for whom no deterministic effects are expected but long term monitoring may be required [28]. For many years, metaphase analysis and frequency of chromosomal aberrations and rearrangements in peripheral blood lymphocytes (PBL) have been used for biodosimetry purposes and the estimation of absorbed doses in real or suspected radiation exposures [4–7]. Specifically, the interpolation of the frequency of dicentrics scored at metaphase to a pre-established dose-effect calibration curve is considered as the «gold standard» method for biological dosimetry that may be also applied for triage biodosimetry in case of a mass-casualty event [29–31]. At present, it is the most validated and widely used method for dose assessment and has been proved to be a very useful biodosimetry tool for acute, protracted, or partial-body exposure [32]. Nevertheless, rapid dose estimates are mainly hampered by the 2-day blood culture required and the delay of lymphocytes carrying dicentrics in their progress throughout the cell cycle. As shown in Figs. 2 and 3, these shortcomings can be now overcome through the use of C/T PNA probes in lymphocyte PCCs in combination with the FISH technique. Specifically, in the present study the applicability of the C/T-PCC-FISH method, based on the yield of dicentrics and centric ring chromosomes scored directly in non-stimulated lymphocyte PCCs, was compared to the methods that use analysis of chromosomal aberrations at metaphase (Fig. 4A and B). Interestingly, since these chromosome aberrations are formed within 8 h post irradiation time, it is plausible that they will be present in the blood sample by the time it arrives for dose estimation. There is no need, therefore, for a 2-day blood culture delay required by the metaphase analysis methods. Based on the dose response curves constructed for the different methods used as presented in Fig. 5, it can be concluded that the application of C/T PNA probes in lymphocyte PCCs in combination with the FISH technique has several advantages and shows a better sensitivity, particularly at high doses, when compared to the conventional chromosome aberration analysis at metaphase. Application of the C/T-PCC-FISH methodology is nevertheless inherently labour intensive and, as an even more rapid

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Fig. 5. Yield of dicentrics and centric ring chromosomes for doses up to 10 Gy using three different approaches: (i) conventional metaphase analysis (ii) metaphase analysis combined with CT staining and FISH and (iii) lymphocyte PCC analysis by using CT and FISH staining.

Fig. 6. Rejoining kinetics of excess PCC fragments after a 4 Gy in vitro irradiation of blood lymphocytes and for up to 24 h repair time.

Fig. 4. Dicentrics and centric ring chromosomes as visualized using the conventional metaphase analysis and Giemsa staining (A). Dicentrics and centric rings detected in lymphocytes at metaphase by means of C/T PNA probes in combination with the FISH technique (B).

alternative approach for triage biodosimetry, here we have proposed and tested a simple PCC-method, based on the scoring of Giemsa stained PCC fragments in excess of 46, as shown in Fig. 1. The objective is to assess the suitability of this simple PCC approach and compare it with the C/T-PCC-FISH methodology for cases of mass casualties with respect to the speed of analysis and the minimum number of cells required to detect and discriminate between exposed and unexposed individuals at different dose levels. The rejoining kinetics of excess PCC fragments after a 4 Gy in vitro irradiation of blood lymphocytes, as shown in Fig. 6, demonstrates that the excess PCC fragments reach a plateau after 8 h post irradiation time period. Therefore, the dose response curve shown in Fig. 7, which is constructed after a 24 h repair period, can be used to obtain dose estimates in case of radiation accidents. The results presented in Table 1 show the dose estimates obtained by applying

Fig. 7. Dose–response curve for excess lymphocyte PCC fragments at 24 h post irradiation repair period and for doses up to 9 Gy.

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the two different PCC approaches following blood sample irradiation at 4 different doses i.e. 1.0, 2.0, 3.5 and 7.0 Gy. Using the t-test, no statistically significant difference was found between the doses estimated by the C/T-PCC-FISH method or the method based on Giemsa stained excess PCC fragments when 10 cells were scored (P = 0.981), when 20 cells were scored (P = 0.731) or when 30 cells were scored (P = 0.757). Although the C/T staining with peptide nucleic acid (PNA) probes enables a rapid detection of dicentrics and centric ring chromosomes in lymphocyte PCCs with accuracy and ease, the procedure is still laborious and time consuming when compared to the analysis of Giemsa stained excess PCC fragments. The analysis of only 10 PCC spreads gives satisfactory dose estimates and comparable results to those obtained from the analysis of 30 spreads, even for the exposure of 1 Gy. Interestingly, the PCC assay based on the yield of excess PCC fragments Giemsa stained, seems also to have many advantages enabling a rapid estimation of absorbed dose within 2 h with a satisfactory level of accuracy and easy, especially for triage biodosimetry.

[7] [8]

[9] [10]

[11]

[12]

[13]

5. Conclusions

[14]

FISH analysis of dicentrics in non-stimulated lymphocyte PCCs by means of C/T PNA probes is shown to be a reliable, sensitive, accurate, and a faster approach for the estimation of absorbed doses, when compared with the analysis of dicentrics at metaphase, which presupposes a 2-day delay for lymphocyte culture. Interestingly, the PCC assay based simply on Giemsa stained excess PCC fragments is also advantageous, and can potentially become a reliable and quick approach for triage biodosimetry, since dose estimates can be obtained within only 2 h after the collection of blood samples. In addition, the analysis of excess fragments is simple and its automation could potentially reduce the number of highly trained individuals needed while increasing the throughput and scoring objectivity of this assay.

[15]

Conflict of interest The authors declare no conflict of interest and no competing interest.

[16]

[17]

[18]

[19]

[20]

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Please cite this article in press as: I. Karachristou, et al., Triage biodosimetry using centromeric/telomeric PNA probes and Giemsa staining to score dicentrics or excess fragments in non-stimulated lymphocyte prematurely condensed chromosomes, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2015), http://dx.doi.org/10.1016/j.mrgentox.2015.06.013