<p>In the April 2013 issue of Haematologica, Lee <em>et al.</em> have described the <em>TET1</em> genomic breakpoints and clinical features of <em>MLL-TET1</em> rearranged cases of acute leukemia. So far, 13 cases have been reported in the literature, 11 in acute myeloid leukemia (AML) patients and 2 in B-cell precursor acute lymphoblastic leukemia (ALL). It was also recently reported that <em>MLL</em> is fused to <em>TET1</em> in only 5 out of 1,590 <em>MLL</em> rearranged <em>AML</em> cases (0.3%). Although those cases are very uncommon, their study can improve our current understanding of leukemogenesis. We report here the first t(10;11) <em>MLL-TET1</em> positive case of T-cell lymphoblastic lymphoma occurring in a 31-year old male patient, with a subsequent transformation to AML.</p> <p>The patient was referred for a large mediastinal mass and right pleural effusion. Blood cell count showed no abnormalities. Mediastinal and bronchus biopsies led to the diagnosis of a precursor-T-cell lymphoblastic lymphoma (pre-T LBL), expressing CD3, CD5, CD4, CD8 and CD10 antigens, together with a high expression of Ki67 (90%). No expression of CD34 or CD79a was observed. The same cells were observed in pleural fluid that expressed CD3, CD4, CD8, CD2, CD7, CD10 antigens but neither CD34 nor myeloperoxidase. Bone marrow examination and central nervous system imaging did not show any other specific localization. The patient was treated following the Groupe d’Etudes des Lymphomes de l’Adulte (GELA) LL03 protocol, and was considered in complete remission after induction and consolidation phases. A 32×22×48 mm residual mediastinal mass remained after treatment, without hypermetabolic abnormality on the FDG-PET scan and was considered to be fibronecrotic scar tissue. Fourteen months after the diagnosis, during the maintenance therapy, a bone marrow examination was performed for thrombopenia (6 g/L) that revealed a myelomonocytic acute leukemia with trilineage dysplasia. The mediastinal mass remained unchanged on the imaging scan. The patient achieved complete remission after intensive chemotherapy based on cytarabine and daunorubicin, followed by a consolidation course with high-dose cytarabine. A non-familial donor allogeneic bone marrow transplant (10/10 match) was performed four months after the diagnosis of the acute myeloid leukemia that was complicated by a Grade IV acute graft-<em>versus</em>-host disease involving digestive tract, liver and skin. The patient died 54 days after the transplant of bacterial sepsis leading to multi-organ failure.</p>

Environmental factors such as solar ultraviolet (UV) radiation and other external aggressors provide an oxidative challenge that is detrimental to skin health. The levels of endogenous antioxidants decrease with age, thus resulting in less protection and a greater potential for skin damage. The NF-E2-related factor-2 (Nrf2) - antioxidant response element (ARE) pathway is a primary defense mechanism against oxidative stress, and induces the expression of antioxidant, detoxification and repair genes. Activation of ARE-Nrf2 can help restore oxidative homeostasis of the skin and play a role in inflammatory response and DNA repair mechanisms.To evaluate the role of a purified parthenolide-depleted Feverfew (PD-Feverfew) extract on the ARE-Nrf2 pathway and DNA repair in skin cells.These studies were undertaken in primary human keratinocytes or KB cells using Luciferase Promoter assay, siRNA transfection studies, Western blot analyses, Immunofluorescence microscopy, comet assay and quantitative real-time PCR.PD-Feverfew was found to induce Nrf2 nuclear translocation and to increase ARE activity in a dose dependent manner. Furthermore, knockdown of Nrf2 resulted in suppression of PD-Feverfew-induced ARE activity. PD-Feverfew was also found to induce phosphorylation of Akt, a kinase downstream of PI3K. Inhibition of PI3K via pre-treatment with the selective pharmacological inhibitor, LY294002, abolished PD-Feverfew-induced Nrf2/ARE activation. PD-Feverfew also reduced UV-induced DNA damage in a PI3K and Nrf2-dependent manner.Therefore, by increasing endogenous defense mechanisms and aid in DNA repair of damaged skin cells via activation of a PI3K-dependent Nrf2/ARE pathway, PD-Feverfew may help protect the skin from numerous environmental aggressors.

Digital object identifier (DOI): 10.1016/j.jdermsci.2013.08.004

Genetic and biochemical sperm integrity is essential to ensure the reproductive competence. However, spermatogenesis involves physiological changes that could endanger sperm integrity. DNA protamination and apoptosis have been studied extensively. Furthermore, elevated rates of aneuploidy and DNA injury correlate with reproductive failures. Consequently, this study applied the conventional spermiogram method in combination with molecular tests to assess genetic integrity in ejaculate from normozoospermic patients with implantation failure by retrospectively analysing aneuploidy (chromosomes 18, X, Y), DNA fragmentation, externalization of phosphatidylserine and mitochondrial membrane potential status before and after magnetic activated cell sorting (MACS). Aneuploid, apoptotic and DNA-injured spermatozoa decreased significantly after MACS. A positive correlation was detected between reduction of aneuploidy and decreased DNA damage, but no correlation was determined with apoptotic markers. The interactions between apoptotic markers, DNA integrity and aneuploidy, and the effect of MACS on these parameters, remain unknown. In conclusion, use of MACS reduced aneuploidy, DNA fragmentation and apoptosis. A postulated mechanism relating aneuploidy and DNA injury is discussed; on the contrary, cell death markers could not be related. An 'apoptotic-like' route could explain this situation. Genetic and biochemical sperm integrity is essential to ensure reproductive success and support the earliest phases of embryo development. Paradoxically, spermatogenesis involves physiological changes that could endanger the DNA and cell integrity. Sperm-specific mechanisms have been studied extensively, and DNA packaging and programmed cell death (apoptosis) are potentially harmful. Also, elevated rates of chromosomal numerical abnormalities and breakage of sperm DNA have been correlated with reproductive failures. In this context, basic sperm examination methods have been combined with molecular tests to assess genetic integrity. On the other hand, magnetic activated cell sorting (MACS) can reduce the number of programmed-to-death spermatozoa. This system retains damaged spermatozoa, thereby improving the sample's quality. The relationships between apoptosis, DNA integrity and chromosomal abnormalities (aneuploidy) as a whole, and the effect of MACS on these parameters remain unknown. We analysed aneuploidy, DNA damage, and biochemical markers of cell death in ejaculate from normozoospermic patients with implantation failures before and after MACS. Aneuploid, apoptotic and DNA-injured spermatozoa decreased significantly after MACS. A positive correlation was detected between the reduction of aneuploidy and DNA damage; on the contrary, no correlation was determined with apoptotic markers. In conclusion, the use of MACS reduced aneuploidy, DNA breakages and apoptosis. A hypothesized mechanism relating aneuploidy and DNA injury is discussed; on the contrary, death cell markers could not be directly related. An 'apoptotic-like' route could explain this situation.

Human tumors using the alternative lengthening of telomeres (ALT) exert high rates of telomere dysfunction. Numerical chromosomal aberrations are very frequent, and structural rearrangements are widely scattered among the genome. This challenging context allows the study of telomere dysfunction-driven chromosomal instability in neoplasia (CIN) in a massive scale. We used molecular cytogenetics to achieve detailed karyotyping in 10 human ALT neoplastic cell lines. We identified 518 clonal recombinant chromosomes affected by 649 structural rearrangements. While all human chromosomes were involved in random or clonal, terminal, or pericentromeric rearrangements and were capable to undergo telomere healing at broken ends, a differential recombinatorial propensity of specific genomic regions was noted. We show that ALT cells undergo epigenetic modifications rendering polycentric chromosomes functionally monocentric, and because of increased terminal recombinogenicity, they generate clonal recombinant chromosomes with interstitial telomeric repeats. Losses of chromosomes 13, X, and 22, gains of 2, 3, 5, and 20, and translocation/deletion events involving several common chromosomal fragile sites (CFSs) were recurrent. Long-term reconstitution of telomerase activity in ALT cells reduced significantly the rates of random ongoing telomeric and pericentromeric CIN. However, the contribution of CFS in overall CIN remained unaffected, suggesting that in ALT cells whole-genome replication stress is not suppressed by telomerase activation. Our results provide novel insights into ALT-driven CIN, unveiling in parallel specific genomic sites that may harbor genes critical for ALT cancerous cell growth.

A variant of del(20q), an isochromosome of the long arm with the loss of an interstitial part of 20q, ider(20q), has been reported in patients with myeloid diseases (Li et al, 2004). About 40 cases with this rearrangement have been reported up to 2012 (reviewed by Mullier et al, 2012). Molecular cytogenetic and array techniques have been used for mapping of the deleted region on 20q (Douet-Guilbert et al, 2009). The proximal breakpoints are consistently located in the 20q11.21 band, and the distal breakpoints span from band 20q13.13 to band 20q13.33.

Meiosis generates haploid cells or spores for sexual reproduction. As a prelude to haploidization, homologous chromosomes pair and recombine to undergo segregation during the first meiotic division. During the entire meiotic prophase of the yeast Saccharomyces cerevisiae, chromosomes perform rapid movements that are suspected to contribute to the regulation of recombination. Here, we investigated the impact of ionizing radiation (IR) on movements of GFP-tagged bivalents in live pachytene cells. We find that exposure of sporulating cultures with >40 Gy (4-krad) X-rays stalls pachytene chromosome movements. This identifies a previously undescribed acute radiation response in yeast meiosis, which contrasts with its reported radioresistance of up to 1,000 Gy in survival assays. A modified 3'-end labeling assay disclosed IR-induced dsDNA breaks (DSBs) in pachytene cells at a linear dose relationship of one IR-induced DSB per cell per 5 Gy. Dihydroethidium staining revealed formation of reactive oxygen species (ROS) in irradiated cells. Immobility of fuzzy-appearing irradiated bivalents was rescued by addition of radical scavengers. Hydrogen peroxide-induced ROS did reduce bivalent mobility similar to 40 Gy X IR, while they failed to induce DSBs. IR- and H2O2-induced ROS were found to decompose actin cables that are driving meiotic chromosome mobility, an effect that could be rescued by antioxidant treatment. Hence, it appears that the meiotic actin cytoskeleton is a radical-sensitive system that inhibits bivalent movements in response to IR- and oxidant-induced ROS. This may be important to prevent motility-driven unfavorable chromosome interactions when meiotic recombination has to proceed in genotoxic environments.

The biological dose of nuclear workers engaged in emergency response tasks at Tokyo Electric Power Company (TEPCO) Fukushima Daiichi Nuclear Power Station was estimated in the present study. As the national core center for radiation emergency medical preparedness in Japan, the National Institute of Radiological Sciences (NIRS) received all individuals who were suspected of being overexposed to acute radiation. In the course of health examinations at NIRS, biological dosimetry was performed by the dicentric chromosome assay (DCA). Twelve individuals were examined from 21 March-1 July 2011. The results indicated that the estimated exposure doses for all individuals were lower than 30 mGy, with the mean value of about 101 mGy. These results by DCA were in accordance with those obtained by physical dosimetry based on personal dosimeter recording assessment. The results corroborate the fact that no acute radiation syndrome was observed among the workers examined.

Pancreatic tumors contain cancer stem cells highly resistant to chemotherapy. The identification of therapies that can eliminate this population of cells might provide with more effective treatments. In the current work we evaluated the potential of oncolytic adenoviruses to act against pancreatic cancer stem cells (PCSC). PCSC from two patient-derived xenograft models were isolated from orthotopic pancreatic tumors treated with saline, or with the chemotherapeutic agent gemcitabine. An enrichment in the number of PCSC expressing the cell surface marker CD133 and a marked enhancement on tumorsphere formation was observed in gemcitabine treated tumors. No significant increase in the CD44, CD24, and epithelial-specific antigen (ESA) positive cells was observed. Neoplastic sphere-forming cells were susceptible to adenoviral infection and exposure to oncolytic adenoviruses resulted in elevated cytotoxicity with both Adwt and the tumor specific AduPARE1A adenovirus. In vivo, intravenous administration of a single dose of AduPARE1A in human-derived pancreatic xenografts led to a remarkable anti-tumor effect. In contrast to gemcitabine AduPARE1A treatment did not result in PCSC enrichment. No enrichment on tumorspheres neither on the CD133(+) population was detected. Therefore our data provide evidences of the relevance of uPAR-controlled oncolytic adenoviruses for the elimination of pancreatic cancer stem cells.

<p>In order to evaluate DNA damage induced by protons at low and radiotherapeutic doses at the therapeutic proton complex at Ružomberok, Slovak Republic, lymphocytes from umbilical cord blood (UCB) of the same four probands were irradiated in the dose range of 1-200 cGy with γ-rays and protons (200 MeV, irradiation in the Bragg peak). DNA repair γH2AX/53BP1 foci were analyzed by fluorescent microscopy and flow cytometry. Statistically significant effects of radiations were detected by fluorescent microscopy at all doses higher 1 cGy. Almost all distributions of foci in irradiated cells fitted to the Poisson distribution. In general, there was no difference in the levels of γH2AX and 53BP1 foci in irradiated cells. Flow cytometry was less sensitive and detected radiation induced effects at doses of 50 cGy and higher. Factorial analysis of variance in the whole studied dose range has shown no significant effect of radiation quality on number of γH2AX and 53BP1 foci. The ratio of proton-induced foci to γ-ray-induced foci was 0.86  ± 0.16 (53BP1) and 0.99  ± 0.34 (γH2AX) as measured by fluorescent microscopy and 0.99 ± 0.16 (γH2AX) as measured by flow cytometry at the radiotherapeutic dose of 2 Gy.Both flow cytometry and fluorescent microscopy indicated that the average value of relative biological efficiency (RBE) at radiation doses ≥ 20 cGy was about 1.0. Our data that RBE increased at low doses ≤ 20 cGy are relevant both to the development of treatment modalities and exposures that take place during space exploration and should be verified by further studies.</p>

Digital object identifier (DOI): 10.3109/09553002.2013.797619

<p>Hydroquinone (HQ) is found in natural and anthropogenic sources including food, cosmetics, cigarette smoke, and industrial products. In addition to ingestion and dermal absorption, human exposure to HQ may also occur by inhaling cigarette smoke or polluted air. The adverse effects of HQ on respiratory systems have been studied, but genotoxicity HQ on human lung cells is unclear. The aim of this study was to investigate the cytotoxicity and genotoxicity of HQ in human lung alveolar epithelial cells (A549). We found that HQ induced a dose response in cell growth inhibition and DNA damage which was associated with an increase in oxidative stress. Cytotoxicity results demonstrated that HQ was most toxic after 24 h (LC₅₀ = 33 μM) and less toxic after 1 h exposure (LC₅₀ = 59 μM). Genotoxicity of HQ was measured using the Comet assay, H2AX phosphorylation, and chromosome aberration formation. Results from the comet assay revealed that DNA damage was highest during the earlier hours of exposure (1 and 6 h) and thereafter was reduced. A similar pattern was observed for H2AX phosphorylation suggesting that damage DNA may be repaired in later exposure hours. An increase in chromosomal aberration corresponded with maximal DNA damage which further confirmed the genotoxic effects of HQ. To investigate whether oxidative stress was involved in the cytotoxic and genotoxic effects of HQ, cellular glutathione and 8-Oxo-deoguanisone (8-Oxo-dG) formation were measured. A decrease in the reduced glutathione (GSH) and an increase oxidized glutathione (GSSG) was observed during the early hours of exposure which corresponded with elevated 8-Oxo-dG adducts. Together these results demonstrate that HQ exerts its cytotoxic and genotoxic effects in A549 lung cells, probably through DNA damage via oxidative stress.</p>

Digital object identifier (DOI): 10.1007/s10565-013-9247-0

The study design and obtained results represent an intercomparison of various laboratories performing dose assessment using the dicentric chromosome analysis (DCA) as a diagnostic triage tool for individual radiation dose assessment. Homogenously X-irradiated (240 kVp, 1 Gy/min) blood samples for establishing calibration data (0.25-5 Gy) as well as blind samples (0.1-6.4 Gy) were sent to the participants. DCA was performed according to established protocols. The time taken to report dose estimates was documented for each laboratory. Additional information concerning laboratory organization/characteristics as well as assay performance was collected. The mean absolute difference (MAD) was calculated and radiation doses were merged into four triage categories reflecting clinical aspects to calculate accuracy, sensitivity and specificity. The earliest report time was 2.4 days after sample arrival. DCA dose estimates were reported with high and comparable accuracy, with MAD values ranging between 0.16-0.5 Gy for both manual and automated scoring. No significant differences were found for dose estimates based either on 20, 30, 40 or 50 cells, suggesting that the scored number of cells can be reduced from 50 to 20 without loss of precision of triage dose estimates, at least for homogenous exposure scenarios. Triage categories of clinical significance could be discriminated efficiently using both scoring procedures.

<p>The genotoxic potential of azidothymidine (Zidovudine, AZT), chosen as a model compound for nucleotide analogs, was comprehensively assessed in vivo for gene mutation, clastogenicity, and DNA breakage endpoints. Male Wistar rats were treated by oral gavage over 7 days with AZT at dose levels of 2×0 (control), 2×250, 2×500, and 2×1000mg/kg/day with a final single dose given on day 8. DNA damage was then evaluated with the comet assay in liver, stomach, and peripheral blood and with the micronucleus test in bone marrow and peripheral blood (by flow cytometry) in the same animals. After a treatment-free period of upto 42 days, the Pig-a gene mutation assay was performed in peripheral blood of the high-dose animals. In the comet assay as well as the micronucleus test, AZT caused a considerable dose-dependent increase in DNA damage in all tissues evaluated and was highly cytotoxic to bone marrow and peripheral blood cells. These data are well in line with published results. Surprisingly, AZT did not significantly increase the number of Pig-a mutant cells. We speculate that two factors likely contributed to this negative result: a predominance of large deletions caused by AZT, and the relatively low statistical power of the first-generation scoring method used for this study.</p>

The focus of the study is an intercomparison of laboratories' dose-assessment performances using the cytokinesis-block micronucleus (CBMN) assay as a diagnostic triage tool for individual radiation dose assessment. Homogenously X-irradiated (240 kVp, 1 Gy/min) blood samples for establishing calibration data (0.25-5 Gy) as well as blind samples (0.1-6.4 Gy) were sent to the participants. The CBMN assay was performed according to protocols individually established and varying among participating laboratories. The time taken to report dose estimates was documented for each laboratory. Additional information concerning laboratory organization/characteristics as well as assay performance was collected. The mean absolute difference (MAD) was calculated and radiation doses were merged into four triage categories reflecting clinical aspects to calculate accuracy, sensitivity and specificity. The earliest report time was 4 days after sample arrival. The CBMN dose estimates were reported with high accuracy (MAD values of 0.20-0.50 Gy at doses below 6.4 Gy for both manual and automated scoring procedures), but showed a limitation of the assay at the dose point of 6.4 Gy, which resulted in a clear dose underestimation in all cases. The MAD values (without 6.4 Gy) differed significantly (P = 0.03) between manual (0.25 Gy, SEM = 0.06, n = 4) or automated scoring procedures (0.37 Gy, SEM = 0.08, n = 5), but lowest MAD were equal (0.2 Gy) for both scoring procedures. Likewise, both scoring procedures led to the same allocation of dose estimates to triage categories of clinical significance (about 83\% accuracy and up to 100\% specificity).

Azidothymidine (Zidovudine, AZT) is part of the standard care of treatment for acquired immunodeficiency syndrome since many years. A great number of studies on the genotoxic potential of AZT have been published, but no comprehensive hypothesis yet explains all observations. We investigated a multitude of genotoxic endpoints, both in vitro and in vivo, with the goal to complete the picture. The mutagenic potential of AZT in bacteria was found to be restricted to strains with an #ochre# target sequence and could be abrogated both by thymidine supplementation and rat liver S9 mix. Single-strand breaks in mammalian cells were detected in the comet assay after short-term treatment (3h) with AZT, which did not induce micronuclei. The latter were mainly seen after prolonged exposure (24 and 48h) and are probably not directly related to AZT incorporation into DNA. Our data demonstrate that short-term exposure to low AZT concentrations does not induce biologically relevant micronucleation. Only treatment with high concentrations of AZT for prolonged time periods manifests in substantial micronucleus induction. Furthermore, we found that high concentrations of thymidine have no effect in the comet assay but increase micronucleus frequency in a manner very similar to AZT. These results lead us to the following hypothesis: AZT is triphosphorylated and then incorporated into DNA strands, leading to mutations and cytotoxicity. Cellular attempts to repair these DNA lesions as well as stalled replication forks due to chain termination are detectable with the comet assay. Increased micronucleus frequency is likely related to nucleotide pool imbalance.

<p>We report the case of an 84 years old prostate cancer patient with severe side effects after radiotherapy in 2006. He was cytogenetically analysed in 2009 and in 2012 in a comparative study for individual radiosensitivity of prostate cancer patients. No other patient had clonal aberrations, but this patient showed ring chromosomes in the range of 21-25% of lymphocytes. He received 5 cycles of 5-fluorouracil/folic acid for chemotherapy of sigmoid colon carcinoma in 2003, three years before radiotherapy of prostate cancer. Blood samples were irradiated ex vivo with Cs-137 γ-rays (0.7Gy/min) in the G0-phase of the cell cycle. 100 FISH painted metaphases were analysed for the control and the irradiated samples each. Multicolour in situ hybridisation techniques like mFISH and mBand as well as MYC locus, telomere and centromere painting probes were used to characterise ring metaphases. Metaphase search and autocapture was performed with a Zeiss Axioplan 2 imaging microscope followed by scoring and image analysis using Metafer 4/ISIS software (MetaSystems). In 2009 chromosome 8 rings were found in about 25% of lymphocytes. Rings were stable over time and increased to about 30% until 2012. The ring chromosome 8 always lacked telomere signals and a small amount of rings displayed up to four centromere signals. In aberrant metaphases 8pter and 8qter were either translocated or deleted. Further analyses revealed that the breakpoint at the p arm is localised at 8p21.2-22. The breakpoint at the q arm turned out to be distal from the MYC locus at 8q23-24. We hypothesise that the ring chromosome 8 has been developed during the 5 FU/folic acid treatments in 2003. The long term persistence might be due to clonal expansion of a damaged but viable hematopoietic stem cell giving rise to cycling progenitor cells that permit cell survival and proliferation.</p>

<p>To investigate whether rotational techniques (Volumetric Modulated Arc Therapy - VMAT) are associated with a higher risk for secondary primary malignancies compared to step-and-shoot Intensity Modulated Radiation Therapy (ss-IMRT). To this end, radiation therapy (RT) induced DNA double-strand-breaks and the resulting chromosomal damage were assessed in peripheral blood T-lymphocytes of prostate cancer (PCa) patients applying γH2AX foci and G0 micronucleus (MN) assays.The study comprised 33PCa patients. A blood sample was taken before start of therapy and after the 1st and 3rd RT fraction to determine respectively the RT-induced γH2AX foci and MN. The equivalent total body dose (<em>D</em>_ETB) was calculated based on treatment planning data. A linear dose response was obtained for γH2AX foci yields versus (<em>D</em>_ETB) while MN showed a linear-quadratic dose response. Patients treated with large volume (LV) VMAT show a significantly higher level of induced γH2AX foci and MN compared to IMRT and small volume (SV) VMAT (p &lt; 0.01). Assuming a linear-quadratic relationship, a satisfactory correlation was found between both endpoints (<em>R</em>² 0.86). Biomarker responses were governed by dose and irradiated volume of normal tissues. No significant differences between IMRT and rotational therapy inherent to the technique itself were observed.</p>

<p>In this pilot study we compared for the first time the radiation sensitivity of mouse lens epithelial cells (LECs) and mouse lymphocytes. We freshly prepared LECs and lymphocytes and irradiated them with γ-rays ((137)Cs; doses ranging from 0.25 to 2 Gy). DNA damage and repair were evaluated by alkaline comet assay and γH2AX foci assay. Using the comet assay, we observed a dose-dependent increase in DNA damage in both cell types. The faster formation of single- and double-strand breaks in LECs of C57BL/6 mice at doses below 1 Gy needs to be confirmed in other mouse strains. Immunofluorescence for γH2AX foci showed a higher degree of lesions in LECs from C57BL/6J mice compared to those of JF1 mice and to lymphocytes of both strains. Correspondingly, repair of DNA damage proceeded faster in LECs of C57BL/6J mice compared to LECs of JF1 mice and lymphocytes of both strains. It is obvious that the lymphocytes of both strains repaired DNA lesions more slowly than the corresponding LECs. In conclusion, our results demonstrate that LECs of C57Bl/6 mice show a steeper dose-response than lymphocytes in both types of experiments. It shows that both test systems are able to be used also at doses below 0.25 Gy. The observed difference in DNA repair between the LECs from C57BL/6J mice compared to the LECs from JF1 mice and to the lymphocytes of both strains warrants further experiments to identify the underlying molecular mechanisms.</p>

Digital object identifier (DOI): 10.1007/s00411-012-0451-8

In this study, we compared conventional sperm selection with high-magnification morphology based on the motile sperm organellar morphology examination (MSOME) criteria, and hyaluronic acid (HA) binding for sperm chromosome aneuploidy and DNA fragmentation rates. Semen from 50 severe male factor cases was processed through density gradient centrifugation, and subjected to sperm selection by using the conventional method (control), high magnification at ?6650 or HA binding. Aneuploidy was detected by fluorescence in situ hybridization with probes for chromosomes 13, 18, 21, X and Y, and DNA fragmentation by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) method. Spermatozoa selected under high-magnification had a lower DNA fragmentation rate (2.6\% vs. 1.7\%; P=0.032), with no significant difference in aneuploidy rate (0.8\% vs 0.7\%; P=0.583), than those selected by the HA binding method. Spermatozoa selected by both methods had much lower aneuploidy and DNA fragmentation rate than the controls (7\% aneuploidy and 26.8\% DNA fragmentation rates, respectively). In the high-magnification group, the aneuploidy rate was lower when the best spermatozoa were selected than when only the second-best spermatozoa were available for selection, but the DNA fragmentation rate was not different. In conclusion, sperm selection under high magnification was more effective than under HA binding in selecting spermatozoa with low DNA fragmentation rate, but the small difference (0.9\%) might not be clinically meaningful. Both methods were better than the conventional method of sperm selection.

Mass casualty scenarios of radiation exposure require high throughput biological dosimetry techniques for population triage in order to rapidly identify individuals who require clinical treatment. The manual dicentric assay is a highly suitable technique, but it is also very time consuming and requires well trained scorers. In the framework of the MULTIBIODOSE EU FP7 project, semi-automated dicentric scoring has been established in six European biodosimetry laboratories. Whole blood was irradiated with a Co-60 gamma source resulting in 8 different doses between 0 and 4.5Gy and then shipped to the six participating laboratories. To investigate two different scoring strategies, cell cultures were set up with short term (2-3h) or long term (24h) colcemid treatment. Three classifiers for automatic dicentric detection were applied, two of which were developed specifically for these two different culture techniques. The automation procedure included metaphase finding, capture of cells at high resolution and detection of dicentric candidates. The automatically detected dicentric candidates were then evaluated by a trained human scorer, which led to the term 'semi-automated' being applied to the analysis. The six participating laboratories established at least one semi-automated calibration curve each, using the appropriate classifier for their colcemid treatment time. There was no significant difference between the calibration curves established, regardless of the classifier used. The ratio of false positive to true positive dicentric candidates was dose dependent. The total staff effort required for analysing 150 metaphases using the semi-automated approach was 2min as opposed to 60min for manual scoring of 50 metaphases. Semi-automated dicentric scoring is a useful tool in a large scale radiation accident as it enables high throughput screening of samples for fast triage of potentially exposed individuals. Furthermore, the results from the participating laboratories were comparable which supports networking between laboratories for this assay.

The identification of severely exposed individuals and reassurance of the 'worried well' are of prime importance for initial triage following a large scale radiation accident. We aim to develop the ã-H2AX foci assay into a rapid biomarker tool for use in accidents. Here, five laboratories established a standard operating procedure and analysed 100 ex vivo ã-irradiated, 4 or 24h incubated and overnight-shipped lymphocyte samples from four donors to generate ã-H2AX reference data, using manual and/or automated foci scoring strategies. In addition to acute, homogeneous exposures to 0, 1, 2 and 4Gy, acute simulated partial body (4Gy to 50\% of cells) and protracted exposures (4Gy over 24h) were analysed. Data from all laboratories could be satisfactorily fitted with linear dose response functions. Average yields observed at 4h post exposure were 2-4 times higher than at 24h and varied considerably between laboratories. Automated scoring caused larger uncertainties than manual scoring and was unable to identify partial exposures, which were detectable in manually scored samples due to their overdispersed foci distributions. Protracted exposures were detectable but doses could not be accurately estimated with the ã-H2AX assay. We conclude that the ã-H2AX assay may be useful for rapid triage following a recent acute radiation exposure. The potentially higher speed and convenience of automated relative to manual foci scoring needs to be balanced against its compromised accuracy and inability to detect partial body exposures. Regular re-calibration or inclusion of reference samples may be necessary to ensure consistent results between laboratories or over long time periods.