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.

Efferent renal sympathetic nerves reinnervate the kidney after renal denervation in animals and humans. Therefore, the long-term reduction in arterial pressure following renal denervation in drug-resistant hypertensive patients has been attributed to lack of afferent renal sensory reinnervation. However, afferent sensory reinnervation of any organ, including the kidney, is an understudied question. Therefore, we analyzed the time course of sympathetic and sensory reinnervation at multiple time points (1, 4, and 5 days and 1, 2, 3, 4, 6, 9, and 12 wk) after renal denervation in normal Sprague-Dawley rats. Sympathetic and sensory innervation in the innervated and contralateral denervated kidney was determined as optical density (ImageJ) of the sympathetic and sensory nerves identified by immunohistochemistry using antibodies against markers for sympathetic nerves [neuropeptide Y (NPY) and tyrosine hydroxylase (TH)] and sensory nerves [substance P and calcitonin gene-related peptide (CGRP)]. In denervated kidneys, the optical density of NPY-immunoreactive (ir) fibers in the renal cortex and substance P-ir fibers in the pelvic wall was 6, 39, and 100\% and 8, 47, and 100\%, respectively, of that in the contralateral innervated kidney at 4 days, 4 wk, and 12 wk after denervation. Linear regression analysis of the optical density of the ratio of the denervated/innervated kidney versus time yielded similar intercept and slope values for NPY-ir, TH-ir, substance P-ir, and CGRP-ir fibers (all R(2) > 0.76). In conclusion, in normotensive rats, reinnervation of the renal sensory nerves occurs over the same time course as reinnervation of the renal sympathetic nerves, both being complete at 9 to 12 wk following renal denervation.

Dicentric assay is the international gold standard for cytogenetic biodosimetry after radiation exposure, despite being very labour-intensive, time-consuming, and highly expertise-dependent. It involves the identification of centromeres and structure of solid-stained chromosomes and the enumeration of dicentric chromosomes in a large number of first-division metaphases of cultured T lymphocytes. The dicentric yield is used to estimate the radiation exposure dosage according to a statistically derived and predetermined dose-response curve. It can be used for population triage after large-scale accidental over-exposure to ionising radiation or with a view to making clinical decisions for individual patients receiving substantial radiation. In this report, we describe our experience in the establishment of a cytogenetic biodosimetry laboratory in Queen Elizabeth Hospital, Hong Kong. This was part of the contingency plan for emergency measures against radiation accidents at nuclear power stations.

<p>Hypothermia during in vitro irradiation of human peripheral blood lymphocytes (PBL) affects the level of chromosome aberrations. The molecular mechanisms of this phenomenon are not fully understood. The aim of our study was to examine the effect of hypothermia on the dose-response relationship for dicentric chromosomes and the level of γ-H2AX (phosphorylated histone H2AX) foci. In addition, the inter- and intra-individual variability was assessed in relation to temperature. PBL were kept at 0.8, 20 and 37°C and then exposed to gamma-rays (from 0-3 Gy). Dicentric chromosomes were scored in first post-treatment mitoses. γ-H2AX foci were scored 15, 30, 60, 120 min and 24 h post irradiation.Our results revealed that the frequency of dicentric chromosomes in cells exposed at 37°C to gamma-rays was higher than after exposure at 0.8 and 20°C. No effect of temperature was observed on the number of γ-H2AX foci as well as on the intra- and inter-individual variations of the dicentric yield and the number of γ-H2AX foci.Temperature at exposure to ionizing radiation has a pronounced effect on the level of cytogenetic damage but not γ-H2AX foci.</p>

Digital object identifier (DOI): 10.3109/09553002.2013.741284

<p>Background Approximately 1% of the spermatozoa found in ejaculate of healthy men are aneuploid and this rate increases in the population of subfertile and infertile men. Moreover, fertilization with these aneuploid sperm can lead to impaired embryo development. Fluorescent In Situ Hybridization (FISH) is the common cytogenetic tool used for aneuploidy screening on sperm. However, it is a time-consuming technique and cytogenetic or in vitro fertilization laboratories cannot routinely use it and face the increasing demand of such analyses before Assisted Reproductive Techniques (ART). As automation can be a clue for routine practice, this study compares manual and automated scoring of sperm aneuploidy rates using a Metafer MetaSystems device. The results obtained also contribute to global data about FISH on sperm cells. Methods We recruited 100 men addressed for sperm cryopreservation. They all signed an informed consent to participate in the study. 29 men were donors or consulted before vasectomy (control group) and 71 were suffering of Hodgkin’s disease or non Hodgkin lymphoma (patient group). One semen sample was collected for each patient, analyzed according to WHO criteria and prepared for a triple-color FISH using centromeric probes for chromosomes 18, X and Y. Automated scoring was performed using a Metafer MetaSystems device. Results 507,019 cells were scored. We found a strong concordance between the automated and the manual reading (d  &lt; 0.01 in Bland-Altman test). We also did not find a statistically significant difference between the automated and the manual reading using Wilcoxon test for total aneuploidy rate (p = 0.06), sex chromosomes disomy (p = 0.33), chromosome 18 disomy (p = 0.39) and diploidy (p = 0.21). Cumulative rate of total aneuploidy was 0.78% ± 0.212% for patient group and 0.54% ± 0.15 for control group and among this, sex chromosome XY disomy rate was of 0.54% for patient group and 0.27% for control group. This study validates the automated reading for FISH on sperm with a Metafer Metasystems® device and allows its use in a laboratory routine.</p>

Recent studies have shown that telomere length was significantly reduced in placentas collected at delivery from pregnancies complicated by intrauterine growth restriction secondary to placental insufficiency. Placental telomere length measurement during ongoing pregnancies complicated by intrauterine growth restriction has never been reported. This was the main objective of our study.

<p>In accidental exposure to ionizing radiation, it is essential to estimate the dose received by the victims. Currently dicentric scoring is the best biological indicator of exposure. The standard biological dosimetry procedure (500 metaphases scored manually) is suitable for a few dose estimations, but the time needed for analysis can be problematic in the case of a large-scale accident. Recently, a new methodology using automatic detection of dicentrics has greatly decreased the time needed for dose estimation and preserves the accuracy of the estimation. However, the capability to detect nonhomogeneous partial-body exposures is an important advantage of dicentric scoring-based biodosimetry, and this remains to be tested with automatic scoring. Thus we analyzed the results obtained with in vitro blood dilutions and in real cases of accidental exposure (partial- or whole-body exposure) using manual scoring and automatic detection of dicentrics. We confirmed that automatic detection allows threefold quicker dicentric scoring than the manual procedure with similar dose estimations and uncertainty intervals. The results concerning partial-body exposures were particularly promising, and homogeneously exposed samples were correctly distinguished from heterogeneously exposed samples containing 5% to 75% of blood irradiated with 2 Gy. In addition, the results obtained for real accident cases were similar whatever the methodology used. This study demonstrates that automatic detection of dicentrics is a credible alternative for recent and acute cases of whole- and partial-body accidental exposures to ionizing radiation.</p>

Tumor biomarkers increasingly provide information for predicting outcomes with chemotherapeutic regimens (personalized medicine). Topo2A is a DNA helicase targeted by anthracyclines, cytotoxic therapeutics used in both adjuvant and palliative treatments of breast cancer. TOP2A gene amplification/deletion is implicated in response to anthracycline-based chemotherapy. We describe an approach for analyzing formalin-fixed, paraffin-embedded breast tumors on tissue microarrays with TOP2A fluorescence in situ hybridization coupled with cytokeratin immunofluorescence to target tumor cells. Stained tissue from patient specimens was imaged and analyzed using Metafer/Metacyte (Metasystems, Waltham, MA, USA), including customized image classifiers. TOP2A/CEN17 ratios of 2.0 or greater (amplified) and 0.8 or less (deleted) were observed for 10.0\% and 6.1\% of the patients, respectively. Patient outcomes for adjuvant chemotherapy (cyclophosphamide-epirubicin-fluorouracil, cyclophosphamide-methotrexate-fluorouracil, no chemotherapy) were evaluated. No statistical significance was achieved for clinical end points regarding TOP2A status in anthracycline-treated patients. However, patients with TOP2A aberrations receiving methotrexate-based therapy exhibited a significant decrease in 5-year distant disease-free survival and breast cancer-specific overall survival, especially for patients with TOP2A deletions (disease-free survival: hazard ratio, 5.31 [P = .001], and breast cancer-specific overall survival: hazard ratio, 6.45 [P ? .001]). No significant differences were seen in patients included in the no-chemotherapy group. Topo2A protein levels were assessed by immunohistochemistry with no correlative statistical relevance to immunofluorescence/fluorescence in situ hybridization-based prognosis for cyclophosphamide-epirubicin-fluorouracil or cyclophosphamide-methotrexate-fluorouracil groups. Interestingly, aberrant (under)expressing patients in the no-chemotherapy group exhibited better 5-year distant disease-free survival (hazard ratio, 0.39; P = .004), trending toward more favorable breast cancer-specific overall survival (hazard ratio, 0.61; P = .11). Our results indicate a strategy by which fluorescence in situ hybridization scoring targeted to cytokeratin-positive tumor cells may provide a tool for added precision and efficiency in TOP2A evaluation from tumor tissue.

Digital object identifier (DOI): 10.1016/j.humpath.2011.08.018

Different fetal cell types have been found in the maternal blood during pregnancy in the past, but fetal cells are scarce, and the proportions of the different cell types are unclear. The objective of the present study was to identify specific fetal cell markers from fetal cells found in the maternal blood circulation at the end of the first trimester.Twenty-three fetal cells were isolated from maternal blood by removing the red blood cells by lysis or combining this with removal of large proportions of maternal white blood cells by magnetic-activated cell sorting. Fetal cells identified by XY fluorescence in situ hybridization and confirmed by reverse-color fluorescence in situ hybridization were shot off microscope slides by laser capture microdissection. The expression pattern of a subset of expressed genes was compared between fetal cells and maternal blood cells using stem cell microarray analysis.Twenty-eight genes were identified as fetal cell marker candidates.Of the 28 fetal marker candidate genes, five coded for proteins, which are located on the outer surface of the cell membrane and not expressed in blood. The protein product of these five genes, MMP14, MCAM, KCNQ4, CLDN6, and F3, may be used as markers for fetal cell enrichment.

<p>The bottleneck in data acquisition during biological dosimetry based on a dicentric assay is the need to score dicentrics in a large number of lymphocytes. One way to increase the capacity of a given laboratory is to use the ability of skilled operators from other laboratories. This can be done using image analysis systems and distributing images all around the world. Two exercises were conducted to test the efficiency of such an approach involving 10 laboratories. During the first exercise (E1), the participant laboratories analysed the same images derived from cells exposed to 0.5 and 3 Gy; 100 images were sent to all participants for both doses. Whatever the dose, only about half of the cells were complete with well-spread metaphases suitable for analysis. A coefficient of variation (CV) on the standard deviation of ?15 \% was obtained for both doses. The trueness was better for 3 Gy (0.6 %) than for 0.5 Gy (37.8 %). The number of estimated doses classified as satisfactory according to the z-score was 3 at 0.5 Gy and 8 at 3 Gy for 10 dose estimations. In the second exercise, an emergency situation was tested, each laboratory was required to score a different set of 50 images in 2 d extracted from 500 downloaded images derived from cells exposed to 0.5 Gy. Then the remaining 450 images had to be scored within a week. Using 50 different images, the CV on the estimated doses (79.2 %) was not as good as in E1, probably associated to a lower number of cells analysed (50 vs. 100) or from the fact that laboratories analysed a different set of images. The trueness for the dose was better after scoring 500 cells (22.5 %) than after 50 cells (26.8 %). For the 10 dose estimations, the number of doses classified as satisfactory according to the z-score was 9, for both 50 and 500 cells. Overall, the results obtained support the feasibility of networking using electronically transmitted images. However, before its implementation some issues should be elucidated, such as the number and resolution of the images to be sent, and the harmonisation of the scoring criteria. Additionally, a global website able to be used for the different regional networks, like Share Points, will be desirable to facilitate worldwide communication.</p>

The zebra mussel Dreissena polymorpha is widely used as sentinel organism for the assessment of environmental contamination in freshwater environments. However, in the River Rhine (Germany), the D. polymorpha population is declining, whereas the closely related quagga mussel D. bugensis is found in high numbers at some sites. In the present laboratory study, D. polymorpha and D. bugensis were exposed to resuspended native sediments for ≤2 weeks. Wet sediments (<63 μm, 100 mg l−1 dry weight) were used as surrogate suspended particulate matter to mimic one of the mussels’ main uptake route for chemicals. The sediments were sampled in (1) the River Elbe in Dessau, a site known to be highly polluted with, e.g., organochlorine (OC) pesticides and (2) at a relatively unpolluted site in Havelberg in the River Havel, one of the Elbe’s tributaries. Chemical analysis of persistent OC compounds (seven polychlorinated biphenyls [PCBs], DDT and its metabolites (DDX), hexachlorocylohexanes [HCHs], and hexachlorobenzene [HCB]) in soft tissue of mussels showed significantly greater values of PCBs 101, 118, 153, 138, 180, the sum of seven PCBs, and p,p′-DDD in D. bugensis compared with D. polymorpha. Fourteen days of exposure to Dessau sediment increased the concentration of p,p′-DDE and p,p′-DDD, as well as the sum of DDX, in both species compared with Havelberg sediment. Interspecific differences were less pronounced when regarding chemical concentrations with lipid content instead of dry-weight of tissue because D. bugensis had greater levels of total lipid than D. polymorpha. DNA damage in gills, as measured with the comet assay, was greater in D. bugensis compared with D. polymorpha. Simultaneously, the content of heat-shock protein (hsp70) in gills was greater in D. polymorpha than in D. bugensis. DNA damage and hsp70 were not induced by exposure time or sediment type. This study shows that D. bugensis and D. polymorpha may differ in their bioaccumulation potential of OC pesticides as well as their levels of DNA damage and hsp70. Therefore, more investigations are needed before quagga mussel can be used as alternative test organism for the zebra mussel.