Manual versus automated γ-H2AX foci analysis across five European laboratories: Can this assay be used for rapid biodosimetry in a large scale radiation accident?

Rothkamm, Kai; Barnard, Stephen; Ainsbury, Elizabeth A.; Al-hafidh, Jenna; Barquinero, Joan Francesc; Lindholm, Carita; Moquet, Jayne; Perälä, Marjo; Roch-Lefèvre, Sandrine; Scherthan, Harry; Thierens, Hubert; Vral, Anne; Vandersickel, Veerle

doi:10.1016/j.mrgentox.2013.04.012

Cited by 62 publications

(57 citation statements)

References 21 publications

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“…However, the mean number of c-H2AX foci/cell/ Gy in lymphocytes incubated at 378C for 30 min and fixed with paraformaldehyde was 11.8. This value is very close to the values reported in other studies [;9-15 c-H2AX foci/ cell/Gy (2,24,30)]. The discrepancies observed between the number of spontaneous and radiation-induced c-H2AX foci in cells fixed with paraformaldehyde and methanol are not easy to explain, as each of the fixatives has properties that may affect obtained results.…”

Section: Discussionsupporting

confidence: 88%

“…These methods differ in their specificity and sensitivity to radiation, stability of signal and processing time. Among them, the c-H2AX assay is one of the most promising for initial triage to support clinical management (1)(2)(3). The methodology of the c-H2AX assay allows the detection of the phosphorylated form of H2AX histone variant as a response to DNA double-strand break (DSB) formation (4)(5)(6).…”

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confidence: 99%

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Defining Blood Processing Parameters for Optimal Detection of γ-H2AX Foci: A Small Blood Volume Method

et al. 2015

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Biodosimetric methods used to measure the effects of radiation are critical for estimating the health risks to irradiated individuals or populations. The direct measurement of radiation-induced γ-H2AX foci in peripheral blood lymphocytes is one approach that provides a useful end point for triage. Despite the documented advantages of the γ-H2AX assay, there is considerable variation among laboratories regarding foci formation in the same exposure conditions and cell lines. Taking this into account, the goal of our study was to evaluate the influence of different blood processing parameters on the frequency of γ-H2AX foci and optimize a small blood volume protocol for the γ-H2AX assay, which simulates the finger prick blood collection method. We found that the type of fixative, temperature and blood processing time markedly affect the results of the γ-H2AX assay. In addition, we propose a protocol for the γ-H2AX assay that may serve as a potential guideline in the event of large-scale radiation incidents.

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Section: Discussionsupporting

confidence: 88%

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confidence: 99%

Defining Blood Processing Parameters for Optimal Detection of γ-H2AX Foci: A Small Blood Volume Method

et al. 2015

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“…We believe that the proposed ex vivo cH2AX assay presented here can overcome these limitations by providing rapid results (within few days) at a very high success rate. In addition, the ex vivo cH2AX assay can further be optimized towards higher clinical practicability such as the use of automated microscope counting software [55,56] and the omission of dose levels without compromising robustness (Suppl. Figs.…”

Section: Discussionmentioning

confidence: 99%

Residual γH2AX foci after ex vivo irradiation of patient samples with known tumour-type specific differences in radio-responsiveness

Menegakis¹,

Colle²,

Yaromina³

et al. 2015

Radiotherapy and Oncology

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known differences in radiation response, i.e. radiosensitive types such as seminomas and resistant types as chondrosarcomas. We hypothesized that the number of residual cH2AX foci corresponds to the expected tumour radiation sensitivity. In addition, in order to enhance clinical practicability for future studies the data were used for simulations to test the robustness of the method when omitting dose levels. Materials and methods Collection and cultivation of patient-derived tumour specimensThe study has been approved by the Ethics Committee of the Medical Faculty of the University of Tübingen (426/2013BO1). All the patients included in the study were untreated prior to surgical procedure. During collection and cultivation tumour tissues were kept in Dulbecco's MEM culture medium supplemented with 2% HEPES, 1% Na-pyruvate, 1% non-essential amino acids, 1% penicillin streptomycin (all Biochrom AG, Berlin, Germany) and 10% FBS (PAN Biotech GmbH, Aidenbach, Germany). Fresh tumour material was retrieved from surgical specimens and placed in 50 ml Falcon tubes (Becton Dickinson International, Heidelberg, Germany) containing 15 ml culture medium. Tumour specimens were transported to the laboratory and subsequently cut manually with the use of surgical forceps (BD027R, B. BRAUN, Aesculap, Tuttlingen, Germany) and scalpels (FEATHER Safety Razor Co., Ltd., Osaka, Japan Number 23) into approximately 2-3 mm slices before being placed in petri dishes (3.5 cm diameter) coated with a 1.5% agarose layer (A9539, Sigma-Aldrich, Germany) containing 3 ml culture media. During all incubation times the petri-dishes were kept in 95% humidified atmosphere at 37 _C and 5% CO2. For the purpose of the study tumour material from a total of 25 patient tumours with 10 different tumour histologies was collected (Table 1; n = 3 seminomas, n = 1 chondrosarcoma, n = 3 urinary bladder carcinomas (Ca), n = 3 colorectal Ca (2 colon Ca + 1 rectum), n = 3 breast Ca, n = 1 hepatocellular Ca, n = 3 renal cell Ca, n = 3 prostate Ca, n = 4 glioblastoma multiforme (GBM) and n = 2 cervix Ca). In one GBM patient, the specimen was removed from the analysis because in the 0 Gy sample there was no viable tumour part (based on morphological criteria and BrdU positivity) and no estimation of the background foci could be performed. Histology and selection of malignant cells for analysis were confirmed by an experienced pathologist (M.S.).Experimental design for evaluation of cH2AX foci in ex vivo irradiated patient-derived tumour specimensThe experimental design has been previously described [45]. Briefly, after initial cultivation for 24 h, the tumour specimens were irradiated typically with 0, 2, 4, 6, 8 Gy single doses (200 kV, RS225 research system Gulmay Medical LTD, Surrey, England; 15 mA; 0.5 mm Cu; dose rate _1 Gy/min). In two tumours, additional doses were delivered to the seminoma sample (#1) doses of 3 and 5 Gy and GBM (#1) 10

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“…Some assays detect the presence of the metabolic products resulting from the effects of radiation (metabolomics). Alternatively, assays can assess indicators produced during the process of repair, especially those related to DNA such as 8-hydroxyguanine or double strand breaks of DNA (gamma-H2AX) (Rothkamm et al 2013a,b). …”

Section: Analysis Of Requirements For Biodosimetry Methods Based On Tmentioning

confidence: 99%

Evaluating the Special Needs of The Military for Radiation Biodosimetry for Tactical Warfare Against Deployed Troops

et al. 2016

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Objectives The aim of this paper is to delineate characteristics of biodosimetry most suitable for assessing individuals who have potentially been exposed to significant radiation from a nuclear device explosion, when the primary population targeted by the explosion and needing rapid assessment for triage is civilians vs. deployed military personnel. Methods We first carry out a systematic analysis of the requirements for biodosimetry to meet the military's needs to assess deployed troops in a warfare situation, which include accomplishing the military mission. We then systematically compare and contrast the military's special capabilities to respond and carry out biodosimetry for deployed troops in warfare, in contrast to those available to respond and conduct biodosimetry for civilians who have been targeted, e.g., by terrorists. We then compare the effectiveness of different biodosimetry methods to address military vs. civilian needs and capabilities in these scenarios and, using five representative types of biodosimetry with sufficient published data to be useful for the simulations, we estimate the number of individuals who could be assessed by military vs. civilian responders within the timeframe needed for triage decisions. Conclusions Analyses based on these scenarios indicate that, in comparison to responses for a civilian population, a wartime military response for deployed troops has both more complex requirements for and greater capabilities to utilize different types of biodosimetry to evaluate radiation exposure in a very short timeframe after the exposure occurs. Greater complexity for the deployed military is based on factors such as a greater likelihood of partial or whole body exposure, conditions that include exposure to neutrons, and a greater likelihood of combined injury. Our simulations showed, for both the military and civilian response, that a very fast rate of initiating the processing (24,000 per day) is needed to have at least some methods capable of completing the assessment of 50,000 people within a 2 or 6 day timeframe following exposure. This in turn suggests a very high capacity (i.e., laboratories, devices, supplies and expertise) would be necessary to achieve these rates. These simulations also demonstrated the practical importance of the military's superior capacity to minimize time to transport samples to offsite facilities and utilize the results to carry out triage quickly. Assuming sufficient resources and the fastest daily rate to initiate processing victims, the military scenario revealed that two biodosimetry methods could achieve the necessary throughput to triage 50,000 victims in 2 days (i.e., the timeframe needed for injured victims) and all five achieved the targeted throughput within 6 days. In contrast, simulations based on the civilian scenario revealed that no method could process 50,000 people in 2 days and only two could succeed within 6 days.

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Manual versus automated γ-H2AX foci analysis across five European laboratories: Can this assay be used for rapid biodosimetry in a large scale radiation accident?

Cited by 62 publications

References 21 publications

Defining Blood Processing Parameters for Optimal Detection of γ-H2AX Foci: A Small Blood Volume Method

Defining Blood Processing Parameters for Optimal Detection of γ-H2AX Foci: A Small Blood Volume Method

Residual γH2AX foci after ex vivo irradiation of patient samples with known tumour-type specific differences in radio-responsiveness

Evaluating the Special Needs of The Military for Radiation Biodosimetry for Tactical Warfare Against Deployed Troops

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