Vandevoorde Charlot, Gomolka Maria, Roessler Ute, Samaga Daniel, Lindholm Carita, Fernet Marie, Hall Janet, Pernot Eileen, El-Saghire Houssein, Baatout Sarah, Kesminiene Ausrele, Thierens Hubert
a Ghent University , UGent , Belgium.
b Federal Office for Radiation Protection , BfS , Germany.
Int J Radiat Biol. 2015 Aug;91(8):653-63. doi: 10.3109/09553002.2015.1047987. Epub 2015 Jul 8.
To conduct a feasibility study on the application of the γ-H2AX foci assay as an exposure biomarker in a prospective multicentre paediatric radiology setting.
A set of in vitro experiments was performed to evaluate technical hurdles related to biological sample collection in a paediatric radiology setting (small blood sample volume), processing and storing of blood samples (effect of storing blood at 4°C), the reliability of foci scoring for low-doses (merge γ-H2AX/53BP1 scoring), as well as the impact of contrast agent administration as potential confounding factor. Given the exploratory nature of this study and the ethical constraints related to paediatric blood sampling, blood samples from adult volunteers were used for these experiments. In order to test the feasibility of pooling the γ-H2AX data when different centres are involved in an international multicentre study, two intercomparison studies in the low-dose range (10-500 mGy) were performed.
Determination of the number of X-ray induced γ-H2AX foci is feasible with one 2 ml blood sample pre- and post-computed tomography (CT) scan. Lymphocyte isolation and fixation on slides is necessary within 5 h of blood sampling to guarantee reliable results. The possible enhancement effect of contrast medium on the induction of DNA DSB in a patient study can be ruled out if radiation doses and the contrast agent concentration are within diagnostic ranges. The intercomparison studies using in vitro irradiated blood samples showed that the participating laboratories, executing successfully the γ-H2AX foci assay in lymphocytes, were able to rank blind samples in order of lowest to highest radiation dose based on mean foci/cell counts. The dose response of all intercomparison data shows that a dose point of 10 mGy could be distinguished from the sham-irradiated control (p = 0.006).
The results demonstrate that it is feasible to apply the γ-H2AX foci assay as a cellular biomarker of exposure in a multicentre prospective study in paediatric CT imaging after validating it in an in vivo international pilot study on paediatric patients.
在一项前瞻性多中心儿科放射学研究中，对γ-H2AX焦点检测作为暴露生物标志物的应用进行可行性研究。
进行了一系列体外实验，以评估儿科放射学环境（血样体积小）中与生物样本采集、血样处理和储存（4℃储存血液的影响）、低剂量焦点评分的可靠性（γ-H2AX/53BP1合并评分）以及造影剂给药作为潜在混杂因素的影响相关的技术障碍。鉴于本研究的探索性质以及与儿科采血相关的伦理限制，这些实验使用了成年志愿者的血样。为了测试在国际多中心研究中不同中心参与时汇总γ-H2AX数据的可行性，在低剂量范围（10 - 500 mGy）内进行了两项比对研究。
在计算机断层扫描（CT）扫描前后采集一份2 ml血样来确定X射线诱导的γ-H2AX焦点数量是可行的。为保证结果可靠，需在采血后5小时内进行淋巴细胞分离并固定在载玻片上。如果辐射剂量和造影剂浓度在诊断范围内，则可以排除患者研究中造影剂对DNA双链断裂诱导的可能增强作用。使用体外照射血样的比对研究表明，成功在淋巴细胞中进行γ-H2AX焦点检测的参与实验室能够根据平均焦点/细胞计数将盲样按辐射剂量从低到高排序。所有比对数据的剂量反应表明，10 mGy的剂量点可与假照射对照组区分开来（p = 0.006）。
结果表明，在对儿科患者进行的体内国际试点研究中验证后，将γ-H2AX焦点检测作为儿科CT成像多中心前瞻性研究中的暴露细胞生物标志物是可行的。
