γ-H2AX foci as in vivo effect biomarker in children emphasize the importance to minimize x-ray doses in paediatric CT imaging

The study population consisted of 51 children undergoing a CT examination of the chest (41) or abdomen (10) in one of the five participating radiology centres (March 2012 through July 2013) (Table 1). The small number of abdomen CT patients was because magnetic resonance imaging (MRI) was the preferred imaging modality of the majority of participating radiologists for abdomen investigations, to avoid ionizing radiation exposure. Exclusion criteria were present or past leukaemia or lymphoma and radiochemotherapy within the last year. The median age of the patient group was 3.8 years (range 0.1–12.5 years). Before blood sampling, an informed consent was signed by one of the parents of the children. The prospective multicentre study was approved by the local review boards of the participating hospitals, and the institutional review board of Ghent University Hospital acted as central ethical committee.

All the participating radiology departments used contemporary state-of-the-art low dose CT systems. The following CT systems were used in this study: Siemens Somatom Definition Flash and Sensation 64 (Siemens Medical Solutions, Germany), Toshiba Aquilion (Toshiba Medical Systems, Japan) and three centres used the GE Discovery CT750 HD (GE Healthcare, USA). Every radiology department used its own, optimized paediatric CT protocol with low kVp settings (median values: chest CT 80 kV, range 80–120 kV; abdomen CT 100 kV, range 100–120 kV), low fixed tube currents or automatic tube current modulation (ATM), adapted pitch values and imaging lengths restricted to the region of interest. Iterative reconstruction technology was applied in two of the institutions, resulting in ultra-low doses (VEO reconstruction from GE Healthcare). Individual CT parameters for the patients of the study are presented in Tables 2 and 3 for chest CT and abdomen CT respectively. The combination of state-of-the-art CT systems with dose reduction techniques and adapted paediatric protocols resulted in low- or even ultra-low CT doses.

Blood samples (2 mL) were collected through the catheter for contrast agent administration. One blood sample was taken before CT, to determine the baseline level of DSBs, and one approximately 5 min after the examination (the catheter was always flushed before sampling). As a result of occlusion, blood sampling through the catheter was not possible for more than half of the patients, especially very young children. For these patients an additional venepuncture after the CT exam was necessary. The blood samples were kept at 37 °C for 30 min to allow DNA damage signalling. Afterwards, DNA repair was arrested by cooling the samples at 0 °C for 15 min. Blood was transported at 4 °C, with an elapsed time no longer than 3 h from collection to processing. Before processing, samples were coded allowing blind scoring later on.

The method is based on the phosphorylation of the histone variant H2AX after DSB formation and follows previously published protocols [11, 16, 17]. In order to be able to work with a homogeneous cell population, T lymphocytes were isolated from the blood with the RosetteSep human T cell enrichment cocktail (StemCell Technologies, France) and resuspended in complete RPMI cell culture medium (84 % RPMI-1640, 15 % foetal calf serum, 1 % l-glutamine, 50 U/mL penicillin and 50 μg/mL streptomycin; Life Technologies, Belgium). For immunofluorescence staining, 250 μL of the resuspended T lymphocytes was centrifuged onto poly-l-lysine-coated slides (VWR International, Belgium). The slides were fixed in 3 % paraformaldehyde (PFA) (Sigma-Aldrich, Belgium) for 20 min and stored overnight in 0.5 % PFA. Fixation should immobilize antigens while retaining the lymphocytes as close to their natural state as possible. The next day, slides were permeabilised by dropping 100 μL of 0.2 % Triton-X-100 (Sigma-Aldrich, Belgium). This permeabilisation step is required because the anti-γ-H2AX antibody binding requires intracellular access to detect the γ-H2AX protein. The immunofluorescence staining was performed with an unlabelled primary mouse monoclonal anti-γ-H2AX antibody (1:500; Biolegend, Belgium) which specifically binds to the target γ-H2AX protein, followed by a secondary rabbit anti-mouse (RAM)–TRITC antibody (1:1,000; DakoCytomation, Denmark). This secondary antibody carries the (TRITC) fluorophore, recognizes the primary anti-γ-H2AX antibody and binds to it. Subsequently, the lymphocyte nucleus was counterstained with 2 % 4′,6-diamidino-2-phenylindole (DAPI) in Slowfade mounting medium (Sigma-Aldrich, Belgium). DAPI is a blue fluorescent stain specific for DNA. Microscopic scoring was performed manually with an Olympus BX60 fluorescence microscope with an Olympus × 100/1.30 oil lens. The images were viewed using Cytovision software and captured with a digital camera (Applied Imaging, USA). Ten optical sections were obtained for each field of vision (Z-stack sections of 1.03 μm). Different images of one blinded slide were stored and on average 250 cells were scored manually for γ-H2AX foci. More than 250 cells were scored for every condition and the number of γ-H2AX foci induced by CT x-rays was obtained by subtracting the pre-scan foci yield from the post-scan foci yield after decoding. The scoring procedure was validated by double immunostaining for both γ-H2AX and p53 binding protein 1 (53BP1), to discriminate between background artefacts and small γ-H2AX foci. The experiments demonstrated that the γ-H2AX-TRITC foci coincide with 53BP1-FITC foci, resulting in a statistical significant agreement between the number of double stained foci (merge) and the number of individual γ-H2AX-TRITC foci (results not shown).

The blood dose was calculated as a weighted sum of doses to the largest blood-containing organs with the percentage of blood pool as weighting factor, namely lungs (12.5 %), heart (10 %), liver (10 %) and remainder (67.5 %) [19]. In order to obtain individual organ dose estimates, an individualized full Monte Carlo patient dose simulation was set up using ImpactMC simulation software 1.3.1 (CT Imaging GmbH, Germany). In this software, patient-specific 3D voxel models are created on the basis of the CT images acquired during CT examination [20]. For the simulation of the CT scan, actual scan parameters, such as tube voltage (kV), tube current–exposure time product (mAs) and pitch, are retrieved from the DICOM header of the CT images. The individual mA values of all the reconstructed CT slices were used in order to take into account of the tube current modulation of the CT scanner. More than 10¹⁰ photons were simulated in order to minimize the uncertainty in the Monte Carlo results. The patient-specific dose distribution was determined by multiplying the air kerma normalized dose distribution from the simulations with the actual air kerma value (mGy) measured free-in-air in the isocentre of the scanner by using a pencil ionization chamber (RaySafe Xi CT detector, Unfors RaySafe, Sweden). In addition to the organ doses needed for the blood dose evaluation, doses to other organs and tissues of interest according to the BEIR VII risk models were also calculated with this Monte Carlo patient dose simulation. Organ volumes were segmented in the original CT images and organ dose deposition was calculated on the basis of the Monte Carlo dose distribution. For the bone marrow, all bony structures in the field of view were segmented and the bone marrow dose was calculated as the weighted sum of the doses to these structures with the percentage of bone marrow in these compartments as weighting factor [21]. To account for differences in photon absorption in bone and bone marrow, the obtained value was corrected for the ratio of mass absorption coefficients in soft tissue and bone [22].

To compare the dose levels of the CT procedures in the current study with similar procedures in different hospitals and countries, the effective dose was calculated for the total group of chest CT and abdomen CT patients. Effective dose is a quantity reflecting the stochastic risk associated with an exposure to ionizing radiation at a population level. The effective doses of the paediatric populations under study were derived from the individual DLP values by using the conversion factors published by Deak et al. as function of kVp, imaging region and age for ICRP publication 103 recommendations [23].