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Calibration of the γ-H2AX DNA double strand break focus assay for internal radiation exposure of blood lymphocytes.

Eberlein U, Peper M, Fernández M, Lassmann M, Scherthan H - PLoS ONE (2015)

Bottom Line: The measured blood doses in our samples ranged from 6 to 95 mGy.A linear relationship was found between the number of DSB-marking foci/nucleus and the absorbed dose to the blood for both radionuclides studied.There were only minor nuclide-specific intra- and inter-subject deviations.

View Article: PubMed Central - PubMed

Affiliation: Department of Nuclear Medicine, University of Würzburg, Würzburg, Germany.

ABSTRACT
DNA double strand break (DSB) formation induced by ionizing radiation exposure is indicated by the DSB biomarkers γ-H2AX and 53BP1. Knowledge about DSB foci formation in-vitro after internal irradiation of whole blood samples with radionuclides in solution will help us to gain detailed insights about dose-response relationships in patients after molecular radiotherapy (MRT). Therefore, we studied the induction of radiation-induced co-localizing γ-H2AX and 53BP1 foci as surrogate markers for DSBs in-vitro, and correlated the obtained foci per cell values with the in-vitro absorbed doses to the blood for the two most frequently used radionuclides in MRT (I-131 and Lu-177). This approach led to an in-vitro calibration curve. Overall, 55 blood samples of three healthy volunteers were analyzed. For each experiment several vials containing a mixture of whole blood and radioactive solutions with different concentrations of isotonic NaCl-diluted radionuclides with known activities were prepared. Leukocytes were recovered by density centrifugation after incubation and constant blending for 1 h at 37°C. After ethanol fixation they were subjected to two-color immunofluorescence staining and the average frequencies of the co-localizing γ-H2AX and 53BP1 foci/nucleus were determined using a fluorescence microscope equipped with a red/green double band pass filter. The exact activity was determined in parallel in each blood sample by calibrated germanium detector measurements. The absorbed dose rates to the blood per nuclear disintegrations occurring in 1 ml of blood were calculated for both isotopes by a Monte Carlo simulation. The measured blood doses in our samples ranged from 6 to 95 mGy. A linear relationship was found between the number of DSB-marking foci/nucleus and the absorbed dose to the blood for both radionuclides studied. There were only minor nuclide-specific intra- and inter-subject deviations.

No MeSH data available.


Related in: MedlinePlus

Vial geometry.Schematic drawing showing a longitudinal section (left) and a cross section along the black dashed line drawn on the vial cap (right) of the geometry used for the Monte Carlo simulation. It represents a vial (black) filled with radionuclide-containing blood (dark grey) surrounded by air (light grey).
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pone.0123174.g001: Vial geometry.Schematic drawing showing a longitudinal section (left) and a cross section along the black dashed line drawn on the vial cap (right) of the geometry used for the Monte Carlo simulation. It represents a vial (black) filled with radionuclide-containing blood (dark grey) surrounded by air (light grey).

Mentions: The set-up for the simulated vial is shown in Fig 1. The left part shows a central plane along the length of the simulated system (vial filled with radioactive blood solution and surrounded by air) and on the right a cross section corresponding to the vial cap. The vial had an internal radius of 0.48 mm and an internal height of 7.49 cm. The radioactive blood solution was considered as a homogeneous mixture of blood with the radionuclide, which was approximated as homogeneous soft tissue-equivalent with density ρ = 1.0 g/cm3. The emission of radiation was considered to be isotropic. The vial and its cap were 1.0 mm thick and made of polypropylene (density ρ = 0.9 g/cm3). The air surrounding the vial was defined as dry air (density = 1.2 10-3 g/cm3). 5 cm of air in each direction from the vial external surface was considered. The atomic composition and density of the materials were taken from the STAR (NIST) database [29].


Calibration of the γ-H2AX DNA double strand break focus assay for internal radiation exposure of blood lymphocytes.

Eberlein U, Peper M, Fernández M, Lassmann M, Scherthan H - PLoS ONE (2015)

Vial geometry.Schematic drawing showing a longitudinal section (left) and a cross section along the black dashed line drawn on the vial cap (right) of the geometry used for the Monte Carlo simulation. It represents a vial (black) filled with radionuclide-containing blood (dark grey) surrounded by air (light grey).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390303&req=5

pone.0123174.g001: Vial geometry.Schematic drawing showing a longitudinal section (left) and a cross section along the black dashed line drawn on the vial cap (right) of the geometry used for the Monte Carlo simulation. It represents a vial (black) filled with radionuclide-containing blood (dark grey) surrounded by air (light grey).
Mentions: The set-up for the simulated vial is shown in Fig 1. The left part shows a central plane along the length of the simulated system (vial filled with radioactive blood solution and surrounded by air) and on the right a cross section corresponding to the vial cap. The vial had an internal radius of 0.48 mm and an internal height of 7.49 cm. The radioactive blood solution was considered as a homogeneous mixture of blood with the radionuclide, which was approximated as homogeneous soft tissue-equivalent with density ρ = 1.0 g/cm3. The emission of radiation was considered to be isotropic. The vial and its cap were 1.0 mm thick and made of polypropylene (density ρ = 0.9 g/cm3). The air surrounding the vial was defined as dry air (density = 1.2 10-3 g/cm3). 5 cm of air in each direction from the vial external surface was considered. The atomic composition and density of the materials were taken from the STAR (NIST) database [29].

Bottom Line: The measured blood doses in our samples ranged from 6 to 95 mGy.A linear relationship was found between the number of DSB-marking foci/nucleus and the absorbed dose to the blood for both radionuclides studied.There were only minor nuclide-specific intra- and inter-subject deviations.

View Article: PubMed Central - PubMed

Affiliation: Department of Nuclear Medicine, University of Würzburg, Würzburg, Germany.

ABSTRACT
DNA double strand break (DSB) formation induced by ionizing radiation exposure is indicated by the DSB biomarkers γ-H2AX and 53BP1. Knowledge about DSB foci formation in-vitro after internal irradiation of whole blood samples with radionuclides in solution will help us to gain detailed insights about dose-response relationships in patients after molecular radiotherapy (MRT). Therefore, we studied the induction of radiation-induced co-localizing γ-H2AX and 53BP1 foci as surrogate markers for DSBs in-vitro, and correlated the obtained foci per cell values with the in-vitro absorbed doses to the blood for the two most frequently used radionuclides in MRT (I-131 and Lu-177). This approach led to an in-vitro calibration curve. Overall, 55 blood samples of three healthy volunteers were analyzed. For each experiment several vials containing a mixture of whole blood and radioactive solutions with different concentrations of isotonic NaCl-diluted radionuclides with known activities were prepared. Leukocytes were recovered by density centrifugation after incubation and constant blending for 1 h at 37°C. After ethanol fixation they were subjected to two-color immunofluorescence staining and the average frequencies of the co-localizing γ-H2AX and 53BP1 foci/nucleus were determined using a fluorescence microscope equipped with a red/green double band pass filter. The exact activity was determined in parallel in each blood sample by calibrated germanium detector measurements. The absorbed dose rates to the blood per nuclear disintegrations occurring in 1 ml of blood were calculated for both isotopes by a Monte Carlo simulation. The measured blood doses in our samples ranged from 6 to 95 mGy. A linear relationship was found between the number of DSB-marking foci/nucleus and the absorbed dose to the blood for both radionuclides studied. There were only minor nuclide-specific intra- and inter-subject deviations.

No MeSH data available.


Related in: MedlinePlus