Lymphocytes of patients receiving irradiation for the treatment of prostate cancer have been analyzed by scoring gamma-H2AX foci. A distribution of delivered dose to the lymphocytes is shown and visualized in the graphics above. Similarity between DLH (dose-lymphocyte-histogram) and DVH (dose-volume-histogram) has been found. The biological measurement on behalf of the human lymphocytes corresponds to the distribution calculated by the physicists: more low-dose-delivery is observed for the SSIMRT compared to the 3D. At the same time, a lower distribution of 30%-90% of the applied dose can be reported for the SSIMRT.
The advantage of this method is an easy and fast access to the required material without any massive medical interventions. The method allows an in vivo estimation respectively proof of the dose distribution calculated by the therapy planning system.
The challenge is that every patient has to be irradiated at a comparable volume and same site of the body. Attention also has to be paid to the repair kinetics and withdraw of gamma-H2AX foci, which make it necessary to stop cell metabolism after a certain duration post irradiation. Due to this context, we fixed all cells 2 h after irradiation (in-vivo and in-vitro) to allow comparability between the samples.
However, the determination of the probability of lymphocytes' presence in the body tissue is difficult, due to the lymphocytes' kinetics (circulation in the blood vessels), migration and adhesion to the vessel wall. These circumstances have been described by Sak et al. in detail . It has to be considered, that lymphocytes in in-field capillaries move slower and receive more dose, than fast moving lymphocytes in larger vessels. Sak et al. described differences in mean numbers of gamma-H2AX foci in lymphocytes depending on irradiated target sites, e.g. brain and thorax. In our study, target site was no variable parameter, since we compared 3D and SSIMRT only in prostate cancer treatment.
The SSIMRT's beam-on-time differed from the 3D's by a factor 5 (Table 2). Assuming a blood circulation time of one minute, this fact causes inaccuracy while measuring the actual dose distribution. On the other hand, table time in both modalities differs by factor 1.4. During 11.5 vs. 16.3 min of table time, lymphocytes in both groups have the chance of being radiated more than one time. The cumulative formation of gamma-H2AX foci can lead to a false high result in evaluating dose distribution. In order to attempt a correction towards real dose distribution in SSIMRT, one would expect even less cells exposed to higher levels of dose. This correction would amplify the differences between 3D and SSIMRT, which again correspond with the physical model.
Statement implying an absolute dose in Gy used for dosimetry, cannot be recommended without doubts, due to the following issues: in the DLH (Figure 4) higher lymphocyte-percentages are plotted, compared to the DVH (Figure 5). The DLH shows a radiation dose of 5% in 7-9% of lymphocytes (DLH), whereas only about 5% of the body volume receives the same dose (DVH). Doses of above 100% can be observed in the DLH, too. This phenomenon can be explained by the possibility of repeated dose exposure of some lymphocytes as explained above.
The linear correspondence between induction of γH2AX foci and the delivered dose has already been verified and practiced especially for low doses [4, 17]. Exceptions from this rule are described and due to different irradiation conditions or different kinds of ionizing irradiation .
The visualization, which is shown for computed tomography examinations of different sites (1), was now extended to the doses of one fraction of radiotherapy for different techniques.
Flow cytometry has also been performed in order to measure delivered dose by γH2AX stain , however, in our case it didn't seem appropriate: The intensity of the gamma-H2AX foci varied and could have led to errors while measuring the background level of fluorescence. In our opinion, a concrete number of foci per nucleus is needed to compare dose distribution exactly.
Jucha et al. evaluated 2-dimentional pictures of the stained lymphocytes using special software , but we set great store by being able to zoom through the slide under the microscope and looking at the complete 3-dimentional nucleus in order to detect every gamma-H2AX foci. For this reason in our experiments foci were counted manually by eye with a fluorescence-microscope.
By creating a dose-lymphocyte histogram (DLH), the gamma-H2AX staining method allows the estimation of the dose distribution after irradiation. One possible application of the present method could also be in radiation-protection for in-vivo dosimetry after accidental exposure to radiation. In case of accidental irradiation, background foci level cannot be determined and therefore cannot be subtracted in the DLH. In this situation background foci level should also not be subtracted in the calibration line. In this manner the error due to background foci level can be reduced, however individual differences of background foci levels remain unconsidered. Another possibility to deal with this limitation is to take blood for background foci level examination several weeks after the exposure, when the circulating lymphocytes have been substituted naturally.