Biodosimetry estimation using the ratio of the longest:shortest length in the premature chromosome condensation (PCC) method applying autocapture and automatic image analysis.

The combination of automatic image acquisition and automatic image analysis of premature chromosome condensation (PCC) spreads was tested as a rapid biodosimeter protocol. Human peripheral lymphocytes were irradiated with (60)Co gamma rays in a single dose of between 1 and 20 Gy, stimulated with phytohaemaglutinin and incubated for 48 h, division blocked with Colcemid, and PCC-induced by Calyculin A. Images of chromosome spreads were captured and analysed automatically by combining the Metafer 4 and CellProfiler platforms. Automatic measurement of chromosome lengths allows the calculation of the length ratio (LR) of the longest and the shortest piece that can be used for dose estimation since this ratio is correlated with ionizing radiation dose. The LR of the longest and the shortest chromosome pieces showed the best goodness-of-fit to a linear model in the dose interval tested. The application of the automatic analysis increases the potential use of the PCC method for triage in the event of massive radiation causalities.

Assessment of simulated high-dose partial-body irradiation by PCC-R assay.

The estimation of the dose and the irradiated fraction of the body is important information in the primary medical response in case of a radiological accident. The PCC-R assay has been developed for high-dose estimations, but little attention has been given to its applicability for partial-body irradiations. In the present work we estimated the doses and the percentage of the irradiated fraction in simulated partial-body radiation exposures at high doses using the PCC-R assay. Peripheral whole blood of three healthy donors was exposed to doses from 0-20 Gy, with 6°Co gamma radiation. To simulate partial body irradiations, irradiated and non-irradiated blood was mixed to obtain proportions of irradiated blood from 10-90%. Lymphocyte cultures were treated with Colcemid and Calyculin-A before harvest. Conventional and triage scores were performed for each dose, proportion of irradiated blood and donor. The Papworth's u test was used to evaluate the PCC-R distribution per cell. A dose-response relationship was fitted according to the maximum likelihood method using the frequencies of PCC-R obtained from 100% irradiated blood. The dose to the partially irradiated blood was estimated using the Contaminated Poisson method. A new D0 value of 10.9 Gy was calculated and used to estimate the initial fraction of irradiated cells. The results presented here indicate that by PCC-R it is possible to distinguish between simulated partial- and whole-body irradiations by the u-test, and to accurately estimate the dose from 10-20 Gy, and the initial fraction of irradiated cells in the interval from 10-90%.

Prematurely condensed chromosome rings after neutron irradiation of human lymphocytes.

Calibration curves for fission spectrum neutrons and other high LET radiations are scarce in cytogenetic dosimetry and particularly for Prematurely Condensed Chromosome Rings (PCC-ring). Here we analyzed the behavior of the PCC-ring frequency and PCC index after neutron irradiation in a broad dose interval from 1 to 26 Gy. PCC-rings were induced in lymphocytes with Calyculin A. 6455 PCC cells in G1, G2/M and M/A stages were analyzed. The best fitting between the frequency of PCC ring (Y) and the Dose (D) was obtained with the equation Y = (0.059 ± 0.003) D. The saturation of the PCC-ring was observed after around 4 Gy, but it was still possible to analyze cells exposed up to 26 Gy. The distribution of rings by cell follows Poisson or Neyman type distribution for all doses. This PCC-ring dose effect curve can be used in case of accidental overexposure to neutron radiation, allowing a dose assessment in a reliable way. Additionally, the PCC index seems to be well correlated with radiation dose and decrease in a dose dependent manner from 13% in non exposed sample down to 0.2%. This observation allows the possibility to perform a quick classification of victims exposed to high doses of both gamma and neutron radiations. The PCC assay can then be used for both neutron dose estimation up to 4 Gy and for the rapid classification of victims exposed to higher doses. This assay could be included in the multiparametric approach developed to optimize the medical treatment of radiation victims.

PCC-ring induction in human lymphocytes exposed to gamma and neutron irradiation

In case of an accidental overexposure to ionizing radiation where the dose received by the victim is over 5 Gy, the conventional biological indicator of dose, the dicentric assay, does not provide an accurate enough dose measurement. A more appropriate technique is to measure ring chromosomes in stimulated lymphocytes. Dose-effect relationships were obtained by plotting the frequencies of Premature Chromosome Condensation (PCC)-rings in PCC lymphocytes obtained by chemical induction with Calyculin A in vitro, irradiated with doses between 5 to 25 Gy. Cells were exposed either to neutron or to gamma rays and the corresponding dose effect curves are presented in this paper for the first time in literature. For the elaboration of these curves, 9 675 PCC cells in G1 G2 and M/A stages were analysed. The results were fitted to a lineal model in gamma irradiation up to 25 Gy. For neutron irradiation the data was fitted to a lineal model up to 10 Gy, and then dose saturation was observed. In conclusion, with this technique it is possible to set up dose effect curves up to 25 or 10 Gy according to the gamma or neutron radiation(AU)

En caso de una sobreexposición accidental a la radiación ionizante que la dosis recibida por la víctima es de más de 5 Gy, el indicador biológico convencional de la dosis, la dicéntricos de ensayo, no proporciona una medición precisa de dosis suficientes. Una técnica es más adecuada para medir los cromosomas en anillo estimulado linfocitos. Relaciones dosis-efecto se obtuvieron mediante el trazado de las frecuencias de Condensación prematura de cromosomas (PCC)-anillos del PCC en linfocitos obtenidos por química de inducción con un Calyculin in vitro, irradiados con dosis de entre 5 a 25 Gy. Las células fueron expuestas a cualquiera de neutrones o rayos gamma y las correspondientes curvas dosis efecto se presentan en este documento por primera vez en la literatura. Para la elaboración de estas curvas, 9 675 células PCC en G1 G2 y M / A se analizaron las fases. Los resultados se ajustaron a un modelo lineal de radiación gamma de hasta 25 Gy. Para los datos de irradiación de neutrones se ajustó a un modelo lineal de hasta 10 Gy, y luego la dosis se observó saturación. En conclusión, con esta técnica es posible la creación de curvas de dosis efecto hasta el 25 o 10 Gy en función de la radiación gamma o de neutrones

PCC-ring induction in human lymphocytes exposed to gamma and neutron irradiation.

In case of an accidental overexposure to ionizing radiation where the dose received by the victim is over 5 Gy, the conventional biological indicator of dose, the dicentric assay, does not provide an accurate enough dose measurement. A more appropriate technique is to measure ring chromosomes in stimulated lymphocytes. Dose-effect relationships were obtained by plotting the frequencies of Premature Chromosome Condensation (PCC)-rings in PCC lymphocytes obtained by chemical induction with Calyculin A in vitro, irradiated with doses between 5 to 25 Gy. Cells were exposed either to neutron or to gamma rays and the corresponding dose effect curves are presented in this paper for the first time in literature. For the elaboration of these curves, 9 675 PCC cells in G1 G2 and M/A stages were analysed. The results were fitted to a lineal model in gamma irradiation up to 25 Gy. For neutron irradiation the data was fitted to a lineal model up to 10 Gy, and then dose saturation was observed. In conclusion, with this technique it is possible to set up dose effect curves up to 25 or 10 Gy according to the gamma or neutron radiation.

Sensitivity and variability of visual scoring in the comet assay. Results of an inter-laboratory scoring exercise with the use of silver staining.

Nineteen scorers from seven Cuban laboratories participated in this slide exercise designed to test the influence of the scorer on the accuracy, sensitivity and variability of the comet assay when a visual method of DNA damage evaluation is used. The assay was performed using human lymphocytes from a single donor exposed in vitro for 5 min at 0 degrees C to doses of 0, 5, 10, 25, 50, 100 and 200 microM of hydrogen peroxide. Each participant scored the same set of 14 coded slides with silver stained comets. The comets were classified visually into five categories according to the appearance resulting from the relative proportion of DNA in the tail. The extent of DNA damage was expressed in arbitrary units. At zero dose the median values of 12 scorers out of 19 were included between the values of the overall 25 and 75 per thousand. This proportion remains practically the same as the dose increases. The lowest dose detected by this method for the majority of scorers (11) was 10 microM. The coefficient of variation at the control dose was the highest (median value 26%), progressively declined to 20%, and starting from 25 microM, values are around 10%. The results of the exercise show the reliability of the silver staining and visual scoring for the comet method.