Skip to main content
European Commission logo print header

New technologies in the automated detection of radiation-induced cytogeneticeffects

Objective


The project, in general, was divided in two broadly overlapping phases. The first phase, lasting about 2 years, was focused on the development of the new methodologies mentioned above, taking place at the MRC, GSF, and Amsterdam. As with most technical research projects, this phase of the investigations proved to be quite laborious and many obstacles had to be overcome. During this period of development an extensive reference data set on aberrations was being generated, at the GSF, by visual scoring using FISH labelling of metaphase spreads.

At the beginning of the project period various ways for the automated analysis of the expression of radiation-induced genomic damage as chromosomal aberrations were open. As none of the possible methods had been investigated in sufficient depth, we chose to pursue the problem from different sides by developing, in parallel, a number of promising methods. This approach required the input from a multi-disciplinary group of highly specialised investigators that could not be found combined in a single institute. By joining the effort of experienced scientists of several institutes from different E.U. member states we expected to advance sufficiently fast to allow a comparison of the merits of the different techniques developed through our common effort before the end of the project period.

The techniques selected for further development were:

- The detection of chromosome damage with automated microscopy and fluorescence in situ hybridisation (FISH) (Green)
- The analysis of micronuclei using flow cytometry and FISH (Nüsse)
- The slit-scanning analysis of dicentric chromosomes by flow cytometry (Aten)
- Investigation by confocal microscopy of the nuclear organisation in relation to radiation sensitivity (Aten).

The development of these techniques for the investigation of radiation-induced cytogenetic effects involved methods:

- To establish reliable, reproducible in situ hybridisation protocols with fluorescent probes for centromeres, telomere marking and for whole
The transition to the second phase, occurring for the different technologies at different periods of the project, was marked by a strongly increased interaction between the participants, as data started being generated by the different automated systems that could used to evaluate their performance. Moreover, during the period covered by the second phase of the project, the exchange of samples between the participating groups became an integral part of several of the measurement programmes. Nüsse and Aten collaborated on the tough problem of centromere labelling of micronuclei and chromosomes in suspension.

At the end of the project the feasibility of the different approaches had been demonstrated in principle. Although it is difficult to predict, at this stage of the development, which of the new techniques will prove to be ultimately the method of choice, it is evident that the data obtained by visual assessment of FISH labelled samples, yield a wealth of quantitative aberration data as well as providing valuable information on the chromosomal processes involved. The quantitative microscopy method promises to provide the same type of information but faster which make it an attractive choice. The micronucleus test is by far the most rapid procedure of all the methods that we investigated but here the background can be problematic. The slit-scanning data also can be obtained at a high rate but the system so far has a limited sensitivity because of background problems. The dynamic organisation of chromosomes in interphase is relevant for the understanding of mechanisms involved in the induction of aberrations.

The aim of this proposal is to develop methods for simplified and automated assessment of radiation induced damage in chromosomes and nuclei, and to make these applicable to radiation
protection purposes. The investigations will involve:
- preparation and staining of chromosomes in metaphase and
interphase and of micronuclei. Reliable, reproducible procedures will be established for fluorescent labeling with DNA probes for centromeres, telomeres and for chromosome painting, in combination with BrdUrd and various immunocytochemical probes.
- Analysis of radiation induced changes in metaphase and interphase nuclei and micronuclei, by two and three colour fluorescence microscopy, confocal scanning laser microscopy and (slit-scanning) flow cytometry. The image analysis analysis aspects will be emphasized.
- Comparison of the results of the automated analysis with data obtained by visual assessment using conventional microscopy
techniques.
The developments of these techniques will achieve results equal to those from visual assessment of dicentrics in human lymphocytes, using transmission or fluorescence microscopy, which is still the most sensitive technique to quantify the exposure of a person to ionizing radiation. The new techniques should satisfy the medical need for dose assessments on large populations in case of an accidental irradiation, and be less time consuming than visual procedures. The new biological dosimetry systems, as well as automation of chromosome analysis have a high potential for this purpose.
These investigations will be carried out jointly by Dr. D. K. Green (MRC, Edinburgh), prof. Dr. M. Bauchinger (GSF, München), Dr. G. Burger (GSF, München), Dr. J. A. Aten and prof. Dr. G. W. Barendsen (UVA, Amsterdam) and Dr. M. Nüsse (GSF, München).

Topic(s)

Data not available

Call for proposal

Data not available

Coordinator

Universiteit van Amsterdam
EU contribution
No data
Address
9,Meibergdreef
1105 AZ Amsterdam
Netherlands

See on map

Total cost
No data

Participants (3)