Objective
The aims of this contract are to develop techniques for the rapid assessment of chromosome aberrations for the purpose of evaluating possible occupational or accidental exposures of human individuals or groups. Protection and intervention measures must be based on dose assessments which are at least partly based on biological methods applied to individuals.
Slit scanning flow cyometry methodology was applied to the analysis of chromosomes. A slit scanning system was developed which is based on commercially available equipment with optical elements also used in standard flow cytometry systems. The method was extended to analyse the shapes of human metaphase choromosomes. The shape of the chromosomes as expressed by their centromeric index (CI) and their deoxyribonucleic acid (DNA) content have been used as parameters in bivariate flow karyotyping. The resolution of the DNA versus CI flow karyogram of the larger chromosomes up to chromosome 13 is much higher than the resolution obtained in the DNA based monovariate flow karyogram. Most of these chromosomes can be distinguished as individual peaks.
The slit scanning analysis was extended to detect dicentric chromosomes by introducing a system forcounting the number of centromere dips in the slit scan profiles. Shape analysis can be performed online allowing chromosomes to be sorted on centromeric characteristics. However, dips in the slit scan profile are not only caused by centromeres. Aggregates of chromosomes or irregularly stretched chromosome arms can also results in trimodal profiles. The relationship between chromosome morphology and corresponding slit scan profile was investigated by sorting chromosomes yielding trimodal profiles on to separate slides for microphotography. Further, the negatives were digitized with a microdensitometer and density profiles were constructed. This data provides valuable clues that help to distinguish between true discentrics and artefacts. This information was used to improve the sensitivity and selectivity of the pulse dip counter.
Chromosome length appears to be an important factor in the resolution of centromeres. Propidium iodide was used for the isolation and staining of the chromosomes followed by incubating the unfixed chromosome suspension with trypsin. The treatment had 2 effects: the total length of the chromosome was increased and the trysin appeared to stretch the centromere more than the chromosome arms. This improves the detection of dicentric chromosomes from irradiated cells.
Various sources of deoxyribonucleic acid (DNA) libraries for chromosome painting were investigated to improve the detection of radiation damage initially on metaphase slide preparations. Chromosome painting uses labelled DNA sequences from a specific chromosome to paint or completely cover that chromosome with a fluorescent signal. The sources of DNA sequence from a specific chromsome were lambda DNA libraries from the American Type Culture Collection (ATCC), somatic cell hydrids containing one single human chromosome and small quantities of sorted chromosomes. The final choice of sources material will depend on both a complete chromosome coverage and low background as well as ease of production.
A second labelling system, digoxygenin, was to be implemented which can be detected by an antibody to digoxygenin coupled to Texas Red or fluorescein isothiocyanate (FITC).
Laser scanning confocal microscopy including a 2 dimensional charge coupled device CCD) camera was used to record digital images of both bright fluorescence objects such as dapi stained chromosomes and weakly labelled fluorescence objects such as those prepared by in situ hybridization of small DNA probes to metaphase chromosomes. The fluorescence intensity of painted chromosomes lies somewhere between bright DNA specific fluorescence and single unique sequence probe fluorescence depending on the quantity and size of the painting probes.
Major changes to the chromosome sorting and analyses flow system have occurred with a view to multicolour exitation and emission anticipated from samples of painted chromosomes. A computer with signal processing hardware was assembled and software written for flow cytometry control. The system was under study.
The induction of micronucelei in cells exposed to ionizing radiation can be used as a measure for both structural and numerical chromosome aberrations. Scoring of micronuclei therefore provides a quantitative measurement for the degree of cytogenetic damage in cells and thus is used for a dose estimation of humans exposed to ionizing radiation. Although scoring of micronuclei should be faster than established chromosome analysis it does not have the capacity needed to screen large human populations. Thus a new flow cytometric technique was developed for scoring micronuclei in human lymphocytes using multiparametric flow cytometry because the high measuring rates of flow cytometers provide the capacity to screen large groups of persons exposed to ionizing radiation. The development of the technique is described in detail. With the new flow cytometric technique the frequencies of radiation induced micronuclei can easily be measured in cell cultures and human lymphocytes irradiated in vitro. In the case of human lymphocytes the fraction of cells in the first and second cell cycle have to be additionly measure by using the flow cytometric bromodeoxyuridine (BrdUrd) Hoechst quenching technique. The results agree with microscopic measurements if the number of micronuclei per main nuclei are calculated and if only those micronuclei are measured that have a deoxyribonucleic acid (DNA) content between 0.5% and 10% of the nucleus. Larger micronuclei are not usually found after irradiation. However, they could be induced by chemicals that would interfere with the spindle apparatus. In this case the flow cytometric technique will give results that do not agree with microscopic observation. This effect is under study.
Compared to conventional dicentric analysis, scoring of micronuclei (MN) is a faster and easier approach of measuring radiation induced chromosome damage in human lymphocytes and has been suggested as an alternative biological dosimeter system. An automated MN scoring system such as flow cytometry would be applicable to large populations if it could be performed with the precision of mircoscopic scoring. To compare data sets derived with flow cytometry experiments were carried out to establish dose effect curves for MN induced by different radiation qualities.
Lymphocyte suspensions obtained from freshly drawn whole blood were exposed to cobalt 60 gamma-rays, 220 kV gamma-rays and fission neutrons. The preliminary results of these dose response experiments show that they are in line with basic radiobiophysical expectations on the effectiveness of different radiation qualities.
Intra-individual and inter-individual variations of background and radiation induced MN frequencies were analyzed. Venous blood was taken from 4 donors aged between 26 and 51 years in at 3 monthly intervals for one year (5 samples from each donor). Lymphocyte suspensions were prepared and one half were exposed to 3 Gy of caesium 137 and one half remained as controls. A significant variation of MN frequencies was found among the 20 control samples as well as among the 20 exposed samples. Intra individual variations revealed a 5-fold background variation in MN levels for donor A and 2-fold variation for induced MN levels of donors A, C and D. To reduce the influence of intra individual and interindividual variations of induced MN frequencies a standard dose effect curve should be based upon data of 35 donors. Due to the considerable variation of background MN frequencies in samples from a single donor at different examination stages it seems difficult to derive a reliable individual low dose estimate below 0.5 Gy.
The experimental objective is the establishment of dose effect curves for chromatin texture assay (CTA) of in vitro exposed human lymphocytes. Initial work was concentrated on the preparation of protocols and the investigation of conceptual relations of chromatin texture features on size and staining density of nuclei.
In order to evaluate the role of chromatin pattern features in cell nuclei for monitoring radiation exposure, work was concentrated on in vitro irradiation of cultured and phytohaemagglutinin (PHA) stimulated human lymphocytes. Although the final goal of the project is to be the direct analysis of nonstimulated lymphocytes from peripheral blood of exposed species more information on the ongoing process in cycling cells was required.
Initial results reported on studies of cellular growth and chromatin assay in G{1} cells were from one single experiment with blood from one human donor.
Human PHA stimulated lymphocytes show changes in growth kinetics in the occurence of highly aneuploid cell components and in the chromatin texture of G{1} cells as a function of radiation dose. Further work is required to validate the findings and to increase the sensitivity of the CT assay.
Investigations, to be carried out at the Laboratory for Radiobiology of the University of Amsterdam, will be aimed at developing a method for the rapid analysis, by automated techniques, of karyotype abnormalities in large numbers of cells using slit-scanning of fluorescent chromosomes in suspensions prepared from irradiated cells.
In the slit-scan flow cytometer the morphology of each chromosome is analyzed separately through the time dependent registration of the fluorescence signal as the chromosome passes through a strongly focused laser beam. Chromosome profiles are characterized by the centromeres that appear as dips in the pulse shapes. Normal chromosomes produce only one centromere dip. Dicentric chromosomes, the most commonly registered type of radiation-induced aberration, show two dips. Using high speed electronics the centromeres are detected and counted in real time (ie during the passage of the chromosome through the flow cytometer). Thus it is possible to activate the cell sorter module incorporated in the system to collect abnormal chromosomes for fixation on microscope slides. These chromosomes are then available for examination by centromere and telomere counting, using visual and automated microscopy techniques. This check is necessary to distinguish chromosome aggregates and other artefacts from chromosome aberrati ns induced by radiation.
The aim of project at the Gesellschaft fur Strahlen und Umweltforschung at Munich is to develop an automated micronucleus (MN) assay applied specifically to human lymphocytes, using flow cytometry.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences genetics DNA
- natural sciences physical sciences nuclear physics
- natural sciences physical sciences optics microscopy confocal microscopy
- natural sciences biological sciences genetics chromosomes
- natural sciences physical sciences optics laser physics
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