International E co G en Incorporated

Flow Cytometry Analysis
-a screening procedure for DNA damage
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APPLICATIONS
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| THE PROBLEM
A mutagen can cause:
When this damage is not repaired, there is an unequal distribution of nuclear DNA passed on to the resultant daughter cells after division. This damage then becomes fixed in the respective daughter cell lines which includes all their subsequent cell descendents. If this damage occurs in the rapidly dividing stem cells or transit cells, then the variation can be greatly bio-magnified in tissues such as human and other mammalian lymphocytes, the nucleated red blood cells of non-mammalian vertebrates, the sperm cells of males, the liver cells of juvenile animals. This increased variation in nuclear DNA content of cells is diagnostic for DNA damage when compared to a control or reference group and can be detected by the flow cytometer as an increase in the coefficient of variation (CV) of the G0/G1 peak. Thus the flow cytometry method detects damage that has not been repaired and that may result in significant effects.
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| THE PROCEDURE
Flow cytometry has been used to detect environmental
mutagenesis in field populations of fish, frogs, birds, turtles
and mice where contaminated sites were compared with reference
sites. Flow cytometry has also been used to detect genetic damage
in chinook salmon under controlled field bioassay exposures to
pulp mill effluent as well as in ocean sampled fish naturally
exposed to sediments and prey species containing PAHs. Our
method detects DNA damage that
To
determine the nature of the contaminant impact, we can examine a range
of tissues from blood to liver, kidney and germ cells. We can examine
any vertebrate animal (i.e. mice, fish, frogs, snakes, birds, humans,
etc.) and most invertebrates. Our only limitation is the tissue freezing
protocol for some invertebrates. The samples can be frozen in the field
and shipped to us on dry ice from any location in the world. We can do
the complete study design for maximum efficiency and cost effectiveness
and then work with consultants located near the study site to implement
the design.
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| We typically examine 10,000 nuclei per animal with each
treatment group being represented by at least 20 individuals. 30 animals of the same sex
per group enables maximum resolution for this method. In
pre-reproductive animals, sexing may
not be possible and so a random sample is necessary. The assay is
sufficiently sensitive to distinguish between populations of
cells whose nuclear DNA content differs by as little as 1 to 2%
and can be performed on a wide array of tissue types, blood being
the tissue of choice. Results of flow cytometry have been
verified by classical cytogenetic methods. We use a weighted
least squares statistical procedure for the analysis of the CV
values. This new procedure which we derived expressly for CV
value analysis is the only valid method presently available for
analyzing data of this kind. An additional benefit of this
statistical method is that the ability to detect differences
between treatment groups is increased by an order of magnitude
compared to historical (and incorrect) methods of statistical
analysis.
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| The service includes advice on statistical design, sample collection, sample preparation for
freezing, flow cytometry analysis, a highly sensitive
statistical analysis of the Coefficient of Variation values
generated by the flow cytometry and an interpretive report. If
necessary, we can also assist in sample collection.
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For further details, please contact |
| Types of Mutation |
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| Consequences of Mutation |
| Our DNA Damage
Detection Method |
© Updated by Michael Easton 2009.