Frequently
Asked
The
Micronucleus Test |
| The in vivo Micronucleus Test is widely accepted by international
regulatory bodies as a way of measuring chromosome damage. New
pharmaceuticals are put through this test prior to Phase II clinical
trials, although it is typically performed before Phase I. |
Questions About MicroFlowPLUS Kits
What is a MicroFlowPLUS Kit?
MicroFlow kits are
used to significantly improve the speed and accuracy of scoring blood
samples during a routine safety evaluation known as the Micronucleus Test.
The
MicroFlowPLUS kit is offered for use with either mouse
or rat studies, and contains all of the quality-assured supplies and reagents
needed to collect and fix up to 60 samples in duplicate. With these kits,
researchers collect and fix blood samples, then perform the sample analysis
using their own in-house flow cytometer.
One of the main benefits
from the MicroFlow kits is the ability to obtain results three to six
weeks faster than is possible using traditional microscopic methods.
In an ultra-competitive environment such as drug discovery, this time
savings can translate into enormous revenue for a company that beats its
competition to the market.
This automated system,
also allows researchers the ability to perform the Micronucleus test earlier
in the screening or discovery process. Since this test must be performed
prior to acceptance by regulatory agencies, it is in a companyâs best
interest to quickly determine if a compound will not pass this standard,
so valuable time and resources can be redirected.
Although MicroFlow
kits are particularly well suited for the pharmaceutical industry, they
offer equally significant benefits to:
- Biotechnology companies
- Universities and Academic
institutions
- Government institutes
& laboratories
- Chemical &
toner industries
- Medical/Dental
device manufacturers
What
materials do I need in order to use the MicroFlowPLUS kit?
Standard laboratory
equipment is needed to use this kit. These include: flow cytometer (capable
of 488 nm excitation), microcentrifuge tubes, pipettes (glass and plastic),
blood collection supplies, centrifuge, flasks, polypropylene centrifuge
tubes, micropipette (20 to 200 µl) and standard tips for fixing the samples,
-75 °C to -85 °C freezer, 20 °C freezer, 4 °C refrigerator.
How
do I know this method is really measuring micronuclei?
1) Microscopic inspection
after flow cytometric sorting shows that those cells presumed to contain
micronuclei, do contain micronuclei. 2) Counts obtained using MicroFlow
kits closely mirror those made by microscopy. 3) A biological standard
(malaria) is used, which has similar DNA size and staining characteristics
to micronucleated cells, allowing researchers to properly and consistently
setup the equipment for MN counts. More specifically, this assures that
the gates and regions are properly set on their flow cytometer.
For a more detailed
description of the rigorous testing that was performed (in order to verify
the accuracy of this method), please contact Litron.
Are
the results obtained using this method reproducible?
Yes. Since the
1980s, Litron Laboratories has been developing and improving the MicroFlow
method. In order to validate the process, the company received NIH funding
to work with 14 different research facilities from around the world.
The results of this research demonstrated very high transferability of
this scoring system. For more information, please refer to the article,
"Flow Cytometric Enumeration of Micronucleuated Reticulocytes: High Transferability
Among 14 Laboratories" Environmental and Molecular Mutagenesis 38:59-68 (2001).
Participants included
such companies and organizations as: Abbott, AstraZeneca (Sweden), Auckland
Cancer Society (New Zealand), Boehringer Ingelheim Pharmaceuticals, CSIRO,
Health Sciences and Nutrition (Victoria, Australia), FDA/CDER (Center
for Drug Evaluation and Research), GlaxoWellcome (UK), GlaxoWellcome (USA),
Health Canada, Janssen Pharmaceutica (Belgium), National Institute of
Hygiene (Warsaw, Poland), Phamacia & Upjohn, SmithKline Beecham (UK),
and Texas Tech University.
Is
the MicroFlow method acceptable for regulatory testing?
Yes, the FDA accepts
MicroFlow data. Additionally, the most current Organization for Economic
Co-Operation and Development (OECD) guideline regarding micronucleus testing
(Guideline 474) indicates that flow cytometry is an acceptable alternative
to manual evaluation. Furthermore, when analyses are performed using
Litron's recommended procedures, the MicroFlow process meets all of the
necessary validation criteria as described by the IWGTP (International
Workshop on Genotoxicity Test Procedures). [Note: Data from Mouse peripheral
blood is acceptable for FDA submission, while data from rat peripheral
blood should be used for screening purposes only.]
Which
species and compartments are compatible with these kits?
Kits are currently
available for both mouse and rat peripheral blood.
Are
the biological standards safe?
Absolutely. The
biological standards are non-infectious, non-hazardous denatured mouse
or rat blood. They are safe and to-date have been transported to locations
throughout the world.
When asked this same
question, officials from the National Institute of Allergy and Infectious
Diseases (at NIH) state that in their professional opinions methanol fixed
blood with parasitized red blood cells is non-infectious. Methanol
is one of many standard fixatives used in preserving blood cells. Methanol
is a poison to all living cellular organisms. It is designed to
kill all cells but preserve them in a state which microscopically resembles
their live state. One scientist went even further, saying that rodent
malaria is not a hazard for humans even following intravenous injection
of live parasites. Since this malaria-infected blood is methanol fixed,
there is no danger to those receiving or working with it.
What
are the temperature requirements for the kit components?
The anticoagulant
must be stored at 4 °C until use. Once aliquoted, the fixed samples must
be maintained at -75 °C to -85 °C. It is imperative that the fixative
and fixed samples do not warm up or be exposed to room temperature conditions
for more than a minute. Increases in temperature will result in cellular
aggregation, a condition that makes the samples unsuitable for flow cytometric
analysis.
What
dosing protocol should be used?
The MicroFlow Kit
is appropriate to use with either single or multiple dosing protocols.
For a more complete dosing discussion, see Organization for Economic Co-Operation
and Development (OECD) guideline 474 (Mammalian Erythrocyte Micronucleus
Test).
How
should blood samples be collected?
The tail vein is
a common site for obtaining peripheral blood. However, the MicroFlow
process may also be used effectively with blood samples obtained from
other sites.
How
much blood should be collected from each animal?
To collect 60-120
µl of blood, 350 µl of anticoagulant must be used. Note: Too much
blood in proportion to anticoagulant compromises the flow cytometric analysis.
Can
blood samples be fixed on dry ice?
Although not recommended,
samples may be fixed on dry ice if care is taken to avoid carbonation
of the fixative reagent (the MicroFlow manuals go into this subject in
great detail).
Can
I substitute my anticoagulant for the one provided in the kit?
Substitutions such
as this are NOT recommended - please use only the anticoagulant provided
with the kit. Litron Laboratories has evaluated many anticoagulants,
and the one provided in the kit is the only one found to perform consistently
and reliably with the MicroFlow process.
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