Frequently
Asked Questions About MicroFlowBASIC Kits
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. |
What
is a MicroFlowBASIC Kit?
MicroFlow kits are
used to significantly improve the speed and accuracy of scoring cells
within a routine safety evaluation known as the Micronucleus Test. These
kits have been specifically created to address the difficulties of time,
data, and subjectivity associated when using traditional microscopic methods
of scoring.
This
patented flow cytometric process is now available in kit format, allowing
researchers to score samples for a complete micronucleus test in just one day, versus the three to six weeks it normally
takes using traditional microscopic methods. In ultra-competitive environments
such as drug discovery, this time savings can translate into enormous
revenue for the company that beats its competition to market.
The MicroFlowBASIC 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. With these kits, researchers collect and fix blood
samples, then ship them on dry ice to Litron Laboratories for analysis.
There is normally a one-week turn around time from the point when samples
are received and the final report is sent (verbal results may be received
sooner).
This automated system
also allows researchers to perform the Micronucleus Test earlier in the
screening or discovery process. Since this test must be completed 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 the MicroFlowBASIC kit is particularly well suited for the pharmaceutical industry, it offers
equally significant benefits to:
- Biotechnology companies
- Universities and Academic
institutions
- Government institutes
& laboratories
- Chemical &
toner industries
- Medical/Dental
device manufacturers
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 each 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.
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?
Only 60-120 µl of
blood is needed. This is collected into 350 µl of anticoagulant. Note:
Too much blood in proportion to anticoagulant can compromise 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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