Calibrating Across Continents

Zucker.Robert@epamail.epa.gov Zucker.Robert at epamail.epa.gov
Tue Jul 5 12:20:11 EST 2005


Hi Norm
ISAC has a new standards initiative to address instrument performance
and quality of flow cytometry data. We hope to increase levels of
performance of confocal microscopes and flow cytometers and other
imaging equipment. We are addressing this issue of proper flow cytometry
QA at a local flow meeting in North Carolina on July 19 2005( see
attachment)

I believe the first calibration procedure should be a check of the
alignment and fluidics of the system with alignments beads. This test is
more sensitive than using the position of multi-intensity beads in a Log
display. It will usually indicate the status of the PMT's, fluidics, and
alignment of the system. Although this test is not routinely done by
many laboratories that measure samples on a flow cytometer, I personally
believe that checking the alignment/fluidics of the system with
alignment beads is the first test that should be done on any flow
cytometer prior to running samples or the companies internal checks for
pass/fail performance. It allows you to understand the real status of
the machine with an unbiased test based on the simple evaluation of CV
of each channel.
Best wishes
Bob

(See attached file: 07-19-05.doc)
Robert M. Zucker, PhD
U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory
Reproductive Toxicology Division, MD 72
Research Triangle Park, North Carolina, 27711
Tel: 919-541-1585; fax 919-541-4017
e-mail: zucker.robert at epa.gov



	     "Jones, Norman						
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								Subject 
				      Calibrating Across Continents	




Hello Cytometrists,
I'm involved in some studies of T-cell activation comparing a population
in the US with
one in Africa. My hope is to be able to analyze both data sets (one
collected on a
Calibur in the US, the other collected on a Calibur in Uganda) using
identical gates.
After optimizing the staining (Ab titrations etc.) on our Calibur in the
US, I ran some
Spherotech Rainbow beads to establish median fluorescence target values
for each channel.
We then ran Rainbow beads on the Calibur in Uganda and adjusted the PMT
voltages so the
median fluorescence matched the target values from our Calibur in the
US. Both cytometers
are calibrated in this way before each acquisition. All the data is
acquired
uncompensated and compensation is done during analysis using FlowJo.
Initially things worked well, but lately the voltages required to get
the Rainbow beads
into the target channels on the Calibur in Uganda are very  low, and
consequently, if we
acquire the samples using those settings the negative peaks are squashed
against the
axes. The Calibur in Uganda was recently moved, but the field service
engineer gave it a
clean bill of health in it's new location.
Was this approach to calibration too simplistic? Perhaps there are other
factors that
need to be taken into consideration (ie, log amplification?). Any ideas
on what might be
going wrong and what we can do to remedy them will be greatly
appreciated.
Regards,
Norm

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