Re: Calibrating Across Continents

Dear Russ
Howard Shapiro	has stated in Practical Flow Cytometry that a 51 micron
particle blocks a 50 micron hole.  It is the basic premise of flow
cytometry. As long as we run flow cytometers,  they will have fluidic
problems that need to be addressed.  You have alluded to one such
problem with the low water pressure effecting the system . Blockage can
also occur during the day. There are a number if other factors	that can
influence  your data. These primarily include the fluidics but can also
include alignment, PMT condition, lasers, electronics and temperature.

I disagree with you that flow cytometers are always stable for the whole
day if the water level is high enough. How are you checking this?  What
is your standard that you are using to make this statement. Is it your
biological sample or the manufacturer's test protocol .  I maintain that
you should be using a procedure that deals with alignment beads,  flow
rates, and CV's. If you know how to interpret this data you may be
really surprised on the condition of your machine,   Data acquired on
machines that are not working correctly may yield bad data that may
result in  bad conclusions. Regulatory agencies want good data.. There
is a reason why we have some regulations and standards. They are
necessary.

I believe that many machines in the field are being run an non optimum
levels due to alignment, bad  PMTs and fluidic problems. This should be
checked and corrected with proper protocols. These issues will be
discussed by an ISAC standards workgroup and at a local flow cytometry
meeting on July 19 in North Carolina at Duke University.
Best wishes
Bob


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@epa.gov



	     anjguy39							
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				      Robert Zucker/RTP/USEPA/US@EPA	
	     07/06/2005 05:21					     cc 
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								Subject 
				      Re: Calibrating Across Continents 




With all do respect, I find all this QC that is done is only to pass
regularatory compliance and nothing to do with the overall instrument
functionality.	There is very little drift of an instrument over a
week.  With the instruments that I have, if the sheath tank is not full
the QC never works, once you fill the tank the QC is right on target,
So what if you pass QC in the morning and the rest of the day the
aligment is off because the shealth tank is low on fluids.  Please note
that instrument manufactures state as part of the operating procedure to

make sure the sheath tank is full before doing Quality Control.  The
reason for this is guarantee that you will pass QC when you do the
initial QC.

Russ

Zucker.Robert@epamail.epa.gov wrote:

>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@epa.gov
>
>
>
>	  "Jones, Norman
>	  (DHS)"
>	  <NJones@dhs.ca.g			  To
>	  ov>		   Cytometry Mailing List
>		       <cytometry@flowcyt.cyto.purdue.ed
>	  07/03/2005 06:12	   u>
>	  AM				  cc
>				 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
>
>
>
>------------------------------------------------------------------------

>
>This attachment - '07-19-05.doc' -  23.04 KBytes - can be viewed at
>http://www.cyto.purdue.edu/MD-parts/6c0192d32471180679e7bfa4385a1da6a694fe3e.doc

>
>
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