Day to day variability.

Marty Bigos mbigos at gladstone.ucsf.edu
Wed Nov 16 17:30:56 EST 2005


Enid-

It is unreasonable to expect that setting a constant voltage on a 
flow cytometer	should guarantee the exact same overall  measurement 
gain on an instrument. Variability due to environmental factors, 
cleanliness of the instrument, etc. will affect this.

In order to maintain consistent measurements a reasonable approach 
would be to run the same standards (Calibrite FITC beads in your 
case) and adjust the PMT voltage to  put the designated peak at the 
same target.

As long as you run the beads at the same flow rate, tracking both the 
PMT voltage and CV of the bead over time will give a good estimate of 
instrument performance. The gain of the type of PMTs used in the 
Caliburs follows a power law - roughly voltage to the 7.6 power. Thus 
the ratio of you Day 1 voltage to your Day X voltage raised to the 
7.6 power will give a "sensitivity" value that can be tracked over 
time. Also the CVs of the beads can be tracked over time; the square 
of their ratio will be inversely proportional to the effective ratio 
of their signals. Although there are no agreed-upon metrics, a 
consistent decrease of 25% in the "sensitivity" by either the PMT or 
CV measurements would justify a service call.

Marty
Marty Bigos
Director, Flow Cytometry Core Laboratory
Gladstone Institute of Virology and Immunology

Mail:
1650 Owens Street
San Francisco CA 94158
(tel) 415-734-4821
(fax) 415-355-0855
(mobile) 415-845-8450

mbigos at gladstone.ucsf.edu
http://www.gladstone.ucsf.edu
http://www.gladstone.ucsf.edu/gladstone/php/?sitename=flowcytometry


>Question:  We are running Calibrite FITC beads on a daily basis just to see
>the day to day variability on our FACSCalibur. The FL1 voltage was set on
>the first bead run and has not been changed since.  The median values for
>fluorescence intensity are the following:
>Day 1: 865.96
>Day 2: 1000
>Day 3: 964.66
>We need to eliminate or reduce, as much as possible, this day to day
>variability.  Any suggestions?
>
>Enid Keyser


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