[Cytometry] Consistent difference between CST vs Manual laser delays

Jaromir Mikes jaromirmikes at yahoo.com
Wed Feb 19 14:44:46 EST 2014

Hello Chuan, 
certainly, the temperature does affect stability of any cytometer and the effect might be especially significant on sorters. Consider also the temperature of your sheath fluid. Is it tempered or do you use a fresh from fridge or freshly autoclaved in the pressure tank? Do you refill the tank with fresh sheath fluid during the day? How long do you run your Aria's stream before you execute CS&T?  I have registered even more significant differences within few hours on closed flow cytometer in permanently air-conditioned (24/7) lab. And the sorters are even more sensitive. 2 degrees do affect laser delays, however the extend depends on settings...exactly how the Ian wrote in previos email - low windows extension value increases sensitivity of the system to any changes. Of course assuming that there is no other technical problem.
What can be done?
1. Considering your data on temperatures ranging 20-24deg, your sorter lab is small or without air-conditioning. It is better to place the sorter in bigger lab - bigger air volume = higher stability. I know you can not change that in most cases. Then check and reset your air-conditioning to control temperature in narrower range and let it work permanently. 
2. Keep the tank with sheath fluid in the lab, let it temper overnight. Fill up the tank with sheath fluid and do not refill it during the day, if possible. You didn't mentioned refilling of the tank, but that can actually have some effect, especially when the temperatures of fluids differ. 
3. Do not move your tank during the day, especially do not change its elevation. Relative elevation of sorter and the sheath fluid tank should  be calibrated by technician during the instrument installation. 
4. Keep the stream on for at least 2-3 hours and execute the CS&T shortly prior to your sorts.
5. And optimize the windows extension values for your assays, especially when the size of particles significantly differ among your assays.

I am used to prepare tank with PBS a day before the sort and keep it in the lab overnight. I start up our Aria in the morning, ASAP, but with the lasers off. I do the fluidics startup and then I leave the stream on for at least 2-3 hours (sometimes, when the work in the lab is not running quite as it should, I leave it stabilize even longer >;o)) ). Meanwhile, I turn on the lasers 45-60 min prior to CS&T and I do it shortly prior to analyses/sorts. I am filling the tank to 3/4 to keep reserve for whole day. The air-conditioning is running the whole time. I also tested that the stream and the sorter itself are more stable when you place the sheath tank off the fluidics cart, but keeping it in the same elevation of course. The 20L BD sheath fluid container (the big blue-gray box) is about the right size, so you just put it size to your cart. If you also place the inline filter aside, you will eliminate the vibration from compressor. Very useful especially
 at low pressures when running the 100 or 130 um nozzle.

Hope you find something that will help you. wish you good luck and  thousands happy samples without accident.


doc. RNDr. Jaromir Mikes, PhD.
Institute of Biology and Ecology
Faculty of Science
P.J. Safarik University in Kosice
Moyzesova 11
040 01 Kosice
tel.: +421(0)55-234-1205

 From: Ian Dimmick <ian.dimmick at newcastle.ac.uk>
To: Chuan En Lam <chuanen.lam at gmail.com>; "cytometry at lists.purdue.edu" <cytometry at lists.purdue.edu> 
Sent: Wednesday, February 19, 2014 3:28 PM
Subject: Re: [Cytometry] Consistent difference between CST vs Manual laser delays

Hello Chuan, firstly I think if you check laser delay settings you should probably use the CS&T beads if they are what your laser delays have been set up on initially. 

I then would perhaps check the laser delays , and expect some minor drift , in fluorescence during the day due to temperature changes , but this will hardly ever affect the analysis of your cells, only in the scenario perhaps where you have applied a very narrow signal window extension, thus seeing a difference which is more proportional to bead/ cell size rather than anything to do with laser/ instrument drift 

Good luck 


>-----Original Message-----
>From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-
>bounces at lists.purdue.edu] On Behalf Of Chuan En Lam
>Sent: 19 February 2014 00:22
>To: cytometry at lists.purdue.edu
>Subject: [Cytometry] Consistent difference between CST vs Manual laser

>Hi all,
>Recently we seem to be seeing consistent differences between CST set laser
>delay values vs Manual laser delay values on our AriaIIu.
>Instrument setup/details:
>AriaIIu (Blue/Red/Violet laser) inside ClassI CAS hood.
>Morning cold temp: 20.0deg,
>Start up to CST: 22.0 deg
>2hrs after start up to end of day: about 24.0 deg
>Mostly, after initial fluidics startup, CST would be run. We then double
>check with manual laser delay setting using single peak beads and 8 peak
>rainbow beads.
>We found a consistent requirement to shift the laser delays by about 0.5
>compared to CST to get optimum position. This is in general consistent
>across nozzles (70um, 100um). We are not sure if temp changes has an
>effect, or was the flow cell dirty (have done flow cell cleans with
>Decon/Coulter cleanz).
>In one scenario, 70um nozzle, after flow cell clean.
>_________CST AM____Manual
>Red           -43.06            -43.50           -43.50      -43.50
>-42.86            -43.36
>Violet          40.64             40.18            39.92
>39.92         40.46             39.86
>Has others seen similar effects or ideas what this might indicate?
>Normal condition sorting (log scales etc) seems to be ok with clean
>Eric Lam
>Flow Cytometry Lab Technician
>Garvan Institute of Medical Research
>Sydney, Australia
>Ph 9295 8431
>e.lam at garvan.org.au <r.salomon at garvan.org.au>

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