FACScalibur maintenance

rozenkov@netscape.net rozenkov at netscape.net
Wed Sep 13 03:11:33 EDT 2006


Dear Uriel,
Just with regards to your points 3 and 5, it will depend on your local regulator or the
one that controls that clinical trial (FDA in the US), but the general GLP/GCP approach
will require you to use all equipment strictly following the manufacturer's instructions,
unless formally validated otherwise. Thus, the previous owners of your FACS used the
original BD fluids, because they were specified in the machine's manual.
The same will apply to your point 5, i.e. DDW vs recommended sheath fluid (if DDW is not
mentioned as an option in the manual).
If the cytometer is just partly used for controlled studies, it still needs to be
maintained to the same standards all the time, including fluids, daily check
up/calibration, user training, etc.
We once had to switch from (free) distilled water to Isoton and adjust all procedures for
a cytometer that was used just for a few clinical CD34 tests per week, apart from other,
experimental work.
Regards.
Vladislav Rozenkov, MD, PhD
-----Original Message-----
From: utk1 at 013.net
To: cyto-inbox
Sent: Thu, 7 Sep 2006 3:05 AM
Subject: FACScalibur maintenance


Dear Friends: 
We just bought a 2nd hand FACScalibur to "bridge" until we get the money for a good, new
cytometer. I will be in charge of its maintenance and QC so I have some questions. 
1) What are your cleaning routines? There are simple daily shutdown procedures, somewhat
longer weekly treatments and monthly thorough cleans. What do you do? 
2) related to the previous is: what fluid do you use to clean? I reviewed what has been
said in the list, and the options are many! routine fluids usually include bleach but it
has been mentioned it is quite corrosive; people mentioned 1, 10 and 50%. Other people
said ethanol 50 or 70%, but said the filters have to bypassed for that, is it so? As for
clogs and thorough clean runs, someone said NaOH, other mentioned 0.1% triton, and even
1-2% SDS. Any comments? I am specially concerned about their corrosive or destructive
effects on the machine itself. What is best in your opinion/experience? 
3) Part of the work done in the machine is for a young company that is developing
procedures and analyzing samples for clinical trials. The company we bought the machine
from said that they used only original BD fluid supplies because "it is needed for
clinical (i.e. FDA approved) procedures". Those of you with experience in these settings,
is this claim for BD original fluid supplies real? All we are going to be doing is
analyzing samples. 
4) Laser life: what is the best strategy for maintaining the laser as long as possible?
This is an argon air-cooled laser. Is the main killer repeated on/off or total hours? do
operational hours count the same as stand by hours? One guy in a lab around here says
that prior to shutdown the machine should be put in "standby" for 5 min for laser "cool
down". is there any truth or logic behind this? 
5) After reviewing the comments on the list, we will be using DDW as sheath. My only
problem is with backflush and residual volumes that might do osmotic damage to the cells.
The machine is not grossly defective, but I assume subtler problems might be going on.
What is the test / procedure to detect if such sheath problems are happening? The
pressure is OK, with both hard and soft stand-by going straight to 10.23. 
We went to this company which is closing, and they had two machines, one from 1999 and
the other from 1998 (alas, both with only one laser). We took some unstained cells and
6-peak pan-fluorescing beads. The FSC-SSC profiles of the cells were somewhat different
in the machines, but I didn't notice much (the PhD that came with me said she thought the
difference was big), and the voltages needed for 1st decade localization were acceptable
(300-400's). But when putting the beads, the differences were striking! the 1999 machine
showed such good peak resolution (separated and tight peaks, including the lowest and
unstained ones) on the 3 detectors I was impressed. the FSC-SSC profile was even and
tight and was in rough relation to the cells. On the 1998 machine, on the other hand, the
lowest 3 peaks were a smear! If the 1999 machine surprised me, this one scared me. Also,
the FSC-SSC profile was somewhat distorted and the FSC detector had to be on a different
setting to detect them. I was amazed at the amount of information that can be obtained
from such simple tests! 
Thanks in advance for your comments, 
Uriel. 
Uriel Trahtemberg, M.Sc. 
MD/PhD student 
The Laboratory for Cellular and Molecular Immunology 
The Hebrew University - Hadassah Medical Organization 
Jerusalem - ISRAEL 
"We shall defend our island, whatever the cost may be, we shall fight on the beaches, we
shall fight on the landing grounds, we shall fight in the fields and in the streets, we
shall fight in the hills; we shall never surrender." 
Winston [Leonard Spencer] Churchill  
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