Summary Document
This document is a summary of message from the Purdue University Cytometry Laboratories Cytometry Discussion list.
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Hello,
Thanks to all for your comments, they have been very helpful. I am posting a summary of the responses (without identification of course).
Regards,
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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Original question about:
FACS_Calibur Maintenence
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Responses:
Lots of questions.........
Just one answer for the cleaning: I use 50% DMSO to clean the lines. So far I have not seen any problems. Just don't store the instument with any cleaning solution then corrosive problems should not come up.
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We have three Caliburs, two purchased new, of nearly the same vintage as the one you've acquired, and one just added as a company "refurb". All are excellent instruments with good solid performance. We've had them serviced so that they run at about the same sensitivity, meaning that the instrument settings are similar for a given standard on all three instruments. This is a matter of laser alignment and is not a trivial undertaking so be certain that your field engineer fully understands your expectations. (we've had excellent luck with our engineers as long as we let them know that we were interested in more than "passing" CVs on our instruments)
1) We follow the daily instrument start-up/shut-down procedures described by the instrument manufacturer and the monthly maintenance schedule. We rack up about 1500 experimental run hours per instrument a year and they've been running without any problems so we'd recommend following BD's suggestions, they really do keep things running well.
2) We use a combination of disinfectants/detergents depending on the situation. We run 10% Bleach between runs and at the end of the day shutdown. We also have been know to run 100% Windex, 100% Coulter Clenz or 10% Contrad for stubborn clogs. We rarely use the "hot" bleach or long clean that BD recommends, we find that the other detergents seem to do a better job cleaning both a clogged nozzle and a dirty flow cell. We run the detergents when we notice a) clogging, b) broadened CV's of bead standards c) shifting of MFI of bead standards.
3) We run Streck Cytometry Concentrate for our sheath fluid. (Streck also manufactures BD's cytometry sheath so we are confident of the quality) It is supplied as a 3x stock which is diluted with water to fill your sheath tanks, takes up far less room in the lab than the old cubetainers and has a bacteriostatic agent to prevent things growing in the system. Many folks swear by running good old DDW but we run the occasional clinical trial sample (and many other strange and wondrous things) so we stick to a product which is least likely to throw aberrant data into the discussion. (if you make your own product or use water that is the first place folks focus their attention when data is less than they expected).
4) We turn our cytometers on in the morning and off at the end of business each evening. They are left on standby in between runs, we never do the laser-cool down that you've described, they simply don't run that hot. The oldest cytometers (1998) are both on their second blue lasers and second or third red lasers (the original red diodes were highly unstable and required replacement within the first couple of years). We run the darlings everyday at least seven hours so I'd say they have excellent laser life, well beyond the manufacturer's guarantee without any miraculous interventions on our part.
5) We've never had osmotic shock problems visible, probably because we don't run anything but cytometry sheath fluid. We have seen gross contamination evidenced by tubes filling up with sheath fluid, dripping from the sip, etc. These are generally due to worn gaskets, bal seals or pump issues or sometimes by clogs in the system. We try and keep the instrument clean and generally don't have many problems, certainly nothing that has ever required an engineer in to service.
6) We run ultra-rainbow beads (Spherotech URP-30-2) as a standard on our instruments each day. We've designed the templates to track MFI and CV's for FSC, SSC, and all the fluorescent channels. (We run with standard instrument settings and monitor whether we see shifts in the means or broadening of the CV's) We find this much more helpful in monitoring instrument performance than running Calibrite beads which don't fail until there is something grossly wrong.
I hope this helps with your new acquisition. We are quite happy with our Caliburs and find them a workhorse in the laboratory.
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We have a FACSCalibur work-horse in our lab and I have to say it powers
along very nicely. I am quite strict with my users and ask that after
every person finishes their run session they pop 10% bleach on the SIP
(leave arm open aspirate approx 1ml, close arm run on high 5 minutes)
then do the same with water. We are like a small core facility and it is
not unusual to have macrophages, dendritic cells, platelet, lymphocyte
and/or tumour cell preps all on the same day (we also run plant,
seawater, acid mining lake water and bacterial samples). At present our
SIP blockage rate is down to approx once every 5-6 weeks and is usually
because someone has either not washed as requested or...not filtered a
sticky clumpy sample! Initially the Calibur was *cleaned* once a month
by placing %10 bleach in the sheath tank (make sure you bypass the
filter or the bleach degrades it) and on the SIP, run on high for 20
minutes, then repeat with water. I now clean once every 2 weeks or once
a week when the lab is reall busy (ie Calibur running 8am-6pm daily). I
have not found the bleach to be corrosive. If there is a stubborn
blockage then I may use 0.1% Triton and syringe it though the flow cell
but this occurs very rarely. I have a PM everything 4 months (BD service
contract) which includes filter/o-ring changes.
As for sheath, I used to buy osmosol from a company here in Australia
(pretty much a saline/EDTA solution), but last year after much
discussion with colleagues and a few trial runs we switched to distilled
filtered water as sheath and have seen no difference in most of our
samples. We have not had any issues with backflushing or residual
volumes so far. I have never used the BD sheath fluid although I know
others that do - bit too pricey for our lab!
As for the laser, we run a lot of lasers in our labs across a variety of
platforms. As a rule of thumb we just try and keep our lasers running
for a while and not switch on/off too quickly (ie on for a couple of
hours or off for a couple of hours), off the top of my head I don't know
how many hours we've run the laser for but I know this laser has been on
the system for at least 5 years possible 6-7 but that predates my
position so I would have to check the installation notes to confirm.
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BD FACSClean is a 5% bleach solution with some stabilisers. If you
are keeping the machine long term then using FACSClean will save you from
lots of problems in the future. We use the branded items simply because it
is easier from a regulatory point of view as we don't have to do all the
validation.
Maintenance we have a low frequency of use but high throughput (1 month on 1
month of type of thing). I find that monthly system wash as explained in the
manual and post run clean of bleach and rinse again in the manual are
enough. We rarely have a breakdown the biggest problem is dirty air intake
filter but these normally go for 6 months.
Laser life - we run morning and afternoon sessions with a good hour between
off and on. replaced one red laser in 5 years, and the machine was second
hand when we got it.
QC - run what you like it! We use FACSComp merely because it gives users a
go no-go signal.
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Hopefully you haven't had a flood of responses and I can be of some help. My lab has 2 Caliburs and this is how I treat them:
1&2)I have users run a tube of 10% bleach for a minute or so at the end of each session, then run and leave a tube of DI H20 on the machine. If there's a clog or the signal looks a little "off," I always prime the machine a few times before doing anything else. If the clog's in the sample probe (no dripping/backflushing) use a syringe with silicon tubing on the tip to blast air through the sample probe (I have one of these "declogulators" stored near every machine and let users do this themselves), then prime it a time or two to make sure there's no air bubbles. If there's a clog or a large amount of junk in the flowcell itself (you can see this by removing the cover over the flow cell and putting a tube of water or sheath fluid on the machine - if the flowcell's clean there shouldn't be much light refracted in the flowcell), clean the flowcell - I can give you directions on how to do this if you need.
3)I'm not involved with clinical trials, but I do use pre-made sheath fluid (Streck Cytometry Sheath) - I think it's available from Fisher - and have good results with it.. In response to number 5 - I recommend using saline sheath fluid with a preservative in it. It seems to keep the machines and samples "happier" and I have heard of "evil black mold" growing in machines that use H20 as sheath fluid. A standing order of sheath fluid is relatively inexpensive.
4)I've found that laser life is most effected by total hours - if there's more than about a two hour gap between users, I turn the machines off. I haven't had any signficant shortening of life due to not allowing the laser to cool off in standby.
If the signal you get from the machine changes or looks ugly, it can usualy be fixed relatively easily with a small adjustment or two - BD might be willing to give advice when this come up.
All in all I have very few problems with my Caliburs - I have users do their part for maintenance and I try to look at the machine's signal with some beads once every week or so to make sure everything's in line. Only very occasionaly does something pop up that can't be fixed quickly.
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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.
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I am managing a (small) FACS Core Facility
since 2001. We are doing only scientific work, so I don´t care about
your points 3+5.
I "force" the users to do a short cleaning routine after each series of
experiment (see attachment shut down routine). From time to time (2-3
times per year) I perform a big cleaning routine how it is claimed by
BD. So far, I had no major problems with the machine (FACSCalibur 4
color). As we also measure CBA and Flex sets, I think there is no
problem with sensitivity or resolution because especially Flex sets
require a "good" maintained machine for measurement. Of course I do the
check up with Beads (mainly Calibrite Beads from BD using FACScomp
software, but also rainbow beads from Polyscience).
I use FACSClean and FACSRinse for my cleaning procedures. But at times,
where I more time, I also setup my own bleach solution (1-5%
hydrochloride for 30-60 min, bypassing the saline filter). For sheath I
use FACSFlow (or, if I have enough time for preparing my own PBS). The
water I used for PBS is filtered and autoclaved.
Point 4: I can only tell you, what I do and what I heared: So I was
told not to switch on and shut down the laser too often a day. The
explanation was, for the laser tube it is not good to warm up and cool
down regulary. So our routine is to switch on the machine on in the
morning and leave it on till early evening (or there are more than 4-5
hours between the next experiments and it is warm, so I shut down the
machine (but not for 2-3 hours standby). I my former lab, where I did
my PhD the FACScans were running sometimes also during the night and
they had a long lifetime.
The laser of our Calibur is several minutes in standby before shut
down, because of the cleaning procedure, but I don´t know, if the laser
really require that cool down circle (On the other hand: At my
FACSVantage machine, I do exactly that, but these lasers are more
powerful and differently cooled (water versus air)).
I hope that my small insight help you a bit but I can say at least our
machine is relatively uncomplicated.
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We use a FACSCalibur for strictly research work, so I can't address
your clinical trial concerns. We use "home-made" sheath fluid (PBS +
0.02% azide, 0.2 micron filtered). The down side is we have to a) make
the sheath fluid and b) collect all fluid as hazardous waste (azide).
We keep bleach in the waste container to give Cf 10% when full. I only
run ethanol or bleach when preparing for or cleaning up after a sort
run - 70% ethanol prior to the sort, followed by sterile PBS; 10%
bleach to decontaminate afterwards, followed by extensive rinsing with
dH2O. The sheath filter must be bypassed for the bleach, but the
ethanol is run through the filter. Otherwise I count on the azide to
keep things from growing. The recommended cleaning procedures are all
in the "User's Guide" - did you get a copy along with your instrument?
We religiously use post-run flow cell cleaning routines, depending on
what has been run:
For live BSL-2 cells or live bacteria, run a tube of 10% bleach on the
flow cell with tube support arm to the side for 1 minute and then for
5' on high; follow with 5% Contrad detergent; follow with distilled
water. For fixed cells or live BSL-1 cells (primary mouse, dicty,
etc), start with the Contrad step. The wash step is required
immediately after any run with PI, even if another user is coming right
in. We've had problems with PI contamination of the flow cell in the
past, and find that immediate post-run cleaning keeps any problems of
this sort from developing.
Another note: never put/leave a sample on the SIP in Standby mode.
This is a sure way to get major backflushing of sheath fluid into the
sample tube. Other than that I haven't noticed any problems, but then
we don't use water as sheath.
