Purdue Cytometry Mailing List: RE: BD bead array software and FC500s -
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Hi Steve, I have never run the BD CBA kit on our FC500 because during
testing I tried to run some FC500 LMD files through the software -
unfortunately the ...
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BD bead array software and FC500s

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From: Novo, David <david.novo@denovosoftware.com>
Date: Fri Aug 17 2007 - 18:52:53 EDT

Hello Kate,

The FC 500 data files are indeed a complicated beast that give you a
lot
of information. There are indeed two separate data sets inside the one
FC500 file. This is allowed by the FCS standard.

The first data set is a FCS 2.0 file. This is quite a typical FCS 2.0
file, stored in 10 bit with the data already saved compensated and
converted to log (if necessary). This is quite clever to put it there
because any analysis software that can read FCS 2.0 files should be
able
to read this file, and not even know that there was a second data set
hiding afterwards. I am not sure why the BD CBA software would not be
able to handle this, maybe there is a hidden setting or something that
allows this.

The second data set is a high resolution data set that is in FCS 3.0
format. This contains all the high resolution linear data, that is
stored uncompensated, and is ideal for performing software
compensation.

I guess our web site is either too high, or too low, and fell out of
the
range of your previous search :-) FCS Express can read both the FCS
2.0
and FCS 3.0 portion of the FCS 500 data files. You can even export
them
as individual files if you like. When loading the files you can either
default to load the FCS 2.0 or FCS 3.0 portion, or be prompted which
to
choose.

I also know that WinList can read both portions of the FC500 file. I
am
not familiar with FlowJo's capabilities in this regard. If you contact
them, I am sure they will be glad to answer this point.

-Dave

-----------------------------------

David Novo

President

De Novo Software

david.novo@denovosoftware.com

________________________________

From: Katherine Pilkington
[mailto:katherine.pilkington@imvs.sa.gov.au]
Sent: Thursday, August 16, 2007 3:16 PM
To: cyto-inbox
Subject: RE: BD bead array software and FC500s

Hi Steve,

I have never run the BD CBA kit on our FC500 because during testing I
tried to run some FC500 LMD files through the software - unfortunately
the BD CBA software can only handle FCS2 files, but even if it could
handle FCS3 files it still wouldn't be able to read the FC500 files as
they are a sort of pseudo FCS2/3.  I have been trying to address this
issue for some time (with both BD and BC) - perhaps Coulter could
think
about including an FCS2 export function in their next version of the
software to make these files more compatible with the standard...?  It
would certainly make life a lot easier if users could run their
samples
through our FC500 instead of having to use our FACSAria!  I have also
hunted high and low for a piece of software that could extract the
FCS2
portion of the file out of the FC500 LMDs, but so far no luck - if
anyone knows of such a program I'd LOVE to hear about it, as I am
sure,
would others.

Cheers,

Kate

         (o o)
---oOO--(_)--OOo----

Katherine Pilkington

Flow Cytometrist and Cell Sorter

Detmold Family Imaging Suite

Institute of Medical and Veterinary Science

Frome Road, Adelaide South Australia 5000

Ph: +61 8 8222 3322

Received on Mon Aug 20 17:58:00 2007

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BD bead array software and FC500s
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From: Stephen Taylor - Porton Down <Stephen.Taylor@hpa.org.uk>
Date: Thu Aug 16 2007 - 07:01:09 EDT
Hi everyone,

I'd like to ask if anyone has any experience using the BD cytokine
bead
array software using LMD files created on a coulter FC500?

One of our users would like to use the BD Th1/Th2 cytokine kit and
we're
wondering if we need to be aware of anything in particular.

Firstly, did it work?! And if so, are there any changes we need to
make
to our settings?

Thanks in advance for any advice

Cheers!

Steve
Received on Thu Aug 16 14:58:00 2007
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RE: BD bead array software and FC500s
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From: Katherine Pilkington <katherine.pilkington@imvs.sa.gov.au>
Date: Thu Aug 16 2007 - 18:15:45 EDT
Hi Steve,

I have never run the BD CBA kit on our FC500 because during testing I
tried
to run some FC500 LMD files through the software - unfortunately the
BD CBA
software can only handle FCS2 files, but even if it could handle FCS3
files
it still wouldn't be able to read the FC500 files as they are a sort
of
pseudo FCS2/3.    I have been trying to address this issue for some time
(with
both BD and BC) - perhaps Coulter could think about including an FCS2
export
function in their next version of the software to make these files
more
compatible with the standard.?    It would certainly make life a lot
easier if
users could run their samples through our FC500 instead of having to
use our
FACSAria!  I have also hunted high and low for a piece of software
that
could extract the FCS2 portion of the file out of the FC500 LMDs, but
so far
no luck - if anyone knows of such a program I'd LOVE to hear about it,
as I
am sure, would others.

Cheers,

Kate

         (o o)
---oOO--(_)--OOo----

Katherine Pilkington

Flow Cytometrist and Cell Sorter

Detmold Family Imaging Suite

Institute of Medical and Veterinary Science

Frome Road, Adelaide South Australia 5000

Ph: +61 8 8222 3322

 _____

From: Stephen Taylor - Porton Down [mailto:Stephen.Taylor@hpa.org.uk]
Sent: Thursday, 16 August 2007 8:31 PM
To: cyto-inbox
Subject: BD bead array software and FC500s

Hi everyone,

I'd like to ask if anyone has any experience using the BD cytokine
bead
array software using LMD files created on a coulter FC500?

One of our users would like to use the BD Th1/Th2 cytokine kit and
we're
wondering if we need to be aware of anything in particular.

Firstly, did it work?! And if so, are there any changes we need to
make to
our settings?

Thanks in advance for any advice

Cheers!

Steve
Received on Fri Aug 17 16:58:00 2007
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Re: BD bead array software and FC500s
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From: WEHICytometry <facs_copy@wehi.EDU.AU>
Date: Sun Aug 19 2007 - 20:08:10 EDT
Katherine,

I'd be surprised if your CBA software issue is due to the unrequited
need for standard FCS2.0 input.  Unless they have changed recently,
Beckman-Coulter .LMD files start out with a perfectly valid FCS2.0
data set.  I can't see that the CBA software would object to there
being other extraneous content following.  The problem is more
likely
a conflict between Becton Dickinson standard and Beckman-Coulter
standard where either some required BD keywords are missing or the
CBA software can't handle the .LMD byte ordering (even though it is
valid FCS2.0).    The only other possible problem I can imagine with
the FCS2.0 standard data set itself is the use of the $NEXTDATA
keyword which, in .LMD files, points to the FCS3.0 data set at the
back end of the file.  I think it's a long shot that it could solve
your problem but you could try ablating the $NEXTDATA key value
using
our De-Identifier program (DeID), downloadable from http://
www.wehi.edu.au/cytometry/freesoftware/ .

If you really wanted to extract the FCS components, you could do
that
with a text editor (Wordpad or TextWrangler).  Search for "FCS3.0".
If you delete everything preceding that you have a valid FCS3.0
file.  If you delete "FCS3.0" and everything following, you have a
valid FCS2.0 file.  In the latter case the file would have some
Beckman-Coulter stuff at the end, pointed to by the "@Acquisition
Protocol Offset" keyword, that most software should just ignore.

Frank Battye.

On 17/08/2007, at 8:15 AM, Katherine Pilkington wrote:

    |     |  << The Cytometry Laboratory
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------!!<<<<<< 1G Royal Parade, Parkville
     /!!\ <<<< Victoria 3050, Australia
    o !! \  << ph: 61_3_9345 2540, fax: 61_3_9347 0852
Received on Mon Aug 20 17:38:00 2007
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RE: BD bead array software and FC500s
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From: Novo, David <david.novo@denovosoftware.com>
Date: Fri Aug 17 2007 - 18:52:53 EDT
Hello Kate,

The FC 500 data files are indeed a complicated beast that give you a
lot
of information. There are indeed two separate data sets inside the one
FC500 file. This is allowed by the FCS standard.

The first data set is a FCS 2.0 file. This is quite a typical FCS 2.0
file, stored in 10 bit with the data already saved compensated and
converted to log (if necessary). This is quite clever to put it there
because any analysis software that can read FCS 2.0 files should be
able
to read this file, and not even know that there was a second data set
hiding afterwards. I am not sure why the BD CBA software would not be
able to handle this, maybe there is a hidden setting or something that
allows this.

The second data set is a high resolution data set that is in FCS 3.0
format. This contains all the high resolution linear data, that is
stored uncompensated, and is ideal for performing software
compensation.

I guess our web site is either too high, or too low, and fell out of
the
range of your previous search :-) FCS Express can read both the FCS
2.0
and FCS 3.0 portion of the FCS 500 data files. You can even export
them
as individual files if you like. When loading the files you can either
default to load the FCS 2.0 or FCS 3.0 portion, or be prompted which
to
choose.

I also know that WinList can read both portions of the FC500 file. I
am
not familiar with FlowJo's capabilities in this regard. If you contact
them, I am sure they will be glad to answer this point.

-Dave

-----------------------------------

David Novo

President

De Novo Software

david.novo@denovosoftware.com

________________________________

From: Katherine Pilkington
[mailto:katherine.pilkington@imvs.sa.gov.au]
Sent: Thursday, August 16, 2007 3:16 PM
To: cyto-inbox
Subject: RE: BD bead array software and FC500s

Hi Steve,

I have never run the BD CBA kit on our FC500 because during testing I
tried to run some FC500 LMD files through the software - unfortunately
the BD CBA software can only handle FCS2 files, but even if it could
handle FCS3 files it still wouldn't be able to read the FC500 files as
they are a sort of pseudo FCS2/3.  I have been trying to address this
issue for some time (with both BD and BC) - perhaps Coulter could
think
about including an FCS2 export function in their next version of the
software to make these files more compatible with the standard...?  It
would certainly make life a lot easier if users could run their
samples
through our FC500 instead of having to use our FACSAria!  I have also
hunted high and low for a piece of software that could extract the
FCS2
portion of the file out of the FC500 LMDs, but so far no luck - if
anyone knows of such a program I'd LOVE to hear about it, as I am
sure,
would others.

Cheers,

Kate

         (o o)
---oOO--(_)--OOo----

Katherine Pilkington

Flow Cytometrist and Cell Sorter

Detmold Family Imaging Suite

Institute of Medical and Veterinary Science

Frome Road, Adelaide South Australia 5000

Ph: +61 8 8222 3322
Received on Mon Aug 20 17:58:00 2007
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"Fl1 drift over time"
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From: Katrina Ann Walsh <kawalsh@unimelb.edu.au>
Date: Tue May 08 2007 - 22:15:35 EDT

Dear Flowers,

We have encountered a perplexing problem with our flow cytometer, a
FC500.
We have noticed that the fluorescence intensity of back ground
controls, such as isotype controls or unlabelled cells appear to
increase rapidly over time, in the order of doubling every 20 seconds.
This is quite troubling when setting negative background, especially
when positive staining is a shoulder rather than a distinct
population.
We notice that it occurs on fl1 and subsequently fl2, however it is
not obvious on fl4. We are told our 488 laser is behaving correctly.
We have tested epithelial cell line, lymphocytes and cyto-comp
(Beckman Coulter) lymphocytes and find large cells, but NOT small
beads, exhibit this "fl1 drift" over time.
We have also tested buffer ph and resuspending cells in isoton. We
find that MFI fluctuates when the same samples are re-read and that
this problem occurs most often in the evening, but not always.
Often we do not seem to have a problem at all.    The flow cytometer room
is air-conditioned  reasonably well and would not get over 27 during
the summer.
What could be the problem? All suggestions welcome.

Thank you

Katrina Walsh
School of Dental Science
University of Melbourne.
email: kawalsh@unimelb.edu.au
Received on Wed May 9 13:38:00 2007
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Antwort: "Fl1 drift over time"
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From: <Hans-Georg.Kreysch@merck.de>
Date: Thu May 10 2007 - 06:38:05 EDT
Hi Katrina,
we have observed such effects when dyes like acridine orange were used
on
our FACS for other experiments. Minimal residual amounts of dye
sticking to
the tubes were sufficient to stain cells during their way in the
sample
tube. Cleaning the sample lines with bleach etc. was helpful in our
case.
Kind regards
Georg

Hans-Georg Kreysch
P R&D / Tech / CADS
Location: A46 - 108
Phone: +49(0)6151 72 7323
Fax:    +49(0)6151 72 917323
Email:    hans-georg.kreysch@merck.de

Merck KGaA
Frankfurter Str. 250
D 64293 Darmstadt
Home:    www.merck.de

Merck KGaA
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Kley
(Vorsitzender), Michael Becker, Bernd Reckmann, Elmar Schnee, Walter
W.
Zywottek - Vorsitzender des Aufsichtsrats: Wilhelm Simson

        Katrina Ann Walsh
        <kawalsh@unimelb.
        edu.au>                            An
                    Cytometry Mailing List
        09.05.2007 04:15        <cytometry@flowcyt.cyto.purdue.edu
                    >
                                    Kopie
                                    Thema
                    "Fl1 drift over time"

Dear Flowers,

We have encountered a perplexing problem with our flow cytometer, a
FC500.
We have noticed that the fluorescence intensity of back ground
controls,
such as isotype controls or unlabelled cells appear to increase
rapidly
over time, in the order of doubling every 20 seconds. This is quite
troubling when setting negative background, especially when positive
staining is a shoulder rather than a distinct population.
We notice that it occurs on fl1 and subsequently fl2, however it is
not
obvious on fl4. We are told our 488 laser is behaving correctly.
We have tested epithelial cell line, lymphocytes and cyto-comp
(Beckman
Coulter) lymphocytes and find large cells, but NOT small beads,
exhibit
this "fl1 drift" over time.
We have also tested buffer ph and resuspending cells in isoton. We
find
that MFI fluctuates when the same samples are re-read and that this
problem
occurs most often in the evening, but not always.
Often we do not seem to have a problem at all.    The flow cytometer room
is
air-conditioned  reasonably well and would not get over 27 during the
summer.
What could be the problem? All suggestions welcome.

Thank you

Katrina Walsh
School of Dental Science
University of Melbourne.
email: kawalsh@unimelb.edu.au

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Received on Thu May 10 14:18:00 2007
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Re: "Fl1 drift over time"
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From: Ray Hicks <rh208@cam.ac.uk>
Date: Thu May 10 2007 - 02:04:18 EDT
Hi - could it be that one of your users is inadverdently loading the
machine with a dye solution that lingers and then stains up subsequent
samples?  I'm not sure that that would explain "fluctuations", but it
may explain why you have a ramping increase in intensity of negative
controls, and the fact that beads aren't affected, and it's more
likely to happen in the afternoon or at least after the machine has
been used.

On our FACSCalibur we find that residual PI from cell-cycle
experiments can linger for quite some time and leap out on
unsuspecting fixed samples for days to come - to the extent that we've
banned use of PI from that machine so that cytokine experiments don't
get ruined so often.    In our case it was only fixed cells that were
affected of course, or dead cells in a non-fixed prep, and the
increases were in fl2 and fl3 (blue excited orange and red) because PI
was the dye being used at high concentration, I'm not sure what dye
might cause a rise in FL1 and FL2 - maybe a rhodamine or fluorescein
derivative? Depending on how contaminated the machine was with PI,
users would see their negatives ramping while trying to set up with
high levels of PI, but the staining would only be obvious on re-
analysed cells at low concentrations.

Hope this helps

Ray

 ----- Original Message -----
 From: Katrina Ann Walsh
 To: Cytometry Mailing List
 Sent: Wednesday, May 09, 2007 3:15 AM
 Subject: "Fl1 drift over time"

 Dear Flowers,

 We have encountered a perplexing problem with our flow cytometer, a
FC500.
 We have noticed that the fluorescence intensity of back ground
controls, such as isotype controls or unlabelled cells appear to
increase rapidly over time, in the order of doubling every 20 seconds.
This is quite troubling when setting negative background, especially
when positive staining is a shoulder rather than a distinct
population.
 We notice that it occurs on fl1 and subsequently fl2, however it is
not obvious on fl4. We are told our 488 laser is behaving correctly.
 We have tested epithelial cell line, lymphocytes and cyto-comp
(Beckman Coulter) lymphocytes and find large cells, but NOT small
beads, exhibit this "fl1 drift" over time.
 We have also tested buffer ph and resuspending cells in isoton. We
find that MFI fluctuates when the same samples are re-read and that
this problem occurs most often in the evening, but not always.
 Often we do not seem to have a problem at all.  The flow cytometer
room is air-conditioned  reasonably well and would not get over 27
during the summer.
 What could be the problem? All suggestions welcome.

 Thank you

 Katrina Walsh
 School of Dental Science
 University of Melbourne.
 email: kawalsh@unimelb.edu.au
Received on Thu May 10 14:38:00 2007
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RE: "Fl1 drift over time"
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From: Kimberly A. Pacella <kimberly.pacella@healthnetworklabs.com>
Date: Wed May 09 2007 - 15:14:40 EDT
Hi Katrina,

I would be interested in hearing your replies to this problem.

We also have an FC500 and have noticed a very high shift in some
samples. We tend to see FL2 (although we have also seen FL1 and once
FL4) shifted out at least a log to a log and a half. It does not
happen
on our unstained sample tube (which we routinely run on every sample
due
to this issue) and does not happen on the isotype control. Sometimes
when we let the sample sit and repeat it the problem disappears and
sometimes it does not. When it happens, it happens for every tube in
that patient's panel but not for every patient either that day or even
on that carousel. We have been in contact with Beckman coulter several
times and have not yet figured this problem out. It does seem to
happen
more in the afternoon when the instrument has been running for quite a
long time. I know that Beckman will tell you that it is temperature
stable, but I think there is some effect.

Thanks,

<mailto:kimberly.pacella@healthnetworklabs.com>

Kimberly A. Pacella, MT(ASCP)QCYM

Manager, Immunology & Flow Cytometry

Health Network Laboratories

ph: (610)402-5593

e-mail:kimberly.pacella@healthnetworklabs.com

________________________________

From: Katrina Ann Walsh [mailto:kawalsh@unimelb.edu.au]
Sent: Tuesday, May 08, 2007 10:16 PM
To: cyto-inbox
Subject: "Fl1 drift over time"

Dear Flowers,

We have encountered a perplexing problem with our flow cytometer, a
FC500.

We have noticed that the fluorescence intensity of back ground
controls,
such as isotype controls or unlabelled cells appear to increase
rapidly
over time, in the order of doubling every 20 seconds. This is quite
troubling when setting negative background, especially when positive
staining is a shoulder rather than a distinct population.

We notice that it occurs on fl1 and subsequently fl2, however it is
not
obvious on fl4. We are told our 488 laser is behaving correctly.

We have tested epithelial cell line, lymphocytes and cyto-comp
(Beckman
Coulter) lymphocytes and find large cells, but NOT small beads,
exhibit
this "fl1 drift" over time.

We have also tested buffer ph and resuspending cells in isoton. We
find
that MFI fluctuates when the same samples are re-read and that this
problem occurs most often in the evening, but not always.

Often we do not seem to have a problem at all.    The flow cytometer room
is air-conditioned  reasonably well and would not get over 27 during
the
summer.

What could be the problem? All suggestions welcome.

Thank you

Katrina Walsh

School of Dental Science

University of Melbourne.

email: kawalsh@unimelb.edu.au

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RE: "Fl1 drift over time"
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From: Phil Marder <pm1@philmarder.com>
Date: Fri May 11 2007 - 08:29:20 EDT
I would first look at fluidics.

A clog in the lines would slow up the sample flow, leaving your cells
(or beads) in the beam and detectors for a longer period and allow for
larger integration of emitted light (FC-500 measures integrated
signals) over time.
Run your sample using a 1-2 minute time stop and check the data
acquisition rate over that period.  Does the data acquisition rate
slow during that time?  If so, you have a partial clog and that is
causing your upward drift of FL1 signal.

Phil Marder

-------- Original Message --------
Subject: "Fl1 drift over time"
From: Katrina Ann Walsh <kawalsh@unimelb.edu.au>
Date: Tue, May 08, 2007 10:15 pm
To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu>

Dear Flowers,

We have encountered a perplexing problem with our flow cytometer, a
FC500.
We have noticed that the fluorescence intensity of back ground
controls, such as isotype controls or unlabelled cells appear to
increase rapidly over time, in the order of doubling every 20 seconds.
This is quite troubling when setting negative background, especially
when positive staining is a shoulder rather than a distinct
population.
We notice that it occurs on fl1 and subsequently fl2, however it is
not obvious on fl4. We are told our 488 laser is behaving correctly.
We have tested epithelial cell line, lymphocytes and cyto-comp
(Beckman Coulter) lymphocytes and find large cells, but NOT small
beads, exhibit this "fl1 drift" over time.
We have also tested buffer ph and resuspending cells in isoton. We
find that MFI fluctuates when the same samples are re-read and that
this problem occurs most often in the evening, but not always.
Often we do not seem to have a problem at all.  The flow cytometer
room is air-conditioned  reasonably well and would not get over 27
during the summer.
What could be the problem? All suggestions welcome.

Thank you

Katrina Walsh
School of Dental Science
University of Melbourne.
email: kawalsh@unimelb.edu.au
Received on Fri May 11 13:38:00 2007
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From: Katrina Ann Walsh <kawalsh@unimelb.edu.au>
Date: Tue May 08 2007 - 22:15:35 EDT

Dear Flowers,

We have encountered a perplexing problem with our flow cytometer, a
FC500.
We have noticed that the fluorescence intensity of back ground
controls, such as isotype controls or unlabelled cells appear to
increase rapidly over time, in the order of doubling every 20 seconds.
This is quite troubling when setting negative background, especially
when positive staining is a shoulder rather than a distinct
population.
We notice that it occurs on fl1 and subsequently fl2, however it is
not obvious on fl4. We are told our 488 laser is behaving correctly.
We have tested epithelial cell line, lymphocytes and cyto-comp
(Beckman Coulter) lymphocytes and find large cells, but NOT small
beads, exhibit this "fl1 drift" over time.
We have also tested buffer ph and resuspending cells in isoton. We
find that MFI fluctuates when the same samples are re-read and that
this problem occurs most often in the evening, but not always.
Often we do not seem to have a problem at all.    The flow cytometer room
is air-conditioned  reasonably well and would not get over 27 during
the summer.
What could be the problem? All suggestions welcome.

Thank you

Katrina Walsh
School of Dental Science
University of Melbourne.
email: kawalsh@unimelb.edu.au
Received on Wed May 9 13:38:00 2007
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over time""
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Antwort: "Fl1 drift over time"
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From: <Hans-Georg.Kreysch@merck.de>
Date: Thu May 10 2007 - 06:38:05 EDT
Hi Katrina,
we have observed such effects when dyes like acridine orange were used
on
our FACS for other experiments. Minimal residual amounts of dye
sticking to
the tubes were sufficient to stain cells during their way in the
sample
tube. Cleaning the sample lines with bleach etc. was helpful in our
case.
Kind regards
Georg

Hans-Georg Kreysch
P R&D / Tech / CADS
Location: A46 - 108
Phone: +49(0)6151 72 7323
Fax:    +49(0)6151 72 917323
Email:    hans-georg.kreysch@merck.de

Merck KGaA
Frankfurter Str. 250
D 64293 Darmstadt
Home:    www.merck.de

Merck KGaA
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6164 -
Sitz der Gesellschaft: Darmstadt
Geschäftsleitung und persönlich haftende Gesellschafter: Karl-Ludwig
Kley
(Vorsitzender), Michael Becker, Bernd Reckmann, Elmar Schnee, Walter
W.
Zywottek - Vorsitzender des Aufsichtsrats: Wilhelm Simson

        Katrina Ann Walsh
        <kawalsh@unimelb.
        edu.au>                            An
                    Cytometry Mailing List
        09.05.2007 04:15        <cytometry@flowcyt.cyto.purdue.edu
                    >
                                    Kopie
                                    Thema
                    "Fl1 drift over time"

Dear Flowers,

We have encountered a perplexing problem with our flow cytometer, a
FC500.
We have noticed that the fluorescence intensity of back ground
controls,
such as isotype controls or unlabelled cells appear to increase
rapidly
over time, in the order of doubling every 20 seconds. This is quite
troubling when setting negative background, especially when positive
staining is a shoulder rather than a distinct population.
We notice that it occurs on fl1 and subsequently fl2, however it is
not
obvious on fl4. We are told our 488 laser is behaving correctly.
We have tested epithelial cell line, lymphocytes and cyto-comp
(Beckman
Coulter) lymphocytes and find large cells, but NOT small beads,
exhibit
this "fl1 drift" over time.
We have also tested buffer ph and resuspending cells in isoton. We
find
that MFI fluctuates when the same samples are re-read and that this
problem
occurs most often in the evening, but not always.
Often we do not seem to have a problem at all.    The flow cytometer room
is
air-conditioned  reasonably well and would not get over 27 during the
summer.
What could be the problem? All suggestions welcome.

Thank you

Katrina Walsh
School of Dental Science
University of Melbourne.
email: kawalsh@unimelb.edu.au

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Received on Thu May 10 14:18:00 2007
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Fw: "Fl1 drift over time"
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From: <marie-ange.e.watson@GSK.COM>
Date: Fri May 11 2007 - 03:04:56 EDT
Hi Katrina,

we had quite a drift problem on our FC500. We have the ability to read
96
well plates and if reading in rows, could see a marked shift in
signal
between the columns. In our case it was due to temperature. We now
have an
additional fan fitted to the back of the machine to cool the machine
down.
Since then we have not seen the problem anymore. Could this be the
same in
your case?

regards,

Marie-Ange Watson
GlaxoSmithKline
Stevenage
United Kingdom

----- Forwarded by Marie-Ange E Watson/PharmRD/GSK on 11/05/2007
08:01
-----

"Kimberly A. Pacella" <kimberly.pacella@healthnetworklabs.com>
09-May-2007 20:14

To
"Cytometry Mailing List" <cytometry@flowcyt.cyto.purdue.edu>
cc

Subject
RE: "Fl1 drift over time"

Hi Katrina,
I would be interested in hearing your replies to this problem.
We also have an FC500 and have noticed a very high shift in some
samples.
We tend to see FL2 (although we have also seen FL1 and once FL4)
shifted
out at least a log to a log and a half. It does not happen on our
unstained sample tube (which we routinely run on every sample due to
this
issue) and does not happen on the isotype control. Sometimes when we
let
the sample sit and repeat it the problem disappears and sometimes it
does
not. When it happens, it happens for every tube in that patient?s
panel
but not for every patient either that day or even on that carousel.
We
have been in contact with Beckman coulter several times and have not
yet
figured this problem out. It does seem to happen more in the
afternoon
when the instrument has been running for quite a long time. I know
that
Beckman will tell you that it is temperature stable, but I think there
is
some effect.
Thanks,

Kimberly A. Pacella, MT(ASCP)QCYM
Manager, Immunology & Flow Cytometry
Health Network Laboratories
ph: (610)402-5593
e-mail:kimberly.pacella@healthnetworklabs.com

From: Katrina Ann Walsh [mailto:kawalsh@unimelb.edu.au]
Sent: Tuesday, May 08, 2007 10:16 PM
To: cyto-inbox
Subject: "Fl1 drift over time"

Dear Flowers,

We have encountered a perplexing problem with our flow cytometer, a
FC500.

We have noticed that the fluorescence intensity of back ground
controls,
such as isotype controls or unlabelled cells appear to increase
rapidly
over time, in the order of doubling every 20 seconds. This is quite
troubling when setting negative background, especially when positive
staining is a shoulder rather than a distinct population.
We notice that it occurs on fl1 and subsequently fl2, however it is
not
obvious on fl4. We are told our 488 laser is behaving correctly.
We have tested epithelial cell line, lymphocytes and cyto-comp
(Beckman
Coulter) lymphocytes and find large cells, but NOT small beads,
exhibit
this "fl1 drift" over time.
We have also tested buffer ph and resuspending cells in isoton. We
find
that MFI fluctuates when the same samples are re-read and that this
problem occurs most often in the evening, but not always.
Often we do not seem to have a problem at all.    The flow cytometer room
is
air-conditioned  reasonably well and would not get over 27 during the
summer.
What could be the problem? All suggestions welcome.

Thank you

Katrina Walsh
School of Dental Science
University of Melbourne.
email: kawalsh@unimelb.edu.au
This message (including any attachments) is intended only for the use
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Received on Fri May 11 14:18:00 2007
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