[Cytometry] 5 color compensation issue

Joe_Trotter@bd.com Joe_Trotter at bd.com
Tue Feb 3 17:12:44 EST 2009

Hi David,

        As the applet author I should add here that the viewer just uses 
"typical" example tandem spectral data (typical spectral data that I could 
locate, rather than either best or worst case for each), and that some 
lots of PE-Cy5.5 may have very little donor (PE) signal while others may 
have more. As Mario said, lot-to-lot variation matters and the PE 
contributions are usually going to be similar for PE-Cy5.5 and PE-Cy5 
using well chosen lots. The PE-Cy5.5 example file in the viewer just 
happens to have significant donor signal - and some lots do while others 
may behave reasonably well in certain panel contexts. Remember that 
compensation is also gain dependent, so it can often be challenging to 
diagnose exactly what the original compensation problem was without more 
information regarding specific tandem reagent properties and the cytometer 
setup & configuration. 


Joseph Trotter
Director, Cytometry / Advanced Technology Group

BD Biosciences
tel: (858) 812-8946  cell: (858) 449-7338  fax: (858) 812-8979
10975 Torreyana Road, San Diego, CA 92121-1106 USA 
E-mail: Joe_Trotter at bd.com   Website: www.bd.com

"Haviland, David L" <David.L.Haviland at uth.tmc.edu> 
Sent by: cytometry-bounces at lists.purdue.edu
02/03/2009 12:28 PM

"Mario Roederer" <roederer at drmr.com>, <cytometry at lists.purdue.edu>

Re: [Cytometry] 5 color compensation issue

Well, I'm not here to start an arugment but I only know from my own
experience which mirrors what I see when comparing PE, PE-Cy5.5, and
PE-Cy5 on the BD spectral viewer.   In closing everything down on the
spectral viewer except the emission profiles of those three, it would
appear that PE-Cy5.5 has a very strong emission, virtually covering the
PE emission making the two very difficult to compensate.  Per the
spectral viewer, PE-Cy5.5 has two emission peaks, one about 550 and the
other short of 700nm.  PE-Cy5, however, only has maybe a 5% emission in
PE making those two workable. 

I don't know if it will come over by I've "snipped" and included the
figure from the BD viewer showing the emission profiles of the three.
I don't know about others but I have the standard filter configurations
in my LSR and Aria (both 10 color) and my user called me over when they
couldn't comp PE and PE-Cy5.5 against each other.   When I had
difficulty, we both went to the viewer to double check after taking the
respective comps to 900+ without resolution.   Said user has since
changed the flours they use. 

Understand that my not "allowing" the pair in the room was pretty much
in jest as well. Seeing the overlap in PE and PE-Cy5.5 in the PE channel
on the spectral viewer pretty much convinced my user to find another
color on their own.   Many users get Cy5 and Cy5.5 confused. 

Won't argue about PE-Cy5/Cy5.5 and APC.   I've dealt with them as well
but at least the combinations were "workable". 


David L. Haviland, Ph.D.
Assistant Professor, Immunology
Institute of Molecular Medicine, R637I
Univ. of Texas, Houston - HSC
1825 Pressler
Houston, TX  77030
713-500-2413 - Voice
713-500-2424 - FAX
-----Original Message-----
From: Mario Roederer [mailto:roederer at drmr.com] 
Sent: Tuesday, February 03, 2009 10:56 AM
To: cytometry at lists.purdue.edu
Cc: Zhugong Liu; Haviland, David L
Subject: Re: [Cytometry] 5 color compensation issue

Actually, PE-Cy5.5 has roughly the same emission overlap into PE as 
does PE-Cy5 (although of course this will be lot-to-lot dependent and 
manufacturer dependent).  PE-Cy5.5 is generally better because it has 
much less compensation into the APC channel.

I'm not sure why you state that it "can't be compensated."  It 
certainly can be compensated, just as easily and "correctly" as PE- 
Cy5; just make sure to use the same reagent in your compensation tube 
as in your experiment.

As a side note, it is important to note that while fluorescence 
emission spectra give us some idea about compensation requirements, 
they are NOT very good -- this is because the FACS uses different 
amplification (gain) settings in different regions of the spectrum, 
where as a fluorescence spectrum collected from a spectrofluorometer 
will have essentially the same gain setting throughout the spectrum.

I hope that you can go back to your user and correct the 
misinformation -- and start "allowing" PE-Cy5.5 into your flow room 
again, as in many cases it is far superior to PE-Cy5.  (Incidentally, 
PerCP-Cy5.5 is even better than PE-Cy5.5 -- having less compensation 
requirement overall).


On Feb 3, 2009, at 10:23 AM, Haviland, David L wrote:

> Yes.   If you compare the emission profile of PE-Cy5.5 to PE you are 
> going to find overlapping emission spectra that can't be 
> compensated.   A better choice for your user would have been PE- 
> Cy5.   I had this once myself and kindly asked the user not to bring 
> that combination in my flow room again.   It was all made clear to 
> my user when I showed her the emmission profiles of the reagents in 
> question.
> Hope this helps...
> David
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu on behalf of Zhugong Liu
> Sent: Mon 2/2/2009 9:29 PM
> To: cytometry at lists.purdue.edu
> Subject: [Cytometry] 5 color compensation issue
> Dear all,
> We are running 5-color flow on FC500 and have some compensation 
> issues.
> The 5-color cocktail includes antibodies with the following
> fluorechromes: FITC, PE, PE-Cy5.5, PE-Cy7 and APC. Do you think there
> might be some reagent interactions in this panel combo, especially
> PE-Cy5.5 and other dyes? Our single controls were well compensated 
> using
> the software-generated matrices, however the fully stained samples 
> were
> not.
> Thanks for any comment/suggestion.
> Best,
> Jason
> -------------------------------------------------------
> Jason Liu, PhD
> Asst. Director, Flow Cytometry
> Tolerance Assays and Data Analysis
> UCSF/Immune Tolerance Network
> 3 Bethesda Metro Center, Suite 400
> Bethesda, MD 20814
> Phone: 240-235-6177
> Fax: 240-235-6198
> Email: zliu at immunetolerance.org
> _______________________________________________
> Cytometry mailing list
> Cytometry at lists.purdue.edu
> https://lists.purdue.edu/mailman/listinfo/cytometry
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