APC-Cy7 and PE-Cy7 compensation

Zeni Alfonso zalfonso at cytoritx.com
Wed Nov 12 18:20:59 EST 2008

Dear All, 
And how about when the sample does contain three or more cell types that present distinct
autofluorescence? That is what I frequently face with samples derived from enzymatically
treated tissues. Your advice is very welcome! 

Zeni Alfonso
zalfonso at cytoritx.com
Cytori Therapeutics

-----Original Message-----
From: Mario Roederer [mailto:Roederer at drmr.com] 
Sent: Tuesday, November 11, 2008 2:32 PM
To: cyto-inbox
Subject: Re: APC-Cy7 and PE-Cy7 compensation

No, no no no no no no!	This is not correct!

The automatic compensation by computer (be it by DiVa or other	
analysis software) is the ONLY way you can get proper compensation.   
It is IMPOSSIBLE to guarantee that manual compensation will be correct	
in any reasonable amount of time (like before the Sun burns up all of  
its fuel).

Single color controls are the ONLY proper compensation controls.  No  
additional controls need to be run with regard to compensation.   
(Other controls are necessary for proper gating, e.g., FMO controls).

Autofluorescence has no impact on the process of compensation.

The only time one needs to "manually" adjust compensation is when you  
have the wrong compensation control for your sample (for example, when	
it's not bright enough or when it's a different lot of a tandem dye).	
And, if you are doing this, do so with the recognition that all you're	
doing is getting closer to proper compensation (IF you know what  
you're doing), but you'll never get there completely.  Better is to  
repeat the experiment with the correct compensation controls.

Please refer to my (highly outdated, yet somehow still accurate) web  
site on compensation:  <http://www.drmr.com/compensation>


On Nov 11, 2008, at 5:05 AM, Oscar Fornas wrote:

> Hi Lynda,
> The phenomenon that you describe is completely normal using single  
> color
> compensation samples.
> For the polychromatic flow cytometry 2 and 3 color compensations  
> controls
> should be performed for proper color compensation.
> The automatic color comp in DiVa software can be good enough for 2  
> colors
> analysis, but using more than 4 colors this tool not always is  
> enough for a
> proper compensation and you must set it manually with additional  
> controls.
> When using several antibodies in a sample, the spreading of negative	
> cells
> for a concrete antigen can be rise since autofluorescence increases  
> giving
> high spillover in other detectors due the addition of a lot of  
> antibodies in
> the same sample.
> This means that compensation with 2 and 3 color comp controls is  
> mandatory,
> otherwise this affect to the spillover and can concludes with a  
> under or
> over compensation. Furthermore, manual color comp must be  
> performed !!!
> This phenomenon that all of us identifies and correct with  
> additional color
> compensation with 2 and 3 color controls was finally fully described	
> and
> illustrated by Mario Roederer (in my opinion) in one of the best  
> technical
> flow cytometry papers. Check it for an amazing guide of color  
> compensation.
> Reference is:
> Optimizing a multicolor immunophenotyping assay
> Clin Lab Med. 2007 Sep;27(3):469-85, v. Review
> Hope this helps.
> Good luck!!!
> Oscar
> ________________________________
> Òscar Fornas, PhD.
> Technical Director, Flow Cytometry Facility.
> DCEXS Universitat Pompeu Fabra.
> Barcelona Biomedical Research Park.
> Doctor Aiguader, 88.
> 08003 Barcelona.
> Spain
> Phone:+ 34 93 316 08 50
> Fax: + 34 93 316 09 01
> E-mail: oscar.fornas at upf.edu
> Web: http://www.upf.es/cexs/sct
> -----Mensaje original-----
> De: Guzik, Lynda [mailto:GuziLJ at ccm.upmc.edu]
> Enviado el: viernes, 07 de noviembre de 2008 22:39
> Para: Cytometry Mailing List
> Asunto: APC-Cy7 and PE-Cy7 compensation
> Hello All,
> I have a researcher who would like to do 6color flow using FITC, PE,	
> PI,
> APC, APC-Cy7, and PE-Cy7.  We are using a FACSVantage DiVa with a  
> 488nm,
> 635nm and UV lasers.	We are having problems with compensation  
> between the
> APC-Cy7 and PE-Cy7 channels.	Using single color positive controls  
> and the
> DiVa algorithm, the compensation looks fine.	However, when we put  
> APC-Cy7
> and PE-Cy7 antibodies in the same tube, the plots look like there is	
> extreme
> overcompensation of PE-C7 against the APC-Cy7 channel.	I've attached a
> couple of plots to illustrate.	I am able to correct for this, but  
> wonder if
> I should be.	And I fail to understand why the single color controls	
> look
> fine but have a problem with the dual stained tube.  Any input would	
> be
> appreciated.
> Thank you,
> Lynda Guzik
> McGowan Institute - Lagasse Labs
> Rm 520A - Bridgeside Point Bldg
> 100 Technology Dr - Suite 200
> Pittsburgh, PA 15219
> 412-235-5213
> guzilj at upmc.edu<mailto:lguzilj at upmc.edu> or
> lyg1 at pitt.edu<mailto:lyg1 at pitt.edu>

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