gating on histogram overlap

Nebe-Von-Caron, G g.nebe-von-caron at
Wed Oct 11 06:09:35 EDT 2006

If you are doing multicolour you have to remember the problem of
'population widening' by the overlap which could screw your gate. Just
as a quick summary on gating in the context of controls and compensation
you might look at a flowjo presentation to appreciate the gating problem
over several colour staining.

The other thing vividly stuck to my mind is a lecture from good old Jim
Watson at Brian Shenton's flow meeting - mainly because I could
understand him whilst I struggled with a guy from Newcastle trying to
explain to me why I could not understand a word from the guy from
Glasgow, both in their most lovely accent. Jim showed us a series of
histograms to identify the % positive he had simulated from independent
distributions. You might read his book on data analysis or use our ISAC
journal to brush up on the info.

As an isac member you should have access to the full journal content.
However, I actually forgot to log in when trying to access the pdf
article above (wouldn't remember my password anyhow), so it looks that
some older material is accessible for free. I hope this is not a glitch
but journal policy, so please use it, cite it and make others aware of
this excellent resource.


> -----Original Message-----
> From: Stevan Lauriault [mailto:stevan at] 
> Sent: 05 October 2006 22:50
> To: Cytometry Mailing List
> Subject: gating on histogram overlap
> Dear All,
> When analyzing histograms of dimly fluorescent samples, and 
> comparing to their specific isotype controls on overlay, 
> there is a significantly large overlap between the two 
> populations (ex. ~10%).  Is there a universally accepted 
> method to statistically quantify the relative proportions of 
> positive vs. negative populations?
> We are unsure whether to gate the entire histogram, or just 
> the positive region when determining relative fluorescence 
> intensity values.
> Any advice would be appreciated.
> Thank you,
> Stevan Lauriault, B.Sc.
> Research Assistant
> Trauma Group
> Operational Medicine Section
> Defence R&D Canada - Toronto
> Stevan.lauriault at
> Work:  416-635-2000 ext. 2303
> Cell:  416-949-4066 
> ________________________________________________________________
> Sent via the WebMail system at

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