[Overton Subtraction]

Nebe-Von-Caron, G g.nebe-von-caron at unipath.com
Fri Jun 22 15:33:32 EDT 2007

Taking the opportunity to state that the article is freely available at

Apart from the isotype control issue which we better do not touch, is
there a problem with log data where sometimes considerable amounts of
data are stuffed in channel 1, particularly when data are compensated. I
would guess they also escape the subtraction.



> -----Original Message-----
> From: W. Roy Overton [mailto:overton at gcatinc.com] 
> Sent: 21 June 2007 15:10
> To: Cytometry Mailing List
> Subject: RE: [Overton Subtraction]
> Christine,
> The 1% positive gating method works well when the positive 
> cells are brightly stained, giving a good separation between 
> the positives and negatives.	In those samples, the 1% method 
> and Overton Subtraction will
> give the same results.	However, if the sample has dim 
> positives, the 1%
> method becomes less reliable and less meaningful, because 
> there may be positive cells that are dimmer than the 1% 
> cutoff.  The Overton Subtraction method finds the maximum 
> difference between the test and control samples and defines 
> that difference as the percentage positive.  Standard 
> histogram subtraction does the same thing, except that it can 
> give false positives due to random channel to channel 
> variations in the data, which are eliminated by Overton 
> Subtraction.	Note that if the sample has both dim and bright 
> positive cells and you only want to know the percentage of 
> bright cells, all of these methods may give you the wrong 
> answer.  In those cases, cytometry becomes an art and you 
> must select the appropriate analysis region manually.
> A more detailed explanation can be found in the original 
> publication of the
> method:
>    Modified histogram subtraction technique for analysis of 
> flow cytometry data
>    Cytometry, Volume 9, Issue 6, Date: November 1988, Pages: 619-626
> If you don't have access to that ancient issue of Cytometry, 
> I think I still have a few dusty reprints left.
> Best regards,
> Roy
> ****************************************************************
> W. Roy Overton, Ph.D.
> GCAT, Inc.
> Tel: 856-478-0019		
> Fax: 856-478-9531
> overton at gcatinc.com
> http://www.gcatinc.com/
> **************************************************************** 
> -----Original Message-----
> From: Christine Dahl [mailto:CHRISTINE.DAHL at KI.AU.DK] 
> Sent: Tuesday, June 19, 2007 5:27 AM
> To: cyto-inbox
> Subject: [Overton Subtraction]
> Dear All!
> I need help in explaning and understanding the difference 
> between 1% positive gating and Overton subtraction of 
> histograms. Thank you in advance and for alle the good 
> answers we can find here! Sincerley Christine Dahl Skejby 
> University Hospital Dep. of Pediatrics Denmark

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