[Overton Subtraction]

W. Roy Overton overton at gcatinc.com
Thu Jun 21 10:10:25 EDT 2007


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
   Modified histogram subtraction technique for analysis of flow cytometry
   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,

W. Roy Overton, Ph.D.
GCAT, Inc.
Tel: 856-478-0019		
Fax: 856-478-9531
overton at 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!
Christine Dahl
Skejby University Hospital
Dep. of Pediatrics

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