Comp beads and negative populations

Mario Roederer roederer at
Wed Nov 30 18:14:06 EST 2005

I should hope that the "school of thought" that the negative 
population should have the same autofluorescent properties as the 
sample is immediately shut down; it should lose any accreditation it 
may have had!

Your positive and negative (bright and dull, whatever) populations 
must be matched for autofluorescence (to each other).  Otherwise, you 
will not get an appropriate measure of the spectral overlap.  This is 
thoroughly discussed on my web site 
(; see specifically

So: use unstained capture beads as your universal negative (at least, 
for the positive comp samples for beads).  If you mix in some cell 
comp controls (like for EMA or PI), then make sure to have positive 
and negatives for that control that are matched (i.e., unstained 
cells and stained cells).

You can mix bead controls on one channel and cell controls on another 
without problem.


At 6:10 PM -0500 11/29/05, Rick Dunham wrote:
>Hello list,
>We recently started using Ig capture beads for compensation and are 
>in a debate over whether to use unstained cells or unstained beads 
>as the negative population in automatic compensation with DiVa or
>FlowJo.  One 'school of thought' is that the positive and negative 
>populations should have the same autofluorescent properties, thus 
>dictating that one should use unstained beads as the universal
>negative population.  The other 'school of thought' is that the 
>negative population should have the same autofluorescent properties 
>as the sample, dictating that one should use unstained cells as the 
>universal negative population. Both arguments seem to have merit, 
>though looking at previous discussions of autofluorescence and
>compensation on this list and in BD and FlowJo literature, 
>autofluorescense is thought to have little effect on compensation.
>However, one can imagine a scenario from the 'old days' of manual 
>compensation when for many, the goal was equalization of the median 
>of the positive and negative populations for a given stained channel 
>vs the other unstained channels, in which different autofluorescent 
>properties of the experimental sample vs the compensation sample 
>would generate over or under compensation, if the mean
>autofluorescence of one channel of the negative events were 
>significantly different with respect to another channel.
>This is where we are in this discussion.  Does anyone out there in 
>the ether have a solution or any comments?  What do you do when 
>using beads?
>Rick Dunham
>Graduate Student
>Emory Vaccine Center
>Emory University
>Atlanta, GA USA
>rdunham at

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