Data Analysis: An old problem revisited

Tom Mc Closkey thomasm at
Thu Feb 13 19:39:03 EST 1997

--- On Wed, 12 Feb 1997 11:15:51 +1100 (EST)  Geoffrey Osborne 
<Geoff.Osborne at> wrote:

>Dear All,
>I'm wondering if any of you can give me some advice regarding a problem a
>user presented me with yesterday. Essentially they have been running a
>series of repeat experiments over a number of months and they want to pool
>all the data. The problem lies in which method of presentation of the pool
>data gives the truest reflect of the the biology of what was occurring on a
>particular day, (given that the negative control can move a little from day
>to day and with aslight voltage change). 
>Here is my example.
>Samples on day 1 for example, were run with the negative control median
>positioned on 5 on the relative fluorescence intensity (rfi) scale 0-10000,
>and the positive sample median falls on 305 rfi.
>Day 2, control negative 9.7, positive 437 rfi. 
>Day 3, control negative 14.7, positive 604 rfi
>What the user wants to do is express the *relative* change in fluorescence,
>so they were dividing the median of the sample by the median of the 
>yielding 61, 45, & 40 respectively. 
>I suggested subtracting the control median from the sample median for each
>day, thus 300, 427.3, 589.1.

>I'd really appreciate your thoughts on this, as I honestly don't know what
>is the "right" answer. 

I think the only accurate way to compare such data collected over months is 
to have standardized the fluorescence of a known quantity [beads] to a 
particular channel number for every experiment.  This procedure eliminates 
variation over time from laser power, alignment, PMT, deterioration of the 
optics, etc.  

Of your two suggested methods, I prefer subtraction.  I think of the test 
sample's fluorescence as a combinaiton of autofluor., background labelling 
with the fluorochrome, and true labelling with the fluorochrome.  To 
eliminate the contribution of the first two, you need to subtract out your 
control value.

Good luck, 

Thomas W. Mc Closkey, Ph. D.
Director, Flow Cytometry
North Shore University Hospital
Biomedical Research Center
350 Community Drive
Manhasset, Long Island, New York 11030
ph: 516-562-4844 [office]; 516-562-1135/4641 [lab]
2/13/97   4:50:32 PM
E-mail: thomasm at 

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