[Cytometry] .fcs standardization/flow accreditation

thomas delohery thomas.delohery at verizon.net
Tue May 17 21:38:10 EDT 2011

okay you guys, 

In recent months I've been working with bronchoalveolar lavage (BAL) samples and the autofluorescence of the various immunocytes   in these samples can extend over 4-decades; i.e., unstained lymphocytes in the first decade and macrophages overlapping the 3rd and 4th decades along with some other interesting populations in between.  Once you add fluorescent markers to the mix it is virtually impossible to get all the populations on a 4+ log scale.  HLA/DR is particularly problematic, esp. when you are attempting to identify subpopulations of dendritic cells other APCs as well as activated T cells.  Running the samples twice after changing PMT voltages, redoing compensation, etc., is something I really want to avoid as it makes the analysis less incredibly difficult, not to mention the analysis.  I think I need more logs.  
Also, how about a 1-log scale for 90' side scatter?  that would be very useful.

now be nice, TomD

On May 9, 2011, at 11:15 PM, Howard Shapiro wrote:

> Mario wrote, in response to Bill Eades:
>> I do completely agree with you on the issue of dynamic range.  Every vendor is striving to "one-up" the next.  Claims of "7 logs of dynamic range" or whatever ... these are hogwash.  I don't care if the electronics IS capable of more than 5 logs of dynamic range (so please, vendor salespeople, do not email me claiming that your cytometer is better than others for this reason).  Biologically, 4-5 decades is the maximum useful range.  Most detectors have this as a useful range.  Just because your instrument goes to 11 doesn't make it better than one which goes to 10.  And... incidentally... let's remember that all the numbers on our axes, all the MFI values .... the magnitude of these numbers is entirely meaningless.  Doesn't matter.  No information.  (Please, don't email me telling me that your company's cytometer's values are relevant.  They're not.)
> Mario is absolutely right; although one can make electronics that will respond over a six- or seven-decade dynamic range, the biology with which we typically deal is what limits the effective dynamic range of our measurements. If the top end of a four-decade log scale represents 1,000,000 MESF or 1,000,000 antibody binding sites, the bottom end represents 100 MESF or 100 antibody binding sites. Although modern flow cytometers may be capable of detecting 100 MESF when measuring a single label that is the only one excited in a given beam, most multilabel staining protocols generate backgrounds of at least several thousand MESF, and relatively few of the antigens measured in most experiments are abundant enough to bind 1,000,000 molecules of antibody. It doesn't seem that we should keep having to make this point, but we apparently do. And to think that I once expected multiparameter flow results would be routinely reported in terms of molecules per cell...
> -Howard
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