[Cytometry] forward-side scatter differences

Howard Shapiro hms at shapirolab.com
Fri Jul 17 10:18:50 EDT 2009

Rudolf Braun wrote:
> FSC-SSC in the analysis of white blood cells, spleen cells or lymph node cells looks different if analyzed by instruments from different vendors.
> My question: how different can they be?
> Do all instruments distinguish granulocytes, monocytes, and lymphocytes from each other using linear settings for FSC and SSC?
All instruments I know of differentiate granulocytes, monocytes, and 
lymphocytes using linear FSC/SSC settings. Side scatter measurements 
don't vary much from instrument to instrument; forward scatter 
measurements do because the geometries used in different systems differ 
considerably. See Practical Flow Cytometry, 4th Edition (hereafter 
PFC4)*, pp. 275-6 and Figure 7-1; FSC signals from various sizes of 
polystyrene beads increase monitonically with size when measured on a 
Beckman Coulter EPICS XL but not when measured on a BD FACSCalibur.

Also bear in mind that a "lymphocyte" gate as defined using FSC/SSC - 
which is used to identify and count lymphocytes in some commercial 
hematology counters - is likely to include some cells which, by the more 
stringent criterion of high CD45 expression, are not lymphocytes (see 
PFC4, pp. 34-5 and Fig. 1-17). That's one reason current CDC and NIAID 
Guidelines for CD4 T cell counting advocate CD45/SSC gating.


*Now that Beckman Coulter has been magnanimous enough to put a free, 
downloadable, printable, searchable .pdf file of PFC4 online at 
http://coulterflow.com/bciflow/research01.php - from which I even get 
some royalties - I see little reason why anybody doing flow cytometry 
shouldn't have a copy accessible. I'm still trying to get BC and Wiley 
to agree to my providing a much nicer copy with more color illustrations 
and some corrections.

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