Absolute Counts in Peripheral Blood

Howard Shapiro (hms@shapirolab.com)
Fri, 15 Nov 1996 08:49:23 -0500 (EST)

>We are trying to generate absolute counts for different cell types in the
>peripheral blood of mice. What are your preferred methods?
The major problem with trying to get absolute counts out of most
fluorescence flow cytometers is that they do not provide a measure of the
sample volume flowing through the apparatus. Ortho's Cytoron Absolute,
which uses precision syringe pumps for sample feed, does; you can (or could)
get an accessory for other instruments from Cytek which would add that
capability. An alternative, available from both B-D and Coulter, employs
beads at known concentration which are added to a known volume of sample;
the ratio of cell counts to bead counts provides absolute count information.

It is established that gradient separations cause differential loss of white
cell types; one would also want to avoid washing, since this could also lead
to differential cell loss.

In clinical hematology systems, sample volume is measured; samples are lysed
but not washed. I'm not sure that there's any published paper examining
diferential cell loss due to lysing agents, because that would require that
a whole-blood counting method be used to determine counts without lysing,
and I'm not aware that any large-scale study comparing whole-blood and
lysing methods has been done. Several non-lysing whole blood counting
methods exist; the oldest is that developed by Adams and Kamentsky in the
1970's using acridine orange in isotonic saline, which gives counts of
lymphocyts, monocytes, and granulocytes, but does not discriminate
eosinophils and basophils. The Block Engineering technology of the
mid-1970's used a mix of fluorescent dyes on glutaraldehyde-fixed cells to
produce a five-part differential, but originally required two- or three-beam
excitation. More recently, (mid 1980's) Leon Terstappen, Mike Loken et al,
then at B-D, described methods using combinations of dyes (Thiazole orange
and LDS751) and monoclonals to provide multipart differentials including
retic counts. Various modifications, permutations and combinations of the
above methods are workable; which one is best for you depends on which cell
types you need to count.


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