Absolute Counts in Peripheral Blood

Jeff_Harvey@bio-rad.com Jeff_Harvey at bio-rad.com
Tue Nov 19 19:48:48 EST 1996

     Just a comment to add to Howard's message: Bio-Rad's flow cytometer, 
     the BRYTE HS, also uses a microsyringe as the sample injection device 
     and can also do absolute counts.
     Jeff Harvey
     Bio-Rad Laboratories

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Subject: Absolute Counts in Peripheral Blood
Author:  Howard Shapiro <hms at shapirolab.com> at Internet
Date:    11/15/96 3:27 PM

>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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