Re: Simple cell count

Sergei Ochkur wrote:
>
> Can we count absolute cell number/concentration by simply dividing 
> total cell count (or number of events collected) by the volume passed 
> through the flow cytometer?
>
> The volume here is:
> (volume of counted cell suspension) = (volume in the tube before flow) 
> - (volume in the tube after flow) - (volume of the dead space the flow 
> Cytometer lines between the intake and the measuring chamber)
>
> If yes, how can we find/measure that dead space volume?
>
With difficulty. And there are some other problems; you can't estimate 
the volume in the tube all that accurately, even if the tube is 
graduated, and the overall strategy assumes that there is never any 
sheath blown back into the tube during sample handling, and also that 
there is no instrument dead time, which may apply to some all-digital 
systems but does not to older flow cytometers.

A method for getting reasionably accurate counts from cytometers with stable flow rates
was described in:
Bergeron M, Lustyik G, Phaneuf S, Ding T, Nicholson JK, Janossy G, Shapiro H, Barnett D,
Mandy F. 
Stability of currently used cytometers facilitates the identification of pipetting errors
and their volumetric operation:
 "time" can tell all. Cytometry B Clin Cytom. 2003; 52:37-9 (free download online). 

The real question is "how good is good enough"? Hematology instruments, which are flow
cytometers, have been
designed to yield accurate and precise counts; manufacturers have, for the most part, not
provided the same capability in
flow cytometers intended for other purposes, including clinical immunophenotyping.

-Howard