I agree that there's virtually no way to reliably measure these volumes you are considering. Another method perhaps to accomplish essentially the same thing would be to use counting beads of some sort (Bangs labs and Invitrogen for example carry these; BD has Trucount tubes for this purpose also.) The advantage is that using beads of a known concentration, once you add beads to a sample in known proportions (ie. 3:1 or 1:1) and count a reliable number of beads to make the count statistically more accurate, your estimate the concentration or absolute count of your cells relative ot the beads regardless of fluidic volumes, additional stuff added to the tubes , etc. This will allow you the single platform count determination that you're looking for with a much more reliable method. Wayne A. C. Harris Bone Marrow Transplant Laboratory Immunology and Flow Cytometry Assays Laboratory Winship Cancer Institute, Emory University 1701 Uppergate Drive WCI, building C, Rm#4032 Atlanta GA 30322 404-727-3086 > > > 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 > > > >Received on Tue May 13 17:38:00 2008
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