absolute counts -Reply

Daniel Vaulot vaulot at sb-roscoff.fr
Fri Feb 28 07:54:38 EST 1997


In response to Keith Bahjat very valid comment (seee below), the key to get
accurate counts is to REMOVE the aspiration needle that is on top of the
FACScan sample needle (of course with samples that allow it, ie non
contaminating samples).  This way the counts from flow rate are extrmely
accurate provided that the sample is inserted rapidly enough so that no drop
has the time to get into it.  The minor draw abck is that water will drip
from the tube when the lower arm is in the open position.  Putting a smalll
petri dish below the needle will prevent spills.

In our hands this method is much more accurate that the bead addition technique.

Daniel Vaulot
>>This is in response to Dave Novo's request for less expensive ways to
>>measure absolute counts.
>>The cheapest way to do absolute counts is to calibrate instrument flow
>>rates at each of the flow settings you commonly use for analysis. I think
>>you'll find flow rate to be very accurate at each setting. The way I do
>>this is to measure the volume of buffer (measured by weight) aspirated
>>per unit time. Repeat this measurement w/o cells a minimum of 6-10
>>times and establish a median flow rate +/- error. Remember to subtract
>>the initial priming volume used to establish stable flow (the measurement
>>of this volume can be tricky). Put a stop count on time, I use 5 minutes to
>>get statistically valid measurements. Then repeat this procedure with
>>buffer containing cells at the cell concentration you use for analysis an
>>see if there is an effect on flow rates, there should not be much effect
>>at [cell] below 10e7 cells/mL.
>>For your experimental cell counts include fluorescent beads (cheap
>>ones) to use as a flow stability indicator and monitor their fluorescence
>>as a function of time. Any break in the fluorescence vs time plot
>>indicates a flow problem during acquisition. Remember to draw a gate
>>around the beads and subtract their count from the final total count.
>>For more details see Rehse, et al Cytometry (Communications in Clinical
>>Cytometry) 22(4): 317-322.
>>MarkRehse at CellPro.com
>Considering that the FACScan/FACSort/FACSCalibur instruments aspirate an
>unknown volume while the tube is being placed on the SIP and again as the
>tube is being taken off, is this method actually clinically accurate
>(i.e.,. comparable to a hematology analyzer)?
>I had always considered this type of measurement only feasable on a syringe
>driven instrument, such as those available from Ortho?
>Wouldn't a fluorescent bead standard be more accurate and reproducable on
>this design of instrument (albeit more expensive)?
>Keith Bahjat
>Northwestern University Medical School
>Comprehensive AIDS Center
>Flow Cytometry Laboratory
>Chicago, IL
>Kbahjat at nwu.edu
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