Re: Calculation of Absolute Count

Is there any studies on the relationship between a size (diameter) of  
the bead and CV ?

Aki


On Mar 1, 2007, at 2:50 PM, Nebe-Von-Caron, G wrote:

> The correct counting formula when using reference beads for  
> enumeration is shown below.
>
> Sample concentration = {[ref volume] x [sample predilution] x  
> [sample count]x [ref concentration ml^-1]}/{ [sample volume] x [ref  
> predilution] x [ref count] }
>
> it does not matter if you dilute your sample in 1 or 50ml as this  
> only alters the flow rate of the sample, but not the ratios of  
> absolute counts.
>
>
> If you add equal volumes of reference beads and sample and nothing  
> is prediluted the formula simplifies to
>
> Sample concentration ml^-1= {[sample count]x [ref concentration  
> ml^-1]}/{[ref count]}
>
>
> The Trucount tube delivers a fixed number of beads trapped in a  
> type of sugar matrix. If you add Xml of sample, your fixed number  
> of beads are therefore diluted in Xml (or else) so you only need to  
> ratio:
>
> Sample concentration ml^-1= [sample count]x [total ref bead  
> number]}/{[ref count] x [Xml]}
>
>
> A similar simplification can be made if your bead volume is much  
> smaller than the sample volume.  If 10ul beads are added to 1ml of  
> sample your total volume is 1010ul which is a 1% volume error in  
> the calculation. Similarly the beads in the true count bead  
> introduce a small volume error which can be corrected for by	
> altering the "reference bead figure" so you don't have to do it in  
> the calculation
>
> There was some discussion with regards to the errors of those  
> methods several years ago on the list. The CV's of all pipetting  
> steps are additive (assume 1% per pipetting step, hoping they are  
> well maintained, fitting tips and good pipetting practice) and the  
> counting errors are also additive (square root of count over	
> count). So if you count 8000 beads but only 100 cells of interest  
> your theoretical counting CV is 11% (1% in the 5000 and 10% in the  
> 100). Other problems come from bead dispersion (we correct for  
> aggregates up to 6 in our aggregation studies) as well as loss of  
> beads or adhesion to cells, tube walls ..
>
>
> Whilst in theory all counting methods should count the same, there  
> seems to be that well known discrepancy between theory and practice.
>
> http://www.affordcd4.com/pdf/Affref07_BB.PDF
>
> good luck
>
>
> Gerhard
>
>
>
> In the case of the
>
>
>
> -----Original Message-----
> From: donny thomas [mailto:donny@reametrix.com]
> Sent: 28 February 2007 13:14
> To: Cytometry Mailing List
> Subject: Re: Calculation of Absolute Count
>
> Sunny
>
> We use the second method you tried, and it works fine with us.
>
> Abslute count = No: of gated cells/No: of gated beads X Total bead  
> count per test(tube)/test volume
>
> Did you try the above mentioned formulae with the Coulter beads? Do  
> you know the no: of beads you use in one test. ?(eg: 50,000 beads  
> per 50uL of blood)
>
> BD FACScount tubes come with presice bead counts per tube.
>
> Donny
>
> On 2/27/07, Xiao Qun Zhai <xqzhai@uclan.ac.uk > wrote:
> Dear All,
>
> I use Absolute Count beads from Beckman Coulter - Product No. 7547053.
>
> On the data sheet, the formula for Absolute Count (cells/uL) is:
>
> Total Number of Cells Counted
> -------------------------------------    x *Beads Concentration (which
> is known from the datasheet).
> Total Number of Beads counted
>
> Beckman Coulter's technician said I don't need to divide this  
> result by
> dilution factor. If so, I would think the *Beads concentration here
> should be the bead conc. in the fianl FACS tube not in the original
> bottle.
>
> However, BD's specilist gave me another formula (here we have BD
> FACSAria), which is
>
> Absolute count = No. of cells in Gate 	     & nbsp;  Bead  
> count (on
> packet)
>	       ;	  -------------------------&nb sp;	 X
> -------------------------------
>	  &n bsp;	       & nbsp; No. of  beads in  
> gate		    & nbsp; Volume of
> FACS tube sample
>
> Which one is right?  I used both of them but neither of the result is
> accordance with my  haemacytometer result.
>
> I am confused.
>
> Thanks for help.
>
> Sunny
>
>