Doublet discrimination : Aria vs Vantage DiVa

Ian Dimmick Ian.Dimmick at newcastle.ac.uk
Sat Apr 15 11:38:15 EDT 2006


I have tested the doublet discrimination on the FACScanto , LSR II ,
FACSAria, FACSCalibur using 

1	Area v width
2	Area v height
3	ratio of width +height

These were compared with conventional area v width on the FACSCalibur
using BD QC particles, all gave exactly the same results, as already
stated doublet discrimination theory relies on your cells, since doublet
detection relies on a perfect spheroid model and cells are usually not
that ,therefore this theory  applied to the cell, and the assumption for
the doublets to be nicely stuck one to the other in a vertical aspect
when traversing the analysis point of the laser beam is not always the
case, the further you get away from this theoretical model then the more
is the probability that you will not detect doublets.

Ian

Ian Dimmick
Flow Cytometry Core Facility Manager
Institute of Human Genetics
Bioscience Centre
Central Parkway
Newcastle Upon Tyne
NE1 3BZ
UK

Ian.Dimmick at ncl.ac.uk
Tel	0044 191 2418831
Fax    0044 1912418666
(mob) 0044 7970344823

-----Original Message-----
From: Yoav Altman [mailto:yoav at burnham.org] 
Sent: 13 April 2006 20:20
To: cyto-inbox
Subject: Re: Doublet discrimination : Aria vs Vantage DiVa

I don't think there's a hard-and-fast rule about whether to use 
height vs. width on the Aria vs. Digital Vantage for doublets.	In my 
experience, both with a Digital Vantage and a FACSCanto, the 
parameters used to discriminated doublets depend more upon the 
cells than the instrument.

I start out with two dotplots, FSC-A vs. FSC-H, and SSC-A vs. SSC-W. 
Sometimes the H vs. A plot gives the best discrimination, 
sometimes the A vs. W plot is better, sometimes either one will 
do, and sometimes I use both, gating first through one, then 
further gating that singlet population through the second plot.

Try it out and see what works for you.	I think you'll find that cell 
morphology dictates your approach more than instrument choice.

Yoav Altman
Manager, High Throughput Cell Analysis
Burnham Institute for Medical Research
10901 North Torrey Pines Road
La Jolla, CA 92037


At 8:25 AM +0200 4/13/06, Roberts Joanna wrote:
>Hello there,
>Could anyone please offer me an explanation about doublet 
>discrimination in DiVa software on the Vantage versus on the Aria? 
>....inasmuch as i have believed that on the Vantage setup in Diva, one 
>uses FSC-A vs FSC-H to best acheive this while on the Aria, width is 
>used.
>Have I got the right end of the stick? Is it important?
>If I have got this right, could somone kindly explain to me 
why this is so.
>Thank-you so much,
>Joanna
>Flow Cytometry
>EPFL
>Lausanne
>Switzerland





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