Re: Aria Sort Yield Question

Dear Matt,

I'd two years ago the same problem that you've. They've been arguing 
that:

1.- I don't run the Aria properly
2.- My costumers doesn't count correctly the cells
3.- Cells are stick to the tube walls
4.- Cells were "stored" the valve that pump the sample on the flow cell
5.- We loose cells on the filtering process
6.- Viability
7.- ...

After some meetings, discussions and hours of hard work, (finally!) 
they recognised that could be a problem ON the FACSAria. Where? As 
you've, we had (and wee still have samples with a high purity = 
99.9%), so machine was the machine was reading everything? so it 
should go to the waste. So we've collected the stream meanwhile we 
were sorting. Clean. We've checked if the sheath goes out of the flow 
cell. Nothing. We've replace the valve. Nothing. BUT, we have changed 
the DAQ Channel 1 and automatically we moved from a 30% of recovery to 
80-90% with the same purity, (which is approximately the same that 
you'll get in another sorters form other companies). I’m curious if 
they have checked that DAQ in another FACSAria and see if the recovery 
has also fallen down. 

There is an easy experiment to check your recovery: accudrops. In 
theory, when you deflect you've to get more than 95% deflected to the 
left square. If that is correct, if you check the amount of beads that 
you run through the lasers and see the amount of beads that you sort, 
in theory should be 95% or more... If that's correct, if you run your 
collection tube with your "sorted accudrop beads", the amount of beads 
that you read should be 95% the original one (less some of them stick 
to the tubes, some conflict aborts,  etc.)... So, probably you'll have 
80-90% of what you expect... Two years ago I'd 30%, 40%, in some cases 
60%. Now I've 80-95%, I'm a very happy BD costumer and I've an easy 
live...  

In summary, my personal suggestion is: ask your engineer to replace 
your DAQ channel for a new one. If it doesn't work, he can always put 
back the old one... 

I hope this can help you. 

Good luck and kind regards,

Alfonso

P.S. if this works, can you let me know? Thanks


------------------------------
 Dr. Alfonso Blanco Fernández

 Flow Cytometry Core Facilities
 UCD - Conway Institute of Biomolecular & Biomedical Research
 University College Dublin
 Belfield, Dublin 4
 IRELAND

 T: 00353(0)1 716 6836/6947

----- Original Message -----
From: Matt Wikstrom <mattw@ichr.uwa.edu.au>
Date: Thursday, February 22, 2007 11:01 pm
Subject: Aria Sort Yield Question
To: cyto-inbox

> Dear Flowers
> 
> We've been using an Aria for about four months and we're happy 
> with it.
> We've done a whole lot of testing to work out how to sort with 
> high purity
> and viability (>97%) and very accurate sort counting. We've now 
> moved on to
> work out how to get as many of the target cells out of the 
> starting sample
> (ie sort yield) while maintaining an acceptable level of purity.
> 
> And this is where our problem lies, because we think we should be 
> able to do
> a lot better. At the moment, we are only recovering about 30-50% 
> of the
> target population. What I mean when I say this, is we take the 
> startingnumber of cells and apply the frequency of the population 
> we're sorting to
> work our theoretical yield. Once we've finished the sort, we take 
> the final
> cell count for the sorted cells and compare that to the 
> theoretical yield
> (and that's where we our figure of 30-50%). This is a big issue 
> one for us
> because most of the sorts we do start with a limited number of cells.
> 
> Can other Aria users tell us what kind of results they get when 
> doing a
> high-speed sort (70 psi sheath pressure, 87k ddf)? We aren't 
> having any
> problems recovering the cells from the sort tubes, because these 
> cell counts
> agree very well with the sort counters on the Aria. We've 
> experimented a
> little with sample rates, but we seem to get the same results at 
> low speeds
> (2000 events/sec) or high speeds (15,000 events/sec), even when 
> the target
> population is around 30% of the start population. Perhaps we could 
> changethe window extension (we're using 2.00 at the moment) or 
> tweak the sort
> masks (we're using the purity setting at the moment) to get closer 
> to a 70%
> yield?
> 
> Any insights will be greatly appreciated.
> 
> Matt
> 
> ___________________________________
> Dr Matthew Wikstrom
> Senior Research Officer
> Division of Cell Biology
> Telethon Institute for Child Health Research
> 100 Roberts Road, Subiaco
> Western Australia, 6008 AUSTRALIA
> 
> Ph 61 8 9489 7777, Fax 61 8 9489 7700
> email mattw@ichr.uwa.edu.au
> __________________________________
> 
> 
>