Sorting Difficulties

Arnold Pizzey a.pizzey at ucl.ac.uk
Mon Oct 21 08:24:16 EST 1996


>To All:
>In the time course of performing a sort (1-2hrs.) my sorter is experiencing
>drifts in droplet breakoff(as observed by the camera screen on the
>sorter).When this is observed,the sort is stopped and the drop delay/droplet
>breakoff is checked by performing the ususal bead count matrix over several
>drop delays.Typically, the drop delay has in fact changed from its original.
>While I want to hear any and all responses as to what the problem may be(my
>performance or the sorter)assume for the time that there is absolutely no
>clogs,no air/fluidic leaks, and all electronics completely in fine tune.
>Thanks.
>Vincent Falco
>New England Medical Center
>vincent.falco at es.nemc.org


Greetings Vincent.

        We run a Coulter ELITE here and I have had similar problems.


 As to the cause of this instability, my bet is sheath temperature (see (3)

        Now I'm sure you do all this stuff but it might be worth setting
out the following tips for others new to the wonderful world of sorting



        1) Set up for sorting using fluorescent beads to get a 'ballpark'
value and *confirm this with the sample to be sorted.*


        2) Periodic monitoring of the sort recovery by spitting 20 or so
sorted particles onto a slide and counting them by eye -I do this every
10-15 minutes or so and find that we can maintain good sorts over the time
period of which you speak

        3) Maintain constant sheath temperature -on the ELITE I leave the
door to the sheath/waste bottles open - even in a temperature controlled
environment there may be local temperature changes around the sheath
container (on the ELITE it is proximal to the compressor assembly)


       4) Minimise sample debris/aggregates -over long periods I suspect
that the region around the sort nozzle becomes contaminated with 'gunge'

       5) (On the ELITE) - sort at reduced sheath pressure - at 9-10 p.s.i
the whole system seems to be more stable, with better recovery and purity
(I use 9 p.s.i)

       6) Strive to reduce 'spray' -by this I refer to that fine mist that
is sometimes seen around the main sort streams, I believe that this
contributes to deflection plate contamination with concomitant irregularity
in the sort trajectories as well as contamination of the sort tip -this is
a tricky one; some days it's there and some days it's not, but the main
culprits seem to be:

        i) dirty/damaged  sample insertion rod (check the tip of this under a
        stereo mike if you have one).

        ii) high crystal drive (in any event, if you find that you need
        more than about 40% drive on the ELITE with the standard tip ,
        the  crystal/flow tip assembly has probably been contaminated with
        sheath fluid at some point).

        iii) loose fitting sort electrode clip -now, I know that at several
        thousand volts the resistance of this connection  should not
        make much difference, but in practice it does seem to. -I give the
        electrode lead a turn before clipping it onto the sample insertion
        rod -this torque seems to help stability!



        I hope this is some help.


                        Arnold.





More information about the Cytometry mailing list