RE: CD34+CD38-

HI Anne - it's not necessarily the answer in your case, but it would fit the
reported results.


The far-red sensitive PMT's fitted in BD and MoFlo machines can give large
numbers of spikes (due to stray scattered laser or thermal photons) when the
voltage is turned up high.  If the spikes coincide with a negative cell
going through the machine then it appears as a dimly positive event,
negative cells that go through out of sync with the single-photon or thermal
spikes stay negative. It may be the case that you have the PMT's turned up
for your cell sorts, then down much lower when sorting beads. Sorting these
(negative cells) then reanalyzing of course gives the same result (negative
cells, the same fraction of which go through at the time of a spike).


You can test this quite easily, turn the PMT to the same voltage used for
sorting and trigger on that parameter (you'll probably get a normal
distribution midway up the graph when triggering just above zero), or just
check the oscilloscope trace for that parameter when running blank beads
(also check the distribution of blank beads with the same PMT settings).


Hope this makes sense and helps - if not drop me a line and I'll send you
some example plots,


Cheers


Ray


Ray Hicks


Cytek Development Inc

http://www.cytekdev.com

tel: +44 (0)208 1337 968 (UK/Europe)

tel: +1 510 931 7294 (USA)

skype: ray.hicks.cytek 




From: Ann Atzberger [mailto:Atzberger@hammer.imm.ox.ac.uk] 
Sent: Monday, April 07, 2008 7:52 AM
To: cyto-inbox
Subject: CD34+CD38-


Hi everybbody,


recently we have been sorting CD34Fitc positive CD38PerCPCY5.5 negative BM
cells on a MoFlo. This population although correct in number of recovered
cells shows no enrichment whatsoever on reanalysis.

This population is part of a 4-way sort. The other 3 populations when
reanalysed have purities above 95%. (Total colours = 6)

We switched to using PE-CY5 instead of PerCP5.5. and saw the same problem.


Using exactly the same voltage and compensation settings we did a 4-way sort
with beads and got 100% purities on all populations...which rules out
malfunction of the instrument.


We reverted back to labelling the CD38 with Fitc and CD34 on PE and found
that the purities were back to normal i.e. above 90%.


Has anyone come across a similar problem with tandem conjugates? I am
reluctant to say that aCD38 cannot be used with a tandem conjugate label and
would greatly appreciate any plausible explanation for this phenomenon.


We are very much aware of the instability of tandem conjugates and the
circumstances that exacerbate this, the tandems mentioned are purchased from
Beckman Coulter - as are most of the tandems we work with and on the whole
our experience with them is good.

regards

Ann