FW: Effect of Lipemia on Side-Scatter

Howard.Gale@med.va.gov Howard.Gale at med.va.gov
Tue Mar 20 08:32:21 EST 2001

-----Original Message-----
From:	Gerhard Nebe-von-Caron [SMTP:Gerhard.Nebe-von-Caron at Unilever.com]
<mailto:[SMTP:Gerhard.Nebe-von-Caron at Unilever.com]>
Sent:	Friday, March 16, 2001 7:57 AM
To:	Cytometry Mailing List
Subject:	RE: Effect of Lipemia on Side-Scatter

The little bubbles upset your side scatter to give it a huge background
signal.  Thus your PMT baseline gets up (constant photon current). Thus if
you raise the voltage you reduce the dynamic range towards saturation of
your PMT signal even further. If you were to look at peak right angle light
scatter (RALS) signals they should all be pretty similar, but your RALS area
still show some variation as it also contains the length of the signal which
is size dependent.

-----Original Message-----
From:	Howard.Gale at med.va.gov <mailto:Howard.Gale at med.va.gov>
[SMTP:Howard.Gale at med.va.gov] <mailto:[SMTP:Howard.Gale at med.va.gov]>
Sent:	Wednesday, March 14, 2001 6:02 PM
To:	Cytometry Mailing List
Subject:	Effect of Lipemia on Side-Scatter

We perform 4-color CD4 panels using BD's lysed, no-wash method on a
FACSCalibur. On heavily lipemic samples the side-scatter signal is reduced
and this lessens the resolution between white cell populations.  Replacing
the plasma with FACSFlow sheath fluid corrects this problem.
We can also correct this problem and avoid centrifugation by raising the
side-scatter gains and LOWERING the side-scatter PMT voltage. Raising the
voltage above its usual setting lowers the white cell signals.	This seems
paradoxical. It is most noticeable in the granulocyte population where even
the proper shape requires a lower voltage.
Eventually lowering the side-scatter voltage does result in a decreasing
signal just as in non-lipemic samples. Can anyone tell me why the white cell
side-scatter signal has a different response to PMT voltage changes in the
presence of lipemia?

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