-----Original Message----- From: Gerhard Nebe-von-Caron [SMTP:Gerhard.Nebe-von-Caron@Unilever.com] <mailto:[SMTP:Gerhard.Nebe-von-Caron@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. Regards Gerhard -----Original Message----- From: Howard.Gale@med.va.gov <mailto:Howard.Gale@med.va.gov> [SMTP:Howard.Gale@med.va.gov] <mailto:[SMTP:Howard.Gale@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? <<ATT58278.ATT>>
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