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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