PBMC isolation from 24hr old patient blood - (Apr/28/2011 )

Hi, I need help.

In my lab, we have to isolate PBMC from 24hr old patient blood, then do Ab staining and FACS afterwards.
Routine procedure: 1000xg, 10min to spin down the whole blood(8-9ml), get most of the plasma to other team, then resuspend the blood with DPBS, total 14 ml. load to 2 Accuspin columns(7ml each), 800xg,15mins, no brake. The PBMCs isolated usually were heavily contaminated with Neutrophil, and not much monocyte enrichment.However,the same method always works with fresh blood and 24hr old blood from healthy subjects.
What should I do? Is it really due to the sick blood?

Fresh blood really is better, and it could depend on what they're sick with.