Comparative Analysis of the Ficoll-Paque and Miltenyi autoMACS® PBMC Isolation Methods.

BACKGROUND: This study compared the isolation of peripheral blood mononuclear cells (PBMCs) using two methods. The first method was our routine Ficoll-Paque density gradient centrifugation isolation, which had been in place for many years. The second method was a new kit produced by Miltenyi on an autoMACS® Pro Separator with the new human Whole Blood PBMC Isolation Kit. The goal was to determine the potential to replace the manual Ficoll-Paque method with this new semi-automated method to improve standardization and efficiency of PBMC isolation, a pivotal step in processing whole blood cells for our biobanking purposes. METHODS: PBMCs were isolated from the peripheral blood of 10 healthy donors by routine Ficoll-Paque density gradient centrifugation and an autoMACS® Pro Separator by Miltenyi. Isolated PBMCs were analyzed by flow cytometry, and the proportions of lymphocyte and monocyte subtypes were determined. In addition, a panel of 16 kinase inhibitors was used to determine differences in drug responses using an assay that determines cell death over time. RESULTS AND CONCLUSION: Compared with Ficoll-Paque, the Miltenyi method yielded approximately 20 percent lower PBMC yield with no significant difference in cell viability and was superior when removing red blood cells. Proportions of some subpopulations (total T cells, B cells, and classical and intermediate monocytes) were altered based on the isolation method. Significant differences in drug responses were observed between the two methods. Moreover, the Miltenyi method introduced additional variability in drug sensitivity assays. These findings demonstrate that the two methods are not equivalent, which may limit the extent to which the Miltenyi method can improve standardization and reduce laboratory technician time in the isolation of PBMCs for biobanking.