Presented at:
Annual Meeting of the Federation of Clinical Immunology Societies
San Francisco, CA, June 1-5, 2006
Comparison of two methods CPT and Ficoll to isolate mononuclear cells from peripheral blood: Effect on viability and function of freshly and cryopreserved PBMCs
Raddassi K, Bourcier K, Estevam J, Gisler T, Hafler DA, Seyfert-Margolis V
Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Institutes of Medicine, Boston MA; Immune Tolerance Network, University of California, San Francisco
Objective: To compare two methods for PBMC isolation from whole blood for detection of antigen specific T cells.
Background: The use of peripheral blood monocytes is a fast and minimally invasive tool to detect antigen specific cells and monitor the effects of potentially immunomodulatory drugs on these cells. Clinical trials performed by the Immune Tolerance Network employ assays that quantify T cell responses to autoantigens. Centralized assay facilities are used to reduce the problem of inter-site variability. However, some inter-site variability can occur during the preparation of PBMC samples at various geographic locations. These steps require PBMC isolation from the whole blood freezing prior to shipping,. We have therefore examined the effects of two different PBMC isolation procedures on the viability, phenotype and function of the cells the aim of this work is to find the method allowing to minimize inter-site variability of the results of T cell assays.
Methods: PBMCs from healthy donors and MS patients were isolated either by using CPT or by ficoll separation. PBMCs obtained using these two methods were compared regarding cell count, viability, phenotype and response to antigens. Parts of the samples were frozen for 3 weeks using conditions that we have optimized previously prior to stimulation in T cell assays. Proliferation ([3H]-Thymidine incorporation), cytokine production (ELISA, flow cytometry (FACS) and elispot) and cell death/apoptosis (FACS) were assayed in parallel in both fresh and frozen samples. Subpopulations of PBMCs were phenotyped by FACS analysis.
Results: Cell counts/ml of blood were 20% lower using CPT compared to ficoll isolation, by contrast, the viability was higher using CPT than ficoll method. This effect was more pronounced after freezing and thawing the PBMCs where CPT method gave a significantly better viability and recovery than ficoll. PBMC proliferation in response to antigens was significantly improved when using CPT isolation method, but cytokine production was similar for both methods as measured by elispot. We did not find significant differences in the cell population percentage between the PBMCs isolated by CPT or ficoll regarding CD3, CD4, CD8, CD14, CD19, activated or memory T cells.
Conclusions: We have found that even though CPT isolation method provide 20% less PBMCs than the classic Ficoll method, the quality of PBMCs isolated by CPT is superior to the one obtained by ficoll as the viability is better and the response to antigen especially after cryopreservation. There was no difference in PBMC population composition. Moreover, this method allows to process quickly and easily PBMCs from the blood with minimal variations among different centers and different operators. It also allows to obtain both PBMCs and autologous plasma from the same subject in one step. This protocol has been adopted as standard operating procedure for studies conducted by the ITN and therefore expands the ITN’s capability to evaluate mechanisms of disease and treatment response in multicenter trials.