Cell-Based Assays Using Leukocyte Populations Isolated Directly From Whole Blood

Abstract 4842

Obtaining pure and unaffected leukocyte populations is of utmost importance in diagnostic as well as research settings. So far, the isolation of functional leukocyte subpopulations from whole blood has been a time-consuming procedure, rendering the performance of downstream assays and analyses a challenging objective.

We have developed a cell isolation technology that allows the purification of immune cells from human whole blood within 20 minutes. This novel technology requires a minimum of laboratory equipment. A cell isolation reagent is added to the anticoagulated blood sample and mixed briefly. While placed in a strong magnetic field, magnetically labeled non-target cells are depleted, while untouched target cells remain in the supernatant. Simultaneously, a reagent-assisted erythrocyte sedimentation phase occurs, which depletes ∼99.7 % of erythrocytes.

Using this novel technology, Natural Killer cells, B cells, T cells, CD4+ T helper cells, CD8+ cytotoxic T cells and naïve B cells were isolated from 30mL of anticoagulated human whole blood. Target cells were recovered in a volume of 25–30 mL of supernatant (67% plasma, 33% Phosphate buffered saline) and average purities among white blood cells were 88.9% for NK cells, 88.2% for B cells, 97.8% for T cells, 93.0% for CD4+ cells, 78.9% for CD8+ T cells and 79.4% for naïve B cells, yields were 75.5%, 84.4%, 54.5%, 63.0%, 59.5% and 96.8% respectively (n >6 each). Red Blood cells were reduced by ∼99.7%, platelets by >99.9%.

Cytotoxicity and proliferative capacity of isolated NK cells were measured in cytotoxicity assays with K562 target cells and proliferation assays with antibody loaded large magnetic beads respectively. Cytotoxicity and proliferation rate were comparable to those assessed using NK cells isolated by Ficoll density gradient separation or magnetic cell sorting (NK cell isolation kit). In vitro proliferation assays with total T cells, CD4+ T cells, CD8+ T cells or B cells revealed that proliferation rate was identical to that of target cells which were isolated by Ficoll density gradient separation and magnetic cell sorting.

We furthermore compared the mRNA yields from cells isolated with either method (new technology vs. Miltenyi's isolation kits). The mRNA samples were subsequently subjected to gene expression analysis. Comparing the results obtained from samples isolated with the two different separation methods, we could not detect any significant differences in gene expression levels.

These results demonstrate, that cells isolated with the novel whole blood cell isolation strategy, can be used for cell-based functional assays, as well as gene expression profiling. Additionally, overall processing time can be significantly reduced, which is highly desirable for sensitive downstream experiments.