Purity was measured by the percentage of CD8 T cells within isolates of PBLs. Due to the lack of cell specificity, purity using Ficoll only reached a high of 20% in normal samples. Purity was further analyzed by the cellular composition of the isolated PBLs by a scatter plot of Forward Scatter and Side Scatter. The cellular constituents were primarily mononuclear cells, dead cells, and lymphocytes, the latter of which consisted of a mix of CD4 and CD8 T lymphocytes and B lymphocytes. The goal of this project was to demonstrate the accuracy of magnetic separation methods in terms of viability, purity and yield of cells. It was our belief that these manual methods should be automated for finer degrees of accuracy and reproducibility for clinically useful whole cell PLX-4720 biomarkers. If successful, the automation process would also increase capacity, accuracy and set the stage for another level of standardizations that would remove the human factor from manually performing the blood separation process. At this time point we were working under the assumption that the standardization of whole blood separations, by the defined parameter of whole cell viability, purity and yield, would also Olaparib strengthen the accuracy of downstream whole cell biomarker assay perfection. One of the early decisions in the design of the whole blood cell separation process was to find a paramagnetically tagged antibody reagent that was compatible with robotic whole blood separations and could bind PBLs of interest, for instance CD8 lymphocytes. After comparing a variety of paramagnetically linked antibody methods, we found that antibodies labeled with 3.5 mm diameter super-paramagnetic beads resulted in lymphocyte subpopulations with the best purity, yield and viability. This is a relatively large bead size for the paramagnetic antibody reagent but it was chosen because larger beads expedite cell migration to the side of the tube. Also, large magnetic beads attached to the blood cells of interest were likely to eliminate the problems inherent in the use of their small counterparts. Small paramagnetic particles attached to antibodies require other blood processing steps such as centrifugation, long time periods for cell separations through viscous blood and complex wash and handling steps due to the longer times to bring a cell to the side of the tube.