Abstract
BackgroundProgress in clinical trials in infectious disease, autoimmunity, and cancer is stymied by a dearth of successful whole cell biomarkers for peripheral blood lymphocytes (PBLs). Successful biomarkers could help to track drug effects at early time points in clinical trials to prevent costly trial failures late in development. One major obstacle is the inaccuracy of Ficoll density centrifugation, the decades-old method of separating PBLs from the abundant red blood cells (RBCs) of fresh blood samples.Methods and FindingsTo replace the Ficoll method, we developed and studied a novel blood-based magnetic separation method. The magnetic method strikingly surpassed Ficoll in viability, purity and yield of PBLs. To reduce labor, we developed an automated platform and compared two magnet configurations for cell separations. These more accurate and labor-saving magnet configurations allowed the lymphocytes to be tested in bioassays for rare antigen-specific T cells. The automated method succeeded at identifying 79% of patients with the rare PBLs of interest as compared with Ficoll's uniform failure. We validated improved upfront blood processing and show accurate detection of rare antigen-specific lymphocytes.ConclusionsImproving, automating and standardizing lymphocyte detections from whole blood may facilitate development of new cell-based biomarkers for human diseases. Improved upfront blood processes may lead to broad improvements in monitoring early trial outcome measurements in human clinical trials.
Highlights
The lack of biomarkers is one of the foremost obstacles to advances in clinical medicine [1,2,3]
Given that many peripheral blood lymphocytes (PBLs) subtypes are exceedingly rare in blood specimens (,161026), yet exert disproportionately large roles in disease, biomarker innovation requires developing an accurate method of separating PBLs from the very abundant red blood cells (RBCs)
With the goal of developing viable whole cell PBL biomarkers, we first sought to create a cell separation method superior to Ficoll density gradients: we developed and tested a new method of magnetic separation
Summary
The lack of biomarkers is one of the foremost obstacles to advances in clinical medicine [1,2,3]. Given that many PBL subtypes are exceedingly rare in blood specimens (,161026), yet exert disproportionately large roles in disease, biomarker innovation requires developing an accurate method of separating PBLs from the very abundant red blood cells (RBCs). PBL separations and biomarker standardizations are difficult to develop partly because of blood’s high viscosity and its high ratio of red to white blood cells (700 RBCs to 1 PBL). Accurate methods of separating the many pathologic PBL subpopulations are central to achieving advances in autoimmunity, infectious disease, and cancer. Progress in clinical trials in infectious disease, autoimmunity, and cancer is stymied by a dearth of successful whole cell biomarkers for peripheral blood lymphocytes (PBLs). One major obstacle is the inaccuracy of Ficoll density centrifugation, the decades-old method of separating PBLs from the abundant red blood cells (RBCs) of fresh blood samples
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