Novel Automated Blood Separations Validate Whole Cell Biomarkers

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.

感染性疾病、自身免疫病及癌症领域的临床试验进展，因缺乏针对外周血淋巴细胞（peripheral blood lymphocytes, PBLs）的有效全细胞生物标志物而受阻。有效的生物标志物可助力在临床试验早期阶段监测药物效应，从而避免研发后期出现代价高昂的试验失败。其中一大主要障碍是菲科勒密度梯度离心法（Ficoll density centrifugation）的精准度不足——这一已有数十年历史的方法，用于从新鲜血液样本的大量红细胞（red blood cells, RBCs）中分离PBLs。
为替代菲科勒密度梯度离心法，我们开发并研究了一种新型血液源性磁性分离方法。该方法在PBLs的细胞活力、纯度及回收率方面显著优于菲科勒密度梯度离心法。为降低人工工作量，我们搭建了自动化分离平台，并对比了两种用于细胞分离的磁体配置方案。这些更精准且节省人力的配置方案，使得淋巴细胞可用于稀有抗原特异性T细胞的生物检测实验。与菲科勒密度梯度离心法全面失败的结果相比，自动化方法成功识别出79%携带目标稀有PBLs的患者。我们对优化后的前期血液处理流程进行了验证，证实其可精准检测稀有抗原特异性淋巴细胞。
对全血来源的淋巴细胞检测进行优化、自动化及标准化，或可助力开发针对人类疾病的新型细胞源性生物标志物。优化后的前期血液处理流程，或可显著改善人类临床试验中早期试验结局指标的监测工作。