Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay
Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay
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DOI：
10.3791/4302
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发表时间：
2012-09-01
影响因子：
1.2
通讯作者：
Andersen-Nissen, Erica
中科院分区：
文献类型：
--
作者：
Hensley, Tiffany R.;Easter, Austin B.;Andersen-Nissen, Erica
外周血白细胞的冷冻保存被广泛用于保存细胞进行临床试验中的免疫反应评估，并为免疫学评估的轻松和标准化提供了许多优势，但是已经观察到了这种过程对某些细胞群的有害影响，例如粒细胞，B细胞，B细胞，B细胞，B细胞和树突状细胞（1-3）。分析新鲜白细胞可以更准确地描绘了细胞的体内状态，但在大型临床试验的背景下通常很难执行。新鲜的细胞分析取决于志愿者的承诺和时间表，如果耗时，由于实验室人员所需的工作时间，它们的应用可能是不切实际的。此外，当在多个中心进行试验时，进行测定所需的资源和培训的实验室可能不足以靠近临床站点。为了解决这些问题，我们开发了一个11色抗体染色面板，可以与Trucount管（Becton Dickinson; CA San Jose，CA）一起使用，以表型并枚举周围血液中的主要白细胞种群特定的信息比完整的血数（CBC）或具有商业上可用面板的测定法相比，专为Trucount试管而设计，仅染色几个细胞类型。染色过程很简单，仅需要100 mu L新鲜的全血，大约需要45分钟，使标准的血液处理实验室可行。它是根据BD Trucount Tube技术数据表（版本8/2010）改编的。染色抗体鸡尾酒可以在中央测定实验室批量提前准备，并运往现场加工实验室。可以固定染色管并将其冷冻以运送到中央测定实验室进行多色流式细胞仪分析。从该染色面板产生的数据可用于跟踪与干预有关的随着时间的流逝的白细胞浓度的变化，并且可以很容易地进一步开发以评估特定细胞类型的激活状态。在本报告中，我们证明了血液处理实验室技术人员对新鲜血液进行染色的程序，以及在支持多中心临床试验的中央测定实验室分析这些染色样品的步骤。该视频详细介绍了在HIV疫苗试验网络（HVTN）中临床试验血液抽血的背景下进行的过程。
Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells (1-3). Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 mu l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (version 8/2010). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).