树突状细胞体外特性的多中心研究。

Multicenter study on in vitro characterization of dendritic cells.

作者信息

Eichler H, Nguyen X D, Roelen D, Celluzzi C M, McKenna D, Pamphilon D, Blair A, Read E J, Takahashi T A, Szczepiorkowski Z M

机构信息

Institute of Transfusion Medicine and Immunology, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Heidelberg, Germany.

出版信息

Cytotherapy. 2008;10(1):21-9. doi: 10.1080/14653240701744263.

DOI:10.1080/14653240701744263

PMID:18202971

Abstract

BACKGROUND

There is growing interest in the use of in vitro-expanded dendritic cells (DC) in cancer immunotherapy as cellular-based vaccines. However, the methods used for in vitro preparation vary widely between institutions. Therefore, a strong need exists for standardization, characterization and quality control (QC) of such vaccines. A first prospective multicenter pilot study was performed to investigate basic QC parameters of frozen/thawed DC. The study design was focused on comparison of test results for cell counts, immunophenotyping and cell viability.

METHODS

CD14+ monocytes were isolated from three healthy volunteers. The cells were expanded in vitro, matured and cryopreserved using a standardized protocol in one laboratory. The aliquots of cryopreserved DC and a panel of reagents were shipped to eight laboratories worldwide. The objective was to compare the results of non-functional QC assays between sites by testing identical DC vaccines and using a pre-defined test protocol.

RESULTS

Measurements of nucleated cell (NC) content of thawed DC vaccines with different types of hematology analyzers (HA) gave similar results for the majority of sites. Immunophenotyping using identical clones of monoclonal antibodies for the detection of surface antigens (i.e. CD1a, CD14, CD16, CD83, CD86 and HLA-DR) provided mostly comparable results between laboratories with an acceptable level of variation. In contrast, highly different results between study sites were generated for measuring the viability of thawed DC by flow cytometry using 7-amino-actinomycin D (7-AAD) dye exclusion.

DISCUSSION

In characterizing frozen/thawed DC vaccines, NC counts generated by HA yielded similar results between different laboratories. Furthermore, immunophenotyping of DC vaccines can be standardized between centers, i.e. by using identical reagents. Because of highly variable results between laboratories, 7-AAD viability testing of thawed DC needs to be studied further to identify potential causes for the observed variability.

摘要

背景

作为基于细胞的疫苗，体外扩增树突状细胞（DC）在癌症免疫治疗中的应用越来越受到关注。然而，各机构用于体外制备的方法差异很大。因此，迫切需要对此类疫苗进行标准化、特性鉴定和质量控制（QC）。开展了第一项前瞻性多中心试点研究，以调查冻融DC的基本QC参数。该研究设计侧重于比较细胞计数、免疫表型分析和细胞活力的测试结果。

方法

从三名健康志愿者中分离出CD14+单核细胞。这些细胞在一个实验室中使用标准化方案进行体外扩增、成熟和冷冻保存。将冷冻保存的DC等分试样和一组试剂运送到全球八个实验室。目的是通过测试相同的DC疫苗并使用预先定义的测试方案，比较不同地点之间非功能性QC分析的结果。

结果

使用不同类型的血液分析仪（HA）测量冻融DC疫苗的有核细胞（NC）含量，大多数地点的结果相似。使用相同克隆的单克隆抗体进行免疫表型分析以检测表面抗原（即CD1a、CD14、CD16、CD83、CD86和HLA-DR），各实验室之间的结果大多具有可比性，变异水平可接受。相比之下，使用7-氨基放线菌素D（7-AAD）染料排除法通过流式细胞术测量冻融DC的活力时，各研究地点产生了高度不同的结果。