与体外细胞因子分泌测定相关的分析和分析前变量的优化。

Optimization of analytical and pre-analytical variables associated with an ex vivo cytokine secretion assay.

作者信息

Ray Chad A, Dumaual Carmen, Willey Mark, Fill Jeffrey, O'Brien Peter J, Gourley Ian, Devanarayan Viswanath, Konrad Robert J

机构信息

Laboratory for Experimental Medicine, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA.

出版信息

J Pharm Biomed Anal. 2006 Apr 11;41(1):189-95. doi: 10.1016/j.jpba.2005.10.013.

DOI:10.1016/j.jpba.2005.10.013

PMID:16321496

Abstract

PURPOSE

Measurements of cytokine release in whole blood after ex vivo stimulation are useful in drug development. The components contributing to variation within such assays have not been clearly defined. Therefore, we characterized the sources of variability within an ex vivo stimulation assay for TNF-alpha release.

METHOD

Fresh whole blood or mononuclear cells from a cell preparation tube were added to silanized, screw-top tubes with a final concentration of 1 microg/mL lipopolysaccharide (LPS). Each tube was purged with 95% air/5%CO2 and incubated 4 or 6 h at 37 degrees C in a metabolic water bath. Plasma TNF-alpha was next measured in supernatants by immunoassay. Total method variability was assessed in 10 normal donors each drawn in the morning and afternoon over 3 days. Four additional samples were pre-treated with dexamethasone to investigate inhibition of TNF-alpha release.

RESULTS

Our analysis indicated precise temperature control, the timing and duration of stimulation, and the surface properties of the stimulation vessel most significantly influenced assay performance. A comparison of multiple anticoagulants indicated that careful consideration should be taken in selecting the optimal anticoagulant. The estimated total assay CV for all anticoagulants tested was less than 33.81%. The analytical variability (stimulation and measurement) was less than 25.88% CV. The one exception was mononuclear cells collected in sodium heparin. The total variability estimate incorporated day-to-day, diurnal, inter-donor, tube-to-tube and immunoassay variability. Using our optimized conditions, TNF-alpha release was inhibited by dexamethasone with a mean IC50 of 33.3 +/- 4.6 nM.

CONCLUSIONS

We have described an optimal set of conditions for collection, storage and processing of an ex vivo cytokine stimulation assay. These conditions were selected for operational feasibility, minimal imprecision and elimination of potential confounding factors. The end result is a more robust method that can be applied to clinical drug development.

摘要

目的

体外刺激后全血中细胞因子释放的测量在药物开发中很有用。此类检测中导致变异的因素尚未明确界定。因此，我们对肿瘤坏死因子-α（TNF-α）释放的体外刺激检测中的变异来源进行了表征。

方法

将新鲜全血或细胞制备管中的单核细胞加入到硅烷化螺口管中，脂多糖（LPS）终浓度为1微克/毫升。每个管充入95%空气/5%二氧化碳，并在37℃的代谢水浴中孵育4或6小时。接下来通过免疫测定法测量上清液中的血浆TNF-α。在3天内，对10名正常供体在上午和下午采集的样本评估总方法变异。另外4个样本用地塞米松预处理以研究对TNF-α释放的抑制作用。

结果

我们的分析表明，精确的温度控制、刺激的时间和持续时间以及刺激容器的表面特性对检测性能影响最为显著。多种抗凝剂的比较表明，选择最佳抗凝剂时应谨慎考虑。所有测试抗凝剂的估计总检测变异系数（CV）小于33.81%。分析变异（刺激和测量）的CV小于25.88%。唯一的例外是用肝素钠收集的单核细胞。总变异估计纳入了日间、昼夜、供体间、管间和免疫测定变异。使用我们优化的条件，地塞米松抑制TNF-α释放，平均半数抑制浓度（IC50）为33.3±4.6纳摩尔。