Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: a multicenter study.
Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: a multicenter study.
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DOI：
10.1021/ac702628q
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发表时间：
2008-06-15
影响因子：
7.4
通讯作者：
Cummins, James E., Jr.
中科院分区：
文献类型：
--
作者：
Fichorova, Raina N.;Richardson-Harman, Nicola;Alfano, Massimo;Belec, Laurent;Carbonneil, Cedric;Chen, Silvia;Cosentino, Lisa;Curtis, Kelly;Dezzutti, Charlene S.;Donoval, Betty;Doncel, Gustave F.;Donaghay, Melissa;Grivel, Jean-Charles;Guzman, Esmeralda;Hayes, Madeleine;Herold, Betsy;Hillier, Sharon;Lackman-Smith, Carol;Landay, Alan;Margolis, Leonid;Mayer, Kenneth H.;Pasicznyk, Jenna-Malia;Pallansch-Cokonis, Melanie;Poli, Guido;Reicholderfer, Patricia;Roberts, Paula;Rodriguez, Irma;Saidi, Hela;Sassi, Rosaria Rita;Shattock, Robin;Cummins, James E., Jr.
阴道分泌物中促炎细胞因子的增加可以作为局部用于预防性传播疾病（包括 HIV-1）的杀微生物剂产品产生的不良炎症反应的替代标志。白细胞介素 (IL)-1β 和 IL-6 已被提议作为炎症和 HIV-1 传播风险增加的指标；然而，缺乏有关阴道液最佳检测平台的信息以及实验室间差异限制了其在杀菌剂评估和其他临床应用中的使用。这项研究检查了与阴道取样技术相关的流体基质变异，并提出了一种用于当前细胞因子检测技术的实验室间比较的模型。 IL-1β 和 IL-6 标准品由四个国家的 12 个实验室使用 14 种免疫测定法和基于吸光度、化学发光、电化学发光和荧光的四个检测平台进行测量。将具有确定生物活性的细胞因子国际参考制剂掺入（1）模拟人阴道液（pH 4.5和7.2）组成的确定培养基，（2）细胞因子临床研究中常用的阴道灌洗取样的生理盐溶液（磷酸盐缓冲盐水和生理盐水），以及（3）人血清中。评估测定的重现性、线性、准确性和细胞因子水平的显着可检测倍数差异。通过 Kruskal-Wallis 方差分析、Dunn 多重比较检验和多元回归模型确定对细胞因子恢复有显着影响的因素。所有测定均显示出可接受的测定内重现性；然而，大多数都与显着的实验室间差异有关。源自实验室间汇总数据的最小可可靠检测的细胞因子差异 (P < 0.05) 根据测定、细胞因子和基质类型的不同，变化范围为 1.5 至 26 倍。与阴道液体基质相比，盐水和磷酸盐缓冲盐水中的 IL-6（而非 IL-1β）测定值较低，且 pH 值没有显着影响。基于（电）化学发光的测定最具辨别力，并且始终检测到每种基质类型内 <2 倍的差异。基于 Luminex 的检测的区分度较低，实验室之间的重现性较低。这些结果表明，在临床试验中验证细胞因子变异的生物学意义之前，需要采用统一的阴道采样技术，并更好地了解免疫测定平台的差异和交叉验证。这项研究提供了第一个评估粘膜细胞因子水平差异的标准化分析方法，并可能改进监测阴道粘膜界面免疫反应的策略。
The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1β and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1β and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal−Wallis analysis of variance with Dunn’s multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences (P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1β determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface.
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影响因子：
3.8
作者：
Al-Harthi, L;Kovacs, A;Landay, A
通讯作者：
Landay, A
影响因子：
2.2
作者：
POOLE, S;DAS, REG
通讯作者：
DAS, REG
影响因子：
7.4
作者：
Wang, WX;Zhou, HH;Becker, CH
通讯作者：
Becker, CH
DOI：
10.1128/cdli.6.1.89-95.1999
发表时间：
1999-01-01
期刊：
CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY
影响因子：
--
作者：
Aziz, N;Nishanian, P;Fahey, JL
通讯作者：
Fahey, JL
影响因子：
3.6
作者：
Fichorova, RN;Bajpai, M;Doncel, GF
通讯作者：
Doncel, GF