Hendrix S W, Miller K H, Youket T E, Adam R, O'Connor R J, Morel J G, Tepper B E
The Procter & Gamble Company, Beauty Care, Sharon Woods Innovation Center, Cincinnati, OH 45241, USA.
Skin Res Technol. 2007 Aug;13(3):330-42. doi: 10.1111/j.1600-0846.2007.00235.x.
BACKGROUND/PURPOSE: This work was performed to optimize extraction conditions for D-Squame tape skin samples for use in the skin multiple analyte profile (SkinMAP) method, a Linco Research Corporation bead-based assay for skin analytes. The experiments were designed to help identify sources of variability during extraction that may be amenable to further control.
Two experimental designs were used to study factors influencing the extraction of skin samples from D-Squame tapes. Visually healthy skin samples were obtained from both female and male adult volar forearms. Factors studied in two experiments included: four surfactant (SDS) levels (0.02-0.2%), two buffer types [Citrate-phosphate buffered saline at pH 5.5, phosphate-buffered saline (PBS) at pH 7.4], two buffer volumes (1.0, 1.5 mL), two propylene glycol (PG) levels (0.1%, 1.0%), two extraction temperatures (7-10 degrees C, 22-30+ degrees C), two extraction times (30, 60 min), and location in sonication bath (two vectors). The response biomarkers were cortisol, fibronectin, human serum albumin, involucrin, keratin-6 and keratins 1, 10. Skin sampling sites were also evaluated as sources of variation.
There was no single set of extraction conditions in our experiments that maximized recovery of all the biomarkers. SDS level had the most consistently significant (P<0.05) and directional effects on biomarker recoveries. In general, higher SDS resulted in higher recovery of all biomarkers. There was less consistency and fewer significant results for the other extraction factors.
These data enable us to better manage SkinMAP studies and interpret their results. The use of 1.5 mL PBS containing 0.2% SDS and 0.5% PG with 30 min sonication at low (near 4 degrees C) temperature is optimal for the quantitation of a range of SkinMAP analytes. In order to protect researchers from obtaining inflated false positive rates, it is crucial to design such studies and analyze the data using appropriate statistical methodology, especially for those studies involving only a small number of subjects.
背景/目的：本研究旨在优化用于皮肤多种分析物谱（SkinMAP）方法的D - 角质层胶带皮肤样本的提取条件，SkinMAP方法是Linco Research Corporation公司基于磁珠的皮肤分析物检测方法。这些实验旨在帮助确定提取过程中可能易于进一步控制的变异性来源。
采用两种实验设计来研究影响从D - 角质层胶带提取皮肤样本的因素。从成年男女的掌侧前臂获取外观健康的皮肤样本。在两个实验中研究的因素包括：四种表面活性剂（SDS）水平（0.02 - 0.2%）、两种缓冲液类型[pH 5.5的柠檬酸 - 磷酸盐缓冲盐水、pH 7.4的磷酸盐缓冲盐水（PBS）]、两种缓冲液体积（1.0、1.5 mL）、两种丙二醇（PG）水平（0.1%、1.0%）、两种提取温度（7 - 10摄氏度、22 - 30 +摄氏度）、两种提取时间（30、60分钟）以及超声浴中的位置（两个向量）。反应生物标志物为皮质醇、纤连蛋白、人血清白蛋白、内聚蛋白、角蛋白 - 6以及角蛋白1、10。皮肤采样部位也作为变异来源进行了评估。
在我们的实验中，没有一组提取条件能使所有生物标志物的回收率最大化。SDS水平对生物标志物回收率具有最一致的显著（P<0.05）且有方向性的影响。一般来说，较高的SDS会导致所有生物标志物的回收率更高。其他提取因素的一致性较差且显著结果较少。
这些数据使我们能够更好地管理SkinMAP研究并解释其结果。使用含0.2% SDS和0.5% PG的1.5 mL PBS在低温（接近4摄氏度）下超声处理30分钟，对于一系列SkinMAP分析物的定量是最佳的。为了保护研究人员避免获得过高的假阳性率，设计此类研究并使用适当的统计方法分析数据至关重要，特别是对于那些仅涉及少数受试者的研究。
