Evaluation of Laboratory Diagnostic Methods

实验室诊断方法评价

• 批准号: 7004851
• 负责人: GLEN HORTIN
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7004851
• 关键词: blood chemistry; blood disorder diagnosis; blood glucose; blood protein disorder; blood proteins; body fluids; cardiovascular disorder diagnosis; diabetes mellitus; diagnosis design /evaluation; diagnosis quality /standard; heart disorder; human tissue; kidney disorder; kidney disorder diagnosis; mass spectrometry; matrix assisted laser desorption ionization; metabolism disorder diagnosis; protein quantitation /detection; proteinuria; sample collection; urinalysis

Goals of this project are to improve clinical laboratory methods for diagnosis of disease. Studies include analysis of clinical laboratory practices, analysis of the accuracy of laboratory tests, and development of new tests and testing technologies. The major effort over the past year has been to analyze complex patterns of peptides and proteins in biological fluids by matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry. This technique allows simultaneous detection of more than 100 peptide and protein components in biological fluids. The primary challenge in applying MALDI TOF mass spectrometry to the analysis of biological fluids is in sample preparation. Biological fluids contain excess salt and a high abundance of several proteins such as albumin and immunoglobulins that interfere with the ability to detect less abundant components. Our efforts have been directed primarily at analysis of peptide and small protein components, which is the mass region yielding highest sensitivity and resolution by MALDI TOF mass spectrometry. Issues of optimal sample collection for analysis of plasma components have been examined. Solid phase extraction of urine was identified as a simple procedure for preparation of specimens for analysis of peptide and small protein components by MALDI TOF mass spectrometry. The relatively simple preparation of urine specimens and lack of overload by albumin may allow diagnostic evaluations of protein components for applications such as identification of kidney tubular defects and of overload proteinurias such as those occurring with myeloma. The potential for blood collection tubes to add extraneous materials that may interfere with mass spectrometric analyses was examined, and many types of tubes were found to add polymeric components that appear in mass spectra. A second major hurdles to the clinical application of MALDI TOF mass spectrometry has been the lack of procedures for internal standardization and for cross-comparison with existing quantitative methods for protein analysis in the clinical laboratory. We have compared the correlation of peak areas by mass spectrometry by investigators in the Department of Transfusion Medicine with quantitative immunoassay results performed in our laboratory, and this may serve as a useful approach for calibration and validation of quantitative measurements by mass spectrometry. Detailed analysis of small peptide components of lipoproteins has been performed. Analysis of purified high-density lipoprotein (HDL) shows the presence of more than 100 peptide components in the mass range of 1,000-5,000 daltons. Sequence analysis of peptides indicates that most of them are peptide fragments of abundant proteins that generally are not considered to be associated with lipoproteins. Many proteins in biological fluids undergo post-translational modifications. Our laboratory has studied optimal procedures for detecting selected post-translational modifications such as sulfation of tyrosine residues.

该项目的目标是改善临床实验室诊断疾病的方法。研究包括临床实验室实践的分析，实验室测试的准确性分析，以及新的测试和测试技术的开发。在过去的一年中，主要的努力一直是通过基质辅助激光解吸/电离飞行时间（MALDI TOF）质谱分析生物液体中的肽和蛋白质的复杂模式。该技术允许同时检测生物流体中的100多种肽和蛋白质组分。将MALDI TOF质谱应用于生物体液分析的主要挑战在于样品制备。生物液体含有过量的盐和高丰度的几种蛋白质，如白蛋白和免疫球蛋白，这些蛋白质干扰检测丰度较低的组分的能力。我们的努力主要是针对肽和小蛋白组分的分析，这是MALDI TOF质谱法产生最高灵敏度和分辨率的质量区域。血浆成分分析的最佳样品采集问题已经过研究。尿液固相萃取被确定为一个简单的程序，用于制备样品，通过MALDI TOF质谱分析肽和小蛋白质组分。尿标本的制备相对简单，白蛋白的超负荷缺乏，可以允许诊断性评价蛋白组分的应用，如鉴别肾小管缺陷和超负荷蛋白尿，如骨髓瘤。检查了采血管添加可能干扰质谱分析的外来物质的可能性，发现许多类型的采血管添加了质谱中出现的聚合物组分。MALDI TOF质谱法临床应用的第二个主要障碍是缺乏内部标准化程序和与临床实验室中现有蛋白质分析定量方法的交叉比较程序。我们比较了由输血医学系的研究者通过质谱法测定的峰面积与我们实验室进行的定量免疫测定结果的相关性，这可能作为质谱法定量测量的校准和验证的有用方法。脂蛋白的小肽成分的详细分析已经完成。对纯化的高密度脂蛋白（HDL）的分析表明，在1000 - 5000道尔顿的质量范围内存在超过100种肽组分。肽的序列分析表明，它们中的大多数是通常不被认为与脂蛋白相关的丰富蛋白质的肽片段。生物体液中的许多蛋白质经历翻译后修饰。我们的实验室已经研究了用于检测选定的翻译后修饰，如酪氨酸残基的硫酸化的最佳程序。