Evaluation of Laboratory Diagnostic Methods

该项目的目标是改进诊断疾病的临床实验室方法。研究包括分析临床实验室实践，分析实验室测试的准确性，以及开发新的测试和测试技术。在过去的一年里，主要的工作是表征具有潜在诊断意义的尿蛋白，主要集中在尿白蛋白的分子形式，确定对临床血磷测定的干扰，评估标本光照对胆红素测定的影响，开发多肽质谱分析的内部标准化的新方法，以及建立高丰度血浆蛋白数据库。1)采用层析、电泳法、Western blotting和质谱法分析尿白蛋白和其他尿蛋白的分子形态。这些分析与尿白蛋白免疫分析相关，以试图了解在这项检测中可能重要的因素，这对糖尿病患者肾脏疾病的早期发现具有重要作用。这些信息应该被证明对国家肾脏疾病教育计划实验室工作组制定尿白蛋白测量实践指南和标准化的努力有价值。2)脂质体两性霉素B被确定为血磷分析中的干扰物质，并将此信息转发给食品和药物管理局，以帮助避免使用该药物治疗的患者出现高磷血症的不当诊断。3)胆红素最近被确定为血浆中的主要抗氧化剂之一，它是由一条可能产生一氧化碳等信号分子的调节途径形成的。因此，最近人们对准确测量健康受试者体内低浓度胆红素的能力感兴趣。在低浓度胆红素的测量中，光的可溶性被确定为一个重要的问题，并确定了样品的可接受的光暴露限度。4)提出了一种新的多肽测定的标准化方法。没有使用稳定的同位素标记的氨基酸，而是进行了有针对性的氨基酸替换，预计对色谱迁移率的影响最小。对于几种多肽，证明了可以在反相柱上制备具有相似色谱迁移率的内标肽，并将其用于多肽的质谱分析。这种方法可能允许更大的灵活性以及更低的成本来开发多肽测量的内部标准。5)建立了丰度最高的血浆蛋白数据库。其中包括许多具有重要生理和诊断价值的蛋白质。该数据库侧重于提供关于多肽浓度的信息，同时链接到现有的关于质量、结构和序列的信息。这些信息是一项初步工作，可以随着新信息的出现而逐步更新。

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 efforts over the past year have been to characterize urinary proteins of potential diagnostic significance with a primary focus on molecular forms of urinary albumin, to identify an interference with clinical assays for serum phosphorus, to assess the effects of light exposure of specimens on bilirubin measurements, to develop new methods for internal standardization of mass spectrometric assays of peptides, and to compile a database of the high-abundance plasma proteins. 1) Chromatographic, electrophoretic, Western blotting and mass spectrometric methods were used to analyze the molecular forms of albumin and other urinary proteins in urine. These analyses were related to immunoassay analysis of urinary albumin to try to understand factors that may be important in this assay which has an important role in the early detection of kidney disease in diabetics. The information should prove of value for efforts by the Laboratory Working Group of the National Kidney Disease Education Program to develop practice guidelines and standardization of urine albumin measurements. 2) Liposomal amphotericin B was identified as an interfering substance in analysis of serum phosphorus and this information was forwarded to the Food & Drug Administration to help avoid inappropriate diagnosis of hyperphosphatemia in patients treated with this drug. 3) Bilirubin recently has been identified as one of the major antioxidants in plasma and it is formed by a regulated pathway that may generate signalling molecules such as carbon monoxide. Therefore, there is recent interest in the ability to accurately measure low concentrations of bilirubin occurring in healthy subjectes. Photolability was identified as a significant issue in the measurement of low concentrations of bilirubin and acceptable limits for light exposure of specimens were determined. 4) A new approach for standardization of peptide measurements by mass spectrometry was developed. Rather than using stable isotope-labeled amino acids, targeted amino acid substitutions were made that were predicted to have minimal effects on chromatographic mobility. For several peptides, it was demonstrated that internal standard peptides with similar chromatographic mobility on reversed-phase columns could be prepared and that these could be used for quantification of peptides by mass spectrometry. This approach may allow greater flexibility as well as lower cost in the development of internal standards for peptide measurement. 5) A database was compiled on the highest abundance plasma proteins. These include many proteins of major physiological and diagnostic interest. This database focused on providing information about polypeptide concentration while linking to existing information on mass, structure, and sequence. This information was an initial effort that can be progressively updated as new information becomes available.