Evaluation of Laboratory Diagnostic Methods

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。过去一年的主要工作是表征具有潜在诊断意义的尿蛋白，主要关注尿白蛋白的分子形式，识别对血清磷临床测定的干扰，评估样本光暴露对胆红素测量的影响，开发肽类质谱测定内部标准化的新方法，并建立高丰度血浆蛋白的数据库。1)采用色谱法、电泳法、蛋白质印迹法和质谱法分析尿中白蛋白和其他尿蛋白的分子形式。 这些分析与尿白蛋白的免疫测定分析相关，以试图了解在该测定中可能重要的因素，该测定在糖尿病患者肾脏疾病的早期检测中具有重要作用。 这些信息应该证明对国家肾脏疾病教育计划实验室工作组制定实践指南和尿白蛋白测量标准化的努力有价值。 2)在血清磷的分析中，脂质体阿替霉素B被确定为干扰物质，该信息被转发给食品和药物管理局，以帮助避免对接受该药物治疗的患者的高磷血症进行不适当的诊断。 3)胆红素最近已被确定为血浆中的主要抗氧化剂之一，它是通过一种受调节的途径形成的，该途径可能产生信号分子，如一氧化碳。 因此，最近人们对准确测量健康受试者中出现的低浓度胆红素的能力感兴趣。 在低浓度胆红素的测量中，光耐受性被确定为一个重要问题，并确定了标本光暴露的可接受限度。 4)本文提出了一种新的多肽质谱标准化方法。 不是使用稳定的同位素标记的氨基酸，而是进行预测对色谱迁移率具有最小影响的靶向氨基酸取代。 对于几种肽，证明可以制备在反相柱上具有相似色谱迁移率的内标肽，并且这些内标肽可用于通过质谱法定量肽。 这种方法可以允许更大的灵活性以及更低的成本，在肽测量的内部标准的发展。 5)编制了关于最高丰度血浆蛋白的数据库。 这些包括许多具有重要生理和诊断意义的蛋白质。 该数据库侧重于提供有关多肽浓度的信息，同时链接到有关质量、结构和序列的现有信息。 这一信息是一项初步工作，可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.