Evaluation of Laboratory Diagnostic Methods

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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.