Standardization of calibration and quality control using surface enhanced laser desorption ionization-time of flight-mass spectrometry.

BACKGROUND: Protein profiling by surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS) is gaining importance as a diagnostic tool for a whole range of diseases. This report describes a QC procedure, which acts prospectively by checking the calibration before starting profiling experiments. METHODS: A well-defined protocol for calibration of the Protein Biosystem IIc instrument was established, using a commercial QC sample containing independent certified standards and by determination of acceptance criteria. Instrument calibration was performed externally every week with the standards provided by the manufacturer. QC was performed for the period of 5 months. RESULTS: According to the acceptance criteria defined in this study, data points should be in the established range of the process mean+/-2 standard deviations for the mass-to-charge ratios (m/z values), peak intensities, signal-to-noise ratios (S/N), and peak resolutions for insulin and apomyoglobin in the QC sample. Moreover, it was demonstrated that the pipetting variability in the handling of the QC sample significantly contributed to systematic errors and that spotting of a larger volume of QC sample resulted in a better reproducibility. CONCLUSIONS: Stringent quality control of the calibration part of SELDI-TOF-MS experiments prevents unreliable data acquisition from the very start.

Eliminating absorbing interference using the H-point standard addition method: case of Griess assay in the presence of interferent heme enzymes such as NOS.

Standard calibration methods used to determine trace analytes usually yield significant deviations from the actual analyte value in the presence of interferents in the assay media. These deviations become of particular concern when the concentration of the analyte is low, and when the results are used to draw mechanistic or kinetic conclusions, for instance in enzyme structure-function studies. In these circumstances, the H-point standard addition method (HPSAM) provides superior precision and accuracy. This method is developed here for the case of the spectrophotometric Griess assay used to determine nitrite in various enzymology investigations, such as nitrite determination in studies of nitrite reductases (NiR), or when determining nitrite as a breakdown product of nitric oxide synthesized by NOS enzymes. The results obtained by HPSAM are contrasted with those of the traditional calibration method.

Standardization of immunoassays for measurement of high-sensitivity C-reactive protein. Phase I: evaluation of secondary reference materials.

BACKGROUND: Inflammation contributes to the development and progression of atherosclerosis, and C-reactive protein (CRP) can be used as a marker to assess risk for cardiovascular diseases. As variability among existing high-sensitivity CRP (hsCRP) assays can lead to misclassification of patients and hamper implementation of population-based medical decision points, standardization of hsCRP assays is needed. METHODS: We evaluated five proposed secondary reference materials, including two diluted preparations of Certified Reference Material 470 (CRM470), two preparations of a serum-based material with recombinant CRP added, and one serum-based material with isolated CRP added. Twenty-one manufacturers participated in the comparison with 28 different assays. We examined imprecision, linearity, and parallelism with these materials and with fresh serum. RESULTS: All materials had similar imprecision; CVs for the undiluted materials were 2.1-3.7%. None of the materials was linear across all assays. Each had between one and three cases of nonlinearity, with one preparation of CRM470 having the fewest cases of nonlinearity. Although none of the materials was parallel across all assays, the differences in slope from fresh serum were similar across all assays. CONCLUSIONS: All materials performed similarly with regard to imprecision, linearity, and parallelism. As one preparation of CRM470 had slightly better characteristics than the other materials and because CRM470 had been certified previously as a reference material for the acute-phase reactant range, it will be used in the next phase to standardize hsCRP assays.

Recent approaches to the standardization of cardiac markers.

The development of commercial assays for the determination of cardiac proteins has been one of the most important innovations in the field of cardiovascular diagnostics in the last decade. Some assays are, however, inadequately appraised prior to their introduction to clinical use. This paper focuses on some important preanalytical, analytical and interpretative problems, and summarizes the status of the ongoing local and international standardization efforts. The most urgent issue at the moment is the development of international reference materials, which can be used for the calibration of different assays, thus decreasing between-assay biases. In order to achieve comparability of test results, another important item is the standardization of the epitopes of the antibodies used for the assay development. Efforts to improve the precision of cardiac marker assays are also warranted. Finally, the effect of storage time and temperature on apparent marker concentration and the possible influence of different anticoagulants on measured marker values should clearly be evaluated.

Human myoglobin: preparation, quantitation and standardization.

The quantitation of myoglobin (Mb) in serum and urine is of clinical importance for the differentiation of myocardial infarction from degenerative cardiac disorders as well as for the detection of traumatic and atraumatic rhabdomyolysis, followed frequently by acute kidney failure. A simple method is described to prepare myoglobin from human muscle extract by negative pressure ultrafiltration and dialysis. By a combination of electrophoretic procedures, this preparation was analysed for purity. Saline myoglobin solutions after deep freezing loose rapidly their immunoreactive Mb content. By addition of pure albumin, not containing heme binding proteins, a stable Mb solution was obtained. This has been used as standard (5--50 microgram/ml) in radial immunodiffusion sensitive for detecting 0.2--1 microgram Mb/ml.