[The ways of harmonization of clinical laboratory measurement techniques].

The results of implementation of different clinical laboratory techniques are to be equal in clinically significant limits to be optimally applied in diagnostics of diseases and treatment of patients. When the results of laboratory tests are not standardized and harmonized for the very same clinical assay the results can be expressed by unmatched numbers. Unfortunately, in some handbooks the values are presented based on the results of application of specific laboratory techniques without considering possibility or likelihood of differences between various techniques. When this is a case, accumulation of data of diferent clinical research studies and working out of clinical handbooks on this basis will be inconsistent. Inadequate understanding of issue that the results of laboratory tests are not standardized and harmonized can lead to incorrect clinical, financial, managerial or technical decisions. The standardization of clinical laboratory techniques was applied to many measurands related to primary referent techniques (standard specimen of pure substance) or/and developed referent measurement techniques. However, harmonization of clinical laboratory techniques for those measurands which are not related any developed measurement techniques is quite problematic due to inadequate determination of measurand, its inadequate analytical specificity, insufficient attention to commutability of referent materials and poor systematic approach to harmonization. To overcome these issues an infrastructure is to be developed to support systematic approach to identification and prioritization of measurands which are to be harmonized on the basis of clinical importance and technical applicability. The management of technical implementation harmonization process for specific measurands.

[Current issues in measurement and reporting of urinary albumin excretion]. / Données actuelles sur le dosage de l'excrétion urinaire de l'albumine.

Urinary excretion of albumin indicates kidney damage and is recognized as a risk factor for progression of kidney disease and cardiovascular disease. The role of urinary albumin measurements has focused attention on the clinical need for accurate and clearly reported results. The National Kidney Disease Education Program and the IFCC convened a conference to assess the current state of preanalytical, analytical, and postanalytical issues affecting urine albumin measurements and to identify areas needing improvement. The chemistry of albumin in urine is incompletely understood. Current guidelines recommend the use of the albumin/creatinine ratio (ACR) as a surrogate for the erro-prone collection of timed urine samples. Although ACR results are affected by patient preparation and time of day of sample collection, neither is standardized. Considerable intermethod differences has been reported for both albumin and creatinine measurement, but trueness is unknown because there are no reference measurement procedures for albumin and no referance materials for either analyte in urine. The recommanded reference intervals for the ACR do not take into account the large intergroup differences in creatinine excretion (e.g., related to differences in age, sex, and ethicity) nor the continuous increase in risk related to albumin excretion. Clinical needs have been identified for standardization of (a) urine collection methodes, (b) urine albumin and creatinine measurements based on a complete reference system, (c) reporting of test results, and (d) reference intervals for the ACR.

Standardization of cardiac troponin I assays: round Robin of ten candidate reference materials.

BACKGROUND: Cardiac troponin I (cTnI) results vary 100-fold among assays. As a step toward standardization, we examined the performance of 10 candidate reference materials (cRMs) in dilution studies with 13 cTnI measurement systems. METHODS: Solutions of 10 cTnI cRMs, each characterized by NIST, were shipped to the manufacturers of 13 cTnI measurement systems. Manufacturers used their respective diluents to prepare each cRM in cTnI concentrations of 1, 10, 25, and 50 microg/L. For the purpose of ranking the cRMs, the deviation of each cTnI measurement from the expected response was assessed after normalization with the 10 microg/L cTnI solution. Normalized deviations were examined in five formats. Parameters from linear regression analysis of the measured cTnI vs expected values were also used to rank performance of the cRMs. RESULTS: The three cRMs demonstrating the best overall rankings were complexes of troponins C, I, and T. The matrices for these three cRMs values differed; one was reconstituted directly from the lyophilized form submitted by the supplier; one was submitted in liquid form, lyophilized at NIST, and subsequently reconstituted; and the third was evaluated in the liquid form received from the supplier. The cRM demonstrating the fourth best performance was a binary complex of troponins C and I supplied in lyophilized form and reconstituted before distribution. CONCLUSIONS: The cRMs demonstrating the best performance characteristics in 13 cTnI analytical systems will be included in subsequent activities of the cTnI Standardization Committee of the AACC.

Heterogeneity in human cardiac troponin I standards.

The LC-MS analysis of recombinant cardiac troponin I (cTnI) and cTnI extracted from human hearts showed a high degree of structural heterogeneity among all samples. The examined recombinant cTnI samples indicated posttranslational modifications, presumably due to their purification (i.e., 2-mercaptoethanol adducts and carbamylation) and related to their expression (i.e., an N-terminal expression tag). The extracted cTnI samples, while having a higher degree of structural heterogeneity, showed less structural variance between samples than the recombinant proteins. The LC-MS analysis of the extracted cTnI samples provided evidence of posttranslational modification by phosphorylation, acetylation, proteolytic cleavage, and intrachain disulfide bond formation.

Characterization of the glycation of albumin in freeze-dried and frozen human serum.

Human serum albumin (HSA) in fresh frozen and freeze-dried serum reference materials was examined by mass spectrometry and a variety of affinity chromatography techniques. The relative molecular mass distribution of HSA in fresh frozen serum was found to be identical to that of an HSA standard. However, the HSA in the freeze-dried reference serum exhibited a relative molecular mass distribution that was shifted to higher mass, broader, and substantially more heterogeneous than that of HSA in fresh frozen serum. A proteolytic cyanogen bromide digestion of the HSA from freeze-dried serum contained adducts approximately 162 u higher in mass than digest fragments 124-298 and 447-548, suggesting glycation. The presence of glycation on fragments 124-298 and 447-548 correlates with the known sites of HSA glycation. Glycation was further confirmed by the mass spectral analysis of the retained and unretained fractions from glycoaffinity chromatography of HSA from freeze-dried serum. The relative molecular weight of the HSA in the retained fraction indicated the presence of a doubly glycated species. The chemical heterogeneity of Cys-34, the site of the only free thiol in HSA, was examined and found not to be a substantial source of molecular mass heterogeneity for HSA from either fresh frozen of freeze-dried serum.

Fragmentation of proteins in the 13- to 29-kDa mass range observed by 252Cf-plasma desorption mass spectrometry.

Results are presented on the observation of sequence-specific fragmentation of proteins in the 13- to 29-kDa mass range (ribonuclease A, papain, and proteinase K) by 252Cf-plasma desorption mass spectrometry. For these proteins, extensive N-terminal an, cn + 2, and dn fragment ions are observed with mass accuracies approaching 200 ppm for the most prominent fragment ions. The fragmentation pattern of these proteins shows the same fundamental behavior (arginine-directed, charge-remote fragmentation) that is observed in the fragmentation of small peptides, indicating that similar processes are occurring when large proteins and small peptides fragment in plasma desorption mass spectrometry.

Fragmentation analysis of bradykinin by (252)cf-plasma desorption mass spectrometry.

The (252)Cf-plasma desorption (PO) mass spectrum of the nonapeptide bradykinin was studied in detail with particular emphasis on the fragmentation pattern resulting from fast metastable decay of the molecular ion. N-acetyl and O-methyl derivatives of bradykinin were used as mass shift species for assignment of N-terminal and C-terminal fragment ions. Thirty-seven sequence-specific fragment ions were identified, including those that are due to cleavage of peptide backbone as well as side chain bonds (i.e., dn, vn, wn fragment ions). The degree of fragmentation correlates with what has been observed by fast atom bombardment tandem mass spectrometry using hi~h energy collisional activation. The high degree of excitation of bradykinin molecules in (252)Cf-POMS is undoubtedly due to the special nature of excitation intrinsic to the (252)Cf_PD process where electronic excitation occurs with a very high probability due to the high electron and photon flux surrounding the nuclear fission fragment track.These results offer further evidence that (252)Cf_PDMS, in addition to providing molecular weight information, can be used to sequence peptides without the need for a second stage of molecular excitation.