Imprecision of high-sensitivity cardiac troponin assays at the female 99th-percentile.

BACKGROUND: An analytical benchmark for high-sensitivity cardiac troponin (hs-cTn) assays is to achieve a coefficient of variation (CV) of ≤ 10.0 % at the 99th percentile upper reference limit (URL) used for the diagnosis of myocardial infarction. Few prospective multicenter studies have evaluated assay imprecision and none have determined precision at the female URL which is lower than the male URL for all cardiac troponin assays. METHODS: Human serum and plasma matrix samples were constructed to yield hs-cTn concentrations near the female URLs for the Abbott, Beckman, Roche, and Siemens hs-cTn assays. These materials were sent (on dry ice) to 35 Canadian hospital laboratories (n = 64 instruments evaluated) participating in a larger clinical trial, with instructions for storage, handling, and monthly testing over one year. The mean concentration, standard deviation, and CV for each instrument type and an overall pooled CV for each manufacturer were calculated. RESULTS: The CVs for all individual instruments and overall were ≤ 10.0 % for two manufacturers (Abbott CVpooled = 6.3 % and Beckman CVpooled = 7.0 %). One of four Siemens Atellica instruments yielded a CV > 10.0 % (CVpooled = 7.7 %), whereas 15 of 41 Roche instruments yielded CVs > 10.0 % at the female URL of 9 ng/L used worldwide (6 cobas e411, 1 cobas e601, 4 cobas e602, and 4 cobas e801) (CVpooled = 11.7 %). Four Roche instruments also yielded CVs > 10.0 % near the female URL of 14 ng/L used in the United States (CVpooled = 8.5 %). CONCLUSIONS: The number of instruments achieving a CV ≤ 10.0 % at the female 99th-percentile URL varies by manufacturer and by instrument. Monitoring assay precision at the female URL is necessary for some assays to ensure optimal use of this threshold in clinical practice.

Development and optimization of simplified LC-MS/MS quantification of 25-hydroxyvitamin D using protein precipitation combined with on-line solid phase extraction (SPE).

25-Hydroxyvitamin D, the most useful marker of the vitamin D status of an individual, has seen an exponential growth of its routine measurement in recent years. Several methods are currently offered but the most specific is LC-MS/MS. However, the routine use of this technique in the clinical laboratory makes it essential to improve key steps of this method for high throughput delivery. Importantly, the preanalytical steps of this assay and the efficacy of the separation system need to be optimized prior to MS detection. In this report we replaced the standard and time consuming liquid-liquid extraction method of vitamin D metabolites with hexane (LLE) combined with centrifugation (LLE/centrifugation) by a simpler protein precipitation with extraction (PPE) in acetonitrile combined with a fast separation process using a 96-well plate filtration system (PPE/filtration). This rapid extraction was then followed by an on-line solid phase extraction (SPE) using a selective chromatographic separation. We also optimized the operational and consumable costs, by using an inexpensive guard column as a trapping column to significantly enhance the lifespan of the analytical column two to three times as compared to conventional chromatography. The LC-MS/MS technique permits the measurement of both 25-hydroxyvitamin D(2) (25-OH D(2)) and the 25-hydroxyvitamin D(3) (25-OH D(3)) metabolites in electrospray ionization (ESI) mode. The chromatographic system consisted of a 2.1 mm × 50 mm C18 3.5 µM column with a 2.1 mm × 20 mm C18 3.5 µM guard column connected with two 6 ports switching valves. Quantifications were done using the isotopic dilution technique with hexadeutered 25-OH D(3) and 25-OH D(2).The ion suppression problem with phospholipids was also evaluated and optimized to minimize this effect through the chromatography process and the on-line SPE trapping. Calibration curves were prepared by diluting a commercial high calibrator Chromsystems (München, Germany) with either pure triple stripped blank serum or diluted in 6% phosphate buffer saline at pH 7.2. Linearity was tested up to 160 nmol/L for 25-OH D(3) and 75 nmol/L for 25-OH D(2). Low limit of quantification (LLOQ) were established at 3 nmol/L for 25-OH D(2) and 4 nmol/L for 25-OH D(3). Inter-assay and intra-assay precision (CV%) was determined using 3 levels of commercial controls (Utak, CA, USA) for 25-OH D(2) and 25-OH D(3). Results obtained for intra-assay and inter-assay precision (CV%) were 1.1-3.4% and 5-8.9% respectively for the PPE/centrifugation technique and 2.0-3.1% and 4.6-6.6% for the PPE/filtration technique. Accuracy was estimated with the same commercial controls: % bias was -11.2 to 4.9% with PPE/centrifugation and -3.2 to 6.1% with PPE/filtration. 25-OH D(2) and 25-OH D(3) concentrations in human serum with LLE were compared to the new extraction methods using either PPE/centrifugation or PPE/filtration. Correlations comparing the two methods revealed a slope approximately 1.0±0.3 with R≥0.98 with a bias<1 nmol/L. In summary, the new LC-MS/MS method described in this report using an on-line SPE technique with a simple off-line pre-treatment is faster, cost-effective, more reliable and more robust than current and widely used LLE/centrifugation methods coupled with LC-MS/MS.

Cloning and functional expression of two plant thiol methyltransferases: a new class of enzymes involved in the biosynthesis of sulfur volatiles.

Glucosinolates are defensive compounds found in several plant families. We recently described five distinct isoforms of a novel plant enzyme, thiol methyltransferase (TMT), which methylate the hydrolysis products of glucosinolates to volatile sulfur compounds that have putative anti-insect and anti-pathogen roles. In the work presented here, two cDNAs encoding these enzymes (cTMT1 and cTMT2) were isolated by screening a cabbage cDNA library with an Arabidopsis EST showing high sequence homology to one TMT isoform. The genomic clone of cTMT1 was subsequently amplified by PCR. Both cDNAs encoded polypeptides of identical lengths (227 amino acids) and similar predicted masses (ca. 25 kDa), but differing in 13 residues. The cDNAs contained the typical methyltransferase signatures, but were otherwise distinct from conventionally known N-, O- or S-methyltransferases. A chloride methyl transferase was the only gene with an assigned function that shared significant similarity with the TMT cDNAs. Southern analysis indicated single copy for each TMT gene. The two cDNAs were expressed in Escherichia coli. The substrate range, kinetic properties and molecular sizes of the purified recombinant proteins were comparable to those of the native enzyme. These data, together with the detection of the sequenced amino acid motif of one native TMT peptide in the cDNAs, confirmed that the latter were authentic TMTs. The expression pattern of the TMTs in various cabbage tissues was consistent with their association with glucosinolates. The cloning of this new class of plant genes furnishes crucial molecular tools to understand the role of this metabolic sector in plant defenses against biotic stress.