Multidisciplinary approach to redefining thyroid hormone reference intervals with big data analysis.

Objectives: This study aimed to employ big data analysis to harmonize reference intervals (RI) for thyroid function tests, with refinement to the TSH upper reference limit, and to optimize the TSH reflex algorithm to improve clinical management and test utilization.

Design & Methods: TSH, free T4, and free T3 results tested in Alberta, Canada, on Roche Cobas and Siemens Atellica were extracted from the laboratory information system (N = 1,144,155 for TSH, N = 183,354 for free T4 and N = 92,632 for free T3). Results from specialists, inpatients, or repeat testing, as well as from positive thyroid disease, autoimmune disease, and pregnancy biomarkers were excluded.

Low partial pressure of oxygen causes significant and unrecognized under-recovery of glucose on blood gas analyzers.

Background: Blood gas analyzers employing glucose-oxidase biosensors under-recover glucose when pO is low. The manufacturer of the GEM®Premier™ series of analyzers introduced an algorithm to detect specimens at risk of low pO interference. We investigated the reliability of this algorithm.

Not T too! False elevations in high-sensitivity cardiac troponin T (hs-TnT) following specimen transport.

Though false elevations attributed to preanalytical specimen handling have been widely reported for Troponin I (TnI), Troponin T (TnT) has appeared more robust to falsely elevated Tn. We describe reproducible false elevations in high sensitivity TnT (hs-TnT) in specimens after courier transport in plasma separator tubes (PST) off-site for testing. Hs-TnT was measured under 5 different conditions: 1) at collection location (N = 24); 2) after transport upright in racks (N = 66); 3) after transport with no control over tube agitation (N = 69); 4) on transported aliquots (N = 84); or 5) immediately after transport with no control over tube agitation (N = 16), followed by keeping the specimen upright and re-measuring at 1hr, 2hr, 4hr, and 20-24hrs (N = 6).

Filling in the GAPS: validation of anion gap (AGAP) measurement uncertainty estimates for use in clinical decision making.

Objectives: We compare measurement uncertainty (MU) calculations to real patient result variation observed by physicians using as our model anion gap (AGAP) sequentially measured on two different instrument types. An approach for discretely quantifying the pre-analytical contributions and validating AGAP MU estimates for interpretation of patient results is proposed.

Methods: AGAP was calculated from sodium, chloride, and bicarbonate reported from chemistry or blood gas analyzers which employ different methodologies and specimen types.

A high-throughput test for diabetes care: An evaluation of the next generation Roche Cobas c 513 hemoglobin A assay.

Objectives: The level of glycated hemoglobin A (HbA) in blood is the preferred marker for diabetes monitoring and treatment. Here, we evaluate the analytical performance of the Roche Diagnostics Cobas c 513, a stand-alone HbA immunoassay analyzer.

Design And Methods: Performance was assessed with regards to imprecision, accuracy, linearity, method comparison against the Roche Cobas Integra 800 CTS, specimen stability, interference from common hemoglobin variants and hemoglobin F, and throughput.

Strategies for mitigating risk posed by biotin interference on clinical immunoassays.

Recently, biotin use as an oral supplement has increased significantly among the general population. Biotin is a water soluble B-vitamin and is marketed to improve the cosmetic appearance of hair, skin, and nails. In addition, high-dose biotin (>5 mg/day) is prescribed to treat inborn errors of metabolism and multiple sclerosis.

Stabilizing specimens for routine ammonia testing in the clinical laboratory.

Background: In vitro deamination generates ammonia in freshly collected blood specimens. To prevent this, samples for ammonia testing are usually collected on ice and run rapidly (e.g.

Erroneous HbA1c results in a patient with elevated HbC and HbF.

Background: HbA1c is used in the diagnosis and monitoring of diabetes mellitus (DM). Interference from hemoglobin variants is a well-described phenomenon, particularly with HPLC-based methods. While immunoassays may generate more reliable HbA1c results in the presence of some variants, these methods are susceptible to negative interference from high concentrations of HbF.

Comparing the calcium binding abilities of two soybean calmodulins: towards understanding the divergent nature of plant calmodulins.

The discovery that plants contain multiple calmodulin (CaM) isoforms of variable sequence identity to animal CaM suggested an additional level of sophistication in the intracellular role of calcium regulation in plants. Past research has focused on the ability of conserved or divergent plant CaM isoforms to activate both mammalian and plant protein targets. At present, however, not much is known about how these isoforms respond to the signal of an increased cytosolic calcium concentration.

Structural analysis of a calmodulin variant from rice: the C-terminal extension of OsCaM61 regulates its calcium binding and enzyme activation properties.

OsCaM61 is one of five calmodulins known to be present in Oryza sativa that relays the increase of cytosolic [Ca(2+)] to downstream targets. OsCaM61 bears a unique C-terminal extension with a prenylation site. Using nuclear magnetic resonance (NMR) spectroscopy we studied the behavior of the calmodulin (CaM) domain and the C-terminal extension of OsCaM61 in the absence and presence of Ca(2+).

Structural characterization of the interaction of human lactoferrin with calmodulin.

Lactoferrin (Lf) is an 80 kDa, iron (Fe(3+))-binding immunoregulatory glycoprotein secreted into most exocrine fluids, found in high concentrations in colostrum and milk, and released from neutrophil secondary granules at sites of infection and inflammation. In a number of cell types, Lf is internalized through receptor-mediated endocytosis and targeted to the nucleus where it has been demonstrated to act as a transcriptional trans-activator. Here we characterize human Lf's interaction with calmodulin (CaM), a ubiquitous, 17 kDa regulatory calcium (Ca(2+))-binding protein localized in the cytoplasm and nucleus of activated cells.

Altered mechanical properties of titin immunoglobulin domain 27 in the presence of calcium.

Titin (connectin) based passive force regulation has been an important physiological mechanism to adjust to varying muscle stretch conditions. Upon stretch, titin behaves as a spring capable of modulating its elastic response in accordance with changes in muscle biochemistry. One such mechanism has been the calcium-dependent stiffening of titin domains that renders the spring inherently more resistant to stretch.

Structural insights into calmodulin-regulated L-selectin ectodomain shedding.

The L-selectin glycoprotein receptor mediates the initial steps of leukocyte migration into secondary lymphoid organs and sites of inflammation. Following cell activation through the engagement of G-protein-coupled receptors or immunoreceptors, the extracellular domains of L-selectin are rapidly shed, a process negatively controlled via the binding of the ubiquitous eukaryotic calcium-binding protein calmodulin to the cytoplasmic tail of L-selectin. Here we present the solution structure of calcium-calmodulin bound to a peptide encompassing the cytoplasmic tail and part of the transmembrane domain of L-selectin.

Fast methionine-based solution structure determination of calcium-calmodulin complexes.

Here we present a novel NMR method for the structure determination of calcium-calmodulin (Ca(2+)-CaM)-peptide complexes from a limited set of experimental restraints. A comparison of solved CaM-peptide structures reveals invariability in CaM's backbone conformation and a structural plasticity in CaM's domain orientation enabled by a flexible linker. Knowing this, the collection and analysis of an extensive set of NOESY spectra is redundant.

Effects of metal-binding loop mutations on ligand binding to calcium- and integrin-binding protein 1. Evolution of the EF-hand?

Calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous, multifunctional regulatory protein consisting of four helix-loop-helix EF-hand motifs. Neither EF-I nor EF-II binds divalent metal ions; however, EF-III is a mixed Mg2+/Ca2+-binding site, and EF-IV is a higher-affinity Ca2+-specific site. Through the generation of several CIB1 mutant proteins, we have investigated the importance of the last (-Z) metal-coordinating position of EF-III (D127) and EF-IV (E172) with respect to the binding of CIB1 to Mg2+, Ca2+, and its biological target, the cytoplasmic domain of the platelet alphaIIb integrin.

Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs.

The 'EF-hand' Ca2+-binding motif plays an essential role in eukaryotic cellular signalling, and the proteins containing this motif constitute a large and functionally diverse family. The EF-hand is defined by its helix-loop-helix secondary structure as well as the ligands presented by the loop to bind the Ca2+ ion. The identity of these ligands is semi-conserved in the most common (the 'canonical') EF-hand; however, several non-canonical EF-hands exist that bind Ca2+ by a different co-ordination mechanism.

Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends.

Non-homologous end joining (NHEJ) is one of the primary pathways for the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in mammalian cells. Proteins required for NHEJ include the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku, XRCC4 and DNA ligase IV. Current models predict that DNA-PKcs, Ku, XRCC4 and DNA ligase IV assemble at DSBs and that the protein kinase activity of DNA-PKcs is essential for NHEJ-mediated repair of DSBs in vivo.