Evaluation of the 25-hydroxy vitamin D assay on a fully automated liquid chromatography mass spectrometry system, the Thermo Scientific Cascadion SM Clinical Analyzer with the Cascadion 25-hydroxy vitamin D assay in a routine clinical laboratory.

Background Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers advantages over immunoassay due to its increased specificity and ability to multiplex metabolites within a single run. Wide scale adoption of LC-MS/MS in routine clinical laboratories is restricted in part due to the high level of technical expertise required. The Thermo Scientific™ Cascadion™ SM Clinical Analyzer is the first fully automated, random access clinical analyser that utilises LC-MS/MS technology. We report an analytical validation of the 25-hydroxy vitamin D2 and D3 assays on the Cascadion Analyzer and an assessment of its performance within a routine clinical laboratory. Methods Analyser usability was assessed by staff with no previous experience of LC-MS/MS. An analytical validation included analysis of 154 patient samples on two different Cascadion Analyzers and a four-way method comparison of 146 patient samples on Roche and Siemens immunoassays and an in-house LC-MS/MS method. Accuracy was assessed using external quality assurance and reference materials. Seven third party IQC materials were tested on Cascadion. Results Cascadion proved easy to use by scientific and non-scientific staff. The assay passed all validation criteria. Excellent agreement was demonstrated between two different Cascadions (y = 0.97x + 3.9 nmol/L, r2 > 0.99). A method comparison demonstrated no significant difference (p > 0.05) between the Cascadion and the Roche immunoassay. A significant difference (p < 0.0001) was observed between the Cascadion and an LC-MS/MS and Siemens methods. Results obtained from EQA and reference material showed a mean bias of +3.09% and all samples were within ±10% of assigned concentrations. All third party IQC samples tested were compatible for use with Cascadion. Conclusions The Cascadion Analyzer is a fully automated LC-MS/MS system that requires no prior LC-MS/MS expertise. The vitamin D assays demonstrated excellent performance with high levels of accuracy.

The amount of self-antigen determines the effector function of murine T cells escaping negative selection.

Autoimmune diseases develop when self-specific T cells that escaped negative selection initiate a harmful immune response against self. However, factors, which influence the initiation and progression of an autoimmune response remain incompletely understood. By establishing a double-transgenic BALB/c mouse system in which different amounts of a cell-surface neo-self-antigen are expressed under the CD11c promoter, we demonstrate that antigen dose dramatically influences T-cell tolerance mechanisms. Moderate antigen expression in both hematopoietic and nonhematopoietic cells favors the development of antigen-specific Treg cells and the establishment of a tolerogenic environment. In marked contrast, a high dose of antigen expression results in very stringent negative selection, in poor development of antigen-specific Treg cells and in the early onset of anemia and splenomegaly and the late development of arthritis and high titers of IgG auto Abs. Disease is initiated by autoreactive T cells, which escape negative selection by expressing a second TCR with a different specificity or an altered affinity. Transfer of Ag-specific Treg cells ameliorates the early onset signs of disease but does not prevent the development of long-term chronic pathologies. Altogether, our results suggest that Ag dose directly affects Treg-cell generation and thus, the set-up of T-cell tolerance.

Seasonal variability of the vitamin D effect on physical fitness in adolescents.

Studies investigating the relationship between vitamin D and physical fitness in youth have provided inconsistent findings. Recent evidence indicates that the expression of receptors and vitamin D-modulated genes in young subjects has a seasonal profile. Therefore, we investigated the role of vitamin D on physical fitness across seasons in a total of 977 male adolescents. Anthropometrics, lifestyle, dietary habits, biochemical profiles and physical fitness were studied. Multiple linear regression models, including pairwise interaction terms involving total 25-OH-vitamin D, were fitted. The interacting effect of season and total 25-OH-vitamin D had a significant influence on physical fitness performance (spring and total 25-OH-vitamin D: ß 0.19, SE 0.07, p = 0.007; summer and total 25-OH-vitamin D: ß 0.10, SE 0.06, p = 0.11; autumn and total 25-OH-vitamin D: ß 0.18, SE 0.07, p = 0.01), whereas the main effect of total 25-OH-vitamin D alone was not significant (p = 0.30). Body fat percentage, recreational physical activity level, time spent per day gaming/TV-watching, smoking, and hemoglobin levels were also related to the physical fitness performance score. Future studies should further explore the role of seasonal-dependent effects of vitamin D on health.

Anatomical 3D fiber-deposited scaffolds for tissue engineering: designing a neotrachea.

The advantage of using anatomically shaped scaffolds as compared to modeled designs was investigated and assessed in terms of cartilage formation in an artificial tracheal construct. Scaffolds were rapid prototyped with a technique named three-dimensional fiber deposition (3DF). Anatomical scaffolds were fabricated from a patient-derived computerized tomography dataset, and compared to cylindrical and toroidal tubular scaffolds. Lewis rat tracheal chondrocytes were seeded on 3DF scaffolds and cultured for 21 days. The 3-(4,5-dimethylthiazol-2yl)-2,5-dyphenyltetrazolium bromide (MTT) and sulfated glycosaminoglycan (GAG) assays were performed to measure the relative number of cells and the extracellular matrix (ECM) formed. After 3 weeks of culture, the anatomical scaffolds revealed a significant increase in ECM synthesis and a higher degree of differentiation as shown by the GAG/MTT ratio and by scanning electron microscopy analysis. Interestingly, a lower scaffold's pore volume and porosity resulted in more tissue formation and a better cell differentiation, as evidenced by GAG and GAG/MTT values. Scaffolds were compliant and did not show any signs of luminal obstruction in vitro. These results may promote anatomical scaffolds as functional matrices for tissue regeneration not only to help regain the original shape, but also for their improved capacity to support larger tissue formation.