Homocysteine measurement by Vitros Microtip homocysteine assay.

BACKGROUND: Elevated circulating total homocysteine is an independent vascular risk factor. Enzymatic homocysteine measurements represent an alternative to HPLC- or immunochemistry-based assays, suitable for automation. Here, we report on analytical performance of a commercial cystathionine beta-synthase-based assay, for use on Vitros automated analyzers. METHODS: Linear range, limit of detection and analytical sensitivity were inferred from duplicate measurements of homocystine standard solutions (1-65 micromol/L). Imprecision was assessed using commercial controls according to NCCLS EP5-A2 and accuracy using NIST-SRM1955 reference material. Agreement with a clinically validated HPLC method was examined on 207 patient samples. RESULTS: The enzymatic assay was linear from 1 to 90 micromol/L homocysteine. Total (within-day) imprecision ranged from 4.5 (3.9)% to 2.8 (1.6)% at homocysteine 9.7-43.2 micromol/L. Accuracy was acceptable at 8.9 and 17.7 micromol/L homocysteine, with +6.4% and -1.2% bias, respectively, but showed substantial negative bias (-20.1%) at 4.0 micromol/L. High triglycerides (19.8 micromol/L) negatively interfered. The enzymatic method was slightly less sensitive than the HPLC method (limit of detection 0.7 and 0.2 micromol/L, respectively) but correlated well with the latter (r2=0.9997, slope=1.04, intercept=-0.66 micromol/L) and was more precise (p<0.05). CONCLUSIONS: The Vitros homocysteine assay met the CLIA Desirable Analytical Quality Specifications at homocysteine > or = 9 micromol/L. Its analytical performance and suitability for automation make the Vitros assay an analytically acceptable alternative to HPLC-based methods.     

Enhanced suppressive capacity of tumor‐infiltrating myeloid‐derived suppressor cells compared with their peripheral counterparts

Although the main site of action for myeloid‐derived suppressor cells (MDSCs) is most likely the tumor microenvironment, so far the study of these cells has been largely restricted to spleen‐derived MDSCs. In this study, we compared the suppressive capacity of splenic and tumor‐derived MDSCs in different subcutaneous mouse tumor models. We investigated which suppressive mechanisms were involved. Finally, we investigated whether MDSCs and regulatory T cells (T_reg) cooperate in the suppression of T‐cell responses. In all models, splenic granulocytic MDSCs (grMDSC) strongly suppress CD4⁺ T‐cell proliferation while the suppressive effect on CD8⁺ T cells is less pronounced. Splenic monocytic MDSCs (moMDSC) have a lower suppressive capacity, compared to grMDSC, on both CD4⁺ and CD8⁺ T‐cell proliferation. Both grMDSC and moMDSC isolated from the tumor have a much stronger suppressive activity compared to MDSCs isolated from the spleen of tumor‐bearing mice, associated with a higher NO₂^? production by the tumor‐derived moMDSC and arginase activity for both subsets. The expression of CD80 is also elevated on tumor‐derived grMDSC compared with their peripheral counterparts. Direct contact with tumor cells is required for the upregulation of CD80 and CD80⁺ MDSCs are more suppressive than CD80^? MDSCs. Coculture of T_reg and MDSCs leads to a stronger suppression of CD8⁺ T‐cell proliferation compared to the suppression observed by T_reg or MDSCs alone. Thus, we showed that tumor‐infiltrating MDSCs possess a stronger suppressive capacity than their peripheral counterparts and that various suppressive mechanisms account for this difference.     

Observ-OM and Observ-TAB: Universal syntax solutions for the integration, search, and exchange of phenotype and genotype information

Genetic and epidemiological research increasingly employs large collections of phenotypic and molecular observation data from high quality human and model organism samples. Standardization efforts have produced a few simple formats for exchange of these various data, but a lightweight and convenient data representation scheme for all data modalities does not exist, hindering successful data integration, such as assignment of mouse models to orphan diseases and phenotypic clustering for pathways. We report a unified system to integrate and compare observation data across experimental projects, disease databases, and clinical biobanks. The core object model (Observ-OM) comprises only four basic concepts to represent any kind of observation: Targets, Features, Protocols (and their Applications), and Values. An easy-to-use file format (Observ-TAB) employs Excel to represent individual and aggregate data in straightforward spreadsheets. The systems have been tested successfully on human biobank, genome-wide association studies, quantitative trait loci, model organism, and patient registry data using the MOLGENIS platform to quickly setup custom data portals. Our system will dramatically lower the barrier for future data sharing and facilitate integrated search across panels and species. All models, formats, documentation, and software are available for free and open source (LGPLv3) at http://www.observ-om.org.     

Image navigation as a means to expand the boundaries of fluorescence-guided surgery

Hybrid tracers that are both radioactive and fluorescent help extend the use of fluorescence-guided surgery to deeper structures. Such hybrid tracers facilitate preoperative surgical planning using (3D) scintigraphic images and enable synchronous intraoperative radio- and fluorescence guidance. Nevertheless, we previously found that improved orientation during laparoscopic surgery remains desirable. Here we illustrate how intraoperative navigation based on optical tracking of a fluorescence endoscope may help further improve the accuracy of hybrid surgical guidance. After feeding SPECT/CT images with an optical fiducial as a reference target to the navigation system, optical tracking could be used to position the tip of the fluorescence endoscope relative to the preoperative 3D imaging data. This hybrid navigation approach allowed us to accurately identify marker seeds in a phantom setup. The multispectral nature of the fluorescence endoscope enabled stepwise visualization of the two clinically approved fluorescent dyes, fluorescein and indocyanine green. In addition, the approach was used to navigate toward the prostate in a patient undergoing robot-assisted prostatectomy. Navigation of the tracked fluorescence endoscope toward the target identified on SPECT/CT resulted in real-time gradual visualization of the fluorescent signal in the prostate, thus providing an intraoperative confirmation of the navigation accuracy.