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Background Very sensitive measurements of serum estrogens and testosterone are important in adult and pediatric endocrinology and immunoassays are known to lack the required performance at very low levels. Our aim was to develop a sensitive HPLC–MS/MS assay for both estradiol (E 2 ) and testosterone (Te) in serum without the need for chemical derivatization and using commercially available calibrators. Methods Serum samples were prepared by the addition of internal standards followed by extraction using hexane:ethyl acetate. Chromatographic separation was achieved using a C 18 column and mass spectrometry was performed in both positive and negative ion modes. Results The lower limits of quantitation (LLOQs) of E 2 and Te were 5 pg/mL and 1 ng/dL, respectively. The analytical measurement range (AMR) for E 2 was 5–600 pg/mL and 1–1,170 ng/dL for Te. Assay accuracy was determined both by comparison with a LC–MS/MS method performed at a national laboratory and the CDC HoSt program. Comparison with samples analyzed by both methods showed excellent correlation. Within-day (N = 10) and between-day (N = 20) CVs at concentrations spanning the AMR were less than 7% for both analytes. Conclusion We have developed an accurate and highly sensitive assay to measure E 2 and Te levels in serum by HPLC–MS/MS without chemical derivatization and using commercially available calibrators. [ABSTRACT FROM AUTHOR]

Ewing sarcoma family of tumors (ESFT) is a group of aggressive pediatric malignancies driven by the EWS-FLI1 fusion protein, an aberrant transcription factor up-regulating specific target genes, such as neuropeptide Y (NPY) and its Y1 and Y5 receptors (Y5Rs). Previously, we have shown that both exogenous NPY and endogenous NPY stimulate ESFT cell death via its Y1 and Y5Rs. Here, we demonstrate that this effect is prevented by dipeptidyl peptidases (DPPs), which cleave NPY to its shorter form, NPY3-36, not active at Y1Rs. We have shown that NPY-induced cell death can be abolished by overexpression of DPPs and enhanced by their down-regulation. Both NPY treatment and DPP blockade activated the same cell death pathway mediated by poly(ADP-ribose) polymerase (PARP-1) and apoptosis-inducing factor (AIF). Moreover, the decrease in cell survival induced by DPP inhibition was blocked by Y1 and Y5R antagonists, confirming its dependence on endogenous NPY. Interestingly, similar levels of NPY-driven cell death were achieved by blocking membrane DPPIV and cytosolic DPP8 and DPP9. Thus, this is the first evidence of these intracellular DPPs cleaving releasable peptides, such as NPY, in live cells. In contrast, another membrane DPP, fibroblast activation protein (FAP), did not affect NPY actions. In conclusion, DPPs act as survival factors for ESFT cells and protect them from cell death induced by endogenous NPY. This is the first demonstration that intracellular DPPs are involved in regulation of ESFT growth and may become potential therapeutic targets for these tumors. [ABSTRACT FROM AUTHOR]

Abstract: Background: Cyclosporine (CsA) monitoring is essential for transplant success. We report a performance study of the recently released, fully automated Siemens ADVIA Centaur® CsA assay. Methods: Whole blood samples from 248 transplant patients were prepared using a new 1-step extraction method. Performance evaluations vs. HPLC–tandem MS (LC-MS/MS), Abbott TDx and AxSYM assays were conducted according to CLSI EP5-A2 and EP9-A2 guidelines. Results: The correlation coefficient for LC-MS/MS and ADVIA Centaur was≥0.97 at each site, and for each transplant type. Regression analysis yielded y =0.94x +19 for all sites: 95% CI=0.91–0.96 (slope) and 10–28 (intercept). Absolute and relative bias was minimal for C0 and C2 sampling. Centaur vs. Abbott TDx and AxSYM assays: y = 0.72x +6, r = 0.98, 95% CI = 0.70–0.73 (slope), 3–9 (intercept); and y = 0.69x +18, r = 0.97, 95% CI = 0.67–0.71 (slope), 8–27(intercept). Within run CVs were 4.5%–7.1%, total CVs were 5.3%–7.7%. Conclusions: The ADVIA Centaur assay compared favorably with LC-MS/MS and Abbott assays, displaying good correlation for all transplant types and methods. [Copyright &y& Elsevier]