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In critically ill patients, the massive release of angiopoietin-2 (Ang-2) from endothelial Weibel–Palade bodies interferes with constitutive angiopoietin-1 (Ang-1)/Tie2 signaling in endothelial cells, thus leading to vascular barrier breakdown followed by leukocyte transmigration and capillary leakage. The use of circulating Ang-1 and Ang-2 as novel biomarkers of endothelial integrity has therefore gained much attention. The preclinical characteristics and clinical applicability of angiopoietin immunoassays, however, remain elusive.    Methods  We developed sandwich immunoassays for human Ang-1 (immunoradiometric sandwich assay/immunoluminometric sandwich assay) and Ang-2 (ELISA), assessed preanalytic characteristics, and determined circulating Ang-1 and Ang-2 concentrations in 30 healthy control individuals and in 94 critically ill patients. In addition, Ang-1 and Ang-2 concentrations were measured in 10 patients during a 24-hour time course with respect to interference by intravenous antibiotic treatment and by extended daily dialysis.    Results  The assays had detection limits of 0.12 ng/ml (Ang-1) and 0.2 ng/ml (Ang-2). Inter-assay and intra-assay imprecision was ≤8.8% and 3.7% for Ang-1 and was ≤4.6% and 5.2% for Ang-2, respectively. Angiopoietins were stable for 24 hours and were resistant to four freeze–thaw cycles. Angiopoietin concentrations were not associated with age, body mass index or renal function in healthy individuals. Ang-1 and Ang-2 concentrations correlated with severity of illness in critically ill patients. Angiopoietin concentrations were not influenced by antibiotic treatment or by extended daily dialysis.    Conclusion  Ang-1 and Ang-2 might serve as a novel class of biomarker in critically ill patients. According to preclinical and clinical validation, circulating Ang-1 and Ang-2 can be reliably assessed by novel immunoassays in the intensive care unit setting.

Circulating angiopoietin-1 and angiopoietin-2 in critically ill patients: development and clinical application of two new immunoassays