Circulating angiopoietin-1 and angiopoietin-2 in critically ill patients: development and clinical application of two new immunoassays.
BACKGROUND
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 < or = 8.8% and 3.7% for Ang-1 and was < or = 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.


CONCLUSIONS
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.
Lukasz
Alexander
A
Department of Nephrology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany.
Hellpap
Julian
J
Horn
Rüdiger
R
Kielstein
Jan T
JT
David
Sascha
S
Haller
Hermann
H
Kümpers
Philipp
P
D000328:Adult; D000368:Aged; D042683:Angiopoietin-1 / Q000097:blood*; D042702:Angiopoietin-2 / Q000097:blood*; D000818:Animals; D015415:Biomarkers / Q000097:blood; D002460:Cell Line; D016638:Critical Illness; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods / Q000639:trends*; D008297:Male; D051379:Mice; D008875:Middle Aged; D012720:Severity of Illness Index
C467630:ANGPT1 protein, human; D042683:Angiopoietin-1; D042702:Angiopoietin-2; D015415:Biomarkers