Fibrinogen levels were derived from the clotting curve of the prothrombin time assay, using Thromborel S (Behringwerke, Marburg, Germany) on the ACL 300 coagulation analyzer (Instrumentation Laboratory). vWF:Ag levels were measured with an in-house ELISA using polyclonal rabbit antihuman VWF antibodies and horseradish-peroxidase-conjugated antihuman VWF antibodies (DakoCytomation, Glostrup, Denmark) to catch and tag vWF. ADAMTS13 activity was measured in a kinetic assay using Fluorescence Resonance Energy Transfer Substrate VWF 73 (FRETS-VWF73), as is thoroughly described in the previous articles [16 (link), 17 (link)].
We determined NET levels by measuring MPO–DNA complexes with an ELISA as reported earlier [18 (link)]. We adjusted the commercial human cell death ELISA kit (Cell death detection ELISAPLUS, Roche Diagnostics Nederland B.V., Almere, The Netherlands). Briefly, as the capturing antibody, we used anti-MPO monoclonal antibody (clone 4A4, ABD Serotec). Patient plasma was added in combination with the peroxidase-labeled anti-DNA monoclonal antibody (from cell death detection ELISA kit; Roche). The absorbance at 405 nm wavelength was measured using Biotek Synergy HT plate reader with a reference filter of 490 nm. The values are expressed as milli-arbitrary units (mAU/mL).
We determined NET levels by measuring MPO–DNA complexes with an ELISA as reported earlier [18 (link)]. We adjusted the commercial human cell death ELISA kit (Cell death detection ELISAPLUS, Roche Diagnostics Nederland B.V., Almere, The Netherlands). Briefly, as the capturing antibody, we used anti-MPO monoclonal antibody (clone 4A4, ABD Serotec). Patient plasma was added in combination with the peroxidase-labeled anti-DNA monoclonal antibody (from cell death detection ELISA kit; Roche). The absorbance at 405 nm wavelength was measured using Biotek Synergy HT plate reader with a reference filter of 490 nm. The values are expressed as milli-arbitrary units (mAU/mL).
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