42 Chapter 3 values were normalized against MFI values of control subject measured on the same day as the patient. VWF antigen levels and VWF ristocetin co-factor activity were measured in PPP on a STA-R Evolution® analyser with the STALIATEST® vWF:ag and the ABP VWF RICOF assay, respectively (Diagnostica Stago, Asnieres, France). The ABP VWF RICOF assay measures the ability of patients’ plasma to agglutinate formalin-fixed platelets in the presence of ristocetin. Reference values for platelet function and VWF function Reference ranges of platelet function values were measured in 123 healthy volunteers, who donated their blood for platelet function measurements in the University Medical Centre Utrecht (UMCU). Reference ranges were determined for each agonist, both for P-selectin expression and for αIIbβ3 activation. Values below mean minus twofold standard deviation were defined as abnormal. We determined the reference range for VWF function in the same blood samples, by stimulating whole blood with ristocetin and measuring the VWF binding to platelets. Like these criteria, we defined patients as having abnormal VWF, if their VWF function falls below the mean minus twofold the standard deviation of the reference group. Prothrombin time and activated partial thromboplastin time Prothrombin time (PT) was determined in platelet-poor plasma obtained from citrate anticoagulated whole blood. Analysis was performed on a STA-R®coagulation analyser (Diagnostica Stago, Asnieres, France) based on electromechanical clot detection. Plasma was incubated with recombinant thromboplastin STA®-Neoplastin (Diagnostica Stago, Asnieres, France), and after addition of CaCl2, PT was measured upon clot formation. According to the similar electro- mechanical detection method, activated partial thromboplastin time (APTT) was determined using the kaolin-based STA Cephascreen® reagents (Diagnostica Stago, Asnieres, France). Thrombin generation Thrombin generation (TG) was measured in a 96-well plate fluorimeter (Ascent reader, Thermo labsystems OY, Helsinki Finland) using the Calibrated Automated Thrombinography (CAT) method as described by Hemker et al.24 All wells contained 80 μL PPP and ei- ther 20 μL of recombinant tissue factor (1 or 5 pmol/L; TF Innovin® from Dade Behring, Germany) and 4 μmol/L phospholipids containing 20 mol% phosphatidylserine, 60 mol% phosphatidylcholine and 20 mol% phosphatidyl-ethanolamine (Avanti Polar Lipids Inc, USA) or 20 μL of calibrator (α2macroglobulin-thrombin (α2M-T) complex prepared in-house).24 Thrombin generation was initiated by the addition of 20 μL purchased from Bachem, Basel, Switzerland) (417 μmol/L) and CaCl2 (16.7 mmol/L). The TG curves and their parameters were derived from the fluorescence curves using the dedicated Thrombinoscope software v3.0 provided with the CAT method.21 TG was adjusted to normal pooled plasma. The signal of NPP was set on 100%, and the signal of the patient sample was presented as a percentage of the NPP signal (23). The reference values for TG were determined in PPP of 126 healthy volunteers, who donated blood to serve as reference for the normal population. The CVs of all TG parameters were ≤5% (23).
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