46 Chapter 3 of VWF and platelets to ristocetin by quantifying the density of VWF on the plate- let surface on a flow cytometer after stimulation of whole blood with ristocetin.25 This test quantifies the binding of activated VWF to the GP-Ib-IX-V receptor on platelets. Figure 3 presents the ristocetin- induced VWF binding to platelets in patients with HMB, regarding the reference ranges, determined in 123 healthy controls as pre- sented in Table 1. In total, we observed 2 patients with reduced VWF function. We also measured VWF levels and VWF ristocetin cofactor activity, and the 2 patients with reduced VWF function had also lower VWF ristocetin co-factor activity (Figure 3). Figure 3: VWF function (VWFact_FC) was measured with binding density of VWF to platelets after stimulation with ristocetin. The values of 58 patients with HMB and 26 controls are plotted, and median values ±2 SD are shown. The grey area represents the reference ranges for VWF function determined in 123 healthy employees of the UMC Utrecht. All patients that fell below the grey area were classified as VWF function disorder. A total of 2 patients of the 58 (3.4%) were defined with a VWF defect. We also measured VWF function with ristocetin co-factor activity (middle graph) and VWF antigen (right graph). For the latter two measurements, we defined a cut-off value of 50%. We used similar strategy to determine the number of HMB patients with impaired coagulation. Reference values were obtained from TG tests in PPP of 126 healthy employees of the MUMC. TG shows the time-dependent dynamics of thrombin, after initiation of coagulation. The TG curves are quantified with the lag time (Figure 4A), the time to reach the maximum amount of thrombin (time to peak; ttPeak; Figure 4B), the maximum amount of thrombin that is formed (Peak; Figure 4C) and the total amount of thrombin that is formed (endogenous thrombin potential; ETP; Figure 4D). All outcome parameters were normalized to NPP,21 and the control plasma samples were collected at the same moment as the plasma samples of the patients with HMB. TG was measured upon stimulation with 1 pmol/L tissue factor and upon stimulation with 5 pmol/L TF. If at least one of the normalized TG values of a patient with HMB falls outside the reference range, then this patient will be classified with a coagulation defect. Using this strategy, we observed that 17 (29%) patients were defined with a coagulation defect. There
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