Summary. Background: Diagnosis of acquired von Willebrand syndrome (AVWS) remains challenging. Diagnostic algorithms suggest the use of factor VIII (FVIII:C), von Willebrand factor antigen (VWF:Ag), ristocetin cofactor (VWF:RCo), and collagen‐binding capacity (VWF:CB), but the sensitivity of these and other laboratory tests for the diagnosis of AVWS is unknown. Objectives: To analyze the capacity of laboratory tests, including point‐of‐care testing (POCT), for the identification of patients with AVWS. Patients/methods: Thirty‐five consecutive patients were enrolled with AVWS diagnosed because of a history of recent onset of bleeding, a negative family history of von Willebrand disease, and abnormal plasma VWF multimers. Results: According to our inclusion criteria, all patients had bleeding symptoms, and the VWF high molecular weight multimers were either decreased or absent. Regarding POCT, PFA‐100 was inconclusive, due to anemia or thrombocytopenia, in 29%; the sensitivity was 80% in the remaining patients. The sensitivity of VWF:Ag (23%), VWF:RCo/Ag ratio < 0.7 (26%), VWF:CB/Ag ratio < 0.7 (46%), anti‐VWF antibodies (15%) and VWF propeptide/Ag ratio (22%) was too low to rule out the disease. A combination of VWF:Ag < 50 IU dL−1, VWF:RCo/Ag ratio < 0.7 and VWF:CB/Ag ratio < 0.8 yielded a sensitivity of 86%. Patients diagnosed only because of abnormal VWF multimers showed similar clinical characteristics as other patients. Conclusions: Early diagnosis of AVWS is difficult, due to lack of sensitivity of the tests used. A substantial number of patients present with normal or increased test results, emphasizing the importance of multimer analysis in all patients with suspected AVWS.
While the procoagulant activity of platelet derived microparticles (PMP) has been widely accepted, knowledge regarding their immunological and adhesive qualities is still limited. It has been shown that murine BM cells covered with PMP engrafted lethally irradiated mice significantly faster than those not covered, indicating that PMPs play an important role in the homing of peripheral blood stem cells (PBSC). Here we studied the impact of PMP on engraftment in human allogeneic PBSC transplants for patients with hematological malignancies. PBSC samples were collected in buffered citrate from transplantation bags after infusion of transplants into patients with hematological malignancies (AML = 5, ALL = 1). Conditioning regimens included busulfan/cyclophosphamide (Bu/Cy), anti-CD66b-radioimmunotherapy (RIT)/Bu/Cy, and reduced intensity regimens with fludarabin/busulfan (Flu/Bu) and FLAMSA. Platelet-poor plasma (PPP) was prepared (1500g for 20min), immediately shock-frozen in liquid nitrogen and stored at −80°C. For further analysis PPP’s were carefully thawed at room temperature (RT). 90μl of PPP was stained with 5μl of CD41-PE and CD62P-FITC each for 15min at RT in the dark (IgG1-FITC and -PE served as negative controls, TRAP-6 (10μM) stimulated whole blood processed in same way as samples as positive control). To stop staining 900μl PBS/BSA 2% was added and 500μl of this solution were transferred into BD Trucount tubes by reverse pipetting giving a final concentration of 100 beads/μl. Samples were analyzed immediately using Coulter FC500 flow cytometer with CXP software. As expected the CD34 cell count (mean=5.1x106/kg body weight, SD=2.0x106/kg) showed a significant correlation (p=0.0197, Pearson r=−0.83) with the time to engraftment (mean=15.7days, SD=2.0d). The amount of CD62P positive microparticles (mean=423/μl, SD=119/μl) and the conditioning regimen showed no significant correlation with CD34 cell count or time to engraftment with leucocytes >1000/μl. In contrast, CD41-PMP count (mean=1223/μl, SD=857μl) correlated significantly with the CD34 cell count (p=0.0086, Pearson r=0.92) and the time to engraftment (p=0.0039, Pearson r = −0.95). Therefore, PBSCT contain significant amounts of PMP which are most likely generated during apheresis. Preliminary results show a stronger correlation with time to engraftment than does CD34 cell count. We conclude that PMP may accelerate engraftment of PBSC in humans. However, this function seems unrelated to P-Selectin expression. Therefore, further studies aiming to identify other adhesion molecules involved in PMP-mediated engraftment of PBSCT are warranted.
Acquired von Willebrand’s syndrome (AVWS) is a rare but probably underdiagnosed hemorrhagic disorder often associated with hematological or cardiovalvular disorders. Diagnostic workup remains challenging, particularly in patients with normal or increased von Willebrand factor antigen (Ag) and ristocetin cofactor (RCo). Here, we present a retrospective single-center study of 35 patients diagnosed with AVWS based on (i) a history of recent onset of bleeding, (ii) a negative family history of von Willebrand’s disease, and (iii) abnormal plasma VWF multimers. AVWS was associated with monoclonal gammopathy (n=11), cardiovalvular disorders (n=16), or other conditions (n=8) including myeloproliferative and autoimmune disorders. The PFA-100® screening test was inconclusive due to anemia (hematocrit <30 %) or thrombocytopenia (<100/nl) in 10 patients (29 %); prolonged closure times were observed using collagen/epinephrine and collagen/adrenalin in 20 of 25 (80 %) and 18 of 25 (72 %) patients, respectively. Factor VIII:C was reduced below 50 IU/dl in 7 of 35 patients (20 %). VWF Ag and RCo were reduced below 50 IU/dl in 8 patients (23 %). VWF Ag and RCo were normal or increased in all patients with cardiovalvular disease and in four of eleven patients with gammopathy. Median VWF Ag was higher in cardiovalvular disease (median 202 IU/dl, range 90 to 608) compared to gammopathy (median 31 IU/dl, range 8 to 468, p<0.02 by Mann Whitney U test). Of 27 patients with normal or increased VWF Ag and RCo, 12 (44 %) had a reduced collagen binding activity (CBA) or CBA to Ag ratio <0.7; 10 (37 %) had a borderline CBA ratio between 0.7 and 0.8; 5 (19 %) had a normal CBA >0.8. A normal or increased VWF Ag together with a CBA ratio >0.7 was observed both in patients with cardiovalvular disease (n=9), gammopathy (n=2) and other disorders (n=4). However, in all patients the largest VWF multimers were decreased (n=14) or absent (n=21). In conclusion, no single test was sufficient to detect all cases of AVWS. The PFA-100® test is of limited use in this population because of its limitation in anemia or thrombocytopenia and because of its low sensitivity. A significant number of patients present with a normal or increased VWF Ag and RCo as well as a CBA ratio >0.7 emphasizing the importance of multimer analysis in all patients with suspected AVWS.
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