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.

Antiplatelet therapy has been the focus of extensive clinical investigations over the last two decades. A variety of agents and regimens have been advanced for the prevention and treatment of vascular disease. Despite the proven life-saving clinical benefits of inhibiting platelets, this therapy is associated with an increased risk of bleeding. The objective of this study was to determine the risk of hemorrhage in the major classes of antiplatelet agents. Data from clinical trials published 1988-2002 in English were retrieved from MEDLINE, OVID, and CARDIOSOURCE. Only those studies in which patients had clinical follow-up for at least 1 month and in which a full description of hemorrhagic complications was reported were included. Information on sample size, study design, duration, agent, patient characteristics, and bleeding severity was independently and blindly reviewed. Data from 51 clinical trials with a total of 338,191 patients were analyzed. The antiplatelet agents were divided into 6 groups: aspirin (ASA) < 100 mg; ASA ‡ 100 mg; dipyridamole, thienopyridines; intravenous and oral GP IIb/IIIa inhibitors. The variance estimate and confidence intervals were calculated for each treatment assignment. Low-dose aspirin and dipyridamole therapy were associated with the lowest risk of bleeding (3.6% and 6.7%, respectively). The highest rate of bleeding complications (44.6%) was associated with the GP IIa/IIIb inhibitors. Despite substantial differences in the reporting patterns of bleeding complications, low-dose ASA and dipyridamole therapy were associated with the lowest risk. Surprisingly, doses of ASA ‡ 100 mg caused a relatively high hemorrhagic event rate, which was comparable to that of ADP-receptor blockers. These findings should be considered when using combination antiplatelet and/or anticoagulant therapy with conventional doses of ASA. Am.

Elevation of serum lipoprotein (a) (Lp[a]) is a known risk factor predisposing to cardiovascular and cerebrovascular disease. However, little is known about the role of increased Lp(a) in venous thromboembolism (VTE). This study evaluated the role of Lp(a) among a panel of established hereditary thrombogenic defects in patients with VTE. A total of 685 consecutive patients with at least one episode of VTE and 266 sex- and age-matched healthy controls were screened with regard to activated protein C resistance, protein C, protein S, and antithrombin deficiency, elevated serum levels of Lp(a), and the factor V G1691A, MTHFR C677T, and prothrombin G20210A mutations. Elevated Lp(a) levels above 30 mg/dL were found in 20% of all patients, as compared to 7% among healthy controls (P &lt; .001, odds ratio [OR] 3.2, 95% confidence interval [CI], 1.9-5.3). The coexistence of FV G1691A and elevated Lp(a) was significantly more prevalent among patients with VTE than in the control group (7% versus 0.8%; P &lt; .001, OR 9.8, 95% CI, 2.4-40.7). No other established prothrombotic risk factor was found to be significantly combined with increased Lp(a). These data suggest that Lp(a) concentrations greater than 30 mg/dL are a frequent and independent risk factor for VTE. Furthermore, elevated Lp(a) levels might contribute to the penetrance of thromboembolic disease in subjects being affected by other prothrombotic defects, such as FV G1691A mutation.

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The amount of residual F8 (FVIII:C) determines the clinical severity of hemophilia A. Recently, we showed that the mutation detection rate in severely affected male patients (FVIII:C<1% of normal) is virtually 100% when testing for the common intron 22-/intron 1- inversions and big deletions, followed by genomic sequencing of the F8 gene. Here we report on the spectrum of mutations and their distribution throughout the F8 gene sequence in 135 patients with moderate (n=23) or mild (n=112) hemophilia A. In contrast to the severe form of the disorder, analysis on the genomic level failed to detect the molecular defect in approximately 4% of the moderately and in approximately 12% of the mildly affected patients. A total of 36 of the mutations identified in this study are novel. The vast majority of the detected changes were missense. The newly detected amino acid substitutions were scored for potential distant or local conformational changes and influence on molecular stability for every single F8 domain with available structures, using homology modeling. Two molecular changes in the promoter region of the factor VIII gene (c.-112G>A and -219C>T), affecting the core segment (minimal promoter) were detected in two patients with mild hemophilia A. To our knowledge this is the first report on promoter mutations in the F8 gene.

Communicated by Arupa GangulyThe amount of residual F8 (FVIII:C) determines the clinical severity of hemophilia A. Recently, we showed that the mutation detection rate in severely affected male patients (FVIII:Co1% of normal) is virtually 100% when testing for the common intron 22-/intron 1-inversions and big deletions, followed by genomic sequencing of the F8 gene. Here we report on the spectrum of mutations and their distribution throughout the F8 gene sequence in 135 patients with moderate (n 5 23) or mild (n 5 112) hemophilia A. In contrast to the severe form of the disorder, analysis on the genomic level failed to detect the molecular defect in 4% of the moderately and in 12% of the mildly affected patients. A total of 36 of the mutations identified in this study are novel. The vast majority of the detected changes were missense. The newly detected amino acid substitutions were scored for potential distant or local conformational changes and influence on molecular stability for every single F8 domain with available structures, using homology modeling. Two molecular changes in the promoter region of the factor VIII gene (c.-112G4A and -219C4T), affecting the core segment (minimal promoter) were detected in two patients with mild hemophilia A. To our knowledge this is the first report on promoter mutations in the F8 gene. Hum Mutat 28(1), [54][55][56][57][58][59][60] 2007.

Platelet satellitism surrounding polymorphonuclear neutrophils has been observed almost exclusively in EDTA-treated blood at room temperature. The mechanism underlying this phenomenon is not understood fully. We report a case of platelet rosetting around atypical lymphocytes in peripheral blood smears made from EDTA-treated and untreated blood. Flow cytometry of the peripheral blood sample and immunohistochemical stains of the subsequent bone marrow biopsy specimen revealed a monoclonal B-cell population positive for CD5, CD20, and cyclin D1 and negative for CD3 and CD23; cytogenetic findings revealed a complex karyotype that included t(11;14). These findings were consistent with mantle cell lymphoma. To our knowledge, the finding of platelet satellitism involving mantle cell lymphoma cells in peripheral blood has not been reported previously.

Consent regarding the correct processing and storage of blood microparticles is lacking and different protocols for the freeze-thaw cycle exist. Therefore, three different thawing procedures were evaluated regarding their influence on recovery and composition of microparticles. Microparticles were prepared by TRAP-6 or A23187 stimulation of platelet-rich plasma from smokers and nonsmokers (n = 8), from an endothelial cell line or directly obtained from platelet-free plasma of septic patients (n = 5). After snap-freezing in liquid nitrogen platelet-free samples were thawed at 37 degrees, at room temperature or on ice and staining of microparticles was carried out with Annexin V-Cy5 as well as fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labelled antibodies or isotype controls. Microparticle concentrations were determined by means of Trucount tubes. Recovery of platelet microparticles was significantly reduced when samples were thawed on ice (P = 0.001 for all antigens) compared with the two other techniques (P = 0.6 for 37 degrees and P = 0.7 for room temperature, respectively) whereas microparticles of endothelial origin appeared to be less influenced. There was a strong trend towards altered microparticle composition as microparticle counts detected by CD41 staining showed a stronger decrease on ice than Annexin V enumeration (P = 0.07). For microparticle detection thawing of snap-frozen, platelet-free plasma samples should be carried out at room temperature or at 37 degrees C in a water bath but not on ice.