F. Stufano

Different bleeding risk in type 2A and 2M von Willebrand disease: a 2-year prospective study in 107 patients.

Summary.  Background:  Type 2A and 2M von Willebrand disease (VWD2A and VWD2M) are characterized by the presence of a dysfunctional von Willebrand factor (VWF) and a variable bleeding tendency. So far, a head‐to‐head comparison of the clinical history and bleeding risk between VWD2A and VWD2M has never been provided in a prospective manner.

A comparative evaluation of a new automated assay for von Willebrand factor activity

The ristocetin cofactor assay (VWF:RCo) is the reference method for assessing von Willebrand factor (VWF) activity in the diagnosis of von Willebrands Disease (VWD). However, the assay suffers from poor reproducibility and sensitivity at low levels of VWF and is labour intensive. We have undertaken an evaluation of a new immunoturbidimetric VWF activity (VWF:Ac) assay (INNOVANCE® VWF Ac. Siemens Healthcare Diagnostics, Marburg, Germany) relative to an established platelet‐based VWF:RCo method. Samples from 50 healthy normal subjects, 80 patients with VWD and 50 samples that exhibited ‘HIL’ (i.e. Haemolysis, Icterus or Lipaemia) were studied. VWF:Ac, VWF:RCo and VWF:Ag were performed on a CS–analyser (Sysmex UK Ltd, Milton Keynes, UK), all reagents were from Siemens Healthcare Diagnostics. The VWF:Ac assay, gave low intra‐ and inter‐assay imprecision (over a 31‐day period, n = 200 replicate readings) using commercial normal (Mean 96.2 IU dL−1, CV < 3.0%) and pathological (Mean 36.1 IU dL−1, CV < 3.5%) control plasmas. The normal and clinical samples exhibited good correlation between VWF:RCo (range 3–753 IU dL−1) and VWF:Ac (rs = 0.97, P < 0.0001), with a mean bias of 5.6 IU dL−1. Ratios of VWF:Ac and VWF:RCo to VWF:Ag in the VWD samples were comparable, although VWF:Ac had a superior lower level of detection to that of VWF:RCo (3% and 5% respectively). A subset (n = 97) of VWD and HIL samples were analysed for VWF:Ac at two different dilutions to assess the effect on relative potency, no significant difference was observed (P = 0.111). The INNOVANCE® VWF Ac assay was shown to be reliable and precise.

Predictors of von Willebrand disease diagnosis in individuals with borderline von Willebrand factor plasma levels

In individuals with borderline von Willebrand factor (VWF) plasma levels, second‐level tests are required to confirm or exclude von Willebrand disease (VWD). These tests are time‐consuming and expensive.

A synonymous (c.3390C>T) or a splice-site (c.3380-2A>G) mutation causes exon 26 skipping in four patients with von Willebrand disease (2A/IIE).

We characterized four unrelated patients with von Willebrand disease type 2A/IIE, sharing the same von Willebrand factor (VWF) in‐frame deletion (p.[P1127_G1180delinsR];[=]) resulting from exon 26 skipping (Δ26).

Evaluation of an heterogeneous group of patients with von Willebrand disease using an assay alternative to ristocetin induced platelet agglutination

Diagnosis of von Willebrand disease (VWD) type 2 usually relies on the discrepancy between the von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) and VWF antigen (VWF:Ag). Type 2B patients can be discriminated from other qualitative VWD variants by using ristocetin‐induced platelet agglutination (RIPA) test. The major limitation of RIPA is the requirement of fresh blood sample.

von Willebrand factor propeptide to antigen ratio in acquired thrombotic thrombocytopenic purpura

von Willebrand factor (VWF) is a large, adhesive, multimeric glycoprotein that plays amajor role in hemostasis bymediating platelet adhesion and aggregation at the sites of vessel wall injury and serving as the carrier of factor VIII [1]. The pre-proVWF, which is synthesized in endothelial cells and megakaryocytes, undergoes intracellular modifications, including signal peptide cleavage, C-terminal dimerization, glycosylation, sulfation, and N-terminal multimerization [2]. Proteolysis then occurs in the trans-Golgi, where the VWF propeptide (VWFpp) is cleaved, but remains stored together with mature VWF in a-granules (megakaryocytes) and Weibel–Palade bodies (endothelial cells). After the secretion of VWFpp and VWF into plasma from endothelial cells as a result of physiologic or pathologic stimuli, VWFpp dissociates from VWF [3,4]. It has been demonstrated that the molar ratio of the propeptide to mature VWF could serve as a tool with which to assess the extent of endothelial cell activation under physiologic and clinical conditions [3,5,6] and assess modified VWF clearance [4]. An increased VWFpp/VWF antigen (VWF:Ag) ratio, which is a marker for enhanced clearance of VWF, has been found in some von Willebrand disease and disseminated intravascular coagulation patients [7–9]. Thrombotic thrombocytopenic purpura (TTP) is a rare, lifethreatening disease, characterized by acute episodes of thrombocytopenia and microangiopathic hemolytic anemia [10]. It is believed that endothelial cell injury may be a triggering mechanism for the onset of thrombosis in TTP. In this regard, we herein evaluate the behavior of the VWFpp/VWF:Ag ratio in patients with acquired TTP at different stages of the disease. The study was conducted on 95 samples collected from 69 patients with acquired TTP (52 females and 17 males, median age 46 years, range 12–73 years), selected from a larger cohort of 136 patients from the Milan TTP registry (http://www. ttpdatabase.org) according to criteria described elsewhere [11]. Samples were also collected from 61 healthy controls (38 males and 23 females, median age 43 years, range 21–64 years). Patient samples were categorized as follows: 29 from patients with severe ADAMTS13 deficiency (< 10%) during their first episodeof acuteTTP; 19 frompatientswith severeADAMTS13 deficiency (< 10%) during their recurrent episode of acute TTP; 20 from patients with severe deficiency of ADAMTS13 (< 10%) during remission after an acute episode of TTP; and 27 from patients with normal plasma levels of ADAMTS13 (> 46%) during remission after an acute episode of TTP. Blood was collected in 3.2% buffered citrate solution (1 : 9, v/v) and EDTA in 5 mM disodium salt (1 : 9, v/v), centrifuged at 3000 · g for 20 min at 4 C, and aliquoted and stored at ) 80 C until assessment. All blood samples were obtained after patients had given informed consent. VWF levels (VWF:Ag) were measured with an immunoturbidometric assay (HemosIL von Willebrand factor antigen) on an ACL Top (IL Instrumentation Laboratory Company, Bedford, TX, USA) [12]. VWFpp was measured with an ELISA, using the MW1939 antibody pair anti-human VWFpp (Sanquin, Amsterdam, The Netherlands). ADAMTS13 activity was measured with the collagen-binding assay, as described previously [13]. VWF multimeric analysis was performed as described by Budde et al. [14], with some modifications. According to Budde et al. [14], the reference plasma lane was divided into small, intermediate and largermultimers of regular size, and the proportion of larger multimers in the sample was calculated by dividing the area corresponding to larger multimers by the total area of the lane. As described by Lotta et al., the ultralarge VWF (ULVWF) multimer ratio was calculated as the ratio of the proportion of large multimers in the sample to the corresponding proportion in the reference plasma within the same gel. A normal range was established by calculating the ULVWF ratio in 36 healthy subjects [15]. Laboratory results for the study groups are summarized in Table 1. There was an equal distribution ofO and non-O blood groups in acute TTP cases (36.6% vs. 36.4%), remission cases Correspondence: Flora Peyvandi, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, IRCCS Cà Granda Ospedale Maggiore Policlinico, Università degli Studi di Milano, Via Pace 9, Milan 20122, Italy. Tel.: +39 255035414; fax: +39 254100125. E-mail: flora.peyvandi@unimi.it

The ISTH Bleeding Assessment Tool and the risk of future bleeding

Essentials ISTH Bleeding Assessment Tool (ISTH‐BAT) is used to assist the diagnosis of bleeding disorders. We examined whether the ISTH‐BAT is capable of predicting the risk of future bleeding. 136 subjects were administered the ISTH‐BAT and followed for up to four years. The ISTH‐BAT score failed to predict the risk of future bleeding.

A comparative evaluation of a new fully automated assay for von Willebrand factor collagen binding activity to an established method

Laboratory diagnosis of von Willebrand disease (VWD) is made by the measurement of von Willebrand factor (VWF) protein level and its activities. Current VWF activity tests include ristocetin cofactor and collagen binding (VWF:CB) assays.

von Willebrand disease type 1 mutation p.Arg1379Cys and the variant p.Ala1377Val synergistically determine a 2M phenotype in four Italian patients

We characterized five patients affected with von Willebrand disease (VWD) carrying the p.Arg1379Cys mutation. One was diagnosed as VWD type 1 and four as type 2M. The 2M patients also have the variant p.Ala1377Val in cis with p.Arg1379Cys.

Evaluation of the Utility of von Willebrand Factor Propeptide in the Differential Diagnosis of von Willebrand Disease and Acquired von Willebrand Syndrome

&NA; An increased von Willebrand factor propeptide (VWFpp) to VWF antigen (VWF:Ag) ratio (VWFpp/VWF:Ag) indicates an enhanced clearance of VWF. This finding has been described in von Willebrand disease (VWD) and in acquired von Willebrand syndrome (AVWS). A distinction between these two diseases, one congenital and the other acquired, is primarily based on family and personal history of bleeding. However, if this information is scanty, the diagnosis might be challenging due to the lack of an effective diagnostic biomarker. In this cross‐sectional study, we assessed the ability of VWFpp/VWF:Ag for the differential diagnosis between VWD and AVWS. VWFpp/VWF:Ag was measured in a group of 153 patients (125 with VWD and 28 with AVWS). Most patients with AVWS and VWD showed an increased VWFpp/VWF:Ag, although to variable degrees. A marked increase of VWFpp/VWF:Ag was mainly associated with the diagnosis of AVWS and VWD type 1 Vicenza. A receiver operating characteristic curve was used to identify the optimal cutoff of VWFpp/VWF:Ag for discrimination of patients with a modestly increased (most VWD cases) versus those with a markedly increased clearance (AVWS and VWD type 1 Vicenza), and this cutoff was identified at the value of 3.9 (sensitivity: 0.70, specificity: 0.97). The ROC curve sorting from a logistic model containing VWFpp/VWF:Ag, age, and sex had an area under the curve (AUC) of 0.88 (95% confidence interval: 0.80‐0.95). A subsequent molecular evaluation discriminated VWD type 1 Vicenza from AVWS. In conclusion, VWFpp/VWF:Ag appears helpful to discriminate patients with a markedly increase VWF clearance (AVWS or VWD type 1 Vicenza) from those with a modestly increased clearance (most VWD patients).