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Acquired von Willebrand disease: potential contribution of the von Willebrand factor collagen‐binding to the identification of functionally inhibiting auto‐antibodies to von Willebrand factor: a rebuttal

We read with particular interest the article by Coleman et al. [1] concerning the detection of auto-antibodies directed against functional domains of von Willebrand factor (VWF) in patients with acquired von Willebrand disease (AVWD) secondary to an immune pathology. Coleman et al. highlighted the poor sensitivity of VWF:ristocetin cofactor (VWF:RCo) mixing studies to detect these antibodies. They reported on one personal case and two cases from the literature [1–3]. VWF collagen-binding (VWF:CB) mixing analyses were used to diagnose the presence of VWF inhibitors in all patients. The authors speculated as to whether VWF:CB mixing studies are more sensitive and more clinically relevant to AVWD-associated bleeding complications than VWF:RCo mixing studies. We wish to report on our own experiences from six patients, all of whom were devoid of both personal and family histories of bleeding. All patients presented with a recent bleeding tendency characterized by easy bruising, recurrent epistaxis, and/or abnormal bleeding following tooth extraction. Five of the patients had a monoclonal gammopathy of IgG subclass, and the other patient had Waldenström s macroglobulinemia with monoclonal immunoglobulin of IgM subclass. All the patients had VWF:RCo values <10 IU dL and ratios of VWF:RCo to VWF:Ag that were below 0.5. Type 2 VWD was confirmed by the decrease or absence of high-molecular weight VWF multimers, as determined by electrophoretic multimer analysis. The diagnosis of AVWD was established using the recommendations of the Scientific and Standardization Committees of the International Society on Thrombosis and Haemostasis [4]. We looked for VWF inhibitors using three neutralization assays: ristocetin-induced platelet agglutination assay (RIPA), VWF:RCo, tested using lyophilized platelets (Helena Biosciences Europe, Gateshead, UK) in an APACT-4 light absorbance-based aggregometer (Greiner BioChemica, Flacht, Germany), and VWF:CB, tested using an in-house test that employed human collagen type III (Southern Biotechnology Associates Inc., Birmingham, AL, USA). The residual VWF:RCo and VWF:CB were measured after 1 h incubation at 37 C of a 1:2 mixture of normal pooled plasma with patient plasma that had previously been heated at 56 C. In both VWF:RCo and VWF:CB assays, values obtained from a 1:2 mixture of normal pooled plasma with phosphate-buffered saline were used to represent 100%. The inhibition of RIPA was tested in the same way using a 1:2 mixture of normal platelet-rich plasma with either phosphate-buffered saline or patient plasma. Plasma from 20 healthy subjects, treated and tested using the same conditions, gave VWF:RCo, VWF:CB or RIPA residual activities that ranged from 80% to 100%. For this reason, the presence of a VWF inhibitor was considered when the residual activity was below 80%. An ELISA method was performed in parallel, using polystyrene plates coated with purified human VWF (Wilfactin, LFB, Les Ulis, France), as previously reported [5]. Inhibitors were classified as weakly positive, positive or strongly positive, when the last dilution that was detected positive was between 1:10 and 1:50, 1:50 and 1:100 or 1:100 and 1:500, respectively. In our study of six patients with AVWD, we found an VWF inhibitor in five patients: three of these (patients 3, 5, and 6) were AVWF IgG-positive by ELISA and were associated with a strongly positive VWF:CB or RIPA mixing studies (Table 1). For the other two positive patients (patients 2 and 4), antibodies were detected either by ELISA (IgG subclass) or the VWF:RCo mixing method. Patient 4 Correspondence: Viviane Guerin, Laboratoire d Hématologie, Hopital Universitaire Pellegrin, Place Amelie Raba – Leon, 33076 Bordeaux, France. Tel.: +33 5 56 79 59 78; fax: +33 5 56 79 60 79. E-mail: viviane.guerin@chu-bordeaux.fr