Multiplate whole blood impedance aggregometry: a new tool for von Willebrand disease

The diagnosis and characterization of von Willebrand disease (VWD) is a well-known challenge. Besides the clinical bleeding tendency, its diagnosis and subtyping require laboratory data, including measurement of PFA-100 closure time (CT), factor VIII:C, von Willebrand factor (VWF) antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and ristocetininduced platelet aggregation (RIPA). RIPA with low doses of ristocetin is crucial for the diagnosis of subtype 2B, and is most commonly performed with light transmission aggregometry (LTA). LTA has remained the reference standard since its introduction in 1962 by Born [1], mostly because of its validated applications, including the diagnosis of VWD. However, LTA remains a technically challenging and timeconsuming method, even with extensive experience, as it requires centrifugation steps, which can lead to artefactual platelet subpopulation selection and activation. It also requires large blood volumes, is difficult to perform in cases of thrombocytopenia, and does not take into account interactions between platelets and other blood cells. In contrast, there is less experience of determining platelet aggregation in whole blood with whole blood impedancemetry (WBI) (a method introduced by Cardinal and Flower in 1980 [2]), although it lacks some of the major drawbacks of LTA [3,4]. WBI is a fast method, and allows the omission of centrifugation steps and the performance of platelet function studies under more physiologic conditions with small sample sizes. It is based on the change of impedance proportional to the number of platelets sticking to two electrodes through which an alternating current is passed. Multiplate (for multiple electrode aggregometry ; Dynabyte, Munich, Germany) is a new generation of WBI aggregometer, using diluted blood and single-use test cells containing twin electrodes that reduce the variation in results. This analyzer has been widely used to evaluate patient responses to platelet aggregation inhibitors [5–7]. However, RIPA assessment with WBI has not been evaluated to date. Using a previously characterized VWD patient population, we aimed to compare the WBI aggregometry Multiplate analyzer and the classic LTA (ThromboAggregometer, Affibio, Nancy, France) RIPA methods. We consecutively tested 30 healthy volunteers (HVs) and 30 patients with VWD. All VWD patients had been previously characterized by extensive laboratory workup, and subtyped according to international criteria [8]. Twenty-six patients had inherited VWD: 12 had type 1, 11 had type 2A or type 2M, and 3 unrelated patients had confirmed type 2B with distinct and recognized mutations in exon 28 of the VWF gene [9]. Patient 26 was classified as type 2B without the use of LTA RL (see below) on the basis of her abnormal laboratory results, which included thrombocytopenia and the same Arg1306 fi Trp mutation [10] as her father (who exhibited an increased LTA RL result). Three patients from a single family, initially diagnosed as type 2B, had a platelet-type (PT) VWD (Gly233 fi Ser mutation in the GPIba gene [11]). One patient had acquired VWD. Table 1 shows the patients results obtained by laboratory measurements performed on the same day as their WBI. The values are indicative of the patients laboratory variables, and were not used for diagnostic purposes (for example, patient 1 had a transient thrombocytopenia and patient 24 had a severe infectious disease on the day of the testing). All HVs and patients gave their written informed consent, and were comparable according to age, sex ratio, and blood group O (data not shown). Coagulation studies were performed on citrated platelet-poor plasma obtained after centrifugation at 2500 · g for 10 min. FVIII:C and VWF:RCo were assessed with an automated BCS coagulometer (Siemens Healthcare Diagnostics, Marburg, Germany), using a one-stage clotting assay and APTT-SP HemosIL (Instrumentation Laboratory, Milan, Italy) and FVIII-deficient plasma (Diagnostica Stago, Asnieres, France), or using BC von Willebrand Reactif (Siemens). VWF:Ag was assessed with an automated STAR coagulometer and Liatest VWF:Ag (Diagnostica Correspondence: Valerie Proulle, Service Hematologie Biologique, Hopital Bicetre, 94275 Le Kremlin Bicetre Cedex, France. Tel.: +33 1 45 21 36 12; fax: +33 1 45 21 28 47. E-mail: valerie.proulle@bct.aphp.fr