Von Willebrand Factor Activity Assays Research Articles

Performance evaluation and multicentre study of a von Willebrand factor activity assay based on GPIb binding in the absence of ristocetin.

The functional activity of von Willebrand factor (VWF) is most frequently measured by using the ristocetin cofactor assay (VWF:RCo). However, the method's drawbacks include unsatisfactory precision, sensitivity and availability of automated system applications. We have developed an alternative assay (INNOVANCE VWF Ac) that is based on the binding of VWF to recombinant glycoprotein Ib (GPIb). Two gain-of-function mutations were introduced into a GPIb fragment, allowing an assay format without ristocetin. Fully automated assay applications are available for the BCS/BCS XP systems and the Sysmex CS-2000i, Sysmex CA-7000, Sysmex CA-1500 and Sysmex CA-560 systems.The INNOVANCE VWF Ac assay measuring range extends from 4 to 600% VWF for all systems except the Sysmex CA-560 system. Within-device precision values were found to be between 2 and 7%. The limit of detection was below 2.2% VWF. In a study on the BCS XP system, a total number of 580 sample results yielded a correlation to the VWF:RCo assay of r equal to 0.99 (slope = 0.96). Very similar results were observed when von Willebrand disease samples type 1, 2A, 2B, 2M, 2N and 3 were investigated with the new assay and the VWF:RCo assay. The excellent performance data and comparability to VWF:RCo, together with the ease of use, led us to the conclusion that the ristocetin cofactor assay can be replaced by the new GPIb-binding assay to reliably diagnosing patients with von Willebrand disease.

Towards improved diagnosis of von Willebrand disease: Comparative evaluations of several automated von Willebrand factor antigen and activity assays

Introductionvon Willebrand disease (VWD) is reportedly the most common bleeding disorder and arises from deficiency and/or defects of von Willebrand factor (VWF). Laboratory diagnosis and typing has important management implications and requires a wide range of tests, including VWF activity and antigen, and involves differential identification of qualitative vs quantitative defects. MethodsWe have assessed several VWF antigen and activity assays (collagen binding [VWF:CB], ristocetin cofactor [VWF:RCo] and the new Siemens INNOVANCE assay [VWF:Ac], employing latex particles and gain of function recombinant glycoprotein Ib to facilitate VWF binding and agglutination without need for ristocetin) using different instrumentation, including the new Sysmex CS-5100, with a large sample test set (n=600). We included retrospective plus prospective study designs, and also evaluated desmopressin responsiveness plus differential sensitivity to high molecular weight VWF. ResultsVWF:Ag and VWF:RCo results from different methods were respectively largely comparable, although some notable differences were evident, including one high false normal VWF:Ag value (105 U/dL) on a type 3 VWD sample, possibly due to heterophile antibody interference in the latex-based CS-5100 methodology. VWF:Ac was largely comparable to VWF:RCo, but VWF:CB showed discrepant findings to both VWF:RCo and VWF:Ac with some patients, most notably patients with type 2M VWD. Conclusions(a) VWF:Ag on different platforms are largely interchangeable, as are VWF:RCo on different platforms, except for occasional (some potentially important) differences, and manufacturer recommended methods may otherwise require some assay optimization; (b) VWF:RCo and VWF:Ac are largely interchangeable, except for occasional differences that may also relate to assay design (differing optimizations); (c) VWF:CB provides an additional activity to supplement VWF:RCo or VWF:Ac activity assays, and is not interchangeable with either.

Comparison of Von Willebrand factor (VWF) activity VWF:Ac with VWF ristocetin cofactor activity VWF:RCo

IntroductionRistocetin cofactor activity of Von Willebrand factor (VWF:RCo) and the ratio VWF:RCo to its antigen VWF:Ag are used as routine screening to estimate VWF function and to detect types of Von Willebrand disease (VWD) caused by loss of high molecular weight multimers. However, the VWF:RCo test is prone to analytic imprecisions due to various reasons. We compared an assay for VWF activity (VWF:Ac) with VWF:RCo putting emphasis on the ratios to VWF:Ag. Materials and MethodsWe evaluated 942 samples from 432 patients and evaluated three groups in detail: normal patients (normal multimers, VWF:Ag and VWF:RCo >0.5 U/ml, VWD type 1 excluded; n=258), VWD type 1 (n=76) and acquired Von Willebrand syndrome (AVWS, n=326). In addition, 30 healthy subjects were analysed. ResultsVWF:Ac and VWF:RCo correlated well (Pearson´s r=0.96, p<0.01), so did their ratios to VWF:Ag (Pearson´s r=0.82, p<0.01). We calculated the normal range of VWF:Ac/VWF:Ag for healthy subjects as 0.8-1.16. In comparison, the reference range (mean±2std) derived from normal patient samples was 0.73-1.14. The corresponding ranges for VWF:RCo/VWF:Ag came to 0.74-1.23 (healthy) and 0.62-1.25 (normal patients). The ratios showed similar results regarding VWD type 1. The sensitivity for AVWS was higher with VWF:Ac/VWF:Ag than with VWF:RCo/VWF:Ag (97.5% versus 84.7%). ConclusionsThe data suggest that the results obtained with the VWF:Ac assay are comparable to that of the VWF:RCo assay. An AVWS was more reliably detected by VWF:Ac/VWF:Ag. We assume that the VWF:Ac assay could replace VWF:RCo for routine screening for AVWS, especially when prompt evaluation is required.

Technological advances in diagnostic testing for von Willebrand disease: new approaches and challenges.

Diagnostic tests for von Willebrand disease (VWD) are important for the assessment of VWD, which is a commonly encountered bleeding disorder worldwide. Technical innovations have been applied to improve the precision and lower limit of detection of von Willebrand factor (VWF) assays, including the ristocetin cofactor activity assay (VWF:RCo) that uses the antibiotic ristocetin to induce plasma VWF binding to glycoprotein (GP) IbIXV on target platelets. VWF-collagen-binding assays, depending on the type of collagen used, can improve the detection of forms of VWD with high molecular weight VWF multimer loss, although the best method is debatable. A number of innovations have been applied to VWF:RCo (which is commonly performed on an aggregometer), including replacing the target platelets with immobilized GPIbα, and quantification by an enzyme-linked immunosorbent assay (ELISA), immunoturbidimetric, or chemiluminescent end-point. Some common polymorphisms in the VWF gene that do not cause bleeding are associated with falsely low VWF activity by ristocetin-dependent methods. To overcome the need for ristocetin, some new VWF activity assays use gain-of-function GPIbα mutants that bind VWF without the need for ristocetin, with an improved precision and lower limit of detection than measuring VWF:RCo by aggregometry. ELISA of VWF binding to mutated GPIbα shows promise as a method to identify gain-of-function defects from type 2B VWD. The performance characteristics of many new VWF activity assays suggest that the detection of VWD, and monitoring of VWD therapy, by clinical laboratories could be improved through adopting newer generation VWF assays.

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 Willebrand's 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.

Differential sensitivity of von Willebrand factor (VWF) ‘activity’ assays to large and small VWF molecular weight forms: a cross‐laboratory study comparing ristocetin cofactor, collagen‐binding and mAb‐based assays

von Willebrand disease (VWD), the most common inherited bleeding disorder, is caused by deficiencies and/or defects in von Willebrand factor (VWF). An effective diagnostic and VWD typing strategy requires plasma testing for factor VIII, and VWF antigen plus one or more VWF 'activity' assays. VWF activity is classically assessed by using VWF ristocetin cofactor activity (VWF:RCo), although VWF collagen-binding (VWF:CB) and VWF mAb-based (VWF activity [VWF:Act]) assays are used by some laboratories. To perform a cross-laboratory study to specifically evaluate these three VWF activity assays for comparative sensitivity to loss of high molecular weight (HMW) VWF, representing the form of VWF that is most functionally active and that is absent in some types of VWD, namely 2A and 2B. A set of eight samples, including six selectively representing stepwise reduction in HMW VWF, were tested by 51 different laboratories using a variety of assays. The combined data showed that the VWF:CB and VWF:RCo assays had higher sensitivity to the loss of HMW VWF than did the VWF:Act assay. Moreover, within-method analysis identified better HMW VWF sensitivity of some VWF:CB assays than of others, with all VWF:CB assays still showing better sensitivity than the VWF:Act assay. Differences were also identified between VWF:RCo methodologies on the basis of either platelet aggregometry or as performed on automated analyzers. We believe that these results have significant clinical implications for the diagnosis of VWD and monitoring of its therapy, as well as for the future diagnosis and therapy monitoring of thrombotic thrombocytopenic purpura.

Detection of Continuous Flow Left Ventricular Assist Device -Associated Acquired Von Willebrand Factor (VWF) Abnormality by An Automated Immunoturbidimetric VWF Activity Assay

Abstract▪2273▪This icon denotes a clinically relevant abstract Background:Continuous flow left ventricular assist device (CF-LVAD) recipients have high tendency of post-surgical gastrointestinal (GI) bleeding. We previously described the loss of high molecular weight VWF multimers (HMWM), due to either an accelerated clearance or enhanced cleavage of the HMWM, in most CF-LVAD recipients (Ann Thorac Surg. 90:1263–9). However, besides the tedious plasma VWF multimer analysis, neither VWF ristocetin cofactor nor collagen binding activity assay provides sufficient sensitivity for detecting such an acquired VWF abnormality (AVWA). In this study, we examined a new automated latex particle-enhanced immunoturbidimetric VWF activity assay (VWF:Lx) for its ability of detecting CF-LVAD-AVWA. We also analyzed the VWF pro-peptide (VWF:pp) to explored the potential mechanism of CF-LVAD-AVWA. Design:As part of an on-going prospective multicenter study, pre- and post-CF-LVAD implantation (7, 30 days and 5–7 months) blood samples were collected from 15 LVAD recipients (median age 52 years; 10 male and 5 female; 2008∼2009). Plasma VWF antigen (VWF:Ag), VWF:Lx activity, VWF:pp/Ag ratios were measured; and plasma VWF multimer analyses were performed on all available plasma samples. Standard statistical analyses were employed. Result:Loss of VWF HMWM was observed in 3 patients prior to CF-LVAD implantation and virtually all patients after surgery. VWF:Ag, VWF:Lx or VWF:pp/Ag ratios of the pre- and post-implantation samples were not significantly different (P> 0.1). However, VWF:Lx/Ag ratios of the post-implantation samples were significantly decreased (P<0.05). At a cut off of 0.8, the VWF:Lx/Ag ratio has a 90% sensitivity and specificity for detecting AVWA. Conclusions:VWF:Lx/Ag ratio has excellent sensitivity and specificity in detecting CF-LVAD-AVWA, and its impact on predicting post-surgical bleeding tendency in CF-LVAD recipients is being investigated. The distinct CF-LVAD-AVWA is most likely caused by enhanced cleavage, rather than clearance, of the HMWM.Table 1:Laboratory Characteristics of VWF in Pre- and Post-CF-LVAD Implantation Patient Plasma Samples.Laboratory Results (Mean±Standard Deviation)Pre-implantation (n=15)Day 7 Post (n=13)Day 30 Post (n=13)5–7 month Post (n=4)VWF:Ag (IU/dL)318 ± 178340 ± 136263.8 ± 122.7224 ± 78VWF:Lx (U/dL)258 ± 145246 ± 87102.5 ± 64.6165 ± 44VWF:Lx/Ag Ratio0.82 ± 0.120.74 ± 0.080.75 ± 0.060.76 ± 0.12VWF:pp/Ag Ratio1.04 ± 0.361.00 ± 0.300.99 ± 0.231.02 ± 0.10Samples with HMWM Loss (Present/Total)3/1510/1312/134/4Agreement between abnormal VWF:Lx/Ag ratio (<0.8) and VWF Multimer Analysis13/1511/1312/134/4 Disclosures:No relevant conflicts of interest to declare.

Validation of an automated latex particle-enhanced immunoturbidimetric von Willebrand factor activity assay.

Laboratory diagnosis of von Willebrand disease (VWD) requires accurate measurement of plasma von Willebrand factor (VWF) activity. To evaluate laboratory characteristics, diagnostic accuracy and testing utilities of an automated latex particle-enhanced immunoturbidimetric VWF assay (VWF:Lx) based on a monoclonal antibody recognizing the VWF-platelet glycoprotein (GP) Ib binding domain. Laboratory characteristics including lower detection limit, linearity, precision, sample stability, and method comparison between VWF:Lx and VWF ristocetin cofactor activity by platelet aggregometry (VWF:RCo) were examined. To assess VWF:Lx diagnostic accuracy, 492 patient plasma samples, including 40 previously characterized VWD patient samples, were tested for VWF antigen (VWF:Ag) and VWF:RCo by either aggregometry or flow cytometry, and VWF:Lx with supplemental VWF multimer analysis when indicated. Based on results of VWF:Ag, VWF:RCo and VWF multimer analysis, and available clinical information, samples were categorized as: normal; VWD types 1, 2A/B, 2M, or severe 1 vs. 2M; or acquired VWF abnormalities (AVWA) due to subtle loss of highest molecular weight multimers. VWF:Lx had excellent laboratory characteristics and linear correlation with VWF:RCo (R(2) = 0.93). VWF:Lx accurately classified virtually all normal and VWD patient samples. Compared with VWF:RCo, VWF:Lx had superior sensitivity and specificity for distinguishing severe type 1 vs. 2M VWD and identifying AVWA. A proposed screening panel comprising VWF:Ag and VWF:Lx had 100% and 83% sensitivity for detecting VWD and AVWA, respectively. VWF:Lx has excellent laboratory characteristics and diagnostic accuracy compared with VWF:RCo, and can be used as part of an initial VWD screening panel and as a supplementary test.

Gain-of-function GPIb ELISA assay for VWF activity in the Zimmerman Program for the Molecular and Clinical Biology of VWD

Abstractvon Willebrand disease (VWD) is a common bleeding disorder, but diagnosis is sometimes challenging because of issues with the current von Willebrand factor (VWF) assays, VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), used for diagnosis. We evaluated 113 healthy controls and 164 VWD subjects enrolled in the T.S. Zimmerman Program for the Molecular and Clinical Biology of VWD for VWF:Ag, VWF:RCo, and a new enzyme-linked immunosorbent assay (ELISA)–based assay of VWF-glycoprotein Ib (GPIb) interactions using a gain-of-function GPIb construct (tGPIbα235Y;239V) as a receptor to bind its ligand VWF in an assay independent of ristocetin (VWF:IbCo ELISA). Healthy controls, type 1, 2A, 2M, and 2N subjects had VWF:RCo/VWF:Ag ratios similar to the ratio obtained with VWF:IbCo ELISA/VWF:Ag. Type 2B VWD subjects, however, had elevated VWF:IbCo ELISA/VWF:Ag ratios. Type 3 VWD subjects had undetectable (< 1.6 U/dL) VWF:IbCo ELISA values. As previously reported, VWF:RCo/VWF:Ag ratio was decreased with a common A1 domain polymorphism, D1472H, as was direct binding to ristocetin for a 1472H A1 loop construct. The VWF:IbCo ELISA, however, was not affected by D1472H. The VWF:IbCo ELISA may be useful in testing VWF binding to GPIb, discrimination of type 2 variants, and in the diagnosis of VWD as it avoids some of the pitfalls of VWF:RCo assays.

Von willebrand disease: An overview

Most commonly inherited bleeding disorder, first described in Aland Islands by Erik von Willebrand. It occurs as a result of decrease in plasma levels or defect in von Willebrand factor which is a large multimeric glycoprotein. Monomers of this glycoprotein undergo N-glycosylation to form dimers which get arranged to give multimers. Binding with plasma proteins (especially factor VIII) is the main function of von Willebrand factor. The disease is of two forms: Inherited and acquired forms. Inherited forms are of three major types. They are type 1, type 2, and type 3; in which type 2 is sub-divided into 2A, 2B, 2M, 2N. Type 1 is more prevalent than all other types. Mucocutaneous bleeding is mild in type 1 whereas it is mild to moderate in types 2A, 2B, and 2M. Type 2N has similar symptoms of haemophilia. The pathophysiology of each type depends on the qualitative or quantitative defects in von Willebrand factor. The diagnosis is based on von Willebrand factor antigen, von Willebrand factor activity assay, FVIII coagulant activity and some other additional tests. Results should be analyzed within the context of blood group. von Willebrand factor multimer analysis is essential for typing and sub typing the disease. The management of the disease involves replacement therapy, non-replacement therapy and other therapies that include antifibrinolytics and topical agents.

Common VWF exon 28 polymorphisms in African Americans affecting the VWF activity assay by ristocetin cofactor

AbstractThe diagnosis of von Willebrand disease relies on abnormalities in specific tests of von Willebrand factor (VWF), including VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo). When examining healthy controls enrolled in the T. S. Zimmerman Program for the Molecular and Clinical Biology of von Willebrand disease, we, like others, found a lower mean VWF:RCo compared with VWF:Ag in African American controls and therefore sought a genetic cause for these differences. For the African American controls, the presence of 3 exon 28 single nucleotide polymorphisms (SNPs), I1380V, N1435S, and D1472H, was associated with a significantly lower VWF:RCo/VWF:Ag ratio, whereas the presence of D1472H alone was associated with a decreased ratio in both African American and Caucasian controls. Multivariate analysis comparing race, SNP status, and VWF:RCo/VWF:Ag ratio confirmed that only the presence of D1472H was significant. No difference was seen in VWF binding to collagen, regardless of SNP status. Similarly, no difference in activity was seen using a GPIb complex-binding assay that is independent of ristocetin. Because the VWF:RCo assay depends on ristocetin binding to VWF, mutations (and polymorphisms) in VWF may affect the measurement of “VWF activity” by this assay and may not reflect a functional defect or true hemorrhagic risk.

Flow-based measurements of von Willebrand factor (VWF) function: Binding to collagen and platelet adhesion under physiological shear rate

Introduction VWF circulates in plasma as a series of heterogeneous multimers, mediating platelet tethering, translocation and finally adhesion to areas of injured endothelium under physiological high arterial blood flow. VWF-platelet binding requires conformational changes in VWF, which are induced by immobilization and shear. Because of unavailability of a simple flow-based measurement system, VWF activity assays are generally performed under static conditions. We describe an easily reproducible in vitro flow-chamber model using commercially available flow devices to examine VWF-collagen binding and VWF-mediated platelet adhesion under physiological flow conditions. Methods The collagen surface of the flow-chamber was analyzed by atomic force microscopy. Collagen-bound VWF was characterized by multimer analysis and multi labelling immunofluorescence detection of exposed GPIb binding domains. Platelet adhesion was captured by time-lapse microscopy. Results The described flow-chamber system facilitates multimer analysis of collagen-bound VWF, whereas all VWF multimers bound to collagen under physiological low to high shear rates. Multi labelling immunofluorescence detection exhibited exposed GPIb binding domains co-localized with VWF molecules. VWF-dependent platelet adhesion using time-lapse microscopy showed values comparable to experiments done with whole blood, and platelet adhesion was dependent on the VWF concentration. Conclusions The established flow-chamber model represents an easy-to-set-up and customized tool for the characterization of VWF-binding to collagen as well as the determination of VWF-dependent platelet adhesion under defined flow conditions in real-time.

Desmopressin therapy to assist the functional identification and characterisation of von Willebrand disease: Differential utility from combining two (VWF:CB and VWF:RCo) von Willebrand factor activity assays?

We performed a retrospective audit of desmopressin (DDAVP) usage to assist in the functional characterisation of von Willebrand disease (VWD). Data was evaluated for 208 patients, comprising those with VWD (Type 1 [n = 160], Type 2A [n = 19], Type 2M [n = 10]), plus 19 individuals with haemophilia or carriers of haemophilia. Laboratory testing comprised pre- and post-DDAVP evaluation of factor VIII (FVIII:C), von Willebrand factor (VWF) antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo) activity, VWF collagen binding (VWF:CB) activity, and in one laboratory an alternate VWF activity assay. In brief, combined usage of VWF:RCo and VWF:CB appears to provide improved functional characterisation and/or ‘classification’ of VWD types, in particular better differentiation of Type 2A and 2M VWD, and clearer validation of a Type 1 VWD diagnosis. Thus, (i) Type 1 VWD displayed generally good absolute and relative rises in all test parameters, although relative rises were greatest for FVIII:C and VWF:CB, and CB/Ag ratio increases overshadowed those for RCo/Ag; (ii) Type 2A VWD patients showed good absolute and relative rises in both FVIII:C and VWF:Ag, but poor absolute rises in both VWF:CB and VWF:RCo; although small rises in both CB/Ag and RCo/Ag were also observed, both ratios tended to remain below 0.7; (iii) finally, Type 2 M VWD patients generally showed good absolute and relative rises in FVIII:C, VWF:Ag and VWF:CB, but a poor absolute and relative rise in VWF:RCo; thus, there were good rises in CB/Ag ratios but little change in RCo/Ag, which tended to remain below 0.7. Future multi-centre prospective investigations are warranted to validate these findings and to investigate their therapeutic implications.

Limitations of Current Plasma VonWillebrand Factor (VWF) Activity Tests: A Comprehensive Comparison of Five VWF Activity Assays.

Accurate measurement of plasma von Willebrand factor (VWF) activity is essential for the laboratory diagnosis and treatment monitoring of von Willebrand disease (VWD).Currently available VWF activity assays include VWF ristocetin cofactor activity by manual light transmission platelet aggregometry (VWF:RCo–Agg) or flow cytometry (VWF:RCo–FL), collagen I and III binding activity (VWF:Co–I and –III) (Technozym), and platelet activity by latex agglutination (VWF:Lx) (Instrumental Laboratory). In this study we evaluated and compared the accuracy and precision of these 5 assay methods. Plasma samples from 11 normal donors and 41 patients categorized as type 1 (n=20) or type 2 (n=21) VWD based on clinical evaluation, fVIII:C activity, VWF:RCo–Agg, VWF antigen (VWF:Ag) level and plasma VWF multimer analysis by agarose gel electrophoresis were assayed for VWF activity by VWF:RCo–FL, VWF:Co–I, VWF:Co–III and VWF:Lx methods. The VWF:Ag/VWF activity ratio by VWF activity assay method was calculated for each sample. For normal donors and type I VWD patients, VWF:RCo–FL and VWF:Lx correlated well with VWF:RCo–Agg (R2=0.87, and 0.97, respectively), while VWF:Co–I and –III were lower compared to VWF:RCo-Agg. For type 2 VWD patients, different VWF:Ag/VWF activity ratio cutoffs (range 0.3–0.7) were used (Figure). Both VWF:RCo–Agg and –FL were sensitive (95%) and specific (97%) for type 2 VWD while the VWF:Lx was slightly less sensitive (81%) but was very specific (100%). VWF:Co–I and –III were the least sensitive (<90%) and specific (<90%); both methods had high false positive and negative rates for type 2 VWD. In Summary, for normals and type 1 VWD patients, VWF:RCo–FL and VWF:Lx correlate well with VWF:RCo–Agg and have similar sensitivities and specificities for type 2 VWD. VWF:Co–I and –III are unreliable for assessing plasma VWF activity.

Rapid quantitative assay of ADAMTS13 activity on an automated coagulation analyzer: Clinical applications and comparison with immunoblot method

Facile assays for ADAMTS13, the metalloprotease that is absent or impaired in thrombotic thrombocytopenic purpura, could allow for timely diagnosis and management of this potentially fatal hematologic disorder; unfortunately, available assays employ methodologies that restrict use to only a few reference laboratories, resulting in reporting delays. Measurement of ADAMTS13 on an automated analyzer could allow widespread accessibility by routine clinical laboratories. We adapted a previously published technique to demonstrate that ADAMTS13 activity could be measured on an Instrumentation Laboratory automated coagulation analyzer after plasma digestion of a commercial source of von Willebrand factor (VWF). Results were obtained using a commercially available immunoturbidimetric assay for residual VWF activity. Samples from 114 patients with suspected thrombotic microangiopathy were analyzed by both standard immunoblot and our new semiautomated method with excellent agreement, particularly at the clinically relevant low levels of ADAMTS13 activity. This new method is semiautomated and offers quantitative results within 2 hr; our modifications allow for widespread applicability on instrumentation already in use by routine clinical laboratories.

VWF Concentrates - Does Size Really Matter - Von Willebrand Factor Function under Physiological Flow Conditions.

Introduction: The Von Willebrand Factor (VWF), is a very large glycoprotein present in human plasma essential for normal thrombus formation at sites of vascular injury. Blood coagulation is initiated by exposure of vessel wall components, e.g. collagen, followed by platelet tethering, activation and adhesion leading to the formation of a stable clot. All steps of blood coagulation occur in flowing blood under various conditions depending on vessel size. It is important to assess the function of a VWF concentrate under physiologic conditions. Under static or low shear conditions, platelets can bind directly to collagen without assistance of VWF, while the VWF is essential for mediation of platelet adhesion under high flow occurring in the arterial circulation. It has been assumed that multimer size plays an important role in this binding and in subsequent platelet adhesion. In this study we evaluated the binding of VWF of different multimeric structures to collagen and determined VWF-mediated platelet binding under low to high shear rates in a flow chamber model.Methods: A flow-chamber coated with human collagen was developed to mimic physiological flow conditions. A high purity VWF/factor VIII (FVIII) concentrate (Wilate®) and two other VWF/FVIII concentrates were tested at shear rates of 400 1700 and 4000 s−1 reproducing shear rates occurring in veins, small arteries and capillaries. Collagen-bound VWF was characterized by antigen determination (VWF:Ag) and multimer (MM) analysis. Binding of labeled platelets was visualized by a fluorescence microscope and surface coverage was quantified.Results: All VWF MMs independent of MM size were found to bind to collagen even under high shear rates. The amount of collagen-bound VWF:Ag and VWF-mediated platelet adhesion at 1700 s−1 differed significantly between the tested concentrates, when equal VWF:Ag amounts were applied.Conclusion: Binding of VWF to collagen does not depend on VWF MM size in this model even when measured under high shear rates. The differences in collagen-bound VWF:Ag and VWF-mediated platelet binding do not seem to depend on the VWF MM distribution of the concentrates. Other structural features than VWF MM size may likely be caused by the differences seen in the binding levels of the different concentrates and should be further explored. Possible differences between VWF activity assays performed under static or flow conditions and their appropriate use for VWD diagnosis and quantification of in-vivo activity need to be further investigated.

Comparison of a new automated von Willebrand factor activity assay with an aggregation von Willebrand ristocetin cofactor activity assay for the diagnosis of von Willebrand disease

von Willebrand disease (VWD) is caused by quantitative and/or qualitative defects of von Willebrand factor (VWF). The HemosIL von Willebrand Factor Activity assay, a new automated immunological test to measure VWF activity, was implemented on STAC and compared with the von Willebrand ristocetin cofactor activity (VWF:RCo) aggregation method. Imprecision and dilution studies were also performed. Within-run imprecision was 17.2% and between-run imprecision was 8.3% (coefficients of variation). Dilution studies showed a linearity between 12.5 and 100%. Passing and Bablok regression comparing the HemosIL von Willebrand Factor Activity assay and the aggregation method yielded a slope of 1.25 (95% confidence interval: 1.11-1.38) and intercept of -1.40 (95% confidence interval: -8.07 to 0.00). The correlation coefficient was 0.84 (95% confidence interval: 0.78-0.89). With a cut-off value of 50% for VWF activity, the assay has a sensitivity of 94.1% and a specificity of 92.8%, compared with the VWF:RCo aggregation assay with a cut-off value of 60% producing a sensitivity of 100.0% (specificity 87.6%). With a cut-off value of 60%, the HemosIL von Willebrand Factor Activity assay on STAC is a reliable assay for VWD. VWF:RCo or other functional testing is still required to confirm the diagnosis and for further classification of VWD.