TR-05528; No of Pages 5 Thrombosis Research xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Regular Article
Comparison of Von Willebrand factor (VWF) activity VWF:Ac with VWF ristocetin cofactor activity VWF:RCo Ulrich Geisen a, Barbara Zieger b, Lea Nakamura b, Andreas Weis c, Jürgen Heinz c, Jan Jacques Michiels d, Claudia Heilmann e,⁎ a
Institute of Clinical Chemistry and Laboratory Medicine, Freiburg, Germany Laboratory for Hemostaseology, Department of Pediatrics and Adolescent Medicine, Freiburg, Germany Dept. of Hematology and Oncology, University Freiburg Medical Center, Freiburg, Germany d Goodheart Institute & Foundation Rotterdam, European Bloodcoagulation Science Center, Rotterdam, The Netherlands e Department of Cardiovascular Surgery, Heart Center University of Freiburg, Freiburg, Germany b c
a r t i c l e
i n f o
Article history: Received 17 December 2013 Received in revised form 24 April 2014 Accepted 27 April 2014 Available online xxxx Keywords: Acquired Von Willebrand syndrome Ristocetin cofactor activity of Von Willebrand factor Von Willebrand factor activity Von Willebrand disease
a b s t r a c t Introduction: Ristocetin 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 Methods: We evaluated 942 samples from 432 patients and evaluated three groups in detail: normal patients (normal multimers, VWF:Ag and VWF:RCo N0.5 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. Results: VWF:Ac and VWF:RCo correlated well (Pearson´s r = 0.96, p b 0.01), so did their ratios to VWF:Ag (Pearson´s r = 0.82, p b 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 0.731.14. 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%). Conclusions: The 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. © 2014 Elsevier Ltd. All rights reserved.
Introduction Ristocetin cofactor activity of Von Willebrand factor (VWF:RCo) and the ratio to the VWF antigen (VWF:Ag) VWF:RCo/VWF:Ag are used worldwide as a routine method to estimate VWF function and as one
Abbreviations: AVWS, acquired Von Willebrand syndrome; BCS, Behring Coagulation System; FVIII:C, coagulation factor VIII; GPIb, platelet receptor glycoprotein Ib; HMW, high molecular weight; U/ml, units per milliliter; VWD, Von Willebrand disease; VWF, Von Willebrand factor; VWF:Ac, VWF activity; VWF:Ag, VWF antigen; VWF:Ac/VWF:Ag, ratio of VWF:Ac to VWF:Ag; VWF:CB, VWF collagen binding capacity; VWF:CB/VWF:Ag, ratio of VWF:CB to VWF:Ag; VWF:RCo, VWF ristocetin cofactor activity; VWF:RCo/VWF: Ag, ratio of VWF:RCo to VWF:Ag. ⁎ Corresponding author at: Department of Cardiovascular Surgery, Heart Center, University of Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany. Tel.: +49 761 270 72200; fax: +49 761 270 72201. E-mail address: [email protected] (C. Heilmann).
step to detect Von Willebrand disease (VWD) type 1 or 3 and some subtypes of VWD type 2, particularly those with loss of high molecular weight (HMW) multimers [1]. The VWF:RCo assay measures the binding of VWF to GPIb receptors of fixed platelets. However, VWF:RCo tests are prone to analytic imprecisions due to interference by hemoglobin, bilirubin and triglyerides, human anti-mouse antibodies or rheumatoid factor, and VWF:Ag excess. Varying platelet preparations and technical difficulties may contribute to changing quality. Moreover, a broad array of genetic variances and polymorphisms at the ristocetin binding site may preclude sensitivity for diagnosis of congenital VWD type 2. [1–3] Thus, the VWF:RCo activity assay shows low accuracy, especially at low VWF:Ag concentrations [4]. A new test for the VWF activity has been introduced in 2011. The assay tests binding of VWF to recombinant GPlb which in turn binds to particle-fixed anti-GPIb antibodies. [5] Due to the dissimilar principles of the two assays, different readings can be assumed. We aimed at the validation of this new parameter VWF:Ac and its ratio to VWF:Ag, VWF:Ac/VWF:Ag in
http://dx.doi.org/10.1016/j.thromres.2014.04.033 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article as: Geisen U, et al, Comparison of Von Willebrand factor (VWF) activity VWF:Ac with VWF ristocetin cofactor activity VWF: RCo, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.033
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U. Geisen et al. / Thrombosis Research xxx (2014) xxx–xxx
Table 1 Descriptive statistics of VWF associated values for all 942 plasma samples of 432 patients.
n mean standard deviation 25% percentile 75% percentile median minimum maximum 1
VWF:Ac (U/ml)
VWF:RCo (U/ml)
VWF:Ag (U/ml)
VWF:Ac/ VWF:Ag
VWF:RCo/ VWF:Ag
VWF:CB (U/ml)
VWF:CB/ VWF:Ag
942 1.70 1.16 0.85 2.28 1.43 0.05 6.60
942 1.70 1.21 0.81 2.16 1.46 0.10 6.56
942 2.33 1.63 0.96 3.21 1.96 0.05 9.42
9161 0.79 0.19 0.64 0.93 0.82 0.10 1.35
9161 0.78 0.22 0.63 0.94 0.78 0.13 1.44
471 1.58 1.34 0.70 1.90 1.14 0.01 9.88
471 0.84 0.46 0.42 1.22 0.82 0.11 3.23
not calculated for 26 data sets due to levels b0.2 U/ml for VWF:Ac, VWF:RCo and/or VWF:Ag.
comparison to VWF:RCo and VWF:RCo/VWF:Ag in routine patients and furthermore at establishing a reference range for VWF:Ac/VWF:Ag. An additional normal range was derived from healthy subjects. Materials and Methods Patients and subjects The patient data set derives of 942 plasma samples from 432 patients. Patients were recruited from the hemostaseologic outpatient clinic and the in-hospital consultant service of our University Medical Center and from the coagulation monitoring program for patients with mechanical circulatory support and heart transplantation at the Department of Cardiovascular Surgery of the Heart Center. VWF:CB was measured in 471 samples simultaneously with VWF:Ac and VWF: RCo analyses. VWF multimer analyses of 462 plasma samples from 264 patients were available. The full set (n = 942) included 258 data sets of 234 patients with normal multimers, VWF:RCo N0.5 U/ml, VWF:Ag N0.5 U/ml and clinical exclusion of VWD type 1 (normal). The reference ranges for the ratios of VWF:Ac, VWF:RCo and collagen binding activity of VWF (VWF:CB) to VWF:Ag were derived from these patients. Moreover, the full set contained 98 data sets of 62 patients with inherited Von Willebrand disease (VWD): 76 data sets of 48 patients with VWD type 1 and 22 data sets of 14 patients with other classifications. The latter were not analysed in detail with respect to the low number. Further, 326 data
A
sets of 89 patients with acquired Von Willebrand syndrome (AVWS) were identified: the causes of AVWS were mechanical circulatory support or severe aortic valve stenosis in 310 data sets of 76 patients and hematologic disorders in 16 data sets of 13 patients. Patients were classified according to the criteria of the International Society of Thrombosis and Haemostasis (ISTH) [1]. The full set comprised also 260 data sets of 47 patients who were tested for various reasons but did not belong to one of the groups described above. In addition, samples from 30 healthy volunteers (15 females, 15 males, age 43.1 ± 10.7 years) were analysed for VWF:Ac, VWF:RCo, VWF:Ag and the ratios VWF:Ac/VWF:Ag and VWF:RCo/VWF:Ag. These subjects reported no previous bleeding tendencies, and c-reactive protein was b5 mg/l. Their data were used to calculate the normal range.
Laboratory methods VWF activity (VWF:Ac, INNOVANCE VWF Ac®), ristocetin cofactor activity (VWF:RCo, BC Von Willebrand Reagent®), VWF antigen (VWF:Ag) and coagulation factor VIII (FVIII:C) (all Siemens Healthcare Diagnostics, Eschborn, Germany) were measured in sodium citrate plasma using the Behring Coagulation System XP® (BCS) according to standard protocols. Standard human plasma (Siemens Healthcare Diagnostics) was used for calibration. Measurements were performed within the routine laboratory analysis. The ratios VWF: Ac/VWF:Ag and VWF:RCo /VWF:Ag were calculated.
B 2
R =0.92
2
R =0.67
Fig. 1. Scatter plot of VWF:Ac against VWF:RCo values (A) and of the ratios VWF:Ac/VWF:Ag against VWF:RCo/VWF:Ag (B). The central lines represent the best linear fit. Outliers are marked: normal, VWD 1, VWD type 1, VWD 2B, VWD type 2B, AVWS m, acquired Von Willebrand syndrome due to mechanical circulatory support, AVWS h, acquired Von Willebrand syndrome due to hematologic disorder.
Please cite this article as: Geisen U, et al, Comparison of Von Willebrand factor (VWF) activity VWF:Ac with VWF ristocetin cofactor activity VWF: RCo, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.033
U. Geisen et al. / Thrombosis Research xxx (2014) xxx–xxx Table 2 Descriptive statistics of VWF associated values of 30 healthy subjects.
n mean standard deviation 25% percentile 75% percentile median minimum maximum calculated normal range
VWF:Ac (U/ml)
VWF:RCo (U/ml)
VWF:Ag (U/ml)
VWF:Ac/ VWF:Ag
VWF:RCo/ VWF:Ag
30 1.07 0.34 0.75 1.31 1.03 0.57 1.97 n. a.
30 1.08 0.37 0.71 1.42 1.03 0.53 1.83 n. a.
30 1.10 0.36 0.82 1.35 1.05 0.57 1.93 n. a.
30 0.98 0.09 0.93 1.04 1.00 0.76 1.13 0.8 - 1.16
30 0.98 0.12 0.92 1.07 1.00 0.61 1.2 0.74 - 1.23
n. a., not applicable.
Collagen binding capacity (VWF:CB) in plasma was determined by a non-commercial ELISA using Collagen type I (Nycomed Pharma, Unterschleissheim, Germany) and VWF:CB/VWF:Ag was calculated. VWF multimers were separated on SDS-agarose low resolution gels (1.0% agarose) and blotted on a PVDF membrane. VWF was detected using the appropriate primary and secondary antibodies (DAKO, Hamburg, Germany) and stained with 3,30-diaminobenzidin/cobalt chloride (Bio-Rad, Munich, Germany) to assess the HMW multimers. Standard human plasma (Siemens Healthcare Diagnostics) was used for control. Platelet counts were determined by employing standard laboratory procedures.
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VWF:Ac and VWF:RCo correlated well with a Pearson´s r of 0.96 (p b 0.01) (Fig. 1A). Pearson´s r of the ratios VWF:Ac/VWF:Ag and VWF:RCo/VWF:Ag was 0.82 (p b 0.01) (Fig. 1B). The VWF data of 30 healthy subjects are presented in Table 2 including VWF:Ac, VWF:RCo, VWF:Ag, the ratios VWF:Ac/VWF:Ag and VWF:RCo/ VWF:Ag and the calculated normal ranges of the ratios. Table 3 contains the same data for normal patients (n = 258) and in addition VWF:CB, VWF:CB/VWF:Ag and its calculated reference range. Comparison of the ratios VWF:Ac/VWF:Ag and VWF:RCo/VWF:Ag (Fig. 2A and B) indicate similar results with regard to classification of VWS type 1, whereas more patients with AVWS were detected by VWF:Ac/VWF:Ag compared to VWF:RCo/VWF:Ag (p = 0.012 for normal range derived from healthy subjects, p b 0.001 for reference range from normal patients) (Table 4). Positive predictive value and specificity were comparable for VWF: Ac/VWF:Ag and VWF:RCo/VWF:Ag considering the normal range of healthy subjects (93.8% versus 93.6% and 91.9% versus 92.6%, respectively). Both positive predictive value and specificity increased with regard to the reference range for normal patients (98.9% versus 97.3% and 98.8% versus 98.1%, respectively) but remained similar. In comparison, the sensitivity of VWF:Ac/VWF:Ag was higher than that of VWF:RCo/ VWF:Ag (97.5% versus 84.7%) with regard to healthy subjects. This difference became even more pronounced with 85.9% versus 55.2%, when the reference range for normal patients was applied. VWF:CB/VWF:Ag is shown for comparison with regard to its reference range (Fig. 2C). Discussion
Statistics The program PASW Statistics 21 (SPSS Inc., Chicago, IL, USA) was used for all calculations. For 26 data sets the ratios VWF:Ac/VWF:Ag and VWF:RCo/VWF:Ag were not calculated. In these samples, at least one of the three parameters VWF:Ac, VWF:RCo or VWF:Ag was at the lower detection limit. A cut-off of b 0.2 U/ml was chosen as this is the lowest calibration point of the VWF:RCo assay according to the manufacturers’ protocol and lower values are not measured but extrapolated. Therefore, the comparison to VWF:Ac and the ratios were considered unreliable in these cases. The normal range and the reference range of the ratios VWF:Ac/ VWF:Ag and VWF:RCo/VWF:Ag were calculated as mean ± 2 standard deviations (std) from healthy subjects and from normal patients, respectively. The Levene test was employed for variance analyses. The numbers of samples outside these ranges of VWF:Ac/VWF:Ag and VWF:RCo/VWF:Ag were compared by the χ2 test. Results Values of VWF:Ac, VWF:RCo, VWF:Ag, VWF:CB and the ratios VWF: Ac/VWF:Ag, VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag for all included data sets are given in Table 1.
We analysed 942 data sets of patients and 30 data sets of healthy subjects for VWF:Ac, VWF:RCo and their ratios to VWF:Ag, i. e., VWF: Ac/VWF:Ag and VWF:RCo/VWF:Ag. The background of our study is the request for prompt diagnostics for patients with bleeding complications. VWF:Ac and VWF:RCo and their ratios to VWF:Ag correlated well. The data suggest comparable performances of VWF:Ac and VWF:RCo at VWF:Ag levels above 0.2 U/ml. Due to very low values for VWF:Ac, VWF:RCo and VWF:Ag, we refrained from calculation of the ratios in some patients. In clinical practice, however, a single severely decreased parameter can already explain an acute bleeding tendency. We used a small group of 30 healthy non-hospitalized voluntary subjects to calculate normal ranges of the ratios VWF:Ac/VWF:Ag and VWF:RCo/VWF:Ag. This range could be appropriately applied to outpatients. However, our study mirrors the daily routine at a tertiary medical center. Data of the “normal” patient population was collected from routine samples at the condition of normal multimers, VWF:RCo and VWF: Ag N0.5 U/ml and clinical exclusion of VWD type 1. These patients were hospitalized for various conditions. VWF is an acute phase protein and most often not within the healthy normal range in such patients. Therefore, increased levels of VWF:Ag and VWF:RCo but a normal ratio could be expected. In our opinion, this population represents a reliable comparator group for severely ill patients with expected or existing bleeding
Table 3 Descriptive statistics of VWF associated values of 258 plasma samples of 234 normal patients.
n mean standard deviation 25% percentile 75% percentile median minimum maximum calculated reference range
VWF:Ac (U/ml)
VWF:RCo (U/ml)
VWF:Ag (U/ml)
VWF:Ac/ VWF:Ag
VWF:RCo/ VWF:Ag
VWF:CB (U/ml)
VWF:CB/ VWF:Ag
258 1.36 0.80 0.82 1.50 1.22 0.44 6.01 n. a.
258 1.37 0.79 0.81 1.59 1.24 0.50 5.67 n. a.
258 1.48 0.92 0.89 1.82 1.24 0.51 7.71 n. a.
258 0.93 0.10 0.87 1.00 0.92 0.66 1.26 0.73 - 1.14
258 0.94 0.16 0.82 1.04 0.93 0.52 1.44 0.62 - 1.25
134 1.31 0.56 0.92 1.53 1.20 0.66 3.65 n. a.
134 1.15 0.28 0.99 1.31 1.11 0.57 2.63 0.60 – 1.70
Normal patients are defined by normal multimers, VWF:RCo and VWF:Ag N0.5 U/ml and clinical exclusion of VWD type 1; n. a., not applicable.
Please cite this article as: Geisen U, et al, Comparison of Von Willebrand factor (VWF) activity VWF:Ac with VWF ristocetin cofactor activity VWF: RCo, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.033
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U. Geisen et al. / Thrombosis Research xxx (2014) xxx–xxx
A
B
n=258
n=76
n=326
n=134
n=46
n=185
n=258
n=76
n=326
C
Fig. 2. Box plots of the ratios VWF:Ac/VWF:Ag (A), VWF:RCo/VWF:Ag (B) and VWF:CB/VWF:Ag (C) for three groups of investigated patients: patients with normal multimers, VWF:Ag N0.5 U/ml, VWF:RCo N0.5 U/ml and clinical exclusion of VWD type 1 (normal) (Tables 3, 4), Von Willebrand disease VWD type 1 and acquired Von Willebrand syndrome (AVWS) (Table 4). The dashed lines delimit the normal range for healthy subjects (Table 2), the black lines mark the reference range for normal patients (Table 3). The boxes contain the middle 50% of the values (25th and 75th percentile), the median is marked. The whiskers indicate the upper and lower non-extreme values. Circles mark outliers (distance from the box between 1.5 and three-fold length of the box), asterisks mark extreme values (distance from the box more than three-fold length of the box).
complications. The lower cut-offs for VWF:RCo/VWF:Ag and VWF:CB/ VWF:Ag derived from our normal patient population are similar to previously published values [6,7]. Our data base largely on non-VWD and acquired VWS cases. They suggest that the results with the VWF:Ac assay are comparable to that of the VWF:RCo assay. The ratio VWF:Ac/VWF:Ag could detect AVWS more reliably then the standard VWF:RCo/WVF:Ag. The VWF:Ac assay
seems to be less susceptible to interference by high bilirubin, free hemoglobin or lipidaemia according to a recent publication [5]. Further, varying ranges of the ratio VWF:RCo/VWF:Ag have been linked to genetic polymorphisms [3]. In our opinion, VWF:Ac/VWF:Ag could contribute to the diagnosis of acquired Von Willebrand syndromes with loss of HMW multimers, which can be associated with diverse diseases like pronounced thrombocythemia, monoclonal gammopathies, heart
Table 4 Ratios VWF:Ac/VWF:Ag and VWF:RCo/VWF:Ag in normal patients, patients with Von Willebrand disease type 1 (VWD 1) and with acquired Von Willebrand syndrome (AVWS). normal range (healthy subjects)
normal patients (n = 258)
VWD 1 (n = 76)
AVWS (n = 326)
reference range (normal patients)
normal patients (n = 258)
VWD 1 (n = 76)
AVWS (n = 326)
VWF:Ac/VWF:Ag
b0.8 N1.16
21 (8.1%) 6 (2.3%)
6 (7.9%) 1 (1.3%)
318 (97.5%) 1 (0.03%)
b0.73 N1.14
3 (1.1%) 7 (2.6%)
0 1 (1.3%)
280 (85.9%) 1 (0.03%)
VWF:RCo/VWF:Ag
b0.74 N1.23
19 (7.4%) 8 (3.1%)
14 (18.4%) 2 (2.6%)
276 (84.7%) 0
b0.62 N1.25
5 (1.9%) 7 (2.6%)
3 (3.9%) 2 (2.6%)
180 (55.2%) 0
Normal patients are characterized by normal multimers, VWF:Ag and VWF:RCo N0.5 U/ml and clinical exclusion of VWD type 1. VWD 1, Von Willebrand disease type 1, AVWS, acquired Von Willebrand syndrome, n, number of data sets (%), n. c., not calculated; differences to 100% due to rounding.
Please cite this article as: Geisen U, et al, Comparison of Von Willebrand factor (VWF) activity VWF:Ac with VWF ristocetin cofactor activity VWF: RCo, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.033
U. Geisen et al. / Thrombosis Research xxx (2014) xxx–xxx
valve defects, or mechanical circulatory support [8–11]. In-depth analysis including more patients is desirable. VWD type 1 is characterized by partial quantitative defects [12]. As expected, the majority of VWF:RCo/VWF:Ag and of VWF:Ac/VWF:Ag values in our study were within the reference range. As a limitation, analyses of collagen binding of VWF were done in all patients for diagnostic purposes, but not always at the same time point as VWF:Ac and VWF:RCo measurement. Therefore, n’s are smaller for VWF:CB/VWF:Ag. According to our data, VWF:Ac could replace VWF:RCo for routine screening for AVWS, especially when prompt evaluation is required. However, a larger study employing more cases of congenital VWD is required to confirm the usefulness of the parameter more broadly.
Conflict of interest statement Ulrich Geisen discloses research funding by competing company (Roche Diagnostics GmbH, Germany, and Roche Diagnostics International AG, Switzerland). All other authors have no conflict of interests to disclose.
Acknowledgements We are indebted to Doris Rockus, Sandra Knackstedt and Marcus Umhau, MD, for expert technical support and to Itumeleng Taunyane for editing.
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References [1] Sadler JE, Budde U, Eikenboom JC, Favaloro EJ, Hill FG, Holmberg L, et al. Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor. J Thromb Haemost 2006;4:2103–14. [2] Gadisseur A, Hermans C, Berneman Z, Schroyens W, Deckmyn H, Michiels JJ. Laboratory diagnosis and molecular classification of von Willebrand disease. Acta Haematol 2009;121:71–84. [3] Flood VH, Gill JC, Morateck PA, Christopherson PA, Friedman KD, Haberichter SL, et al. Common VWF exon 28 polymorphisms in African Americans affecting the VWF activity assay by ristocetin cofactor. Blood 2010;116:280–6. [4] Cunningham MT, Brandt JT, Chandler WL, Eby CS, Hayes TE, Krishnan J, et al. Quality assurance in hemostasis: the perspective from the College of American Pathologists proficiency testing program. Semin Thromb Hemost 2007;33:250–8. [5] Lawrie AS, Stufano F, Canciani MT, Mackie IJ, Machin SJ, Peyvandi F. A comparative evaluation of a new automated assay for von Willebrand factor activity. Haemophilia 2013;19:338–42. [6] Adcock DM, Bethel M, Valcour A. Diagnosing von Willebrand disease: a large reference laboratory's perspective. Semin Thromb Hemost 2006;32:472–9. [7] Flood VH, Gill JC, Christopherson PA, Wren JS, Friedman KD, Haberichter SL, et al. Comparison of type I, type III and type VI collagen binding assays in diagnosis of von Willebrand disease. J Thromb Haemost 2012;10:1425–32. [8] Michiels JJ, Budde U, van der Planken M, van Vliet HH, Schroyens W, Berneman Z. Acquired von Willebrand syndromes: clinical features, aetiology, pathophysiology, classification and management. Best Pract Res Clin Haematol 2001;14:401–36. [9] Tiede A. Diagnosis and treatment of acquired von Willebrand syndrome. Thromb Res 2012;130(Suppl. 2):S2–6. [10] Heilmann C, Geisen U, Beyersdorf F, Nakamura L, Benk C, Berchtold-Herz M, et al. Acquired von Willebrand syndrome in patients with ventricular assist device or total artificial heart. Thromb Haemost 2010;103:962–7. [11] Heilmann C, Geisen U, Beyersdorf F, Nakamura L, Benk C, Trummer G, et al. Acquired von Willebrand syndrome in patients with extracorporeal life support (ECLS). Intensive Care Med 2012;38:62–8. [12] Branchford BR, Di Paola J. Making a diagnosis of VWD. Hematol Am Soc Hematol Educ Program 2012;2012:161–7.
Please cite this article as: Geisen U, et al, Comparison of Von Willebrand factor (VWF) activity VWF:Ac with VWF ristocetin cofactor activity VWF: RCo, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.033
Contents lists available at ScienceDirect
Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
Regular Article
Comparison of Von Willebrand factor (VWF) activity VWF:Ac with VWF ristocetin cofactor activity VWF:RCo Ulrich Geisen a, Barbara Zieger b, Lea Nakamura b, Andreas Weis c, Jürgen Heinz c, Jan Jacques Michiels d, Claudia Heilmann e,⁎ a
Institute of Clinical Chemistry and Laboratory Medicine, Freiburg, Germany Laboratory for Hemostaseology, Department of Pediatrics and Adolescent Medicine, Freiburg, Germany Dept. of Hematology and Oncology, University Freiburg Medical Center, Freiburg, Germany d Goodheart Institute & Foundation Rotterdam, European Bloodcoagulation Science Center, Rotterdam, The Netherlands e Department of Cardiovascular Surgery, Heart Center University of Freiburg, Freiburg, Germany b c
a r t i c l e
i n f o
Article history: Received 17 December 2013 Received in revised form 24 April 2014 Accepted 27 April 2014 Available online xxxx Keywords: Acquired Von Willebrand syndrome Ristocetin cofactor activity of Von Willebrand factor Von Willebrand factor activity Von Willebrand disease
a b s t r a c t Introduction: Ristocetin 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 Methods: We evaluated 942 samples from 432 patients and evaluated three groups in detail: normal patients (normal multimers, VWF:Ag and VWF:RCo N0.5 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. Results: VWF:Ac and VWF:RCo correlated well (Pearson´s r = 0.96, p b 0.01), so did their ratios to VWF:Ag (Pearson´s r = 0.82, p b 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 0.731.14. 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%). Conclusions: The 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. © 2014 Elsevier Ltd. All rights reserved.
Introduction Ristocetin cofactor activity of Von Willebrand factor (VWF:RCo) and the ratio to the VWF antigen (VWF:Ag) VWF:RCo/VWF:Ag are used worldwide as a routine method to estimate VWF function and as one
Abbreviations: AVWS, acquired Von Willebrand syndrome; BCS, Behring Coagulation System; FVIII:C, coagulation factor VIII; GPIb, platelet receptor glycoprotein Ib; HMW, high molecular weight; U/ml, units per milliliter; VWD, Von Willebrand disease; VWF, Von Willebrand factor; VWF:Ac, VWF activity; VWF:Ag, VWF antigen; VWF:Ac/VWF:Ag, ratio of VWF:Ac to VWF:Ag; VWF:CB, VWF collagen binding capacity; VWF:CB/VWF:Ag, ratio of VWF:CB to VWF:Ag; VWF:RCo, VWF ristocetin cofactor activity; VWF:RCo/VWF: Ag, ratio of VWF:RCo to VWF:Ag. ⁎ Corresponding author at: Department of Cardiovascular Surgery, Heart Center, University of Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany. Tel.: +49 761 270 72200; fax: +49 761 270 72201. E-mail address: [email protected] (C. Heilmann).
step to detect Von Willebrand disease (VWD) type 1 or 3 and some subtypes of VWD type 2, particularly those with loss of high molecular weight (HMW) multimers [1]. The VWF:RCo assay measures the binding of VWF to GPIb receptors of fixed platelets. However, VWF:RCo tests are prone to analytic imprecisions due to interference by hemoglobin, bilirubin and triglyerides, human anti-mouse antibodies or rheumatoid factor, and VWF:Ag excess. Varying platelet preparations and technical difficulties may contribute to changing quality. Moreover, a broad array of genetic variances and polymorphisms at the ristocetin binding site may preclude sensitivity for diagnosis of congenital VWD type 2. [1–3] Thus, the VWF:RCo activity assay shows low accuracy, especially at low VWF:Ag concentrations [4]. A new test for the VWF activity has been introduced in 2011. The assay tests binding of VWF to recombinant GPlb which in turn binds to particle-fixed anti-GPIb antibodies. [5] Due to the dissimilar principles of the two assays, different readings can be assumed. We aimed at the validation of this new parameter VWF:Ac and its ratio to VWF:Ag, VWF:Ac/VWF:Ag in
http://dx.doi.org/10.1016/j.thromres.2014.04.033 0049-3848/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article as: Geisen U, et al, Comparison of Von Willebrand factor (VWF) activity VWF:Ac with VWF ristocetin cofactor activity VWF: RCo, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.033
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U. Geisen et al. / Thrombosis Research xxx (2014) xxx–xxx
Table 1 Descriptive statistics of VWF associated values for all 942 plasma samples of 432 patients.
n mean standard deviation 25% percentile 75% percentile median minimum maximum 1
VWF:Ac (U/ml)
VWF:RCo (U/ml)
VWF:Ag (U/ml)
VWF:Ac/ VWF:Ag
VWF:RCo/ VWF:Ag
VWF:CB (U/ml)
VWF:CB/ VWF:Ag
942 1.70 1.16 0.85 2.28 1.43 0.05 6.60
942 1.70 1.21 0.81 2.16 1.46 0.10 6.56
942 2.33 1.63 0.96 3.21 1.96 0.05 9.42
9161 0.79 0.19 0.64 0.93 0.82 0.10 1.35
9161 0.78 0.22 0.63 0.94 0.78 0.13 1.44
471 1.58 1.34 0.70 1.90 1.14 0.01 9.88
471 0.84 0.46 0.42 1.22 0.82 0.11 3.23
not calculated for 26 data sets due to levels b0.2 U/ml for VWF:Ac, VWF:RCo and/or VWF:Ag.
comparison to VWF:RCo and VWF:RCo/VWF:Ag in routine patients and furthermore at establishing a reference range for VWF:Ac/VWF:Ag. An additional normal range was derived from healthy subjects. Materials and Methods Patients and subjects The patient data set derives of 942 plasma samples from 432 patients. Patients were recruited from the hemostaseologic outpatient clinic and the in-hospital consultant service of our University Medical Center and from the coagulation monitoring program for patients with mechanical circulatory support and heart transplantation at the Department of Cardiovascular Surgery of the Heart Center. VWF:CB was measured in 471 samples simultaneously with VWF:Ac and VWF: RCo analyses. VWF multimer analyses of 462 plasma samples from 264 patients were available. The full set (n = 942) included 258 data sets of 234 patients with normal multimers, VWF:RCo N0.5 U/ml, VWF:Ag N0.5 U/ml and clinical exclusion of VWD type 1 (normal). The reference ranges for the ratios of VWF:Ac, VWF:RCo and collagen binding activity of VWF (VWF:CB) to VWF:Ag were derived from these patients. Moreover, the full set contained 98 data sets of 62 patients with inherited Von Willebrand disease (VWD): 76 data sets of 48 patients with VWD type 1 and 22 data sets of 14 patients with other classifications. The latter were not analysed in detail with respect to the low number. Further, 326 data
A
sets of 89 patients with acquired Von Willebrand syndrome (AVWS) were identified: the causes of AVWS were mechanical circulatory support or severe aortic valve stenosis in 310 data sets of 76 patients and hematologic disorders in 16 data sets of 13 patients. Patients were classified according to the criteria of the International Society of Thrombosis and Haemostasis (ISTH) [1]. The full set comprised also 260 data sets of 47 patients who were tested for various reasons but did not belong to one of the groups described above. In addition, samples from 30 healthy volunteers (15 females, 15 males, age 43.1 ± 10.7 years) were analysed for VWF:Ac, VWF:RCo, VWF:Ag and the ratios VWF:Ac/VWF:Ag and VWF:RCo/VWF:Ag. These subjects reported no previous bleeding tendencies, and c-reactive protein was b5 mg/l. Their data were used to calculate the normal range.
Laboratory methods VWF activity (VWF:Ac, INNOVANCE VWF Ac®), ristocetin cofactor activity (VWF:RCo, BC Von Willebrand Reagent®), VWF antigen (VWF:Ag) and coagulation factor VIII (FVIII:C) (all Siemens Healthcare Diagnostics, Eschborn, Germany) were measured in sodium citrate plasma using the Behring Coagulation System XP® (BCS) according to standard protocols. Standard human plasma (Siemens Healthcare Diagnostics) was used for calibration. Measurements were performed within the routine laboratory analysis. The ratios VWF: Ac/VWF:Ag and VWF:RCo /VWF:Ag were calculated.
B 2
R =0.92
2
R =0.67
Fig. 1. Scatter plot of VWF:Ac against VWF:RCo values (A) and of the ratios VWF:Ac/VWF:Ag against VWF:RCo/VWF:Ag (B). The central lines represent the best linear fit. Outliers are marked: normal, VWD 1, VWD type 1, VWD 2B, VWD type 2B, AVWS m, acquired Von Willebrand syndrome due to mechanical circulatory support, AVWS h, acquired Von Willebrand syndrome due to hematologic disorder.
Please cite this article as: Geisen U, et al, Comparison of Von Willebrand factor (VWF) activity VWF:Ac with VWF ristocetin cofactor activity VWF: RCo, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.033
U. Geisen et al. / Thrombosis Research xxx (2014) xxx–xxx Table 2 Descriptive statistics of VWF associated values of 30 healthy subjects.
n mean standard deviation 25% percentile 75% percentile median minimum maximum calculated normal range
VWF:Ac (U/ml)
VWF:RCo (U/ml)
VWF:Ag (U/ml)
VWF:Ac/ VWF:Ag
VWF:RCo/ VWF:Ag
30 1.07 0.34 0.75 1.31 1.03 0.57 1.97 n. a.
30 1.08 0.37 0.71 1.42 1.03 0.53 1.83 n. a.
30 1.10 0.36 0.82 1.35 1.05 0.57 1.93 n. a.
30 0.98 0.09 0.93 1.04 1.00 0.76 1.13 0.8 - 1.16
30 0.98 0.12 0.92 1.07 1.00 0.61 1.2 0.74 - 1.23
n. a., not applicable.
Collagen binding capacity (VWF:CB) in plasma was determined by a non-commercial ELISA using Collagen type I (Nycomed Pharma, Unterschleissheim, Germany) and VWF:CB/VWF:Ag was calculated. VWF multimers were separated on SDS-agarose low resolution gels (1.0% agarose) and blotted on a PVDF membrane. VWF was detected using the appropriate primary and secondary antibodies (DAKO, Hamburg, Germany) and stained with 3,30-diaminobenzidin/cobalt chloride (Bio-Rad, Munich, Germany) to assess the HMW multimers. Standard human plasma (Siemens Healthcare Diagnostics) was used for control. Platelet counts were determined by employing standard laboratory procedures.
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VWF:Ac and VWF:RCo correlated well with a Pearson´s r of 0.96 (p b 0.01) (Fig. 1A). Pearson´s r of the ratios VWF:Ac/VWF:Ag and VWF:RCo/VWF:Ag was 0.82 (p b 0.01) (Fig. 1B). The VWF data of 30 healthy subjects are presented in Table 2 including VWF:Ac, VWF:RCo, VWF:Ag, the ratios VWF:Ac/VWF:Ag and VWF:RCo/ VWF:Ag and the calculated normal ranges of the ratios. Table 3 contains the same data for normal patients (n = 258) and in addition VWF:CB, VWF:CB/VWF:Ag and its calculated reference range. Comparison of the ratios VWF:Ac/VWF:Ag and VWF:RCo/VWF:Ag (Fig. 2A and B) indicate similar results with regard to classification of VWS type 1, whereas more patients with AVWS were detected by VWF:Ac/VWF:Ag compared to VWF:RCo/VWF:Ag (p = 0.012 for normal range derived from healthy subjects, p b 0.001 for reference range from normal patients) (Table 4). Positive predictive value and specificity were comparable for VWF: Ac/VWF:Ag and VWF:RCo/VWF:Ag considering the normal range of healthy subjects (93.8% versus 93.6% and 91.9% versus 92.6%, respectively). Both positive predictive value and specificity increased with regard to the reference range for normal patients (98.9% versus 97.3% and 98.8% versus 98.1%, respectively) but remained similar. In comparison, the sensitivity of VWF:Ac/VWF:Ag was higher than that of VWF:RCo/ VWF:Ag (97.5% versus 84.7%) with regard to healthy subjects. This difference became even more pronounced with 85.9% versus 55.2%, when the reference range for normal patients was applied. VWF:CB/VWF:Ag is shown for comparison with regard to its reference range (Fig. 2C). Discussion
Statistics The program PASW Statistics 21 (SPSS Inc., Chicago, IL, USA) was used for all calculations. For 26 data sets the ratios VWF:Ac/VWF:Ag and VWF:RCo/VWF:Ag were not calculated. In these samples, at least one of the three parameters VWF:Ac, VWF:RCo or VWF:Ag was at the lower detection limit. A cut-off of b 0.2 U/ml was chosen as this is the lowest calibration point of the VWF:RCo assay according to the manufacturers’ protocol and lower values are not measured but extrapolated. Therefore, the comparison to VWF:Ac and the ratios were considered unreliable in these cases. The normal range and the reference range of the ratios VWF:Ac/ VWF:Ag and VWF:RCo/VWF:Ag were calculated as mean ± 2 standard deviations (std) from healthy subjects and from normal patients, respectively. The Levene test was employed for variance analyses. The numbers of samples outside these ranges of VWF:Ac/VWF:Ag and VWF:RCo/VWF:Ag were compared by the χ2 test. Results Values of VWF:Ac, VWF:RCo, VWF:Ag, VWF:CB and the ratios VWF: Ac/VWF:Ag, VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag for all included data sets are given in Table 1.
We analysed 942 data sets of patients and 30 data sets of healthy subjects for VWF:Ac, VWF:RCo and their ratios to VWF:Ag, i. e., VWF: Ac/VWF:Ag and VWF:RCo/VWF:Ag. The background of our study is the request for prompt diagnostics for patients with bleeding complications. VWF:Ac and VWF:RCo and their ratios to VWF:Ag correlated well. The data suggest comparable performances of VWF:Ac and VWF:RCo at VWF:Ag levels above 0.2 U/ml. Due to very low values for VWF:Ac, VWF:RCo and VWF:Ag, we refrained from calculation of the ratios in some patients. In clinical practice, however, a single severely decreased parameter can already explain an acute bleeding tendency. We used a small group of 30 healthy non-hospitalized voluntary subjects to calculate normal ranges of the ratios VWF:Ac/VWF:Ag and VWF:RCo/VWF:Ag. This range could be appropriately applied to outpatients. However, our study mirrors the daily routine at a tertiary medical center. Data of the “normal” patient population was collected from routine samples at the condition of normal multimers, VWF:RCo and VWF: Ag N0.5 U/ml and clinical exclusion of VWD type 1. These patients were hospitalized for various conditions. VWF is an acute phase protein and most often not within the healthy normal range in such patients. Therefore, increased levels of VWF:Ag and VWF:RCo but a normal ratio could be expected. In our opinion, this population represents a reliable comparator group for severely ill patients with expected or existing bleeding
Table 3 Descriptive statistics of VWF associated values of 258 plasma samples of 234 normal patients.
n mean standard deviation 25% percentile 75% percentile median minimum maximum calculated reference range
VWF:Ac (U/ml)
VWF:RCo (U/ml)
VWF:Ag (U/ml)
VWF:Ac/ VWF:Ag
VWF:RCo/ VWF:Ag
VWF:CB (U/ml)
VWF:CB/ VWF:Ag
258 1.36 0.80 0.82 1.50 1.22 0.44 6.01 n. a.
258 1.37 0.79 0.81 1.59 1.24 0.50 5.67 n. a.
258 1.48 0.92 0.89 1.82 1.24 0.51 7.71 n. a.
258 0.93 0.10 0.87 1.00 0.92 0.66 1.26 0.73 - 1.14
258 0.94 0.16 0.82 1.04 0.93 0.52 1.44 0.62 - 1.25
134 1.31 0.56 0.92 1.53 1.20 0.66 3.65 n. a.
134 1.15 0.28 0.99 1.31 1.11 0.57 2.63 0.60 – 1.70
Normal patients are defined by normal multimers, VWF:RCo and VWF:Ag N0.5 U/ml and clinical exclusion of VWD type 1; n. a., not applicable.
Please cite this article as: Geisen U, et al, Comparison of Von Willebrand factor (VWF) activity VWF:Ac with VWF ristocetin cofactor activity VWF: RCo, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.033
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U. Geisen et al. / Thrombosis Research xxx (2014) xxx–xxx
A
B
n=258
n=76
n=326
n=134
n=46
n=185
n=258
n=76
n=326
C
Fig. 2. Box plots of the ratios VWF:Ac/VWF:Ag (A), VWF:RCo/VWF:Ag (B) and VWF:CB/VWF:Ag (C) for three groups of investigated patients: patients with normal multimers, VWF:Ag N0.5 U/ml, VWF:RCo N0.5 U/ml and clinical exclusion of VWD type 1 (normal) (Tables 3, 4), Von Willebrand disease VWD type 1 and acquired Von Willebrand syndrome (AVWS) (Table 4). The dashed lines delimit the normal range for healthy subjects (Table 2), the black lines mark the reference range for normal patients (Table 3). The boxes contain the middle 50% of the values (25th and 75th percentile), the median is marked. The whiskers indicate the upper and lower non-extreme values. Circles mark outliers (distance from the box between 1.5 and three-fold length of the box), asterisks mark extreme values (distance from the box more than three-fold length of the box).
complications. The lower cut-offs for VWF:RCo/VWF:Ag and VWF:CB/ VWF:Ag derived from our normal patient population are similar to previously published values [6,7]. Our data base largely on non-VWD and acquired VWS cases. They suggest that the results with the VWF:Ac assay are comparable to that of the VWF:RCo assay. The ratio VWF:Ac/VWF:Ag could detect AVWS more reliably then the standard VWF:RCo/WVF:Ag. The VWF:Ac assay
seems to be less susceptible to interference by high bilirubin, free hemoglobin or lipidaemia according to a recent publication [5]. Further, varying ranges of the ratio VWF:RCo/VWF:Ag have been linked to genetic polymorphisms [3]. In our opinion, VWF:Ac/VWF:Ag could contribute to the diagnosis of acquired Von Willebrand syndromes with loss of HMW multimers, which can be associated with diverse diseases like pronounced thrombocythemia, monoclonal gammopathies, heart
Table 4 Ratios VWF:Ac/VWF:Ag and VWF:RCo/VWF:Ag in normal patients, patients with Von Willebrand disease type 1 (VWD 1) and with acquired Von Willebrand syndrome (AVWS). normal range (healthy subjects)
normal patients (n = 258)
VWD 1 (n = 76)
AVWS (n = 326)
reference range (normal patients)
normal patients (n = 258)
VWD 1 (n = 76)
AVWS (n = 326)
VWF:Ac/VWF:Ag
b0.8 N1.16
21 (8.1%) 6 (2.3%)
6 (7.9%) 1 (1.3%)
318 (97.5%) 1 (0.03%)
b0.73 N1.14
3 (1.1%) 7 (2.6%)
0 1 (1.3%)
280 (85.9%) 1 (0.03%)
VWF:RCo/VWF:Ag
b0.74 N1.23
19 (7.4%) 8 (3.1%)
14 (18.4%) 2 (2.6%)
276 (84.7%) 0
b0.62 N1.25
5 (1.9%) 7 (2.6%)
3 (3.9%) 2 (2.6%)
180 (55.2%) 0
Normal patients are characterized by normal multimers, VWF:Ag and VWF:RCo N0.5 U/ml and clinical exclusion of VWD type 1. VWD 1, Von Willebrand disease type 1, AVWS, acquired Von Willebrand syndrome, n, number of data sets (%), n. c., not calculated; differences to 100% due to rounding.
Please cite this article as: Geisen U, et al, Comparison of Von Willebrand factor (VWF) activity VWF:Ac with VWF ristocetin cofactor activity VWF: RCo, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.033
U. Geisen et al. / Thrombosis Research xxx (2014) xxx–xxx
valve defects, or mechanical circulatory support [8–11]. In-depth analysis including more patients is desirable. VWD type 1 is characterized by partial quantitative defects [12]. As expected, the majority of VWF:RCo/VWF:Ag and of VWF:Ac/VWF:Ag values in our study were within the reference range. As a limitation, analyses of collagen binding of VWF were done in all patients for diagnostic purposes, but not always at the same time point as VWF:Ac and VWF:RCo measurement. Therefore, n’s are smaller for VWF:CB/VWF:Ag. According to our data, VWF:Ac could replace VWF:RCo for routine screening for AVWS, especially when prompt evaluation is required. However, a larger study employing more cases of congenital VWD is required to confirm the usefulness of the parameter more broadly.
Conflict of interest statement Ulrich Geisen discloses research funding by competing company (Roche Diagnostics GmbH, Germany, and Roche Diagnostics International AG, Switzerland). All other authors have no conflict of interests to disclose.
Acknowledgements We are indebted to Doris Rockus, Sandra Knackstedt and Marcus Umhau, MD, for expert technical support and to Itumeleng Taunyane for editing.
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References [1] Sadler JE, Budde U, Eikenboom JC, Favaloro EJ, Hill FG, Holmberg L, et al. Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor. J Thromb Haemost 2006;4:2103–14. [2] Gadisseur A, Hermans C, Berneman Z, Schroyens W, Deckmyn H, Michiels JJ. Laboratory diagnosis and molecular classification of von Willebrand disease. Acta Haematol 2009;121:71–84. [3] Flood VH, Gill JC, Morateck PA, Christopherson PA, Friedman KD, Haberichter SL, et al. Common VWF exon 28 polymorphisms in African Americans affecting the VWF activity assay by ristocetin cofactor. Blood 2010;116:280–6. [4] Cunningham MT, Brandt JT, Chandler WL, Eby CS, Hayes TE, Krishnan J, et al. Quality assurance in hemostasis: the perspective from the College of American Pathologists proficiency testing program. Semin Thromb Hemost 2007;33:250–8. [5] Lawrie AS, Stufano F, Canciani MT, Mackie IJ, Machin SJ, Peyvandi F. A comparative evaluation of a new automated assay for von Willebrand factor activity. Haemophilia 2013;19:338–42. [6] Adcock DM, Bethel M, Valcour A. Diagnosing von Willebrand disease: a large reference laboratory's perspective. Semin Thromb Hemost 2006;32:472–9. [7] Flood VH, Gill JC, Christopherson PA, Wren JS, Friedman KD, Haberichter SL, et al. Comparison of type I, type III and type VI collagen binding assays in diagnosis of von Willebrand disease. J Thromb Haemost 2012;10:1425–32. [8] Michiels JJ, Budde U, van der Planken M, van Vliet HH, Schroyens W, Berneman Z. Acquired von Willebrand syndromes: clinical features, aetiology, pathophysiology, classification and management. Best Pract Res Clin Haematol 2001;14:401–36. [9] Tiede A. Diagnosis and treatment of acquired von Willebrand syndrome. Thromb Res 2012;130(Suppl. 2):S2–6. [10] Heilmann C, Geisen U, Beyersdorf F, Nakamura L, Benk C, Berchtold-Herz M, et al. Acquired von Willebrand syndrome in patients with ventricular assist device or total artificial heart. Thromb Haemost 2010;103:962–7. [11] Heilmann C, Geisen U, Beyersdorf F, Nakamura L, Benk C, Trummer G, et al. Acquired von Willebrand syndrome in patients with extracorporeal life support (ECLS). Intensive Care Med 2012;38:62–8. [12] Branchford BR, Di Paola J. Making a diagnosis of VWD. Hematol Am Soc Hematol Educ Program 2012;2012:161–7.
Please cite this article as: Geisen U, et al, Comparison of Von Willebrand factor (VWF) activity VWF:Ac with VWF ristocetin cofactor activity VWF: RCo, Thromb Res (2014), http://dx.doi.org/10.1016/j.thromres.2014.04.033
