Accepted Article
Received Date : 27-Jun-2014 Accepted Date : 12-Nov-2014 Article type : Original Article - Coagulation
Predictors of von Willebrand disease diagnosis in individuals with borderline von Willebrand factor plasma levels Running head: VWD diagnosis in individuals with borderline VWF Paolo Bucciarelli,1 Simona M. Siboni,1 Francesca Stufano,1 Eugenia Biguzzi,1 Maria T.
Canciani,1 Luciano Baronciani,1 Maria T. Pagliari,1 Silvia La Marca,1 Claudia Mistretta,1 Frits R. Rosendaal,2,3 Flora Peyvandi1
1
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’
Granda Ospedale Maggiore Policlinico and University of Milan, Milan, Italy
2
Department of Clinical Epidemiology, and 3 Department of Thrombosis and Hemostasis,
Leiden University Medical Center, Leiden, The Netherlands
Correspondence to: Paolo Bucciarelli, MD Angelo Bianchi Bonomi Hemophilia and Thrombosis Center Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Via Pace, 9 - 20122 Milan (Italy) tel.: +39-02-55035274 fax: +39-02-55034439 e-mail: [email protected]
Abstract Background. 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. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jth.12799 This article is protected by copyright. All rights reserved.
Accepted Article
Aim. To assess which parameters can predict VWD diagnosis in individuals with borderline VWF levels (30 to 60 IU/dL).
Methods. 950 individuals with bleeding episodes or abnormal coagulation tests were investigated with first level tests (blood count, PT, APTT, factor VIII:C, VWF:RCo and VWF:Ag), and 93 (62 females and 31 males; median age: 28 yrs [IQR: 15-44]) had borderline VWF:RCo levels. All underwent second level investigations to confirm or exclude VWD. A multivariable logistic regression model was fitted with sex, age, bleeding score, family history, VWF:RCo and ABO blood group as predictors, and used to predict VWD diagnosis.
Results. Forty-five of the 93 individuals (48%) had VWD (84% type 1). A negative linear relationship between VWF:RCo levels and risk of VWD diagnosis was present, particularly evident with blood group non-O (adjusted odds ratio: 7.00 [95%CI: 1.48-33.11] for every 5 IU/dL decrease in VWF:RCo). The other variable clearly associated with VWD diagnosis was female sex (adjusted odds ratio: 5.76 [95%CI: 1.47-22.53]). The AUC of the full logistic model was 0.89 (95%CI: 0.82-0.95).
Conclusions. In individuals with borderline VWF, the two strongest predictors of VWD diagnosis are low VWF:RCo levels (particularly in blood group non-O) and female sex. This predictive model has a promising discriminative capability to identify patients with borderline VWF levels who are likely to have VWD.
Keywords: blood coagulation , von Willebrand disease , von Willebrand factor , ABO Blood-Group System , Bayesian Prediction
This article is protected by copyright. All rights reserved.
Accepted Article
characterize the disease (VWF collagen binding [VWF:CB], VWF multimeric analysis, ristocetin-induced platelet agglutination [RIPA], FVIII:C binding to VWF, and VWF intraplatelet analysis) are time-consuming, expensive and not easily available in all laboratories. Advance knowledge on which individual parameters can predict the diagnosis of VWD would allow performing second level tests only in a subset of individuals with borderline VWF, thus saving time and money and avoiding a useless stress of testing in some individuals. This approach could have significant implications for medical practice.
The aim of the present study was to assess which individuals with borderline VWF
plasma levels are likely to have VWD and are therefore candidate to go through more detailed examination.
Methods Study population Between 2007 and 2011, 950 individuals who attended the Angelo Bianchi Bonomi Hemophilia and Thrombosis Center in Milan for a first evaluation after a bleeding episode (n=785) or detection of a prolonged APTT (n=165) were investigated with first level tests (blood count, PT, APTT, FVIII:C, VWF ristocetin cofactor activity [VWF:RCo] and VWF antigen [VWF:Ag]). Among them, 96 (10%) were found to have borderline VWF plasma levels, defined as VWF:RCo between 30 and 60 IU/dL. One of them had mild hemophilia A (VWF:RCo=49%, FVIII:C=10% with normal test of binding to VWF), and 2 (both females with VWF:RCo levels between 30 and 40 IU/dL) had an acquired von Willebrand syndrome. These 3 patients were excluded from further analyses. Among the 70 individuals out of the remaining 93 with borderline VWF (75%), who were investigated after a bleeding episode, mucocutaneous bleeding was present in 35, while 25 patients bled after surgery and 10 after dental procedures. Ten patients experienced more than one
This article is protected by copyright. All rights reserved.
Accepted Article
characterize the disease (VWF collagen binding [VWF:CB], VWF multimeric analysis, ristocetin-induced platelet agglutination [RIPA], FVIII:C binding to VWF, and VWF intraplatelet analysis) are time-consuming, expensive and not easily available in all laboratories. Advance knowledge on which individual parameters can predict the diagnosis of VWD would allow performing second level tests only in a subset of individuals with borderline VWF, thus saving time and money and avoiding a useless stress of testing in some individuals. This approach could have significant implications for medical practice.
The aim of the present study was to assess which individuals with borderline VWF
plasma levels are likely to have VWD and are therefore candidate to go through more detailed examination.
Methods Study population Between 2007 and 2011, 950 individuals who attended the Angelo Bianchi Bonomi Hemophilia and Thrombosis Center in Milan for a first evaluation after a bleeding episode (n=785) or detection of a prolonged APTT (n=165) were investigated with first level tests (blood count, PT, APTT, FVIII:C, VWF ristocetin cofactor activity [VWF:RCo] and VWF antigen [VWF:Ag]). Among them, 96 (10%) were found to have borderline VWF plasma levels, defined as VWF:RCo between 30 and 60 IU/dL. One of them had mild hemophilia A (VWF:RCo=49%, FVIII:C=10% with normal test of binding to VWF), and 2 (both females with VWF:RCo levels between 30 and 40 IU/dL) had an acquired von Willebrand syndrome. These 3 patients were excluded from further analyses. Among the 70 individuals out of the remaining 93 with borderline VWF (75%), who were investigated after a bleeding episode, mucocutaneous bleeding was present in 35, while 25 patients bled after surgery and 10 after dental procedures. Ten patients experienced more than one
This article is protected by copyright. All rights reserved.
Accepted Article
symptom. All the 93 individuals with borderline VWF underwent the second level tests to confirm and better characterize VWD. Before blood collection, all patients were interviewed by trained doctors, and the following information was collected into a database: age, sex, personal and family history of bleeding, ABO blood group. A positive family history was defined as having at least one first- or second-degree family member with hemorrhagic diathesis. In all patients the bleeding diathesis was evaluated by means of a bleeding score (BS) previously validated in VWD patients, [9,10] calculated from a standardized questionnaire administered by a trained doctor.
The study was approved by the Hospital Institutional Review Board and all patients (or their parents) gave written informed consent to participate to the study.
Laboratory tests Blood was collected in a 3.2% buffered citrate solution (for all coagulation tests) and ethylenediaminetetraacetic acid in 5 mM disodium salt (EDTA, for VWF multimeric analysis), and centrifuged at 3,000 g for 20 minutes at room temperature. All samples were tested in the same Coagulation Laboratory at the Hemophilia and Thrombosis Center in Milan. The first step of laboratory testing consisted in the first level tests: FVIII:C, VWF:Ag and VWF:RCo. FVIII:C was measured by a one-stage assay (Factor VIII deficient-plasma, HemosIL, IL Instrumentation Laboratory Company, Bedford, USA). The measurement of VWF:Ag was performed using an in-house enzyme-linked immunosorbent assay (ELISA) using polyclonal antibodies against VWF (DAKO, Glostrup, Denmark) [11] and VWF:RCo was measured with a previously described method. [12] The coefficient of variability (CV) for the VWF:RCo test in the range between 30 and 60 UI/dl was 9.3%, as previously reported. [12] This value is consistent with our laboratory intraand inter-assay CVs. After this step, plasma samples were aliquoted and stored at –80°C.
This article is protected by copyright. All rights reserved.
Accepted Article
As a second step, some of the second level tests (VWF:CB, VWF multimeric analysis and FVIII binding to VWF) were performed on frozen samples in order to confirm or exclude VWD. VWF:CB was measured in plasma using a solution of 95% of type I and 5% of type III collagens previously diluted in Horm buffer (Horm Nycomed Austria GmbH, Linz, Austria). [11] Plasma VWF multimeric analysis was performed by sodium dodecylsulfate gel electrophoresis [13] under non-reducing conditions in low-resolution (1.2% HGT agarose) and intermediate-resolution (1.6% LGT agarose) gels. The FVIII binding activity of VWF was investigated using an ELISA method. [14] The remaining assays of the second level tests (RIPA and VWF intra-platelet analysis, both requiring the use of fresh blood samples) were carried out on a new blood sample, which was also used to repeat VWF:RCo and VWF:Ag measurements (the mean value of these 2 separate tests was used in this study). RIPA was performed in platelet-rich plasma by aggregometry and values were expressed in terms of the minimal concentration of ristocetin giving at least a 30% increase in transmission (normal reference range: 0.7 to 1.2 mg/mL ristocetin). [15] Platelet lysates were obtained using a density gradient (Histopaque 1119; Sigma-Aldrich, St Louis, MO, USA) and Triton X-100. [16] On the basis of second level tests, VWD was confirmed if at least one of these conditions was met: a) low VWF values in the intraplatelet content; b) any of the VWF:RCo to VWF:Ag, VWF:CB to VWF:Ag or FVIII:C to VWF:Ag ratios was
Received Date : 27-Jun-2014 Accepted Date : 12-Nov-2014 Article type : Original Article - Coagulation
Predictors of von Willebrand disease diagnosis in individuals with borderline von Willebrand factor plasma levels Running head: VWD diagnosis in individuals with borderline VWF Paolo Bucciarelli,1 Simona M. Siboni,1 Francesca Stufano,1 Eugenia Biguzzi,1 Maria T.
Canciani,1 Luciano Baronciani,1 Maria T. Pagliari,1 Silvia La Marca,1 Claudia Mistretta,1 Frits R. Rosendaal,2,3 Flora Peyvandi1
1
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’
Granda Ospedale Maggiore Policlinico and University of Milan, Milan, Italy
2
Department of Clinical Epidemiology, and 3 Department of Thrombosis and Hemostasis,
Leiden University Medical Center, Leiden, The Netherlands
Correspondence to: Paolo Bucciarelli, MD Angelo Bianchi Bonomi Hemophilia and Thrombosis Center Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Via Pace, 9 - 20122 Milan (Italy) tel.: +39-02-55035274 fax: +39-02-55034439 e-mail: [email protected]
Abstract Background. 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. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jth.12799 This article is protected by copyright. All rights reserved.
Accepted Article
Aim. To assess which parameters can predict VWD diagnosis in individuals with borderline VWF levels (30 to 60 IU/dL).
Methods. 950 individuals with bleeding episodes or abnormal coagulation tests were investigated with first level tests (blood count, PT, APTT, factor VIII:C, VWF:RCo and VWF:Ag), and 93 (62 females and 31 males; median age: 28 yrs [IQR: 15-44]) had borderline VWF:RCo levels. All underwent second level investigations to confirm or exclude VWD. A multivariable logistic regression model was fitted with sex, age, bleeding score, family history, VWF:RCo and ABO blood group as predictors, and used to predict VWD diagnosis.
Results. Forty-five of the 93 individuals (48%) had VWD (84% type 1). A negative linear relationship between VWF:RCo levels and risk of VWD diagnosis was present, particularly evident with blood group non-O (adjusted odds ratio: 7.00 [95%CI: 1.48-33.11] for every 5 IU/dL decrease in VWF:RCo). The other variable clearly associated with VWD diagnosis was female sex (adjusted odds ratio: 5.76 [95%CI: 1.47-22.53]). The AUC of the full logistic model was 0.89 (95%CI: 0.82-0.95).
Conclusions. In individuals with borderline VWF, the two strongest predictors of VWD diagnosis are low VWF:RCo levels (particularly in blood group non-O) and female sex. This predictive model has a promising discriminative capability to identify patients with borderline VWF levels who are likely to have VWD.
Keywords: blood coagulation , von Willebrand disease , von Willebrand factor , ABO Blood-Group System , Bayesian Prediction
This article is protected by copyright. All rights reserved.
Accepted Article
characterize the disease (VWF collagen binding [VWF:CB], VWF multimeric analysis, ristocetin-induced platelet agglutination [RIPA], FVIII:C binding to VWF, and VWF intraplatelet analysis) are time-consuming, expensive and not easily available in all laboratories. Advance knowledge on which individual parameters can predict the diagnosis of VWD would allow performing second level tests only in a subset of individuals with borderline VWF, thus saving time and money and avoiding a useless stress of testing in some individuals. This approach could have significant implications for medical practice.
The aim of the present study was to assess which individuals with borderline VWF
plasma levels are likely to have VWD and are therefore candidate to go through more detailed examination.
Methods Study population Between 2007 and 2011, 950 individuals who attended the Angelo Bianchi Bonomi Hemophilia and Thrombosis Center in Milan for a first evaluation after a bleeding episode (n=785) or detection of a prolonged APTT (n=165) were investigated with first level tests (blood count, PT, APTT, FVIII:C, VWF ristocetin cofactor activity [VWF:RCo] and VWF antigen [VWF:Ag]). Among them, 96 (10%) were found to have borderline VWF plasma levels, defined as VWF:RCo between 30 and 60 IU/dL. One of them had mild hemophilia A (VWF:RCo=49%, FVIII:C=10% with normal test of binding to VWF), and 2 (both females with VWF:RCo levels between 30 and 40 IU/dL) had an acquired von Willebrand syndrome. These 3 patients were excluded from further analyses. Among the 70 individuals out of the remaining 93 with borderline VWF (75%), who were investigated after a bleeding episode, mucocutaneous bleeding was present in 35, while 25 patients bled after surgery and 10 after dental procedures. Ten patients experienced more than one
This article is protected by copyright. All rights reserved.
Accepted Article
characterize the disease (VWF collagen binding [VWF:CB], VWF multimeric analysis, ristocetin-induced platelet agglutination [RIPA], FVIII:C binding to VWF, and VWF intraplatelet analysis) are time-consuming, expensive and not easily available in all laboratories. Advance knowledge on which individual parameters can predict the diagnosis of VWD would allow performing second level tests only in a subset of individuals with borderline VWF, thus saving time and money and avoiding a useless stress of testing in some individuals. This approach could have significant implications for medical practice.
The aim of the present study was to assess which individuals with borderline VWF
plasma levels are likely to have VWD and are therefore candidate to go through more detailed examination.
Methods Study population Between 2007 and 2011, 950 individuals who attended the Angelo Bianchi Bonomi Hemophilia and Thrombosis Center in Milan for a first evaluation after a bleeding episode (n=785) or detection of a prolonged APTT (n=165) were investigated with first level tests (blood count, PT, APTT, FVIII:C, VWF ristocetin cofactor activity [VWF:RCo] and VWF antigen [VWF:Ag]). Among them, 96 (10%) were found to have borderline VWF plasma levels, defined as VWF:RCo between 30 and 60 IU/dL. One of them had mild hemophilia A (VWF:RCo=49%, FVIII:C=10% with normal test of binding to VWF), and 2 (both females with VWF:RCo levels between 30 and 40 IU/dL) had an acquired von Willebrand syndrome. These 3 patients were excluded from further analyses. Among the 70 individuals out of the remaining 93 with borderline VWF (75%), who were investigated after a bleeding episode, mucocutaneous bleeding was present in 35, while 25 patients bled after surgery and 10 after dental procedures. Ten patients experienced more than one
This article is protected by copyright. All rights reserved.
Accepted Article
symptom. All the 93 individuals with borderline VWF underwent the second level tests to confirm and better characterize VWD. Before blood collection, all patients were interviewed by trained doctors, and the following information was collected into a database: age, sex, personal and family history of bleeding, ABO blood group. A positive family history was defined as having at least one first- or second-degree family member with hemorrhagic diathesis. In all patients the bleeding diathesis was evaluated by means of a bleeding score (BS) previously validated in VWD patients, [9,10] calculated from a standardized questionnaire administered by a trained doctor.
The study was approved by the Hospital Institutional Review Board and all patients (or their parents) gave written informed consent to participate to the study.
Laboratory tests Blood was collected in a 3.2% buffered citrate solution (for all coagulation tests) and ethylenediaminetetraacetic acid in 5 mM disodium salt (EDTA, for VWF multimeric analysis), and centrifuged at 3,000 g for 20 minutes at room temperature. All samples were tested in the same Coagulation Laboratory at the Hemophilia and Thrombosis Center in Milan. The first step of laboratory testing consisted in the first level tests: FVIII:C, VWF:Ag and VWF:RCo. FVIII:C was measured by a one-stage assay (Factor VIII deficient-plasma, HemosIL, IL Instrumentation Laboratory Company, Bedford, USA). The measurement of VWF:Ag was performed using an in-house enzyme-linked immunosorbent assay (ELISA) using polyclonal antibodies against VWF (DAKO, Glostrup, Denmark) [11] and VWF:RCo was measured with a previously described method. [12] The coefficient of variability (CV) for the VWF:RCo test in the range between 30 and 60 UI/dl was 9.3%, as previously reported. [12] This value is consistent with our laboratory intraand inter-assay CVs. After this step, plasma samples were aliquoted and stored at –80°C.
This article is protected by copyright. All rights reserved.
Accepted Article
As a second step, some of the second level tests (VWF:CB, VWF multimeric analysis and FVIII binding to VWF) were performed on frozen samples in order to confirm or exclude VWD. VWF:CB was measured in plasma using a solution of 95% of type I and 5% of type III collagens previously diluted in Horm buffer (Horm Nycomed Austria GmbH, Linz, Austria). [11] Plasma VWF multimeric analysis was performed by sodium dodecylsulfate gel electrophoresis [13] under non-reducing conditions in low-resolution (1.2% HGT agarose) and intermediate-resolution (1.6% LGT agarose) gels. The FVIII binding activity of VWF was investigated using an ELISA method. [14] The remaining assays of the second level tests (RIPA and VWF intra-platelet analysis, both requiring the use of fresh blood samples) were carried out on a new blood sample, which was also used to repeat VWF:RCo and VWF:Ag measurements (the mean value of these 2 separate tests was used in this study). RIPA was performed in platelet-rich plasma by aggregometry and values were expressed in terms of the minimal concentration of ristocetin giving at least a 30% increase in transmission (normal reference range: 0.7 to 1.2 mg/mL ristocetin). [15] Platelet lysates were obtained using a density gradient (Histopaque 1119; Sigma-Aldrich, St Louis, MO, USA) and Triton X-100. [16] On the basis of second level tests, VWD was confirmed if at least one of these conditions was met: a) low VWF values in the intraplatelet content; b) any of the VWF:RCo to VWF:Ag, VWF:CB to VWF:Ag or FVIII:C to VWF:Ag ratios was
